Natural killer cell protocols: cellular and molecular methods (volume 612)

ME T H O D S

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MO L E C U L A R BI O L O G Y

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For other titles published in this series, go to www.springer.com/series/7651

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Natural Killer Cell Protocols Cellular and Molecular Methods Second Edition

Edited by

Kerry S. Campbell Fox Chase Cancer Center, Philadelphia, PA, USA

Editor Kerry S. Campbell Fox Chase Cancer Center Institute for Cancer Research 333 Cottman Avenue Philadelphia PA 19111-2497 USA [email protected]

ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-60761-361-9 e-ISBN 978-1-60761-362-6 DOI 10.1007/978-1-60761-362-6 Library of Congress Control Number: 2009939929 © Humana Press, a part of Springer Science+Business Media, LLC 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper springer.com

Preface It is becoming increasingly clear that natural killer (NK) cells are critical sentinels of the innate immune response. NK cells play important roles in protecting the body from numerous pathogens and cancer in addition to contributing to normal pregnancy and impacting the outcomes of transplantation. They have the unique capacity to detect and immediately respond to abnormal cells in the body without prior exposure. NK cell responses include the classical tumor cytolytic activity for which they were named and for the production of a number of cytokines that directly contribute to or potentiate the immune response. Although efficient tolerance mechanisms appear to prevent NK cells from causing autoimmune diseases, a great deal of genetic evidence suggests that match or mismatch of certain NK cell regulatory receptors [namely killer cell Ig-like receptors (KIR)] and their MHC class I ligands can influence a wide variety of human pathological conditions, including altering outcomes of viral infections, transplantations, pregnancies, and tumor therapies. The second edition of Natural Killer Cell Protocols provides a broad collection of some of the most important methods currently being used to study NK cells both in vitro and in vivo. The authors are international leaders in the field, who are directly using these methods to advance our understanding of this fascinating subset of lymphocytes. While the first edition provided a valuable collection of classical cellular and in vivo techniques to study NK cell functions, the chapters in the second edition focus on more recently developed methods, more refined techniques, and protocols designed to study NK cells within specialized tissue sites. These include protocols to analyze the various stages of NK cell development/maturation, to assess NK cell interactions with target cells and dendritic cells, to evaluate signal transduction by NK cell receptors, and to define KIR expression profiles by genotyping or flow cytometry. Additional chapters describe methods for the study of unique subsets of NK cells within the uterus during pregnancy and at intestinal mucosal surfaces, as well as techniques to evaluate NK cell responses to viral infections and malaria. The collection also includes specialized techniques to identify ligands for NK cell receptors, to define promoters regulating human KIR expression, to map receptors encoded within the murine NK cell gene locus that are responsible for resistance to pathogens, and to introduce cDNAs and shRNAs into NK cells using recombinant retrovirus or lentivirus. Finally, the book’s appendix provides a rich resource summarizing available reagents to study NK cells, cross-referencing KIR nomenclature, and detailing the many HLA ligands for various KIR family members. NK cells in rodents and humans are regulated by very different repertoires of receptors. Therefore, protocols from both perspectives are provided in this volume, with the species noted in the title or abstract. Furthermore, we have emphasized the “Notes” sections, which provide important details within each protocol, thereby extending the longstanding tradition of the Methods in Molecular Biology series. NK cells play unique roles in the immune response, but despite several decades of study, there is still much to learn about their functions, maturation, and regulation. The goal of Natural Killer Cell Protocols is to provide open access to important techniques

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written by key researchers in the field. I am indebted to the authors who have contributed their time and energies to provide high quality protocols. Their commitment to providing detailed descriptions of the methods was essential in making this project a success. I hope that this collection of methods will make significant contributions to your research and thereby further advance our collective understanding of these fascinating cells for many years to come. Kerry S. Campbell

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1.

2.

Purification of Human NK Cell Developmental Intermediates from Lymph Nodes and Tonsils . . . . . . . . . . . . . . . . . . . . . . . . . . Aharon G. Freud and Michael A. Caligiuri

1

In Vitro Development of Human Killer–Immunoglobulin Receptor-Positive NK Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . Frank Cichocki and Jeffrey S. Miller

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3.

Subset Analysis of Human and Mouse Mature NK Cells . . . . . . . . . . . . . Yoshihiro Hayakawa, Daniel M. Andrews, and Mark J. Smyth

27

4.

Assessing Licensing of NK Cells . . . . . . . . . . . . . . . . . . . . . . . . . . A. Helena Jonsson and Wayne M. Yokoyama

39

5.

Use of Stem Cell Radiation Chimeras to Analyze How Domains of Specific Proteins Impact on Murine NK Cell Development In Vivo . . . . . . Rebecca H. Lian and Vinay Kumar

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6.

Use of Transfected Drosophila S2 Cells to Study NK Cell Activation . . . . . . . Michael E. March, Catharina C. Gross, and Eric O. Long

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7.

Natural Killer Cell Conjugate Assay Using Two-Color Flow Cytometry . . . . . Deborah N. Burshtyn and Chelsea Davidson

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8.

Studying NK Cell/Dendritic Cell Interactions . . . . . . . . . . . . . . . . . . Mathias Lucas, Cedric Vonarbourg, Peter Aichele, and Andreas Diefenbach

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9.

Analysis of the NK Cell Immunological Synapse . . . . . . . . . . . . . . . . . 127 Keri B. Sanborn, Gregory D. Rak, Ashley N. Mentlik, Pinaki P. Banerjee, and Jordan S. Orange

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Measuring Intracellular Calcium Signaling in Murine NK Cells by Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Alexander W. MacFarlane IV, James F. Oesterling, and Kerry S. Campbell

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Intracellular Staining for Analysis of the Expression and Phosphorylation of Signal Transducers and Activators of Transcription (STATs) in NK Cells . . . . 159 Takuya Miyagi, Seung-Hwan Lee, and Christine A. Biron

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A Model System for Studying NK Cell Receptor Signaling . . . . . . . . . . . . 177 Lukasz K. Chlewicki and Vinay Kumar

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Expression of cDNAs in Human Natural Killer Cell Lines by Retroviral Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 S. M. Shahjahan Miah and Kerry S. Campbell

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Lentiviral Gene Transduction in Human and Mouse NK Cell Lines . . . . . . . 209 Ram Savan, Tim Chan, and Howard A. Young

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Introduction of shRNAs into Human NK-Like Cell Lines with Retrovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 Amanda K. Purdy and Kerry S. Campbell

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Introduction of shRNAs into Primary NK Cells with Lentivirus . . . . . . . . . 233 Sam K.P. Kung

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Methods to Identify and Characterize Different NK Cell Receptors and Their Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Dikla Lankry, Roi Gazit, and Ofer Mandelboim

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Generating NK Cell Receptor-Fc Chimera Proteins from 293T Cells and Considerations of Appropriate Glycosylation . . . . . . . . . . . . . . . . . 275 Alon Zilka, Michal Mendelson, Benyamin Rosental, Oren Hershkovitz, and Angel Porgador

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Identification of NK Cell Receptor Ligands Using a Signaling Reporter System . 285 Yoshie-Matsubayashi Iizuka, Nikunj V. Somia, and Koho Iizuka

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Determining Ligand Specificity of Ly49 Receptors . . . . . . . . . . . . . . . . 299 Kerry J. Lavender and Kevin P. Kane

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Probing the Interactions of NK Cell Receptors with Ligand Expressed in trans and cis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 Jonathan Back, L´eonardo Scarpellino, and Werner Held

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A Simple Method to Measure NK Cell Cytotoxicity In Vivo . . . . . . . . . . . 325 Aurore Saudemont, Shannon Burke, and Francesco Colucci

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Functional Analysis of Human NK Cells by Flow Cytometry . . . . . . . . . . . 335 Yenan T. Bryceson, Cyril Fauriat, Jo˜ ao M. Nunes, Stephanie M. Wood, Niklas K. Bj¨orkstr¨om, Eric O. Long, and Hans-Gustaf Ljunggren

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Analysis of the KIR Repertoire in Human NK Cells by Flow Cytometry . . . . . 353 Niklas K. Bj¨orkstr¨om, Cyril Fauriat, Yenan T. Bryceson, Johan K. Sandberg, Hans-Gustaf Ljunggren, and Karl-Johan Malmberg

25.

KIR Genotyping by Multiplex PCR-SSP . . . . . . . . . . . . . . . . . . . . . 365 Smita Kulkarni, Maureen P. Martin, and Mary Carrington

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Identification and Analysis of Novel Transcripts and Promoters in the Human Killer Cell Immunoglobulin-like Receptor (KIR) Genes Hongchuan Li, Paul W. Wright, and Stephen K. Anderson

. . . . . 377

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Use of Inbred Mouse Strains to Map Recognition Receptors of MCMV Infected Cells in the NK Cell Gene Locus . . . . . . . . . . . . . . . . . . . . 393 Nassima Fodil-Cornu, Michal Pyzik, and Silvia M. Vidal

28.

Studying NK Cell Responses to Ectromelia Virus Infections in Mice . . . . . . . 411 Min Fang and Luis Sigal

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29.

Activation of Human NK Cells by Malaria-Infected Red Blood Cells . . . . . . . 429 Amir Horowitz and Eleanor M. Riley

30.

Natural Killer Cells in Human Pregnancy . . . . . . . . . . . . . . . . . . . . . 447 Victoria Male, Anita Trundley, Lucy Gardner, Jacquie Northfield, Chiwen Chang, Richard Apps, and Ashley Moffett

31.

Analysis of Uterine Natural Killer Cells in Mice . . . . . . . . . . . . . . . . . . 465 B. Anne Croy, Jianhong Zhang, Chandrakant Tayade, Francesco Colucci, Hakim Yadi, and Aureo T. Yamada

32.

Isolation of NK Cells and NK-Like Cells from the Intestinal Lamina Propria . . . 505 Stephanie L. Sanos and Andreas Diefenbach

Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545

Contributors PETER AICHELE • Institute of Medical Microbiology & Hygiene, University of Freiburg Medical Center, Freiburg, Germany STEPHEN K. ANDERSON • Cancer and Inflammation Program, Laboratory of Experimental Immunology, SAIC-Frederick Inc., National Cancer Institute-Frederick, Frederick, MD, USA DANIEL M. ANDREWS • Peter MacCallum Cancer Centre, Parkville, Victoria, Australia RICHARD APPS • Department of Pathology, University of Cambridge, Cambridge, UK JONATHAN BACK • Ludwig Institute for Cancer Research Ltd., Lausanne Branch, and University of Lausanne, Epalinges, Switzerland PINAKI P. BANERJEE • Division of Immunology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA CHRISTINE A. BIRON • Department of Molecular Microbiology and Immunology, Division of Biology and Medicine, Brown University, Providence, RI, USA NIKLAS K. BJo¨ RKSTRo¨ M • Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden YENAN T. BRYCESON • Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden SHANNON BURKE • Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, UK DEBORAH N. BURSHTYN • Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada MICHAEL A. CALIGIURI • The Comprehensive Cancer Center and The James Cancer Hospital & Solove Research Institute, The Ohio State University, Columbus, OH, USA KERRY S. CAMPBELL • Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA, USA MARY CARRINGTON • Cancer and Inflammation Program, Laboratory of Experimental Immunology, SAIC-Frederick Inc., National Cancer Institute-Frederick, Frederick, MD, USA TIM CHAN • Cancer and Inflammation Program, Laboratory of Experimental Immunology, National Cancer Institute-Frederick, Frederick, MD, USA CHIWEN CHANG • Department of Pathology, University of Cambridge, Cambridge, UK LUKASZ K. CHLEWICKI • Department of Pathology, The University of Chicago, Chicago, IL, USA FRANK CICHOCKI • University of Minnesota Cancer Center, Minneapolis, MN, USA FRANCESCO COLUCCI • Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, UK B. ANNE CROY • Department of Anatomy and Cell Biology, Queen’s University, Kingston, ON, Canada CHELSEA DAVIDSON • Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada

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ANDREAS DIEFENBACH • Institute of Medical Microbiology & Hygiene, University of Freiburg Medical Center, Freiburg, Germany MIN FANG • Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA, USA CYRIL FAURIAT • Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden NASSIMA FODIL-CORNU • Department of Human Genetics, McGill University, Montreal, QC, Canada AHARON G. FREUD • Department of Pathology, Stanford University, Stanford, CA, USA LUCY GARDNER • Department of Pathology, University of Cambridge, Cambridge, UK ROI GAZIT • The Hebrew University – Hadassah Medical School, Jerusalem, Israel CATHARINA C. GROSS • Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, Rockville, MD, USA YOSHIHIRO HAYAKAWA • The University of Tokyo, Bunkyo-Ku, Tokyo, Japan WERNER HELD • Ludwig Institute for Cancer Research Ltd., Lausanne Branch, and University of Lausanne, Epalinges, Switzerland OREN HERSHKOVITZ • Department of Microbiology and Immunology, Ben Gurion University, Beer Sheva, Israel AMIR HOROWITZ • Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK KOHO IIZUKA • Department of Medicine and Center for Immunology, Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA YOSHIE-MATSUBAYASHI IIZUKA • Department of Medicine and Center for Immunology, Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA A. HELENA JONSSON • Medical Scientist Training Program, Rheumatology Division, Departments of Medicine, Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA KEVIN P. KANE • Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada SMITA KULKARNI • Cancer and Inflammation Program, Laboratory of Experimental Immunology, SAIC-Frederick Inc., National Cancer Institute-Frederick, Frederick, MD, USA VINAY KUMAR • Department of Pathology, University of Chicago, Chicago, IL, USA SAM K.P. KUNG • Laboratory of Innate Immunobiology, Department of Immunology, University of Manitoba, Winnipeg, MB, Canada DIKLA LANKRY • The Hebrew University – Hadassah Medical School, Jerusalem, Israel KERRY J. LAVENDER • The Edward Jenner Institute for Vaccine Research, University of Oxford, Oxford, UK SEUNG-HWAN LEE • Department of Molecular Microbiology and Immunology, Division of Biology and Medicine, Brown University, Providence, RI, USA HONGCHUAN LI • Cancer and Inflammation Program, Laboratory of Experimental Immunology, SAIC-Frederick Inc., National Cancer Institute-Frederick, Frederick, MD, USA REBECCA H. LIAN • Department of Pathology, University of Chicago, Chicago, IL, USA HANS-GUSTAF LJUNGGREN • Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden ERIC O. LONG • Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA

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MATHIAS LUCAS • Institute of Medical Microbiology & Hygiene, University of Freiburg Medical Center, Freiburg, Germany ALEXANDER W. MACFARLANE IV • Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA, USA VICTORIA MALE • Department of Pathology, University of Cambridge, Cambridge, UK KARL-JOHAN MALMBERG • Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden OFER MANDELBOIM • The Hebrew University – Hadassah Medical School, Jerusalem, Israel MICHAEL E. MARCH • Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, Rockville, MD, USA MAUREEN P. MARTIN • Cancer and Inflammation Program, Laboratory of Experimental Immunology, SAIC-Frederick Inc., National Cancer Institute-Frederick, Frederick, MD, USA MICHAL MENDELSON • Department of Microbiology and Immunology, Ben Gurion University, Beer Sheva, Israel ASHLEY N. MENTLIK • Cell Biology and Physiology Graduate Group, Division of Immunology, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA, USA S. M. SHAHJAHAN MIAH • Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA, USA JEFFREY S. MILLER • University of Minnesota Cancer Center, Minneapolis, MN, USA TAKUYA MIYAGI • Department of Molecular Microbiology and Immunology, Division of Biology and Medicine, Brown University, Providence, RI, USA ASHLEY MOFFETT • Department of Pathology, University of Cambridge, Cambridge, UK JACQUIE NORTHFIELD • Department of Pathology, University of Cambridge, Cambridge, UK JO˜aO M. NUNES • Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden JAMES F. OESTERLING • Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA, USA JORDAN S. ORANGE • The Joseph Stokes Jr. Research Institute, Division of Immunology, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine and School of Veterinary Medicine, Philadelphia, PA, USA ANGEL PORGADOR • Department of Microbiology and Immunology, Ben Gurion University, Beer Sheva, Israel AMANDA K. PURDY • Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA, USA MICHAL PYZIK • Department of Human Genetics, McGill University, Montreal, QC, Canada GREGORY D. RAK • Cell Biology and Physiology Graduate Group, Division of Immunology, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine and School of Veterinary Medicine, Philadelphia, PA, USA ELEANOR M. RILEY • Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK BENYAMIN ROSENTAL • Department of Microbiology and Immunology, Ben Gurion University, Beer Sheva, Israel

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KERI B. SANBORN • Immunology Graduate Group, Division of Immunology, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA, USA JOHAN K. SANDBERG • Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden STEPHANIE L. SANOS • Institute of Medical Microbiology & Hygiene, University of Freiburg Medical Center, Freiburg, Germany AURORE SAUDEMONT • Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, UK RAM SAVAN • Cancer and Inflammation Program, Laboratory of Experimental Immunology, National Cancer Institute-Frederick, Frederick, MD, USA L´eONARDO SCARPELLINO • Ludwig Institute for Cancer Research Ltd., Lausanne Branch, and University of Lausanne, Epalinges, Switzerland LUIS SIGAL • Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA, USA MARK J. SMYTH • Peter MacCallum Cancer Centre, Parkville, Victoria, Australia NIKUNJ V. SOMIA • Department of Genetics, Cell Biology and Development, Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA CHANDRAKANT TAYADE • Department of Anatomy and Cell Biology, University of Guelph, Guelph, ON, Canada ANITA TRUNDLEY • Department of Pathology, University of Cambridge, Cambridge, UK SILVIA M. VIDAL • Department of Human Genetics, McGill University, Montreal, QC, Canada CEDRIC VONARBOURG • Institute of Medical Microbiology & Hygiene, University of Freiburg Medical Center, Freiburg, Germany STEPHANIE M. WOOD • Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden PAUL W. WRIGHT • Cancer and Inflammation Program, Laboratory of Experimental Immunology, SAIC-Frederick Inc., National Cancer Institute-Frederick, Frederick, MD, USA HAKIM YADI • Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, UK AUREO T. YAMADA • Laboratory of Histochemistry and Cytochemistry, Institute of Biology, UNICAMP, Campinas, Brazil WAYNE M. YOKOYAMA • Howard Hughes Medical Institute, Rheumatology Division, Departments of Medicine, Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA HOWARD A. YOUNG • Cancer and Inflammation Program, Laboratory of Experimental Immunology, National Cancer Institute-Frederick, Frederick, MD, USA JIANHONG ZHANG • Department of Anatomy and Cell Biology, Queen’s University, Kingston, ON, Canada ALON ZILKA • Department of Microbiology and Immunology, Ben Gurion University, Beer Sheva, Israel

Chapter 1 Purification of Human NK Cell Developmental Intermediates from Lymph Nodes and Tonsils Aharon G. Freud and Michael A. Caligiuri Abstract Accumulating data indicate that human natural killer (NK) cells undergo terminal maturation in secondary lymphoid tissues (SLTs) including lymph nodes (LNs) and tonsils. In addition, recent studies have revealed that maturing NK cells progress through at least five functionally discrete stages of development within SLTs. These discoveries provide unique possibilities for researchers to investigate the natural processes governing human NK cell development, as they exist in vivo, through analysis of NK cell maturational intermediates found in situ. Herein we describe a detailed, yet simple, four-step protocol for the viable enrichment and purification of human NK cell developmental intermediates from LNs and tonsils. Key words: CD34, lymph node, tonsil, secondary lymphoid tissue, lymphopoiesis, NK development, stages of NK development, NK maturation, purification, isolation, sorting.

1. Introduction Like all other leukocyte populations, natural killer (NK) cells derive from pluripotent hematopoietic progenitor cells (HPCs) through a complicated process of development that involves both differentiation toward the NK cell lineage and maturation into functional competence (1). Traditionally, the process of human NK cell development has been studied almost exclusively in vitro, with researchers culturing purified CD34(+) HPCs in medium plus cytokines in order to derive functionally competent NK cells as well as their immediate precursors. Although such culture systems have provided invaluable insight into numerous aspects of K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 1, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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human NK cell development, they are nonetheless limited by the very fact that it is essentially impossible to wholly recapitulate in vitro what naturally occurs in vivo. Recent discoveries from our laboratory and from others indicate that human NK cells undergo terminal maturation in secondary lymphoid tissues (SLTs) including lymph nodes (LNs) and tonsils (2, 3). Moreover, it is now clear that maturing NK cells progress through at least five functionally discrete stages of development within SLTs: stage 1 CD34(+)CD117(−)CD94(−)CD16(−), stage 2 CD34(+) CD117(+)CD94(−)CD16(−), stage 3 CD34(−)CD117(+) CD94(−)CD16(−), stage 4 CD34(−)CD117(+/−)CD94(+)CD 16(−), and stage 5 CD34(−)CD117(−)CD94(+/−)CD16(+) (4, 5). These findings provide unique possibilities for researchers to investigate the natural processes governing human NK cell development as they exist in vivo, through analysis of NK cell maturational intermediates found in situ. The process of isolating human NK cell developmental intermediates is relatively straightforward, and we have developed a simple, four−step protocol for the viable enrichment and purification of these cells directly from LNs and tonsils using commercially available reagents. The first step in this protocol involves the generation of SLT mononuclear cell (SLTMC) single-cell suspensions from gross tissue. Subsequently, the NK-lineage cells, which are all, by definition, CD3(−)CD19(−), are greatly enriched from SLTMCs via the depletion of total CD3(+) T cells and CD19(+) B-lineage cells that generally constitute >90% of the SLTMCs. Following the depletion step, stage 1 and stage 2 CD34(+) populations are separated from stage 3–5 cells via positive selection of CD34 HPCs. Lastly, each of the stage 1–5 cells may be sorted to purity, and we provide a useful staining procedure and sorting scheme for this purification.

2. Materials 2.1. Generation of SLTMC Single-Cell Suspensions

1. Dulbecco’s phosphate buffered saline (PBS) 1× (without calcium chloride or magnesium chloride) at room temperature (Invitrogen). 2. Ficoll-PaqueTM Plus density gradient at room temperature (GE Healthcare BioSciences). 3. 100− × 20 mm-style polystyrene cell culture dish (Corning Inc.). 4. 70−␮m cell strainers (BD Biosciences).

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5. Small surgical scissors and forceps. 6. 3-ml syringe (Becton Dickinson). 7. 50-ml centrifuge tubes (Greiner Bio-One). 2.2. Depletion of T and B Cells from SLTMCs

1. Midi selection magnet(s) with stand (Miltenyi Biotech). 2. LD depletion columns (Miltenyi Biotech). 3. Anti-human CD3 microbeads (Miltenyi Biotech). 4. Anti-human CD19 microbeads (Miltenyi Biotech). 5. De-gassed PBS+: PBS plus 0.5% fetal bovine serum (FBS) plus 2 mM EDTA (Invitrogen). Keep at 4◦ C or on ice (see Note 1). 6. 50-ml centrifuge tubes. 7. 15-ml centrifuge tubes (Greiner Bio-One). 8. 70-␮m cell strainers.

2.3. Positive Enrichment of CD34(+) HPCs

1. Mini selection magnet with stand (Miltenyi Biotech). 2. MS selection columns packaged with plungers (Miltenyi Biotech). 3. PBS+. 4. CD34 progenitor isolation kit including Fc-blocking antibody (Ab), anti-CD34 hapten Ab, and anti-hapten microbeads (Miltenyi Biotech). 5. 5-mL round-bottom tubes with caps (Becton Dickinson) (see Note 2).

2.4. Immunofluorescent Staining for Viable Cell Sorting

1. PBS+. 2. Culture medium consisting of RPMI-1640 + Glutamax plus 10% FBS and antibiotics (Invitrogen). 3. Direct fluorochrome-conjugated antibodies: CD45RA fluorescein isothiocyanate (FITC) (clone HI100), CD94 FITC (clone HP-3D9), CD117 phycoerythrin (PE) (clone 104D2), CD34 allophycocyanin (APC) (clone 581), CD3 APC (clone UCHT1), CD16 APC-Cy7 (clone 3G8) (BD Biosciences). 4. 5-mL round-bottom tubes with caps.

3. Methods It is important to appreciate the fact that human NK cell developmental intermediates, while relatively enriched among total HPCs and NK-lineage cells in SLTs compared to the blood or bone

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marrow compartments, nonetheless collectively constitute only a very minor fraction of the total cellularity of tonsils and LNs (4). Indeed these tissues contain not only a predominance of T and B cells but also numerous other cell types, including mast cells, dendritic cells, myeloid cells, endothelial cells, and nonhematopoietic stroma, which normally reside in SLTs. Therefore, although tonsil specimens may provide an average of one billion total mononuclear cells (with LNs providing far less depending upon size), it is common to obtain only a few thousand stage 1 and stage 2 cells and less than 105 stage 3–5 cells each after each isolation. Such low cell yields may preclude some molecular studies. 3.1. Generation of SLTMC Single-Cell Suspensions

1. The bottom and the lid of the Petri dish are separated from each other and each is filled with 20 mL room temperature PBS. The tonsil or the LN is placed into the bottom of the dish and cut into roughly 1 cm3 pieces using the forceps and scissors. Note that as soon as cuts are made into the tissue, cells can be seen diffusing into the surrounding PBS. If the tissue is encroached by fat (as is often the case with LN specimens), the latter should be removed prior to cutting and discarded as much as possible to avoid losing cells sticking to the fat. 2. A 70-␮m cell strainer is placed into the PBS within the lid of the Petri dish, and the first piece of lymphoid tissue (LT) is then cut as finely as possible over the strainer (being careful not to cut the strainer with the scissors). Using the flat top of the 3-ml syringe plunger, the tissue is gently, yet firmly, mashed against the bottom of the strainer in circular motions so that individual cells pass through into the PBS, whereas fibroadipose pieces of tissue remain within the strainer. The strainer is then discarded (see Note 3). 3. Step 2 is repeated with three more pieces of LT using a new cell strainer for each. 4. The 20mL of PBS-containing strained cells (from a total of four LT pieces) is carefully mixed within the lid with a pipette and then evenly distributed across three 50-ml tubes. 5. The lid of the Petri dish is refilled with 20 mL fresh PBS and Steps 2–4 are repeated until all LT pieces have been mashed and strained. The PBS contained within the bottom of the dish (where the numerous pieces of LT were kept in queue) is then strained and evenly distributed across the three 50ml tubes. All LT cells are now in suspension and divided evenly across three 50-ml tubes.

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6. The cells in each 50-ml tube are split across two tubes (thus, there will be six tubes altogether). The volume within each tube is then brought up to 40 mL total with room temperature PBS. 7. Fifteen milliliters of room temperature Ficoll is added to 12 separate 50-ml tubes (see Note 4). Twenty milliliters of cells is then gently and slowly overlaid onto the Ficoll in each of the 12 tubes (see Note 5). 8. The 12 tubes containing cells and Ficoll are centrifuged for 30 min at room temperature, 740 × g with the brake OFF (see Note 6). 9. The mononuclear layers are removed and each is added to a new 50-ml tube (12 tubes altogether). 10. The cells in each tube are washed of the Ficoll by dilution to a total volume of 50 mL with PBS followed by centrifugation for 8 min at 15◦ C, 385 × g with the brake on. 11. The supernatants are aspirated and the cell pellets are combined into one of the 50-ml tubes with a total of 10 mL (see Note 7). The 11 other tubes are then serially washed with 30 mL PBS that is finally added to the 10 mL of combined cells so that all of the SLTMCs are in suspension within a total volume of 40 mL. 12. The cells are thoroughly mixed and a small aliquot is removed for enumeration on a hemocytometer (see Note 8). The SLTMCs can be placed on ice while counting. 3.2. Depletion of T and B Cells from SLTMCs

1. CD3/CD19 depletion involves a one-step staining process followed by depletion across LD columns. 2. The SLTMCs are centrifuged for 8 min at 15◦ C, 385 × g. 3. Following centrifugation, the supernatant is discarded and the cells are resuspended and stained as follows within the 50-ml tube: For every one million cells in the pellet, add 8 ␮L PBS+ plus 1 ␮L CD3 microbeads plus 1 ␮L CD19 microbeads. Thus, the cells will be in a total volume of 10 ␮L/106 cells (see Note 9). 4. The cells are incubated in a 4◦ C refrigerator or cold room (rather than on ice, as per manufacturer’s instructions) for at least 15 min. For total volumes greater than 10 mL, the 50-ml tube should be placed on a nutator mixer or some similar device within a cold room to keep the cells thoroughly mixed in suspension with the microbeads during the incubation period. For samples less than 10 mL, it is sufficient to gently mix the cells via brief vortex or swirling

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the tube in a circular motion by hand for 15 s halfway through the incubation period. 5. During the incubation period, LD columns are placed into Midi magnets as per the manufacturer’s instructions, and the columns are equilibrated with 3 mL PBS+ each(to calculate the total number of LD columns needed for the entire sample, see Note 10). It usually takes 10–12 min for 3 mL to completely flow through each LD column. The LD columns are ready for use once all PBS+ is gone from the reservoir above the magnetic particles within the column and droplets have ceased to form below the column. The magnets and columns should be set up so that a 15-ml centrifuge tube can be placed underneath each column in order to catch the flow-through negative fraction. Every part of this step should be done at room temperature inside a tissue-culture hood to maintain sterility. 6. After the incubation of the cells with the microbeads, it is necessary to wash the cells to remove any unbound microbeads. The cells are diluted with additional PBS+ to a total volume of 50 mL within the 50-ml tube, and then they are centrifuged for 8 min at 15◦ C, 385 × g. The supernatant is discarded and the cells are resuspended in 50 mL PBS+ for a repeat wash step with centrifugation as described above. 7. Following the second wash, the cells are resuspended in ice cold PBS+ for depletion over the equilibrated LD columns. The total volume for depletion is 3 mL multiplied by the total number of LD columns. For example, for 10 LD columns (one billion cells in total), the cells should be resuspended in 30 mL PBS+. Prior to adding the cells to the columns, it is beneficial to filter out any clumps that may have formed during the incubation period. This is done by passing the cells over a 70-␮m cell strainer into a new 50-ml tube. In this case, to avoid losing cells leftover in the old tube, the cells can first be resuspended in a volume less than the final volume needed (for example, 20 mL rather than 30 mL as in the case above), then passed over the cell strainer, and finally the old tube can be washed with 10 mL PBS+ and passed over the same cell strainer to give a total volume of 30 mL (3 mL per LD column). 8. Once the cells are resuspended in the appropriate volume of PBS+, the cells are mixed thoroughly and distributed across the depletion columns. If there are more cells than columns available, the cells may be placed on ice until the next round of depletion using new LD columns.

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9. After the cells have completely passed through the columns (i.e., no cells are left in the LD column reservoirs), the 50-ml tube (if empty) is washed with cold PBS+ (3 mL per LD column) and then 3 mL is added per column. This is the first wash, and the 50-ml tube can now be discarded. It is important to not let the columns go dry, so care should be taken to ensure that the wash steps occur very soon after the cells pass through the columns. 10. A second wash of 3 mL PBS+ per LD column is added to each column after the first wash is complete. 11. After the second wash goes through and no more droplets fall from the bottom of the columns, the 15-ml tubes (now containing approximately 11 mL of cells in PBS+) are removed from the magnet apparatus, capped, and centrifuged for 8 min at 15◦ C, 385 × g. 12. During centrifugation, the LD columns can be discarded unless the T and/or B cells within the columns are to be used. In this case, the columns are removed from the magnets and repeatedly plunged with 5 mL PBS+ (four plunges altogether) into a collection tube. 13. After centrifugation of the 15-ml tubes containing Tand B-cell-depleted SLTMCs (Section 3.2, Step 11), the supernatants are removed and the cells are combined into one 15-ml tube. A small aliquot is then taken for enumeration on a hemocytometer and the rest of the cells are placed on ice (see Note 11). 3.3. Positive Enrichment of CD34(+) HPCs

1. The CD34 enrichment involves a two-step staining process followed by positive selection over MS columns. 2. The T-/B-cell-depleted SLTMCs in the 15-ml tube (Section 3.2, Step 13) are centrifuged for 8 min at 15◦ C, 385 × g. The supernatant is discarded, and the cell pellet is resuspended within the 15-ml tube in 2 ␮L PBS+, 1.5 ␮L Fc-blocking Ab, and 1.5 ␮L anti-CD34 hapten Ab, each per million cells in that order. If the total cell number is less than 10 million cells, they should be stained as if there were 10 million (i.e., not less than 20 ␮L PBS+ plus 15 ␮L each antibody in total). 3. The cells are incubated at 4◦ C in a refrigerator for 15 min, with gentle mixing by swirling the tube for 15 s halfway through the incubation. 4. The cells are then washed by adding 5 mL PBS+ followed by centrifugation for 8 min at 15◦ C, 385 × g. 5. After the spin, the supernatant is discarded and the cells are resuspended in 3.5 ␮L PBS+ plus 1.5 ␮L anti-hapten microbeads per million cells in that order.

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6. The cells are again incubated for 15 min at 4◦ C in a refrigerator, as per Section 3.3, Step 3. 7. During this second incubation, one MS column per sample is equilibrated by placing the column on the Mini magnet and adding 1 mL PBS+ to the reservoir. A sterile 5-mL tube is placed directly underneath the column in order to catch the flow-through. This is all done inside a sterile tissueculture hood. 8. Following incubation, the cells are again washed as in Section 3.3, Step 4. 9. After discarding the supernatant, the cells are resuspended in 1 mL PBS+ and placed into the reservoir of the equilibrated MS column that is on the Mini magnet (the 15ml tube is saved for the next step). Because the CD34(+) HPCs are labeled with microbeads, stage 1 and 2 NK cell developmental intermediates are retained within the column (while it is on the magnet), whereas stage 3–5 cells flow through the column and are collected in the 5-mL tube. 10. Once all the fluid has passed through the column, the bottom of the 15-ml tube is rinsed with 0.5 mL PBS+ that is then added to the reservoir above the column. 11. After the PBS+ rinse has passed through the column (ensuring first that drops have ceased to form under the column tip), the 5-mL tube with the CD34(−) fraction is set aside and replaced with a new sterile 5-mL tube, labeled “NK presort.” 12. The CD34(−) cell fraction in the first 5-mL tube is mixed by gentle pipetting and then passed over the column for a second round in order to retrieve any residual CD34(+) cells that may have initially passed through the column. We have observed this to significantly increase overall yields of CD34(+) HPCs. 13. After the cells have passed through the column, the first 5-mL tube is rinsed with 1 mL PBS+ that is then passed over the column as a wash step. 14. After the last of the drops have fallen from beneath the column, the “NK presort” tube is capped and set aside on ice. The MS column is removed from the magnet and plunged with 1 mL PBS+ into a new 5-mL tube, labeled “34 presort.” For best results, the PBS+ should be forced through the column immediately after it has been added to the column. The plunger is removed from the reservoir of the column and another 1 mL PBS+ is plunged through the column into the “34 presort” tube. This is again repeated

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twice for a total of four plunges. These plunges should rapidly follow one after the other, and it is recommended that if working with more than one sample, the first column should be plunged all four times before moving to subsequent columns. 15. At this step there are two tubes per sample (“CD34 presort” and “NK presort”), each with ∼4 mL total. The “34 presort” and “NK presort” tubes are then centrifuged for 8 min at 15◦ C, 385 × g. 16. Following the spin, the supernatants are carefully aspirated, leaving ∼100 ␮L liquid. The cell pellets are resuspended in this residual volume, and the tubes can be placed on ice until proceeding with the immunofluorescent staining protocol. 3.4. Immunofluorescent Staining for Viable Cell Sorting

1. Eight new 5-mL tubes are labeled as follows: “34 unstained,” “34 FITC,” “34 PE,” “NK unstained,” “NK FITC,” “NK PE,” “NK APC,” and “NK APC-Cy7.” These

Table 1.1 Antibodies and volumes for immunofluorescent staining prior to sorting Fluorochrome (volume added) Tube label

FITC

PE

APC

APC-Cy7

34 Unstained

–

–

–

–

34 FITC

CD45RA FITC (2.5 ␮L)

–

–

–

34 PE

–

CD117 PE (5 ␮L)

–

–

34 Presort

CD45RA FITC (2.5 ␮L)

CD117 PE (5 ␮L)

CD34 APC (2.5 ␮L)

–

NK unstained

–

–

–

–

NK FITC

CD94 FITC (10 ␮L)

–

–

–

NK PE

–

–

–

NK APC

–

CD117 PE (5 ␮L) –

CD3, CD34 APC (2.5 ␮L each)

–

NK APC-Cy7

–

–

–

CD16 APC-Cy7 (2.5 ␮L)

NK presort∗

CD94 FITC

CD117 PE

CD3, CD34 APC

CD16 APC-Cy7

∗ The volume of each antibody added to the “NK presort” tube depends upon the total cell number (for simplicity, the total cell number from Section 3.2, Step 13 can be used). For every 2 million cells, add 10 ␮L CD94 FITC, 5 ␮L CD117 PE, 2.5 ␮L CD16 APC-Cy7, 2.5 ␮L CD3 APC, and 2.5 ␮L CD34 APC.

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Fig. 1.1. Representative gating scheme for sorting NK cell developmental intermediates from human SLTs. (A) Enriched tonsil CD34(+) cells were stained as indicated in Table 1.1 and analyzed on a FACSVantage flow cytometer. The dot plots are gated as indicated with the following regions: R1, live cells with size and granularity characteristics typical of progenitor cells; R2, CD45RA(+) events; R3, CD34(+)CD117(−) events; R4, CD34(+)CD117(+) events. (B) Tonsil CD3(−)CD19(−)CD34(−) cells were stained as indicated in Table 1.1 and analyzed on a FACSVantage. The dot plots and histogram are gated as indicated with the following regions: R1, live cells within the typical “lymphocyte” gate (small size, low granularity); R2, CD3(−)CD34(−) events; R3, CD117(+)CD94(−) events; R4, CD117(dim/−)CD94(+) events; R5, CD16(−) events; R6, CD16(+) events.

are the control tubes for setting the voltage, amplitude, and compensation parameters on the cell sorter (see Note 12). 2. One hundred microliters of ice cold PBS+ are added to each of the control tubes. 3. Three microliters of cells from the “34 presort” and “NK presort” tubes are then added to each of the corresponding control tubes listed in Section 3.4, Step 1. 4. The cells in each of the tubes are stained with fluorochromelabeled Abs as shown in Table 1.1. We provide the volumes of antibodies typically used in our laboratory, but it is rec-

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ommended that these Abs be titrated for optimal staining prior to use as there may be variations from lot to lot. After adding the appropriate volume of each of the Abs, the samples are incubated on ice and covered from light for at least 15 min. 5. Following incubation, the cells are washed with 2 mL PBS+ and then centrifuged for 8 min at 15◦ C, 385 × g.

3.5. Gating Scheme for Sorting Stages 1–5

6. The supernatants are discarded, and the cell pellets are resuspended in the following volumes of culture medium: 300 ␮L for each of the control tubes; 1 mL for the “34 presort” tube; and 3 mL for the “NK presort” tube (see Note 13). The cells are kept on ice and covered from light until they are sorted. 1. Figure 1.1 shows dot plots from a sort for human NK cell developmental intermediates using the staining protocol outlined in Section 3.4. 2. Using the staining protocol outlined herein, stage 1–5 cells are sorted as follows: a. Stage 1 cells: R1 + R2 + R3 (Fig. 1.1A) b. Stage 2 cells: R1 + R2 + R4 (Fig. 1.1A) c. Stage 3 cells: R1 + R2 + R3 (Fig. 1.1B) d. Stage 4 cells: R1 + R2 + R4 + R5 (Fig. 1.1B) e. Stage 5 cells: R1 + R2 + R4 + R6 (Fig. 1.1B) (see Note 14)

4. Notes 1. The SLTMCs are kept in PBS+ solution for most of the protocol following the Ficoll centrifugation step during which the PBS and Ficoll are used at room temperature (see Note 6). For better viability and less chance that the cells will become activated through the procedure, the PBS+ should be kept cold even though many steps of the protocol, aside from incubations and centrifugations, are performed in a sterile hood at room temperature. In addition, to prevent microbubbles from clogging the magnetic columns, the manufacturers recommend that the PBS+ be “de-gassed” prior to use. This can be done by filtering the freshly made PBS+ through a 0.22−␮m vacuum filter (e.g., Steriflip products by Millipore) and leaving the vacuum attached for 30 min after the liquid has been filtered. 2. Both polystyrene and polypropylene 5-mL round-bottom tubes are commercially available. Either can be used for this

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protocol, but compatibility with the cell sorter may depend on the make and model of the latter. Researchers should determine which kind of tube is to be used at their institution. 3. Most of the LTs consist of hematopoietic cells that will go into suspension. Therefore, a good rule of thumb is to continue gently grinding the tonsil or the LN piece against the strainer until all that is left is white, fibroadipose tissue. 4. In our experience, the majority of tonsils provide between 0.5 and 2 × 109 SLTMCs in total. For this number of cells, 12 Ficoll tubes is an appropriate number so as not to overwhelm the Ficoll and have potentially inefficient separation of the mononuclear cells from the fat, debris, endothelial cells, and other non-mononuclear cells. However, for small LN specimens or very large tonsil specimens, individuals may choose to use fewer or more Ficoll tubes, respectively. 5. One must take care to pipette very slowly when initially overlaying the cells upon the Ficoll so as not to mix the cells with the former prior to centrifugation. For those with experience in overlaying blood upon Ficoll, note that blood is much thicker and, thus, less likely to mix with the Ficoll during the overlay compared to LT cells in suspension in PBS. A helpful technique is to angle the 50-ml Ficoll tube at 45◦ and to disperse the cells near the top of the tube very slowly so that the cells run down the side and gently lay over the Ficoll. 6. It is very important to leave the centrifuge break off so that the mononuclear cell layers are not disrupted by an abrupt stop. In addition, the centrifuge should be set at room temperature in order to increase the probability of obtaining very distinct mononuclear layers above the Ficoll. In our experience, centrifugation at cold temperatures results in suboptimal results with hazy mononuclear layers. This is why room temperature PBS and Ficoll are used prior to the centrifugation step. 7. For this step, 10 mL PBS is used to resuspend the cell pellet in the first tube. This cell suspension is then used to resuspend the next cell pellet and so on until all cell pellets have been resuspended and combined in a total of 10 mL within one 50-ml tube. 8. For counting the cells, we generally start with a 40-fold dilution of the cells by first removing 10 ␮L from the mixed cell sample, diluting this 1:10 with PBS, and then diluting the cells again 1:4 with trypan blue. This prevents an excess waste of trypan blue, which is also toxic to cells at high exposure.

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9. With so many cells in one 50-ml tube, the cell pellet is very large. To ensure proper resuspension, it is recommended to first use a 5-mL pipette (smaller volume pipettes have smaller diameter tips through which the cells are forced) with 3–5 mL PBS+ and to repeatedly spray the cells against the side of the tube multiple times until there are no visible clumps. Subsequently, additional PBS+ can be added up to the desired volume. This procedure is recommended in general for resuspending large cell pellets. 10. As per the manufacturer’s instructions, each LD column can efficiently deplete up to a maximum of 1 × 108 cells in total. In our laboratory, to determine the number of LD columns to use, we round the total number of cells to the nearest 108 and divide by 108 . For example, for a sample with 9.3 × 108 cells, nine LD columns are used. Note: Because one Midi magnet can hold only one LD column, it is beneficial to purchase multiple Midi magnets and stands so that cells can be passed over the LD columns in parallel rather than in series. This is very important in terms of saving time, because one round of depletion over an LD column, including the initial cell suspension followed by two sequential washes, takes roughly 45 min. With 10 Midi magnets, one can deplete a total of one billion SLTMCs in 45 min, whereas it would take 7.5 h with the use of only one magnet for the same total number of SLTMCs. 11. It is common for the cell yield following T- and B-cell depletion to be approximately 1% of the initial SLTMC count. As such, the cell pellets are much smaller (1–2 mm in diameter) after the depletion step. Nonetheless, the NKlineage cells are greatly enriched within this cell mixture. 12. This staining protocol is applicable for sorting human NK cell developmental intermediates using a FACSVantage or a FACSAria cell sorter from BD Biosciences. Alternative staining protocols may be required if using different sorting machines. 13. In our practice, we prefer to put the cells in culture medium (on ice) prior to the sort in the event that there may be unforeseen delays in sorting the cells. If it is anticipated that the cells will be on ice only for a short amount of time prior to sorting, it is fine to put the cells in PBS+ rather than medium. In addition, we have found that it is preferable to dilute the cells as much as reasonably possible so that there is less chance of cell clumping that could clog the sorter. 14. The minimal CD3(−)CD16(+) phenotype is not sufficient to identify stage 5 NK cells, because non-NK-lineage cells,

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including monocytes and granulocytes, can express CD16 within SLTs. In contrast, the CD3(−)CD94(+)CD16(+) phenotype does unequivocally identify CD56(+) NK cells in these tissues (data not shown). Given that the vast majority of stage 5 cells express CD94, for practical purposes we sort stage 5 cells as CD3(−)CD34(−)CD94(+)CD16(+) and forgo the rare stage 5 cells with background level fluorescent intensity staining of CD94.

Acknowledgments The authors would like to thank Tamra Brooks for her assistance with the chapter. Funding for the Caligiuri laboratory is provided by the National Cancer Institute (P30 CA16059, CA68458 and CA95426) (M.A.C.). References 1. Colucci F., Caligiuri M. A., Di Santo J. P. (2003) What does it take to make a natural killer? Nat Rev Immunol 3, 413–25. 2. Ferlazzo G., Thomas D., Lin S. L., Goodman K., Morandi B., Muller W. A., Moretta A., M¨unz C. (2004) The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic J Immunol 172, 1455–62. 3. Freud A. G., Becknell B., Roychowdhury S., Mao H. C., Ferketich A. K., Nuovo G. J., Hughes T. L., Marburger T. B., Sung

J., Baiocchi R. A., Guimond M., Caligiuri M. A. (2005) A human CD34(+) subset resides in lymph nodes and differentiates into CD56bright natural killer cells Immunity 22, 295–304. 4. Freud A. G., Caligiuri M. A. (2006) Human natural killer cell development Immunol Rev 214, 56–72. 5. Freud A. G., Yokohama A., Becknell B., Lee M. T., Mao H. C., Ferketich A. K., Caligiuri M. A. (2006) Evidence for discrete stages of human natural killer cell differentiation in vivo J Exp Med 203, 1033–43.

Chapter 2 In Vitro Development of Human Killer–Immunoglobulin Receptor-Positive NK Cells Frank Cichocki and Jeffrey S. Miller Abstract The in vitro culture system outlined in this chapter allows for the delineation of events that occur during the development of CD34+ hematopoietic precursor cells into mature KIR+ human NK cells. This system can also be utilized to study the effects of gene overexpression or knockdown on the process of NK cell differentiation through retroviral transduction and long-term culture. The necessary soluble factors and contact-dependent conditions for in vitro human NK cell development have been worked out in our laboratory over the past 16 years. Key words: Human NK cell development, lymphocyte differentiation from hematopoietic precursors

1. Introduction Both mouse and human natural killer cells recognize transformed and virally infected cells and influence the direction of the adaptive immune response in infectious settings (1). However, there are several notable differences between mouse and human NK cells with respect to differentiation markers, making developmental comparisons between the species difficult (2–4). To evaluate and manipulate human natural killer cell differentiation, a robust ex vivo cell culture system is necessary. To this end, our laboratory has developed a long-term culture system for studying human NK cell development from primitive progenitor cells (5–9). In 1992, we were the first to show that primitive progenitors from adult bone marrow can give rise to functional NK cells K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 2, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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when cultured in contact with human bone marrow stroma (5). In 1994, we found that in the absence of factors known to support NK cell differentiation, direct contact with human allogeneic stroma is critical for NK cell differentiation (6). In 1998, after an explosion of new data describing novel NK cell receptors recognizing class I MHC, we found that our in vitro model of NK cell differentiation supported the acquisition of KIR (specifically KIR3DL1 in this initial description) (7). Limitations of these initial studies included the heterogeneity and variability of primary human stroma, low cloning frequency when plating primitive stem cell populations, and the inability to support differentiation at the single cell level. These issues raise the possibility of starting progenitor contamination. Therefore, definitive study of NK cell precursors was technically difficult. In 1999, we pioneered the use of a novel murine stromal cell line, called AFT024, which was derived from day 14 gestational fetal liver cells immortalized by a retrovirus containing a temperature-sensitive SV40 T antigen. The most important finding for the success of NK cell development cultures is that AFT024 could officially support NK cell differentiation of human cells at the single cell level. Using this system, we have shown that KIR and NKG2/CD94 receptors are acquired late in NK cell development (8). In 2008, we further improved this system by comparing NK cell development on a novel murine cell line called EL08-1D2 cloned from a culture of embryonic liver at day 11 of gestation. EL08-1D2 was chosen for these studies because of its ability to support generation of human hematopoietic progenitors from CD34+ umbilical cord blood cells without the addition of any cytokines. Use of EL08-1D2 identified several novel properties of this stromal feeder. First, when IL-15 was eliminated from the culture medium, there was an accumulation of CD56− NK cell precursors defined as CD34+ /CD7− , CD34+ /CD7+ , and CD34− /CD7+ . The role of IL-3 and IL-3 plus Flt3 ligand was established, and c-kit ligand and IL-7 appear to add efficiency to the system but are not absolutely required for NK cell differentiation. EL08-1D2 was superior to AFT024 for supporting differentiation of NK cell precursors, NK cell commitment, the acquisition of KIR, and overall proliferation (9). EL08-1D2 has been the stromal feeder of choice in our laboratory based on its ability to recapitulate the acquisition of class I recognizing receptors and developmental intermediates which may be important in NK cell maturation (10). There has been support in the literature for NK cell differentiation cultures in the absence of stroma. In review of this literature, our stromal-based cultures seemed to better allow the acquisition of NK cell receptors where NK cell differentiation in the absence of stroma but in the presence of high concentrations of human cytokines allows NK cell commitment with poor expression of

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17

KIR (11). Our data support the notion that unique signals from stroma are important in the acquisition of NK cell receptors. Further proof is the finding that CD56bright KIR− cells can transition into CD56dim KIR+ cells on EL08-1D2 and human IL-15 (12). In summary, methods for NK cell differentiation cultures using EL08-1D2 will be described here. Other stromal cell lines, such as OP9 and MS5, have been used in the literature for similar purposes and could possibly be substituted here but direct comparisons with EL08-1D2 have not been performed in our laboratory (13, 14). CD34+ hematopoietic progenitors from umbilical cord blood are highly efficient in NK cell development in terms of both cloning efficiency and proliferation. Other hematopoietic stem cell sources such as bone marrow, peripheral blood progenitors, and fetal liver have been tested in our laboratory and can be substituted here if desired, but their efficiency may differ from the bulk of our work using human umbilical cord blood progenitors.

2. Materials This protocol includes the experimental procedures that our laboratory has developed to retrovirally transduce CD34+ cells prior to long-term culture in order to study the effects of gene overexpression or knockdown. If the user does not desire to carry out retroviral transduction, Section 2.3 and steps 24–42 in Section 3.3 can be excluded from the protocol. 2.1. EL08-1D2 Cell Culture and Irradiation

1. EL08-1D2 cells: mouse embryonic liver cells. These cells were obtained from E. Dzierzak at Erasmus University MC, Rotterdam, the Netherlands. 2. EL08-1D2 medium: 202.5 ml ␣-MEM medium (Gibco, R M5300 medium (StemCarlsbad, CA), 250 ml Myelocult Cell Technologies Inc., Vancouver, BC, Canada), 37.5 ml fetal calf serum (see Note 1), 5 ml Penicillin + Streptomycin, 5 ml 100× Glutamax (Gibco), 35.0 ␮l ␤-mercaptoethanol (0.143 M stock: 1:100 in H2 O), and hydrocortisone at a final concentration of 10−6 M. Hydrocortisone must be added fresh at the time of use. This medium (without hydrocortisone) can be stored at 4◦ C for up to 1 month. Note that these additives should be included on top of what is already R M5300 medium. present in the Myelocult 3. Ultrapure water with 0.1% gelatin (Chemicon Intl., Billerica, MA). 4. Trypsin/EDTA (Gibco).

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5. Flat-bottom 96-well cell culture plates (Becton Dickinson, Franklin Lakes, NJ). 6. Multi-channel pipetter. 7. Cesium irradiator. 2.2. CD34+ Cell Isolation from Umbilical Cord Blood

R 1. Histopaque -1077 (Sigma-Aldrich, St. Louis, MO).

2. Ammonium chloride solution (StemCell Technologies Inc.). R (Beckman Coulter, Fullerton, 3. Coulter Particle Counter CA).

4. PBS/0.3% BSA. 5. Direct CD34 Progenitor Cell Isolation Kit, human (Miltenyi Biotech, Oberlin, CA). R LS separation columns (Miltenyi Biotech). 6. MACS R pre-separation filters (Miltenyi Biotech). 7. MACS R magnetic stand and magnets (Miltenyi Biotech). 8. MACS

9. Cycling Medium: Iscove’s Modified Dulbecco’s Medium (with L-glutamine), 20% fetal bovine serum, 1% Penicillin + Streptomycin with 20 ng/ml IL-7, 20 ng/ml c-kit ligand, 20 ng/ml Flt3 ligand, and 20 ng/ml thrombopoietin. This medium can be stored at 4◦ C for up to 1 month. 10. 24-well cell culture plates (Becton Dickinson). 2.3. Retroviral Transduction and Cell Sorting of CD34+ Cells

1. Four separate 3 ml aliquots of retroviral supernatant (see Note 2). 2. Transwells with sterile 6-well culture plates − 0.4 ␮M PTFE membrane, 24 mm insert (Corning Incorporated, Corning, NY). 3. Tweezers. 4. RetroNectin (r-Fibronectin) (Otsu, Shiga, Japan). 5. PBS/2% BSA. 6. Iscove’s Modified Dulbecco’s Medium (with L-glutamine) without fetal bovine serum (Gibco). 7. Cycling medium (see Section 2.2). 8. APC-conjugated anti-CD34 monoclonal antibody (clone 8G12, mouse IgG1 ; BD Biosciences) 9. APC-conjugated IgG1 isotype control monoclonal antibody (BD Bioscience). 10. 5 ml polystyrene round-bottom tubes with cell-strainer cap (BD Biosciences). 11. A fluorescence-activated cell sorter.

In Vitro Development of Human Killer–Immunoglobulin Receptor-Positive

2.4. Long-Term Culture of Sorted CD34+ Cells on the EL08-D12 Stromal Line

19

1. Basal Culture Medium: A 2:1 (vol:vol) mix of Dulbecco’s Modification of Eagle’s Medium (DMEM) with 4.5 g/l glucose, L-glutamine, and sodium pyruvate/Ham’s F12 Medium. This medium mixture is then supplemented with 24 ␮M 2-mercaptoethanol, 50 ␮M ethanolamine, 20 mg/l ascorbic acid, 50 ␮g/l sodium selenite, 1% penicillin + streptomycin and 20% heat-inactivated human AB serum (Valley Biomedical, Inc., Winchester, VA). The following cytokines must also be added: 10 ng/ml IL-15, 5 ng/ml IL3, 20 ng/ml IL-7, 20 ng/ml c-kit ligand, and 10 ng/ml Flt3 ligand. This medium can be stored at 4◦ C for up to 1 month. 2. Multi-channel pipetter.

3. Methods 3.1. General Culture Conditions for EL08-1D2 Cells

1. Coat sterile cell culture flasks (75 cm2 or 150 cm2 ) with enough sterile ultrapure water with 0.1% gelatin to cover the bottom of the flask. Let the flask sit in the culture hood at room temperature for 10 min. The gelatin water is necessary to promote adherence of the EL08-1D2 cells. 2. After 10 min, remove the ultrapure water with 0.1% gelatin and gently rinse the bottom of the culture flask with PBS. Remove the PBS. 3. Plate cells at approximately 4000 cells/cm2 on gelatincoated flasks in an appropriate volume of EL08-1D2 medium (12 ml for a 75 cm2 flask or 24 ml for a 150 cm2 flask) containing 20% conditioned EL08-1D2 medium (see Note 3). 4. Culture cells in an incubator set to 32◦ C 5% CO2 . This is important, as the cells grow in a temperature-sensitive manner. 5. Once the cells are 95–99% confluent, they can be split into new flasks. 6. To split cells, remove the spent medium, filter, and store at −20◦ C. Add enough 1× trypsin/EDTA to cover the bottom of the flask and incubate at room temperature until the cells start to lift off the bottom of the flask. Add 5 ml of 0.2 ␮m-filtered EL08-1D2 conditioned supernatant to neutralize the trypsin and collect cells in a 15 ml centrifuge tube. 7. Count cells using a hemocytometer. Remove the desired number of cells (approximately 4000 cells/cm2 ) to seed a new flask(s).

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8. Centrifuge at 550 × g for 4 min and decant supernatant. 9. Resuspend cells in freshly prepared EL medium and gently transfer to a flask(s) freshly coated with gelatin water. 10. Return flask(s) to a 32◦ C 5% CO2 incubator. 3.2. Irradiation of EL08-1D2 Cells

1. Use a multi-channel pipette to add 50 ␮l of ultrapure water with 0.1% gelatin per well into the desired number of flatbottom 96-well cell culture plates. Let plates incubate for 10 min at room temperature. 2. After 10 min, remove the ultrapure water with 0.1% gelatin and gently rinse the bottom of the culture plates with PBS. Remove the PBS. 3. Resuspend EL08-1D2 cells in fresh EL08-1D2 medium (with 20% conditioned medium and hydrocortisone) at a concentration of 104 cells/ml. 4. Add 103 cells per well (100 ␮l) to the gelatin-coated and rinsed plates. Incubate plates at 32◦ C 5% CO2 for 3–5 days until the plates are 95–99% confluent. 5. Irradiate confluent plates with 30 Gy of radiation and let the plates incubate at 32◦ C 5% CO2 for at least 2 h before use. Irradiated plates can be kept for up to 1 week before use in an NK cell developmental assay.

3.3. CD34+ Cell Isolation and Retroviral Transduction

Day 1 R -1077 and PBS to room temperature. 1. Warm Histopaque 2. Transfer umbilical cord blood into a sterile container and add 50 ml PBS to dilute the blood. R -1077 to the necessary number of 3. Add 20 ml Histopaque 50 ml centrifuge tubes. R 4. Slowly layer 30 ml diluted blood over the Histopaque 1077.

5. Centrifuge at 550 × g for 30 min at room temperature with the brake off. 6. Slowly aspirate the plasma layer leaving approximately 1/2 R inch of liquid above the lymphocyte/Histopaque -1077 interface. 7. Harvest the lymphocytes from the lymphocyte interface and transfer to a new 50 ml centrifuge tube. Fill up to 50 ml with PBS and centrifuge at 850 × g for 5 min. 8. Gently decant the supernatant and resuspend cells in 10 ml ice-cold ammonium chloride solution. Incubate the cells on ice for 10 min. 9. Centrifuge for 5 min at 850 × g. Gently decant supernatant and resuspend cells in 20 ml PBS.

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10. Determine total cell number using a Coulter Particle R (see Note 4). Counter 11. Wash the cells by adding another 30 ml of PBS to the cell suspension. Centrifuge for 5 min at 850 × g. 12. Gently decant the supernatant and resuspend cells in 300 ␮l PBS/0.3% BSA per 108 cells. Keep the PBS/0.3% BSA cold throughout the isolation procedure. 13. Label cells for isolation by adding 100 ␮l FcR blocking reagent for every 108 cells. Next, add 100 ␮l CD34 Microbeads for every 108 cells. Incubate for 30 min on ice in the dark. 14. Add 30 ml of PBS/0.3% BSA and centrifuge for 5 min at 850 × g. 15. Gently decant the supernatant and resuspend the cells in 0.5 ml of PBS/0.3% BSA. R LS column per cord blood donor on 16. Set up one MACS R R  pre-separation a MACS Magnetic Stand. Place a MACS filter on top of each column and equilibrate the column with 3 ml of PBS/0.3% BSA. R pre-separation 17. Pipette the labeled cells into the MACS filter and let the cells pass through the column. There is no need to save the effluent, as the CD34+ cells will be retained within the column.

18. Wash the column three times with PBS/0.3% BSA. R 19. Remove the MACS pre-separation filter, pull the column off the magnet, and transfer the column to a 15 ml centrifuge tube. Apply 5 ml of PBS/0.3% BSA and forcefully plunge the liquid through the column to purge the labeled cells. R LS column. It is 20. Repeat steps 16–19 with a new MACS necessary to double-column the labeled cells in order to obtain a CD34+ population with a high level of purity.

21. Count the total number of cells in the CD34+ fraction using a hemocytometer. 22. Centrifuge cells for 4 min at 550 × g at 4◦ C (see Note 5). 23. Gently decant the supernatant and resuspend the cell pellet in Cycling Medium at a concentration of 5 × 105 cells/ml. Transfer the cells in Cycling Medium to a 6-well or 24-well cell culture plate and place in an incubator set to 37◦ C 5% CO2 . If cells are resuspended in less than 2 ml of medium (1 × 106 cells or less), they should be added to a well of a 24-well plate. Larger volumes should be added to a 6-well plate. The number of cells isolated will vary widely between

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umbilical cord blood donors. Let the cells proliferate in the Cycling Medium for 3 days (see Note 6). Day 4 24. Three days after the CD34+ cell isolation, reconstitute one vial of RetroNectin at a concentration of 0.05 mg/ml according to the manufacturer’s instructions. 25. Transfer two transwells per umbilical cord donor to a 6-well culture plate using a pair of sterilized tweezers. 26. Coat transwells in the 6-well plate with 0.1 mg of RetroNectin (2 ml of 0.05 mg/ml RetroNectin per well). Incubate plate for 2 h at room temperature. 27. Remove RetroNectin from both the top and the bottom of the transwells. 28. Wash by adding 2 ml of PBS/2% BSA to the transwell. Let the transwells sit for 1/2 h at room temperature (see Note 7). 29. Remove PBS/2% BSA from both the top and the bottom of the transwells. 30. Add 2 ml of Iscove’s Medium (without FBS) to each transwell and let the transwell sit for 1/2 h at room temperature. 31. Remove all Iscove’s Medium from the top and the bottom of the transwells. 32. Add 2 × 105 CD34+ cells (at a concentration of 4 × 105 cells per ml) to each transwell along with 3 ml of the desired viral supernatant. Do not let the virus sit at room temperature longer than necessary before adding it to the transwell. 33. Place plates in an incubator set to 37◦ C 5% CO2 for 6 h. 34. Remove the viral supernatant from the bottom of the transwells, taking care to disturb the cells on the top of the transwell as little as possible. 35. Place the transwell plate back into the incubator for 5 min to allow the rest of the viral supernatant to pass through the transwell. 36. Remove the remaining viral supernatant from the bottom of the transwells. 37. Slowly add 3 ml Cycling Medium to the top of each transwell and place the transwell plate back in the incubator overnight. Day 5 38. Remove the Cycling Medium from the bottom of the transwells and allow all of the medium to pass through. Remove the remaining medium from the bottom of the transwells. 39. Add 3 ml of the same type of viral supernatant used during the previous day for transduction to each transwell.

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40. Place plates in an incubator set to 37◦ C 5% CO2 for 6 h. 41. Repeat steps 34–37 in this section. Day 7 42. Harvest the cells from each transwell by pipetting up and down a few times and washing the top of the transwells with 1 ml of medium taken from the bottom of the transwells. Place the cells into a 15 ml centrifuge tube. 43. Remove 100 ␮l from each GFP control sample and put aside on ice for an isotype control. 44. Count cells using a hemocytometer. Generally, you should expect to observe a two- to threefold increase in cell number over the course of the retroviral transduction due to expansion in the Cycling Medium. 45. Centrifuge cells at 550 × g for 4 min. 46. Gently decant the supernatant and add 10 ␮l of anti-CD34 APC-conjugated monoclonal antibody per 1 × 106 cells. 47. Add 1.5 ␮l of APC-conjugated mouse IgG1 antibody to the 100 ␮l of cell set aside for the isotype control. 48. Incubate samples in the dark on ice for 30 min. 49. Wash off excess antibody by adding 5 ml of PBS/0.3% BSA and centrifuging at 550 × g for 4 min. 50. Gently decant supernatant and resuspend stained cells in approximately 0.3 ml of PBS/0.3% BSA. 51. Pass the labeled cells through the strainer cap and into a 5 ml FACS tube. 52. Collect the double-positive GFP+ /CD34+ population using a fluorescence-activated cell sorter (see Note 8 and Fig . 2.1A). 53. Resuspend sorted GFP+ CD34+ cells at a concentration of 500 cells per ml in Basal Culture Medium. 54. Remove the 96-well plates containing irradiated EL08D12 cells from the incubator and gently remove the EL08D12 medium using a multi-channel pipette. 55. Add 100 ␮l of sorted cells in Basal Culture Medium to each well containing irradiated EL08-D12 cells. This will result in a plating of 50 transduced CD34+ cells per well. 56. Place plates in an incubator set to 37◦ C and 5% CO2 . 3.4. Long-Term Culture of Sorted CD34+ Cells on the EL08-1D2 Stromal Line

1. After 7 days have passed, add 100 ␮l of Basal Culture Medium to each well and place the plates back into the incubator. 2. After 14 days have passed, carefully remove 150 ␮l of medium from each well without disturbing the cells on the bottom of the wells. Add 150 ␮l of Basal Culture Medium to

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Fig. 2.1. (A) Representative phenotypes of retrovirally transduced CD34+ cells harvested at the time of sorting. A fairly conservative CD34+ /GFP+ gate should be selected to collect this population for long-term culture. The CD34− cells in this plot represent cells that have already begun to differentiate during the cycling phase of the protocol. These cells should not be collected. (B) Representative phenotypes of long-term cultures of CD34+ cells differentiated on EL08-1D2 stroma in the presence of 10 ng/ml IL-15, 5 ng/ml IL-3, 20 ng/ml IL-7, 20 ng/ml c-kit ligand, and 10 ng/ml Flt3 ligand. CD56 and KIR (mixture of DX9, EB6, GL183, and FES172 monoclonal anti-KIR antibodies conjugated with a common fluorochrome) expressions on cells harvested from day 14 and day 21 cultures are shown. As this figure illustrates, the major transition of hematopoietic cells into mature NK cells occurs between 2 and 3 weeks after plating.

each well. At this time point, a proportion of the cells in culture begin to express CD56, but do not express significant levels of KIR as determined by FACS analysis. 3. After 21 days, change the medium again by removing 150 ␮l of medium from each well and adding back 150 ␮l of Basal Culture Medium. At this time point, a significant number of cells in culture express both CD56 and KIR as determined by FACS analysis (see Note 9 and Fig. 2.1B). 4. Full NK cell maturation will be observed after 21 or 28 days in culture, and this is usually used as an endpoint for in vitro NK cell development experiments (see Note 10).

4. Notes 1. The fetal calf serum used for EL08-D12 cell culture is specially designed for the maintenance of stromal cell lines from mouse embryonic tissues and is distributed by StemCell Technologies, Inc. Other types of serum should not be substituted without comparative testing. 2. For this protocol, it is necessary to prepare ahead of time two 3 ml aliquots of retroviral supernatant prepared using a control GFP construct for each umbilical cord blood donor. You will also need two 3 ml aliquots of retroviral supernatant prepared using a construct containing GFP and your gene-of-interest for each umbilical cord donor.

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We have consistently used Murine Stem Cell Virus (MSCV) promoter constructs for gene overexpression in CD34+ cells. We package MSCV retroviral particles in the 293kj cell line using the PCL packaging plasmid as previously described (15). 3. The “conditioned” EL08-1D2 medium refers to the supernatant from EL08-1D2 culture flasks that are at or near confluency. This medium should be harvested, 0.2 ␮mfiltered, and frozen at −20◦ C for storage up to 6 months. Conditioned medium should be added to the EL Medium immediately before use at a 20% concentration. 4. Because of the high cell counts per unit of umbilical cord blood, counting with a hemacytometer will be less accurate. We also recommend that the user add 5–6 drops of Zap-oglobinTM II Lytic Reagent (Beckman Coulter) to the Isotone reagent before counting to obtain a more accurate lymphocyte count. 5. There is considerable variability in CD34+ cell yields between umbilical cord donors. This may be because of age from procurement to use or due to individual unit variability. Final cell counts generally fall between 3 × 105 cells and 1.5 × 106 cells. It is our experience that fresh umbilical cord blood units are more efficient in NK cell differentiation cultures than cryopreserved cells which work but with slightly less efficiency. 6. If gene modification with an EGFP marker is considered, it is important to setup the experiment so that you can sort the cells using a fluorescence-activated cell sorter 6 days after the CD34+ cell isolation. 7. Because of variability in the manufacturing of the transwells, the RetroNectin solution may or may not pass through individual transwells. This will not affect the experiment. However, if the RetroNectin does not pass through, we recommend adding 1 ml of PBS/0.3% BSA to the bottom of the transwell and 1 ml of PBS/0.3% BSA to the top of the transwell during the washing step to soak the transwell so that viral supernatant can pass through. 8. The percentage of GFP+ /CD34+ cells in culture depends on the size of the retroviral construct and the potency of the virus. We routinely observe that, when using conservative gating, 1–5% of cells are GFP+ /CD34+ at the time of the sort. 9. A reduction in the number of EL08-D12 cells will usually be observed after 21 days in culture due to their lysis by NK cells. The EL08-D12 stromal layer will disappear completely at later time points.

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10. There is considerable variability between umbilical cord donors with respect to the rates of differentiation and proliferation in culture, which may be due to the intrinsic sensitivity of individual donors to the cytokines used in culture. Therefore, we recommend that at least four to six donors are analyzed and compared for each experiment. References 1. Colucci, F., Caligiuri, M.A., and Di Santo, J.P. (2003) What does it take to make a natural killer? Nat. Rev. Immunol. 3, 413–425. 2. Huntington, N.D., Vosshenrich, C.A., and Di Santo, J.P. (2007) Developmental pathways that generate natural-killer-cell diversity in mice and humans. Nat. Rev. Immunol. 7, 703–714. 3. Kim, S., Iizuka, K., Kang, H.P., Dokun, A., French, A.R., Greco, A., and Yokoyama, W.M. (2002) In vivo developmental stages in murine natural killer cell development. Nat. Immunol. 3, 523–528. 4. Freud, A.J., Yokohama, A., Becknell, A., Lee, M.T., Mao, H.C., Ferketich, A.K., and Caligiuri, M.A. (2006) Evidence for discrete stages of human natural killer cell differentiation in vivo. J. Exp. Med. 203, 1033–1043. 5. Miller, J.S., Verfaille, C., and McGlave, P. (1992) The generation of human natural killer cells from CD34+ /DR– primitive progenitors in long-term bone marrow culture. Blood. 80, 2182–2187. 6. Miller, J.S., Alley, K.A., and McGlave, P. (1994) Differentiation of natural killer (NK) cells from human primitive marrow progenitors in a stroma-based long-term culture system: identification of a CD34+ 7+ NK progenitor. Blood. 9, 2594–2601. 7. Miller, J.S., McCullar, V., and Verfaillie, C.M. (1998) Ex vivo culture of CD34+ /Lin− /DR− cells in stromaderived soluble factors, interleukin-3, and macrophage inflammatory protein-1␣ maintains not only myeloid but also lymphoid progenitors in a novel switch culture assay. Blood. 12, 4516–4522. 8. Miller, J.S., and McCullar, V. (2001) Human natural killer cells with polyclonal lectin and immunoglobulinlike receptors develop from single hematopoietic stem cells with preferential expression of NKG2A and KIR2DL2/L3/S2. Blood. 98, 705–713.

9. McCullare, V., Oostendorp, R., PanoskaltsisMortari, A., Yun, G., Lutz, C.T., Wagner, J.E., and Miller, J.S. (2008) Mouse fetal and embryonic liver cells differentiate human umbilical cord blood progenitors into CD56negative natural killer cell precursors in the absence of interleukin-15. Exp. Hematol. 36, 598–608. 10. Grzywacz, B., Kataria, N., Sikora, M., Oostendorp, R.A., Dzierzak, E.A., et al. (2006) Coordinated acquisition of inhibitory and activating receptors and functional properties by developing human natural killer cells. Blood. 108, 3824–3833. 11. Yu, H., Fehniger, T.A., Fuchshuber, P., Thiel, K.S., Vivier, E., et al. (1998) Flt3 ligand promotes the generation of a distinct CD34+ human natural killer cell progenitor that responds to interleukin-15. Blood. 92, 3647– 3657. 12. Cooley, S., Xiao, F., Pitt, M., Gleason, M., McCullar, V. et al. (2007) A subpopulation of human peripheral blood NK cells that lacks inhibitory receptors for self-MHC is developmentally immature. Blood. 110, 578–586. 13. Nakayama, N., Fang, I., and Elliott, G. (1998) Natural killer and B-lymphoid potential in CD34+ cells derived from embryonic stem cells differentiated in the presence of vascular endothelial growth factor. Blood. 1, 2283–2295. 14. Giarratana, M.C., Verge, V., Schmitt, C., Tertho, J.M., et al. (2000) Presence of primitive lymphoid progenitors with NK or B potential in ex vivo expanded bone marrow cell cultures. Exp. Hematol. 28, 46–54. 15. Chiorean, E.G., Dylla, S., Olsen, K., Lenvik, T., Soignier, Y., et al. (2003) BCR/ABL alters the function of NK cells and the acquisition of killer immunoglobulin-like receptors (KIR). Blood. 101, 3327–3533.

Chapter 3 Subset Analysis of Human and Mouse Mature NK Cells Yoshihiro Hayakawa, Daniel M. Andrews, and Mark J. Smyth Abstract Identification of natural killer (NK) cell subsets has gained attention with the recent discovery that mature mouse NK cells comprise two distinct stages. Delineation of the stages is performed using the markers CD27 and CD11b on gated NK cells. The significance of this finding is underpinned by recent discoveries that mature human NK cells can also be discriminated by differential expression of CD27. This chapter will describe the methods required for the purification of lymphocytes from blood and other organs and the delineation of NK cell subsets by flow cytometry. Key words: Flow cytometry, natural killer, murine, human, CD27, CD11b, CD56.

1. Introduction Peripheral NK cells mediate numerous functions including the cytotoxic control of tumors and virus-infected cells as well as the production of cytokines such as IFN-␥ and TNF-␣ (1). Principally, NK cells have been regarded as mediators of natural host immune resistance. More recently it has been demonstrated that NK cells can act to regulate the generation of the adaptive immune response. Clearly, a high level of complexity underlies mature NK cells in secondary lymphoid and peripheral organs. The first dissection of NK cells into subsets arose through the work of Lewis Lanier in 1983 (2). However, it was not until 1989 that a universal method of human NK cell subset identification was identified (3). It is now widely accepted that human mature NK cells are comprised of CD56dim and CD56high cells; however, this antigen is not expressed on mouse K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 3, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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NK cells, and thus the translation of biological information concerning NK cells in the mouse has been problematic. While the development of mouse NK cells from precursors has been quite widely studied (4), the identification of mature stages has been largely ignored. Developmentally, mouse NK cells were defined as mature once expression of the integrin CD11b (Mac1) was detected (5). Subsequently, work from our laboratory showed that NK cells could be further identified on the basis of TRAIL expression in adult livers (6). This underlying suggestion of heterogeneity within mature murine NK cells was ultimately demonstrated by delineating two stages of mature NK cells on the basis of CD27 and CD11b (7). Significantly, human NK cells have also been shown to differentially express CD27 (8, 9), making comparative interpretations of the subsets functionality more clear. In the mouse, CD161+ (NK1.1) CD3− (or TCR␣␤− ) NK cells can be divided into CD27lo CD11blo , CD27hi CD11blo , CD27hi CD11bhi , and CD27lo CD11bhi stages. The CD27lo CD11blo stage remains poorly characterized and may be the most immature, but differentiation has been shown to proceed from CD27hi CD11blo through CD27hi CD11bhi to CD27lo CD11bhi (7). The most mature CD27lo NK cell subset possesses a higher threshold to stimulation and appears to be tightly regulated by the expression of NK cell inhibitory receptors (Ly-49/NKG2A). Comparatively, the CD27hi NK cell subset displays a greater effector function, exhibits a distinct tissue distribution and responsiveness to chemokines, and interacts productively with dendritic cells. Importantly, we and others have verified that CD27hi and CD27lo subsets with distinct cell surface phenotypes also exist in human peripheral blood (8, 9). These findings clearly reclassify mature NK cells into distinct stages and begin to discern their specific role in immune responses. The protocol for enriching lymphocytes from specific organs and delineating mature NK cell subsets by flow cytometry is described below.

2. Materials 2.1. Human PBMC Enrichment

1. Ficoll-Paque PLUS (Amersham Biosciences, Piscataway, NJ, USA). Store at 4◦ C. 2. Phosphate-buffered saline (PBS): Prepare stocks at 3.2 mM Na2 HPO4 , 0.5 mM KH2 PO4 , 1.3 mM KCl, and 135 mM NaCl. Adjust to pH 7.4 with HCl if necessary. Store at room temperature. R . 13 × 75 mm, 4 ml. 3. BD Vacutainer

4. 15 ml centrifuge tubes (BD Falcon, Bedford, MA, USA).

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1. Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 2% fetal bovine serum (FBS, Mt Wellington, Auckland, NZ). Store at 4◦ C. 2. Type IV collagenase (Worthington Biochemicals, Lakewood, NJ, USA) dissolved in PBS at 1 mg/ml. Store at 4◦ C. 3. Extraction buffer: RPMI-2% FBS supplemented with Type IV collagenase (10 ␮g/ml). 4. ACK lysis buffer: 8.29 g/L NH4 Cl, 1 g/L KHCO3 , 0.0367 g/L Na2 -EDTA. Adjust to pH 7.2–7.4 with HCl. Store at room temperature. 5. Percoll (Amersham Biosciences, Piscataway, NJ, USA). Store at 4◦ C. 6. 10× PBS: Prepare stocks at 32 mM Na2 HPO4 , 5 mM KH2 PO4 , 13 mM KCl, and 1.35 M NaCl. Store at room temperature. 7. 15 and 50 ml centrifuge tubes (BD Falcon, Bedford, MA, USA). 8. 40 ␮m cell strainers (BD Falcon, Bedford, MA, USA). 9. Spoon sieves (see Note 1).

2.3. Flow Cytometry Reagents

1. PBS 2% FBS: PBS supplemented with 2% fetal bovine serum (FBS, Mt Wellington, Auckland, NZ). Store at 4◦ C. Keep sterile. 2. Blocking buffer: Add at 2 ␮g/ml anti-Fc␥RII/Fc␥RIII monoclonal antibody (clone 2.4G2) to PBS 2% FBS. Prepare fresh each time. Store at 4◦ C. 3. 7AAD or Fluorogold (Sigma, St Louis, MO). Store at 4◦ C. Keep in darkness to prevent photobleaching. 4. 24-well tissue culture plates and 96-well U-bottom plates (BD Falcon). 5. 1.2 ml microtiter tubes (Molecular BioProducts, USA). 6. Monoclonal antibodies: Anti-human: CD161 (DX12, BD Pharmingen), CD3 (SK7, BD Pharmingen), CD27 (LG.3A10 or LG.7F9, eBioscience), CD56 (MEM188 or B159, BD Pharmingen). Anti-mouse: NK1.1 (PK136, eBioscience), CD3 (145-2C11, BD Pharmingen), CD27 (LG.7F9, eBioscience), CD11b (M1/70, eBioscience). Store at 4◦ C. Keep in darkness to prevent photobleaching.

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3. Methods NK cells are lymphocytes and thus have some characteristics of T and B cells. In studying NK cells by flow cytometry it is critically important to exclude any populations that may share a common marker. For instance, NKT cells in the mouse also express NK1.1 (albeit at a lower level than T cells) making it possible to accidentally include this cell population during analysis. The following protocol will outline how to ensure analysis is restricted to NK cells only and will provide details for their enrichment from peripheral organs. For obvious reasons our understanding of mouse NK cells in lymphoid and peripheral organs is much better, thus the detection of human NK cells in this chapter will be restricted to blood. Although the process for isolation of lymphocytes from mouse organs is, for the most part, generic, several aspects require some attention and will be addressed in the notes section. 3.1. Enrichment of Human PBMC from Whole Blood

R 1. 2 ml of whole blood is drawn into a BD vacutainer and supplemented with 2 ml of PBS (see Note 2).

2. The blood and PBS should be mixed. 3. Invert the Ficoll-Paque to ensure that it is mixed. Remove 3 ml and add to a 15 ml centrifuge tube. 4. Overlay the diluted blood sample onto the Ficoll-Paque ensuring that the blood and Ficoll-Paque do not mix. 5. Centrifuge the cells at 400× g for 30 min at room temperature. 6. Aspirate the supernatant to leave the lymphocytes intact (see Note 3). 7. Transfer the lymphocytes to a clean centrifuge tube (see Note 4). Add at least three volumes of PBS to the lymphocytes. 8. Centrifuge at 300× g for 7 min at room temperature. 9. Aspirate supernatant and wash as per steps 7 and 8. 10. Aspirate supernatant and resuspend cells in PBS 2% FBS.

3.2. Isolation of Lymphocytes from Mouse Organs

1. Pre-warm extraction buffer (see Section 2.2). Add extraction buffer to 24-well plate appropriate to organ. For spleen and lung add 1 ml, for lymph nodes add 200 ␮l. Livers should not be extracted in the presence of collagenase (see Note 5). 2. Enrichment of lymphocytes from the liver requires perfusion of this organ. Expose the femurs (upper leg bones), viscera,

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and chest of euthanized mice and cut the left ventricle of the heart. Expose the portal vein by moving the ventral liver lobe up toward the chest. Insert a 25G needle into the portal vein and perfuse the liver with 5–10 ml of PBS. Successful perfusion will result in the liver becoming beige in color. 3. Remove all organs and chop into small pieces (excluding the femurs) in extraction buffer (remembering not to use collagenase for the liver). Spleen, lung, and lymph nodes should be incubated at 37◦ C for 25 min with agitation. 4. For livers, chop into small pieces and wash through a metal spoon sieve by using the barrel of a 10 ml syringe to disrupt the liver across the sieve. Transfer cells into a 50 ml centrifuge tube. 5. The ends of the femurs should be cut with a scalpel. Using a 25 g syringe, aspirate the marrow with 5 ml of PBS. Aspirate through a 40 ␮m cell strainer inserted into a 50 ml tube to remove large chunks and wash through with 4 ml of PBS. 6. For spleen, lymph node, and lung transfer digested tissue and media into a 40 ␮m cell strainer inserted into a 50 ml tube. Wash wells of 24-well plate with 1 ml of PBS and add to tube containing organ. Disrupt organ through sieve using barrel of 10 ml syringe. Wash cells through strainer using 8 ml of PBS (2 × 4 ml at a time). 7. Centrifuge cells from all organs at 300× g for 7 min. 8. For the liver aspirate supernatant, add 10 ml of PBS and wash again as per step 7. 3.2.1. Preparation of Lymphocytes from Spleen, Lymph Node, and Bone Marrow

1. Once centrifuged, cells from the lymph node can be resuspended in blocking buffer (see Note 6). Maintain at 4◦ C. 2. Lyse red blood cells in bone marrow and spleen using ACK lysis buffer. Resuspend spleen or marrow cells in 1 ml of buffer for 2 min at room temperature. Stop lysis by addition of 4 ml of PBS and immediately centrifuge at 300× g for 7 min. 3. Resuspend cells in blocking buffer (see Note 6). Maintain at 4◦ C.

3.2.2. Preparation of Lymphocytes from Liver

1. Prepare a 37.5% Percoll solution. Adjust Percoll to 90% (mouse osmolarity) using 10× PBS. (i.e., add 5 ml of 10× PBS to 45 ml of Percoll). Dilute 90% Percoll to 37.5% using PBS (i.e., add 37.5 ml of 90% Percoll to 62.5 ml of PBS. Adjust volumes accordingly for scaling up) (see Note 7). 2. Resuspend aspirated liver preparations (Section 3.2, step 8) in 1 ml of 37.5% Percoll using a P1000 (see Note 8). Transfer cells from 50 ml centrifuge tube into a 15 ml centrifuge tube.

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Top up to 15 ml with remaining Percoll and centrifuge at 690× g for 12 min at room temperature. 3. Following centrifugation lymphocytes will pellet, while hepatocytes will float to the top. Aspirate the hepatocytes and supernatant, leaving the lymphocytes intact. 4. Lyse red blood cells in ACK lysis buffer as per Section 3.2.1, step 2 (see Note 9). 5. Resuspend cells in blocking buffer (see Note 6). Maintain at 4◦ C.

3.2.3. Preparation of Lymphocytes from Lung

1. Prepare a 90% Percoll solution as described in Section 3.2.2, step 1. Use this solution to prepare 45% and 67.5% solutions (i.e., for the 45% solution add 4 ml of 90% Percoll to 4 ml of PBS and for the 67.5% solution add 6 ml of 90% Percoll to 2 ml of PBS. Adjust volumes accordingly for scaling up) (see Note 7). 2. Resuspend aspirated lung preparations in 1 ml of 67.5% Percoll using a P1000 (see Note 8). Transfer cells from 50 ml centrifuge tube into a 15 ml centrifuge tube. Add further 2 ml of 67.5% Percoll to make a final volume of 3 ml. Carefully overlay 2 ml of 45% Percoll onto the cell suspension. Avoid mixing of the 45% and 67.5% layer. 3. Centrifuge at 800× g for 15 min at room temperature. Ensure that brake and acceleration are turned to minimum. 4. After centrifugation, lymphocytes will form a band at the interface of the 45% and 67.5% gradients. Aspirate the 45% gradient, leaving the lymphocyte band intact. 5. Transfer the lymphocytes to a 15 ml centrifuge tube and add PBS to fill the tube. 6. Centrifuge at 300× g for 7 min. 7. Lyse red blood cells in ACK lysis buffer as per Section 3.2.1 part 2. 8. Resuspend cells in blocking buffer (see Note 6). Maintain at 4◦ C.

3.3. Preparation/ Blocking of Cells for Staining with Fluorescent Antibodies

1. Cells should be counted and aliquoted in blocking buffer at 106 cells per stain (see Note 10). 2. For multiple samples use a 96-well U-bottom plate. Add 106 cells per well and maintain at 4◦ C for at least 30 min to ensure appropriate blocking of the Fc receptors. 3. Centrifuge plate at 200× g for 4 min. and aspirate supernatant.

Subset Analysis of Human and Mouse Mature NK Cells

3.4. Staining of Cells with Fluorescent Antibodies to Detect NK Cells

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1. Prepare a cocktail of antibodies comprising CD161, CD3, CD27, and CD56. Add an appropriate volume (see Note 11) per well and resuspend cells using a pipette. 2. Maintain cells at 4◦ C for at least 1 h.

3.4.1. Detecting Human NK Cell Subsets

3.4.2. Detecting Mouse NK Cell Subsets

1. Prepare a cocktail of antibodies comprising NK1.1, CD3, CD27, and CD11b. Add an appropriate volume (see Note 11) per well and resuspend cells using a pipette. 2. Maintain cells at 4◦ C for at least 1 h.

3.4.3. Labeling Dead Cells for Exclusion on the Flow Cytometer

1. Centrifuge labeled cells at 200× g for 4 min. 2. Aspirate supernatant and resuspend cells in 50–100 ␮l of either PBS 2% FBS with 2 ␮g/ml 7AAD or PBS 2% FBS with 2 ␮g/ml Fluorogold. Leave for 1 min at room temperature. FACS instruments with a 355 nm laser (such as the LSR-II) can use Fluorogold. Most bench top instruments (such as the CANTO) do not have this laser and must use the 488 nm laser to excite 7AAD. 3. Centrifuge cells at 200× g for 4 min. 4. Resuspend cells in 200–400 ␮l of PBS 2% FBS. Transfer to 1.2 ml microtiter tubes (see Note 12). Maintain in the dark and keep cool before acquiring samples on the flow cytometer.

3.5. Acquisition and Gating of Human and Mouse NK Cells by Flow Cytometry

1. Ensure that correct PMT voltages and compensation are applied to the cytometer. 2. In the first instance set an acquisition plot of forward scatter height (FSC-H) vs. forward scatter area (FSC-A) and place a gate around singlet cells (Fig. 3.1A). 3. Within the singlet population set an acquisition plot of FSCA vs. 7AAD or Fluorogold (consult with your FACS operator for correct channels on appropriate instruments). Set a gate around cells that have excluded the viability die. 4. On the live cells, set an acquisition plot of NK1.1 vs. CD3 (for mouse) or CD161 vs. CD3 (for human). In each instance set a gate around the NK1.1+ /CD3− or CD161+ /CD3− cells (Figs. 3.1 and 3.2). 5. On the NK cells set an acquisition plot of CD27 vs. CD11b (for mouse) or CD27 vs. CD56 (for human). Mouse NK cell subsets are identified as immature (CD27− /CD11b− , CD27+ /CD11b− ) and mature and can be subdivided into CD27+ /CD11b+ and CD27− /CD11b+ (Fig. 3.1).

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Fig. 3.1. The gating procedure and use of fluorescent minus one staining (FMO) to detect mouse NK cell subsets. (A) Singlet cells (to exclude cell doublets, which can appear as cells expressing both fluorochromes) are gated on the 45◦ angle of a plot between FSCA and FSC-H prior to gating on live cells (represented here are fluorogold (FG)-negative cells). The live population is then shown with NK1.1 and CD3, NK cells are gated as NK1.1+ and CD3− . (B) In order to set the gates properly for the subsets FMO are used. The first panel shows gated NK cells (NK1.1+ /CD3− ) on which only CD11b is stained, while in the second panel only CD27 is stained. This allows the quadrant gates to be set appropriately on CD11b+ and CD27+ . The final panel in part B shows the four major subsets (CD27− CD11b− a, CD27+ /CD11b− b; CD27+ /CD11b+ c; CD27− /CD11b+ d). The quadrant gates have been set correctly by the use of the FMO setup tubes.

Human NK cell subsets are identified as CD56dim /CD27− , CD56hi /CD27− , and CD56hi /CD27+ (Fig. 3.2). See Note 13 for choice of fluorochromes and setting gates appropriately.

Fig. 3.2. The gating procedure and use of fluorescent minus one staining (FMO) to detect human NK cell subsets. Singlet cells (to exclude cell doublets, which can appear as cells on the line of identity between FSC-A and FSC-H) are gated on the 45◦ angle prior to gating on live cells (represented here are fluorogold-negative cells) as shown in Fig. 3.1. The live population is then shown in a plot of CD161 vs. CD3, and NK cells are gated as CD161+ and CD3− . In order to set the gates properly for the subsets, FMO are used. The second panel shows gated NK cells (CD161+ /CD3− ) on which only CD56 is stained, while in the third panel only CD27 is stained. This allows the gates to be set appropriately on CD56+ and CD27+ subsets. The final panel shows the three major subsets (CD56dim /CD27− ; CD56hi /CD27− ; CD56hi /CD27+ ). Box gates have been set correctly by the use of the FMO setup tubes. CD27hi /CD56hi cells are gated by aligning the vertical edges of the CD27 box with the CD56hi box.

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6. Set the cytometer to acquire samples. For analysis of NK cell subsets it is appropriate to collect at least 2000 NK cell events from each organ (Fig. 3.3).

Fig. 3.3. Representative density plots showing mouse NK cells and their subsets in the spleen, liver, lung, bone marrow, and lymph node. (A) Identification of NK cells by using NK1.1 and CD3 on preparations from the spleen, liver, lung, and bone marrow and lymph node. (B) Identification of the NK cell subsets in the different organs. The subsets of CD3− NK1.1+ NK cells are differentially represented across a range of lymphoid and non-lymphoid organs.

4. Notes 1. Spoon sieves with a pore size of around 1–2 mm are most appropriate. Any larger than this and the liver in particular will not be sufficiently homogenized prior to centrifugation. 2. Although mouse blood can be treated in the same manner as human blood, in order to enrich murine lymphocytes this protocol is not necessary. The simpler method for mouse blood is to harvest blood directly into a 1 ml centrifuge tube into which 20 ␮l of 50 mM EDTA has been added. The blood/EDTA is then mixed to prevent coagulation and the cells pelleted by centrifugation. At this point it is possible to then process as per Section 3.2.1, step 2. 3. Always aspirate gently and leave around 500 ␮l of serum remaining above the lymphocyte band. 4. When harvesting lymphocytes from a band within a gradient it is best to aspirate from slightly above the cells. Try to remove as little of the gradient mixture (in this case Ficoll) as this will add to the density of the subsequent wash. If too much gradient and not enough PBS is in the wash step then the lymphocytes will not pellet.

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5. Although it is possible to remove lymphocytes from spleen and lymph node by mechanical disruption between glass slides we have found this process to be less efficient and more damaging to the cells than collagenase digestion. Digesting organs in collagenase removes cells from the stroma, thereby increasing yields. Although collagenase may increase yields from liver and lung, collagenase should never be used when analyzing liver NK cells for CD27. CD27 expression in the liver is extremely sensitive to collagenase digestion resulting in NK cell preparations in which CD27 cannot be detected. 6. Lymph nodes are free from red blood cells and thus do not need ACK lysis. Naive lymph nodes and lungs have very few cells (˜106 ), thus volumes for resuspending should be carefully controlled in order to have accurate cell counts. The most appropriate volume for resuspending of bone marrow, spleen, and livers for accurate counting is 1 ml. Our laboratory normally resuspends lymph nodes in 200 ␮l of blocking buffer. For trypan blue exclusion counting a 1:10 to 1:20 dilution is acceptable while a 1:500 dilution is fine for use in a Coulter Counter. Using the volumes described here will result in high cell densities. This is important when transferring the cells to a 96-well U-bottom plate. The maximum volume in these wells is around 200 ␮l. Thus, resuspending the organs in a higher density in blocking buffer allows their transfer directly from the centrifuge tube to the 96-well plate without having to spin the cells and increase their density before transferring to the 96-well plate. 7. Percoll needs to be at room temperature when spinning. Always prepare fresh the morning of the experiment, prior to organ collection. As stock Percoll is kept at 4◦ C it is advisable to place the prepared gradients in a 37◦ C water bath while collecting organs. 8. The use of a P1000 pipette is more appropriate as this will better homogenize the pellet than larger pipettes (i.e., a 5–10 ml pipette). A more homogenized pellet will allow better separation of the lymphocytes from the hepatocytes or lung parenchyma. 9. When the lymphocytes are in ACK lysis buffer transfer them to a fresh tube. If the cells are pelleted in the same tube that the Percoll gradient was used for, some hepatocytes that can stick to the top of the tube may pellet with the lymphocytes following centrifugation of the ACK lysed cells. These hepatocytes can block the flow cytometer.

Subset Analysis of Human and Mouse Mature NK Cells

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10. Cell numbers used in the FACS protocol must always be equivalent in order to ensure reproducibility of the FACS staining. If doing multiple stains it may only be possible to stain 105 cells (especially in the lung where only 106 total cells are recovered). 11. While most commercial outlets claim that 100 ␮l per well of 106 cells/cocktail is necessary we routinely use 20 ␮l per well on 106 cells. This is beneficial as it reduces reagent use by fivefold. It is always best to titrate amounts of antibodies used to optimize staining intensity and thereby assure that adequate gating can be achieved. 12. The use of microtiter tubes can greatly facilitate large experiments. These tubes can be inserted into a 96-well plate or 200 ␮l tip box. In contrast to the larger centrifuge tubes, the 1.2 ml tubes are small enough for all 12 ends of a multichannel pipette to fit. Thus, transfer of 12 wells at a time is possible. 13. When analyzing NK cell subsets the choice of fluorochromes is important. Our laboratory routinely uses NK1.1-PE (2 ␮g/ml), CD3-PECy5.5 (2 ␮g/ml), CD27APC (1 ␮g/ml), and CD11b-FITC (1 ␮g/ml). It is important to note that many mouse strains do not have NK1.1, including BALB/c. In order to identify NK cells in BALB/c mice it is necessary to use DX5 in replacement of NK1.1. DX5 should be used quite concentrated as the antigen is expressed weakly and DX5lo cells (which are also CD3− ) are not mature NK cells and may make analysis difficult. More recently an antibody to NKp46 has become available. This antigen is restricted to NK cells and is found in all mouse strains; however, it is yet to replace NK1.1 for FACS identification. The use of NK1.1-PE results in a bright signal for NK cells and is then easily discriminated from T cells and NKT cells (can be confirmed with CD1d tetramer). We do not encourage the use of similar fluorochromes for analysis of CD27 and CD11b. For example the use of CD27-APC and CD11b-APC-Cy7 is problematic. As these fluorochromes have very similar excitation and emission characteristics it is difficult to compensate them accurately. This makes it difficult to accurately discriminate the three NK cell stages. While APC-Alexa750 does not bleed into the APC channel as much, a similar problem regarding the subsets is still observed. In order to gate the subsets accurately it is necessary to include Fluorescent Minus One tubes (FMO). These tubes will contain either NK1.1/CD3/CD27 or NK1.1/CD3/CD11b. The use of these FMO tubes will allow quadrant gates to be set accurately.

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Acknowledgments The authors would like to thank Ms. Anabel Silva for her assistance with developing the protocols for identifying human NK cell subsets with CD27. This work was supported by a National Health and Medical Research Council of Australia Program Grant and Doherty Fellowship (DMA, MJS). References 1. Smyth, M. J., Hayakawa, Y., Takeda, K., and Yagita, H. (2002) New aspects of naturalkiller-cell surveillance and therapy of cancer. Nat Rev Cancer 2, 850–61. 2. Lanier, L. L., Le, A. M., Phillips, J. H., Warner, N. L., and Babcock, G. F. (1983) Subpopulations of human natural killer cells defined by expression of the Leu-7 (HNK1) and Leu-11 (NK-15) antigens. J Immunol 131, 1789–96. 3. Ellis, T. M., and Fisher, R. I. (1989) Functional heterogeneity of Leu 19”bright”+ and Leu 19”dim”+ lymphokineactivated killer cells. J Immunol 142, 2949–54. 4. Huntington, N. D., Vosshenrich, C. A., and Di Santo, J. P. (2007) Developmental pathways that generate natural-killer-cell diversity in mice and humans. Nat Rev Immunol 7, 703–14. 5. Kim, S., Iizuka, K., Kang, H. S., Dokun, A., French, A. R., Greco, S., and Yokoyama, W. M. (2002) In vivo developmental stages

6.

7.

8.

9.

in murine natural killer cell maturation. Nat Immunol 3, 523–8. Takeda, K., Cretney, E., Hayakawa, Y., Ota, T., Akiba, H., Ogasawara, K., Yagita, H., Kinoshita, K., Okumura, K., and Smyth, M. J. (2005) TRAIL identifies immature natural killer cells in newborn mice and adult mouse liver. Blood 105, 2082–9. Hayakawa, Y., Huntington, N. D., Nutt, S. L., and Smyth, M. J. (2006) Functional subsets of mouse natural killer cells. Immunol Rev 214, 47–55. Silva, A., Andrews, D. M., Brooks, A. G., Smyth, M. J., and Hayakawa, Y. (2008) Application of CD27 as a marker for distinguishing human NK cell subsets. Int Immunol 20, 625–30. Vossen, M. T., Matmati, M., Hertoghs, K. M., Baars, P. A., Gent, M. R., Leclercq, G., Hamann, J., Kuijpers, T. W., and van Lier, R. A. (2008) CD27 defines phenotypically and functionally different human NK cell subsets. J Immunol 180, 3739–45.

Chapter 4 Assessing Licensing of NK Cells A. Helena Jonsson and Wayne M. Yokoyama Abstract Natural killer (NK) cells express receptors to detect and kill target cells based on expression of target cell surface molecules. Through a process termed NK cell licensing, only NK cells that express inhibitory receptors (e.g., Ly49 receptors in the mouse) for self-major histocompatibility complex (MHC) class I molecules become functionally competent to be triggered through their activation receptors. To determine the licensing status of particular Ly49+ murine NK cell subsets, splenocytes are stimulated with plate-bound anti-NK1.1 monoclonal antibody in the presence of brefeldin A and then assessed for NK cell activation on a single-cell basis using intracellular cytokine interferon-␥ staining and flow cytometry. Key words: Natural killer cells, NK cells, NK cell licensing, NK cell education, Ly49, inhibitory receptors, self-tolerance, NK cell activation.

1. Introduction Natural killer (NK) cells express receptors to detect and kill target cells that display cell surface ligands for these receptors (1). To accomplish this task, NK cells use activation and inhibitory receptors to balance the ability to recognize “missing” or “induced” self with the need to maintain self-tolerance (2, 3). Through an NK cell education process termed licensing, only NK cells that express inhibitory receptors for self-major histocompatibility complex (MHC) class I molecules become functionally competent to be triggered through their activation receptors (Fig. 4.1) (4–6). Conversely, NK cells that lack inhibitory receptors for selfMHC or develop in an environment devoid of MHC class I are unresponsive to most conventional NK cell activation receptor stimuli and hence are termed “unlicensed” (4). While the initial K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 4, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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Fig. 4.1. Licensing of NK cells. A developing NK cell stochastically expresses some but not all inhibitory NK cell receptors. In a host with an MHC environment where the NK cell is able to bind MHC class I via one or more of its inhibitory receptors (shown as matching black Ly49 and MHC class I molecules), the NK cell receives a licensing signal. This licensing signal requires an intact cytoplasmic Ly49 tail, including the ITIM. An NK cell that cannot engage MHC class I with any of its inhibitory receptors remains unlicensed, i.e., hyporesponsive. Upon stimulation via their activation receptors ex vivo, a higher percentage of licensed NK cells produce cytokines as compared to unlicensed NK cells.

studies of licensing were performed in mice, recent studies suggest a similar process occurs in humans (7, 8). In mice, the major NK cell inhibitory receptors that bind selectively to specific classical MHC class I alleles include members of the C-type lectin-like Ly49 family, such as Ly49A, C, I, and G2. For example, Ly49A binds H-2Dd but not H-2Kb , whereas the more promiscuous Ly49C binds H-2Kb well and H2Dd relatively weakly (9). Furthermore, inhibitory Ly49 receptors are expressed in a stochastic or probabilistic fashion such that each individual NK cell expresses a random assortment of the genome-encoded Ly49 molecules (10, 11). Because of these expression and binding patterns of Ly49 receptors, a significant portion of the NK cell population in any given mouse may be unable to engage self-MHC class I expressed on normal cells (4, 12). The unlicensed state of these cells ensures

Assessing Licensing of NK Cells

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their self-tolerance since they are unable to respond to “missing self” stimuli. Notably, unlicensed cells are not hyporesponsive due to exhaustion because phorbol ester and ionomycin stimulation induces abundant cytokine production by these cells comparable to licensed NK cell populations (4). Finally, unlicensed NK cells can be induced to become functionally responsive in an MHCindependent manner by other stimuli, such as cytokine exposure in vivo or in vitro (4). In the laboratory, a licensed NK cell population is currently revealed by response to activation receptor cross-linking. AntiNK1.1 (Nkrp1c) has been most widely used though licensed NK cells can also respond to cross-linking of Ly49D, NKG2D, CD16, and YAC-1 targets (4). The use of brefeldin A and intracellular cytokine staining enables single-cell detection of activation and surface receptor expression using flow cytometry. The frequency of cytokine production by a given subset reflects its licensing status (Fig. 4.1).

2. Materials 1. Mice, including suitable positive and negative control strains (see Note 1). 2. R10 culture medium: RPMI-1640 supplemented with 10% heat-inactivated (45 min, 56◦ C) fetal bovine serum (FBS), 2 mM supplemental L-glutamine, 10 U/mL penicillin (optional), 10 ␮g/mL streptomycin (optional), 0.1 mM 2mercaptoethanol. Store at 4◦ C. 3. Phosphate-buffered saline (PBS): 137 mM NaCl, 8 mM Na2 HPO4 , 2.7 mM KCl, 1.5 mM KH2 PO4 , 0.5 mM MgCl2 , pH 7.2. Store at room temperature. 4. FACS staining buffer: 1× PBS (see above) supplemented with 1% FBS and 0.2% sodium azide (see Note 2). Store at 4◦ C. 5. Tissue culture-treated 6-well plates (see Note 3). 6. Purified PK136 (anti-NK1.1) antibody (#HB-191 at American Type Culture Collection (ATCC), Manassas, VA). 7. Brefeldin A – GolgiPlug (BD Biosciences, San Jose, CA) contains 0.1% BFA in DMSO (see Note 4). Store in 50 ␮L aliquots at 4◦ C (optional). 8. Phorbol ester and ionomycin – as optional positive control for cytokine production. Store aliquoted stock solutions of phorbol ester and ionomycin in DMSO at 0.5 and 1 mg/mL, respectively, at –20◦ C. Aliquot volumes of 10 and 20 ␮L, respectively, are convenient.

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9. BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences). Includes a formaldehyde- and saponin-based fixation and permeabilization reagent as well as a 10x concentrate of Perm/Wash buffer containing saponin to maintain permeabilization. 10. Staining tubes or plates (see Note 5). 11. Cell lineage antibodies (Table 4.1). These should include antibodies to gate out CD3+ cells and ideally also CD19+ cells. There should also be an antibody to positively gate on NK cells, such as a monoclonal antibody directed at NKp46, NK1.1 (in C57BL/6 mice), or integrin ␣2 (i.e., mAb clone DX5).

Table 4.1 Recommended fluorochrome selections for four-color flow cytometry Channel (FACSCalibur)

Staining setup #1

Staining setup #2

FL1

FITC-conjugated anti-Ly49 mAb

FITC-conjugated anti-IFN␥ mAb

FL2

PE-conjugated anti-IFN␥ mAb

Biotinylated anti-Ly49 mAb; PE-conjugated streptavidin

FL3

PerCP Cy5.5-conjugated anti-CD3 and anti-CD19 mAbs

PerCP Cy5.5-conjugated antiCD3 and anti-CD19 mAbs

FL4

APC-conjugated anti-NK1.1 mAb

APC-conjugated anti-NK1.1 mAb

12. Antibody monospecific for the inhibitory NK cell receptor of interest (see Note 6). 13. Antibody against murine interferon-␥ (clone XMG1.2). 14. Fc␥RII/III block, e.g., 2.4G2 supernatant (#HB-197 at ATCC). 14. 40 ␮m nylon mesh through which to filter cells after staining.

3. Methods 3.1. Preparation of Reagents 3.1.1. Prepare Splenocyte Suspension 3.1.2. Prepare Antibody-Coated Plates

1. For murine NK cells, harvest spleens and prepare RBC-lysed single-cell suspensions at a concentration of 107 cells/mL in cold, fresh R10 medium. 1. Dilute purified PK136 (anti-NK1.1) antibody to concentrations of 5, 2, and 0.5 ␮g/mL, respectively, in PBS (see Notes 7 and 8).

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2. Add 1 mL of antibody dilution to each well of polystyrene 6-well plates, making one well per stimulation dose per mouse. For example, for 10 mice, make 10 wells of 5 ␮g/mL PK136, 10 wells of 2 ␮g/mL PK136, and 10 wells of 0.5 ␮g/mL PK136. 3. Also make uncoated wells (one per mouse) by adding PBS alone or purified isotype control antibody diluted in PBS, for unstimulated control samples. 4. Incubate the plates in 37◦ C 5% CO2 incubator for 90 min. 5. Rinse the plate 3× with PBS. Do not let the wells dry at any time. Immediately add cells to the wells as described below. 3.2. Stimulation of NK Cells 3.2.1. Plate-Bound Monoclonal Antibody Stimulation of Murine Splenocytes

1. Add 107 splenocytes (i.e., 1 mL cell suspension) to each well. Gently swirl the plates to assure uniform distribution of cells over the bottom of the well. 2. If including a phorbol ester and ionomycin positive control, dilute them together in R10 medium to concentrations of 0.5 and 4 ␮g/mL, respectively. For example, dilute 5 ␮L of 0.5 mg/mL phorbol ester and 20 ␮L of 1 mg/mL ionomycin into 5 mL of R10 medium. Add 1 mL of this dilution to 1 mL of cell suspension. 3. Incubate the cells in a 37◦ C 5% CO2 incubator for 1 h (see Note 9). 4. After 1 h of stimulation, add Brefeldin A (BD GolgiPlug) as follows: dilute 50 ␮L of 0.1% BFA stock into 450 ␮L of R10 medium for 100× working stock, and add 12 ␮L working stock to each sample to each sample for a final concentration of 1.2×. Mix well by gently swirling the plate. 5. Incubate the cells for an additional 7 h in a 37◦ C 5% CO2 incubator (see Note 10). 6. Stop the stimulation by adding 5 mL cold FACS staining buffer and storing the plates at 4◦ C until ready to proceed to cell harvest (usually overnight) (see Note 11).

3.2.2. Cell Harvest

1. Harvest cells by pipeting up and down repeatedly with a 5 or 10 mL pipet. Collect the cells into a 15 mL tube. Wash the well with an additional 5 mL FACS staining buffer. Collect the wash into the same 15 mL tube. 2. Spin tubes at 500g for 5 min at 4◦ C. Discard supernatant. 3. Resuspend pellet in the remaining buffer and transfer the cell suspension to a FACS tube or deep-well plate (see Note 5). 4. Also set up unstained and single-stain control tubes or wells using pooled residual stimulated cells or fresh splenocytes (see Note 12).

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3.3. Immunofluorescent Staining of Stimulated Cells 3.3.1. Surface Staining

1. If using a biotinylated anti-Ly49 receptor monoclonal antibody, dilute this antibody in Fc ␥R block (e.g., 2.4G2 supernatant) and add to the cells (see Note 6). If the anti-Ly49 receptor antibody is directly conjugated, proceed to Step 4. Incubate the cells on ice for 30 min. 2. Wash twice in 1 mL FACS staining buffer. Spin tubes or plates at 500g after each wash. After second wash, resuspend cells in 100 ␮L FACS buffer. 3. Make antibody master mix by combining anti-CD3, CD19, and -NKp46 antibodies and either the directly conjugated anti-Ly49 receptor or a secondary reagent (e.g., streptavidin-PE) in Fc␥R block (2.4G2 supernatant) in a total volume of 70 ␮L per tube or well. Aliquot the master mix to each tube and pipet or vortex to mix. Also make single-stain controls. Incubate the cells on ice for 30 min. 4. Wash twice in 1 mL FACS staining buffer. Spin tubes or plates at 500g after each wash.

3.3.2. Intracellular Cytokine Staining

1. After aspirating or decanting the supernatant from the last wash of the surface staining, vortex the plate or tubes to loosen the pellets. 2. Add 250 ␮L Cytofix/Cytoperm to each sample and pipet repeatedly to mix. Incubate on ice for 25 min, shaking the tubes or plate after 12 min. 3. Add 1 mL of Perm/Wash buffer to each sample and incubate 5 min on ice. 4. Spin at 500g for 5 min and discard supernatant. 5. Add 1 mL of Perm/Wash buffer and resuspend the pellet by pipeting. Spin for 5 min at 500g and discard supernatant. 6. Make a master mix of anti-IFN␥ antibody diluted in Perm/Wash buffer to a final volume of 50 ␮L per sample. Aliquot the master mix to each of the wells and pipet or vortex to mix. Incubate the cells 30 min on ice. 7. Wash twice in 1 mL Perm/Wash buffer. 8. Add 300 ␮L of FACS staining buffer, resuspend the pellet by pipeting, and filter each sample through 40 ␮m nylon mesh into new FACS tubes. 9. Store FACS tubes at 4◦ C, protected from light, until ready to analyze by flow cytometry (see Note 13).

3.4. Flow Cytometric Data Collection and Analysis 3.4.1. Data Collection

1. Arrange the flow cytometer settings for post-collection compensation (see Notes 14 and 15). 2. For best results, collect flow cytometry data for at least 106 live cells (approximately 15,000 NK cells, gated as NKp46+

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Fig. 4.2. Analysis of licensing assay data. To quantify licensing, graph NK cells (NK1.1+ CD3− CD19− ) on a dot plot with Ly49 on the vertical axis and IFN␥ on the horizontal axis and make a quadrant gate to frame the four populations. In this example, the middle plot displays NK cells from a B10.D2 (H-2d ) mouse, whereas the bottom plot displays NK cells from a C57BL/10 (H-2b ) mouse. The frequency of IFN␥ production by Ly49+ NK cells is calculated using the formula UR/(UR+UL)∗ 100. A high frequency of IFN␥ production (usually 12–25%) indicates licensing of that Ly49+ population. The “licensing ratio” describes the increased propensity of Ly49A+ NK cells to produce IFN-␥ relative to Ly49A− NK cells and is the result of dividing the percentage of Ly49A+ NK cells that produce IFN␥ by the percentage of Ly49A− NK cells that produce IFN␥. If the Ly49 receptor does not mediate licensing, the licensing ratio is approximately 1 (usually 0.8– 1.2). If the Ly49 receptor mediates licensing, the licensing ratio will be over 1.5.

CD3− CD19− ). This often means collecting all events until the sample runs out. 3.4.2. Analyze Flow Cytometry Data

1. Gate on live cells using a forward scatter and side scatter dot plot. Subsequently, gate on NK cells (NKp46+ CD3− CD19− ) (see Note 16).

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2. Display NK cells on a dot plot with Ly49 on the vertical axis and IFN␥ on the horizontal axis and make a quadrant gate to frame your four populations (Fig. 4.2). The quadrants will henceforth be referred to as UR (upper right), UL (upper left), LL (lower left), and LR (lower right). 3. Calculate the frequency of IFN␥ production by Ly49+ NK cells using the formula UR/(UR+UL)∗ 100. A high frequency of IFN␥ production (usually 12–25%) indicates licensing of that Ly49+ population. 4. Calculate the “licensing ratio” of the Ly49+ NK cell population using the formula (UR/(UR+UL))/(LR/(LR+LL)). If the Ly49 receptor does not mediate licensing, the licensing ratio is approximately 1 (usually 0.8–1.2). If the Ly49 receptor mediates licensing, the licensing ratio will be over 1.5 (see Note 17). Positive and negative control samples are very important in these calculations (see Note 1).

4. Notes 1. Including appropriate positive and negative control strains is very important. For Ly49C+ NK cells, a C57BL/6 control mouse is a good positive control for licensing. For experiments addressing licensing through Ly49A, a B10.D2 mouse (H-2d , Stock #000463, Jackson Labs, Bar Harbor, ME) or a H-2Dd -transgenic mouse is an appropriate positive control. In most cases, a MHC class I-deficient mice (e.g., ␤2 m−/− ) is a good negative control for cytokine production: NK cells from these mice should produce little if any cytokine in this assay, although they can respond to high concentrations of cross-linking antibody. Nonetheless, licensed NK cells respond better than unlicensed NK cells regardless of strength of activation (4). It is also often useful to include a MHC-sufficient strain that is known not to license the Ly49+ population of interest. For example, a B10.D2 mouse (H-2d ) can serve as an unlicensed control for studies of Ly49C+ NK cells, as can a C57BL/6 mouse (H-2b ) for studies of Ly49A+ NK cells. In addition, the use of age-matched mice is important as NK cell activity changes with age in early adult mice. Dates of birth should ideally be matched to within 2–3 weeks. Mice younger than 7 weeks and older than 16 weeks may have poor NK cell responses and should be avoided. 2. In addition to preventing contamination, azide in the FACS staining buffer blocks internalization or shedding of antibody–receptor complexes.

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3. This assay is normally performed in 6-well plates using 107 splenocytes in a 1 mL reaction volume. However, the assay can be scaled down if necessary for other applications where cell number is limited. 4. Monensin can be used as an alternative to brefeldin A, if desired. 5. For staining of stimulated cells, conventional 12 × 75 mm round bottom culture tubes work well. For processing of larger numbers of samples, deep-well conical-bottom plates (e.g., 2.2 mL Mark II plates (#AB-0932), ABgene, Surrey, UK) may be more convenient. Washes can then be performed quickly and easily with a P1000 multichannel pipetman. Traditional 96-well conical bottom plates are less ideal for staining several million cells per well as they can only hold small volumes and therefore will require extra wash steps. 6. The anti-Ly49 monoclonal antibody (mAb) must be selected with care. For best results, the mAb should be monospecific, such as clones 4LO33 (anti-Ly49C) and JR9 (anti-Ly49A). Certain mAbs, such as clone A1 (antiLy49A), are very sensitive to cis engagement of Ly49 with MHC class I expressed on the same cell, which results in decreased mAb reactivity (4, 13). These mAb clones should be avoided because it may be difficult or impossible to distinguish the relevant Ly49+ and Ly49– populations. If alternative mAb clones are not available, carefully optimize the choice of fluorochromes and staining conditions. For example, biotinylated 4LO33 (anti-Ly49C) followed by PE-conjugated streptavidin produces a Ly49C+ population that is clearly distinguishable from the Ly49C− cells, while staining with FITC-conjugated 4LO33 does not. 7. The cell suspensions should be ready to go by the time the plates are washed. The timing usually works out right if the purified PK136 antibody is added to the plates immediately after RBC lysis of the splenocytes. The 90-min incubation of the plates provides enough time to count the cells and resuspend them to 107 cells/mL. 8. If enough cells are available, do one well of each antibody concentration for each mouse. If cell number is a limitation, omit the 0.5 and 5 ␮g/mL PK136 stimulations. 9. Incubation of less than 1 h before adding BFA results in significantly lower yield of intracellular cytokines. 10. After the addition of BFA, each hour of incubation increases the amount of intracellular cytokines. However, incubations longer than 8 h may increase cell death due to

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BFA toxicity. Seven hours of incubation with BFA allows for ample accumulation of intracellular cytokines without any noticeable cytotoxic effects. 11. The overnight incubation at 4◦ C is not a necessary step but is merely for convenience. 12. Single-stain controls should be made for each fluorochrome. Since NK cells are rare among splenocytes, avoid anti-NK1.1 or anti-Ly49 antibodies for these controls. Instead, use fluorochrome-conjugated antibodies directed at more common markers such as CD19 or CD8 that are expressed at similar levels to markers of interest in NK cells. The unstained control should consist of cells pooled from the remainder of the stimulation. The single-stained controls can be made of these cells as well or from fresh splenocytes. Alternatively, compensation control beads (BD Biosciences) can be used in place of cells. 13. Since the cells are fixed, flow cytometric data collection can be postponed for up to 1 week. However, best results are obtained by analyzing the cells within 24 h of staining. 14. Licensing assays require the use of a flow cytometer that can distinguish at least four colors. Table 4.1 lists two fluorochrome combinations that work well on BD FACSCalibur machines. If simultaneous analysis of multiple Ly49 populations is desired, the fluorochromes must be selected with care to avoid compensation issues since the NK cell subset populations are so rare. 15. If possible, perform compensation after data collection (14). Many contemporary flow cytometry analysis software programs (e.g., FlowJo, Treestar, Ashland, OR) feature easy-to-use post-collection compensation functions. 16. If using NK1.1 to define the NK cell population, be aware that plate-bound PK136 stimulation partially inhibits subsequent staining with fluorochrome-conjugated antiNK1.1 antibodies. In this situation, use a free-form polygon gate to gate on all NK1.1+ cells, even those exhibiting intermediate staining. (NK cells with lower NK1.1 expression are often the most robust cytokine-producing cells.) Also, stimulating with a lower concentration (e.g., 2 ␮g/mL or lower) of plate-bound PK136 may make the NK1.1+ population more distinct. Alternatively, use antibodies against a different marker, such as NKp46 or integrin ␣2 (DX5), to identify NK cells. 17. The licensing ratio is dependent on the “background” IFN-␥ production by NK cells, i.e., the percentage of IFN␥+ cells among NK cells that lack the receptor of inter-

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est. This ratio may therefore falsely indicate no licensing of a given Ly49 subset if the Ly49-negative subset expresses another receptor capable of self-MHC recognition. Care must be taken when using licensing ratios, especially between murine strains and Ly49/MHC combinations that differ from the Ly49A-H-2Dd and Ly49C-H2 Kb interactions already documented. Absolute frequencies of IFN␥ production may be preferable in these situations. It is also possible that if the NK cells had been previously stimulated in vivo or in vitro with cytokines or other stimuli, then it may be difficult to show an MHCdependent licensing effect because the stimuli may induce unlicensed NK cells to become functionally competent (4).

References 1. Yokoyama, W. M. (2008) Chapter 17. Natural killer cells, in Fundamental Immunology. (Paul, W. E., ed.), Lippincott-Raven, New York. pp. 483–517. 2. Diefenbach, A., and Raulet, D. H. (2003) Innate immune recognition by stimulatory immunoreceptors. Curr Opin Immunol 15, 37–44. 3. Ljunggren, H. G., and Karre, K. (1990) In search of the ‘missing self’: MHC molecules and NK cell recognition. Immunol Today 11, 237–244. 4. Kim, S., Poursine-Laurent, J., Truscott, S. M., Lybarger, L., Song, Y. J., Yang, L., French, A. R., Sunwoo, J. B., Lemieux, S., Hansen, T. H., and Yokoyama, W. M. (2005) Licensing of natural killer cells by host major histocompatibility complex class I molecules. Nature 436, 709–713. 5. Yokoyama, W. M., and Kim, S. (2006) How do natural killer cells find self to achieve tolerance? Immunity 24, 249–257. 6. Yokoyama, W. M., and Kim, S. (2006) Licensing of natural killer cells by self-major histocompatibility complex class I. Immunol Rev 214, 143–154. 7. Anfossi, N., Robbins, S. H., Ugolini, S., Georgel, P., Hoebe, K., Bouneaud, C., Ronet, C., Kaser, A., DiCioccio, C. B., Tomasello, E., Blumberg, R. S., Beutler, B., Reiner, S. L., Alexopoulou, L., Lantz, O., Raulet, D. H., Brossay, L., and Vivier, E. (2004) Expansion and function of CD8+ T cells expressing Ly49 inhibitory receptors specific for MHC class I molecules. J Immunol 173, 3773–3782. 8. Kim, S., Sunwoo, J. B., Yang, L., Choi, T., Song, Y. J., French, A. R., Vlahiotis,

9.

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11.

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A., Piccirillo, J. F., Cella, M., Colonna, M., Mohanakumar, T., Hsu, K. C., Dupont, B., and Yokoyama, W. M. (2008) HLA alleles determine differences in human natural killer cell responsiveness and potency. Proc Natl Acad Sci U S A 105, 3053–3058. Hanke, T., Takizawa, H., McMahon, C. W., Busch, D. H., Pamer, E. G., Miller, J. D., Altman, J. D., Liu, Y., Cado, D., Lemonnier, F. A., Bjorkman, P. J., and Raulet, D. H. (1999) Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors. Immunity 11, 67–77. Raulet, D. H., Vance, R. E., and McMahon, C. W. (2001) Regulation of the natural killer cell receptor repertoire. Annu Rev Immunol 19, 291–330. Pascal, V., Stulberg, M. J., and Anderson, S. K. (2006) Regulation of class I major histocompatibility complex receptor expression in natural killer cells: one promoter is not enough! Immunol Rev 214, 9–21. Fernandez, N. C., Treiner, E., Vance, R. E., Jamieson, A. M., Lemieux, S., and Raulet, D. H. (2005) A subset of natural killer cells achieves self-tolerance without expressing inhibitory receptors specific for selfMHC molecules. Blood 105, 4416–4423. Doucey, M. A., Scarpellino, L., Zimmer, J., Guillaume, P., Luescher, I. F., Bron, C., and Held, W. (2004) Cis association of Ly49A with MHC class I restricts natural killer cell inhibition. Nat Immunol 5, 328–336. Herzenberg, L. A., Tung, J., Moore, W. A., Herzenberg, L. A., and Parks, D. R. (2006) Interpreting flow cytometry data: a guide for the perplexed. Nat Immunol 7, 681–685.

Chapter 5 Use of Stem Cell Radiation Chimeras to Analyze How Domains of Specific Proteins Impact on Murine NK Cell Development In Vivo Rebecca H. Lian and Vinay Kumar Abstract Although the use of mutant mice has been extremely useful in identifying those proteins and molecules specifically required for the development of NK cells, the establishment of a well-defined protocol to replicate in vitro the major steps corresponding to the process of NK cell differentiation and maturation has enabled us to dissect the molecular events governing certain aspects of NK cell development. This chapter describes a protocol that combines both the use of mutant mice and the in vitro bone marrow (BM) culture system for examining the role of proteins and their putative signaling domains in NK cell development. BM-derived Lin–c-kit+ stem cells expressing the protein of interest are first cultured for 6 days in a cocktail of cytokines that promote lymphoid development. The semi-differentiated cells are then transplanted into mice to complete their development in vivo. While all hematopoietic lineages can develop from these transplanted cells, we focus primarily on assessing the effect of the protein on the production of NK cells, as well as the acquisition of Ly49 receptors. The most prevalent advantage of this method is the ability to potentially link signaling regulators to known aspects of NK cell development. Key Words: Stem cell chimeras, natural killer cells, Ly49, IL-15R␣.

1. Introduction The biological importance of certain protein receptors or signaling molecules is most often revealed through studies using mutant mice (1). Likewise, in the process of identifying the necessary components needed for natural killer (NK) cell development, molecules such as lymphotoxin, IL-15, IL-15R, IL-2R, Id2, IRF1, Ets-1, and many others were, through the use of genetically deficient mice, categorically described as indispensable for the normal production and maturation of NK cells (2). However, K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 5, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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linking the biochemical nature of a particular region within a surface receptor or intracellular protein to its specific developmental function in vivo would require generating multiple mutant mice, each transgenically expressing a point mutation or truncation in the protein of interest. Although a viable option, we have instead employed the use of generating radiation chimeras in which donor whole bone marrow cells are substituted with genetically modified stem cells. Traditional radiation chimeras are generated by transplanting whole bone marrow (BM) cells from one donor mouse into a lethally irradiated recipient mouse and then monitoring the developmental path of the donor cells in the milieu of the recipient. In this chapter, we describe a method to study how mutated forms of a protein that is known to be essential in NK development affect the process of differentiation and development of NK progenitors that express those proteins. For obvious reasons, the BM cells for transplantation must originate from a donor that is null for the protein of interest. Then, because whole BM cells contain a mixed population of cells undergoing various stages and lineages of development, purified undifferentiated stem cells are a preferred choice as donors for transplantation. Also, if the protein being analyzed is required for NK cell production, the BM population of these mice would, therefore, most likely lack NK progenitors, leaving the stem cells as the best candidates for study. Finally, the use of stem cells lets us manipulate the system to enrich for only those cells that are successfully expressing cDNA encoding the protein of interest. In this protocol Lin–c-kit+ stem cells are purified from the whole bone marrow of a knock-out (KO) mouse, cultured in vitro under conditions that initiate NK cell differentiation (3, 4), retrovirally transduced with cDNA encoding various mutated forms of the knock-out protein, and consequently re-established in a recipient wild-type mouse. The NK cell progenitors whose development was promoted by the in vitro differentiation system will continue their maturation in vivo in a normal bone marrow milieu, but under the influence of a mutated receptor that is expressed on the progenitors. Because fully matured NK cells acquire various NK lineage markers, including Ly49 receptors (5, 6), the effect of the mutated receptor on proper NK cell development and production can be easily assessed by analyzing the splenocytes of the chimeric mice by flow cytometry for these markers.

2. Materials 1. RNA extraction reagent such as Trizol (www.invitrogen. com) or the RNeasy Plus kit (www1.qiagen.com).

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2. A first-strand cDNA synthesis kit (www.fermentas.com). 3. pCR2.1-TOPO cloning kit (Invitrogen). 4. pMIGR1-GFP retroviral vector is available from most labs that routinely perform retrovirus-based transfection methods. Use the standard protocol for requesting and obtaining reagents from other research labs – e-mailing a request usually suffices. 5. Retrovirus packaging cell line: we use PlatE cells, but any 293-based packaging cell line (such as GP293, 293FT, or Phoenix) can be used as long as the vectors encoding the gag, pol, or env genes are available for co-transfection with the retroviral vector. 6. Reagents for PlatE cell maintenance and transfection: 0.25% Trypsin-1 mM EDTA solution, blastocidin, and puromycin (both from InvivoGen, www.invivogen.com); ExGen500 In Vitro Transfection Reagent (Fermentas), sterile 150 mM NaCl. 7. Mice: Mice that are deficient in the protein of interest (knock-out, KO) that is on the C57BL/6J background (CD45.2, wild type), 4–6 weeks old (JAX, stock no. 000664) to provide donor stem cells; B6.SJLPtprca Pepcb /BoyJ congenic mice (CD45.1), approximately 6 weeks old (JAX, stock no. 002014) to serve as recipient animals. After transplantation, donor cells of KO mice can be differentiated from host congenic mouse cells by their respective CD45.2 or CD45.1 protein marker. 8. Flavored pediatric Bactrim (Trimethoprim-Sulfoxaxole) can be obtained from the animal housing facility veterinarian or staff and administered to mice before and after irradiation. 9. QuadroMACSTM and MiniMACSTM cell separation units and the MACS MultiStand (Miltenyi Biotec; www.miltenyibiotec.com). 10. LS and MS columns (Miltenyi Biotec). 11. Streptavidin (SA) magnetic microbeads or particles for binding to biotinylated antibodies (Miltenyi Biotec, cat# 130-048-102 or BD Biosciences, cat# 557811) and antiFITC magnetic microbeads (Milteny Biotec). 12. MACS buffer: 1× Dulbecco’s phosphate-buffered saline (DPBS) containing 2 mM EDTA and 0.5% bovine serum albumin. 13. DMEM-10 medium: Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4,500 mg/L D-glucose with 10% fetal bovine serum, 100 units/ml penicillin,

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100 ␮g/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 0.1 mM MEM nonessential amino acids, and 50 ␮M 2-mercaptoethanol. 14. Differentiation medium: DMEM-10 medium containing 1 ␮g/ml indomethacin, 0.5 ng/ml murine IL-7, 30 ng/ml murine stem cell factor (SCF), and 20 ng/ml human Flt3ligand. 15. Antibodies: Conjugated monoclonal antibodies (mAbs) recognizing CD45.1, CD45.2, NK1.1 (clone PK136), CD3 (clone 2C11), Ly49A (any clone), Ly49C/I (clone 5E6), Ly49G2 (clone 4D11), and Ly49D (clone 4E5). Other mAbs used for purification of bone marrow (BM)-derived stem cells include biotinylated forms of anti-CD11b (Mac-1), anti-Gr-1, anti-B220, antierythroid cells (TER-119), and anti-CD2. All mAbs can be purchased from BD Biosciences or eBioscience (www.ebioscience.com) at stock concentrations of 0.5 mg/ml. Also needed will be a biotinylated mAb that recognizes the protein of interest if it is expressed on the cell surface. 16. Cytokines and growth factors: Recombinant murine IL7, Flt3-Ligand (Flt3L), and stem cell factor (SCF) can be purchased from Invitrogen or from R&D Systems (www.rndsystems.com). 17. Flow cytometry: Cell acquisition is performed using the FACS Diva software on the FACSCanto (BD Biosciences). Sorting of BM stem cells is accomplished using the MoFlo (Beckman Coulter, Miami, FL) and data are analyzed using FlowJo software (www.flowjo.com). 18. 2.4G2 hybridoma supernatant (cell line from ATCC, cat# HB-197). 19. Other reagents: 0.2% NaCl and 1.6% NaCl for lysis of erythrocytes; hexadimethrine bromide (Sigma-Aldrich) for enhancing retroviral infection.

3. Methods 3.1. Cloning Your Gene into the Retroviral Expression Vector pMIGR1-GFP

The pMIGR1-GFP retroviral vector contains an IRES sequence directly upstream of the GFP gene. Therefore, subcloning your gene upstream of the IRES sequence will permit the expression of the GFP marker in tandem with your gene of interest (see Fig. 5.1). Due to this bi-cistronic system of expression, transfected or transduced cells that are GFP+ are, therefore, more than

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Fig. 5.1. The pMIGR1-GPF vector for transfection of purified Lin–c-kit+ stem cells. It is 6056 bp in size, contains an internal ribosomal entry site (IRES), and a multiple cloning site at BglII (1406) to EcoRI (1424).

likely to also express the gene that is subcloned upstream of the IRES-GFP sequence. The GFP marker also facilitates easy and rapid monitoring of transfection and transduction efficiencies, as well as permits enrichment of positive cells by cell sorting using GFP fluorescence without the need for antibody staining. 1. Amplify your gene of interest (wild type or mutated) by PCR and insert the cDNA into any sub-cloning vector such as pCR3.2-TOPO (Invitrogen) using TA cloning methods according to the manufacturer’s instructions. To generate point or deletion mutants, it is easiest to incorporate the mutation sites into the PCR primers used for cloning. 2. Sequence the cDNA to verify that the gene has been correctly cloned with respect to length and PCR proofreading accuracy. 3. Excise the gene and insert into the pMIGR1 expression vector containing an IRES-controlled GFP gene. 4. Digest the vector and gene at the appropriate sites and sequence the final construct to verify that the gene has been correctly inserted with respect to orientation. 3.2. Preparing Viral Supernatant Using PlatE Packaging Cells

PlatE cells have been genetically manipulated and already contain vectors encoding all the genes necessary for virus particle packaging – gag, pol, and env(7). To maintain only a population of useful (positive) cells, they are cultured in the presence of antibiotics until just 24 h prior to transfection.

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3.2.1. Culture of PlatE Cells

1. To maintain cultures, grow cells in DMEM-10 medium containing 1 ␮g/ml of puromycin and 10 ␮g/ml blastocidin to keep cells under selective pressure. These antibiotics are necessary to prevent the outgrowth of vector-negative PlatE cells. Prolonged maintenance of these cells beyond 6 weeks is not recommended since the cultures may phenotypically and/or functionally drift. 2. When the cells are confluent, divide them at a 1:10 dilution.

3.2.2. Transfection of cDNA into PlatE Cells Using the ExGen500 Reagent

1. 24 h before transfection, plate 2 × 106 PlatE cells in 10 ml of DMEM-10 medium without puromycin or blastocidin into a 100 mm tissue culture dish. Optimum density should be achieved 12–16 h postplating. 2. The next day, remove the medium from the dish containing the cells and add 3 ml of fresh DMEM-10 medium. 3. For each dish of PlatE cells to be transfected, prepare one sterile Eppendorf (microfuge) tube containing a mixture of 5 ␮g of cDNA and 300 ␮l sterile 150 mM NaCl. Add 16.45 ␮l of ExGen500 reagent into the DNA/salt solution and mix gently for 10 s. Let the mixture incubate for 10 min at room temperature. Following this, add the DNA/salt/ExGen500 mixture dropwise and evenly onto the PlatE cells. Gently rock the plate back and forth to evenly distribute the DNA solution. If possible, spin the plate for 5 min at 280 × g at room temperature. Incubate these cells overnight in 37◦ C/5% CO2 . For production of mock virus supernatant, add nuclease-free water instead of cDNA. 4. The day after transfection, remove the medium from the cells and add 3 ml of fresh DMEM-10 medium to the culture dish. Return cells to the incubator. 5. To verify that the transfected PlatE cells are expressing your gene of interest, harvest a small sample of the cells 48–72 h posttransfection and analyze for GFP expression by flow cytometry (see Note 1). 6. Harvest supernatant from cells whose GFP expression levels are equivalent to or more than 90%. This supernatant contains virus particles and will be used for infection of mouse stem cells (see Note 2). 7. Filter the supernatant through a sterile 45 ␮m syringe filter to remove any cellular debris, aliquot into sterile tubes, and store at −80◦ C to −135◦ C. Supernatants can be thawed just prior to each use. Repeat freezing and thawing is not recommended.

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3.3. Preparing NK Progenitors from Mouse Bone Marrow for Retroviral Infection and Transplantation 3.3.1. Harvesting Whole Bone Marrow Cells from Mice

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1. Euthanize mice whose bone marrow cells are to be harvested for transplanting into a recipient. The donor mice are those that are deficient for the protein of interest and usually KO mice of the CD45.2 genotype. 2. Remove both the large femoral bones from the mice. Scrape away all muscle tissue and place all the cleaned bones into a sterile ceramic mortar containing 5–10 ml of DMEM-10 medium. 3. Using a sterile pestle, carefully grind the bones to release the cells in the marrow space. 4. Place a 70 ␮m cell strainer (BD Falcon) over the mouth of a sterile 50-ml tube, and using a pipet, transfer the cell solution into the tube through the strainer. This step filters out the bone debris. 5. Add 5–10 ml of sterile 1X PBS to the mortar dish and swirl to rinse the bones. Transfer the cell solution into the same 50-ml tube, again filtering through the cell strainer. 6. Continue adding fresh 1× PBS to the 50-ml tube through the strainer until the tube is filled. 7. Centrifuge at 500 × g for 10 min at 4◦ C to pellet the cells. 8. Loosen the pellet by vortexing. Lyse the erythrocytes by adding 10 ml of sterile 0.2% NaCl solution followed immediately by equal volumes of 1.6% NaCl solution. The first salt solution is hypotonic and quickly lyses the erythrocytes, whereas the second salt solution is hypertonic and is required to restore osmotic balance to the cell environment. Delay in adding the hypertonic salt solution may result in lysis of the other cell types, thus lowering your yield. 9. Fill the remaining volume in the tube with DMEM-10 medium. 10. Centrifuge to pellet the cells. Resuspend the pellet in 5–10 ml of sterile azide-free 2.4G2 hybridoma supernatant and remove the cell debris by filtering through a cell strainer. 11. Count cells to determine total cell count. 12. Incubate the cells (resuspended in the 2.4G2 supernatant) at 4–8◦ C for 20 min.

3.3.2. Purifying Lin– c-kit+ Stem Cells

1. Centrifuge to pellet the whole BM cells that were incubating in 2.4G2 hybridoma supernatant. 2. Decant the 2.4G2 supernatant and loosen the cell pellet. To the pellet add 2–3 ␮l of each of the following biotinylated mAbs (taken directly from the stock without

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dilution): anti-CD2, anti-CD3, anti-Gr-1, TER119, antiMac-1 (CD11b), anti-NK1.1, and anti-B220. At this time, 2–3 ␮l of FITC-labeled anti-c-kit mAb is also added to the cells. 3. Incubate cells with mAbs on ice for 30 min. 4. Wash cells 1× with sterile ice-cold MACS buffer by centrifugation. 5. Resuspend the pellet with 90 ␮l of MACS buffer for every 1 × 107 (or fewer) total cells. Add 10 ␮l of SA microbeads (Miltenyi Biotec) or SA particles (BD Biosciences) for every 1 × 107 (or fewer) total cells. 6. Mix thoroughly and incubate cells with beads on ice for 20–30 min or at 4◦ C for 12–15 min. 7. During this incubation, prepare the LS column (use more than 1 column if cell number is >3 × 108 total) by placing it on the QuadroMACSTM magnet and rinsing it with 5 ml of de-gassed ice-cold MACS buffer. Discard the flowthrough buffer. 8. When the cells have completed their incubation, wash them once with excess MACS buffer and resuspend the pellet in 3 ml of de-gassed MACS buffer. 9. Add the cell solution to the previously rinsed LS column(s) and allow the cell mixture to flow through the column bed. Collect the flow-through – this fraction contains the Lin– cells. 10. Wash the column 3× with MACS buffer and collect the flow through from each wash. 11. To collect the c-kit+ population, centrifuge the flowthrough to pellet the Lin– cells. 12. To the pellet, add 90 ␮l of MACS buffer and 10 ␮l of antiFITC microbeads for every 1 × 107 (or fewer) total cells. 13. Mix thoroughly and incubate at 4◦ C for 20 min. 14. While cells are incubating, prepare the MS column (may need to use two columns depending on the total cell number) by attaching it to the MiniMACSTM separation magnet and rinsing it with 1 ml of de-gassed MACS buffer. Discard the flow-through. 15. After incubation, collect the cells by washing with excess volumes of MACS buffer. 16. Resuspend the pellet in 0.5 ml MACS buffer and transfer the mixture to the previously rinsed MS column(s). Allow the cell mixture to flow through the column bed. Discard the flow-through – it contains the c-kit− cells. 17. Wash the column 3× with MACS buffer and discard the flow-through from each wash.

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18. Transfer the MS column to a clean (sterile) 5-ml tube, add 1 ml of DMEM-10 medium and immediately elute the c-kit+ cells using the column plunger. 19. Centrifuge and resuspend in DMEM-10 medium. Count and resuspend cells at 8 × 105 –1.5 × 106 cells/ml of Differentiation medium. Distribute 2 ml of the cell solution into each well of a 24-well sterile tissue culture plate with lid and incubate overnight at 37◦ C with 5% CO2 . 3.3.3. Retroviral Infection of Lin–c-kit+ Stem Cells

1. Carefully remove 1 ml of medium from each well of stem cells. 2. Add 1 ml of viral supernatant stock to each well of cells. As a control, add 1 ml of mock virus (no particles) supernatant to a few wells. 3. To each well also add 10 mM HEPES and 2 ␮g/ml hexadimethrine bromide, also known as polybrene, and mix gently. Polybrene is a cationic compound that enhances the interaction between virus particles and the cell surface membrane by neutralizing repulsion charges between the virus and the cell membrane. 4. Carefully wrap the plate and centrifuge at 1,300 × g for 90 min at 30◦ C (spinfection step). 5. Resuspend cells by gentle mixing and incubate at 37◦ C and 5% CO2 for 2–4 h. 6. Harvest cells, pooling them according to their condition, and centrifuge as usual. 7. Resuspend the cells in Differentiation medium and reculture them in a clean 24-well plate at 2 ml per well. 8. Incubate at 37◦ C and 5% CO2 overnight. 9. Repeat the virus spinfection steps the next day, and if desired, the following day as well for a total of three rounds of infection (see Note 3). 10. On day 5, GFP expression can be assessed by flow cytometry. GFP+ cells can be enriched by FACS sorting (see Note 4). 11. After enrichment, culture the cells for one additional day in Differentiation medium prior to transplantation into lethally irradiated recipients.

3.4. Generating Radiation Chimeras 3.4.1. Transplanting Whole BM Cells

Since the idea is to use stem cells instead of whole bone marrow cells for transfer, it is important to make a side-by-side comparison of mouse chimeras generated from each donor cell type. The goal here is to show that using Lin–c-kit+ stem cells that had been cultured in vitro for 6 days (see Fig. 5.2A) is equivalent

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Fig. 5.2. Reciprocal radiation chimeras whose donor cells are obtained from cultured stem cells are equivalent to those from total bone marrow preparations. FACS analysis of freshly isolated spleen cells obtained after 2, 4, or 8 weeks after transplantation. Data are shown as percent positive of cells that were gated on the CD3− NK1.1+ lymphocyte population. Insets show the percent splenic NK cells of each chimera examined. (A) A total of 10 × 106 Lin–c-kit+ stem cells purified from whole BM of WT or IL-15R␣KO mice were transduced with “mock” virus supernatant and cultured for 6 days in SCF, IL-7, and Flt3L before transplanting into lethally irradiated recipient mice. In (B), 10 × 106 total BM cells were harvested and immediately transplanted into recipients.

to using freshly isolated whole BM preparations as donor cells (see Fig. 5.2B). These experiments conclusively eliminate the possibility that a failure to reconstitute NK cells is caused by the method of using cultured stem cells as donor cells. Also, to verify that the mutated proteins are effectively and properly expressed in the cell, transduce the cDNA constructs encoding the mutations into purified stem cells (or a cell line deficient for your protein) and assay by flow cytometry (surface protein) or Western Blotting (intracellular protein). For example, in our own experiments, we were able to demonstrate that our transduced cells correctly expressed

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Fig. 5.3. FACS analysis of 58−/− cells expressing cloned WT or cytoplasmic mutants of IL-15R␣ (Y227F, 16 and 30). Successful transduction was measured by GFP fluorescence with surface expression of WT and mutant IL-15R␣ also detected by staining with anti-mouse IL-15R␣ mAb.

either wild-type or various cytoplasmic mutant forms of the IL15R␣ gene on the cell surface and at fairly equivalent levels (see Fig. 5.3). 1. 7–10 days before transplantation, begin a regimen of Bactrim suspension (5–250 ml of drinking water) for the intended recipient mice, which in most cases will be the CD45.1 congenic mice. All aspects of drug administration should be approved by your institution’s veterinarian and comply with all guidelines specified by your animal facility. 2. 24 h before transplantation, lethally irradiate (10 Gy) recipient mice. Prepare at least three mice per experimental condition, including three control mice (to receive PBS instead of cells) (see Note 5). 3. On the day of transplantation, harvest donor whole nucleated BM cells (depleted of erythrocytes), which, in most cases will be the gene KO mice expressing the CD45.2 marker. 4. Resuspend the cells in sterile PBS to a concentration of 5 × 107 –1 × 108 cells/ml. 5. Using a tuberculin syringe and 27-gauge needle, inject 100 ␮l of the cell solution into the tail veins of the lethally irradiated recipient mice. The absolute number of cells injected into each mouse will be 5 × 106 –1 × 107 cells. Equivalent numbers should be injected for each

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experimental condition. Continue with treatment of Bactrim for two more weeks after transplantation. 6. Analyze the chimeric mice 8 weeks post-BM transfer, at which time >96% of the splenic leukocytes are donor derived. 7. For details on analysis, see Section 3.5. 3.4.2. Transplanting BM-Derived Stem Cells

1. Prepare recipient mice as before (see previous section). 2. On the day of transplantation, harvest Lin–c-kit+ stem cells previously isolated from donor mice that have been cultured with SCF, Flt3L, and IL-7 for 5 days, infected with virus particles and enriched for GFP expression (see Note 6). 3. Wash the cells several times with sterile PBS to remove traces of cytokines. 4. Resuspend the cells in sterile PBS to a concentration of 1 × 107 –5 × 107 cells/ml. 5. Inject 100 ␮l of the cell solution into the tail veins of lethally irradiated recipient mice. The absolute number of cells injected into each mouse will be 1 × 106 –5 × 106 cells. Equivalent numbers should be injected for each experimental condition. 6. Analyze the chimeric mice 8 weeks after stem cell transfer, at which time >96% of the splenic leukocytes are donor derived. 7. For details on analysis, see Section 3.5.

3.5. Analyzing the Radiation Chimeras by Flow Cytometry

1. Harvest spleen cells from the chimeric mice and lyse erythrocytes as usual. 2. Pre-incubate nucleated cells with the 2.4G2 (anti-FcR) hybridoma supernatant and aliquot 5 × 105 cells into each analysis tube. 3. Follow the standard protocol of antibody staining for flow cytometry. 4. To each tube of cells, add the following fluorochromeconjugated mAbs: anti-CD3, anti-NK1.1, anti-CD45.2, and one anti-Ly49 mAb. For example, a combination of antiCD3 PE-Cy7, anti-NK1.1 PE, anti-CD45.2 PE-Cy5.5, and anti-Ly49G2 FITC can be used for one of the tubes. One tube of cells should only contain the first three mAbs, and instead of an anti-Ly49 mAb, use an isotype-matched control mAb. 5. For analysis, quantitate Ly49 expression on cells after gating on the population that is CD3− , NK1.1+ , and CD45.2+ (see Note 7). Figure 5.4 shows results that are representative of

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Fig. 5.4. FACS analysis of freshly isolated splenocytes harvested from chimeric mice that were transplanted 8 weeks prior with Lin–c-kit+ stem cells transduced with vector alone, wild-type, or mutated forms of IL-15R␣. Data show percent splenic NK cells (inset) and Ly49 expression of cells gated on CD3− NK1.1+ CD45.2+ for each mouse. The results here indicate that IL-15R␣-mediated signals are important for NK cell production as well as the expression of certain Ly49 molecules.

these experiments. Here, the flow cytometric data indicate the importance of the cytoplasmic domains of the IL-15R␣ protein in NK production and Ly49 expression.

4. Notes 1. Harvesting a small sample of the transfected PlatE cells can be easily done by using a 200–1000 ␮l pipetter and sterile tip to draw 500 ␮l of culture medium while scraping across the bottom of the dish. Since these cells are used as a quick and rough measure to survey the extent of transfection, there is no need to count the cells prior to flow cytometric analysis. Just a quick wash in PBS containing 2% FBS and filtration through a 70 ␮m sieve is sufficient preparation for analysis. 2. The efficiency of transduction can sometimes fluctuate and, therefore, result in the production of insufficient numbers of GFP+ progenitor cells for successful transfer into mice. One extremely important factor that should not be overlooked is the “potency” of the virus supernatant for transduction. If supernatant is collected on days when fewer than 70% of transfected PlatE cells are GFP+ , the supernatant may not contain high enough titers of virus particles to be effective.

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Therefore, transduction efficiency and quality is determined by the concentration of virus particles in the supernatant. Given this, supernatants can be concentrated using special filters. However, three easier and less expensive methods would be (1) to harvest the supernatant when more than 90% of PlatE cells are GFP+ , (2) to enrich the supernatant by incubating the cells for a longer period with medium prior to harvesting, and (3) to enrich the supernatant by reducing the volume of medium added to the PlatE cells following transfection. In terms of quality, freshly harvested supernatants are the most effective, followed by freshly thawed stocks of supernatant. Supernatant stocks kept at 4◦ F will quickly lose their potency. Repeated freezing and thawing of stocks are also not recommended. 3. Three rounds of infection typically yield 50–65% GFP+ cells. However, we have in the past obtained improved results by following a more labor-intensive protocol involving the use of fibronectin (FN) (8, 9). For that method, pre-coat the 24well plates with 20 ␮g of sterile FN (from BD Biosciences) per well and incubate 2–3 h at room temperature. After that, remove the FN solution and add PBS containing 2% BSA to the same wells for 30 min at room temperature to block nonspecific binding sites. Remove the PBS/BSA solution, wash once with DMEM-10 medium, followed by adding 1 ml of virus supernatant stock to each treated well. Incubate 1 h at room temperature. Remember to add 1 ml of “mock” virus supernatant to one or more wells as control. Finally add 1 ml of cells that were pre-washed and resuspended in 2X Differentiation medium containing 20 mM HEPES and 40 ␮g/ml of polybrene. The rest of the protocol is the same as described in Section 3.3.3, step 4. 4. Magnetic-based methods can be used only if the protein of study is a receptor (i.e., expressed on the surface) for which antibodies are available. 5. Lethally irradiated mice that only receive PBS and not BM cells will usually die within 10 days. 6. To obtain the results in Fig. 5.2A, transplant with stem cells previously infected with mock virus supernatant, i.e., contains no virus particles. An even better control is to use cells infected with virus particles expressing the empty pMIGR1GFP vector. 7. Except when performing reciprocal transfers, in most cases the KO mouse-derived donor cells express the CD45.2 marker, and the recipient cells express the CD45.1 marker of the congenic mouse strain. As such, staining the splenocytes with anti-CD45.2 will identify the donor cells co-expressing

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your wild-type or mutated protein. Although all the donor cells presumably originated from GFP+ stem cells, the differentiated population has undergone several cycles of division, during which time GFP expression is extensively diluted and often undetectable in the final analysis. References 1. Williams, N. S., Klem, J., Puzanov, I. J., Sivakumar, P. V., Schatzle, J. D., Bennett, M., and Kumar, V. (1998) Natural killer cell differentiation: insights from knockout and transgenic mouse models and in vitro systems Immunol Rev 165, 47–61. 2. Lian, R. H., and Kumar, V. (2002) Murine natural killer cell progenitors and their requirements for development Semin Immunol 14, 453–60. 3. Williams, N. S., Moore, T. A., Schatzle, J. D., Puzanov, I. J., Sivakumar, P. V., Zlotnik, A., Bennett, M., and Kumar, V. (1997) Generation of lytic natural killer 1.1+, Ly49- cells from multipotential murine bone marrow progenitors in a stroma-free culture: definition of cytokine requirements and developmental intermediates J Exp Med 186, 1609–14. 4. Williams, N. S., Klem, J., Puzanov, I. J., Sivakumar, P. V., Bennett, M., and Kumar, V. (1999) Differentation of NK1.1+, Ly49+ NK cells from flt3+ multipotent marrow progenitor cells J Immunol 163, 2648–56.

5. Dorfman, J. R., and Raulet, D. H. (1998) Acquisition of Ly49 receptor expression by developing natural killer cells J Exp Med 187, 609–18. 6. Roth, C., Carlyle, J. R., Takizawa, H., and Raulet, D. H. (2000) Clonal acquisition of inhibitory Ly49 receptors on developing NK cells is successively restricted and regulated by stromal class I MHC Immunity 13, 143–53. 7. Morita, S., Kojima T., and Kitamura, T. (2000) Plat-E: an efficient and stable system for transient packaging of retroviruses Gene Ther 7, 1063–6. 8. Moritz, T., Dutt, P., Xiao, X., Carstanjen, D., Vik, T., Hanenberg, H., and Williams, D. A. (1996) Fibronectin improves transduction of reconstituting hematopoietic stem cells by retroviral vectors: evidence of direct viral binding to chymotryptic carboxy-terminal fragments Blood 88, 855–62. 9. Bajaj, B., Behshad, S., and Andreadis, S. T. (2002) Retroviral gene transfer to human epidermal keratinocytes correlates with integrin expression and is significantly enhanced on fibronectin Hum Gene Ther 13, 1821–31.

Chapter 6 Use of Transfected Drosophila S2 Cells to Study NK Cell Activation Michael E. March, Catharina C. Gross, and Eric O. Long Abstract Determining the contribution of individual receptors to natural killer (NK) cell function is complicated by the multiplicity of activating and inhibitory NK cell receptors. Mammalian target cells typically express a variety of ligands for NK cell receptors. Engagement of NK cell receptors by antibodies may not mimic activation by natural ligands. To define requirements for activation and dissect the contribution of receptors to NK cell function, we have generated Drosophila Schneider line 2 (S2) cell transfectants expressing ligands for NK cell receptors. The evolutionary distance between Drosophila and mammals greatly reduces the potential of recognition of insect cell molecules by mammalian NK cells. Here, we present methods for maintenance and transfection of S2 cells, as well as protocols for their use in NK cell assays. Key words: Drosophila, S2, insect cells, NK cells, activation, inhibition, transfection.

1. Introduction The activation of natural killer (NK) cells is controlled by combinations of activating and inhibitory receptors expressed by NK cells. Integration of the signals downstream from a wide variety of receptors determines whether or not the NK cells respond (1– 4). Higher expression of activating ligands on target cells induces activation of NK cells, whereas higher expression of inhibitory ligands prevents it. Human NK cells express a wide variety of activating receptors, which respond to a wide variety of ligands. The natural cytotoxicity receptors (NCRs) NKp46, NKp44, and NKp30 and receptors NKG2D, 2B4 (CD244), CD2, LFA1 (CD11a/CD18), and DNAM-1 (CD226) are some of the K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 6, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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receptors known to deliver activating signals to NK cells. Engagement of combinations of these receptors can activate NK cell functions such as adhesion, cytotoxicity, and cytokine secretion (5–7). NK cells are also potent mediators of antibody-dependent cellular cytotoxicity (ADCC) through the low-affinity Fc ␥RIII (CD16). Inhibitory receptors are expressed on NK cells in a clonally distributed manner, where individual NK cells may express single or multiple inhibitory receptors in an essentially random manner. Inhibitory receptors in humans include members of the killer cell Ig-like receptor (KIR) family, which recognize alleles of HLA class I, members of the CD94/NKG2 family that recognize HLA-E, and several other inhibitory receptors that bind to non-MHC ligands (4, 8). The response of NK cells to signals from individual receptors, or combinations of receptors, is often studied by masking the receptors with antibodies in order to block a response to a given target cell (9–13) or by cross-linking the receptors on the surface of NK cells with antibodies in order to provoke a response in the absence of target cells (7, 14). Such experiments can be very informative; however, antibodies typically have much higher affinities for receptors than the natural ligands do and may fail to stimulate those receptors in the physiological way. This is particularly true for receptors such as integrins that undergo conformational changes upon ligand binding (15, 16), which may not occur following antibody cross-linking. Cell lines that are susceptible or resistant to NK cell cytotoxicity are also used to probe NK cell responses. Ectopic expression of ligands in cell lines has revealed much about the contributions of both activating and inhibitory receptors to the regulation of NK cells (9, 13, 17, 18). Interpretation of such experiments can be complicated by the expression of many, often uncharacterized, ligands on target cell lines. For example, the EBV-transformed B cell line 721.221 expresses ICAM-1 (CD54), ICAM-2 (CD102), and ICAM-3 (CD50) (ligands for the integrin LFA-1), CD48 (ligand for 2B4), and B7 (CD80, a ligand of CD28) (19). The erythroblastoid cell line K562 expresses ULBP2 and MICA/B (ligands for NKG2D) (20, 21) along with ICAM-1 and ICAM-2 (22). Additionally, the Fc␥R+ mouse cell line P815, which is often used to stimulate NK cells with antibodies via redirected lysis (10, 11), expresses the mouse ICAM-1, which binds to human LFA-1 (23). In addition, P815 cells are more sensitive to lysis by NK cells that express high levels of NKp46, suggesting that they may express ligands for this NCR (24). Ligands for the NCRs have not been fully characterized, and the pattern of expression on many NK target cell lines is unknown. These uncertainties created a need for a target cell line in which expression of ligands is more easily characterized and controlled. A cell line derived from an evolutionarily distant species,

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which would also be easy to transfect and maintain, would be a useful surrogate target cell. Schneider line 2 (S2) cells fulfill these requirements. The S2 cell line was derived from latestage embryos of Drosophila melanogaster (25). The cells grow in a loose monolayer. S2 cells are easily transfected. They can be adapted to growth in serum-free medium, suitable for the purification of recombinant secreted proteins. S2 cells have been used to produce and purify proteins in sufficient quantities for structural and biochemical studies (26). Expression of exogenous proteins is often accomplished by transfection with the pRmHa3 plasmid, which uses the Drosophila metallothionein gene promoter for inducible expression, allowing high expression even for proteins that prove deleterious to the growth of S2 cells (27). Transfected S2 cells have been used previously to investigate the minimal requirements for T-cell activation and costimulation. S2 cells expressing peptide-loaded MHC class I, either alone or in combination with ICAM-1 and B7, were used as antigen-presenting cells (APCs) for the stimulation of na¨ıve T cells expressing the 2C transgenic TCR (28, 29). The results demonstrated that signaling through the TCR alone is not sufficient to activate na¨ıve CD8+ T cells and that B7 and ICAM-1 could provide the required co-stimulation for activation. Optimal co-stimulation occurred with both B7 and ICAM-1 expressed on the same MHC class I+ S2 cell. Besides confirming the two-signal hypothesis for T-cell activation, these data also demonstrated that co-engagement of the TCR with a co-stimulatory receptor is sufficient for T-cell activation. The use of S2 cells as APCs for T cells indicated that this Drosophila cell line could be used to reconstitute a sensitive target cell for NK cells. We have successfully used S2 cells to investigate the response of primary human NK cells to stimulation and inhibition through individual receptors (5, 30, 31). A notable advantage of this system is that resting NK cells, freshly isolated from human blood, can be used directly in functional assays with S2 cell transfectants, without further manipulation. In most assays examining degranulation or cytokine secretion, untransfected S2 cells induce minimal or no response from either primary NK cells or NK cell lines. Expression of individual ligands by stable transfection is sufficient to induce responses such as adhesion (5) and granule polarization (30, 31), whereas multiple ligands may be required to induce other responses such as degranulation (Y. Bryceson, unpublished). Transmembrane proteins expressed in S2 cells are glycosylated, which may be important for recognition by some NK cell receptors. S2 cells are easily transfected, either transiently or stably, and can be substituted for mammalian target cells in many NK cell assays with little or no modification. We will present here methods for stable transfection of S2 cells, methods for isolation of cells expressing the transfected protein,

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as well as specific protocols for the use of S2 cells in assays for NK cell function.

2. Materials 2.1. Care and Culture of S2 Cells 2.1.1. Standard Culture

1. S2 culture medium: Schneider’s Drosophila medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT; complement inactivated by heating at 56◦ C for 30 min). 2. 75 cm2 tissue culture flasks (Costar, Corning, Lowell, MA). 3. Freezing medium: Fresh S2 cell culture medium mixed with an equal volume of conditioned medium (the medium in which S2 cells have been grown) supplemented with 10% dimethyl sulfoxide (DMSO) (Sigma Aldrich, St. Louis, MO). Alternatively, freezing medium can be composed of 90% FBS supplemented with 10% DMSO. 4. Reagent for inducible expression: Expression from metallothionein promoter-based plasmids is induced by the addition of cupric sulfate (CuSO4 , Sigma Aldrich) to the cell culture to a final concentration of 1 mM. A 100 mM CuSO4 stock solution is made in deionized water and then sterilized by filtration through a 0.45 ␮m filter.

2.1.2. Adaptation of S2 Cells to Culture in Serum-Free Conditions

1. Bio Whittaker Insect-Xpress medium (Lonza, Basel Switzerland). 2. Iscove’s Modified Dulbecco’s Medium (Invitrogen) supplemented with 0.2% BSA (see Note 1). 3. Fetal Bovine Serum (Hyclone). 4. 75 cm2 tissue culture flasks (Costar, Corning). 5. 125 ml Disposable Erlenmeyer Flask with 0.2 ␮m PTFE Vented Closure (Bellco Glass Inc, Vineland, NJ). 6. Orbital shaker capable of handling 125 ml flasks, at 27◦ C.

2.2. Transfection of S2 Cells 2.2.1. Calcium Phosphate Transfection

1. HEPES buffered saline (HBS) for calcium phosphate transfection: 50 mM HEPES, 1.5 mM Na2 HPO4 , and 280 mM NaCl, in water, adjusted to pH 7.1, sterilized by filtration through a 0.45 ␮m filter. 2. CaCl2 for calcium phosphate transfection: 2 M CaCl2 (Sigma Aldrich) in water, sterilized by filtration through a 0.45 ␮m filter. 3. 6-well tissue culture plates (Costar, Corning).

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4. pAc5.1-V5-His: Plasmid for constitutive expression in S2 cells using the Drosophila Actin 5 promoter (requires a licensing agreement with Invitrogen). 5. pRmHa3: Plasmid for inducible expression in S2 cells using the metallothionein promoter (available to not-for-profit labs from the Drosophila Genome Research Center, Bloomington, IN). 6. Plasmids for selection of transfectants, which include pNeoFly (27), pCoBlast (Invitrogen), and pCoHygro (Invitrogen). 7. Agents for selection of stable transfectants: Geneticin (G418 sulfate, Invitrogen), Puromycin dihydrochloride (Sigma Aldrich), Hygromycin B (Roche Diagnostics GmbH, Basel Switzerland), Blasticidin S HCl (Invitrogen). 8. 6 ml polypropylene, round bottom tubes, sterile (Falcon, BD Biosciences, San Jose, CA). 9. 15 ml polystyrene conical screw cap tubes (Sarstedt, Newton, NC). 2.2.2. Transfection with Cellfectin

1. Schneider’s Drosophila medium (Invitrogen) ± 10% FBS. 2. Cellfectin Transfection Reagent (Invitrogen). 3. Expression and drug resistance plasmids and agents for selection of stable transfectants (see Section 2.2.1). 4. 6-well tissue culture plates (Costar, Corning). 5. 6 ml polypropylene, round bottom tubes, sterile (Falcon, BD Biosciences).

2.2.3. Enrichment of Expressing Cells

1. Antibodies for the transfected proteins. For fluorescenceactivated cell sorting (FACS), antibodies directly coupled to fluorescent labels may be used when available. Alternatively, unlabeled primary antibodies can be stained with fluorescently labeled secondary antibodies. For enrichment of cells expressing a given protein using magnetic beads coated with goat anti-mouse antibodies, unlabeled primary antibodies are recommended. 2. Buffer for FACS sort: PBS supplemented with 2% heatinactivated FBS, 2 mM EDTA, 100 U/ml Penicillin, and 100 ␮g/ml Streptomycin. 3. FACS tubes: 5 ml polystyrene tubes, 12 × 75 mm (Falcon, BD Biosciences). 4. Dynabeads Goat anti-mouse IgG and selection magnet (Invitrogen). 5. 14 ml Polypropylene Round Bottom Tubes (Falcon, BD Biosciences).

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6. Flat bottomed 96-well plates (Costar, Corning). 2.3. S2 Cells as Targets in NK Cell Assays

1. Anti-CD56 mAb (clone NCAM 16.2) PE (BD Bioscience).

2.3.1. CD107a Degranulation Assay

1. PKH26 Red Fluorescent Cell Linker Kit (Sigma Aldrich).

2.3.2. Conjugate Assay

3. Hank’s Buffered Saline Solution (Invitrogen) supplemented with 5% FBS.

2. Anti-CD107a mAb (clone H4A3) FITC (BD Bioscience).

2. CellTracker Green CMFDA (Invitrogen).

4. IMDM + 10% FBS. 5. FACS tubes: 5 ml polystyrene tubes, 12 × 75 mm (BD Biosciences). 6. 0.5% Paraformaldehye (16% solution from Electron Microscopy Sciences, Hatfield, PA, diluted in phosphatebuffered saline (PBS)). 2.3.3. Perforin Polarization Assay

1. 15 ml polystyrene conical screw cap tubes (Sarstedt). 2. Hank’s Buffered Saline Solution (HBSS) (Invitrogen) supplemented with 3% FBS. 3. Poly-D-Lysine coated 2-well Culture Slides (BD BioCoat, BD Biosciences). 4. 4% Paraformaldehye (16% solution Microscopy Sciences diluted in PBS).

from

Electron

5. Normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA) reconstituted in 10 ml water. 6. Triton X-100 (Sigma Aldrich). 7. Permeablization buffer: 10% normal donkey serum and 0.5% Triton X-100 in PBS. 8. Staining buffer: 3% normal donkey serum and 0.5% Triton X-100 in PBS. 9. CellTracker Green CMFDA (Invitrogen). 10. Anti-perforin antibody, clone ␦G9 (Thermo Scientific, Rockford, IL). 11. Alexa Fluor 568 conjugated goat anti-mouse secondary antibody (Invitrogen). 12. Prolong Gold antifade reagent (Invitrogen). 2.3.4. Flow Cytometry-Based Cytotoxicity Assay

1. PKH67 Green Fluorescent Cell Linker Kit (Sigma Aldrich). 2. 15 ml polystyrene conical screw cap tubes (Sarstedt). 3. Iscove’s Modified Dulbecco’s Medium with and 25 mM HEPES (Invitrogen).

L -Glutamine

4. Propidium iodide solution, 1 mg/ml (Sigma Aldrich).

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5. FACS tubes: 5 ml polystyrene tubes, 12 × 75 mm (BD Biosciences)

3. Methods 3.1. Care and Culture of S2 Cells 3.1.1. Standard Culture

1. S2 cells grow at or slightly above room temperature. Additional carbon dioxide (CO2 ) is not required. Incubators, without CO2 injection, can be used to maintain an optimal temperature of approximately 27◦ C. Alternatively, S2 cells will grow reasonably well in a drawer or any location at room temperature that is protected from light. 2. S2 cells should be thawed at room temperature. Since S2 cells undergo a heat shock at 37◦ C, thawing the cells in a warm water bath will reduce viability. 3. Cell cultures should be maintained between 1 × 106 and 2.5 × 106 cells/ml in Schneider cell medium supplemented with 10% FBS. Cells grow best when diluted no more than 1:3 at each passage. Maintenance of a healthy culture of growing cells requires a 1:2 passage every other day (see Note 2). 4. Freezing of S2 cells is most successful when large numbers of cells from a healthy culture are frozen, such as 20–30 million cells per vial. S2 cells can be frozen in a variety of freezing media. We have used mainly 45% fresh culture medium combined with 45% conditioned S2 cell medium (the medium in which the cells have been grown) supplemented with 10% DMSO. Alternatively, a simple mixture of 90% FBS supplemented with 10% DMSO has been used. 5. Proteins can be expressed by transfection with either a constitutive or an inducible promoter-containing plasmid. Plasmid pAc5.1 (Invitrogen) drives expression with a Drosophila Actin 5 promoter, which results in constitutive expression. Plasmid pRmHa3 uses a metallothionein promoter, the activity of which is induced with 1 mM CuSO4 . A total of 48 h of induction with 1 mM CuSO4 is sufficient to yield maximal expression of transfected proteins. A fraction of the cell culture is reserved, without induction, for continued culture. Constitutive expression is more convenient, as the transfected proteins are always expressed and variability introduced by induction is avoided. Expression from a constitutive promoter is often more uniform over time than induced expression, which often yields heterogeneous expression levels. Constitutive expression can also be beneficial in transient transfections, where cells already dealing with the stress of the transfection may be adversely affected

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even further by the induction in cupric sulfate. Inducible expression is advantageous when a transfected protein is toxic to the S2 cells. Induced expression is also often used for large-scale protein production in S2 cells, where constitutive expression may slow the expansion of the cultures. The cells can be expanded without expressing the protein, and expression can be induced in 24–48 h. 6. Co-expression of multiple proteins in transfected S2 cells can be achieved either by co-transfection of all the required plasmids at once or by sequential transfections (see Notes 3 and 4). 7. Expression of MHC class I molecules in S2 cells requires co-transfection with ␤2 -microglobulin. Furthermore, exogenous peptides have to be loaded onto MHC class I because insect cells do not have the endogenous peptide loading machinery (e.g., TAP transporter, chaperones) (32). Peptide loading is very efficient because “empty” class I–␤2 microglobulin dimers are readily expressed at the surface of S2 cells at 27◦ C. Appropriate peptides are added to the S2 cell culture at a final concentration of 1 ␮m 1 day prior to the assay with NK cells. 8. Using S2 cells in NK cell assays is often a matter of simply replacing the conventional target cells in the assay with S2 cells. However, there are two important considerations. First, insect cells undergo heat shock and will eventually die at 37◦ C. Second, S2 cells exposed to human serum that has not been heat inactivated are killed in less than 1 h. Each of those issues can be addressed as follows. The viability of S2 cells following incubation at 37◦ C is not significantly reduced for at least 3 h. The assays described here using S2 cells at 37◦ C do not exceed 3 h. S2 cells grow happily at 27◦ C in heat-inactivated bovine serum. However, our primary human NK cells are usually cultured in human serum, which is not routinely heat inactivated. To avoid exposure of S2 cells to human serum, the simplest approach is to wash and resuspend NK cells in medium containing FBS prior to the assays with S2 cells. Although, in our hands, primary human NK cells survive and proliferate best in medium containing 10% human serum, they remain functional in medium containing FBS in short-term assays (see Note 1). 3.1.2. Adaptation of S2 Cells to Culture in Serum-Free Conditions

S2 cells used for production of proteins are often cultured in serum-free conditions in order to simplify the purification of the protein of interest. Serum-free culture of S2 cells has an additional advantage, of interest when using S2 cells as target cells in NK cell assays. Drosophila does not synthesize sterols. Its sole source of cholesterol is from the diet. Culture of S2 cells in serum-free

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medium is therefore a method to deplete the cells of cholesterol without using compounds such as ␤-methylcyclodextrin. Therefore, S2 cells grown in serum-free conditions may be a useful tool to investigate the role of cholesterol-dependent membrane microdomains on the behavior of ligands of NK cell receptors. A previous report indicated that the inclusion of MICA in lipid rafts on target cells is necessary for the NKG2D-dependent response of NK cells (33). 1. S2 cells to be adapted should be thawed from frozen stocks and cultured in 75 cm2 flasks in Insect-Xpress medium supplemented with 5% FBS. Cells should be expanded until a healthy growing culture at 2 × 106 cells/ml is obtained. 2. Pellet 150 × 106 cells at 135g. Resuspend in 30 ml InsectXpress + 5% FBS and transfer the cell suspension to a 125 ml disposable Erlenmeyer Flask. 3. Expand the cells by culturing on a shaker at 140 rpm at 27◦ C for 2–3 days. 4. Pellet 150 × 106 cells at 135g. Resuspend in 30 ml InsectXpress + 2.5% FBS and transfer the cell suspension to a 125 ml disposable Erlenmeyer Flask. 5. Again, expand the cells by culturing on a shaker at 140 rpm at 27◦ C for 2–3 days. 6. Perform three more cycles of pelleting 150 × 106 cells, resuspending in Insect-Xpress medium, and expanding the cells in shaking culture for 2–3 days, using medium that contains 1% FBS, then 0.5% FBS, and finally no serum. 7. Cells should be cultured in serum-free medium for a further 2 weeks before use in an assay (see Note 1). 3.2. Transfection of S2 Cells

For most NK cell assays, it is preferable to use S2 cells that express ligands at a fairly uniform level. To this end, stable transfectants of S2 cells are usually generated, from which the desired expressing cells can be enriched or cloned, when necessary. Two protocols for the generation of stable S2 cell lines are presented here. In some cases, transient transfections of S2 cells can be used. Transient expression is advantageous to test expression from new plasmids, for preliminary functional assays (where appropriate), and for the rapid screening of a very large number of transfectants. However, there is less control over protein expression levels in transient transfections, and there will usually be a sizeable fraction of the S2 cells that do not express the transfected proteins (see Note 5).

3.2.1. Calcium Phosphate Transfection

1. For transfection, S2 cells should be growing logarithmically, at a density around 2 × 106 /ml, with high viability. Cells can be counted on a hemacytometer with trypan blue staining.

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2. The day before transfection, spin down 3 × 106 cells per transfection in a 15 ml conical tube at 215g. Resuspend the cells at 1 × 106 cells/ml and plate 3 ml (3 × 106 cells) in a well of a 6-well plate. Incubate overnight at 27◦ C. 3. Set up the transfection mix. In one 6 ml tube, aliquot 300 ␮l of HBS. In a second 6 ml tube, mix 36 ␮l of 2 M CaCl2 with 19 ␮g of sterile DNA and sterile water to a total volume of 300 ␮l. This mixture should contain 18 ␮g of the plasmid expressing the protein of interest and 1 ␮g of the desired selection plasmid (see Notes 6 and 7). Vortex the tube containing the HBS at a moderate setting. While vortexing, slowly add the CaCl2 /DNA mixture dropwise to the HBS. This process should take approximately 1 min. Incubate the mixture at room temperature for 30 min. 4. Add the DNA mixture to the S2 cells that had been plated the day before. The mixture should be added to the medium on the cells in a dropwise manner. Gently swirl the plate in order to mix the medium and DNA. Incubate the cells overnight with the transfection mixture at 27◦ C. 5. Pipet the cells into a 15 ml conical tube. Wash the well with 5 ml of S2 medium in order to recover adherent cells. Pellet the cells at 215g and wash the cells once with 10 ml of medium. Plate the cells into a fresh well in a 6-well plate in 3 ml medium and incubate for 48 h at 27◦ C. 6. Add the appropriate selection (see Note 8). Incubate the cells for up to 2 weeks, until the resistant cells expand through the selection. The drug-resistant cells should be assayed for protein expression by flow cytometry. 3.2.2. Cellfectin Transfection

1. The day before the transfection, plate 1 × 106 cells into a well of a 6-well plate in 3 ml of Schneider’s Drosophila medium + 10% FBS. Culture the cells overnight. 2. Prepare the transfection mixture. a. In a 6 ml polypropylene tube, prepare a mixture consisting of 0.5 ␮g of the drug resistance plasmid and 5 ␮g of each protein expression plasmid in 100 ␮l of serum-free Schneider’s Drosophila medium (see Notes 6 and 7). The DNA should be sterile. b. In a second tube, add 10 ␮l Cellfectin Reagent to 90 ␮l serum-free Schneider’s Drosophila medium. c. Add the Cellfectin-containing medium to the tube containing the DNA mixture. Mix gently. d. Incubate the combined mixture at room temperature for 15 min.

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e. Add 800 ␮l serum-free Schneider’s Drosophila medium to the mixture. 3. Gently aspirate the medium from the S2 cells. Carefully wash the cells once with 2 ml serum-free Schneider’s Drosophila medium. Aspirate the wash medium. 4. Add the Cellfectin/DNA mixture to the cells. Incubate the cells for 18 h at 27◦ C. 5. Replace the DNA-containing medium with Schneider’s Drosophila medium + 10% FBS. Incubate for 48 h at 27◦ C. 6. At this point, the cells can be analyzed by flow cytometry for the expression of proteins from constitutively expressing plasmids, and protein expression from inducible plasmids can be induced with 1 mM CuSO4 and examined after another 48 h. 7. To select stable transfectants, the cells from Step 5 should be harvested and collected by centrifugation and resuspended in 3 ml Schneider’s Drosophila medium + 10% FBS containing the required selection reagent (see Note 8). Cells can be replated into the same well of the 6-well plate in which the transfection was performed. The cells should be incubated at 27◦ C, and the medium exchanged for fresh growth medium every 7 days, until stably transfected cells begin to expand. 3.2.3. Enrichment of Expressing Cells

Cells that have been selected through drug resistance often contain only a subpopulation of cells that express the protein(s) of interest. Although this amount of expression is sometimes sufficient for use in certain assays, it is usually preferable to obtain a more uniform profile of expression. 1. Expressing cells can be enriched with magnetic beads. We have had success with Dynabeads from Invitrogen, simply following the manufacturer’s protocol. This type of enrichment works best with relatively high levels of expression. The S2 cells must be stained with a saturating amount of primary antibody and must be kept cold (preferably on ice) for the duration of the procedure. S2 cells are phagocytic. If the S2 cells are allowed to phagocytose magnetic beads at room temperature, the enrichment for expression will be contaminated with non-expressing cells. 2. Expressing cells can be isolated by FACS. Again, it is critical to obtain the brightest antibody staining possible to ensure the removal of non-expressing cells. When expression of a protein is marginal, it is recommended that only the brightest cells are selected. 3. After enrichment of expressing cells by magnetic beads or FACS, it is necessary to regularly monitor expression of the transfected proteins. We have found that these enriched pop-

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ulations tend to lose expression during culture, presumable due to selective expansion of cells with lower expression. More stable, uniform expression can be obtained by deriving clones from the resistant populations, immediately following transfection or after enrichment by beads or sorting. To clone S2 cell transfectants, plate cells in medium with selection in 96-well plates at decreasing cell concentrations beginning at 10 cells per well. Threefold dilutions of the cells down to a concentration of 0.3 cell per well can be plated. Proliferating clones will usually be visible within 2 weeks. Clones should be expanded into larger volumes of medium until sufficient cells are available to screen by flow cytometry. Multiple clones, often with varying amounts of protein expression, should be selected for experimentation (see Note 9). 3.3. S2 Cells as Targets in NK Cell Assays

The usefulness of the S2 cells relies on the ability to use them in a variety of assays investigating NK cell function. We have successfully performed a variety of flow cytometry- and microscopybased assays, using the S2 cells as target cells to investigate responses of human NK cells to a variety of ligands for NK cell receptors. We have used S2 cells to monitor NK cell degranulation as measured by the surface expression of lysosome-associated membrane protein 1 (LAMP-1, also called CD107a) (31), binding of NK cells to target cells in a conjugation assay (5), clustering of activating and inhibitory receptors at NK–S2 cell synapses (34, 35), signaling induced in NK cells by contact with S2 cells (36), and the intracellular polarization of perforin-containing granules to the site of contact with target cells (31). We have also used S2 cells as target cells in a modified cytotoxicity assay. Of particular note, we have generated, through Anaspec (San Jose, CA), a rabbit polyclonal antiserum against an S2 cell membrane preparation. Addition of this serum to S2 cells provides a potent, physiological stimulus to human NK cells for ADCC through CD16 (31). Untransfected S2 cells provide negative controls for each of these assays.

3.3.1. CD107a Assay

The detection of NK cell degranulation through monitoring of the surface expression of CD107a has rapidly become the method of choice. This assay works well with S2 target cells. For a thorough description of the CD107a assay, please refer to the chapter in this issue by Bryceson et al., as well as our previous work (31). Due to a relatively high auto-fluorescence of S2 cells, it is important to obtain a bright staining of NK cells with the CD56 antibody in order obtain a stringent gate for NK cells. This may require a two-step staining protocol, using a secondary antibody against the CD56 antibody. Note that the E:T ratio used with S2 cells is 1:5 instead the 1:2 used for other target cells.

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1. This assay has been adapted from Burshtyn et al. (37) (see Chapter 7 in this volume), which is also described for use with mammalian target cells in a chapter in this issue. If necessary, induce expression in S2 cells transfected with a metallothionein-based plasmid with 1 mM CuSO4 48 h before the conjugation assay. If the S2 cells express MHC molecules, those molecules will need to be loaded with a compatible peptide. Peptides should be added to the cells 1 day before the assay to a final concentration of 1 ␮M. Expression of ligands on the S2 cells should always be monitored by flow cytometry on the day of the assay (see Note 10). 2. Each combination of NK cell and target cell should be assayed for conjugation at a series of time points. We routinely perform 0, 5, 10, 20, and 40 min time points. With S2 cell targets, each point in the assay will include 1 × 105 NK cells and 4 × 105 target cells, for a final E:T ratio of 1:4. In the following steps, be sure to label sufficient cells for the assay. 3. Label the S2 cells with the red PHK26 dye, according to the manufacturer’s protocol, summarized here (see Note 11). a. Pellet the required number of S2 cells by centrifugation at 300g. b. Count the cells. Use about 5 × 106 for labeling (scale up as necessary); put 5 × 106 cells into a new tube and bring them up to 10 ml total volume with serum-free medium. Pellet the cells again. c. While the cells are in the centrifuge, prepare the fluorescent dye. For labeling, you need 100 ␮l of “2× dye” for every 106 cells; 2× dye for 5 × 106 cells is 588 ml of Diluent C (supplied with the labeling kit) + 12 ␮l of PKH26. d. Aspirate the medium from the cells. Resuspend the target cells in 100 ␮l of Diluent C for every 106 cells. Add the 2× dye to the cells, 100 ␮l for each 1 × 106 cells. e. Incubate 5 min at room temperature. Add an equal volume of serum to the cells and incubate 1 min at room temperature. f. Wash the cells twice with Schneider cell medium + 10% FBS. After the second wash, resuspend the cells in 5 ml of medium + 10% FBS. Incubate the cells at 27◦ C for 2 h. 4. Label the NK cells with Celltracker Green. The cells should be incubated, in their standard medium at a concentration of 10 × 106 cells/ml, with 1 ␮g/ml of the Celltracker Green for 30 min. The cells should be washed twice with their standard medium and then allowed to rest in 10 ml of medium in a 15 ml tube for 30 min at 37◦ C.

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5. Wash both the NK and the S2 cells twice with HBSS + 5% FBS. Resuspend the cells in HBSS with 5% FBS and count them. Pellet the cells and resuspend the NK cells at a concentration of 1 × 106 cells/ml and the S2 cells at a concentration of 4 × 106 cells/ml using ice cold HBSS with 5% FBS. 6. To a series of FACS tubes (one tube for each time point), add 1 × 105 NK cells and 4 × 105 of the appropriate S2 cells (100 ␮l each), for an E:T ratio of 1:4. Vortex the cells quickly to mix. 7. Pellet the cells, at 4◦ C, for 3 min at 20g. 8. Incubate the tubes in a 37◦ C water bath for the desired lengths of time. When each time point is reached, vortex the appropriate tubes and then fix the cells by adding 1 ml of 0.5% paraformaldehyde. 9. Acquire samples on a flow cytometer, using stained NK and S2 cells alone as compensation controls. Conjugate pairs are represented by green and red double-stained events. Results are typically represented as the percentage of all NK cells that are in conjugates relative to the total number of NK cells. 3.3.3. Perforin Polarization Assay

NK cells respond to contact with sensitive target cells by polarizing their perforin-containing lytic granules to the site of contact with the target cell. This polarization can be detected through fluorescent microscopy. Certain S2 cell transfectants have been shown to induce polarization of granules in human NK cells (30, 31). 1. NK cells and S2 cells will ultimately be mixed at a 1:1 E:T ratio, with 1 × 106 of each cell type per sample. The required number of each transfected S2 cell should be labeled, in standard medium at a concentration of 10 × 106 cells/ml, with 1 ␮g/ml of Celltracker Green for 30 min. The cells should be washed twice in standard medium and then allowed to rest in 1 ml of medium in a 15 ml tube for 30 min. Wash twice with 10 ml HBSS + 3% FBS. Resuspend at 10 × 106 cells/ml. 2. Pellet the required number of NK cells. Wash twice with 10 ml HBSS + 3% FBS. Resuspend at 10 × 106 cells/ml. 3. In a 15 ml conical tube, combine 1 × 106 NK cells and 1 × 106 target cells (100 ␮l each). Vortex briefly to mix the cells. 4. Pellet the cells at 20g for 3 min. 5. Incubate in a 37◦ C water bath for 20 min. 6. Transfer the cells to poly-D-lysine coated culture slides. Resuspend the cells gently to preserve cell:cell contacts.

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Incubate the cells on the slides for 60 min at room temperature. 7. Aspirate the medium. Wash the cells gently twice with 1 ml PBS. Fix the cells by adding 1 ml 4% paraformaldehyde. Incubate for 20 min at room temperature. 8. Wash the fixed cells twice with PBS. 9. Permeablize the cells with 0.5 ml of 10% normal donkey serum (NDS) and 0.5% Triton X-100 in PBS. Incubate at room temperature for 30 min with gentle shaking. 10. Stain the cells with 3 ␮g/ml anti-perforin antibody (clone ␦G9) in 3% NDS, 0.5% Triton X-100 in PBS. Incubate for 1 h at room temperature with gentle shaking. 11. Wash three times, for 5 min each, with 1 ml PBS. 12. Stain with a fluorescently labeled goat anti-mouse secondary antibody in 3% NDS and 0.5% Triton X-100 in PBS. We typically use an Alexa Fluor 568 conjugated secondary antibody at a concentration of 1 ␮g/ml. Incubate for 1 h at room temperature with gentle shaking. 13. Wash three times, for 5 min each, with 1 ml PBS. 14. Aspirate the PBS and allow the slides to air dry. 15. Seal the samples on slides under a coverslip, using a drop of Prolong Gold antifade reagent as a mounting medium. 16. The cells should be imaged with a fluorescent microscope. We have acquired images on a Zeiss LSM510 confocal microscope. Z-stacks are acquired and used to create 3dimensional reconstructions of the cell contacts. Contacts between a perforin-containing NK cell and a Celltracker Green labeled target cell are identified, and the perforin is scored as polarized or not polarized by the visual appearance of the cell:cell conjugate. In most experiments, NK cells in which granules have polarized are obvious. Large numbers of cell contacts must be analyzed for rigorous analysis. In order to acquire sufficient data, acquire microscope images under relatively low magnification (e.g., multiple 40× fields). We typically analyze 150–200 contacts for each transfected S2 cell analyzed. 3.3.4. Cytotoxicity Assay by Flow Cytometry

One situation where S2 cells cannot be simply substituted for conventional target cells is cytotoxicity assays. Most cytotoxicity assays require that target cells be loaded with a radioactive or fluorescent vital label, like chromium-51, Europium, or calcein. Lysis of target cells is monitored by the release of the vital label into the medium. S2 cells label poorly with these reagents, and they have a high rate of spontaneous release. These factors combine to leave only a very small window between spontaneous

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and maximal release, making it very difficulty to detect specific NK cell-mediated cytotoxicity. Killing of S2 cells is therefore best measured using a flow cytometry-based assay, in which the S2 cells are labeled with a green lipophilic dye, mixed with NK cells, and then stained with propidium iodide to detect cytotoxicity. 1. If necessary, induce expression in S2 cells transfected with a metallothionein-based plasmid with 1 mM CuSO4 48 h before the cytotoxicity assay. If the S2 cells express MHC molecules, those molecules will need to be loaded with a compatible peptide. Peptides should be added to the cells the day before the assay to a final concentration of 1 ␮M. Expression of ligands on the S2 cells should always be monitored by flow cytometry on the day of the cytotoxicity assay (see Note 10). Untransfected S2 cells must be included as a control in the assay (see Note 12). 2. Label S2 cells with the green PKH67 dye, according to the manufacturer’s protocol, as described in Step 3 of Section 3.3.2 (see Note 11). 3. During the incubation, spin down the NK cells and wash with Iscove’s medium containing 10% FBS. The number of NK cells required will depend on the effector to target ratios being used. Each point requires 20,000 targets cells and is done in duplicate. Typically, for S2 cells, the following E:T ratios are performed: 30:1, 10:1, 3:1, 1:1, and target cells alone (spontaneous release). Set up the dilutions of NK cells. Ultimately, the target cells will be resuspended at a concentration of 20,000 cells in 100 ml, and the NK cells will be set up in dilutions containing the appropriate number of NK cells in 100 ml. For example, for the 30:1 points, the NK cells will need to be at a concentration of 6 × 105 cells per 100 ml. 4. Prepare FACS tubes. The cytotoxicity assay will be conducted directly in the FACS tubes. Label 2 tubes for duplicates of every target/dilution combination. 5. Pipet 100 ␮l of the NK cell dilutions into appropriately labeled tubes. Place these tubes in the 37◦ C incubator until the 2-h incubation of the labeled target cells is complete. 6. When the 2-h incubation of the labeled target cells is complete, bring the volume of the target cells up to 10 ml with Schneider’s medium containing 10% FBS, and spin down the cells at 215g. Wash the cells twice more with 10 ml of serum-containing medium. 7. Resuspend the target cells in 4 ml of Schneider cell medium with 10% FBS. Count the cells.

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8. Transfer 800,000 cells to a new tube and bring the volume up to 4 ml, resulting in a concentration of 20,000 cells in every 100 ␮l. 9. Add 100 ␮l (20,000 cells) to each tube with the NK cells. Vortex each tube. Then spin the tubes at 20g for 5 min. 10. Put the tubes at 37◦ C for 3 h. 11. Add propidium iodide to each tube to a final concentration of 50 ␮g/ml and vortex gently to mix. 12. On the flow cytometer, acquisition should be set up so that the green target cells, very bright in FL1, are gated on and the PI profile for those cells is displayed. Acquire at least 2000 gated target cells. 13. The percentage of cells that are positive for both the PKH67 dye and propidium iodide is the percentage of S2 cells lysed in each sample. Values from duplicate samples are usually averaged. The percentage specific lysis is calculated according to the formula [100 × (% PI -positive cells – % Spontaneous Death)/(100 – % Spontaneous Death)] where spontaneous death is determined with a sample containing only labeled S2 cells.

4. Notes 1. In functional assays involving S2 cells adapted to serumfree conditions, medium supplemented with 0.2% BSA must be used in place of serum-containing medium. Addition of serum to adapted cells will allow the recovery of cholesterol-containing microdomains. 2. S2 cells in a healthy culture are semi-adherent. When passaged into new tissue culture plates or flasks, most of the cells will adhere to the plastic fairly firmly. The cells can be detached from the plastic by repeated washes with medium using a pipet. After several passages in the same flask, the cells will not adhere as much, although they will proliferate and remain trypan blue negative. When in suspension, healthy S2 cells will grow individually, with minimal clustering or clumping. Expression of transfected proteins can be greatly affected by the health of the cells. Cells in overgrown cultures will often express lower levels of protein, whether expression is driven from a constitutive or an inducible promoter. S2 cultures in poor health can often be rescued by seeding cells into fresh medium at 1 × 106 cells/ml and allowing the cells to grow

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to 2.5 × 106 cells/ml. Once the cells are proliferating at a normal rate, protein expression will often recover to previous levels. 3. We have successfully co-transfected S2 cells with six different proteins simultaneously. It is likely that an even greater number of proteins could be simultaneously transfected. 4. Any given protein is usually expressed at a similar level in independently transfected S2 cells. For instance, expression of ICAM-1 is almost always high, whereas other molecules have proven more difficult to express. In the latter case, it is preferable to isolate S2 cells expressing a decent level of the troublesome protein after a first round of transfection and to “super-transfect” plasmids for other proteins in a second round of transfection using a different drug resistance plasmid. For example, S2 cells expressing CD48 were isolated first, prior to sequential transfections for expression of additional proteins. 5. Transient expression of proteins can be achieved by transfection with either calcium phosphate or Cellfectin method. Proteins will be expressed almost immediately following the addition of the transfection mixtures, with maximal expression occurring at 48–72 h. Either protocol should be followed to Step 5 (Section 3.2.1 for calcium phosphate transfection or Section 3.2.2 for Cellfectin transfection), where the transfection mixtures are removed by washing, and the cells are cultured for 48 h. If induction is required, 1 mM CuSo4 should be added 24 h after the washes, and induction should proceed for a further 24–48 h. 6. There are a number of plasmids using different promoters that can be used to obtain expression of exogenous proteins in Drosophila cells. We have used pRmHa3 and pAc5.1 plasmids successfully, as described in Step 5 of Section 3.1.1. Our cDNA constructs for expression in S2 cells have contained the entire coding sequence of the protein of interest, including the signal sequence. Where possible, 5’and 3’-untranslated regions of the mRNA were included in the expression constructs. It has not been necessary to alter the cDNA sequences in order to get good expression of the various human and mouse proteins in S2 cells. 7. The plasmids used for expression of selectable drug resistance must have promoters for constitutive expression. Commercial plasmids exist for selection of S2 cells with blasticidin or hygromycin (Invitrogen), as well as plasmids made by individual researchers for selection with puromycin and neomycin. It is not necessary to use expres-

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sion vectors that carry both a selectable marker and the coding region of the protein of interest. It is much more convenient to co-transfect plasmids containing the selectable marker together with the plasmids for protein expression. S2 cells are transfected by the calcium phosphate method with 19 ␮g of DNA, of which 18 ␮g is the expression plasmid(s) and 1 ␮g is the selection plasmid. In cases where expression of multiple exogenous proteins is desired, the plasmids are mixed in equal amounts to a total of 18 ␮g of DNA (for example, 6 ␮g each of three different plasmids). 8. The appropriate drug concentration for killing of untransfected S2 cells and selection of resistant S2 cells must be determined experimentally by serial dilutions. We have had success selecting resistant S2 cells with 1 mg/ml G418, 6 ␮g/ml puromycin, and 300 ␮g/ml hygromycin. Puromycin and hygromycin kill untransfected cells within 7 days. After their addition, the transfected S2 cell culture can be left alone until resistant cells grow, which usually occurs in 2 weeks. As G418 kills cells much more slowly, it is recommended to monitor live cell numbers. If the number reaches 3 × 106 cells/ml the cells should be removed from the medium by centrifugation and re-plated at 1 × 106 cells/ml in fresh medium containing G418. Protein expression may not be detectable for 2 weeks in G418 selection. 9. Selection of transfected clones is a time-consuming process, usually requiring an additional month after resistant cells have been obtained. To accelerate the process by several weeks, one can isolate expressing cells by FACS or by enrichment with antibodies coupled to magnetic beads. However, cell sorting will usually result in a broader range of expression than cell cloning and will often result in the loss or a reduction of protein over time. Therefore, it is necessary to regularly monitor expression in a sorted population and to repeat cell sorting when necessary. 10. Expression of proteins on transfected S2 cells should always be verified on the day of an assay. Expression level is dependent on the condition of the cells and can vary widely. 11. Alternatively, the S2 cells can be labeled with Celltracker dyes (Celltracker Green as a replacement for PKH67 and Celltracker Orange for PKH26). The cells should be incubated, in their standard medium at 10 × 106 cells/ml, with 4 ␮g/ml of the Celltracker reagent for 30 min at room temperature. The cells should be washed twice with their standard medium and then allowed to rest in 10 ml of medium

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in a 15 ml tube at room temperature for 30 min. Celltracker dyes are more convenient to use than the lipophilic PKH dyes. Additionally, the S2 cells occasionally display reduced viability after labeling with the PKH reagents. However, S2 cells labeled with the PKH dyes often display more uniform fluorescence than cells labeled with Celltracker, simplifying the FACS analysis. The appropriate label for a given application should be determined experimentally. 12. It is common to encounter problems with high killing of untransfected S2 cells in cytotoxicity assays. To minimize such occurrences, it is essential to seed S2 cells at optimal numbers and to allow them to proliferate for at least a week prior to the assay. However, nonspecific killing of untransfected S2 cells may also be due to constitutive release of perforin and granzymes by activated NK cells. We have not found a reliable solution to this problem. Assays for NK cell degranulation and for granule polarization in NK cells are very reliable and focus on the functions of interest (i.e., NK cell responses) rather than the fate of S2 cells. Nevertheless, target cell lysis may occasionally be a useful parameter of NK cell function, as it requires the combination of granule polarization and degranulation (31).

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ization in NKG2D-mediated NK cell cytotoxicity. Proc. Natl. Acad. Sci. U S A 105, 3017–22. Akella, R., and Hall, R. E. (1992) Expression of the adhesion molecules ICAM-1 and ICAM-2 on tumor cell lines does not correlate with their susceptibility to natural killer cell-mediated cytolysis: evidence for additional ligands for effector cell beta integrins. Eur. J. Immunol. 22, 1069–74. Johnston, S. C., Dustin, M. L., Hibbs, M. L., and Springer, T. A. (1990) On the species specificity of the interaction of LFA-1 with intercellular adhesion molecules. J. Immunol. 145, 1181–7. Sivori, S., Pende, D., Bottino, C., Marcenaro, E., Pessino, A., Biassoni, R., Moretta, L., and Moretta, A. (1999) NKp46 is the major triggering receptor involved in the natural cytotoxicity of fresh or cultured human NK cells. Correlation between surface density of NKp46 and natural cytotoxicity against autologous, allogeneic or xenogeneic target cells. Eur. J. Immunol. 29, 1656–66. Schneider, I. (1972) Cell lines derived from late embryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morphol. 27, 353–65. Matsumura, M., Saito, Y., Jackson, M. R., Song, E. S., and Peterson, P. A. (1992) In vitro peptide binding to soluble empty class I major histocompatibility complex molecules isolated from transfected Drosophila melanogaster cells. J. Biol. Chem. 267, 23589–95. Bunch, T. A., Grinblat, Y., and Goldstein, L. S. (1988) Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res. 16, 1043–61. Cai, Z., Brunmark, A., Jackson, M. R., Loh, D., Peterson, P. A., and Sprent, J. (1996) Transfected Drosophila cells as a probe for defining the minimal requirements for stimulating unprimed CD8+ T cells. Proc. Natl. Acad. Sci. U S A 93, 14736–41. Cai, Z., Brunmark, A. B., Luxembourg, A. T., Garcia, K. C., Degano, M., Teyton, L., Wilson, I., Peterson, P. A., Sprent, J., and Jackson, M. R. (1998) Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells. Immunol. Rev. 165, 249–65. Barber, D. F., Faure, M., and Long, E. O. (2004) LFA-1 contributes an early signal for NK cell cytotoxicity. J. Immunol. 173, 3653–9.

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31. Bryceson, Y. T., March, M. E., Barber, D. F., Ljunggren, H. G., and Long, E. O. (2005) Cytolytic granule polarization and degranulation controlled by different receptors in resting NK cells. J. Exp. Med. 202, 1001–12. 32. Jackson, M. R., Song, E. S., Yang, Y., and Peterson, P. A. (1992) Empty and peptidecontaining conformers of class I major histocompatibility complex molecules expressed in Drosophila melanogaster cells. Proc. Natl. Acad. Sci. U S A 89, 12117–21. 33. Eleme, K., Taner, S. B., Onfelt, B., Collinson, L. M., McCann, F. E., Chalupny, N. J., Cosman, D., Hopkins, C., Magee, A. I., and Davis, D. M. (2004) Cell surface organization of stress-inducible proteins ULBP and MICA that stimulate human NK cells and T cells via NKG2D. J. Exp. Med. 199, 1005–10.

34. Faure, M., Barber, D. F., Takahashi, S. M., Jin, T., and Long, E. O. (2003) Spontaneous clustering and tyrosine phosphorylation of NK cell inhibitory receptor induced by ligand binding. J. Immunol. 170, 6107–14. 35. Schleinitz, N., March, M. E., and Long, E. O. (2008) Recruitment of activation receptors at inhibitory NK cell immune synapses. PLoS ONE 3, e3278. 36. Riteau, B., Barber, D. F., and Long, E. O. (2003) Vav1 phosphorylation is induced by beta2 integrin engagement on natural killer cells upstream of actin cytoskeleton and lipid raft reorganization. J. Exp. Med. 198, 469–74. 37. Burshtyn, D. N., Shin, J., Stebbins, C., and Long, E. O. (2000) Adhesion to target cells is disrupted by the killer cell inhibitory receptor. Curr. Biol. 10, 777–80.

Chapter 7 Natural Killer Cell Conjugate Assay Using Two-Color Flow Cytometry Deborah N. Burshtyn and Chelsea Davidson Summary This flow cytometry-based method is a quick way to detect adhesion of NK cells to target cells. The two cell types are labeled with distinct fluorescent dyes and following co-incubation, the number of NK cells firmly adhered to target cells is quantified using two-color flow cytometry. Key words: Natural killer cells, adhesion, cell conjugates, flow cytometry.

1. Introduction Intercellular adhesion is required for NK cells to specifically lyse target cells. The formation of a stable conjugate between the NK cell and its target cell allows for signal transduction and ultimately polarized degranulation at the point of contact with the target cell. The adhesion of NK cells to target cells requires the specific interaction of adhesion molecules such as the integrin LFA-1 on NK cells and ICAM on target cells (1). Integrins such as LFA-1 can provide the initial tethering of one cell to another as well as produce stronger binding following activation of the integrin by various stimuli (chemokines, antigen receptor signaling). The latter occurs when signals provided by receptors that lead to cellular activation also modify the ability of integrins to bind to their ligands by enhancing their affinity and/or avidity for their ligands. This process of an activating receptor upregulating the binding of an integrin is known as inside-out signaling and leads to tightly bound cell:cell conjugates. The assay described here measures the formation of such tightly bound conjugates between NK cells K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 7, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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and their targets. Although we originally developed this assay as a means to optimize conditions for performing live cell imaging on NK:target cell conjugates, the assay has proved useful for studying the molecules involved in adhesion and for detecting signals involved in forming and inhibiting conjugation. We and others have performed this assay, or variations of this assay, with a variety of natural killer cells including human NK cell lines (YTS, NK92), ex vivohuman-derived IL-2-activated NK populations and clones, mouse NK cells, and rat RNK cells (2–7). The flow cytometry method described herein is versatile, avoids the tedious task of counting conjugates by microscopy required by older methods, and does not require specialized equipment beyond the flow cytometer. The cell populations under study are labeled using nonspecific fluorescent membrane dyes. Once labeled, the NK and target cells are incubated together to allow for conjugation, agitated to disperse loosely associated cells, and then fixed. Finally, the conjugates are analyzed by twocolor flow cytometry to quantitatively measure the degree of conjugation.

2. Materials 2.1. Fixative Solution

1. Add 0.5 g of paraformaldehyde to 100 ml PBS. 2. Gently warm on a heating plate while gently stirring with stir bar until completely dissolved. Do not boil. 3. Allow to cool to room temperature. 4. Store at 4◦ C for up to 1 week. Prior to experiment, chill on ice.

2.2. Cell Labeling

1. SIGMA Fluorescent cell linker kits: PKH67-GL (Green) and PKH26-GL (Red) (see Note 1). The kits contain dye in ethanol and a diluent (Diluent C) and are stored at 4◦ C protected from the light according to the manufacturer’s instructions. 2. Serum-free medium. 3. Assay medium: 5% FBS in NK cell culture medium supplemented with 1 mM L-glutamine (see Note 2). 4. FBS. 5. 15 ml conical polypropylene centrifuge tubes.

2.3. Conjugation

1. Standard 3 ml polystyrene flow cytometry tubes (Falcon 2052). 2. 0.5% paraformaldehyde/PBS. 3. Flow cytometer.

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3. Methods 3.1. Cell Labeling

1. Prewarm Diluent C and dye to room temperature. 2. If crystals have formed in the dye stock, warm in 37◦ C water bath until dissolved. 3. Pellet 4 × 106 NK cells and 4 × 106 target cells separately in 15 ml conical centrifuge tubes (see Note 3). 4. Resuspend in 10 ml of serum-free medium. 5. For each cell type, retain 2 × 105 unlabeled cells in a FACs tube. 6. Pellet remaining cells. 7. Resuspend cells in 0.4 ml of Diluent C (room temperature). 8. Prepare 0.4 ml of 20 ␮M of each dye in Diluent C (8 ␮l of stock to 392 ␮l of Diluent C). 9. Add 0.4 ml of NK cells to the PKH67 dye and pipette up and down gently to mix. 10. Add 0.4 ml of target cells to the PKH26 dye and pipette up and down gently to mix. 11. Incubate 5 min at room temperature (22–25◦ C), resuspending the cells at 2 and 4 min by gently flicking the tube. 12. Add 2 ml FBS to stop the labeling and incubate 1 min at room temperature. 13. Wash cells twice by adding 10 ml room temperature assay medium and centrifuging 4 min at 300g. 14. Resuspend in 10 ml assay medium. 15. Incubate with the lid loose for at least 1 h at 37◦ C/5% CO2 to allow the excess dye to bleed out. 16. Perform a viable cell count. Cells should be greater than 90% viable at this stage. 17. Pellet and resuspend target cells in assay medium at 2 × 106 live cells/ml and the NK cells at 1 × 106 live cells/ml. For more information regarding the E:T, see Note 4. 18. Proceed immediately to conjugation assay.

3.2. Cell Conjugation

1. Dispense 100 ␮l of each labeled cell type into a FACs tube to be used for setting up the flow cytometry parameters. 2. Add 300 ␮l of ice cold 0.5% paraformaldehyde to the unlabeled and labeled control samples and store on ice. 3. For each desired time point (e.g., 0, 30 s, 1, 2, 5, and 10 min) aliquot 100 ␮l of target cells into FACs tubes (see Note 5).

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4. Add 100 ␮l of NK cells. 5. Spin at 20 × g for 1 min (see Note 6). 6. Incubate at 37◦ C in a water bath for desired time points (see Notes 7 and 8). 7. At the end of each time point, vortex sample at high speed for 3 s (see Note 9). 8. Immediately fix by adding 300 ␮l of ice-cold 0.5% paraformaldehyde. 9. Store samples at 4◦ C protected from light until analysis. 10. Analyze by flow cytometry as soon as possible (see Note 10). Use untreated NK and target cells to set up the for-

Fig. 7.1. Illustration for gating on cell:cell conjugates. YTS cells were labeled with PKH67 and 721.221 cells with PKH26. The top panels shows the forward and side scatter plots for the mixture of the two cells at the 0 and 10 min time points. The large viable gate shown was used to capture the conjugates that have shifted to the right and up for the corresponding analysis shown in the lower panels. The numbers in the upper and lower right quadrants are the event counts that are used to calculate the % YTS cells in conjugates. For this example, the background conjugation works out as 72/(4679+72)∗ 100% = 1.5% which increased at 10 min to 1271/(2594+1271) = 33%. Note that the apparent decrease in total “YTS” events is likely due to aggregates forming that are counted as a single event and leads to an underestimate of the actual % conjugation.

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ward and side scatter parameters and set an acquisition gate to collect at least 20,000 live cell events. Be sure to use a wide forward scatter gate to capture conjugates, which may have higher forward scatter (see Fig. 7.1). Use the untreated and labeled cells to set up the sensitivity and compensation for the FL-1 (PKH67) and FL-2 (PKH26) channels correspondingly and acquire the samples. 11. Set up the analysis leaving a generous margin for single color events when setting the quadrants. Calculate the % of conjugated NK cells = 2-color events/(2-color events + FL-1+ events) × 100%. If desired, the background value can be subtracted from each experimental point which for some cell combinations can be a significant value.

4. Notes 1 Our experience has been extensively with the PKH series of dyes from Sigma and EGFP expressing cells (2, 3, 6, 8). However, this assay is adaptable to many dye pairs (such as a variety of CellTracker dyes from Molecular Probes/Invitrogen) or live cell fluorochromes (i.e., EGFP) that are compatible with flow cytometry, provided they are stably associated with the cells (5, 7, 9, 10). It is important when using the PKH dyes not to shorten the “bleed out” incubation time as this will lead to nonspecific transfer of dye from one cell type to the other through the medium. Also, after long time points of conjugation (e.g., >1 h), we do observe some transfer of the dye from one cell type to another which may result from conjugates that have dissociated. As these are dim events, allowing a wide window for the single color events will exclude these from being scored as bona fide two-color events. 2. We find the cells perform best when they are not shocked by any changes. Therefore, we use the same medium to perform the washes and assay as used to culture the cells. Typically we culture our NK lines in Iscoves medium. If you are using RPMI or other media, it is best to supplement with HEPES to prevent the pH from rising sharply when working in small volumes on the bench. The assays were optimized in medium similar to that typically used in our laboratory for cytotoxicity assays, hence 5% serum. The rate and plateau values of conjugation may be affected by higher or lower amounts of serum but we have not investigated this ourselves.

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3. The NK and target cells should be taken from log phase growth cultures with high viability. When certain NK cell lines become overgrown, the degree of conjugation falls off in parallel to their cytotoxicity. This is particularly obvious with NK92 and YTS cells beyond 7 × 105 cells/ml. Experiment to experiment reproducibility is best when the cells used have been cultured to a similar density each time. Primary NK cells from very dense cultures work fine as long as they are still in rapid growth. If using NK populations or NK clones, the extent of binding will vary with the donor as well as for each clone from a given donor due to the heterogenous nature of these cells. Extremely large target cells can be problematic as it is difficult to properly gate on the NK cells and NK cells in conjugates by forward and side scatter at the same time. 4. The E:T outlined in this protocol is 1:2 as we have found this to be optimal for several NK:target cell combinations. However, titration of the E:T from 2:1 to 1:5 will reveal where the binding is saturated. This should be done by altering the concentration of the targets while holding the concentration of the effectors constant and performing the assay in the same volume. Be careful to keep E:T ratios consistent between samples since small differences in ratios can greatly impact upon the results. 5. Some combinations of NK and targets will produce a considerable amount of background binding at the “zero” time point without incubation at 37◦ C. This binding is occurring before and during the centrifugation step that in reality provides considerable time for binding. This background can be reduced dramatically by incubating the cells on ice for 10 min prior to mixing the cells. However, many NK cell lines and primary NK cells temporarily lose their lytic function when exposed to low temperatures. We found that YTS cells and RNK cells were able to function well in standard chromium release assays following incubation on ice, and therefore, we do incubate these cells on ice prior to beginning the assay, while we avoid this for NK92 and primary NK cells and alternatively prepare them at room temperature. 6. The rationale for this step is to facilitate contact between the cells by concentrating them at the bottom of the tube. The spin is done very gently to avoid shocking the membranes by collision with the plastic or form a tightly packed pellet. 7. Each NK–target cell combination may exhibit a slightly different rate of binding. Generally we find that maximal conjugation has occurred by 10 min and then after 1 h

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begins to subside particularly when the target cells are lysed by the NK cell. When performing long time course experiments, we find it convenient to perform the assay in reverse by mixing all the samples on ice, transferring them all to the 37◦ C water bath, and then removing the samples from shortest to longest time points. Also, handling of too many samples will increase variability at the early time points. 8. The kinetics of conjugation are affected by temperature, occurring more rapidly at 37◦ C than at lower temperatures (2). For reproducibility between samples, we found round bottom tubes to be the best to maximize cell-to-cell contact and allow rapid and even heating of the sample when warmed to 37◦ C. Similarly, it is best to use a recirculating water bath to ensure even heating in order to obtain the most reproducible results particularly for early time points. 9. The vortexing step following the co-incubation prior to fixation is done to break apart loosely associated cells. Without the vortex we observe high binding for many cell:cell combinations. However, too strong or too long a vortex can cause the cells to shear resulting in patches of effector or target cell membrane attached to the other cell type that can confound the flow cytometric analysis. These can readily be visualized by fluorescence microscopy. Consistent vortexing is important to obtaining reproducible results and therefore when learning the technique it is helpful to perform duplicate samples to ensure reproducibility. 10. Typically we analyze the samples promptly. However, we have stored samples in the fixative overnight without a loss of signal or resolution.

Acknowledgments This work was supported by CIHR and AHFMR. References 1. Matsumoto, G., Nghiem, M. P., Nozaki, N., Schmits, R., and Penninger, J. M. (1998) Cooperation between CD44 and LFA1/CD11a adhesion receptors in lymphokineactivated killer cell cytotoxicity. J Immunol 160, 5781–5789. 2. Burshtyn, D. N., Shin, J., Stebbins, C., and Long, E. O. (2000) Adhesion to target cells is disrupted by the killer cell inhibitory receptor. Curr Biol 10, 777–780.

3. Standeven, L., Carlin, L. M., Borszcz, P., Davis, D. M., and Burshtyn, D. N. (2004) The actin cytoskeleton controls the efficiency of Killer cell Ig-like Receptors (KIR) accumulation at inhibitory Natural Killer cell immune synapses. J Immunol 173, 5617–5626. 4. Vyas, Y. M., Maniar, H., Lyddane, C. E., Sadelain, M., and Dupont, B. (2004) Ligand binding to inhibitory killer cell Ig-like

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receptors induce colocalization with Src homology domain 2-containing protein tyrosine phosphatase 1 and interruption of ongoing activation signals. J Immunol 173, 1571–1578. 5. Yusa, S., and Campbell, K. S. (2003) Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) can play a direct role in the inhibitory function of killer cell Iglike receptors in human NK cells. J Immunol 170, 4539–4547. 6. Osman, M. S., Burshtyn, D. N., and Kane, K. P. (2007) Activating Ly-49 receptors regulate LFA-1-mediated adhesion by NK cells. J Immunol 178, 1261–1267. 7. Back, J., Chalifour, A., Scarpellino, L., and Held, W. (2007) Stable masking by H-2Dd cis ligand limits Ly49A relocalization to the site of NK cell/target cell contact. Proc Natl Acad Sci U S A 104, 3978–3983.

8. Borszcz, P. D., Peterson, M., Standeven, L., Kirwan, S., Sandusky, M., Shaw, A., Long, E. O., and Burshtyn, D. N. (2003) KIR enrichment at the effector-target cell interface is more sensitive than signaling to the strength of ligand binding. Eur J Immunol 33, 1084–1093. 9. Yusa, S., Catina, T. L., and Campbell, K. S. (2004) KIR2DL5 can inhibit human NK cell activation via recruitment of Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2). J Immunol 172, 7385–7392. 10. Li, C., Ge, B., Nicotra, M., Stern, J. N., Kopcow, H. D., Chen, X., and Strominger, J. L. (2008) JNK MAP kinase activation is required for MTOC and granule polarization in NKG2D-mediated NK cell cytotoxicity. Proc Natl Acad Sci U S A 105, 3017– 3022.

Chapter 8 Studying NK Cell/Dendritic Cell Interactions Mathias Lucas, Cedric Vonarbourg, Peter Aichele, and Andreas Diefenbach Abstract Although NK cells were originally identified as “naturally” active cells believed to follow a cellautonomous activation program, it is now widely accepted that NK cells need to interact with dendritic cells for their full functional activation and for their homeostasis. In this chapter, we will provide an experimental guide to the analysis of NK cell/DC interactions in vitro and in vivo. We have put special emphasis on the recently developed mouse models allowing the inducible and specific ablation of various subsets of DCs and other myeloid cells. Key words: LCMV, Listeria monocytogenes , toll-like receptors, dendritic cells, macrophages, diphtheria toxin receptor.

1. Introduction Natural killer (NK) cells were discovered as lymphocytes that can spontaneously kill certain tumor target cells (1–3). These early findings have led to the proposal that NK cells are innate immune cells which follow a cell-autonomous activation and effector program once confronted with an appropriate target cell expressing stimulatory ligands. In contrast to T cells of the adaptive immune system, NK cells were widely believed to be independent of signals provided by other cell types for activation or priming. However, freshly isolated NK cells from mice and humans show only minimal effector functions (cytotoxicity, cytokine production) when incubated in vitro with tumor target cells or when directly trigK.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 8, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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gered via their stimulatory receptors (4, 5). These data suggested that resting NK cells might depend on additional signals for their activation. Indeed, most investigators treat mice with tolllike receptor (TLR) ligands, ligands for the cytoplasmic helicase protein melanoma differentiation-associated gene 5 (MDA5) or cytokines (e.g., type I interferons, IFN-I or their synthetic inducers) or culture NK cells ex vivo in the presence of cytokines (e.g., IL-2, IL-15) to elicit detectable effector functions prior to in vitro analyses of NK cell responses (4, 6, 7). These findings have led to more detailed investigations into whether NK cells might be dependent on signals from other cells for their full functional activation. A role for myeloid cells in the induction of NK cell responses in vivo has been considered by many reports (8–16). These initial studies showed that in vitro co-cultures of stimulated or infected bone marrow (BM)-derived dendritic cells (DCs) or macrophages with NK cells resulted in NK cell activation. In other studies, BMderived DCs were injected into mice which led to enhanced NK cell activity against tumors or virally infected cells (12, 17, 18). Until recently, it was not possible to study NK cell activation in the absence of DCs or macrophages in vivo because mouse models specifically lacking DCs or macrophages were unavailable. Studies using depletion of myeloid cells from mice by injection of depleting antibodies were hard to interpret as most of the phenotypic markers used are also expressed by other hematopoietic cells. The systemic depletion of DCs can be achieved by the injection of clodronate liposomes (19). However, uptake of clodronate liposomes requires phagocytic activity of the cell resulting in the depletion of both DC and macrophage populations. In the last years, various genetically modified mouse models became available allowing for the inducible and specific ablation of myeloid cell subsets. All these models employ tissue-specific expression of the avian diphtheria toxin receptor (DTR) (20). As mouse cells are insensitive to diphtheria toxin (DT) from Corynebacterium diphtheriae, only those cells expressing the ectopic DTR are sensitive to DT. By now, various studies are available that have analyzed NK cell function in mice lacking DCs. Collectively these data demonstrate that NK cell function in response to TLR-mediated stimulation and to various pathogens strictly depends on the presence of conventional DCs (5, 21–26). On the following pages, we will provide protocols for the analysis of NK cell priming or activation by myeloid cells in vitro. Furthermore, we discuss how the available mouse models allowing for the ablation of various myeloid cell subsets can be employed to analyze NK cell function in vivo and how they can be further manipulated to reveal the molecular pathways required for the priming of NK cells by myeloid cell populations in vivo.

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2. Materials 2.1. Mice

1. C57BL/6 (B6; expressing the CD45.2 antigen) 2. B6-Ly5.2/Cr (C57BL/6-Ly-5a congenic mice, expressing the CD45.1 antigen) 3. CD11c DTR transgenic mice (B6.FVB-Tg(ItgaxDTR/EGFP)57Lan/J) (27) or other mouse strains allowing for the depletion of myeloid cell subsets (Table 8.1). 4. H-2 K-BCL-2: Bcl-2 transgenic mice under the control of the H-2 K promoter (28). 5. Il15 −/− (C57BL/6NTac-IL15tm1Imx )(29) 6. All mice are maintained under specific pathogen-free conditions and used at 8–16 weeks of age.

2.2. Cell Lines

1. MC57 (H-2b ) is a C57BL/6-derived methylcholanthreneinduced fibrosarcoma cell line used to titer LCMV (Section 3.6) (30). 2. NCTC clone 929 (L-929; Connective tissue, mouse; ATCC CCL-1) 3. YAC-1 (ATCC TIB-160) is a T-cell lymphoma used for the NK cell activation assays (Section 3.8) (31). 4. RMA-S is a Tap-2-defective cell line used for the NK cell activation assays (Section 3.8) (32). 5. RMA-S-H60 cells are RMA-S cells retrovirally transduced with a ligand of the NKG2D receptor, H60 (5, 33). These cells are used as target cells in the NK cell activation assays (Section 3.8).

2.3. Solutions

1. Minimum essential medium (MEM) cell culture medium: MEM (Invitrogen) containing Earle’s Salts and nonessential amino acids (NEAA) is complemented with 5% fetal calf serum (FCS), 200 mg/l glutamine (Sigma), 50,000 U/l penicillin, and 50 mg/l streptomycin (Invitrogen), filter sterilized (0.22 ␮m), and stored a 4◦ C. 2. 2× MEM: MEM powder (Invitrogen) is dissolved in 5 l ddH2 O (instead of 10 l) to obtain a 2× concentration. 2× MEM is complemented with 10% FCS, 400 mg/l glutamine, 100,000 U/l penicillin, and 100 mg/l streptomycin, filter sterilized (0.22 ␮m), and stored at 4◦ C. 3. Dulbecco’s Modified Eagle Medium (DMEM) cell culture medium: DMEM (Invitrogen) containing 4.5 g/l glucose and L-glutamine is complemented with 5–10% FCS (this percentage may vary depending on the cultured

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cells), 200 mg/l glutamine, 50,000 U/l penicillin, 50 mg/l streptomycin, 10 mg/l gentamycin sulfate (Biowhittaker), and 50 ␮M ␤-mercaptoethanol (Sigma), filter sterilized (0.22 ␮m), and stored at 4◦ C. 4. Brain Heart Infusion (BHI) medium: 1 l deionized water; 37 g BHI powder (Oxoid). Heat at 100◦ C for 15–20 min in order to completely dissolve the powder. Autoclave and store at 4◦ C. 5. BHI Agar: 1 l deionized water supplemented with 16 g Bacto Agar (BD); 37 g BHI powder. Heat at 100◦ C for 15– 20 min in order to completely dissolve the powder. Autoclave and store at 4◦ C. 6. Methylcellulose solution: Mix 1 vol. of 2% methylcellulose (Sigma) in ddH2 O with 1 vol. 2× MEM. 7. BHI containing 0.05% Triton X-100 (Amresco). 8. Dulbecco’s phosphate buffered saline (DPBS; Invitrogen) containing 0.5% Triton X-100. 9. Trypsin-EDTA: 0.05% Trypsin-EDTA (Invitrogen). 10. ACK lysis buffer (red blood cell lysing buffer): 1 l deionized water supplemented with 8.025 g NH4 Cl, 1 g KHCO3 , and 200 ␮l 0.5 M EDTA (pH 8.0); sterilize using 0.22 ␮m filter and store at 4◦ C. 11. BD Pharm Lyse buffer (BD Biosciences). 12. FACS buffer: DPBS (Invitrogen) complemented with either 2% FCS or 0.5% BSA (Sigma), filter sterilized (0.22 ␮m), and stored a 4◦ C. 13. MACS buffer: DPBS (Invitrogen) complemented with 0.5% BSA and 2 mM EDTA (Invitrogen), filter sterilized (0.22 ␮m), and stored a 4◦ C. 14. PBS/EDTA: DPBS containing 5 mM EDTA (pH 8.0). 15. Monensin (GolgiStop, BD Bioscience). 16. Brefeldin A (Sigma): stock solution of 5 mg/ml in ethanol. 17. Cytofix/Cytoperm solution (BD Bioscience). 18. FACS permeabilization buffer: FACS buffer containing 0.5% saponin (Sigma). 19. FACS fixing solution: DPBS (Invitrogen) containing 4% formaldehyde (dilution of a 37% formaldehyde solution in PBS). 20. 4% Paraformaldehyde: Heat 50 ml ddH2 O to 60◦ C (glass beaker); add 4 g paraformaldehyde (Merck). Stir (stirring bar) and add few drops (2–3) of 1 N NaOH until the solution clears (if not, check 7
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21. Percoll: Percoll (GE Healthcare) is not an isotonic solution. Thus, 1 vol. DPBS 10× (Invitrogen) has to be added first to 9 vol. of Percoll in order to have an isotonic Percoll solution (Percoll 90%). Dilute the 90% percoll with medium in order to obtain 40% and 60% Percoll solutions used for the separation gradient. 22. Lympholyte-M (Cedarlane). 23. Digestion buffer (for preparation of lung lymphocytes): Hank’s balanced salt solution (HBSS) containing CaCl2 and MgCl2 but no phenol red (Invitrogen) and complemented with (final concentrations) 1 mg/ml collagenase D (Roche). 24. Diphtheria toxin (DT): DT (Sigma or Calbiochem) is reconstituted in 1 ml sterile distilled water (1 mg/ml). After reconstitution, aliquot and freeze at -80◦ C. Repeated freezing and thawing is not recommended (see Note 1). 25. Cytokines: IL-12 (R&D Systems), type I IFN: IFN-␣A and IFN-␤ (PBL Biomedical Laboratories), GM-CSF (Peprotech) M-CSF (Peprotech), IL-2 (Peprotech) and IL-15 (Peprotech). All cytokines were reconstituted and stored following the manufacturer’s protocol. 26. Peroxidase substrate: SIGMAFAST TM OPD (Sigma). 27.

2.4. Abs

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Cr: sodium chromate [51 Cr] (Perkin Elmer), 5 mCi/ml (needs institutional approval).

1. CD3␧ (145-2C11) monoclonal antibody (mAb) (eBioscience). 2. CD11b (M1/70) mAb (eBioscience) reacts macrophages, neutrophils, and some NK cells.

with

3. CD11c (N418) mAb (eBioscience) reacts with different subsets of cells and mostly with DCs and NK cells. 4. CD16/CD32 (2.4G2) mAb (BD Bioscience) is used to block the Fc␥RIII/II. 5. CD19 (MB19-1) mAb (eBioscience) reacts with a coreceptor on B cells. 6. CD25 (PC61.5) mAb (eBioscience) reacts with the IL-2 receptor ␣ chain. 7. CD45R/B220 (RA3-6B2) mAb (BD Biosciences) reacts with different cell subsets but mostly with B cells. 8. CD45.1 (A20) mAb (eBioscience) is used to detect adoptively transferred cells. 9. CD45.2 (104) mAb (eBioscience) is used to detect adoptively transferred cells.

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10. CD49b (DX5) mAb (eBioscience), which reacts with a pan-NK cell marker. 11. CD69 (H1.2F3) mAb (eBioscience) is a marker for lymphocyte activation. 12. CD122 (TM-␤1) mAb (eBioscience), which reacts with the IL-2/15 receptor ␤ chain. 13. Class II MHC (M5/114.15.2) mAb (eBioscience). 14. Goat anti-rat IgG (H+L)-peroxidase Ab (Jackson Immunoresearch) used in the plaque assay experiment after LCMV infection. 15. Anti-Granzyme A rabbit serum (34). 16. Granzyme A (3G8.5) mAb (Santa Cruz) (35). 17. Anti-Granzyme B rabbit serum (34). 18. Granzyme B (GB12) mAb (Caltag) (35). 19. IFN-␥ (XMG1.2) mAb (eBioscience). 20. IL-15R␣ Ab (goat IgG; R&D Systems). 21. Ly49A (A1) mAb (eBioscience). 22. Ly49C/I (5E6) mAb (eBioscience). 23. Ly49D (4E5) mAb (eBioscience). 24. Ly49G2 (Cwy-3) mAb (eBioscience). 25. NKG2A/C/E (20d5) mAb (eBioscience). 26. NKR-P1B/C (PK136) mAb (eBioscience) reacts with a pan-NK cell marker (NK1.1) only in certain mouse strains, such as C57BL/6 (NKR-P1C) and SJL (NKR-P1B). 27. PDCA-1 (Miltenyi), which reacts with pDCs. 28. Perforin 1 (OMAK-D) mAb (eBioscience) (35). 29. Biotin-conjugated rabbit anti-goat IgG (H+L) (Zymed). 30. Rat anti-LCMV nucleoprotein (VL4) used in the plaque assay experiment after LCMV infection (30). 31. Fluorochrome-labeled goat anti-rabbit mAb (Molecular Probes) used as secondary Abs for the anti-Granzyme A and B rabbit sera. 2.5. Magnetic Beads and Columns

1. Anti-PE Microbeads (Miltenyi Biotec).

2.6. Stimulation of Myeloid Cells

1. TLR3/MDA5 agonist: poly(I:C) (Sigma) (see Note 2)

2. LS Columns (Miltenyi Biotec).

2. TLR4 agonist: ultrapure LPS from E. coli O111:B4 (Invivogen) (see Note 2) 3. TLR7 agonist: R837/imiquimod (Invivogen) (see Note 2) 4. TLR9 agonist (see Note 2)

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a. CpG1668 (B class) G∗ G∗ GGGACGATCGTC∗ G∗ G∗ G∗ G∗ G∗ G b. CpG2216 (A class) T∗ C∗ G∗ T∗ C∗ G∗ T∗ T∗ T∗ T∗ G∗ T∗ C∗ G∗ T∗ T∗ T∗ T∗ G∗ T∗ C∗ G∗ T∗ T (∗ : phosphorothioate modification; both synthesized by ThermoFisher). 5. Anti-CD40 in vitro stimulant: CD40 (FGK45) mAb (36). 6. Anti-CD40 in vivo stimulant: CD40 (1C10) mAb (eBioscience). 7. Listeria monocytogenes strain EGD (needs institutional approval) (37). 8. LCMV-WE (needs institutional approval) (38). 2.7. Organ Homogenization

1. Mechanical Stirrer/grinder (Heidolph). 2. Teflon pestle and 10 ml glass tubes (e.g., Milian International). 3. Polystyrene dishes without further surface modifications (e.g., Greiner Bio-One). 4. 40 ␮m nylon mesh or a cell strainer (BD Biosciences). 5. Frosted glass slides.

3. Methods 3.1. Generation of Bone Marrow-Derived DC or Macrophages

Bone marrow (BM) cells can be used to generate BM-DCs or macrophages required for in vitro studies of NK/DC interactions (Section 3.4) or for in vivo rescue of mice depleted of all DCs (Section 3.6). 1. Femurs and tibias are surgically dissected from mice and remaining muscle and tissue are removed with sterile gauze. It is important to isolate the entire bone that should not be damaged during the preparation procedure. 2. Bones are then placed for 3–5 min in a Petri dish containing pure ethanol (it is very important to not extend the soaking time in ethanol). 3. Sterilized bones are rinsed in DMEM medium containing 10% FCS (referred to as “medium” for the rest of this protocol) and are cut open at the epiphyses using scissors. BM cells are obtained by repeated flushing with a 27 g needle attached to a 1 ml syringe filled with medium. Alternatively, whole bones can be crushed with a sterile mortar in a Petri dish with 5 ml medium. Once bones have been entirely crushed, the cell preparation must be passed through a 40–100 ␮m nylon mesh to remove any pieces of bone. This is very important if the cells have to be injected into mice.

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4. BM cells are pelleted by centrifugation (300 × g for 5 min), resuspended in fresh medium and counted. 5. BM cells are seeded at 4 × 105 /ml medium in 6-well plates (2 × 106 cells per well) or in Petri dishes (12 × 106 for one 100 × 15 mm dishes). 6. For the generation of BM-derived DCs, BM cells are cultured in medium containing 10 ng/ml GM-CSF. Every other day, half of the medium is replaced with fresh GMCSF-containing medium. After 5–6 days immature DCs can be harvested and used in the experiments. It should be noted that immature DCs are not strongly adherent and can be easily detached. 7. For the generation of BM-derived macrophages, it is strongly recommended NOT to use tissue culture dishes in which macrophages become too adherent and cannot be easily detached for further use. We recommend the use of polystyrene dishes without further surface modifications like those widely used in bacteriology. BM cells are cultured in medium containing 10 ng/ml M-CSF. Every other day, half of the medium is replaced with fresh M-CSF-containing medium. After 7–10 days macrophages can be harvested with PBS containing EDTA and used in experiments. 3.2. Lymphocyte Extraction from Peripheral Organs 3.2.1. Blood

In this section, we describe methods used to prepare lymphocytes from peripheral organs such as spleen, liver, and lungs as a source of NK cells. 1. Blood samples are collected by cardiac puncture. 2. Red blood cells are lysed using the BD PharmLyse solution (BD Biosciences) according to manufacturer’s instructions.

3.2.2. Spleen

1. Spleens are surgically removed from mice. 2. Splenocyte suspensions are prepared by passing the spleen through a 40 ␮m nylon mesh or a cell strainer using the flat side of a syringe plunger. 3. Flush out remaining cells with 5–10 ml DMEM medium complemented with 10% FCS (referred to as “medium” for the rest of this protocol). 4. Pellet cells (300 × g, 5 min). 5. For red blood cell lysis, pelleted cells are resuspended in 1 ml ACK buffer per spleen and incubated for 1–2 min. ACK is quenched with an equal volume of medium and cells are pelleted again. 6. Washed cells are resuspended in the appropriate buffer and can be processed further.

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1. Lymph nodes are surgically removed from mice using fine forceps. 2. Lymph nodes are crushed between the frosted ends of two glass slides in DMEM medium. 3. Cells are filtered through a nylon mesh, pelleted, and resuspended in the appropriate buffer.

3.2.4. Liver

1. Mice are euthanized and perfused through the left ventricle with 20 ml PBS. 2. Perfused livers are surgically removed. 3. Single cell suspensions are prepared by passing the liver through a 40 ␮m nylon mesh or a cell strainer using the flat side of a syringe plunger. 4. Cells are pelleted at 400 × g for 5 min and resuspended in 10 ml 40% isotonic Percoll solution per liver. 5. 5 ml of this suspension is carefully layered on top of 5 ml 60% isotonic Percoll (i.e., two 15 ml centrifuge tubes per liver). 6. Centrifuge 20 min at 900 × g at room temperature without brake. 7. After centrifugation, red cells are located at the bottom of the tube, most of the lymphocytes are located at the interface between the two different gradients of Percoll, and hepatocytes are floating on the top of the solution forming a dense layer of cells. 8. Carefully remove the hepatocytes with a pipette. 9. Harvest the interphase and the rest of the upper phase containing the lymphocytes. Add 5 ml of fresh DMEM medium and pellet cells by centrifugation (400 × g, 5 min). 10. Washed cells are resuspended in the appropriate buffer and can be processed further.

3.2.5. Lungs

1. Mice are euthanized and perfused through the right ventricle with 20 ml PBS. 2. Lungs are surgically removed. 3. Lung tissue is cut into small pieces using a scalpel and incubated with agitation for 45 min at 37◦ C in HBSS containing collagenase D. 4. The enzymatic reaction is stopped by the addition of 0.1 vol. of 5 mM EDTA. 5. Lung pieces are passed through a nylon mesh using the flat side of a syringe plunger. 6. Cells are pelleted at 400 × g for 5 min.

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7. The pellet is resuspended in 5 ml PBS (at room temperature) and carefully layered upon 3 ml of Lympholyte-M (also at room temperature). 8. After centrifugation (1000 × g, 20 min, room temperature, without brake), lymphocytes are recovered at the interphase using a Pasteur pipette. 9. Lymphocytes are washed in PBS, pelleted at 800 × g for 10 min, and resuspended in the appropriate buffer for further processing. 3.3. NK Cell Purification

It is very important to obtain highly purified NK cell populations for the in vitro co-culture assays. To this end, we have developed a method combining the use of magnetic beads and flow cytometry-based sorting. 1. NK cells are enriched from the various lymphocyte preparations using magnetic beads. To this end, lymphocytes (prepared as described in Section 3.2) are stained in microfuge tubes (maximally 50 × 106 cells/ml) with 2 ␮g/ml of PEconjugated CD49b antibody (clone DX5) for 20 min on ice (staining is best performed in the dark) (see Note 3). 2. After washing two times (300 × g, 5 min, 4◦ C) with MACS buffer, cells are incubated with anti-PE microbeads and positively sorted on LS columns following the manufacturer’s instructions (Miltenyi Biotec). 3. After counting, CD49b-enriched cells are labeled with 5 ␮g/ml of FITC-labeled CD3⑀ antibody (2C11), washed two times (300 × g, 5 min, 4◦ C), and CD49b+ CD3− NK cells are highly purified using a cell sorter. 4. Expected yield from one spleen is 1 × 106 NK cells at a purity of 95–99%.

3.4. Assays for Studying NK Cell Priming by DCs or Macrophages In Vitro

NK cell priming by myeloid cells can be analyzed in vitro by coculture of stimulated or infected myeloid cells with highly purified NK cells. Stimulation of myeloid cells is obtained by addition of TLR ligands, bacteria, viruses, cytokines, or by cross-linking of activating receptors expressed by myeloid cells (e.g., CD40). 1. BM-derived DCs or macrophages and NK cells are mixed in DMEM supplemented with 10% FCS. In our experience, various DC/macrophage: NK cell ratios can be employed. We usually use 1 × 106 BM-DC or BM-macrophages and 2 × 106 NK cells in 24 well plates. NK cells without the addition of any myeloid cells are used as a control. Several stimulation options are possible, including one or more of the following: a. If stimulation by anti-CD40 is desired, cells are added to 24-well plates previously coated at 4◦ C with 10 ␮g/ml

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anti-CD40 (clone FGK45) in PBS for 18–24 h (wash the plate three times with PBS before using it). b. Add the required stimulus (cytokine or TLR ligand) to the co-cultures. Usually, the effective concentrations of the respective stimuli need to be experimentally established. c. If desired, blocking antibodies to soluble factors or cell surface receptors involved in the activation of NK cells by myeloid cells can be added to the co-cultures. Usually, the effective concentrations of these antibodies need to be experimentally established but often are between 10–50 ␮g/ml. d. Another way to address the molecular pathways involved in the activation or priming of NK cells by myeloid cells is through the use of myeloid cells or NK cells obtained from mice genetically deficient for gene products of interest. 2. After 18–24 h of co-culture (37◦ C in a humidified atmosphere containing 5% CO2 ), NK cell activation can be assessed as described in Section 3.8 (see Note 4). 3.5. Diphtheria Toxin-Based In Vivo Mouse Models for Depleting Myeloid Cells

The in vivo analysis of immune responses in the absence of myeloid cells has been hampered by the absence of experimental mouse models lacking myeloid cells. Recently, various mouse strains became available that allow for the inducible and specific ablation of various myeloid cell populations. These model systems are based on the tissue-specific expression of the simian diphtheria toxin receptor (DTR) under the control of a tissue-specific promoter (20). The most commonly used strategy was to express the DTR as a transgene under the control of a cell type-specific promoter or to “knock-in” the DTR cassette into the translational start of a tissue-specific gene (Table 8.1) (39). Another highly versatile strategy employed a mouse strain in which the DTR cassette is “knocked into” the ubiquitously expressed Rosa26 locus (iDTR mice) (40). In a very similar approach, the toxic subunit A of DT (DTA) was “knocked into” the Rosa26 locus (R-DTA mice) (41). Expression of DTR or DTA is prevented by a stop codon preceding the coding sequence of DTR or DTA flanked by two loxP sites. Crossing of iDTR or DTA mice with mice expressing Cre recombinase under the control of a tissue-specific or cell type-specific promoter allows for the excision of the stop sequence and expression of DTR or DTA. It should be noted that there are significant differences between the Cre/lox and the above mentioned conventional approaches. In the conventional strategy, all cells in which the promoter is active at the time of DT injection express DTR and will be depleted. In contrast, the iDTR and DTA strategies result in the expression of DTR or DTA

DTR-OVA-EGFP cassette inserted into a BAC (RPCI-24-7812) containing the full Cd11c promoter

DTR-EGFP inserted into the second exon of the Langerin gene

CD11c DTR BAC

Langerin-DTR

DTR-EGFP cassette expressed under the control of the Cd11b promoter

DTR under the control of the Rosa26 promoter (loxP) STOP (loxP)

DTA under the control of the Rosa26 promoter (loxP) STOP (loxP)

CD11b DTR

iDTR

R-DTA

Langerin-DTA

DTR-EGFP cassette expressed as a transgene under the control of the Cd11c promoter

CD11c DTR

IRES-DTR-EGFP inserted into the sixth exon (3’ UTR) of the Langerin gene Transgenic mice expressing a modified human BAC (RP11-504o1) containing the gene for Langerin (Langerin-IRES-DTA)

Constructs used

DTR model

– Depends on the Cre-expressing mouse strains used

– Depends on the Cre-expressing mouse strains used

– Macrophages

– Epidermal LC

– Lymphoid Langerin+ cells

–

4 (3–7 inj. every 24 h)

25 (2 inj. every 48 h)

–

2–16 (1 inj.)

41

40

50, 51

49

48

47

46 25

– Activated CD8 T cells

– Epidermal LC

45

– Plasma B cell blasts 8–64 (1–21 inj. every 24 h)

44

– Alveolar macrophages

27, 42 43

2–4 (1 inj.)

– CD11chigh DC

Reference

− Metallophilic and marginal zone macrophages

DT dose (ng/g body weight)

Cell-type(s) depleted

Table 8.1 Overview of genetically modified mouse strains for the depletion of myeloid cell subsets

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by all cells that are currently able to activate the promoter and, in addition, by all cell types that had previous promoter activity during their lineage commitment. Thus, if the promoter driving Cre expression is active in a precursor subset and is subsequently silenced, the progeny of these cells will still express DTR and will be depleted following DT injection. Similarly, transient Cre expression in the DTA model will lead to DTA expression and irreversible loss of the respective population (Notes 5 and 6). 3.5.1. CD11c DTR Transgenic (tg) Mice

The best-characterized strain is the CD11c DTR tg line (27) in which DTR expression is controlled by the Cd11c promoter (52). In these mice, a single DT injection (4 ng/g body weight) results in specific ablation of conventional CD11chigh DCs (including myeloid, lymphoid, and subsets of dermal DCs) whereas pDCs and most Langerhans cells (LC) are not depleted (5, 27, 39). Although NK cells express intermediate to high levels of CD11c (53), we and others demonstrated that NK cells are not affected after DT injection (5, 22). Thus, CD11c DTR tg mice allow for the analysis of NK cell responses in the absence of DCs in vivo. Ablation is not entirely DC-specific as two distinct splenic macrophage populations (marginal zone macrophages, MZM and metallophilic macrophages, MM) (5, 43) and alveolar macrophages (44) are also depleted after injection of DT. It is important to note that after a single DT injection, DC ablation is transient and does not last more than 2–3 days. In contrast, ablation of MZM and MM lasts longer (at least 7–9 days). These different regeneration kinetics of DCs and macrophages can be exploited to distinguish between DC-mediated and macrophagemediated effects on NK cell responses. Specifically, NK cell responses should be analyzed 24–48 h after DT injection (depletion of DCs, MZM, MM) and at day 5 or 6 (DC compartment restored, ongoing depletion of MZM, MM) (5). The transient nature of DC depletion makes it impossible to analyze NK cell responses at later time points of an infection and it has been observed by various groups that a second injection of DT is lethal. Lefrancois and colleagues presented a solution to this problem by generating BM chimeric mice (lethally irradiated B6 mice are reconstituted with BM of CD11c DTR tg mice) (42). These mice can then be repeatedly injected with DT, facilitating the analysis of DC-dependent immune responses at later time points. These data also imply that the lethality observed after a second injection of DT is due to the expression of the DTR by radioresistant, likely non-hematopoietic cells (42). A concern was that repeated DT injections might lead to the generation of neutralizing antibodies. However, Waisman and colleagues showed that repeated injections of DT did not result in ineffectiveness of the DT treatment (40).

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Very recently, another CD11c DTR tg mouse line was developed using a BAC transgenic approach that reportedly allows multiple injections of DT and might be suitable to study NK cell responses and homeostasis in more long-term experiments without the need to generate BM chimeric mice (25). Initial reports are also available regarding the suitability of the R-DTA mice for DC depletion studies. Crossing CD11cCre mice (54) with R-DTA mice led to depletion of conventional DCs but also of pDCs and thus seems to be less specific than the CD11c DTR tg mice (26). This report also confirmed previous studies by showing that relevant NK cell responses to L. monocytogenes require the presence of DCs (5). 3.5.2. Langerin DTR tg Mice

To specifically deplete LC in vivo, two groups have generated mice with a “knock-in” of the DTR into the translational start (47) or as an IRES-DTR sequence into the 3’ untranslated region of the Langerin gene (48). Depending on the dose of DT injected, both types of Langerin-DTR mice show depletion of all LCs and of Langerin-positive lymphoid organ-resident DCs (16 ng DT/g body weight: depletion of both cell populations; 2 ng DT/g body weight: selective ablation of LCs). Eradication of these DC populations lasts for up to 4 weeks after a single injection of DT. Repeated injections of DT are well tolerated by these animals. A third group generated a transgenic mouse model in which the toxic subunit A of the DT is over-expressed under the Langerin promoter (49). In these mice, LCs are constitutively absent throughout life. The role of LCs for NK cell function or homeostasis has not yet been probed.

3.5.3. CD11b DTR tg Mice

To probe the role of macrophages for NK cell responses, a CD11b DTR tg mouse line might be helpful. After injection of DT (25 ng/g body weight), CD11b+ F4/80+ macrophages and monocytes in the peripheral blood were transiently depleted (restoration after day 4), whereas neutrophil numbers did not significantly change despite their high-level expression of CD11b (50, 51). An analysis of NK cell responses and of any effects of DT injection on NK cells using CD11b DTR tg mice have yet to be performed.

3.6. Assays for Studying NK Cell Priming In Vivo

The following is a basic protocol for the analysis of NK cell responses to infections in the absence of DCs employing the CD11c DTR tg mice. This protocol can be easily modified to use other stimuli and/or other mouse strains using the DT/DTR system. 1. At day –1, CD11c DTR tg mice are injected i.p. with 2–4 ng DT/g body weight. Control mice receive PBS injections. To control for DT effects other than DC ablation, it might be

3.6.1. In Vivo Analysis of NK/DC Interactions Following TLR Ligand Stimulation

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advisable to have a group of B6 mice injected with DT (see Note 7). 2. At day 0, mice are injected i.p. or s.c. (for studying NK cell responses in the local draining lymph node) with TLR ligands (TLR3: 50 or 5 ␮g poly(I:C), TLR4: 25 or 1 ␮g ultrapure LPS from E. coli O111:B4, TLR7: 250 or 25 ␮g R837, TLR9: 150 or 10 ␮g CpG1668) or with an agonistic antibody specific for CD40 (100 ␮g 1C10; eBioscience) Control mice receive injections of PBS or 100 ␮g isotype control antibody (rat IgG2a), respectively. 3. At day 1, (18–24 h after injection) lymphocytes are harvested from the desired organs (see Section 3.2) and activation of NK cells activity can be tested in the desired assay (see Section 3.8 and Notes 5 and 6). 3.6.2. In Vivo Analysis of NK/DC Interactions Following Listeria Infection

The following is a basic protocol for the analysis of NK cell responses to a bacterial pathogen in mice ablated of all DCs (see Note 7). It can be easily modified to study NK cell responses to other pathogens. All waste generated during this experiment must be autoclaved. All experiments are performed in a BSL2 laboratory or animal facility. 1. Preparation of Listeria monocytogenes a. The day before the infection, bacteria (inoculated from frozen stocks) are cultured overnight (O/N) in 3 ml BHI culture medium with agitation (37◦ C, shaking incubator at 200 rpm). b. Add 500 ␮l of the O/N culture to 50 ml BHI medium and grow until they reach 0.2 OD600 (0.1 OD600 ≈ 2 × 108 bacteria/ml). c. Wash 1 ml of the culture twice in PBS (microfuge tube, 750 × g, 5 min) (see Note 8). d. Dilute bacteria at the desired concentration for injection. 2. At day −1, mice are injected i.p. with 2–4 ng DT/g body weight. Control mice are given i.p. injections of PBS. To control for DT effects other than DC ablation, it might be advisable to have a group of B6 mice injected with DT. 3. At day 0, mice are injected with 104 cfu L. monocytogenes strain EGD i.v. 4. At day 2, lymphocytes are harvested from the desired organs (see Section 3.2) and activation of NK cells can be tested in the desired assay (see Section 3.8). 5. Analysis of bacterial titers in LN and spleen: a. Organs are homogenized separately in 5 ml of BHI containing 0.05% Triton X-100 (use stainless steel wire mesh, nylon mesh, or cell strainers and 50 mm Petri dishes).

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b. Perform three serial 1:10 dilutions of the organ homogenates in 1.5 ml microfuge tubes by adding 100 ␮l of homogenate in 900 ␮l BHI containing 0.05% Triton X-100, mixing well, transferring 100 ␮l of this first dilution into 900 ␮l BHI containing 0.05% Triton X-100, mixing well again, and repeating the previous step one more time for the last dilution. c. Serial dilutions of the homogenates are plated on BHI agar plates. d. Colonies are counted after incubation at 37◦ C in a humidified atmosphere containing 5% CO2 for 24 h. 3.6.3. In Vivo Analysis of NK/DC Interactions Following LCMV Infection

The following is a basic protocol for the analysis of NK cell responses to a viral pathogen in mice ablated of all DCs (see Note 7). It can be easily modified to study NK cell responses to other pathogens. All waste generated during this experiment must be autoclaved. All experiments are performed in a BSL2 laboratory or animal facility. 1. Preparation of LCMV a. 1 × 107 mouse fibroblast cells L929 are seeded about 14 h before infection (O/N) in a 150 cm2 tissue culture flask (in DMEM culture medium containing 5% FCS). b. The next day, medium is removed and cells are infected with LCMV-WE at an MOI (multiplicity of infection) of 0.01 (in 5 ml DMEM containing 2% FCS). This should allow the infection of 1 × 105 cells. c. Incubate for 1.5 h at 37◦ C in a humidified atmosphere containing 5% CO2 . d. Remove the medium (dispose as autoclave waste). e. Add 30 ml of DMEM culture medium containing 5% FCS. f. Harvest the supernatant 48 h later (harvest I), centrifuge at 650 × g for 20 min at room temperature in a table-top centrifuge and freeze harvest I at −80◦ C (see Notes 9 and 10). g. Add 30 ml of DMEM culture medium containing 5% FCS to the infected cells. h. Harvest the supernatant 24 h later (harvest II, 72 h after infection), centrifuge at 650 × g for 20 min at room temperature in a table-top centrifuge and freeze harvest II at −80◦ C. i. Before any further use, experimentally determine virus titers of harvest I and II using a modified version of the focus formation assay described in Step 6 below (30).

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Replace the serial dilutions of the organ homogenates by serial dilutions of harvest I and II (start at 1:1000). j. Freeze aliquots containing the desired viral titer for mouse injection. 2. At day −1, mice are injected i.p. with 2–4 ng DT/g body weight. Control mice are given i.p. injections of PBS. To control for DT effects other than DC ablation, it might be advisable to have a group of B6 mice injected with DT. 3. At day 0, mice are injected with 200 pfu of LCMV i.v. 4. At day 2, lymphocytes are harvested from the desired organs (see Section 3.2) and activation of NK cells can be tested in the desired assay (see Section 3.8). 5. Analysis of viral titers in spleen and organs: a. Spleens from infected mice are homogenized in 10 ml glass tubes with a Teflon pestle coupled to a grinder/mechanical stirrer (2 ml MEM + 2% FCS; 2000 rpm for approximately 30 s per organ; see Notes 11 and 12). b. Organ homogenates are centrifuged (650 × g; 20 min) to remove cell debris. Supernatants are used to determine viral titers (see Note 11). c. Six serial 1:10 dilutions of the organ homogenates are performed in culture medium containing only 2% FCS (1 ml final). d. Add 200 ␮l of cell culture medium containing 5% FCS and 8 × 105 cells/ml MC57 cells to each well of a 24well plate. e. Add 200 ␮l of the organ homogenate dilutions. f. Incubate 4–5 h at 37◦ C in a humidified atmosphere containing 5% CO2 . g. Prepare a 2% methylcellulose solution in 2 × MEM (prepare more than 12 ml per 24-well plate). h. Add 400 ␮l of pre-warmed (37◦ C) 1% methylcellulose to each well of a 24-well plate. Incubate 48 h at 37◦ C in a humidified atmosphere containing 5% CO2 . i. Remove medium and fix cells by addition of 200 ␮l of 4% formaldehyde in PBS (the formaldehyde must be gently pipetted down the side of the well). Incubate 20 min at room temperature and then discard the supernatant (dispose as autoclave waste). j. Lyse the cells in 200 ␮l of 0.5% Triton X-100 in PBS (5 ml/plate). Incubate 30 min at room temperature. k. Discard the supernatant and wash wells with PBS (you can soak the entire plate in a receptacle containing PBS).

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l. Block with 200 ␮l PBS containing 10% FCS per well. Incubate 1–1.5 h at room temperature. m. Flick the plate (do not wash it) and add 200 ␮l of the PBS diluted anti-LCMV nucleoprotein Ab (VL4) per well (the dilution factor of the VL4 supernatant has to be first established; prepare about 5 ml/plate). Incubate 1 h at room temperature. n. Wash wells with PBS. o. Add 200 ␮l of a 1:400 dilution (in PBS 1% FCS) of the goat anti-rat IgG-peroxidase secondary Ab. Incubate 30– 60 min at room temperature. p. Wash wells three times with PBS. q. Prepare and add the Peroxidase substrate (SIGMAFASTTM OPD) following the manufacturer’s protocol. r. Once colored spots appear, stop the reaction by replacing the substrate solution (discard it in the toxic waste) with tap water. s. Flick and dry the plate on paper towels. t. Count the plaques. 3.7. Studies of the Molecular Program of NK Cell/DC Interactions

In order to analyze the cytokines, receptors, or signaling molecules involved in the priming of NK cells by DCs a tissuespecific gene targeting strategy can be employed. A limitation of this approach is that the availability of floxed cytokine or cytokine receptor loci is rather limited. To this end, we and others have developed a mixed bone marrow chimera system in which lethally irradiated mice are reconstituted with a 1:1 mixture of BM cells from a mouse strain genetically deficient of cytokine, cytokine receptor, or a signaling molecules and from CD11c DTR tg mice (carrying a different congenic marker) or any other DTR strain (5, 24). After injection of DT, DCs (or other DTR+ cells) are removed as the source of the molecule of interest and NK cell responses can be tested. This strategy is required for the analysis of molecules such as IL-15 and IL-15R␣ that are permissive for NK cell development (29, 55). Another approach in this scenario is the adoptive transfer of wild-type NK cells into genetically deficient mice. As IL-15 and IL-15R␣ are required for the survival of NK cells, either shortterm experiments need to be performed (< 12 h after NK cell transfer) or Bcl-2 tg NK cells should be employed, which survive independent of IL-15 or IL-15R␣ (56). In the first scenario, the survival and functional competence of the transferred NK cells should be controlled for.

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1. Mice are lethally irradiated (e.g., for C57BL/6 background: 12 Gy are applied in two sessions of 6 Gy each separated by 4 h in order to avoid collateral organ damage). 2. A carefully filtered (40 ␮m) 1:1 mixture of 1–2 × 106 BM cells derived (see Section 3.1, steps 1–4) from the desired mouse strains (CD11c DTR tg mice and mice genetically deficient of gene of interest) are injected i.v. (the two bone marrow grafts should have different congenic markers (e.g., CD45.1 vs. CD45.2) in order to distinguish cells of different origin later). 3. BM chimeric mice are analyzed 8–12 weeks after transplantation in order to evaluate the chimerism (FACS analysis of blood leukocytes for the congenic markers). 4. Mice are then depleted of DCs and stimulated as described above (Sections 3.6.2 and 3.6.1). It is important to control these experiments with groups of chimeric mice that do not receive DT. 5. NK cell function can then be assessed as described in Section 3.8.

3.7.2. Adoptive Transfer of NK Cells into Mice Genetically Lacking Genes Implicated in NK Cell Priming

1. Purify splenic NK cells from C57BL/6 mice as described in Section 3.3. It is important to ensure that the transferred NK cell population is free of contaminating DCs. 2. 2–5 × 106 purified NK cells are injected i.v. into the host mice. 3. Four hours post-transfer, mice are stimulated as discussed above (Sections 3.6.1 and 3.6.2). Stimulation can be performed 18–24 h following NK cell injection if NK cells can survive in the new environment (see Note 13). 4. NK cell function can be assessed after 4–6 h post stimulation (18–24 h if the injected NK cells can survive in the host environment; see Note 14).

3.8. NK Cell Activation Assays

3.8.1. Analysis of NK Cell Activation Markers (CD69, CD25)

All the experiments described so far require the analysis of NK cell function as a final readout. We are providing four protocols to assess NK cell activation (up-regulation of “activation markers,” cytokine production, cytotoxic effector molecule expression, and cell-mediated cytotoxicity). 1. Cells from the desired organs or cells from in vitro co-culture experiments are prepared as a single cell suspension in icecold FACS buffer. 2. Immunofluorescent labeling of cell suspensions can be performed in U- or V-bottom 96-well plates containing 0.1–2 × 106 cells per well in 25–100 ␮l of FACS buffer containing Abs on ice (see Notes 3 and 15).

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3. Spin plates at 400–500 × g for 5 min at 4◦ C. Remove supernatant by flicking plates. 4. Resuspend cells in 50 ␮l FACS buffer containing 5–10 ␮g/ml 2.4G2 Ab to block non-specific binding to Fc receptors. 5. Wash the cells (by filling the wells with 150 ␮l FACS buffer followed by centrifugation at 400–500 × g, 3–5 min, 4◦ C). Remove the supernatant by simply flicking the plate above the sink and briefly dry any liquid outside of the wells by blotting the inverted plate on a paper towel. 6. Activation of NK cells is measured by staining of cells with 50 ␮l of FACS buffer containing fluorescently conjugated antibodies specific for CD25 and CD69. In order to detect CD25 and CD69 expressed by NK cells, cells are co-stained with a combination of anti-NKp46 (or anti-NK1.1 or antiDX5) and anti-CD3⑀ Abs. Additional antibodies visualizing various NK cell subsets can be added (e.g., Ly49C/I for “armed” NK cells of B6 mice, CD11b or KLRG1 for “mature” NK cells). Usually, 1–5 ␮g/ml of fluorescently conjugated antibodies are used for cell surface staining. It is a good idea to experimentally establish the amount of antibody required for a decent stain. 7. Staining is performed for > 15 min on ice protected from light. 8. Cells are washed 3× in ice-cold FACS buffer. 9. Cells can either be directly analyzed on a flow cytometer or fixed in FACS buffer containing 1% formaldehyde and can be stored protected from light (aluminum foil) for < 3–4 days at 4◦ C. 3.8.2. Intracellular Cytokine Stain

1. In order to assess cytokine production by NK cells, NK cells are re-stimulated for 4–6 h in vitro in the presence of 2 ␮M monensin (GolgiStop diluted at 1:1500) or 10 ␮g/ml brefeldin A in order to block protein export and facilitating cytokine accumulation inside cells. 2. In vitro restimulation of NK cells can be performed by either of these methods: a. Adding cells to flat-bottom 96-well plates coated with antibodies specific for activating NK cell receptors (e.g., NKG2D, NKp46, NKR-P1B/C, Ly49D, Ly49H). Plates should be pre-coated overnight at 4◦ C with the desired concentration of the antibody in PBS. Before use, coated wells are washed three times with PBS (see Note 16). Cell suspensions can then be directly added to the wells. The optimal concentration of antibody for NK cell stimulation should be experimentally determined. In our experience, 10 ␮g/ml of most

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commercially available antibodies work quite well. After stimulation, lymphocytes are transferred to a U- or Vbottom 96-well plate for intracellular cytokine staining. b. Co-culturing of NK cell-containing cell suspensions with target cells (e.g., YAC-1, RMA-S or RMA-S-H60 cells) is generally performed at a 1:1 ratio (e.g., 1 × 106 splenocytes for 1 × 106 target cells) in cell culture medium and U-bottom 96-well plates (200 ␮l final volume). 3. At the end of the incubation period, cells are spun down and resuspended in 50 ␮l FACS buffer containing 5– 10 ␮g/ml 2.4G2 Ab to block non-specific binding to Fc receptors. 4. Wash the cells by filling the wells with 150 ␮l FACS buffer, followed by centrifugation at 400–500 × g, 3–5 min, 4◦ C. Remove the supernatant by simply flicking the plate above the sink and briefly dry any liquid outside of the wells by blotting the inverted plate on a paper towel. 5. Resuspend the cells in 50 ␮l FACS buffer containing 5– 10 ␮g/ml of Ab to markers defining the NK cells or NK cell subsets (see Section 3.8.1, Step 6; see Notes 3 and 15). 6. After the extracellular staining step, cells are permeabilized and fixed using 100 ␮l Cytofix/Cytoperm) for 20 min and protected from light on ice. 7. Cells are washed twice in FACS buffer containing 0.5% saponin. 8. The intracellular staining is performed in 50 ␮l FACS buffer containing 0.5% saponin and the desired antibodies (usually anti-IFN-␥ at 1–2 ␮g/ml) for 20–30 min (on ice, protected from light). In addition to the intracellular staining antibody, a separate sample should be stained with an isotype control antibody conjugated with the same fluorescent dye. 9. Cells are washed two times with FACS buffer containing 0.5% saponin and two times with regular FACS buffer (no saponin). 10. Cells can either be directly analyzed on a flow cytometer or fixed in FACS buffer containing 1% formaldehyde and can be stored protected from light (aluminum foil) for <3– 4 days at 4◦ C. 3.8.3. Intracellular Stain for Effector Molecules (Granzymes and Perforin)

Detection of cytotoxic effector molecules (granzymes, perforin) in NK cells and CD8 T cells from mice has been problematic as specificity of some of the commercially available antibodies has been questioned. We used antibodies specific for granzymes A

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and B generated by the group of Dr. M. Simon (Max-PlanckInstitute of Immunobiology, Freiburg, Germany) that have been carefully tested for their specificity with the help of mouse models genetically deficient for those molecules (34). Recent reports have successfully employed commercially available antibodies specific for perforin and granzymes A and B (35). All those antibodies have been listed in Section 2.4. 1. Cells from the desired organs or cells from in vitro coculture experiments are prepared as a single cell suspension in ice-cold FACS buffer. 2. Immunofluorescent labeling of cell suspensions can be performed in U- or V-bottom 96-well plates containing 0.1–2 × 106 cells per well in 25–100 ␮l of FACS buffer containing Abs on ice. 3. Spin plates at 400–500 × g for 5 min at 4◦ C. Remove supernatant by flicking plates. 4. Resuspend cells in 50 ␮l FACS buffer containing 5– 10 ␮g/ml 2.4G2 Ab to block non-specific binding to Fc receptors. 5. Wash the cells (by filling the wells with 150 ␮l FACS buffer followed by centrifugation at 400–500 × g, 5 min, 4◦ C). Remove the supernatant by simply flicking the plate above the sink and briefly dry any liquid outside of the wells by blotting the inverted plate on a paper towel. 6. Cells are extracellularly stained with Abs to markers defining the NK cells or NK cell subsets (see Section 3.8.1, Step 6, and Notes 3 and 15). 7. After the extracellular staining step, cells are permeabilized and fixed using 100 ␮l Cytofix/Cytoperm for 20 min and protected from light on ice. 8. Cells are washed twice in FACS buffer containing 0.5% saponin. 9. The intracellular staining is performed in 50 ␮l FACS buffer containing 0.5% saponin and the desired antibodies for 20–30 min (on ice, protected from light). If not specified by the manufacturer, the antibody dilution is usually 1–5 ␮g/ml for a monoclonal Ab. Concerning sera, the concentration has to be established by the user (e.g., we used the rabbit sera against granzymes A and B at a 1:700–1:800 dilution). In addition to the intracellular staining antibody, a separate sample should be stained with an isotype control antibody conjugated with the same fluorescent dye. 10. Cells are washed two times with FACS buffer containing 0.5% saponin. 11. Optional: If a secondary Ab is used, incubate cells in 50␮l FACS buffer containing 0.5% saponin and the desired

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secondary antibody for 20–30 min (on ice, protected from light). Repeat step 10. 12. Cells are washed two times more in regular FACS buffer (no saponin). 13. Cells can now be analyzed on the flow cytometer. 3.8.4. Cytotoxicity Assay

3.8.4.1. Target Cell Labeling

Cytotoxicity of NK cells against target cells (e.g., YAC-1, RMAS, or RMA-S-H60 cells) is determined in a modified standard 4 h 51 chromium release assay (5). We and others have observed that following priming of NK cells by DCs, the fraction of NK cells of all splenocytes is decreased (5). Thus, it is very important to normalize the assay based on the total number of NK cells present in each organ suspension. To this end, the percentage of NKp46+ CD3− cells in the lymphocyte populations should be determined prior to the cytotoxicity assays and lymphocyte numbers should be adjusted to contain the same absolute number of NK cells. The starting NK cell:target ratio should be 6:1 (roughly equivalent to a 100–200:1 splenocyte:target ratio). Threefold serial dilutions of the effector cells should be performed. Usually four E:T ratios (6:1, 3:1, 0.8:1, 0.2:1) are sufficient. It should be considered that 10,000 target cells per well or 1 × 106 target cells per 96-well U-bottom plate are needed for the assay. Maximally eight different experimental groups or conditions can be assayed per plate with four different E:T ratios and triplicate determinations. Since some cells will be lost during the labeling procedure, it is necessary to start with double the amount of cells needed for the assay (i.e., if one 96-well plate will be used, 1 × 106 target cells are necessary and labeling should be started with 2 × 106 cells). 1. Split target cell(s) the day before the experiment (1:3) into fresh medium. This procedure will result in a lower spontaneous release by the target cells. 2. Harvest target cells, count cells, pellet cells, and resuspend them at 1–5 × 106 per 470 ␮l of tissue culture medium. 3. From this step onward, target cell handling needs to performed behind leaded shielding in a designated area for radioactive work. Add 30 ␮l (ca. 150 ␮Ci) 51 Cr (see Note 17). 4. Incubate cells on a rotating platform for 90 min at 37◦ C in a humidified atmosphere containing 5% CO2 .

3.8.4.2. Effector Cell Preparation

1. Prepare/count Section 3.2.

cells

from

organs

as

described

in

2. Stain an aliquot of cells from each organ (or each condition if an in vitro assay has been performed) with Abs and

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analyze by FACS to determine the fraction of NK cells (see Section 3.8.1). 3. Prepare U-bottom 96-well plates. Add 100 ␮l of culture medium to each well, except those of row A and row E. 4. Add 150 ␮l of effector cells in triplicates to each well in row A and/or row E. Usually, we start with a 6:1 NK:T ratio, which means that 60,000 NK cells per well will be needed. To facilitate the threefold dilutions, we add to the first well 150 ␮l tissue culture medium containing 90,000 NK cells (6 × 105 NK cells/ml). Serial dilutions are performed using a multichannel pipette by taking 50 ␮l from the initial cell suspension and transferring them to the next well containing 100 ␮l medium. This procedure is repeated until the desired lowest E:T ratio is reached. The remaining 50 ␮l at the end will be discarded. 3.8.4.3. Target Cell Preparation

1. Wash labeled target cells three times with 10 ml of 5% FCS DMEM culture medium (300 × g, 5 min, room temperature) 2. After the final wash, target cells are resuspended in ca. 5 ml of medium. Incubate the cells on a rotating platform for 30 min at 37◦ C in a humidified atmosphere containing 5% CO2 . This step decreases spontaneous release by the target cells. 3. Pellet target cells (300 × g, 5 min, room temperature). 4. Resuspend the cells in 1–2 ml of 10% FCS DMEM culture medium and count them. 5. Dilute target cells to the desired concentration (1 × 105 cells/ml) in 10% FCS DMEM culture medium.

3.8.4.4. Assay SetUp

1. Add 100 ␮l of target cells from Section 3.8.4.3 to the effector cells from Section 3.8.4.2. 2. Fill 12 more wells with target cells only and 100 ␮l of medium for the negative (spontaneous release) and the positive controls (maximal release). 3. Optional: Centrifuge plate for 5 min at 400 × g at room temperature. 4. Incubate plates for 4 h at 37◦ C in a humidified atmosphere containing 5% CO2 . 5. Centrifuge the plate (450 × g, 5 min, room temperature) and carefully harvest 100 ␮l of the supernatant with filtered tips into tubes that can be used in the ␥-counter. 6. For determination of the spontaneous release, 100 ␮l of supernatant from six of the target cell-only wells is harvested. 7. For determination of the maximal release, pipette up and down the six remaining wells containing target cells only

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and transfer 100 ␮l for determination of the total label. We found that this procedure is safer than using detergent lysis of target cells to determine the maximal release and the error introduced is negligible. 8. Determine radioactivity contained in supernatants in a ␥-counter (measure each sample for 1 min). 9. Calculate specific lysis using the formula: %specific lysis = {[mean (experimental release) −mean (spontaneous release)] / [mean (total label) −mean (spontaneous release)]} × 100.

4. Notes 1. We and others have observed that depending on its origin or depending on the lot, the DT can cause side effects (wasting syndrome) even in wild-type mice. Thus, it is very important to test the DT preparation in normal mice (weight, survival, behavior) prior to any experimental usage. 2. It now widely recognized that TLR-ligand preparations by many vendors are potentially contaminated by other pathogen-derived substances (e.g., LPS). Thus, it is highly recommended to work with ultrapure chemicals. We often test for contamination of TLR-L by injecting them in mice genetically deficient for the respective TLR and analyzing for NK cell and DC activation. Only those compounds that do not activate NK cells and DCs in the genetically deficient mouse strains should be used in the experiments. 3. For immunofluorescence staining, we label a maximum of 2 × 106 cells in 50 ␮l (40 × 106 cells/ml). It is OK to increase the concentration of cells to 50 × 106 cells/ml when large numbers of cells need to be stained, such as DX5 staining for magnetic sorting. 4. It is important to note that the CD49b antibody used for the NK cell purification procedure might still be detectable at the cell surface. Thus, it is not recommended to use a PE-conjugated antibody of another specificity for consecutive analysis. 5. Ablation of DCs should be confirmed in each experiment by staining for CD19− CD11chigh MHC-II+ cells in spleen and LN. Routinely, we observed a >95% reduction of CD11chigh DCs.

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6. DT injection into CD11c DTR tg mice leads to the depletion of conventional DC, MZM and MM (see above). To prove that any observed effects are indeed reflecting the absence of conventional DCs, TLR, or pathogen injection is performed at day 4 or 5 after DT injection and NK cell responses are evaluated 24 h later. At this time point, the DC compartment is already restored whereas MZM and MM are still lacking. Thus, if a DC-dependent stimulus has been used NK cell function should be restored. 7. In order to confirm the role of DCs during the priming of NK cells, 3–10 × 106 BM-derived DCs can be injected i.v. into CD11c DTR mice 1 day prior to DT injection. 8. Listeria can be stored at −80◦ C at high concentration (at least 1 × 108 bacteria/ml) in BHI. 9. During LCMV production, it is very important to keep and titer both harvest I and harvest II since their titers may vary from experiment to experiment. 10. When determining the LCMV titer, use thick (e.g., leather) gloves under the latex gloves when you use the glass homogenizer. The glass tubes can break and you want to be protected against shards of glass contaminated with high titers of LCMV. 11. Spleens or organ homogenates can be frozen at −80◦ C if the experiment cannot be performed the same day. 12. When preparing the BM chimeras involving injection of CD11c DTR BM into lethally irradiated B6 mice, we have observed that 8 weeks after transfer a few remaining “radioresistant” DCs from the host (resistant to DT) were still detectable. Thus, it is sometimes necessary to wait at least 12 weeks (or even more) before doing any further experiment. 13. Recombinant cytokines can be injected i.p. at the time of TLR stimulation to rescue NK cell function. For example, we have used a single i.p. injection of 4 ␮g recombinant IL-15 per mouse to rescue NK cell function in Il15 −/− mice. 14. Recombinant cytokines can be added to the respective assays of NK cell function (see Section 3.4). For example, we have used 10 ng/ml recombinant IL-15 to rescue function of NK cells adoptively transferred into Il15 −/− mice. 15. In order to stain NK cells, antibodies against different panNK cells markers are used. Unfortunately, none of them are entirely NK cell-specific and some of them are not expressed by all inbred strains of mice (e.g., DX5 and NK1.1 also stain subpopulations of T cells; NK1.1 does

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not stain NK cells in some strains such as BALB/c). Very recently, a new antibody was introduced specific for another NK cell marker, NKp46 (57). Many reports have shown that NKp46 is specifically expressed by NK cells in all mouse strains investigated (57). We recommend to use the NKp46 antibody instead of other pan-NK cell markers. 16. For experiments employing plate-bound antibodies, all antibodies described in this chapter efficiently bind to normal tissue culture plates. Nevertheless, it is possible that other antibodies may be less efficient in binding to normal plates. In our experiments, tissue culture plates with high protein binding capacity (e.g., Costar, Catalog No. 9018) resolve such problems. 17. The half-life of the 51 Cr is ca. 1 month. Batches older than one half-life can still be used in experiments but the volume of 51 Cr used in the assay should be increased. References 1. Herberman, R. B., Nunn, M. E., and Lavrin, D. H. (1975) Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. I. Distribution of reactivity and specificity. Int J Cancer 16, 216–229. 2. Kiessling, R., Klein, E., and Wigzell, H. (1975) “Natural” killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Eur J Immunol 5, 112–117. 3. Trinchieri, G. (1989) Biology of natural killer cells. Adv Immunol 47, 187–376. 4. Bryceson, Y. T., March, M. E., Ljunggren, H. G., and Long, E. O. (2006) Synergy among receptors on resting NK cells for the activation of natural cytotoxicity and cytokine secretion. Blood 107, 159–166. 5. Lucas, M., Schachterle, W., Oberle, K., Aichele, P., and Diefenbach, A. (2007) Dendritic cells prime natural killer cells by transpresenting interleukin 15. Immunity 26, 503–517. 6. Gidlund, M., Orn, A., Wigzell, H., Senik, A., and Gresser, I. (1978) Enhanced NK cell activity in mice injected with interferon and interferon inducers. Nature 273, 759. 7. Djeu, J., Heinbaugh, J., Holden, H., and Herberman, R. (1979) Augmentation of mouse natural killer cell activity by interferon and interferon inducers. J Immunol 122, 175. 8. Cudkowicz, G., and Yung, Y. P. (1977) Abrogation of resistance to foreign bone marrow grafts by carrageenans. I. Studies

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24. Mortier, E., Woo, T., Advincula, R., Gozalo, S., and Ma, A. (2008) IL-15Ralpha chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation. J Exp Med 205, 1213–1225. 25. Hochweller, K., Striegler, J., Hammerling, G. J., and Garbi, N. (2008) A novel CD11c.DTR transgenic mouse for depletion of dendritic cells reveals their requirement for homeostatic proliferation of natural killer cells. Eur J Immunol 38, 2776–2783. 26. Kang, S. J., Liang, H. E., Reizis, B., and Locksley, R. M. (2008) Regulation of hierarchical clustering and activation of innate immune cells by dendritic cells. Immunity 29, 819–833. 27. Jung, S., Unutmaz, D., Wong, P., Sano, G., De los Santos, K., Sparwasser, T., Wu, S., Vuthoori, S., Ko, K., Zavala, F., Pamer, E. G., Littman, D. R., and Lang, R. A. (2002) In Vivo Depletion of CD11c+ dendritic cells Abrogates Priming of CD8+ T Cells by Exogenous Cell-Associated Antigens. Immunity 17, 211–220. 28. Domen, J., Gandy, K. L., and Weissman, I. L. (1998) Systemic overexpression of BCL-2 in the hematopoietic system protects transgenic mice from the consequences of lethal irradiation. Blood 91, 2272–2282. 29. Kennedy, M. K., Glaccum, M., Brown, S. N., Butz, E. A., Viney, J. L., Embers, M., Matsuki, N., Charrier, K., Sedger, L., Willis, C. R., Brasel, K., Morrissey, P. J., Stocking, K., Schuh, J. C., Joyce, S., and Peschon, J. J. (2000) Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. J Exp Med 191, 771–780. 30. Battegay, M., Cooper, S., Althage, A., Banziger, J., Hengartner, H., and Zinkernagel, R. M. (1991) Quantification of lymphocytic choriomeningitis virus with an immunological focus assay in 24- or 96-well plates. J Virol Methods 33, 191–198. 31. Cikes, M., Friberg, S., Jr., and Klein, G. (1973) Progressive loss of H-2 antigens with concomitant increase of cell-surface antigen(s) determined by Moloney leukemia virus in cultured murine lymphomas. J Natl Cancer Inst 50, 347–362. 32. Karre, K., Ljunggren, H. G., Piontek, G., and Kiessling, R. (1986) Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defense strategy. Nature 319, 675–678. 33. Diefenbach, A., Jamieson, A. M., Liu, S. D., Shastri, N., and Raulet, D. H. (2000) Ligands for the murine NKG2D receptor: expression by tumor cells and activation of

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homing and proliferation. Immunity 9, 669–676. 56. Cooper, M. A., Bush, J. E., Fehniger, T. A., VanDeusen, J. B., Waite, R. E., Liu, Y., Aguila, H. L., and Caligiuri, M. A. (2002) In vivo evidence for a dependence on interleukin 15 for survival of natural killer cells. Blood 100, 3633–3638. 57. Walzer, T., Blery, M., Chaix, J., Fuseri, N., Chasson, L., Robbins, S. H., Jaeger, S., Andre, P., Gauthier, L., Daniel, L., Chemin, K., Morel, Y., Dalod, M., Imbert, J., Pierres, M., Moretta, A., Romagne, F., and Vivier, E. (2007) Identification, activation, and selective in vivo ablation of mouse NK cells via NKp46. Proc Natl Acad Sci U S A 104, 3384–3389.

Chapter 9 Analysis of the NK Cell Immunological Synapse Keri B. Sanborn, Gregory D. Rak, Ashley N. Mentlik, Pinaki P. Banerjee, and Jordan S. Orange Abstract Since NK cells specialize in contact-dependent functions including cytotoxicity, interest has focused on the direct study of the interface between the NK cell and the cell with which it is interacting. This interface is also known as the immunological synapse and is characterized by an extraordinary number of dynamic molecular events that have the potential to result in NK cell function. Here we describe microscopy-based methods for evaluating and quantifying the NK cell immunological synapse that can be useful in enabling experimental studies. Key words: Immunological synapse, confocal microscopy, colocalization, fluorescence.

1. Introduction The NK cell immunological synapse can be defined as the dynamic arrangement of molecules at the interface between an NK cell and a target cell (reviewed in (3)). During activation and cytotoxicity, the immunological synapse is the site of the directed secretion of lytic granule contents and thus enables cytotoxicity. The synapse undergoes a number of dynamic changes and is also the site of regulation for critical NK cell functions. Although biochemical studies can provide insight into the NK cell immunological synapse, direct visualization is essential. This is typically performed through the use of a variety of microscopic technologies. The most productive to date has been fluorescence microscopy, which enables the identification of key molecular structures through the use of fluorescently labeled molecules K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 9, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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expressed in cells as well as in fluorescent dyes, binding proteins, and immunological reagents. This has allowed the direct and indirect visualization and localization of cellular elements critical to synapse function and control. Although there are many different types of fluorescence microscopy that have been used in the study of NK cells, the most productive in terms of visualizing the synapse has been confocal microscopy. Confocal microscopy enables the visualization of fluorescence at a single two-dimensional plane within a cell and prevents the interference provided by fluorescence emanating from other planes within the cell. This provides very accurate information as to the arrangement of molecules at the immunological synapse and elsewhere in the cell. Because single planes can be imaged without significant interference from adjacent planes, confocal microscopy can also enable the three-dimensional reconstruction of fluorescence signal in an NK cell with great resolution. This allows accurate information to be obtained throughout the full volume of the cell. Confocal microscopes can also be equipped to visualize living cells, and thus the synapse can be studied accurately over time to facilitate the appreciation of dynamic aspects of synapse formation and function. While a picture may be worth a thousand words, a picture demonstrating a biological phenomenon at the NK cell immunological synapse is not sufficient as independent data. Experiments need to be carefully controlled, repeated, and the microscope specifically calibrated to exploit meaningful data from multiple images of a single phenomenon. When acquired appropriately, images of the synapse can be quantified to yield objective and highly quantitative data. In this chapter, we review some approaches to generating, imaging, and quantifying the immunological synapse in NK cells. Most are written with confocal microscopes in mind, but can be also applied to other microscopy technologies. The quantitative aspects of the approaches described utilize specific imaging and quantification software algorithms. A large number are available commercially, such as Volocity (Improvision) and SlideBook (3i), as well as the flexible and customizable ImageJ algorithm available as shareware from the National Institutes of Health (http://rsweb.nih.gov/ij/).

2. Materials 1. Culture medium: RPMI 1640 (Gibco), supplemented with 10% Fetal Bovine Serum (FBS), 10 mM HEPES, 1× Penicillin/Streptomycin, 100 ␮M MEM nonessential amino acids, 1 mM MEM sodium pyruvate, 2 mM L-glutamine

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(all supplements from Gibco) (R-10). Dye-free RPMI is available for imaging. 2. RPMI 1640, supplemented as above, with 20% FCS (R-20). 3. DMEM, supplemented as above or with varying amounts of FCS. 4. G418 sulfate – used at 1.6 g/L (Cellgro) – for transduced cells only. 5. Amaxa Nucleofector Kit R. 6. Amaxa Nucleofector. 7. PLAT-E retroviral packaging cells or Phoenix retroviral packaging cells. 8. YTS cell line expressing the mouse ecotropic retrovirus receptor – YTSeco. Alternatively, other human immortalized NK cell lines may be used if using amphotropic retrovirus. Examples include NK-92, NKL, and NK3.3. 9. Target cell lines that elicit desired response from the NK cell line chosen for the experiment. One example is the 721.221 EBV-transformed B cell line, which activates most NK cell lines to mediate cytotoxicity. Another example is the K562 erythroleukemia cell line, which can be killed by most NK cell lines but not YTS. 10. Fugene (Roche). 11. Hexadimethrine bromide (Polybrene, e.g., Sigma Cat# H9268). 12. Poly-L-lysine-coated glass slides (Sigma Polyprep precoated slides Cat #P0425 or manually coated using PolyL -lysine solution Sigma Cat #P8920). 13. Glass-bottom live cell imaging dishes (e.g., T dishes, clear or black – Bioptechs). 14. Hydrophobic ink pen (e.g., Pap Pen, Sigma). 15. Fixative and permeabilization agent (e.g., Cytofix/Cytoperm, BD) containing 0.1% (v/v) Triton X-100. 16. Phosphate-buffered saline (PBS). 17. PBS containing 1% bovine serum albumin (BSA). 18. PBS containing 1% BSA and 0.1% saponin (PBS-S). 19. Specific antibodies to perforin (mouse anti-human perforin clone ␦G9 – BD Cat #556434), ␣-tubulin (biotinylated mouse anti-␣-tubulin – Molecular Probes Cat #A21371), and any other desired molecules. 20. Fluorescently conjugated phalloidin (Invitrogen).

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21. Mounting medium with a fluorescent protectant (e.g., Vectashield, Vector laboratories) or ProLong Antifade, Invitrogen. 22. Lysotracker dyes (Invitrogen). 23. Parafilm M.

3. Methods 3.1. Preparation of NK Cells and Target Cells for Imaging 3.1.1. Preparation of NK and Target Cells for Fixed Cell Imaging

1. Culture target cells (and effector cells in separate culture if using an NK cell line) in culture medium (see Section 2) at a low density, preferably under 5 × 105 cells/mL, to enable their optimal conjugation and function. Wash effector and target cells 3× prior to use. 2. Mix NK cells and target cells at a ratio of 2:1 in up to 200 ␮L medium in a 15 mL polypropylene conical tube at approximately 2.5 × 106 cells/mL for NK cells and 1.25 × 106 cells/mL for target cells. 3. Allow conjugates to form in suspension for approximately 15 min prior to adding cells to slides.

3.1.2. Preparation of NK and Target Cells for Live Cell Imaging

1. Pre-coat a 0.15 mm-thick glass-bottom imaging dish with a monoclonal antibody diluted to approximately 8 ␮g/mL with PBS that will recognize the target cell. Allow the antibody to adhere to the glass overnight at 4◦ C or for 1 h at 37◦ C. As an alternative, an antibody directed against an NK cell surface receptor can be used. This can enable the evaluation of NK cells in the absence of target cells. 2. Rinse the imaging dish 3× with PBS. 3. Prepare target cells and NK cells separately, washing each 2× with PBS by spinning at 225 × g for 10 min. 4. Count cells and resuspend in culture medium at 3 × 106 (effector) and 1.5 × 106 (target) cells/mL (to enable a 2:1 effector to target ratio). 5. Aliquot 500 ␮L of the target cell suspension onto the precoated, washed imaging dishes, and incubate at 37◦ C for 15 min to allow the target cells to adhere. 6. Prepare the confocal microscope and place the chamber slides into a heated stage adapter at 37◦ C (such as the Bioptechs T dish stage adapter). 7. Aliquot 100 ␮L of the effector cell suspension onto the chamber slide with the target cells in the general area of the objective at the start of the assay (time = 0).

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3.2. Visualizing the Immunological Synapse

The immunological synapse can be visualized using several imaging techniques. While fixed cell imaging allows for easy simultaneous visualization of a number of proteins at the synapse, it provides only a static image of what is a dynamic process. Live cell imaging enables the monitoring of cells over time; however, imaging of more than one or two proteins or organelles in live cells can be difficult. Often, combining the two methods is the best approach to a comprehensive visualization project.

3.2.1. Fixed Cell Imaging

Imaging of fixed cells allows for visualization of several components of the immunological synapse at once and also enables the easy analysis of the entire volume of the cell. Kinetic insight into the synapse can be obtained by varying the conjugation time between NK cells and target cells. In the procedure here we have recommended 30 min of conjugation time (15 min in Section 3.2.1 and 15 min on the slide – Step 7 below) as we have found this to represent an optimal time for maximal NK cell cytolytic synapse maturity. For fixed cell imaging, lytic granules can be visualized using antibodies against lytic granule components, such as perforin or granzymes, or against lysosomal membrane proteins (e.g., LAMP-1). The microtubule organizing center (MTOC) can be visualized with antibodies to ␣-tubulin or pericentrin. The visualization of lytic granules and the MTOC helps to indicate whether the NK cell cytolytic machinery is polarized toward the target cell. Actin can be best visualized using phalloidin, which can be commercially obtained directly conjugated to a variety of fluorescent molecules.

3.2.1.1. Slide Preparation

1. Prepare a covered humidified chamber containing moistened paper towels for incubating slides during the staining process. 2. Prepare antibodies at desired concentration in PBS with 1% BSA and 0.1% saponin (PBS-S, see Note 1). 3. On poly-L-lysine-coated slides, use a hydrophobic pen to mark an individual section for each staining performed. Circular regions with a 1.5 cm diameter should hold up to 100 ␮L of liquid. At this time, also label the slides with a pencil or detergent-insoluble ink. 4. Keep slides in the humidified chamber at 37◦ C until NK and target cells have conjugated. 5. Following conjugation of NK and target cells (see Section 3.1.1), gently resuspend the conjugates using a 20–200 ␮L pipette by slowly withdrawing and replacing 100 ␮L of suspension three times. 6. Gently pipette 60–80 ␮L of the conjugates inside each hydrophobic ink region on the slides.

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7. Allow the conjugates to adhere to the poly-L-lysine-coated slides in the humidified chamber for 15 min at 37◦ C. 8. Hold the labeled end of the slide and gently dip the entire region of the slide containing cells into a 50 mL conical tube containing PBS 3× to remove non-adherent cells. 9. Add 100 ␮L of Cytofix/Cytoperm-0.1%Triton mix (see Note 1) within the hydrophobic ink circle and incubate in the humidified chamber for 15 min at room temperature. 10. Gently dip the slide 3× in a 50 mL conical tube containing PBS-S solution. 11. Gently wipe off the droplets of PBS-S which form on the hydrophobic ink using a long cotton swab to restore the hydrophobicity of the ink. Be sure not to touch the cells within the encircled region. 12. Add 100 ␮L of primary antibody solution or isotype control (diluted in PBS-S) to each staining region and incubate in the humidified chamber at room temperature for 1 h. 13. Gently dip the slide 3× in the PBS-S solution and wipe excess liquid off the hydrophobic ink as per Step 11. 14. Add 100 ␮L of fluorophore-conjugated secondary antibody solution (if the primary antibody is unconjugated) or fluorophore-conjugated streptavidin (if the primary antibody is biotinylated) diluted in PBS-S to each hydrophobic ink encircled region and incubate in the humidified chamber at room temperature for 1 h. If the primary antibody is directly fluorophore conjugated, skip to Step 17. 15. Gently dip the slide 3× in the PBS-S solution and wipe droplets of PBS-S off the hydrophobic ink as per Step 11. 16. If more than one molecule needs to be visualized, repeat Steps 12–15 for each subsequent antibody. Between any two different antibody staining procedures, perform a blocking step with an isotype control, IgG, or heatinactivated serum from the species of the previously used primary antibody at approximately 10 times higher concentration than used for the primary antibody staining. This will ensure that binding sites of the previously used secondary antibody will be occupied before a new primary antibody is added. 17. Add directly conjugated fluorescent phalloidin and/or an additional directly fluorophore-conjugated antibody. Additional staining using a directly fluorophore-conjugated nonspecific protein such as BSA or streptavidin (if a biotinylated antibody was not used in the experiment) should be prepared as a control for phalloidin staining. A directly fluorophore-conjugated isotype control should

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be used in an additional independent staining to control for any directly conjugated primary antibodies used. 18. Gently dip the slide 3× in the PBS-S solution and remove excess moisture from the slide surface outside of the hydrophobic ink region using a Kimwipe or swab. 19. Add one drop of mounting medium per staining region. 20. Place a coverslip on the slide using forceps. Anchor one end of the coverslip with a gloved finger and gently press on the coverslip to remove the excess mounting medium between the coverslip and the slide and dry with a Kimwipe. 21. Keep the slides with coverslips facing up at room temperature in the dark overnight. 22. Seal the edges of the coverslip with acrylic nail polish. 3.2.1.2. Imaging

1. Adjust fluorescence detection using control-stained slides. The exact details of how the controls are used will depend on the type of microscope used. That said, the maximal detection settings on the microscope (i.e., camera exposure, camera intensification) resulting in minimal signal for individual fluorophores should be set using negative control slides. Settings should not be raised above these levels for any purpose. 2. Further adjust fluorescence detection using cells that have been stained for a molecule of interest with only one fluorophore (these are similar to compensation standards for flow cytometry). Here the detection settings obtained in Step 1 should be adjusted downward so that reasonable positive and efficient fluorescence signal can be obtained. The other fluorescent settings used to detect the additional fluorophores utilized in the experiment should also be examined to be sure that any bleed-through into these is minimal. This is also a feature of the titration of the antibody being used (see Note 2). Ideally there will be a full range of fluorescence intensities with a minimal number of pixels fully saturated (see Fig. 9.1). In order to ensure accuracy, however, detection needs to be optimized based upon the actual endpoint (e.g., accumulated F-actin). Once obtained, the detection settings should not be raised above these levels. 3. Repeat Step 2 for slides singly stained for each fluorophore to be used in the experiment. 4. Detection settings that maintain the biologically relevant fluorescence structures within the dynamic fluorescence scale and not under – or, more challengingly, over – range will enable quantitative analysis of images. Once established, fluorescence settings should not be changed within an experiment. Subsequent alterations will negate the ability to obtain

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Fig. 9.1. Examples of acceptable and unacceptable detection of fluorescence at the NK cell immunological synapse. The figure shows two NK cells conjugated with target cells. The IS is marked with a white arrow. The scale on the left demonstrates the range of pixel intensities. The image on the left demonstrates a variety of fluorescence intensities all within the 0–255 intensity scale. This is acceptable and will allow accurate quantification of fluorescence. The image on the right demonstrates fluorescence at the synapse that is off-scale, and thus the ability to quantify accumulation or localization is reduced largely to a binary factor. This image is therefore unacceptable.

quantitative results and can even constitute a misinterpretation of data. 5. Image slides individually and acquire multiple fields (full three-dimensional acquisitions of fields are preferable). Fields should be chosen without bias toward fluorescent data and should be selected using brightfield transmitted light. Once a reasonable field of cells has been chosen based on the uniformity and distribution of cells, then fluorescence imaging should be performed without further adjustment. 6. If a high magnification objective is being used to directly image conjugates, then conjugates should be selected using brightfield transmitted light and imaged for fluorescence only after the conjugate was chosen. 3.2.2. Live Cell Imaging of Components of the Immunological Synapse – Transfection

In order to image components of the immunological synapse in live cells, introduction of fluorescent elements is necessary. In NK cells, standard transfection has proven difficult, and thus in recent years, Nucleofection (Amaxa) has become popular as it generally provides high efficiency while maintaining cell viability. An advantage of Nucleofection is the ability to introduce the expression of a protein of interest fused to fluorescent protein only transiently. To obtain appropriate results, it is necessary to standardize the amount of DNA, cell number, Nucleofection solution, nucleofection program, and time point after Nucleofection at which to analyze fluorescence. Nucleofection can also be used to introduce

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small-interfering RNA molecules or other oligonucleotides that will interfere with gene expression. These can facilitate the consideration of specific genes and proteins on the function of existing fluorescent elements within an NK cell. For troubleshooting tips for Nucleofection, see Note 3. This protocol describes Nucleofection of YTS cells for which we routinely Nucleofect at an efficiency of 50–80%. 1. Culture YTS cells at a concentration of 2.5 × 105 cells/mL in culture medium. 2. On the day of Nucleofection, warm Nucleofection Reagent R to room temperature. 3. Add 3 mL of R-20 medium to each well of a 6-well tissue culture plate and place in a 37◦ C, 5% CO2 incubator. 4. Add the required amount of DNA/RNA onto the inner wall of a Nucleofection cuvette. Generally, 2–5 ␮g of DNA, or 1–5 ␮M siRNA, in 1–30 ␮L of volume is sufficient for one Nucleofection. 5. Set the Nucleofector to program O-017 (or other desired program). 6. Pellet 2 × 106 YTS cells per Nucleofection, remove all medium, and add 100 ␮L of Nucleofection Reagent R for each 2 × 106 cells. 7. Add 100 ␮L of cell suspension to each cuvette. Exercise caution to ensure that the DNA/RNA drop on the cuvette wall is included when adding the cells. 8. Tap the cuvette gently to mix but avoid generating bubbles. 9. Place the cuvette into the Nucleofector and Nucleofect. 10. Immediately after Nucleofection, add 0.75–1 mL of prewarmed R-20 medium to each cuvette. Using the transfer pipette provided with the kit, transfer cuvette contents into one well of the 6-well plate containing 3 mL of medium. Do not disrupt cell aggregates. 11. Assess Nucleofection efficiency and cell viability within 24– 72 h by FACS, Trypan blue exclusion, or Western blot as appropriate. 3.2.3. Live Cell Imaging of Components of the Immunological Synapse – Transduction

An alternative method of imaging components of the immunological synapse in live cells is to use cells transduced with a retroviral construct that has become stably integrated into their genome. This has the advantage of creating cells having constitutive and stable expression of the gene of interest. For tips on transduction, see Note 4.

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Day 1 1. Plate packaging (e.g., Phoenix or Plat-E cells) cells in a 60 mm tissue culture dish at a concentration of 5–10 × 105 cells/mL in 3 mL of DMEM with 10% FCS and supplements (D-10). The plate should be at least 75% confluent before proceeding. The recommended number of cells should allow for 75% confluence in about 24 h. Day 2 2. Remove medium and feed cells with fresh D-10 medium for 1 h at 37◦ C. 3. Add 4–8 ␮g of retroviral vector DNA to 1.5 mL sterile microfuge tubes. 4. Add 180 ␮L of RPMI-1640 medium without FCS or antibiotics to a separate sterile 1.5 mL tube. 5. Add 21.6 ␮L of Fugene drop by drop to the medium from Step 3. Do not let the Fugene solution directly contact the walls of the tube. 6. Maintain mixture at room temperature for 5 min. 7. Add mixture dropwise to the DNA-containing tubes and maintain at room temperature for 30 min. 8. Remove all but 0.5 mL of medium from the tissue culture dishes containing packaging cells. 9. Add the DNA mixture from Step 6 to the Plat-E cells and swirl the plate slowly. 10. Add 2 mL of DMEM without FCS and incubate in a humidified incubator at 37◦ C with 5% CO2 for 24 h. 11. If Plat-E cells are not being used, plasmids containing retroviral packaging genes (helper plasmids) will need to be introduced into appropriate cells in addition to adding retroviral vector. Day 3 12. Remove packaging cell culture medium and exchange with 2 mL fresh D-10 per culture dish. Day 4 13. Equilibrate a centrifuge with a microtiter plate adaptor to 32◦ C. 14. Pipette 1.7 ␮L of Polybrene (from stock at 8 mg/mL) into a 1.5 mL polypropylene microfuge tube. 15. Remove medium from lipofected packaging cells (Step 9) by withdrawing it into a 5 mL syringe and filter it through a 0.22 ␮m syringe filter into the Polybrene-containing tube and place on ice. 16. Resuspend 1 × 106 washed YTSeco cells in the Polybrenecontaining supernatant from Step 15 and transfer to a

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single well within a 6-well tissue culture-treated microplate. Secure the cover with Parafilm. 17. Centrifuge the microplate at 1000×g for 90 min at 32◦ C. 18. Remove the Parafilm and incubate at 32◦ C with 5% CO2 for 6 h. 19. Transfer microplate into a 37◦ C, 5% CO2 incubator. Day 5 20. 12–24 h after Step 17, discard the medium and add 2 mL R-10 medium. Subsequent days 21. Cells should be maintained in log phase and monitored by flow cytometry for expression of the transduced fluorophore. 22. Ultimately, cultures should be sorted by FACS to obtain cells with similar expression levels of the transgene. These can be maintained as stable cultures without the need for additional selection but should be monitored regularly. 3.2.4. Live Cell Imaging of Lytic Granules and Fluorescent Fusion Proteins

Live cell imaging of lytic granules may be performed either in untransfected and untransduced cells or in cells that have an added fluorescent construct. The latter allows for the simultaneous visualization of both lytic granules and another protein of interest. 1. Wash NK cells twice with PBS and resuspend in R-10 medium at 2 × 106 cells/mL. 2. Add 4 ␮L Lysotracker dye for every 5 × 105 cells (16 ␮M) and incubate for 30 min at 37◦ C. 3. Wash cells at least 3× with PBS and resuspend in dye-free culture medium. 4. Begin by focusing upon target cells adherent to the glass surface. 5. Add labeled cells to live cell imaging chamber as per Section 3.1.2. Add NK cells and begin imaging as they approach target cells. 6. Considerations for fluorescence detection in live cell imaging are fairly similar to those outlined for fixed cells in Section 3.2.1.2 with several variations: a. Cells not containing fluorescent molecules and not loaded with dyes should be used as a control for adjusting initial fluorescence detection (akin to Step 1 in Section 3.2.1.2). b. Cells containing Lysotracker alone or fluorescent fusion proteins alone should be used for further adjustment of fluorescent detection and to prevent detection of

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fluorescence bleed-through (akin to Steps 2 and 3 in Section 3.2.1.2). c. Cells containing the fluorescent protein alone – not fused to the protein of interest – are an essential control and will help determine any specific localization or accumulation of the fluorescent fusion protein. 7. The time-lapse interval used will depend upon the biological phenomenon under study. Some will require rapid streams of images taken immediately, while others will require long imaging sequences. Preliminary observation of the effector/target cell combination of choice will aid in choosing an appropriate time-lapse interval. 8. If confocal imaging is being performed, some type of Z-axis locking device or recentering guide can be helpful in preventing drift from the initial focal plane (also see Note 5). 3.2.5. Live Cell Imaging of the Immunological Synapse by TIRF

Total internal reflection fluorescence (TIRF) microscopy can be used to image lytic granules at the activated NK cell surface. By selectively illuminating lysosome-localized fluorophores within 200 nm of the adhered membrane, TIRF allows for imaging of only those granules that are immediately below the cell surface (1, 2). This technique reduces background fluorescence and provides insight into granule activity immediately at or near plasma membrane fusion sites. 1. Incubate 250 ␮L of YTS cells at 1–2 × 106 cells/mL with 10 ␮M Lysotracker Green (see Note 6) as described in Section 3.2.4. After the final wash, discard supernatant and resuspend the cells in the small volume of PBS remaining in the tube. 2. Incubate a live cell microscopy dish with 5 ␮g/mL activating antibody (e.g., anti-CD28 for YTS cells) or control antibody (IgG) for 1 h at 37◦ C. Wash dish 3× with PBS. Add 1 mL of culture medium and place the dish into incubator until use. 3. Place pre-warmed live cell microscopy dish containing 1 mL dye-free medium into stage adapter. Add cells. 4. Using settled cells, adjust focus to the glass surface and then align 488 nm TIRF laser so that it is completely reflected away from the sample (see Note 7). 5. Using appropriate exposure time, acquire images as time lapses or streams depending on the biological question under study (see Note 5).

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3.3. Analysis of the Immunological Synapse in Fixed Cells 3.3.1. F-Actin Accumulation at the Immunological Synapse

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Accumulation of F-actin in NK cells at the interface of the NK cell and its target cell is an early step denoting the formation of an immunological synapse (reviewed in (3)). Absence of F-actin accumulation at the immunological synapse generally indicates an inhibitory interaction where F-actin accumulation has been defined not to occur. While this protocol is written to analyze the accumulation of F-actin at the synapse, it may be applied to other molecules as well. A detailed quantitative method for measuring F-actin accumulation at the immunological synapse is described elsewhere (4). In essence, this method involves measuring the area and fluorescence intensity of F-actin in defined regions at the synapse which are then compared to cells not forming synapses as well as those forming homotypic synapses. There are, however, useful alternative methods which have been employed. Some general considerations are provided. 1. Select images of NK cell–target cell conjugates which have been stained with fluorescently conjugated phalloidin (see Note 8). 2. To enable quantitative microscopy, it is useful to use exact size/distance measurements instead of pixels or voxels. Thus, specific pixel dimensions can be determined using a micrometer to calibrate the number of pixels per unit measure. Some microscope acquisition interfaces assimilate this information fluidly. 3. The area of accumulation of F-actin can be determined using an intensity threshold for phalloidin fluorescence. A number of different algorithms can be employed to help define the brightest regions and obtain their area/volume, dimensions, and mean fluorescence intensity (see Note 9). When fluorescence detection for an experiment is optimized (see Section 3.2.1.2), the intensity of a region is reflective of the quantity of fluorescent molecules present. 4. The accumulated F-actin in the NK cell can be separated from that in the target cell and also be determined in a number of ways. One is to use a fluorescently labeled target cell (such as with CFSE) and to ignore all phalloidin fluorescence that colocalizes with CFSE fluorescence. This will yield only the accumulated phalloidin fluorescence in the NK cell. 5. The accumulated fluorescence in the NK cell at the synapse can then be separated from accumulated phalloidin fluorescence at other points in the cell in several ways. One way is to divide the cell longitudinally into quadrilles and consider the quadrille closest to the target cell as synaptic. Another is to drop perpendicular lines from the edges of the NK cell phalloidin fluorescence where it no longer contacts the CFSE

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fluorescence of the target cell and crop to only that region of accumulated fluorescence in the NK cell (which will be roughly rectangular). 6. The selected region of phalloidin fluorescence at the NK cell synapse (representing accumulated synaptic F-actin) can then be compared to the total accumulated F-actin region in the NK cell. It can also be presented as a region with dimensions, area/volume, and a mean intensity and can be compared to similar regions obtained from NK cells under different experimental conditions (see Note 10). 3.3.2. Lytic Granule and MTOC Polarization to the Immunological Synapse

The existing model of NK cell activation and formation of a mature cytolytic synapse is characterized by the polarization of the MTOC and associated lytic granules to the immunological synapse (reviewed in (3)). Thus, in addition to using a cytotoxicity assay to determine if a particular interaction is cytolytic, a cytolytic or non-cytolytic synapse can also be characterized by measuring the distance of MTOC from the synapse. This value can provide some indication of how poised an NK cell may be for cytotoxicity. Shorter distances define MTOC polarization and correlate with increased NK cell cytotoxic activity (5). 1. Follow the method as described in Section 3.2.1 to stain NK cells conjugated with target cells for ␣-tubulin, which will identify all microtubules, or pericentrin, which is a more defined marker for the MTOC (a centrosomal protein). 2. Obtain images so that control antibody-stained slides are uniformly negative and there is not any bleed-through from adjacent fluorescent channels as outlined in Section 3.2.1.2. 3. Define the number of ␮m per pixel using a micrometer. The number of pixels contained within the measured distance can be identified using analysis software. Some (i.e., Volocity) have a properties entry screen which will accept this conversion and apply it to all subsequent measurements. 4. Determine the “centroid” of the ␣-tubulin-defined MTOC or pericentrin-defined MTOC which represents the coordinates of the center of the region of highest ␣-tubulin fluorescence or pericentrin fluorescence. Imaging software such as Volocity has algorithms to define the centroid of any given object. 5. Generate a straight line from the centroid of the MTOC to the synapse using the shortest distance (Fig. 9.2). This can be performed mathematically using coordinates of the centroid and those of the closest point of the synapse or by actually superimposing the shortest line using a line tool in analysis software (5). Either approach provides a single linear

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distance which can then be compared to that derived from other experimental conditions. 6. Lytic granule accumulation at the synapse can be objectively determined by the percentage of lytic granules (as defined by perforin staining or similar) that colocalize with accumulated F-actin (see Section 3.3.1) at the synapse (Fig. 9.2). Colocalization methods are outlined in Section 3.3.3 and can be applied to both lytic granules and F-actin. a. In a mature synapse, there is fairly extensive colocalization between lytic granules and accumulated F-actin at the synapse. This colocalization, however, represents a fairly terminal step in lytic synapse formation. It requires lytic granule and MTOC polarization, but provides only a binary measure of polarization (i.e., if there is colocalization between synaptic F-actin and lytic granules, then there has been polarization). b. To define lytic granule polarization, a method similar to that described in Steps 4 and 5 for the MTOC can be used. Here, the lytic granules can be identified and grouped into a single region (in Volocity this can be performed using the “Join Objects” command). This

Fig. 9.2. Schema of polarization measurements at the immunological synapse. The diagram on the left demonstrates a mature lytic synapse with polarized MTOC and perforin-containing lytic granules. The dark gray region represents F-actin and is intended to show accumulation at the synapse. The enlargement of the MTOC and synapse on the right denotes the measured distance from the MTOC to the synapse “D” as well as the positioning of the lytic granules. Although all of the granules pictured are polarized to the synapse, only some have penetrated into the actin cortex. Aside from granule polarization, this represents another measure of synapse maturity prerequisite for lytic function. The granules outlined with a dashed line would have 100% colocalization with accumulated F-actin at the synapse, while the granule outlined with a solid line has only partial colocalization with F-actin at the synapse.

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combined region will have a single centroid that can then be used to determine a physical distance from the synapse. This distance can then be compared to that obtained in other cells and experimental conditions. 3.3.3. Analysis of Molecular Colocalization

While colocalization of two or more molecules does not prove that the molecules interact, it provides preliminary information to direct further studies defining an interaction. Colocalization can also suggest the involvement of several molecules in the same cellular function or process. Thus, colocalization can be a useful starting point for further studies as well as an additional means of extending biochemical results. For the immunological synapse, it is particularly useful to examine the colocalization of a protein of interest with F-actin, lytic granules, or other signaling molecules known to accumulate at the synapse. While the protocol below defines colocalization of two molecules, the same process can be used for the colocalization of three or more molecules within the cell. This analysis can also be applied to three-dimensional images, where colocalization of fluorescent intensities is determined not for pixels but for voxels (cubic pixels). 1. Generate a duplicate of the image of interest and crop to the conjugate or cell being analyzed. This will preserve the original image, as well as reduce the requirements for computer processor power and increase the speed of analysis. 2. Identify and select a region of interest in your image which you would like to analyze. This can be done manually, but is easily done objectively if the cell of interest is labeled with a fluorescent dye or has been stained with an antibody which identifies a large portion of the cell. If identifying the region of interest based on fluorescence signal is desired, the region of interest can be chosen by creating a low fluorescence intensity threshold that allows it to be just detectable over background. 3. Measure the areas within the region of interest containing a specific fluorescent molecule (e.g., FITC). This can be done by creating a threshold of intensity for the fluorescent molecule. In most cases, this should be done using a threshold of one or more standard deviations above the mean intensity of the image or region of interest (see Note 7). At this time, other measurements of the same regions (such as mean intensity of the region) may be made. 4. Repeat the measurement of area for another fluorescent signal in the image (e.g., DAPI). 5. Identify the area containing the signal of both fluorescent molecules (e.g., both DAPI and FITC). This can be done by setting thresholds for both fluorescent signals in the same

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analysis. The area of this region that falls above threshold and contains both fluorophores is the colocalized area (see Note 11). 6. To determine the percent of one molecule colocalized with another, divide the colocalized area by the area occupied by the fluorescent molecule. As an example: • Percent Colocalization of FITC with DAPI = AreaFITC+DAPI /AreaFITC • Percent Colocalization AreaFITC+DAPI /AreaDAPI 3.4. Analysis of the Immunological Synapse in Live Cells 3.4.1. Measuring MTOC Polarization in Live Cells

of

DAPI

with

FITC

=

A lytic NK cell synapse is mature when the MTOC and lytic granules are polarized toward the synapse, and therefore analysis of MTOC polarization in live cells can determine the nature of an interaction, whether cytolytic or non-cytolytic, and can also be useful in determining the stage of an NK cell interaction with its target. Additionally, the location of the MTOC can be used to define the relative location of other molecules within the cell. 1. Obtain a time-lapse image sequence which has the MTOC clearly within the plane of view. 2. As directed in part 5 of Section 3.3.1, define the MTOC, determine its centroid and determine the shortest distance between the MTOC and the immunological synapse (see Note 12). 3. Repeat for all images in the image sequence. 4. The cumulative measurements should be tabulated to then obtain a timeline of MTOC polarization to the immunological synapse. 5. To circumvent the need for individually analyzing each image in the time-lapse sequence, certain software packages have object-tracking features that will enable the automated determination of centroid coordinates at each time point. If the synapse can be defined as a fixed point such as a bead or the glass slide, then the distance between the two coordinates can be calculated rapidly.

3.4.2. Measuring Lytic Granule Approximation to the Membrane Using TIRF

1. Using image analysis software (such a Volocity), identify lytic granules in Lysotracker-loaded cells using an intensity threshold (see Note 12). 2. When analyzing image streams, software can be instructed to track objects over the full series of image frames. 3. Measurements to be performed include: granule area, number of granules, granule path length, granule velocity, granule displacement, granule displacement rate, and duration of time granule is present in image.

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4. Export data to a data processing program such as Microsoft Excel for further analysis. 3.4.3. Measuring Colocalization in Live Cells

While the analysis of molecular colocalization is fundamentally similar in live cells to that in fixed cells, it presents the challenge of simultaneously monitoring two molecules which are in motion inside a live cell. Because of the variability of live cell imaging, modifications must be made in an attempt to accurately measure the sequence of events that occurs (see Note 12). 1. Determine which method to use to analyze colocalization in your images (see Note 9). 2. To reduce the quantity of data obtained, it may reasonable to select specific intervals from within a set of time-lapse images to evaluate. 3. For each time point to be analyzed, perform area and colocalization analysis as described in Section 3.3.3. 4. Analyze changes in colocalization between molecules over time (see Note 10).

4. Notes 1. Make PBS-S solution fresh before each use. Ensure that the solution is at room temperature before use in experiments. Always prepare Cytofix/Cytoperm-Triton solution before each use. For best results, pre-warm the Cytofix/Cytoperm to 37◦ C prior to adding the Triton X-100. 2. For antibody titration a. Adequate antibody titration is essential to ensure efficient fluorescent detection of a molecule while minimizing “bleed-through” of fluorescence into that meant to detect other fluorophores. b. Each primary antibody being used should be individually titrated and compared to an isotype control. c. Secondary antibodies also need to be titrated but should be done using a fixed concentration of a primary antibody with known effectiveness. d. In an initial titration experiment, a wide range of antibody concentrations should be utilized. The ideal concentration will provide efficient fluorescence at reasonable microscope settings and will result in a good signal-to-noise ratio. For example, an antibody dilution that gives saturated fluorescence signal using 10 ms of exposure would limit the ability to acquire meaningful quantitative data and should be avoided. Conversely, an antibody concentration that provides signal

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only minimally detectable above background after 1 s of exposure will similarly limit ability to acquire meaningful data. e. If needed, a second, narrower titration can be performed to identify the exact concentration needed. 3. For troubleshooting Nucleofection: a. Abundant cell death: (A) Culture the cells as described in Step 1 of Section 3.2.2 and also prepare fresh culture medium before Nucleofection. (B) Transfer the Nucleofected cells to pre-warmed medium immediately after Nucleofection. b. Low Nucleofection efficiency: (A) Check the purity of DNA/RNA. Impurity adversely affects efficiency. (B) Nucleofection solutions expire within 3 months and solutions older than 3 months can adversely affect efficiency. (C) Ensure that the Nucleofection solution is made by properly mixing both components according to the Amaxa recommended protocol. (D) Using more than 3 × 106 cells per reaction reduces Nucleofection efficiency. c. Successful introduction of control GFP plasmid or FITC-linked oligonucleotides, but no results with the experimental nucleic acids: (A) Evaluate Nucleofection at different time points. (B) Check purity of nucleic acid being Nucleofected. d. Large inter-experiment variability: (A) Maintain cells at low density in culture. (B) Use new Nucleofection solution. e. Control siRNA downregulates the gene of interest: (A) Reduce the amount of siRNA used for the control and target genes. (B) Use a pool of several siRNA fragments against the target gene, but at lower concentrations. 4. For transduction: a. If Plat-E cells are used they should be cultured in DMEM with 10% FCS, in the presence of puromycin (1 mg/mL) and blasticidin (10 ␮g/mL) to maintain expression of retroviral helper genes. b. If the gene of interest is not expressed as a direct fusion to a fluorescent protein, it should be cloned into a viral vector that uses an internal ribosomal entry site (IRES) followed by a fluorescent protein gene (such as EGFP). This will enable the identification of transgeneexpressing cells and the level of fluorescence will correlate with the expression of the transgene. Either approach will enable the use of FACS to select the

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population of cells expressing the gene of interest. Cultures can then also be regularly monitored for transgene expression using flow cytometry. c. If transduction efficiency is low, fluorescent cells should be enriched by FACS or by limiting dilution. d. Polybrene has toxic organ (blood and kidney) effects as documented by OSHA and thus caution should be exercised. 5. For extended time-lapse acquisitions, the focal plane may drift. There are compensation systems available, e.g., Olympus offers the Zero Drift Compensation (ZDC) component and Advanced Imaging Systems (AIS) manufactures the C-RIFF system. Alternatively, one can maintain the focus on a fluorescent bead; this eliminates the need to focus on a cell that is potentially moving z-axially. 6. Optimal Lysotracker Green concentration needs to be titrated for laser output power and cell number optimized for size of dish and desired density. 7. We use 100 nm fluorescent beads (Invitrogen FluoSpheres carboxylate-modified microspheres, 0.1 ␮m) to align the TIRF laser prior to addition of cells. This minimizes the amount of time between cell addition and image acquisition. Briefly, after dishes are coated with antibody and washed, they are incubated with an appropriate dilution of fluorescent beads in PBS for 1 h at 37◦ C. After washing 2× with PBS, 1 mL of culture medium is added. The glass surface is found using widefield fluorescence before switching to TIRF and aligning the laser. 8. It is very important to select only the conjugates where an NK cell is conjugated with a target cell. NK cells touching two target cells should not be taken under consideration as there are a separate set of considerations that apply to multiply conjugated NK cells. 9. Because the signal generated by fluorescent molecules occurs in a spectrum of intensities, in order to correctly identify areas containing fluorescent molecules, it is necessary to set a specific intensity threshold for measurements. All image analysis software has this functionality, although there are a number of options. Similar to FACS, all events (which in the case of microscopy are individual pixels or voxels and not whole cells) have a numerical intensity value assigned to them that is generally >0. That said, very low numerical intensity values are generally not reflective of true signal (similar to “autofluorescence” in FACS). Options for creating a threshold over which fluorescence intensities are to be included in analyses include:

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(1) selecting an objective florescence intensity value; (2) selecting a percentage of fluorescence intensity (i.e., the top 40% of fluorescence intensities; and (3) selecting a specified number of standard deviations above the mean fluorescence intensity. Although there is some degree of relativity present in the latter two approaches, they are especially useful in time-lapse experiments, where the fluorescence may decay over time. In this case, the relative intensity differences will generally be maintained and can be still exploited for quantitative analysis. In all cases comparison to controls is essential in quantitation. These include staining controls as well as biological controls. By using each, true increases, decreases, and alterations in intensity, as well as area/volume containing those intensities can be measured. Note that during colocalization analysis, the threshold for measuring one molecule may be different than the threshold for measuring another. 10. In comparing fluorescent regions as described in the procedures and above in Note 9, it is important that sufficient numbers of cells be evaluated. There is no set number of cells that must be evaluated for an experiment to be valid. What is appropriate, however, is that the number of cells included for analysis represents a sample that is sufficiently powered to enable statistical testing of the hypothesis. In order to decide upon the number of cells that should be evaluated for a given experiment, it is best to perform a sample size calculation using values obtained from a pilot or related experiments. 11. Importantly, once a threshold is set it needs be applied to all analyses within a given experiment uniformly. Different software packages have different options for identifying the pixels that contain fluorescence of multiple fluorophores that fall above the user-defined threshold (in Volocity, this can be performed using the “Intersect Objects” command). These algorithms will also provide the mean intensity of each fluorophore in that colocalized region. As there will likely be multiple colocalized regions within a single cell, all of the individual regions can be joined into a single region to contain the total colocalized area along with its mean intensity (in Volocity this can be performed using the “Join Objects” command). 12. When defining intensity thresholds in live cell experiments, it can be helpful to use percentage intensity- or standard deviation-based thresholds, as in longer experiments the absolute fluorescence of a fluorophore can fade, but the differences remain intact.

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References 1. Axelrod, D. (2001) Total internal reflection fluorescence microscopy in cell biology. Traffic 2, 764–774. 2. Axelrod, D. and Davidson, M.W. (2008) Specialized Microscopy Techniques – Total Internal Reflection Fluorescence Microscopy. Available online at www. olympusmicro.com/primer/techniques/ fluorescence/tirf/tirfintro.html. 3. Orange, J. S. (2008) Formation and function of the lytic NK-cell immunological

synapse. Nat Rev Immunol. 2008 Sep; 8(9): 713–725. 4. Banerjee, P.P. and Orange, J.S., submitted. 5. Banerjee, P. P., Pandey, R., Zheng, R., Suhoski, M. M., Monaco-Shawver, L., and Orange, J. S. (2007) Cdc42-interacting protein-4 functionally links actin and microtubule networks at the cytolytic NK cell immunological synapse. J Exp Med 204, 2305–2320.

Chapter 10 Measuring Intracellular Calcium Signaling in Murine NK Cells by Flow Cytometry Alexander W. MacFarlane IV, James F. Oesterling, and Kerry S. Campbell Abstract This chapter describes a method by which activating receptor-mediated calcium signaling can be measured in individual murine NK cells using a flow cytometer fitted with a UV laser. One major advantage of this method is that the calcium response of the minority NK cell population and even smaller NK cell subpopulations can be measured simultaneously from a mixture of freshly prepared total splenocytes without resorting to prior cell sorting or expansion in culture. Briefly, cells are harvested and stained to mark the populations of interest, then loaded with indo-1 AM dye and analyzed on the flow cytometer. After an appropriate baseline is established, the cells are treated with a biotinylated antibody to activating receptors, which are subsequently cross-linked by addition of streptavidin. The increase in intracellular calcium is quantified by measuring a shift in the indo-1 emission spectrum that takes place when the dye becomes bound to calcium. Key words: Calcium signaling, indo-1, flow cytometry, NK1.1.

1. Introduction Increased levels of cytosolic calcium are critical to numerous lymphocyte functions including proliferation, metabolism, apoptosis, migration, cytotoxicity, and the formation of an immunological synapse (1). This protocol builds on the work of Valittuti and Dessing, who previously described a methodology for measuring calcium signaling in individual cells within a T-cell population in response to target cell engagement in this series (2). Here we expand upon the method to demonstrate NK cell calcium mobilization in response to soluble ligands and demonstrate how the relative responsiveness of various developmental subpopulations K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 10, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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can be resolved. Cytosolic calcium concentrations are normally tightly regulated in resting cells. The increased intracellular calcium concentration in response to activating receptor engagement takes place in two stages. Calcium is initially released from the endoplasmic reticulum over a short period of time, followed by a sustained influx from the extracellular environment. The data collected is a convolution of these two processes, but since the early time points are dominated by release of intracellular calcium stores and the later time points by influx from the extracellular medium, it should be possible to see a specific effect on either of the two processes. In this method, the cells are first stained with antibodies to cell surface markers that enable the identification of NK cells and any subsets of interest, then loaded with indo1 AM dye and stimulated through activating receptors. Calcium flux is initiated by cross-linking activating receptors that are first engaged by a biotinylated antibody and subsequently clustered together by addition of streptavidin. Changes in the intracellular calcium concentration are quantified by a shift in the indo1 emission peak from 485 nm (indo-blue) for unbound dye to 405 nm (indo-violet) when the indo-1 molecule is bound to calcium. Mean intracellular calcium concentration is quantified in terms of the ratio of 405/485 nm indo-1 emission peaks. Measuring the change in emission ratio allows comparisons between individual cells within the population that may not be loaded with equivalent amounts of indo-1 dye.

2. Materials 1. Erythrocyte lysis buffer: 125 mM KHCO3 , 1 mM Na2 EDTA.

NH4 Cl,

10 mM

2. Serum-free RPMI-1640 medium (Life Technologies, Rockville, MD). 3. Complete RPMI medium: RPMI-1640 medium, 10% FBS (Hyclone), 100 ␮g/ml penicillin/streptomycin, 2 mM Lglutamine, 10 mM HEPES, pH 7.4, 1 mM MEM sodium pyruvate, and 50 ␮M 2-mercaptoethanol (all from Life Technologies). 4. Cell permeant indo-1 AM dye (Invitrogen, Eugene OR): prepare a 2.5 mM stock in DMSO. Store protected from light at –20◦ C (see Note 1). 5. Pluronic F-127 20% solution in DMSO (Invitrogen). 6. Conjugated monoclonal antibodies: anti-CD3␧PerCP/Cy5.5, anti- anti-CD122-FITC [or anti-CD49b-

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FITC (DX5)], anti-CD11b-APC/Cy7, and biotinylated anti-NK1.1 (PK136) (all from BioLegend, San Diego, CA). 7. Propidium iodide 1 mg/ml (Invitrogen) (see Note 2). 8. Purified streptavidin (Sigma). 9. 40 ␮m cell strainer (Falcon, BD Biosciences). 10. 10 ml syringe. 11. Flow cytometer: Our studies used a BD FacsVantage SE flow cytometer with the FACSDiVa Option fitted with a Coherent Innova Model 302C Krypton Laser. The laser is operated in multiline UV mode with emission lines ranging from 337.5 to 356.4 nm and a Chroma 440 DCLP splitter/indo-1 filter set measuring emission peaks at 405 and 485 nm. 12. FlowJo software, version 8.7.1 (TreeStar) and appropriate computer.

3. Methods 3.1. Cell Preparation

1. Prepare single cell suspensions of mouse splenocytes by mashing spleens through a 40 ␮m nylon cell strainer with a rubber-tipped 10 ml syringe plunger and rinse the cell strainer with a total of 10 ml of complete RPMI culture medium. 2. Spin the cells at 500 g for 5 min and resuspend in ice cold erythrocyte lysis buffer. Incubate for 3 min on ice, then pellet cells at 500 g for 5 min. 3. Immediately resuspend in 10 ml of cold complete RPMI medium and count. 4. Spin down the cells and resuspend at a dilution of 20 million cells in 1 ml of complete RPMI-1640. 5. Add appropriate staining titer of each of the fluorochromeconjugated antibodies and incubate on ice for 20 min. Single color compensation controls and an unstained control should be prepared at this time as well (see Notes 3 and 4). 6. While the cells are being stained, add 1 ␮l of 20% Pluronic F-127 solution to every 9 ␮l of indo-1 AM stock solution needed and warm at 37◦ C for 5–10 min in a shaking heat block to ensure that it is mixed properly (see Note 1). Protect from light during all preparation procedures.

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7. Rinse cells twice with serum-free RPMI-1640 medium and resuspend in 4 ml of serum-free RPMI-1640 medium that has been pre-warmed to 37◦ C and place in a 37◦ C water bath. Cell concentration should be about 5 million/ml. 8. Allow 10 min for the cells to warm at 37◦ C, then add 1– 1.5 ␮l of the indo/Pluronic solution prepared in Step 5 to each ml of cell suspension and mix thoroughly by inverting the tube several times. If comparing different cell populations, it is critical to assure uniform indo-1 loading between the different cell samples, so exactly the same conditions should be used for each sample and cells should be mixed thoroughly after addition of the dye. 9. Incubate the cells for 30 min at 37◦ C under foil to protect from light. Resuspend the cells every 10 min during this incubation. Then centrifuge the cells at 500 g for 5 min and resuspend in 2 ml of complete RPMI-1640 medium. If propidium iodide is being used to exclude dead cells, then add at a final concentration of 200 ng/ml. 3.2. Flow Cytometry

Maintain labeled cells in the dark at room temperature (not on ice) at all times prior to analysis on the flow cytometer. Do not store labeled cells on ice, since this will diminish the intensity of subsequent calcium responses. 1. Prewarm a 500 ␮l cell sample of cell suspension in a 5 ml FACS tube at 37◦ C for 5 min prior to analysis on the flow cytometer. 2. Run the 500 ␮l sample through the flow cytometer at about 2000–2500 events/s for 1–2 min to establish the baseline indo-1 signal that represents the basal intracellular calcium concentration. The cell sample should be maintained at 37◦ C throughout the time course of analysis. Although calcium mobilization can be measured at room temperature, the response will be suboptimal as compared to analysis at 37◦ C. 3. Remove the sample and add 4 ␮g of biotinylated anti-NK1.1 (PK136) mAb and return the sample to the instrument (see Note 5). 4. Allow the cells to run for another minute to determine if the antibody stimulates the cells in the absence of streptavidin. We have not found this to be true for PK136, but it may occur when other receptors or antibodies are used. 5. Remove the sample once again, add 8 ␮g of streptavidin, and return the sample to the instrument to collect data for an additional 4–5 min (see Note 6). An increase in indoviolet (405 nm) signal and a decrease in indo-blue (485 nm) signal are indicative of a stimulation-induced increase in

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intracellular calcium concentration. If longer analysis is desired, an increased starting volume of cells (750–1000 ␮l) should be used and antibody/streptavidin concentrations should be adjusted accordingly. 3.3. Data Analysis

We have used FlowJo software (version 8.7.1) to analyze our data. It is necessary to create a derived parameter to display the indo-violet (405 nm) intensity divided by the indo-blue (485 nm) intensity. This is accomplished by navigating through the following menu tabs: Platform -> Derive Parameters -> Define new or change. Here you can create the new parameter and give it a name. Make sure the “display with linear scale” box is checked and start with lower and upper axis limits of 0.4 and 1.2, respectively. Once the parameter is created you can view the mean relative cytosolic calcium concentration within the gated population by highlighting the desired NK cell gate in the FlowJo work space and choosing Platform -> Kinetics from the menu tabs. In Fig. 10.1A, we set the axis limits to 0.5 and 0.9, while choosing to display the mean fluorescent intensity acquired over the integration time of each data point. Since the actual signal is changing slowly compared with the fluctuations in the data, it is reasonable to increase the signal–to-noise ratio by averaging adjacent points. Figure 10.1B shows the same data with a moving average applied. This replaces each data point with the average value of the data point with several points on either side. The Gaussian smoothing shown in Fig. 10.1C is a moving average that weights adjacent points more heavily than those that are further away. This results in less smoothing than the unweighted moving average, but is also less likely to obscure rapidly changing details or distort the actual rates of ascent and decline. Another approach is to plot the percent of cells with an indo-violet to indo-blue ratio above a baseline threshold value. This provides information about what fraction of a given population or subpopulation is undergoing calcium flux. Figure 10.1D shows this with a threshold of 0.65 and moving average smoothing. One of the important advantages of this method is the ability to distinguish populations and subpopulations of cells without sorting or other mechanical fractionation. Figure 10.2 shows how NK cells can be distinguished from other cell types and the different magnitudes of calcium flux within each gated subpopulation. The first gate, shown in Fig. 10.1A, is applied to a plot of forward scatter height versus forward scatter area. This is referred to as a singlet gate because it excludes instances where more than one cell is contained in a droplet that is analyzed by the cytometer. Figure 10.2B shows the lymphocyte gate, which excludes cell fragments, the majority of dead and dying cells, and large aggregates. As shown in Fig. 10.2C, NK cells are gated as CD122+ (or

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Fig. 10.1. Mean calcium flux data from NK cells stimulated with biotinylated antiNK1.1 mAb (added at 60 s) streptavidin (at 120 s) are visualized by several smoothing methods. Plotting the ratio of mean indo-violet/indo-blue emission values at individual time points provides values corresponding to relative calcium concentration of cells within the population over the time course of the experiment. (A) No smoothing. (B) Moving average. (C) Gaussian smoothing. (D) Plot of indo-violet (V)/ indo-blue (B) events that are above a threshold ratio value of 0.65. Time in seconds is shown in the x-axis.

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Fig. 10.2. Electronic gating to distinguish NK cells and subsets of NK cells from other lymphocytes and cellular debris. (A) To assure a single cell analysis, events containing one cell are selected based on linear correlation between forward scatter area (FSC-A) and forward scatter height (FSC-H). (B) Predominantly viable cells are distinguished from dead cells and small particles based on their forward scatter height and side scatter area (SSC). (C) NK Cells are gated as CD3 (TCR)– , CD122+ . (D) Subpopulations of NK cells are defined by their expression levels of CD11b immature (CD11blow ) and mature (CD11bhigh ). (E) Calcium flux measurements of the subsets defined in (D) above with the thin line representing immature NK cells and the thicker line representing mature NK cells.

CD49b+ if substituted), CD3− . Once the NK population has been selected, the cells can be further subdivided according to expression of CD11b into successive maturation stages that are shown in Fig. 10.2D. A comparison of the kinetics of these two subpopulations shown in Fig. 10.2E reveals greater functionality in the mature subset.

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4. Notes 1. The acetoxymethyl (AM) ester derivative of indo-1 is uncharged and can cross the plasma membrane into cells. Once inside the cells, esterases cleave the side chain to create a charged form of the parent indo-1 molecule that is retained in the cytoplasm. Pluronic F-127 is non-ionic detergent with low cytotoxicity that aids solubility of indo-1AM in aqueous solutions for improved loading of cells. 2. Propidium iodide or 7AAD can be used to exclude dead cells from the analysis, but if the emission channel is needed for another antibody it is also possible to gate populations of cells that have greater than 98% viability by using the forward scatter and side scatter gates. This is possible because dead and dying cells tend to be smaller and more granular, so they exhibit less forward scatter and greater side scatter than viable cells. 3. Avoid staining with antibodies directed toward NK cellactivating receptors that can be triggered by antibody engagement. 4. One should be cautious if considering the use of Cascade Blue, Alexafluor 405, or other fluorophores in the violet/blue emission range as gating antibodies, because their emission spectra may overlap with that of indo-1. 5. NK1.1 is a member of the NKR-P1 family of receptors and is only expressed on NK cells from certain mouse strains, such as C57Bl/6, but not 129 or Balb/c. 6. The balance of biotinylated antibody to streptavidin determines the degree of receptor aggregation, which is critical for the experiment to succeed. Biotinylated antibody and streptavidin concentrations should be titrated to determine the optimal concentrations whenever a new activating receptor is chosen to initiate the calcium flux. This may require significant effort to achieve the proper combination. If not enough streptavidin is used, then the receptors will be poorly cross-linked and fail to stimulate the cells. Using too much streptavidin can also result in poor cross-linking if each biotin moiety binds a single streptavidin molecule, instead of multiple biotin molecules binding to each streptavidin tetramer. Another option is to use fluorophore-conjugated streptavidin as a cross-linking agent to assess degree of cross-linking and surface levels of receptor being engaged. The conditions for biotinylated anti-NK1.1 and streptavidin have been optimized in our hands using this experimental design, in which the NK cells make up on average about 3% of the total splenocytes being analyzed.

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Acknowledgments We would like to thank Dr. Richard (Randy) Hardy for assistance in setup of the flow cytometry equipment and for suggestions to improve the manuscript. Supported by National Institutes of Health grants R01-CA083859, R01-CA100226 (K.S.C.), T32AI007492 (A.W.M.), and Centers of Research Excellence grant CA06927 (FCCC). The research was also supported in part by the FCCC Blood Cell Development and Cancer Keystone Program and an appropriation from the Commonwealth of Pennsylvania. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute. References 1. Clapham, D.E. (2007) Calcium Signaling. Cell 131, 1047–1058. 2. Valitutti, S. and Dessing, M. (2000) Measurement of Calcium Mobilization

Responses in Killer Cell/Target Conjugates by FACS Analysis. Methods Mol Biol 121, 305–311.

Chapter 11 Intracellular Staining for Analysis of the Expression and Phosphorylation of Signal Transducers and Activators of Transcription (STATs) in NK Cells Takuya Miyagi, Seung-Hwan Lee, and Christine A. Biron Abstract Cytokines stimulate biological responses by activating intracellular signaling pathways. We have been adapting flow cytometric techniques to measure the levels of expression and activation of signaling molecules within mixed populations containing NK cells and to characterize their differences within NK cell subpopulations. Approaches for evaluating the total levels of the signal transducers and activators of transcription STAT1 and STAT4, of STAT1 in cells expressing IFN␥, and of the type 1 interferon (type 1 IFN) activation by phosphorylation, i.e., induction of pSTAT1 and pSTAT4, have been developed. The results of experiments using these techniques have demonstrated that an unusual feature of NK cells is high basal expression of STAT4 but reduced STAT1 levels. The condition predisposes for pSTAT4 activation by type 1 IFNs. The work has also shown, however, that total STAT1 levels are induced during viral infections as a result of IFN exposure, and that this change acts to promote the activation of STAT1 but limit both the activation of STAT4 and IFN␥ expression. The intracellular staining approaches used for the studies described here have utility in characterizing other mechanisms regulating cytokine-mediated signaling, and defining additional pathways shaping cellular responses to cytokines. Key words: STAT1, STAT4, pSTATs, intracellular staining, type 1 IFN.

1. Introduction 1.1. Cytokine Signaling

A range of cytokines use signal transducers and activators of transcription molecules (STATs) as intracellular intermediaries for eliciting cellular responses. A class of cytokines using STATs to signal is the type 1 interferon (IFN) family comprised of a single ␤ and multiple ␣s. The factors are elicited in response to a variety of stimuli including viral infections (1, 2). As a result of binding to specific receptors, type 1 IFNs induce particular

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kinases to activate STATs by phosphorylation (3, 4). The best understood signaling pathway downstream of the type 1 IFN receptor uses STAT1 and STAT2, and this pathway is linked to the induction of antiviral defense mechanisms. Type 1 IFNs, however, have been reported to induce a wide range of biological functions and have important immunoregulatory effects (1, 2). Some of these are paradoxical, including enhancing and inhibiting IFN␥ production as well as blocking and promoting cell proliferation (1, 5–8), but the mechanisms regulating selection of the subset functions are poorly understood. There are a total of seven STATs, 1 through 6 with two different 5s. Type 1 IFNs have been reported to conditionally activate all of these, including STAT4 (1, 9). Their negative effects on IFN␥ expression and cell proliferation are dependent on STAT1 (5, 6, 10), whereas STAT4 contributes to IFN␥ expression (7, 11, 12). Thus, conditions affecting the relative accessibility of STAT4 and STAT1 have the potential to contribute to the shaping of cellular responses to type 1 IFNs. 1.2. Approaches for Evaluating Access to Signaling Pathways

Until recently, evaluation of the availability and activation of intracellular signaling pathways has been limited to biochemical Western blot analyses using proteins extracted from cell populations. The technique does allow determination of total protein and phospho-protein levels within samples and has the advantage of revealing the molecular weights of proteins detected. Using this approach, we have analyzed samples from mouse splenic leukocytes to show that there is an inverse correlation between type 1 IFNs’ ability to activate STAT4 with total STAT1 levels (7). This method, however, does not allow characterization of differences in responses within mixed cell subpopulations, and the numbers of purified cells required can present a challenge for certain NK cell studies. Flow cytometric or fluorescent-activated cell sorting (FACS) techniques can evaluate multiple parameters within mixed cell subsets. The approach has become very powerful because of the development of (1) specific monoclonal antibodies against a variety of cell determinants and cytokines; (2) an increasing range of fluorochromes with different excitation and emission spectra for coupling to the monoclonal antibodies; and (3) improved instrumentation (13). In addition to being used to evaluate the molecules expressed on the surfaces of cell subsets, flow cytometry is being used to characterize mixed cell subsets in regard to the range and levels of cytokines they can be induced to express in their cytoplasm. This is possible because of the development of different permeabilization protocols allowing intracellular access of antibodies detecting cytokines. Thus, there are indications that flow cytometric techniques may provide opportunities for studying intracellular expression of signaling molecules in different cells within mixed populations.

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1.3. Technical Challenges to Flow Cytometric Approaches for Measuring STATs

Because of our interest in defining the intracellular signaling pathways activated by type 1 IFNs in NK cells, this group committed to developing protocols to evaluate the levels of STAT1, STAT4, pSTAT1, pSTAT4, IFN␥, and/or combinations of these within NK cells (14). This approach required identification of multiple cell surface determinants with multiple intracellular molecules. Challenges included the availability of specific antibodies and/or specific antibodies for use with fluorochromes having emission spectra that could be separated. In addition, depending on their activation state, STATs can be found in both the cytoplasm and the nucleus. Therefore, staining and permeabilization/fixation methods that could be used in combination needed to be identified to allow detection of the different STATs or pSTATs within cells without destroying the fluorescent function of the fluorochromes and/or the antigenic determinants being detected by the monoclonal antibodies. The methods developed are based on the following: commercially available protocols for staining incorporated nuclear analogues (BD Biosciences), earlier work from our group examining STAT1 levels in T cells (10), reports for detecting pSTATs (15–20), testing using wild type (WT), STAT1-deficient and STAT4-deficient cells (14), and comparisons of results to those obtained with Western blotting (14). Commercially available antibodies to detect pSTATs facilitated the work (BD Biosciences). Antibodies from a variety of sources were screened for intracellular staining of total STAT1 and STAT4 proteins. Monoclonal antibodies detecting full-length STAT1 and STAT4 were being commercially produced and identified (BD Biosciences) and were first used with specific secondary antibodies. Eventually custom reagents with directly conjugated fluorochromes were made. Methanol permeabilization was optimal for intracellular detection of the STATs. It did, however, present problems for detection of cytoplasmic cytokines and the use of particular fluorochromes to identify cell surface markers.

1.4. Methods Developed

The protocols were developed with the goal of characterizing the signaling pathways and responses to type 1 IFN exposure in vivo. The focus was on measuring total STATs, pSTATs, and IFN␥ levels immediately after isolation of the cells from uninfected mice and/or from mice at different times after viral infection. It became clear, however, that optimal protocols for detecting STATs were not compatible with detecting intracellular cytokines. Thus, a second method was developed for identifying STAT1 and IFN␥ within the same cells. Finally, the detection of pSTAT activation in vivo was possible in only low frequencies of cells from immunocompetent mice. This is likely to be in part as a result of the need to capture cells just after cytokine exposure because the biochemical evidence indicates that phosphorylated forms of

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the STATs are short lived. In addition, however, the experiments carried out also demonstrated that a dynamic regulation of the STAT levels contributed to the selection of particular STATs for activation in vivo. This latter point was proven by examining cellular responsiveness for type 1 IFN activation of STAT1 or STAT4 under control ex vivo conditions after isolation, and by using cells from mice mutated in STAT1 or STAT2. The three methods are presented below: (1) detecting total STATs or total STAT1 with pSTAT4 within freshly isolated NK cells; (2) detecting STAT1 with IFN␥ immediately after isolation; and (3) detecting ex vivo responsiveness to type 1 IFNs with the examination of single parameter pSTATs or total STAT1 with pSTAT4.

2. Materials 2.1. Mice

2.2. General

All protocols require the preparations of splenic leukocytes from wild-type (WT) mice and mice genetically deficient for STAT1 or STAT2. They are available on the 129 background (21, 22) (Taconic Labs). For NK studies, it is best to use mice at 4–9 weeks of age. As indicated, mice can be treated to induce an immunological response. For the protocols detailed below, mice were either uninfected (D0) or infected intraperitoneally with lymphocytic choriomeningitis virus (LCMV) (10, 14). 1. 6-well tissue culture plate (BD Biosciences). 2. 96-well-V-bottom assay plate (Costar). 3. 15 ml polypropylene conical tubes (BD Falcon). 4. 24-well tissue culture plate (BD Biosciences). 5. FACS tube: 1.2 ml polypropylene U-bottom tube (Costar). 6. Sterile frosted glass microscope slides (Fisher Scientific). 7. Nylon mesh (Sefar America). 8. Red Blood Cell Lysing Buffer (Sigma). 9. RPMI-1640 medium (GIBCO). 10. Assay Medium: RPMI-1640 containing 10% FBS, with 1× Penicillin–Streptomycin and 10 mM Hepes Buffer (GIBCO) at pH 7.4. 11. Brefeldin A (Sigma), dissolved in DMSO at 10 mg/ml, aliquoted and stored at −20◦ C. 12. Staining Buffer: PBS containing 2% fetal bovine serum (FBS, Hyclone).

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13. Goat Block: PBS containing 20% FBS and 10% goat serum (Sigma). 14. 2.4G2 antibody (anti-Fc␥RIII/II; BioXcell) at a working concentration of 1 mg/ml. 15. Cytofix/Cytoperm Buffer (BD Biosciences). 16. Perm Wash Buffer (BD Biosciences). 17. DNase I (Sigma), dissolved in PBS, aliquoted at 1 mg/ml concentration of stock solution and stored at −80◦ C. Dilute with PBS to 300 ␮g/ml for use. 18. Methanol (Fisher Scientific). 2.3. Stimulation

1. In vivo: 2 × 104 plaque-forming units (PFUs) of LCMV. 2. Ex vivo: Recombinant murine IFN␤ (a gift from Biogen Idec, specific activity of 2 × 109 U/mg). Other type 1 IFNs, i.e, IFN␤ or IFN␣, can be used (PBL InterferonSource).

2.4. Flow Cytometric Application and Fluorochromes

There are a number of instruments available for FACS, and these have increasing flexibility in extending parameters for measurement. The methods described here, however, were developed using a FACSCalibur (BD Biosciences) with two lasers having outputs at 15 mW of 488 and 635 nm wavelengths. The results were analyzed using the CellQuest Pro Software (BD Biosciences). The experiments required staining with either three or four different antibodies identified by three or four fluorochromes that could be (1) excited at these wavelengths, (2) result in emission wavelengths distinguishable using available filters, and (3) purchased from commercial sources either directly conjugated to, or available for use in secondary detection steps with, the antibodies needed. In the end, five different fluorochromes were used: fluorescein (FITC), Alexa Fluor 647 (Alexa 647), phycoerythrin (PE), allophycocyanin (APC), and peridinin chlorophyll protein (PerCP). The combinations of four that can be used in individual tests are FITC, PerCP, PE, and Alexa 647; or FITC, PerCP, PE, and APC (see Note 1).

2.5. Antibodies

The reagents detecting mouse leukocyte cell surface markers were developed against mouse determinants. Because NK cells were being identified, two reagents are required for cell surface staining to identify the NK cells and exclude T cells. In the detailed methods, CD49b expression is used to identify NK cells and CD3ε expression is used to exclude T cells. As discussed in the Notes, however, other combinations of antibodies can be used, including expression of NK1.1 to identify NK cells from appropriate strains of mice and expression of TCR␤ to exclude T cells. In contrast to the reagents identifying murine leukocyte subsets, many of the reagents being developed to detect total STATs or pSTATs

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were prepared against the human molecules, but cross-react with mouse because of the strong homologues between the species. Care should be taken to verify specific reactivity with mouse STATs or pSTATs when new reagents are used. The detailed methods use a custom prepared anti-STAT1 conjugated to PE to detect total STAT1. An anti-STAT4 mouse monoclonal antibody of the IgG1 isotype is identified using a monoclonal antimouse IgG1. This secondary reagent specifically identifies the primary anti-STAT4 when (1) the other reagents used are as listed in the methods because no other is a mouse IgG1 and (2) the splenic leukocytes are prepared at times during infection that precede induction of B-cell isotype switching. The methods describe the use of antibodies recognizing STAT forms phosphorylated at particular tyrosine residues. STATs can be phosphorylated at different tyrosine or serine residues, and the targets of phosphorylation may vary depending on the conditions of stimulation. Thus, modifications of methods to evaluate other sites of phosphorylation maybe of interest to investigators, but will require careful consideration of the biochemical literature. All of the antibodies purchased for flow cytometry and used in the methods are available from BD Biosciences. Some of them can also be purchased from eBioscience. The amounts identified are based on the lots used for developing the protocols. Generally, 30–500 ngs of a particular antibody are used for a single test with 2 × 106 splenic leukocytes. All new lots should be evaluated by titration. 2.5.1. Detection of Total STAT1, Total STAT4, or Total STAT1 with pSTAT4

1. Commercial-specific antibodies: FITC-anti-CD49b (clone DX5); Biotin-anti-CD49b (clone DX5); PerCP-anti-CD3ε (clone 145-2C11); Streptavidin-APC; purified anti-STAT4 (clone 8); FITC-anti-mouse IgG1 (clone A85-1); Alexa 647-anti-STAT4 pY693 (clone 38/pSTAT4); all purchased from BD Biosciences. 2. Customized antibody: PE-anti-STAT1 (C-terminal clone 42), prepared and conjugated with fluorescence dye by BD Biosciences, at a working stock of 2 ␮g/ml. 3. Isotype Controls: PE-Mouse IgG2b (clone 27–35) for PEanti-STAT1; purified mouse IgG1 (clone MOPC-21) for purified anti-STAT4; Alexa 647-Mouse IgG2b (clone 27– 35) for Alexa 647-anti-STAT4 pY693; all purchased from BD Biosciences.

2.5.2. Detection of Total STAT1 with IFN␥

1. Commercial-specific antibodies: FITC-anti-CD49b (clone DX5); PerCP-anti-CD3ε (clone 145-2C11); APC-antiIFN␥ (clone XMG1.2); all antibodies purchased from BD Biosciences.

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2. Customized antibody: PE-anti-STAT1 (C-terminal clone 42), prepared and conjugated with fluorescence dye by BD Biosciences, at a working stock of 2 ␮g/ml. 3. Isotype Controls: PE-Mouse IgG2b (clone 27–35) for PESTAT1; APC-Rat IgG1 (clone R3-34) for APC-anti-IFN␥; from BD Biosciences. 2.5.3. Detection of Type 1 IFN Responsiveness for pSTAT1 or pSTAT4 Activation or Total STAT1 with pSTAT4 Activation

1. Commercial-specific antibodies: FITC-anti-CD49b (clone DX5); PerCP-anti-CD3ε (clone 145-2C11); PE-antiSTAT1 pY701 (clone 4a); Alexa 647-anti-STAT4 pY693 (clone 38/p-STAT4); all purchased from BD Biosciences. 2. Customized Antibody: PE-anti-STAT1 (C-terminal clone 42), prepared and conjugated with fluorescence dye by BD Biosciences, at a working stock of 2 ␮g/ml. 3. Isotype controls: PE-Mouse IgG2a (clone MOPC-173) for PE-anti-STAT1 pY701; Alexa 647-Mouse IgG2b (clone 27– 35) for Alexa 647-anti-STAT4 pY693; all purchased from BD Biosciences.

3. Methods 3.1. Cell Preparation

Prepare splenic leukocytes (see Note 2) by a regular method (see Note 3). Spleen processing is carried out in a 6-well tissue culture plate, with RPMI medium 1640. Sterile frosted glass microscope slides and nylon mesh, 15 ml polypropylene conical tubes, and Red Blood Cell Lysing Buffer are used.

3.2. Detection of Total STAT1, Total STAT4, or Total STAT1 with pSTAT4

The methods provided below evaluate intracellular levels of total STATs or both STAT1 and pSTAT4 expression immediately after isolation of cells from mice (Fig. 11.1A). They all use methanol permeabilization. The methanol permeabilization is required for detection of STAT4 or pSTATs. The steps detailed will result in samples having been stained with (1) FITCanti-CD49b, PerCP-anti-CD3ε, and PE-anti-STAT1 antibodies; (2) biotin-anti-CD49b antibody detected with streptavidin-APC, PerCP-anti-CD3ε antibody, anti-STAT4 antibody detected with FITC-conjugated anti-mouse IgG1 antibody; and (3) FITC-antiCD49b, PerCP-anti-CD3ε, PE-anti-STAT1, and Alexa 647-antiSTAT4 pY693 antibodies. 1. Resuspend the cells to 2 × 107 cells/ml in cold Staining Buffer. 2. Use 96-well-V-bottomed plate and load 100 ␮l of cell suspension per well (or 2 × 106 cells per test). 3. Centrifuge at 700 g for 3 min with low brake. Remove buffer from plate by flicking plate.

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4. To prevent non-specific binding of antibodies to Fc receptors during staining, add 200 ␮l of Goat Block with 0.25 ␮l of 2.4G2 antibody to each well and mix well (e.g., mix up and down 10 times) (see Note 4). Incubate for 15 min at 4◦ C. Centrifuge as described above and remove buffer from plate. 5. To label the cells with NK cell surface marker, add 50 ␮l of Staining Buffer containing 0.5 ␮l of FITC-anti-CD49b (for test leading to STAT1 detection) or biotin-conjugated anti-CD49b antibody (for test leading to STAT4 detection) (see Notes 1 and 5) to each well and mix well. Incubate for 15 min at 4◦ C. 6. To wash the cells, add 150 ␮l of Staining Buffer to each well, mix up and down four times. Then centrifuge and remove buffer. 7. To fix the cells, add 100 ␮l of Cytofix/Cytoperm to each well and mix well under the fume hood (see Note 6). Incubate for 20 min at 4◦ C. 8. Wash once with freshly prepared Perm Wash Buffer. 9. To permeabilize the cell, add 200 ␮l of pre-chilled (−20◦ C) pure methanol (see Note 7) to each well and mix well under the fume hood. Incubate for 15 min on ice under the fume hood (see Note 8). 10. Spin down the cells by centrifugation. 11. Flick plate and wash two times with Staining Buffer. 12. Add 50 ␮l of Staining Buffer containing 0.5 ␮l of PerCPconjugated anti-CD3ε antibody (see Notes 1 and 9), with 15 ␮l of PE-conjugated anti-STAT1 antibody (for STAT1 detection), or 0.5 ␮l of streptavidin-APC and 2 ␮l of antiSTAT4 antibody (for STAT4 detection), to each well. For control staining, add the same amount of corresponding isotype controls. In the case of simultaneous staining for STAT1 and pSTAT4, use 15 ␮l of PE-conjugated antiSTAT1 antibody and Alexa 647-conjugated anti-STAT4 pY693 antibody or the same amount of corresponding isotype controls to tests that have been first labeled with FITC-anti-CD49b antibody. 13. Mix well and incubate for 20 min at room temperature. For STAT1 staining, proceed to the step 16. 14. Wash once with Staining Buffer. 15. For STAT4 staining, add 50 ␮l of Staining Buffer containing the secondary antibody, 0.5 ␮l of FITC-conjugated anti-mouse IgG1 antibody to each well and mix well. Incubate for 15 min at 4◦ C.

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16. Wash once with Staining Buffer. 17. Resuspend in 250 ␮l of Staining Buffer and then transfer to FACS tubes. The samples are ready to be acquired using FACSCalibur (BD Biosciences) with the CellQuest Pro software (BD Biosciences) (see Note 10). An example result in the single staining of total STAT1 or STAT4 is shown in Fig. 11.1B. Another example result in the staining of both total STAT1 and pSTAT4 is shown in Fig. 11.1C.

Fig. 11.1. Methods for identifying intracellular total STATs or total STAT1 with pSTAT4. (A) Leukocytes are prepared from spleens as defined in Section 3.1 and stained to identify the CD49b+CD3ε− NK cells as defined in Section 3.2. Intracellular levels of STAT1, STAT4, or pSTAT4 are evaluated using methanol permeabilization. Total cells are evaluated based on gates set by forward and side scatter. Flow cytometric gates set on CD49b+CD3ε− cell subset allows examination of the NK cell subset. Representation of results with STAT levels in histograms can reveal either general shifts in intensity or mixed populations of positive and negative cells. Dot plots of total STAT1 levels with pSTAT4 reveals diminishing STAT4 activation associated with increasing concentrations of STAT1. (B) Histograms show total levels of STAT1 and STAT4 evaluated separately in total and NK cells prepared from WT mice either uninfected (D0) or LCMV infected for 1.5 or 2.5 days (D1.5 or D2.5). (C) Dot plots show levels of STAT1 and pSTAT4 in NK cell subsets at D0, D1.5, and D2.5 after infection as evaluated in WT, STAT2-, and STAT1-deficient mice. (Panels B and C were originally published in The Journal of Experimental Medicine, 2007, 204, 2382–2396. © Miyagi et al., 2007. doi:10.1084/jem.20070401.)

3.3. Detection of Total STAT1 with IFN␥

The methods presented here evaluate the levels of total STAT1 as compared to IFN␥ expression in individual cells (Fig. 11.2A). Because detection of intracellular cytokines is not possible after treatment with methanol, the fixation and permeabilization method used is dependent on Cytofix/Cytoperm (BD Biosciences) (see Note 11). At this time, it appears that the only signaling molecule detectable by this approach is STAT1. The steps detailed will result in samples having been stained with FITC-antiCD49b, PerCP-anti-CD3ε, PE-anti-STAT1, and APC-anti-IFN␥ antibodies.

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Fig. 11.2. Methods for detecting total STAT1 and IFN␥ within individual cells. (A) Leukocytes are prepared from spleens, treated with brefeldin A, and stained to identify the CD49b+CD3ε− NK cells as defined in Section 3.3. Intracellular levels of STAT1 along with IFN␥ are evaluated using fixation/permeabilization with Cytofix/Cytoperm. Flow cytometric gates are set on CD49b+CD3ε− cell subset to identify the NK cell subset. Dot plots of total STAT1 levels with IFN␥ reveals increases in STAT1 expression associated with decreasing cytokine expression. (B) Dot plots show NK cells levels of STAT1 and IFN␥ expression at D0, D1.5, and D2.5 after infection as evaluated in WT, STAT2-, STAT1-deficient mice. (Panels B was originally published in The Journal of Experimental Medicine, 2007, 204, 2382–2396. © Miyagi et al., 2007. doi:10.1084/jem.20070401.)

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1. Prepare cells and resuspend at 1 × 107 cells/ml in Assay Medium containing brefeldin A (see Note 12) in a 15 ml conical tube. 2. Incubate for 4 h at 37◦ C in a CO2 incubator, with mixing after 2 h by tapping the bottom of the tube. 3. To prepare cells for flow cytometry, use a 96-well-Vbottomed plate and load 200 ␮l of cell suspension per well (or 2 × 106 cells per test). Centrifuge and then flick buffer from plate. 4. Wash once with Staining Buffer. 5. To prevent non-specific binding of antibodies to Fc receptors, add 200 ␮l of Goat Block with 0.25 ␮l of 2.4G2 Ab to each well and mix well (see Note 4). Incubate for 15 min at 4◦ C. Centrifuge and then flick buffer from plate. 6. To label the cells with surface markers, add 50 ␮l of Staining Buffer containing 0.5 ␮l of FITC-conjugated antiCD49b antibody and 0.5 ␮l of PerCP-conjugated antiCD3ε antibody to each well and mix well. Incubate for 15 min at 4◦ C. 7. Wash once with Staining Buffer. 8. To fix and permeabilize the cells, add 100 ␮l of Cytofix/Cytoperm to each well and mix well under a fume hood. Incubate for 20 min at 4◦ C. 9. Wash once with freshly prepared Perm Wash Buffer. 10. Add 100 ␮l of DNase solution to each well and mix well. Incubate (preferably with a lid such as aluminum foil) for 1 h at 37◦ C in an incubator. 11. Wash once with the Perm Wash Buffer. 12. Add 50 ␮l of the Perm Wash Buffer containing 0.5 ␮l of APC-conjugated anti-IFN␥ antibody and 0.5 ␮l of PEconjugated anti-STAT1 antibody, or corresponding isotype controls, to each well. Mix well and incubate for 20 min at room temperature. 13. Wash once with the Perm Wash Buffer. 14. Resuspend in 250 ␮l of Staining Buffer and then transfer to FACS tubes. The samples are ready to be acquired using FACSCalibur with the CellQuest Pro software. An example result is shown in Fig. 11.2B. 3.4. Detection of Type 1 IFN Responsiveness for pSTAT1 or pSTAT4 Activation or Total STAT1 with pSTAT4 Activation

The methods presented below evaluate changes in responsiveness to type 1 IFN for activation of STAT1 or STAT4 resulting from conditioning during different ex vivo treatments (Fig. 11.3A). Thus, they require the ex vivo exposure of isolated populations to cytokines prior to staining. The steps detailed will result in samples having been stained with (1)

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FITC-anti-CD49b, PerCP-anti-CD3ε, and PE-anti-STAT1 pY701 antibodies; (2) FITC-anti-CD49b, PerCP-anti-CD3ε, and Alexa 647-anti-STAT4 pY693 antibodies; and/or (3) FITCanti-CD49b, PerCP-anti-CD3ε, PE-anti-STAT1, and Alexa 647-anti-STAT4 pY693 antibodies. 1. Resuspend the prepared cells to 2 × 107 cells/ml in Assay Medium. 2. Use 24-well-flat-bottomed plate and load 500 ␮l of cell suspension per well. To clear receptors of cytokines bound in vivo and allow cells to return to basal states, incubate for 4 h at 37◦ C in an incubator before testing for responsiveness to type 1 IFNs for STAT1 or STAT4 activation. 3. Add 500 ␮l of Assay Medium with or without a type 1 IFN (e.g., recombinant murine IFN␤ at a final concentration of 10,000 U/ml) for stimulated or unstimulated cells, respectively, to each well. Mix and incubate with a lid for 90 min at 37◦ C in a CO2 incubator. 4. To prepare cells for transfer and staining, mix the content in each well by pipetting up and down 10 times on ice. 5. For the analysis of flow cytometry, use 96-well-V-bottomed plate and load 200 ␮l of medium with stimulated or unstimulated cells to each well (or 2 × 106 cells per test). Centrifuge and then flick buffer from plate. 6. Wash once with cold Staining Buffer. 7. To prevent non-specific binding of antibodies for surface staining, add 200 ␮l of Goat Block with 0.25 ␮l of 2.4G2 Ab to each well and mix well (see Note 4). Incubate for 15 min at 4◦ C. Centrifuge and then flick buffer from plate. 8. To label the cells with NK cell surface marker, add 50 ␮l of Staining Buffer containing 0.5 ␮l of FITC-conjugated anti-CD49b antibody to each well and mix well. Incubate for 15 min at 4◦ C. 9. Wash once with Staining Buffer. 10. To fix the cells, add 100 ␮l of Cytofix/Cytoperm to each well and mix well under a fume hood. Incubate for 20 min at 4◦ C. 11. Wash once with freshly prepared Perm Wash Buffer. 12. To permeabilize the cells, add 200 ␮l of pre-chilled (−20◦ C) pure methanol to each well and mix well under a fume hood. Incubate for 15 min on ice. 13. Spin down the cells by centrifugation. 14. Flick and wash two times with Staining Buffer. 15. Add 50 ␮l of Staining Buffer containing 0.5 ␮l of PerCP-conjugated anti-CD3ε antibody with 15 ␮l of

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PE-conjugated anti-STAT1 pY701 antibody and/or Alexa 647-conjugated anti-STAT4 pY693, or the same amount of corresponding isotype controls, to each well. If the experiment is to detect total STAT1 and pSTAT4, add 50 ␮l of Staining Buffer containing 0.5 ␮l of PerCP-conjugated anti-CD3ε antibody with 15 ␮l of PE-conjugated antiSTAT1 antibody and Alexa 647-conjugated anti-STAT4 pY693, or the same amount of corresponding isotype controls, to each well. Mix well and incubate for 20 min at room temperature. 16. Wash once with Staining Buffer. 17. Resuspend in 250 ␮l of Staining Buffer and then transfer to FACS tubes. The samples are ready to be acquired using FACSCalibur with the CellQuest Pro software. Examples of single parameter analyses of pSTAT1 and pSTAT4 are shown in Fig. 11.3B and of total STAT1 with pSTAT4 analysis are shown in Fig. 11.3C.

Fig. 11.3. Methods for evaluating type 1 IFN responsiveness with pSTAT1 or pSTAT4 activation. (A) Leukocytes are prepared from spleens, rested in culture to clear cytokine receptors, and treated with type 1 IFN to induce STAT activation. The cells are then stained to identify the CD49b+CD3ε− NK cells as per Section 3.4. Intracellular levels of pSTAT1 or pSTAT4 are evaluated using methanol permeabilization. Total cells are evaluated based on gates set by forward and side scatter. Flow cytometric gates set on CD49b+CD3ε− cells allows examination of the NK cell subset. Dot plots of total STAT1 levels with pSTAT4 reveals differences in STAT4 activation associated with increasing concentrations of STAT1. (B) Ex vivo responsiveness of total and NK cells prepared from WT mice on D0, D1.5, and D2.5 of LCMV infection for STAT1 or STAT4 activation in response to type 1 IFN treatment. (C) Evaluation of ex vivo responsiveness of NK cells to type 1 IFN treatment in association with STAT1 levels on D0 and D2.5 of LCMV infection. Populations for analysis were prepared from WT, STAT2-, and STAT1-deficient mice. (Panels B and C were originally published in The Journal of Experimental Medicine, 2007, 204, 2382–2396. © Miyagi et al., 2007. doi:10.1084/jem.20070401.)

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4. Notes 1. The PE and PerCP fluorochromes are sensitive to methanol and cannot be used to label cell surface determinants prior to this treatment for cell permeabilization. This may require staining of cell determinants with PE- or PerCPconjugated antibodies after methanol treatment, but it is not always possible to do so because antigenic determinants can also be sensitive to fixation and permeablization treatments. Thus, it is necessary to evaluate expression before and after treatments. 2. This protocol can be adapted for leukocytes from murine liver, and also for human mononuclear cells from peripheral blood with appropriate surface markers (data not shown). 3. The basic protocol for preparation of leukocytes from murine spleen is as follows. Sacrifice mice and remove spleen. Place spleens in a 6-well tissue culture plate containing 5 ml of RPMI medium on ice. Grind spleen between the rough surfaces of frosted glass slides. Filter cell suspension through a nylon mesh and transfer into a 15-ml centrifuge tube. Wash the well with additional 5 ml of cold RPMI medium. Centrifuge at 300 g for 10 min at 4◦ C, and discard supernatant. Disturb pellet by tapping the bottom of the tube, add 1 ml of Red Blood Cell Lysing buffer and vortex briefly. Incubate for 1 min at room temperature. Fill the tube to 10 ml with cold RPMI medium. Filter cell suspension again through nylon mesh and transfer into a 15ml centrifuge tube and count cells. Centrifuge at 300 g for 10 min at 4◦ C, discard supernatant, and resuspend cells at 2 × 107 cells/ml in cold RPMI medium. 4. The combination of goat serum and the 2.4G2 antibody, with directed against receptors for immunoglobulins (FcRs), in the Goat Block is required because it is difficult to inhibit non-specific binding of the antibodies for flow cytometry to the FcRs, particularly when the cells are isolated from infected mice. 5. The APC- as well as FITC- and biotin-conjugated but not PE-conjugated anti-CD49b antibodies (eBioscience) were resistant to methanol exposure. Therefore, APCconjugated anti-CD49b antibody can be adapted in this protocol. Using cells from C57BL/6 background mice, the FITC-, APC-, or biotin-conjugated but not PEconjugated anti-NK1.1 antibodies (PK136) were also resistant to methanol exposure. Moreover, a FITC-, APC-,

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or biotin-conjugated but not PE-conjugated anti-TCR␤ (clone H57-597) antibodies were found to be resistant to methanol exposure. 6. To fix cells, we take advantage of the Cytofix/Cytoperm from BD because it contains paraformaldehyde (PFA). We found that 4% PFA provided almost identical results with those by Cytofix/Cytoperm so that 4% PFA can be used to fix cells at this step instead of Cytofix/Cytoperm. As PFA is an evaporating toxin, this step must be performed under a fume hood. 7. The treatment with cold pure methanol for the permeabilization of cells was found to provide the best results for intracellular staining of total STATs and pSTATs. The treatment with Cytofix/Cytoperm alone, which is normally used for intracellular staining of cytokines, did not demonstrate a sufficient level of intracellular total STAT4 or pSTATs. 8. Pure methanol is also an evaporating toxin. Thus, this step must be performed under a fume hood. 9. In the methods, anti-CD3ε staining is done after methanol treatment because the fluorochromes available for the combinations required in individual tests limits the commercially available regent to PerCP-anti-CD3ε, and because the CD3ε determinant is still detectable after methanol treatment. 10. At least 100,000 events should be collected within the leukocytes gated for analysis of intracellular molecules in NK cells because the cells are generally at lower than 5% of the total populations. 11. The treatment of methanol following Cytofix/Cytoperm treatment did not allow detection of intracellular IFN␥ expression. It was possible, however, to analyze both STAT1 and IFN␥ because in contrast to STAT4 or the pSTATs, STAT1 levels could be identified following fixation/permeabilization with Cytofix/Cytoperm and DNase treatment. This treatment was already reported to be useful to identify intracellular STAT1 level (10). 12. The treatment with brefeldin A is required for detecting the intracellular cytokine under these conditions. The best final concentration and time of incubation was found to be 5–10 ␮g/ml and 4–6-h of incubation, respectively, in this system.

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Acknowledgments The authors thank D. Ashley Feldman for review of the manuscript. This work was supported by RO1 grants CA041268 and AI055677 from the National Institutes of Health, a Canadian Institutes of Health Research Fellowship, and funding from the Shinya Foundation. T.M.’s current address is Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan. References 1. Garcia-Sastre, A., and Biron, C.A. (2006) Type 1 interferons and the virus-host relationship: a lesson in detente. Science 312, 879–882. 2. Biron, C.A. and Sen, G.C. (2007) Innate Immune Responses to Viral Infection. In: Fields Virology Fifth Edition. Knipe D.M. and Howley P.M., eds. Walter Kluwer/Lippincott, Williams & Wilkins, pp. 249–278. 3. Stark, G.R., Kerr, I.M., Williams, B.R., Silverman, R.H., and Schreiber, R.D. (1998) How cells respond to interferons. Annu Rev Biochem 67, 227–264. 4. Platanias, L.C. (2005) Mechanisms of type-Iand type-II-interferon-mediated signalling. Nat Rev Immunol 5, 375–386. 5. Bromberg, J.F., Horvath, C.M., Wen, Z., Schreiber, R.D., and Darnell, Jr., J.E. (1996) Transcriptionally active Stat1 is required for the antiproliferative effects of both interferon alpha and interferon gamma. Proc Natl Acad Sci U S A 93, 7673–7678. 6. Nguyen, K.B., Cousens, L.P., Doughty, L.A., Pien, G.C., Durbin, J.E., and Biron, C.A. (2000) Interferon alpha/beta-mediated inhibition and promotion of interferon gamma: STAT1 resolves a paradox. Nat Immunol 1, 70–76. 7. Nguyen, K.B., Watford, W.T., Salomon, R., Hofmann, S.R., Pien, G.C., Morinobu, A., Gadina, M., O Shea, J.J., and Biron, C.A. (2002) Critical role for STAT4 activation by type 1 interferons in the interferongamma response to viral infection. Science 297, 2063–2066. 8. Tanabe, Y., Nishibori, T., Su, L., Arduini, R.M., Baker, D.P., and David, M. (2005) Cutting edge: role of STAT1, STAT3, and STAT5 in IFN-alpha beta responses in T lymphocytes. J Immunol 174, 609–613.

9. Brierley, M.M., and Fish, E.N. (2002) Review: IFN-alpha/beta receptor interactions to biologic outcomes: understanding the circuitry. J Interferon Cytokine Res 22, 835–845. 10. Gil, M.P., Salomon, R., Louten, J., and Biron, C.A. (2006) Modulation of STAT1 protein levels: a mechanism shaping CD8 Tcell responses in vivo. Blood 107, 987–993. 11. Kaplan, M.H., Sun, Y.L., Hoey, T., and Grusby, M.J. 1996. Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice. Nature 382:174–177. 12. Lawless, V.A., Zhang, S., Ozes, O.N., Bruns, H.A., Oldham, I., Hoey, T., Grusby, M.J., and Kaplan, M.H. (2000) Stat4 regulates multiple components of IFN-gammainducing signaling pathways. J Immunol 165, 6803–6808. 13. Herzenberg, L.A., and De Rosa, S.C. (2000) Monoclonal antibodies and the FACS: complementary tools for immunobiology and medicine. Immunol Today 21, 383–390. 14. Miyagi, T., Gil, M.P., Wang, X., Louten, J., Chu, W.M., and Biron, C.A. (2007) High basal STAT4 balanced by STAT1 induction to control type 1 interferon effects in natural killer cells. J Exp Med 204, 2383–2396. 15. Fleisher, T.A., Dorman, S.E., Anderson, J.A., Vail, M., Brown, M.R., and Holland, S.M. (1999) Detection of intracellular phosphorylated STAT-1 by flow cytometry. Clin Immunol 90, 425–430. 16. Uzel, G., Frucht, D.M., Fleisher, T.A., and Holland, S.M. (2001) Detection of intracellular phosphorylated STAT-4 by flow cytometry. Clin Immunol 100, 270–276. 17. Krutzik, P.O., and Nolan, G.P. (2003) Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry A 55, 61–70.

Intracellular Staining for Analysis of the Expression and Phosphorylation 18. Krutzik, P.O., Irish, J.M., Nolan, G.P., and Perez, O.D. (2004) Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications. Clin Immunol 110, 206–221. 19. Krutzik, P.O., Clutter, M.R., and Nolan, G.P. (2005) Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry. J Immunol 175, 2357–2365. 20. Irish, J.M., Hovland, R., Krutzik, P.O., Perez, O.D., Bruserud, O., Gjertsen, B.T., and Nolan, G.P. (2004) Single cell profiling

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of potentiated phospho-protein networks in cancer cells. Cell 118, 217–228. 21. Meraz, M.A., White, J.M., Sheehan, K.C., Bach, E.A., Rodig, S.J., Dighe, A.S., Kaplan, D.H., Riley, J.K., Greenlund, A.C., Campbell, D., Carver-Moore, K., DuBois, R.N., Clark, R., Aguet, M., and Schreiber, R.D. (1996) Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 84, 431–442. 22. Park, C., Li, S., Cha, E., and Schindler, C. (2000) Immune response in Stat2 knockout mice. Immunity 13, 795–804.

Chapter 12 A Model System for Studying NK Cell Receptor Signaling Lukasz K. Chlewicki and Vinay Kumar Abstract Study of NK cell receptor signaling in mouse NK cells has been difficult since there are no clones of murine NK cells. We describe here a model system that overcomes this problem. This system allows the study of many aspects of NK cell receptor function with complete control over the variables that may affect activity such as cis versus trans ligand engagement, homotypic interactions, multiple target types, receptor number, receptor-ligand affinity, and signaling adaptor molecule expression. Although we give examples only for 2B4, Ly49C, and CD48, any NK cell receptors could be studied using these methods. Since many NK cell receptors such as 2B4, CD48, and the Ly49 family can be expressed in T cells, this model system allows the study of not only NK cells but also T cells with NK cell receptors. A standardized system for determining the regulation of NK cell receptor signaling can be important for understanding the anti-tumor activities of NK cells. Key words: Natural killer (NK) cells, T-cell receptor (TCR), target cell, interleukin-2 (IL-2), signaling, activation, inhibition, cross-linking, cis, trans, homotypic.

1. Introduction Natural Killer (NK) cells are lymphocytes which can kill target cells that have become infected with viruses or those that are tumorigenic (1). Lysis operates in a manner similar to cytotoxic T cells (CD8+), where cytolytic molecules from granules including various serine proteases play the predominant role. NK cell surface receptors have been known to share signaling molecules with T cells (reviewed in (2)). Unlike humans, there are no clones of mouse NK cells; hence the mechanism of signaling in NK cells at the clonal level is not completely understood. Activating receptors can recruit small adaptor molecules, such as DAP12, that K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 12, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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contain immunoreceptor tyrosine-based activation motifs (ITAMs) (3−9). NK cell ITAMs are then phosphorylated by Lck or Fyn, which initiates T-cell-like signaling pathways (10–15). Inhibitory receptors can express immunoreceptor tyrosine-based inhibition motifs (ITIMs) that can recruit negative regulators of NK cell function such as SHP-1, SHP-2, and SHIP (15–17). The 58−/− cell line is a derivative of the DO-11.10.7 mouse T-cell hybridoma. It does not express TCR-␣ and -␤ chains (18) and is negative for CD4, CD8, CD2, 2B4, CD48, and NKG2D, but it expresses CD3 and is H-2K positive (data not shown). Many different NK cell receptors such as 2B4 and the Ly49 family can be expressed on T cells (19, 20), and since 58−/− cells seem to be devoid of many different NK cell receptors, they can be used as a tool to investigate NK cell receptor signaling. The mechanisms involving TCR signaling have been studied extensively; therefore the approach we have taken is to transfect TCR into 58−/− cells along with various NK cell receptors. This allows the investigator to evaluate the function of NK cell receptors when the activating or triggering receptor is TCR. Activation using the TCR, as opposed to some other NK triggering receptor, allows for precise control over stimulation with either cross-linking antibodies, peptide MHC expressed on target cells, or immobilized soluble monomers (21). Target cells can consist of other transfected 58−/− cells (that normally express Kk and Dk , unable to stimulate the 2C TCR) or EL4 cells (Kb with loaded 2C agonist SIYRYGGL peptide, which can stimulate the 2C TCR). Activation through the 2C TCR in this model system results in the secretion of IL-2, which we have shown can be modulated by the stimulation of co-transfected NK cell receptors such as 2B4, CD48, and Ly49C (21–23). Here we outline the generation of fully functioning NK cell receptor expressing 58−/− effector cells and provide several methods for testing their function. 1.1. Generation of Stable Cell Lines in 58−/− and EL4 by Transfections

In order to study the receptor of choice, stable cell lines should be generated in 58−/− cells since this ensures more consistent expression of receptors when compared to transient transfections. We outline the generation of stable cell lines in both 58−/− and EL4 by transfections using cationic lipid reagents.

1.2. Testing Receptor Function by Cross-Linking and Target Cells

Transfected 58−/− cell lines should be screened for the ability to secrete IL-2 by stimulating the triggering receptor, in this case TCR, using either a cross-linking antibody or target cells expressing the TCR ligand. Use of the immobilized ligands allows precise control of local concentration of ligands, which is otherwise difficult to control in purely cell-based systems. Anti-CD3 and anti-CD28 cross-linking have been used extensively in TCR signaling (numerous published reports). Anti-V␤8 (anti-TCR) cross-linking is used in this method instead of anti-CD3 since the

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anti-TCR antibody is more specific for the transfected triggering receptor. A protocol outlining the use of peptide-loaded target cells is also described. 1.3. IL-2 Cytokine ELISA

In addition to killing infected cells, T cells have the ability to secrete the cytokine IL-2. Changes in levels of IL-2 secretion can be monitored by a standard sandwich ELISA to determine the function of the NK cell receptor of interest under various conditions. We expect inhibitory receptors to decrease IL-2 secretion and activating or co-receptors to increase IL-2 secretion.

1.4. Cis Versus Trans Stimulation

An NK cell can encounter multiple cells simultaneously, yet can differentiate these encounters to perform the appropriate functions to each target. The methods presented in this section allow an investigator to test several possible scenarios that an effector cell may encounter in vivo. Under cis conditions, the triggering receptor and the receptor of interest are ligated near each other spatially, mimicking the situation when ligands for both receptors are expressed closely on the same target cell or surface. A subset of cis stimulation, referred to as shared-cis, occurs when the ligand for both triggering receptor (TCR) and protein to be tested is the same molecule, such as class I MHC. Under trans-conditions, the ligand for the TCR or triggering receptor is presented on one surface and the ligand for the receptor to be studied is presented on a different surface. This scenario more closely resembles an encounter that an NK cell may have two target cells, each bearing different ligands.

1.5. Homotypic Interactions

NK cell receptors of the SLAM family, such as SLAM (CD150) (24), engage themselves in a homotypic manner on target cells (25, 26). By using a soluble antibody co-incubation step, homotypic interactions can be identified.

1.6. Intracellular Signaling Experiments

Given the robust growth and ease of transduction of 58−/− , various intracellular signaling studies can be performed. We outline a method using a retroviral transduction system based on the PlatE packaging cell line (27), which both guarantees a higher expression than standard stable cell lines (over-expression) and enables a faster throughput when screening many different signaling adaptor molecules. Over-expression is confirmed using RT-PCR.

2. Materials 2.1. Generation of Stable Cell Lines in 58−/− and EL4 by Transfections

1. Effector cell line: 58−/− (provided by Dr. David Kranz, University of Illinois) or other cell line capable of IL-2 cytokine secretion. 2. Target cell lines: EL4 (ATCC # TIB-39), RMA/S (available from Dr. Vinay Kumar, University of Chicago), or other appropriate target cell line.

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3. RPMI growth medium: RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS, Sigma), 5 mL non-essential amino acids solution (NEAA, HyClone, Logan, UT), 5 mL sodium pyruvate solution (HyClone), 5 mL Penicillin–Streptomycin solution (HyClone), 5 mL 1 M HEPES (HyClone). 4. Plasmid-containing hEF1␣ promoter (see Note 1), pEF6, or pBUDCE4.1 (Invitrogen, Carlsbad, CA). 5. Optimem-I serum-free medium (Invitrogen). 6. Cationic lipid reagent such as Lipofectamine 2000 (Invitrogen) for 58−/− and EL4 cells. 7. Blasticidin (Invivogen, San Diego, CA), ready to use liquid (10 mg/mL), final working concentration is 5–10 ␮g/mL (1:2000–1:1000). Aliquot and freeze at −20◦ C, do not freeze thaw, and do not store in a frost-free freezer. An enzyme box can be used to store in standard freezer. Shortterm storage at 4◦ C. 8. Puromycin (Invivogen), ready to use liquid (10 mg/mL), final working concentration is 1 ␮g/mL (1:10,000). Longterm storage −20◦ C, short-term storage 4◦ C. 9. G418 (Gemini Biosciences, Woodland, CA), reconstitute to final potency of 50 mg/mL in 1X Dulbecco’s phosphate buffered saline (D-PBS) and filter sterilize (0.22 ␮M). Typical G418 powders are usually about 0.725–0.75 mg potency per mg powder. Final working concentration of active product should be 500 ␮g/mL to 1 mg/mL. This needs to be optimized for each cell line. Store at 4◦ C. 10. Freeze Medium: 45% final FBS, 45% final culture medium for cell line, 10% final DMSO; store at 4◦ C. 2.2. Testing Receptor Function by Cross-Linking and Target Cells

1. Stable 58−/− transfectants, EL4, or RMA/S targets. 2. SIYRYGGL peptide (custom synthesis). 3. RPMI growth medium (see above). 4. Coat Buffer (1X): 26.807 g Na2 HPO4 -7H2 O in 1 L of water, pH 9.0 (No NaCl). 5. PBS (10X): 2.57 g NaH2 PO4 -H2 O, 22.49 g Na2 HPO4 7H2 O, and 87.65 g NaCl per liter of water, pH 7.4. 6. Blocking Buffer: 1X PBS with 1% BSA (10 mg/mL), filter sterilize and store in aliquots at −20◦ C. 7. Wash Buffer: 1X PBS with 0.05% Tween-20, filter sterilize half for coating and store at 4◦ C, the rest is used as an ELISA wash buffer and does not need sterilization (see Note 2)

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8. EIA/RIA-certified high binding 96-well assay plates, sterile with lid (#07-200-721, Corning), these plates work best for coating, since each well is raised and separated from the next by gaps, which greatly reduces cross-contamination between wells. 9. Purified anti-V␤8 cross-linking antibody clone F23.1 (BD Pharmingen, San Jose, CA) or other appropriate purified cross-linking antibody such as 2C11, the anti-CD3 antibody. 10. Mouse IgG control antibody (Jackson Immunoresearch, West Grove, PA). 11. 5 mL polypropylene tubes (BD Falcon). 2.3. IL-2 Cytokine ELISA

1. Coat, wash, and blocking buffers from Section 2.2. 2. EIA/RIA-certified 96-well assay plates non-sterile, no lid (#07-200-39, Corning). 3. Streptavidin-HRP (Jackson Immunoresearch). 4. 1X TMB substrate solution (eBiosciences, San Diego, CA). 5. Stop solution: 3 M Sulfuric acid. 6. Plate reader that reads an absorbance at or near 450 nm. 7. IL-2 matched pair ELISA antibodies: Purified anti-mouse IL-2 clone JES6-1A12 and biotin-conjugated anti-mouse IL-2 clone JES6-5H4 (eBiosciences).

2.4. Cis Versus Trans Stimulation

1. TCR+ 2B4+ , TCR+ Ly49C+ 58−/− effector cell lines. CD48− EL4, CD48+ EL4 target cell lines. 2. RPMI growth medium from Section 2.1. 3. 5 mL polypropylene tubes (BD Falcon). 4. Coat, wash, and blocking buffers from Section 2.2. 5. EIA/RIA-certified high binding 96-well plates with lids (#07-200-721, Corning). 6. Purified anti-V␤8 cross-linking antibody, clone F23.1 (BD Pharmingen), mouse anti-2B4 antibody clone 2B4 (BD Pharmingen). Anti-Ly49C blocking antibody clone 5E6 (BD Pharmingen). 7. Purified class I MHC monomers SIYR/Kb , OVA/Kb (custom synthesis). 8. SIYR (SIYRYGGL) and OVA (SIINFEKL) peptides (custom synthesis).

2.5. Homotypic Interactions

1. TCR+ CD48+ 58−/− effector cell lines, CD48− EL4, CD48+ EL4, 2B4+ EL4 target cell lines. 2. RPMI growth medium from Section 2.1.

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3. 5 mL polypropylene tubes (# 14-959-11A, BD Falcon). 4. Coat, wash, and blocking buffers from Section 2.2. 5. EIA/RIA-certified high binding 96-well plates (Corning). 6. Purified anti-V␤8 cross-linking antibody, clone F23.1 (BD Pharmingen), mouse anti-CD48 antibody clone HM48-1 (BD Pharmingen). 2.6. Intracellular Signaling Experiments

1. Retroviral transduction: pMIG, pMIGR1 (addgene plasmid# 9044 or available from Dr. Vinay Kumar, University of Chicago) or other retroviral expression plasmid, packaging cell line Plat-E, Polybrene (stock 4 mg/mL, Sigma), 1 M HEPES, cDNA of signaling molecules to be tested. 2. Dulbecco’s modified eagle medium (DMEM, Sigma) supplemented in the same manner as complete RPMI-1640 medium (Section 2.1) with the addition of 50 ␮M 2mercaptoethanol (final concentration). 3. Phosphate buffered saline (PBS). 4. RNA extraction: TRIzol Reagent (Invitrogen), 2-propanol, chloroform, and RNase, DNase-free water. 5. RT-PCR primers for signaling molecules to be tested. 6. First strand cDNA synthesis kit (Fermentas). 7. KOD Hot starts DNA polymerase (EMD Biosciences, Gibbstown, NJ). 8. TAE buffer, Agarose.

3. Methods The 58−/− cell line is quick growing and has been previously grown in RPMI 1640, DMEM, and Advanced-MEM (Invitrogen) media. Optimal transfection results have always been obtained using RPMI 1640 medium. If murine Ly49C function is to be studied, Advanced-MEM medium (Invitrogen) can be used and supplemented with 2% murine serum isolated from B6 mice. Avoid growing 58−/− cells to confluency, since this can adversely affect both cytokine secretion and transfection/transduction efficiencies. 58−/− usually grows in suspension, but can adhere lightly to cell culture plates; it should be passaged between 1:10 and 1:50. As with all receptor work, trypsin−EDTA should never be used to dislodge adherent cells. EL4 target cells grow quicker and to higher densities (2–4 million cells/mL) than 58−/− and should be maintained in complete RPMI 1640 medium. EL4 can be passaged very thin

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and tolerate overcrowding better than 58−/− . As with all transfected cell lines, care should be taken to freeze back as many early passages as possible and not culture cells longer than 6–8 weeks before thawing an earlier stock. The use of this model system depends on the transfected receptor of interest to modulate TCRmediated IL-2 secretion, so check the parental cell line for IL-2 secretion before commencing transfections. 3.1. Generation of Stable Cell Lines in 58−/− and EL4 by Transfection

A stable variant of 58−/− expressing the 2C T-cell receptor was previously generated (21) and requires 1 mg/mL final concentration of the G418 antibiotic for selection. However, any TCR or known triggering receptor capable of activating the T-cell receptor signaling machinery can be used (see Note 3). The following protocol can also be used to transfect proteins into EL4. EL4 requires double the concentration of Blasticidin to kill nonexpressing cells and has higher stable transfection efficiency than 58−/− (personal observation). 1. Check 58−/− cells for expression of receptors of interest by flow cytometry. 2. On the day of transfection, plate 2 million 58−/− cells per transfection in 2 mL of fresh RPMI (complete) medium in each well of a 6-well plate or a 35 mm dish (see Note 4). 3. Following manufacturer’s suggestions, mix 4 ␮g plasmid DNA (see Note 5) with 250 ␮L Optimem-I in a sterile microfuge tube. Mix 10 ␮L of Lipofectamine 2000 with 250 ␮L Optimem-I in a separate tube. Let it stand for 5 min at R.T. 4. Combine contents of two tubes, mix gently, and incubate 20 min at R.T. 5. Add mixture drop wise on top of cells. 6. Gently rock plate back and forth several times to spread transfection medium. 7. Incubate plate/s in cell culture incubator at 37◦ C and 5% CO2 overnight. 8. The following day, expand each well (from 6-well plate) into a new 100 mm plate using 10 mL fresh complete RPMI medium, and incubate overnight. 9. 45–48 h post-transfection, expand the 12.5–100 mL with complete RPMI medium and add selective antibiotics for final 1 mg/mL G418 (for TCR expression) and 5–10 ␮g/mL Blasticidin (for gene to be tested, see Note 6). Aliquot 1 mL into each well of four 24-well plates. Incubate in cell culture incubator until colonies are visible (usually 6–10 days, Note 7).

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10. Circle and pick colonies (100 ␮L setting on pipette) when densely packed colonies (1–2 mm in size) appear and transfer to wells of a new 24-well plate in 1 mL selection medium. 11. Analyze for expression either by flow cytometry or by western blot after 24–48 h. 12. Expand positive colonies to 6-well plates and test for receptor signaling by receptor cross-linking or with target cells (see Note 8). 13. Clones testing positive for both surface expression and cytokine secretion should be expanded and several aliquots should be frozen in freeze medium and stored at −80◦ C for several days, followed by transfer to liquid nitrogen (see Note 9). 3.2. Testing Receptor Function Using a Cross-Linking Antibody or Target Cells

3.2.1. Receptor Cross-Linking

The following protocol assumes that the murine 2C TCR is used as the triggering receptor for IL-2 secretion. For 2C, antiV␤8.1/2 (F23.1) and anti-V␤8.2 (F23.2) provide a robust signal, comparable to the anti-CD3 antibody 2C11 and are more specific for the transfected TCR (Fig. 12.1A). As with all quantitative experiments, full titrations should be performed with each batch of purified antibody/reagents to determine concentrations required to achieve a 1/2 maximal IL-2 secretion (see Note 10). 1. Dilute the anti-V␤8 (or anti-CD3) antibody to a final concentration of 0.1–0.5 ␮g/mL (determined previously to be the 1/2 maximal concentration) in coat buffer (see Note 11). 2. Aliquot 50 ␮L per well, incubate at least 2 h or preferably overnight at 4◦ C. 3. Block using 100 ␮L sterile PBS-1% BSA, at least 30 min, but not more than 2 h. 4. During blocking step, count cells. 5 × 104 cells are needed per well for optimal IL-2 secretion. A higher number of cells will decrease the availability of ligand in a cross-linking experiment. However, low cell numbers may not yield sufficient IL-2 for detection by ELISA (personal observation). 5. Aliquot appropriate amount of cells, spin down cells, and decant supernatant. 6. Resuspend cell pellet in RPMI growth medium (no selection antibiotics) to 500,000 cells/mL density, which yields 5 × 104 cells for every 100 ␮L per well, and set aside. Small 5 mL polypropylene tubes are ideal when working with multiple cell lines.

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7. Dump out coat and block solution and smack dry on paper towels. 8. Wash plate with sterile wash buffer and smack dry. 9. Add 100 ␮L complete RPMI medium (no selection antibiotics) to each well of plate, use 50 ␮L instead for each well if using a blocking antibody. 10. If using a blocking antibody, add 50 ␮L of a blocking antibody solution (at 40 ␮g/mL, 4× strength) to each well. To wells not receiving blocking antibody, add isotype control IgG antibody. Omit control IgG if no blocking antibody is being used (see Note 12). 11. Aliquot 100 ␮L of each cell suspension per well, for a final volume of 200 ␮L. 12. Incubate plate at 37◦ C and 5% CO2 in cell culture incubator for 26–30 h. 13. Harvest 50 ␮L supernatants (see Note 13) and place directly on ELISA plates pre-coated with capture antibody, and follow protocol in Section 3.3. 14. Plate can be frozen and stored at −20◦ C until ready to assay. 3.2.2. Using Target Cells

Since the triggering receptor is a T-cell receptor, target cells must first be loaded with the appropriate concentration of peptide, which must be determined using serial dilution titrations (Fig. 12.1B). Experiments should always be performed in triplicates. 1. Count target cells and aliquot out the appropriate number. We will initially use a 1:1 effector:target ratio for 58−/− to EL4, therefore 5 × 104 targets per well will be required.

Fig. 12.1. TCR+ 58−/− transfectants secrete IL-2 (measured as Absorbance at 450 nm) (A) TCR+ 58−/− effector cells were stimulated by plate-bound anti-TCR antibodies. Anti-CD3 (2C11), anti-V␤8 (F23.1), and anti-V␤8.2 (F23.2) antibodies were immobilized on plates at various concentrations and TCR+ effectors were added and monitored for IL-2 secretion. (B) TCR+ 58−/− effector cells were stimulated by EL4 target cells loaded with SIYRYGGL peptide titrated at various concentrations and assayed for IL-2 secretion.

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2. The final assay plate volume will be 200 ␮L per well as with cross-linking. Resuspend target cells in 50 ␮L RPMI growth medium per 5 × 104 cells. This leaves room for peptide, blocking antibodies, and target cells for the remainder 150 ␮L/well. 3. Load EL4 target cells with peptide: Dilute SIYR peptide to 4 × 10−5 M (40 ␮M, this is 4× strength since the sample will be diluted fourfold final in the well) in a fresh polypropylene tube and serially dilute to desired concentrations in RPMI growth medium (see Note 11). 4. Add 50 ␮L of the 4× peptide solution to each well. 5. Add 5 × 104 target cells per well (50 ␮L). 6. If using blocking antibody, add 50 ␮L of a blocking antibody solution (at 40 ␮g/mL, 4×, diluted in RPMI growth medium) to each well. To wells not receiving blocking antibody, add the same quantity of isotype control IgG antibody. Omit control IgG if no blocking antibody is being used. 7. Incubate plate at 37◦ C and 5% CO2 in cell culture incubator for 1 h. 8. Take plate out from incubator and add 5 × 104 effector cells diluted in 50 ␮L of RPMI growth medium per well. 9. Incubate plate at 37◦ C and 5% CO2 in cell culture incubator for 26–30 h. 10. Harvest supernatants and perform ELISA as with crosslinking experiment. 3.3. IL-2 Cytokine ELISA

To detect the secretion of IL-2 by the effector cell lines, perform a standard IL-2 sandwich ELISA. Since the absorbance scale is not linear, a twofold serial dilution of soluble murine IL-2 should be performed on the ELISA plate to quantify. 1. Dilute the IL-2 capture antibody to 2 ␮g/mL final (1:250 of commercial stock) in coat buffer and add 50 ␮L to each well of a 96-well ELISA/EIA/RIA plate. Incubate at RT for 2 h or overnight at 4◦ C (see Note 14). 2. Add 100 ␮l Blocking buffer to each well (see Note 15). Incubate at RT for 1 h. 3. Dump out coat and block solution and smack dry on paper towels. 4. Wash plate with wash buffer and smack dry. 5. Add 50 ␮L of supernatants from experiment to each well. Incubate 2–4 h at RT or overnight at 4◦ C. 6. Remove supernatants from all the wells and smack dry on paper towels. Be careful not to cross-contaminate between wells.

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7. Add 150–200 ␮l wash buffer to each well and then remove as previously. Wash three times. 8. Dilute biotinylated IL-2 sandwich antibody 1:250 in blocking buffer and add 50 ␮l per well. Incubate for 1 h at RT 9. Remove secondary antibody and wash three times. 10. Dilute Streptavidin-HRP 1:1000 and add 50 ␮l per well. Incubate 45 min to 1 h at RT. 11. Remove Streptavidin-HRP and wash four times. 12. Add 50 ␮l 1X TMB substrate solution to each well. Gently tap plate to mix. 13. Put plate on white sheet of paper and wait for blue color to develop (length of time will vary from 10 s−10 min depending on samples, shorter for cross-link, longer for targets). 14. Stop reaction using 50 ␮l of stop solution per well. Gently tap plate to mix. Color will then turn yellow instantly. 15. Read plate on plate reader at 450 nm and analyze data. 16. Since not all cell lines secrete the same level of IL-2, data should be normalized to percent change of IL-2 secretion (of control) when comparing different cell lines. When a single cell line or multiple cell lines with similar IL-2 secretion are used, it is fine to express the data as just raw IL-2 secretion or absorbance at 450 nm. 3.4. Cis Versus Trans Stimulation 3.4.1. Cis Stimulation

Cis stimulation can be replicated experimentally by using target cells bearing ligands for TCR and NK cell receptors or by immobilizing the ligands, either stimulating antibodies or soluble proteins, on the same surface of a plate as indicated in Fig. 12.2A. As an example, we generated 58−/− effector cell lines expressing both 2B4 and 2C TCR and co-cultured the cells with EL4 target cells expressing SIYR/Kb and CD48, which are ligands for the TCR and 2B4, respectively (28–30). This example shows that 2B4 can inhibit TCR-mediated IL-2 secretion in cis with target cells (Fig. 12.2B). The use of a target cell line negative for CD48 and a blocking antibody shows that these effects are specific and are recommended controls. Since immobilization of ligands allows more precise control over ligand concentration compared to using target cells, it is preferred when quantification is important (see Note 16). An example using an anti-TCR antibody along with MHC monomers (for Ly49C+ , TCR+ effectors) is given in Fig. 12.2C. In this example, stimulation of Ly49C, by OVA/Kb monomers leads to a decrease in TCR-mediated IL-2 secretion using the anti-V␤8 antibody F23.1. The following method is similar to method 3.2.1 with an added ligand for the receptor to be tested.

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Fig. 12.2. Cis stimulation. (A) Cis presentation of two ligands using either a target cell or by immobilization on the surface of a plate. (B) TCR+ 2B4+ 58−/− or control TCR+ 58−/− cells were stimulated by CD48− or CD48+ EL4 target cells loaded with SIYRYGGL peptide in the presence or absence of blocking antibody and assayed for changes in IL-2 secretion. IL-2 secretion is measured as a percentage change of control of IL-2 secreted by CD48− EL4 targets. (C) TCR+ Ly49C+ 58−/− or control TCR+ 58−/− cells were stimulated by 0.3 ␮g/mL anti-TCR and increasing concentrations of OVA/Kb monomers immobilized on the surface of plates and assayed for changes in IL-2 secretion. (D) In sharedcis stimulation, TCR+ Ly49C+ 58−/− or control TCR+ 58−/− cells were stimulated by SIYRYGGL peptide-loaded EL4 cells in the presence or absence of 5E6 blocking antibody and assayed for IL-2 secretion.

1. Dilute the anti-V␤8 antibody to final concentration determined previously to yield a 1/2 maximal IL-2 secretion (usually 0.1–0.5 ␮g/mL) in coat buffer; we will use 0.5 as our example. Be sure to make enough to cover all the wells that will be used to titrate the antibody/protein specific for the receptor that is being studied. Since an ELISA plate has 8 × 12 rows, decide on 7 or 11 different concentrations of the other antibody or protein to use, leave 1 row aside to use as a control having only triggering receptor (anti-TCR) antibody and IgG control. Check manufacturer’s specifications for the total amount of IgG/well that can bind to your plates. If you exceed this number, triggering receptor antibody will compete for binding sites in the wells, altering your results (see Note 17). 2. Using coat buffer containing anti-TCR antibody at a fixed concentration, perform serial dilutions of antibody or protein to test (see Note 18). For this example, we use 4 ␮g/mL as our high point and perform twofold serial dilutions. Leave the last tube only with anti-TCR antibody.

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3. Make up the difference to ∼4.5 ␮g/mL total (anti-TCR plus antibody/protein to test) using isotype IgG control antibody so that anti-TCR antibody in each well has an equal chance of adhering to bottom of well. 4. Add 50 ␮L solution to each well, let incubate at least 2 h RT or overnight at 4◦ C. 5. Follow from step 7 of standard cross-linking method 3.2.1. The 2C TCR and Ly49C bind at different sites of SIYR/Kb (31, 32), thus the same ligand could potentially be presented to both Ly49C and the TCR simultaneously. In our example, Ly49C (on TCR+ , Ly49C+ effectors) can inhibit TCR-mediated IL-2 secretion when engaging SIYR/Kb loaded EL4 targets (Fig. 12.2D). This method of presentation uses the target cell protocol in Section 3.2.2. As with all the methods outlined here, it is recommended to use blocking antibodies to confirm the specificity of the interaction. 3.4.2. Trans Stimulation

Although it is possible to use two target cells simultaneously (Fig. 12.3A), we feel that the difficulty in controlling the growth rate of three cell types could present additional variables

Fig. 12.3. Trans stimulation. (A) Trans presentation of two ligands using either two target cells or by expressing one ligand on a target cell and the other immobilized on the surface of a plate. (B) TCR+ 2B4+ 58−/− or control TCR+ 58−/− cells were stimulated by immobilized anti-TCR cross-linking antibody and CD48+ or CD48− EL4 target cells (no peptide loaded) in the presence or absence of blocking antibodies and assayed for changes in IL-2 secretion. (C) TCR+ Ly49C+ 58−/− or control TCR+ 58−/− cells were stimulated by immobilized anti-TCR cross-linking antibody and OVA peptideloaded EL4 cells and assayed for changes in IL-2 secretion. Control IL-2 secretion was about 3000 pg/mL or an A450 of about 1.2 for all the samples tested.

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making analysis more difficult. Removing serum to arrest the growth phase also inhibits the secretion of IL-2 (personal observation). To get around this problem the stimulating anti-TCR antibody can be immobilized on plates, while the ligand for either NK cell receptor 2B4 or Ly49C can be expressed on target cells, therefore spatially separating the two ligands. This method combines the protocols of both the cross-linking and the target cell stimulation methods from Sections 3.2.1 and 3.2.2. An example of this method for both 2B4 and Ly49C expressing transfectants is given in Fig. 12.3B and C. In both of these scenarios, no inhibitory signal is generated, even at high concentrations of ligand (see Note 19). 3.5. Homotypic Interactions

To study homotypic interactions, stable cell lines expressing TCR along with the receptor of interest are cultured in the presence of cross-linking anti-TCR antibody and blocking antibodies for this other receptor. This assures that interactions occur only amongst neighboring effector cells. When TCR+ effector cell lines were generated with stable CD48 expression, blocking antibody alone (devoid of any targets) yielded a decrease in IL-2 secretion. Since 58−/− does not normally express ligands for CD48, this suggests homotypic interactions from neighboring effector cells are responsible for this effect (Fig. 12.4A). To confirm this effect and analyze how this homotypic interaction compares to the known heterotypic interaction of 2B4−CD48, we generated separate EL4 cell lines expressing high levels of either CD48 or 2B4 and cultured them with the TCR+ CD48+ effectors. Under these conditions the CD48−CD48 homotypic interaction is not as strong as the CD48−2B4 heterotypic interaction (using 2B4+ EL4 targets) (Fig. 12.4B).

Fig. 12.4. Homotypic interactions. (A) TCR+ CD48+ 58−/− or control TCR+ 2B4+ 58−/− cells were stimulated by immobilized anti-TCR and anti-IgG control antibodies in the presence or absence of blocking antibodies and assayed for changes in IL-2 secretion. (B) TCR+ CD48+ 58−/− cells were cultured with CD48+ EL4, 2B4+ EL4, or control EL4 cells (all loaded with SIYR peptide) in the presence or absence of blocking antibodies and assayed for changes in IL-2 secretion. IL-2 secretion was normalized to treatment with control EL4 targets.

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Transient transfections of 58−/− are not recommended for overexpression studies since 58−/− divides rapidly and does not transiently express constructs well (personal observation). The common pMIG/MIGR1 retroviral transduction system using the PlatE packaging cell line produces excellent over-expression results, with typical transduction efficiencies from 45 to 95% and at higher levels than transfection. These can easily be sorted for GFP+ populations by FACS. 1. Maintain PlatE cells in complete DMEM supplemented with 50 ␮M 2-mercaptoethanol, 1 ␮g/mL Puromycin and 10 ␮g/mL Blasticidin. 2. 24 h before transfection, plate ∼2–3 million cells per 100 mm dish in complete DMEM medium, incubate overnight at 37ºC and 5% CO2 . 3. Remove medium and add 3 mL fresh complete DMEM medium without selection antibiotics. 4. In each microfuge tube, combine 5 ␮g DNA in 300 ␮L 150 mM NaCl and mix. 5. Add 16.45 ␮L ExGen 500, vortex for 10 s, and incubate for 10 min at RT. 6. Add mixture on top of cells drop wise, rock back and forth to spread out. 7. Incubate overnight at 37ºC and 5% CO2 . 8. Remove supernatant and add 3 mL of fresh complete DMEM medium and incubate overnight at 37ºC and 5% CO2 . 9. Harvest supernatant in the morning (now 41–46 h posttransfection), add 3 mL fresh medium, and incubate until the end of the day. 10. Harvest supernatant, add fresh medium, and incubate overnight at 37ºC and 5% CO2 . 11. Harvest supernatant in the morning and discard the cells. 12. Filter supernatant with a 0.45 ␮M syringe filter and freeze at −80◦ C (long term) or store at 4◦ C for no longer than several days if to be used soon. 13. Prepare 58−/− cells: Keep cells in log growth phase before cells are overcrowded and before medium turns yellow. Otherwise passage cells again. 14. Plate 1 million cells/mL in each well of a 24-well plate (see Note 20). 15. Add 2 ␮L of Polybrene (stock at 4 mg/mL) and 20 ␮L of 1 M HEPES. 16. Add 1 volume of viral supernatant (1:1 mL cells) and mix.

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17. Centrifuge at 30ºC at 1300 g for 90 min. 18. Loosen cells by pipetting. 19. Incubate overnight at 37ºC and 5% CO2 . 20. Replace medium with fresh medium and expand culture to 6-well plates. 21. Check for GFP expression at 48 h post-transduction and sort if necessary. 22. Perform experiments as outlined earlier. 3.6.2. RT-PCR for Expression

After transduction, the expression of the transduced gene should be confirmed either by western blot for protein expression or by RT-PCR for transcript level and compared to the parental cell line with a mock transduction of GFP-containing vector control. 1. Pellet 1 × 107 cells by centrifugation, aspirate medium, wash once with 1 × PBS. 2. Add 1 mL TRIzol for every 1 × 107 cells, pipette up and down to shear precipitate and obtain smooth, wellsuspended solution. 3. Incubate 5 min at RT or freeze cells at −80◦ C for up to 1 month. 4. Add 0.2 mL chloroform per 1 mL TRIZOL. 5. Shake tubes vigorously by hand for 15 s and incubate at RT for 2–3 min. 6. Centrifuge samples at 12,000 g for 15 min at 4◦ C. 7. Recover upper phase into a new tube. 8. Add 0.5 mL 2-propanol per 1 mL TRIZOL (to upper phase in new tube) and incubate for 10 min at RT. 9. Centrifuge at 12,000 g for 10 min at 4◦ C. 10. Remove supernatant (aspirate). 11. Wash with 1 mL 75% ethanol. 12. Centrifuge at no more than 7500 g for 5 min at 4◦ C. 13. Aspirate very carefully. 14. Air dry pellet for 5–15 min. 15. Dissolve RNA in 20–60 ␮L DEPC-water or RNase-, DNase-free water. 16. Measure absorbance at 260 nm. 17. Perform reverse transcription according to Fermentas kit instructions. Usually we use about 2 ␮g of total RNA for each reaction. 2 ␮L of this will subsequently be used for the PCR (see Note 21).

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Fig. 12.5. Cytoplasmic signaling studies. TCR+ 2B4+ 58−/− cells were stimulated by immobilized anti-TCR cross-linking antibody and increasing concentrations of anti-2B4 cross-linking antibody and assayed for changes in IL-2 secretion. Clones were transduced to over-express EAT-2A (EAT-2A ex) or the control GFP plasmid. Equal quantities of cDNA were amplified to estimate relative level of EAT-2A transcript, GAPDH was amplified as a control.

18. Determine conditions for PCR and perform according to the protocol provided with the KOD polymerase, trying to optimize the annealing temperature that is best for the primer pair. The user should optimize the annealing temperature and primers to achieve one solid band, also the number of cycles may need to be adjusted to 30 or fewer so, as not to reach saturation (see Note 22). 19. Make serial dilutions of PCR product and resolve on TAEAgarose gel. After expression of the adaptor molecule/construct is confirmed, proceed with experiments as outlined earlier. In our example we over-expressed the SAP-like adaptor molecule EAT-2A and showed that it helped increase TCR-mediated IL-2 secretion by receptor cross-linking (Fig. 12.5). This effect is similar, but weaker in magnitude than what we reported previously using the adaptor molecule SAP, which is very similar to EAT-2A (33).

4. Notes 4.1. Generation of Stable Cell Lines in 58−/− and EL4 by Transfections

1. A number of different promoters have been used in 58−/− cells. However, the hEF1␣ promoter offers the most reliability. Expression profiles with this promoter can range several orders of magnitude as measured by flow cytometry. CMV-based promoters from non-retroviral vectors are not reliable in this cell line (personal observation). Always include a Kozak initiation sequence before your ATG to assure expression. Epitope tags such as myc, 6×-His, HA, etc., are not recommended initially for receptors, since they may interfere with ligand binding. Unless you are certain, do not use them. A myc-tag on Ly49C does not interfere

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with MHC binding on target cells (personal observation). Caution should be used on cytoplasmic proteins if the tag is placed near any functional domains. A myc-tag, however, enables rapid isolation of proteins by immunoprecipitation. 2. 1X PBS with either BSA or with Tween contaminates quickly. Both of these should be kept sterile for use in cell culture coating plates. However, PBST used for ELISA washes does not need to be sterile. DO NOT add Na-Azide to any diluted reagent that will be used for ELISA, as this will completely deactivate the HRP. 3. 2C is chosen since it recognizes EL4 target cells loaded with the superagonist peptide SIYRYGGL. Any TCR capable of recognizing a target cell loaded with a peptide of choice that can be controlled by the user may be used, including activating NK cell receptors. 4. This protocol uses a cationic lipid reagent, but 58−/− can also be transfected using electroporation; however, cells should be cultured in RPMI medium with 1% DMSO for 24 h post-electroporation. Cationic lipid transfections are used in lieu of retroviral transduction so as to leave the cells GFP negative to allow later over-expression of adaptor molecules. One should also use unique antibiotics for each gene or simply inactivate one of the resistance genes with a restriction enzyme if a co-transfection is to be used. For co-transfections with two unique drugs, use 2 ␮g of each DNA for each well of a 6-well format. For co-transfections with an inactivated gene, use 4 ␮g total DNA with a 10:1 ratio of inactivated: active resistance gene. ExGen500 (Fermentas) may also be used with reduced transfection efficiency. 5. Plasmid DNA should have a 280/260 ratio of at least 1.8. It is not necessary to perform a Maxi-prep (Qiagen), since several mini-preps will provide ample, highly purified DNA for multiple transfections. Mini-prep kits from Qiagen, Fermentas, and Eppendorf all yield highly purified DNA suitable for transfections. 6. The exact concentration of selection antibiotics varies greatly with culture media composition and cell density and should first be determined by plating about 100,000 cells per well in a 24-well plate at various concentrations of antibiotics. In addition, 2-mercaptoethanol in the medium (at 50–100 ␮M final) usually requires double the concentration of Blasticidin and is omitted in our formulation. The lowest concentration of antibiotic that kills all cells within 24 h (for Blasticidin and Puromycin) is the amount to use for selection. 7. Avoid picking colonies that appear after 12–14 days, since the effectiveness of Blasticidin diminishes in culture after this

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period. Also avoid letting the medium turn yellow at any time during the course of culture since this may dramatically decrease the ability of the cells to secrete IL-2. 8. As with any cell line for generating stables, multiple lines should be isolated and tested before any conclusions are drawn. Always attempt to use the largest pool possible of stable cell lines to assure accuracy. We usually test between 15 and 30 separate cell lines at various surface expression profiles before proceeding with experiments. 9. Not all cell lines will secrete IL-2, even if the parental line did. If no cell lines secrete IL-2, check for triggering receptor expression and another batch of cross-linking antibody. If still no luck, thaw a new aliquot of parental cell line. Secretion of IL-2 is lost from time to time and certain cell lines may lose and reacquire the ability to secrete IL-2. 4.2. Testing Receptor Function by Cross-Linking and Target Cells

10. A 1/2 maximal concentration of antibody for triggering receptor should be used when one wants to study the effects that treatment has on IL-2 secretion since this will give the highest signal-to-noise ratio in the experiment. Maximal concentrations should not be used since a receptor or adaptor molecule providing a co-activating signal may show no response, likewise too low of a concentration will mask an inhibitory signal. Anti-CD3 should not be used when EL4 targets are employed, since these cells also stain positive for CD3. Antibody purity/activity is critical and should be analyzed for every new batch of antibody. Custom-purified antibodies (in-lab) have consistently performed better than commercial antibodies (two- to fourfold) in these sensitive assays and are preferred (personal observation). 11. Twofold serial dilutions give more points; however, many wells are required. ∼3.16-fold serial dilutions can be used to give two points per log (i.e., 3.162 = ∼10). 12. Blocking antibodies can be added to any of the steps, as long as the blocking antibody is added at least before or with the effector cells. Blocking antibody may be preincubated with effector cells if specific for transfected receptor or with target cells if specific for a target cell ligand. 10 ␮g/mL final blocking concentration is usually sufficient to block. Not all blocking antibodies are effective at blocking their respective receptor completely. For example, the commercially available anti-2B4 antibody does not completely block 2B4−CD48 interactions (34). Higher concentrations may be required or blockade of the ligand may be necessary.

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13. It is not necessary to centrifuge the supernatants since cells will have settled to the well bottom during the experiment; however, it will not hurt to do so or to transfer supernatants to new plates before freezing. 4.3. IL-2 Cytokine ELISA

14. A capture antibody concentration of 0.2–10 ␮g/mL is usually used. Most standard binding ELISA plates will saturate closer to 5 ␮g/mL (check each spec sheet), therefore higher concentrations can actually lower signal since they can bind soluble antigen left over and be washed away during the wash steps. 15. It is not necessary to dump out the coat step, since the concentration of soluble proteins in 1% BSA of 10 mg/mL is much higher than the single well binding capacity. However, the user can choose to dump this out before blocking.

4.4. Cis Versus Trans Stimulation

16. It is very important to only titrate one antibody or protein at a time, otherwise data analysis is very difficult. Usually one would want to use a constant concentration of crosslinking antibody for the triggering receptor and then vary the concentration of the proteins or cross-linking antibodies for the receptor to be tested. One should aim to stimulate the triggering receptor with a concentration that is near the 1/2 maximal required for stimulation, as this concentration allows for a very sharp rise or fall in IL-2 secretion indicative of activation or inhibition, respectively. Always have proper controls for every parameter tested, including a control for spontaneous IL-2 release. 17. Each well must contain the same amount of total IgG or protein, otherwise differences may appear in the wells simply arising from competition for binding spots. If the manufacturer states that only 4.5 ␮g/mL can bind per well, each well should contain 4.5 ␮g/mL total IgG maximum, this includes both antibody for triggering receptor (TCR) and the antibody or protein that will be tested along with control IgG to make up the difference. 18. Antibodies should be at the highest purity and activity possible since these experiments use low concentrations to see dramatic effects, batch differences have been detected before in antibodies of up to —three- to fourfold. To achieve the highest possible replication of results, always test each batch of antibody. If growing hybridoma supernatants yourself, be sure to use serum that has been stripped of any possible bovine IgG. 19. Trans stimulation can also be achieved by immobilizing an antibody/ligand for the receptor of interest and target cells for TCR.

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20. A higher density of cells helps increase retroviral transduction. Transductions should be performed when cells are still actively dividing. 21. It is far cheaper to purchase the kit components separately; however, we list it here to make it easier for novices. 22. One should aim to have as clean a PCR product as possible, with one intense band on the gel. By using 30 or fewer cycles, the largest difference between samples can be obtained, which helps data analysis. In samples with around 35 cycles, the difference between high and low expressers is not as dramatic, since PCR reactions have plateaued.

Acknowledgments We would like to thank David M Kranz for providing the 58−/− hybridoma and the F23.2 antibody, Cox Terhorst for providing the EAT-2 cDNA, and Hans Schreiber for providing SIYRYGGL peptide. References 1. Trinchieri, G. (1989) Biology of natural killer cells. Adv Immunol 47, 187–376. 2. Lanier, L. L. (2003) Natural killer cell receptor signaling. Curr Opin Immunol 15, 308–314. 3. Lanier, L. L., Corliss, B. C., Wu, J., Leong, C., and Phillips, J. H. (1998) Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 391, 703–707. 4. Smith, K. M., Wu, J., Bakker, A. B., Phillips, J. H., and Lanier, L. L. (1998) Ly-49D and Ly-49H associate with mouse DAP12 and form activating receptors. J Immunol 161, 7–10. 5. Lanier, L. L., Corliss, B., Wu, J., and Phillips, J. H. (1998) Association of DAP12 with activating CD94/NKG2C NK cell receptors. Immunity 8, 693–701. 6. McVicar, D. W., Taylor, L. S., Gosselin, P., Willette-Brown, J., Mikhael, A. I., Geahlen, R. L., Nakamura, M. C., Linnemeyer, P., Seaman, W. E., Anderson, S. K., Ortaldo, J. R., and Mason, L. H. (1998) DAP12mediated signal transduction in natural killer cells. A dominant role for the Syk protein-tyrosine kinase. J Biol Chem 273, 32934–32942. 7. Campbell, K. S., and Colonna, M. (1999) DAP12: a key accessory protein for relaying signals by natural killer cell receptors. Int J Biochem Cell Biol 31, 631–636.

8. Gosselin, P., Mason, L. H., Willette-Brown, J., Ortaldo, J. R., McVicar, D. W., and Anderson, S. K. (1999) Induction of DAP12 phosphorylation, calcium mobilization, and cytokine secretion by Ly49H. J Leukoc Biol 66, 165–171. 9. Wu, J., Cherwinski, H., Spies, T., Phillips, J. H., and Lanier, L. L. (2000) DAP10 and DAP12 form distinct, but functionally cooperative, receptor complexes in natural killer cells. J Exp Med 192, 1059–1068. 10. Lowin-Kropf, B., Kunz, B., Schneider, P., and Held, W. (2002) A role for the src family kinase Fyn in NK cell activation and the formation of the repertoire of Ly49 receptors. Eur J Immunol 32, 773–782. 11. Gadue, P., Morton, N., and Stein, P. L. (1999) The Src family tyrosine kinase Fyn regulates natural killer T cell development. J Exp Med 190, 1189–1196. 12. Marti, F., Xu, C. W., Selvakumar, A., Brent, R., Dupont, B., and King, P. D. (1998) LCKphosphorylated human killer cell-inhibitory receptors recruit and activate phosphatidylinositol 3-kinase. Proc Natl Acad Sci U S A 95, 11810–11815. 13. Brumbaugh, K. M., Binstadt, B. A., Billadeau, D. D., Schoon, R. A., Dick, C. J., Ten, R. M., and Leibson, P. J. (1997) Functional role for Syk tyrosine kinase in natural killer cell-mediated natural cytotoxicity. J Exp Med 186, 1965–1974.

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14. Adunyah, S. E., Wheeler, B. J., and Cooper, R. S. (1997) Evidence for the involvement of LCK and MAP kinase (ERK-1) in the signal transduction mechanism of interleukin15. Biochem Biophys Res Commun 232, 754–758. 15. Binstadt, B. A., Brumbaugh, K. M., Dick, C. J., Scharenberg, A. M., Williams, B. L., Colonna, M., Lanier, L. L., Kinet, J. P., Abraham, R. T., and Leibson, P. J. (1996) Sequential involvement of Lck and SHP-1 with MHC-recognizing receptors on NK cells inhibits FcR-initiated tyrosine kinase activation. Immunity 5, 629–638. 16. Binstadt, B. A., Billadeau, D. D., Jevremovic, D., Williams, B. L., Fang, N., Yi, T., Koretzky, G. A., Abraham, R. T., and Leibson, P. J. (1998) SLP-76 is a direct substrate of SHP-1 recruited to killer cell inhibitory receptors. J Biol Chem 273, 27518–27523. 17. Tangye, S. G., Lazetic, S., Woollatt, E., Sutherland, G. R., Lanier, L. L., and Phillips, J. H. (1999) Cutting edge: human 2B4, an activating NK cell receptor, recruits the protein tyrosine phosphatase SHP-2 and the adaptor signaling protein SAP. J Immunol 162, 6981–6985. 18. Letourneur F. M. B. (1989) Derivation of a T cell hybridoma variant deprived of functional T cell receptor alpha and beta chain transcripts reveals a nonfunctional alpha-mRNA of BW5147 origin. Eur J Immunol 12, 2269–2274. 19. McMahon, C. W., and Raulet, D. H. (2001) Expression and function of NK cell receptors in CD8+ T cells. Curr Opin Immunol 13, 465–470. 20. Ugolini, S., and Vivier, E. (2000) Regulation of T cell function by NK cell receptors for classical MHC class I molecules. Curr Opin Immunol 12, 295–300. 21. Holler, P. D., Chlewicki, L. K., and Kranz, D. M. (2003) TCRs with high affinity for foreign pMHC show self-reactivity. Nat Immunol 4, 55–62. 22. Holler, P. D. and Kranz, D. M. (2003) Quantitative Analysis of the Contribution of TCR/pepMHC Affinity and CD8 to T Cell Activation. Immunity 18, 255–264. 23. Holler, P. D., Lim, A. R., Cho, B. K., Rund, L. A., and Kranz, D. M. (2001) CD8(-) T cell transfectants that express a high affinity T cell receptor exhibit enhanced peptidedependent activation. J Exp Med 194, 1043–1052.

24. Wang, N., Morra, M., Wu, C., Gullo, C., Howie, D., Coyle, T., Engel, P., and Terhorst, C. (2001) CD150 is a member of a family of genes that encode glycoproteins on the surface of hematopoietic cells. Immunogenetics 53, 382–394. 25. Engel, P., Eck, M. J., and Terhorst, C. (2003) The SAP and SLAM families in immune responses and X-linked lymphoproliferative disease. Nat Rev Immunol 3, 813–821. 26. Sidorenko, S. P., and Clark, E. A. (2003) The dual-function CD150 receptor subfamily: the viral attraction. Nat Immunol 4, 19–24. 27. Morita, S., Kojima, T., and Kitamura, T. (2000) Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 7, 1063–1066. 28. Udaka, K., Wiesmuller, K. H., Kienle, S., Jung, G., and Walden, P. (1996) Self-MHCrestricted peptides recognized by an alloreactive T lymphocyte clone. J Immunol 157, 670–678. 29. Latchman, Y., McKay, P. F., and Reiser, H. (1998) Identification of the 2B4 molecule as a counter-receptor for CD48. J Immunol 161, 5809–5812. 30. Brown, M. H., Boles, K., van der Merwe, P. A., Kumar, V., Mathew, P. A., and Barclay, A. N. (1998) 2B4, the natural killer and T cell immunoglobulin superfamily surface protein, is a ligand for CD48. J Exp Med 188, 2083– 2090. 31. Degano, M., Garcia, K. C., Apostolopoulos, V., Rudolph, M. G., Teyton, L., and Wilson, I. A. (2000) A functional hot spot for antigen recognition in a superagonist TCR/MHC complex. Immunity 12, 251–261. 32. Dam, J., Guan, R., Natarajan, K., Dimasi, N., Chlewicki, L. K., Kranz, D. M., Schuck, P., Margulies, D. H., and Mariuzza, R. A. (2003) Variable MHC class I engagement by Ly49 natural killer cell receptors demonstrated by the crystal structure of Ly49C bound to H-2 K(b). Nat Immunol 4, 1213– 1222. 33. Chlewicki, L. K., Velikovsky, C. A., Balakrishnan, V., Mariuzza, R. A., and Kumar, V. (2008) Molecular basis of the dual functions of 2B4 (CD244). J Immunol 180, 8159– 8167. 34. Clarkson, N. G., Simmonds, S. J., Puklavec, M. J., and Brown, M. H. (2007) Direct and indirect interactions of the cytoplasmic region of CD244 (2B4) in mice and humans with FYN kinase. J Biol Chem 282, 25385– 25394.

Chapter 13 Expression of cDNAs in Human Natural Killer Cell Lines by Retroviral Transduction S. M. Shahjahan Miah and Kerry S. Campbell Abstract Human NK-like cell lines are difficult to transfect using standard mammalian expression vectors and conventional transfection protocols, but they are susceptible to retroviral transduction as a means to introduce cDNAs. Our laboratory has exploited this technique to study a number of receptors in human NK cell lines. The method utilizes a bicistronic retroviral vector that co-expresses either drug resistance or enhanced green fluorescent protein (EGFP) in parallel with the gene of interest. After a single infection with recombinant retrovirus, transduced NK cells can be sorted for expression of EGFP or the transduced cell surface marker. Alternatively, cells expressing the transduced cDNAs can be selected for by treatment with neomycin, puromycin, or hygromycin. Using this method, the sorted/selected cells uniformly express the gene of interest and the expression is stable for many weeks of culture. Key words: Retroviral transduction, NK cell lines, EGFP.

1. Introduction A number of transformed human natural killer (NK)-like cell lines have been adapted to culture and provide valuable models for studying NK cell function and signal transduction. Most of these cell lines lack expression of many normal NK cell surface receptors, especially killer cell Ig-like receptors (KIR), CD94/NKG2 heterodimers, and CD16. Therefore, it is very attractive to express these receptors in the transformed NK cell lines to examine molecular functions. Unfortunately, the available NK cell lines are highly resistant to transfection with standard mammalian expression vectors. K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 13, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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In the late 1990s, several groups successfully expressed cDNAs in NK-like cell lines by retroviral transduction. Amphotropic retroviral transduction was first successfully used to introduce the IL-2 cDNA into the human NK-92 cell line (1). Cohen et al. subsequently introduced the ecotropic receptor into the human YTS cell line, which permitted susceptibility to transduction with mouse ecotropic retrovirus [(2) and further described in chapter 17 by Mandelboim in this volume]. Amphotropic retroviruses can infect most mammalian cells, including human, and can therefore be biohazardous to laboratory personnel. On the other hand, ecotropic virus can only infect mouse and rat cells, and hence, working with ecotropicsensitive YTS cells has the advantage of avoiding some biosafety issues. Nonetheless, many versions of replication-incompetent amphotropic retrovirus have been engineered, and these strains are not particularly dangerous if carefully handled under BSL2 biosafety conditions, which are achievable in most modern biology laboratories (see Note 1). Our laboratory has exploited amphotropic retroviral transduction to introduce a number of cDNAs into a variety of NK-like cell lines, including NK-92, NKL, NK3.3, and KHYG-1 (3–7), and our optimized transduction protocol is detailed in this chapter (see Note 2). Retroviral vectors derived from Moloney murine leukemia virus (MMLV) are the most widely used and allow the delivery of genes to dividing mammalian cells. The expression of a cloned gene of interest is strongly promoted by the flanking longterminal repeat (LTR) elements within these vectors, and the vectors integrate into the cell’s chromosomes, thereby establishing long-term, stable protein expression after a single transduction procedure. Retroviral infection generates a polyclonal transduced population, since the distinct random chromosomal integration events occur in multiple clones. The polyclonal nature of the transduced population thereby dilutes potential bias that may be introduced by influencing an integrated vector’s promoter on genes adjacent to the integration site when studying monoclonal transfected populations. To allow purification of transduced cells that express the gene of interest, retroviral vectors may also encode selectable markers, such as neomycin-, puromycin-, or hygromycin-resistance genes, or a fluorescent marker, especially enhanced green fluorescence protein (EGFP). Standard mammalian expression vectors contain independent transcription units for the selectable marker and the gene of interest. A number of bicistronic retroviral vectors have been developed; however, that contain an internal ribosome entry site (IRES), which allows both the marker and the gene of interest to be expressed independently from a single transcript. Our retroviral transduction of NK cell lines has utilized a system developed and made readily available by Dr. Garry Nolan

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(Stanford University, Stanford, CA). Detailed information about this system can be found at www.stanford.edu/group/nolan/. This system utilizes the retroviral vector pBMN-IRES-EGFP, which co-expresses EGFP and the Phoenix-amphotropic packaging cell line. The Phoenix-amphotropic packaging lines encode three major retroviral elements: (1) pol, which functions as a reverse transcriptase, RNase H, and integrase, (2) gag, which is a large protein that is processed into viral matrix and core structures, and (3) the envelope (env) protein, which exists in the lipid layer and determines viral tropism. When Phoenix cells are transfected with the pBMN plasmid, these elements package a replication-incompetent retrovirus that is secreted into the culture medium and used to infect the NK cell lines.

2. Materials 1. Phoenix-amphotropic retroviral packaging cell line: The cell line can be obtained from Dr. Garry Nolan. It is based on the 293T cell line, a human embryonic kidney fibroblast that is transformed with adenovirus E1a and carries a temperature-sensitive T antigen co-selected with neomycin. The Phoenix-ampho cell line was created by introducing genes producing gag-pol and env for infection of most mammalian cells, including human. Gag-pol and envelope use different promoters to minimize their interrecombination potential (see Note 3). 2. NK-like cell lines: NK-92 (ATCC #CRL-2407), NKL (obtained from Dr. Marco Colonna, Washington University, St. Louis, MO), NK3.3 (obtained from Dr. Jacki Kornbluth, St. Louis University School of Medicine, St. Louis, MO), and KHYG-1 (obtained from Health Science Research Resources Bank, Japan Health Sciences Foundation, Osaka, Japan, #JCRB0156). 3. pBMN-IRES-EGFP retroviral vector (also obtained from Dr. Garry Nolan) with cDNA of interest sub-cloned into appropriate restriction sites. 4. Stbl2 bacteria (Invitrogen) (see Note 4). 5. Complete RPMI medium: RPMI 1640 medium (Life Technologies, Rockville, MD) containing 10% heatinactivated fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT), 2 mM L-glutamine, 100 ␮g/ml penicillin, 100 ␮g/ml streptomycin, 10 mM HEPES (pH 7.4), 1 mM sodium pyruvate (all supplements from Life Technologies or Mediatech, Manassas, VA), and 50 ␮M 2mercaptoethanol.

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6. Folic acid stock solution (2 mM): Mix 0.221 g folic acid (Gibco) in 250 ml ddH2 O. Autoclave, wrap in foil, and store at 4◦ C. The final preparation will be a suspension and needs to be warmed to 70◦ C for 10 min prior to addition to the complete ␣-MEM preparation. 7. Myo-inositol stock solution (20 mM): Mix 0.901 g myoinositol (Sigma-Aldrich, St. Louis, MO) in 250 ml ddH2 O, sterile filter, and store at 4◦ C. 8. Complete ␣-MEM: ␣-minimal essential medium (MEM; Life Technologies, Rockville, MD) containing 10% heatinactivated FBS (HyClone Laboratories, Logan, UT), 10% heat-inactivated horse serum (Life Technologies), 2 mM Lglutamate, 100 ␮g/ml penicillin, 100 ␮g/ml streptomycin, 1 mM sodium pyruvate, 100 ␮M 2-mercaptoethanol, 2.5 ␮M folic acid (Sigma-Aldrich), 200 ␮M myo-inositol (Sigma-Aldrich), and 100–500 U/ml recombinant human IL-2 (Roche). 9. OPTI-MEM reduced serum medium (Gibco/Invitrogen). 10. Recombinant human IL-2, available from a variety of commercial sources, such as Roche (Basel, Switzerland) that was generously provided by the National Cancer Institute Biological Resources Branch (Frederick, MD). 11. Freezing medium: 94% heat-inactivated FBS supplemented with 6% DMSO. 12. Cryogenic freezing vials (Nalgene). 13. NALGENETM cryo freezing container (Nalgene). 14. LipofectamineTM reagent and PlusTM reagent (Invitrogen). 15. Polybrene (1,5-dimethyl-1,5-diazaundecamethylene polymethobromide and hexadimethrine bromide; Sigma): Polybrene is dissolved at a stock concentration 5 mg/ml in PBS, subsequently filtered through a 0.2 ␮m filter, and stored for several weeks at +4◦ C or long term at −80◦ C. The working concentration of polybrene is 10 ␮g/ml. 16. Plasticware: T25 and T75 culture flasks (Nunc, Denmark), 15 ml conical centrifuge tubes (BD Falcon, USA), and sterile disposable 5 and 10 ml pipettes (FisherBrand). 17. Neomycin (Fisher Scientific) was dissolved in HEPES buffer, pH 7.2, at a concentration of 50 mg/ml and used to select transduced cells at a final concentration of 1.25 mg/ml. 18. Puromycin (EMD/Calbiochem, San Diego, CA) dissolved in DMSO at 5 mg/ml stock concentration and was used to select transduced cells at a concentration of 2.5 ␮g/ml.

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3. Methods 3.1. Cell Culture 3.1.1. Culture of Phoenix-Amphotropic Cells (see Note 5) 3.1.2. Culture of NK-Like Cell Lines

1. Phoenix cells are cultured in complete RPMI medium in 25 mm flasks (set horizontally) maintained at 37◦ C in humidified 7% CO2 atmosphere. 2. When cells reach about 80% confluence, they should be split 1:10 into 1:20 every 3–4 days with fresh culture medium. Several different NK cell lines, including KHYG-1, NK-92, NK3.3, and NKL, can be cultured under the following conditions. Cultures should be replaced with freshly thawed stocks every 4–6 weeks to maintain biological uniformity that can drift upon long-term culture. 1. NK cell lines are cultured in 50 ml of fresh complete ␣MEM medium in T75 flasks (standing vertically) at 37◦ C in a humidified 7% CO2 atmosphere. 2. Cells are passaged between 1:5 and 1:10 into fresh medium with human IL-2 every 4 days. Optimal growth is achieved by seeding new cultures with 4 million cells per 50 ml.

3.1.3. Freezing Cell Lines

To assure the optimal viability of cell lines, they should be harvested from log-phase cultures prior to freezing. NK cell lines grow in suspension, whereas Phoenix cells are adherent and easily detach from the tissue culture flask by gentle shaking. 1. Collect cells in a centrifuge tube and centrifuge the cells at 500 × g for 5 min. 2. Remove the medium, resuspend in fresh medium, and count the cells. 3. Centrifuge again at 500 × g for 5 min. 4. Remove supernatant and resuspend in freezing medium at 2–3 × 106 cells per ml. 5. Transfer 1 ml to a 2 ml cryogenic freezing vial, put the vial in a NALGENETM cryo freezing container at room temperature, and transfer to −70◦ C overnight. 6. Transfer vials to liquid nitrogen on the following day for long-term storage.

3.1.4. Thawing Cells

1. Remove vial from liquid nitrogen and thaw rapidly in a 37◦ C water bath. 2. Immediately after complete thaw, add 1 ml of warm culture medium (37◦ C) to the freezing vial and transfer this solution to 15 ml sterile conical screw cap tube containing 13 ml of warm culture medium.

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3. Centrifuge tube at 500 × g for 5 min. 4. Remove the supernatant and resuspend the cell pellet by flicking the tube. Add warm culture medium and transfer to a culture flask. Expand the culture at 37◦ C in humidified 7% CO2 atmosphere. 3.2. Retroviral Transduction of Human NK-Like Cell Lines (see Note 2) 3.2.1. Transfection of Phoenix-Amphotropic Cells

The first step of retroviral transduction is to clone your gene of interest into the pBMN-IRES-EGFP vector (see Note 4) and use this engineered vector to prepare recombinant retrovirus by transfecting into the Phoenix-amphotropic packaging cell line. The Phoenix-amphotropic cell line should be maintained at less than 80% confluence, and cultures should be replaced with freshly thawed stocks every 6–8 weeks (see Note 3). Day 1: 1. Plate 0.1–0.2 × 106 Phoenix-amphotropic cells per 6 ml complete RPMI medium per well in a 6-well plate 24 h prior to transfection. Phoenix cells should be about 70–80% confluent on the day of transfection (see Note 5). Day 2: 2. In a 1.5 ml microfuge tube, mix at least 4 ␮g pBMN-IRESEGFP vector containing cDNA of interest with 10 ␮l Plus Reagent and bring total volume to 100 ␮l with the addition of pre-warmed reduced serum OPTI-MEM. 3. In a separate microfuge tube, add 8 ␮l Lipofectamine to 92 ␮l pre-warmed reduced serum OPTI-MEM. 4. Incubate both samples at RT for 15 min. 5. After incubation, mix the contents of both tubes together for a total volume of 200 ␮l and incubate at RT for another 15 min. 6. Wash Phoenix-amphotropic cells once by aspirating the culture medium and gently adding 6 ml pre-warmed reduced serum OPTI-MEM along the side of the well. Add the medium slowly, because Phoenix cells do not adhere tightly and the added medium should not detach the cells. 7. After the DNA and transfection reagents have finished incubating, add 800 ␮l pre-warmed reduced serum OPTIMEM for a total volume of 1 ml. 8. Gently aspirate the wash medium from the culture well and gently add the 1 ml transfection reaction slowly to the Phoenix cells by releasing along the side of the well with a pipette. 9. Incubate the plate at 37◦ C in 7% CO2 atmosphere for at least 3 h.

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10. After 3 h of incubation add 4 ml pre-warmed complete RPMI medium to the transfected well and incubate plate at 37◦ C, 7% CO2 overnight. Day 3: 11. Remove medium by aspiration and wash cells once with 5 ml pre-warmed reduced serum OPTI-MEM. 12. Add 2 ml reduced serum OPTI-MEM and incubate at 37◦ C, 7% CO2 for 24 h (add 1.3 ml OPTI-MEM if using 2 wells for single transfection). 3.2.2. Collection of Virus and Transduction of NK Cell Line

This section describes the generation and manipulation of biohazardous retrovirus. Therefore, BSL2 biosafety procedures should be followed throughout the following steps. Upon finishing this section, incubate all disposed plasticware (including pipettes) under the UV light of a biohazard hood for at least 1 h to destroy the retroviral contamination. 1. On day 4, the retroviral supernatant is ready for harvesting. Collect supernatant (containing virus) into a 15 ml centrifuge tube. 2. Centrifuge the tube at 500 × g for 5 min to remove any remaining cells or filtering through a 45 ␮m filter (see Note 6). 3. Transfer virus into a new 15 ml centrifuge tube. 4. Add 20 ␮l Plus Reagent to virus suspension and incubate at RT for 15 min. 5. After incubation add 8 ␮l Lipofectamine to virus suspension and incubate at RT for another 15 min. 6. During the virus/Plus/Lipofectamine incubation, wash NK cells once with OPTI-MEM by spinning at 500 × g for 5 min. Count the cells and transfer 0.5 × 106 cells to a 15 ml tube. 7. Resuspend NK cells to be transduced with 2 mL viruscontaining supernatant and transfer to a single well of a 12-well plate. Centrifuge the plate at 700 × g for 30 min (see Note 7). 8. Incubate the plate at 37◦ C in a humidified 7% CO2 atmosphere for at least 3 h. 9. Centrifuge the plate again at 700 × g for 30 min, incubate again for 3 h at 37◦ C, and then proceed to step 10 or 11. 10. Transfer cells from each well of the 12-well plate to a separate T25 flask and add 8 mL of fresh IL-2-containing ␣-MEM culture medium to each flask. Incubate the culture at 37◦ C in a humidified 7% CO2 atmosphere until cells are confluent and proceed to step 12.

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11. Alternatively after step 9, to maximize transduction efficiency, add 2 mL of fresh IL-2-containing ␣-MEM culture medium to the virus-infected NK cells in well(s) of the 12well plate (total 4 ml of medium) and incubate overnight at 37◦ C in a humidified 7% CO2 atmosphere. Then transfer cells from the plate to the T25 culture flask (most of the cells will detach from plastic and can be easily transferred) and add another 6 ml of fresh complete ␣-MEM containing IL-2. Incubate the culture at 37◦ C in a humidified 7% CO2 atmosphere until cells are confluent and proceed to step 12. 12. When cells are ready (∼6 days after transduction, see Note 8) sort the transduced population for EGFP or for the expression of the transduced surface marker by flow cytometry (see Note 9). Alternatively, select transduced cells by treatment with antibiotics (if using a vector containing an antibiotic resistance gene; see Note 10). The fraction of transduced cells depends on the cell line used. The resulting cell population will retain expression for many weeks of culture. 13. Expand the transduced NK cell population and freeze several vials as described in Section 3.1.3. Discard growing transduced NK cell populations after 4–6 weeks of culture and replace with a newly thawed aliquot of frozen stock (see Note 11).

4. Notes 1. Importantly, constructs encoding potential oncogenes should be avoided when working with retroviral, adenoviral, or lentiviral expression systems. 2. This is a general protocol for transducing human NK cell lines but can be applied with little variation to other cell lines. 3. An important feature of the Phoenix cell lines is hightransfection efficiency using conventional transfection methods (e.g., including calcium phosphate or lipidbased techniques). In our hands, approximately 60–90% of Phoenix-amphotropic cells can be transiently transfected with Lipofectamine reagents, depending on the construct introduced. 4. The Stbl2 bacterial cells are suitable for the cloning of unstable inserts such as LTR-containing retroviral sequences or direct repeats, and for optimal performance, bacteria should be grown at 30◦ C.

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5. The transfection efficiency of Phoenix-amphotropic retroviral packaging cells depends on their health and growth status, which must be maintained by regular passage. If the cells are 100% confluent, transfection is very inefficient, so never let the cells reach confluence. 6. The retroviral supernatant can be used immediately for transduction of target cells or kept on ice if used within several hours. Otherwise, retroviral supernatant may be frozen at −80◦ C, resulting in a minimal loss of viral titer. 7. Our usual centrifugation period is 30 min, but increasing the centrifugation time up to 90 min can increase transduction efficiency in some cell lines. 8. Depending on growth rate, cells are generally sorted 6–8 days after transduction. Transduction efficiency can depend upon the number of cells infected, the construct used, and the NK cell line to be transduced. Starting with a higher number of cells usually requires relatively shorter times to be ready for sorting. 9. We have successfully used this transduction protocol to express cDNAs in the following human NK-like cell lines at the indicated efficiency of transduction: KHYG-1 (20– 50%), NK-92 (5–15%), NKL (15–30%), and NK3.3 (5– 15%). KHYG-1 cells are highly susceptible to retroviral transduction and can be successfully transduced by adding polybrene (10 ␮g/ml) instead of using Lipofectamine reagents at steps 4–6 in Section 3.2.2. To improve transduction efficiencies, more NK-92 or NK3.3 cells can be infected in steps 6–7 of Section 3.2.2. 10. Starting on day 2 after transduction with retrovirus containing an antibiotic resistance gene, the transduced cells should be selected with antibiotics for 5 days. 11. For testing any biological effect in transduced cells, the results should always be compared in cells from separate transduction procedures using the same construct. This will assure that the impact is not unique to the cells derived from a specific transduced population.

Acknowledgments We would like to thank all previous members of the Campbell Laboratory for establishing and optimizing this technique, Dr. Amanda Purdy for review of the chapter, and Dr. Garry Nolan for reagents. Supported by National Institutes of Health grants R01-CA083859, R01-CA100226 (K.S.C.), T32CA009035 (S.M.S.M.), and Centers of Research Excellence grant

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CA06927 (FCCC). The research was also supported in part by an appropriation from the Commonwealth of Pennsylvania. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute. References 1. Nagashima, S., R. Mailliard, Y. Kashii, T. E. Reichert, R. B. Herberman, P. Robbins, and Whiteside, T. L. (1998) Stable transduction of the interleukin-2 gene into human natural killer cell lines and their phenotypic and functional characterization in vitro and in vivo. Blood 91:3850–3861. 2. Cohen, G. B., Gandhi, R. T., Davis, D. M., Mandelboim, O., Chen, B. K., Strominger, J. L., and Baltimore, D. (1999) The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity 10:661–671. 3. Yusa, S., Catina, T. L., and Campbell, K. S. (2002) SHP-1- and phosphotyrosineindependent inhibitory signaling by a killer cell Ig-like receptor cytoplasmic domain in human NK cells. J Immunol 168: 5047–5057. 4. Kikuchi-Maki, A., Yusa, S., Catina, T. L., and Campbell, K. S. (2003) KIR2DL4 is an

IL-2-regulated NK cell receptor that exhibits limited expression in humans but triggers strong IFN-gamma production. J Immunol 171:3415–3425. 5. Yusa, S., Catina, T. L., and Campbell, K. S. (2004) KIR2DL5 can inhibit human NK cell activation via recruitment of Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2). J Immunol 172:7385–7392. 6. Alvarez-Arias, D. A., and K. S. Campbell. (2007) Protein kinase C regulates expression and function of inhibitory killer cell Iglike receptors in NK cells. J Immunol 179: 5281–5290. 7. Miah, S. M., Hughes, T. L., and Campbell, K. S. (2008) KIR2DL4 differentially signals downstream functions in human NK cells through distinct structural modules. J Immunol 180: 2922–2932.

Chapter 14 Lentiviral Gene Transduction in Human and Mouse NK Cell Lines Ram Savan, Tim Chan, and Howard A. Young Abstract Natural killer (NK) cells play a vital role in the control of cancer and microbial infections. A major hinderance in studying NK cells is the resistance of these cells to gene transfer. Considering over-expression and gene knockdown studies are crucial tools to study the biology of cells, technologies suitable for transfering genes into NK cells are invaluable. Among various technologies available for gene transfer, lentiviral-mediated transduction has been successful in introducing genes into NK cells. We have standardized methods of lentiviral infection in human and mouse NK cell lines. We obtain transduction efficiencies of 15% in the NK-92 cell line and 30–40% in LNK, YT, and DERL7 cell lines. This method allows efficient and stable introduction of genes and shRNAs into NK cell lines. Key words: Natural killer cells, lentivirus, transduction, flow cytometry, viral titration, human, mouse, NK-92, LNK.

1. Introduction Natural killer (NK) cells are large, granular lymphocytes which play a vital role in tumor immunosurveillance and combating microbial infections (1). These cells eliminate targets using multiple mechanisms and also recruit and amplify inflammatory responses. NK cells are able to lyse class I MHC-negative targets by releasing perforin, granzymes, and TNF ligands upon stimulation. These effector functions of NK cells are gradually acquired during their development and maturation. These cells are also known to recruit other immune cells to the sites of infection by releasing chemokines and cytokines. To understand the biology of NK cells and dissect specific gene functions, tools facilitating over-expression of genes or knockdown of genes by siRNA are invaluable. Resistance of NK cells to exogenous gene transfer is K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 14, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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a major hinderance in understanding NK cell biology and the potential use of these cells in immunotherapy. Human and mouse NK cells (primary cells and cell lines) are refractory to gene transfer. Methods like calcium phosphate precipitation (CaPO4 ), liposome reagents, and electroporation techniques have resulted in very low rates of gene transfer (2). Nucleofection technology (Amaxa, Inc.) has been the most promising among nonviral technologies that has shown some success in gene transfer in NK cell lines (3, 4). Several viral-mediated gene transfer protocols have been reported, but these approaches have had only variable success (5–7). For example, gene introduction with vaccinia virus altered the phenotype of NK cells (6) while adenoviral vectors have shown to be ineffective because NK cells do not possess appropriate receptors to mediate attachment of the virus to the cell. In contrast, Schroers et al. (8) showed that chimeric fiber-modified Ad5/F35 adenoviral vector efficiently infected primary human NK cells. Recently, ALAK (IL2-activated NK) cells transduced by adenovirus have been used in tumor therapy in mice (9, 10). Use of retroviral transduction in primary NK cells has resulted in partial success, primarily because of the requirement of multiple rounds of infection to introduce genes (11). Among the viral-mediated gene transfer systems, lentivirus transductions have been the most efficient gene delivery systems available for hematopoietic cells and hard to transfect cells. Recently, Tran and Kung (7) demonstrated an average of 40% transduction efficiency with primary murine NK cells. Furthermore, they show that lentiviral transduction does not affect the viability, function, and phenotype of NK cells. Lentiviral vectors (LV) are increasingly being utilized as a tool for introducing and obtaining stable expression of transgenes in both dividing and nondividing cells. Self-inactivating replicationincompetent lentiviral particles are generated by co-expressing the packaging elements of the virus along with the vector genome in the most commonly used virus-producing cell line, human embryonic kidney cell 293 or 293T (containing the SV40 large T antigen). The packaging elements of the HIV-1-based lentiviral vectors are from the HIV-1 genome. These HIV vector systems can be divided into three generations based on the progressive deletions of the packaging system from the parent vector. The first LV generation system contained all the HIV-1 genes except for the envelope in the parent vector. In the second generation, HIV genes including vpu and nef were deleted and the LV packing elements are provided as separate vectors. The third generation LV system offers maximum biosafety features by having the gag, pol, and rev genes provided as separate vectors. For this latest system, the envelope gene is from a heterologous virus, vesicular stomatitis virus (VSV), thus resulting in a pseudotyped virus with a broad host range.

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The first sucessful report utilizing lentiviral vectors in NK cells described the transduction of primary murine NK cells (7). As the lentiviral transduction system allows efficient and stable integration of transgenes, NK cell lines can be utilized to develop in vitro models to study and further understand the biology of NK cells. Several human and mouse cell lines have been developed that can be used to study the function of NK cells. Among the human NK cell lines, NK-92 (12), YT (13), NKL (14), and DERL7 (15) are the most commonly used. NK-92 was originally derived from a male non-Hodgkin s lymphoma patient (12). This IL-2dependent NK cell line kills target cells and displays characteristics of activated NK cells. NK-92 produces copious amounts of IFN-␥ upon stimulation by cytokines individually or in combination (16), making it an ideal model for studying gene regulation in NK cells. DERL7 (CD56+ , CD3− , TCR␥␦− ) is a newly described cell line from a non-Hodgkin’s lymphoma patient, which posses both NK and T-cell surface markers (15). There is only one NK cell line derived from mice, designated LNK (CD132+ , CD16+ , CD3− , IgM− ), which is an IL-2-dependent NK line derived from liver lymphocytes of BALB/c nude mice (17). We have developed protocols which facilitate efficient and stable introduction of genes and short-hairpin RNA (shRNAs) into NK cell lines. The transduction efficiencies obtained are around 15% in NK-92 cells and 30–40% in LNK, YT, and DERL7 cells.

2. Materials 2.1. Plasmid Preparation

1. Plasmids: pLKO.1, pGIPZ, and pTRIPZ (Open Biosystems, www.openbiosystems.com); pMD2.G (Plasmid 12259; Addgene, www.addgene.org); and psPAX2 (Plasmid 12260; Addgene). 2. Bacterial strains: Vectors from Open Biosystems are generally transformed in Escherichia coli DH5␣. To prevent recombination of the plasmid with genomic DNA, use E. coli strains that lack the recombinase gene (recA). We recommend the use of commercially available electro- or chemicalcompetent E. coli strains like Stbl2 or Stbl4. 3. Luria–Bertani (LB) medium (1 L): Tryptone 10 g, yeast extract 5 g, and NaCl 10 g. Adjust volume to 1 L distilled water, sterilize by autoclaving, and store at room temperature (RT). 4. SOC medium (1 L); tryptone 20 g, yeast extract 5 g, 5 M NaCl 2 mL, 1 M KCl 2.5 mL, 1 M MgCl2 10 mL, 1 M

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MgSO4 10 mL, and 1 M glucose 20 mL. Adjust volume to 1 L distilled water, sterilize by autoclaving, and store at RT. 5. Antibiotics: Prepare ampicillin stock solution at 50 mg/mL in distilled water and store at −20◦ C. 6. Plasmid purification kits: Purchase mini- and maxi-prep kits from any commercial source. Endo-free maxi-prep plasmid purification kits are recommended to obtain transfection quality DNA free of endotoxin. 2.2. Transfection Reagents

1. 2 M calcium chloride (CaCl2 ; Invitrogen, Walkersville, MD, USA). 2. 2× HEPES (2.5 M, Invitrogen).

2.3. Cell Lines and Culture Media

1. NK-92: This human NK cell line can be purchased from ATCC (American Type Culture Collection, Rockville, MD, USA; CRL-2047). NK-92 cells are cultured in RPMI 1640 medium (Cambrex, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, 50 ␮g/mL streptomycin, and 2 mM L-glutamine (PSG, Cambrex), recombinant human IL-2 (1000 IU/mL; PeproTech, Rocky Hill, NJ), and recombinant human IL-15 (100 ng/mL; PeproTech). 2. DERL7 (CD56+ , CD3− , TCR␥␦− ): This human cell line was kindly provided by Dr. L. Del Vecchio (A Cardarelli Hospital, Italy) and cultured in RPMI 1640 medium supplemented with 20% FBS, 100 IU/mL penicillin, 50 ␮g/mL streptomycin and 2 mM L-glutamine, recombinant human IL-2 (1000 U/mL), and stem cell factor (250 ng/mL; PeproTech). 3. LNK cell line: LNK is cultured in RPMI 1640 medium supplemented with 10% FBS, 100 IU/mL penicillin, 50 ␮g/mL streptomycin and 2 mM L-glutamine, 5% HEPES, 5% sodium pyruvate and 1× beta-mercaptoethanol, and recombinant human IL-2 (1000 U/mL). 4. 293FT cell line (Invitrogen): This human embryonic kidney line transfected with the SV40 large T antigen is cultured in DMEM supplemented with 10% FBS, 100 IU/mL penicillin, 50 ␮g/mL streptomycin and 2 mM L-glutamine, 5% HEPES, 5% sodium pyruvate, and 1% gentamicin. 5. 3T3 cell line: This mouse fibroblast cell line is cultured in DMEM supplemented with 10% FBS, 100 IU/mL penicillin, 50 ␮g/mL streptomycin and 2 mM L-glutamine, 5% HEPES, and 5% sodium pyruvate. 6. Trypsin−EDTA: 2.5% trypsin and 0.5 M ethylenediaminetetraacetic acid (EDTA).

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7. PBS−EDTA: Phosphate-buffered saline (pH 7.4) and 0.1 M EDTA. 8. Antibiotics: (a) Puromycin: prepare stock solution at 10 mg/mL in PBS and store at −20◦ C. (b) Geneticin (G418): prepare stock solution at 50 mg/mL in PBS and store at −20◦ C. 9. Polybrene (Sigma): Prepare stock solution at 8 mg/mL in distilled water and filter sterilize. Aliquot (200 ␮L) and store at −20◦ C. 2.4. Virus Production and Purification

1. 10 cm collagen-coated tissue culture grade plates (we have routinely utilized BD Biocoat plates, but other brands may also be suitable) 2. Syringe-driven filter unit: Use low protein binding 0.45 ␮M pore size filters 3. 10 mL disposable syringe 4. 1.8 mL cryo-vials

3. Method 3.1. Preparation of Plasmids

1. Mix 100 ng of plasmid DNA with chemical-competent E. coli (DH5␣, Top10, Stbl2, or Stbl4) in a 1.5 mL polypropylene tube and incubate on ice for a minimum of 30 min. 2. Heat shock the bacteria−plasmid mixture at 42◦ C for 30 s and immediately incubate on ice for 2 min. 3. Add 500 ␮L of SOC medium to the bacteria−plasmid mixture and incubate in a shaker incubator (≈300 rpm) for 1 h. 4. Spread 100 ␮L of transformed bacteria onto LBA (LBagar) containing ampicillin (100 ␮g/mL) and incubate overnight at 37◦ C bacterial incubator. 5. Pick at least 10 colonies from each plate and inoculate in 5 mL LB medium containing ampicillin (100 ␮g/mL) and incubate in a shaker incubator for 6 h. 6. Aliquot 3 mL of the bacteria from above (step 5) and extract plasmid using any commercially available mini-prep kit and verify plasmid by restriction enzyme digestion. 7. Aliquot 1 mL from step 5 and grow in 300 mL of LB medium (supplement with 100 ␮g/mL ampicillin) for 12−16 h at 37◦ C. 8. Centrifuge the bacteria at 6000×g for 15 min.

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9. Extract plasmid using any commercial endotoxin-free maxi-prep kit. 10. Verify by restriction digestion and store plasmid at 4◦ C at a final concentration of 1 mg/mL in TE buffer. 3.2. Virus Production (see Note 1)

Day 1 (seeding 293FT cells) 1. Seed 10 mL of 2.5 × 105 /mL of 293FT cells in complete DMEM medium on a collagen-coated 10 cm plate and incubate overnight at 37◦ C in a CO2 incubator (see Note 2). Day 2 (calcium phosphate transfection) 2. The cells should be 60–80% confluent the following day (100% confluency must be avoided). Gently remove medium and replace with pre-warmed 3T3 complete DMEM medium and return the culture dish to the incubator for a minimum of 4 h. 3. Mix lentiviral vector, gag/pol (psPAX2), and VSVG (pMD2.G) DNAs in a ratio of 2:2:1. We use 21 ␮g of the parent lentiviral vector, 21 ␮g of psPAX2, and 10 ␮g of VSVG (see Note 3) in a 14 mL polypropylene tube (Falcon). 4. Add 36 ␮L of 2 M CaCl2 solution and cell culture grade water to the plasmid mix to a final volume of 300 ␮L and vortex it for a second or gently flick the mixture. 5. It is necessary to next bubble air slowly into the mixture. We routinely do so with a Pasteur pipette. While the bubbling is ongoing, add 300 ␮L of 2× HBSS into the tube in a drop-wise manner. This process should be slow and lasts 1–2 min. 6. Incubate the above mixture for 30 min at RT (see Note 4). 7. Add the mixture onto the 293FT plate drop-wise and mix the contents on the plate by gently swirling the plate. Return the plate to the CO2 incubator at 37◦ C. 8. After 4–6 h, gently remove the medium from the plate and wash with 1× PBS. Add 10 mL of complete DMEM and incubate for 30–48 h at 37◦ C in a CO2 incubator. Day 3 (viral harvest) 9. Harvest the supernatant from the dish in a 15 mL capped polypropylene tube (Falcon). Centrifuge the tube at 360×g for 5 min to pellet cellular debris. 10. Pass the supernatant through 0.45 ␮M syringe-driven filter unit (see Note 5). 11. Aliquot the filtered supernatants into 1.8 mL cryo-vials and store them at −80◦ C.

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Day 1 1. The day before titering, plate 3T3 cells at a density of 2 × 105 cells/well of a 6-well plate and incubate in a CO2 incubator overnight (see Note 7). Cell confluency should be approximately 30–50% the following day. Day 2 2. Thaw the lentiviral sample (see Note 8) and prepare 10-fold serial dilutions in complete DMEM medium of the LV supernatant from 10−2 to 10−6 in sterile 5 mL polypropylene tubes (Falcon) with a minimal final volume of 2 mL for each dilution. Mix by gently pipetting the solution; DO NOT VORTEX. 3. Aspirate the medium from the 6-well overnight cultured plates and add 1 mL of the diluted virus-containing medium to their respective wells. Also include a control well. 4. Add polybrene to final concentration of 8 ␮g/mL to each well and incubate overnight. Day 3 5. Remove the infection medium from each well and replace with 2 mL of complete medium containing 10% FBS. Day 4 6. Remove the medium from the well and replace with complete medium containing 2 ␮g/mL puromycin to begin selecting transduced cells (see Note 9). Days 5–13 7. Every 3–4 days, remove the medium and replace with fresh complete medium containing 2 ␮g/mL puromycin Day 14 8. After 10 days on selection medium, the control well should have no cells left and the sample well should have some discrete puromycin-resistant colonies observed. 9. Remove the medium and wash the wells with PBS twice. 10. Add 1 mL Crystal Violet solution (1% w/v in 10% ethanol) and incubate at room temperature for 10 min. 11. Remove crystal violet solution and wash the wells with PBS twice. 12. Record the number of blue colonies observed in each of the wells with respect to the appropriate dilution. 13. Calculate the titer as transducing units (TU/mL) as follows: TU/mL = (number of discrete colonies/dilution factor)/volume of infection medium used.

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3.3.2. qPCR-Based Titering (see Note 10)

14. Utilizing 150 ␮L of the viral supernatant, purify the viral RNA using the Nucleospin RNA Virus kit (Macherey-Nagel), according to the manufacturer’s protocol. Elute the RNA from the column using 50 ␮L RNasefree water. 15. Remove contaminating vector DNA from the sample by treating the sample with DNAse I (8 U/reaction) and incubating the tube at 37◦ C for 30 min followed by 5 min at 70◦ C. 16. Meanwhile, prepare 10-fold dilutions of the RNA Control template (Clontech, Mountain View, CA, USA) and your samples. You will use 2 ␮L for each reaction done in duplicates. 17. Prepare a Master Reaction mix containing 50 nM of the forward and reverse primer, 1× SYBR green, 1× QTaq DNA polymerase, 1× reaction buffer, and 1× qRT Mix (Clontech) with a final volume of 23 ␮L/sample. Add 2 ␮L of the diluted RNA control template and sample to the reaction. 18. Using a 7300 ABI Prism qPCR thermocycler, program the reaction as per manufacturer’s protocol and run the reaction/analysis. 19. Determine the copy number based upon the raw copy number obtained from the qRT-PCR standard and factoring the dilutions and amount of sample used to obtain the copy number/mL. Utilize an infectivity coefficient (as determined by a biological titering assay) to relate the copies/mL to obtain the transforming units/mL.

3.4. Transduction of Human NK Cell Lines (NK-92 and DERL7)

Day 1 1. Plate the NK cell line in appropriate complete culture medium at a density under 1.0 × 106 cells/mL (see Note 11). Day 2 2. Count the cells and resuspend in appropriate medium at 1.0 × 106 cells/mL. 3. Stimulate the lines with human IL-2 (100 U/mL) and human IL-12 (PeproTech; 100 ng/mL) for 2 h before transduction (see Note 12). 4. Following cytokine stimulation, readjust the concentration of the cells to 1.0 × 105 cells/mL using NK cell medium and plate 1 mL in each well of a 12-well plate. 5. Add viral supernatant for a multiplicity of infection between 20 and 100 of the titered virus to the medium-containing

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cells and polybrene (8 ␮g/mL; see Note 13) and incubate overnight at 37◦ C. 6. Alternatively, spinoculation by centrifugation of cells and virus-containing supernatant at 360×g for 90 min at 32◦ C yields good transduction efficiency. Day 3 7. Remove the medium by spinning the cells at 360×g for 5 min at RT and resuspend the cells in 0.5 mL of NK cell medium and 0.5 mL of conditioned NK cell medium (see Note 14). Days 5–10 (selection of transgene-positive cells) 8. Cells can be selected by mammalian antibiotic resistance or YFP-positive cells depending on the marker used (see Fig. 14.1). We have titrated the required puromycin and hygromycin concentrations to be 2 ␮g/mL when using NK-92 or DERL7 cells (see Note 9).

Fig. 14.1. Flow cytometric analyses of yellow fluorescent protein (YFP)-positive cells in NK-92 and DERL7 cells postlentiviral transduction compared to controls.

9. Once selected for positive selection marker, choose up to five clones and check for knockdown (for shRNA) or transgene expression and select the clone with the desired expression level (see Note 15).

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3.5. Transduction of Mouse NK Cell Line (LNK)

Day 1 1. Plate the LNK cell line in appropriate medium at a density under 1.0 × 105 cells/mL on a 6-well plate (see Note 16). Day 2 2. Stimulate the lines with high dose of IL-2 (1000 U/mL) for 2 h before transduction. 3. Add viral supernatant for a multiplicity of infection between 20 and 100 of the titered virus to the medium-containing cells and polybrene (8 ␮g/mL) and incubate overnight at 37◦ C. Day 3 4. Remove the medium and add 0.5 mL of fresh NK cell medium and 0.5 mL of conditioned NK cell medium (see Note 14). Days 5–10 (selection of transgene-positive cells) 5. Cells can be selected by mammalian antibiotic resistance or green fluorescent protein (GFP)-positive cells or other criteria depending on the marker used. We have titrated puromycin and hygromycin to be effective at killing non-transduced cells at a concentration of 2 ␮g/mL (see Note 9). 6. Once selected for the positive selection marker, select up to five clones and check for knockdown (for shRNA) or transgene expression and select the clone with desired expression level (see Note 15).

4. Notes 1. You must obtain approval from your institutional biosafety committee (IBC) before producing and/or utilizing lentivirus in the laboratory. All the vectors and the packaging systems should be listed in the IBC. Safety hoods, incubators, and centrifuges used for lentivirus work should be clearly indicated in the laboratory. 2. Refer to the manual from the commercial supplier for subculturing conditions for 293FT cells. Always use low passage number of 293FT cells for virus production. 293FT with high passage number will result in significantly lower viral titers. Do not allow the cell line to become overconfluent at any time. 3. The ratio and amount of plasmids are variable and should be optimized based on the titers obtained.

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4. The mixture will form fine precipitates. However, if they form large precipitates, disperse the precipitates using a Pasteur pipette. 5. You could further concentrate the virus by ultracentrifugation to get a higher titer of virus (18). 6. The method for titering will depend upon the marker available within the LV system utilized. Biological titering will only be useful for LV systems with a selectable marker such as puromycin or blasticidin. If a reporter gene (e.g., green fluorescent protein or DsRed) is present, a flow cytometry-based method is needed. Titering, using qPCR-based methods, will work on all systems regardless of the selection or reporter gene present, but only provides the number of copies of genes present in the sample, and does not reflect the biological infectivity of the lentivirus. Therefore, if qPCR-based methods are utilized, an infectivity coefficient needs to be determined with the addition of a biological titering assay to obtain a ratio between the qPCR titer and the biological titer. 7. Various cell lines may be used for titering the lentivirus such as HeLa (human cervical cancer; ATCC CCL-2) or HT1080 (human fibrosarcoma; ATCC CCL-121). These cells are easy to work with, grow attached to the plate/flask, and rapidly divide. It is generally not recommended to use the packaging cell line (i.e., 293T cells) for titering, as carryover plasmid from the transfection may result in an overestimate of the titer obtained. 8. Lentiviruses should not be thawed more than three times. Viral titers are known to decrease with each thaw. 9. The actual concentration for puromycin selection will need to be determined for the different cell lines used as the sensitivity of alternate cell lines to puromycin may differ from that reported here. 10. This protocol shown below is based upon Clontech’s protocol for determining the lentivirus titer via RNA isolation. There are also other qPCR-based methods that can be done by isolating DNA and/or RNA at various steps through virus production and using gene-specific primers for the lentiviral vectors such as LV1, LV2, GAG, etc. (18, 19). 11. NK cells grow well at a density of 0.5–1.0 × 106 cells/mL. In our experience, we see that they do not transduce well if they are plated at a lower or higher density.

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12. Stimulation of DERL7 cell line with human IL-2 (100 U/mL) and stem cell factor (250 ng/mL) results in better transduction efficiencies. 13. Retronectin can be used as an alternative to polybrene as human NK cells express VLA-4 and VLA-5, cell surface molecules which mediate adhesion to fibronectin (20). 14. Conditioned NK cell medium is filter-sterilized medium from log-phase culture of NK-92 or DERL-7 cell lines. This medium provides additional factors for the growth and survival of infected NK cells. 15. Expression or knockdown of the gene can be assayed by western blot or real-time PCR. 16. LNK cells are adherent cells and can be dislodged from the culture dish by washing once with PBS and then incubating the cells in PBS containing 1 mM EDTA for 5 min at 37◦ C in a CO2 incubator. The cells should then be washed with PBS to remove EDTA from the cells.

Acknowledgments This research was supported by the Intramural Research Program of the National Cancer Institute − Center for Cancer Research (NCI-CCR), National Institutes of Health. We thank Dr. Morihiro Watanabe for his help and advice in the development of lentivirus protocols for infection of NK cells. We thank Dr. Geraldine O’Connor for critical review of the chapter. The authors do not endorse any particular commercial products mentioned in this chapter nor should the specific product designations be interpreted as an endorsement of the products by the US Government.

References 1. Trinchieri, G. (1989) Biology of natural killer cells. Adv Immunol 47, 187–376. 2. Grund, E. M. and Muise-Helmericks, R. C. (2005) Cost efficient and effective gene transfer into the human natural killer cell line, NK92. J Immunol Methods 296, 31–36. 3. Maasho, K., Marusina, A., Reynolds, N. M., Coligan, J. E., and Borrego, F. (2004) Efficient gene transfer into the human natural killer cell line, NKL, using the Amaxa nucleofection system. J Immunol Methods 284, 133–140.

4. Schoenberg, K., Trompeter, H. I., and Uhrberg, M. (2008) Delivery of DNA into natural killer cells for immunotherapy. Methods Mol Biol 423, 165–172. 5. Jiang, W., Zhang, J., and Tian, Z. (2008) Functional characterization of interleukin15 gene transduction into the human natural killer cell line NKL. Cytotherapy 10, 265–274. 6. Kirwan, S., Merriam, D., Barsby, N., McKinnon, A., and Burshtyn, D. N. (2006) Vaccinia virus modulation of natural killer cell function by direct infection. Virology 347, 75–87.

Lentiviral Gene Transduction in Human and Mouse NK Cell Lines 7. Tran, J., and Kung, S. K. (2007) Lentiviral vectors mediate stable and efficient gene delivery into primary murine natural killer cells. Mol Ther 15, 1331–1339. 8. Schroers, R., Hildebrandt, Y., Hasenkamp, J., Glass, B., Lieber, A., Wulf, G., and Piesche, M. (2004) Gene transfer into human T lymphocytes and natural killer cells by Ad5/F35 chimeric adenoviral vectors. Exp Hematol 32, 536–546. 9. Goding, S., Yang, Q., Mi, Z., Robbins, P. D., and Basse, P. H. (2007) Targeting of products of genes to tumor sites using adoptively transferred A-NK and T-LAK cells. Cancer Gene Ther 14, 441–450. 10. Goding, S. R., Yang, Q., Knudsen, K. B., Potter, D. M., and Basse, P. H. (2007) Cytokine gene therapy using adenovirally transduced, tumor-seeking activated natural killer cells. Hum Gene Ther 18, 701–711. 11. Guven, H., Konstantinidis, K. V., Alici, E., Aints, A., Abedi-Valugerdi, M., Christensson, B., Ljunggren, H. G., and Dilber, M. S. (2005) Efficient gene transfer into primary human natural killer cells by retroviral transduction. Exp Hematol 33, 1320–1328. 12. Gong, J. H., Maki, G., and Klingemann, H. G. (1994) Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 8, 652–658. 13. Yodoi, J., Teshigawara, K., Nikaido, T., Fukui, K., Noma, T., Honjo, T., Takigawa, M., Sasaki, M., Minato, N., Tsudo, M., et al. (1985) TCGF (IL 2)-receptor inducing factor(s). I. Regulation of IL 2 receptor on a natural killer-like cell line (YT cells). J Immunol 134, 1623–1630. 14. Robertson, M. J., Cochran, K. J., Cameron, C., Le, J. M., Tantravahi, R., and Ritz, J. (1996) Characterization of a cell line,

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NKL, derived from an aggressive human natural killer cell leukemia. Exp Hematol 24, 406–415. Di Noto, R., Pane, F., Camera, A., Luciano, L., Barone, M., Lo Pardo, C., Boccuni, P., Intrieri, M., Izzo, B., Villa, M. R., Macri, M., Rotoli, B., Sacchetti, L., Salvatore, F., and Del Vecchio, L. (2001) Characterization of two novel cell lines, DERL-2 (CD56+/CD3+/Tcry5+) and DERL-7 (CD56+/CD3-/ TCRgammadelta-), derived from a single patient with CD56+ nonHodgkin s lymphoma. Leukemia 15, 1641–1649. Hodge, D. L., Schill, W. B., Wang, J. M., Blanca, I., Reynolds, D. A., Ortaldo, J. R., and Young, H. A. (2002) IL-2 and IL-12 alter NK cell responsiveness to IFN-gammainducible protein 10 by down-regulating CXCR3 expression. J Immunol 168, 6090–6098. Tsutsui, H., Nakanishi, K., Matsui, K., Higashino, K., Okamura, H., Miyazawa, Y., and Kaneda, K. (1996) IFN-gammainducing factor up-regulates Fas ligandmediated cytotoxic activity of murine natural killer cell clones. J Immunol 157, 3967–3973. Salmon, P., and Trono, D. (2006) Production and titration of lentiviral vectors. Curr Prot Neurosci Chapter 4, Unit 4 21. Sastry, L., Johnson, T., Hobson, M. J., Smucker, B., and Cornetta, K. (2002) Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods. Gene Ther 9, 1155–1162. Gismondi, A., Morrone, S., Humphries, M. J., Piccoli, M., Frati, L., and Santoni, A. (1991) Human natural killer cells express VLA-4 and VLA-5, which mediate their adhesion to fibronectin. J Immunol 146, 384–392.

Chapter 15 Introduction of shRNAs into Human NK-Like Cell Lines with Retrovirus Amanda K. Purdy and Kerry S. Campbell Abstract Natural killer (NK) cell lines are difficult to transfect using standard techniques, which limits the ability to establish long-term knockdown of proteins with short-hairpin (sh)RNAs. We have developed a method to stably knockdown protein expression in human NK-like lines by introducing shRNAs in retroviral vectors. After a single transduction with retrovirus, shRNA-containing cells can be selected with drug treatment or sorted for enhanced green fluorescent protein (EGFP) expression. With this method, protein expression can be stably decreased to less than 10% of wild-type levels. Key words: Retroviral transduction, shRNAs, NK-like cell lines.

1. Introduction Primary NK cells are difficult to transfect with standard vectors under a variety of conditions proven successful in other cell types (1). These cells are also not generally amenable to viral or retroviral infection (2), although recent successes with lentivirus (see protocols by Kung and Savan/Young in this volume) are overcoming this technical hurdle. In contrast, NK-like cell lines are more permissive to gene transfer by transfection (3, 4) and especially by retroviral transduction (2), thereby allowing for overexpression and knockdown of genes of interest. The technique of RNA interference (RNAi) has revolutionized modern cell biology by enabling researchers to selectively eliminate the expression of specific mRNAs. To achieve shortterm knockdown of the target mRNA expression (and subsequent K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 15, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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protein expression), double-stranded short-interfering (si)RNAs of 21–23 nucleotides (nt) matching specific sequences in the target mRNA are introduced into cells by electroporation or lipofection. Alternatively, short-hairpin (sh)RNAs can be expressed in cells (see below) and processed by the Dicer endonuclease complex to generate sustained expression of double-stranded 21–23-nt siRNAs, achieving long-term, stable knockdown of target mRNAs (5). The siRNAs hybridize with target mRNAs to tag them for degradation by the RNAi-induced silencing complex (RISC), which contains an endoribonuclease (6). The stable expression of shRNAs in cell lines can be achieved by transfection with certain vectors or transduction with retroviral or lentiviral vectors. Constructs cloned into these vectors generally consist of a 60-nt oligo that, when expressed in cells, is processed to generate a 19-nt siRNA with uridine overhangs (Fig. 15.1). Expression of the shRNA construct is driven by the polymerase III HI promoter, which produces a small RNA transcript, lacking a poly-A tail, and can be processed into a standard siRNA molecule.

Fig. 15.1. Schematic of a 60-nt siRNA-generating oligo. The ds oligo, flanked by restriction sites for cloning at the 5’ and 3’ ends, is composed of a 19 nucleotide sense sequence of the siRNA, a 9 nucleotide hairpin spacer sequence followed by the complementary 19 nucleotide anti-sense sequence of the siRNA. This figure was reproduced with permission from Oligoengine.

Here, we describe a method for knocking down proteins of interest in NK-like cell lines by the co-expression of two different retroviral vector-based shRNAs. This protocol can be modified to include co-expression of up to three distinct shRNAs simultaneously in the same NK cell line.

2. Materials 2.1. Generating shRNA-Containing Retroviral Vectors

1. Buffered saline: 100 mM NaCl and 50 mM HEPES, pH 7.4, in water 2. shRNA oligos: design an oligo targeting a specific sequence in the mRNA of interest. We have successfully designed

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shRNAs that knocked down the expression of several proteins using the online software provided by Oligoengine (www.oligoengine.com; Seattle, WA). The software scans an input mRNA sequence to predict the optimal target siRNAs. Upon choosing a particular target sequence, the software designs a double-stranded oligo consisting of a sense and an anti-sense strand with an intervening hairpin sequence. Each oligo also contains appropriate restriction site overhangs (Bgl II and Hind III) for cloning. These oligos can be purchased directly from Oligoengine. 3. pSuperior.retro.puromycin, pSuperior.retro.neomycin, and pSuperior.retro.neomycin.gfp vectors (Oligoengine) (see Note 1) 4. Agarose (Invitrogen, Carlsbad, CA) 5. SV Gel and PCR Clean Up System (Promega, Madison, WI) 6. Restriction enzymes: Bgl II, Hind III, EcoRI, and associated buffers (Invitrogen) 7. T4 ligase and 5× ligase buffer (Invitrogen) 8. Stbl2-competent bacterial cells (Invitrogen) 9. Liquid LB supplemented with 0.4% glucose and 50 ␮g/ml ampicillin 10. Wizard Plus SV Miniprep kit (Promega) 11. High-purity plasmid purification system (Marlingen, Ijamsville, MD) 2.2. Generating Retrovirus and Transduction of NK-Like Cell Lines

1. Phoenix-amphotropic retroviral packaging cell line (a gift from Dr. Garry Nolan, Stanford University, Stanford, CA) 2. Complete RPMI medium: RPMI-1640 medium (Mediatech, Herndon, VA) containing 10% heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT), 2 mM Lglutamate, 100 IU/ml penicillin (Mediatech), 100 ␮g/ml streptomycin (Mediatech), 50 mM HEPES, and 50 ␮M 2mercaptoethanol (ME) 3. Complete ␣-MEM: ␣-minimum essential medium (MEM; Life Technologies, Rockville, MD) containing 10% heat-inactivated FBS, 10% heat-inactivated horse serum (Invitrogen), 2 mM L-glutamate, 100 IU/ml penicillin, 100 ␮g/ml streptomycin, 1 mM sodium pyruvate (SigmaAldrich, St. Louis, MO), 200 ␮M myoinositol (SigmaAldrich), 2.5 ␮M folic acid (Sigma-Aldrich), 1× nonessential amino acids (Mediatech), and 100 ␮M 2-ME 4. OPTI-MEM reduced serum medium (Invitrogen) 5. Recombinant IL-2, available from a variety of commercial sources, such as Roche (Basel, Switzerland) was

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generously provided by the NCI Biological Resources Branch (Frederick, MD). 6. 6- and 12-well culture plates (Fisher Scientific) 7. Plus Reagent and lipofectamine (Invitrogen) 8. 15 ml conical tubes (BD Falcon, USA) 9. Puromycin (Calbiochem, San Diego, CA) dissolved in DMSO at 5 mg/ml stock concentration 10. Neomycin dissolved in HEPES buffer, pH 7.2, at a stock concentration of 50 mg/ml (Fisher Scientific)

3. Methods 3.1. Designing shRNAs

There are numerous free programs available for designing shRNAs (Oligoengine, Dharmacon, etc.). For this protocol, Oligoengine software was used, since the interface was user-friendly, GenBank sequences could be uploaded directly, and both the secondary structure and the nucleotide usage were considered in the selection algorithm. We suggest designing a minimum of four shRNAs for each gene of interest, since we encountered an approximate 50% success rate. For knocking down SHP-2 phosphatase in the human NK-like cell line, KHYG-1, the expression of a single shRNA resulted in ∼50% decrease in wild-type protein levels, while co-expression of two shRNAs decreased levels by >90% (7). Avoid designing shRNAs that target common domains within gene families (e.g., phosphatase domain), as these will be less specific for the gene of interest and could nonspecifically suppress other members of the gene family. The final sequence should be tested for homology with other mRNAs with the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and sequences with high homology to other known sequences should be abandoned. In addition, do not limit shRNAs to just one region of the mRNA. Finally, generate scrambled versions of each shRNA (having the same nucleotides as a shRNA targeting the gene of interest, in a randomly scrambled order) for use as controls. Scrambled control sequences should also not crossreact with known mRNAs in the targeted species, as assessed by a BLAST search. Alternatively, predesigned/pretested shRNAs of many genes cloned into retroviral vectors can be purchased from a commercial provider (e.g., Santa Cruz Biotechnology Inc., Santa Cruz, CA; Sigma-Aldrich, St. Louis, MO; Invitrogen).

3.2. Generating shRNA-Containing Vectors

The procedure for generating shRNA constructs was adapted from the Oligoengine pSuperior protocol. The protocol can be adapted for other specific vector and restriction enzyme combinations.

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1. To generate double-stranded shRNAs, combine 1 ␮l of each single-stranded oligo (both sense and anti-sense strands with compatible restriction site overhangs at 3 mg/ml) in 48 ␮l of buffered saline. Heat the oligo mix to 95◦ C for 10 min, and then slowly cool to RT (see Note 2). Once RT is reached, shRNAs can be immediately used or stored at 4◦ C for future use. 2. Digest pSuperior.retro vectors with BglII and Hind III restriction enzymes for 1 h at 37◦ C. 3. Separate the digested vector on a 1% agarose gel and purify with Promega Gel Extraction kit according to the manufacturer’s instructions. 4. Ligate shRNAs (1 ␮l of 1:100 dilution) into the purified linearized vector at 14◦ C overnight (see Notes 3 and 4). 5. Transform Stbl2 recombination-deficient competent cells (see Note 5) with 8 ␮l of the ligation reaction. 6. Plate the bacteria on LB culture plates containing 50 ␮g/ml ampicillin and grow at 30◦ C overnight. 7. Pick at least six colonies and culture each in 5 ml liquid LB with 0.4% glucose and 50 ␮g/ml ampicillin at 30◦ C overnight (see Note 6). 8. Isolate bacterial DNA with the Promega Miniprep kit, digest with Eco RI and Hind III for 1 h at 37◦ C, and separate digests on 1% agarose gels. Positive colonies are identified by the presence of a 300 bp band upon digestion (see Note 7). 9. Confirm the orientation of the shRNA insert by sequencing. 3.3. Transfection of Phoenix-Ampho Cells

It is important for both the Phoenix-amphotropic and the NKlike cell lines to be freshly passed a day prior to transfection and transduction, respectively. This protocol works well for KHYG-1, NKL, NK3.3, and NK-92 cell lines (7). Day 0: 1. One day prior to transfection, plate 5 ml Phoenixamphotropic cells into a 6-well culture plate (9 ml of a confluent Phoenix culture +21 ml of complete RPMI medium) and culture overnight in a 37◦ C incubator with 7% CO2 atmosphere (see Notes 8 and 9). Day 1: 2. For each transfection reaction, combine 4 ␮g of each shRNA-containing plasmid, 10 ␮l Plus Reagent, and OPTI-MEM to a final volume of 100 ␮l and incubate for 15 min at RT.

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3. In a separate tube, combine 8 ␮l lipofectamine with 92 ␮l OPTI-MEM for each reaction and incubate for 15 min at RT. 4. After 15 min, combine the plasmid and lipofectamine solutions and incubate for 15 min more. 5. During the incubation, wash Phoenix-amphotropic cells once with 6 ml OPTI-MEM (see Note 10). 6. Add 800 ␮l of OPTI-MEM to the plasmid/lipofectamine solution (total volume is now ∼1 ml). Remove the wash solution from Phoenix-amphotropic cells and cover the cells with the plasmid/lipofectamine solution. 7. Transfect cells for at least 3 h at 37◦ C and then cover with 4 ml complete RPMI medium and incubate overnight. Day 2: 8. On the next day (afternoon), remove the medium, wash once with 6 ml OPTI-MEM, and cover cells with 2 ml OPTI-MEM. Day 3: 9. Collect the Phoenix cell supernatant containing the retrovirus and transfer into a 15 ml conical tube (see Note 11). Remove any cells by centrifugation at 500×g for 3 min. Transfer the viral supernatant to a new tube containing 20 ␮l of Plus Reagent. 10. Incubate the viral supernatant for 15 min at RT. 11. After 15 min, add 8 ␮l of lipofectamine and incubate 15 min longer (see Note 12). 12. During incubation, prepare NK cells for transduction by washing once with OPTI-MEM. 13. Transfer 5 × 105 NK cells to a 15 ml conical tube and pellet cells by centrifugation 500×g to remove the medium. 14. Resuspend the NK cells in the viral supernatant (about 2 ml), transfer to a 12-well cell culture plate, and spin at 700×g for 30 min at RT. 15. Incubate cells for at least 6 h at 37◦ C. 16. After incubation, cover cells with ∼2 ml complete ␣MEM with 50 U/ml recombinant IL-2 and incubate cells overnight at 37◦ C (see Note 13). Day 4: 17. Remove supernatant and continue to culture in fresh complete ␣-MEM with 50 U/ml recombinant IL-2 (see Note 14). 3.4. shRNA Selection

Selection scheme will depend upon which vectors (puromycin, neomycin, or gfp) were used.

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Day 6: 1. After resting cells for 2 days following transduction, add fresh complete ␣-MEM with 50 U/ml recombinant IL-2 and/or 2.5 ␮g/ml puromycin, 1.25 mg/ml neomycin (see Notes 15–17). Day 7: 2. Remove the old medium and add fresh complete ␣MEM with 50 U/ml recombinant IL-2 and/or 2.5 ␮g/ml puromycin, 1.25 mg/ml neomycin (see Notes 18). Day 9: 3. Remove the old medium and add fresh, drug-free complete ␣-MEM with 50 U/ml recombinant IL-2. 4. Check knockdown at the protein level by Western blotting or at the mRNA level by RT-PCR. For SHP-2 and SHP-1, protein knockdown was observed after 3 days of drug treatment (earliest time point analyzed), but knockdown did not become stable and consistent until 7 days after drug treatment was concluded. 5. Compare cellular protein or mRNA levels in cells transduced to express single shRNAs versus cells expressing the scrambled shRNAs or empty pSuperior vector.

4. Notes 1. Depending upon your selection scheme (selection with antibiotics versus GFP expression), it is important to choose the appropriate retroviral vector(s). With this system, one can express and select for up to three separate shRNAs at the same time (one shRNA in puromycin, neomycin, and gfp vectors). We have had success using each shRNA-containing vector singly or all together. 2. It is easiest to use a heat block for this step, turning it off after the 95◦ C incubation and allowing the block to cool to RT. 3. It is recommended to have a 1:3 ratio of vector to insert for the ligation reaction. Some PCR and gel extraction kit elution buffers disrupt accurate determinations of the DNA concentration by absorbance spectroscopy. To avoid this issue, measure the concentration of the insert and digested vector on an agarose gel using a DNA ladder for the concentration control. 4. To decrease the number of false-positive colonies, digest the ligation reaction with Bgl II for 1 h at 37◦ C. The Bgl II

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site is destroyed upon successful cloning of the shRNA pair; therefore, vectors containing the shRNA insert will not be cut. 5. To prevent recombination at retroviral LTRs, always use recombination-defective bacteria (e.g., Stbl2) when working with retroviral vectors, and always culture bacteria at 30◦ C. 6. Grow colonies for longer times to compensate for the reduced growth temperature. 7. False positives that stem from re-ligation of empty vector will have a ∼1 kb band in the Oligoengine system. 8. Phoenix cells should be ∼80% confluent for optimal transfection. Never allow the cells to reach full confluence or transduction efficiency will suffer. 9. Several varieties of the Phoenix packaging cells are available (e.g., amphotropic, ecotropic, polytropic), which differ primarily in the expression of the viral envelope proteins that mediate viral entry into target cells (8). Use Phoenixamphotropic cells when generating virus for transduction of human cells, Phoenix-ecotropic for murine cells. 10. Phoenix cells are semi-adherent and can easily detach from the plate. Exercise caution when manipulating the cells or when changing the medium. Add medium very slowly to the side of the 6-well plate, keeping the pipette tip horizontal to the plane of the cells. Never add medium directly on top of the cells. 11. Although the retrovirus is replication defective and should be noninfectious, be sure to UV treat all contaminated glass and plasticware for at least 1 h before discarding. The use of retroviral technology requires standard class BSL2 biohazard safety precautions and approval by the local biohazard safety committee in your institution. 12. Retrovirus can be used immediately or can be stored at −80◦ C for up to 4 months with an estimated potency loss of ∼50%. We have had success using virus that was frozen to co-transduce KHYG-1 cells with two vectors simultaneously when subsequently selected in medium containing the appropriate combination of antibiotics. 13. To increase viability, let cells detach from the 12-well plate overnight instead of forcing cell detachment with pipetting. 14. Transduction efficiency varies widely among known NK-like cells lines. For example, NK-92 cells had ∼4% transduction efficiency with this protocol, while KHYG-1 had almost 30% efficiency for single vector transductions.

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15. To improve drug selection, expand cells to multiple wells if overgrown. 16. Determine the optimal concentration of drug needed to kill each NK-like cell line being used. For transduction of KHYG-1, NK-92, and NKL cells, puromycin was used at 2.5 ␮g/ml and G418 at 1.25 mg/ml. 17. To ensure proper drug selection, include a well of nontransduced cells during selection. 18. Puromycin-induced death was observed by days 1–2 of drug treatment and days 3–5 for neomycin.

Acknowledgments We would like to thank Drs. Lauren O’Donnell and S. M. Shahjahan Miah for helpful comments on the chapter. Supported by National Institutes of Health grants R01-CA083859, R01CA100226 (K.S.C.), T32-CA009035 (A.K.P.), and Centers of Research Excellence grant CA06927 (FCCC). The research was also supported in part by an appropriation from the Commonwealth of Pennsylvania. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute. References 1. Goldfarb, R. H., Whiteside, T. L., Basse, P. H., Lin, W. C., Vujanovic, N., and Herberman, R. B. (1994) Natural killer cells and gene therapy: potential of gene transfection for optimizing effector cell functions and for targeting gene products into tumor metastases. Nat Immunol 13, 131–140. 2. Nagashima, S., Mailliard, R., Kashii, Y., Reichert, T. E., Herberman, R. B., Robbins, P., and Whiteside, T. L. (1998) Stable transduction of the interleukin-2 gene into human natural killer cell lines and their phenotypic and functional characterization in vitro and in vivo. Blood 91, 3850–3861. 3. Liu, J. H., Wei, S., Blanchard, D. K., and Djeu, J. Y. (1994) Restoration of lytic function in a human natural killer cell line by gene transfection. Cell Immunol 156, 24–35. 4. Tran, A. C., Zhang, D., Byrn, R., and Roberts, M. R. (1995) Chimeric

5. 6. 7.

8.

zeta-receptors direct human natural killer (NK) effector function to permit killing of NK-resistant tumor cells and HIVinfected T lymphocytes. J Immunol 155, 1000–1009. Tijsterman, M., Ketting, R. F., and Plasterk, R. H. (2002) The genetics of RNA silencing. Annu Rev Genet 36, 489–519. Hannon, G. J. (2002) RNA interference. Nature 418, 244–251. Purdy, A. K. and Campbell, K.S. (2009) SHP-2 expression negatively regulates NK cell function. J Immunol in press. Danos, O. and Mulligan, R. C. (1988) Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc Natl Acad Sci U S A 85, 6460–6464.

Chapter 16 Introduction of shRNAs into Primary NK Cells with Lentivirus Sam K.P. Kung Abstract Natural killer (NK) cells are lymphocytes that provide an important line of defense against viruses and tumors. Technical hurdles in genetic modifications of primary NK cell ex vivo had limited our studies of protein function(s) in NK cell differentiation, acquisition of self-tolerance, and induction of antitumor responses. We used VSV-G-pseudotyped, EGFP-expressing lentiviral vectors to develop an efficient gene transfer system to modify gene expression in primary murine NK cells with or without prior IL-2 activation. Lentiviral vector transduction did not impair NK cellular viability, phenotype, or functions. We also demonstrated the use of this system in modifying differentiating NK cells derived from lentiviral-transduced murine hematopoietic progenitor cells. Furthermore, the same transduction protocol is amendable to delivery of short-hairpin RNA (shRNA) for specific gene silencing. Collectively, our approach in genetic engineering of primary murine NK cells will prove useful in studying basic NK cell biology and in exploring therapeutic potentials of NK cells in inbred and transgenic mouse models. Key words: Lentiviral vectors, natural killer cells, LAK, mouse, transduction, gene therapy.

1. Introduction The availability of a simple and efficient methodology that supports long-term and stable transduction of genetic materials into primary NK cells and NK progenitors will empower us the ability to acquire greater mechanistic understanding of NK cell differentiation and receptor function(s) both in vitro and in vivo. From a therapeutic point of view, the ability to manipulate NK cells directly to express novel receptors or genetically engineered NK receptors will allow us to enhance NK target recognition and redefine NK target specificities.

K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 16, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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A major challenge in genetically engineering primary NK cells is to introduce stable genetic modification of NK cells efficiently with minimal in vitro manipulation, and without any impairment in cellular viability, phenotype, or functions. Our laboratory used replication-incompetent HIV-1 lentiviral vectors to develop a complementary strategy to the existing methods to deliver either a transgene (1) or a short-hairpin RNA (shRNA) into primary NK cells. Lentiviral vectors transduce and integrate into host genomes without the need of cell division (2–4). They will therefore support persistent expression of introduced genetic materials in modified NK cells (progenitors). In addition, as they can transduce both nondividing and dividing cells in vitro and in vivo (4–6), they may also be used to circumvent the need of an in vitro activation step in NK cell transduction and/or to improve transduction efficiency of primary human and mouse NK cells. Here, we describe a single-step lentiviral transduction of primary murine NK cells that supported efficient transduction and stable expression of the transferred genetic materials without any apparent compromise in the cellular viability, phenotype, or functions (1).

2. Materials 2.1. DNA Plasmids

1. Second-generation lentiviral vector packaging system (Fig. 16.1): Three-plasmid components are needed to produce replication-incompetent lentiviral particles. It includes a gene transfer vector of choice to transduce NK cells to either over-express a transgene [e.g., FUGW or pRRLSIN.cPPT.PGK-GFP, Addgene, Cambridge, MA; SIN18RhMLV-Cppt2E (7, available from our laboratory upon request)] or to down-regulate endogenous gene expression via RNA interference (e.g., pLKO or FG12) (Addgene), a packaging plasmid (pCMV-dR8.2dvpr) (Addgene), and an envelope plasmid (pCMV-VSVG) (Addgene) for pseudotyping. 2. Stbl3 (Invitrogen, Carlsbad, CA)-competent cells for transformation. 3. Plasmids are isolated using commercial plasmid purification kits such as Qiagen Plasmid Midi Kit (Qiagen, Valencia, CA) and Nucleobond Plasmid Midi Kit (Clontech, Mountain View, CA).

2.2. Viral Vector Preparation, Concentration, and Transduction

1. Iscove’s Modified Dulbecco’s Medium (IMDM) (HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS) (HyClone) and 1% penicillin/streptomycin/ glutamate (PSG) (Invitrogen).

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Fig. 16.1. Lentiviral vector production. A Second-generation three-plasmid transfection system for replicationincompetent lentiviral vector productions. Three plasmid components are needed to produce replication-incompetent lentiviral particles in 293T cells: (1) gene transfer vector of choice, (2) a packaging plasmid, and (3) an envelope plasmid for pseudotyping (see Section 2.1). Viral particles are collected in the culture supernatant and can be further concentrated by ultracentrifugation. B Examples of lentiviral vectors for NK cell transduction. Lentiviral vectors that use internal promoters RhMLV (rhesus-adapted Murine Leukemia Virus LTR) (SIN18-RhMLV-cppt2E), PGK (phosphoglycerate kinase) (pRRL.SIN.cPPT.PGK.-GFP.WPRE), or UbiC (Ubiquitin-C) (FUGW) are used in transgene over-expression in NK cells. In shRNA-expressing lentviral vectors (FG12 and pLKO.1), U6 promoter is used to direct RNA Polymerase III transcription of the shRNA. Reporter genes (EGFP or puromycin-resistant gene) are expressed under a separate transcription cassette driven by an internal UbiC or PGK promoter. Note that the use of FG12 requires cloning of an expression cassette that contains U6 RNA pol III promoter and the shRNA target sequence of interest. Suggested restriction sites for cloning are indicated. C Schematic representation of a 5 −3 oligonucleotide that contains a shRNA target sequence for cloning (see Section 3.5). Synthesize the complementary 3 −5 oligonucleotide for annealing. Selection of G as the initiating nucleotide facilitates U6 promoter transcription. The shRNA is followed by a polyT termination sequence for RNA Polymerase III.

2. Phosphate-buffered saline (PBS), pH 7.4, autoclaved. 3. Chloroquine (Sigma, St. Louis, MO) is reconstituted in autoclaved PBS at 10 mM stock concentration. The solution is sterilized by 0.22 ␮m filter cup, stored in aliquots at −20◦ C. 4. Hexadimethrine bromide (Polybrene) (Sigma) is reconstituted in autoclaved PBS at 1 mg/ml stock concentration, sterilized using 0.22 ␮m filter cup, and stored in aliquots at −20◦ C. 5. 2 M CaCl2 (Tissue culture grade, Sigma) solution is prepared by dissolving CaCl2 salt in autoclaved distilled water. ◦ It is sterilized by 0.22 ␮m filter and stored at 4 C.

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6. 2× HEPES-buffered saline (HBS) (100 ml): 50 mM HEPES (1.19 g) 280 mM NaCl (1.63 g) 1.5 mM Na2 HPO4 Pre-prepared 0.25 M Na2 HPO4 stock (0.6 ml) NaOH (5 M) and (1 M) for pH adjustment to 7.12 All chemicals are purchased from Sigma. Bring volume up to 100 ml. Filter solution with 0.22 ␮m filter. Store at ◦ 4 C. 7. Beckman ultracentrifuge with a SW41 or SW28 rotor. Polyallomer centrifuge tubes (Beckman, Palo Alto, CA). 8. Puromycin (Calbiochem, San Diego, CA, USA) is dissolved in PBS at 2 mg/ml stock solution. 9. 0.05% trypsin−EDTA (Invitrogen) in autoclaved PBS. 10. Sterilized 50 ml polypropylene conical tubes, T175 flask (BD Falcon, Franklin Lakes, NJ) 11. 293T cells (GenHunter, Nashville, TN, USA). 12. HIV-1 p24 ELISA (PerkinElmer Life Sciences, Inc., Waltham, Massachusetts, USA). 2.3. Natural Killer Cells Purification, Activation, and Expansion In Vitro

1. Roswell Park Memorial Institute 1640 (RPMI 1640) culture medium: RPMI 1640 (HyClone) supplemented with 10% FBS, 1% PSG, and 1.6 mM 2-mercaptoethanol (Sigma). 2. EasySep Mouse NK Negative Selection Kit (StemCell Technologies, Vancouver, British Columbia, Canada). 3. IL-2 (Peprotech, Rocky Hill, NJ, USA) is dissolved in autoclaved distilled water at 0.1 mg/ml, further diluted with RPMI 1640 culture medium to obtain a stock solution of 100 U/␮l. The cytokine is stored in aliquots at −70◦ C until use. A final concentration of 1000 U/ml is used in NK cultures.

2.4. Isolation of Progenitor Stem Cells (PSCs) and NK Differentiation In Vitro

1. Progenitor stem cells are enriched using an EasySep Mouse Progenitor Stem Cell Negative Selection kit (StemCell Technologies). 2. Cytokines IL-2, IL-7, and IL-15, stromal cell factor (SCF), and Flt3L (Peprotech) are reconstituted according to manufacturer’s instruction. 3. NK differentiation-conditioned medium: RPMI 1640 supplemented with 10% FBS, 1% PSG, 1.6 mM 2-ME, 0.5 ng/ml of mIL-7, 30 ng/ml of SCF, and 50 ng/ml Flt3L.

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4. RPMI 1640 culture medium containing 30 ng/ml of IL-15. 5. 24-, 48-well tissue culture plate (BD Falcon).

3. Methods 3.1. VSV-GPseudotyped Lentiviral Vector Production 3.1.1. Preparing 293T Cells for Transfection on Day 0 (see Note 1)

Adherent 293T cells are collected by trypsin treatment and gentle tapping of the flask (Fig. 16.1). 1. Aspirate and discard the IMDM culture medium from the T175 flask. 2. Add 15 ml of PBS to rinse the adherent cells. 3. Aspirate and discard PBS. 4. Add 3 ml trypsin−EDTA to the flask to cover all the adherent 293T cells. Leave the flask at room temperature for 5 min. 5. Gently tap the bottom of the flask to dislodge the cells into suspension. 6. Add 10 ml of IMDM culture medium to the flask. 7. Pipette up and down a few times to prepare single-cell suspension. 8. Centrifuge cells at 400 × g for 5 min to collect cells. 9. Resuspend cells in 10 ml IMDM culture medium. Count cells. 10. Culture 2 × 107 293T cells in 25 ml IMDM culture medium in a T-175 flask on the day before transfection (see Note 2).

3.1.2. Three Plasmids Transfection of 293T Cells on Day 1

1. Aspirate to remove the IMDM culture medium from the 293T cell culture flask. Feed them with 25 ml fresh warm medium. 2. Prepare the DNA mix in a 50 ml conical tissue culture centrifuge tube. A total of 30 ␮g of plasmid DNA should be used for each T175 flask (VSVG 5 ␮g, packaging 12.5 ␮g, and vector 12.5 ␮g). Add autoclaved ddH2 O to a final volume of 977 ␮l (see Notes 3 and 4). 3. Add 133 ␮l of 2 M CaCl2 into the DNA mix, vortex well, and leave on ice for 5 min. 4. Add 1110 ␮l of 2× HBS solution drop by drop, while simultaneously mixing the solution by vortex. Mix again and rest it on ice for 20 min. 5. Flip the T175 flask upside down to drain the culture medium to the other side of the flask that does not have

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cells (to avoid detaching 293T cells during the mixing of DNA precipitates). Add the mixed DNA precipitates into the culture medium drop by drop and mix gently (see Note 5). 6. Add 100 ␮l of chloroquine (10 mM) to the culture. 7. Incubate the culture for 6–8 h at 37◦ C, 5% CO2 . 8. After 6–8 h incubation, change medium with 35 ml of fresh medium to remove the transfection mix. Do not touch cells for 36 h (see Note 6). 9. On Day 3, harvest the culture supernatant as virus and keep at 4◦ C. Feed the culture again with 25 ml of fresh IMDM culture medium. 10. On day 4, collect supernatant again. 3.1.3. Concentrating VSV-Pseudotyped Lentiviral Vectors (to 100 ×)

1. Centrifuge viral supernatant at 500 × g for 5 min, 4◦ C, to remove cell debris. 2. Filter the supernatant through 0.45 ␮m filter cups. 3. Transfer 35 ml of viral supernatant into a polyallomer ultracentrifuge tube for SW28 rotor (see Note 7). 4. Centrifuge at 50,000×g (16,000 rpm) at 4◦ C for 90 min in a Beckman ultracentrifuge to collect viral particles at the bottom of the ultracentrifuge tube. 5. Remove the supernatant by decantation and aspirate residual supernatant with pipetman. Add IMDM culture medium to a total volume of 350 ␮l. 6. Use parafilm to seal the ultracentrifuge tubes and leave them at 4◦ C overnight for the viral vector pellet to resuspend. 7. Use a P-1000 pipetman to pipette up and down the viral particles three times to resuspend the viral particles in the next morning. Store the viral particles in aliquots at −70◦ C. Avoid freeze-and-thaw. 8. Viral particles and wastes should be handled according to the biosafety regulation and guidelines from your institution (see Note 8).

3.2. Determination of Viral Titers

For the lentiviral vectors that express a puromycin-resistant gene or proteins (surface or cytosolic) detectable by flow cytometry. 1. Plate 5 × 104 cells/well of 293T cells in a 24-well tissue culture dish a day before titration. With single doubling time, it will become 105 cells on the day of transduction. 2. On day of titration dilute virus (keep virus on ice as much as possible): 100× → 10× (10 ␮l virus in 90 ␮l IMDM medium)

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10×→ 1× (40 ␮l 10× in 360 ␮l IMDM medium) 1×→ 1/10× (30 ␮l 1× in 270 ␮l IMDM medium) 3. Add polybrene to the diluted viruses (typically 1×, 1/10×) at a final concentration of 8 ␮g/ml. Mix well. 4. Remove tissue culture medium from the wells. Add 250 ␮l of the diluted viruses to each well of 293T cells. Include 1–2 mock transduction control wells (tissue culture medium plus polybrene). 5. Incubate at 37◦ C in a tissue culture incubator for 2 h. 6. Remove virus-containing medium. Add 1 ml fresh IMDM medium and continue to culture for 3 days. 7. For the vectors that express a puromycin-resistant gene, add puromycin-containing tissue culture medium at 8 ␮g/ml on day 2 for an additional 2 days. Leave a mocktransduced control well without puromycin treatment to determine total cell number. Determine the number (and percentage) of cells that are resistant to puromycin for each viral dilution tested. 8. For vectors that express surface or intracellular proteins detectable by flow cytometry. Harvest cells and determine the percentage of the transduced cells that express the gene of interest: For surface proteins, use specific mAb in standard surface staining procedures for flow cytometry. For intracellular proteins, use specific mAb in intracellular staining procedures for flow cytometry. For fluorescence proteins, such as EGFP, the transduced cells can be analyzed in flow cytometry directly (see Note 9). 9. Use the data from the dilution that provided 5–20% transduced cells. Calculate the titer based on this equation: Titer (IU/ml) = (1 × 105 cells) × (% of the transduced cells) × (4) × (dilution factor). 10. For vectors that cannot be titered directly in the above assays, a HIV p24 ELISA is recommended. The p24 concentration is not a direct measurement of the infectious particles but will be useful in normalizing the same amount of viral particles (single or multiple) to be used in the transduction. 3.3. Primary NK Cell Transduction

1. Isolate primary NK cell from splenocytes that are free of T and NKT cell (see Note 10).

3.3.1. Adding Viral Vector Particles to NK Cells

2. Purified NK cells can be used directly for transduction or cultured as LAK cells in IL-2 (1000 U/ml) before transduction. 3. Place 2.0 × 105 cells into a 1.5 ml screw-cap centrifuge tube.

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4. Pellet cells at 1700 × g for 10 min in a microcentrifuge. 5. Remove supernatant. 6. Resuspend cells in 0.25 ml viral mixture [virus + 8 ␮g/ml polybrene + complete RPMI medium (10% FBS, 1% PSG, 1.6 mM 2-mercaptoethanol)] at MOI of 20 (see Note 11). 7. Transduction can be carried out by using either the “spin” or the “no-spin” protocol as described below (see Notes 12–15). 3.3.2. No-Spin Protocol

1. Loosely tighten the cap on centrifuge tube (from step 6). 2. Incubate cells at 37◦ C and 5% CO2 for 2 h. 3. Pellet cells at 1700 × g for 10 min. 4. Remove viral supernatant. 5. Resuspend in 1 ml 1,000 U/ml IL-2.

complete

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containing

6. Transfer to 48-well culture plate. 7. Incubate cells at 37◦ C and 5% CO2 for 3 days before analysis. 3.3.3. Spin Protocol

1. Transfer the resuspended cells into a 48-well culture plate. (see Note 16). 2. Centrifuge plate at 900 × g for 1 h at room temperature. 3. Remove viral supernatant. 4. Resuspend in 1 ml of complete RPMI medium containing 1,000 u/ml IL-2. 5. Incubate cells at 37◦ C and 5% CO2 for 3 days before analysis. Examples of primary NK LAK transduction are shown in Fig. 16.2. Lentiviral vector transduction of primary NK cells is efficient, stable, and applicable to different mouse strains (e.g., B6, SJL/J) (see Note 17).

3.4. Transduction of Bone Marrow Progenitor Stem Cells (PSCs) for NK Differentiation In Vitro

1. Obtain single-cell suspension of bone marrow cells from C57BL/6 mice. 2. Enrich for progenitor stem cells using an EasySep Mouse Progenitor Stem Cell Negative Selection kit (StemCell Technologies). 3. 0.3 × 106 PSCs are placed into each well of a 48-well culture plate (BD Falcon). Transduction is conducted at a MOI of 20 immediately after enrichment of progenitor stem cells using the spin protocol. 4. After transduction, remove virus-containing supernatant. Culture the transduced progenitor stem cells in NK differentiation-conditioned medium (RPMI 1640 supplemented with 10% FBS, 1% PSG, 1.6 mM 2-ME, 0.5 ng/ml of mIL-7, 30 ng/ml of stromal cell factor (SCF), and 50 ng/ml Flt3L.

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Fig. 16.2. Stable and efficient transduction of primary NK LAKs. A Day 3 IL-2-activated B6 LAK or Day 7 IL-2activated SJL/J LAK cells were transduced with SIN18-RhMLV-cppt2E lentiviral vectors at MOI of 20 with the spin protocol. The transduced cells were analyzed for EGFP expression under fluorescence microscopy on days 3, 6, and 10 post-transduction. Mock-transduced NK LAK cells were negative controls for EGFP expression. B A representative flow cytometric analysis of the percentage of EGFP-expressing B6 NK cells on day 6 post-transduction. Percentage of EGFP-expressing NK cells remained relatively unchanged despite their active proliferation during this 10 day posttransduction period. C Down-regulation of SHP-1 phosphatase protein expression in B6 LAK. Day 5 IL-2-activated LAK cells were transduced with EGFP shRNA control or SHP-1 shRNA vectors at MOI = 40 IU/cell on two consecutive days using the “spin” protocol as described. Transduced cells were incubated in IL-2-supplemented medium for 3 days posttransduction. Cells were selected for puromycin resistance (at 24 mg/ml) for 24 h. The selected cells were stained intracellularly with anti-SHP-1/anti-rabbit-AlexaFluor antibodies and then analyzed by flow cytometry 3 days post-puromycin selection.

5. On day 3 post-transduction, add 0.5 ml of fresh NK differentiation-conditioned medium. 6. On day 5, pellet cells to remove old medium from the culture. Add 0.5 ml of complete RPMI medium containing 30 ng/ml of IL-15 to culture the cells. 7. On day 8, add 0.5 ml of fresh IL-15-containing medium. 8. On day 10, replace old medium with fresh RPMI medium containing 30 ng/ml of IL-15 and 1000 U/ml of IL-2. 9. On day 14, analyze the differentiating NK cells in flow cytometry. These NK cells can be further differentiated in the presence of a stromal cell line. 3.5. A Variation of the Common Theme − Introduction of shRNA

RNA interference (RNAi) is an innate cellular process that involves multiple RNA−protein interactions (8, 9). Its gene silencing activity is activated when a double-stranded RNA

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molecule of greater than 19 duplex nucleotides enters the cells, causing degradation of both the dsRNA and the single-stranded RNA (endogenous mRNA) of identical sequences (8, 9). We and others have previously established a single lentiviral vector system to stably express simultaneously both a reporter gene (EGFP or puromycin-resistant gene) and a short-hairpin RNA (shRNA) to induce RNA interference (RNAi) in cell lines and primary T cells (10–12). Using the lentiviral transduction protocol we described in Section 3.3, we are successful in introducing shRNA into primary NK cells for specific gene silencing. We use the U6 promoter to direct RNA Polymerase III transcription of the shRNA. The sequence encoded in the dsRNA contributes to the potency of the dsRNA in degradation of target mRNA (13). 1. Determine the “optimal” 21-mer target sequence in your gene (see Note 18). 2. Synthesize forward and reverse shRNA oligonucleotides that contain the overhanging restriction enzyme sites for cloning, 21 “sense” bases identical to the target gene, a loop, and 21 “antisense” bases that are complementary to the “sense” bases. The shRNA is followed by a polyT termination sequence for RNA Polymerase III (see Note 19, Fig. 16.1C). 3. Anneal the oligonucleotides and clone into the restriction enzyme sites of lentiviral vectors containing a U6 pol III promoter (e.g., pLKO) (see Note 20). 4. Produce VSV-G-pseudotyped lentiviral particles as described in Section 3.1. 5. Isolate primary NK cells and culture them in IL-2supplemented RPMI medium (1000 U/ml) for 3–5 days. 6. Transduce NK LAK cells with the shRNA-containing lentiviral particles at a MOI of 20, using the “spin” protocol as described in Section 3.3. Use a lentiviral vector that contains an irrelevant target gene sequence (e.g., luciferase) or scrambled RNA sequence for specificity control. Culture the transduced cells in IL-2-supplemented RPMI medium. 7. Repeat the transduction on the next day to increase the transduction efficiency if necessary. 8. For lentiviral vectors that express EGFP reporter protein (e.g., FG12), the transduced LAK cells were cultured in IL2-supplemented medium (1000 U/ml) for 2–3 days before carrying out the analyses or cell sorting in flow cytometry. 9. For lentiviral vectors that express puromycin resistance gene, culture the transduced LAK in IL-2-supplemented medium (1000 U/ml) for 3 days before the addition of selection medium (i.e., puromycin) at a concentration of 24 ␮g/ml for an additional 24 h. Analyze the puromycin-resistant

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LAK cells for phenotype and functions or expand these selected cells further in IL-2-supplemented medium (see Note 20). An example of the shRNA-mediated silencing of SHP-1 phosphatase in primary NK LAK is shown in Fig. 16.2C.

4. Notes 1. The condition of the cells is very important for optimal transfection, which in turn determines the viral titers obtained in the preparation. They should be growing exponentially and evenly distributed. We do not use 293T cells that have been in passage for more than 3 months even though they look “normal.” 2. We aim at obtaining 70% confluency of 293T cells on the day of transfection (and determined that seeding 18–20 × 106 293T cells a day before the transfection would yield 70% confluency). As culture condition (such as the FCS) used in different laboratories may vary, the number of 293T cells used to seed a T175 the day before transfection may need to be adjusted empirically. 3. 30 ␮g of total DNA mix is used in transfecting a T175 flask of 293T cells. In making a few flasks of the same virus, we simply multiply the amount of each DNA plasmid by the number of flasks (up to 3 flasks) to be mixed in a single 50 ml conical centrifuge tube. Multiple 50 ml centrifuge tubes are used if we will prepare more than 3 flasks of the same virus. 4. New lentiviral vectors for more specific or tighter control in transgene or shRNA expression have been developed in different laboratories. Packaging systems (such as the third-generation packaging system which involves four plasmids transfection for maximal biosafety) or commercial lentiviral packaging mix are also available. It is important to check the compatibility of the packaging system and the gene transfer lentiviral vector that you are interested in. 5. 293T cells are highly susceptible to transfection. We used calcium phosphate and 2× HBS transfection protocol because they are cheap and easy to prepare in the laboratory. It can be replaced by other commercially available transfection reagents.

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6. On days 2 and 3 post-transfection, VSVG-induced syncytium formation may be observed in the transfected 293T cultures. This is normal. 7. We do not sterilize polyallomer centrifuge tubes and parafilm before use. We open a new box inside a biosafety cabinet and handle it under standard aseptic tissue culture practice. We do not observe any contamination. 8. We transplanted autologous G-CSF/SCF-mobilized CD34+ cells that were transduced with lentiviral vectors expressing EGFP into myeloablated rhesus macaques. The safety of these replication-incompetent vectors was assessed in vivo for more than 4 years post-transplant. The animals remain healthy with no evidence of circulating HIV-1 viruses nor hematopoietic abnormalities/malignancies (14). 9. Viral titers: Using EGFP-expressing lentiviral vectors in flow cytometric analysis, we routinely obtained 105 –106 Infectious Units/ml viral titer (before ultracentrifugation). 10. NK cells can be isolated by either negative selection or positive selection kits (StemCell, AutoMACS). If a DX5positive selection kit is used, a complement depletion of Thy1.2+ cells is needed to remove contaminating NKT cells. 11. MOI (multiplicity of infection) is defined as a ratio of the number of infectious virus particles to target cells. We observed that MOIs of 20–40 are usually optimal for a single round of NK transduction; however, we suggested a titration of MOI be performed in each laboratory. 12. We found that primary NK cells can be transduced efficiently either by the “no-spin” or by the “spin” protocol. However, the “spin” protocol is more effective when a lower MOI is used (1). 13. Lentiviral vectors can transduce ex vivo-purified NK cells, as well as lymphokine-activated cells; however, we observed that cytokine activation of NK cells enhanced transgene expression (1). 14. Lentiviral vectors use internal promoters to drive transgene expression or shRNA expression. Their promoter activities in primary NK cells are therefore critical in determining the efficiency of transgene expression (1) or shRNA knockdown. We found that the CMV promoter is weak in mouse NK cells. We recommended PGK, Ubiquitin, or a rhesus-derived MLV promoter (7) for trans-

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gene expression in primary NK cells. U6 promoter is a promoter of choice in driving shRNA expression. 15. If needed, transduction can be repeated on the second day to improve transduction efficiency (1). 16. Transduction can also be done in 24-well culture plates. Up to 4.0 × 105 cells per well can be used in the transduction. 17. The genetically modified NK cells maintained stable EGFP transgene expression in vitro and can be further expanded in IL-2-supplemented culture medium. Lentiviral transduction does not affect NK surface phenotypes or functions (apoptosis, cytokine production, and cytotoxicity) (1). The transduction protocol is amendable to human NK cells. 18. Design of shRNA sequence. Selection of suitable 21-mer targets in your gene is the first step toward efficient gene silencing. 18.1. Guideline for designing siRNAs with effective gene silencing (adapted from www.rnaiweb.com/RNAi/siRNA Design): 1. Targeted regions on the cDNA sequence of a targeted gene should be located at least 25 nucleotide downstream of the start codon (ATG). 2. Search for sequence motif AA(N19)TT or NA(N21), or NAR(N17)YNN, where N is any nucleotide, R is purine (A, G), and Y is pyrimidine (C, U). 3. Avoid sequences with >50% G+C content. 4. Avoid stretches of 4 or more nucleotide repeats. 5. Avoid sequences that share a certain degree of homology with other related or unrelated genes. 18.2. Examples of commercial companies and web sites available to assist the design of siRNA target sequences. 1. http://www.ambion.com/techlib/misc/ siRNA finder 2. http://jura.wi.mit.edu/bioc/siRNAext/ 18.3. Examples of commercial companies that offer a collection of ready-to-test shRNA-containing lentiviral vectors.

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1. Sigma (www.sigmaaldrich.com/life-science/ functional-genomics-and-rnai/shrna/trcshrna-products) 2. OpenBiosystems (www.openbiosystems.com) 19. We used 5 -TTCAAGAGA-3 or 5 -CTCGAG-3 loop sequence in the constructs (see Fig. 16.1C). 20. Under the optimal transduction protocol, the choice of shRNA sequences determines the potency of shutdown. We usually generate a few lentiviral constructs that express different shRNA sequences against the target gene and test them on NK cells to determine their knockdown potency (flow, western blots, qPCR). 21. We found that 24 ␮g/ml of puromycin is the minimal concentration of puromycin that results in complete NK cell death within 24 h of culture in our laboratory. However, depending on NK cultures and batch to batch preparations of puromycin stock, the concentration of puromycin used in the selection may vary. We suggest that an optimal concentration of puromycin be predetermined in each laboratory.

Acknowledgments This work has been supported by the Establishment and Operating grants from Manitoba Health Research Council, Manitoba Institute of Child Health, Canada Foundation for Innovation and Natural Sciences and Engineering Research Council (to S.K.P.K). Some of the constructs were obtained from the Biomedical Functionality Resource established under the support of Dean Strategic Research Fund at University of Manitoba. S.K.P.K. is a Basic Science Career Development Research Awardee of the Manitoba Medical Service Foundation supported with funds provided by the Manitoba Blue Cross. The author declares there is no conflict of interest. References 1. Tran, J. and Kung, S. K. (2007). Lentiviral vectors mediate stable and efficient gene delivery into primary murine natural killer cells. Mol. Ther. 15:1331–1339. 2. Case, S. S., Price, M. A., Jordan, C. T., Yu, X. J., Wang, L., Bauer, G., Haas, D. L., Xu, D., Stripecke, R., Naldini, L., Kohn, D. B., and Crooks, G. M. (1999). Stable transduction of quiescent CD34(+)CD38(-) human

hematopoietic cells by HIV-1-based lentiviral vectors. Proc. Natl. Acad. Sci. U S A 96:2988–2993. 3. Miyoshi, H., Smith, K. A., Mosier, D. E., Verma, I. M., and Torbett, B. E. (1999). Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors. Science 283:682–686.

Introduction of shRNAs into Primary NK Cells with Lentivirus 4. Sutton, R. E., Wu, H. T., Rigg, R., Bohnlein, E., and Brown, P. O. (1998). Human immunodeficiency virus type 1 vectors efficiently transduce human hematopoietic stem cells. J. Virol. 72:5781–5788. 5. Blomer, U., Naldini, L., Kafri, T., Trono, D., Verma, I. M., and Gage, F. H. (1997). Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J. Virol. 71:6641–6649. 6. Kafri, T., Blomer, U., Peterson, D. A., Gage, F. H., and Verma, I. M. (1997). Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat. Genet. 17:314–317. 7. Kung, S. K., An, D. S., and Chen, I. S. (2000). A murine leukemia virus (MuLV) long terminal repeat derived from rhesus macaques in the context of a lentivirus vector and MuLV gag sequence results in highlevel gene expression in human T lymphocytes. J. Virol. 74:3668–3681. 8. Hannon, G. J. (2002). RNA interference. Nature 418:244–251. 9. Fire, A. (1999). RNA-triggered gene silencing. Trends Genet. 15:358–363. 10. Qin, X. F., An, D. S., Chen, I. S., and Baltimore, D. (2003). Inhibiting HIV-1 infec-

11.

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tion in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc. Natl. Acad. Sci. U S A 100: 183–188. An, D. S., Xie, Y., Mao, S. H., Morizono, K., Kung, S. K., and Chen, I. S. (2003). Efficient lentiviral vectors for short hairpin RNA delivery into human cells. Hum. Gene Ther. 14:1207–1212. Stewart, S. A., Dykxhoorn, D. M., Palliser, D., Mizuno, H., Yu, E. Y., An, D. S., Sabatini, D. M., Chen, I. S., Hahn, W. C., Sharp, P. A., Weinberg, R. A., and Novina, C. D. (2003). Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9: 493–501. Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W. S., and Khvorova, A. (2004). Rational siRNA design for RNA interference. Nat. Biotechnol. 22(3):326–330. Kung, S. K., An, D. S., Bonifacino, A., Metzger, M. E., Ringpis, G. E., Mao, S. H., Chen, I. S., and Donahue, R. E. (2003). Induction of transgene-specific immunological tolerance in myeloablated nonhuman primates using lentivirally transduced CD34+ progenitor cells. Mol.Ther. 8: 981–991.

Chapter 17 Methods to Identify and Characterize Different NK Cell Receptors and Their Ligands Dikla Lankry, Roi Gazit, and Ofer Mandelboim Abstract Different cellular immune responses are modulated by the cross talk between activating and inhibitory signaling pathways initiated via different cell surface receptors. Similarly, the killing of NK cells is controled by multiple activating and inhibitory surface receptors. In humans, the major NK triggering receptors, identified so far, include NKp80, 2B4 NKG2D, and CD16 and the natural cytotoxic receptors (collectively named NCRs) include NKp46, NKp44, and NKp30. The two major families of MHC-specific inhibitory receptors identified in humans are the Ig superfamily (KIR and LIR) and the C-type lectin (CD94/NKG2A) receptor superfamily. The different inhibitory receptors show diverse specificity and discriminate between different class I MHC proteins. Much is known about the function and expression patterns of the different NK cell receptors, but the ligand identity of several of the activating NK cell receptors is yet to be discovered. This chapter introduces several research tools that can be used to uncover the identities of different ligands for NK cell receptors. Key words: NK cell clones, Ig-fused proteins, BW assay, cytotoxicity assays, point mutations.

1. Introduction Human natural killer (NK) cells are bone marrow-derived lymphocytes that comprise 5–15% of the peripheral blood lymphocytes (1). As part of the innate immune system, NK cells are designed to kill a broad spectrum of infectious agents and tumors without prior specific stimulation. Each NK cell is capable of recognizing and destroying multiple targets. This broad specificity is mediated by multiple activating and inhibitory surface receptors and intracellular signal transduction molecules (2). However, the ligands for many of the NK cell receptors are still unknown. To K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 17, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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study the NK cell receptors, we must first separate the NK cells from the other peripheral blood mononuclear cells (3). Once we obtained high purity of NK cells, we can identify the different receptors expressed by the different NK cell clones and further study the variations in the different killing potentials of these specific clones. Two different methods described in this section help us identify target cells that express potential NK cell receptor ligand(s): (1) The fusion Ig protein approach (4) allows us to easily scan many target cells for a potential ligand(s). (2) The BW assay (5, 6), which is a cell-to-cell interaction assay, utilizes the NK cell receptor expressed on the cell surface in its accurate conformation to detect presence of a ligand on target cells. In addition, to study the function of a specific NK cell receptor with regard to killing, we can use NK tumor lines such as the YTS cell line (6). Using this system we can study the function of the native NK cell receptor or generate mutations and study their effects on the activity of a particular receptor. In vitro methods are essential tools to study NK cell receptors, but an in vivo system is more accurate and informative. Therefore, we constructed a knockout mouse lacking the NCR1 receptor, and its entire NK cell repertoire is marked with GFP. Using this system, we can easily isolate the NK cells and study the importance of the NCR1 receptor for the immune response executed by NK cells (7)

2. Materials 2.1. General Reagents

1. RPMI-1640 medium (GIBCO, Invitrogen) 2. Dulbecco’s Modified Eagle Medium (DMEM; GIBCO, Invitrogen) 3. Supplements for complete media preparations: 1% sodium pyruvate solution (Biological Industries),1% penicillin/streptomycin solution (Biological Industries), 1% L-glutamine solution (Biological Industries), 1% nonessential amino acids solution (Biological Industries), and 10% fetal bovine serum (FBS; PERBIO) 4. 96 U-well/96 V-well/96 flat (F)-well/6-well/24-well culture plates (no particular manufacturer necessary) 5. PCR primers (SIGMA) 6. PBS (1× prepared from 10× stock; Biological Industries)

2.2. Culturing of Primary NK Clones

1. Heparin (ROTEXMEDICA) 2. Ficoll-Paque PLUS (GE Healthcare) 3. RPMI-8866 cell line (ATCC)

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4. Gamma irradiation source 5. PHA−L (phytohemagglutinin-L; Roche) 6. AutoMACS separator (Miltenyi Biotec) 7. Human NK cell isolation kit: biotin Ab cocktail and antibiotin microbeads (Miltenyi Biotec) 8. AutoMACS Medium (1 L): 4 mL EDTA (final concentration of 2 mM), pH 8 and 5 mL FBS in PBS 9. NK cell medium: 350 mL DMEM, 150 mL F-12 Ham‘s medium (GIBCO, Invitrogen), 50 mL human serum (SIGMA), 5 mL of sodium pyruvate solution, penicillin/streptomycin solution, L-glutamine solution and nonessential amino acids solution (Biological Industries), and 2 mL IL-2, final concentration of 500 U/mL (500,000 U/mL stock; Boehringer Mannheim GmbH) 10. Human anti-CD56/PE mAb (BioLegend) 11. Human anti-CD3/FITC mAb (BioLegend) 12. Negative control mouse IgG1/RPE (Dako) 13. Negative control mouse IgG1/FITC (Dako) 14. Anti-CD3 mAb (T3D Hybridoma) 2.3. Producing Fusion Ig Proteins

1. Restriction enzymes: Hind III, Bam HI (Fermentas) 2. TOP10/P3 bacteria (Invitrogen) 3. HiYield Plasmid mini kit (RBC Bioscience) 4. PureLink HiPure Plasmid maxi prep kit (Invitrogen) 5. Ampicillin 100 mg/mL solution (Roche) 6. Tetracycline 1 mg/mL solution (Boehringer Mannheim GmbH) 7. LB broth base (Invitrogen) 8. COS-7 cells (ATCC) 9. TransIT − LT1 Transfection reagent (MIRUS Bio.) 10. 500 mL Filter system − 0.22 ␮m CA (Corning Incorporated) 11. LPM medium, Low Protein Medium BSA-Free (Biological Industries) 12. 10% sodium azide stock solution 13. Protein A/G column − HiTrap Healthcare)

TM

Protein A/G HP (GE

14. Pharmacia P-1 peristaltic pump with adjustable flow rate up to 500 mL/h (Pharmacia Biotech) 15. 0.1 M glycine stock solution, pH 2.7 16. 1 M Tris−HCl, pH 8.8

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17. 20% ethanol solution 18. SnakeSkin Pleated Dialysis Tubing (PIERCE) 2.4. BW Zeta Assay

1. BW cells (ATCC) 2. Electroporation instrument: BioRad Gene Pulser 3. Gene Pulser cuvettes − 0.4 cm (BioRad) 4. Neomycin 10 mg/mL solution: Geneticin G-418 sulfate (GIBCO) 5. MaxiSorp plate (Nunc) 6. Binding buffer: 0.1 M Na2 HPO4 , pH 9.0 (0.1 M NaH2 PO4 ) 7. PBS/Tween20 0.05% solution: 0.05% polyoxyethylene 20 sorbitan monolaurate (Tween20; J.T. Baker) in PBS 8. Blocking buffer: PBS containing 10% FBS or 3% BSA 9. Purified rat anti-mouse IL-2 antibody, 0.5 mg/mL (BioLegend) 10. Biotin rat anti-mouse IL-2, 0.5 mg/mL (BioLegend) 11. Streptavidin−HRP, 1 mg/mL (Jackson Immunoresearch) 12. Chromogen mation)

tetramethylbenzidine

(TMB;

Dakocyto-

13. Optic reader PowerWave ×S (BioTek) 2.5. Killing Assay

1.

S-methionine, 7.5 mCi/500 ␮L (Institute of Isotopes Co. Ltd) 35

2. P815 mastocytoma cell line (ATCC) 3. Modified RPMI-1640 (SIGMA)

medium,

lacking

methionine

4. Opaque Opti-plates (Perkin Elmer) 5. Scintillation liquid: MicroSCINT

TM

40 (Perkin Elmer)

6. Top seal A (Perkin Elmer) 7. Microplate scintillation and luminescence TM (␤ counter): Packard Top Count − NXT 2.6. Mutations and Transfection into YTS Cells

counter

1. YTS-ECO cells (ATCC) 2. BOSC cell line (ATCC) 3. 100% ethanol supplemented with 10% of 3 M sodium acetate solution, pH 5.3 4. Puromycin diHCl (Calbiochem) 5. Hexadimethrine bromide (polybrene; Aldrich)

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3. Methods 3.1. Culturing of Primary NK Cell Clones

In order to study the NK cell receptors and their interactions with different target cells, it is important to separate the NK cells from the peripheral blood mononuclear cells (PBMC). Working with NK populations of high purity will help to define the specific NK cell receptor interactions and activities.

3.1.1. Isolation of Human PBMCs from Peripheral Blood

Work in a sterile environment. 1. Use a 30 mL syringe washed with heparin to obtain 10 mL of fresh blood. Transfer the blood into 50 mL tube and dilute 1:2 with pre-warmed RPMI medium, 37◦ C (see Note 1). 2. Add 13 mL of Ficoll-Paque Plus into a new 50 mL tube and gently load 30 mL of the diluted blood on top of it (Ficoll:medium ratio approximately 1:2). Centrifuge for 30 min at 515×g at room temperature, brake off. PBMC will be localized between the Ficoll layer and the plasma layer as a white ring. 3. Take out most of the plasma layer, leave about 5 mL above the PBMC ring, and use 10 mL pipette to collect the PBMC ring and transfer into a new 50 mL tube. 4. Wash the PBMC with pre-warmed RPMI medium, 37◦ C, centrifuge for 5 min at 515×g at 4◦ C, and resuspend the cells with 10 mL pre-warmed RPMI medium, 37◦ C.

3.1.2. Preparation of Feeder Cells

Work with 96 U-well plates 1. Use Ficoll gradient in order to separate PBMC from two different donors (see Section 3.1.1 and Note 1). 2. Mix together 5×106 cells/plate from each donor (total of 10×106 ) and 5×105 cells/plate of the EBV-transformed Bcell lymphoma RPMI-8866 (ATCC). 3. Irradiate the cells with 6000 rad. 4. Wash the cells with pre-warmed RPMI medium, 37◦ C, and centrifuge for 5 min at 515×g at 4◦ C. 5. Discard supernatant, resuspend the pellet with NK medium, 100 ␮L/well (total of 10 mL/plate), and add 1 mg/mL PHA-L (final concentration of 0.1 ␮g/well). Seed 100 ␮L/well of the feeder cells in 96 U-well plates and incubate in a 37◦ C, 5% CO2 incubator until the NK separation process is completed (see Section 3.1.3). Seed about 8−12 96 U-well plates.

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3.1.3. Separation of Human NK Cells from PBMCs by Using the AutoMACS Instrument.

Keep working on ice! These steps are performed according to the manufacturer’s instructions (Miltenyi). 1. Collect 1×106 PBMCs and wash with cold autoMACS medium. Centrifuge for 5 min at 515×g at 4◦ C (see Note 2). 2. Discard supernatant and gently resuspend the pellet with 40 ␮L of cold autoMACS medium. Add 10 ␮L biotin Ab cocktail, gently pipette, and incubate on ice for 10 min. 3. Add 30 ␮L of cold autoMACS medium and 20 ␮L of antibiotin microbeads, gently pipette, and incubate on ice for 15 min. 4. Wash the cells by adding 5 mL of cold autoMACS medium and centrifuge for 5 min at 515×g at 4◦ C. 5. Discard all supernatant by using a 200 ␮L micropipette and resuspend the pellet with 500 ␮L of cold autoMACS medium. 6. Separate the NK cells by using the autoMACS instrument based on a negative selection (according to the manufacturer’s instructions); select the “depletes” program and use one tube to collect the negative fraction (NK cells) and another to collect the positive fraction (mainly T cells). 7. Count the number of NK cells in the negative fraction. About 10% NK cells should be obtained. 8. Validate NK cell phenotype (CD56+ CD3− ) by FACS after staining the cells with anti-CD56/PE and anti-CD3/FITC. In a separate sample, use mouse IgG1/RPE and mouse IgG1/FITC as negative controls. Alternatively, identify the NK cells with anti-NKp46 mAb.

3.1.4. NK Cell Cloning and Culturing

NK cells are cloned and cultured in 8–12 96 U-well plates 1. Dilute the NK cells in NK cell medium according to the desirable number of cells per well. Seed different amounts of NK cells: two plates with 8 cells/well, 2 plates with 4 cells/well, 2 plates with 2 cells/well, 2 plates with 1 cells/well, and 2 plates with all of the remaining NK cells (the total plate). Add 100 ␮L NK cell medium per well. 2. Incubate the plates for 1 week in a 37◦ C, 5% CO2 incubator. 3. Following 1 week incubation, repeat step 3.1.2 and add irradiated feeder cells in 50 ␮L/well instead of 100 ␮L. 4. Two options to obtain polyclonal NK cell lines: a. After 2–3 days from the second addition of feeder cells, plates that were seeded with a large number of NK cells (the total plates) will become yellow, which represents cell growth. Collect the medium from the well by using

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a multichannel micropipette into a 10 mL culture dish. Then, by using a 10 mL pipette, reseed the cells into 24well plates, 2 mL/well. Validate NK cell phenotype by FACS staining (see Section 3.1.3 and step 8). b. After 2–3 days from the second addition of feeder cells, single wells in the different plates will become yellow. Collect each clone into a new 96-U plate and validated NK cell phenotype by FACS staining, using anti-CD56 and anti-CD3 or anti-NKp46 antibodies. To obtain NK cell lines, at least 20 CD3− and CD56+ clones should be mixed and grown together in 24-well plates containing 2 mL NK cell medium. 5. Once every 3–4 days, NK cells should be split by mixing each well with a 1000 ␮L micropipette and transferring 1 mL into a new 24-well plate. Then add 1 mL NK cell medium to each well for a final volume of 2 mL/well (see Note 3). 6. In order to obtain NK cell clones: a. Mix the desired NK cell clone well and split the well into two new wells of a 96 U-well plate by transferring 100 ␮L to each well. Then add 100 ␮L NK cell medium to each well for a final volume of 200 ␮L/well. b. After 2–3 days, split each well until you have 4–8 wells of each clone. c. Validate NK cell phenotype by FACS staining, using antiCD56 and anti-CD3 or anti-NKp46 antibodies. 3.2. Producing Fusion Ig

3.2.1. Generation of the Genetic Constructs

COS-7 cells are transfected with an expression vector carrying a gene encoding the extracellular domain of a desired receptor attached to the Fc portion of an IgG1 antibody. This method allows us to create an easy screening method for target cells that express the appropriate ligand(s) for a particular receptor. 1. Use PCR in order to amplify the leader peptide and the entire extracellular portion of the relevant receptor by using primers containing restriction enzyme sites for Hind III at the 5 and Bam HI at the 3 ends (see Notes 4 and 5 and Fig. 17.1). 2. Insert the fragment by ligation into an Ig expression plasmid (Homemade plasmid Cs -4Fc by Brian Seed, Harvard, Boston), which contains the Fc portion of human IgG1 (see Notes 6 and 7). Following ligation, transform the plasmid into TOP10/P3-competent bacteria and seed on LB plates containing ampicillin and tetracycline solutions (1:4000 and 1:100 for final concentrations of 0.025 and 0.01 mg/mL, respectively) (see Note 8).

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Fig. 17.1. Fusion Ig protein production. A. Schematic representation of the fusion Ig protein production. B. Generation of the genetic constructs: amplification of the extracellular portion by PCR and insertion into the Ig fusion vector.

3. Collect 5–10 bacteria colonies and extract plasmids by using a mini prep kit. 4. Analyze gene insertion by restriction enzymes and gel electroporation. 5. Upon identifying the desired colony, produce the plasmid in large quantities by using a Maxi prep kit. 6. Send the plasmid for sequence analysis in order to ensure correct sequence and reading frame coordinates with the Fc portion. 3.2.2. Generation of the Ig-Fused Receptors in COS-7 Cells

1. Seed COS-7 cells in 6-well plates (75,000– 100,000 cells/well) in complete DMEM medium (2 mL /well) and incubate overnight in a 37◦ C, 5% CO2 incubator (see Note 9). 2. DNA precipitation: a. Precipitate 180 ␮g DNA by adding 2.5× DNA volume of 100% ethanol containing 10% sodium acetate 3 M, pH=5.3. Vortex in order to homogenize the fluids. b. Incubate overnight at −20◦ C or 2 h at −70◦ C. c. Centrifuge for 30 min at 20,000×g at 4◦ C. d. Discard the supernatant and add 1 mL of 70% ethanol. e. Centrifuge for 30 min at 20,000×g at 4◦ C. f. Work in a sterile environment. Discard the supernatant with a 1000 ␮L micropipette, and avoid touching the

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DNA sediment. Leave the tube open in order to dry ethanol remnants. g. Resuspend the DNA with 360 ␮L pre-warmed 37◦ C DMEM medium. h. Transfer the resuspended DNA into a new 50 mL tube. 3. DNA transfection: Work under sterile conditions. a. Transfer 36 mL DMEM medium into a new 50 mL tube and gently drop 720 ␮L of TransIT − LT1 Transfection reagent on top of it, incubate at room temperature for 5 min. b. Following incubation, resuspend the medium, and by using a 10 mL pipette, gently drop the entire 36 mL on top of the resuspend DNA. Incubate at room temperature for 15 min. c. Following incubation, resuspend the medium and use a 200 ␮L micropipette to gently drop 100 ␮L of the DNAcontaining medium on top of each well containing COS7 cells. Upon completing this procedure for each 6-well plate, gently swirl the plate in circular movements in order to evenly distribute the transfection reagent. d. Incubate for 6 h or overnight in a 37◦ C, 5% CO2 incubator. e. Following incubation, discard the medium from each well by using a 10 mL pipette. Gently add 2 mL of complete LPM medium lacking serum to each well (see Note 10). f. Incubate for 48 h in a 37◦ C, 5% CO2 incubator. g. Following incubation, collect the medium from each well into 50 mL tubes and gently add to each well 2 mL of new complete LPM medium lacking serum. Work under sterile conditions (see Note 11). h. Incubate for an additional 48 h in a 37◦ C, 5% CO2 incubator. i. Following incubation, collect the medium from each well into 50 mL tubes and discard the 6-well plates. Working in a sterile environment is no longer necessary! 4. Filtering the collected medium. a. Centrifugate the collected medium for 5 min at 515×g at 4◦ C to discard remaining cells. b. Following centrifugation, filter the medium with a 0.22 ␮m 500 mL filter and add a final concentration of 0.05% sodium azide. c. Transfer the medium into a 1 L beaker and keep at 4◦ C (see Note 12).

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5. Protein purification using a protein A/G column. These steps should be performed in a 4◦ C room and all media/buffers should also be at 4◦ C. a. Pump properties: speed − 5 mL/min, rate: ×10. b. Apply 30 mL of 0.1 M glycine HCl, pH 2.7, elution buffer onto the column. c. Apply 30 mL of DMEM medium onto the column. d. Apply the medium containing the fusion protein onto the column e. Apply 30 mL of DMEM medium onto the column. f. Protein elution: transfer 10 mL of 0.1 M glycine HCl, pH 2.7, elution buffer collect the eluate into a 50 mL tube containing 1 M Tris−HCl, pH 8.8 (25 ␮L/1 mL of elution buffer). Collect at least three fractions of 10 mL each, depending on column size. Column preservation: g. Apply 30 mL of 0.1 M glycine HCl, pH 2.7. h. Apply 30 mL of double-distilled water (DDW). i. Apply 30 mL 20% ethanol. 6. Protein dialysis: a. Transfer the three fractions into three different dialysis bags (5–10 cm of dialysis tubing each) and place them into a beaker containing 2 L of cold PBS buffer in a room at 4◦ C overnight under stirring conditions. The following day, discard the PBS, add a new 2 L of PBS, and repeat the same procedure. b. The following day, transfer each fraction into a new 50 mL tube and check if the pH = 7–7.5. Determine protein concentration (by spectrophotometer, or any other appropriate protein method). c. Aliquot the proteins to 0.5–1 mL in microfuge tubes and store at −20◦ C. 3.3. BW Zeta Assay

3.3.1. Generation of Receptor-CD3-Zeta Fusion Constructs

The mouse T lymphoma cell line, BW, is transfected by electroporation with an expression vector carrying genes encoding the extracellular domain of a desired receptor and transmembrane and cytoplasmic domains of the mouse CD3-zeta chain. This method enables the generation of an in vitro system in which the activity of the receptor is measured. 1. Use two-step PCR reactions in order to amplify the entire extracellular (EC) portion of the relevant receptor fused to the transmembrane and tail of mouse CD3-zeta chain (see Fig. 17.2 and Note 13).

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Fig. 17.2. Generation of CD3-zeta-fusion constructs. A two-step PCR reaction is used in order to construct a receptor − mouse CD3-zeta fusion protein. In the first PCR reaction, amplify the receptor extracellular portion and the mouse CD3-zeta TM and cytoplasmic tail. In the second PCR reaction, attach the two segments together and the construct is inserted into the appropriate vector.

a. In the first step, amplify each of the fragments (EC portion of the receptor and the transmembrane and tail of CD3-zeta). The EC portion of the receptor should be amplified by using a 5 primer containing the appropriate restriction site and beginning of the leader sequence and a 3 primer corresponding to the last 20 bp of the EC domain of the receptor and the first 9 bp of the transmembrane of CD3-zeta in the same codon reading frame. The CD3-zeta portion should be amplified similarly by using a 3 primer containing the appropriate restriction site and a 5 primer containing the last 9 bp of the EC domain of the receptor and the first 20 bp of the transmembrane region of CD3-zeta. b. In the second step, these two fragments are mixed and amplified by PCR using the 5 primer of the receptor and the 3 primer of CD3-zeta. 2. Insert the fragment by ligation into the pCDNA3 plasmid or any other expression plasmid (see Note 14). 3. Following ligation, transform appropriate competent bacteria with the plasmid and seed them onto LB plates containing the appropriate antibiotics (in accordance with the plasmid used), such as ampicillin solution (1:1000) for final concentrations of 0.1 mg/mL. 4. Collect 5–10 bacteria colonies and extract plasmids by using a mini prep kit. 5. Analyze the gene insert by restriction enzyme digest and gel electrophoresis. 6. Once the desired colony is identified, produce the plasmid in large quantities and purify using a Maxi prep kit.

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7. Sequence the plasmid in order to ensure correct reading frame and sequence of the EC − CD3-zeta fusion construct. 3.3.2. Stable Transfections by Electroporation

Perform these steps under sterile conditions. 1. Precipitate 100 ␮g of the CD3-zeta fusion construct DNA (see Section 3.3.1) and resuspend the DNA with 800 ␮L RPMI medium. 2. Collect 10×106 BW cells into a new 50 mL tube. 3. Centrifuge for 5 min at 515× at 4◦ C. 4. Discard the supernatant and resuspend cells in the 800 ␮L of DNA-containing RPMI medium. 5. Incubate the cells and electroporation cuvette separately for 5–10 min on ice. 6. Transfer the cells into the electroporation cuvette and perform electroporation (250 V, 500 ␮F). 7. Transfer the cells from the electroporation cuvette into a new 50 mL tube. Add 50 mL of RPMI medium and centrifuge for 5 min at 515×g at 4◦ C. 8. Discard supernatant and resuspend cells with 40 mL complete RPMI medium. 9. Seed the cells into 24-well plates at 1 mL/well. Also seed one control well which contains 250,000 untransfected BW cells (these should die under antibiotic selection). 10. Incubate for 24 h in a 37◦ C, 5% CO2 incubator. 11. Following incubation, add 1 mL/well of complete RPMI medium containing neomycin, selection medium, reaching a final concentration of 5 mg/mL G418 (see Note 15). If using a plasmid containing a different antibiotic selection gene, calibrate the antibiotic concentration required to kill the untransfected BW cells. 12. Every 2–3 days, remove 1 mL medium from each well and add 1 mL of fresh selection medium to achieve a final concentration of 5 mg/mL G418 (see Note 16). Medium refreshment should be performed until no sign of growing cells is evident (about 3–4 times). 13. Upon emergence of neomycin-resistant clones, analyze cells for protein expression by using FACS staining or any other appropriate method. 14. Grow the cells in the appropriate selective medium until stable expression is achieved (see Note 17).

3.3.3. IL-2 Secretion Assay

1. Collect transfected BW cells (effector cells; 50,000 cells/well) into a new 50 mL tube (see Note 18). 2. Centrifuge for 5 min at 515×g at 4◦ C.

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3. Resuspend cells with complete RPMI medium (100 ␮L containing 50,000 cells/well) and keep on ice until obtaining target cells. 4. Collect target cells into a new 50 mL tube. 5. Irradiate target cells with 3000 rad. 6. Add 40 mL of RPMI medium to each target cell and centrifuge for 5 min at 515×g at 4◦ C. 7. Resuspend target cells with complete RPMI medium (100 ␮L/well). 8. Seed transfected BW cells and target cells [maintaining consistent effector cell concentration, alter target cell concentration to achieve different effector to target (E:T) ratios, either 1:1 or 2:1, are recommended] in 96 F-well plates and incubate in a 37ºC, 5% CO2 incubator for 24 or 48 h (see Note 19). 9. Following incubation, centrifuge for 5 min at 515×g at 4◦ C and collect 100 ␮L of the supernatant for cytokine analysis (see Note 20). 3.3.4. ELISA for Cytokines

1. Coat MaxiSorp plate with 0.1 ␮g/well of purified rat antimouse IL-2 in 50 ␮L/well binding buffer. 2. Incubate overnight at 4ºC or 1 h at 37ºC. 3. Discard the supernatant and wash plate four times with PBS/Tween20 0.05%. 4. Add 200 ␮L/well blocking buffer (PBS/10% FCS or 3% BSA). 5. Incubate at room temperature for 2 h. 6. Discard the supernatant. 7. Add the collected supernatant from Section 3.3.3 and incubate for 4 h at room temperature or overnight at 4◦ C. 8. Discard the supernatant and wash plate four times with PBS/Tween20 0.05%. 9. Add 100 ␮L/well of biotinylated rat anti-mouse IL-2 (0.1 ␮g/well) diluted in blocking buffer containing 0.05% Tween20 and incubate for 1 h at room temperature (see Note 21). 10. Discard the supernatant and wash plate x6 with PBSx1/Tween20 0.05%. 11. Add 100 ␮L/well of streptavidin−HRP (0.1 ␮g/well) diluted in blocking buffer and incubate for 30 min at room temperature. 12. Discard the supernatant and wash plate six times with PBS/Tween20 0.05%.

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13. Add 100 ␮L/well of TMB substrate and read the results using an Optic reader at a wavelength of 650 nm. 3.4. Cytotoxicity Assays

3.4.1. 35 S-Methionine Release Cytotoxicity Assays

NK cells are incubated with radioactive-labeled target cells. This method allows us to monitor the killing of the target cells by NK cells. By measuring the radioactivity levels released into the supernatants, we can determine efficiency of the killing. Furthermore, by using redirected killing, which is mediated by antibodies and an Fc-receptor-expressing target cell, we can define the function of a specific receptor and whether this is an inhibitory, activating, or co-stimulatory receptor. 1. Radioactive labeling of target cells: a. Collect target cells (1×106 cells/well of a 6-well plate). b. Centrifuge for 5 min at 515×g at 4◦ C. c. Resuspend cells with complete modified RPMI-1640 medium (lacking methionine), which contains [35 S]Methionine (1 ␮L of [35S]-methionine stock/1 mL complete modified RPMI medium). d. Seed the cells into a 6-well plate and incubate overnight in a 37◦ C, 5% CO2 incubator. e. Following incubation, collect the labeled target cells into a new 50 mL tube and add 40 mL of RPMI medium (normal RPMI-1640 medium from here onward). f. Centrifuge for 5 min at 515×g at 4◦ C. g. Resuspend the cells with 2–5 mL complete RPMI medium and determine cell number. 2. Seed target cells (5000 cells/well) diluted in complete RPMI medium (100 ␮L/well) into a 96 U-well plate. Seed 3 wells of each target cell alone (5000 cells/well) to determine spontaneous radioactive release and another 3 wells with target cells alone (5000 cells/well) to determine the total radioactive release (see Notes 22 and 23). 3. Seed different amounts of effector NK cells (various E:T ratios) diluted with complete RPMI medium (100 ␮L/well) to the same wells into which the target cells were seeded (see Note 24). Each E:T ratio condition should be seeded in triplicate wells. Do not seed effector cells into the wells dedicated for spontaneous and total release. 4. Incubate in a 37◦ C, 5% CO2 incubator for at least 5 h. 5. Following incubation, add 100 ␮L of 0.1 M NaOH to each total release well (medium color turns red) (see Note 25). 6. Centrifuge for 5 min at 515×g at 4◦ C.

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7. Collect 50 ␮L of cell supernatants (be careful not to touch the cells at the bottom of the well) and transfer to opaque Opti-plates. 8. Add 150 ␮L scintillation liquid to each well, cover with plastic sticker (Top seal A), and incubate in the dark at room temperature overnight. 9. Analyze results by using a ␤-counter (see Note 26). 3.4.2. Redirected Cytotoxicity Assays

1. Use the Fc-receptor-expressing P815 mouse mastocytoma cell line as target cells (see Fig. 17.3).

Fig. 17.3. Redirected cytotoxicity assays. P815 cells (mouse lymphoblast-like mastocytoma cell line), which express Fc-␥ receptors, are incubated on ice for 1 h with 0.2 ␮g/␮L antibodies which recognizes an activating receptor (gray) or an inhibitory receptor (black). The Fc portion of the IgG antibody binds to the Fc-␥ receptor expressed on the P815 cells. Once NK cells are added, the appropriate receptor on the NK cell is cross-linked and thus is activated. This allows the specific examination of a particular receptor.

2. Label target cells as described in Section 3.4.1. 3. Seed target cells (5000 cells/well) diluted with complete RPMI medium (50 ␮L/well) into a 96 U-well plate. Seed 3 wells with target cells (5000 cells/well) for spontaneous radioactive release and another 3 wells with target cells (5000 cells/well) for total radioactive release (see Notes 22 and 23). 4. Prior to NK cell addition, incubate P815 cells with the relevant mouse IgG antihuman NK cell receptor mAb (0.2 ␮g/␮L diluted in 50 ␮L/well complete RPMI medium) for 1 h on ice (this allows the binding of antibodies to the P815 cells via their Fc-receptors and thus mediates cross-linking of the desired receptor on the NK cell’s surface) (see Notes 27 and 28). 5. Continue working according to Section 3.4.1 and steps 3–9.

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3.5. Expression of Receptors in YTS Cells

3.5.1. Generation of Point Mutant Constructs

YTS cell lines are transformed NK-like cells, which do not express all NK cell receptors. Hence, we can express a specific NK cell receptor and study its function in these cells. The insertion of a point mutation can determine the importance and function of a single amino acid in the receptor’s sequence. 1. Two-step PCR reactions can be used in order to insert a point mutation into the desired receptor (see Fig. 17.4 and Notes 29):

Fig. 17.4. Generation of point mutated constructs. A two-step PCR reaction is used in order to insert a point mutation. In the first PCR reaction, amplify the 5 and 3 portion of the gene with the appropriate primers, which contain the mutation. In the second PCR reaction, attach the two segments together, and the construct is inserted into the appropriate vector. If the mutation insertion is a part of the 5 primer or the 3 primer, a one-step PCR reaction can be performed.

a. In the first step, amplify each of the fragments by PCR (upper and lower portion of the receptor). The upper portion of the receptor should be amplified by using a 5 primer containing the appropriate restriction site and a 3 primer corresponding to the 20 bp surrounding the targeted mutation. The lower portion should be amplified similarly by using a 3 primer containing the appropriate restriction site and a 5 primer corresponding to the 20 bp of the area with the targeted mutation. b. In the second step, these two fragments are mixed and amplified by PCR using the 5 - and 3 -end primers of the receptor. 2. Insert the fragment by restriction digestion and ligation into a retroviral plasmid, such as pBabe (see Note 30). 3. Following ligation, transform the plasmid into appropriate competent bacteria, such as Stbl2 (Invitrogen) to avoid vector recombination events. Seed transformed bacteria onto LB plates containing the appropriate antibiotics (in accordance with the plasmid used), such as ampicillin (1:1000 for final concentrations of 0.1 mg/mL).

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4. Collect 5–10 bacteria colonies and extract plasmids by using a mini prep kit. 5. Analyze the gene insertion by restriction enzyme digestion and gel electrophoresis. 6. Once the desired colony is identified, produce the plasmid in large quantities by growing a large volume culture and purify using a Maxi prep kit. 7. Send the plasmid for sequence analysis in order to ensure correct reading frame, sequence and introduction of mutation. 3.5.2. Retroviral Transduction into YTS Cells

YTS cells cannot be transfected via electoporation or with other conventional methods of transfection. To avoid using human retroviruses, these cells were transfected with an ecotropic receptor and hence were given the name YTS-Eco. Protein expression in YTS-Eco cells is now also possible by using mouse retroviruses produced in BOSC packaging cells. 1. Seed BOSC cells in 10 mL culture dish (1.7×106 cells/plate in 10 mL complete DMEM medium). 2. Incubate overnight in a 37ºC, 5% CO2 incubator. 3. DNA precipitation: a. Precipitate 10 ␮g DNA by adding 2.5× of DNA volume of 100% ethanol containing 10% sodium acetate 3 M, pH 5.3 (see Note 31). b. Incubate overnight at −20◦ C or 2 h at −70◦ C. c. Centrifuge for 30 min at 20,000×g at 4◦ C. d. Discard the supernatant and add 1 mL of 70% ethanol. e. Centrifuge for 30 min at 20,000×g at 4◦ C. f. Working under sterile conditions, discard the supernatant with a 100 ␮L micropipette and avoid touching the DNA sediment. Leave the tube open in order to air dry ethanol remnants. g. Resuspend the DNA with 100 ␮L DMEM medium prewarmed to 37◦ C (see Note 32). 4. DNA transfection performed under sterile conditions: a. Transfer 400 ␮L DMEM medium into a new microfuge tube and gently drop 30 ␮L of TransIT−LT1 transfection reagent on top of it. Incubate at room temperature for 5 min. b. Using a 200 ␮L micropipette, resuspend and gently drop the 100 ␮L of DNA on top of the 400 ␮L of medium containing the transfection reagent (see Notes 33 and 34). Incubate at room temperature for 15 min.

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c. Following incubation, resuspend the medium and use a 200 ␮L micropipette in order to transfect the BOSC cells. Gently drop all 500 ␮L of the DNA-containing medium on top of the 10 mL medium in the culture dish. Use circular movements while dropping in order to cover the entire plate. Before incubation gently swirl the plate further in circular movements in order to evenly distribute the transfection reagent in each dish. d. Incubate the cells for 48 h in a 37ºC, 5% CO2 incubator. 5. Mouse retrovirus collection and YTS-Eco cell transduction: a. Following 48 h of culture, gently collect the culture medium into a new 50 mL tube. Try to avoid collecting cells! The supernatant contains the ecotropic retrovirus. b. Centrifuge for 30 min at 14,000 rpm at 4◦ C. c. Using a 10 mL pipette, gently transfer the supernatant into a new 50 mL tube and avoid touching the cell sediment. d. Add 400,000 YTS-Eco cells into a new 15 mL tube. e. Centrifuge for 5 min at 515×g at 4◦ C. f. Discard the supernatant, resuspend cell pellet with 1.5–2 mL of virus-containing medium, add 1 ␮L of polybrene solution (5 ␮g/␮L stock), and mix. g. Seed the cells in a 96 U-well plate by adding 150 ␮L of cell suspension/well and centrifuge for 1.5 h at 515×g at 32◦ C (see Note 35). h. Incubate the cells for 5–6 h in a 37ºC, 5% CO2 incubator. i. Following incubation, collect the cells into a new 15 mL tube. j. Centrifuge for 5 min at 515×g at 4◦ C. k. Discard the supernatant, resuspend the cells in 1 mL of complete RPMI medium, seed into a well of a 24-well plate, and culture for 24 h in a 37ºC, 5% CO2 incubator. l. After 24 h incubation, add 1 mL complete RPMI medium containing G418 and puromycin (for final concentrations of 1.6 mg/mL G418 and 0.7 ␮g/mL puromycin, add 1 mL of medium containing 3.2 mg/mL G418 and 1.4 ␮g/mL puromycin, for the pBabe plasmid; for other plasmids, the antibiotic concentrations need to be established). m. Every 2–3 days, remove 1 mL medium from each well and add 1 mL of fresh selection medium (final concentrations of 1.6 mg/mL G418 and 0.7 ␮g/mL puromycin). Medium refreshment should be performed until no sign of growing cells is evident (about 3–4 times).

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n. Upon emergence of G418- and puromycin-resistant clones, analyze cells from individual wells for protein expression by using FACS staining or other appropriate method. o. Transfer positive clones into 6-well plates, and after 2–3 days, transfer the cells into 10 mL culture dishes. Grow the cells in the appropriate selection medium (final concentrations of 1.6 mg/mL G418 and 0.7 ␮g/mL puromycin) until stable expression is achieved (see Note 36). 3.6. Gene Targeting in Mice for Study of NK Cells(7)

The ability to directly target a gene of interest is one of the most powerful tools for in vivo research, as recently recognized with the Nobel prize to Mario R. Capecchi, Martin J. Evans, and Oliver Smithies (http://nobelprize. org/nobel prizes/medicine/laureates/2007/adv.html). Knocking a gene out (KO) is an ultimate test for its essential role; introduction of a reporter further allows precise detection of the cells that express it, and more sophisticated manipulations involving introducing specific recombinase sites and inducible elements open additional options to study the functional roles of genes in specific cell types and at defined times. In this section we describe a general strategy toward designing and generating a genetic model that can help in the study of NK cells in vivo.

3.6.1. Choose Your Gene

Simply said, this is the most important decision one has to take in this procedure, as one gene may yield a significant and interesting phenotype, while another might be redundant. Deep search into the literature is essential; notably many genes have multiple names that make this search a bit tricky.

3.6.2. Has This Gene Been Targeted Before?

As the number of genes is finite, and more genes are being targeted every day, it is highly possible that the gene you have picked was already targeted. If a paper was published, you may yet decide to take a different strategy [e.g., target different part of the gene, introduce a mutation (knock-in), or make a conditional KO]. Importantly, there are several large-scale efforts that have and continue to target many genes in ES cells, finding targeted cells may save you time.

3.6.3. Learn the Locus

Some genes are small and simple, while some contain many exons and spread over a long genomic region. Nevertheless, it is possible to target any gene with the right plan. Genome browsers at the NCBI (http://www.ncbi.nlm.nih.gov/ sites/entrez?db=genomeprj&cmd=Retrieve&dopt=Overview& list˙uids=169), UCSC (http://genome.ucsc.edu/cgi-bin/ hgGateway?org=mouse), and Sanger institute EMBL-EBI

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(http://www.ensembl.org/Mus musculus/index.html) are of great help. We personally found the UCSC to have wide-ranging data nicely organized and an incorporated BLAST algorithm, while different advantages might be found in the others. Be sure to consider the known and the possible alternative splicing forms that might be expressed. If you choose to target just part of a gene, consider the possibility that the remaining parts will still be expressed and functional (either normal or abnormal). In particular, targeting only initial exons may fail due to skipping over them or use of an alternative start codon, thus resulting in no detectable phenotype. Avoid repeat elements that appear many times in the genome, as they will reduce the frequency of targeting and may prevent southern analysis. Be sure to plan precise analysis and confirm it experimentally. 3.6.4. Making a Targeting Construct

A general schematic construct with homologous arms, positive and negative selections, is shown (Fig. 17.5). Use genomic DNA from the same strain as your ES cells to assure a perfect match (different strains have variations in their genomes that may reduce efficiency of homologous recombination and complicate southern analysis). Classical cloning, PCR, and restriction enzymes are well established and sufficient, while more advanced recombineering and BAC approaches may have advantages, once familiarized. In any case, the size of the homologous arms should be in the order of several thousand bp. Within this range, shorter fragments are easier to handle, but longer ones are believed to increase the frequency of homologous recombination. Utilization of pre-made plasmids that already have positive and negative selection (and other elements if desired) will save some time. Sequencing of the whole construct is highly recommended.

Fig. 17.5. Gene targeting scheme. A gene locus, targeting vector and targeted locus are presented schematically. Restriction sites for one enzyme are presented above the original locus by the letter R and the fragment lengths by the black lines between them. Additional restriction sites for the same enzyme are present in the targeting vector and, upon correct targeting, will change the sizes of fragments detected by the 3 and 5 probes (shown above the targeted represent exons (numbered above in the original locus) and dashed lines indicate the area of homologous locus). is for a possible reporter, and is for antibiotic selection cassette (which also has recognirecombination. tion sequences for a recombinase before and after it, which would allow its removal in the mice by expression of the recombinase, such as Cre).

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3.6.5. Targeting the Gene in Mouse ES Cells

First make sure that you start with ES cells that are capable of potently establishing germ line chimeric mice (7). If your laboratory is not experienced with ES cell culturing, it is better to collaborate with someone with experience or use a professional service. The linearized construct is electroporated into the ES cells, which are then cultured under antibiotic selection. Positive clones are picked and expanded to allow screening for correctly targeted ones. It is important to maintain proper culture conditions throughout, since even a slight differentiation may impair the ability of ES cells to generate germ line chimeric mice.

3.6.6. Generating Chimeric Mice

Confirmed ES cell transfectants bearing the correctly targeted construct are either injected into blastocytes or aggregated with early morula. Both techniques require practical experience, and getting help from specialized centers is important. Using different mouse strains that differ in coat color from that of the ES cells allows easy identification of chimeric mice.

3.6.7. Obtaining Germ Line Transmitted Offspring

Founder chimeras are mated with divergent coat color mice to identify ES cell-derived offspring. However, one must also consider the genetic background necessary for further experiments. It is possible to first mate chimeric founders with another strain to identify germ line-positive mice and then mate them with the strain of choice. Backcrossing onto a desired genetic background should be started as soon as possible (literally by mating the chimeric founder with the strain of interest).

3.6.8. Verify the Correct Gene Targeting in the Mice

Characterization of newly generated mice is essential. Southern analysis for correct targeting of the gene locus should be performed, as well as northern analysis or RT-PCR for mRNA expression and protein detection by western or any additional method that applies. There are numerous examples of reasonable approaches that generated a mouse with no phenotype that were later realized to suffer from a technical problem. Having mice with verified targeting should be established as soon as possible in your new model.

3.6.9. Backcross onto a Pure Genetic Background

For NK cell studies, it is essential to backcross at least —seven to nine generations onto a chosen background mouse strain to assure homozygosity in both the NK cell gene locus (especially the Ly49 genes, which are polymorphic between mouse strains and can account for variations in NK cell responsiveness between mouse strains) and the MHC class I locus (to assure Ly49 ligands are also consistent). Several published reports of NK cell defects in knockout mouse studies have subsequently been attributed to lack of adequate backcross to a homozygous background in these gene loci, thereby preventing adequate comparisons with wild-type control mice of that background.

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4. Notes 1. When using a buffy coat, dilution should be 1:3. 2. If separating more than 1×106 cells, multiply all added amounts accordingly, for example, for 2×106 , add 80 ␮L of cold autoMACS medium and 20 ␮L biotin Ab cocktail (multiply by 2). 3. NK cell splitting should be performed when the well becomes yellow. If cell splitting is done ahead of time, NK cell division will be stopped. 4. cDNA of the relevant gene should be constructed from mRNA of cells that express the gene. Expression should be validated by FACS staining or by any other appropriate method. 5. Restriction enzymes should be chosen according to the enzyme restriction map of the relevant gene and plasmid. Other restriction sites can be used if different vectors are utilized. 6. Design your construct so that the extracellular portion of the desired receptor will be in frame with the Fc portion. 7. The Ig plasmid is a low-copy vector; therefore, the large prep should be cultured in a large volume of 500 mL LB medium containing ampicillin and tetracycline solutions (1:4000 and 1:100, respectively). [It is now possible to work with a high copy Ig plasmid, which can be transfected into regular competent bacteria (see Fig. 17.3).] 8. TOP10 Escherichia coli cells carrying the p3 plasmid are designed for transformation of vectors that encode the synthetic supF gene (tyrosine tRNA suppressor). The p3 plasmid is a low-copy number, 60 kb plasmid that carries the drug resistance markers for kanamycin, tetracycline, and ampicillin. The kanamycin gene is fully active and is used to select for cells carrying p3. The tetracycline and ampicillin genes carry amber mutations that render the genes inactive during normal growth and replication of the bacteria. Upon transformation of a vector carrying the suppressor F gene (such as pcDNA1.1 or pCDM8), the amber mutations in the tetracycline and ampicillin genes on the p3 plasmid are suppressed and the E. coli are resistant to these antibiotics. 9. To obtain a sufficient amount of a single-fusion Ig protein, harvest from 60- × 6-well plates, for a total of 360 wells, is recommended. All calculations are for 360 wells.

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10. Medium should be gently dropped on the well’s side walls and not directly on top of the cells, since the cells are easily detached from the plate. 11. The medium at this step contains the Ig fusion protein. The collected medium should be yellow, which indicates cell growth and fusion Ig secretion. 12. Medium from the first and second collection should be pooled together. 13. Restriction enzymes should be chosen according to the enzyme restriction map of the relevant gene and plasmid. 14. The pcDNA3 plasmid is a high-copy vector; therefore, the large prep should be in a volume of 200–250 mL LB containing ampicillin (1:1000) for final concentrations of 0.1 mg/mL. 15. For final concentration of 5 mg/mL add 1 mL of medium containing 10 mg/mL G418. 16. For final concentration of 5 mg/mL add 1 mL of medium containing 5 mg/mL G418. 17. Analyze cell for protein expression after each cell transfer from one plate to another since cells sometimes tend to lose expression once transferred to larger volumes. 18. Use normal BW cells as a negative control. 19. Prepare triplicates for each E:T ratio of each target cell. 20. Supernatant can be transferred onto an ELISA plate coated with the relevant antibody or transferred to a new 96 F-well plate and kept at −20◦ C until further analysis. Once analysis is possible, thaw the supernatant at room temperature and transfer onto a MaxiSorp plate coated with the relevant antibody. 21. Importantly, the two anti-IL-2 mAb should recognize different epitopes. 22. Work with triplicates for each target cell, for each effector to target ratio, and for each experiment. 23. Spontaneous and total radioactive release determinations are important for the result analysis and calculations. It enables the comparison between different experiments. 24. Do not add NK cells to the spontaneous and total release wells!!! Equalize the volume in each spontaneous release well to 200 ␮L by adding complete RPMI medium. The volume in each total well will be normalized to 200 ␮L later on by adding 100 ␮L of 0.1 M NaOH. 25. 0.1 M NaOH should be prepared fresh while plates are being centrifuged (use 1 M NaOH stock solution).

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26. The experiment is considered to be valid if the spontaneous release does not exceed 10–30% of the total release, meaning that the target cells were in good condition. Results are calculated as percentages according to the following equation: Target − Spontaneous  ∗ 100 = % Specific Lysis Total − Spontaneous 27. If adding two different antibodies or more, dilute each antibody so that total/sum antibodies volume/well will be 50 ␮L. 28. Depending on killing levels, one can determine if the receptor is activating, inhibitory, or co-stimulatory. An activating receptor is expected to exhibit high killing percentage following antibody cross-linking. Inhibitory receptors are expected to cause low killing percentage following combined addition of both activating and inhibitory receptor antibodies as compared to addition of activating receptor antibody alone. A co-stimulatory receptor is expected to cause low killing percentage following antibody crosslinking alone, which resembles killing levels of control antibody cross-linking or no antibody. On the other hand, co-stimulatory receptor antibody should increase killing percentage when added in combination with an activating receptor antibody, as compared to adding the activating receptor antibody alone. 29. Restriction enzymes should be chosen according to the enzyme restriction map of the relevant gene and plasmid. 30. The pBabe plasmid is a high-copy plasmid; therefore, the large prep bacterial culture should be grown in a volume of 200–250 mL LB containing ampicillin (1:1000), for final concentrations of 0.1 mg/mL. pBabe can be obtained from Dr. Garry Nolan, Stanford University. 31. It is best to work with triplicates. Therefore, 40 ␮g should be precipitated, which would include 10 ␮g for each individual transfection and 10 ␮g extra. 32. If triplicate transfections are being performed, resuspend the 40 ␮g of DNA with 300 ␮L of DMEM medium (100 ␮L/10 ␮g DNA). Ignore the extra 10 ␮g and consider as triplicates only. Also prepare three different microfuge tubes with 400 ␮L medium containing the transfection reagent, which would include one for each individual sample. 33. In contrast to the previously described transfections, be sure to drop the DNA on top of the medium containing the transfection reagent here and not the other way around.

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34. If 40 ␮g of DNA was resuspended with 300 ␮L DMEM medium, mix well and add 100–400 ␮L of DMEM medium plus transfection reagent ×3. 35. This is an important step for ensuring high transfection efficiency. 36. Analyze cells for protein expression following each cell transfer from one plate to another. Cells tend to lose expression once transferred to larger volumes. References 1. Katz, G., Gazit, R., Arnon, T. I., GonenGross, T., Tarcic, G., Markel, G., Gruda, R., Achdout, H., Drize, O., Merims, S., and Mandelboim, O. (2004) MHC class I-independent recognition of NK-activating receptor KIR2DS4. J Immunol 173, 1819–1825. 2. Biassoni, R., Cantoni, C., Pende, D., Sivori, S., Parolini, S., Vitale, M., Bottino, C., and Moretta, A. (2001) Human natural killer cell receptors and co-receptors. Immunol Rev 181, 203–214. 3. Mandelboim, O., Reyburn, H. T., ValesGomez, M., Pazmany, L., Colonna, M., Borsellino, G., and Strominger, J. L. (1996) Protection from lysis by natural killer cells of group 1 and 2 specificity is mediated by residue 80 in human histocompatibility leukocyte antigen C alleles and also occurs with empty major histocompatibility complex molecules. J Exp Med 184, 913–922. 4. Mandelboim, O., Malik, P., Davis, D. M., Jo, C. H., Boyson, J. E., and Strominger, J.

L. (1999) Human CD16 as a lysis receptor mediating direct natural killer cell cytotoxicity. Proc Natl Acad Sci U S A 96, 5640–5644. 5. Richardson, J., Reyburn, H. T., Luque, I., Vales-Gomez, M., and Strominger, J. L. (2000) Definition of polymorphic residues on killer Ig-like receptor proteins which contribute to the HLA-C binding site. Eur J Immunol 30, 1480–1485. 6. Baba, E., Erskine, R., Boyson, J. E., Cohen, G. B., Davis, D. M., Malik, P., Mandelboim, O., Reyburn, H. T., and Strominger, J. L. (2000) N-linked carbohydrate on human leukocyte antigen-C and recognition by natural killer cell inhibitory receptors. Hum Immunol 61, 1202–1218. 7. Nagy, A., Rossant, J., Nagy, R., AbramowNewerly, W., and Roder, J. C. (1993) Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci U S A 90, 8424–8428.

Chapter 18 Generating NK Cell Receptor-Fc Chimera Proteins from 293T Cells and Considerations of Appropriate Glycosylation Alon Zilka, Michal Mendelson, Benyamin Rosental, Oren Hershkovitz, and Angel Porgador Abstract The use of recombinant receptors as a scientific tool has become widespread in many research fields. Of particular interest are the natural killer (NK) receptors that play a major role in the immune response against tumors and virus-infected cells. We present here (i) a detailed protocol for the production and purification of soluble recombinant NK cell receptors tagged with human IgG1-Fc (thus termed receptorFc chimera or receptor-Ig fusion protein) and (ii) a protocol for cell staining with these recombinant receptor-Fc chimeras. As these recombinant proteins are produced in eukaryotic cells, we further discuss the glycosylation pattern of these receptors that might interfere with their ligand-binding phenotype. Key words: Natural killer (NK), natural cytotoxicity receptors (NCRs), recombinant receptor-Fc chimera, transient transfection, glycoprotein.

1. Introduction Employment of recombinant immunoreceptor-Fc chimeras as a scientific tool was first reported nearly two decades ago [e.g., the elucidation of the CD28/CTLA4 and B7 interactions (1, 2)]. Today, these chimeras have become imperative for basic and applied scientific research in various fields including studies of natural killer (NK) cell receptors. When compared to anti-ligand mAbs, the receptor-Fc chimera approach can present advantages for studies involving unknown ligands, receptors that recognize a multitude of ligands, and agonist studies. We have employed the chimera approach for the identification of ligands to natural K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 18, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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cytotoxicity receptors (NCRs). Several companies (e.g., R&D, Autogen bioclear) sell recombinant NCR-Fc chimeras, yet we prefer to produce them in-house. This is not simply because of financial reasons but mostly due to a need for stringent quality and batch control; in particular, we consider the glycosylation state of these recombinant glycoproteins to be very important and therefore produce them in eukaryotic expression systems (see Notes 1 and 2). For the NCR-Fc chimera, we have utilized (i) the ectodomains of the NCRs mostly with the membrane linker region (3–6) and (ii) a human (h) IgG1 Fc (CH3 + CH2 + hinge sequences) as published previously (7) (see Note 3 for the amino acid sequence) and also used within commercial products. We have employed transient transfection (HEK-293T) or have generated stable transfectants (CHO-K1), using pCDNA3.1-based vectors (4, 5, 8, 9). For transient transfections we have also employed pDC409- or piSV-based vectors. Below, we describe a protocol for production of NCR-Fc chimeras in HEK-293T cells, purification of the chimeric protein, and use in flow cytometrybased experiments. The yield ranges between 1 and 5 mg of purified product per 1 L of supernatant from transiently transfected HEK-293T cells.

2. Materials 1. Plasticware: 25 and 10 mL pipettes, 100 mm tissue culture plates, 0.22 ␮m filters (1 L, 500 and 250 mL), and T25, T75, and T175 tissue culture flasks. 2. 5% CM DMEM: 460 mL DMEM medium containing glutamine, 25 mL fetal bovine serum (FBS; heat-inactivated), 5 mL 1 M HEPES, 5 mL nonessential amino acids, and 5 mL sodium pyruvate (all solutions from Gibco BRL, Carlsbad, CA, USA). We do not add antibiotics. 3. HEK-293T cells (American Type Culture Collection). 4. BIO-CHO (+): 500 mL BIO-CHO-1 (Biological Industries, Beit-Haemek, Israel), 5 mL BIOGRO-CHO (Biological Industries), and 5 mL L-glutamine ×100 (Gibco BRL). This is a medium without serum that is intended for collection of the supernatant from transfected cells. 5. 2 M CaCl2 : resuspend 5.88 g of CaCl2 ·2H2 O in 20 mL sterile DDW. Sterilize by passing through a 0.2 ␮m syringe filter (Corning, Acton, MA, USA). Keep in a 50 mL tube sealed with parafilm. 6. HBS (HEPES-buffered saline) ×2: in 90 mL sterile DDW, dissolve 1.6 g NaCl, 0.074 g KCl, 0.027 g Na2 HPO4 ·2H2 O, 0.2 g dextrose, and 5 mL 1 M HEPES

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solution (Gibco BRL). Mix thoroughly until dissolved, add sterile DDW up to 100 mL, and filter through a 0.22 um filter. The final concentrations of the ingredients are as follows: 280 mM NaCl, 10 mM KCl, 1.5 mM Na2 HPO4 ·2H2 O, 12 mM dextrose, and 50 mM HEPES. This solution is stable for only 3 months, so make sure to note the date on the bottle. pH of the solution should be 7.05, otherwise titer (important). 7. 20% ETOH. 8. D-PBS (Dulbecco’s phosphate-buffered saline; Gibco BRL). 9. 25% trypsin-EDTA (Gibco BRL). 10. Akta Explorer FPLC system (Pharmacia Biotech, GMI Inc., Ramsey, MN, USA). 11. HitrapTM protein G HP column (GE Healthcare, Uppsala, Sweden). 12. Amicon Ultra-15 dialyzer (Millipore, Carrigtwonhill, Ireland). 13. 0.1 M glycine (pH 2.7). 14. Tris−HCL (1 M solution, pH 9). 15. PBA×10: dissolve BSA (Sigma, cat. A-7030) and sodium azide in D-PBS to a final concentration of 5 and 0.5%, respectively. Dilute PBA×10 in D-PBS to get PBA×1 (final concentrations: 0.5% BSA and 0.05% sodium azide). 16. D-PBS + 1 mM EDTA. 17. APC/PE-conjugated F(ab )2 -goat-antihuman IgG-Fc secondary antibody with minimal cross-reaction to bovine and mouse IgG (Jackson Immuno Research, West Grove, PA, USA). 18. PI (propidium iodide) stock (1 mg/mL).

3. Methods 3.1. Transient Transfection Method for Production of Recombinant Receptor-Fc 3.1.1. Preparation for Transfection

All media and solutions are sterile; use sterile technique throughout the following procedures. Log all activities from medium preparation onto the batch record of the specific production. 1. Defrost an aliquot of HEK293T cells (P2-passage freeze). Centrifuge cells and resuspended in 10 mL of 5% CM DMEM. Then add to a T25 flask and transfer to a 37◦ C, 5% CO2 incubator for cell growth.

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2. Cell expansion: upscale from a T25 flask to T75 and T175 flasks: a. During cell expansion, harvest flasks with a confluence below 80%, thus keeping the cells in a log phase. b. Carefully decant the used medium from the flask. c. Add 10 mL D-PBS to the flask without pouring it on the cells so that they will not fall. d. Wash carefully and decant the D-PBS. Add trypsin solution for 1 min (1 mL to each T75 or 2 mL to each T175) to detach the cells from the flask without tapping. e. Add 10 mL 5% CM DMEM and transfer to the bigger flask (1:3 ratio from T25 to T75 and from T75 to T175; 25 mL in T75, and 50 mL in T175). 3.1.2. Day 1 of Transfection

1. Trypsinize the cells in the T175 flasks as above. Transfer cells (about 12.5 mL of trypsin and medium) from each T175 flask to a different 50 mL tube. Using a different tube will prevent cross-contamination, should it happened in one of the flasks. 2. Count the cells in a hemocytometer. Add 5% CM DMEM to the 50 mL tube, so the cells will be at a final concentration of 0.75×106 cells/mL. After counting the cells from all flasks (each flask in a different 50 mL tube) decide how many 100 mm plates to use. For large-scale protein prep, use fifty to sixty 100 mm plates. Take into account the available medium, plates, cells, and DNA per plate: for each plate you need 20 mL BIO-CHO (+), 2 ␮g DNA, and 0.75×106 cells. 3. Prepare the needed number of 100 mm plates and apply 9 mL of 5% CM DMEM to each plate with a 25 mL pipette. Then add 1 mL cells, stir the plate gently and allow the cells to adhere overnight.

3.1.3. Day 2 of Transfection

All calculations listed are for fifty 100 mm plates. 1. Check the 100 mm plates for a confluence of 40–50%, not more. 2. In a 50 mL tube, mix 3.1 mL 2 M CaCl2 with 22.75 mL sterile DDW (∼0.25 M CaCl2 final concentration). Then add 105.5 ␮g of plasmid DNA and mix. Let it stand for a minimum of 1 min. 3. Prepare 10 sterile 1.5 mL microfuge tubes on a stand inside the sterile hood. 4. Aliquot 490 ␮l HBS×2 to each microfuge tube. 5. Then add 490 ␮l of the CaCl2 −plasmid mix to the HBS×2: very slowly − drop by drop to create precipitation. Do so for all of the 10 microfuge tubes.

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6. Collect the CaCl2 + plasmid + HBS without pipetting, using a 1 mL pipetman and add it to the cells drop by drop. 7. Stir the medium gently and incubate the plates in the 37◦ C, 5% CO2 incubator. 3.1.4. Day 3 of Transfection

1. Take out the BIO-CHO (+) medium and warm it to 37◦ C. (If you want to change medium in the morning, leave it overnight on the bench.) 2. Take out the plates from the incubator and organize them for efficient work. Raise a plate at one side and aspirate the DMEM from the other side. Remove the medium from 10 plates before applying the BIO-CHO (+). Discard the used DMEM medium. 3. Apply 10–13 mL of BIO-CHO (+) onto the side of the raised empty plate. Do not perform it too quickly, as cells can detach from the plate, and they will not re-adhere under these conditions. Gently return to the incubator.

3.1.5. Days 5 and 8 of Transfection

1. Take out the BIO-CHO (+) medium and warm it to 37◦ C. (If you want to change medium in the morning, leave it overnight on the bench.) 2. Take out the plates from the incubator and organize them for efficient work. Raise the plate at one side and aspirate the BIO-CHO (+). Filter the collected medium through a 500 mL 0.22 ␮m filter apparatus. Collect the medium from 10 plates before applying the fresh BIO-CHO (+). 3. Apply 10–13 mL of fresh BIO-CHO (+) onto the one sideraised empty plate. Do not perform it too quickly, as cells can detach from the plate and they will not re-adhere under these conditions. Gently return to the incubator. 4. Take the collected and filtered BIO-CHO (+) medium, seal the cap with parafilm, and label the bottle as collection I with date and protein name. Store at 4◦ C for the purification step. 5. On day 8, collect the medium from the plates as above, but filter the collected medium through a 1 L 0.22 ␮m filter apparatus and re-filter the day 5-collected medium (collection I) into the same 1 L apparatus. Store at 4◦ C for the purification step, yet better to purify ASAP. After collecting the medium, discard the plates.

3.2. Protein G-Based Purification of NCR-Fc Chimera on FPLC

The following protocol is generalized for a Akta Explorer FPLC (Pharmacia Biotech), although other equipment can be substituted. 1. Filter the protein-containing medium in a 0.2 um filter onto the same day it is loaded on the FPLC. As a rule, all

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solutions (except the 20% ETOH) should be sterilized by filtration before they are loaded on the FPLC. 2. Install a 5 mL HitrapTM protein G HP column on the FPLC. 3. Wash the system and the column with 10 column volumes (CV) of DDW. 4. Change the loading buffer to D-PBS and the elution buffer to glycine 0.1 M pH 2.7 and rewash the system and the column with 10CV D-PBS. 5. Load the filtered protein-containing medium onto the column at a max flow of 5 mL/min. 6. Wash the column with 10CV of D-PBS to remove unbound proteins. 7. Install cap-less eppendorfs containing 80 ␮L of Tris−HCL 1 M pH 9 on the fraction collector. 8. Elute the recombinant protein with a linear gradient of glycine 0.1 M pH 2.7. 9. Collect the fractions containing the recombinant protein (as seen on the 280 uv OD graph) and dialyze on an Amicon Ultra-15 with the appropriate molecular weight cutoff (MWCO) against D-PBS. 10. Wash the FPLC system and the column with DDW and then with 20% ETOH. 11. Measure protein concentration and keep in aliquots at −20o C. 3.3. Cell Staining with Fusion Proteins (Receptor-Fc Chimera) Using Flow Cytometry

Keep cells and reagents on ice throughout all this procedure. 1. For floating/loosely attached cell, remove cells from the flask by pipetting with D-PBS or tapping the flask. For adherent cells, remove the medium and incubate the cells with 10 mL D-PBS + EDTA 1 mM for a few minutes. Tap the flask lightly to remove the cells. 2. Transfer the cells to a 15 mL tube and centrifuge 300×g for 6 min at 4◦ C. 3. Re-suspend the pellet in PBA, count the cells, and dilute to final concentration of 105 cells/100 ␮L. 4. Plate the cells in 96-well U-bottom plate, 100 ␮L in each well. 5. Centrifuge the plate 1100×g for 3 min at 4◦ C. 6. Discard the supernatant by flipping and gently blotting the inverted plate onto a paper towel. 7. Add 20 ␮L PBA to each well to prevent cells from drying.

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8. Gently vortex the plate while holding the cover until the cell pellet breaks. 9. Prepare the fusion protein as follows: for each well, use 1–10 ␮g of fusion protein (depend upon the recombinant receptor), diluted in D-PBS to a final volume of 72 ␮L, plus 8 ␮L of PBA×10. 10. Add fusion proteins to the wells with a multichannel pipetman. Incubate on ice for 2 h. 11. Add 130 ␮L of PBA with a multi-pipetman to each well and centrifuge 1100×g for 3 min at 4◦ C. Discard the supernatant by flipping and gently blotting the inverted plate onto a paper towel. Do not vortex the plate! 12. Add a secondary antibody at 1:60 or 1:75 dilution in 50 ␮L PBA and incubate on ice for 30 min (cover with aluminum foil). We recommend APC/PE-conjugated F(ab )2 -goatantihuman IgG-Fc with minimal cross-reaction to bovine and mouse IgG as a secondary reagent. 13. Add 150 ␮L of PBA with a multichannel pipetman to each well and centrifuge 1100×g for 3 min at 4◦ C. Discard the supernatant by flipping and gently blotting the inverted plate onto a paper towel. 14. Add 200 ␮L of PBA with a multichannel pipetman to each well, transfer the samples to FACS tubes, and analyze the samples using a flow cytometry instrument. If staining the cells with PI, use 5 ␮L of PI stock +195 ␮L of PBA

4. Notes 1. The utilization of recombinant proteins including receptorFc chimeras for scientific research and biotechnology industry is associated with the concern for proper glycosylation (10). The importance of the posttranslational modification of proteins with N- or O-linked oligosaccharides is well documented by their implication in numerous biological phenomena (11, 12). In particular, the phenomenon of altered glycosylation interfering with ligand binding of a glycoprotein is well established (13, 14). The recombinant glycoproteins produced by the pharmaceutical industry are analyzed regularly for their glycan content in accordance with FDA regulations. However, insufficient analysis of glycan content is performed for recombinant glycoproteins produced solely for research purposes. We have shown that for NCRs, conjugated glycans can be involved (i) directly − e.g., the interaction of influenza hemagglutinin

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with NKp46 and NKp44 (3, 6, 15) and (ii) indirectly − e.g., excessive N-glycosylation of NKp30 inhibits its binding to tumor membrane-associated heparan sulfate (8). The effect of N-glycosylation on the ability or NKp30 to bind to cellular ligands is not clearly evident for NKp46 and NKp44 (data not shown). This could be due to the location of the N-glycans, which are restricted to the membrane linker hinge region in NKp44 and NKp46, yet are located within the Ig-like domain for NKp30 (8). Therefore, excessive N-glycosylation within the domain could induce nonreversible glycan structuredictated changes in protein folding that take place in the ERGolgi (16). Alternatively, excessive N-glycosylation within the domain could mask the site for binding of the cellular ligands. 2. We showed that the choice of cell line for production of the NCR-Fc chimera has a prominent effect on the conjugated glycoforms (8). Moreover, the growth conditions of the producing cell line could alter the glycosylation phenotype. We compared NKp30-Fc produced in CHO-K1 cells grown in bioreactor or in tissue culture flasks and observed excessive glycosylation for the NKp30-Fc from the bioreactor-grown cells. That growth conditions induce changes of glycosylation in recombinant glycoproteins is well documented (17). Therefore, we recommend checking and comparing each new batch for size, purity, and monomer−dimer ratio by SDS-PAGE analysis (with and without ␤-ME) and for sialic acid content by 2D (IEF/SDS-PAGE) electrophoresis. If we have indications for excessive N-glycosylation, we further assess the batch glycoforms in the Center for Glycobiology at Ben Gurion University. 3. Amino acid sequence of the hIgG1 Fc region (CH3 + CH2 + hinge sequences): DPEPKSSDKTHTCPPCPAPEFEGAPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK

Acknowledgments This work was supported by a grant from the United States-Israel Binational Science Foundation (AP).

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References 1. Linsley, P. S., Brady, W., Grosmaire, L., Aruffo, A., Damle, N. K., and Ledbetter, J. A. (1991) Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. J Exp Med 173, 721–730. 2. Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L. S., Damle, N. K., and Ledbetter, J. A. (1991) CTLA-4 is a second receptor for the B cell activation antigen B7. J Exp Med 174, 561–569. 3. Arnon, T. I., Lev, M., Katz, G., Chernobrov, Y., Porgador, A., and Mandelboim, O. (2001) Recognition of viral hemagglutinins by NKp44 but not by NKp30. Eur J Immunol 31, 2680–2689. 4. Bloushtain, N., Qimron, U., Bar-Ilan, A., Hershkovitz, O., Gazit, R., Fima, E., Korc, M., Vlodavsky, I., Bovin, N. V., and Porgador, A. (2004) Membrane-Associated Heparan Sulfate Proteoglycans Are Involved in the Recognition of Cellular Targets by NKp30 and NKp46. J Immunol 173, 2392–2401. 5. Hershkovitz, O., Jivov, S., Bloushtain, N., Zilka, A., Landau, G., Bar-Ilan, A., Lichtenstein, R. G., Campbell, K. S., Kuppevelt, T. H., and Porgador, A. (2007) Characterization of the Recognition of Tumor Cells by the Natural Cytotoxicity Receptor, NKp44. Biochemistry 46, 7426–7436. 6. Mandelboim, O., Lieberman, N., Lev, M., Paul, L., Arnon, T. I., Bushkin, Y., Davis, D. M., Strominger, J. L., Yewdell, J. W., and Porgador, A. (2001) Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells. Nature 409, 1055–1060. 7. Aruffo, A., Stamenkovic, I., Melnick, M., Underhill, C. B., and Seed, B. (1990) CD44 is the principal cell surface receptor for hyaluronate. Cell 61, 1303–1313. 8. Hershkovitz, O., Jarahian, M., Zilka, A., Bar-Ilan, A., Landau, G., Jivov, S., Tekoah, Y., Glicklis, R., Gallagher, J. T., Hoffmann, S. C., Zer, H., Mandelboim, O., Watzl, C., Momburg, F., and Porgador, A. (2007) Altered glycosylation of recombinant NKp30

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hampers binding to heparan sulfate: a lesson for the use of recombinant immunoreceptors as an immunological tool. Glycobiology, epub Nov 15. Zilka, A., Landau, G., Hershkovitz, O., Bloushtain, N., Bar-Ilan, A., Benchetrit, F., Fima, E., van Kuppevelt, T. H., Gallagher, J. T., Elgavish, S., and Porgador, A. (2005) Characterization of the heparin/heparan sulfate binding site of the natural cytotoxicity receptor NKp46. Biochemistry 44, 14477–14485. Jenkins, N., Parekh, R. B., and James, D. C. (1996) Getting the glycosylation right: implications for the biotechnology industry. Nat Biotechnol 14, 975–981. Gabius, H. J. (2006) Cell surface glycans: the why and how of their functionality as biochemical signals in lectin-mediated information transfer. Crit Rev Immunol 26, 43–79. Rudd, P. M., Elliott, T., Cresswell, P., Wilson, I. A., and Dwek, R. A. (2001) Glycosylation and the immune system. Science 291, 2370–2376. Kaneko, Y., Nimmerjahn, F., and Ravetch, J. V. (2006) Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313, 670–673. Krapp, S., Mimura, Y., Jefferis, R., Huber, R., and Sondermann, P. (2003) Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity. J Mol Biol 325, 979–989. Ho, J. W., Hershkovitz, O., Peiris, M., Zilka, A., Bar-Ilan, A., Nal, B., Chu, K., Kudelko, M., Kam, Y. W., Achdout, H., Mandelboim, M., Altmeyer, R., Mandelboim, O., Bruzzone, R., and Porgador, A. (2008) H5-type influenza virus hemagglutinin is functionally recognized by the natural killer-activating receptor NKp44. J Virol 82, 2028–2032. Molinari, M. (2007) N-glycan structure dictates extension of protein folding or onset of disposal. Nat Chem Biol 3, 313–320. Werner, R. G., Noe, W., Kopp, K., and Schluter, M. (1998) Appropriate mammalian expression systems for biopharmaceuticals. Arzneimittelforschung 48, 870–880.

Chapter 19 Identification of NK Cell Receptor Ligands Using a Signaling Reporter System Yoshie-Matsubayashi Iizuka, Nikunj V. Somia, and Koho Iizuka Abstract NK cell responses are regulated by a balance of inhibitory and activating signals, reflecting the net effect of interactions between receptors and ligands on target and effector cell surfaces. The identification of ligands for orphan NK cell receptors is key to enhancing our understanding of NK cell biology. Here we describe a strategy (protocol) for the identification of ligands for orphan NK cell receptors using signaling reporter cells in combination with a virus rescue system. Key words: expression cloning, NK cell receptor, retroviral expression vector, retrovirus rescue, repackaging of retrovirus.

1. Introduction Expression cloning requires screening and detection procedures with as little background noise in the system as possible. Where this is unavoidable, multiple rounds of screening and detection may reduce noise (reiterated screening). Success further depends on the specificity and sensitivity of baits (or assays) that may also contribute to reducing background noise. Here we describe a protocol for the identification of orphan NK cell receptor ligands using a signaling reporter cellular assay as bait. The expression cloning protocol is composed of two components: screening candidate cells expressing putative ligand(s) with subsequent expression cloning and enriching candidate cDNA by repackaging the integrated cDNA in a retrovirus vector (retrovirus rescue). The success of T cell expression cloning methodology has established the feasibility of using cellular reporter assays as highly specific and sensitive baits. This methodology was developed to K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 19, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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identify a specific peptide for a T cell receptor (TCR) by using a T cell hybridoma harboring a reporter construct with a tandem repeat of the NFAT-binding site from the IL-2 promoter region following a LacZ sequence (NFAT-LacZ reporter gene) (1). The cytoplasmic tail of the CD3␨ chain was shown to be sufficient for TCR signaling by crosslinking of CD8/zeta chimeric receptors (2). Many NK cell receptors contain immunoreceptor tyrosinebased inhibitory motifs (ITIMs) or immunoreceptor tyrosinebased activating motifs (ITAMs). NK cells utilize many of the same signaling molecules involved in the TCR activation pathway. In this context, a reporter cell expressing a chimeric receptor consisting of the ectodomain of an orphan receptor with a CD3␨ cytoplasmic domain and an integrated NFAT-LacZ reporter gene can interact with cells expressing putative ligand and induce LacZ expression with specificity and high sensitivity (3) (Fig. 19.1). This reporter system allows us to detect ligand expression on a target cell surface as a positive functional readout, regardless of the native receptor function or structure. The specificity of signaling is confirmed by the negative responses of reporter cells expressing the orphan receptor without a cytoplasmic signaling chain. A signaling reporter system utilizing a chimeric receptor achieves functional specificity through cellular binding followed by a functional signaling readout. Thus, reporter cells serve as an effective screening tool for cells expressing putative ligands in expression cloning strategies. Upon identification of a cell line or cell types that stimulate reporter cells, a cDNA library within a retrovirus expres-

Fig. 19.1. Reporter assay system. Reporter cell (J7) harbors a reporter construct with three tandem repeats of the NFAT-binding sites from the IL-2 promoter followed by a lacZ sequence (1). The chimeric reporter construct − Nkrp1f ectodomain, Ly49A transmembrane (TM), and mouse CD3␨ cytoplasmic domain − is transduced into J7 reporter cells. Upon ligand (Clrg) recognition, reporter cells induce lacZ expression.

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sion vector can be generated from ligand-expressing cells. During screening, negative cell lines that fail to stimulate reporter cells will also be identified. These ligand-negative cell lines can be transduced with the cDNA library generated from ligand-positive cells, hence enabling ligand display and cloning using a reporter cell line. One of the most important and critical steps in expression cloning using a retroviral expression vector is to control the number of cDNA-containing vectors integrated into a recipient cell. Knowing this number enables an evaluation of how many clones should be screened from a given size of the cDNA library. As a rule you need to screen three times the base number of the library to get coverage, i.e., for a library of 106 clones, 3 × 106 integrants need to be analyzed. An estimate of the number of cDNA vector integrates is important not only for the evaluation of library coverage but also for recovering the candidate cDNA. Single integrants make the recovery and re-analysis of potential ligand cDNAs a facile process. Multiple integrations can be easily avoided by lowering the titer of infection. However, this strategy is not ideally applied to screening a cDNA library in a ligand-negative and a reporter cell assay, because lowering the multiplicity of infection (MOI) significantly increases the number of plates required for screening. On the other hand, higher MOI increase the number of integrants per cell and reduces the number of screening plates. How-

Fig. 19.2. Schematic representation of recovering stably integrated helper-free retrovirus by repackaging. Stably integrated packaging signal ()-containing helper-free viruses (GFP in this scheme) in NIH 3T3 or BW5147 (3T3-GFP and BW-GFP, respectively) were rescued by introduction of MMLV env and gag-poleither by transient transfection or by stable transduction with a lentivirus vector. IP, IRES-Puro; IB, IRES-blasticidin.

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ever, this approach requires a greater effort to establish which cDNA is responsible for reporter signaling. We address this problem by recovering multiple-integrated cDNAs from infected cells by repackaging them as infectious virions. To this end, we introduce Moloney murine leukemia virus (MMLV) env and MMLV gag-pol genes by either transient transfection or transduction of these genes by lentiviral vectors (Fig. 19.2). By repeating the screening and enriching the pool of cDNA for the ligand, candidate cDNA can be identified. Due to unknown nature of putative ligand and orphan receptor, a specific description of the protocol for each receptor and ligand is not applicable. Thus, in Section 3, we describe a general procedure for this cloning strategy. In Section 4, we describe an established library and reporter for Nkrp1f and the library containing its ligand cDNA, Clrg (3). We also describe potential caveats and troubleshooting related to this example receptor and ligand that may be instructive for other receptor–ligand pairs.

2. Materials

2.1. Cell Culture and Reagents for Transduction and Transfection

1. RPMI-1640 medium supplemented with 2 mM glutamine, 1 mM pyruvate, 50 ␮M 2-mercaptoethanol (2-ME), 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 10% fetal bovine serum. 2. Appropriate medium for target cells expressing the candidate ligand. 3. Polybrene (Sigma-Aldrich). 4. FuGENE 6 (Roche)

2.2. CPRG Assays

1. Z-buffer (PBS pH 7.4 containing 100 mM 2-ME, 9 mM MgCl2 , and 0.125% NP-40). Store at room temperature. Stable at least 3 weeks. 2. 200 × CPRG stock solution (30 mM chlorophenol red galactoside; Calbiochem). Cover with foil, store at 4◦ C. 3. Z-buffer containing CPRG: dilute CPRG stock solution 200-fold in Z-buffer for a final concentration of 150 ␮M CPRG. 4. Stop Buffer (H2 O, 300 mM glycine, 15 mM Na2 EDTA)

2.3. Microplate Reader 2.4. Cell Lines

Several commercial options are available. We use ␮Quant (BIOTEK Instruments). 1. Reporter cell line expressing orphan receptor-CD3␨ chimeric construct and reporter cell line expressing orphan

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receptor without the cytoplasmic signaling motifs as negative control (see Note 1). We use BWZ-36 (mouse reporter cell line) or J7 (Jurkat cell-derived reporter cell line) as reporter cells (1, 4). 2. Slate of cell lines or primary cells to screen with the reporter cells (see Note 2). 3. Ecotropic and/or amphotropic packaging cell lines (see Note 3). We use 293T cell-based package cell lines. We use Plat-E for ecotropic virus production and Nick-A for amphotropic virus production (4, 5). 2.5. cDNA Library and Expression Vectors Containing gag-pol and env Genes

1. cDNA library generated from mRNA from the cell line stimulating reporter cells but not control reporter cells. For cDNA library construction, please refer to the protocol in each cDNA library construction kit. A MLV-derived retroviral expression vector should be used for this protocol. Here, we use the pMX vector (5). 2. pMX-GFP to use as positive control and to estimate the average number of integrated viral vector in recipient cells. 3. Mammalian expression vectors coding for MMLV env and gag-pol. We prepared eco-env and gag-pol cDNAs from MMLV genome (NC 001501) by PCR and inserted a Kozak sequence to the cDNAs. Similarly, we prepared ampho-env (M33470) with a Kozak sequence. We inserted these cDNAs into pEF-BOS vector (6) (see Note 4).

3. Methods 3.1. Screening the Slate of Cell Lines and Primary Cells

1. Culture a total of 1 × 105 reporter cells overnight with 1 × 105 target cells in a 96-well plate. Incubate them in a CO2 incubator overnight. 2. Spin down the plate for 5 min at 500 g. Stop centrifuge with minimal or no brakes. 3. Remove medium by flipping the plate. Place the plate on paper towels for 5 s to remove extra medium. 4. Add 100 ␮l of 1 × Z-buffer containing CPRG. 5. Place the plate for 4 h at 37◦ C in a regular and non-culture incubator. 6. Add 100 ␮l of stop buffer to terminate the enzyme reaction. 7. Measure the quantitative accumulation of chlorophenol red on the microplate reader by determining the absorption at 595 nm, using 635 nm as reference wavelength.

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8. During the screening process, cell lines negative for ligand will produce a negative readout on co-cultivation with reporter cells. One of these cell lines will be the recipient cell for the cDNA library (see Note 5). 3.2. Characterization of Ligand Expression by the Cell Line Stimulating the Reporter Cells

Before construction of the cDNA library from a ligand-positive cell line, we suggest careful examination of the amplitude of reporter response in the reporter cell assay. If augmented reporter cell responses are observed, i.e., a greater amplitude, it suggests that ligand expression is increased at the cell surface, possibly due to increased mRNA for the ligand or possibly co-expression of another ligand that can stimulate reporter cell responses. Therefore, a cDNA library generated from these cells may increase the chance of identifying the ligand. Various stimulating reagents that may induce or increase ligand expression, such as cytokines and LPS, may also need to be tested. Special caution must be paid to the reagents that can directly stimulate TCR signaling because the remaining reagents in the system may directly stimulate reporter cell responses. This can be easily controlled by applying the reagent onto the reporter cell and assaying for reporter expression. Construct a cDNA library using mRNA from the identified ligand-positive cells line. The cDNA should be cloned into a retroviral expression vector and the base number of independent cDNA clones should be quantified.

3.3. Determine the Infection Efficiency of Recipient Cells with Marker Viruses

Since the infection efficiency of retroviral vectors can vary depending on the cell type, it is important to quantify the infection efficiency of the ligand-negative recipient cells (see Note 6). 1. Prepare supernatant containing retroviral vectors made from pMX-GFP. 2. Infect the recipient cells with undiluted and serial dilution pMX-GFP supernatant in the presence of polybrene at 10 ␮g/ml on day 0. Incubate for 6 h. Add the same amount of fresh medium into the wells. 3. On day 1, harvest cells, wash out virus supernatant, and incubate cells in fresh medium. 4. On day 2, analyze the GFP expression by FACS. 5. With serially diluted viral vector supernatants, we observe a condition where infection efficiency (i.e., the number of cells infected) correlates with the dilution of the vector (i.e., the linear responses), but the mean fluorescence intensity (MFI) of GFP is constant. We assume that these cells harbor a single GFP vector. In our experience, most cell lines have single GFP integration when the infection efficiency is less than 5–15%. By comparing MFI of GFP between single

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virus integration and multiple integrations, one can estimate the approximate number of GFP virus integrations. For a 20-fold increase in MFI compared to single GFP cell populations, we estimate that this cell population has a corresponding 20-fold increase in integrated cDNA. 3.4. Determine the Sensitivity of Reporter Cell Assay

Determine the sensitivity of reporter cell assay by serially diluting the percentage of ligand-positive and recipient cells in a total of 1 × 105 target cells in a 96-well plate. If you can detect one positive cell out of 100 recipient cells (1% positive cells), the sensitivity is 100.

3.5. Expression Cloning of Ligand with Reporter Cells

1. On day 0, transfect the cDNA library into the packaging cell line. As a control virus for vector production and to track infection efficiency, transfect with pMX-GFP in a duplicate plate of packaging cells. 2. On day 2, harvest the supernatant containing the library viral vectors and infect recipient cells as in Section 3.3. Infect GFP virus into recipient cells as control. 3. On day 4, analyze the GFP expression on a per-cell basis by fluorescence cytometry and calculate the number of cells infected. Assume a similar infection for the library vectors. 4. Plate the number of library-infected cells in each well of a 96-well plate corresponding to the number for reporter cell sensitivity determined in Section 3.4. Determine the number of plates to cover the library size (see Note 7). 5. Maintain library-infected cells in 96-well plates by changing 50% of the medium (see Note 8). When cells become 80% confluent, duplicate all the plates. 6. When duplicated plates are semi-confluent, perform reporter cell assays on one set of the duplicated plates by adding 1 × 105 reporter cells. It is not necessary to count the cell number of each recipient cell well. 7. Positive wells will be identified as described in Section 3.1. 8. Cells from the duplicated positive well will be expanded and applied to the reporter assays in a quantitative manner (i.e., 1 × 105 cells in a 96-well plate) together with noninfected recipient cells as a negative control. Once the positive well is confirmed by the reporter cells, make backup frozen vials of these bulk populations. 9. Establish a positive clone or oligo-clonal cell population by limiting dilution. 10. Introduce into the positive clonal cells the MMLV env and gag-pol genes either by transient transfection or stable transduction with a lentivirus vector.

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11. Harvest the supernatant of positive clonal cells that have been either transfected or transduced with the env and gagpol genes. The supernatant should contain the retrovirus encoding cDNA for the ligand. Infect the next round of recipient cells with the supernatant (see Note 9). 12. Two days after the infection, perform the reporter cell assay with infected cells. Confirm that the infected cells can stimulate reporter cells. Plate cells into 96-well plates and perform a second screening. 13. After establishing positive clonal cells in the second round of screening, isolate the integrated cDNA by PCR using primers specific to the expression vector. If oligo-bands are observed, sequence them and confirm the specificity with reporter cells after transducing the candidate cDNA(s) into recipient cells. If multiple PCR bands are observed, further repackaging of cDNA and a third round of screening may be required to simplify the downstream analysis.

4. Notes 1. The Nkrp1f chimeric receptor cell was generated with the Nkrp1 ectodomains and CD3␨ cytoplasmic domains. To avoid difficulties in expression of putative activation receptors and a confounding association with signaling adaptor molecules that contain charged transmembrane (TM) residues, we replace the TM domain with that from Ly49A (Fig. 19.1). For orphan receptors with a type I structure, we replace the TM domain with that from CD8. A bulk population of reporter cells is perfectly suitable for the initial screening of ligand-expressing cells. We sometimes observe that reporter cells with low levels of receptor expression become dominant after a long period of culture. Therefore, it is advisable to establish clonal reporter cells expressing a high level of receptor for use in expression cloning procedures following the identification of ligand-positive cells. Such clonal reporter cells will make the assay condition stable, in addition to increasing the sensitivity of the assay. 2. Cell lines are preferable as a source of mRNA for cDNA library construction. The size of the cDNA library from heterogeneous cell populations will not reflect the distribution and abundance of mRNA from a homogeneous cell population. 3. We utilize FuGENE 6 for plasmid transfection according to the manufacturer’s instruction. We utilize 2 × 106

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packaging cells, 9 ␮l of FuGENE6, and 3.5 ␮g of plasmid DNA in a 10 cm dish with 8 ml medium. We strongly recommend establishing a transfection condition using GFP expression in pMX or the vector utilized for the cDNA construction, because we have observed significant variability in transfection efficiency among laboratories. We use freshly prepared supernatant containing the virus because the freezing of supernatant decreases the viral titer. Filtration of virus supernatant also decreases the titer. Unless the packaging cell line is capable of stimulating reporter cells, we utilize the unfiltered supernatant after a brief centrifuge (500 g, 10 min in a table top centrifuge). 4. Alternatively, recombinant lentiviruses or adenoviruses expressing Env and Gag-Pol can be utilized. For lentivirus expression, an expression plasmid for VSV-G and the delta8.9 plasmid (7) are required. The choice of the method for retrovirus rescue is dependent on several factors related to the recipient cells: efficiency of the gene delivery method and expression level of Env and Gag-Pol achieved by the vector system. When recipient cells are adherent cells or harbor large T antigen, we utilize env and gag-pol in a pEF-BOS vector (6). When we repackaged pMX-GFP from NIH3T3 cells by transfecting these vectors, we observed that more than 40% of 3T3 cells expressed GFP after infection from the day 2 supernatant (following transfection) (Fig. 19.2A and data not shown). When cells are non-adherent and refractory to conventional transfection, we prefer to use a lentivirus vector, pEF-SIN (8), containing env-IRES-puro and gag-pol-IRESblasticidin (Fig. 19.2A). Puromycin and blasticidin selection is required for efficient repackaging of the pMX-GFP when we utilize BW5147 cells as recipient cells (data not shown). When we repackaged pMX-GFP from BW-GFP by sequential infection and selection, we observed that 5% of BW5147 cells expressed GFP from the day 2 supernatant (data not shown). These data indicate that each cell type can repackage the integrated virus cDNA with different efficiency. Please note that a regular retrovirus vector cannot be used in this strategy unless the expression vector has been modified to a “self-inactivating” (SIN) form (9). Similar repackaging methodology using adenovirus (Ad) has also been described and high virus titer recovery is reported (10). Infection efficiency of adenovirus is dependent on the expression of coxsackie-adeno receptor (CAR). When CAR expression is low in the recipient cells, Ad containing an

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Arg-Gly-Asp (RGD) motif mutant in the fiber-knob region (11, 12) may be an efficient alternative vector to overcome this problem. 5. The choice of recipient cell is critical. Ideal characteristics of a recipient cell line for this expression cloning strategy are (1) the cell line can integrate many virus cDNAs without compromising proliferation ability and (2) the cell line can be efficiently transfected or virally infected for expression of env and gag-pol genes for repackaging. Please refer to Notes 4, 7, and 9 for further detail. The biggest caveat in expression cloning is that the ligand may be a complex of molecules. To account for this possibility, a parental cell line capable of stimulating reporter cells and a derivative mutant cell line incapable of stimulating reporter cells can be searched for or generated. The derivative mutant cell line can be used as recipient cell due to the prediction that simultaneous mutations in the genes of the ligand complex are unlikely, unless the complex genes are on the same loci. Alternatively, using a similar cell type to the ligand-expressing cell may resolve this problem. Successful expression cloning of a ligand complex has been reported for the DX-5 antigen with a mAb in this manner (13). 6. According to Poisson distribution, when MOI of 1 (1 viral particle per cell) is used to infect a population of cells, the probability that a cell will not get infected is 37%, and the probability that it be infected by a single particle is 37%, by two particles is 18%, by three particles is 6%, and so on. However, we observe variability among cells utilized for infection. As a practical approach to estimate the average number of integrated viral vector in recipient cells, we utilize FACS with a GFP vector as surrogate marker. Hence, we assume that the titer of GFP vector preparation is similar to the one of retroviral cDNA library vector generated under the same conditions. Please note that our estimate is based on the assumption that the number of integrated GFP vector and the MFI of GFP expression are in a linear phase when measured on the FACS. Clonal analysis followed by a Southern blotting or quantitative PCR may be an alternative method (14). 7. Estimate the number of cDNA that can be screened in a 96-well plate. For example, if the sensitivity is 100 and the estimated number of integration is 10, then approximately 96,000 cDNA clones can be screened in a 96-well plate (100/sensitivity per well × 10/integration per cell × 96 well). Another consideration is the doubling time of recipient cells. If the doubling time is 24 h, it should be assumed that the 100 cells on day 2 represent the 25 cells on the day

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of infection. Plate the number of cells corresponding to the sensitivity into each well in the number of 96-well plates required to cover the library size. We utilized the cDNA library for Nkrp1f ligand, which contained 2.8 × 106 independent inserts (bi-directional insertion). When we utilized 293T cells as recipient cells, we observed that a 293T cell can harbor four pMX-GFP inserts with our supernatant containing pMX-GFP viruses (data not shown). When we plated 100 cells per well in 20 of 96-well plates, we observed two positive wells with J7Nkrp1f reporter cells and confirmed the insertion of Clrg cDNA by PCR and sequencing (data not shown). When we utilized BW-5147 cells as recipient cells, we observed that a BW-5147 cell can harbor eight pMX-GFP inserts with our supernatant containing pMX-GFP viruses (data not shown). When we plated 100 cells per well in 20 of 96-well plates, we observed 13 positive wells with J7-Nkrp1f reporter cells and confirmed the insertion of Clrg cDNA by PCR and sequencing (data not shown). 8. We utilize a multi-channel pipette for changing the medium. We change tips between removing and adding medium for each plate but not for each well. We set a constant ori-

Fig. 19.3. Recovery of stably integrated helper-free retrovirus as infectious helper-free virus. Clonal 293T cells stimulating Nkrp1f reporter cells were established from a positive well in the first round of screening and were transfected with pBOS-AmphoEnv and pBOS-gp. Two days after the transfection, supernatant were harvested, serially diluted, and used for infection with 293T cells. Two days after the infection, 1 × 105 of infected 293T cells were applied to Nkrp1f reporter cells assays.

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entation for this procedure. By doing so, we are able to detect potential cross-contamination if we observe a gradual decline of reporter responses in the orientation. 9. As was already mentioned in Note 4, the titer of repackaged viruses varies depending on the recipient cells. A high repackaging efficiency (i.e., high virus titer) may be observed: We established oligo-clonal 293T cells capable of stimulating Nkrp1f reporter cells from the first screening process described in Note 6. To rescue integrated virus in an amphotropic envelope, we transfected them with ampho-env and gag-pol in a pEF-BOS vector. Supernatant was harvested 2 days after transfection. We infected 293T cells with serially diluted supernatant. We observed that Nkrp1f reporter cells detected ligand-expressing cells infected even with 16 times diluted supernatant (Fig. 19.3). When low repackaging efficiency (i.e., low virus titer) is observed, we recommend the following protocol to concentrate retroviruses (15): spin filtered supernatant at 6000×g for 16 h at 4◦ C and resuspend in a cell culture medium or Hank’s balance salt solution (HBSS). With this method, we observed at least 10 times the concentration of the virus supernatant. References 1. Sanderson, S., and Shastri, N. (1994) LacZ inducible, antigen/MHC-specific T cell hybrids. Int Immunol 6, 369–376. 2. Irving, B. A., and Weiss, A. (1991) The cytoplasmic domain of the T cell receptor zeta chain is sufficient to couple to receptorassociated signal transduction pathways. Cell 64, 891–901. 3. Iizuka, K., Naidenko, O. V., Plougastel, B. F., Fremont, D. H., and Yokoyama, W. M. (2003) Genetically linked C-type lectinrelated ligands for the NKRP1 family of natural killer cell receptors. Nat Immunol 4, 801–807. 4. Ito, D., Iizuka, Y. M., Katepalli, M.P., Iizuka, K. (2009) Essential role of the Ly49A stalk region for immunological synapse formation and signaling. Proc Natl Acad Sci USA. 106(27), 11264–11269. 5. Morita, S., Kojima, T., and Kitamura, T. (2000) Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 7, 1063–1066. 6. Mizushima, S., and Nagata, S. (1990) pEFBOS, a powerful mammalian expression vector. Nucleic Acids Res 18, 5322. 7. Lois, C., Hong, E. J., Pease, S., Brown, E. J., and Baltimore, D. (2002) Germline transmis-

8.

9.

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11.

sion and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868–872. Cui, Y., Golob, J., Kelleher, E., Ye, Z., Pardoll, D., and Cheng, L. (2002) Targeting transgene expression to antigenpresenting cells derived from lentivirustransduced engrafting human hematopoietic stem/progenitor cells. Blood 99, 399–408. Yu, S. F., von Ruden, T., Kantoff, P. W., Garber, C., Seiberg, M., Ruther, U., Anderson, W. F., Wagner, E. F., and Gilboa, E. (1986) Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc Natl Acad Sci U S A 83, 3194–3198. Bhattacharya, D., Logue, E. C., Bakkour, S., DeGregori, J., and Sha, W. C. (2002) Identification of gene function by cyclical packaging rescue of retroviral cDNA libraries. Proc Natl Acad Sci U S A 99, 8838–8843. Dmitriev, I., Krasnykh, V., Miller, C. R., Wang, M., Kashentseva, E., Mikheeva, G., Belousova, N., and Curiel, D. T. (1998) An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus

Identification of NK Cell Receptor Ligands Using a SignalingReporter System receptor-independent cell entry mechanism. J Virol 72, 9706–9713. 12. Nagi, P., Vickers, S. M., Davydova, J., Adachi, Y., Takayama, K., Barker, S., Krasnykh, V., Curiel, D. T., and Yamamoto, M. (2003) Development of a therapeutic adenoviral vector for cholangiocarcinoma combining tumor-restricted gene expression and infectivity enhancement. J Gastrointest Surg 7, 364–371. 13. Arase, H., Saito, T., Phillips, J. H., and Lanier, L. L. (2001) Cutting edge: the mouse NK cell-associated antigen recognized by DX5 monoclonal antibody is CD49b (alpha 2 integrin, very late antigen-2). J Immunol 167, 1141–1144.

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14. Kustikova, O. S., Wahlers, A., Kuhlcke, K., Stahle, B., Zander, A. R., Baum, C., and Fehse, B. (2003) Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population. Blood 102, 3934–3937. 15. Bowles, N. E., Eisensmith, R. C., Mohuiddin, R., Pyron, M., and Woo, S. L. (1996) A simple and efficient method for the concentration and purification of recombinant retrovirus for increased hepatocyte transduction in vivo. Hum Gene Ther 7, 1735–1742.

Chapter 20 Determining Ligand Specificity of Ly49 Receptors Kerry J. Lavender and Kevin P. Kane Abstract Ly49 receptors in rodents, like KIR in humans, play an integral role in the regulation of NK cell activity. Some inhibitory Ly49 are known to interact with specific MHC I alleles to maintain tolerance to self tissues, and NK activation is triggered upon the loss of inhibitory signals due to pathological downregulation of self MHC I. Although a virally encoded ligand has been identified that can trigger NK cytotoxicity through an activating Ly49, some activating Ly49 also recognize MHC I and the role of most activating receptors in NK effector function remains poorly defined. As many Ly49 remain orphan receptors, we describe methods to unambiguously discern receptor–ligand pairs. Additionally, we describe a method for the mutagenesis of Ly49 and MHC ligands that can be used to define the motifs conferring receptor specificity for their ligands. Further elucidation of Ly49 ligands is required to continue to define the role of Ly49 in regulating NK cell effector function and may give vital clues to the role of KIR in human health and disease. Key words: Rodent, MHC I, Ly49, mutagenesis, transfection, 51 Cr release assay.

1. Introduction Ly49 are C-type lectin-like receptors found in mice and rats that regulate the balance of NK cell activation and tolerance. These receptors are functional homologues to the immunoglobulinlike KIR receptors that regulate NK cell activity in humans and other primates. Ly49 are encoded as a multi-gene family and inbred rodent strains can differ with respect to their complement of genes encoding activating and inhibitory type Ly49 (1–3). To date, more than 20 Ly49 genes have been described in the mouse, 13 of which are inhibitory and 8 that are activating (4). Furthermore, 25 Ly49 have been identified in the rat including 13 inhibitory, 8 activating, and 5 bi-functional receptors (1). K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 20, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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Ligands for some Ly49 have been identified (5–10) and like the few ligands identified for KIR receptors (11–15), these primarily consist of a single or small repertoire of specific MHC I alleles. Additionally, a mouse cytomegalovirus-encoded MHC I homologue, m157, has been identified as the ligand for the activating Ly49H and inhibitory Ly49I receptors of specific mouse strains and may suggest that similar viral ligands could exist in human systems (16). Despite some progress in identifying Ly49 ligand specificity, many Ly49, particularly those in the rat, remain orphan receptors. As there are many parallels between Ly49 and KIR functionality in NK cell biology, continued elucidation of Ly49 receptor−ligand pairs in rodents can provide valuable models to further understand the significance of KIR receptors in human health and disease. Although identifying ligands for Ly49 can be approached broadly using activated effector and target cells from inbred or congenic strains, it is nearly impossible to deduce specific interactions from this system as most targets will express more than one potential MHC I ligand. Additionally, the complex signals derived through the interaction of MHC I and the variegated Ly49 repertoires expressed by NK cells in any individual animal can result in complicated and potentially ambiguous interpretations of ligand specificity. To remove such ambiguity, we describe a system involving the transfection of potential receptor−ligand pairs into a specific RNK-16 NK cell effector−YB2/0 target system followed by a description of a 51 Cr release assay for direct and specific recognition. The RNK-16/YB2/0 system was originally described by J. Ryan et al. (17), and our description here is an update and expansion of the approach. The effector−target system displays a moderate level of background cytotoxicity that allows the detection of increased cytotoxicity upon ligand recognition by an activating receptor, as well as suppressed cytotoxicity upon ligand recognition by an inhibitory receptor. Furthermore, the isolation of MHC I and Ly49 DNA for transfection allows modifications of the sequence, including the addition of tags to facilitate or confirm the successful transfection and potentially the surface expression of molecules that cannot be identified by specific antibody. We give one example here of the C-terminal fusion of EGFP to MHC I that we have used to facilitate screening for positive transfectants. Additionally, we describe a method for mutagenesis of either the ligand or Ly49 to identify the epitopes conferring specificity, as such knowledge can aid in the description of motifs that can lead to the identification of other potential ligands for Ly49. Finally, slight alterations to the methods we discuss here can allow the identification of virally encoded or induced ligands for Ly49, by modifying the assays to include infected target cells or target cells transfected to express virally encoded ligands.

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2. Materials 2.1. Mutagenesis of Ly49 and MHC I

1. DNA plasmid containing the MHC I or Ly49 insert to be mutated (see Note 1). 2. Mutagenesis primers (1.25 ␮g/␮l in 10 mM Tris−Cl, pH 8.5 (see Note 2). 3. PfuTurbo DNA polymerase (2.5 U/␮l) and 10× buffer (Stratagene, La Jolla, CA). 4. dNTP mix (100 mM) (Stratagene, La Jolla, CA) (see Note 3). 5. RNAse/DNase-free H2 O (Invitrogen, Carlsbad, CA). 6. DpnI restriction enzyme (10 U/␮l) (New England BioLabs, Ipswich, MA). 7. Max-efficiency DH5␣ competent cells (Invitrogen, Carlsbad, CA). 8. Agar plates containing antibiotic selection appropriate to your vector, for example, ampicillin (50 ␮l/ml) for pC1-neo and BSR␣EN or kanamycin (30 ␮l/ml) for pEGFP-N3. 9. Luria-Bertani (LB) Broth. 10. 50% glycerol in RNase/DNase-free H2 O.

2.2. Transfection of YB2/0 and RNK-16 Cells

1. Endo-free DNA preps of the plasmid containing the molecule to be transfected (see Notes 1 and 4). 2. RNK-16 growth medium: RPMI-1640 (Invitrogen/Gibco, Carlsbad, CA) supplemented with 10% FBS (Hyclone, Logan, UT), 1% of penicillin/streptomycin, 1% Lglutamine, and 0.1% mercaptoethanol (Invitrogen/Gibco, Carlsbad, CA). 3. YB2/0 growth medium: Dulbecco’s modified Eagle’s medium, (DMEM) (Invitrogen/Gibco, Carlsbad, CA) supplemented with 10% FBS (Hyclone, Logan, UT), 1% L-glutamine, 1% sodium pyruvate, and 1% penicillin/streptomycin (Invitrogen/Gibco, Carlsbad, CA). 4. Unsupplemented DMEM and RPMI-1640 medium (Invitrogen/Gibco, Carlsbad, CA). 5. Geneticin (G418) (50 mg/ml) (Invitrogen/Gibco, Carlsbad, CA). 6. Electroporation cuvettes (4 mm) and electroporator (BioRad, Hercules, CA). 7. Black-walled, flat and clear-bottomed, tissue culture-treated 96-well plates (Thermo Scientific, Asheville, NC).

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2.3. Screening MHC I−EGFP and Ly49 Transfectants

1. FLA-5100 imaging system (Fujifilm). 2. Phosphate-buffered Carlsbad, CA).

saline

(PBS)

(Invitrogen/Gibco,

3. ChromPure Rat or Mouse IgG (Jackson Immunoresearch, West Grove, PA) or serum can be obtained directly from animals and maintained as frozen stocks. 4. Purified primary antibody specific to the transfected molecule. 5. FITC- or PE-conjugated AffiniPure F(ab’)2 fragment donkey anti-mouse (or rat) secondary antibody (Jackson Immunoresearch, West Grove, PA). 6. 37% formaldehyde solution. 7. 10% fetal bovine serum (FBS) (Hyclone, Logan, UT). 8. Dimethyl sulfoxide (DMSO) (Pierce Biotechnology, Inc., Rockford, IL). 2.4. Assay of Recognition

1. RNK-16 growth medium: RPMI-1640 (Invitrogen/Gibco, Carlsbad, CA) supplemented with 10% FBS (Hyclone, Logan, UT), 1% of penicillin/streptomycin, 1% Lglutamine, and 0.1% mercaptoethanol (Invitrogen/Gibco, Carlsbad, CA). 2. YB2/0 growth medium: Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen/Gibco, Carlsbad, CA) supplemented with 10% FBS (Hyclone, Logan, UT), 1% L-glutamine, 1% sodium pyruvate, and 1% penicillin/streptomycin (Invitrogen/Gibco, Carlsbad, CA). 3. Wash medium: RPMI-1640 (Invitrogen/Gibco, Carlsbad, CA) supplemented with 3% fetal bovine serum (FBS) (Hyclone, Logan, UT). 4. Lead shielding (Marshield, Burlington, ON) (see Note 11). 5. 1 mCi/ml Na51 CrO4 (51 Cr) (Perkin Elmer, Woodbridge, ON, Canada). 6. 25 and 100 ␮l multichannel pipette(s). 7. Scintillation fluid (OptiPhase SuperMix, Perkin Elmer, ON, Canada). 8. TriLux Microbeta and flexible 96-well microplates (Perkin Elmer, Waltham, MA).

3. Methods 3.1. Mutagenesis of Ly49 and MHC I

1. Thaw your DNA template, primers, 10× PfuTurbo reaction buffer and dNTPs (see Note 3). Centrifuge briefly and place on ice.

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2. In a PCR tube add a. 5 ␮l of 10× PfuTurbo reaction buffer b. 50 ng of DNA template c. 1 ␮l of each mutagenesis primer d. 1 ␮l of dNTP mix e. RNase/DNase-free H2 O to a final volume of 50 ␮l f. 1 ␮l PfuTurbo DNA polymerase (see Note 3) g. Overlay with mineral oil if required for your thermocycler Place in thermocycler and cycle as follows. Segment

Cycles

Temperature, ºC

Time

1

1

95

30 s

95

30 s

55

1 min

68

2 min/kb of plasmid length

2

12–18

∗

∗ 12

cycles for point mutations, 16 for single amino acid changes, and 18 for multiple amino acid insertions or deletions.

3. After cycling, cool the reaction to ≤37ºC and add 1 ␮l of DpnI (see Note 3) to the reaction tube. Ensure to insert the pipette tip below the mineral oil overlay, if used. 4. Mix gently by pipetting up and down several times, spin for 1 min in a microcentrifuge, and incubate at 37ºC for 2 h to overnight. 5. The next day, transform max-efficiency DH5␣ cells using 4 ␮l of the DNA reaction mixture. Streak on plates containing the appropriate selection antibiotic for your vector and incubate overnight at 37ºC. 6. Select two to three colonies for overnight expansion in 5 ml of LB broth plus selection antibiotic (for example, 30 ␮l/ml kanamycin or 50 ␮l/ml ampicillin). 7. Next day create glycerol stocks in a 1.5 ml microtube using 700 ␮l of culture and 300 ␮l of 50% glycerol, store at −80ºC. 8. Purify plasmid from the remainder of the culture and perform DNA sequencing to ensure the correct mutation has been introduced. 9. Make Endo-free preps of successful mutants using the stored glycerol stock. 3.2. Transfection of YB2/0 and RNK-16 Cells

1. Culture YB2/0 cells in appropriate growth medium. 2. Culture RNK-16 cells in appropriate growth medium (see Note 5).

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3. On the day of transfection remove cells in log-phase growth from 100 mm tissue culture plates using cell scrapers for YB2/0 or gentle washing for RNK-16 cells (see Note 6). 4. Harvest 2×106 cells per transfection for YB2/0 and 4×106 cells for RNK-16. 5. Resuspend each transfection in 0.4 ml cold unsupplemented DMEM (YB2/0) or RPMI-1640 (RNK-16). 6. Add 10 ␮l (YB2/0) or 20 ␮l (RNK-16) of sterile plasmid (see Note 7) per transfection, mix gently, and transfer into a 4 mm electroporation cuvette. Place briefly on ice and proceed immediately to transfection. 7. Turn the Bio-Rad electroporator off and on a few times. Set the electroporator to 0.18 V and the capacitance extender to 960 ␮F. 8. Insert each cuvette snugly into the cuvette holder ensuring good metal-to-metal contact. Depress the double red buttons on the front of the machine simultaneously until a beep is heard indicating electroporation is complete. 9. Replace the cuvette onto ice and as soon as possible add the contents of the cuvette to 7 ml of growth medium dropwise in a 100 mm tissue culture-treated plate. 10. Aspirate the medium the day after transfection and resuspend the cells in 10 ml growth medium supplemented with 1 mg/ml G418. Count and dilute YB2/0 cells to 2×104 /ml in G418-supplemented growth medium, making enough for two 96-well plates (25 ml) though more plates may be added for stubborn transfections. RNK-16 cells are left undiluted. 11. Plate cells at 100 ␮l/well into flat-bottomed, tissue culturetreated 96-well plates: RNK-16 cells into standard plates and YB2/0 cells into black-walled plates. 12. After 4 days, add an additional 100 ␮l of G418supplemented growth medium to each well. 13. After an additional 5 days check wells for growth. Up to 30% of YB2/0 wells will show growth in a successful transfection; additional growth suggests the wells may not be clonal. RNK-16 cell transfection is much more difficult and successful transfection efficiency can be 1–3% or less. RNK-16 cells often take 2–4 weeks to exhibit growth and additional G418 medium may be added to avoid excessive evaporation. 3.3. Screening for MHC I–EGFP and Ly49 Transfectants

1. Aspirate medium from wells containing actively growing YB2/0 clones that have become confluent in the well (see Note 8) and proceed to the FLA-5100 Imaging System.

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2. Scan the plate for the presence of EGFP fluorescence. Positive wells will appear in a uniform dark color if cells are evenly distributed and confluent in the well. 3. Transfer EGFP-positive YB2/0 cells and actively growing RNK-16 cells from 96-well plates into 1 ml of G418supplemented growth medium (see Section 3.2) in 24-well plates. Ensure clones have reached sufficient density prior to transfer (see Note 5). 4. When cells are confluent in the 24-well plates transfer three-fourths of the cells from each well into 96well V-bottom plates for antibody staining. Resuspend the remaining cells in G418 medium for continued growth. 5. Prepare a single well of 1×106 untransfected cells to serve as a negative control. 6. Wash the cells to be stained once with 100 ␮l/well PBS. Centrifuge plates at 450×g for 4 min using low brake. Gently flick the liquid from plates into an appropriate receptacle to remove PBS from the pellet. 7. Add 50 ␮l/well of blocking serum or IgG. Use serum/IgG from the species that will not react with your secondary antibody. For example, if your secondary antibody is an anti-mouse antibody you need to block with rat serum/IgG. Dilute commercial IgG 1:250 in PBS. Serum is used as a 4% solution in PBS. 8. Incubate at room temperature for 15 min then wash two times as described in step 6. 9. Add antibody specific to the transfected molecule. Most antibody preparations are highly concentrated and are diluted in cold PBS as recommended by the manufacturer just prior to use. Prepare enough antibody to allow for 50 ␮l/well. 10. Incubate for 15 min on ice then wash two times as described in step 6. 11. Add fluorochrome-conjugated secondary antibody diluted 1:100 in cold PBS making enough for 50 ␮l/well. Since MHC I−EGFP constructs will fluoresce in the FITC channel regardless of surface expression, a fluorochrome other than FITC must be used to detect surface expression of the transfected molecule. Most flow cytometers are capable of detecting FITC and PE fluorescence making PE the recommended choice. 12. Incubate in the dark at 4ºC for 15 min then wash two times as described in step 6.

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13. Fix cells in 400 ␮l of a freshly made 2% formaldehyde−PBS solution and transfer to FACS tubes. 14. Proceed to FACS analysis. Compare fluorescence (surface expression) of each transfectant to the negative control. Successful MHC I transfectants will have expression between 1 and 2 (or more) logs higher than the negative control. Successful Ly49 transfectants are lower, around 1 log higher than the negative control (see Note 9). 15. Continue to grow transfectants with good surface expression in selection medium and discard those with no or inadequate expression. 16. Freeze down 10 vials of successfully transfected clones at 4×106 cells in 1 ml of 5% DMSO in FBS. Choose 3–5 MHC I clones with differing levels of surface expression to allow comparisons with other clones of interest and with your positive control. Not all RNK-16 clones will necessarily exhibit similar levels of cytotoxicity despite similar surface expression of the transfected Ly49. Therefore, all successful RNK-16 transfections should be preserved until they can be tested for cytotoxicity. 3.4. Assay of Recognition

1. Thaw RNK-16 effectors and YB2/0 targets. Include untransfected RNK-16 and YB2/0 cells as negative controls. Also include a known Ly49/MHC I recognition pair as a positive control; preferably a known ligand for each Ly49 receptor being used. 2. Grow transfected target and effector cells in G418supplemented growth medium as described in Section 3.2. Change to G418-free growth medium 48 h prior to assay. Exactly 2.4×105 target cells are required for each effector in the assay and 1.5×106 effector cells are required for each target in the assay. Prepare a slight excess. Ideally RNK16 cells will be 90% confluent on the day of the assay (see Note 5). 3. Harvest target cells (see Note 10) and resuspend in 85 ␮l of FBS. 4. In a designated area for radioactive work add Na51 CrO4 following the handling protocols for gamma radiation at your facility (see Notes 11 and 12). Be careful to shield yourself appropriately and to properly contain and dispose of radioactive material. 5. Incubate for 1 h in a 37ºC water bath gently agitating every 15 min. 6. While the targets are incubating prepare your effector cells. Harvest RNK-16 cells by gently washing them off the plate. Wash and resuspend in 5 ml of wash medium.

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7. Count cells and resuspend at 2.5×106 /ml in RNK-16 growth medium. 8. Prepare 1:2 dilutions in RNK-16 growth medium from 25×105 /ml to 1.56×104 /ml. Set aside until targets are ready. RNK-16 growth medium containing 1.5×106 cells/600 ␮l is required for each target cell type in the assay, always prepare slightly more than is required. 9. Once the target cell incubation is complete wash targets four times in 10 ml of wash medium. 10. After the final wash carefully aspirate all remaining wash medium using a pipetman and resuspend target cell pellet in 2 ml of RNK-16 growth medium. 11. Count targets using a hemocytometer designated for radioactive work, being careful to contain and properly dispose of all radioactive materials. 12. Calculate the number of target cells required and resuspend at 1×105 targets/ml in RNK-16 growth medium. Twenty-four wells of 1×104 targets/100 ␮l are required for each effector in the assay, prepare slightly more than that is required. 13. Plate each target into two rows of a V-bottom 96-well plate, aliquoting 100 ␮l/well (1×104 cells/well). 14. Plate each effector cell dilution, as prepared in step 8, into triplicate wells for each target following the final effector:target ratios as shown in the plate layout in Table 20.1. Aliquot 100 ␮l/well of growth medium into the spontaneous (S) and total (T) release wells. 15. Centrifuge plates at 250×g for 3 min with low brake and incubate for 4 h at 37ºC. 16. After incubation, centrifuge plates at 450×g for 5 min using no brake. 17. Harvest 25 ␮l of supernatant from each well into microbeta plates. Total release (T) wells are resuspended prior to harvest. 18. Add 100 ␮l of scintillation fluid to harvested samples.

Table 20.1 Format of effector to target cell ratios of individual wells in a 96-well plate 25:1

25:1

25:1

12:1

12:1

12:1

6:1 6:1 6:1 3:1 3:1 3:1

1.5:1 1.5:1 1.5:1 0.78:1 0.78:1 0.78:1 S

S

S

T

T

T

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19. Seal plates and shake for 10 min on a plate shaker. 20. Place plates in the microbeta counter and count for 1 min/sample. 21. Calculate % specific lysis using the formula % specific lysis =

observed release−spontaneous release × 100 total release − spontaneous release

22. Reduced or increased specific lysis in a set of wells would indicate that the MHC class I molecule in those target cells effectively engaged with the inhibitory or activating Ly49, respectively, on the effector cells to function as a ligand. Specific lysis equivalent to that of untransfected effector cells would indicate that the MHC class I molecule on the targets was not an effective ligand.

4. Notes 1. We recommend cloning of MHC I into either pCIneo (Promega, Madison, WI) or into pEGFP-N3 (Clontech, Mountain View, CA). The pEGFP-N3 vector allows for the creation of a MHC I molecule that is Cterminally fused to EGFP and facilitates screening of positive transfectants. Mutagenesis of the start codon of EGFP to isoleucine (ATG to ATA) aids the translation of an intact fusion molecule. Cloning Ly49 into BSR␣EN (Dr. A. Shaw, Washington University, St. Louis, MO) is highly recommended for successful transfection of RNK-16 cells. 2. Design primers containing the desired mutation and that anneal to the identical sequence on opposite strands. Ideally primers should a. contain the desired mutation in the middle of the primer with about 10–15 bases of correct sequence on either side, b. have a GC content of ≥40% and terminate at each end in one or more C or G bases, c. have a Tm ≥ 78ºC (Tm = 81.5 + 0.41(%GC) − 675/primer length in base pairs), d. be FPLC or PAGE purified. 3. We suggest aliquoting dNTPs into smaller lots to reduce the number of freeze−thaw cycles. All enzymes (polymerases, restriction enzymes, etc.) should be removed

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from −20◦ C just prior to use and immediately replaced. Do not vortex enzymes. 4. RNK-16 and YB2/0 are devoid of mouse and human molecules but YB2/0 naturally express MHC I of the rat LOU strain and RNK-16 express NK receptors of the F344 rat strain. This must be considered when choosing receptor/ligand combinations for testing. 5. RNK-16 cells are very sensitive to cell density. When passaging, plate at 3×105 cells/ml and passage every 2–3 days. Over-dilution can result in cell death. Conversely, allowing RNK-16 to become too dense can reduce both viability and cytotoxic function. 6. Actively growing log-phase cells that have not been passaged for more than a week transfect most efficiently. Do not use confluent cells. 7. Transfections are more successful with Endo-Free DNA, particularly for RNK-16 cells. Inhibitory Ly49 are typically easier to transfect than activating receptors. We usually set up —two to three transfections for each inhibitory Ly49 and —five to six for each activating Ly49. A single MHC I transfection using YB2/0 cells is sufficient. 8. Wells containing growing YB2/0 clones often contain a single large clump of cells. It is important to resuspend these clumps the day before screening to redistribute the cells on the well floor and maximize the fluorescence intensity across the well. All wells will appear positive if the culture medium is not sufficiently removed. 9. Assessing whether surface expression of ligand and receptor is adequate for recognition is an important factor to consider. Matching expression levels of potential ligands on YB2/0 with those of known ligands on YB2/0 cells is one possibility. This cannot be done when assessing Ly49 expression levels on RNK-16 cells. A better technique for assessing the triggering capacity of a MHC I or Ly49 transfectant is to use ADCC or reverse ADCC, respectively. This is particularly important for RNK-16 transfectants, which can vary widely in their level of cytotoxicity independent of receptor expression. 10. Do not use trypsin for adherent cells as this will remove the surface ligand. Use cells scrapers or Versene. 11. Most institutions have guidelines and regulations that must be met before investigators can be licensed for radionuclide use. Obtain information from your institution on proper shielding, waste management, and regular monitoring for contamination.

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12. The half-life of 51 Cr is 27 days. For a 1 mCi/ml stock less than 2 weeks old 100 ␮l is sufficient for 5–25×106 target cells, if more than 2 weeks old 150 ␮l is required. Using 51 Cr that is greater than 1 month old is not recommended.

Acknowledgments This work was supported by Canadian Institutes of Health Research grant 43864. References 1. Nylenna, O., Naper, C., Vaage, J., Woon, P., Gauguier, D., Dissen, E., Ryan, J., and Fossum, S. (2005) The genes and gene organization of the Ly49 region of the rat natural killer cell gene complex. Eur. J. Immunol. 35, 261–272. 2. Proteau, M., Rousselle, E., and Makrigiannis, A. (2004) Mapping of the BALB/c Ly49 cluster defines a minimal natural killer cell receptor gene repertoire. Genomics 84, 669–677. 3. Wilhelm, B., Gagnier, L., and Mager, D. (2002) Sequence analysis of the ly49 cluster in C57BL/6 mice: a rapidly evolving multigene family in the immune system. Genomics 80, 646–661. 4. Carlyle, J., Mesci, A., Fine, J., Chen, P., B´elanger, S., Tai, L., and Makrigiannis, A. (2008) Evolution of the Ly49 and Nkrp1 recognition systems. Semin. Immunol. 20, 321–330.. 5. Lian, R., Y. Li, S. Kubota, Mager, D., and Takei, F. 1999. Recognition of class I MHC by NK receptor Ly-49C: identification of critical residues. J. Immunol. 162, 7271–7276. 6. Silver, E., Gong. D., Chang, C., Amrani, A., Santamaria, P., and Kane, K. (2000) Ly-49P activates NK-mediated lysis by recognizing H-2Dd. J. Immunol. 165, 1771–1781. 7. Karlhofer, F., Ribaudo, R., and Yokoyama, W. (1992) MHC class I alloantigen specificity of Ly-49+ IL-2-activated natural killer cells. Nature 358, 66–70. 8. Silver, E., Gong, D., Hazes, B., and Kane, K. (2001) Ly-49 W, an activating receptor of nonobese diabetic mice with close homology to the inhibitory receptor Ly-49G, recognizes H-2D(k) and H-2D(d). J. Immunol. 166, 2333–2341. 9. Nakamura, M., Linnemeyer, P., Niemi, E., Mason, L., Ortaldo, J., Ryan, J., and Sea-

10.

11.

12.

13.

14.

15.

man, W. (1999) Mouse Ly-49D recognizes H-2Dd and activates natural killer cell cytotoxicity. J. Exp. Med. 189, 493–500. Silver, E., Lavender, K., Gong, D., Hazes, B., and Kane, K. (2002) Allelic variation in the ectodomain of the inhibitory Ly-49G2 receptor alters its specificity for allogeneic and xenogeneic ligands. J. Immunol. 169, 4752–4760. Pende, D., Biassoni, R., Cantoni, C., Verdiani, S., Falco, M., di Donato, C., Accame, L., Bottino, C., Moretta, A., and Moretta, L. (1996) The natural killer cell receptor specific for HLA-A allotypes: a novel member of the p58/p70 family of inhibitory receptors that is characterized by three immunoglobulinlike domains and is expressed as a 140-kD disulphide-linked dimer. J. Exp. Med. 184, 505–518. Vitale, M., Sivori, S., Pende, D., Augugliaro, R., Di Donato, C., Amoroso, A., Malnati, M., Bottino, C., Moretta, L., and Moretta, A. (1996) Physical and functional independency of p70 and p58 natural killer (NK) cell receptors for HLA class I: their role in the definition of different groups of alloreactive NK cell clones. Proc. Natl. Acad. Sci. U S A 93, 1453–1457. Litwin, V., Gumperz, J., Parham, P., Phillips, J., and Lanier, L. (1994) NKB1: a natural killer cell receptor involved in the recognition of polymorphic HLA-B molecules. J. Exp. Med. 180, 537–543. D¨ohring, C., Scheidegger, D., Samaridis, J., Cella, M., and Colonna, M. (1996) A human killer inhibitory receptor specific for HLAA1, 2. J. Immunol. 156, 3098–3101. Moretta, A., Vitale, M., Bottino, C., Orengo, A., Morelli, L., Augugliaro R., Barbaresi M., Ciccone E., and Moretta, L. (1993) P58 molecules as putative receptors for major histocompatibility complex (MHC) class I

Identifying Ly49 Ligands molecules in human natural killer (NK) cells. Anti-p58 antibodies reconstitute lysis of MHC class I-protected cells in NK clones displaying different specificities. J. Exp. Med. 178, 597–604. 16. Arase, H., Mocarski E., Campbell A., Hill A., and Lanier, L. (2002) Direct recogni-

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tion of cytomegalovirus by activating and inhibitory NK cell receptors. Science 296, 1323–1326. 17. Ryan, J., Niemi E., and Nakamura, M. (2000) Functional analysis of natural killer cell receptors in the RNK-16 rat leukemic cell line. Methods Mol. Biol. 121, 283–295.

Chapter 21 Probing the Interactions of NK Cell Receptors with Ligand Expressed in trans and cis ´ Jonathan Back, Leonardo Scarpellino, and Werner Held Abstract Certain receptors on natural killer (NK) cells, which are specific for MHC class I (MHC-I) molecules, do not only interact with ligand expressed on opposing cell membranes (in trans) but also interact with those on the same cell membrane (in cis). Cis interactions have been demonstrated for only a small number of cell surface receptors. However, this has not been tested systematically, raising the possibility that additional receptors may be able to bind ligand expressed in cis. Here we describe a number of approaches to evaluate trans and cis binding of the Ly49A NK cell receptor to its H-2Dd ligand. These procedures should facilitate the investigation of cis/trans interactions of other receptor–ligand pairs and simplify the analysis of NK cell receptor variants. Key words: Ly49, MHC class I, cis interaction, receptor masking, tetramer, cellular adhesion, ligand transfer.

1. Introduction NK cells use activating and inhibitory receptors to scan host cells for altered expression of various self-ligands such as MHC-I molecules. MHC-I receptors include the C-type lectin-like Ly49 receptor family in mice and the killer cell immunoglobulin-like receptor (KIR) family in humans (1). Many of these receptors are inhibitory and they ensure that NK cells do not attack host cells expressing MHC-I at a normal surface density while attacking those with low levels of MHC-I. Ly49A represents the prototype MHC-I receptor on mouse NK cells. This receptor binds to H-2Dd (Dd ) or Dk but not to K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 21, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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Db expressed on potential target cells to inhibit NK cell-mediated effector functions (2). Besides binding ligand expressed on other cells (in trans), Ly49A can also bind ligand expressed in the plane of the NK cells’ membrane (in cis) (3). Cis binding reduces the number of Ly49A receptors that can functionally interact with Dd expressed on potential target cells (4). Hence, masking of Ly49A lowers the threshold at which NK cell activation exceeds inhibition. This renders Ly49A NK cells more useful to detect diseased host cells. Besides Ly49A, several other Ly49 family receptors can bind MHC-I ligand expressed in cis (5). Other examples include the structurally distinct, immunoglobulin-like, inhibitory human LILRB2 and the homologous mouse PIR-B receptors that also interact with MHC-I ligand expressed in trans as well as in cis (6) or sialic acid-binding Ig-like lectins (Siglecs), which associate with sialic acid modifications of glycoproteins expressed in the same cell membrane (reviewed in (7)). We have developed a number of tests to determine to what extent the Ly49A receptor is masked by Dd ligand expressed in the plane of the same membrane. Here, we detail the use of soluble ligand to determine Ly49A receptor accessibility prior to and following the destruction of tri-molecular MHC-I ligand complexes, which unmasks Ly49A (see Note 1). In addition to soluble ligand, we describe two approaches to determine whether ligand expression in cis affects ligand binding to membrane-associated trans ligand. Collectively, these procedures should be useful to rapidly determine whether receptor variants retain their ability to bind ligand in trans and in cis. Moreover, they may be adapted to investigate cis/trans interactions of other receptor/ligand pairs.

2. Materials 1. Cells: C1498 (ATCC #TIB-49) (H-2b ) is an immature NK T cell line (8). C1498 cells can be efficiently transfected using electroporation or infected with lentiviruses. Stable cell lines are rapidly established using multiple rounds of magnetic cell sorting (MACS, Miltenyi Biotec, Gladbach, Germany), or using expression vectors, which include a puromycin resistance gene (select using 10 ␮g/mL of puromycin). 2. Stable C1498 transfectants used here express Ly49A, Dd ; Ly49A together with Dd ; or a Dd -EYFP fusion protein (3, 4).

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3. Cell culture medium: RPMI 1640 containing glutamax (Invitrogen, Carlsbad, CA, USA) supplemented with HEPES (10 mM), 2-mercaptoethanol (5×10−5 M), penicillin (50 ␮g/mL), streptomycin (50 ␮g/mL) (all from Invitrogen) and 10% foetal calf serum (FCS). 4. Plasticware: 96-well V-bottom plates (such as Costar); 96-well U-bottom plates (such as Costar); FACS tubes (5 mL round bottom polystyrene tubes, BD Biosciences, San Jose, CA, USA) and 1.4 mL U-bottom tubes (such as #M32022 plus rack from Micronic, Lelystad, The Netherlands); 15 mL V-bottom polypropylene tubes and 25 cm2 cell culture flasks. 5. Tetramers and monoclonal antibodies (mAbs): Dd tetramers were refolded using mouse ␤2 m and the HIV-derived peptide RGPGRAFVTI using standard techniques (9) (see Note 2) and labelled with phycoerythrin (PE); anti-Ly49A (JR9-318) either purified or labelled with fluorescein isothiocyanate (FITC) or biotin; anti-Dd (34-2-12), either PE or FITC labelled; anti-␤2m (19.11) biotinylated (all available from BD Biosciences); streptavidin−PE (Invitrogen Molecular Probes) to detect biotinylated mAbs. 6. FACS buffer: Phosphate-buffered saline (PBS) supplemented with 2% FCS. 7. Flow cytometer (such as FACScan BD Biosciences) together with analysis software (such as CellQuest (BD Biosciences) or FlowJo (Tree Star, Ashland, OR, USA)). 8. Stripping buffer: Citric acid 0.133 M, di-sodium hydrogen phosphate dihydrate (Na2 HPO4 (2H2 O)) 0.066 M. The pH is adjusted to 3.3 before sterile filtration (0.22 ␮m, Millipore, Billerica, MA, USA). The solution is stable for 2–3 months at room temperature; however the pH should be checked before use. Aliquots can be stored at −20◦ C. 9. Neutralization buffer: PBS supplemented with 1% bovine serum albumin (BSA), fraction V (Sigma-Aldrich, Munchen, Germany) and 10 mM HEPES. 10. Fixing solution: Paraformaldehyde (PFA) (1%) in PBS, made fresh. Alternatively, aliquots of 4% PFA in PBS can be stored at −20◦ C. 11. Cell labelling reagents: 5-(and-6)-(((4-chloromethyl) benzoyl) amino) tetramethylrhodamine)-mixed isomers (CMTMR) (Invitrogen Molecular Probes); carboxyfluorescein diacetate succinimidyl ester (CFSE) (SigmaAldrich).

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3. Methods 3.1. Determining Ly49A Receptor Masking Using Soluble Dd Ligand

This procedure allows an estimation of receptor masking by comparing Dd tetramer binding to Ly49A in the presence or absence of Dd ligand expressed in cis. In addition, Dd tetramer binding is assessed prior to and following Ly49A unmasking. For MHC-I ligands, this is achieved by exposing cells to an acidic buffer, which disrupts the tri-molecular MHC-I complexes (see Note 1). 1. Harvest C1498 cells. Half the cells will be treated with acidic buffer, the rest are kept for control staining in cold FACS buffer. 2. Centrifuge 3–10 × 106 cells in a 15 mL polypropylene tube for 5 min at 500×g. Wash the cells twice with 10 mL of PBS, completely remove the remaining PBS. 3. Resuspend the cells in 500 ␮L of stripping buffer, leave for 4 min (not longer!) at room temperature. 4. Add 14 mL of neutralization buffer and immediately centrifuge at 500×g for 5 min. 5. Completely remove the remaining liquid and resuspend the cells in FACS buffer, count cells and determine viability (which should not be affected by acid treatment). Wash once more and resuspend the cells at 5 × 106 cells/mL in FACS buffer. 6. Distribute 5 × 105 cells (stripped or non-stripped) per well into V-bottom 96-well plates. After centrifugation (3 min at 500×g), the cells are stained for flow cytometry in a final volume of 50 ␮L for 30 min on ice. We routinely use 10–20 ␮g/mL of PE-labelled Dd tetramer, but the optimal tetramer concentration has to be determined for each batch. Independent samples are stained with anti-Ly49A mAb (to determine the cell surface density of Ly49A) and with anti-␤2m mAb (to determine whether ␤2m was quantitatively removed by acid treatment) (Fig. 21.1). Background staining is determined using untransfected C1498 cells. Non-stripped, transfected cells are used as positive controls. 7. Add 200 ␮L of FACS buffer, centrifuge 3 min at 500×g, remove the liquid by flicking the plate. 8. Resuspend the cells in 400 ␮L of cold FACS buffer, transfer to 5 mL FACS tubes and run samples on a flow cytometer. 9. Data analysis and quantification: All flow cytometry analysis is done on live cells (identified based on their forward and

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Fig. 21.1. Ly49A accessibility probed with soluble Dd ligand. Histograms show nontransfected (-) C1498 cells or cells transfected with Ly49A or co-transfected with Ly49A and Dd , stained for Ly49A (mAb JR9), ␤2m (mAb 19.11) and with Dd tetramers. Open histograms depict cells that had been treated with an acidic buffer and grey-filled histograms show cells that were left untreated. Numbers depict the mean fluorescence intensity (MFI) of staining with the indicated reagent.

side scatters (FSC/SSC)). Determine the mean fluorescence intensity (MFI) of staining using ␤2m mAb, Ly49A mAb and Dd tetramer. a. Determine the “relative tetramer binding” by calculating the ratio of MFI (tet)/MFI (mAb) for Ly49A transfectants that lack and that express Dd ligand (Fig. 21.1) (see Note 3). b. Determine “receptor masking” by calculating the ratio of MFI (tet, strip)/MFI (tet, non-strip) for Ly49A transfectants that lack and that express Dd ligand (Fig. 21.1) (see Note 4). 3.2. Determining Ly49A Receptor Accessibility Using MembraneAssociated Dd Ligand

While soluble Dd ligand is useful to determine receptor masking, the use of multimeric ligands is not likely to completely reflect receptor binding to monomeric ligands that are expressed on a target cell membrane. This is of importance when analysing receptor variants in which either cis and/or trans binding may be impaired. The assays shown below are used to assess the ability of Ly49A to interact with membrane-bound ligand and to see to what extent Dd expression in cis impacts this interaction. In Section 3.2.1 we describe a FACS-based cell−cell adhesion assay

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(based on (10)) and in Section 3.2.2 we exploit Dd ligand capture from neighbouring cells (11) to assess the capacity of Ly49A to interact with membrane-bound ligand. 3.2.1. Cell Adhesion Assay

1. Prepare a fresh solution of 1 ␮M CMTMR in PBS and of 0.1 ␮M CFSE in PBS. Keep the solutions protected from light. 2. Harvest and count C1498 transfectants. Take 5–10 × 106 cells per labelling and wash once with PBS. 3. Discard the PBS and resuspend in 1 ␮M CMTMR solution (107 cells/mL) or in 0.1 ␮M of CFSE solution (107 cells/mL), incubate for 10 min at 37◦ C. 4. Add 14 mL of cold cell culture medium, centrifuge 5 min at 500×g. 5. Wash the cells two more times and resuspend at 0.5 × 106 cells/mL in cold cell culture medium. 6. Mix 2.5 × 104 (50 ␮L) of CMTMR-labelled cells with 2.5 × 104 (50 ␮L) of CFSE-labelled cells in a U-bottom 96-well plate. 7. Centrifuge the plate for 1 min at 75×g and incubate for 10 min at 37◦ C. Do not resuspend the pellet! 8. Transfer the plate on ice and add directly 100 ␮L/well of 1% PFA in PBS. Gently resuspend the cells by pipetting. In order not to disrupt conjugates use tips where the ends have been cut off. 9. Transfer the resuspended cells to 1.4 mL collection tubes, which contain 200 ␮L of 1% PFA fixation solution. 10. Run the cells immediately on a flow cytometer. 11. Data analysis: Ly49A-expressing cells (CMTMR+) are detected in FL2, Dd cells (CFSE+) are detected in FL1. Conjugates are double positive for CMTMR and CFSE (Fig. 22.2). Determine the percentage of Ly49A cells (CMTMR+) conjugated with CFSE+ cells as a percentage of all CMTMR+ cells (see Note 5).

3.2.2. Ligand Capture Assay

Upon interaction, Ly49A cells acquire Dd ligand from neighbouring cells. In fact acquisition is bi-directional as Dd cells also acquire Ly49A receptor from neighbouring cells (Fig. 21.3A). Ligand transfer can easily be quantified using C1498 cells expressing a Dd -EYFP fusion protein (Dd -EYFP cells) and measuring green (EYFP) fluorescence of Ly49A+ cells. 1. Harvest and count transfectants, include C1498 cells expressing the Dd -EYFP fusion protein.

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Fig. 21.2. Ly49A-mediated cell−cell adhesion. Formation of conjugates between Ly49A-expressing and Dd -expressing C1498 transfectants. C1498 transfectants were labelled with CMTMR or CFSE, mixed and incubated for 10 min at 37◦ C before fixation. Numbers in the density plots indicate the percentage of CMTMR+ cells conjugated with CFSE+ cells as a percentage of all CMTMR+ cells.

2. Mix 105 Ly49A cells with 105 Dd -EYFP cells in a final volume of 8 mL of cell culture medium in a 25 cm2 culture flask. Independently set up 2 × 105 Ly49A, Ly49A Dd cells or Dd EYFP cells. As a specificity control for ligand transfer set up a co-culture of non-transfected C1498 cells with Dd -EYFP cells. 3. Culture cell mixtures in a CO2 incubator (37◦ C, 5% CO2 ). 4. After 48 h harvest the co-cultures (they must be confluent), count, wash and resuspend cells at 5 × 106 cells/mL in FACS buffer. The co-culture time may be reduced. However, the initial cell density must be increased accordingly to reach confluence more rapidly. 5. Distribute 5 × 105 cells per well in V-bottom 96 well plates. After centrifugation (3 min, 500×g), flick the plate and stain the cells with anti-Ly49A−biotin in a final volume of 50 ␮L of FACS buffer for 30 min on ice. 6. Samples are washed once in FACS buffer, and further stained with streptavidin−PE in 50 ␮L of FACS buffer for 20 min on ice. 7. After washing, the cells are resuspended in a total volume of 400 ␮L of cold FACS buffer, transferred to FACS tubes and run on a flow cytometer. 8. Determine the mean fluorescence intensity (MFI) of (Dd -) EYFP (FL1) on gated Ly49A cells (FL2) (see Note 6).

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Fig. 21.3. Ly49A-mediated ligand uptake from environmental cells. (A) C1498 transfectants (acceptor cells) were co-cultured with transfectants expressing a Dd -EYFP fusion protein (donor cells) (lower panel) or were cultured alone (upper panel). The cell mixture was stained for Ly49A and analysed by flow cytometry. Density plots show that Ly49Aexpressing cells (rectangular gate) acquired green fluorescence (Dd -EYFP). Dd -EYFP transfer was not observed when acceptor cells lacked Ly49A and transfer was strongly reduced when Ly49A cells co-expressed Dd . Numbers indicate the mean FL1 (EYFP) fluorescence intensity (MFI) in the indicated gate. The net Dd -EYFP transfer (MFI) was calculated by subtracting the FL1 MFI of acceptor cells cultured alone from that of acceptor cells co-cultured with Dd -EYFP-expressing cells. (B) Confocal microscopy pictures of Dd -EYFP fluorescence, Ly49A staining and transmitted light (maximum intensity projections of series of Z-stack confocal acquisitions) show a Ly49A transfectant conjugated with a Dd -EYFP transfectant. Arrows highlight Dd -EYFP acquired by the Ly49A cell.

4. Notes 1. Acid stripping disrupts tri-molecular MHC-I complexes. Whereas bound peptide and ␤2m are removed, the MHC-I heavy chain remains membrane bound. This procedure was initially developed to extract antigenic peptides bound to MHC molecules from living cells (12). In the case of Siglecs, sialic acid cis ligands can be removed enzymatically using sialidase treatment (13). 2. Binding of several Ly49 receptors to MHC-I is influenced by species-specific residues in ␤2m (5, 14). It is

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therefore important to use mouse ␤2m to refold MHC-I tetramers. While Ly49A binding to Dd is not influenced by the peptide, Ly49I binding to Kd is strongly influenced by bound peptide (5, 15). For commercially available MHC-I tetramers see Beckman Coulter (Fullerton, CA, USA), ProImmune (Oxford, UK), and http://www.niaid. nih.gov/reposit/TETRAMER/overview.html 3. “Relative tetramer binding” takes into account a possible difference in the Ly49A cell surface density between transfectants. A reduction of the MFI (tet)/MFI (mAb) ratio in the presence of ligand indicates that ligand expression masks the receptor (Fig. 21.1). In the example shown (Fig. 21.1), the ratio of MFI (tet) to MFI (mAb) for cells expressing Ly49A in the absence of ligand is 3.5, while it is 1.4 for cells co-expressing Ly49A and Dd . Tetramer binding is thus specifically reduced (2.5-fold) in the presence of Dd ligand, indicating that approximately 60% of Ly49A receptors are masked. The accurate determination of MFI (tet)/MFI (mAb) in the absence and presence of ligand is based on the assumption that antibody binding is not influenced by the expression of ligand in cis. Ly49A staining with mAb JR9-318 (or YEI-48) is actually slightly reduced (1.2- to 1.5-fold) in the presence of Dd . While true receptor downmodulation cannot be completely excluded (16) we believe that this effect is predominantly based on receptor masking. Indeed, mAb staining improves upon acid stripping (Fig. 21.1). This method may thus somewhat underestimate receptor masking. The binding of some mAbs, such as A1, is much more sensitive to the presence of Dd as compared to mAb JR9318. This is almost completely due to the masking of the A1 epitope by Dd expression in cis (unpublished data). Thus what may appear as a reduction of Ly49 cell surface density is actually due to receptor masking and the extent of the effect is dependent on which mAb is used for detection. As another example, the binding of mAb 5E6 to Ly49C is strongly reduced by the presence of Kb in cis, while the binding of mAb 4D12 is much less affected. 4. Acid stripping is controlled by a reduction of staining for ␤2m: as shown in Fig. 21.1, the MFI of ␤2m staining is 66 prior to stripping and 4 after stripping. Next, the MFI of tetramer staining of acid−stripped cells (MFI (tet, strip)) is compared to that of non-stripped cells (MFI (tet, no strip)) (Fig. 21.1). In our example, the ratio of MFI (tet, strip)/MFI (tet, no strip) for Ly49A cells is 0.9. The corresponding ratio for Ly49A Dd cells is 3.0 (Fig. 21.1),

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indicating that the access to the receptor has improved due to acid stripping. The extent of improvement suggests that 66% of Ly49A receptors were masked by Dd . It is important to determine the ratio between MFI (tet, strip)/MFI (tet, no strip) in the absence of known ligand. In this case, a ratio of >1.0 indicates that Ly49A may be masked by an additional ligand. Conversely, a ratio significantly <1.0 suggests that the receptor itself is acid sensitive. While this is not the case for Ly49A or other Ly49 receptors, we have noted that besides MHC-I molecules, LFA-1 is sensitive to acid treatment. 5. In this system, adhesion independent of Ly49A and Dd (using non-transfected C1498 cells) is low (9.8% of C1498 are conjugated) (Fig. 21.2). There is no Dd -independent adhesion to Ly49A (10.5 and 11.6% conjugated) and there is also no Ly49A-independent adhesion to Dd (11.0%). A large fraction of Ly49A cells (46.2%) and fewer Ly49A Dd cells (21.5%) formed conjugates with Dd cells. Thus, specific adhesion mediated by the interaction between Ly49A and Dd was 35.7% for Ly49A cells and 9.9% for cells coexpressing Ly49A and Dd . This represents a reduction of approximately 3.6-fold (72%) due to cis-masking of Ly49A. 6. In the example shown in Fig. 21.3, the FL1 MFI of Dd -EYFP cells is 128, which indicates that <10% of the Dd EYFP molecules transfer from Dd -EYFP to Ly49A cells. The net Dd -EYFP uptake by Ly49A cells (=MFI) is obtained by subtracting the FL1 MFI of Ly49A cells that were cultured alone from that of Ly49A cells that were co-cultured with Dd -EYFP cells (Fig. 21.3) (MFI=9.5). Ligand transfer is Ly49A dependent as no Dd -EYFP is detectable on C1498 cells that lack Ly49A (MFI=−0.1). Finally, Dd -EYFP transfer is strongly reduced when Ly49A cells co-express Dd (MFI=3.8) (see Fig. 21.3). In our example, this reduction is 2.5-fold indicating that 60% of Ly49A receptors are masked.

References 1. Lanier, L.L. (2005) NK cell recognition. Annu Rev Immunol, 23, 225–274. 2. Karlhofer, F.M., Hunziker, R., Reichlin, A., Margulies, D.H. and Yokoyama, W.M. (1994) Host MHC class I molecules modulate in vivo expression of a NK cell receptor. J Immunol, 153, 2407–2416. 3. Doucey, M.A., Scarpellino, L., Zimmer, J., Guillaume, P., Luescher, I.F., Bron, C. and Held, W. (2004) Cis association of Ly49A

with MHC class I restricts natural killer cell inhibition. Nat Immunol, 5, 328–336. 4. Back, J., Chalifour, A., Scarpellino, L. and Held, W. (2007) Stable masking by H-2Dd cis ligand limits Ly49A relocalization to the site of NK cell/target cell contact. Proc Natl Acad Sci U S A, 104, 3978–3983. 5. Scarpellino, L., Oeschger, F., Guillaume, P., Coudert, J.D., Levy, F., Leclercq, G. and Held, W. (2007) Interactions of Ly49 family

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7.

8.

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receptors with MHC class I ligands in trans and cis. J Immunol, 178, 1277–1284. Masuda, A., Nakamura, A., Maeda, T., Sakamoto, Y. and Takai, T. (2007) Cis binding between inhibitory receptors and MHC class I can regulate mast cell activation. J Exp Med, 204, 907–920. Held, W. and Mariuzza, R.A. (2008) Cis interactions of immunoreceptors with MHC and non-MHC ligands. Nat Rev Immunol, 8, 269–278. LaBelle, J.L. and Truitt, R.L. (2002) Characterization of a murine NKT cell tumor previously described as an acute myelogenous leukemia. Leuk Lymphoma, 43, 1637–1644. Altman, J.D., Moss, P.A., Goulder, P.J., Barouch, D.H., McHeyzer-Williams, M.G., Bell, J.I., McMichael, A.J. and Davis, M.M. (1996) Phenotypic analysis of antigenspecific T lymphocytes. Science, 274, 94–96. Burshtyn, D.N., Shin, J., Stebbins, C. and Long, E.O. (2000) Adhesion to target cells is disrupted by the killer cell inhibitory receptor. Curr Biol, 10, 777–780. Zimmer, J., Ioannidis, V. and Held, W. (2001) H-2D ligand expression by Ly49A+ natural killer (NK) cells precludes ligand uptake from environmental cells: implications for NK cell function. J Exp Med, 194, 1531–1539.

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12. Storkus, W.J., Zeh, H.J., 3rd, Maeurer, M.J., Salter, R.D. and Lotze, M.T. (1993) Identification of human melanoma peptides recognized by class I restricted tumor infiltrating T lymphocytes. J Immunol, 151, 3719–3727. 13. Collins, B.E., Blixt, O., DeSieno, A.R., Bovin, N., Marth, J.D. and Paulson, J.C. (2004) Masking of CD22 by cis ligands does not prevent redistribution of CD22 to sites of cell contact. Proc Natl Acad Sci U S A, 101, 6104–6109. 14. Michaelsson, J., Achour, A., Rolle, A. and Karre, K. (2001) MHC class I recognition by NK receptors in the Ly49 family is strongly influenced by the beta 2-microglobulin subunit. J Immunol, 166, 7327–7334. 15. Hanke, T., Takizawa, H., McMahon, C.W., Busch, D.H., Pamer, E.G., Miller, J.D., Altman, J.D., Liu, Y., Cado, D., Lemonnier, F.A. et al. (1999) Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors. Immunity, 11, 67–77. 16. Andersson, K.E., Williams, G.S., Davis, D.M. and Hoglund, P. (2007) Quantifying the reduction in accessibility of the inhibitory NK cell receptor Ly49A caused by binding MHC class I proteins in cis. Eur J Immunol, 37, 516–527.

Chapter 22 A Simple Method to Measure NK Cell Cytotoxicity In Vivo Aurore Saudemont, Shannon Burke, and Francesco Colucci Abstract Natural killer (NK) cells were discovered in the 1970s and named after their naturally occurring cytotoxic activity against tumor cells. It has recently become clear that NK cells are not just killers and that malignancy is unlikely to be the selective pressure driving the evolution of NK cells. Indeed, NK cells secrete a host of cytokines and chemokines that contribute to tissue remodeling at the feto-maternal interface and to both innate and adaptive immunity during infection. Moreover, in certain conditions, they cannot deliver functions cell autonomously, as they require priming from other cells, namely dendritic cells. Nevertheless, natural cytotoxicity is still considered an important parameter used to evaluate NK cell biology, both in the clinic and in the research lab. In this chapter we describe a simple method to quantify spontaneous NK cell cytotoxicity in vivo. Key words: Lymphoma, melanoma, CFSE, peritoneal exudate.

1. Introduction Cellular cytotoxicity is usually measured in classical 51 Cr release assays, in which tumor target cells are labeled with the radioisotope and co-incubated at various ratios with the effector cells for 4 h. The extent of specific lysis is calculated based on the amount of free radioactivity in the supernatant. Originally tailored to study the activity of cytotoxic T cells (1), the 51 Cr release assay has been instrumental to define several aspects of the biology of cytotoxicity, including its genetic control (2). The assay helped in the discovery of NK cells (3) and has since become the gold standard for NK cell biologists. While the 51 Cr release assay is sensitive, it has some shortcomings, including the use of inflated effector-to-target ratios and the NK cell culture conditions, such K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 22, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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as supraphysiological levels of growth or activation stimuli, which may lower their activation threshold. Moreover, as in vitro killing assays require the effector and target cells to be cultured in the same well, this negates the need for NK cells to actively locate target cells. Thus, in vivo assays have been developed to study NK cell immunosurveillance. The first assays to measure NK cell activity in vivo were based on the use of tumor target cells labeled with [125 I]5-iodo2 -deoxyuridine (4). In these assays, the labeled target cells are injected intravenously and, generally after 4–6 h, various organs are explanted. The amount of residual radioactivity in the organs (typically lungs) inversely correlates with NK cell activity. The use of radioactivity poses several constraints and therefore alternative methods have been developed to measure cytotoxicity in vitro (5–7) and in vivo (8, 9). The in vivo method is based on fluorometric techniques for detection of target cells labeled with an intracellular vital dye, usually carboxyfluorescein succinimidyl ester (CFSE). The use of CFSE was originally introduced to track migrating cells (10) and to quantify the number of cell divisions in a population of transferred leukocytes (11). We and others have used similar approaches (described in details below) to study the rejection of lymphoma cells (12–15) and have also had some success in tracking the elimination of melanoma cells (16). A similar method can be applied to the study of hematopoietic cell rejection (17). We typically use lymphoma cells to measure NK cell cytotoxicity and the capacity to discriminate between NK cell-resistant RMA cells and NK-susceptible RMA-S cells. We inject an equal number of the two cell types (typically 2×105 cells of each type) in the peritoneal cavity and use a simple readout at day 2 after the injection. The readout is the enumeration of the two cell types in the peritoneal lavage. Normally we recover high numbers of RMA cells (roughly 2–3×105 cells, regardless of the NK cell activity), because they are not rejected, and much fewer RMA-S cells (roughly 0.05–0.1×105 cells). The assay is therefore based on the inverse correlation between NK cell activity and the number of RMA-S cells recovered. For example, we have recovered 2.88 ± 2.42×105 RMA cells and 0.085 ± 0.077 × 105 RMA-S cells from wild-type C57BL/6 mice (n =20), whereas from NK cell-deficient Rag2−/− Il2rg−/− mice (n =20), we have recovered 3.44 ± 2.7× 105 RMA cells and 3.31 ± 2.66×105 RMA-S cells. This assay is suited to analyze innate responses due to its short-term duration, which makes it unlikely for T or B cells to mount adaptive responses. Indeed the rejection of RMA-S cells in this assay is independent of adaptive lymphocytes, as shown in Rag2−/− mice (15). The assay can be also used to assess NK cell extravasation (13) and trafficking (18) to the tumor site.

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Other, less rapid, and non-radioactive in vivo assays to measure NK cell activity in mice are based on the administration of various doses of tumor cells subcutaneously and the measurement of either tumor mass or tumor incidence over several weeks (19). NK cell activity to control metastatic progression in mice is typically measured by quantifying the numbers of lung metastases 2 weeks after the injection of various doses of melanoma cells intravenously (20).

2. Materials 2.1. Mice

1. C57BL/6 WT mice. 2. Rag2−/− Il2rg−/− mice (C57BL/6 background) (21). This strain was established by crossing the Rag2-deficient mice (22) with mice that lack the common gamma chain of the IL-2 receptor (23). The latter mutation prevents signaling of, among others cytokines, IL-15, which is essential for development of mature NK cells (see Note 1). Therefore, these mice lack mature T, B, and NK cells.

2.2. Solutions

1. Culture medium: RPMI 1640 medium containing L -glutamine (2 mM) and 2-mercaptoethanol (50 ␮M), penicillin (100 U/ml), streptomycin (100 ␮g/ml), and 5% fetal bovine serum (FBS). Use this medium to grow tumor cells and for peritoneal lavage. For labeling tumor cells with CMTMR, the medium needs to be FBS free. 2. Phosphate-buffered saline (PBS), pH 7.4, containing bovine serum albumin (BSA; Sigma-Aldrich): Supplementation with 1% BSA is used for resuspending the tumor cells for flow cytometry analysis, and supplementation with 0.1–0.5% BSA is used for resuspending the tumor cells for CFSE labeling and for flow cytometry analysis of infiltrating NK cells in the lavage collection.

2.3. Dyes

1. CFSE:[5-(and-6)-carboxyfluorescein diacetate, succinimidylester (5, 6)]-CFDA, SE (Invitrogen) stock prepared at 5 mM in DMSO and stored at −20 ◦ C. 2. CellTracker Orange CMTMR (Invitrogen): stock prepared at 10 mM in DMSO and stored at –20◦ C. 3. PKH26 (Sigma-Aldrich): stock prepared at 5 mM in buffer provided and stored at room temperature. PKH26 can be used as an alternative dye to CMTMR. In our experience, we obtain more consistent labeling of cell lines but failed to obtain satisfactory labeling of primary cells with PKH26.

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2.4. Equipment

1. 10 ml syringes (BD Biosciences) for injection and collection of fluid. 2. 21 or 23 g needles (BD Biosciences) for fluid injection in the peritoneal cavity. The same needle can be used for injecting the fluid in all mice. 3. 25 or 26 g needles for tumor injection and fluid collection. Large gauge should be used in order to prevent damage to cells. 4. 3 ml Pasteur pipettes for fluid collection in cases where a more concentrated lavage fluid is desired or for subsequent analysis of the peritoneal membranes. 5. 15 ml conical centrifuge tubes (BD Biosciences) for collecting the lavage fluid. 6. Hemocytometer. 7. Benchtop centrifuge.

3. Methods

3.1. Cell Preparation/Labeling and Injection

1. Collect the number of tumor cells needed for the experiment (see Notes 2 and 3). We routinely inject 2×105 tumor cells per mouse, but this may be adjusted depending on the cell type and experimental setting. 2. Centrifuge the cells at 300×g for 10 min. Now proceed to desired labeling technique below.

3.1.1. CFSE Labeling (see Note 4)

1. Resuspend the cells in PBS 0.5% BSA at 107 cells/ml. 2. Add 1 ␮l/ml of CFSE dye (stock solution at 5 mM) to obtain a final concentration of 5 ␮M. 3. Incubate at 37ºC for 10 min. 4. Stop the reaction by adding cold culture medium containing 5% FBS, or PBS 0.1% FBS (10×reaction volume) or pure FBS (same volume as reaction volume). 5. Wash twice with culture medium containing 5% FBS.

3.1.2. CMTMR Labeling

1. Resuspend the cells in culture medium without FBS at 107 cells/ml. 2. Add 0.5 ␮l/ml of CMTMR dye (stock solution at 10 mM) to obtain a final concentration of 5 ␮M. 3. Incubate at 37ºC for 10 min. 4. Stop the reaction by adding either cold culture medium containing 5% FBS, or PBS 0.1% FBS (10×reaction volume), or pure FBS (same volume as reaction volume).

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Fig. 22.1. Elimination of tumor cells from the peritoneal cavity. (A) A total of 2×105 PKH26-labeled live RMA cells and 2×105 CFSE-labeled live RMA-S cells were injected intraperitoneally into C57BL/6 WT mice or Rag2−/− Il2rg−/− mice. Forty-eight hours later, mice were culled and a peritoneal lavage performed. The percentage (number in gate) of labeled cells within the peritoneal lavage was determined by flow cytometry. (B) A total of 2×104 CFSE-labeled live RET-tg melanoma cells were injected intraperitoneally into C57BL/6 WT mice or Rag2−/− Il2rg−/− mice. Forty-eight hours later, mice were culled and a peritoneal lavage performed. The percentage (number in gate) of CFSE-labeled melanoma cells within the peritoneal lavage was determined by flow cytometry. The absolute numbers of residual tumor cells within the peritoneal cavity was enumerated as described in the Sections 3 and 4.

5. Wash twice with culture medium containing 5% FBS. 3.1.3. PKH26 Labeling

1. Resuspend the cells in the buffer provided at 107 cells/ml. 2. Add 1 ␮l/ml of PHK26 dye (stock solution at 5 mM) to obtain a final concentration of 5 ␮M. 3. Incubate at room temperature for 10 min. 4. Stop the reaction by adding either cold culture medium containing 5% FBS, or PBS 0.1% FBS (10×reaction volume), or pure FBS (same volume as reaction volume). 5. Wash twice with PBS 0.5% BSA.

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3.1.4. Cell Injection

1. Perform a cell count before mixing different cell lines prior to injection. 2. If two cell lines have been mixed in a 1:1 ratio, the mixture should be analyzed by flow cytometry to make sure of this ratio prior to injection (see Note 5). 3. Mice are challenged intraperitoneally with the tumor cells in 500 ␮l PBS using a 25 g needle.

3.2. Collection and Analysis of Peritoneal Fluid 3.2.1. Collection of Peritoneal Fluid (see Note 6)

1. Mice can be killed by cervical dislocation or by CO2 inhalation. 2. The peritoneal cavity is washed with 10 ml cold culture medium containing 5% FBS. This injection can be done with a 23 g needle. Gently massage the abdomen prior to collection of the fluid. 3. Collect the peritoneal fluid in a 15 ml centrifuge tube using a 25 g needle or a Pasteur pipette. 4. Keep the peritoneal fluid on ice and protect from light.

3.2.2. Analysis of Peritoneal Fluid

1. Centrifuge the peritoneal fluid at 300×g for 10 min. 2. Count cells. 3. Cells are then analyzed by flow cytometry for the detection of the dye(s) corresponding to the tumor cell line(s) (Fig. 22.1A). 4. Quantification of tumor cells: total cell number × % tumor cells × 10 (if the lavage was performed with 10 ml fluid and regardless of the volume recovered) (see Notes 7 and 8).

4. Notes 1. If alymphoid Rag2−/− Il2rg−/− mice are not available, mice in which NK cells are depleted can be used as controls. Typically NK cells are depleted in C57BL mice by anti-NK1.1 antibody treatment. Alternatively, antibodies specific for asialo-GM1 or CD122 (i.e., IL2R␤) can be used to deplete most NK cells in NK1.1-negative strains of mice. We routinely use also Rag2−/− mice (of the C57BL/6 background) to have a strain of NK-sufficient mice that have no other lymphocyte subset. These mice lack functional recombinase activating gene 2 and thus do not develop mature B and T lymphocytes due to an inability to undergo V(D)J recombination (22). Natural killer cell development and function are largely normal in these mice, although in some

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assays NK cells can be more effective than that in WT mice, due to the lack of the suppressive action of regulatory T cells. 2. For optimum results, cells need to be in the exponential growth phase on the day of the assay. 3. It should be noted that high concentrations of membranepermeable dyes may be toxic. Therefore, the concentration of dye and the time of the labeling period should be determined for each cell type (10, 24). For the three dyes we use, the final concentration is typically 0.5–10 ␮M. Within these parameters, we have not observed compromised survival of the cells. 4. Concurrent flow cytometry analysis of red fluorescent dyes with CFSE can be problematic due to the very high CFSE signal. This is particularly true for freshly labeled cells which contain a substantial quantity of unincorporated dye and, as a result, exhibit a very intense signal. Therefore, it is recommended that multi-parametric analysis not be attempted within the first 24 h as compensation against CFSE can be almost impossible. After this time, compensation between CFSE and PE can be facilitated by ensuring that the voltage on the PE channel is reduced together with the use of a bright PE dye or marker. 5. If possible, an aliquot of the labeled tumor cells or mixture of labeled tumor cells injected should be put in culture and checked for labeling and mixture ratio the day of analysis. 6. Peritoneal lavages require practice and care to ensure thorough removal of the injected wash volume and to avoid rupturing blood vessels and collection of blood cells, which may confound analysis. This will improve the consistency of results between mice and ensure a significant proportion of the residual tumor cells are retrieved. We have found that including a group of mice which have not been injected with tumor cells can be useful to determine the baseline above which the tumor cells fall in the scatter profile of peritoneal lavage and to determine the fluorescence background against which the analysis gate should be positioned. Tumor cells can be distinguished from host cells by virtue of allelic polymorphism (e.g., CD45, MHC class I). Moreover, mice that have received unlabeled tumor cells may be an important control for determining fluorescence background in cases where there is a significant cellular infiltrate in response to the tumor cells. In most cases, however, the latter controls are not necessary if the level of fluorescent dye is first optimized. 7. There are some important caveats when using this assay with adherent cells lines or cell lines with novel characteristics. We

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have found that the number of residual tumor cells retrieved is considerably less with melanoma cells compared to lymphoma, irrespective of their susceptibility to immune control. It is possible that melanoma and lymphoma cells display differential propensities to migrating out of the peritoneal cavity or to adhere to it. Although we have not tested these possibilities, we suspect that melanoma cells adhere to the peritoneal membrane. While a subcutaneous site may represent a more physiological site for assessing the growth of melanoma cells, the peritoneal clearance assay offers the advantages to more easily quantify the number of residual tumor cells. We have used a melanoma cell line derived from the RET transgenic mouse model of spontaneous melanoma (16). Briefly, melanoma cells are harvested during the log phase of growth, labeled with 5 ␮M CFSE and 2×104 cells are injected in the peritoneal cavity of C57BL/6 or Rag2−/− Il2rg−/− mice. The number of residual tumor cells in the peritoneal cavity is then determined after 48 h. Results from one such experiment are represented in Fig. 22.1B. Clearance of RET melanoma cells in this model is dependent on NK cells as there is an increased tumor burden in the peritoneal cavity of mice depleted of NK cells (not shown), or in Rag2−/− Il2rg−/− mice (4.1×104 ± 2.9×104 , n=11), compared to control mice (1.1×104 ± 0.7×104 , n=11). 8. In parallel to the analysis of tumor elimination, an analysis of the composition of specific lymphocyte subsets can also be done in the peritoneal lavages recovered from tumor bearing mice. To more specifically measure extravasation of NK cells to the tumor site, we inject 107 CFSE-labeled splenocytes (which contain 2–5% NK cells) intravenously into mice that have received tumor cells intraperitoneally and 2 days after we quantify the numbers of NK cells that have extravasated into the peritoneal cavity (13).

Acknowledgments We would like to thank past and present members of the lab for suggestions and discussions. Research in our lab is funded by BBSRC, CR-UK, and MRC. References 1. Brunner, K. T., Mauel, J., Cerottini, J. C., and Chapuis, B. (1968) Quantitative assay of the lytic action of immune lymphoid cells

on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 14, 181–196.

A Simple Method to Measure NK Cell Cytotoxicity In Vivo 2. Nabholz, M., Vives, J., Young, H. M., Meo, T., Miggiano, V., Rijnbeek, A., and Shreffler, D. C. (1974) Cell-mediated cell lysis in vitro: genetic control of killer cell production and target specificities in the mouse. Eur J Immunol 4, 378–387. 3. Kiessling, R., Klein, E., and Wigzell, H. (1975) Natural killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Eur J Immunol 5, 112–117. 4. Riccardi, C., Puccetti, P., Santoni, A., and Herberman, R. B. (1979) Rapid in vivo assay of mouse natural killer cell activity. J Natl Cancer Inst 63, 1041–1045. 5. Betts, M. R., Brenchley, J. M., Price, D. A., De Rosa, S. C., Douek, D. C., Roederer, M., and Koup, R. A. (2003) Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 281, 65–78. 6. Liu, L., Chahroudi, A., Silvestri, G., Wernett, M. E., Kaiser, W. J., Safrit, J. T., Komoriya, A., Altman, J. D., Packard, B. Z., and Feinberg, M. B. (2002) Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates. Nat Med 8, 185–189. 7. Sheehy, M. E., McDermott, A. B., Furlan, S. N., Klenerman, P., and Nixon, D. F. (2001) A novel technique for the fluorometric assessment of T lymphocyte antigen specific lysis. J Immunol Methods 249, 99–110. 8. Hermans, I. F., Silk, J. D., Yang, J., Palmowski, M. J., Gileadi, U., McCarthy, C., Salio, M., Ronchese, F., and Cerundolo, V. (2004) The VITAL assay: a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo. J Immunol Methods 285, 25–40. 9. Oehen, S., Brduscha-Riem, K., Oxenius, A., and Odermatt, B. (1997) A simple method for evaluating the rejection of grafted spleen cells by flow cytometry and tracing adoptively transferred cells by light microscopy. J Immunol Methods 207, 33–42. 10. Weston, S. A., and Parish, C. R. (1990) New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy. J Immunol Methods 133, 87–97. 11. Lyons, A. B., and Parish, C. R. (1994) Determination of lymphocyte division by flow cytometry. J Immunol Methods 171, 131–137. 12. Johansson, S. E., Hall, H., Bjorklund, J., and Hoglund, P. (2004) Broadly impaired NK

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cell function in non-obese diabetic mice is partially restored by NK cell activation in vivo and by IL-12/IL-18 in vitro. Int Immunol 16, 1–11. Saudemont, A. (2009) The p110g and p110d isoforms of phosphoinositide 3kinase differentially regulate natural killer cell migration in health and disease. Proc Natl Acad Sci U S A,106, 5795–5800 Saudemont, A., Okkenhaug, K., and Colucci, F. (2007) p110delta is required for innate immunity to transplantable lymphomas. Biochem Soc Trans 35, 183–185. Zompi, S., Hamerman, J. A., Ogasawara, K., Schweighoffer, E., Tybulewicz, V. L., Di Santo, J. P., Lanier, L. L., and Colucci, F. (2003) NKG2D triggers cytotoxicity in mouse NK cells lacking DAP12 or Syk family kinases. Nat Immunol 4, 565–572. Lakshmikanth, T. (2009) NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo.J Clin Invest 119, 1251–1263 Oberg, L., Johansson, S., Michaelsson, J., Tomasello, E., Vivier, E., Karre, K., and Hoglund, P. (2004) Loss or mismatch of MHC class I is sufficient to trigger NK cellmediated rejection of resting lymphocytes in vivo - role of KARAP/DAP12-dependent and -independent pathways. Eur J Immunol 34, 1646–1653. Smyth, M. J., Kelly, J. M., Baxter, A. G., Korner, H., and Sedgwick, J. D. (1998) An essential role for tumor necrosis factor in natural killer cell-mediated tumor rejection in the peritoneum. J Exp Med 188, 1611–1619. Karre, K., Ljunggren, H. G., Piontek, G., and Kiessling, R. (1986) Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature 319, 675–678. Kim, S., Iizuka, K., Aguila, H. L., Weissman, I. L., and Yokoyama, W. M. (2000) In vivo natural killer cell activities revealed by natural killer cell-deficient mice. Proc Natl Acad Sci U S A 97, 2731–2736. Colucci, F., Soudais, C., Rosmaraki, E., Vanes, L., Tybulewicz, V. L., and Di Santo, J. P. (1999) Dissecting NK cell development using a novel alymphoid mouse model: investigating the role of the c-abl proto-oncogene in murine NK cell differentiation. J Immunol 162, 2761–2765. Shinkai, Y., Rathbun, G., Lam, K. P., Oltz, E. M., Stewart, V., Mendelsohn, M., Charron, J., Datta, M., Young, F., Stall, A. M.,

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and et al. (1992) RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68, 855– 867. 23. Di Santo, J. P., Kuhn, R., and Muller, W. (1995) Common cytokine receptor gamma chain (gamma c)-dependent cytokines: understanding in vivo functions

by gene targeting. Immunol Rev 148, 19–34. 24. Quah, B. J., Warren, H. S., and Parish, C. R. (2007) Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat Protoc 2, 2049–2056.

Chapter 23 Functional Analysis of Human NK Cells by Flow Cytometry ˜ M. Nunes, Stephanie M. Wood, Yenan T. Bryceson, Cyril Fauriat, Joao ¨ ¨ Eric O. Long, and Hans-Gustaf Ljunggren Niklas K. Bjorkstr om, Abstract Natural killer (NK) cells are a subset of lymphocytes that contribute to innate immunity through cytokine secretion and target cell lysis. NK cell function is regulated by a multiplicity of activating and inhibitory receptors. The advance in instrumentation for multi-color flow cytometry and the generation of specific mAbs for different epitopes related to phenotypic and functional parameters have facilitated our understanding of NK cell responses. Here, we provide protocols for flow cytometric evaluation of degranulation and cytokine production by human NK cells from peripheral blood at the single-cell level. In addition to offering insight into the regulation of human NK cell responses, these techniques are applicable to the assessment of various clinical conditions, including the diagnosis of immunodeficiency syndromes. Key words: Human, natural killer cells, immunophenotyping, polychromatic flow cytometry, CD107a, lysosomal-associated membrane protein-1, chemokines, IFN-␥, MIP-1␤, TNF-␣.

1. Introduction Natural killer (NK) cells are a subset of lymphocytes that participate in innate resistance to infected and neoplastic cells (1). Moreover, NK cells interface with adaptive immunity through physical interactions with dendritic cells and T cells and by their ability to secrete specific cytokines (2, 3). NK cell function is regulated by a multiplicity of activating and inhibitory receptors (4). Upon activation, NK cells kill sensitive target cells by directed exocytosis of perforin-containing secretory lysosomes, also called cytotoxic granules. In addition to their cytolytic function, NK cells may secrete chemokines and cytokines. The soluble factors secreted K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 23, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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by NK cells, such as MIP-1␤, TNF-␣, and IFN-␥, recruit other immune cells, promote cellular resistance to infection, and influence adaptive immunity. Due to the important role of NK cells in immunity, analysis of NK cell responses is proving valuable in the assessment various clinical conditions, including post-stem cell transplantation to estimate infection risk, and in the diagnosis of immunodeficiency syndromes. In this section, we present protocols and provide advice regarding flow cytometric assessment of NK cell function. Flow cytometry represents a sensitive and quantitative technique readily applicable to phenotypic and functional characterization of lymphocytes. Several well-characterized mAbs to receptors and other surface molecules that facilitate assessment of NK cell responses and discrimination of NK cell subsets have been generated. Specifically, secretory lysosome release can be quantified by the induced cell surface expression of transmembrane proteins that usually reside in secretory lysosomes. In unactivated cytotoxic lymphocytes, CD63 (lysosomal-associated membrane protein 3, LAMP-3), CD107a (LAMP-1), CD107b (LAMP-2), and CD178 (Fas ligand) reside in secretory lysosomes (5, 6). In NK cells from healthy donors, CD107a co-localizes with perforin and appearance of CD107a at the NK cell surface occurs upon lysis of susceptible target cells (7, 8). Defective induction of CD107a surface expression is associated with certain subtypes of hyper-inflammatory immunodeficiency syndromes (9, 10). Combined assessment of degranulation and de novo transcriptional responses, such as chemokine and cytokine synthesis promises to unravel novel signaling components associated with human immunodeficiencies affecting transcriptional responses or globally impairing lymphocyte effector functions. In addition, these tools should provide insight into how NK cell responses are regulated during other clinical and pathological conditions.

2. Materials 2.1. Cells, Media, and Solutions

1. Whole blood collected in sodium heparin vials (3–10 mL is sufficient for multiple functional experiments), or buffy coats. 2. Lymphoprep (Axis-Shield, Oslo, Norway) stored at room temperature and protected from light. 3. Optional: NK cell-negative isolation kit (Miltenyi, Bergisch Gladbach, Germany). 4. Complete culture medium: RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1 mM L -glutamine (all Invitrogen, Carlsbad, CA).

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5. Target cells: The human erythroleukemia cell line K562 and the murine Fc receptor+ mastocytoma cell line P815 (both American Tissue Type Collection, Manassas, VA) are maintained in complete culture medium. 6. Staining solution: Phosphate-buffered saline (PBS) supplemented with 2% heat-inactivated FBS and 2 mM ethylenediaminetetraacetic acid (EDTA). 7. Fixation solution: PBS supplemented with 2% (w/v) paraformaldehyde (Sigma, St. Louis, MO). 8. Permeabilization solution: PBS supplemented with 2% heatinactivated FBS, 2 mM EDTA, and 0.5% saponin (Sigma). 2.2. Antibodies and Fluorescent Reagents

1. Anti-CD3 mAb (clone SK7 for stimulation of T-cell responses) purified (BD Bioscience, Franklin Lakes, NJ).

2.2.1. Stimulating mAbs for Cellular Assays

3. Anti-CD16 mAb (clone 3G8) purified (BD Bioscience).

2.2.2. Staining mAbs for 2-h Degranulation Assay

1. Anti-CD3 mAb (clone SK7) PerCP (BD Bioscience).

2. Anti-CD3 mAb (clone SK7) PerCP (BD Bioscience).

2. Anti-CD56 mAb (clone NCAM 16.2) PE (BD Bioscience). 3. Anti-CD107a mAb (clone H4A3) FITC (BD Bioscience). 4. Optional: Anti-CD8 mAb (clone SK1) APC (BD Bioscience).

2.2.3. Staining mAbs and Fluorescent Reagents for 6-h Multiple Response Assay

1. Anti-CD3 mAb (clone UCHT1) Cascade Yellow (Dako, Glostrup, Denmark). 2. Anti-CD14 mAb (clone MP3) APC-Cy7 (BD Bioscience). 3. Anti-CD19 mAb (clone SJ25C1) APC-Cy7 (BD Bioscience). 4. Anti-CD56 mAb (clone NCAM 16.2) PE-Cy7 (BD Bioscience). 5. Anti-CD107a mAb (clone H4A3) biotin (BD Bioscience). 6. Anti-IFN-␥ mAb (clone 25723.11) FITC (BD Bioscience). 7. Anti-MIP-1␤ mAb (clone D21-1351) PE (BD Bioscience). 8. Anti-TNF-␣ mAb (clone MAb11) Pacific Blue (eBioscience, San Diego, CA). 9. Qdot 605 streptavidin conjugate (Invitrogen). 10. LIVE/DEAD Fixable Far Red Dead Cell Stain Kit (Invitrogen).

2.3. Flow Cytometry Hardware and Software

1. For analysis of degranulation with the three-color flow cytometry panel outlined in this chapter, a FACS Calibur

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Table 23.1. Instrument configuration and antibody panel Laser (nm)

Detector Filter (nm)

Fluorochrome

Utilized panel

Dilution

488

FL1

530/30

FITC

CD107a

1:50

488

FL2

585/42

PE

CD56

1:50 1:50

488

FL3

670/LP

PerCP

CD3

635

FL4

661/16

APC

Available

(BD Bioscience) with a 488 nm laser is sufficient. Table 23.1 provides a detailed description of the filter setup and utilized detectors. 2. Analysis of multiple functional responses with the seven-color flow cytometry staining panel described here is optimized for a CyAn ADP 9 Color Analyzer (Beckman Coulter, Fullerton, CA) equipped with a 405 nm laser, a 488 nm laser, and a 635 nm laser. Table 23.2 provides a detailed description of the filter setup and utilized detectors (11).

Table 23.2. Instrument configuration and antibody panel Laser (nm)

Detector

FluoroFilter (nm) chrome

Utilized panel Dilution

488

FL1

530/40

FITC

IFN-␥

5:50

488

FL2

575/25

PE

MIP-1␤

2:50

488

FL3

613/20

Qdot 605

CD107a

2:50

488

FL4

630/80

PerCP

Available

488

FL5

750LP

PE-Cy7

CD56

405

FL6

450/50

PacB

TNF-␣

3:50

405

FL7

550/30

CasY

CD3

1:50

635

FL8

665/20

APC

Available

635

FL9

750LP

APC-Cy7

CD14/19/ DCM

1:50

2:50

3. FlowJo software (version 8.7, TreeStar, Ashland, OR) for analysis of acquired raw data. 4. Simplified Presentation of Incredibly Complex Evaluations (SPICE) software (version 4.1.6, courtesy of Mario Roederer, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD) for processing and presentation of analyzed raw data.

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1. GolgiPlug (protein transport inhibitor containing brefeldin A, BD Bioscience). 2. GolgiStop (protein transport inhibitor containing monensin, BD Bioscience). 3. Anti-mouse Ig ␬ compensation beads (BD Bioscience).

3. Methods Upon stimulation by sensitive target cells, NK cells rapidly release cytotoxic proteins by polarized fusion of secretory lysosomes with the plasma membrane. Secretion of chemokines and cytokines is a slower process, requires transcription and de novo protein synthesis, and follows different vesicular pathways. Thus, the temporal kinetics of responses must be taken into consideration when designing experiments that assess distinct NK cell functional parameters. As a note of caution, although NK cell degranulation is a prerequisite for NK cell cytotoxicity, assessment of degranulation does not necessarily correlate with target cell lysis. Target cell lysis depends not only on the extent of effector cell degranulation but also on the content of secretory lysosomes, target cell structures that facilitate adhesion, and polarized secretion of secretory granules, in addition to the target cell’s intrinsic sensitivity to NK cellmediated death pathways (8). Here we provide detailed instructions for a 2-h assay that quantifies human NK cell degranulation by assessing CD107a surface expression, which is based on the use of peripheral blood mononuclear cells (PBMCs) and standard target cell lines. This assay has been used for the differential diagnosis of defects in cellular cytotoxicity (10, 12). Furthermore, instructions are presented for a comprehensive 6-h assay in which human NK cell degranulation, as evaluated by CD107a surface expression, is assessed simultaneously with chemokine and cytokine production, detected by intracellular staining of MIP-1␤, TNF-␣, and IFN-␥. Induction of MIP-1␤ production is generally robust in NK cells and can be measured as early as 30 min after target cell mixing, whereas TNF-␣ and IFN-␥ are delayed. Depending on the available flow cytometer, additional antibodies can successfully be combined, facilitating increasingly detailed analysis of other functional parameters or responses in specific NK cell subsets. Although PBMCs are used as effector cells in the assays described here, the assays are also applicable to purified NK cells.

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3.1. Preparation of Peripheral Blood Mononuclear Cells

1. Isolate PBMCs from heparinized whole blood samples by density gradient centrifugation with lymphoprep according to the manufacturer’s instructions (see Note 1). 2. After centrifugation, harvest the PBMCs in a Falcon tube and add PBS for washing. 3. Centrifuge the cells at 450×g for 10 min. 4. Discard the supernatant and wash the cell pellet twice in PBS. 5. Optional: Purify NK cells by negative selection using a NK cell isolation kit according to the manufacturer’s instructions. 6. Resuspend cells in culture medium at a concentration equal to or less than 5 × 106 cells/mL. 7. After isolation, maintain effector cells overnight in complete culture medium in an incubator set at 37◦ C and 5% CO2 .

3.2. Stimulation and Staining of Effector Cells

1. Spin down the PBMC and resuspend in complete medium at 2 × 106 cells/mL (or 1 × 106 cells/mL if purified NK cells are used) (see Note 2).

3.2.1. Two-Hour Degranulation Assay

2. Spin down target cells and resuspend at 2 × 106 cells/mL in complete medium. For redirected antibody-dependent cellular cytotoxicity, add stimulating mAbs at a concentration of 5 ␮g/mL to target cell suspensions. Mix well. Pipette 100 ␮L of target cell suspension per well into a V-bottomed 96-well plate, as indicated. Table 23.3 provides a schematic representation of how effector cells and target cells are distributed in an assay designed to evaluate cytotoxic lymphocyte degranulation induced by receptors for natural cytotoxicity, Fc receptors, and the T-cell receptor in a diagnostic setting (see Note 3). 3. Add 100 ␮L of the effector cell suspension per well to the V-bottomed 96-well plate, as indicated. Table 23.3 provides a schematic representation of the assay.

Table 23.3. Layout of effector and target cells in wells for diagnostic evaluation of cytotoxic lymphocyte degranulation P815 Medium K562 P815 anti-CD16

P815 anti-CD3

P815 anti-CD3∗

Control Patient ∗ PerCP-conjugated

mAb. The top row indicates the target cells mixed with stimulating mAbs, as indicated. The left column indicates the effector cells.

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4. Centrifuge the cells at 30×g for 3 min. 5. Place the cells in an incubator at 37◦ C for 2 h (see Note 4). 6. During the incubation period, prepare a master mix containing fluorochrome-conjugated anti-CD3, anti-CD56, and anti-CD107a mAbs in staining solution. Table 23.1 provides a summary of the suggested antibody panel including recommended dilutions of the fluorochromeconjugated mAbs (see Notes 5–8). 7. Centrifuge the cells at 450×g for 3 min. 8. Promptly flick off the supernatant. 9. Resuspend the cells in 50 ␮L staining solution containing the appropriate combination and dilution of fluorochromeconjugated mAbs. Incubate samples for 30 min in the dark at 4◦ C. 10. Centrifuge the cells at 450×g for 3 min. 11. Discard the supernatant and wash the cells once with 200 ␮L staining solution. 12. Resuspend the cells in 200 ␮L staining solution and transfer the cells to cytometer tubes. 13. Analyze cells on a flow cytometer, e.g., a FACS Calibur (see Note 9). 3.2.2. Six-Hour Multiple Response Assay

1. For cell stimulation, follow Methods 3.2.1, steps 1 through 4. 2. Place the cells in an incubator at 37◦ C for 1 h. 3. To each well, add 20 ␮L of culture medium supplemented with GolgiPlug diluted 1:100 and GolgiStop diluted 1:150. Immediately return the cells to the incubator at 37◦ C for 5 more hours (see Notes 6 and 10). 4. After a total of 6 h, centrifuge the cells at 450×g for 3 min (see Note 11). 5. Promptly flick off the supernatant. 6. Prepare a master mix of staining solution for cell surface staining containing fluorochrome-conjugated anti-CD3, anti-CD14, anti-CD19, and anti-CD56, in addition to biotinylated anti-CD107a mAbs and LIVE/DEAD Cell Stain diluted 1:200. Table 23.2 provides a summary of the suggested antibody panel including recommended dilutions of the fluorochrome-conjugated mAbs. (see Notes 6, 7, 12, and 13). 7. Resuspend the cells in 50 ␮L staining solution containing an appropriate combination and dilution of fluorochromeconjugated mAbs. Incubate samples for 30 min in the dark at 4◦ C.

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8. Centrifuge the cells at 450×g for 3 min. 9. Discard the supernatant and wash the cells twice with 200 ␮L staining solution. 10. Resuspend the cells in 50 ␮L staining solution supplemented with streptavidin-Qdot 605 (recommended dilution 1:500). Incubate samples for 30 min at 4◦ C in the dark. 11. Centrifuge the cells at 450×g for 3 min. 12. Discard the supernatant and wash the cells twice with 200 ␮L staining solution. 13. Fix the cells in 100 ␮L fixation solution for 10 min at room temperature in the dark. 14. Centrifuge the cells at 450×g for 3 min. 15. Discard the supernatant and wash the cells once with 200 ␮L staining solution. 16. Resuspend the cells in 25 ␮L permeabilization solution. Incubate the cells for 10 min at 4◦ C in the dark. 17. Prepare a master mix of permeabilization solution for intracellular staining containing fluorochrome-conjugated anti-IFN-␥, anti-MIP-1␤, and anti-TNF-␣ mAbs (see Note 13). 18. Add 25 ␮L permeabilization solution supplemented with an appropriate combination and dilution of fluorochromeconjugated mAbs for intracellular staining. Mix well. Incubate the cells for 30 min at 4◦ C in the dark. 19. Centrifuge the cells at 450×g for 3 min. 20. Discard the supernatant and wash the cells once with 200 ␮L staining solution. 21. Resuspend the cells in 200 ␮L staining solution and transfer the cells to cytometer tubes. 22. Analyze cells on a flow cytometer. The panel is optimized for a CyAn ADP 9 Color Analyzer (see Note 8). However, the staining panel can be adapted to most multi-color flow cytometry instruments with three or four lasers and at least nine fluorescence detectors (see the chapter by Bj¨orkstr¨om et al. for means of adapting staining panels to a four-laser BD LSR II System). 3.2.3. Control Samples for Compensation of Spectral Overlap

1. Either cells or anti-mouse Ig ␬ compensation beads can be used for compensation of spectral overlap among different fluorophores (see Note 14). Cells or compensation beads are plated in a 96-well V-bottomed plate and saturating amounts of fluorochrome-conjugated mAb are added followed by a 30 min incubation at 4◦ C in the dark. Typically, for

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compensation beads, 1␮L of mAb per staining is sufficient. Incubate the cells or beads for 30 min at 4◦ C in the dark. 2. Follow Methods 3.2.1, steps 9–11, for instructions on the washing procedure. 3. Acquire the stained anti-mouse Ig ␬ beads on the flow cytometer instrument used for acquisition of the cellular assay. Identical photo multiplier tube (PMT) voltages should be used for acquisition of PBMC samples and compensation control samples. Acquisition of compensation control samples should typically be performed in the same session as the functional assay. 3.3. Flow Cytometry Compensation and Analysis 3.3.1. Two-Hour Degranulation Assay

1. Compensation matrixes can be generated automatically in FlowJo after acquisition of data, or manually with CellQuest software upon acquisition on a FACS Calibur instrument. For a more extensive discussion of compensation for multiparameter flow cytometry, we refer the reader to previously published guidelines on standard operating procedures (13). 2. After compensation of all samples, the cell population of interest is identified and data are acquired on parameters of interest. In this example, the functional response of CD56dim NK cells has been assessed. Figure 23.1A shows a schematic representation of a gating strategy. Briefly, create a “lymphocyte gate” on a forward scatter/side scatter plot. Apply the “lymphocyte gate” to all samples. Thereafter, gate on CD56+ CD3− cells on an anti-CD3/anti-CD56 plot. Apply this “NK cell gate” to all samples. 3. To quantify the percentage of degranulating cells, gate on CD107a+ cells on a histogram displaying anti-CD107a staining from a sample of effector cells with no target cells added. Without target cells, the frequency of CD107a+ NK cells should be less than 1%. Figure 23.1B exemplifies NK cell degranulation after stimulation with different target cells.

3.3.2. Six-Hour Degranulation Assay

1. For generation of a compensation matrix, software-based compensation is recommended for multi-color experiments, here performed with FlowJo [see (11) for a discussion on how selection of fluorochromes can influence degree of compensation]. 2. Following compensation of all samples, the cell population of interest is identified. In this example, the functional response of CD56dim NK cells has been assessed. Figure 23.2A provides a schematic representation of a gating strategy. Briefly, create a “lymphocyte gate” on a forward scatter/side scatter plot. Apply the “lymphocyte gate” to all

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Fig. 23.1. Analysis of human NK cell degranulation by flow cytometric analysis. PBMCs were incubated with target cells for 2 h at 37◦ C and surface stained with fluorochromeconjugated anti-CD3, anti-CD56, and anti-CD107a mAbs. (A) Profiles demonstrate the gating strategy [side scatter (SSC) versus forward scatter (FCS) and CD56 versus CD3] for identification of CD3– CD56+ NK cells with FlowJo software. (B) Profiles show CD56 versus CD107a staining on CD3– CD56+ NK cells for one representative donor after incubation of PBMC with indicated target cells.

samples. Exclude cells adhering to each other by creating a gate on single cells on a FSC Lin/FSC Area plot. Further, eliminate dead cells and cells that potentially may bind mAbs nonspecifically by gating on LIVE/DEAD Cell Stain− CD14− CD19− cells. Finally, gate on CD56dim CD3− cells on an anti-CD3/anti-CD56 plot. Apply this “CD56dim NK cell gate” to all samples. Alternatively, by different gating strategies CD56bright CD3− or CD56+ CD3− NK cell can be assessed. 3. To quantify the percentage of differentially responding CD56dim NK cells, a preferable method to identify the 16 (24 ) possible combinations generated with the four markers is to apply a Boolean gating strategy by identifying the positive population for each of the molecules/receptors of interest. Figure 23.2B exemplifies CD56dim NK cell responses after stimulation with K562 cells. An algorithm in FlowJo

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Fig. 23.2. Analysis of multiple human NK cell responses by flow cytometric analysis. PBMCs were incubated with target cells for 6 h at 37◦ C, surface stained with fluorochrome-conjugated anti-CD3, anti-CD14, anti-CD19, anti-CD56, and antiCD107a mAbs, followed by intracellular staining with fluorochrome-conjugated anti-IFN-␥, anti-MIP-1␤, and anti-TNF-␣ mAbs. (A) Gating strategy for identification of CD3− CD56dim NK cells. (B) Profiles show staining for multiple responses on CD3− CD56dim NK cells from one representative donor after incubation with target cells as indicated for 6 h at 37◦ C. One representative donor is shown. (C) Pies represent the distribution of cells responding with different numbers of distinct responses, as indicated. Surrounding the pies, lines indicate the nature of individual responses.

allows for automatic generation of the different subpopulations of interest. 4. The Boolean gating strategy presented above simplifies analysis of raw data. Software such as SPICE can be used for processing, organizing, and visualizing data from Boolean analysis. Figure 23.2C provides an example of presentation of data with SPICE (see Note 15). SPICE enables the investigator to navigate through complex data sets allowing simplified data interpretation and presentation (14).

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4. Notes 1. Vials for the collection of blood must contain an anticoagulant. Sodium heparin vials are recommended for collecting blood samples to be analyzed for cellular function. Other anticoagulants such as EDTA and citrate deplete intracellular calcium stores. Therefore, cells collected in sample vials with such anticoagulants may display functional hyporesponsiveness, unless the cells are sufficiently rested in culture medium to replenish intracellular calcium stores. If the samples require transport to a laboratory for diagnostic evaluation, we recommend shipping them at room temperature. Furthermore, for obtaining comparable results of cellular responses from shipped blood relative to results of cells isolated directly from fresh blood, the duration from sample acquisition to isolation of PBMCs should not exceed 24 h. 2. The assays presented in this chapter are designed for analysis of human NK cells in PBMC populations. However, the assays are applicable to, and similar results are obtained with, freshly isolated, purified NK cells. To exclude the possibility of NK cell cross talk with other cells that might confound interpretation of data on NK cell responses, investigators may wish to use purified NK cells in certain experimental settings. The use of purified NK cells also eliminates the need to stain with anti-CD3 mAbs. Thus, in the place of anti-CD3 mAbs, mAbs facilitating analysis of other parameters of NK cell responses or phenotype can be added to the panel when purified NK cells are used as effectors. Moreover, these assays are applicable to frozen PBMC samples, the use of which may reduce inter-assay variation when performing a clinical investigation of different patient cohorts. For consistent results, we recommend that frozen PBMC samples be thawed 1 day prior to stimulation and rested overnight in culture medium. Efficient expression of DNA delivered into primary NK cells is very difficult to achieve. Similarly, expression knockdown with siRNA is inefficient. Therefore, to study mechanisms of NK cell activation, many investigators use NK celllike cell lines, which are more receptive to DNA or RNA delivery. Unfortunately, we have not been able to obtain satisfactory results with the anti-CD107a-based degranulation assay using NK cell lines NK92, NKL, or YTS. Instead, assays quantifying release of secretory lysosome contents, such as perforin and granzyme, can be used to assess the response of cell lines.

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3. In addition to experiments addressing basic questions of NK cell biology, these assays are applicable to several clinical settings. In regards to diagnosis of immunodeficiencies, we routinely evaluate NK cell function based on their responses to the prototypical NK cell-sensitive cell line K562, in addition to redirected antibody-dependent responses with P815 cells and anti-CD16 mAbs. As outlined in this chapter, anti-CD3 mAbs can also be used to evaluate the function of effector T cells. CD8+ effector T cells are the predominant T-cell subset that expresses surface CD107a in response to anti-CD3 stimulation (10). In addition, subsets of CD16+ CD8+ effector T cells may also respond to Fc receptor stimulation (10). Thus, the capacity of NK cells and T cells to respond to triggers of natural, Fc receptor-dependent, and T-cell receptor-dependent degranulation can be quantified with the suggested assays. In the foreseeable future, tumor cells from cancer patients might be evaluated for susceptibility to autologous or allogeneic NK cells in clinical settings (15, 16). The assays presented here could provide useful tools to evaluate tumor cell recognition and to assess the potential efficacy of various NK cell-mediated immunotherapies in individual patients. 4. Upon stimulation of cytotoxic cells by sensitive target cells, secretory lysosomes rapidly fuse with the plasma membrane, as detected by CD107a surface expression (7, 8, 17). With freshly isolated NK cells, CD107a surface expression typically peaks at 1–2 h after mixing NK cells with sensitive target cells (8). 5. A number of transmembrane proteins are confined to secretory lysosomes and appear at the cell surface upon secretory lysosome exocytosis. These proteins include CD63, CD107a, CD107b, and FasL. Comparing intracellular staining of a panel of fluorochrome-conjugated mAbs to CD63, CD107a, and CD107b, we find that anti-CD107a mAbs provide the highest staining intensity (Fig. 23.3A). Both CD107a and CD107b expression correlates with intracellular expression of perforin and granzymes, whereas CD63 expression does not (10). In accord with the intracellular staining intensities, surface staining for CD107a provides the most sensitive marker for NK cell degranulation (Fig. 23.3B and C). To enhance the sensitivity for detection of degranulating cells, mAbs to different secretory lysosome proteins may be combined (18, 19). However, in diagnostic settings, combining mAbs for assessment of cytotoxic lymphocyte degranulation capacity may confound results. Tentatively, a mutation that selectively affects the binding of

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Fig. 23.3. Comparison of different lysosomal markers for the identification of degranulating NK cells. (A) PBMCs were surface stained with fluorochrome-conjugated anti-CD3 and anti-CD56 mAbs, followed by intracellular staining with fluorochrome-conjugated anti-CD63, anti-CD107a, anti-CD107b, anti-perforin, or anti-granzyme A mAbs, as indicated. Profiles show CD56 versus intracellular lysosomal protein staining, as indicated on CD3− CD56+ NK cells. PBMCs were incubated either alone (B) or with K562 cells (C) for 2 h at 37◦ C, surface stained with fluorochrome-conjugated anti-CD3 and anti-CD56 mAbs, in addition to anti-CD63, anti-CD107a, or anti-CD107b mAbs, as indicated. Profiles show CD56 versus surface staining of lysosomal proteins, as indicated on CD3− CD56+ NK cells.

one mAb epitope can be misinterpreted as a diminished capacity for degranulation. To avoid such misinterpretations and unequivocally ascertain degranulation deficits in patients, we recommend using anti-CD107a mAbs exclusively for assessment of degranulation and supplementing such assays with intracellular staining of CD107a to verify the presence of the CD107a epitope. 6. CD107a, once exposed on the cell surface after degranulation, becomes internalized by endocytosis. A strategy to enhance the sensitivity for detection of degranulating cells is to include fluorochrome-conjugated anti-CD107a mAb during the stimulation assay, allowing internalization of complexes of CD107a and fluorochrome-conjugated mAb (7, 19). To prevent degradation of internalized CD107a, monensin can be added to such assays (7, 19). Monensin is a polyether ionophore that blocks the acidification of endocytic vesicles. Arguably, in experiments studying NK cell function, it is desirable to avoid the use of such ionophores, which may perturb cellular signaling and function. Moreover, in assays lasting 2 h or less, the internalization of surface expressed CD107a on resting NK cells is negligible (8). 7. Investigators are advised to titrate each fluorochromeconjugated mAb for optimal staining. The most desirable

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mAb concentration is the one that provides the brightest signal of a positive subset together with the dimmest background signal of the negative subset. The titration procedure is to be repeated for each new batch of fluorochromeconjugated mAb. Prepare the mAbs (as listed in Tables 23.1 and 23.2) by mixing them in a total volume of 50 ␮L of staining solution for each sample to be stained. This can, if necessary, be prepared the day before. To limit interexperimental variability, a master mix covering several days of experiments can be prepared. Store the mAb master mix at 4◦ C in the dark until usage, and for a maximum of 5 days. 8. For analysis on a FACS Calibur, the suggested mAb panel has one available fluorescence channel. To assess degranulation by CD8+ T cells, APC-conjugated anti-CD8 mAb can be added to the panel. Alternatively, this channel can be used for markers that discriminate NK cell subsets, such as inhibitory receptors that can influence the education of human NK cells (20), or mAbs that quantify functional parameters, such as MIP-1␤ that can be detected at early time points after stimulation of NK cells. 9. We recommend acquiring samples the same day as running the cell stimulation and staining experiments. However, samples may be fixed in staining solution supplemented with 1% paraformaldehyde and acquired at later time points. 10. Flow cytometric detection of cellular cytokine production is enhanced by inhibitors of the constitutive pathway for secretion of newly synthesized proteins. Brefeldin A is an inhibitor of the guanine exchange activity of ADP-ribosylation factor (ARF) proteins and interferes with anterograde protein transport from the endoplasmic reticulum to the Golgi apparatus. Thus, addition of Brefeldin A retains cytokines within the responding cells, facilitating sensitive detection of cytokine-producing cells. 11. As previously alluded to in Note 4, the duration of the assay is critical in determining the frequency of cells responsive to distinct functional parameters. Whereas CD107a surface expression and MIP-1␤ synthesis are rapidly induced upon NK cell activation, the synthesis of TNF-␣ and particularly IFN-␥ is protracted. Therefore, for analysis of IFN-␥ by NK cells, we recommend at least 6 h of stimulation. For production of IFN-␥ induced by exogenous cytokines, secretion cannot be detected until more than 12 h after stimulation (Y.T.B, unpublished observation).

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12. Dead cells can nonspecifically bind mAb conjugates, leading to erroneous conclusions. Recently developed aminereactive viability dyes are useful dead cell exclusion markers, as they reproducibly identify dead cells even after subsequent fixation and permeabilization of the cells (21). Dissolve the LIVE/DEAD Cell Stain in 500 ␮L of dehydrated DMSO. Aliquot and store at –20◦ C. Dilute the aliquoted LIVE/DEAD Cell Stain at 1:200 in staining solution prior to cell staining. LIVE/DEAD Cell Stain aliquots can be repeatedly freeze–thawed four times. The LIVE/DEAD Cell Stain must be protected from light. 13. For analysis on a CyAn ADP 9 Color Analyzer, the suggested mAb panel has two available fluorescence channels, which can be used for antibodies conjugated to the fluorochromes PerCP and APC. These are commonly used fluorochromes and a variety of mAbs to different cellular markers that are directly conjugated to these fluorochromes are available. Thus, these channels can be used for markers that discriminate NK cell subsets, such as inhibitory receptors, markers of cellular activation or senescence, or markers that provide measures of additional functional parameters. Alternatively, these channels can be used to discriminate other lymphocyte subsets, e.g., with fluorochrome-conjugated anti-CD8 to identify cytotoxic T lymphocytes, so that the responses of such subsets can be assessed concurrent with the responses of NK cells. 14. Compensation for spectral overlap among different fluorophores is required for analysis of any multi-color flow cytometry experiment. Controls should encompass single staining of all fluorochromes used, in addition to stainings where all aside from one fluorochrome is included. The signal intensity of compensation controls should be at least as high as for the signals obtained with cellular stainings. Ideally, the compensation controls comprise two equal populations of stained and unstained particles. We recommend using anti-mouse Ig ␬ compensation beads for spectral compensation staining that includes an equal number of positive and negative beads. For a more extensive discussion of compensation for multi-parameter flow cytometry, we refer the reader to previously published standard operating procedures (13). 15. Considerable donor variation in NK cell responses is to be expected, both in terms of the magnitude and the kinetics of responses. We have also noted that the relationship among responses may vary among donors. In most donors (approximately 75%), after 6 h of stimulation, IFN-␥ expression is confined to a subset of CD107a+ cells,

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Fig. 23.4. Donor variation in NK cell responses. NK cells were stimulated with (A) K562 cells or (B) P815 cells supplemented with anti-CD16 mAb and incubated for 6 h at 37◦ C. Profiles show CD107a versus IFN-␥ staining on CD3−CD56dim NK cells from two different donors.

whereas in some donors IFN-␥ expression does not correlate with CD107a surface expression (Fig. 23.4). Similar variations in TNF-␣ responses are also observed (J.M.N. and Y.T.B., unpublished observations). Such variations in different cellular responses could be attributed to previous stimulation of cells in vivo. Cells that have been subject to inflammatory stimuli may produce cytokines more readily upon ex vivo stimulation.

Acknowledgments Our research is supported by the Swedish Foundation for Strategic Research, Research Council, and Cancer Society (to H.-G.L.) and the Intramural Research Program of the NIH, NIAID (to E.O.L.). Y.T.B is supported by a grant from Mary Beve’s Foundation. References 1. Vivier, E., Tomasello, E., Baratin, M., Walzer, T., and Ugolini, S. Functions of natural killer cells. (2008) Nat Immunol 9, 503–10. 2. Raulet, D. H. Interplay of natural killer cells and their receptors with the adaptive immune response. (2004) Nat Immunol 5, 996–1002. 3. Strowig, T., Brilot, F., and Munz, C. Noncytotoxic functions of NK cells: direct pathogen

restriction and assistance to adaptive immunity. (2008) J Immunol 180, 7785–91. 4. Bryceson, Y. T., and Long, E. O. Line of attack: NK cell specificity and integration of signals. (2008) Curr Opin Immunol 20, 344–352. 5. Peters, P. J., Borst, J., Oorschot, V., Fukuda, M., Krahenbuhl, O., Tschopp, J., Slot, J. W., and Geuze, H. J. Cytotoxic T lymphocyte granules are secretory lysosomes, containing

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Bryceson et al. both perforin and granzymes. (1991) J Exp Med 173, 1099–109. Bossi, G., and Griffiths, G. M. Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells. (1999) Nat Med 5, 90–6. Alter, G., Malenfant, J. M., and Altfeld, M. CD107a as a functional marker for the identification of natural killer cell activity. (2004) J Immunol Methods 294, 15–22. Bryceson, Y. T., March, M. E., Barber, D. F., Ljunggren, H. G., and Long, E. O. Cytolytic granule polarization and degranulation controlled by different receptors in resting NK cells. (2005) J Exp Med 202, 1001–12. Marcenaro, S., Gallo, F., Martini, S., Santoro, A., Griffiths, G. M., Arico, M., Moretta, L., and Pende, D. Analysis of natural killer-cell function in familial hemophagocytic lymphohistiocytosis (FHL): defective CD107a surface expression heralds Munc134 defect and discriminates between genetic subtypes of the disease. (2006) Blood 108, 2316–23. Bryceson, Y. T., Rudd, E., Zheng, C., Edner, J., Ma, D., Wood, S. M., Bechensteen, A. G., Boelens, J. J., Celkan, T., Farah, R. A., Hultenby, K., Winiarski, J., Roche, P. A., Nordenskjold, M., Henter, J. I., Long, E. O., and Ljunggren, H. G. Defective cytotoxic lymphocyte degranulation in syntaxin-11 deficient familial hemophagocytic lymphohistiocytosis 4 (FHL4) patients. (2007) Blood 110, 1906–15. Gonzalez, V. D., Bj¨orkstr¨om, N. K., Malmberg, K. J., Moll, M., Kuylenstierna, C., Micha¨elsson, J., Ljunggren, H. G., and Sandberg, J. K. Application of nine-color flow cytometry for detailed studies of the phenotypic complexity and functional heterogeneity of human lymphocyte subsets. (2008) J Immunol Methods 330, 64–74. Rudd, E., Bryceson, Y. T., Zheng, C., Edner, J., Wood, S. M., Ramme, K., Gavhed, S., Gurgey, A., Hellebostad, M., Bechensteen, A., Ljunggren, H. G., Fadeel, B., Nordenskjold, M., and Henter, J. I. Spectrum, and clinical and functional

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implications of UNC13D mutations in familial haemophagocytic lymphohistiocytosis. (2008) J Med Genet 45, 134–41. Roederer, M. Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. (2001) Cytometry 45, 194–205. Lamoreaux, L., Roederer, M., and Koup, R. Intracellular cytokine optimization and standard operating procedure. (2006) Nat Protoc 1, 1507–16. Bryceson, Y. T., March, M. E., Ljunggren, H. G., and Long, E. O. Activation, coactivation, and costimulation of resting human natural killer cells. (2006) Immunol Rev 214, 73–91. Ljunggren, H. G., and Malmberg, K. J. Prospects for the use of NK cells in immunotherapy of human cancer. (2007) Nat Rev Immunol 7, 329–39. Betts, M. R., Brenchley, J. M., Price, D. A., De Rosa, S. C., Douek, D. C., Roederer, M., and Koup, R. A. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. (2003) J Immunol Methods 281, 65–78. Enders, A., Zieger, B., Schwarz, K., Yoshimi, A., Speckmann, C., Knoepfle, E. M., Kontny, U., Muller, C., Nurden, A., Rohr, J., Henschen, M., Pannicke, U., Niemeyer, C., Nurden, P., and Ehl, S. Lethal hemophagocytic lymphohistiocytosis in Hermansky-Pudlak syndrome type II. (2006) Blood 108, 81–7. Andre, P., and Anfossi, N. Clinical analysis of human natural killer cells. (2008) Methods Mol Biol 415, 291–300. Anfossi, N., Andre, P., Guia, S., Falk, C. S., Roetynck, S., Stewart, C. A., Breso, V., Frassati, C., Reviron, D., Middleton, D., Romagne, F., Ugolini, S., and Vivier, E. Human NK cell education by inhibitory receptors for MHC class I. (2006) Immunity 25, 331–42. Perfetto, S. P., Chattopadhyay, P. K., Lamoreaux, L., Nguyen, R., Ambrozak, D., Koup, R. A., and Roederer, M. Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry. (2006) J Immunol Methods 313, 199–208.

Chapter 24 Analysis of the KIR Repertoire in Human NK Cells by Flow Cytometry ¨ ¨ Cyril Fauriat, Yenan T. Bryceson, Niklas K. Bjorkstr om, Johan K. Sandberg, Hans-Gustaf Ljunggren, and Karl-Johan Malmberg Abstract Human natural killer (NK) cells are regulated by a diverse receptor repertoire. This makes multi-color flow cytometry-based approaches highly attractive for detailed phenotypical evaluation of NK cells. Several functional parameters can also be evaluated using this technology. In the present chapter, we demonstrate the applicability of this technology for the analysis of the human killer cell Ig-like receptor (KIR) repertoire. We present an antibody panel allowing simultaneous assessment of the four major inhibitory KIRs and NKG2A. We further provide guidance on how to apply standard operating procedures to multi-color flow cytometry experiments. Finally, we discuss possibilities as well as limitations with the application of multi-color flow cytometry techniques to future studies of human NK cells. Key words: Human, natural killer cells, immunophenotyping, multi-color flow cytometry, killer cell immunoglobulin-like receptors.

1. Introduction Over the past decade an increasing number of receptors and other surface molecules have been defined that regulate the function, and help to discriminate subsets, of human immune cells. Flow cytometry has greatly facilitated our understanding of the immune response serving as a tool to accurately characterize and isolate lymphocytes with defined properties (1). The means to investigate co-expression of multiple phenotypic markers and receptors at a single-cell level has been hampered by limitations in instrumentation and availability of fluorochrome-conjugated reagents for multi-color flow cytometry analysis. Recent progress, K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 24, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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however, has made this technology more user-friendly, and with an expanding number of fluorochrome-conjugated reagents available, the technology has become increasingly valuable in many laboratories. Multi-color flow cytometry approaches are well suited for detailed phenotypic and functional characterization of human natural killer (NK) cells, since these cells are regulated by highly complex receptor repertoires and perform a multitude of effector functions (2, 3). Here, we demonstrate how this technology can be used to analyze the full killer cell Ig-like receptor (KIR) repertoire on human NK cells (4, 5). To make such an analysis possible, we describe a panel of mAbs allowing assessment of the four major inhibitory KIRs and NKG2A (4). Primarily, we discuss this within the context of analyzing human NK cells from KIR haplotype A donors. However, we also provide advice on how to address similar questions in KIR haplotype B donors. We discuss the possibilities and limitations associated with the current technology. Furthermore, we emphasize throughout the chapter the need to follow standard operating procedures for multicolor flow cytometry. Finally, we discuss the feasibility of moving beyond present standard eight-color flow cytometry for the detailed phenotypical and functional assessment of highly defined subsets of human NK cells.

2. Materials 2.1. Media and Solutions

1. Culture medium: RPMI-1640 supplemented with 10% heatinactivated (45 min, 56◦ C) fetal bovine serum (FBS) and 1 mM L-glutamine (all Invitrogen). 2. Staining solution: Phosphate-buffered saline (PBS) with 2% heat-inactivated FBS and 2 mM ethylenediaminetetraacetic acid (EDTA). 3. Fixation solution: PBS with 2% (w/v) formaldehyde (Sigma).

2.2. Antibodies and Fluorescent Reagents

1. Anti-CD56 (clone NCAM 16.2) PE-Cy7 (BD Bioscience). 2. Anti-CD3 (clone UCHT1) Cascade Yellow (Dako).

2.2.1. Lineage mAbs and Dead Cell Exclusion Markers

3. Anti-CD14 (clone MP3) APC-Cy7 (BD Bioscience).

2.2.2. Anti-KIR and Anti-NKG2A mAbs

1. Anti-KIR2DL1/S1 (clone EB6) PE (Beckman Coulter).

4. Anti-CD19 (clone SJ25C1) APC-Cy7 (BD Bioscience). 5. LIVE/DEAD Fixable Far Red Dead Cell Stain Kit (Invitrogen).

2. Anti-KIR2DL2/3/S2 (clone GL183) APC (Beckman Coulter).

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3. Anti-KIR3DL1 (clone DX9) FITC (BD Bioscience). 4. Anti-KIR3DL2 (clone DX31) purified (hybridoma kindly provided by Dr. J. Phillips, DNAX Research Institute, Palo Alto, CA, USA) (see Note 1). 5. Anti-NKG2A (clone Z199) purified (Beckman Coulter). 1. Qdot 605 Antibody Conjugation Kit (Invitrogen) to label purified anti-KIR3DL2 (clone DX31).

2.2.3. Fluorescent Labeling of mAbs

2. Pacific Blue Monoclonal Antibody Labeling Kit (Invitrogen) to label purified anti-NKG2A (clone Z199). 2.3. Flow Cytometry Hardware and Software

1. The eight-color flow cytometry staining panel outlined in this chapter is optimized for a CyAn ADP 9 Color Analyzer (Beckman Coulter) equipped with a 25 mW 405 nm laser, a 20 mW 488 nm laser, and a 25 mW 635 nm laser (see Table 24.1 for a detailed description of filter setup and detectors utilized) (6). The staining panel can easily be adapted to most multi-color flow cytometry instruments with three or four lasers and at least nine fluorescent detectors (see Note 2 for a description on how to use the staining panel on a four-laser BD LSR II System).

Table 24.1 Instrument configuration Laser (nm)

Detector

Filter (nm)

Fluorochrome

Utilized panel

Alternative panel

488

FL1

530/40

FITC

KIR3DL1

Availableb

488

FL2

575/25

PE

KIR2DL1

Available

488

FL3

613/20

Qdot 605

KIR3DL2

KIR3DL2

488

FL4

680/30

PerCP

Available

KIR2DL1

488

FL5

750LP

PE-Cy7

CD56

CD56

405

FL6

450/50

PacBa

NKG2A

NKG2A

405

FL7

550/30

CasY

CD3

CD3/DCM

635

FL8

665/20

APC

KIR2DL3

KIR2DL3

635

FL9

750LP

APC-Cy7

CD14/19/DCM

KIR3DL1

a PacB,

Pacific Blue; CasY, Cascade Yellow, DCM, dead cell marker. channel for other mAbs of interest.

b Available

2. FlowJo software version 8.7 (TreeStar) for analysis of acquired raw data. 3. Simplified Presentation of Incredibly Complex Evaluations (SPICE) software version 4.1.6 (courtesy of Mario Roederer, Vaccine Research Center, NIAID, NIH) for processing and presentation of analyzed raw data.

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2.4. Other Material

1. Ficoll-Na metrizoate density gradient (1.007 g/mL), such as Ficoll-Hypaque (GE Healthcare Biosciences AB) stored in the dark at room temperature. 2. Anti-mouse IgG␬ beads (BD Biosciences). 3. Standard kit for isolation of genomic DNA from whole blood, buffy coats, tissue, or purified PBMC. Here we have used DNeasy Blood & Tissue Kit (Qiagen). 4. KIR genotyping kit (Olerup-SSP, Qiagen or Invitrogen). 5. Optional: KIR HLA ligand kit (Olerup-SSP, Qiagen), HLAA low-resolution kit (Olerup-SSP, Qiagen).

3. Methods 3.1. Preparation of Human NK Cells and Donor Selection

1. Freshly isolated PBMC or purified NK cells from PBMC, propagated in culture medium, can be used in the procedures described below. The protocol is also applicable for the analysis of cryo-preserved PBMC/purified NK cells. Detailed instructions on how to purify PBMC and NK cells have been published in a previous issue of this series (7). 2. To identify haplotype A KIR donors, it is necessary to isolate genomic DNA and determine the presence of inhibitory and activating KIR genes with a KIR typing kit. Standard kits for isolation of genomic DNA and KIR typing kit contain all necessary reagents, including detailed protocols and troubleshooting information. To minimize the number of performed genotypings in the screening for haplotype A donors, it is possible to perform a FACS-based prescreening with a standard fourcolor staining including antibodies against CD56, CD3, KIR2DL2/3/S2 (clone GL183), and KIR2DL3 (clone 180701). Haplotype A KIR donors uniformly lack the KIR2DL2 and KIR2DS2 genes and will consequently stain double positive with the two KIR mAbs (see Fig. 24.1) (8) (http://www.ebi.ac.uk/ipd/kir/introduction.html) (see Notes 3 and 4). 3. (Optional) Determine the presence of ligands to the KIR genes using genomic DNA with the KIR HLA ligand kit and the HLA-A low-resolution kit according to the manufacturer’s instructions (see Note 5).

3.2. Eight-Color Flow Cytometry Staining 3.2.1. Preparative Work: mAb Conjugations (see Note 6)

Purified anti-KIR3DL2 (clone DX31) and anti-NKG2A (clone Z199) mAbs need to be fluorescently labeled with Qdot 605 and Pacific Blue, respectively, according to the manufacturer’s instructions.

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Fig. 24.1. Screening for potential KIR haplotype A donors by flow cytometry (see Methods 3.1.2).

3.2.2. Flow Cytometry Staining (see Note 7)

1. Prepare the mAbs (Table 24.1) by mixing them in a total volume of 50 ␮L of staining solution for each sample to be stained (see Note 8). 2. Plate cells in 96-well V-bottomed plates. For staining of PBMC samples, 1×106 cells/well is recommended. 3. Centrifuge the plate at 450×g for 3 min and discard the supernatant. 4. Add the mAb mix (50 ␮L) to the samples and incubate for 30 min on ice in the dark. 5. Add 150 ␮L of staining buffer to the wells and resuspend. 6. Centrifuge the plate at 450×g for 3 min and discard the supernatant. 7. Add 200 ␮L of staining buffer to the wells and resuspend. 8. Centrifuge the plate at 450×g for 3 min and discard the supernatant. 9. Resuspend samples in 150 ␮L of fixation solution and incubate for 10 min in the dark at 4◦ C. Keep the plate at 4◦ C in the dark until analysis. Sample acquisition during the same day is recommended but not absolutely necessary depending on the stability of fluorochromes used. 10. Acquire samples on a desired flow cytometer instrument, in this case the CyAn ADP Analyzer. In order to later perform a multi-parametric analysis simultaneously assessing five heterogeneously expressed surface markers on the NK cells, we recommend the acquisition of at least 50,000 CD56dim NK cells on the instrument (see Methods 3.4.2 for comments on events collected in relation to feasibility to perform multi-parametric assessments).

3.3. Eight-Color Flow Cytometry Compensation

1. Compensation for spectral overlap is a requirement for analysis of any multi-color experiment. In this chapter, FlowJo

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version 8.7 was used to generate compensation matrices. Cells or beads designed to specifically capture mAb may be used. Single-stained and one unstained bead sample should be included. Compensation controls should be at least as bright as the stained sample. Identical PMT voltages for PBMC samples and compensation samples should be used. For further details on compensation for multi-parameter flow cytometry, we refer to previously published standard operating procedures (9). 2. Anti-mouse IgG␬ beads are recommended by us as compensation controls and all single stainings include an equal number of positive and negative beads. An alternative option is to use a universal negative bead together with single stainings of positive beads. 3. Anti-mouse IgG␬ beads are plated in a 96-well V-bottomed plate and saturating amounts of mAb are added followed by a 30 min incubation on ice in the dark. Typically, 1 ␮L of mAb per staining is enough. 4. See Methods 3.2.2, steps 5–9, for instructions on washing procedure. 5. Acquire the stained anti-mouse IgG␬ beads on the flow cytometer instrument that was used for acquisition of the NK cells with identical PMT voltages. The acquisition of the beads should typically be performed in the same session as the NK cells. 6. For generation of the final matrix, software-based compensation is recommended for multi-color experiments, here performed with FlowJo version 8.7 (see (6) for a discussion on how selection of fluorochromes can influence the degree of compensation). 3.4. Eight-Color Flow Cytometry Analysis 3.4.1. Analysis of Acquired Raw Data

1. The cell population of interest is identified; in this case CD56dim NK cells. To comply with standard operating procedures, precautions have to be undertaken to eliminate the risk of analyzing inaccurate events (see Fig. 24.2A). Cells adhering to each other are removed by doublet event exclusion [FSC linear (lin; may also be referred to as FSC height) vs. FSC area]. Furthermore, cells with the potential to bind nonspecifically, including CD14+, CD19+, and apoptotic and dead cells, here identified with an amine-reactive dye (dead cell marker, DCM) (10), are excluded from the analysis. 2. After identifying CD56dim NK cells, we now have the option to investigate surface expression of four KIRs and NKG2A on the cells. A preferable method to identify the 32 (25 ) possible combinations generated with these five markers is

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to apply a Boolean gating strategy by identifying the positive population for each of the molecules/receptors of interest (see Fig. 24.2B) (11, 12). An algorithm created in FlowJo enables automatic analysis of the different subpopulations of interest. 3.4.2. Data Processing and Visualization

The amount of data generated by multi-parameter flow cytometry is often extensive. The Boolean gating strategy presented above aids in simplifying the analysis process of raw data. Recently developed software, such as SPICE, are now available for processing, organization, and visualization of data from Boolean analysis. SPICE will enable the user to navigate through complex

Fig. 24.2. Analysis of the KIR repertoire in human NK cells by polychromatic flow cytometry (see Methods 3.4). (A) Gating scheme to identify CD56dim NK cells. Doublet cells were excluded based on FSC area vs. FSC lin parameters, and CD14 and CD19+ events were excluded together with dead cells (DCM). (B) Representative stainings for KIR2DL1, KIR2DL3, KIR3DL1, KIR3DL2, and NKG2A on CD56dim NK cells defining the positive subset used for Boolean analysis. (C) Boolean analysis to illustrate the frequency of the 32 NK cell subsets identified using the gating scheme described above.

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data sets allowing simplified data interpretation and presentation (see SPICE documentation for tutorials, protocols, and troubleshooting information) (13). The Boolean gating analysis generated 32 distinct KIR- and NKG2A-expressing subsets of human CD56dim NK cells, including cells expressing all five receptors as well as those expressing only one or none of the five evaluated markers. The five receptors are listed along the x-axis with each of the possible 32 combinations (Fig. 24.2C). In the current example, where NK cells from a healthy donor were used, the size of the subsets ranged from 0.15% for 2DL1+2DL3+3DL1+3DL2−NKG2A+ cells to 23.9% for NKG2A single positive cells of the total CD56dim NK cell population. In order to accurately analyze rare subpopulations, here exemplified by 2DL1+2DL3+3DL1+3DL2−NKG2A+ cells, the acquisition of at least 50,000 CD56dim NK cells is recommended. In the current example, this yielded an absolute event count ranging from 107 to 14,151 events for the subsets studied. 3.5. Prospects to Move Beyond Eight-Color Flow Cytometry

This chapter has described a method to simultaneously determine the expression pattern of five major inhibitory receptors on NK cells. It is conceivable that a substantial degree of heterogeneity exists within the 32 defined NK cell subsets. Having identified different KIR- and NKG2A-expressing subsets, one may want to extend the analysis to explore their differentiation status, chemokine expression patterns, as well as their functional properties. Thus, the presented panel might serve as a platform for further phenotypical and functional assessments of these 32 visualized NK cell subsets. The CyAn ADP Analyzer has the potential to simultaneously detect emitted light in nine detectors, where eight detectors in the current setup have been dedicated to detecting KIRs, linage markers, and markers for eliminating potentially inaccurate events. Consequently, this offers one free channel to be used for further functional and/or phenotypical evaluation of the distinct subsets (12). An alternative panel (Table 24.1) would allow for the concurrent investigation of two additional markers. This can be achieved by replacing the KIR3DL1 (DX9) mAb in FITC with a KIR3DL1 (DX9) mAb in Alexa Fluor 700 and by biotinylating purified KIR2DL1 for detection with streptavidin-PerCP in order to increase the signaling strength from the weakly emitting PerCP fluorochrome. The usage of an instrument with more detectors would greatly facilitate the proposed analyses because the number of detectors clearly serves as a limiting factor. The relatively scarce current availability of commercially produced anti-KIR mAbs conjugated with other fluorochromes besides FITC, PE, and APC represents a hurdle for the analysis of the human KIR repertoire.

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4. Notes 1. To our knowledge, there are no commercially available antiKIR3DL2 mAbs. If access to this mAB is not granted, another suitable anti-KIR3DL2 mAb is the Q66 clone mAb (14). 2. The BD LSR II System is probably the most widespread instrument for multi-color flow cytometry. A four-laser BD LSR II System with 405, 488, 532, and 635 nm lasers without modifications can be utilized as an instrument for the mAb − fluorochrome panels presented in this chapter. In fact, this instrument has advantages to the CyAn ADP Analyzer offering greater flexibility in mAb panel design as well as high-energy lasers aiding in the emission intensity from weak fluorochromes or molecules/receptors expressed at low levels. With the BD LSR II System, moving the PE-based conjugates (FL2-5, Table 24.1) to detectors on the 532 laser, will yield better separation of these markers (15). We also advise investigators to move the Qdot 605conjugated mAb to a detector on the 405 nm laser line since Qdot fluorochromes are better excited by a 405 nm laser as compared to a 488 nm laser. Further, with additional detectors available both for the 405 nm and for the 635 nm lasers, new fluorochromes can be introduced allowing investigators to move beyond eight-color flow cytometry for detailed studies of highly defined subsets of NK cells (see Methods 3.5 and Note 4). 3. Both inhibitory and activating KIR genes exist. Most available mAbs against KIRs cross-react between inhibitory and activating forms of the receptors, e.g., the EB6 clone binding to inhibitory KIR2DL1 and activating KIR2DS1. In order to perform a stringent analysis, information on which KIR genes are present is considered a prerequisite. Briefly, two groups of human KIR haplotypes exist, group A and group B haplotypes. Donors having the group A KIR haplotype mainly express KIR genes with inhibitory function, whereas individuals with the group B KIR haplotype have variable numbers of both activating and inhibitory KIR genes present (8). 4. The availability of mAbs with a defined specificity for only one inhibitory KIR has improved over the last couple of years. This includes the KIR3DL1 (clone DX9)specific mAb, as well as the KIR2DL1 (clone 143211)- and KIR2DL3 (clone 180701)-specific mAbs from R&D Systems. In theory, this now presents three tentative pairs of

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mAb to be combined for the detailed studies of inhibitory and activating KIRs in haplotype B KIR donors [KIR2DL1 (143211) vs. KIR2DL1/S1 (EB6), KIR2DL3 (180701) vs. KIR2DL2/3/S2 (GL183), and KIR3DL1 (DX9) vs. KIR3DL1/S1 (Z27)]. The panel presented in this chapter encompasses the usage of eight out of the available nine channels on the CyAn ADP Analyzer, thus making it difficult to include additional markers for the specific assessment of inhibitory and activating KIR within a KIR receptor group, i.e., analysis of 3DL1+3DS1− and 3DL1−3DS1+ NK cells (16) (see Methods 3.5 describing options still available). However, the transition to multi-color instruments allowing analysis of more than 10 conjugates should greatly facilitate such analysis. 5. It has recently been shown that NK cells, in order to gain functional competence, need to express at least one inhibitory receptor specific for self MHC class I, a functional maturation process also referred to as licensing, arming, or education (17, 18). Since most donors have subsets of NK cells lacking the expression of inhibitory receptors, and some donors have NK cells expressing inhibitory receptors in the absence of a self MHC class I ligand (18), it is for some types of studies necessary to determine the presence of cognate HLA ligands. Here, we briefly present a rapid method to do this without having to perform a full high-resolution HLA typing (see Methods 3.1.3). 6. Investigators are advised to titrate the mAbs before use in experiments. The optimal concentration is the concentration that gives the brightest specific signal of the positive subset together with the dimmest background signal of the negative subset. The titration procedure is typically to be repeated for each new batch of “in-house” fluorescently conjugated mAb produced. 7. The cellular expression levels of receptors and other cellular proteins vary widely. Fluorochromes can be bright or dim with respect to emitted light. Usually one combines bright fluorochromes with cell surface molecules expressed at low levels and vice versa (6, 19). APC, PE, and Pacific Blue are brightly emitting fluorochromes whereas PerCP and Cascade Yellow are weaker. Tandem dyes, including APC-Cy7 and PE-Cy7, make up good alternatives when expanding the panel. Recent progress has enabled the usage of Qdots that have narrow emission peaks and consequently reduce the need to compensate for spectral overlap (20). The panel presented in this chapter utilized, for instance, a weak fluorochrome (Cascade Yellow) for the detection of CD3, a highly expressed and readily detectable protein. All the

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anti-KIR mAbs were detected with brightly emitting fluorochromes to maximize separation in the described panel (see Table 24.1 and Methods 3.5. for alternative approaches). 8. If a series of experiments are scheduled within a shorter period of time, it is conceivable to prepare a large cocktail of antibodies to minimize inter-experimental variability. Keep the mAb mix at 4◦ C in the dark until usage, but for a maximum of 5 days. References 1. Herzenberg, L. A., and Herzenberg, L. A. (2004) Genetics, FACS, immunology, and redox: a tale of two lives intertwined. Annu Rev Immunol 22, 1–31. 2. Bryceson, Y. T., March, M. E., Ljunggren, H. G., and Long, E. O. (2006) Activation, coactivation, and costimulation of resting human natural killer cells. Immunol Rev 214, 73–91. 3. Lanier, L. L. (2005) NK cell recognition. Annu Rev Immunol 23, 225–274. 4. Fauriat, C., Andersson, S., Bjorklund, A. T., Carlsten, M., Schaffer, M., Bjorkstrom, N. K., Baumann, B. C., Michaelsson, J., Ljunggren, H. G., and Malmberg, K. J. (2008) Estimation of the size of the alloreactive NK cell repertoire: studies in individuals homozygous for the group A KIR haplotype. J Immunol 181, 6010–6019. 5. Yawata, M., Yawata, N., Draghi, M., Partheniou, F., Little, A. M., and Parham, P. (2008) MHC class I-specific inhibitory receptors and their ligands structure diverse human NK-cell repertoires toward a balance of missing self-response. Blood 112, 2369–2380. 6. Gonzalez, V. D., Bj¨orkstr¨om, N. K., Malmberg, K. J., Moll, M., Kuylenstierna, C., Micha¨elsson, J., Ljunggren, H. G., and Sandberg, J. K. (2008) Application of ninecolor flow cytometry for detailed studies of the phenotypic complexity and functional heterogeneity of human lymphocyte subsets. J Immunol Methods 330, 64–74. 7. Bennett, I. M., and Perussia, B. (1996) Purification of peripheral blood natural killer cells. Methods in Molecular Medicine: Hum Cell Cult Protoc 2, 161–177. 8. Uhrberg, M., Valiante, N. M., Shum, B. P., Shilling, H. G., Lienert-Weidenbach, K., Corliss, B., Tyan, D., Lanier, L. L., and Parham, P. (1997) Human diversity in killer cell inhibitory receptor genes. Immunity 7, 753–763.

9. Roederer, M. (2001) Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry 45, 194–205. 10. Perfetto, S., Chattopadhyay, P., Lamoreaux, L., Nguyen, R., Ambrozak, D., Koup, R., and Roederer, M. (2006) Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry. J Immunol Methods 313, 199–208. 11. Betts, M. R., Nason, M. C., West, S. M., De Rosa, S. C., Migueles, S. A., Abraham, J., Lederman, M. M., Benito, J. M., Goepfert, P. A., Connors, M., Roederer, M., and Koup, R. A. (2006) HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells. Blood 107, 4781–4789. 12. Fauriat, C., Andersson, S., Bj¨orklund, A., Carlsten, M., Schaffer, M., Bj¨orkstr¨om, N. K., Baumann, B. C., Micha¨elsson, J., Ljunggren, H. G., and Malmberg, K. J. (2008) J Immunol 181, 6010–6019. 13. Lamoreaux, L., Roederer, M., and Koup, R. (2006) Intracellular cytokine optimization and standard operating procedure. Nat protoc 1, 1507–1516. 14. Pende, D., Marcenaro, S., Falco, M., Martini, S., Bernardo, M. E., Montagna, D., Romeo, E., Cognet, C., Martinetti, M., Maccario, R., Mingari, M. C., Vivier, E., Moretta, L., Locatelli, F., and Moretta, A. (2008) Anti-leukemia activity of alloreactive NK cells in KIR ligand-mismatched haploidentical HSCT for pediatric patients: evaluation of the functional role of activating KIR and redefinition of inhibitory KIR specificity. Blood. 15. Perfetto, S., and Roederer, M. (2007) Increased immunofluorescence sensitivity using 532 nm laser excitation. Cytometry A 71, 73–79. 16. Alter, G., Martin, M. P., Teigen, N., Carr, W. H., Suscovich, T. J., Schneidewind, A., Streeck, H., Waring, M., Meier, A., Brander, C., Lifson, J. D., Allen, T. M.,

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Carrington, M., and Altfeld, M. (2007) Differential natural killer cell-mediated inhibition of HIV-1 replication based on distinct KIR/HLA subtypes. J Exp Med 204, 3027–3036. 17. Kim, S., Poursine-Laurent, J., Truscott, S., Lybarger, L., Song, Y., Yang, L., French, A., Sunwoo, J., Lemieux, S., Hansen, T., and Yokoyama, W. (2005) Licensing of natural killer cells by host major histocompatibility complex class I molecules. Nature 436, 709–713. 18. Anfossi, N., Andr´e, P., Guia, S., Falk, C. S., Roetynck, S., Stewart, C. A., Breso, V., Frassati, C., Reviron, D., Middleton,

D., Romagn´e, F., Ugolini, S., and Vivier, E. (2006) Human NK cell education by inhibitory receptors for MHC class I. Immunity 25, 331–342. 19. Maecker, H., Frey, T., Nomura, L., and Trotter, J. (2004) Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 62, 169–173. 20. Chattopadhyay, P., Price, D., Harper, T., Betts, M., Yu, J., Gostick, E., Perfetto, S., Goepfert, P., Koup, R., De Rosa, S., Bruchez, M., and Roederer, M. (2006) Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nat Med 12, 972–977.

Chapter 25 KIR Genotyping by Multiplex PCR-SSP Smita Kulkarni, Maureen P. Martin, and Mary Carrington Abstract Diversity across KIR haplotypes stems from differences in numbers of inhibitory and activating receptors, as well as allelic polymorphism of individual genes. The KIR locus has undergone large expansions and contractions over time and is believed to be coevolving with genes encoding its HLA class I ligands located within the MHC locus. KIR and HLA compound genotypes have been associated with susceptibility to or protection from infectious, autoimmune, reproductive, and malignant disorders. We describe here a simple and reliable multiplex PCR-SSP (sequence-specific priming) method for relatively rapid and inexpensive genotyping of 15 KIR genes using standard agarose gel electrophoresis. Key words: KIR genotyping, NK cell, KIR haplotypes, multiplex PCR, PCR-SSP.

1. Introduction Natural killer (NK) cells are central to the innate immune system and they represent the first line of defense against bacteria, parasites, viruses, and malignant cells (1). NK cells comprise 5–15% of circulating lymphocytes and are also found in tissues including the liver, peritoneal cavity, and placenta. They can mediate spontaneous killing of infected or transformed target cells and produce immunoregulatory cytokines and chemokines that stimulate the adaptive immune response (2, 3). In addition, they engage in interactions at the maternal−fetal interface that are critical for successful pregnancy (4). NK cell activity is tightly controlled by activating and inhibitory interactions through numerous cell surface receptors. They respond to targets that express aberrant levels of MHC class I, and this involvement of MHC class I molecules in NK cell K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 25, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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recognition of self vs nonself was originally described over two decades ago (5). MHC class I molecules are the essential signature of self that NK cell inhibitory receptors engage in order to avoid NK cell attack of normal cells (5–7). The original ‘missing self’ hypothesis (8) proposed that downregulation of class I expression on target cells leads to spontaneous destruction by NK cells. Current knowledge of NK cell biology suggests that NK cells not only patrol for abnormal cells that lack MHC class I but also for those that over-express ligands for activating receptors such as altered MHC (‘altered self’) and non-MHC ligands (‘induced self’ or ‘nonself’) (9). The killer cell immunoglobulin-like receptors (KIR) are one of the main types of MHC class I-specific receptors utilized by human NK cells, and they are comprised of both activating and inhibitory counterparts that are encoded by highly homologous sequences. The KIR locus maps to chromosome 19q13.4 within the leukocyte receptor complex (LRC) (10). The high sequence similarity of the KIR genes may facilitate the occurrence of nonallelic homologous recombination (NAHR) (11, 12) and probably partially explains expansion and contraction of KIR haplotypes (10). KIR haplotypes have been broadly classified into two groups referred to as A and B, both of which are found in most populations but sometimes at very different frequencies. Haplotype A is invariant in terms of gene content while haplotype B is quite variable (13) (Fig. 25.1). Currently, more than 40 different B haplotypes based on gene content have been described

Fig. 25.1. The leukocyte receptor complex on chromosome 19q13.4. The KIR gene cluster is located within the leukocyte receptor complex. The KIR locus is expanded to show the order of the genes on chromosome 19. Haplotypes A and B are labeled as such. The A haplotype is fixed in terms of gene content, while B haplotype is characterized by variable gene numbers. Black boxes represent inhibitory receptors, hatched boxes represent activating receptors, open boxes represent pseudogenes, and gray boxes represent framework loci present on virtually all haplotypes.

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[summarized in (14)], and further diversity is afforded by allelic polymorphism of individual KIR genes such that it is unlikely that any two randomly selected individuals have identical KIR genotypes (15). The highly polymorphic and genetically unlinked KIR and MHC class I loci appear to have coevolved to give selective advantage to higher order mammals with respect to reproduction and resistance against infections. Thus, it is not surprising that an ever-increasing number of genetic studies have shown that combinations of KIR and HLA confer either protection against or susceptibility to infectious, reproductive, autoimmune, and malignant disorders (16). KIR genotypic variation across populations is also important from an evolutionary standpoint since differences may in part reflect a response to differences in exposure to infectious agents across populations (17). This chapter will describe a method for multiplex KIR genotyping based on our previously published methodology (18). We have used the same primers but in multiplex combinations that can be resolved by gel electrophoresis. This method detects the presence/absence of KIR genes, thus providing KIR gene profiles.

2. Materials 2.1. PCR Amplification

1. Good-quality DNA from tissues or cells isolated using standard protocols at a concentration of 50–100 ng/␮l (see Notes 1 and 2). 2. Oligonucleotide primer mixes containing forward and reverse primers (see Table 25.1 for primer sequences and Table 25.2 for list of primer mixes and primer concentrations). 3. 50 mM MgCl2 . 4. dNTP mix: 25 mM each of dATP, dTTP, dGTP, and dCTP. 5. 10× PCR buffer: 200 mM Tris−HCl, pH 8.4, and 500 mM KCl. 6. Taq DNA polymerase (see Note 4). 7. 384-Well PCR plates. Each sample will require 12 wells, thus allowing genotyping of 32 samples per plate. 8. Multichannel pipettors (8 and 16 channels).

2.2. Gel Electrophoresis

1. Orange G loading buffer: 0.5% Orange G, 20% Ficoll, and 100 mM ethylenediaminetetraacetic acid (EDTA).

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Table 25.1 KIR genotyping primers Primers Sequences

Exon

Size (bp)

2DL1 A

F1

4

146

R1

GGTCCCTGCCAGGTCTTGCG

4

2DL1 B

F2

TGGACCAAGAGTCTGCAGGA

8

R2

TGTTGTCTCCCTAGAAGACG

3’UTR

2DL2 A

F1

CTGGCCCACCCAGGTCG

4

R1

GGACCGATGGAGAAGTTGGCT

4

2DL2 B

F2

GAGGGGGAGGCCCATGAAT

5

R2

TCGAGTTTGACCACTCGTAT

5

F1

CTTCATCGCTGGTGCTG

7

R1

AGGCTCTTGGTCCATTACAA

8

2DL3 B

F2

TCCTTCATCGCTGGTGCTG

7

R2

GGCAGGAGACAACTTTGGATCA

9

2DL4 A

F1

CAGGACAAGCCCTTCTGC

3

R1

CTGGGTGCCGACCACT

3

2DL4 B

F2

ACCTTCGCTTACAGCCCG

5

R2

CCTCACCTGTGACAGAAACAG

5

F1

TTCTGCACAGAGAGGGGAAGTA

4

R1

GGGTCACTGGGAGCTGACAA

4

F2

CGGGCCCCACGGTTT

5

R2

GGTCACTCGAGTTTGACCACTCA

5

F1

TGGCCCACCCAGGTCG

4

Gene 2DL1

2DL2

2DL3

2DL4

2DS2

2DL3 A

2DS2 A 2DS2 B

2DS3

2DS4

2DS5

2DS3 A

GTTGGTCAGATGTCATGTTTGAA

R1

TGAAAACTGATAGGGGGAGTGAGG 4

2DS3 B

F2

CTATGACATGTACCATCTATCCAC

5

R2

AAGCAGTGGGTCACTTGAC

5

2DS4 A

F1

CTGGCCCTCCCAGGTCA

4

R1

TCTGTAGGTTCCTGAAAGGACAG

4

2DS4 B

F2

GTTCAGGCAGGAGAGAAT

5

R2

GTTTGACCACTCGTAGGGGAC

5

F1

TGATGGGGTCTCCAAGGG

4

R1

TCCAGAGGGTCACTGGGC

4

F2

ACAGAGAGGGGACGTTTAACC

4

R2

ATGTCCAGAGGGTCACTGGG

4

2DS5 A 2DS5 B

Comments

∼330 Misses∗ 005, Sees 2DL2∗ 004

173

Misses∗ 004

151 ∼550 Misses∗ 007 ∼800 254 288 175 240

Misses ∗ 00104

242 190 204 197/219 126

Misses ∗ 003

178

(continued)

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Table 25.1 (continued) Gene 3DL1

3DL1 A 3DL1 B

3DL2

3DL2 A 3DL2 B

3DS1

3DL3

DRB1

Comments

F1

CGCTGTGGTGCCTCGA

3

191

Misses ∗ 009

R1

GGTGTGAACCCCGACATG

3

F2

CCCTGGTGAAATCAGGAGAGAG 4

R2

TGTAGGTCCCTGCAAGGGCAA

4

F1

CAAACCCTTCCTGTCTGCCC

3

R1

GTGCCGACCACCCAGTGA

3

CCCATGAACGTAGGCTCCG

5

CACACGCAGGGCAGGG

5

3DS1 A

F1

AGCCTGCAGGGAACAGAAG

8

R1

GCCTGACTGTGGTGCTCG

3’UTR

3DS1 B

F2

CCTGGTGAAATCAGGAGAGAG

4

3DL3 A

2DL5 A

2DP1 A 2DP1 B

2DS1

Size (bp)

F2

2DL5 B 2DP1

Exon

R2

3DL3 B 2DL5

Primers Sequences

2DS1

R2

GTCCCTGCAAGGGCAC

4

F1

GTCAGGACAAGCCCTTCCTC

3

R1

GAGTGTGGGTGTGAACTGCA

3

F2

TTCTGCACAGAGAGGGGATCA

4

R2

GAGCCGACAACTCATAGGGTA

4

F1

GCGCTGTGGTGCCTCG

3

R1

GACCACTCAATGGGGGAGC

3

F2

TGCAGCTCCAGGAGCTCA

5

R2

GGGTCTGACCACTCATAGGGT

5

F1

GTCTGCCTGGCCCAGCT

3

R1

GTGTGAACCCCGACATCTGTAC 3

F2

CCATCGGTCCCATGATGG

R2

CACTGGGAGCTGACAACTGATG 4

F1

CTTCTCCATCAGTCGCATGAA

4

F2

CTTCTCCATCAGTCGCATGAG

4

R1

AGAGGGTCACTGGGAGCTGAC

4

4

186 242

Misses ∗ 013, ∗ 014

130 ∼300 180 232 165 214 191 205 89 102

F1

TGCCAAGTGGAGCACCCAA

intron 3 796

R1

GCATCTTGCTCTGTGCAGAT

Intron 3

Internal control

2. 10× TAE electrophoresis buffer: 400 mM Tris, 200 mM acetic acid, and 10 mM EDTA. 3. 100 bp DNA ladder. 4. Electrophoresis grade agarose. 5. Horizontal electrophoresis chamber.

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Table 25.2 Primer mixes for multiplex PCR Mix #

Primers

Product size (bp)

Primer concentrations (␮M)

1

2DL1 B

330

5

2DL2 A

173

5

2DL2 B

151

4

2 3

4

5 6

7

8 9

2DS3 B

190

5

2DP1 B

89

5

3DL1 A

191

5

3DS1 A

300

5

DRB1

800

4

2DL5 A

214

5

2DL5 B

191

5

2DS1

102

5

2DL3 B

800

5

2DL4 A

254

5

2DL3 A

550

5

2DS3 A

242

5

3DS1 B

180

5

2DS4 A

204

4

3DL1 B

186

5

2DL1 A

146

5

2DS4 B

197/219

5

2DS5 A

126

5

3DL3 A

232

5

2DS5 B

178

5

3DL2 B

130

5

2DP1 A

205

5

2DS2 A

175

5

11

2DS2 A

240

5

12

2DL4 B

288

5

3DL2 A

242

10

3DL3 B

165

5

10

6. Gel casting tray with 50-well combs having teeth appropriately spaced to allow for loading with a multichannel pipettor. The CentipedeTM horizontal model D3–D14 wide gel system (Woburn, MA) will accommodate two combs per

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gel allowing for electrophoresis of eight DNA samples, each with 12 PCR reactions. 7. 10 mg/ml ethidium bromide solution. 8. High-voltage power supply. 9. Photographic means of gel documentation (ultraviolet light source, camera).

3. Methods (KIR Genotyping by Gel Electrophoresis) 3.1. PCR

1. Using a multichannel pipette, dispense 1 ␮l of each primer mix into separate wells of the 384-well PCR plate. Each sample will require 12 wells in a horizontal row (e.g., A1A12) such that 32 samples can be accommodated on one plate. 2. Prepare PCR cocktail of 60 ␮l (enough for 15 wells) for one sample as follows: 150–200 ng DNA, 7.5 ␮l 10× PCR buffer, 2.25 ␮l MgCl2 (final PCR concentration 1.5 mM), 0.6 ␮l dNTP (final PCR concentration 200 ␮M), and 0.375 ␮l Platinum taq polymerase. Add water to make up the volume to 60 ␮l. Mix well by vortexing at low speed. 3. Dispense 4 ␮l of PCR cocktail for each DNA sample to each primer mix in the plate for a total PCR reaction volume of 5 ␮l. 4. Cover the plate with acetate film and centrifuge briefly to pull the contents of the well to the bottom of the plate. 5. The amplification is carried out in a programmable PCR thermal cycler with heated lid to minimize evaporation using the following program: 3 min at 94◦ C, 5 cycles of 15 s at 94◦ C, 15 s at 65◦ C, 30 s at 72◦ C; 21 cycles of 15 s at 94◦ C, 15 s at 60◦ C, 30 s at 72◦ C; 4 cycles of 15 s at 94◦ C, 1 min at 55◦ C, 2 min at 72◦ C followed by a final 7 min extension step at 72◦ C.

3.2. Agarose Gel Electrophoresis

1. For one gel, prepare 150 ml of 3% w/v agarose in 1× TAE by heating until the agarose is completely solubilized. 2. Cool the mixture to 65◦ C and add 5 ␮l ethidium bromide and gently mix to avoid bubble formation. 3. Pour the gel mixture into the gel casting tray and insert the 50-well combs. Allow the gel to solidify (∼20 min).

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4. Submerge the solidified gel in the electrophoresis chamber filled with 800 ml of 1× TAE buffer. Remove the combs. 5. Add 5 ␮l of Orange G loading buffer to each PCR product and centrifuge briefly. 6. Load 2 ␮l of the 100 bp DNA ladder to the first and last well of each row of wells. 7. Using a 16-channel pipettor, load 10 ␮l of each PCR product into the gel. 8. Electrophorese for 70 min at 100 V or until the Orange G has migrated 6 cm. 9. Visualize the gel using a UV light source and photograph the gel for a permanent record (Fig. 25.2).

Fig. 25.2. KIR genotyping by multiplex PCR-SSP and agarose gel electrophoresis. (A) Agarose gel electrophoresis depicting the presence of all the KIR genes tested. (B) Gel electrophoresis results from an individual homozygous for haplotype A. This individual possesses one truncated (197 bp) and one full-length copy (219 bp) of KIR2DS4 as shown by the presence of the appropriately sized bands in primer mix 8. (C) Gel electrophoresis results from an individual homozygous for haplotype B. The order of primer mixes (see Table 25.2) used for (B) and (C) is as shown in (A). (D) Genotyping results for the samples shown in (A–C) (samples 1–3). M = 100 bp molecular weight marker.

3.3. Interpretation of Result

Determination of KIR genotype using this method depends on presence or absence of detectable amplification product for each gene-specific primer pair (see Notes 9–11). It is important to have a good quality gel picture for accurate judgment of the product sizes, as each well contains multiple products of varying size. The most important product is the positive internal control amplicon, which should be present in all the samples. Each KIR gene is amplified using two pairs of specific primers and products should

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be detected for both sets of primers. If the amplification by the two gene-specific primer pairs is discrepant for any gene, the reaction should be repeated. Detectable bands as well as absence of product can be recorded in a spreadsheet such as Microsoft Excel. Some alleles of the KIR2DS4 gene have a 22 bp deletion in exon 5 that can be detected by the KIR2DS4 primers in primer mix 8. The full-length product is 219 bp while the deletion variant is 197 bp (see Fig. 25.2B).

4. Notes 1. The quality of DNA is the most important requirement for reliable PCR-SSP results. DNA extracted from heparinized blood should be avoided, as heparin is a PCR inhibitor (19). 2. A total of 100–200 ng (gel electrophoresis method) DNA is required for reliable amplification using the primer mixes described here. Increasing the number of cycles is likely to increase false-positive reactions. Therefore, if the amplification is not satisfactory because of DNA quality, it is recommended that each primer mix be amplified individually, as described in (18). 3. All primer mixes should be tested with positive and negative controls when new primer mixes are made from the stock primers. 4. Platinum Taq (Invitrogen) polymerase gives the most reliable and consistent results for this protocol. 5. A fast ramping thermal cycler with a heated lid is recommended for best results. The ‘step-down’ program was designed for this protocol to increase specificity of the amplifications. It might be necessary to alter the PCR conditions slightly depending on the PCR machine being used. 6. PCR machines should be checked regularly to ensure that all wells are amplifying uniformly. It is also important to ensure that the PCR plates fit snugly into the machine. 7. The quality of dNTPs is important for reliable amplifications. Avoid repeated freeze−thawing; instead freeze in small aliquots after reconstitution. 8. The highly homologous sequences of the KIR loci can lead to nonspecific amplification especially with increased numbers of cycles (>30) used for PCR. 9. Some primers miss a couple of alleles of a given gene as noted in Table 25.1 and others amplify one allele

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of another gene. Furthermore, there are likely to be yet undiscovered alleles of a gene that might not amplify with the current set of primers and alternatively some of these might cross-react with alleles of other KIR genes. We strongly recommend routine checks of the KIR database (http://www.ebi.ac.uk/ipd/kir) for new alleles which might require designing new primers. It should also be remembered that most of the available KIR gene sequences have been obtained from Caucasians and thus may not include alleles that are prevalent in other populations. This highlights the importance of using more than one pair of primers for amplification of each gene. 10. The gene content of some KIR haplotypes and sequence of some KIR alleles strongly suggest that unequal crossingover in the region accounts for a substantial amount of the diversity at this locus (20–23). This could result in the production of unusual profiles where, for example, one or more of the framework genes are missing or chimeric genes are present. 11. Some of the KIR loci exhibit strong negative and positive linkage disequilibrium between them. For example, some gene pairs such as KIR2DL2/2DS2, KIR2DL1/2DP1, and KIR3DL1/2DS4 are almost always found together across different haplotypes. This information is very useful in validation of the genotyping results.

Acknowledgments This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. References 1. Trinchieri, G. (1989) Biology of natural killer cells. Adv Immunol 47, 187–376. 2. Cooper, M. A., Fehniger, T. A., and Caligiuri, M. A. (2001) The biology of

human natural killer-cell subsets. Trends Immunol 22, 633–40. 3. Nguyen, K. B., Salazar-Mather, T. P., Dalod, M. Y., Van Deusen, J. B., Wei, X. Q.,

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5.

6.

7.

8.

9. 10.

11. 12.

13.

Liew, F. Y., et al. (2002) Coordinated and distinct roles for IFN-alpha beta, IL12, and IL-15 regulation of NK cell responses to viral infection. J Immunol 169, 4279–87. Trowsdale, J., and Moffett, A. (2008) NK receptor interactions with MHC class I molecules in pregnancy. Semin Immunol 6, 317–20. Karre, K., Ljunggren, H. G., Piontek, G., and Kiessling, R. (1986) Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature 319, 675–8. Shimizu, Y., and DeMars, R. (1989) Demonstration by class I gene transfer that reduced susceptibility of human cells to natural killer cell-mediated lysis is inversely correlated with HLA class I antigen expression. Eur J Immunol 19, 447–51. Storkus, W. J., Alexander, J., Payne, J. A., Dawson, J. R., and Cresswell, P. (1989) Reversal of natural killing susceptibility in target cells expressing transfected class I HLA genes. Proc Natl Acad Sci U S A 86, 2361–4. Ljunggren, H. G., and Karre, K. (1990) In search of the  missing self : MHC molecules and NK cell recognition. Immunol Today 11, 237–44. Lanier, L. L. (2005) NK cell recognition. Annu Rev Immunol 23, 225–74. Wilson, M. J., Torkar, M., Haude, A., Milne, S., Jones, T., Sheer, D., et al. (2000) Plasticity in the organization and sequences of human KIR/ILT gene families. Proc Natl Acad Sci U S A 97, 4778–83. Lupski, J. R. (1998) Charcot-Marie-Tooth disease: lessons in genetic mechanisms. Mol Med 4, 3–11. Carrington, M., and Cullen, M. (2004) Justified chauvinism: advances in defining meiotic recombination through sperm typing. Trends Genet 20, 196–205. Uhrberg, M., Valiante, N. M., Shum, B. P., Shilling, H. G., Lienert-Weidenbach, K., Corliss, B., et al. (1997) Human diversity in

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killer cell inhibitory receptor genes. Immunity 7, 753–63. Khakoo, S. I., and Carrington, M. (2006) KIR and disease: a model system or system of models? Immunol Rev 214, 186–201. Shilling, H. G., Young, N., Guethlein, L. A., Cheng, N. W., Gardiner, C. M., Tyan, D., et al. (2002) Genetic control of human NK cell repertoire. J Immunol 169, 239–47. Kulkarni, S., Martin, M. P., and Carrington, M. (2008) The Yin and Yang of HLA and KIR in human disease. Semin Immunol 6, 343–52. Carrington, M., and Martin, M. P. (2006) The impact of variation at the KIR gene cluster on human disease. Curr Top Microbiol Immunol 298, 225–57. Martin, M. P., and Carrington, M. (2008) KIR locus polymorphisms: genotyping and disease association analysis. Methods Mol Biol 415, 49–64. Satsangi, J., Jewell, D. P., Welsh, K., Bunce, M., and Bell, J. I. (1994) Effect of heparin on polymerase chain reaction. Lancet 343, 1509–10. Shilling, H. G., Lienert-Weidenbach, K., Valiante, N. M., Uhrberg, M., and Parham, P. (1998) Evidence for recombination as a mechanism for KIR diversification. Immunogenetics 48, 413–6. Martin, M. P., Bashirova, A., Traherne, J., Trowsdale, J., and Carrington, M. (2003) Cutting edge: expansion of the KIR locus by unequal crossing over. J Immunol 171, 2192–5. Gomez-Lozano, N., Estefania, E., Williams, F., Halfpenny, I., Middleton, D., Solis, R., et al. (2005) The silent KIR3DP1 gene (CD158c) is transcribed and might encode a secreted receptor in a minority of humans, in whom the KIR3DP1, KIR2DL4 and KIR3DL1/KIR3DS1 genes are duplicated. Eur J Immunol 35, 16–24. Shilling, H. G., Lienert-Weidenbach, K., Valiante, N. M., Uhrberg, M., and Parham, P. (1998) Evidence for recombination as a mechanism for KIR diversification. Immunogenetics 48, 413–6.

Chapter 26 Identification and Analysis of Novel Transcripts and Promoters in the Human Killer Cell Immunoglobulin-like Receptor (KIR ) Genes Hongchuan Li, Paul W. Wright, and Stephen K. Anderson Abstract This chapter describes the techniques our lab has used to find the multiple promoters present in individual KIR genes. Our previous studies in the murine Ly49 gene family led us to expect the presence of distal promoters, antisense transcripts, and bi-directional promoters in the KIR gene cluster. We present here all of the techniques used to systematically determine if a gene possesses these types of control elements. Key words: 5 -RACE, 3 -RACE, RPA, reporter assays, antisense transcripts.

1. Introduction NK cells make use of large families of polymorphic receptors for class I MHC to distinguish normal healthy cells from infected or cancerous cells (1). Mice use members of the Ly49 family of lectin-related receptors to recognize class I MHC, whereas humans use the killer cell immunoglobulin-like receptor (KIR) family. These two gene families are highly polymorphic and the number of genes present varies in individual humans and mouse strains (reviewed in (2–4)). The human KIR and mouse Ly49 class I MHC receptors provide an excellent model system for the study of selective activation of gene expression. Individual NK cells select only a subset of the available receptor genes for expression by a seemingly random K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 26, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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process (5–7). The probability of co-expression of two distinct inhibitory receptors is equal to the product of their individual frequencies, and NK cells appear to turn on class I MHC receptors until a self-reactive inhibitory receptor is present (8, 9). Multiple promoters are present within each gene in both the KIR and Ly49 clusters, including bi-directional promoters that are proposed to function as probabilistic switches controlling the probability of gene activation (10–13). In order to gain a complete understanding of the mechanisms involved in the variegated expression of the KIR and Ly49 genes, a large array of molecular techniques are necessary, and several techniques have to be modified to detect rare transcripts and identify the sense and antisense promoters that function during NK cell development. The first step in understanding the control of a gene or a gene cluster is the identification of all transcripts and promoter elements. This can be achieved by RT-PCR analysis and mapping of transcriptional start sites using probes and primers spanning the entire gene. There are two methods that can be used to reliably identify transcriptional start sites. The classic approach is the direct determination of the 5 end of the transcript by RNase protection of a labeled RNA probe spanning an expected site of transcript initiation. The more current approach uses 5 -RACE on RNA that has been de-capped and ligated to a 5 linker, allowing specific cloning of the 5 start site. The drawback of the 5 -RACE approach is the reliance of this technique on subsequent PCR, cloning, and sequencing to identify products. The bias toward cloning smaller products prevents this technique from identifying the major start site of transcription if there are multiple start sites. Once promoter regions have been identified, the use of reporter systems allows the fine mapping of transcriptional control regions into specific enhancer, promoter, and silencer regulatory elements. The two most commonly used reporter systems are based on the chloramphenicol acetyltransferase (CAT) gene and the firefly (Photinus pyralis) luciferase gene (14, 15). The luciferase reporter system is currently the preferred approach. The main advantages of the luciferase system are the nonisotopic assay of bioluminescence, the high degree of sensitivity, the absence of endogenous enzyme activity in eukaryotic cells, a wide linear range of quantitation, the presence of an internal control with the dual-luciferase system, and the ease and speed of performance. In this chapter, we describe the methods used in our laboratory to identify and analyze the multiple KIR promoters that we have studied. Techniques used for the analysis of KIR3DL1 gene promoters in the YT-Indy human NK cell line as compared to the HEK293 embryonic kidney cell line are used as an example. The

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protocols provided in this chapter are easily adapted to the study of other gene families controlled by multiple promoters.

2. Materials 2.1. Identifying KIR Transcripts Initiating Upstream of the Known Promoter

1. Ficoll-Hypaque (Sigma Diagnostics, St. Louis, MO). 2. RNeasy Mini Kit with RNase-Free DNase Set (Qiagen, Valencia, CA). 3. Superscript First-Strand System for RT-PCR (Invitrogen, Carlsbad, CA). 4. Oligonucleotide primers, 10 nM scale, purified by desalting (Operon, Huntsville, AL). Resuspend primers at a final concentration of 100 ␮M. Primers should be 21–24 bp in length and have a melting temperature of greater than 60◦ C. R PCR SuperMix (Invitrogen). 5. Platinum R 6. Polymerase chain reaction (PCR) machine: GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) or equivalent.

7. UltrapureTM Agarose (Invitrogen). 8. UltrapureTM 10× TBE Buffer (Invitrogen). 9. GelRed, nucleic acid gel stain, 10,000× (Biotium, Hayward, CA). 10. StrataClone PCR Cloning Kit with StrataClone SoloPack Competent Cells (Stratagene, La Jolla, CA). 11. LB-agar plates containing 50 ␮g/mL of ampicillin (KD Medical, Columbia, MD). 12. LB Broth (Invitrogen). 13. QuickLyse Miniprep Kit (Qiagen). 14. RNAqueous-4PCR Kit (Applied Biosystems/Ambion, Austin, TX), total cellular RNA isolation system. 2.2. Identification of Antisense KIR Transcripts

2.3. Mapping Transcription Start Sites

1. ThermoscriptTM RT-PCR System (Invitrogen). 2. ChargeSwitch PCR Clean-Up Kit (Invitrogen). R R R 3. TOPO TA Cloning Kit (pCR 2.1-TOPO vector) with R  TM One Shot Mach1 T1 Phage-Resistant Chemically Competent E. coli (Invitrogen).

1. RPA II RNase Protection Kit (Ambion). 2. pCR4-TOPO vector (Invitrogen).

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3. PmeI and FspI restriction England Biolabs, Beverly MA).

enzymes

(New

4. QIAquick Gel Extraction Kit (Qiagen). 5. ␣-32 P-UTP (PerkinElmer, Waltham, MA). 6. MAXIscript in vitro transcription kit (Ambion). 7. 10-Base RNA ladder: DecadeTM Marker System (Ambion). 8. PhosphorImager System (Molecular Dynamics, Sunnyvale, CA). 9. FirstChoice RLM-RACE kit (Ambion). 2.4. Generation of Luciferase Reporter Plasmids

1. QIAamp DNA Blood Mini Kit (Qiagen). 2. Restriction enzymes: SacI, XhoI, and HindIII (New England Biolabs). 3. Luciferase Reporter Vector: pGL3-basic firefly luciferase reporter vector (Promega, Madison, WI). 4. T4 DNA Ligase (Invitrogen). 5. HiSpeed Plasmid Midi Kit (Qiagen). 6. RV3 sequencing primer (Promega).

2.5. Cell Transfection, Electroporation, and Luciferase Assay

1. HEK293 human embryonic kidney cells are cultured in Dulbecco s modified Eagle s medium (DMEM; GIBCO/Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Hyclone, Logan, UT), 100 U/mL each of penicillin and streptomycin antibiotics, sodium pyruvate, and 2 mM L-glutamine (GIBCO/Invitrogen). 2. Internal Control Reporter Vector: pRL-SV40 Renilla luciferase vector (Promega). 3. Fugene (Roche Diagnostics, Indianapolis, IN). R Reporter Assay System (Promega). 4. Dual-Luciferase

5. YT-Indy cells (16) are cultured in RPMI-1640 (GIBCO/Invitrogen) medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 U/mL each of penicillin and streptomycin antibiotics, and 2 mM L -glutamine (GIBCO/Invitrogen). 6. 1× Phosphate-buffered saline (GIBCO/Invitrogen). 7. Electroporation system (pulse generator): BTX ECM-830 electroporator (Harvard Apparatus, Holliston, MA). 8. Sterile electroporation cuvettes with an electrode gap of 0.4 cm: Gene Pulser/MicroPulser Cuvette, 0.4 cm (BioRad, Hercules, CA). 9. Luminometer.

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3. Methods 3.1. Identifying KIR Transcripts Initiating Upstream of the Known Promoter

1. Mononuclear cells are obtained by Ficoll-Hypaque (Sigma Diagnostics) density-gradient centrifugation of peripheral blood from healthy donors. 2. Total RNA is isolated with the RNeasy Mini Kit (Qiagen), including the removal of genomic DNA contamination with the Qiagen RNase-Free DNase Set. 3. Two micrograms of NK cell total RNA is subjected to reverse transcription for 1 h at 42◦ C in a total volume of 20 ␮L with an oligo-dT primer using the Invitrogen SuperscriptII first-strand cDNA synthesis kit. A Parallel reaction lacking reverse transcriptase is performed to control the possible amplification of contaminating DNA in the subsequent PCR reaction. 4. One microliter of the cDNA product is used in a 50 ␮L PCR reaction using Invitrogen PlatinumTM PCR SuperMix (see Note 1), with an exon 4 reverse primer at position +512 relative to the start codon (ATCTGTCCAACGAGGCGTGAG), together with a series of 5 forward primers spaced approximately 200 bp apart, and spanning the entire intergenic region from the start codon to the polyA signal of the previous gene (see Notes 2 and 3). Thirty-five cycles of amplification of 30 s at 94◦ C, 30 s at 56◦ C, and 30 s at 73◦ C are performed and the products are separated on a 0.8% agarose gel cast in 0.5× TBE buffer containing 1× GelRed DNA stain. 5. Two microliters of the PCR reaction is used to clone the products with the StrataClone PCR Cloning Kit, since this system is better suited to the cloning of large PCR products (see Note 4). Two microliters of the cloning reaction is transformed into competent bacteria and 20 ␮L of bacteria are spread on an LB–ampicillin plate. 6. Six individual bacterial clones are picked, grown in 2 mL of LB with 50 ␮g/mL of ampicillin overnight and then the plasmid is purified from 1.5 mL with the Qiagen QuickLyse Miniprep Kit. 7. Plasmid minipreps are sequenced with the m13 reverse and T7 primers, and the presence of spliced KIR exons 1–4 preceded by intergenic sequence indicates that an RNA transcript of intergenic origin has been cloned.

3.2. Identification of Antisense KIR Transcripts

Since antisense transcripts may not produce spliced messenger RNA, it is important to confirm the strand of origin. This can be

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achieved in any of the three ways. The first approach is through the use of a cDNA primer specific for antisense transcripts together with a thermostable reverse transcriptase to increase the specificity of cDNA synthesis by performing a high-temperature reverse transcription reaction. The second approach uses a 3 RACE technique to clone polyadenylated reverse transcripts. The third approach is 5 -RACE with the oligo-capping method. This method has the advantage of identifying the transcription start site which will indicate the 3 boundary of the antisense promoter. The 5 -RACE method is described in Section 3.3.2. 3.2.1. Cloning Antisense Transcripts by High-Temperature Reverse Transcription

1. Total RNA is isolated with the RNeasy Mini Kit (Qiagen), including the removal of genomic DNA contamination with the RNase-Free DNase Set. 2. The Invitrogen ThermoscriptTM RT-PCR System is used to generate cDNA from 2 ␮g of RNA using a primer specific for KIR3DL1 antisense transcripts located 416 nucleotides upstream from the start codon of the KIR3DL1 protein (TGGTTTATTGTCACAATTG). This primer was chosen because it contains the first consensus polyA addition site (AATAAA) on the antisense strand, therefore the antisense transcript is expected to continue to at least this point. 3. Two micrograms of RNA is mixed with 1 ␮L of 10 mM dNTP, 1 ␮L of antisense primer, and adjusted to a final volume of 10 ␮L. 4. The RNA is denatured at 65◦ C for 5 min and then immediately placed at 55◦ C rather than on ice as indicated in the manufacturer s protocol. It is very important that the mix be kept at or above 55◦ C to maintain the specificity of the cDNA synthesis. 5. Add 9 ␮L of a pre-warmed reaction mixture containing 2 ␮L of RT buffer, 2 ␮L of 0.1 M DTT, 4 ␮L of 25 mM MgCl2 , and 1 ␮L of RNaseOUTTM Recombinant RNase Inhibitor. 6. Add 1 ␮L of ThermoScriptTM Reverse Transcriptase (15 U/␮L) and incubate at 55◦ C for 1 h. Set up a duplicate sample without added reverse transcriptase as a negative control. 7. Inactivate the RT enzyme by incubation at 85◦ C for 5 min. 8. Cool the reaction to room temperature and add 1 ␮L of RNaseH to digest RNA. Incubate at 37◦ C for 20 min. 9. Two microliters of the cDNA product is used for PCR with a primer pair spanning the region between the RT primer and the predicted location of the antisense promoter. 10. PCR products are cloned and sequenced as described in Sections 3.1.3–3.1.5.

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1. Generate cDNA from 0.5–2.0 ␮g of NK cell RNA using an oligo-dT primer with an added 5 unique anchor sequence (CTTGACCTCATCTGACCACTCACCTCACTTTTTTTTTTTTTTT; see Note 5). 2. Add 4 ␮L of the cDNA reaction to 50 ␮L of PCR SuperMix and prepare a linear amplification reaction by adding 1 ␮L of a KIR antisense primer (TTCTACCTTGCATGAGGCCCAGTG) downstream of the predicted antisense promoter (see Note 6). 3. Perform 17 cycles of 94◦ C for 20 s, 62◦ C for 20 s, 73◦ C for 5 s, pause PCR machine at 94◦ C, and add 1 ␮L of an outer primer corresponding to the anchor sequence of the oligodT primer for the final three cycles (CTTGACCTCATCTGACCACTC) so that the other strand will be synthesized for next step of linear amplification. 4. Purify product using the ChargeSwitch PCR Clean-Up Kit (Invitrogen). 5. Take 10% of the purified product and perform 20 cycles of linear Amp (94◦ C for 20 s, 62◦ C for 20 s, 73◦ C for 5 s) with a nested antisense KIR primer (GACACCTCGCGTCCTTCACTATGAC). Purify product as described in Step 4. 6. Use 10% of product for PCR with an inner primer corresponding to the anchor sequence (TCATCTGACCACTCACCTCAC) and a third nested antisense primer (CTCAAAACACGTCTCAGATCCAACCTC). Perform PCR for 35 cycles of 94◦ C for 20 s, 58◦ C for 20 s, and 73◦ C for 5 s. 7. Cloning and sequencing of PCR products. The amplified cDNA is cloned into the Invitrogen TOPO TA vector. Clones are isolated and sequenced as described in Section 3.1.4. 8. A polyadenylated antisense transcript should have a consensus polyA signal (AATAAA or ATTAAA) approximately 15 nucleotides prior to the polyA tract. If the beginning of the polyA tract overlaps with an A-rich segment in the genomic sequence and no polyA signal is present, the cloned product is likely an artifact derived from genomic DNA contamination.

3.3. Mapping Transcription Start Sites 3.3.1. Identification of Transcription Start Sites by RNase Protection Assay

1. The RNase protection assay (RPA) is performed using the RPA II Kit (Ambion). The probe region is chosen so that the expected region of transcript initiation is approximately 30–50 nucleotides from the 3 end of the probe. The probe fragment is generated from genomic DNA by PCR and cloned into the pCR4-TOPO vector. Clones containing the

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probe region in both the sense and the antisense orientations are isolated, and plasmid DNA is isolated. 2. Insert containing the flanking T7 polymerase-binding site is obtained by digesting 20 ␮g of plasmid DNA with PmeI and FspI restriction enzymes and separating the fragments on a 1.4% TBE-agarose gel. The probe fragment is visualized under low-intensity UV light, cut from the gel with a clean razor blade, and purified using the QIAquick Gel Extraction Kit (Qiagen). 3. One microgram of insert DNA is used in a 20-␮L labeling reaction containing ␣-32 P-UTP (see Note 7) together with unlabeled ATP, CTP, GTP, and T7 polymerase in transcription buffer provided in the MAXIscript In Vitro Transcription Kit (Ambion). The [␣-32 P]-labeled RNA probe is separated on a 6% denaturing polyacrylamide gel and the full-length probe is excised and eluted by overnight incubation at 37◦ C in gel elution buffer (Ambion). 4. Approximately 1×105 cpm of the gel-purified probe is added to 20 ␮g of total RNA in each reaction mixture. Two additional tubes containing 20 ␮g of yeast RNA and probe should be set up as controls. The probe/target RNA mixes are precipitated by adding ammonium acetate to 0.5 M followed by 2.5 volumes of ethanol. Centrifuge at maximum speed in a refrigerated microcentrifuge for 20 min and carefully remove the supernate. The pellet is dissolved in 20 ␮L of hybridization buffer and incubated at 90◦ C to dissolve and denature the RNA. 5. The hybridization is carried out overnight (>16 h) at 42◦ C. After hybridization, 200 ␮L of RNase digestion buffer containing a 1:100 dilution of RNase A/RNase T1 enzyme is added to each tube. Digestion buffer without enzyme is added to one of the two tubes containing probe and yeast RNA as a control for probe integrity. Digestion is performed at 37◦ C for 30 min. The reactions are then precipitated with RNase inactivation/precipitation solution and resuspended in gel loading buffer. 6. The protected RNA products are separated on an 8% denaturing polyacrylamide gel alongside a labeled RNA marker (DecadeTM Marker System, Ambion). The gel is dried and exposed for ∼24 h in a PhosphorImager cassette (Molecular Dynamics). The image is visualized using PhosphorImager SI analysis and ImageQuaNT (Molecular Dynamics). 3.3.2. Identification of Transcription Start Sites by 5 -RACE

1. 5 -RACE is performed with the FirstChoice RLM-RACE Kit (Ambion). Ten micrograms of total RNA is dephosphorylated with 2 ␮L calf Intestine Alkaline Phosphatase in 1× CIP buffer for a total volume of 20 ␮L.

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2. After 1 h incubation at 37◦ C, the volume is adjusted to 150 ␮L with 115 ␮L of water and 15 ␮L of ammonium acetate solution. The RNA solution is extracted once with acid phenol/chloroform, and the upper aqueous phase is extracted a second time with 150 ␮L of chloroform. 3. The RNA is precipitated by the addition of 150 ␮L of isopropyl alcohol, chilled on ice for 10 min, and then centrifuged for 20 min at top speed in a refrigerated microcentrifuge. The pellet is carefully washed with 70% ethanol and air dried. 4. The RNA is resuspended in 7 ␮L of water and the decapping reaction is performed with tobacco acid pyrophosphatase (TAP) in a total volume of 10 ␮L containing 1 ␮L of 10× TAP buffer and 2 ␮L of TAP enzyme. The reaction is incubated at 37◦ C for 1 h and then 2 ␮L of the reaction is used immediately for ligation of a 5 RNA linker. 5. The linker ligation reaction contains 2 ␮L processed RNA, 1 ␮L RNA adaptor, 1 ␮L 10× RNA ligase buffer, 2 ␮L T4 RNA ligase, and 4 ␮L water. The reaction is incubated at 37◦ C for 1 h. 6. Reverse transcription is performed with either random decamers or a gene-specific primer. cDNA generated with random decamers can be used to map start sites of several transcripts; however, the increased sensitivity of specific priming is necessary when mapping the start site of rare transcripts. 7. The cDNA is then amplified with a gene-specific 3 primer together with a 5 primer corresponding to the linker added to the 5 end of the RNA. 8. The PCR products are cloned using the StrataClone PCR Cloning System, plated on ampicillin plates, and at least 24 individual clones are selected for sequencing. The sequence of the cDNA clones is compared to the genomic sequence, and the junction between the 5 linker and the homology to KIR genomic sequence indicates the start site of transcription. 3.4. Generation of Luciferase Reporter Plasmids

1. Mononuclear cells are obtained by Ficoll-Hypaque (Sigma Diagnostics) density-gradient centrifugation of peripheral blood from healthy donors. Genomic DNA is isolated using QIAamp DNA Blood Mini Kit (Qiagen). 2. A fragment of the KIR3DL1 core bi-directional promoter (see Note 8) is amplified from human genomic DNA using a gene-specific forward primer starting at −229 (ATAGTGAAGGACGCGAGGTGTC) and a reverse primer

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starting at −1 relative to the start codon of the gene (GGTGCTGCCGGTGCAGAC). 3. The PCR reactions are carried out in a 50 ␮L final volume containing 100 ng of genomic DNA and 20 pmol of each R PCR SuperMix (Invitrogen). The primer using Platinum thermal cycling conditions were 35 cycles of 94◦ C for 20 s, 59◦ C for 30 s, and 73◦ C for 5 s. 4. PCR products are cloned into the Invitrogen TOPO TA vector when promoter fragments less than 500 bp are studied (see Note 4). Clones are isolated and sequenced as described in Sections 3.1.3–3.1.5. 5. Bacterial clones containing the desired promoter fragment are grown in 70 mL of LB with 50 ␮g/mL ampicillin shaking at 37◦ C overnight, and plasmid DNA is isolated using a Midi Plasmid Kit (Qiagen). 6. The inserts are excised with either SacI/XhoI to produce one orientation or XhoI/HindIII for the opposite orientation (see Note 9). Forty micrograms of plasmid DNA is digested for 1 h at 37◦ C in a total volume of 100 ␮L containing 80 units of enzyme (4 ␮L of 20 units/␮L enzyme) and 1 ␮g/mL of BSA in reaction buffer compatible with both enzymes used. NEB buffer 1 is used for SacI/XhoI digestion and NEB buffer 2 is used for XhoI/HindIII digestion (all reagents are provided with the enzyme from New England Biolabs). 7. The digested DNA is separated by electrophoresis in a 1.4% agarose gel in 0.5× TBE buffer containing 1× GelRed, nucleic acid gel stain, using a well comb large enough to accommodate the entire 100 ␮L of digested DNA. A 30-mm-wide well is adequate when using a 1-mm-thick gel comb if the agarose gel is poured to a depth of at least 5 mm. 8. The DNA fragments corresponding to KIR3DL1 promoter are visualized under low-intensity UV light and cut from the gel with a clean razor blade, and purified using the QIAquick Gel Extraction Kit (Qiagen). 9. The purified inserts are cloned into the pGL3-basic vector (Promega) digested with the appropriate enzymes to generate constructs in both forward and reverse orientations. A typical ligation reaction for SacI/XhoI cloning contains 2 ␮L of 5× T4 DNA ligase buffer (Invitrogen), 2 ␮L of 50 ng/␮L SacI/XhoI insert, 1 ␮L of 50 ng/␮L SacI/XhoIdigested pGL3-basic vector, 1 ␮L of 20 units/␮L T4 DNA ligase, and 4 ␮L of ddH2 O. The reaction is incubated for 15 min at room temperature.

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10. Chemically competent Escherichia coli cells are transformed with 3 ␮L of the pGL3/insert ligation. Twenty microliters of bacteria are plated on LB–ampicillin and grown overnight. Individual colonies are picked and grown in 70 mL of LB with 50 ␮g/mL of ampicillin shaking at 37◦ C overnight, and plasmid DNA is isolated using a Midi Plasmid Kit (Qiagen). All constructs are verified by sequencing with the pGL3-specific RV3 primer (Promega). 3.5. Cell Transfection, Electroporation, and Luciferase Assays 3.5.1. HEK293 Cell Transfection

1. HEK293 cells are plated at 0.5—1.0 ×105 cells/well in a sixwell plate on the day before transfection (see Notes 10–13). Incubate the cells at 37◦ C in a CO2 incubator overnight. The cell density will reach 3–4 ×105 cells/well when the cells are harvested. 2. For each well, mix 1 ␮g of an individual reporter construct plus 0.01 ␮g of the Renilla luciferase pRL-SV40 control DNA in a 1.5-mL microcentrifuge tube. 3. For each well, add 5 ␮L of Fugene (Roche Diagnostics) to 95 ␮L of DMEM medium without serum. Wait for 5 min before using this mixture. 4. Add the Fugene mixture dropwise to the tube containing DNA. 5. Wait for 15 min and then add directly to cells in a six-well plate. 6. Harvest cells at the desired time after transfection (usually 48 h) and assay luciferase expression using the DualLuciferase Reporter Assay System (Promega).

3.5.2. YT-Indy Cell Electroporation

1. Culture YT-Indy cells to reach a cell density of 5–10 ×105 cells/mL of culture at the time of transfection (see Notes 10–13). 2. Centrifuge the cells in a conical centrifuge tube at 400×g for 5 min at 4◦ C. 3. Resuspend the cell pellet in 10 mL of ice cold PBS, re-pellet. 4. Remove the supernatant by aspiration and resuspend the cell pellet at a concentration of 1 × 107 cells/mL in RPMI medium without added serum, and place on ice. 5. Add 10 ␮L of 1 ␮g/␮L pGL3 construct plus 10 ␮L of freshly prepared 10 ng/␮L Renilla luciferase pRL-SV40 vector to a 1.5-mL microcentrifuge tube and place on ice. 6. Add 0.5 mL of YT-Indy cells to each tube, mix by pipetting. Transfer the mixture into an electroporation cuvette (BioRad) and place on ice.

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7. Transfect YT-Indy cells using electroporation with a BTX ECM 830 (Harvard Apparatus) set at 250V, with three pulses of 7 ms at an interval of 100 ms. 8. Remove cells from the cuvette with a sterile pipette and place in a six-well culture plate with 5 mL of culture medium. 9. Incubate at 37◦ C in a CO2 incubator for 8–48 h (48 h gives optimal activity). Luciferase activity is assayed using the Dual-Luciferase Reporter Assay System (Promega). 3.5.3. Preparation of Cell Lysates for Luciferase Assay

1. After 48 h of transfection, transfer cells to a 15-mL conical centrifuge tube and centrifuge the cells at 400×g for 5 min at 4◦ C. 2. Remove the medium from cells and wash once with 10 mL of cold PBS. 3. Use a micropipette to remove any residual PBS from the cell pellet. 4. Dilute 5× passive lysis buffer (Promega) 1:5 using dH2 O to generate 1× passive lysis buffer. 5. Add 500 ␮l of 1× passive lysis buffer and vortex samples. 6. Transfer the mixture to a 1.5-mL microcentrifuge tube, place at room temperature for 15 min. Centrifuge for 30 s to pellet debris.

3.5.4. Luciferase Activity Assay

1. Prepare the Luciferase Assay Reagent II (LAR II) by adding 10 mL of luciferase assay buffer II to a bottle of lyophilized luciferase assay substrate (Promega). Vortex gently for 10 s and put on ice. The LAR II can be stored at −20◦ C for 1 month. 2. Prepare Stop & Glo reagent by adding 20 ␮L of 50× Stop & Glo substrate to 980 ␮L of Stop & Glo buffer (Promega), vortex gently, and put on ice. This reagent must be freshly prepared. 3. Add 100 ␮L of the LAR II reagent to a luminometer tube. 4. Add 20 ␮L of cell lysate, pipetting up and down three to five times to mix, and measure luciferase activity on the luminometer. 5. Take tube out of the luminometer and add 100 ␮L of Stop & Glo solution, vortex 2–3 s. Put the tube back into the luminometer and measure again. Try to be consistent with the timing of this step for each sample. 6. The luciferase activity of the promoter constructs is normalized relative to the activity of the Renilla luciferase produced by the pRL-SV40 control vector. The Renilla activity of the pGL3-basic negative control is divided by the Renilla activity

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of each construct and the firefly luciferase activity is multiplied by this ratio to normalize it relative to the pGL3-basic control. 7. The fold promoter activity relative to empty vector (pGL3basic) is calculated as the ratio of the corrected firefly luciferase activity of each construct relative to the pGL3 activity. 8. Each construct should be tested in at least three independent experiments for both forward and reverse orientations. In all experiments, values are given as mean ±SD (n≥3).

4. Notes 1. The PCR SuperMix is very convenient for rapidly performing PCR reactions under defined conditions. We have found, however, that some lots of SuperMix have low Taq activity; therefore we routinely add 3 ␮L of purified Platinum Taq enzyme (Invitrogen) to each tube of SuperMix when it is thawed and subsequently store it at 4◦ C to avoid repeated cycles of freeze-thawing. 2. When choosing primers for PCR of potential intergenic transcripts, it is important to avoid making primers that span possible RNA splice sites. Therefore, primers should not span CT-rich regions that contain a potential AG splice acceptor or regions that correspond to splice donors (GTNAGT). 3. All PCR primers should be tested for specificity with the NCBI BLAST program (http://blast.ncbi.nlm. nih.gov/Blast.cgi) before ordering. 4. PCR products less than 500 bp are cloned into the Invitrogen TOPO TA vector, whereas the Stratagene system is used for cloning larger fragments. The Stratagene system is designed to accommodate larger fragments, whereas the Invitrogen system is designed such that the open ends of the vector are held together, thereby increasing the efficiency of cloning small fragments but decreasing the cloning of large fragments. 5. The anchor provides an efficient primer binding site at the 3 end of the message and avoids the continuous elongation of the PCR product that occurs when an oligo-dT primer is used to amplify cDNA. 6. When performing 3 -RACE on rare transcripts, we find that it is absolutely necessary to add a linear amplification step, since there is no gene specificity provided by the 3 primers.

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7. Radioactive nucleotides and solutions containing them should always be kept behind plexiglass shields to avoid unnecessary exposure to radiation. Lab workers should be properly trained in the safe use of radioisotopes. 8. When choosing fragments for reporter assays, it is important to avoid insertion of competing ATG start codons in front of the luciferase start codon, especially those contained within a Kozak consensus (ACCATGG), since it will decrease the observed promoter activity (17). In addition, the presence of consensus splice donor (GTNAGT) or acceptor (YNYYYNCAG) sequences can affect transcript stability or bypass the luciferase start codon (18). 9. Our laboratory routinely clones all promoter fragments into pGL3 in both orientations in order to determine whether bi-directional promoter activity is present. 10. The efficiency of the transfection should be optimized and reproducible. Individual experiments should contain sufficient cells to transfect all reporter plasmids being analyzed in the study in a single experiment. The use of the Renilla internal control in each transfection allows normalization of transfection efficiency within each experiment and provides a more reliable result for comparison of subsequent experiments. 11. For good transfection efficiency it is crucial that the plasmid DNA is of high quality; the A260/A280 ratio should be at least 1.8. 12. To ensure a high degree of reproducibility, a single large batch of each control vector (pGL3-basic and pRL-SV40) should be produced and used for all experiments. 13. Transfected cells can be analyzed several hours to several days after electroporation. Times shorter than 8 h are usually not long enough for reasonable expression of the protein. Over a period of several days, the cells lose transfected DNA and thus expression from the plasmid will gradually decrease. We have found that 48 h is optimal for most reporter experiments.

Acknowledgments This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services nor does men-

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tion of trade names, commercial products, or organizations imply endorsement by the US government. This research was supported in part by the Intramural Research Program of the NIH, the National Cancer Institute, the Center for Cancer Research. References 1. Lanier, L. L. (2005) NK cell recognition. Annu Rev Immunol 23, 225–274. 2. Moretta, L., and Moretta, A. (2004) Killer immunoglobulin-like receptors. Curr Opin Immunol 16, 626–633. 3. Parham, P. (2005) MHC class I molecules and KIRs in human history, health and survival. Nat Rev Immunol 5, 201–214. 4. Yokoyama, W. M., and Plougastel, B. F. (2003) Immune functions encoded by the natural killer gene complex. Nat Rev Immunol 3, 304–316. 5. Moretta, A., Bottino, C., Pende, D., Tripodi, G., Tambussi, G., Viale, O., Orengo, A., Barbaresi, M., Merli, A., Ciccone, E., and Moretta, L. (1990) Identification of four subsets of human CD3-CD16+ natural killer (NK) cells by the expression of clonally distributed functional surface molecules: correlation between subset assignment of NK clones and ability to mediate specific alloantigen recognition. J Exp Med 172, 1589–1598. 6. Valiante, N. M., Uhrberg, M., Shilling, H. G., Lienert-Weidenbach, K., Arnett, K. L., D Andrea, A., Phillips, J. H., Lanier, L. L., and Parham, P. (1997) Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors. Immunity 7, 739–751. 7. Held, W., and Kunz, B. (1998) An allelespecific, stochastic gene expression process controls the expression of multiple Ly49 family genes and generates a diverse, MHCspecific NK cell receptor repertoire. Eur J Immunol 28, 2407–2416. 8. Raulet, D. H., Vance, R. E., and McMahon, C. W. (2001) Regulation of the natural killer cell receptor repertoire. Annu Rev Immunol 19, 291–330. 9. Parham, P. (2006) Taking license with natural killer cell maturation and repertoire development. Immunol Rev 214, 155–160.

10. Saleh, A., Davies, G. E., Pascal, V., Wright, P. W., Hodge, D. L., Cho, E. H., Lockett, S. J., Abshari, M., and Anderson, S. K. (2004) Identification of probabilistic transcriptional switches in the Ly49 gene cluster: a eukaryotic mechanism for selective gene activation. Immunity 21, 55–66. 11. Pascal, V., Stulberg, M. J., and Anderson, S. K. (2006) Regulation of class I major histocompatibility complex receptor expression in natural killer cells: one promoter is not enough! Immunol Rev 214, 9–21. 12. Stulberg, M. J., Wright, P. W., Dang, H., Hanson, R. J., Miller, J. S., and Anderson, S. K. (2007) Identification of distal KIR promoters and transcripts. Genes Immun 8, 124–130. 13. Davies, G. E., Locke, S. M., Wright, P. W., Li, H., Hanson, R.J., Miller, J. S., and Anderson, S. K. (2007) Identification of bi-directional promoters in the human KIR genes. Genes Immun 8, 245–253. 14. Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982) Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2, 1044–1051. 15. Wood, K. V. (1991) in Chemiluminescence: Current Status (Stanley, P. and Cricka, L., eds.), Wiley, New York, p. 543. 16. Yodoi, J., Teshigawara, K., Nikaido, T., Fukui, K., Noma, T., Honjo, T., Takigawa, M., Sasaki, M., Minato, N., Tsudo, M., Uchiyama, T., and Maeda, M. (1985) TCGF (IL 2)-receptor inducing factor(s). I. Regulation of IL 2 receptor on a natural killerlike cell line (YT cells). J Immunol 134, 1623–1630. 17. Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283. 18. Sharp, P. A. (1981) Speculation on RNA splicing. Cell 42, 643–646.

Chapter 27 Use of Inbred Mouse Strains to Map Recognition Receptors of MCMV Infected Cells in the NK Cell Gene Locus Nassima Fodil-Cornu, Michal Pyzik, and Silvia M. Vidal Abstract Genetically distinct inbred strains of mice that differ in their susceptibility to mouse cytomegalovirus (MCMV) are invaluable for dissecting complex host–pathogen interactions. Their study has allowed the identification of host-resistance loci, including several activating NK cell receptors of major histocompatibility complex (MHC) class I. In this chapter, we provide a practical guide to the genetic mapping and functional characterization of NK cell receptors that control innate immunity against MCMV via specific recognition of infected cells. Key words: MCMV, host resistance, cross, genotyping, mapping, retroviral expression, reporter cell assay.

1. Introduction The complex interaction between NK cells and cytomegaloviruses (CMVs) has been appreciated since the recognition that patients (1) and mutant mice (2) lacking functional NK cells are particularly vulnerable to infection. Shortly after, experimental depletion of NK cells using anti-NK cell-specific antibodies confirmed the role of this lymphocyte subset in host defense. Since then, infection of mice with mouse CMV (MCMV) has provided overwhelming evidence of the critical role of NK cells in the innate anti-viral response. Two major approaches have been used: “reverse genetics,” whereby mice carrying targeted Nassima Fodil-Cornu and Michal Pyzik contributed equally.

K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 27, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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mutations in known genes are tested for the response against MCMV. This approach has shown that mutations in genes involved in NK cell maturation, stimulation, or function (cytokine secretion, cytotoxicity, receptor-mediated apoptosis) dramatically alter the response to MCMV and render mutant mice highly susceptible compared to control littermates. “Forward genetics,” whereby natural phenotypic differences in mice are exploited to isolate and understand the genetic determinants of the NK cellmediated response against MCMV; the goal of “forward genetics” is to link a change in phenotype with a change in genotype in mouse candidate genes. Two major genetic loci have been linked to innate resistance to MCMV: NKC (natural killer cell complex) and the H2 (Histocompatibility 2 locus or mouse MHC) on chromosome 6 and 17, respectively. Among others, the NKC encodes for the C-type lectin receptor family expressed at the surface of NK cells, whereas the H2 encodes for the MHC class I molecules which constitute the natural ligands of these NK cell receptors. Among several phenotypically defined loci mapped at the NKC (3), three MCMV resistance loci exist, including Cmv1 and Cmv3, which have been resolved at the gene level as members of the Ly49 NK cell receptor family. Ly49 receptors bind to classical MHC class I or MHC class I-like proteins and present varied repertoire in different inbred mice, delivering inhibitory or activating signals. Activating Ly49 receptors, contrary to inhibitory ITIM-bearing Ly49 receptors, associate with the ITAM-containing DAP12 adapter protein. Cmv1, originally described by Scalzo and co-workers in C57BL/6 mice, is inherited as a single dominant H2-independent locus (4). The resistance allele (Cmv1r ) encodes the activating receptor Ly49H capable of binding an MHC class I-like viral product, m157 (5). In contrast, Cmv3-mediated resistance present in MA/My mice depends on a specific combination of alleles at Ly49 and H2 loci (6). In the MA/My mouse strain, the activating Ly49P receptor is able to recognize target cells but only if the cells are MCMV infected and present the H2k haplotype. Although the details of the Ly49P ligand remain to be fully elucidated, Ly49P and the H2Dk molecules seem to work in a synergistic manner in which both participants are required for viral control. Those two modes of inheritance of MCMV resistance correspond to two different modes of Ly49-mediated recognition of the infected cell. Thus, the “forward genetics” approach allowed to assign a function to activating Ly49 receptors and to identify NK cell-specific mechanisms for the recognition and control of MCMV infection. In this chapter, we provide a practical guide to the identification of Ly49 receptors that define innate resistance to MCMV via specific recognition of MCMV-infected cells.

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In inbred strains resistant to MCMV, NK cells control MCMV growth and eliminate the virus from target organs within the first few days of infection. Such NK cell control is absent in MCMV-susceptible strains, including BALB/c, BALB.K, NOD, and 129S, leading to high viral growth and eventually death. Therefore, a typical forward genetic study involves the mating of MCMV-resistant (R) and MCMV-susceptible (S) mouse strain and the evaluation of progeny cohorts for viral titer in target organs, as was done in an informative (MA/My × BALB/c)F2 cross (Fig. 27.1 and Sections 3.1 and 3.2). To test a possible role of Ly49 genes and/or H2 genes in the resistance phenotype, first polymorphic markers at these loci (Table 27.1 ) are used to genotype progeny mice (Section 3.2). Subsequently, the association between the phenotype and the genotype data is tested statistically using ANOVA (Section 3.3 ). With such analysis it is possible to determine the mode of inheritance of MCMV resistance, which may segregate as an H2-independent or H2-dependent locus. Upon the confirmation of the role of Ly49 and/or H2 genes in the MCMV-resistant phenotype, a functional assay is required to determine the Ly49-mediated mechanism of recognition of the infected cell. One method originally described in Dr. Lewis Lanier’s group involves the expression of Ly49 cDNAs in reporter cells, which are co-cultured with MCMV-infected cells. The triggering of reporter cells, which is monitored by FACS, indicates

 Fig. 27.1. Investigation of the inheritance mode of Ly49-mediated resistance to MCMV: the Cmv3 example. (A) Segregation of MCMV resistance. MCMV titers (expressed as log10 PFU) in the spleens of MCMV-resistant mice (MA/My), MCMV-susceptible mice (BALB/c), and their F1 and F2 progenies. In this cross, 120 F2 mice were generated. The distribution of the spleen viral titer in the F2 progeny is continuous, i.e., characterized by a wide span ranging from resistant to susceptible mice. Such phenotypic distribution indicates that MCMV resistance is complex in this cross, i.e., determined by more than one gene. (B) Genotyping of the Ly49 and H2 loci using the Ly49e and IAA1 markers. Ly49e and IAA1 were amplified by PCR and digested by HincII and PstI (Table 27.1) to detect polymorphisms within the Ly49 and H2 loci between BALB/c and MA/My paternal strains and the heterozygote progenitors. The three possible genotypes for the Ly49 and H2 loci are shown. (C) Contribution of Ly49 and H2 loci to the resistance phenotype. Ly49e genotypes are shown at the bottom; ‘m’ and ‘c’ represent inheritance of MA/My and BALB/c alleles, respectively. H2 genotypes (obtained with the IAA1 genetic marker) are shown at the top; H2k and H2d represents inheritance of MA/My and BALB/c alleles, respectively. Box plots indicate the interquartile range and median (horizontal line) of log10 PFU spleen viral load for each of the nine possible allelic combinations at Ly49 and H2 loci. Using ANOVA and Bonferroni post hoc analysis, we found that there is a significant effect on spleen viral load only when both the H2kk haplotype and homozygosity at the Ly49mm locus from the MA/My strain are present. Likewise, only when the Ly49mm is homozygous for the MA/My genotype, the H2kk has an effect. This result indicates that MCMV resistance depends on the interaction of two loci, Ly49 and H2.

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Fig. 27.1. (continued).

the ability of the specific Ly49 receptor to recognize the MCMVinfected cell (Fig. 27.2 and Section 3.5)

2. Materials 2.1. Investigation of the Inheritance Mode of Ly49-Mediated Resistance to MCMV

1. Animals: Mice are purchased from Jackson Laboratories. The strain name, the MCMV phenotype, and the catalog number are listed in Table 27.1.

128468912

128784912 129544491

129849145

D6mit61

D6mit135 Nkg2d

Ly49e

PCR

47707105

29405253 34137861

D17mit68

D17mit101 D17MIT28

PCR PCR

RFLP or PstIa HindIIIb

RFLP HinFI

RFLP HincII

PCR RFLP XbaI

PCR

PCR

143 120

134

167, 96b

374

980

140 300, 300, 100

136

147

Methodology C57Bl/61R

IAA1

D6Ott11

127788235

D6mit52

pb MGI

137

140

ND ND

ND

263a

-

<374 263a/b

980

ND 300, 300, 100

ND

ND

145 104

146

206, 57a

<<374

850, 150

148 600, 100

146

143

PWK/Pas3R BALB/c4S

980

144 350, 320

ND

147

MA/My2R

Jackson Laboratory Stock numbers: 0006641 ; 0006772 ; Pasteur Institute3 ; 0037154 ; 0006514 ; 0019515 ; 0018006 ; 0024487 R, MCMV resistant; S, MCMV susceptible; ND, not detected a Band size obtained by PstI digestion b Band size obtained by HindIII digestion

H2

NKC

ID

PCR or RFLP product size

Table 27.1 Genetic markers at NKC and H2 in MCMV-resistant and -susceptible inbred mice strains

145 92?

146

263a/b

<<374

850, 150

148 600, 100

146

143

BALB.K5S

ND ND

ND

167, 96b

<374

980

ND ND

ND

125

FVB6S

ND 120

ND

167, 96b

>374

980

ND ND

146

143

129S17S

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Fig. 27.2. Cloning and functional analysis of Ly49 candidate gene(s). (A) Retrovirus-mediated expression: Following transduction of 2B4 reporter cells with Ly49-encoding retrovirions, Ly49 receptor expression is monitored by FACS in the whole 2B4 cell population to determine transduction efficiency. In the experiment shown on the left panel, 60% of cells have been successfully transduced. Expression levels of the Ly49 receptor vary across a large range (101 to 5× 102 ). The experiment in the right panel shows Ly49 expression on sorted high-expressing cells. All 2B4 cells express the Ly49 receptor across a narrower range. (B) Cell reporter assay: Examples of a negative (top) and a positive result from a cell reporter assay are shown. MEF cells that have been infected with MCMV (cartoon on the left) are co-cultured with 2B4 cells that express a candidate Ly49 receptor (cartoon in the middle). Upon recognition of the MCMV-infected cell by the Ly49 receptor, DAP12 initiates a signaling cascade, leading to GFP production: 2B4 cells fluoresce green. 2B4 cell fluorescence is acquired by FACS. Absence of fluorescence is analyzed as absence of recognition. A shift in the fluorescence intensity is analyzed as Ly49-mediated recognition of the infected cells.

2. MCMV: Smith mouse salivary gland (VR-1399) from ATCC is passaged twice in BALB/c mice (7). 3. Mouse embryonic fibroblasts (MEF) are prepared from BALB/c mice at 14–16 days of pregnancy (7). 4. DMEM-10% or DMEM-2% media: Dulbecco’s Modified Eagle Medium (DMEM; Wisent) containing penicillin/streptomycin (dilution of 100× solution; Wisent), 25 mM HEPES (1 M stock; Invitrogen), and 10% or 2% heat-inactivated fetal bovine serum (FBS; Wisent).

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5. Opti-MEM reduced serum medium (Invitrogen). 6. 0.05% Trypsin–EDTA (Wisent). 7. Low-melting agarose (LMA; Invitrogen). 8. 1× PBS (Wisent). 9. DMEM containing 10% FBS and 2% LMA. 10. 10% Formalin solution. 11. 1% (w/v) methylene blue (Harleco) in 70% ethanol. 12. DNA lysis buffer: 0.5% SDS; 0.1 M NaCl; 50 ml Tris–base, pH 8; 2.5 mM EDTA, and 150 ␮g/ml proteinase K (Fermentas). 13. RNase A 10 mg/ml solution (Fermentas). 14. 8 M Potassium acetate solution. 15. Chloroform. 16. 100% and 80% ethanol. 17. PCR buffer mix: containing Taq polymerase, 10× Taq polymerase buffer, dNTPs (Fermentas), and specific primers. 18. Enzymes: XbaI, HincII, HinF1, Pst1, and HindIII (Fermentas). 19. Ethidium bromide solution. 20. High-resolution agarose, separation <1000 bp (USB). 21. 48-Well culture plates. 22. 15-ml centrifuge tubes. 2.2. Candidate Gene Cloning

1. RNeasy Kit (Qiagen). 2. Reverse transcription reactions: SuperScript II Reverse Transcriptase (Invitrogen), Random Primers (Invitrogen). 3. Advantage cDNA PCR Kit (Clontech) for high-fidelity PCR reactions. 4. T4 DNA ligase (Invitrogen). 5. pGem-T Easy Vector System (Promega). 6. Calf intestinal alkaline phosphatase (Invitrogen). 7. Subcloning efficiency DH5␣-competent cells (Invitrogen). 8. Ampicillin (Invitrogen). 9. X-Gal (Invitrogen). 10. IPTG (Invitrogen). 11. Illustra PlasmidPrep Mini Spin Kit (GE Healthcare). 12. HiSpeed Plasmid Midi Kit (Qiagen). 13. M13 sequencing primers.

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14. pMX-puromycin retroviral vector (L. Lanier laboratory, UCSF USA). 15. PLAT-E retroviral packaging cell line. 16. FLAG-DAP12 NFAT-GFP 2B4 reporter cells (L. Lanier laboratory, UCSF USA). 17. Transfection reagents: Lipofectamine 2000 (Invitrogen), DOTAP (Roche). 18. DMEM-10% medium: DMEM containing penicillin/streptomycin (dilution of 100× solution; Wisent), 25 mM HEPES (1 M stock solution; Invitrogen), and 10% heat-inactivated FBS (Wisent). 19. Opti-MEM reduced serum medium (Invitrogen). 20. Antibodies: Anti-Ly49H (clone 3D10) (eBioscience), antiLy49P (clone 4D11) (eBioscience), and anti-FLAG (M2; Sigma-Aldrich). 21. Cell-associated MCMV obtained from supernatant of MCMV-infected BALB/c MEF cells (7). 22. MEF prepared as in Section 2.1. 23. Complete RPMI-10% medium: RPMI-1640 medium (Wisent) containing 10% FBS (Hyclone). 24. 0.05% Trypsin–EDTA solution (Wisent). 25. Carbonate-coating buffer: 0.1 M Na2 CO3 and 0.1 M NaHCO3 , pH=9.5. 26. FACS Buffer: PBS 1X, 2% FBS 2.3. Equipment

1. PCR machine 2. Spectrophotometer 3. Agarose gel electrophoresis apparatus 4. FACS machine, such as FACSCalibur 5. Polytron homogenizer 2100

3. Methods 3.1. Investigation of the Inheritance Mode of Ly49-Mediated Resistance to MCMV 3.1.1. Breeding an F2 Cross and MCMV Phenotyping

To evaluate the genetic factor/s responsible for the resistance trait to MCMV infection, strains with MCMV-resistant (R) and MCMV-susceptible (S) phenotype have to be identified first (see Table 27.1). This is done by determining the viral load in spleen and liver 3 days post-infection using a plaque-forming assay (see Note 1). Subsequently, F1 and F2 progenies derived from a cross between R and S mice (R × S) are generated. In general, a gene mapping study requires approximately 150 F2 progenies.

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As illustrated in Fig. 27.1A, a two-log difference distinguishes MCMV titers in the spleens of BALB/c (S) and MA/My (R) strains. Once F2 mice reach adulthood, they are tested for the response to infection. In order to avoid any confounding variables, it is important to use mice of identical age (between 7 and 8 weeks) and the same virus stock. The plaque assay technique (described below) is used to assay MCMV titer in various organs of infected mice (7, 8). For each experiment of the phenotypic survey, we use about 30–40 F2 mice together with parental controls. The viral load distributions of the F1 and F2 progenies are an indicator of the inheritance mode of the phenotype. A discontinuous F2 distribution is an indicator of a major locus effect, such as Cmv1. A continuous distribution is indicative of a more complex inheritance, as seen in the (MA/My × BALB/c)F2 cross used to map Cmv3 (Fig. 27.1A). In this case, the resistance trait depends on allelic combinations at the Ly49 and the H2 loci. 1. Set up five breeding cages of reciprocal crosses between R and S mouse strains to produce F1 progeny (see Note 2). 2. Set up 5–10 breeding cages of reciprocal F1 intercrosses to produce F2 progeny. 3. Infect F1 , F2 , and parental control mice intraperitoneally with 5×103 PFU of salivary gland MCMV/200 ␮l (see Note 3). 4. At day 2 PI, plate 105 MEF cells/well in a 48-well plate for 24 h. 5. Sacrifice mice 3 days post-infection by cervical dislocation or CO2 exposure. 6. Harvest spleens and livers in 5 ml of ice-cold DMEM-2% medium. Collect tails (about 2–4 cm) and store at −20◦ C for DNA extraction. 7. Homogenize the collected organs for 5–10 s using a Polytron 2100 set at speed 15. Keep on ice (see Note 4). 8. Centrifuge at 1000×g for 15 min at 4◦ C; the supernatant contains the virus. 9. Perform serial dilutions of the supernatant in DMEM-2% medium. 10. Plate 100 ␮l of the diluted homogenate in duplicate in a 48-well plate with a confluent monolayer of BALB/c MEF. 11. Incubate for 1 h at 37◦ C, 5% CO2 . 12. Aspirate the supernatant and overlay the cells with 1 ml of DMEM containing 10% FBS and 2% LMA.

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13. Incubate the plates for 3 days to allow plaque formation at 37◦ C, 5% CO2 . 14. Overlay wells with 0.5 ml 10% formalin for 5 min to fix the cells. 15. Stain the cell monolayer with solution of 1% (m/v) methylene blue in 70% ethanol for 10 min. 16. Count the plaques under a low-magnification microscope and express the viral titer as MCMV log10 PFU per organ (see Note 5). 3.1.2. Isolation of Genomic DNA and Genotyping with Markers Within Ly49 and H2 Loci

Different methods of DNA extraction can be used to prepare genomic DNA from tail tips. In our laboratory, we have been using proteinase K/chloroform extraction and found this method to be very efficient and low cost for genomic DNA preparation. In order to analyze the genetic variations at Ly49 and H2, genetic markers such as microsatellites, simple sequence repeats (SSRs), and restriction fragment length polymorphisms (RFLPs) (Table 27.1) are used for PCR-based genotyping (see Note 6). RFLPs require an additional step of enzymatic digestion to differentiate between alleles. Routinely we have used the Ly49e and IAA1 markers to detect the Ly49 and H2 loci, respectively. These markers are polymorphic between different strains of mice as described in Table 27.1. However, if these markers do not show difference for the strains under study, other markers in the Ly49 (within the NKC) and the H2 (Table 27.1) regions are tested. 1. Digest 0.5 cm sample of each mouse tail (see Step 6 above) at 56◦ C in DNA lysis buffer for 3–8 h in 1.5-ml tubes. 2. Add 1 ␮l of RNase A and incubate at 37◦ C for 15 min. 3. Precipitate the DNA with 70 ␮l of 8 M potassium acetate. 4. Add 500 ␮l of chloroform, mix, and incubate for 1 h at −20◦ C. 5. Centrifuge for 10 min at maximum speed at RT. 6. Collect the aqueous phase and re-precipitate the DNA by adding 2 volumes of 100% ethanol. 7. Centrifuge at maximum speed for 10 min. 8. Wash the DNA pellet with 70% ethanol and dry. 9. Dissolve the pellet in 100–200 ␮l of ddH2 O by incubation at 56◦ C for 20 min. The stock DNA is quantified by OD260 . A working stock is made for genotyping needs (25 ng/␮l) (see Note 7). 10. Combine 2 ␮l of DNA (25 ng/␮l) with PCR reaction mix containing NKC or H2 primers (0.4 ␮M), from Table 27.2, for the PCR.

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Table 27.2 List of PCR primers and conditions for genotyping and cloning Primers Cloning

Ly49 Ly49h Ly49k Ly49n

Genotyping Ly49e D6Ott11 IAA1

Sequence

Tannealing (◦ C)

Forward

CCCAAGATGAGTGAGCAGGAGG

55

Reverse

GAGAGTCAATGAGGGAATTTATCC

Forward

AGCCTCTTAGGGGATACAGAC

Reverse

TGTCAAGATAGATAGGAGAGG

Forward

GATGGGTGAGCAGGAAGTCG

Reverse

CCACAAATACAGTAGTAGGGAA

Forward

TTCCCAACTATGAGATTCCAC

Reverse

GCTTTAGATAAAAATAAACATCCTA

Forward

GGCAGCTCACTGTGAGATCG

Reverse

GTTCCTCACCTGGACTGCAA

Forward

GAAGTCATACTGCTTCAGTC

Reverse

ACTCTCTGCTTGCCACTTTG

Forward

GAAGACGACATTGAGGCCGACCACGTAGGC 60

Reverse

ATTGGTAGCTGGGGTGGAATTTGACCTCTT

60 60 61 60 55

Ly49e and D6Ott11 primers are routinely used to detect Ly49p and Ly49h genes within the NKC. IAA1 primers are usually used to differentiate between the H2 of MA/My, BALB/c, FVB and 129 strains

11. Run a PCR with adequate annealing temperature (Table 27.2) for 40 cycles. 12. Digest the PCR product with the adequate enzyme (Table 27.1) for 90 min at 37◦ C for RFLP. 13. Run microsatellite PCR products in 3% high-resolution agarose and RFLPs in 1.5% regular agarose to resolve genotypes. Gels contain 5 ␮l of EtBr/100 ml and 0.5% TBE. 14. Visualize DNA products by UV and score genotypes according to parental and F1 strain controls (Fig. 27.1B).

3.1.3. Gene Interaction Analysis Using Statistical Test (ANOVA)

At this stage, each animal is defined by a viral load in the spleen and its genotype at the Ly49 and H2 loci. In an F2 population, nine different genotype combinations exist, as shown in Fig. 27.1C. A statistical test is used to determine possible genotype/phenotype associations. The two-way ANOVA (for ANalysis Of VAriance) is one of the statistical tests that can be used to analyze a dataset that is determined by two independent variables, in this case the Ly49 and the H2, and one dependent variable, spleen viral load. Analysis of this dataset by the ANOVA method can be separated into two functions: (i) the first function analyses the main effect of the Ly49 or the H2 on spleen viral load and (ii)

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the second test analyses the interaction between the two loci. In the case of the (MA/My × BALB/c)F2 cross, no main effect was associated with the H2 or the NKC; however, a significant interaction was found between the H2 and the Ly49. To identify where each interaction lies within the F2 dataset, post hoc tests must be conducted. For example, multiple t-tests can be used in conjunction with the Bonferroni method, a conservative procedure used to divide the significance value of each post hoc t-test by the total number of tests conducted in order to lower the amount of false statistical significance seen. The Bonferroni method is widely available in many software packages including GraphPad Prism or Statistical Package for the Social Sciences (SPSS). Several different strategies can be employed to characterize this interaction: simple main effects analyses or interaction analyses. As an example, an examination of the simple main effects within the significant interaction would identify at which H2 genotype the Ly49 has an effect, and vice versa. These simple analyses show that the significant interaction seen in the two-way ANOVA arises from the contribution of H2kk and Ly49mm of MA/My strain (Fig. 27.1C). 3.2. Cloning and Functional Analysis of Ly49 Candidate Gene(s)

3.2.1. Cloning of Candidate Ly49 Receptor cDNA

From the mapping analysis, Ly49 receptors in combination with H2 emerged as the candidate genes underlying the MCMV resistance in MA/My. To date, four sequenced Ly49 haplotypes (C57BL/6, NOD, 129, BALB/c) showing variation in gene number and repertoire are available (9). However, in the context of MCMV infection, a functional approach is required to characterize Ly49 receptor–ligand interactions (Table 27.2 and Section 3.4). 1. Extract total RNA from the spleen of founder animals with the RNeasy Kit. 2. Perform a reverse transcription reaction to obtain cDNA using SuperScript II reverse transcriptase and random primers according to the manufacturer’s instructions. 3. Use degenerate primers based on consensus sequence of published Ly49 receptors (10) (Table 27.2) to amplify the candidate Ly49 cDNA using high-fidelity PCR with the Advantage cDNA PCR Kit for 18 cycles, as described in the manufacturer’s instructions. 4. Run the PCR reaction on 0.8% agarose gel in 0.5× TBE at 120 mA for 30 min. 5. Purify the Ly49 cDNA PCR fragment from the gel (around 900 bp).

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6. Ligate into pGEM-T Easy Vector according to the manufacturer’s instructions (see Note 8) and transform competent cells. 7. Grow bacterial colonies and extract pGEM-T easy plasmid DNA using an Illustra PlasmidPrep Mini Spin Kit. 8. Sequence the Ly49 cDNA insert using M13 primers. 9. Design Ly49-specific primers carrying restriction sites at 3 - and 5 -termini with suitable restriction enzyme for cloning full-length cDNA into the packaging vector pMxpuromycin (see Note 9). 10. Amplify Ly49 cDNA with newly designed primers using high-fidelity Taq polymerase as above in step 2. 11. Digest the Ly49 PCR product with the appropriate restriction enzymes. 12. Ligate into pMx-puromycin vector (pre-treated with calf alkaline phosphatase) as above. 13. Verify Ly49 cDNA sequence using pMx-specific 5 primer and the Ly49 3 primer used for cloning and listed in Table 27.2. 14. Prepare high-concentration plasmid DNA (1 mg/ml) using HiSpeed Plasmid Midi Kit. 3.2.2. Retrovirus-Mediated Expression of Candidate Ly49 Receptors

To determine the ability of Ly49 receptor(s) to recognize infected cells, the receptors are stably expressed in reporter cells using a retrovirus-mediated expression cloning method. The production of a retrovirus involves transient transfection of a retroviral vector (pMx) containing packaging signal and lacking viral structural genes into a packaging cell line (Plat-E). The packaging cell line expresses gag-pol-env and complements vector deficiency (11). Retrovirus particles containing the Ly49 construct are transduced into the 2B4 reporter T cell hybridoma. These cells contain the reporter GFP gene under the control of the NFAT promoter and stably express the FLAG-tagged DAP12 protein. Therefore, the successful expression of activating Ly49 receptor can be detected either by specific anti-Ly49 antibody or by the anti-FLAG antibody (Fig. 27.2A). 1. Plate 5×105 PLAT-E cells/well in 5 ml of DMEM-10% medium in a six-well plate (see Note 10). Incubate for 24 h at 37◦ C, 5% CO2 . 2. Replace the medium with 4 ml of fresh medium. 3. Dilute 4.5 ␮g of plasmid DNA in a final volume of 250 ␮l of Opti-MEM. 4. Add 12.5 ␮l of Lipofectamine 2000 to 237 ␮l of OptiMEM in another tube.

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5. Mix solutions by gently flicking tubes, do not mix by vortexing or pipetting up and down. Incubate both tubes at RT for 5 min. 6. Carefully add 250 ␮l of the Lipofectamine–Opti-MEM medium to the plasmid DNA solution. 7. Incubate for 20 min at RT. 8. Add 0.5 ml of the Lipofectamine–Opti-MEM–plasmid DNA mix to the monolayer of the packaging cells. Rock gently to redistribute the solution evenly. Incubate at 37◦ C for 24 h. 9. Replace the medium with 5 ml of fresh medium. Incubate cells at 37◦ C for 24 h. At this time point, infectious retrovirions start to be produced. 10. Carefully collect the retrovirion-containing supernatant from the packaging cells. 11. Centrifuge the supernatant at 800×g for 5 min at 4◦ C to pellet out the cell debris. This supernatant is immediately used for transduction, but it can be kept for 3 days at 4◦ C for additional infections. 12. For the transduction, two wells of a 48-well plate are used. In the first well, plate 1×105 2B4 cells in a total volume of 100 ␮l of fresh RPMI-10% medium. In the second well, prepare the transduction mix by adding 10 ␮l of DOTAP to 900 ␮l of the retrovirion supernatant and incubate for 5 min at RT. 13. Transfer the transduction mix to the wells containing 2B4 cells. 14. Incubate for 24 h at 37◦ C. 15. Carefully replace the medium without disrupting the nonadherent 2B4 cells with 1 ml of fresh medium. Incubate for 24 h at 37◦ C (see Note 11). 16. Collect a small sample of cells for FACS analysis and plate the remaining cells on day 2 post-transduction. 17. Stain for 15 min 1×105 cells with anti-FLAG antibody or specific anti-Ly49 receptor antibody in FACS buffer to analyze by FACS. 18. Collect the 2B4 cells and stain as before after 4 days of expansion. 19. Sort the cells expressing the highest level of Ly49 (∼20% of the transduced population) (Fig. 27.2A). 20. Expand the sorted cells and freeze stock aliquots of 2× 106 cells/ml in FBS containing 10% DMSO.

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The reporter cell assay consists of co-culture of 2B4 reporter cells expressing the Ly49 activating receptor and MCMV-infected MEF cells. In the case of Ly49 recognition of the MCMVinfected cell, DAP12 initiates a stimulatory cascade leading to activation of NFAT and ultimately expression of the GFP reporter gene, which can be analyzed by FACS (Fig. 27.2B) (see Note 12). 1. Prepare two T25 flasks with 2×106 MEF cells/flask. 2. Plate 7×106 2B4 cells in a T75 flask (Note 11). 3. Wash confluent MEF cells with PBS. 4. Overlay MEF cells with 1 ml of MCMV diluted in DMEM2% medium to obtain an MOI of 1 or with medium alone for uninfected MEF cells. 5. Incubate for 1 h at 37◦ C, 5% CO2 . 6. Add 4 ml of DMEM-10% medium and incubate O/N at 37◦ C, 5% CO2 . 7. Detach MEF cells with trypsin–EDTA solution, spin at 320g for 5 min and resuspend at 106 /ml in RPMI-10% medium. 8. Add 100 ␮l/well of MCMV-infected or uninfected MEF cells into wells of a 48-well plate. 9. Collect and resuspend Ly49-expressing 2B4 reporter cells at 106 /ml in RPMI-10% medium. 10. Add 100 ␮l/well of 2B4 cells to MCMV-infected and uninfected MEF cells. 11. Add 100 ␮l/well of 2B4 cells to wells containing platebound anti-FLAG antibody (see Note 13) as a positive control, or to untreated wells as a negative control. 12. Add RPMI-10% medium to achieve a final volume of 1 ml/well and incubate for 18–24 h at 37◦ C, 5% CO2 . 13. Resuspend and collect the 2B4 cells for FACS analysis. This functional approach can be extended to activating Ly49 receptors present in MCMV-susceptible mouse strains to evaluate a possible Ly49P-like H2-dependent recognition of MCMVinfected cells. Such strains may have a Ly49 receptor capable of recognizing infection in different H2 contexts. Moreover, a similar functional approach could be used to evaluate the ability of activating Ly49 receptors to recognize cells in the context of other pathogens, an important question suggested by the presence of host resistance to ectromelia virus (Rmp1) and herpes simplex virus 1 (Hsv1) loci linked to the Ly49 region.

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4. Notes 1. Another method used to evaluate MCMV burden is quantitative PCR (12). 2. To determine the possible sex effect on the resistance trait, it is important to monitor the gender of the parental origin of F1 and F2 mice. For this, a reciprocal breeding is conducted in which the F1 population is generated through crossing an R male to an S female and vice versa [(R) ♂ × (S) ♀ or (R) ♀ × (S) ♂]. 3. We initially determined the titer of MCMV infectious stock by inoculating resistant and susceptible mice (for example, BALB/c vs. C57BL/6) with a range of virus doses. We selected a dose giving at least 2 log10 MCMV PFU difference in the spleens of resistant and susceptible mice. 4. Be careful to avoid overheating and excessive homogenization, which may destroy the virus. 5. The liver viral load provides a control of infection, since MCMV replicates productively in livers of both R and S mice. Therefore, absence of viral load indicates that the mouse has not been infected and should be removed from the experiment. 6. The Mouse Genome Informatics (MGI) web site (http://www.informatics.jax.org/) and the Mouse Microsatellite Data Base of Japan (MMDBJ) web site (http://www.shigen.nig.ac.jp/mouse/mmdbj/top.jsp) are helpful web sites to find genetic markers. 7. DNA master stock can be kept for several months at −20◦ C. 8. The high-fidelity Taq polymerase introduces dA overhangs in the PCR products which can then be introduced into the pGEM-T vector that has dT overhangs. 9. The 5 end of the cDNA should be cloned as close to the promoter as possible to obtain optimal expression. 10. Packaging cell line should be in their log phase of growth (80–90% confluent) on the day of transfection. 11. Overgrowing of 2B4 reporter cells may result in nonspecific activation. Therefore, it is recommended to split 2B4 cells 1:5 every day. 12. Other tools have been developed for cell reporter assays using ␤-galactosidase expression as readout by colorimetric detection (13, 14).

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13. Positive control wells in plates coated with anti-FLAG antibody are prepared in advance by incubating 200 ␮l of a solution containing 10 ␮g/ml of M2 antibody in carbonate-coating buffer for 24 h at 4◦ C. Wash coated wells three times with PBS on the day of the experiment. References 1. Biron CA, Byron KS, Sullivan JL. Severe herpes virus infections in an adolescent without natural killer cells. N Engl J Med 1989;320:1731–5. 2. Shellam GR, Allan JE, Papadimitriou JM, Bancroft GJ. Increased susceptibility to cytomegalovirus infection in beige mutant mice. Proc Natl Acad Sci U S A 1981;78:5104–8. 3. Yokoyama WM, Plougastel BF. Immune functions encoded by the natural killer gene complex. Nat Rev Immunol 2003;3:304–16. 4. Scalzo AA, Lyons PA, Fitzgerald NA, Forbes CA, Yokoyama WM, Shellam GR. Genetic mapping of Cmv1 in the region of mouse chromosome 6 encoding the NK gene complex-associated loci Ly49 and musNKRP1. Genomics 1995;27:435–41. 5. Arase H, Mocarski ES, Campbell AE, Hill AB, Lanier LL. Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors. Science 2002;296: 1323–6. 6. Desrosiers MP, Kielczewska A, Loredo-Osti JC, et al. Epistasis between mouse Klra and major histocompatibility complex class I loci is associated with a new mechanism of natural killer cell-mediated innate resistance to cytomegalovirus infection. Nat Genet 2005;37:593–9. 7. Brune W, Hengel H, Koszinowski UH. A mouse model for cytomegalovirus infec-

8.

9. 10.

11.

12.

13.

14.

tion. Curr Protoc Immunol 2001; Chapter 19:Unit 19 7. Scalzo AA, Farrell HE, Karupiah G. Techniques for studying murine natural killer cells in defense against viral infection. Methods Mol Biol 2000;121:163–77. Makrigiannis AP, Parham P. The evolution of NK cell diversity. Semin Immunol 2008;20:309–10. Silver ET, Gong D, Hazes B, Kane KP. Ly-49 W, an activating receptor of nonobese diabetic mice with close homology to the inhibitory receptor Ly-49G, recognizes H-2D(k) and H-2D(d). J Immunol 2001;166:2333–41. Kitamura T, Koshino Y, Shibata F, et al. Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics. Exp Hematol 2003;31:1007–14. Wheat RL, Clark PY, Brown MG. Quantitative measurement of infectious murine cytomegalovirus genomes in real-time PCR. J Virol Methods 2003;112:107–13. Smith HR, Heusel JW, Mehta IK, et al. Recognition of a virus-encoded ligand by a natural killer cell activation receptor. Proc Natl Acad Sci U S A 2002;99:8826–31. Carlyle JR, Mesci A, Ljutic B, et al. Molecular and genetic basis for strain-dependent NK1.1 alloreactivity of mouse NK cells. J Immunol 2006;176:7511–24.

Chapter 28 Studying NK Cell Responses to Ectromelia Virus Infections in Mice Min Fang and Luis Sigal Abstract Here we describe methods for the in vivo study of antiviral NK cell responses using the mouse Orthopoxvirus ectromelia virus as a model, the agent of mousepox. The methods include those specific for the preparation and use of ectromelia virus such as the production of virus stocks in tissue culture and in live mice, the purification of virus stocks, the titration of virus stocks and virus loads in organs, and the infection of mice. The chapter also includes methods for the specific study of NK cell responses in infected mice such as the preparation of organs (lymph nodes, spleen, and liver) for analysis, the study of NK cell responses by flow cytometry, the adoptive transfer of NK cells, the measurement of NK cell cytolytic activity ex vivo and in vivo, and the determination of NK cell proliferation by bromodeoxyuridine loading or by dilution of carboxyfluorescein diacetate succinimidyl ester (CFSE). Key words: NK cells, virus, ectromelia virus, innate immunity, In Vivo mouse studies.

1. Introduction Natural killer (NK) cells are innate effector cells serving as a first line of defense against certain viral infections and tumors (1,2). In the mouse, NK cells are known to be essential for resistance to mouse cytomegalovirus (MCMV; a herpes virus) (3–5) and to mousepox (mouse smallpox) caused by the mouse-specific Orthopoxvirus ectromelia virus (ECTV) (6–8). These two models are very useful for studying the mechanisms of NK cell-mediated resistance to viral disease including the role of cytokines and effector molecules, the identification of activating receptors, the effect of NK cells in the overall immune response, and how the NK cell response is affected by immune evasion proteins encoded by the virus. K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 28, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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ECTV is highly infectious through any route. However, studies in mice housed in the same cage have indicated that it normally enters through micro-abrasions in the skin and also through the respiratory tract. To mimic the natural route, most experimental procedures employ the inoculation of 102 –104 PFU ECTV into the footpad. While all strains of mice become infected with ECTV, some strains of mice such as C57BL/6 and 129 are resistant to mousepox following footpad infection, while other mouse strains such as DBA2/J and BALB/c are highly sensitive with over 80% lethality when exposed to as little as five plaque-forming units (PFU) virus. The two most notorious symptoms of mousepox are the high lethality due to acute liver failure and the resulting toxemia, and the severe skin rash with macular, pustular, and scabbing stages in the surviving animals. These symptoms have strong resemblance to smallpox in humans, which was also characterized by severe rash and 30% mortality due to toxemia (9). Importantly, a gene for resistance to mousepox has been mapped to the NK complex in the distal arm of chromosome 6 (10). Recent work in ours and other laboratories has identified several of the mechanisms involved in the NK cell-mediated resistance to mousepox (7,8). In particular we now know that NK cells are essential during the first 5 days of postinfection (PI). We also know that NK cells curb virus spread from the draining lymph node to the liver using direct effector mechanisms involving perforin and IFN-␥, and indirectly by enhancing the T-cell response. Furthermore, we have identified the NK cell-activating receptor NKG2D as an important costimulatory signal for optimal NK cell responses to ECTV. Because our laboratory has been working with ECTV for several years, this chapter focuses on this virus. Therefore, some of the methods described (e.g., production and purification and titration of virus stocks) are specific for the ECTV model. However, the methods that we describe for the direct study of NK cell responses should be broadly applicable to many other viral models and useful to many researchers studying other infectious models. These include methods for depleting NK cells in vivo; blockade of NK cell receptors during virus infection; monitoring of NK cells by flow cytometry; and assays to determine NK cell activation, such as production of IFN-␥ and granzyme B and ex vivo and in vivo killing of targets.

2. Materials 2.1. Cell Lines and Virus

1. Cell lines: BSC-1 [American Type Culture Collection (ATCC) no. CCL-26], MC57G (ATCC no. CRL-2295), A9 (ATCC no. CCL-1.4), and YAC-1 (ATCC no. TIB160). 2. Virus: ECTV (Moscow strain; ATCC no. VR-1374).

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1. RPMI-10 medium for tissue culture: RPMI-1640 tissue culture medium (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone), 4 mM L-glutamine (Mediatech), 100 IU/ml penicillin and 100 ␮g/ml streptomycin (Mediatech), 10 mM Hepes buffer (Sigma), and 0.05 mM 2-mercaptoethanol (Sigma). 2. RPMI-2.5 medium for testing virus titers: RPMI-1640 tissue culture medium (Invitrogen) supplemented with 2.5% fetal bovine serum (Hyclone), 100 IU/ml penicillin, and 100 ␮g/ml streptomycin (Mediatech).

2.3. Buffers, Reagents, and Equipments

1. Tris–HCl: 1 mM and 10 mM solution, pH 9.0, sterile. 2. Sucrose solutions: Prepare 36% (w/v) sucrose solution in 10 mM Tris–HCl, pH 9.0. Prepare 40%, 36%, 32%, 28%, and 24% (w/v) sucrose solutions in 1 mM Tris–HCl. 3. 0.1% crystal violet solution: Add 200 ml ethanol to 1 g crystal violet, swirl to dissolve the stain, and add additional ddH2 O to a final volume of 1000 ml. 4. Ammonium chloride (NH4 Cl ) red cell lysis buffer: Add 800 ml ddH2 O to 8.4 g NH4 Cl, dissolve thoroughly, and add additional ddH2 O to 1000 ml, aliquot 100 ml/bottle, sterilize in the autoclave (liquid cycle, 20 min). 5. 35% Percoll: 35% Percoll (MP Biomedicals) in Hanks’ Balanced Salt Solution (HBSS). 6. Brefeldin A (BFA): BFA (Sigma) is prepared as a 20 mg/ml stock in dimethyl sulfoxide (DMSO). For in vitro cytokine staining, further dilution to 1 mg/ml is made in DMSO. For in vivo injection, further dilution to 0.5 mg/ml is made in PBS, and 500 ␮l is injected into each mouse i.v. 7. FACS staining buffer: PBS buffer containing 2% fetal bovine serum and 0.04% sodium azide (NaN3 ). 8. Fixation and permeabilization solution: BD Cytofix/ Cytoperm solution (BD Biosciences). 9. Perm/Wash buffer: BD Perm/Wash buffer (BD Biosciences). 10. 0.5% paraformaldehyde (PFA) fixing solution: 0.5% PFA in PBS. 11. Carboxyfluorescein diacetate succinimidyl ester (CFSE) solution: CFSE (Invitrogen) is prepared as a 4 mM stock in DMSO. 12. NK cell isolation kit: Isolation of untouched NK cells from single cell suspensions of mouse lymphoid tissues (Miltenyi Biotec).

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13. CD49b (DX5) microbeads: Positive selection or depletion of mouse NK cells (Miltenyi Biotec). 14. Radioisotopes: Sodium chromate (51 Cr) solution (GE Healthcare). 15. Lab equipment: Water bath, sonicator, tissue culture hood, tissue homogenizer (Tissuemizer Homogenizer, Fisher Scientific), Packard TopCount instrument (for reading radioactivity, PerkinElmer), MACS separator and LS column (Miltenyi Biotec).

3. Methods All procedures involving mice require a protocol pre-approval by the Institutional Animal Care and Use Committee (IACUC). Consult with your institution for the procedure to submit a protocol. Ectromelia is transmissible from mouse to mouse and can also be transmitted through the hands and clothing of those handling virus and infected mice. Thus, precautions must be made to avoid spread of the virus to the rest of the mouse colony. In particular, working with ECTV-infected mice requires dedicated facilities where mice and cell cultures can be housed in isolation. These facilities are referred to here as the infection suite which in our institution follows the guidelines of a biosafety level 3 (BSL3) room and consists of a personnel changing room, an animal room with a high density ventilated rack and a tissue culture room with dedicated biohazard tissue culture hood, CO2 incubators, and centrifuge. The suite also has a two-door autoclave where all wastes and animal cages are sterilized before leaving the room (see Note 1). 3.1. Preparation and Use of ECTV 3.1.1. Preparation of Tissue Culture Stock of ECTV

For the preparation of ECTV stocks, we normally use A9 cells. BSC-1 cells may also be used with comparable virus yields. The advantage of using A9 cells is that they grow faster than BSC-1 cells. It may be convenient to grow cells initially in 175 cm2 flasks before transferring them to roller bottles. Virus yield is improved when the cells are in log growth phase. All procedures are performed in sterile conditions using a biosafety tissue culture hood. 1. Grow A9 cells in 1750 cm2 roller bottles to 80–90% confluence (∼5×108 cells/bottle) with RPMI-10 medium. Maintain cultures on rollers in a 37◦ C incubator. 2. Just prior to use, combine the required amount of ECTV stock with an equal volume of 0.25% trypsin and mix by vortexing vigorously. Incubate 30 min at 37◦ C, vortexing at 5- to 10-min intervals. Infecting the cells with a low multiplicity of infection (MOI) is important to avoid

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3. 4. 5. 6. 7. 8.

9.

10. 11. 12. 13. 14. 15. 16. 17.

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the formation of non-replicative viral particles. Thus, we usually infect A9 cells with 0.05–0.2 PFU/cell. At this dose, we generally obtain high titers of virus stock (∼ 1× 108 PFU/ml). Thus, for one 1750-cm2 roller bottle at 90% confluency, the virus dose used for infection should be 2.5– 10 × 107 PFU. Add RPMI-10 to the virus stock to a final volume of 25 ml. Remove the growth medium from the roller bottles by aspiration. Rinse the bottle with 100 ml PBS. Add 25–50 ml of 0.04% trypsin to detach the cells. When most of the cells are detached, add 200 ml medium and transfer the cells to 50-ml tubes. Centrifuge (800×g for 5 min), decant the supernatant and resuspend the cells to 2×107 cells/ml in RPMI-10 medium (25 ml/roller bottle). Transfer the cells to one 175-cm2 flask, then add the diluted virus. Incubate at 37◦ C in a 5% CO2 incubator for 2 h. Mix the cells by shaking the flask every 15 min. Seed 5 ×107 cells into one 175-cm2 flasks (∼10 flasks/ roller bottle). Add a total of 25 ml RPMI-2.5 medium to each flask. Incubate at 37◦ C in a 5% CO2 incubator for 4–5 days. Using a disposable sterile cell scraper, detach the cells from the flask into the medium. Collect the cell suspension into sterile centrifuge tubes. Centrifuge the cell suspension at 1,000×g for 5 min at 4◦ C. Discard the supernatant. Resuspend the pellet in 5–10 ml PBS. Lyse the cells to release the virus by freezing and thawing three times, using a bucket of dry ice and a 37◦ C water bath. Virus yield can be improved by sonicating the lysate at 40 W for 2 min with 10 s intervals in a water bath sonicator. Aliquot and store at –80◦ C. Use one aliquot to determine the virus titers.

ECTV stock can also be prepared from the organs of infected mousepox-susceptible mice. We usually use BALB/c mice because their spleens and livers contain very high titers of virus 7–8 days PI. 1. Infect five BALB/c mice (see Section 3.1.5). 2. At 7 days PI, euthanize the mice by halothane inhalation. 3. Remove the spleen and the liver.

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4. Homogenize the tissues in 5 ml RPMI-2.5/mouse using a tissue homogenizer. 5. Release the virus by freezing and thawing three times using a bucket of dry ice and a 37◦ C water bath. Virus yield can be improved by sonicating the lysate at 40 W for 2 min with 10-s intervals in a water bath sonicator. 6. Centrifuge at 3000×g for 10 min at 4◦ C. Collect the supernatant. Aliquot and store at –80◦ C. Use one aliquot to determine the virus titers. 3.1.3. Purification of ECTV Stocks

Both the ECTV stocks produced from tissue culture and infected mice can be purified by sucrose gradient centrifugation (11,12). 1. One day before the procedure, prepare a sterile 24–40% continuous sucrose gradient in a sterile SW-27 centrifuge tube by carefully layering 6.8 ml of each of the following sucrose solutions in the tube in the following order: 40%, 36%, 32%, 28%, and 24% (all in 1 mM Tris–HCl, pH 9.0). Allow the gradient to sit overnight at 4◦ C. 2. Layer 3 ml of ECTV stock onto a cushion of 17 ml 36% sucrose solution (in 10 mM Tris–HCl, pH 9.0) in a sterile SW-27 centrifuge tube. Centrifuge for 80 min at 32,900×g at 4◦ C. 3. Aspirate and discard the supernatant. Resuspend the viral pellet in 1 ml 1 mM Tris–Cl, pH 9.0. 4. Sonicate once for 30 s in a water bath sonicator. 5. Overlay the sucrose gradient from step 1 with 1 ml sonicated viral pellet. 6. Centrifuge for 50 min at 26,000×g (12,000 rpm in an SW-27 rotor) at 4◦ C. The virus will appear as a milky band at the middle of the tube. 7. Carefully collect the virus band (∼10 ml) into a sterile SW27 tube, fill the tube with 1 mM Tris–Cl, pH 9.0. 8. Centrifuge the tube for 60 min at 32,900×g at 4◦ C. 9. Discard the supernatant and add 1 ml of 1 mM Tris–Cl, pH 9.0, to the pellet. Sonicate as previously in a water bath sonicator. Divide into aliquots and freeze at –80◦ C. Use one aliquot to determine the virus titers.

3.1.4. Plaque Assay for Quantifying Ectromelia Virus

ECTV infectivity is quantified using BSC-1 cell monolayers (see Note 1). 1. One day before the plaque assay, seed 3 × 105 BSC-1 cells/2 ml RPMI-10 medium in wells of six-well plates. Rock the plates very gently to ensure that the cells are seeded evenly.

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2. If determining virus titers in organs, go to step 3. If determining titers in virus stocks, go to step 6. 3. Weigh the organs individually and place into 5–10 ml RPMI-2.5 medium. 4. Disrupt the organs individually using a tissue homogenizer. 5. Freeze and thaw three times to release the virus. Centrifuge for 5 min at 3,000×g and transfer the supernatant to a new tube. 6. Make several 10-fold serial dilutions of the organ supernatant or the virus stock (using a separate pipette tip each time) in RPMI-2.5 medium. 7. Remove the medium from monolayers of BSC-1 cells. Add 500 ␮l of each dilution to individual wells of BSC-1 cell monolayers and incubate in a CO2 incubator for 1 h. Rock the plates very gentle every 15 min. 8. Add 2 ml RPMI-2.5 medium to each well. 9. Incubate in a CO2 incubator at 37◦ C for 5 days. 10. Aspirated the medium. Fix and stain the cells with 0.1% crystal violet solution for 10 min. 11. Aspirate the crystal violet solution and allow to dry. 12. Count the number of plaques and calculate the titer as follows: Virus titer (PFU/mL) = number of plaques × dilution factor × 2 (we normally calculate average of two duplicated wells). When calculating the titers in mouse organs, the percentage of homogenate must be taken into account and the titer is expressed as PFU/g tissue or PFU/organ. 3.1.5. Footpad Infection of Mice with ECTV

For ECTV infection, sex-matched mice are transferred to an infection room approved by the institutional animal facility (see Note 2). 1. Anesthetize mice by methoxyflurane inhalation by placing them in a jar containing cotton embedded with 100 ␮l methoxyflurane. Retrieve the mice when they are under deep anesthesia. 2. Using a 1-ml syringe with a 27-g needle, inoculate the mice in the left rear footpad with 25 ␮l PBS containing 0.1–3 103 PFU of ECTV (see Notes 3 and 4).

3.1.6. Determination of Resistance to ECTV Infection

1. Individually weigh groups of 5–10 mice. 2. Infect mice with ECTV as in Section 3.1.5. 3. Observe mice daily. Weigh daily starting on day 3 PI. Euthanize moribund mice and count as dead (see Note 5).

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3.2. Study of NK Cell Responses

Footpad infection with ECTV results in the recruitment and activation of NK cells in the draining popliteal lymph node, which peaks on day 2 PI. This initial activation is not accompanied by cell proliferation. As the virus spreads to and replicates in the liver and the spleen, NK cells also become activated in these organs, but in this case, peak activation is accompanied by strong proliferation. NK cell activation can be determined by flow cytometry by looking for changes in the expression of cell surface and intracellular molecules or by determining the ability of NK cells to kill targets ex vivo and in vivo. Proliferation can be determined by incorporation of bromodeoxyuridine (BrdU) or by dilution of a fluorescent marker into daughter cells. All this can be modified by depleting NK cells or blocking their cell surface molecules using specific antibodies. This may result in lack of virus control, which is evidenced by lethality and increased virus titer changes in different organs.

3.2.1. Preparation of Organs

Organs can be obtained to study NK cell responses or to purify NK cells.

3.2.1.1. To Obtain Lymphocytes from Spleen

1. Add 10 ml RPMI-10 medium to individual 60-15-mm ´ tissue culture dishes. 2. Place one spleen into each dish. 3. Make single cell suspensions by crushing spleen between two frosted slides. Using a transfer pipette, transfer to 15-ml tubes. 4. Centrifuge at 800×g for 4 min, discard the supernatant. 5. Add 10 ml 0.84% NH4 Cl buffer at room temperature to lyse the red blood cells (no need to incubate, mix the cells very well in the lysis buffer and centrifuge immediately). Centrifuge and wash the cells once with 10 ml RPMI-10 medium. 6. Count cells by trypan blue exclusion.

3.2.1.2. To Obtain Lymphocytes from LNs

1. For cell surface staining, place LNs in 10 ml RPMI-10 medium in a 60-15-mm ´ tissue culture dish. For intracellular cytokine staining, place the intact LNs in 2 ml RPMI10 medium containing 10 ␮g/ml brefeldin A and 10 IU/ml IL-2 in a well of a 24-well plate. Incubate for 1 h at 37◦ C and then transfer the intact organs to a 60-15-mm ´ tissue culture dishes containing 10 ml RPMI-10 medium (see Note 6). 2. Make single cell suspensions by crushing between two frosted slides. 3. Using a transfer pipette, transfer to 15-ml tubes. Centrifuge and wash the cells once with RPMI-10 medium.

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1. Add 10 ml HBSS buffer to one 60-15-mm ´ tissue culture dish/mouse. 2. Mice are exsanguinated from the orbital cavity to decrease the amount of blood in the liver (because we are working with ECTV-infected mice, we perform all the processing in a laminar flow hood making it inconvenient to perform a PBS perfusion although this can be done). 3. Remove the whole liver from the mouse, rinse the liver once in the dish. 4. Push the liver through a cell strainer (BD Falcon 40 ␮m) to make a single cell suspension. 5. Wash once with HBSS by centrifugation. 6. In one 15-ml centrifuge tube, resuspend the cells of one liver in 10 ml 35% Percoll (in HBSS) solution containing 100 U/ml heparin. Centrifuge for 15 min at 830×g at room temperature. 7. Aspirate the supernatant. Resuspend the lymphocyte pellets in 10 ml of 0.84% NH4 Cl solution and incubate at RT for 10 min to lyse the red blood cells. 8. Wash twice with RPMI-10 medium by centrifugation at 830×g at room temperature. 9. Count by trypan blue exclusion. The yield of lymphocytes from an uninfected mouse is ∼1–3 million cells/liver and may increase up to 10-fold in infected mice.

3.2.2. Analysis of NK Cells and their Functions by Flow Cytometry

Fluorochrome-conjugated mAbs are used for surface staining of NK cells. Typical markers used for distinguishing NK cells are anti-NK1.1 (e.g., PK136) or anti-CD49b (e.g., DX5). Because NKT cells also express these molecules, cells should also be stained with anti-CD3, which is expressed by NKT but not NK cells. Additional markers that can be used are those directed to activating and inhibitory receptors, maturation markers, etc. (e.g., Ly49s, NKG2D, CD94, CD11b, CD27). Cells can also be stained for intracellular molecules such as granzyme B or IFN-␥. In addition, cells can also be stained intracellularly for bromodeoxyuridine (BrdU) incorporation to determine proliferation. In addition to NK cells, other cell populations such as CD4 T, CD8 T, and NKT cells can also be labeled to determine the effects of NK cells in these populations (e.g., during NK cell depletion experiments) (see Note 7. 8) 1. Staining of lymphocytes. 2. Resuspend spleen, lymph node, or liver lymphocytes (prepared as described in Section 3.2.1) to 2×107 cells/ml. 3. Add 100 ␮l per well to V-bottom 96-well plates.

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4. To each well add 50 ␮l/well anti-Fc␥RII/III mAb (Clone 2.4G2) to block non-specific binding to Fc receptors. Incubate for 15 min at 4◦ C. 5. Centrifuge at 830×g for 3 min, flick off the medium. 6. Vortex to resuspended the pellet. Add fluorochromelabeled surface antibodies in 50 ␮l of staining buffer (e.g., anti-NK1.1, -CD49b, -CD3) to each sample. Appropriate isotype controls should also be used (in general, we use 0.1 ␮g of Ab/sample but titering each Ab is strongly recommended). 7. Incubate for 30 min at 4◦ C. 8. Add 150 ␮l PBS to each well, centrifuge at 830×g for 3 min, flick off the supernatant, vortex, and wash the cells two more times with 200 ␮l PBS. 9. Centrifuge, flick the supernatant, and vortex. For intracellular staining, go to Step 10. For surface staining only, go directly to step 17. 10. Add 50 ␮l Cytofix/Cytoperm solution (BD) to fix and permeabilize the cells. Incubate the cells for 15 min at 4◦ C. 11. Wash cells twice with 200 ␮l Perm/Wash buffer (BD). 12. Centrifuge, flick the supernatant, and vortex. For BrdU incorporation, go to step 13. For other intracellular staining, go to step 15. 13. Resuspend cells in 100 ␮l DNase (300 ␮g/ml in PBS), incubate the plates at 37◦ C for 1 h. 14. Wash twice with 200 ␮l Perm/Wash buffer. 15. Add one or more labeled Abs to intracellular molecules (e.g., anti-IFN-␥, -granzyme B, -BrdU) or appropriate isotype controls to each well (0.1 ␮g/sample in 50 ␮l Perm/Wash buffer) and incubate at 4◦ C for 30 min. Mix gently by tapping the plate. 16. Wash two times with Perm/Wash buffer. 17. Centrifuge, flick the supernatant, and vortex. Resuspend in 200 ␮l 0.5% PFA in PBS. 18. Transfer the cells to 5-ml polystyrene tubes. Analyze by flow cytometry, collecting at least 500,000 events. 3.2.3. Adoptive Transfer of Lymphocytes to Track NK Cell Migration and Proliferation

All leukocytes in B6 mice express CD45.2, while congenic B6.SJL mice express CD45.1. By adoptive transferring of NK cells from B6 mice to B6.SJL mice (or vice versa), CD45.1 and/or CD45.2 staining and flow cytometry can be used to track the transferred cells. Cells can be transferred as unlabeled whole lymphocyte populations or labeled with CFSE to monitor proliferation (13,14).

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Cells can also be transferred as purified NK cells or even FACSsorted purified subpopulations of NK cells (see Notes 9 and 10). 1. Prepare single cell suspension from pooled spleens and LNs from B6 or B6.SJL mice as described in Section 3.2.1. 2. If transferring unlabeled whole lymphocytes, go to step 8. If transferring purified NK cells, go to step 3. If transferring lymphocytes stained with CFSE, go to step 4. 3. Purify NK cells using anti-CD49b conjugated microbeads (positive selection, Miltenyi Biotec) or NK cell isolation kit (negative selection, Miltenyi Biotec) and an LS column (Miltenyi Biotec) according to the manufacturer’s instructions. If purification of subpopulations is necessary, label purified NK cells with antibodies to desired surface markers and use a flow cytometer to sort the populations of interest. If cells need to be labeled with CFSE, go to step 4, otherwise, go to step 8. 4. Wash the cells once with PBS/0.1% BSA buffer. 5. Aspirate and resuspend the pellet of cells in the residual buffer by flicking, quickly add pre-warmed (37◦ C) PBS/0.1% BSA buffer containing 4 ␮M CFSE to the cells to a final concentration of 1×107 cells/ml. Rapidly mix the cells in the buffer by pipetting. 6. In a water bath, incubate the cells at 37◦ C for 10 min, mix the cells every 2–3 min for even labeling. Wash the cells twice with PBS –0.1% BSA. 7. Wash the cells once with PBS. 8. To transfer unlabeled or labeled whole lymphocytes, resuspend cells to 2–4×107 /ml (1–2×107 lymphocytes will be transferred to the recipient mouse) in PBS. To transfer unlabeled or labeled purified NK cells or NK cell subpopulations, resuspend cells to 1–6 × 106 /ml in PBS (5×105 – 3×106 NK cells will be transferred). 9. Inject 500 ␮l into the tail vein of the recipient mice. Rest the mice for 1 day. 10. Infect the mice with ECTV by footpad inoculation. 11. The distribution and proliferation (CFSE dilution) of transferred NK cells can be determined at different times post ECTV infection by FACS in LN, spleen, and liver as described in Section 3.2.2. 3.2.4. Determination of Cytolytic Activity Using the 51 Cr-Release Assay

The use of 51 Cr requires a license to work with radioactivity. 51 Cr emits gamma radiation and is blocked only with lead shielding. Manipulate your 51 Cr stocks behind a lead shield. When using the 51 Cr stock, work quickly and try to keep the container shielded and away from your body. Dispose of all materials as radioactive.

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Different target cells can be used to measure the cytolytic activity of NK cells. The assay is carried out in triplicate using various effector:target ratios with the number of 51 Cr-labeled targets kept constant at 5×103 per well in U-bottom 96-well tissue culture plates. This recipe is for two 96-well plates. If you have more samples, increase volumes accordingly. 1. Infect mice with ECTV. 2. Prepare single cell suspension from spleens as described in Section 3.2.2.1. For footpad inoculation, the peak of NK cell killing activity in the spleen occurs on day 5 PI. 3. Purify NK cells from spleens using the NK cell isolation kit and an LS column (Miltenyi Biotec) according to the manufacturer’s instructions. 4. Resuspend NK cells in RPMI-10 medium for the desired highest effector:target ratio [e.g. target cells are always diluted to 1×105 cells/ml (5×103 cells in 50 ␮l medium to each well)]. Thus, for a 100:1 effector:target ratio, resuspend the NK cells at 5×106 cells/ml). 5. In U-bottom 96-well plates, make 1:3 serial dilutions of the NK cells to a final volume of 100 ␮l/well (for a 100:1 effector:target ratio, the first effector sample should contain 5×105 cells). Reserve six wells without effectors for maximal and spontaneous release controls. 6. In a 15-ml conical tube, resuspend 106 target cells in 100 ␮l RPMI-10 medium. 7. Add 100 ␮l fetal bovine serum. Tap the bottom of the tube to mix. 8. Using a disposable 1-ml syringe, add 100 ␮l (0.1 mCi) 51 Cr to the bottom of the tube. Tap the bottom of the tube to mix. 9. Incubate at 37◦ C for 1 h. 10. Wash cells four times with 3 ml RPMI-10 medium. Do not fill the tube to avoid contaminating the centrifuge with 51 Cr. Spent radioactive supernatant should be disposed according to institutional regulations. We normally drop the supernatant into cat litter to make solid rather than liquid waste and keep the waste behind lead until it decays. 11. Resuspend target cells in 10 ml RPMI-10 medium (105 cells/ml). 12. Add 50 ␮l (5×103 cells) to the 96-well plate containing NK cell effectors. 13. Centrifuge at 200×g for 1 min to favor cell–cell contact. The plates are incubated at 37◦ C for 4 h.

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14. Add 100 ␮l 1% Triton X-100 in water or 100 ␮l RPMI-10 medium to the maximal and spontaneous release control wells, respectively. 15. Centrifuge at 830×g for 3 min. 16. Being careful not to disrupt the pellet, collect 50 ␮l culture supernatant from each well and measure radioactivity in a gamma counter. For reading radioactivity, we use a Packard TopCount instrument (PerkinElmer), as described in steps 17–19. The method should be adapted to your instrument. If measuring radioactivity with another instrument, measure cpm and go to step 20. 17. When using a TopCount instrument, transfer 50 ␮l culture supernatant from each well with a 12-well channel pipette to white 96-well plates containing 75 ␮l Microscint40 scintillation fluid (PerkinElmer) or the scintillation fluid that your instrument requires. The same tips can be used for the whole plate, provided the lowest E:T ratios are harvested first and the maximal release controls last. 18. Seal the plate with microplate press-on adhesive sealing film (PerkinElmer). Vortex the plates to mix and briefly centrifuge. 19. Read the plates using the TopCount instrument 20. Average the values of the three replica wells and calculate specific lysis according to the formula: specific lysis = [(experimental release–spontaneous release)/(maximal release–spontaneous release)]×100. 3.2.5. In Vivo NK Cell Cytotoxicity Assay

NK cell cytolytic function can also be determined in vivo using MHC class I-deficient target cells, which trigger NK cell killing due to loss of inhibitory ligands. For this purpose, we usually use lymphocytes from TAP-deficient mice (TAP KO) that have low-level expression of class I MHC at the cell surface. 1. Obtain total lymphocytes from spleen and LNs from B6 mice and TAP KO mice as described in Section 3.2.1. 2. Label B6 and TAP KO cells with CFSE as described in Section 3.2.3, with the exception that the B6 and TAP KO lymphocytes are labeled for 10 min at 37◦ C with 0.8 and 4 ␮M CFSE, respectively, and resuspended in PBS. 3. Count live cells by trypan blue exclusion and adjust to 2–4×107 viable cells/ml. 4. Mix equal volumes of both cell types. Inject 500 ␮l cell suspension (1–2×107 cells of each population) into the tail vein of na¨ıve or ECTV-infected mice. Wait for 2 h.

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5. Euthanize the mice by halothane inhalation. 6. Collect lymph nodes and spleens and make single cell suspensions. 7. Analyze by flow cytometry gating on CFSE-positive cells. High (TAP KO) and low (B6) populations should have wellseparated peaks. 8. Calculate specific lysis using the following formula: percentage specific lysis = [1 – (ratio uninfected/ratio infected)] x 100, where ratio = (percentage CFSElow /percentage CFSEhigh ). 3.2.6. BrdU Incorporation Assay

In addition to CFSE dilution, NK cell proliferation can be determined by measuring BrdU incorporation. BrdU is a uridine derivative that can be incorporated into DNA in place of thymidine. Cells synthesizing DNA (in S-phase of the cell cycle) will incorporate BrdU into the DNA. Intracellular staining with antiBrdU antibodies can identify cells that are proliferating (undergo DNA synthesis during exposure to BrdU) after virus infection. In this assay, mice are injected with BrdU IP and euthanized 3 or 4 h later. Thus, the assay determines the cells that divided during this brief time. 1. Inject mice (infected or na¨ıve control) IP with 200 ␮l 10 mg/ml BrdU in PBS. 2. Euthanize mice 3–4 hours later, obtain desired organs (lymph nodes, spleen, liver), and stain as in Section 3.2.2 using Abs to distinguish NK cells (i.e. NK1.1 or CD49b and CD3) and anti-BrdU, and other surface or intracellular molecules, if desired.

3.2.7. Treatment of Mice with NK Cell-Depleting or Receptor-Blocking Antibodies

To determine the role of NK cells and their receptors in mediating resistance against virus infection, the NK cell responses can be modulated by treatment with Abs. In our laboratory we have used this procedure to deplete NK cells and to block the activating receptor NKG2D, which is described here, but other Abs could also be used. However, the dose, time, and number of inoculations and the effectiveness of the treatment must be tested (see Notes 11–14). 1. Dilute the required amount of depleting/blocking or appropriate control Ab (e.g., rat IgG, rabbit serum) in 500 ␮l PBS/mouse. 2. Inoculate mice IP using a 3-ml syringe and a 25-g needle. 3. Infect the mice with ECTV by footpad inoculation. 4. Repeat Ab inoculation as necessary. Determine lethality, as described in Section 3.1.6, or study their responses, as described in Section 3.2.2.

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4. Notes 1. Plaque assays can be performed with agarose overlay. For this purpose, aspirate the medium from step 3 in Section 3.1.5, then add 2 ml 1% agarose in RPMI-2.5 [by mixing equal volumes of 2% agarose in H2 O (sterile by autoclaving) and 2× RPMI medium containing 5% FBS and 2× antibiotics] to the plates. Make sure that the temperature of the agarose overlay is not too high or too low (around 45◦ C). Allow the overlay to solidify at room temperature and place the plates in the CO2 incubator at 37◦ C for 4–5 days before staining and counting the plaques. 2. The Moscow strain of ECTV is highly virulent and may require euthanizing all the mice in a colony should outbreaks occur, with tremendous economical losses. To avoid this, we have a biosafety level 3 (BSL3)-type suite where we perform all the work involving live ECTV. This suite has a tissue culture room and a mouse infection room. Cells and tissues are retrieved only after the virus has been inactivated by fixation or autoclaving. No mouse entering the suite can be retrieved. All waste and mouse cages are autoclaved prior to exiting the suite. It is important to avoid entering the regular mouse colony on the same day after entering the infection room. Good animal husbandry and respect for these rules are critical when working with ECTV. 3. For virus infection of mice, we usually do not use purified virus since the crude virus stock will be diluted many times, thereby reducing any impact from cell debris. For virus infection of cell lines, we use purified virus stock, since they have higher virus titers and are free of any cell debris. 4. ECTV is quite stable at –80◦ C; however, repeated freeze and thaw will reduce the titers. We usually prepare aliquots ready for infection and freeze them at –80◦ C, discard any leftovers after single use to avoid repeat freeze and thaw. 5. Variations in lethality can be determined using the logrank test. Usually the use of 300–3,000 PFU will give a good indication of an increase in susceptibility using 5–10 mice/group. However, when the increase in susceptibility is mild, it may be necessary to increase the sample size to increase the potency of the assay. After a pilot experiment, it is recommended to consult with a statistician to determine the appropriate group size. It may also be useful to determine the dose that is lethal for 50% of the animals (LD50). This can be done by using groups of 6–10 mice and by testing various PFU (from less than 1 to ∼107 PFU).

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6. To obtain single cell suspensions for the intracellular cytokine staining of LNs, we normally use the method described in Section 3.2.1.2. Alternatively, the LNs can be prepared as single cell suspensions before BFA treatment. Another alternative that we have used is to inoculate mice with 250 ␮g BFA in PBS i.v. (15) and collect the organs after 2–3 hours. However, the IFN-␥ staining of NK cells is lower when using this approach than with the two other methods. 7. For each experiment, it is very important that you have na¨ıve control mice and also isotype antibody controls. 8. We usually perform cell staining in V-bottom 96-well plates because of lower cell loss during washing, especially for intracellular staining where fixation alters the density of the cells. 9. It is a good practice to set aside some cells to analyze the transferred cell population. Controls should include uninfected mice transferred with the relevant lymphocyte population and a na¨ıve mouse that did not receive transferred cells. 10. For short-term NK cell migration and distribution assays, instead of using NK cells from B6.SJL mice, NK cells from B6 mice can be labeled with 4 ␮M CFSE and then transferred to recipient B6 mice. 11. NK cell depletion in mouse strains that express NK1.1 (e.g., B6) can be accomplished with anti-NK1.1 mAb (200 ␮g) PK136 (mouse IgG2a), or anti-asialo GM1 (Wako, 20 ␮l rabbit polyclonal). In strains that do not express NK1.1 (e.g., BALB/c, 129, DBA2/J), only antiasialo GM1 can be used. Normally, one dose of PK136 or anti-asialo-GM1 is sufficient to deplete NK cells up to 7 days after treatment and induce susceptibility in B6 mice when treated 24 h before the infection. NK cells can be depleted 1 day before to several days PI to determine when NK cells are required for resistance. If constant NK cell depletion is needed, treat the mice with depleting Ab every 7 days. NKG2D is blocked with 200 ␮g of the nondepleting anti-NKG2D mAb CX5 (rat IgG1 obtained from Dr. Lewis Lanier, UCSF) 1 day before and 2 days postinfection. Variations can be used to determine when NKG2D signaling is required. 12. Depletion of NK cells requires large amounts of Abs. Some can be purchased (e.g., anti-asialo GM1). For others, buying the purified Abs would be too expensive and hybridomas should be obtained to produce large batches of Ab. For this purpose, we grow hybridomas in protein-free medium in CELLine CL 1000 flasks (Wilson Wolf) according to

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manufacturer’s instructions. We then purify the Abs by ammonium sulfate precipitation according to standard procedures (16). 13. For each new batch of Ab, it is always necessary to test that the procedure is effective. To test depletions, perform flow cytometry analysis (see Section 3.2.2) detecting NK cells with Abs to a molecule different from that used for the depletion (e.g., when depleting with antiNK1.1, stain samples with anti-CD49b (DX5) and antiCD3). When testing for blockade, stain NK populations and the molecule of interest with the same Ab used for blockade (e.g., for NKG2D blockade, use fluorochromeconjugated anti-NK1.1, anti-CD3, and CX5 to label cells). 14. It is useful to bleed the mice and perform a FACS stain 1 day after the mice are given the depleting Ab to make sure that NK cells are depleted.

References 1. French, A. R., and Yokoyama, W. M. (2003) Natural killer cells and viral infections. Curr Opin Immunol 15, 45–51. 2. Biron, C. A., Nguyen, K. B., Pien, G. C., Cousens, L. P., and Salazar-Mather, T. P. (1999) Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol 17, 189–220. 3. Arase, H., Mocarski, E. S., Campbell, A. E., Hill, A. B., and Lanier, L. L. (2002) Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors. Science 296, 1323–6. 4. Daniels, K. A., Devora, G., Lai, W. C., O Donnell, C. L., Bennett, M., and Welsh, R. M. (2001) Murine cytomegalovirus is regulated by a discrete subset of natural killer cells reactive with monoclonal antibody to Ly49H. J Exp Med 194, 29–44. 5. Lee, S. H., Girard, S., Macina, D., Busa, M., Zafer, A., Belouchi, A., Gros, P., and Vidal, S. M. (2001) Susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the C-type lectin superfamily. Nat Genet 28, 42–5. 6. Jacoby, R. O., Bhatt, P. N., and Brownstein, D. G. (1989) Evidence that NK cells and interferon are required for genetic resistance to lethal infection with ectromelia virus. Arch Virol 108, 49–58.

7. Parker, A. K., Parker, S., Yokoyama, W. M., Corbett, J. A., and Buller, R. M. (2007) Induction of natural killer cell responses by ectromelia virus controls infection. J Virol 81, 4070–9. 8. Fang, M., Lanier, L. L., and Sigal, L. J. (2008) A role for NKG2D in NK cellmediated resistance to poxvirus disease. PLoS Pathog 4, e30. 9. Fenner, F.,Henderson, D. A., Arita, I., Jezek, Z., Ladnyi, D., and Organization, W. H. (1988) Smallpox and its eradication, World Health Organization, Geneva. Chapter 1, 2–68. 10. Delano, M. L., and Brownstein, D. G. (1995) Innate resistance to lethal mousepox is genetically linked to the NK gene complex on chromosome 6 and correlates with early restriction of virus replication by cells with an NK phenotype. J Virol 69, 5875–7. 11. Earl, P. L., Moss, B., Wyatt, L. S., and Carroll, M. W. (2001) Generation of recombinant vaccinia viruses. Curr Protoc Mol Biol Chapter 16, Unit 16 17. 12. Scalzo, A. A., Farrell, H. E., and Karupiah, G. (2000) Techniques for studying murine natural killer cells in defense against viral infection. Methods Mol Biol 121, 163–77. 13. Lyons, A. B., and Parish, C. R. (1994) Determination of lymphocyte division by flow cytometry. J Immunol Methods 171, 131–7.

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14. Barber, D. L., Wherry, E. J., and Ahmed, R. (2003) Cutting edge: rapid in vivo killing by memory CD8 T cells. J Immunol 171, 27–31. 15. Liu, F., and Whitton, J. L. (2005) Cutting edge: re-evaluating the in vivo cytokine

responses of CD8+ T cells during primary and secondary viral infections. J Immunol 174, 5936–40. 16. Kent, U. M. (1999) Purification of antibodies using ammonium sulfate fractionation or gel filtration. Methods Mol Biol 115, 11–8.

Chapter 29 Activation of Human NK Cells by Malaria-Infected Red Blood Cells Amir Horowitz and Eleanor M. Riley Abstract This chapter describes a protocol to assess activation of human NK cells following in vitro stimulation with malaria-infected red blood cells. Activation is assessed by flow cytometry, staining for cell surface expression of CD69 and accumulation of intracellular IFN-␥. Procedures are described for in vitro propagation and purification of Plasmodium falciparum parasites, separation of peripheral blood mononuclear cells from heparinised blood by density centrifugation, in vitro culture of PBMC and for staining and analysis of PBMC by flow cytometry. Some examples of typical FACS plots are shown. Key words: Malaria, Plasmodium falciparum, CD69, IFN-␥, intracellular cytokine staining, flow cytometry, human.

1. Introduction Although natural killer (NK) cells are typically thought of as cells that kill neoplastic, virally infected, stressed or otherwise diseased cells as a result of their abnormal expression of NK cell ligands, such as MHC class I and class I-like molecules, it is now widely recognised that NK cells are an important (and sometimes essential) source of cytokines during the innate immune response to many types of infection, including infections in which there is no marked change in MHC class I expression. The various means by which NK cells are activated during bacterial, protozoal, fungal and viral infections have recently been reviewed (1). Importantly, in almost all infections studied to date, NK cells are absolutely dependent upon signals from accessory cell populations (including dendritic cells and macrophages) for their ability to respond to K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 29, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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pathogens or pathogen-infected cells (2–5). It is essential therefore to use mixed cell populations [e.g. human peripheral blood mononuclear cells (PBMCs) or murine splenocytes] rather than NK cell lines or purified NK cells when examining the ability of NK cells to respond to pathogens. If for some reason it is necessary to use purified NK cells for such experiments, it is important to add adherent accessory cells to the cultures to provide a source of the essential cytokines (IL-12, IL-18, possibly IL-2 and IFN-␣) and cell contact-mediated signals (ICAM-1 and others). Note, however, that it has proved difficult to recapitulate the full NK cell response that is seen with PBMCs or splenocytes using NK cell lines plus accessory cells (3). A number of markers have been described for assessing NK cell activation during infection including cell surface expression of the early activation marker CD69, the high-affinity IL-2R␣ chain (CD25), the lysosomal-associated membrane protein LAMP-1 (CD107a) and intracellular accumulation of effector molecules including granzymes, perforin, IFN-␥ and TNF-␣. There is also evidence for altered expression of NK cell regulatory receptors such as CD94/NKG2A on human NK cells stimulated with malaria-infected red blood cells (6), CD94/NKG2C on NK cells from human cytomegalovirus-infected individuals (7) and NKG2C and NKG2C2 on SIV-infected simian NK cells (8). All of these markers are typically upregulated within 12–24 h of in vitro co-culture with the pathogen (9). In the case of malaria infection, NK cells have been shown to be an essential early source of IFN-␥ in experimental Plasmodium chabaudi chabaudi AS and P. yoelii infections of mice, containing the first wave of infection and allowing time for the animal to develop an effective adaptive response which eventually clears the infection (10–13). It has recently been reported that activated NK cells flood the peripheral circulation during the first 18 h of P. chabaudi infection, suggesting that peripheral blood mononuclear cells rather than splenocytes may be the optimal tissue for the analysis of NK cell responses to this infection in mice (14). In vitro studies where human PBMCs (from malaria na¨ıve blood donors) are cultured with P. falciparum-infected red blood cells (iRBCs) have indicated that NK cells are among the first cells to respond, upregulating CD69, CD25, LAMP-1 and making IFN-␥ (9, 15, 16). Importantly however, these responses are not seen in all donors; the proportion of NK cells producing IFN-␥ after in vitro culture with iRBCs varies from less than 0.1% in some individuals to greater than 70% in others (6, 9, 15). It is essential therefore to include a good positive control in these in vitro assays to ensure that NK cells from “non-responding” donors are indeed viable. We have found that a combination of rIL-12 and rIL-18 serves as an excellent positive control, reliably inducing a substantial proportion of NK cells to produce IFN-␥ within 18 h

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in all subjects studied to date. Also, whilst IFN-␥ is produced only in highly activated NK cells, we have found that CD69 is significantly upregulated even on NK cells which do not produce IFN-␥. Thus, we now routinely include staining for CD69 in our flow cytometry panels as another good positive control. Human NK cells are defined as large, granular, CD3− CD56 (NCAM)+ lymphocytes. This population can be divided into CD56bright and CD56dim subsets. CD56bright NK cells bear the surface phenotype CD16− CCR7+ CD62L+ , are largely killer cell immunoglobulin-like receptor (KIR)− , secrete large amounts of cytokines (IFN-␥ and TNF) and can traffic to lymph nodes (LN). CD56dim NK cells express high levels of CD16 (Fc␥RIIIA receptor), are KIR+ , produce large amounts of cytokines, possess cytotoxic properties and are often recruited into inflamed tissues (17, 18). Flow cytometry has been an invaluable tool in the phenotypic and functional analysis of NK cell responses to infections, including malaria. In this chapter, we describe methods for phenotyping in vitro NK cell responses to iRBCs by characterising the frequency and absolute number of CD3− CD56bright/dim NK cells that produce IFN-␥ and upregulate the activation marker CD69. Suitable reagents for detecting the expression of IL-2R␣ (CD25), LAMP-1 (CD107a), KIR and CD94/NKG2 receptors can be found in our published research papers (6, 9). For NK cell activation by malaria, it is necessary to use mature schizont-stage parasites and − because the NK assay is run over a period of 12–24 h − it is crucial to start the experiment with the parasites at just the right stage of development. Excellent illustrations of the P. falciparum life cycle that provide a useful tool for identifying specific stages of the life cycle under the microscope can be found at http://www.who.int/malaria/docs/hbsm diagnosis.htm

2. Materials 2.1. Parasite Culture

1. Parasite culture medium: for in vitro propagation of P. falciparum iRBCs (pH adjusted to 7.2–7.4): RPMI1640 medium (Gibco) supplemented with 25 mM HEPES (Sigma), 28 mM sodium bicarbonate (BDH), 20 ␮g/L hypoxanthine (Sigma) and 10% normal human AB serum (UK National Blood Service or equivalent) (see Note 1) and gentamicin (0.08%, v/v) (Sigma) (see Note 2). 2. Solutions for thawing parasites: – Solution 1: NaCl (12%) in deionised water

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– Solution 2: NaCl (1.6%) in deionised water – Solution 3: NaCl (0.9%) + glucose (0.2%) in deionised water 3. Gas for culturing P. falciparum: 3% O2 , 4% CO2 and 93% N2 (see Note 3) 4. 5% sorbitol solution for parasite synchronisation: 2.5 g of sorbitol in sterile water and then 0.2 ␮m filter sterilised (see Note 4). 5. RPMI-1640 medium for washing red blood cells for adding to cultures. 6. MACS “LD” separation columns (Miltenyi Biotec) for magnetic isolation of schizont-stage iRBCs. 7. Giemsa stain solution (10×) (BDH). 8. Giemsa buffer: 1.00 g/L Na2 HPO4 ; 0.70 g/L KH2 PO4 ; dissolve slowly in water and use to dilute Giemsa stain to 1×. 9. Glass microscope slides (1.0–1.2 mm thick/twin-frost; VWR). 10. Immersol 518N immersion oil (Zeiss). 2.2. Isolating and Culturing Peripheral Blood Mononuclear Cells (PBMCs)

1. Histopaque-1077 (Sigma) for density-gradient separation of whole blood. 2. RPMI-1640 medium (Gibco) + heat-inactivated human AB serum (1%) for washing cells (keep on ice or at 4◦ C until ready for use). 3. Leucocyte culture medium: RPMI-1640 medium (Gibco) + heat-inactivated autologous plasma (10%) + 100 IU/ml penicillin/streptomycin (Gibco) + 2 mM L-glutamine (Gibco). 4. Trypan blue solution (0.4%) filtered through a 0.2-␮m filter, using a syringe; for microscopic determination of PBMC viability (see Note 5). 5. 96-Well U-bottom plates (Sterile) (any supplier). 6. Cytokines: recombinant human IL-12 (Peprotech) (2 ␮g/ml); recombinant human IL-18 (MBC) (2 ␮g/ml). 7. Brefeldin A (eBioscience): stock solution of 3 mg/ml in methanol (which is equal to 1000 ´final working concentration). Dilute this 1:100 in RPMI-1640 medium to give a 30 ␮g/ml (10×) solution.

2.3. Flow Cytometry Reagents

1. Antibodies: anti-human CD3-peridinin chlorophyll (PerCP) (e.g. SK7, mouse IgG1 , BD Biosciences, cat. No. 345766), anti-human CD56-allophycocyanin (APC) (e.g. N901, mouse IgG1 , Beckman Coulter cat. No. IM2474),

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anti-human CD69-phycoerythrin (PE) (e.g. CH/4, mouse IgG2A , Caltag, cat. No. MHCD6904), anti-human IFN-␥fluorescein (FITC) (e.g. 4S.B3, mouse IgG1 , Serotec, cat. No. MCA1581F). 2. Cold wash buffer: phosphate-buffered saline (PBS) with 1% heat-inactivated fetal calf serum (FCS) (heat inactivation at 56◦ C for 30 min − invert gently every 10–15 min). 3. 1.2-ml microtubes (alpha laboratories) for storage of cells to be acquired on the flow cytometer. 4. Paraformaldehyde (Pfa) solution (1% diluted in 1 × PBS) prepared weekly and stored at 4◦ C protected from light using an aluminium foil. 5. Sterile disposable 3-ml pastettes (Fisher Scientific).

3. Methods Use sterile technique throughout the following procedures. 3.1. Culturing P. falciparum (Starting with a Frozen Aliquot)

P. falciparum parasites (strain 3D7) are grown in ORh− human erythrocytes in parasite culture medium (see Note 6). 1. Prepare materials in advance, allowing 15 min to warm thawing solutions and culture medium at 37◦ C. Label tubes and flasks. 2. Place cryotube of frozen iRBCs in a water bath preset to 37◦ C. Gently swirl cryotube in the water bath until the contents are just thawed. Do not submerge the vial in water to prevent (non-sterile) water entering the cryotube. Wipe outside of cryotube with 70% ethanol. 3. Transfer cryotube contents into a sterile 15-ml tube, measure the volume and add 20 vol.% (i.e. 200 ␮l/1 ml blood) of 12% NaCl (solution 1). Add the solution dropwise using a pastette and shake gently or mix with pastette between each drop. Allow to stand without agitation for 3–5 min at RT. 4. Add 10 × the original volume (i.e. 9 ml per 1 ml of frozen iRBCs) of 1.6% NaCl (solution 2) dropwise with mixing as before. Centrifuge at 500 × g for 5 min at 25◦ C with the brakes on and discard supernatant. 5. Add 5 ml of 0.9% NaCl (solution 3) dropwise with mixing as before. Centrifuge at 500 × g for 5 min at 25◦ C with the brakes on. 6. Discard the supernatant. Add fresh ORh− human erythrocytes to bring the volume of packed blood to 0.5 ml. Gently

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resuspend iRBC pellets in 4.5 ml of complete parasite culture medium to a haematocrit level of 10% and transfer to a T25-cm2 tissue culture flask. Gas the flask with 3% O2 , 4% CO2 and 93% N2 for 30 s using a sterile, blunt-ended needle connected via a filter to the gas cylinder by plastic tubing. Tightly seal the tissue culture flask and place in an incubator set to 37◦ C (see Note 7). 7. For optimal growth of iRBCs, the culture medium should be replaced every 24 h (see Note 8). Aspirate the spent medium using a sterile pastette and replace with an equal volume of pre-warmed complete culture medium. Spent medium should be treated to render it non-infectious. 8. Every 1–2 days, prepare a Giemsa-stained smear of the culture and count the proportion of parasite-infected red blood cells (“parasitaemia”) (see Note 9). Use 20 ␮l of the iRBC suspension to prepare a smear on a clean glass microscope slide. Stain with Giemsa (10-fold diluted in freshly prepared Giemsa buffer) for 10–15 min. Examine at 100× magnification using immersion oil. For routine propagation of parasites, when parasitaemia reaches approximately 3–4%, split the culture and add fresh medium and freshly washed uninfected red cells to reduce the parasitaemia to approximately 0.5%. 9. Ideally, for an NK assay, the parasite culture will be at a parasitaemia > 5% and the parasites will be at the “early segmented” stage as seen in the first two examples of the “schizont” series in the upper panel, or the first example of a schizont in the lower panel, of the illustrations referred to above (http://www.who.int/malaria/docs/hbsm diagnosis.htm). Synchronisation will be needed in order to get parasites at high schizont parasitaemia. 3.2. Isolating PBMCs and Set-Up of NK Cell Stimulation Assays

It is important to prepare the PBMCs and/or purified human cell populations before beginning to prepare the iRBCs. However, since the assay depends critically on having iRBCs at the appropriate stage of development, it is wise to check the status of the parasite culture (by microscopy) prior to preparing the PBMCs. 1. Collect 50 ml of blood from a donor into sodium heparin (10 IU/ml blood) and dilute with an equal volume of RPMI-1640 medium (room temperature, RT). 2. In a 50-ml conical centrifugation tube, gently layer 35 ml of diluted blood on top of 15 ml of Histopaque-1077 using a 25-ml pipette (i.e. you will need three tubes to process the original 50 ml sample). Centrifuge for 25–30 min at 940×g with the brakes off at RT (see Note 10).

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3. Gently remove 10 ml of diluted plasma from the upper layer of each Histopaque-1077 tube into a new, labelled 50-ml conical centrifugation tube and heat inactivate the plasma by incubating it in a water bath preset to 56◦ C for 30×min. Every 10–15 min, remove the tube from water bath and gently invert a few times. 4. Using a sterile disposable 3-ml plastic pastette, collect the PBMCs localised at the interface between the Histopaque1077 and the medium/plasma and transfer the cells to a new, labelled 50-ml conical centrifugation tube. Raise the volume to 50 ml using washing medium (RPMI-1640 plus 1% autologous heat-inactivated AB serum). Centrifuge at 500 × g for 10 min at 4◦ C with the brakes on. 5. Discard the supernatant and gently resuspend the white cell pellet in 1 ml of washing medium trying to avoid creating air bubbles as this can affect the viability of cells (see Note 11). Raise the volume to 50 ml with washing medium 6. Transfer 20 ␮l of cell suspension into a microtube and set on ice for cell counting. Wash the cells for a second time by centrifuging at 500×g for 10 min at 4◦ C with the brakes on. 7. During the centrifugation, count the cells using a haemocytometer and record the number of viable cells. 8. Prepare leucocyte culture medium. 9. After centrifugation of PBMCs, discard the supernatant. Gently resuspend the cell pellet in a sufficient volume of leucocyte culture medium to give a cell concentration of 2.0 × 106 PBMCs/ml. Place tube on ice (or at 4◦ C). 3.3. Isolation of iRBCs by Magnetic LD Separation Columns

iRBCs are fragile and should be prepared only once all the PBMCs or purified leucocytes are ready. Set-up a sterile LD column onto its fixed position on the magnet. 1. Run 3 ml of RPMI-1640 (RT) through the column and allow it to drain into a tube. Repeat this step (see Note 12). 2. Remove the tissue culture flask containing iRBCs from the incubator and harvest the medium into a conical centrifugation tube (medium is to be disposed of in biohazard waste), being careful not to disrupt the layer of RBCs resting on the bottom of the flask. Remove most of the medium using either a 10-ml or a 25-ml serologic pipette, trying to leave behind approximately 10 ml of medium in the flask. 3. Close the lid of the flask and gently shake the flask to detach the layer of iRBCs. Once all cells have detached from the flask, transfer the entire contents into a new sterile 15-ml

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centrifuge tube. Centrifuge at 500×g with brakes on at RT for 5 min. 4. Discard the remainder of the medium from the tube by pipetting without disturbing the iRBC pellet. Leave approximately 1 ml of medium in the tube and gently resuspend. Once the cell pellet has been thoroughly resuspended, add 2 ml of fresh RPMI-1640 medium and transfer all 3 ml onto the LD separation column (see Note 13). 5. Once all of the solution has drained through the column (approximately 15 min), wash the column with 3 ml of fresh RPMI-1640 medium (RT). Repeat this washing step twice until medium emerges clear. 6. Once the column has fully drained, remove it from the magnet and set it gently on top of a sterile 15-ml centrifuge tube. Add 7 ml of fresh RPMI-1640 medium to the column. To dislodge the bound iRBCs, flush the medium quickly through the column and into the centrifuge tube using the plunger supplied with the column (see Note 14). 7. Count the cells using a haemocytometer (see Note 15). 8. Centrifuge at 500 × g for 5 min at RT with brakes on. 9. Discard the supernatant being careful not to disrupt the cell pellet. 10. Resuspend the iRBCs at 3 × 108 cells/ml in RPMI-1640 medium and set aside at RT. 11. Use 20 ␮l of the iRBC suspension to prepare a smear on a glass microscope slide. Stain with Giemsa (10-fold diluted in freshly prepared Giemsa buffer) for 10–15 min. 12. Use a light microscope at 100 × magnification (with oil immersion) to assess purity. The cells should comprise more than 95% of late-stage trophozoites to mature schizonts. 3.4. Preparation of NK Cell Stimuli

1. Leucocyte growth medium (LGM) = negative control. 2. uRBCs = uninfected red blood cells; negative control for the iRBCs (we use ORh− human erythrocytes that are cultured in parasite culture medium under the same conditions as flasks containing iRBCs). Bring concentration of uRBCs to 108 cells/ml in RPMI-1640 medium. 3. iRBCs = P. falciparum schizont-infected red blood cells (we use strain 3D7 grown in ORh− human erythrocytes that are cultured in parasite culture medium). Bring concentration of iRBCs to 108 cells/ml in RPMI-1640. 4. rHuman IL-12 + rHuman IL-18 = positive control. Reconstitute contents of each vial in RPMI-1640 medium

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containing 0.01% BSA to generate stock solutions containing 2 ␮g/ml of each cytokine. Combine equal volumes of stock solutions of rHuman IL-12 and rHuman IL-18 to generate a stock solution containing 1 ␮g/ml of each cytokine. Store on ice at all times. 3.5. Addition of Cells and Antigens to Culture

Thoroughly resuspend PBMCs and add 200 ␮l to each well in a 96-well U-bottom tissue culture plate (see Note 16). 1. Negative control wells: Add nothing; PBMCs are already suspended in LGM. 2. uRBC negative control wells: Gently but thoroughly resuspend uRBCs by pipetting up and down. Add 2.4 ␮l of uRBC suspension to wells. 3. iRBC wells: Gently but thoroughly resuspend iRBCs by pipetting up and down and add 2.4 ␮l to wells. 4. rHuman IL-12/rHuman IL-18 wells: Thoroughly resuspend cytokine solution by pipetting and add 20 ␮l to wells. 5. Cover the tissue culture plate with its lid and incubate for 18–21 h at 37◦ C with 5% CO2 (see Note 17). 6. Add 22 ␮l of 10 ␮M brefeldin A to each well to achieve a final concentration of 3 ␮g/ml. (Brefeldin A interferes with intracellular protein transport, allowing any IFN-␥ being secreted to accumulate in the endoplasmic reticulum.) 7. Incubate for an additional 5–6 h at 37◦ C with 5% CO2 .

3.6. Surface and Intracellular Cytokine Staining for Phenotyping NK Cells

1. Centrifuge the 96-well U-bottom plate at 500 × g for 2 min with the brakes on [the temperature is not so critical at this step (4◦ C−25◦ C)]. 2. If you wish to save cell culture supernatants (for example, for assaying secreted cytokines), using a multichannel pipette, transfer approximately 150 ␮l of the supernatant to a new pre-labelled 96-well plate. Wrap the sides of the new plate containing the supernatant with laboratory film (Parafilm “M”, Pechiney Plastic Packaging) to prevent spillage and then freeze the plate at −80◦ C until required. 3. To wash the cells, add 150 ␮l of cold wash buffer and resuspend the cell pellet. Centrifuge the plate again at 500 × g for 3–5 min at 4◦ C. After centrifugation, gently invert the plate over a sink to discard the wash solution supernatant. 4. Repeat the washing step. 5. Stain the cells for surface markers by adding antibodies as shown in Table 29.1(see Note 18). 6. Incubate the plate at 4◦ C in the dark for 30 min.

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Table 29.1. Suggested antibodies for surface staining NK cell panel

Volume added (␮l)

␣-Human CD3 PerCP

2.5

␣-Human CD56 APC

2.5

␣-CD69 RPE

2.5

FACS staining buffer

42.5

7. Wash the cells by adding 150 ␮l of FACS wash buffer to each well, using a multichannel pipette, and then centrifuge the plate at 500×g for 3–5 min at 4◦ C with the brakes on. 8. During centrifugation, prepare the cytofix/cytoperm solution (BD PharMingen) (see Note 19). 9. After centrifugation, gently invert the plate over a sink to discard the supernatant. 10. Resuspend the cell pellets in 100 ␮l of cytofix/cytoperm solution. Incubate in the dark at 4◦ C for 45–60 min. 11. Wash out cytofix/cytoperm solution by adding 100 ␮l of permeabilisation buffer to each well using a multichannel pipette. Centrifuge at 500×g for 3–5 min at 4◦ C with the brakes on. 12. After centrifugation, gently invert the plate over a sink to discard the supernatant. 13. Add 200 ␮l of permeabilisation buffer to each well using a multichannel pipette. Centrifuge at 500×g for 3–5 min at 4◦ C with the brakes on. 14. During the centrifugation, prepare the anti-IFN-␥ antibody for intracellular staining by diluting the antibody 1:20 in permeabilisation buffer to result in a total volume of 50 ␮l/well. 15. After centrifugation, gently invert the plate over a sink to eject the supernatant. 16. Add 50 ␮l of diluted anti-IFN-␥ Ab to each well and resuspend gently, but thoroughly, using a multichannel pipette. 17. Incubate in the dark for approximately 45 min at 4◦ C. 18. Wash the cells by adding 150 ␮l of permeabilisation buffer to each well using a multichannel pipette. Centrifuge at 500×g for 3–5 min at 4◦ C with the brakes on. 19. After centrifugation, gently invert the plate over a sink to discard the supernatant.

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20. Repeat the washing step using 200 ␮l of permeabilisation buffer per well. 21. Resuspend the cell pellets in 150 ␮l of FACS buffer containing 1–2% paraformaldehyde and transfer into FACS tubes. Now the cells are ready for analysis on the flow cytometer. 22. Typical FACS plots for this experiment are shown in Figs. 29.1–29.3.

Fig. 29.1. Demonstration of the gating strategy for CD3− CD56+ NK cells. PBMCs were incubated in either lymphocyte growth medium (LGM) or in rIL-12+IL-18 for 24 h. Lymphocytes are gated on FSC and SSC (A) and then on CD56 and CD3 (B). Intracellular IFN-␥ is then examined for the NK cells (CD3− CD56+ ) cultured in LGM (C) or cultured in the presence of rIL-12+IL-18 (D).

4. Notes 1. For safety reasons and to avoid confounding co-infections in the assays, it is important to use human AB serum that has been screened and shown to be free of blood-borne viruses (e.g. HIV, hepatitis B and C). We use serum from AB blood group individuals because they are very unlikely to contain anti-A or anti-B antibodies. Human serum can be purchased from commercial companies but it is typically easier, cheaper and safer to obtain blood products

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Fig. 29.2. Demonstration of the gating strategy for CD3− CD56+ CD69+ NK cells. PBMCs were incubated in either lymphocyte growth medium (LGM) or in rIL-12+IL-18 for 24 h. Lymphocytes are gated on FSC and SSC (data not shown) and then on CD56 and CD3 (A). CD69 expression is then examined for the NK cells (CD3− CD56+ ) cultured in LGM (B) or cultured in the presence of rIL-12+IL-18 (C).

from a registered and licensed blood service centre. Most importantly, blood products must be screened routinely for the absence of Mycoplasma spp. which, if present, release copious amounts of lipopolysaccharides into the medium, which causes such high background levels of IFN-␥ production that any effect of the experimental stimulus is overwhelmed. Ideally, an aliquot from each batch of culture medium and each batch of parasites is saved and stored and periodically these aliquots are tested for the presence of Mycoplasma spp. by commercial PCR kits (VWR bioMarke PCR Mycoplasma test kit). 2. Gentamicin is primarily used during long-term propagation of P. falciparum.However, growing cultures continuously with gentamicin can mask the build up of culture contaminants such as Mycoplasma spp. and bacteria by inhibiting

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Fig. 29.3. Demonstration of the gating strategy for CD3− CD56+ IFN-␥+ NK cells and CD3− CD56+ CD69+ NK cells. Inter-individual heterogeneity in the production of IFN-␥ or the upregulation of CD69 by NK cells is then examined for the NK cells after incubation with iRBCs for 24 h by comparing the responses between a high-responding individual (donor 1) (panels A, C) and a low-responding individual (donor 2) (panels B, D).

their growth; however, this treatment does not eliminate them completely from culture. So, it is recommended that cultures are grown for at least two to three cycles (where a cycle is the seeding, growth and splitting of a culture) in the absence of any antibiotics, prior to harvesting iRBCs for use in NK cell assays. 3. This is an unusual gas mixture and needs to be specially ordered. It can take some time to obtain and you are advised to keep up to 3 months supply available if you are culturing parasites routinely. 4. For synchronising parasites, sorbitol solutions are sometimes made up as 15% stock solutions in RPMI; however, sorbitol diluted to 5% in water works as efficiently and we have found that the cells recover faster upon being returned to culture. Sorbitol treatment causes all except the most immature parasites (the ring stages) to lyse, leaving behind

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highly synchronised cultures. Resuspend the packed RBC pellet in 5–10× volume of sorbitol solution, shake the tube vigorously for 15–30 s and then pellet the cells by centrifugation. Examine a Giemsa-stained smear of the resulting pellet to make sure that all the trophozoites and schizonts have lysed and that all the remaining parasites are at the ring stage of development. If the treatment was not effective, then the procedure should be repeated. Sorbitol solutions should keep for 1–2 months if properly stored at 4◦ C. 5. Counting PBMCs under a microscope using the 40× objective makes it easier to differentiate red blood cells (which appear as slightly smaller cells with a reddish tint in their centre) from the larger, uncoloured PBMCs. 6. P. falciparum is a human pathogen; infection with infected red blood cells can be fatal if not treated. In the laboratory, the most likely route of infection is through a needle-stick injury or contamination of an open wound. Depending on local regulations, P. falciparum is either a biohazard safety level II or III organism and appropriate safety measures should be observed, following home institution policies. The 3D7 clone of P. falciparum is widely used for in vitro assays because it is highly sensitive to most anti-malarial drugs including chloroquine, a very safe and well-tolerated drug. 7. If many flasks are to be cultured, the gas mixture can be used to aerate the entire incubator, but this will obviously use much more gas. Tissue culture flasks with a 0.2-␮m membrane filtered cap can be used if there are many cultures to maintain at one time; this reduces the chance of contamination between flasks as well as minimises the chance of contamination from the outside environment. Cultures can also be adapted to grow under aerobic conditions (19). If the gas mixture is unavailable, cultures can be grown in candle jars: place the flask − with the cap loosely in place − in a large sealable glass or a plastic container, place a lighted candle in the container and seal it, making sure it is airtight. The candle will continue to burn until the level of oxygen falls, generating a suitable environment for the parasites to grow. 8. As schizont-stage iRBCs burst, they release a plethora of proteins, lipids and haeme-containing molecules into solution. These by-products of parasite growth are toxic and will retard parasite development. It is thus important to keep parasitaemia below 3% for routine parasite propagation. 9. In order to obtain an accurate measurement of parasitaemia, the cultures need to be brought to a

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haematocrit level of 50% before making the smear. Transfer cultures into 15-ml centrifuge tubes and centrifuge at 500×g for 5 min at RT with brakes on. Aspirate off most of medium, leaving behind 1× the volume of packed blood. Resuspend the cell pellet and transfer 20 ␮l onto the end of a glass slide. Using another glass slide, hold it at approximately 30◦ angle and gently smear the blood across the slide in one smooth motion. This is a technique that requires practice and experience. This is a very important technique as it is used routinely in malaria culture labs. 10. After centrifugation, the RBCs and granulocytes can be seen as a pellet at the bottom of the tube. Directly above the red cell pellet will be the Histopaque-1077 layer. Resting on top of the Histopaque-1077 will be the PBMC layer, which appears as a fluffy, white (opaque) layer. Above the buffy coat will be the diluted plasma. This plasma layer should be harvested as a source of autologous plasma for the cell cultures. The plasma has been diluted 1:1 with RPMI-1640 medium, and as the final concentration of autologous plasma in the culture medium should be 10%, the diluted plasma needs to be added at 20% in order to achieve the correct final concentration. 11. Sometimes you will observe contamination of the buffy coat by RBCs. If this happens, harvest the buffy coat as normal, spin it down (the pellet will appear pink or red depending on the extent of the RBC contamination) and resuspend in 5–10 ml of red cell lysing solution. Centrifuge at 500×g for 10 min at 4◦ C with the brakes on. The pellet should now be white or only very pale pink. Discard the supernatant and gently resuspend the pellet in wash buffer. Wash the cells once more by adding 50 ml of cold wash medium and centrifuge again. 12. It is important to allow the fluids to pass through the column slowly, i.e. by gravity alone. This will take approximately 15 min each time. In total, preparing the column will take about 30 min, so get the column fully prepared before removing the iRBC culture from the incubator. This protocol assumes that the investigator has already checked that the cultures are at the correct stage by regularly making and reading Giemsa-stained smears. It can take some time to gain sufficient experience to know exactly when the culture will be at the optimal stage for harvesting and use in the experiments. 13. It is very important to add the iRBC solution to the column very gently in order to avoid creating air bubbles as this will interfere with the efficiency of the purification. A good technique to avoid this is to add the cell solution at an angle

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so that it falls onto the wall of the column and runs down onto the top of the filtration matrix. 14. During elution of the iRBCs, the flow-through will first appear red but translucent; however, once the iRBCs begin to elute from the column, the solution will become brown. At this point it is safe to assume that column is not and will not become blocked. If at any point the column does become blocked, resuspend the solution in the top of the LD separation column, being very careful not to create any air bubbles. 15. Set microscope to 40× magnification. If the iRBC culture is properly synchronised, over 80% of RBC should have a clearly identifiable food vacuole containing hemozoin aggregates. This will appear as a dark brown spot within the cell. 16. Ideally, three or four replicate wells would be made for each stimulus to allow true biological variation and technical reliability of the data to be assessed. In practice, however, numbers of PBMC may be limited and the cost of processing numerous replicates for flow cytometry is high. As a compromise, we tend to carry out assays in duplicate and we discard data (or repeat assays) where the results of the two duplicate wells differ by more than a pre-determined amount. Since the cells we are looking for (NK cells) are not rare cells, we obtain robust data by analysing 100,000 lymphocytes per stimulus; we find that it is typically very easy to acquire 100,000 lymphocytes from a single well and thus there is no need to pool cells from multiple wells. 17. Prior to harvesting the cells for FACS analysis, it is a good idea to quickly view the plate under an inverted microscope. The cells in the negative control wells should form a small, regular pellet in the bottom of the well. By contrast, in the positive control wells, the cell pellet is typically larger and slightly fuzzy around the edges, indicating that the cells have proliferated and increased in number. 18. We have optimised this experiment to be cost effective whilst not compromising the quality of staining. We have managed to reduce the total staining volume to 50 ␮l and we typically use commercial antibodies at a dilution of 1:20. However, whenever a new stock of antibody is used, it is necessary to titrate the antibody to find the optimum concentration required. 19. There are numerous commercial kits that are frequently used for intracellular cytokine staining. We have found subtle differences between the various kits. Primarily, they differ with respect to incubation times for the

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cytofixation/cytopermeabilisation steps and the intracellular staining steps. Paraformaldehyde plus saponin is a good alternative to the commercial kits. Also, we find that saponin diluted to between 0.1% and 1% saponin in PBS is ideal for optimal permeabilisation of cells for intracellular staining. The optimal concentration of saponin to be used depends on the duration of antibody staining (longer staining requires a lower saponin concentration, and vice versa). This requires optimisation by the investigator. References 1. Newman, K. C., and Riley, E. M. (2007) Whatever turns you on: accessory-celldependent activation of NK cells by pathogens. Nat Rev Immunol 7, 279–291. 2. Haller, D., Serrant, P., Granato, D., Schiffrin, E. J., and Blum, S. (2002) Activation of human NK cells by staphylococci and lactobacilli requires cell contact-dependent costimulation by autologous monocytes. Clin Diagn Lab Immunol 9, 649–657. 3. Newman, K. C., Korbel, D. S., Hafalla, J. C., and Riley, E. M. (2006) Cross-talk with myeloid accessory cells regulates human natural killer cell interferon-gamma responses to malaria. PLoS Pathog 2, e118. 4. Gorak, P. M., Engwerda, C. R., and Kaye, P. M. (1998) Dendritic cells, but not macrophages, produce IL-12 immediately following Leishmania donovani infection. Eur J Immunol 28, 687–695. 5. Dalod, M., Hamilton, T., Salomon, R., Salazar-Mather, T. P., Henry, S. C., Hamilton, J. D., and Biron, C. A. (2003) Dendritic cell responses to early murine cytomegalovirus infection: subset functional specialization and differential regulation by interferon alpha/beta. J Exp Med 197, 885–898. 6. Artavanis-Tsakonas, K., Eleme, K., McQueen, K. L., Cheng, N. W., Parham, P., Davis, D. M., and Riley, E. M. (2003) Activation of a subset of human NK cells upon contact with Plasmodium falciparuminfected erythrocytes. J Immunol 171, 5396–5405. 7. Guma, M., Cabrera, C., Erkizia, I., Bofill, M., Clotet, B., Ruiz, L., and Lopez-Botet, M. (2006) Human cytomegalovirus infection is associated with increased proportions of NK cells that express the CD94/NKG2C receptor in aviremic HIV-1-positive patients. J Infect Dis 194, 38–41. 8. LaBonte, M. L., McKay, P. F., and Letvin, N. L. (2006) Evidence of NK cell dysfunction in

9.

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13. 14.

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SIV-infected rhesus monkeys: impairment of cytokine secretion and NKG2C/C2 expression. Eur J Immunol 36, 2424–2433. Korbel, D. S., Newman, K. C., Almeida, C. R., Davis, D. M., and Riley, E. M. (2005) Heterogeneous human NK cell responses to Plasmodium falciparum-infected erythrocytes. J Immunol 175, 7466–7473. Mohan, K., Moulin, P., and Stevenson, M. M. (1997) Natural killer cell cytokine production, not cytotoxicity, contributes to resistance against blood-stage Plasmodium chabaudi AS infection. J Immunol 159, 4990–4998. Choudhury, H. R., Sheikh, N. A., Bancroft, G. J., Katz, D. R., and De Souza, J. B. (2000) Early nonspecific immune responses and immunity to blood-stage nonlethal Plasmodium yoelii malaria. Infect Immun 68, 6127–6132. De Souza, J. B., Williamson, K. H., Otani, T., and Playfair, J. H. (1997) Early gamma interferon responses in lethal and nonlethal murine blood-stage malaria. Infect Immun 65, 1593–1598. Stevenson, M. M., and Riley, E. M. (2004) Innate immunity to malaria. Nat Rev Immunol 4, 169–180. Kim, C. C., Parikh, S., Sun, J. C., Myrick, A., Lanier, L. L., Rosenthal, P. J., and DeRisi, J. L. (2008) Experimental malaria infection triggers early expansion of natural killer cells. Infect Immun 76, 5873–5882. Artavanis-Tsakonas, K., and Riley, E. M. (2002) Innate immune response to malaria: rapid induction of IFN-gamma from human NK cells by live Plasmodium falciparuminfected erythrocytes. J Immunol 169, 2956–2963. Baratin, M., Roetynck, S., Lepolard, C., Falk, C., Sawadogo, S., Uematsu, S., Akira, S., Ryffel, B., Tiraby, J. G., Alexopoulou, L., Kirschning, C. J., Gysin, J., Vivier, E., and Ugolini, S. (2005) Natural killer cell

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and macrophage cooperation in MyD88dependent innate responses to Plasmodium falciparum. Proc Natl Acad Sci U S A 102, 14747–14752. 17. Maghazachi, A. A. (2003) G protein-coupled receptors in natural killer cells. J Leukoc Biol 74, 16–24. 18. Fehniger, T. A., Cooper, M. A., Nuovo, G. J., Cella, M., Facchetti, F., Colonna, M., and

Caligiuri, M. A. (2003) CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2: a potential new link between adaptive and innate immunity. Blood 101, 3052–3057. 19. Miyagami, T., and Waki, S. (1985) In vitro cultivation of Plasmodium falciparum under aerobic atmosphere in a CO2 incubator. J Parasitol 71, 262–263.

Chapter 30 Natural Killer Cells in Human Pregnancy Victoria Male, Anita Trundley, Lucy Gardner, Jacquie Northfield, Chiwen Chang, Richard Apps, and Ashley Moffett It is quite surprising how many children survive in spite of their mothers. Norman Douglas

Abstract Natural killer (NK) cells account for 70% of the leukocytes in the mucosal lining of the uterus (the decidua) in the first trimester of pregnancy. They are CD56superbright granulated cells expressing a repertoire of Killer-cell Immunoglobulin-like Receptors (KIR) skewed towards recognising HLA-C, which is the only classical class I MHC found on placental trophoblast cells. The function of decidual NK cells is not yet known, but there is evidence to suggest that they are involved in mediating trophoblast invasion into the decidua and modifying maternal spiral arteries to increase blood flow to the placenta. In order to characterise decidual NK cells and to understand their interactions with other cells at the maternal–foetal interface, it is useful to be able to isolate these cells. Here, we describe methods for the isolation and culture of decidual NK cells, decidual stromal cells and trophoblast cells from human first trimester tissue samples. Key words: Human, pregnancy, isolation, trophoblast, decidua, leukocytes, natural killer cells, stromal cells.

1. Introduction During pregnancy, the intimate juxtaposition of the foetal and maternal circulations allows the exchange of nutrients and gases between mother and child. In humans, the foetus achieves this by forming a highly invasive haemochorial placenta, which invades K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 30, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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into the wall of the uterus and erodes maternal blood vessels (1). This invasion into the uterine mucosa, which in pregnancy is called the decidua, occurs during the first trimester of pregnancy and is critical for its later success. The placental cells that form the barrier between mother and foetus are known as trophoblast. Villous trophoblast cells are noninvasive and form chorionic villi, which function as the site of exchange between the maternal and foetal circulations. Extravillous trophoblast cells invade into the decidua and transform the maternal spiral arteries allowing greater blood flow to the placental bed (2). Villous trophoblast does not express any MHC molecules and is therefore considered immunologically inert (3). Extravillous trophoblast does not express class II MHC but does express the non-classical class I MHC molecules HLA-E and HLA-G and the classical class I molecule HLA-C, but not HLA-A or -B (4–9). The decidua is thought to be critical for controlling the depth to which trophoblast cells are allowed to invade. It contains specialised stromal cells, glands and endothelial cells, all of which could play some role in regulating the extent of trophoblast invasion. The decidua also contains a unique complement of immune cells, comprising approximately 10% T cells, 20% macrophages and 70% NK cells (10). Decidual NK cells differ from blood NK cells both phenotypically and functionally. Decidual NK cells are CD56superbright but unlike CD56bright blood NK cells they contain cytotoxic granules and express KIR, primarily those recognising HLA-C, which is the only classical class I MHC found on human trophoblast (11–13). They express the activating receptors NKG2C, NKG2D, NKG2E, NKp30, NKp44 and NKp46 and are capable of killing class I MHC-deficient targets, although less efficiently than blood NK cells due to their inability to polarise cytotoxic granules towards class I-deficient target cells (14–16). Decidual NK cells cannot kill trophoblast unless they are activated in vitro by treatment with IL-2, which is not present in the uterus (17, 18). Although no function has been definitively proven for decidual NK cells, they are most numerous during the period of implantation and establishment of the placenta, and thus the first 3 months of gestation is the time when they must exert their function. A number of studies suggest that decidual NK cells have a role in mediating trophoblast invasion and that they must be activated to fulfil this function. They produce cytokines including IFN␥, GM-CSF, CSF-1 and LIF for which trophoblast has receptors (19–22). Particular combinations of maternal KIR genotypes lacking activating receptors with foetal genotypes possessing HLA-C group 2 alleles are associated with pre-eclampsia, a

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disease of pregnancy characterised by insufficient trophoblast invasion (23). Furthermore, NK clones expressing inhibitory KIR produce lower levels of trophoblast chemoattractants than those expressing activating KIR (24). Studies in mice have suggested that decidual NK cells may be able to modify placental blood flow directly by acting on maternal vessels (25). In humans, evidence for this function comes from immunohistochemisty showing decidual NK cells clustered around the spiral arteries and their production of angiogenic factors, such as angiopoietin 2, VEGF and PDGF (26, 27). Isolation of decidual NK cells is necessary in order to study their function. Similarly, in order to investigate their interaction with trophoblast or decidual stromal cells, these cells must also be isolated from the foetal–maternal interface. In this chapter, we describe methods for the isolation and culture of decidual leukocytes, trophoblast and decidual stromal cells from first trimester elective termination samples. These methods are not suitable for the isolation of cells from term placenta samples, in which decidual NK cells are sparse and the anatomical organisation of both the placenta and the decidua is different. Ethical approval for the use of first trimester termination samples in our laboratory comes from the Local Research Ethics Committee, Cambridge Health Authority, Addenbrooke’s Hospital, Cambridge, UK and informed consent is obtained from the patients. Using unscreened human material has health and safety implications, which necessarily affect the way in which work is carried out. All work on human samples in our laboratory is performed in a designated containment level I tissue culture room inside class II microbiological safety cabinets. Researchers working on human material must be immunised against, and shown to be protected against, Hepatitis B. Tissue sorting is carried out in a dedicated plastic tray lined with paper to absorb spillages and researchers adhere strictly to wearing of lab coats, two pairs of gloves and plastic aprons. The possibility of infectious pathogens also has implications for waste disposal, spillages and culture of isolated cells. It is important that laboratories undertaking these isolation procedures comply with their local rules for health and safety and waste disposal.

2. Materials All plasticware and instruments that come into contact with the tissue or cells are sterile. Unless otherwise indicated, cell culture media are produced in-house to standard specifications. 2.1. Tissue Sorting

1. 150 mm diameter petri dishes (Falcon). 2. Ham’s F12 medium.

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3. RPMI 1640 medium (in-house preparation used for washing). 4. Large metal sieve with waste collection tray (Endecotts Ltd., London, UK). We use an 80 ␮m mesh sieve, which serves to support the gauze. 5. Gauze. Butter muslin cut to 200 mm squares to go in large metal sieve and then autoclaved to sterilise. 6. Forceps. 7. Scissors. 8. Magnetic stirrer. 9. Sterile magnetic fleas. 10. 250 mL sterile bottles. 2.2. Enzymatic Isolation of Decidual Leukocytes and Stromal Cells

1. Foetal Calf Serum (FCS) (GibcoTM , InvitrogenTM Corporation, Carlsbad, CA, USA). Heat inactivated at 56◦ C for 60 min, cooled quickly in cold water and then stored in aliquots at −20◦ C and after opening at 4◦ C for 1 month. 2. RPMI 10% FCS: RPMI 1640 medium supplemented with L -glutamine (Sigma-Aldrich D5025, Poole, UK), 10% Foetal Calf Serum (FCS) and antibiotics (penicillin/streptomycin 100 Units/mL and amphotericin-B 2.5Units/mL, Sigma). 3. Scalpels. 4. Forceps. 5. Collagenase Type V (10 mg/mL) solution: Dissolve 1 g of Collagenase C 9263 (Sigma-Aldrich, Poole, UK) in 100 mL RPMI 10% FCS. Make 5 mL aliquots and store at −20◦ C. 6. 37◦ C-incubator with roller apparatus to turn the tube. R 7. 100, 70 and 40 ␮m cell filters (BD Falcon ). 8. PBS with 2% FCS (as above) and 0.1% azide (Sigma-Aldrich, Poole, UK). 9. LymphoprepTM (Nycomed, Oslo, Norway).

2.3. Mechanical Isolation of Decidual Leukocytes

1. 2. 3. 4.

2.4. Magnetic Purification of Decidual NK Cells

1. Ice.

Scalpels. Forceps. RPMI 10% FCS (see above) Large metal sieve (75 ␮M mesh) with waste collection tray (as above) 5. Large rubber bung 6. LymphoprepTM (as above)

R buffer: PBS with 0.5% BSA (Sigma-Aldrich, Poole, 2. MACS UK) and 2 mM EDTA (BDH Laboratory Supplies, Poole UK). Filter sterilised through 0.2 ␮m filter, degas under a

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vacuum and autoclave to sterilise. Store at 4◦ C for a month (see Note 1). 3. Human ␥-globulins (Sigma-Aldrich, Poole, UK): Made up in PBS to 1 mg/mL. 1 mL aliquots stored at −20◦ C until use and then at 4◦ C for a month. 4. CD56 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). 5. 40 ␮m cell filter (as above). 6. LS, MS or MiniMACS separation columns and collection tubes (Miltenyi Biotec). 7. VarioMACSTM magnet (Miltenyi Biotec). 8. RPMI 10% FCS (see above). 9. Mouse anti-human CD9 PerCp-Cy5.5 antibody, clone ML13 (BD Biosciences, Erembodegem-Aalst, Belgium). 2.5. Decidualisation of Decidual Stroma

1. Charcoal stripped Foetal Calf Serum (FCS) (GibcoTM , InvitrogenTM Corporation, Carlsbad, CA, USA). Heat inactivated and stored as for FCS, above. 2. DMEM-F12 10% charcoal stripped FCS: DMEM-F12 medium supplemented with L-glutamine (Sigma), antibiotics (penicillin/streptomycin 100 Units/mL and amphotericin-B 2.5 Units/mL, Sigma) and 10% charcoal stripped FCS. 3. DMEM-F12 2% charcoal stripped FCS: DMEM-F12 medium without indicator, supplemented with L-glutamine (Sigma), antibiotics (as above) and 2% charcoal stripped FCS. 4. Insulin (10 mg/mL) stock: Add 150 ␮L glacial acetic acid to 9.85 mL sterile distilled water. Dilute 100 mg insulin (Sigma) in 10 mL acidified water and store at 4◦ C for up to a year. 5. Estradiol (1 mM) stock: Dissolve 1 mg estradiol (Sigma) in 3.67 mL 100% ethanol, aliquot and store at −20◦ C. Stock may be diluted 1000 ´in medium to give a 1 ␮M working dilution. 6. Medroxyprogesterone 17-acetate (MPA; 1 mM) working dilution: Dissolve 1 mg MPA (Sigma) in 2.51 mL 100% ethanol, aliquot and store at −20◦ C. 7. 8-Bromo-cAMP (100 mM) working dilution: Dissolve 100 mg 8-bromo-cAMP (Sigma) in 2.325 mL serum-free medium, aliquot and store at −20◦ C.

2.6. Isolation of Trophoblast Cells

1. 0.2% trypsin, 0.02% EDTA solution: Glucose 0.3 g (Sigma), NaCl 12 g, KCl 0.3 g, disodium hydrogen

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orthophosphate 1.725 g, potassium dihydrogen orthophosphate 0.3 g. Make up to 1 L with sterile water. Mix thoroughly on stirrer. Adjust pH to 7.4. Add 2 g trypsin (trypsin 250, Difco) and 0.2 g EDTA (BDH Laboratory Supplies, Poole UK). Stir until dissolved. Leave overnight at 4◦ C. Then filter-sterilise 75 mL aliquots into 100 mL bottles using 0.2 ␮m filters. Store at −20◦ C. 2. 150 mm diameter petri dishes. 3. Scalpels. 4. Forceps. 5. Newborn Calf Serum (NCS) (GibcoTM , InvitrogenTM Corporation, Carlsbad, CA, USA). Heat inactivated and stored as for FCS above. 6. Ham’s F12/20% NCS: Ham’s-F12 medium supplemented with 20% NCS. 7. Plastic funnel. 8. Gauze (as above). Cut to go in plastic funnel. 9. LymphoprepTM (as above). 10. Sterile 35 mm diameter petri dishes. 11. Fibronectin solution: Add 1 mL of sterile water to 1 mg vial of fibronectin (BD Biosciences). Leave to stand for 30 min (do not mix). 20 ␮L aliquots stored at −70◦ C. 12. 35 mm fibronectin-coated dishes: Dilute 20 ␮L of fibronectin solution in 1 mL Ham’s F12 and place in 35 mm petri dish. Leave at room temperature for 45 min. For immediate use, pour off the fibronectin solution; otherwise, the dishes can be stored at −20◦ C for 2 weeks. 13. Mouse anti-human HLA-G antibody, clone G233 (5) 14. Mouse anti-human EGF receptor antibody, clone EGFD1 (Insight Biotechnology, Wembley, UK)

3. Methods Several stages in these methods require the use of sharp instruments on primary human tissue. Investigators must therefore take particular care to avoid injury and potential infection with human pathogens such as HIV and Hepatitis C. 3.1. Tissue Sorting

1. Prepare several large petri dishes for tissue collection. For collection of trophoblast the dish should contain approximately 20 mL of Ham’s F12 medium and for decidua the same volume of RPMI 1640 medium.

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2. Place a metal sieve over a waste collection vessel and line with double thickness sterile gauze. Carefully empty the contents of the tissue collection bag into the gauze. Gently disaggregate large clots with sterile forceps and wash the tissue with large amounts of RPMI 1640 medium. Pick the pieces of decidua or villi into the appropriate dish of media. The villi are identifiable by their light pink colour and when washed will have a frond-like/fluffy appearance. Pieces of decidua parietalis are generally greyer, smoother and more solid in appearance. Membranes and pieces of tissue that are very bloody are avoided; it is important to minimise as much maternal blood contamination as possible, especially when isolating decidual leukocytes. Pieces of decidua that are more ragged, have flecks of yellow and haemorrhagic areas, are likely to be decidua basalis containing trophoblast cells. These tissues are generally discarded because they are too bloody; however, we do collect them if tissue sections from the implantation site are required. 3. Tissues can be pooled or retained as single samples depending on the experiment and how many cells are required (see Note 2). Samples are then placed in 250 mL bottles with a sterile magnetic flea and are covered in the appropriate media. Samples are stirred at moderate speed for 15 min in order to wash off as much residual blood as possible. 3.2. Enzymatic Isolation of Decidual Leukocytes and Stromal Cells

1. Clean decidual tissue is finely minced (see Note 3) using surgical blades and residual blood clots are removed. 2. 10 g of the minced tissue is placed in 19 mL of RPMI 10% FCS with 4.8 mL of Collagenase solution. Digestion of the tissue is allowed to proceed for 1 h on a roller at 37◦ C (see Note 4). 3. At the end of the incubation 30 mL of RPMI 10% FCS is added and the mixture is left to stand for 5 min to allow undigested tissue to sediment. 4. The supernatant is decanted by aspiration and is passed sequentially through 100, 70 and 40 ␮m filters. 5. The filtrate is centrifuged at 450 × g for 5 min and the resultant cell pellet is resuspended in 40 mL of RPMI containing 2% FCS and 0.1% azide (see Note 5). This mixture is then overlaid onto 4 × 8 mL LymphoprepTM in universal tubes and centrifuged at 710 × g for 20 min with no brake. (see Note 6). 6. The cells at the interface are collected, diluted in RPMI 10% FCS and pelleted by centrifugation at 550×g for 5 min. This yields a mixture of both stromal cells and leukocytes, which

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may be used immediately, separated by plating down on plastic (see Steps 7 and 8) or further purified by magnetic selection (see Section 3.4). 7. Decidual stromal cells are adherent whereas leukocytes are not. This means that the two fractions may be crudely separated by plating down on plastic. The cells are resuspended in RPMI 10% FCS and cultured in 75 cm2 flasks for at least 2 h and not longer than overnight. (see Note 7). The supernatant contains the leukocytes, which usually consist of 60–80% NK cells (CD56+ CD16− ), 5–15% CD14+ macrophages and 10–20% T cells plus some non-leukocyte cells (Fig. 30.1).

Fig. 30.1. Total decidual leukocytes are gated by scatter (A) and examined for CD56 and CD3 staining (B). CD56+ CD3- (NK) cells usually account for approximately 70%, and CD56- CD3+ (T) cells for 10%, of leukocytes before magnetic selection.

8. The adherent layer consists mostly of decidual stromal cells, but also contains small numbers of glandular epithelial cells, endothelial cells and decidual leukocytes. Decidual stromal cells may be further cultured in RPMI 10% FCS and remain viable for up to five passages in culture. Decidual stromal cells may also be “decidualised” by treatment with steroid hormones (see Section 3.5). 3.3. Mechanical Isolation of Decidual Leukocytes

Mechanical isolation produces a lower yield of decidual leukocytes than enzymatic digestion but may be desirable for some downstream applications, such as staining for flow cytometry with an antibody whose epitope is sensitive to collagenase. Indeed, CD56superbright staining is only seen on mechanically isolated cells. Note that, unlike enzymatic digestion, mechanical digestion does not yield decidual stromal cells as a product. 1. Clean decidual tissue is finely minced (see Note 3) using surgical blades and residual blood clots are removed. 2. Cells are pushed through a 75 ␮m sieve using the flat surface of a large rubber bung. RPMI 10% FCS is used to wash cells through the sieve to the collection tray beneath.

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3. Cells and RPMI 10% FCS are collected and centrifuged at 450 × g for 5 min. The resultant cell pellet is resuspended in 20–40 mL of RPMI containing 2% FCS and 0.1% azide (see Note 5). This cell suspension is then overlaid onto 2–4 × 8 mL LymphoprepTM and the tube is centrifuged at 740 × g for 20 min with no brake. 4. The cells at the interface are collected, washed in RPMI 10% FCS and pelleted by centrifugation at 550 × g for 5 min. 3.4. Magnetic Purification of Decidual NK Cells

CD56+ decidual NK cells are further purified from decidual leukocytes by positive selection using CD56 antibody-coated magnetic MicroBeads and a Magnet assisted cell separation R ) protocol. (MACS 1. Decidual leukocytes are washed once in 20 mL ice-cold PBS centrifuged at 450 × g for 5 min and resuspended in R buffer with additional 0.05% human ␥-globulins. MACS R 7 buffer. 10 cells are resuspended in 100 ␮L of MACS 7 CD56 MicroBeads are added at 20 ␮L per 10 cells and the mixture is incubated on ice for 20 min. 2. After the incubation the cells are washed in 20 mL PBS, centrifuged at 450 × g for 5 min and resuspended in cold R buffer at a concentration of 107 cells/mL. The cell MACS suspension is then passed through a 40 ␮m cell filter to remove clumps, which might block the column, and then kept on ice during the purification. 3. A separation column is positioned within the magnetic field of a VarioMACSTM magnet and a collection tube is placed R beneath it. The column is equilibrated with 3 mL of MACS buffer. 4. The cell suspension is applied to the column 1 mL at a time (see Note 8). The column is then washed with 15 mL of iceR buffer. cold MACS 5. CD56- cells flow through the column and into the collection tubes. CD56+ cells are retained in the magnetic matrix of the column. The cells are eluted out of the column by R buffer followed by the addition of 6 mL of ice-cold MACS removal of the column from the magnet. The elution buffer is flushed gently into a collection tube using the plunger supplied (see Note 9). The suspension is then centrifuged at 450 × g for 5 min in order to pellet the cells. The cells are finally resuspended in RPMI 10% FCS. CD56+ cells purified in this way are typically >95% pure (see Notes 10–14). 6. Decidual NK cells uniformly express CD9, whereas blood NK cells are uniformly CD9 negative (15). Therefore, the decidual NK cells may be stained with mouse anti-human CD9 and examined by flow cytometry to assess the extent of contamination with blood NK cells (Fig. 30.2).

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Fig. 30.2. Approximately 10% of total blood leukocytes (A) are CD56+ , and the majority of these are CD56dim . Decidual leukocytes (B) not only have a greater proportion of CD56+ cells, but these cells are CD56superbright . Panels (C) and (D) show CD9 staining (bold line) on CD56+ leukocytes. The broken line represents the isotype control. Blood CD56+ cells (C) are CD9- , whereas decidual CD56+ cells are uniformly CD9+ . Thus CD56+ CD9- decidual leukocytes are contaminants from the blood.

3.5. Decidualisation of Decidual Stroma

Decidual stromal cells “decidualise” when cultured with the appropriate steroid hormones (28). This technique recapitulates in vitro the changes that occur to the stroma in vivo during the secretory phase of the menstrual cycle and in the first trimester of pregnancy. 1. Decidual stromal cells are grown to 80% confluence in DMEM-F12 10% charcoal stripped FCS with 2 ␮g/mL insulin and 1 nM estradiol. 2. When cells have reached 80% confluence, the medium is changed to DMEM-F12 2% charcoal stripped FCS with 1 ␮M medroxyprogesterone 17-acetate (MPA) and 0.5 mM 8-bromo-cAMP. Cells grown in DMEM-F12 2% charcoal stripped FCS without MPA and cAMP may be used as a negative control. 3. The cells are cultured for a further 3–6 days. Decidualised stromal cells produce prolactin and the extent of decidualisation may be assessed by ELISA of supernatant for prolactin.

3.6. Isolation of Trophoblast Cells

1. Pre-warm 75 mL of 0.2% trypsin/0.02% EDTA to 37◦ C. 2. The washed villous tissue is placed in a clean large petri dish and the cells are carefully separated from the denser,

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supporting connective tissue by gentle scraping with a scalpel blade. Any residual blood clots should either be cut from the villous tissue or removed with forceps. Membranous remnants are then discarded. 3. The tissue is transferred to the warmed trypsin and the suspension is placed in a sterile 100 mL bottle and stirred on a heater block at 37◦ C for 8–9 min (see Note 15). The digestion is stopped by the addition of 30 mL Ham’s F12 20% NCS. 4. The digested tissue is then filtered through double thickness gauze and the cells recovered as a pellet by centrifugation at 450 × g for 5 min. The pellet is soft at this stage and care must be taken not to decant the pellet as well. 5. The cell pellet is resuspended in 10 mL of Ham’s F-12 medium and the cell suspension is layered onto 2 × 8 mL of LymphoprepTM in universal tubes. The tubes are then centrifuged at 710 × g for 20 min with no brake. 6. After centrifugation cells at the interface are collected and washed with 10 mL of Ham’s F12 medium. At this stage the preparation contains approximately 50% HLA-G+ cells (see Note 16 and 17). 7. To remove placental macrophages (Hofbauer cells), the cell pellet is resuspended in 3 mL of Ham’s F12 medium and incubated for 20 min at 37◦ C in a 35 mm petri dish. 8. Non-adherent cells are collected and may be examined immediately or seeded into a 35 mm fibronectin-coated dish overnight at 37◦ C. Cells examined immediately are mostly EGFR+ and MHC class I- (a villous trophoblast phenotype), whereas cells recovered the next day have an extra-villous phenotype, typically consisting of ∼70–90% HLA-G+ cells (Figs. 30.3 and 30.4)(see Notes 18 and 19).

4. Notes 1. The manufacturer recommends degassing the buffer to prevent the formation of air bubbles, which may block the column. 2. The decision whether to pool samples will depend on the nature of the experiments to be undertaken. Single samples are ideal for studies of immune recognition as different individuals will express different HLA and immune receptor phenotypes, which can be defined by keeping aside a piece of tissue for PCR analysis. However, pooled

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Fig. 30.3. Trophoblast cells are gated by scatter (A) and to exclude macrophages, which are LILRB3+ (B). Cells examined immediately (C and D) are predominantly EGFR+ and BBM.1 (MHC class I)- , which represents a villous phenotype. After overnight culture (E and F), trophoblast exhibits predominantly an extravillous phenotype, in which most of the cells are HLA-G+ .

samples are useful for the analysis of the general features of a certain cell population and also will provide larger numbers of cells. Typically, we find that from 1 g of decidua we can obtain 3–5 × 106 decidual leukocytes and 1–3 × 106 purified decidual NK cells. The yield of trophoblast cells is very variable and less predictable. This may be related to different ages of samples or batches of trypsin. Typically, we obtain 4 × 106 trophoblast cells per sample. 3. Fine mincing should take at least 10 min using two scalpels. We have found that placing a damp paper towel under the dish prevents it slipping and makes chopping easier. At

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Fig. 30.4. Trophoblast cells isolated from a normal first trimester placenta, plated on fibronectin-coated dishes and cultured overnight. Non-adherent cells and debris have been washed off. These cells are predominantly HLA-G+ .

the required consistency for a 1 h digest, the tissue should appear granular but homogenous with no visible lumps. For an overnight digest half the amount of chopping is required: small pieces of tissue (approximately 2 mm3 ) will still be visible. 4. For practical purposes it may be useful to digest the tissue overnight so that the cells can be extracted the next morning. Overnight digestion of the whole tissue does not obviously adversely affect the viability of the recovered cells compared to the 1 h digest. In this case the tissue is chopped into small pieces but not as finely as for a 1 h digest. 10 g of tissue is digested in 20 mL of RPMI 10% with 0.6 mg/mL collagenase. The digestion is performed on a roller for 18 h at room temperature. 5. This solution is used to remove dead cells from the preparation. The dead cells take on azide and are sedimented to the bottom of the LymphoprepTM . The azide is washed off after this step and in our experience is not detrimental to cell functions. However the azide can be left out. 6. LymphoprepTM is a ready-made solution that is most often used for the purification of peripheral blood mononuclear cells by density gradient centrifugation (there are similar

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products which can be used, such as Ficoll from Amersham Biosciences). It contains a chemical that aggregates red blood cells, which cause them to sediment faster than the white blood cell fraction. This technique can also be used to separate other cells from red blood cells. For those unfamiliar with this technique, there is a knack. The heterogeneous cell suspension is very gently overlaid onto the LymphoprepTM solution so that there is no mixing of the layers. There are several ways to approach this: one is to place the LymphoprepTM in the tube and then, holding the tube at a 45◦ angle, very slowly pipette the cell suspension down the side of the tube with the pipette tip fairly close to the LymphoprepTM . This addition should start drop-wise and then gradually build up, keeping the flow steady. The tubes should be handled very carefully thereafter to prevent mingling of the layers and also the tubes should be balanced so that the centrifugation is as smooth as possible. After centrifugation there should be four visible layers in the tube: red blood cells in a pellet at the bottom, LymphoprepTM clear solution, a thin white cloudy layer (the interface) and a top layer of medium supernatant. Holding the tube to eye level, use a Pasteur pipette to suck the cells from the interface. It is important to avoid sucking up too much LymphoprepTM as this can prevent your recovered cells from pelleting. If this has happened, cell recovery may be improved by further dilution of your aspirated solution at the washing step. 7. Decidual stromal cells require at least 2 h to adhere to the plastic. Glandular epithelial cells adhere to plastic less quickly than stromal cells and so contamination of stromal cell cultures with glandular epithelium can be reduced by changing the medium between 2 and 3 h after plating down on plastic. Culturing decidual stromal cells with decidual leukocytes longer than overnight adversely affects the survival and growth of the stromal cells, although decidual leukocytes survive better when cultured in the presence of stromal cells. 8. One of the problems that can occur with this method is that the column blocks, which is why it is best to only add a small amount of cells at a time. If the column blocks it should be washed and flushed to elute the cells. The procedure is then restarted using a new column. Blockage can also be avoided by increasing the dilution of the cells before they are applied to the column, although this does make the procedure longer. 9. Removing the plunger and reflushing the column is not recommended as this generates a lot of cellular debris,

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which may adversely affect later analysis. The recovered cells can be stored on ice and recombined later. It is also important to prevent the column from running dry. 10. The presence of CD56 MicroBeads on the CD56 antigen does not prevent labelling with Becton Dickinson CD56PE antibody for FACS analysis. However, they will interfere with indirect labelling with mouse antibodies as the secondary anti-mouse antibody can bind to the CD56 antibody on the beads. We have found that the MicroBeads are lost after a few days in culture. 11. Some investigators may be concerned that the positive selection procedure may alter the decidual NK cell phenotype. However, CD56 is of unknown function and crosslinking this receptor has never been shown to have an effect on known functions of NK cells. Indeed, anti-CD56 is regularly used as a negative control antibody in NK cell functional assays. MicroBead cocktails are also available for the isolation of dNK cells by negative selection, although these are more costly than the CD56 MicroBeads. 12. This method can easily be adapted for the recovery of other leukocytes such as CD14+ macrophages or CD3+ T cells using different sets of MicroBeads. 13. The MACS procedure generally takes about 90 min. From tissue sorting to purified decidual NK cells will probably take 3–4 h. 14. Purified decidual NK cells do not survive long in culture so they should be used immediately. In our experience only 30% of purified decidual NK cells will be viable after overnight culture in RPMI 10% FCS unless 1 ng/mL of IL-15 is added. Decidual NK cells can be made to proliferate by culturing with a combination of 50 ng/mL SCF and 5 ng/mL IL-15 (both R&D Systems, Oxford, UK). We have then cultured these cells over a period of 4 days. 15. In our experience, each batch of trypsin gives different results and may require a variation of a minute either way for optimal digestion time. When tissues are available it is advantageous to empirically determine the best timing when each new batch of trypsin is made. In the hope of avoiding this problem we have, in the past, attempted to use commercially available, ready-made, pure trypsin solutions. For reasons we do not understand these do not work. 16. In freshly purified trophoblast before overnight culture on fibronectin there are approximately 20% HLA negative cells, which are, therefore, of the villous phenotype.

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17. The extraction takes around 2.5 h from scraping to plating of the trophoblast on fibronectin. 18. It is not yet known whether this increase in HLA-G+ cells is due to preferential sticking of HLA-G+ cells to the matrix or due to a switching of phenotype. 19. We usually use these cells within 3 days but they can be plated on fibronectin for a further 4–6 days. References 1. Boyd, J.D., and Hamilton, W. J. (1970). The Human Placenta. W. Heffner and Sons, Cambridge, UK 2. Kam, E.P., Gardner, L., Loke, Y.W., and King, A. (1999). The role of trophoblast in the physiological change of the decidual spiral arteries. Hum. Reprod. 14, 2131–2138 3. Moffet, A., and Loke, C. (2006). Immunology of placentation in eutherian mammals. Nat. Rev. Immunol. 6, 584–94 4. Kovats, S., Main, E.K., Librach, C., Stubblebine, M., Fisher, S.J., and DeMars, R. (1990). A class I antigen, HLA-G, expressed in human trophoblasts. Science 248, 220–3 5. Loke, Y.W., King, A., Burrows, T., Gardner, L., Bowen, M., Hiby, S., Howlett, S., Holmes, N., and Jacobs, D. (1997). Evaluation of trophoblast HLA-G antigen with a specific monoclonal antibody. Tissue Antigens 50, 135–46 6. King, A., Burrows, T.D., Hiby, S.E., Bowen, J.M., Joseph, S., Verma, S., Lim, P.B., Gardner, L., LeBouteiller, P., Ziegler, A., Uchanska-Ziegler, B., and Loke, Y.W. (2000). Surface expression of HLA-C antigen by human extravillous trophoblast. Placenta 21, 376–87 7. King, A., Allan, D.S., Bowen, M., Powis, S.J., Joseph, S., Verma, S., Hiby, S.E., McMichael, A.J., Loke, Y.W., and Braud, V.M. (2000). HLA-E is expressed on trophoblast and interacts with CD94/NKG2 receptors on decidual NK cells. Eur. J. Immunol. 30, 1623–31 8. Apps, R., Gardner, L., Hiby, S.E., Sharkey, A.M., and Moffett, A. (2008). Conformation of human leukocyte antigen-C molecules at the surface of human trophoblast cells. Immunology 124, 322–8 9. Apps, R., Murphy, S.P., Fernando, R., Gardner, L., Ahad, T., and Moffett, A. (2009). Human leukocyte antigen (HLA) expression by normal trophoblast cells and placental cell lines, determined using single antigen beads to characterize allotype specificities of anti-HLA antibodies. Immunology 127, 26–39

10. King, A., Balendran, N., Wooding, P., Carter, N.P., and Loke, Y.W. (1991). CD3- leukocytes present in the human uterus during early placentation: phenotypic and morphologic characterization of the CD56++ population. Dev. Immunol. 1, 169–90 11. King, A., Wooding, P., Gardner, L., and Loke, Y.W. (1993). Expression of perforin, granzyme A and TIA-1 by human uterine CD56+ NK cells implies they are activated and capable of effector functions. Hum. Reprod. 8, 2061–7 12. Verma, S., King, A., and Loke, Y.W. (1997). Expression of killer cell inhibitory receptors on human uterine natural killer cells. Eur. J. Immunol. 27, 979–83 13. Sharkey, A.M., Gardner, L., Hiby, S., Farrell, L., Apps, R., Masters, L., Goodridge, J., Lathbury, L., Stewart, C.A., Verma, S., Moffett, A. (2008). Killer Ig-like receptor expression in uterine NK cells is biased toward recognition of HLA-C and alters with gestational age. J Immunol. 181, 39–46. 14. King, A., Birkby, C., and Loke, Y.W. (1989). Early human decidual cells exhibit NK activity against the K562 cell line but not against first trimester trophoblast. Cell Immunol. 118, 337–4415. 15. Koopman L.A., Kopcow, H.D., Rybalov, B., Boyson, J.E., Orange, J.S., Schatz, F., Masch, R., Lockwood, C.J., Schachter, A.D., Park, P.J., and Strominger, J.L. (2003). Human decidual natural killer cells are a unique NK subset with immunomodulatory potential. J. Exp. Med. 198, 1201–12 16. Kopcow H.D., Allan, D.S., Chen, X., Rybalov, B., Andzelm, M.M., Ge, B., and Strominger, J.L. (2005). Human decidual NK cells form immature activating synapses and are not cytotoxic. Proc. Natl. Acad. Sci. U S A. 102, 15563–8 17. King, A., Kalra, P., and Loke, Y.W. (1990). Human trophoblast cell resistance to decidual NK lysis is due to lack of NK target structure. Cell Immunol. 127, 230–7

Natural Killer Cells in Human Pregnancy 18. King, A., and Loke, Y.W. (1990). Human trophoblast and JEG choriocarcinoma cells are sensitive to lysis by IL-2-stimulated decidual NK cells. Cell Immunol. 129, 435–48 19. Hampson, J., McLaughlin, P.J., and Johnson, P.M. (1993). Low-affinity receptors for tumour necrosis factor-alpha, interferongamma and granulocyte-macrophage colonystimulating factor are expressed on human placental syncytiotrophoblast. Immunology 79, 485–90 20. Saito, S. Nishikawa, K., Morii, T., Enomoto, M., Narita, N., Motoyoshi, K., and Ichijo, M. (1993). Cytokine production by CD16CD56bright natural killer cells in the human early pregnancy decidua. Int. Immunol. 5, 559–63 21. Jokhi, P.P., King, A., Jubinsky, P.T, and Loke, Y.W. (1994). Demonstration of the lowaffinity alpha subunit of the granulocytemacrophage colony-stimulating factor receptor (GM-CSF-R alpha) on human trophoblast and uterine cells. J. Reprod. Immunol. 26, 147–64 22. Sharkey, A.M., King, A., Clark, D.E., Burrows, T.D., Jokhi, P.P., Charnock-Jones, D.S., Loke, Y.W., and Smith, S.K. (1999). Localization of leukaemia inhibitory factor and its receptor in human placenta throughout pregnancy. Biol. Reprod. 60, 355–64 23. Hiby, S.E., Walker, J.J., O’Shaughnessy, K.M., Redman, C.W., Carrington, M., Trowsdale, J., and Moffet, A. (2004). Com-

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binations of maternal KIR and fetal HLAC genes influence the risk of preeclampsia and reproductive success. J. Exp. Med. 200, 957–65 Hanna, J., Goldman-Wohl, D., Hamani, Y., Avraham, I., Greenfield, C., NatansonYaron, S., Prus, D., Cohen-Daniel, L., Arnon, T.I., Manaster, I., Gazit, R., Yutkin, V., Benharroch, D., Porgador, A., Keshet, E., Yagel, S., and Mandelboim, O. (2006). Decidual NK cells regulated key developmental processes at the human fetal-maternal interface. Nat. Med. 12, 1065–74 Ashkar, A.A., DiSanto, J.P., and Croy, B.A. (2000). Interferon gamma contributes to initiation of uterine vascular modification, decidual integrity, and uterine natural killer cell maturation during normal murine pregnancy. J. Exp. Med. 192, 259–70 Li, X.F., Charnock-Jones, D.S., Zhang, E., Hiby, S., Malik, S., Day, K., Licence, D., Bowen, J.M., Gardner, L., King, A., Loke, Y.W., and Smith, S.K. (2001). Angiogenic growth factor messenger ribonucleic acids in uterine natural killer cells. J. Clin. Endocrinol. Metab. 86, 1823–34 Trundley, A., and Moffett, A. (2004). Human uterine leukocytes and pregnancy. Tissue Antigens 63, 1–12 Tabanelli, S., Tang, B., and Gurpide, E. (1992). In vitro decidualization of human endometrial stromal cells. J. Steroid Biochem. Mol. Biol. 42, 337–44

Chapter 31 Analysis of Uterine Natural Killer Cells in Mice B. Anne Croy, Jianhong Zhang, Chandrakant Tayade, Francesco Colucci, Hakim Yadi, and Aureo T. Yamada Abstract The term uterine natural killer (uNK) cell is applied in mice to an abundant but transient NK cell population that undergoes unique, terminal differentiation within embryo implantation sites during endometrial decidualization and pregnancy. In mice, decidualization is induced by attachment and implantation of hatched, blastocyst-stage embryos. Within each implantation site, uNK cells proliferate and rapidly differentiate into highly restricted regions called decidua basalis and the mesometrial lymphoid aggregate of pregnancy (MLAp). uNK cells begin to die within healthy decidua basalis by day 8 of the 19–20 day pregnancy of mice. By gestation day 12, uNK cell numbers have peaked and most uNK cells show in situ nuclear fragmentation indicative of disintegration. Morphological studies (standard histology, ultrastructure, immunohistochemistry, in situ hybridization, and RNA analyses from laser capture microdissected uNK cells) have provided most of the current understanding regarding this cell lineage. These approaches identified the special angiogenic properties of uNK cells and their regulatory relationships with normal physiological changes to the uterine (endometrial) arterial tree that accompany successful pregnancy. This chapter highlights key information needed for successful dissection of the dynamically changing decidua basalis that is enriched in uNK cells and special morphological procedures used for uNK cell study. Preparation of viable mouse uNK cell suspensions is difficult but can be achieved. This chapter includes techniques for isolation of uterine leukocyte suspensions and their enrichment for uNK cells that permit immediate downstream applications such as culture, isolation of high quality RNA, or flow cytometry. Key words: Angiogenesis, DBA lectin, Decidua, Endometrium, Flow cytometry, Laser capture microdissection, Magnetic bead-based uNK cell enrichment, Pregnancy.

1. Introduction During early pregnancy in mice, uterine natural killer (uNK) cells accumulate in abundance on the mesometrial sides of the uterine horns at each implantation site. Late on gestation day (gd)3.5 (counting the morning of copulation plug detection as gd0.5), K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 31, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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free-floating mouse embryos hatch from the zona pellucida and immediately implant on the anti-mesometrial (see Note 1) side of the uterus. This induces the transient, gestational transformation of uterine stromal cells at each implantation site into large cells called decidual cells. Over the next ∼4 days, decidual development progresses from the relatively avascular, anti-mesometrial side of the uterus to the mesometrial side where major blood vessels enter and exit the uterus. Coincident with induction of mesometrial decidua is the appearance of unique, terminally differentiating cells called uNK cells. uNK cells are numerous by gd6.5 (Fig. 31.1A), proliferate rapidly, and increase in number (Fig. 31.1B) until midgestation (gd10.5–12.5; Fig. 31.1C). In normal mice, most uNK cell nuclei show DNA fragmentation from gd12.5 and the numbers decrease (Fig. 31.1D) until only a small, residual population remains at term. These residual uNK cells are shed with the placenta (1, 2). Early postpartum uterus is essentially devoid of terminally differentiated uNK cells. Cells expressing NK markers first appear in the infant mouse uterus ∼2 weeks postpartum (3). These should be considered

Fig. 31.1. Photomicrographs of DBA lectin-stained midsagittal sections from C57BL/6 J implantation sites from gd6.5 (A), gd8.5 (B), gd10.5 (C), gd13.5 (D). Facing arrows in (C) indicate the midgestation maternal–fetal interface formed by the decidua basalis and trophoblast giant cells. There are dynamic changes in the uNK cell population during normal mouse pregnancy. M, mesometrial side; L, remainder of the uterine lumen; T, ectoplacental cone trophoblast; MLAp, mesometrial lymphoid aggregate of pregnancy; DB, decidua basalis; F, fetus; P, placenta; SA, spiral artery; SM, smooth muscle.

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endometrial (e)NK cells because they do not show the characteristics of terminally differentiated uNK cells that appear with uterine decidualization (4, 5). Differentiation of mouse uNK cells does not require an embryo, fetus, or placenta. Rather, uNK cell differentiation appears to be driven by ovarian progesteroneinduced changes to the endometrial stroma (see Note 2). In human endometrium, decidua-associated NK cells, recognized as CD56bright , CD16− , CD3− , differentiate initially between days 3 and 8 after the menstrual cycle surge in luteinizing hormone, whether or not intercourse or conception occurred. These cells, now called “eNK cells of the menstrual cycle,” have no equivalent in rodents. If a woman conceives, NK cells that appear analogous to mouse uNK cells become abundant in the decidua and are called either dNK for decidual NK cells (6) or uNK cells. It is not yet clear how the two human cell populations (eNK and dNK/uNK) are related to each other but they are distinct from systemic NK cells (7, 8). In both humans and mice, the origins and relationships of NK cells found within the virgin or pregnant uterus to systemic NK cells remain unclear and under study (9). Current information suggests mouse gestational uNK cells are unlike NK cells in lymphoid tissues (10, 11), although the lineage is readily established from any lymphoid organ of a normal male or female mouse by transplantation into mated females co-deficient in NK and T cells (12). Additionally, uterine segments from normal mice engrafted into normal mice but not into mice deficient in NK and T cells differentiate uNK cells upon mating and decidualization. These data suggest self-renewing progenitors of uNK cells do not reside within mouse uterus. uNK cells differentiating within mouse decidua change dramatically in morphology (Fig. 31.2A,B) and phenotype. The cells rapidly acquire membrane-bound, cytoplasmic granules and increase to become gigantic in size (a change from ∼10 ␮M to >80 ␮M in diameter). The granules contain many enzymes and glycoproteins including perforin and granzymes and appear to be stored within the cytoplasm, eventually filling it. The number of granules/cell is used to estimate uNK cell maturity. Four morphologically different uNK cell subtypes can be recognized using this approach (13). By ultrastructure and immunoelectron microscopy, the cytoplasmic granules in mouse uNK cells were found to be composed of two segregated compartments (Fig. 31.2C,D), a peripheral multi-vesicular electron dense lysosomal domain (Fig. 31.2E) and a central homogeneous core (Fig. 31.2F) that contains secretory, lytic proteins, and proteoglycans (14–16). Similar granules are present in cytotoxic T cells but are unusual in most other mammalian cell types (17). Secretory pathways are more generally studied in uNK cells than the degradative pathways, leaving a deficit in information regarding the biological functions of these

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Fig. 31.2. Ultrastructure of uNK cells. Immature uNK cells with few cytoplasmatic granules predominate in early pregnancy (gd5.5 to 7.5) (A). (B) From gd8.5 onward, fully mature uNK cells with huge numbers of granules predominate. The most characteristic morphological feature found in uNK cells is the secretory-lysosomal granule. These are concentrated around the Golgi complex, their site of origin (C). The double compartment secretory-lysosome granules (D) have an outer, electron-dense cap formed by microvesicles and central homogenous core. (E) The peripheral cap region is a lysosomal domain as shown by immunoelectron microscopy for cathepsin D expression. (F) The central homogenous domain contains secretory proteins and proteoglycans as is shown by cuprolinic blue ultrastructural cytochemistry. N, nucleus; G, granules; GC, golgi complex; M, mitochondria; SM, smooth muscle cells of myometrium; ∗ , uNK granule secretory compartment; #, uNK granule lysosome compartment.

cells. uNK cells are usually regarded as being weakly lytic in vivo. From midgestation onward, the very large sizes of uNK cells, their noxious granule content, and their widespread in situ death program, make the success of culture-based studies challenging.

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Heavily granulated uNK cells are found throughout the decidua basalis (see Fig. 31.1 to appreciate this region) and, in some strains, as infrequent cells within the placenta. uNK cells are not normally found in anti-mesometrial decidua or in non-decidualized endometrium between implantation sites. As pregnancy progresses, a thickening of the uterine wall occurs at each implantation site. From ∼gd8.5, this is seen by histological examination to be a lymphocyte-driven separation between the two layers of uterine smooth muscle. This thickened, donut-like microenvironment contains the smallest, proliferating, least granulated uNK cells and encircles the major maternal vessels to form portals for vessel entry to and exit from implantation sites. Experimental studies suggest that mouse and human uNK cells make pivotal contributions to the unique, gestation-induced structural changes in maternal arteries that supply each developing placenta (1, 8, 18), although human uNK cells have not been shown to have such rigorous positioning within implantation sites. The changes to vascular structure that appear to be uNK cell-initiated result in loss of vascular smooth muscle and lumen dilation. These changes greatly reduce/prevent vasoconstriction in the major placental “feed arteries” during pregnancy and are reversed postpartum. In women, pre-eclamptic pregnancies are associated with limited-to-absent changes in the analogous vessels named spiral arteries. Classically, mouse uNK cells have been identified histologically by staining with Periodic Acid Schiff’s (PAS) reagent (5), a reagent that detects the glycoproteins found in the cytoplasmic granules of mature uNK cells. This approach has several limitations. First, cells other than uNK cells react to this stain and must be discriminated morphologically as non-lymphoid cells. Second, only uNK cells with cytoplasmic granules are identified, which leaves lineage-committed, immature cells unmarked. Advantages of PAS staining are its ease and low cost. This makes PAS staining useful when an experiment requires screening or study of a serial section series. Use of Dolichos biflorus (DBA) lectin staining has widely replaced PAS staining for histological recognition of mouse uNK cells (see Note 3). This lectin which recognizes terminal N-acetyl−D-galactosamine, reacts with the plasma membranes of both uNK cells and their granules, permitting identification of early uNK cells prior to their acquisition of cytoplasmic granules (13). DBA lectin surface-reactive lymphocytes are not found in peripheral lymphoid tissues of virgin or gd0.5-7.5 mice (9). Within mouse implantation sites, DBA lectin reactivity is also seen on yolk sac (fetal) and endometrial (maternal) endothelium (Fig. 31.1C,D). Protocols for either histochemical or lectin reactivity can be dramatically shortened to stain frozen tissue sections for successful RNA recovery and subsequent analysis of uNK cells using laser capture microdissection (LCM). DBA lectin is

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additionally useful for isolation of uNK cells from decidual cell suspensions and for flow cytometry. Our recent histochemical and cytometric studies indicate that DBA lectin does not mark all uNK cells and that a significant (20–25%) PAS positive /DBA lectin negative population exists. Definition of uNK cell subsets based on functional and/or phenotypic differences rather than granularity, size, and nuclear properties is in its infancy. Such studies have required development of culture and/or holding media that sustain viability and differentiation of this hormone- and stromal cell-dependent lineage over the analysis interval. Here we provide protocols for (i) mating of mice, perfusion fixation, and preparation of paraffin-embedded, frozen, or ultrathin histological sections; (ii) routine DBA lectin staining, uNK cell quantification and spiral arterial evaluation in paraffinembedded tissue sections; (iii) brief staining with hematoxylin and eosin (H&E) for LCM, the LCM process followed by RNA isolation, amplification, reverse transcription, and real-time PCR; (iv) dissection of implantation sites and preparation of uNK cell suspensions; (v) culture of mouse uNK cells; and (vi) flow cytometry of uNK cells. There are no special features for studies of uNK cells by immunohistochemistry, in situ hybridization, or ultrastructure beyond collection/study of the correct region of the implantation site. Other protocol sources should be sought by readers requiring guidance in these techniques. Similarly, once uNK cell suspensions are prepared, their downstream handling for molecular or protein studies employing cell lysis is not distinct. Because uNK cells are exceptionally large and fragile cells committed to a cell death pathway, cytotoxicity studies must include incubated controls of uNK effector cells only and subsequent transfer of the post-incubation culture supernatants to target cells. This will establish whether true cytolytic effector function was measured in test wells or spontaneous release of effector molecules from uNK cells degenerating during short-term culture.

2. Materials 2.1. Mouse Mating and Preparation of Histological Materials from Post-Implantation Uteri

1. Randombred or inbred female mice at 8–12 weeks of age, cycling naturally and selected for estrus. 2. Adult but not overweight male mice, caged individually as stud males. 3. Microspatula (Fisher Scientific, #21-401-25A) for use as a vaginal speculum. 4. CO2 cylinder and chamber for euthanasia, if perfusion of the mouse is not required.

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5. Small dissection tray lined with disposable adsorbent material and dissection pins. 6. Kimwipes or sterile gauze squares for removing fur and blood from dissection sites and instruments. 7. Heparin (0.1%) in PBS. 8. Dissection instruments (sterilized if cell culture is planned, RNase free if molecular studies are planned). Fine forceps – one pair for skin, two pairs for organ holding (Fine Science Tools, #11241-30); several pairs of ultrafine #5 watchmaker’s forceps (Fine Science Tools, #11252-00); one pair curved, blunt-ended forceps (Fine Science Tools, #1100312); fine dissection scissors – one pair for skin, one pair for internal organ dissection (Fine Science Tools, #1408011); Vannas spring scissors – one pair (Fine Science Tools, #91501-09); curved fine iris scissors – one pair (Fine Science Tools, #14061-09). 9. Alcohol (70%) as a fur moistening agent, skin disinfectant, and instrument wipe. 10. Butterfly infusion set (Fisher Scientific, #NC9175100) and 50 mL syringe if animal is to be perfusion fixed prior to tissue dissection. Use 30 mL freshly prepared 4% paraformaldehyde (PFA), 0.1 M sucrose in 0.1 M phosphate buffered saline (PBS) pH 7.4 to perfuse for light microscopy. Use 30 mL of a freshly prepared solution of 2% paraformaldehyde, 1.5% glutaraldehyde (GTA, Polysciences, Inc., Electron Microscopy grade), and 0.1 M sucrose in 0.1 M PBS, pH 7.4 to perfuse for standard ultrastructural studies or 4% PFA + 0.2% GTA and 0.1 M sucrose in 0.1 M PBS, pH 7.4 to perfuse for ultrastructural immunocytochemistry. 11. Timer. 12. Anesthetic and apparatus for its administration, according to investigator’s preferences and approved animal utilization protocols. 13. Small disposable sterile petri dishes, beakers, and conical tubes (different sizes). 14. Holding medium for dissection. This will vary with the purpose of the study. PBS is used for histology, RPMI 1640 or Hank’s (Invitrogen) medium with supplementation (may include BSA or serum and IL-15 or OP-9 conditioned medium) for longer procedures or culture. 15. A dissection microscope with a long working distance may be useful, especially when learning the dissections or conducting them before midgestation (gd10.5). With experience, this may be less necessary.

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16. Frosted-end, positively charged glass microscope slides, 60×22 mM glass coverslips (Thermo Shandon), Permount (Fisher Scientific) or other compound appropriate for mounting coverslips onto stained slides. 17. DAKO marking pen (DAKO, #S2002). 18. For preparation and cutting of paraffin sections: a. histological embedding rings (Thermo Shandon, #58993) b. embedding molds (Thermo Shandon, #6401015/ 6401016) c. tissue processor d. chemicals for paraffin embedding (see Note 4) e. embedding station, microtome with disposable blades, warming plate for spreading of sections, and jars for staining series as appropriate. 19. For preparation and cutting of cryostat (frozen) sections: a. isopentane (Fisher Scientific) in a nalgene beaker cooled in liquid N2 for rapid freezing (Caution: never use a glass beaker which will shatter at the ultralow temperature) b. small, 10×10×5 mM cryomolds (Tissue Tek, Miles Inc., #NC9806558) for gd5.5-8.5 specimens; larger, 15×15×5 mM cryomolds for specimens older than gd8.5 (Tissue Tek, Miles Inc., #NC9008138) c. aluminum foil and small, ziplock freezer bags, cryomarking pens to label freezer bags d. optimal cutting compound (OCT; Tissue Tek, #4583) e. long, sturdy forceps (25 cm or longer) to remove specimens from liquid N2 , freezer safety gloves f. Cryostat g. −80o C freezer for storage of frozen samples. 20. For preparation and cutting of ultrathin sections for electron microscopy: a. oscillating tissue slicer (Electron Microscopy SciencesEMS, Hatfield, USA) b. quick polymerizing glue (Super-bonder, 3 M) c. 4% PFA + 0.2% GTA + 0.1 M sucrose in 0.1 M PBS pH7.4 d. ultra-pure water e. forceps for tissue processing or thin glass rods f. dissection microscope for observation and handling of thin tissue slices

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g. 3–5 mL glass vials; fume hood h. gloves; safety goggles i. 1% osmium tetroxide in 0.1 M PBS (Caution: osmium is highly toxic and volatile – avoid inspiration or contact with skin or mucosal surfaces) j. graded ethanol series in water (50%, 70%, 85%, 95%); absolute ethanol k. propylene oxide (Caution: this is highly volatile – close the vial!) l. N,N, dimethyl formamide (Sigma) m. epoxy resin (PolyBed812, Polysciences) or LR-white resin (Polysciences) n. beem capsules (size 00, flat button, EMS) o. oven for holding 60◦ C temperature p. razor blades, specimen rotator (Bal-Tec, #B7925), ultramicrotome (Ultracut, Leica, Sweden), glass histology slides q. 0.5% toluidine blue r. knife maker (Leica, Sweden), glass (6 or 8 mM thick), and 3.5 mM diamond knives (Diatome, EMS) s. 150 mesh cooper grids (Veco, #0150-Cu); 200 mesh Nickel grids-(Veco, #0200-Ni); large petri dishes to hold grids t. Reynold’s lead citrate solution (Caution: avoid air contact to prevent precipitation. Keep aliquots in syringes at 4◦ C) u. 2% uranyl acetate (Caution: Keep this solution in a dark bottle and centrifuge it at 1000×g for 5 min before use) v. carbon coating (Bal-Tec, SCD050 Sputter Coating) w. freeze substitution unit (Bal-Tec, FSU030). 2.2. Basic DBA Lectin Staining and Quantification of uNK Cells and Spiral Arterial Modification

1. Prepared tissue sections mounted on glass slides. Paraffin sections should be cut at 4–7 ␮M and deparaffinized before use. Cryostat sections may be cut at 5–10 ␮M, fixed briefly (10 s) in acetone and air dried before use. 2. PBS 0.1 M and 0.05 M pH7.4. (Comment: For reaction steps, 0.1 M PBS is recommended for effective buffering. For washing steps, 0.05 M PBS is typically used). 3. Tris-buffered saline (TBS; 0.1 M Tris mixed with 0.3 M NaCl, 1:1, pH7.4 for reactions; 0.05 M for washes). 4. Biotinylated Dolichos biflorus agglutinin (DBA; Vector Laboratories, # B-1035).

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5. 0.1 M N-acetyl-D-galactosamine (Fisher Scientific) 6. ExtrAvidin peroxidase (Sigma). 7. Diaminobenzidine (DAB; Sigma). (see Note 5). 8. 3% H2 O2 (see Note 6). 9. Harris’s Hematoxylin (Sigma). 10. Compound photomicroscope with image analysis system. Fluorescence detection is useful for immunohistochemical studies. 11. Xylene (Fisher Scientific). 12. Graded ethanol series including 100% (anhydrous) ethanol. 2.3. Implantation Site Dissection, Preparation of Frozen Tissue Sections for Laser Capture Microdissection (LCM), Rapid Staining for H&E that Permits Subsequent RNA Analysis of Dissected uNK Cells (see Note 7)

1. RNase inhibitor (such as SUPERase, Ambion) for preparing dissection instruments, areas to be used for dissections when LCM is planned, microscope stages, and for preparation of staining solutions.

2.3.1. Supplies Unique For LCM

5. New disposable blades for Leica CM1850 UN (Leica Microsystems) or other available cryostat.

2. RNase-, DNase-free water (Arcturus or Gibco BRL). 3. Dry ice. 4. Frozen tissue block. This can be used for RNA or DNA isolation. Paraffin-embedded, formalin-fixed blocks can also be used for DNA isolation but must be pre-tested (Paradise Plus Reagent System, Molecular Devices) before LCM to determine if RNA isolation is possible.

6. Uncharged glass histology slides or membrane-coated slides (PEN Membrane Glass Slide, Molecular Devices, #LCM0522) depending on the downstream applications and tissue lifting success during dissections. Coverslips are not used. 7. Desiccator (DRIERITE Co) containing slide storage box. 8. Capsure HS LCM caps (Molecular Devices). 9. Tissue preparation strips (Fisher Scientific) or clean-up pads. 10. ExtracSure extraction device (Molecular Devices). 11. 0.5 mL capacity microfuge tubes. 12. Incubation block (Molecular Devices, #LCM0505) and incubator. 13. Nucleic acid extraction reagents such as Picopure RNA or DNA isolation kits (Molecular Devices). 14. LCM dissection microscope (a Molecular Devices infrared system is recommended for this work rather than a laser cutting system since RNA transcripts are longer (better

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quality) when single cells or tissue <30 ␮M are to be dissected). 15. −80◦ C freezer for RNA sample storage. 16. UV spectrophotometer such as Nanodrop (Thermo Scientific, Nanodrop 1000) and bioanalyzer (Agilent Technologies). 17. Latex disposable gloves which will be worn at all steps in sample preparation. 2.3.2. Reagents for Hematoxylin and Eosin Staining for LCM

1. All staining reagents (ethanol (75%, 95%), Harris’s hematoxylin (Sigma, #HHS-16), eosin (Fisher Scientific, #22220-104)) are prepared using RNase-/DNase-free water and RNase inhibitor. Staining jars are prepared to be RNasefree. 2. Fresh, 100% ethanol stored in small aliquots to prevent environmental hydration. 3. 100% Xylene. 4. Dako pen (DAKO, #S2002).

2.3.3. Reagents for RNA Analyses Following LCM

1. Picopure RNA extraction kit (Molecular Devices). 2. RNase-, DNase-free water (Invitrogen). 3. First strand cDNA synthesis kit (GE Healthcare). 4. Filter tips (10 ␮L, 100 ␮L, 200 ␮L, Ultident). 5. 0.5 mL capacity microfuge tubes (Ultident). 6. Quantitect SYBR Green I PCR mix kit (Qiagen). Reagents mentioned in the protocol are from this kit. 7. MessageAmp II aRNA kit (Ambion or similar kits available from MDS Analytical Technologies or others). Reagents mentioned in the protocol are from this kit. 8. Primers for genes of interest. 9. Roche Real-Time PCR Light Cycler (Roche Diagnostics, Laval, QU, Canada) or other system.

2.4. Preparation of Decidual Cell Suspensions (see Note 8) 2.4.1. Tissue Dissociation and uNK Cell Enrichment Using Density Gradient Centrifugation

1. Ice bucket and small plate, each containing crushed ice to keep the cell isolation procedure below ambient temperature and increase viable cell yields. 2. Sterile scissors and forceps for dissections (see Section 2.1.8). Sterile scalpel blades (Fisher Scientific) or razor blades to remove unwanted tissues. 3. Dissection microscope with long working distance. 4. Cell culture dishes to prepare single cell suspensions (BD Biosciences).

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5. Stainless steel mesh or nylon tissue sieves with 80–200 ␮M screens. 6. 0.1 M PBS, pH7.4 (Gibco). 7. Pasteur pipettes or other large bore liquid transfer tools. 8. Hematocytometer such as Neubauer’s chamber and 0.1% trypan blue in PBS or other viability stain. 9. RPMI medium 1640 (Gibco) with 10% fetal bovine serum (Gibco). 10. Plungers from syringes (1–10 mL sizes are suitable). 11. Centrifuge tubes (15 mL, 1.5 mL). R X-12R) 12. Centrifuges (such as Beckman Coulter, Allegra ◦ adjusted to working temperatures of 4 C, except during the density gradient centrifugation step when ambient temperature provides optimal step gradient separation. R -M density gradient (CEDARLANE Labora13. Lympholyte tories Ltd.).

2.4.2. Tissue Dissociation and uNK Cell Purification Using DBA lectin-Coated Magnetic Beads

1. Materials 1–6, 8 from Section 2.1. 2. Hank’s balanced salt solution (Sigma). 3. Hank’s solution containing 1000 IU DNase I (Fisher Scientific). 4. Distilled water and 3.5% NaCl for erythrocyte lysis or supplies for any alternate method. 5. 0.1 M PBS pH7.4 containing 2% BSA. 6. Refrigerated centrifuge and tubes or microfuge and tubes (our laboratories differ over this equipment but all obtain successful cell isolation). 7. M450 CelLection-Biotin Binder magnetic beads (Invitrogen) and biotinylated DBA lectin (Sigma) to coat the beads. 8. Magnetic particle concentrator (Dynal Inc.). 9. 0.1 M N-acetyl-D-galactosamine (Sigma). 10. RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum and 1 mg/mL gentamycin. 11. 0.1% Tween (Sigma) in 0.1 M PBS pH7.4.

2.5. Microspot Culture of Mouse uNK Cells

1. Slide culture chambers with four or eight chambers/slide (Fisher Scientific). 2. 2 ␮g/mL Fibronectin (Invitrogen). 3. RPMI 1640 medium supplemented with 10% fetal bovine serum, 200 ng/mL IL-15 (Invitrogen), and 100 ng/mL CXCL10 (Invitrogen).

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4. Sterile mineral oil tested on mouse pre-implantation embryos and found to be non-toxic (Sigma). 5. Humidified, 5% CO2 incubator at 37◦ C. 6. Dissection microscope. 7. 1.0 mM inner diameter glass capillary tube and micromanipulator arm apparatus. 2.6. Flow Cytometry

1. uNK cell suspension. 2. Flow cytometry system. 3. Culture tubes for sample preparation and for loading samples into the flow cytometer (VWR International). 4. FACS buffer (0.1 M PBS with 5% fetal bovine serum or 1% BSA). 5. Various antibodies or lectins (10, 19). Those we have used are given in Table 31.1.

Table 31.1 Antibodies or lectins used to characterize uNK cells Antibodies (clone)

Cat.#

Manufacturer

FITC-anti-mouse pan-NK cells (CD49b) (DX5)

#11-5971

eBioscience

PE-Cy5 anti-mouse CD3 (145-2C11)

#15-0031

eBioscience

PE anti-mouse TCR␤ (H57-597)

#12-5961

eBioscience

Biotin conjugated DBA lectin

#L6533

Sigma

FITC-CD122 (TM-b1)

#553361

BD Pharmingen

FITC-NK1.1 (PK136)

#553164

BD Pharmingen

PE-CD27 (LG.3A10)

#558754

BD Pharmingen

FITC anti-mouse NK cell (Ly-49C/I/F/H) (14B11)

#12-5991

eBioscience

PE anti-mouse Ly-49A/D(Ly49A/D) eBio12A8 (12A8)

#12-5783

eBioscience

PE anti-mouse CD11b (M1/70)

#12-0112

eBioscience

3. Methods 3.1. Preparation of Mice

1. Toward the end of the mouse colony lighting period (see Note 9), female mice should be gently picked up and held vertically against a stable surface such as the investigator’s chest, to examine their vaginal opening for signs of estrus (dilation, axial ridges on the dorsal mucosa, and just after a stage of moist redness when the mucosa is strongly pink but

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not too shiny). Estrous females should be placed into the cage of a stud male who is individually housed. One to three females may be used per male. If copulation occurs, the male should be rested 1–2 nights before being used again. 2. The morning after co-habitation with the male, each female is removed from the mating cage and examined in a stabilized, vertical position for a hard, occlusive plug within the vagina. The position of the plug can range from very deep against the cervix to very superficial in position. The plug will fall out naturally 12–15 h after mating. 3. The success of each stud male in producing a pregnancy should be recorded. Unsuccessful or older, overweight males should be replaced quickly by young males ∼7 weeks of age. 3.2. Tissue Fixation and Processing for Histological Studies

1. Mated animal(s) at the desired gestational stage. Most work is done between gd6.5-12.5. 2. If cell suspensions are to be prepared, the investigator should employ her/his preferred and approved method of euthanasia, then wet the cadaver with 70% alcohol, and open the skin and ventral abdominal wall using two instrument sets and move to Section 3.6. For most histological studies, perfusion-induced euthanasia is used. The investigator should deeply anesthetize the mouse with her/his approved method of choice. Steps 3–5 in this section describe the perfusion process. Select and prepare the medium to be used for perfusion (paraformaldahyde for light microscopy, paraformaldehyde with glutaldehyde for electron microscopy) and load it into a 50 mL syringe for each animal. Attach a butterfly needle system to a 5cc syringe filled with 0.1% heparin in PBS. 3. Place the deeply anesthetized mouse on its back in the adsorbent-lined tray and wet the animal liberally with 70% alcohol to prevent hair contamination of the dissection. With a Kimwipe or gauze square, wipe the thoracic and abdominal areas in the direction of fur growth to remove any excess alcohol from the intended incision site. Pin the mouse to the tray to immobilize it and open the abdominal cavity along the ventral midline, to confirm pregnancy. Separate sets of instruments are used for cutting skin, which can then be peeled away and for incision of the abdominal wall. This reduces hair contamination of histological work and chemical and molecular contamination of RNA preparations. If the animal is not pregnant, remove it from the tray and euthanize it. If the animal is pregnant, continue immediately with the perfusion.

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4. With heavier dissection scissors, transect the sternum and cut ribs to access the heart. Introduce the needle of the butterfly system into the left ventricle chamber of the still beating heart and start perfusion with 5 mL PBS-heparin. Then cut a notch in the right side of the heart for perfusate outflow, attach the large syringe of fixative to the butterfly apparatus and deliver 30 mL of fixative (always use exactly the same mount) as follows. The first 5 mL of fixative solution should be delivered with uniform pressure and quickly, taking ∼1 min. The remaining fixative should be delivered at a much reduced pressure that will take 15 min to completely deliver. Time these steps of the perfusion to assure identical and comparative fixation for all animals in and between experiments. Terminal respiratory distress will be seen and it is essential for humane reasons that any animal being used for perfusion be under deep anesthesia. Any study involving blood vessel measurements must employ well-perfused specimens. 5. After the fixative solution has been delivered, let the animal rest in the tray for an additional 15 min before removing the uterus by transecting at the oviducts and cervix. If ovarian study is a component of the work, the first transection can be above the ovary at the ovarian ligament. The ovaries are then separated from the pregnant uterus and processed independently. 6. The pregnant uterus is scored for implantation site number and color for each horn. We find assigning our experimental animal number to the mother and then subcoding each implantation site, the simplest method. (For example, a histology sample #1003 would be all members of the litter and #1003A−1003F would indicate six implantation sites from the same mother). Record your observations with special care to annotate smaller, paler, or darker red implantation sites which may be failing members of a healthy litter that you might wish to exclude from your study or process as a separate study group. Then, transect between each implantation site. It is most important for any histological studies of uNK cells that the implantation site is not separated from the uterine wall (Fig. 31.3)!!! If embedding of late gestation specimens proves difficult, the perfusion-fixed uterus can be trimmed to remove the fetus and leave the placenta attached to the uterine wall. uNK cells will then remain undisturbed. If an experimental design requires removal of the fetus from a non-fixed uterus, cut rather than pull it away, to reduce extremely misleading artifacts in subsequent histology of the mesometrial uterus.

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Fig. 31.3. Drawings to illustrate the dissection and embedding of mouse implantation sites at gd6.5 (upper panel) and gd10.5 (lower panel). Fixed implantation sites were put on a cutting plate, and cut carefully along the line indicated as b–b’. This line is perpendicular to the long axis of the uterus (line a–a’) in (A, D). The tissue is then processed for paraffin-embedding. After tissue processing, the cut surface is placed facing the bottom of an embedding mold (B, E). Cut sections will have the final histological appearance of (C) or (F) once stained by DBA lectin. Many sections need to be cut to reach the center of the implantation site as shown in (C). The first sections to be cut in specimens prepared as (D, E) will have the appearance shown in (F). MLAp, mesometrial lymphoid aggregate of pregnancy; DB, decidual basalis; UA, uterine artery; P, placenta.

3.2.1. For Paraffin Embedding

1. All trimmed implantation sites for paraffin embedding are postfixed in 4% PFA using ∼50 mL/litter so the samples are well covered. This is timed. If the samples are up to gd10.5, postfixation is 15 min. If the samples are gd10.5 or older (i.e., larger), postfixation is 60 min. 2. Implantation sites are washed in 0.1 M PBS pH7.4 containing 0.1 M sucrose. For some studies, it may be desirable to trim the implantation site (Fig. 31.3A) or, for larger implantation sites, to cut them mid-sagittally into two equal parts (Fig. 31.3D) to reduce the labor in microtome cutting into the region of interest. Specimens are placed individually into labeled embedding cassettes and automatically processed into paraffin using a commercial tissue processor. 3. Positioning of the samples at the embedding station is a crucial step. While uNK cells are likely to be found regardless of tissue orientation, investigators wishing to compare their studies with those in the literature (Fig. 31.3C,F) or to undertake critical quantitative comparisons must ensure precise orientation. For intact implantation sites, the two cut ends of the uterus should be held directly over each other in

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a vertical plane (Fig. 31.3B,C). Putting a thin paraffin layer on the bottom of the mold to initiate solidification prior to inserting the cut tissue end may be helpful in obtaining this orientation, which is aligned against gravity. Then, place an embedding ring on top of the mold and fill it with liquefied paraffin. Hold the upper end of the specimen while filling the mold and during the first stages of paraffin solidification. If the implantation site was cut into halves, place the two pieces from the same implantation site cut face down on the bottom of the mold (Fig. 31.3E), place the embedding ring on and gently fill the mold. It is desirable to have the two placenta halves at the same depth in the block for cutting. After chilling on the embedding station refrigeration platform to solidify the paraffin, remove the mold. This exposes the cutting face of the block which faced the bottom of the mold. 4. Cut sections using a rotary microtome set to the desired thickness (7 ␮M are common). Since the uNK cells are found toward the center of the decidua basalis, it may be necessary to cut a large number of sections to reach the appropriate area (Fig. 31.3B). The largest part of the implantation site is the desired area of study in properly oriented specimens. This can be recognized while serially cutting the block and verified by placing an unstained section on a histology slide and examining it under a microscope at low power. The embryonic crypt should be present in the section as well as the mesenteric vessels in the mesometrial myometrium (see Fig. 31.1). Once the region of interest has been reached (this is immediate in larger specimens that were halved (Fig. 31.3E), although the first few sections may show artifacts due to the halving process that would require their discard), standard processes are used for mounting tissue sections to slides. 3.2.2. For Standard Cryostat Sections with Preserved uNK Cell Cytoplasmic Granules

1. All trimmed implantation sites for preparation of standard cryostat sections are postfixed in ∼200 mL of 4% PFA in 0.1 M PBS with 0.1 M sucrose for 24 h. 2. Implantation sites are transferred to 30% sucrose in 0.1 M PBS at room temperature (20◦ C) for 72 h. This step may be shortened but is essential for preserving uNK cell granules during the freezing process. 3. Implantation sites are tapped to a Kimwipe to remove excess moisture, mounted in OCT compound using one implantation site/mold, and the orientation described in Fig. 31.3, and frozen by dropping into isopentane chilled on dry ice. Using very long forceps, remove the cryomolds as soon as the OCT turns white. Place on small squares of aluminum

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foil and wrap tightly to limit air access to the specimen. Overcooling will crack the OCT and yield specimens that cannot be cut. 4. Frozen tissues are placed into labeled freezer bags and held at −80o C (see Note 10) until cut at 5–10 ␮M. Standard procedures for cryostat sectioning and immunohistology are subsequently used. 3.2.3. For Electron Microscopic Plastic Resin Embedding

1. Implantation sites fixed with 2% PFA + 1.5% GTA (for conventional ultrastructural study) or 4% PFA + 0.2% GTA (for ultrastructural immunocytochemistry) in 0.1 M PBS are rinsed in 0.1 M PBS pH7.4, then glued into the tissue-slicer tray with Super-bonder, positioned to get transverse sections and the chamber is filled with 0.1 M PBS. Cut 300–500 ␮M thick slices. Handle these carefully with forceps (do not pinch) or thin glass rods and select 2–3 mM of the middle portion of each implantation site (see Figs. 31.1 and 31.3) for uNK cell prospecting aided by dissection microscope observation. Transfer the desired 3–4 sections to a 3–5 mL glass vial. 2. For conventional ultrastructural study, wash the thick sections with 2 mL 0.1 M PBS twice for 10 min each, then replace with 1 mL 1% osmium tetroxide in 0.1 M PBS for 60 min at room temperature in a fume hood and continue standard processes for dehydration in ethanol and propylene oxide for epoxy resin embedding. Polymerize the block at 60◦ C (48–72 h). Collect the ultrathin sections on copper grids, stain with uranyl citrate, lead citrate, and carbon-coating using the reagents and equipment listed in Section 2.1.20. 3. For ultrastructural immunocytochemistry, wash the slices with 0.1 M PBS (Comment: Do not postfix with osmium tetroxide). Dehydrate with graded ethanol (50% to absolute) 1 h each at 4ºC. Transfer to ethanol/N,N-dimethyl formamide mix (1:1) then to pure N,N-dimethyl formamide (twice for 1 h each at −20◦ C) and then to LR-White resin for 12 h (−20◦ C). Transfer the slices to a beem capsule with LR-White resin and polymerize with UV radiation (FSU30, Bal-Tec, Germany) at −20◦ C for 48–72 h. Collect ultrathin sections (80–100 nM) thick on Ni grids and proceed to standard post-embedding ultrastructural immunocytochemistry.

3.2.4. For Cryostat Sections Being Used for Laser Capture Microdissection

1. Put on disposable gloves before beginning. If they become nicked or contaminated in any way that might reduce RNA quality, change them immediately before proceeding.

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2. Collect the tissue samples using sterile, RNAse-free surgical instruments (see Section 2.1.8.) and freeze immediately as in Section 3.2.2., Steps 3 and 4. 3. Pre-cool the cryostat. Remove and discard old blade. Wipe down the knife holder and anti-roll plate (if present) with 100% ethanol. Install a new disposable blade in the cryostat for each set of tissue. Place a microslide box on dry ice beside the cryostat. 4. Move the cryomold containing your specimen from the freezer to the dry ice. 5. Remove the block from the cryomold and attach it to the chuck in the cryostat with OCT. The cutting face of the block should be as parallel as possible to the knife. 6. Wait for 10 min for your specimen to reach its appropriate cutting temperature. Each cryostat will differ. We set ours at −20 to −22o C. Then, cut 7–8 ␮M sections. If you are dealing with specimens from different experimental groups, discard the blade and replace it with a new one for each group. 7. Place sections on room temperature LCM slides and move these immediately into the microslide box and cover. Do not allow the sections to air-dry!!! Only 6–8 slides are usually prepared at a single time. 8. If LCM is to be performed that day, keep the covered slide box in the cryostat or on dry ice and proceed immediately for staining. RNA integrity will be compromised within ∼45 min. 9. Alternatively, slides may be stored in paper slide boxes at −80◦ C until needed. 3.3. Basic DBA Lectin-Peroxidase Staining Protocol

1. Bake the slides containing the paraffin sections of interest at 50◦ C in an oven for 2 h. 2. Deparaffinize, rehydrate, and wash as below in coplin jars using accurate timing. i. Xylene 10 min for each of 2 baths ii. 100% ethanol

5 min for each of 2 baths

iii. 95% ethanol

5 min

iv. 70% ethanol

5 min

v. distilled water

3 min

3. Tap slides to Kimwipes to drain them; then encircle sections carefully using a DAKO pen to retain the experimental solutions directly over the tissue. 4. Cover sections with PBS (0.05 M, pH7.4) 5 min and then with PBS (0.1 M, pH7.4) + 1% H2 O2 for 30 min at room temperature to quench endogenous tissue enzymes. Slide

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incubations can be conducted over a sink, in humidified plastic boxes, or in other ways. It is essential that the solutions never dry out on the tissue. A water tap vacuum aspiration system is very convenient for removing treatments and washes. 5. Wash slides thrice with PBS (0.05 M, pH7.4) then apply a blocking buffer of 1% BSA in 0.1 M PBS for 30 min to block nonspecific binding sites. 6. Remove blocking buffer and load biotinylated DBA lectin diluted at 1:150–1:600 (see Note 11) in 0.1 M PBS with 1% BSA over the test sections. Negative control sections should include some covered with buffer only and some covered with lectin solution that was pre-mixed with 0.1 M N-acetyl-D-galactosamine, the competitive ligand of DBA lectin. Positive control sections for any run could be gd10.5 implantation sites from normal mice. Put the test and control slides into a humidified box with a lid, close the lid, and incubate overnight at 4◦ C. 7. The next morning, wash the slides thrice with PBS (0.05 M, pH7.4). Then cover the sections with Extravidin peroxidase (1:200) for 30 min at room temperature. 8. Wash the slides thrice with PBS (0.05 M, pH7.4) and then thrice with TBS (0.05 M, pH7.4). 9. Cover the sections with 0.1% diaminobenzidine (DAB) in TBS (0.05 M, pH7.4 containing 0.3% H2 O2 ). DAB is light sensitive and must be kept in the dark until immediately before use. Monitor the color development reaction under the light microscope (usually 1–5 min, over-staining will obliterate details of subcellular organelles). Once a brown color has developed, stop the reaction by dipping the slides into tap water then wash them in distilled water 3 min. 10. Counter stain ∼10 s in Harris’s Hematoxylin and rinse in running tap water for 5 min. 11. Dehydrate and clear the slides using the following steps: a. 70% ethanol 5 min b. 95% ethanol

5 min for each of 2 baths

c. 100% ethanol

5 min for each of 2 baths

d. Xylene

5 min for each of 2 baths

12. Mount coverslips onto the slides and view microscopically. 3.4. Quantitative Histological Assays 3.4.1. uNK Cell Enumeration

1. Slides are mounted using a ribbon of serial sections from the middle of an implantation site. Try to identify the precise middle section. This section will be quantified. Because most uNK cells are <50 ␮M in diameter, the remaining sections

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to be scored must be this distance apart. From the center section being scored, we score five sections before the middle and five sections past the middle. Thus, if you have cut sections at 5 ␮M, you need a ribbon with ∼100 sections (early implantation sites however usually give <10 useful sections). We strongly recommend mounting sections individually using the pattern shown in Fig. 31.4A. While this

Fig. 31.4. Drawings to illustrate preparation of serial sections for multiple studies on the same implantation site (A) and use of the micrometer scoring grid for uNK cell enumeration (B). A set of glass slides is serially numbered from 1 to 7 (or other value depending on thickness you are cutting and space you wish to leave between sections to prevent duplicate counting of the same large uNK cell. This may vary by the stage of gestation). The first section cut (example uses 7 ␮M with scoring 49 ␮M apart) is placed at the top of slide 1, the second at the top of slide 2 and so on until slide 7. The 8th section is placed in the middle of slide 1, the 9th section in the middle of slide 2 and so on until the section 14 on slide 7. The 15th section is placed at the bottom of slide 1, the 16th at the bottom of slide 2 and so on until 21st section is placed at the bottom of slide 7. This can then be repeated with a new set of slides labeled 8–14 as required. This process prepares 7 slides with 3 sections each with a total of 21 sections. It represents ∼150 ␮M of serial sections and any slide chosen has 3 sections 50 ␮M apart, suitable for quantitative analysis. The remaining slides are already prepared for alternate staining procedures and have the great advantage of being sequential serial (mirror) sections to compare with the first stain protocol used. Two replicate experiments for three different protocols of the same tissue area can thus be conducted. This procedure takes more time and technical attention but optimizes the use of important or rare specimens. The alternate approach of cutting the same block again fails to give replicate cellular /tissue components of small areas like the implantation crypt or MLAp in early mouse implantation sites. For uNK cell enumeration, all uNK cells (DBA lectin staining positive) inside the grid area should be counted. To adjust counts to cells/mM2 of tissue, counts should be adjusted to exclude the areas measured that were vascular spaces (indicated as SA, spiral artery), particularly if the specimen was perfused, which would have washed out intravascular uNK cells.

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method is more time consuming than mounting a ribbon, it improves staining consistency for sections that will be scored and pre-prepares adjacent serial section slides for subsequent histo- or immunohistochemistry. The center of each implantation site is confirmed by the presence of the uterine mesenteric vessels. DBA lectin positive cells will include mature uNK cells with positive surface membranes and positive granules, immature uNK cells with positive surface membranes and no granules, endothelial cells, and fetal yolk sac islands. In mice, the separation of the decidua basalis from the MLAp should be recognized in samples >8.5 days of gestation. uNK cells are enumerated separately in these two regions at 400× magnification on the 11 sections selected for study. Only uNK cells with nuclei in the plane of section are counted to prevent duplicate counting in other sections of a single, very large cell. We still employ a 1 mM2 ocular grid for manual scoring and adjust the tissue area calculation for vascular spaces that contain no uNK cells due to the perfusion process (Fig. 31.4B; Comment: If intravascular uNK cells are of interest, the animal should not be perfused. Sections from such an animal should not be used for measurements of blood vessel parameters, however, due to postmortem constriction of vascular smooth muscle permitted by the lack of perfusion). 2. Similar measurements are ideally made on three implantation sites from a single pregnant female and three females pregnant at the same gd or in the same treatment group are used. 3. We find it essential that one observer conduct all of the counting for a study. A full complement of independent counts can be made by a second observer. Partial counts by two observers should not be mixed in a study as some judgments are required in this scoring. 3.4.2. Spiral Artery Evaluation

1. Spiral arteries occur in the central decidua basalis from ∼ gd9.5 (Fig. 31.1D). They can be identified in hematoxylin and eosin stained sections or with special stains described in standard microscope techniques books (20, 21). Spiral arteries are usually seen as vessel cross section clusters with somewhat thick muscular walls. Locate such vessels, selecting those closest to a round shape. 2. We use an AxioVision 4 software-equipped Zeiss M1 microscope to draw the contours of both the limit of the spiral artery outer wall (V) and of the endothelial surface at the lumen (L) and calculate surface areas. This is done for the central section and five preceding and following sections as outlined in Section 3.4.1. Ratios of these measures are then

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calculated to assess the extent of spiral arterial modification. This type of study requires perfusion fixation to maintain in vivo vessel size. Again, we find it essential that calculations from different observers not be pooled but reported as replicate observations. 3.5. Molecular Analyses of LCM-collected uNK Cells 3.5.1. Rapid Hematoxylin and Eosin Staining for LCM (see Section 2.3.2 and Notes 12 and 13)

1. Leave slides at room temperature for 30 s to 1 min. 2. Move to 75% ethanol for 30 s. 3. Rinse sections with ultra-pure water (RNase, DNase free). 4. Place slides on paper toweling. 5. Add hematoxylin (enough to cover the sections) for 15 s. 6. Rinse sections with ultra-pure water (RNase, DNase free). 7. Add eosin (enough to cover the sections) for 15 s. 8. Rinse sections with ultra-pure water (RNase, DNase-free). 9. Move to 75% ethanol for 30 s (see Note 14). 10. Move to 95% ethanol for 30 s. 11. Move to 95% ethanol for 30 s. 12. Move to 100% ethanol for 30 s. 13. Move to Xylene (100%) for 5 min. 14. Air-dry slides for 5 min. 15. After staining, proceed immediately with LCM.

3.5.2. Dissection of uNK Cells from Frozen Tissue Sections and Cell Lysis for Nucleotide Extraction

1. Wipe down the LCM instrument, especially the stage with RNase inhibitor solution just before use. Turn on the LCM instrument, camera, and computer (see Notes 7 and 15). Check the settings on the controller. Load Capsure HS LCM caps onto the dovetail. You will manipulate the microscope stage using the joystick. Avoid using hydrophobic wax pens for writing on the glass slides. Pencil or diamond pen writing should be used. 2. Place your slide on the stage and find your area of interest. This takes practice because the frozen, weakly stained tissue lacking a coverslip is very different to coverslipped, paraffinembedded tissue. Use of fluorescent-tagging systems is recommended as a superior way to identify cells to be collected for LCM. Turn on the vacuum on the controller to hold the field of interest in position. 3. Swing the arm, loaded with caps, over the slide and place it onto the field of interest. Remember, you will not be able to see the laser spot unless you put the cap over the section. This is to protect your eyes from the infrared beam. 4. Select the laser spot size. Turn on the laser key and enable the laser beam by pushing the button on the controller.

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Focus the laser beam. Focusing at 10× magnification gives the best focal point. 5. Make fine adjustments to the power and duration settings to get the desired laser spot. Check the laser spot in a tissue-free area of the slide to ensure it is the desired size. 6. Proceed to capture the desired number of cells. We normally capture 200 or 500 individual cells on one cap to make a sample. 7. Unload the LCM cap with the cap insertion tool and immediately conduct RNA or DNA extraction. 3.5.3. RNA Isolation and Linear Amplification

1. Place LCM cap into the ExtraSure assembly in an alignment tray. Follow Picopure RNA Extraction Kit instructions (summarized below for current product) for RNA extraction. 2. Add 10 ␮L of extraction buffer. 3. Place 0.5 mL microfuge tube on top of the assembly and incubate at 42◦ C for 30 min. 4. Centrifuge the microfuge tube mounted by cap-ExtraSure assembly to collect cell extract. This is the first place you can stop in the LCM protocol by storing the cell extract at −80◦ C. 5. To continue with the fresh or a thawed sample, precondition the RNA purification column with extraction buffer (50 ␮L). 6. Centrifuge the column. 7. Add 10 ␮L of 100% ethanol to the cell extract. Pipette this mixture to the pre-conditioned column and centrifuge. 8. Wash the column by centrifugation at 8000×g for 1 min with 50 ␮L of wash buffer 1 followed by 50 ␮L of wash buffer 2. 9. Repeat the wash using only wash buffer 2 and centrifuge at 16000×g for 2 min. 10. Elute the RNA with 10 ␮L of nuclease-free water. 11. This RNA can be immediately used for downstream applications like reverse transcription (RT) or anti-sense (a)RNA amplification or can be stored at −80◦ C. If you have sufficient RNA, quantify it using a UV spectrophotometer or evaluate its quality by bioanalyzer (see Note 16).

3.5.4. Linear Anti-sense RNA (aRNA) Amplification, an Optional Step

This process is used only if the RNA yield from the LCM harvested cells is very low. RNA amplification is carried out using MessageAmp II aRNA kit as per manufacturer’s instructions, summarized below.

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1. Take 11 ␮L of total RNA isolated using the picopure extraction kit and add 1 ␮L of T7 oligo (dT) primer. Incubate this mixture at 70◦ C for 10 min in a thermal cycler and then place it on ice. 2. Add 8 ␮L of reverse transcription master mix (2 ␮L of 10× first strand buffer, 1 ␮L of RNase inhibitor, 4 ␮L of dNTP mix, 1 ␮L of reverse transcriptase) to each sample and incubate for 2 h at 42◦ C. 3. Follow immediately with second strand cDNA synthesis using a master mix comprised of 20 ␮L of the cDNA sample just obtained, 63 ␮L nuclease-free water, 10 ␮L of 10× second strand buffer, 4 ␮L of dNTP mix, 2 ␮L of DNA polymerase, and 1 ␮L of RNA H and incubate for 2 h at 16◦ C. 4. Filter this cDNA using a cDNA filter cartridge and elute in 20 ␮L of nuclease-free water. 5. Perform in vitro transcription to synthesize anti-sense RNA and pass this mixture through aRNA filter cartridge as per kit instructions. 6. The resultant aRNA can be eluted in 100 ␮L of nucleasefree water and stored at −80◦ C until further use or can be utilized for reverse transcription to make cDNA. 7. Avoid repeated freezing and thawing of the RNA. 3.5.5. cDNA Synthesis

1. One ␮g of total RNA or 200 ng of aRNA is brought to a total volume of 20 ␮L using nuclease-free water and is reverse transcribed using the First Strand cDNA synthesis kit (GE Healthcare). 2. Incubate the RNA samples in a thermal cycler (10 min, 65◦ C) then mix with 1 ␮L of DTT, 1 ␮L of Not-1 d (T) primer, and 11 ␮L of bulk first strand cDNA mix. 3. Incubate this reaction mixture at 37o C for 60 min followed by 90o C for 5 min and then place it on ice. 4. The resulting cDNA can be used in downstream applications such as real-time PCR or stored at −20◦ C for further use.

3.5.6. Quantitative Real-Time Polymerase Chain Reaction

1. Design sets primers using Primer 3 (http://frodo. wi.mit.edu/cgi-bin/primer3/primer3 www.cgi) or other software programs. 2. A wide variety of real-time PCR equipment is available. We use Roche Real-Time PCR Light Cycler (Roche Diagnostics, Laval, QU, Canada). The methods and PCR program described below are only applicable to this instrument. We routinely use Quantitect SYBR Green I PCR mix kit for gene expression quantification.

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3. LightCycler reactions are performed in 20 ␮L total reaction. Each reaction mixture contains 2 ␮L cDNAs, 10 ␮L SYBR Green I master mix, 1 ␮L sense, and anti-sense primers (0.5 ␮M each), and 6 ␮L PCR-grade water supplied with the kit. 4. The LightCycler program for each gene is denaturation (94◦ C, 15 min), PCR amplification and quantification (95◦ C, 10 s; 58◦ C, 5 s; 72◦ C, 20 s), and the fluorescence measurement at specific acquisition temperatures for 5 s, repeated for 45 cycles. Relative gene expression data are quantified using RelQuant LightCycler analysis software. 3.6. Tissue Dissociation and uNK Cell Enrichment Using Density Gradient Centrifugation (for Flow Cytometry)

1. The euthanized, non-perfused mouse is placed on its back in a dissection tray, moistened with 70% ethanol, wiped to remove excess alcohol, and the abdominal cavity is opened with a long, ventral midline laparotomy incision. A skin incision is made with the first set of dissection instruments and the abdominal muscles are incised with clean new instruments. 2. Using sterilized forceps, retract the uterus gently (it may be partly hidden by a full urinary bladder) until the ovaries are seen. Transect the uterine suspensory tissue below each ovary; then lift the uterus to identify the uterus–cervix junction and transect the uterus at this junction to remove it by cutting the mesentery. Leave a small edge of mesentery attached to the uterus for orientation during dissection. Place the intact uterus into a petri dish containing the cold medium to be used in the study. If several mice are to be dissected for study, it is optimal to completely dissect one uterus before euthanasia of the next mouse. 3. Dissections at gd6.5-8.5 differ to those at later times. The early times are prior to placental differentiation; the later times are after placental development. It is common to obtain 6–10 healthy implantation sites/uterus. Regions with resorbing or retarded fetuses are excluded from study or studied separately. For gd6.5-8.5 specimens, transfer the uterus to a dry petri dish to improve ease of dissection but you will need to work quickly to prevent tissue drying. Open the uterus along the anti-mesometrial side using Vannas scissors. If these are not available, use straight iris scissors or two pairs of number 5 watchmaker’s forceps at 90◦ angles to each other for a scissor-like cutting action (see Note 17). 4. Using curved, blunt-ended forceps, gently stroke the mesometrial tissue to remove each implantation site off the uterus and place all conceptuses of a single group (age or manipulation or genotype, etc.) into a dish of medium to hold them until the full group of uteri is dissected. Discard

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the uterus (progenitor cells may be present in this tissue but not uNK cells). 5. Transfer the dissected conceptuses enclosed in decidua to the dissection microscope. For gd6.5-7.5, the primitive streak embryo lies in the center of the decidua in a crypt that is visibly red due to its content of erythrocytes. The poles of the decidua at either end of this red streak are different. The anti-mesometrial pole is smoother, narrower, and pinker; the mesometrial decidua basalis is white, wider, and uneven where it detached from the uterus. With two pairs of #5 watchmaker’s forceps or one forceps and very fine scissors, remove the decidua basalis by transecting just beyond the end of the red streak of the embryonic cavity and place the decidua basalis into medium. Proceed to dissect all of the conceptuses in this way. Again, doing the dissection on a dry dish makes the task much easier by elimination of sample floating. This is highly recommended but work quickly. 6. For dissections at gestation stages with a placenta (gd9.5 or later), instructions 1–3 in this section are the same. The differences come in retaining the uterus, where the MLAp has now developed and in the more difficult dissection to remove decidua basalis. Begin using curved forceps to pull the conceptus off the uterus. Try to keep the conceptus and placenta together at this stage and place into holding medium. Loss of amniotic fluid is not of concern. Return to the uterus with the lumen surface facing up and apply traction to thin out the uterus. You will see a linear series of raised white pebble-like structures. These are the MLAps, one at each place a healthy conceptus was removed. MLAps are usually covered by remnants of decidua basalis that can be scrapped off and reserved for study before MLAp dissection. It is impossible to dislodge the MLAp when scraping off the residual decidua, so considerable force can be used in this clean up. Then, using curved iris scissors, cut out each MLAp with a scoop-like cut. Try not to collect the non-decidualized endometrium around the MLAp and not to cut through the uterine wall, since these actions will unnecessarily contaminate your lymphocyte preparation with uNK cell-devoid endometrium from between implantation sites, muscle cells, and visceral peritoneum. As the MLAps are removed, place them into fresh medium in a tube or dish labeled MLAp. 7. Return to the conceptuses. Move each in turn to a dry dissection dish, remove, and discard the fetus, being careful to note which side of the placental disc (red) faced the fetus. It is best when learning, to complete dissection of each conceptus fully, before continuing to ensure orientation. Place

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the fetal-facing side down and the maternal side up and view under a dissection microscope. The fluffy whiter material facing you is the decidua basalis, a covering over the fetal trophoblast layers of the placenta. Decidua cannot be dissected as a pure tissue but is significantly enriched by dissection. Decidua should be carefully under-excavated with #5 watchmaker’s forceps and taken off like the peel on a navel orange with the recessed “button” being where the decidua basalis tore away and stayed over the MLAp. It sometimes helps to cut out a small, full thickness wedge to the center of the placenta and view the layers of tissue [there are three different colors with the labyrinthine region (bottom most and central in this dissection the reddest)] before undertaking the “peeling” dissection. Alternatively, some investigators find it sufficient to pinch off the whiter surface. Others may use the intact placenta (both maternal and fetal parts) but this approach again yields very high levels of extraneous material. 8. Once all of the decidua basalis is harvested (pool samples dissected from the placenta with those scraped off the MLAp), sterile scissors are used to finely mince it into ∼1 mM3 pieces in a small volume of medium (∼1–3 mL). To isolate leukocytes, these pieces are pressed through a stainless steel mesh into medium using the syringe plunger; then transferred to tubes. Cells are pelleted by centrifugation (750×g), re-suspended in 3 mL 0.1 M PBS, loaded over 3 mL Lympholyte-M density gradient, and centrifuged (1350×g; 30 min; 20◦ C). Cells are aspirated from the interface, harvested by centrifugation (1200×g), and washed twice with PBS before enumeration of a trypan blue-suspended aliquot in a hematocytometer. Usually, about 1–2×104 lymphocytes can be obtained from one gd9.5 implantation site. Multiple implantation sites from one or more pregnant mice of the same genotype and treatment group at the same gestation stage can be pooled together unless the investigator is using each pregnancy as a replicate experiment. 3.7. Tissue Dissociation and uNK Cell Purification Using DBA lectin-Coated Magnetic Beads for Cell Culture or RNA Isolation (see Note 18)

1. To prepare DBA lectin-coated beads, set a 1.5 mL tube of 50 ␮L CelLection M450 magnetic beads into a magnetic particle concentrator (MPC, Dynal Inc). Wash the beads thrice with PBS/Tween. Remove the MPC, resuspend the beads in 100 ␮L PBS/Tween, add 4 ␮L biotinylated DBA lectin, and incubate 30 min at room temperature. Reload the tube to MPC, wash thrice with PBS/Tween, then resuspend the beads in 0.1 M PBS. 2. Dissect uNK cell-enriched tissues by following Section 3.6 using Hank’s balanced salt solution as the medium.

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3. Chop the MLAp and decidua into small pieces with sterile razor blades in 1mL Hank`s solution containing 1000 IU DNase I in a sterile petri dish and dissociate further by delicate pipetting for 2 min. Remove the undissociated tissue debris by passing the sample through an 80 ␮M mesh nylon filter membrane and collect the homogenate containing uNK cells in a 10 mL centrifuge tube (see Note 19) 4. Add 2 mL distilled water to the cell suspension. After 20 s, restore the osmolality by adding 1.25 mL of 3.5% NaCl. Invert the tube rapidly but gently mix and centrifuge (250×g for 5 min at 4◦ C). Resuspend the cell pellet with 1.5 mL 0.1 M PBS pH7.4 containing 2% BSA. 5. Enumerate a trypan blue-suspended aliquot in a hematocytometer. 6. Add DBA lectin-coated magnetic beads in a volume to give 1 bead for each 2 cells in the cell suspension. Mix 15 min on an orbital rotating platform (∼20 rpm) at 4◦ C. (see Note 20). Set the MPC into the orbital rotor and immobilize the magnetic beads on one side of the microfuge tube for 20 s. 7. Remove the cell suspension carefully without disturbing the immobilized cells. Transfer the cell suspension to a new, labeled microfuge tube (non-uNK cell-1) and keep it in the ice bath. 8. Remove the microfuge tube from the MPC and resuspend the magnet-retained cells (Fig. 31.5A) in 500 ␮L of 0.1 M PBS/2% BSA containing 0.1 M N-acetyl-D-galactosamine for 5 min. Set the microfuge tube in the magnetic particle concentrator and again immobilize the magnetic beads, collect the cell suspension to a new labeled microfuge tube (uNK cell-1; see Note 21; Fig. 31.5B). 9. Centrifuge (750×g) 5 min and resuspend the cell pellet with RPMI 1640 medium supplemented with 10% fetal bovine serum and 1 mg/mL gentamycin. 10. To increase cell yields, the negatively selected cell suspension (non-uNK cell-1) needs to be reloaded with DBA lectin-coated magnetic beads and Steps 6–9 repeated. Place the first cell suspension removed into a new microfuge labeled “non-uNK cell-2” and the enriched cells into a new tube labeled “uNK cell-2”. 11. Now starting with the tube labeled “non-uNK cell-2” repeat Steps 6–9 placing the first cell suspension removed into a new microfuge tube labeled “non-uNK cell-3” and the enriched cells into a new microfuge tube labeled “uNK cell-3”.

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Fig. 31.5. (A) Differential interference contrast (DIC) image of uNK cells freshly isolated using DBA lectin-coated magnetic beads and (B) after detachment using the competitive sugar N-acetyl-D-galactosamine (see Section 3.7). The small cells (arrows) are contaminating red blood cells that can be removed by hypotonic lysis. (C) illustrates a four chamber culture glass slide with two droplets of culture medium (pink dots) in each chamber covered with mineral oil. (D) detail illustrating the side view of one chamber with two culture droplets (shaded domes) completely covered with mineral oil. (E, F) uNK cells attached on a fibronectin coated surface and cultured with RPMI 1640 medium supplemented with IL-15 and CXCL10 at 24 and 96 h, respectively.

12. Pool “uNK cell-1, cell-2 and cell-3” tubes and count an aliquot suspended in trypan blue in a hematocytometer. Up to 90% cell viability can be achieved with 0.7–1.2×104 uNK cells recovered per implantation site at gd7.5 or 8.5. 3.8. Microspot Culture of Mouse uNK Cells

1. Coat the desired number of slide culture chambers (Fig. 31.5C,D) with 2 ␮g/mL sterile fibronectin. 2. Place 0.2 to 2×104 cells in 50 ␮L sterile RPMI 1640 medium supplemented with 10% fetal bovine serum, 0.2 ␮g/mL IL-15, and 100 ng/mL CXCL10 onto the

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center of each culture chamber, taking care not to allow the dot to spread on the surface. Fill the chamber carefully with sterile mineral oil, covering the dot of culture medium entirely. The aqueous culture medium becomes a round dot surrounded by hydrophobic paraffin oil. 3. Rotate the slide gently to concentrate (swirl) the medium and cells to the center of droplet. 4. Place the cultures into a humidified atmosphere with 5% CO2 at 37◦ C. 5. After 6 h, using trans-illuminated stereomicroscopic observation, remove half (∼25 ␮L) of the culture medium and any cell debris (Comment: Viable uNK cells will be firmly attached to the fibronectin-coated surface. Floating cells and cell debris must be removed as lytic enzymes from damaged uNK cells appear to reduce the viability of healthy uNK cells). A microcapillary needle mounted on a micromanipulator arm is required to control the slow flow rate and precise measurement of volumes needed for feeding these cultures. Replace the medium with an equal volume of fresh medium. This should be done twice so that 75% of the medium in each culture droplet is fresh every 24 h. 6. uNK cell integrity can be followed by daily microscopic observation. This microspot culture system maintains purified uNK cells suitable for microscale in vitro assays (such as cytokine stimulation) for 5–7 days. About 2×103 uNK cells from these microspot cultures yield sufficient mRNA for evaluation by conventional RT-PCR. The conditioned medium supplemented with IL-15 and CXCL10 is adequate for uNK cell maintenance and differentiation, but does not induce cell proliferation. Studies to improve these cultures for expansion of uNK cell numbers continue. 3.9. Flow Cytometry

1. Conduct all steps in the cold and keep samples protected from light when working with fluorescent antibodies. Because uNK cell viability is sensitive to microenvironmental change and many uNK cells are very large and fragile, we highly recommend performing flow cytometry as soon as the cell suspension is available. 2. uNK cells should be freshly isolated without enzymatic digestion. Cells should initially be incubated with anti-CD16 (clone 2.4G2) for 10 min at 4◦ C to block FcRs on various leukocytes, including uNK cells, then washed by centrifugation (750×g) in FACS buffer. 3. Dilute previously titered primary and isotype control antibodies or DBA lectin to their optimal concentrations in FACS buffer. Dispense antibodies or lectins into pre-labeled

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microfuge tubes (1.5 mL). If performing multiple color staining, add all of the fluorochrome-labeled antibodies simultaneously. Use 50 ␮L of PBS for the unstained negative control. 4. Add 50 ␮L of prepared cell suspension (equal to 106 cells) to each tube, mix gently, and incubate 30 min in the dark at 4◦ C. (Note: Some antibodies may require longer incubation times, which would be determined in preliminary experiments. The DBA lectin incubation must be terminated at 5 min to prevent cell lysis). 5. After the incubation period, add 1000 ␮L PBS to each tube. Centrifuge the cells for 5 min (750×g) at 4◦ C. Aspirate the supernatant and repeat an additional two times for a total of three washes. If you are using unconjugated- or biotin-labeled antibodies, you would now add the proper second step reagent (a fluorochrome- or Avidin-conjugated secondary antibody) in 50–100 ␮L of PBS to each sample. This would be incubated in the dark for 30 min at 4◦ C and the cells recovered by two washes. 6. Resuspend the stained cell pellet in 200 ␮L FACS buffer and analyze the samples using a flow cytometer. Typically, 10,000–50,000 lymphocytes can be evaluated but when working with uNK cells, the investigator must anticipate having limited total cell numbers. 7. At Queen’s University, we use a CytomicsTM FC500 Flow Cytometer and perform data analyses with FlowJo software. At The Babraham Institute, a DB LSR-II benchtop cytometer is used with the same software. Figure 31.6 shows an analysis of uNK cells prepared from gd9.5 C57BL/6J implantation sites by Section 3.6.8. The first gates were set around the lymphocytes in the forward scatter and side scatter dot plots. This excludes other cell types from the analyses. First, a gate was set around the DAPI− cells to exclude DAPI+ dead cells (not shown in the figure; see Note 22). Then gates were set around two partially overlapping populations defined by their forward and side scatter profiles: small cells (R1, which contain lymphocytes found in lymphoid organs, i.e., spleen, lymph nodes, thymus, and bone marrow) and large granular cells (R2). Live uNK cells are found in both R1 and R2 but further analysis was carried out on the small cells in R1, because they can be directly compared for size and granularity to NK cells found in lymphoid organs. The cells in R1 were further divided into two subsets, one that is NK1.1+ and DX5+ (S1), and another that is negative for both (S2). These latter cells are the DBA+ cells (see Notes 23 and 24).

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Fig. 31.6. Leukocyte suspensions were prepared from gd9.5 C57BL/6 mice as described in the text. Dead cells were excluded by DAPI (not shown). The scatter plots highlight two populations in the uterus, one that is similar to the lymphoid population in the spleen (R1, 20–25%), and the other that contains larger and more granular cells (R2, 10–15%). After gating on R1, NK cells are gated as CD3− CD122+ cells and, among these cells, NK1.1 and DX5 differentiate two subsets. One minor subset (S1, about 30% of the uNK cells) is positive for both markers and therefore similar to peripheral NK cells, whereas the other subset (S2, about 70% of the uNK cells) does not express either marker. Most of the NK1.1− DX5− cells in S2 are DBA lectin reactive, whereas both splenic and uterine NK1.1+ DX5+ NK cells are negative for DBA lectin staining. The frequency of the R2 population varies between experiments; however, the NK1.1+ DX5+ and the NK1.1− DX5− subsets are also found in R2 and their frequency as well as their reactivity with DBA lectin are comparable to those in R1. To date, there are no reports on functional assessment of these cells as individual subsets. NA = not applicable.

4. Notes 1. It is essential to understand the anatomical meaning of “anti-mesometrial” and “mesometrial” for successful study of uNK cells. The mesometrium is the mesentery suspending the uterus in the abdominal and pelvic cavities. It is also called the “broad ligament.” The mesentery carries the arteries, veins, and sympathetic nerve fibers that service the uterus and are only found at one point on the circumference of mouse uterine horns, the mesometrial side. Sympathetic nerve fibers cross the mouse uterine wall (myometrium) but do not enter the mucosa (endometrium) or decidualized mucosa. Uterine arteries and veins cross the uterine wall and become finely branched in the endometrium. Vessels in the mesentery that lead to an implantation site are noticeably dilated compared to vessels servicing uterine tissue between implantation sites. The normal mouse placenta is always on the mesometrial side of the implantation site while the amnion-enclosed fetus proper always lies towards the anti-mesometrial side. Dissection to open the uterine lumen for uNK cell study is

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always conducted from the anti-mesometrial side to prevent damage to the uNK cell-rich regions. 2. Endometrial decidualization in the absence of a conceptus (conceptus is a term encompassing and implying both fetus and placenta) is referred to as deciduoma (singular) or deciduomata (plural). When deciduoma is present, the mouse is described as pseudopregnant. Study of this “artificial” decidua is limited to early events because, for most induction protocols, the tissue breaks down earlier than the decidua of pregnancy. The stages of development and distribution of uNK cells within the deciduomata may not be equivalent to those in the pregnant uterus (22–24). Women with ectopic pregnancies do not develop decidua at the ectopic blastocyst implantation site. However, their uterus decidualizes as if it contained the conceptus. In such cases, the decidua is retained as long as the woman carries the ectopic pregnancy. This uNK cellcontaining human decidua, lacking fetal cell invasion, is called “eutopic” decidua. 3. DBA lectin is not a universally successful stain for uNK cells in all species. There is no membrane reaction with rat uNK cells, while in pig endometrium many cell types react and the lectin lacks useful specificity. The usefulness of DBA lectin as a marker for uNK cells in species other than mice must not be assumed. 4. Tissue processor chemicals: 70% alcohol; 80% alcohol; 95% alcohol × 2 baths; 100% alcohol × 2 baths; Xylene × 2 baths; Paraffin (Fisher Scientific, #23-021-401) × 3 baths. Several less hazardous and less toxic naphthalene or petroleum-derived solvents like Paraclear (Polysciences, Inc), Clear Advantege (Polysciences, Inc), UltraClear (JT Baker) can be used as substitutes for xylene or toluene during histoprocessing for paraffin embedding, deparaffinization, and permanent mounting of paraffin sections. For standard morphological evaluations or DBA lectin staining using peroxidase detection, no differences are apparent with these alternative solvents. For immunocytochemistry, this can be antigen/antibody dependent. For example, antigens localized close to the plasma membrane or expressed by membranous organelles can be masked by the use of ParaClear, a less effective solvent for lipid solubilization. 5. DAB is a suspected carcinogen. Wear the appropriate protective clothing. Deactivate DAB with chlorine bleach in a sealed container overnight in a fume hood. Noxious fumes are produced when the bleach is added. Dispose of this solution according to local safety guidelines.

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6. H2 O2 is unstable. It should be stored cold and in the dark. We find it most effective to aliquot freshly opened product ◦ into 1.5 mL volumes and freeze it at −20 C. Once thawed, the product is used only once and discarded. 7. The methods described relate to our current system, the Arcturus Pixcell IIe. This instrument has been replaced by newer models but most of the concepts and protocols remain valid. Recently, Molecular Devices was purchased by MDS Analytical Technologies, Toronto, Canada. Readers should anticipate changes in ordering information for supplies we have listed as coming from Molecular Devices. 8. It is possible to collect migrating uNK cells from tissue explants of the MLAp (25) but it is recommended that the original overnight incubation be replaced with a 3.5 h incubation and that IL-15 supplementation of the medium be used. Early explants of mesometrial tissue (gd3.5–5.5) provide outgrowth of apparent uNK progenitor cells (Croy, unpublished). None of the authors currently use this method. 9. Mice are nocturnal animals and breed in the dark part of their day–night cycles. Most animal colonies use 12 h light and 12 h dark. If possible for breeding studies, a 10 h light and 14 h dark cycle should be used. Mating is estimated as occurring at the midpoint of the dark interval. The morning of the vaginal plug is thus properly called gd0.5. Some investigators prefer to call it gd0 while others call it gd1. It is essential that authors define their gestation dating nomenclature in publications to permit data comparisons between laboratories since rapid (<24 h) changes occur in conceptuses and in the endometrial immune system during early gestation. 10. For long time storage (>1 week), −80◦ C is recommended. Long-term freezing at −20◦ C dries out the OCT and tissue and results in tissue shrinkage. 11. DBA lectin of different manufacturing lots and from different manufacturers will not show equal reactivity. A titration experiment is needed to establish optimal staining from each lot. 12. We also developed a rapid DBA lectin staining method (<40 min) for identification of mouse uNK cells in frozen sections being used for LCM from the protocol given in Section 3.3. Rapid DBA lectin staining requires the same rigorous RNase-free methods described for H&E staining for LCM. Thaw cut frozen sections at room temperature (30 s) without drying and fix immediately (30 s) in 70% ethanol. Rehydrate the sections in nuclease-free

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water (45 s). Cover the sections for 5 min with 1:10 hydrogen peroxide and Tris-Borate solution pH7.5 (TBS; 0.1 M, boric acid 0.1 M). To avoid nonspecific binding, block the sections with 4% bovine serum albumin in TBS (5 min), then incubate the slides on an orbital shaker (37◦ C, 10 min) with DBA lectin (1:25 dilution in TBS). Wash sections with TBS, and apply streptavidin (1:50) in TBS (10 min). After rinsing with TBS, apply 3-3 diaminobenzene for 5 min. Rinse the sections in nucleasefree water and counterstain for 15 s with hematoxylin, followed by dehydration for 15 s in each of 75%, 95%, and 100% ethanol and move into xylene for 5 min. Air-dry these slides (5 min) and place into a slide box over desiccator sand and perform LCM immediately (Comment: RNA cannot be isolated from PAS-stained sections). 13. QUICK STAINING KITS are also available from DAKO. Incubate slides with primary, secondary, and tertiary reagents for 90–120 s each, rinse briefly with PBS at each step. Develop color with DAB for 3–5 min and counterstain with Hematoxylin. 14. If you experience difficulty in lifting cells from the tissue, incomplete dehydration of the tissue is often the problem. One approach to reduce this difficulty is to double each dehydrating alcohol step. Then, dip the slide in the first bath and immediately move it to the second bath of the same concentration for the timed step. This reduces carryover of the more aqueous solution between reagents. 15. These comments apply to an Arcturus Pixcell IIe. Newer instruments are more automatic, do not use a joystick, do not require focusing of the laser, do not use a vacuum slide holding system, and can be loaded with more than one slide. We describe the basic system that we have used for our published work (26–28). 16. If RNA cannot be recovered from the LCM sample, the starting material may contain poor quality RNA, the RNA may have degraded during extraction, or the RNA may not have been extracted from the cells on the LCM Cap. If the RNA isolated is of poor quality, check the source tissue of the LCM sample for RNA quality, use proper rapid staining procedures, perform LCM immediately after preparing the LCM slides, or use only frozen or alcohol fixed tissue. Some fixatives, such as formalin or PFA, diminish RNA quality. If the RNA yield is too low, RNA integrity in the sample is compromised. Poor quality RNA may not bind effectively to the purification column. You should check buffer concentrations of the extraction kit and ensure timing of incubation during the extraction step was not too short.

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17. It is essential to keep the delicate points of #5 watchmaker’s scissors in high quality condition. They must not be burred, bent, or misaligned when closed. Look at the tips under the dissection scope to assess damage. Repairs can be achieved using a sharpening stone (Fine Science Tools, #29008-22) or #6-0 diamond sand paper under dissection microscope observation. Most mouse embryologists keep their own forceps closely guarded and several pairs should be available for each investigator requiring them. 18. NK cell suspensions can be isolated from the non-pregnant uterus using a similar approach. Usually, we dissect the uterus longitudinally in half for its full length by cutting along both lateral sides. We retain the mesometrial side and discard the anti-mesometrial side. Neither decidua nor MLAps are present. You may use the entire uterus should your research question require this. 19. For studies employing flow cytometry, enzymatic digestion is omitted because it reduces surface expression of some receptors. Minced decidua is pushed through a 100 ␮M screen with a plunger from a syringe (Colucci laboratory) and the resulting suspension is transferred to LympholyteM for purification (Section 3.6.8). The purely mechanical method was discarded as too harsh by the Croy laboratory who found it reduced the validity of 51 Cr-release assays. Investigators are advised to test both protocols and to modify them to define the optimal cell suspension protocol for their own investigational purposes. 20. Do not increase the association time beyond 15 min because longer incubations cause uNK cell lysis. 21. N-acetyl-D-galactosamine is the specific competitive sugar binding to DBA lectin and is used to remove magnetic beads from uNK cell surfaces. N-acetyl-D-galactosamine should be added as soon as possible to increase the disassociation rate of the magnetic beads from the cells and increase the viability of uNK cells in subsequent vitro assays. If the beads are not removed within 30 min, uNK cell disruption occurs. 22. Uterine leukocyte suspensions contain a considerably higher proportion of dead cells than lymphoid organ suspensions. Dilacetate DAPI (D3571) can be used to exclude dead cells from the analysis if the cytometer is equipped with a 355 nM UV laser. 23. To identify uNK cells, monoclonal antibodies specific for the following antigens can be used CD3ε (1452C11), CD122 (IL2␤B, TM␤1), NK1.1 (PK136), CD49b (DX5), and DBA lectin (10). At gd9.5, about 30% of the

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cells found in the R1 and R2 gates are CD3− CD122+ . Within the CD3− CD122+ cells, the majority are negative for markers expressed by peripheral NK cells such as NK1.1 and DX5, whereas a smaller subset of cells is NK1.1+ DX5+ . DBA+ cells are confined to the CD3− CD122+ NK1.1− DX5− subset. Thus, not all uterine NK cells are DBA+ and those that are DBA+ do not express usual NK cell markers. 24. For strains of mice that do not express NK1.1, a combination of CD3, DX5 (or CD122), and DBA will be sufficient to distinguish the two subsets of NK cells found in the uterus.

Acknowledgments Funding support for the collaborations that have led to these studies came from NSERC, Ontario Pork, the Ontario Veterinary College Bull Travel Fellowships Awards Program (BAC and CT), the Canada Research Chairs Program (BAC), China 2001 Award (JHZ), CNPq and FAPESP Brazil (AY), the BBSRC, and the MRC UK (FC). We thank Drs. Sandra Peel, Southampton, UK and Ian Stewart, Aberdeen, Scotland for first engaging us with their ideas about rodent “granulated metrial gland cells,” now called uNK cells, and for sharing friendship and their wealth of scientific and technical knowledge that significantly advanced our studies. References 1. Croy BA, van den Heuvel MJ, Borzychowski AM, Tayade C. (2006) Uterine natural killer cells: a specialized differentiation regulated by ovarian hormones. Immunol. Rev. 214, 161–185. 2. Croy BA, He H, Esadeg S, Wei Q, McCartney D, Zhang J, Borzychowski A, Ashkar AA, Black GP, Evans SS, Chantakru S, van den Heuvel M, Paffaro VA, Jr., Yamada AT. (2003) Uterine natural killer cells: insights into their cellular and molecular biology from mouse modelling. Reproduction. 126, 149–160. 3. Kiso Y, McBey BA, Mason L, Croy BA. (1992) Histological assessment of the mouse uterus from birth to puberty for the appearance of LGL-1+ natural killer cells. Biol. Reprod. 47, 227–232. 4. Parr EL, Parr MB, Young JD. (1987) Localization of a pore-forming protein (perforin) in granulated metrial gland cells. Biol. Reprod. 37, 1327–1335.

5. Peel S. (1989) Granulated metrial gland cells. Adv. Anat. Embryol. Cell Biol. 115, 1–112. 6. Manaster I, Mizrahi S, Goldman-Wohl D, Sela HY, Stern-Ginossar N, Lankry D, Gruda R, Hurwitz A, Bdolach Y, Haimov-Kochman R, Yagel S, Mandelboim O. (2008) Endometrial NK cells are special immature cells that await pregnancy. J. Immunol. 181, 1869–1876. 7. Koopman LA, Kopcow HD, Rybalov B, Boyson JE, Orange JS, Schatz F, Masch R, Lockwood CJ, Schachter AD, Park PJ, Strominger JL. (2003) Human decidual natural killer cells are a unique NK cell subset with immunomodulatory potential. J. Exp. Med. 198, 1201–1212. 8. Hanna J, Goldman-Wohl D, Hamani Y, Avraham I, Greenfield C, Natanson-Yaron S, Prus D, Cohen-Daniel L, Arnon TI, Manaster I, Gazit R, Yutkin V, Benharroch D, Porgador A, Keshet E, Yagel S, Mandelboim O. (2006) Decidual NK cells regulate

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key developmental processes at the human fetal-maternal interface. Nat. Med. 12, 1065–1074. Bianco J, Stephenson K, Yamada AT, Croy BA. (2008) Time-course analyses addressing the acquisition of DBA-lectin reactivity in mouse lymphoid organs and uterus during the first week of pregnancy. Placenta. 29, 1009–1015. Yadi H, Burke S, Madeja Z, Hemberger M, Moffett A, Colucci F. (2008) Unique receptor repertoire in mouse uterine NK cells. J. Immunol. 181, 6140–6147. Zhang J, Dong H, Wang B, Zhu S, Croy BA. (2008) Dynamic changes occur in patterns of endometrial EFNB2/EPHB4 expression during the period of spiral arterial modification in mice. Biol. Reprod. 79, 450–458. Chantakru S, Miller C, Roach LE, Kuziel WA, Maeda N, Wang WC, Evans SS, Croy BA. (2002) Contributions from self-renewal and trafficking to the uterine NK cell population of early pregnancy. J. Immunol. 168, 22–28. Paffaro VA, Jr., Bizinotto MC, Joazeiro PP, Yamada AT. (2003) Subset classification of mouse uterine natural killer cells by DBA lectin reactivity. Placenta. 24, 479–488. Burkhardt JK, Hester S, Lapham CK, Argon Y. (1990) The lytic granules of natural killer cells are dual-function organelles combining secretory and pre-lysosomal compartments. J. Cell Biol. 111, 2327–2340. Bossi G, Trambas C, Booth S, Clark R, Stinchcombe J, Griffiths GM. (2002) The secretory synapse: the secrets of a serial killer. Immunol. Rev. 189, 152–160. Griffiths GM, Argon Y. (1995) Structure and biogenesis of lytic granules. Curr. Top. Microbiol. Immunol. 198, 39–58. Dongfang L, Liang X, Fan Y, Dongdong L, Feili G, Tao X. (2005) Rapid biogenesis and sensitization of secretory lysosomes in NK cells mediated by target-cell recognition. Proc. Natl. Acad. Sci. U. S. A. 102, 123–127. Leonard S, Murrant C, Tayade C, van den Heuvel M, Watering R, Croy BA. (2006) Mechanisms regulating immune cell

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contributions to spiral artery modification – facts and hypotheses – a review. Placenta. 27 Suppl A, S40–46. Hayakawa Y, Smyth MJ. (2006) CD27 Dissects mature NK cells into two subsets with distinct responsiveness and migratory capacity. J. Immunol. 176, 1517–1524. Sheehan DC, Hrapchak B. (1980) Theory and Practice of Histotechnology. Battelle Press. Columbus, OH. Prophet EB, Mills R, Arrington JB, Sobin LH. (1992) Laboratory Methods in Histotechnology. American Registry of Pathology. Washington, D.C. Vassiliadou N, Bulmer JN. (1998) Characterization of tubal and decidual leukocyte populations in ectopic pregnancy: evidence that endometrial granulated lymphocytes are absent from the tubal implantation site. Fertil. Steril. 69, 760–767. Bianco J, Andrade CGTJ, Yamada AT. (2009) Pseudopregnant mouse uterus revisited for uterine natural killer cell evaluation. Submitted. Herington JL, Bany BM. (2007) Effect of the conceptus on uterine natural killer cell numbers and function in the mouse uterus during decidualization. Biol. Reprod. 76, 579–588. Mukhtar DD, Stewart I. (1988) Migration of granulated metrial gland cells from cultured explants of mouse metrial gland tissue. Cell Tissue Res. 253, 413–417. Tayade C, Hilchie D, He H, Fang Y, Moons L, Carmeliet P, Foster RA, Croy BA. (2007) Genetic deletion of placenta growth factor in mice alters uterine NK cells. J. Immunol. 178, 4267–4275. Tayade C, Fang Y, Hilchie D, Croy BA. (2007) Lymphocyte contributions to altered endometrial angiogenesis during early and midgestation fetal loss. J. Leukoc. Biol. 82, 877–886. Tayade C, Fang Y, Black GP, V AP, Jr., Erlebacher A, Croy BA. (2005) Differential transcription of Eomes and T-bet during maturation of mouse uterine natural killer cells. J. Leukoc. Biol. 78, 1347–1355.

Chapter 32 Isolation of NK Cells and NK-Like Cells from the Intestinal Lamina Propria Stephanie L. Sanos and Andreas Diefenbach Abstract Being exposed to food products, pathogens and harmless commensal bacteria, the mucosal immune system faces a constant challenge. Therefore, maintenance of a homeostatic balance is required to achieve tolerance to harmless bacteria and their products and to induce potent immunity to infection with pathogenic bacteria. Until recently, the literature on mucosal natural killer (NK) cells residing in the intestinal lamina propria was scarce and phenotype and function of gut mucosal NK cells did not receive much attention. Recently, data have become available identifying two distinct subsets of mucosal NKp46+ lymphocytes based on the expression of the orphan transcription factor ROR␥t. In many ways, the ROR␥t− subset resembled “classical” NK cells in that it was developmentally dependent on IL-15 but not on ROR␥t and displayed NK cell function (e.g., cell-mediated cytotoxicity, IFN-␥ production). In contrast, the ROR␥t+ subset developed independent of IL-15 but required ROR␥t, suggesting that this subset may be related to lymphoid tissue inducer (LTi) cells. Interestingly, these ROR␥t+ NKp46+ NK-LTi cells constitutively produced large amounts of IL-22, a cytokine regulating antimicrobial protection and regeneration of epithelial cells. In this chapter, we provide experimental procedures to isolate “classical” NK cells from the intestinal lamina propria as well as the newly described lymphoid tissue inducer-like (LTi-like) cells producing IL-22 and co-expressing NK cell receptors. Key words: Lymphoid tissue inducer cells, ROR␥t, IL-22, IL-15, mucosal immunity, cryptopatches.

1. Introduction NK cells are important effector cells of the innate immune system required for a first line of defence against transformed and infected cells (1–3). NK cell function has been studied in a vast array of disease models but their function in mucosal immunity has not been explored.

K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6 32, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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The mucosal immune system is the site of entry for potential pathogens, food products and commensal bacteria (4). Being exposed to such a complex environment, the mucosal immune system has evolved strategies to simultaneously achieve a state of tolerance against both food products and commensal bacteria, whilst eliciting immunity when encountering pathogens (5, 6). Recently, it has become clear that the interaction between the luminal microflora, epithelial cells and immune cells promotes epithelial homeostasis and the expression of antimicrobial molecules by epithelia (7–9). Indeed, the breach of such homeostatic conditions can lead to the development of inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn’s disease (7, 10). The organized lymphoid structures of the mucosal immune system of the intestine (also known as the gut-associated lymphoid tissue, GALT) include the mesenteric lymph nodes (MLN), Peyer’s patches (PP) (11), cryptopatches (CP) (12) and isolated lymphoid follicles (ILF) (13). The latter three structures are localized within the lamina propria of the intestine. In addition to these structured lymphoid organs of the lamina propria, a large number of immune cells are scattered throughout the lamina propria including NK cells, regulatory T cells, Th17-like CD4 T cells, ␥␦ T cells, macrophages and various populations of dendritic cells (DCs) (14–19). Lamina propria-resident NK cells have not been thoroughly investigated, and their biological function within this immune compartment has remained elusive. Previous studies have reported NK cell-mediated natural cytotoxicity in lamina propria lymphocyte preparations (20–22). Recent work has provided a phenotypic analysis of lamina propria lymphocytes expressing the NKp46 receptor (15–17), a marker believed to be specific for the NK cell lineage (23, 24). Although these lymphocytes were CD122 (IL-2/15R ␤-chain)+ cells and expressed activating NK cell receptors (i.e., NKG2D, NKp46, NK1.1), only very low levels of inhibitory Ly49 receptors or of the maturation markers CD49b (DX5), CD11b, or KLRG1 were found. Instead, these cells expressed markers of lymphoid progenitors such as CD127 (IL-7R␣), CD117 (c-Kit) and CD51 (␣v integrin). Table 32.1 summarizes the phenotype of lamina propriaresident NKp46+ CD3− CD19− lymphocytes. A key finding was that a significant subpopulation of intestinal NKp46+ cells expressed the orphan transcription factor ROR␥t (15, 16). ROR␥t is required for the lineage commitment of lymphoid tissue inducer cells (LTi cells) and, consequently, mice lacking ROR␥t fail to develop lymph nodes and cryptopatches (25–27). Of note, the majority of NKp46+ ROR␥t+ cells were localized within the cryptopatches of the small intestine and ROR␥t was required for their development, whereas ROR␥t−

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Table 32.1 Phenotype of NKp46+ ROR␥t+ and NKp46+ ROR␥t–/low cells of the intestinal lamina propria Lamina propria Spleen

ROR␥t−/low

ROR␥thigh

NKp46

+

+

+

CD122

+

+

+

NK1.1 (NKR-P1B/C)

High

High

Intermediate

NKG2D

+

+

+

NKG2A

+

+

+

CD49b (DX5)

+

−/low

−

CD11b

Subpopulation

−

−

CD27

Subpopulation

−

−

KLRG1

Subpopulation

−

−

CD51 (␣v integrin)

−

+

+

Ly49C/I

Subpopulation

−/low

−

Ly49G2

Subpopulation

−/low

−

c-Kit

−

+

+

CD127 (IL-7R␣)

−

+

+

Perforin

+++

+

−

Granzyme B

+++

+

−

Cytotoxicity

+++

+

−

IFN-␥

+++

+

−

IL-22

−

−

++

IL-17A

−

−

−

NK cells developed in the absence of this transcription factor (15, 16). Similar to “classical” NK cells, ROR␥t− NK cells required IL-15 for their development, whereas ROR␥t+ NKp46+ cells did not. The developmental dichotomy between these lymphocyte subsets was also reflected by their functional properties. Specifically, NKp46+ ROR␥t− cells but not NKp46+ ROR␥t+ cells mediated cytotoxicity and produced IFN-␥ albeit at lower levels than did mature splenic NK cells (15, 16). These data suggest that NKp46+ ROR␥t− cells are the LP-resident NK cell population, whereas the NKp46+ ROR␥t+ cells might have derived from LTi cells. Figure 32.1 summarizes our current understanding of the developmental program of these NKp46+ lymphocyte populations. The differentiation of NKp46+ ROR␥t+ cells depends on the presence of commensal microflora (15, 17). However, the function of NKp46+ ROR␥t+ cells is still to be revealed. Available data

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Fig. 32.1. NK and LTi-like cells in the intestinal mucosa. Summary of developmental, phenotypic and functional characteristics of the two NK cell-like (left) and LTi cell-like (right) lymphocyte populations within the lamina propria of the small intestine.

demonstrate that NKp46+ ROR␥t+ cells constitutively produce IL-22, a cytokine regulating epithelial expression of antimicrobial proteins (e.g., RegIII␤ and RegIII␥) and of proteins involved in epithelial regeneration (15, 16, 28). It was also shown that IL23 produced in response to Citrobacter rodentium-induced colitis further upregulated the production of IL-22 which ameliorated the clinical course of the colitis (17, 29, 30). Mucosal immunity is beginning to be an important area of research, most particularly the understanding of the acquisition and disruption of homeostatic conditions within the intestinal environment. In this chapter, we will describe methods to isolate lamina propria lymphocytes and to identify both NKp46+ ROR␥t− intestinal NK cells and IL-22-producing NKp46+ ROR␥t+ LTi-derived cells.

2. Materials 2.1. Mice

1. C57BL/6 2. ROR␥tgfp/+ mice (C57BL/6 Rorctm2Litt ; Jackson Laboratories) (31)

2.2. Solutions

1. Hank’s balanced salt solution (HBSS) without calcium/magnesium (Invitrogen) (see Note 1). 2. Cell dissociation solution: HBSS (Ca/Mg-free) supplemented with 5 mM EDTA and 10 mM Hepes (Invitrogen).

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3. Digestion solution: HBSS (Ca/Mg-free) containing 2% FCS, 50 U/ml dispase (Becton Dickinson), 0.5 mg/ml collagenase (Roche) and 0.5 mg/ml DNase (Sigma). 4. 10× Dulbecco’s phosphate buffered saline (DPBS; Invitrogen). 5. Cell culture medium: Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen) containing 4.5 g/l glucose, 10% FCS, 200 mg/l glutamine, 50,000 U/l penicillin, 50 mg/l streptomycin (all from Invitrogen), 10 mg/l gentamicin sulphate (Biowhittaker), 50 ␮M ␤-mercaptoethanol (Sigma), 1 mM sodium pyruvate (Invitrogen) and 0.1 mM non-essential amino acids (Invitrogen), filter-sterilized (0.22 ␮m) and stored at 4◦ C. 6. Percoll (GE Healthcare): Percoll is not an isotonic solution. Thus, 1 vol. of 10× DPBS is added to 9 vol. of Percoll in order to obtain an isotonic 90% Percoll solution. Dilute 90% Percoll with the appropriate amount of cell culture medium in order to obtain 40% and 80% Percoll solutions used for the separation gradient. 7. Staining buffer: 1× DPBS complemented with either 2% FCS or 0.5% BSA (Sigma), filter-sterilized (0.22 ␮m) and stored at 4◦ C. 8. PBS/EDTA: DPBS containing 5 mM EDTA (pH 8.0). 9. 4 ,6-Diamidino-2-phenylindole dihydrochloride (DAPI; Sigma) is reconstituted and stored according to the manufacturer’s protocol at a concentration of 2 mg/ml. 10. Foxp3 Staining Kit (eBioscience): This set includes the wash/permeabilization buffer and the fixation/permeabilization buffer. 2.3. Abs

1. CD3ε (145-2C11) mAb (eBioscience). 2. CD16/CD32 (2.4G2) mAb (BD Bioscience) is used to block Fc␥RIII/II. 3. CD45 (30F11) mAb (eBioscience) is used to detect haematopoietic cells. 4. NKR-P1B/C (PK136) mAb (eBioscience) reacts with a panNK cell marker (NK1.1) in certain mouse strains, such as C57BL/6 (NKR-P1C) and SJL (NKR-P1B). 5. NKp46 (29A1.4) mAb (eBioscience) specifically reacts with mouse NK cells in all inbred mouse strains tested. 6. ROR␥t (B2D) mAb (eBioscience): This is an unconjugated mAb that we conjugate with AlexaFluor-488 (Molecular Probes, Invitrogen) following the manufacturer’s protocol (see Note 2).

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3. Methods It is important to ensure that all the buffers and solutions as well as tubes and petri dishes used during the procedure are placed on ice, before starting to work. Lamina propria lymphocytes are very fragile, sensitive cells, hence this will minimize the amount of dead cells at the end of the isolation procedure. The protocol below describes the isolation of lamina propria lymphocytes from the small intestine of a single mouse. If large numbers of lamina propria lymphocytes from multiple mice are needed, the small intestines should not be pooled during the isolation procedure, as this will lead to a reduced yield of cells at the end of the procedure. Thus, we recommend treating each small intestine individually. In our experience, colons from up to three mice can be pooled without a significant decrease in efficiency (see Note 3). 3.1. Lamina Propria Lymphocyte Isolation

1. Euthanize mouse and open the peritoneal cavity. Using forceps, hold the intestine below the stomach and pull out gently, taking care not to damage the intestine, and cut before the caecum. For the colon, cut after the caecum. 2. Place small intestine and colon in separate petri dishes containing ice-cold DPBS and keep moist at all times. 3. Using fine forceps, remove the mesenterium, residual fat and connective tissue. The better the cleaning procedure, the cleaner the lymphocyte preparation, which will ensure a nice staining. 4. Using curved scissors, remove the Peyer’s patches of the small intestine. There are between 8 and 10 Peyer’s patches per small intestine. Start from the distal end of the intestine. It might be slightly difficult to visualize the Peyer’s patches of the distal end, as they are small. Nevertheless, they become bigger towards the proximal end of the small intestine. They can be easily recognized, as they are of whitish colour. Colons do not have Peyer’s patches. 5. The intestine is then cut open longitudinally using angled scissors while gently holding the tissue with forceps. It is best to cut the intestine on the mesenteric side. Once the intestine is opened, it should be shaken gently in the petri dish containing ice-cold DPBS in order to remove most of the faecal material. 6. Transfer the tissue into a 50 ml centrifuge tube containing ice-cold DPBS. Shake the tube and carefully discard DPBS. Refill the tube with fresh DPBS and repeat this procedure

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at least 4–5 times until the DPBS in the tube is clear and devoid of faecal material. 7. Place the intestine in a fresh petri dish and cut into pieces of 1–1.5 cm. 8. Place into a 50-ml centrifuge tube and add 5–10 ml of cell dissociation solution. This solution is used to separate lamina propria lymphocytes from the epithelial layer containing both epithelial cells and intra-epithelial lymphocytes. Each small intestine should be treated separately, while up to three colons can be combined at this step. 9. Incubate for 15–20 min at 37◦ C with slow rotation at 100 rpm on a shaker. 10. Vortex vigorously for 15 s, discard the SN containing epithelial cells and intra-epithelial lymphocytes. 11. With the remaining pieces of tissue, Steps 8–11 are repeated. 12. Collect the tissue fragments and cut into fine pieces using a scalpel or razor blades in a small petri dish. Transfer into a 50-ml centrifuge tube. 13. Add 5 ml of digestion solution. This is a collagenase−DNase-containing solution, which allows the digestion of the lamina propria facilitating the release of lymphocytes from this tissue (see Note 4). 14. Digest for 20 min at 37◦ C with slow rotation on a bacterial rotator (side to side) at 100 rpm. 15. Vortex well for 15 s. 16. Collect the supernatant (SN) by filtering through a 40-␮m cell strainer into a 50-ml centrifuge tube, which is placed on ice. 17. Add 5 ml of fresh enzyme solution and repeat Steps 14–16 two or three more times. Combine the SNs from one small intestine into one tube. 18. Pellet the cells for 10 min at 860 × g at 4◦ C. 19. Prepare 90% Percoll solution (9 vol. of Percoll and 1 vol. of 10× DPBS). Prepare the 40% and 80% Percoll solutions with cell culture medium. You will need 10 ml of 40% Percoll and 5 ml of 80% Percoll for each intestine. 20. Resuspend the cell pellet from Step 19 in 1 ml of 40% Percoll and top up with another 9 ml of 40% Percoll (10 ml/mouse intestine). 21. Place 5 ml of 80% Percoll in a fresh 15-ml centrifuge tube and gently overlay the 10 ml of 40% Percoll with cells (from Step 21) on top of the 80% Percoll slowly and with constant

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speed. The slower the speed used when one overlays the 40% cell suspension onto the 80% Percoll layer, the better the interphase obtained at the end. 22. Optional: Let stand for 10 min at room temperature. 23. Centrifuge the tubes at 900 × g for 20 min at 20◦ C without break. 24. Collect and discard the top layer of epithelial cells using a serum pipette. 25. Collect the lymphocytes at the interface between the 40% and 80% layers using a small serum pipette. An opaque ring of lymphocytes should be visible at the interface between the two Percoll layers. 26. Collect the cells in a fresh 15- or 50-ml tube (now suspensions of multiple intestines can be combined). Add 5– 10 vol. of ice-cold staining buffer and pellet by centrifugation for 5–10 min at 800 × g at 4◦ C. Resuspend in staining buffer and keep on ice. The cells are now ready for staining. 3.2. Staining Procedure for NKp46+ ROR␥t+ LTi-Like Cells and of “Classical” Mucosal NK Cells

3.2.1. Staining Procedure for Rorc(␥t)gfp/+ Mice

Two protocols are provided for detecting and analyzing the various NKp46+ lymphocyte populations in the intestinal lamina propria that can be distinguished by their expression of ROR␥t. One protocol uses genetically modified mice that carry a green fluorescent reporter gene knocked into the transcriptional start of the Rorc(γ t) gene (31). The alternative protocol employs an intracellular staining procedure using an ROR␥t-specific MAb (see Note 2). 1. Prior to surface staining, cells are blocked for 20 min on ice with purified CD16/CD32 (2.4G.2) MAb. 2. Spin down and discard the supernatant. 3. Surface staining is performed for 20 min on ice using the desired cocktail of fluorescently labelled antibodies. We have found that co-staining with PE-Cy7-labelled anti-CD3, PElabelled anti-NKp46 (or anti-NK1.1), APC-Cy7-labelled anti-CD45 allows for a good separation of the cell populations (Figs. 32.2 and 32.3). This staining protocol minimally allows the use of APC- and PerCP-labelled antibodies to assess other surface markers on lamina propria cells. In our experience, it is advised not to use PerCP-labelled anti-CD3. 4. Wash stained cells twice in staining buffer. 5. Resuspend in 200 ␮l of staining buffer containing 1 ␮g/ml DAPI (1:2000 of stock). 6. The cells are now ready for analysis on a flow cytometer.

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Fig. 32.2. Analysis of NKp46+ lamina propria lymphocytes using Rorc(␥t)gfp/+ mice. Flow cytometry analysis of lamina propria lymphocytes of Rorc(␥t)gfp/+ mice stained with the DNA dye DAPI and antibodies to CD45, CD3, NKp46 and NK1.1. (A–C) Gating procedure for the analysis of CD3− CD45+ lamina propria lymphocytes. Stained cells are first analyzed for granularity (SSC-H) and size (FSC-H) and an electronic gate is applied including all lymphocytes (A). Dead cells are excluded by electronically gating on DAPI− cells (B). In order to visualize NK cells and LTi-like cells, electronic gating on all CD3− CD45+ cells is performed (C). (D, E) Analysis of ROR␥t expression by NKp46+ (D) or NK1.1+ (E) lymphocytes within the CD3− CD45+ lamina propria lymphocyte population. Four populations can be easily distinguished. (a) NKp46+ ROR␥t− NK cells; (b) NKp46+ ROR␥tlow cells; (c) NKp46+ ROR␥thi cells; (d) NKp46− ROR␥thi LTi cells. (a’) NK1.1hi ROR␥t− NK cells; (b’) NK1.1hi-int ROR␥tlow cells; (c’) NK1.1int ROR␥thi cells; (d’) NK1.1− ROR␥thi LTi cells.

Analysis of NKp46+ lamina propria lymphocytes using intracellular staining for ROR γ t

Fig. 32.3. Analysis of NKp46+ lamina propria lymphocytes using intracellular staining of ROR␥t. Flow cytometry analysis of lamina propria lymphocytes stained with antibodies to CD45, CD3, ROR␥t, NKp46 and NK1.1. (A–C) Gating procedure for the analysis of CD3− CD45+ lamina propria lymphocytes. Stained cells are first analyzed for granularity (SSC-H) and size (FSC-H) and an electronic gate is applied including all lymphocytes (A). In order to visualize NK cells and LTi-like cells, electronic gating on all CD3− CD45+ cells is performed (B). (C, D) Analysis of ROR␥t expression by NKp46+ (C) or NK1.1+ (D) lymphocytes within the CD3− CD45+ lamina propria lymphocyte population. Three populations can be easily distinguished. (a) NKp46+ ROR␥t− NK cells; (c) NKp46+ ROR␥thi cells; (d) NKp46− ROR␥thi LTi cells. (a’) NK1.1hi ROR␥t− NK cells; (c’) NK1.1int ROR␥thi cells; (d’) NK1.1− ROR␥thi LTi cells.

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3.2.2. Staining Procedure Using Intracellular Staining of ROR␥t

1. Prior to surface staining, cells are blocked for 20 min on ice with purified CD16/CD32 (2.4G2) mAb. 2. Spin down and discard the supernatant. 3. Surface staining is performed for 20 min on ice using the desired cocktail of fluorescently labelled antibodies. We have found that co-staining with PE-Cy7-labelled anti-CD3, PElabelled anti-NKp46 (or anti-NK1.1), APC-Cy7-labelled anti-CD45 allows for a good separation of the cell populations (Fig. 32.3). This staining protocol minimally allows the use of APC- and PerCP-labelled antibodies to assess other surface markers on lamina propria cells. In our experience, it is advised not to use PerCP-labelled anti-CD3. 4. Wash stained cells twice in staining buffer. 5. After the last wash, cells are permeabilized using the fixation/permeabilization buffer of the Foxp3 Staining Kit for 45 min at 4◦ C in the dark according to the protocol provided by the manufacturer. 6. Wash twice using the wash/permeabilization buffer provided with the Foxp3 Staining Kit. 7. Intracellular staining is performed with Alexa-488conjugated anti-ROR␥t diluted in wash/permeabilization buffer solution (provided with the Foxp3 Staining Kit) for 1 h at 4◦ C in the dark. We found it beneficial to add anti-NKp46 or anti-NK1.1 used in Step 3 for the surface stain also during the intracellular stain. 8. Wash twice with wash/permeabilization buffer provided with the Foxp3 Staining Kit. 9. Resuspend stained cells in regular staining buffer. The cells are now ready for analysis on a flow cytometer.

3.3. Flow Cytometric Analysis of Classical Mucosal NK Cells and ROR␥t+ NKp46+ NK1.1+ LTi-Like Cells

The flow cytometric analysis of lamina propria lymphocytes is more difficult than is the analysis of lymphocyte preparations from lymphoid organs such as spleen, thymus or lymph nodes. This is partly due to residual epithelial cells and often a considerable amount of dead cells in the lamina propria cell preparation. Thus, we found it very helpful to gate out all dead cells and to include staining with a marker that identifies all haematopoietic cells (CD45). Such a procedure can be easily employed when no intracellular stain is performed. When intracellular staining for ROR␥t (or other markers) is performed, an exclusion of dead cells is not possible. Lamina propria lymphocytes are first recorded in FSC-H and SSC-H (Figs. 32.2A and 32.3A) and a similar gate as that used when gating live splenocytes is applied. It might be a good idea to run some splenocytes in parallel to the lamina propria

Isolation of NK Cells and NK-Like Cells from the Intestinal Lamina Propria

515

preparations if unsure how to set the gate. Unlike splenocytes, which generally form a uniform population of small, non-granular cells, cells isolated from the lamina propria are more heterogeneous. Therefore, the use of DAPI to gate out the dead cells is recommended and necessary for the analysis of such a preparation (Fig. 32.2B). DAPI cannot be used if the intracellular staining of ROR␥t or cytokines is employed (Fig. 32.3). Similarly, DAPI− lymphocytes were further gated on cells expressing CD45, a marker expressed by all haematopoietic cells, allowing the exclusion of epithelial cells. As the analysis of both LTi cells and NK cells requires the exclusion of CD3+ cells, we co-stain with CD3 and electronically gate on CD45+ CD3− cells (Fig. 32.2C and 32.3B). DAPI− CD45+ CD3− cells can now be further analysed for ROR␥t expression and NK cell markers (Figs. 32.2D,E and 32.3C,D). It is pivotal to co-analyze the expression of ROR␥t together with those of NK cell receptors, as only this will allow to distinguish between the resident NK cells of the lamina propria (ROR␥t−/low : Fig. 32.2D,E, populations a, b or a’, b’) and LTi-like cells that upregulate NK cell receptors (ROR␥thigh : Fig. 32.2D,E population c or c’). Both populations express NKp46 and NKG2D and the levels of expression seem to be very similar. Of note, only the reporter mice (ROR␥tgfp/+ ) allow to distinguish between NKp46+ ROR␥t− NK cells (Fig. 32.2D,E populations a and a’) and NKp46+ ROR␥tlow NK cells (Fig. 32.2D,E populations b and b’) whereas the intracellular ROR␥t strain allows only to visualize the ROR␥thigh cells whereas the ROR␥tlow cells cannot be discriminated (Fig. 32.3C,D). Whereas the expression of NKG2D and NKp46 is comparable between lamina propria NK cells and NK-LTi cells, the expression of NK1.1 (NKR-P1B/C receptors) further distinguishes between these two populations. While lamina propria NK cells express high levels of the NK1.1 antigen, NK-LTi cells express only low levels (Fig. 32.2E and 32.3D). These populations of NKp46+ cells also display distinct functional properties. While NKp46+ NK1.1high ROR␥t−/low NK cells produce IFN-␥ and display cell-mediated cytotoxicity, NKp46+ NK1.1int ROR␥thigh NK-LTi cells produce high levels of IL-22 but fail to produce IFN-␥ and are not cytotoxic.

4. Notes 1. It is very important to use Ca/Mg-free HBSS as Ca/Mgcontaining HBSS will not allow for good isolation and separation, resulting in the loss of lamina propria lymphocytes.

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2. For the purpose of these studies, we used a purified ROR␥t monoclonal antibody, which we conjugated to Alexa Fluor488 (Invitrogen). Now a directly PE-conjugated ROR␥t monoclonal antibody (Clone AFKJS-9) is available (eBioscience). 3. We have observed that in order to obtain the best isolation of LP cells possible, a limited number of mice should be used. In our experience, intestines from up to 10 mice can be handled by one person. 4. It is important to make the digestion solution fresh every time. References 1. Herberman, R. B., Nunn, M. E., Holden, H. T., and Lavrin, D. H. (1975) Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. II. Characterization of effector cells. Int J Cancer 16, 230–239. 2. Kiessling, R., Klein, E., Pross, H., and Wigzell, H. (1975) “Natural” killer cells in the mouse. II. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Characteristic of the killer cell. Eur J Immunol 5, 117–121. 3. Trinchieri, G. (1989) Biology of natural killer cells. Adv Immunol 47, 187–376. 4. Mowat, A. M., and Viney, J. L. (1997) The anatomical basis of intestinal immunity. Immunol Rev 156, 145–166. 5. Newberry, R. D., and Lorenz, R. G. (2005) Organizing a mucosal defense. Immunol Rev 206, 6–21. 6. Artis, D. (2008) Epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut. Nat Rev Immunol 8, 411–420. 7. Rakoff-Nahoum, S., Paglino, J., EslamiVarzaneh, F., Edberg, S., and Medzhitov, R. (2004) Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118, 229–241. 8. Cash, H. L., Whitham, C. V., Behrendt, C. L., and Hooper, L. V. (2006) Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science 313, 1126–1130. 9. Rescigno, M., Urbano, M., Valzasina, B., Francolini, M., Rotta, G., Bonasio, R., Granucci, F., Kraehenbuhl, J. P., and Ricciardi-Castagnoli, P. (2001) Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol 2, 361–367.

10. Nenci, A., Becker, C., Wullaert, A., Gareus, R., van Loo, G., Danese, S., Huth, M., Nikolaev, A., Neufert, C., Madison, B., Gumucio, D., Neurath, M. F., and Pasparakis, M. (2007) Epithelial NEMO links innate immunity to chronic intestinal inflammation. Nature 446, 557–561. 11. Dobson, G. E. (1884) On the presence of Peyer’s patches (Glandulae Agminatae) in the caecum and colon of certain mammals. J Anat Physiol 18, 388–392. 12. Kanamori, Y., Ishimaru, K., Nanno, M., Maki, K., Ikuta, K., Nariuchi, H., and Ishikawa, H. (1996) Identification of novel lymphoid tissues in murine intestinal mucosa where clusters of c-kit+ IL-7R+ Thy1+ lympho-hemopoietic progenitors develop. J Exp Med 184, 1449–1459. 13. Hamada, H., Hiroi, T., Nishiyama, Y., Takahashi, H., Masunaga, Y., Hachimura, S., Kaminogawa, S., Takahashi-Iwanaga, H., Iwanaga, T., Kiyono, H., Yamamoto, H., and Ishikawa, H. (2002) Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine. J Immunol 168, 57– 64. 14. Ivanov, II, McKenzie, B. S., Zhou, L., Tadokoro, C. E., Lepelley, A., Lafaille, J. J., Cua, D. J., and Littman, D. R. (2006) The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126, 1121–1133. 15. Sanos, S. L., Bui, V. L., Mortha, A., Oberle, K., Heners, C., Johner, C., and Diefenbach, A. (2009) RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells. Nat Immunol 10, 83–91. 16. Luci, C., Reynders, A., Ivanov, II, Cognet, C., Chiche, L., Chasson, L., Hardwigsen,

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J., Anguiano, E., Banchereau, J., Chaussabel, D., Dalod, M., Littman, D. R., Vivier, E., and Tomasello, E. (2009) Influence of the transcription factor RORgammat on the development of NKp46+ cell populations in gut and skin. Nat Immunol 10, 75–82. Satoh-Takayama, N., Vosshenrich, C. A., Lesjean-Pottier, S., Sawa, S., Lochner, M., Rattis, F., Mention, J. J., Thiam, K., CerfBensussan, N., Mandelboim, O., Eberl, G., and Di Santo, J. P. (2008) Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense. Immunity 29, 958–970. Zhou, L., Lopes, J. E., Chong, M. M., Ivanov, II, Min, R., Victora, G. D., Shen, Y., Du, J., Rubtsov, Y. P., Rudensky, A. Y., Ziegler, S. F., and Littman, D. R. (2008) TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. Nature 453, 236–240. Iwasaki, A. (2007) Mucosal dendritic cells. Annu Rev Immunol 25, 381–418. Tagliabue, A., Befus, A. D., Clark, D. A., and Bienenstock, J. (1982) Characteristics of natural killer cells in the murine intestinal epithelium and lamina propria. J Exp Med 155, 1785–1796. Hogan, P. G., Hapel, A. J., and Doe, W. F. (1985) Lymphokine-activated and natural killer cell activity in human intestinal mucosa. J Immunol 135, 1731–1738. Gibson, P. R., and Jewell, D. P. (1985) The nature of the natural killer (NK) cell of human intestinal mucosa and mesenteric lymph node. Clin Exp Immunol 61, 160–168. Walzer, T., Blery, M., Chaix, J., Fuseri, N., Chasson, L., Robbins, S. H., Jaeger, S., Andre, P., Gauthier, L., Daniel, L., Chemin, K., Morel, Y., Dalod, M., Imbert, J., Pierres, M., Moretta, A., Romagne, F., and Vivier, E. (2007) Identification, activation, and selective in vivo ablation of mouse NK cells via NKp46. Proc Natl Acad Sci U S A 104, 3384–3389.

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24. Sivori, S., Vitale, M., Morelli, L., Sanseverino, L., Augugliaro, R., Bottino, C., Moretta, L., and Moretta, A. (1997) p46, a novel natural killer cell-specific surface molecule that mediates cell activation. J Exp Med 186, 1129–1136. 25. Sun, Z., Unutmaz, D., Zou, Y. R., Sunshine, M. J., Pierani, A., Brenner-Morton, S., Mebius, R. E., and Littman, D. R. (2000) Requirement for RORgamma in thymocyte survival and lymphoid organ development. Science 288, 2369–2373. 26. Kurebayashi, S., Ueda, E., Sakaue, M., Patel, D. D., Medvedev, A., Zhang, F., and Jetten, A. M. (2000) Retinoid-related orphan receptor gamma (RORgamma) is essential for lymphoid organogenesis and controls apoptosis during thymopoiesis. Proc Natl Acad Sci U S A 97, 10132–10137. 27. Eberl, G., and Littman, D. R. (2004) Thymic origin of intestinal alphabeta T cells revealed by fate mapping of RORgammat+ cells. Science 305, 248–251. 28. Zheng, Y., Valdez, P. A., Danilenko, D. M., Hu, Y., Sa, S. M., Gong, Q., Abbas, A. R., Modrusan, Z., Ghilardi, N., de Sauvage, F. J., and Ouyang, W. (2008) Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nat Med 14, 282–289. 29. Cella, M., Fuchs, A., Vermi, W., Facchetti, F., Otero, K., Lennerz, J. K., Doherty, J. M., Mills, J. C., and Colonna, M. (2008) A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity. Nature. [epub ahead of print] 30. Zenewicz, L. A., Yancopoulos, G. D., Valenzuela, D. M., Murphy, A. J., Stevens, S., and Flavell, R. A. (2008) Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease. Immunity 29, 947–957. 31. Eberl, G., Marmon, S., Sunshine, M. J., Rennert, P. D., Choi, Y., and Littman, D. R. (2004) An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells. Nat Immunol 5, 64–73.

Appendix

I. Antibodies to Human Natural Killer Cell Receptors The listed sources of antibodies are original authors, company, or hybridoma bank resource (American Type Culture Collection, ATCC, Manassas, VA; www.atcc.org).

Anti-Killer Cell Immunoglobulin-Like Receptor (KIR, CD158) KIR constitutes a family of polymorphic gene products that exhibit variable inter-individual and inter-clonal expression on NK cells and some subsets of T cells. Expression is genetically determined and does not correlate with MHC class I haplotype. Antibodies to some isoforms of KIR have not been reported, and the cross-reactivities of most antibodies should be appreciated when typing receptor expression on NK cells. Some additional cross-reactivities of these antibodies have been reported on KIR transfectants, which should also be appreciated.

Clone

Specificity

Species/Isotype

Source/Reference

EB6

KIR2DL1, 2DS1

Mouse IgG1

Beckman Coulter (1)

GL183

KIR2DL3, 2DS2, 2DL2

Mouse IgG1

AbD Serotec, Beckman Coulter (1)

DX9

KIR3DL1

Mouse IgG1

BD Pharmingen, R&D Systems, LifeSpan BioSciences, Biolegend, Abcam, ATCC (2)

Z27.3.7

KIR3DL1, 3DS1

Mouse IgG1

Beckman Coulter (3, 4)

177407

KIR3DL1

Mouse IgG2a

R&D Systems

HP-3E4

KIR2DL1, 2DS1, 2DS4

Mouse IgM

BD Pharmingen (5, 6, 7)

5.133

KIR3DL1, 3DL2, 2DS4

Mouse IgG1

M. Colonna, Washington University, St. Louis (7)

180704

KIR2DL2, 2DL3, 2DS2, 2DS4

Mouse IgG2b

R&D Systems

K.S. Campbell (ed.), Natural Killer Cell Protocols, Methods in Molecular Biology 612, DOI 10.1007/978-1-60761-362-6, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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520

Appendix

(continued) Clone

Specificity

Species/Isotype

Source/Reference

NKVFS1

pan KIR2D

Mouse IgG1

Novus Biologicals, LifeSpan BioSciences, AbD Serotec, Accurate, Acris, GeneTex, Genway

143211

KIR2DL1

Mouse IgG1

R&D Systems

2F9

KIR2DL1

Mouse IgG2a

Abnova, Santa Cruz Biotechnology

3H1905

KIR2DL1

Mouse IgG1

Santa Cruz Biotechnology

4j52

KIR2DL1

Mouse IgG1

LifeSpan BioSciences

180701

KIR2DL3

Mouse IgG2a

R&D Systems

190IIC311

KIR2DL3

Mouse IgG2a

Abnova, Santa Cruz Biotechnology, AbD Serotec, Accurate, GeneTex

DX27

KIR3DL2

Mouse IgG2a

LifeSpan BioSciences, Biolegend, Pharmingen, Biolegend (6)

179315

KIR2DS4

Mouse IgG2a

R&D Systems

BD

5F2

KIR2DS4

Mouse IgG2b

Abnova, Santa Cruz Biotechnology, GenWay

FES172

KIR2DS4

Mouse IgG2a

Beckman Coulter (8)

181703

KIR2DL4

Mouse IgG2a

R&D Systems

2H6

KIR2DL4

Mouse IgG2b

Abnova, Santa Cruz Biotechnology, Abcam, GenWay

Anti-CD94/NKG2 (CD159) CD94 is expressed as a heterodimer with various isoforms of NKG2 family of polypeptides. The NKG2 isoforms are expressed variably between clones within an individual on most NK cells and a subset of T cells. NKG2A is an inhibitory receptor, while NKG2C, -E, and -F facilitate association with the transmembrane DAP12 signaling adapter to activate cells. NKG2D is found on most NK cells and some T cells, exists as a homodimer, and transduces activating signals through physical association with either DAP10 or DAP12.

Clone

Specificity

Species/Isotype

Source/Reference

HP-3B1

CD94 (KLRD1)

Mouse IgG2a

Beckman Coulter, AbD Serotec (9, 10)

HP-3D9

CD94 (KLRD1)

Mouse IgG1

BD Pharmingen, Accurate (9)

DX22

CD94 (KLRD1)

Mouse IgG1

AbD Serotec, Accurate, eBiosciences, Biolegend

Z199

NKG2A (KLRC1)

Mouse IgG2b

Beckman Coulter (10)

131411

NKG2A (KLRC1)

Mouse IgG2a

R&D Systems

2C3

NKG2A (KLRC1)

Mouse IgG1

Abnova

14F09

NKG2A (KLRC1)

Mouse IgG2a

Santa Cruz Biotechnology

134522

NKG2C (KLRC2)

Mouse IgG2b

R&D Systems

134591

NKG2C (KLRC2)

Mouse IgG1

R&D Systems

L45

NKG2C (KLRC2)

Mouse IgG1

Santa Cruz Biotechnology

Appendix

521

(continued) Clone

Specificity

Species/Isotype

Source/Reference

49B18

NKG2C (KLRC2)

Mouse IgG2b

Santa Cruz Biotechnology

3D5

NKG2E (KLRC3)

Mouse IgG2a

Abnova

1D10

NKG2F (KLRC4)

Mouse IgG2a

Abnova

149810

NKG2D (KLRK1)

Mouse IgG1

R&D Systems

3.1.1.1

NKG2D (KLRK1)

Mouse IgG1

Millipore

1D11

NKG2D (KLRK1)

Mouse IgG1

Santa Cruz Biotechnology, eBiosciences, Biolegend

5C6

NKG2D (KLRK1)

Mouse IgG2a

Santa Cruz Biotechnology, eBiosciences

Anti-CD161 (NKR-P1A) NKR-P1A is found on most human NK cells and a subset of T cells. The NKR-P1A gene is the only NKR-P1 gene identified in man.

Clone

Specificity

Species/Isotype

Source/Reference

DX12

NKR-P1A

Mouse IgG1

BD Pharmingen (11)

191B8

NKR-P1A

Mouse IgG2a

Beckman Coulter (12)

B199.2

NKR-P1A

Mouse IgG2b

AbD Serotec, Accurate (13)

Anti-CD16 (Fcγ RIIIA) The transmembrane (and signaling competent) form of CD16 is expressed on human NK cells, a subset of T cells, macrophages, and mast cells. Fc␥RIIIA is an activating Fc receptor for IgG that triggers antibody-dependent cellular cytotoxicity through association with FcεRI-␥ and/or TCR-␨. A glycosylphosphatidylinositol (GPI)-linked form of CD16 (Fc␥RIIIB) has also been identified on neutrophils.

Clone

Specificity

Species/Isotype

Source/Reference

3G8

CD16

Mouse IgG1

BD Pharmingen, Invitrogen, Accurate, AbD Serotec, Beckman Coulter, Biolegend (14)

B73.1

CD16

Mouse IgG1

BD Pharmingen (15)

GRM1

CD16

Mouse IgG2a

Southern Biotechnology

522

Appendix

Anti-CD56 (N-CAM) CD56 is an isoform of the neural cell adhesion molecule (N-CAM) which is expressed in the brain. The leukocyte isoform of CD56 is expressed on NK cells and subsets of T cells in humans (but not mice).

Clone

Specificity

Species/Isotype

Source/Reference

MEM-188

CD56

Mouse IgG2a

Invitrogen, AbD Serotec, eBiosciences, Biolegend, Southern Biotechnology

B159.5

CD56

Mouse IgG1

BD Pharmingen, Accurate (16, 17)

N901 (NKH-1)

CD56

Mouse IgG1

Beckman Coulter (18)

T-199

CD56

Mouse IgG1

Accurate (19)

C218

CD56

Mouse IgG1

Beckman Coulter

NKI-nbl-1

CD56

Mouse IgG1

Accurate

Activating Receptors These receptors contribute significantly to NK cell activation during natural cytotoxicity through recognition of ligands on target cells.

Clone

Specificity

Species/Isotype

Source/Reference

C1.7

2B4 (CD244)

Mouse IgG1

Beckman Coulter, eBiosciences, Biolegend (20)

BAB281

NKp46 (NCR1, CD335)

Mouse IgG1

Beckman Coulter (21)

195314

NKp46 (NCR1, CD335)

Mouse IgG2b

R&D Systems

9E2

NKp46 (NCR1, CD335)

Mouse IgG1

Biolegend

Z231

NKp44 (NCR2, CD336)

Mouse IgG1

Beckman Coulter (22)

253415

NKp44 (NCR2, CD336)

Mouse IgG2a

R&D Systems

P44-8

NKp44 (NCR2, CD336)

Mouse IgG1

Biolegend

210845

NKp30 (NCR3, CD337)

Mouse IgG2a

R&D Systems

210845

NKp30 (NCR3, CD337)

Mouse IgG2a

R&D Systems

Z25

NKp30 (NCR3, CD337)

Mouse IgG1

Beckman Coulter

P30-15

NKp30 (NCR3, CD337)

Mouse IgG1

Biolegend

239127

NKp80 (KLRF1)

Rat IgG2a

R&D Systems

References 1. Moretta, A., Vitale, M., Bottino, C., Orengo, A. M., Morelli, L., Augugliaro, R., Barbaresi, M., Ciccone, E., and Moretta, L. (1993) p58 molecules as putative receptors for major histocompatibility complex (MHC) class I molecules in human natural

killer (NK) cells: anti-p58 antibodies reconstitute lysis of MHC class I-protected cells in NK clones displaying different specificities. J. Exp. Med., 178:597–604. 2. Litwin, V., Gumpery, J., Parham, P., Phillips, J. H., and Lanier, L. L. (1994) NKB1: a nat-

Appendix

3.

4.

5.

6.

7.

8.

9.

ural killer cell receptor involved in the recognition of polymorphic HLA-B molecules. J. Exp. Med., 180:537–543. Vitale, M., Sivori, S., Pende, D., Augugliaro, R., Di Donato, C., Amoroso, A., Malnati, M., Bottino, C., Moretta, L., and Moretta, A. (1996) Physical and functional independency of p70 and p58 natural killer (NK) cell receptors for HLA class I: their role in the definition of different groups of alloreactive NK cell clones. Proc. Natl. Acad Sci. USA, 93:1453–1457. Albi, N., Ruggeri, L., Aversa, F., Merigiola, C., Tosti, A., Tognellini, R., Grossi, C. E., Martelli, M. F., and Velardi, A. (1996) Natural killer (NK)-cell function and antileukemic activity of a large population of CD3+ /CD8+ T cells expressing NK receptors for Major Histocompatibility Complex class I after “three-loci” HLA-incompatible bone marrow transplantation. Blood, 87: 3993–4000. ´ Melero, I., Salmeron, A., Balboa, M. ´ A., Aramburu, J., and Lopez-Botet, M. (1994) Tyrosine kinase-dependent activation of human NK cell functions upon stimulation through a 58-kDa surface antigen selectively expressed on discrete subsets of NK cells and T lymphocytes. J. Immunol., 152: 1662–1673. S¨oderstr¨om, K., Corliss, B., Lanier, L. L., and Phillips, J. H., (1997) CD94/NKG2 is the predominant inhibitory receptor involved in recognition of HLA-G by decidual and peripheral blood NK cells. J. Immunol., 159:1072–1075. D¨ohring, C., Samaridis, J., and Colonna, M. (1996) Alternatively spliced forms of human killer inhibitory receptors. Immunogenetics, 44:227–230. Bottino, C., Sivori, S., Vitale, M., Cantoni, C., Falco, M., Pende, D., Morelli, L., Augugliaro, R., Semenzato, G., Biassoni, R., Moretta, L., and Moretta, A. (1996) A novel surface molecule homologous to the p58/p50 family of receptors is selectively expressed on a subset of human natural killer cells and induces both triggering of cell functions and proliferation. Eur. J. Immunol., 26:1816–1824. Aramburu, J., Balboa, M. A., Ramirez, A., Silva, A., Acevedo, A., Sanchez-Madrid, F., ´ De Landazuri, M. O., and Lopez-Botet, M. (1990) A novel functional cell surface dimer (Kp43) expressed by natural killer cells and T cell receptor–gamma/delta+ T lymphocytes. I. Inhibition of the IL-2-dependent proliferation by anti-Kp43 monoclonal antibody. J. Immunol., 144:3238–3247.

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10. Perez-Villar, J. J., Carretero, M., Navarro, F., Melero, I., Rodriguez, A., Bottino, C., ´ Moretta, A., and Lopez-Botet, M. (1996) Biochemical and serologic evidence for the existence of functionally distinct forms of the CD94 NK cell receptor. J. Immunol., 157:5367–5374. 11. Lanier, L. L., Chang, C., and Phillips, J. H. (1994) Human NKR-P1A. A disulfidelinked homodimer of the C-type lectin superfamily expressed by a subset of NK and T lymphocytes. J. Immunol., 153: 2417–2428. 12. Poggi, A., Costa, P., Morelli, L., Cantoni, C., Pella, N., Spada, F., Biassoni, R., Nanni, L., Revello, V., Tomasello, E., Mingari, M. C., Moretta, A., and Moretta, L. (1996) Expression of human NKRP1A by CD34+ immature thymocytes:NKRP1A-mediated regulation of proliferation and cytolytic activity. Eur. J. Immunol., 26:1266–1272. 13. Bennett, I. M., Zatsepina, O., Zamai, L., Azzoni, L., Mikheeva, T., and Perussia, B. (1996) Definition of a Natural Killer NKRP1A+/CD56-/CD16- functionally immature human NK cell subset that differentiates in vitro in the presence of interleukin 12. J. Exp. Med., 184:1845–1856. 14. Fleit, H. B., Wright, S. D., and Unkeless, J. C. (1982) Human neutrophil Fc gamma receptor distribution and structure. Proc. Natl. Acad. Sci. USA, 79: 3275–3279. 15. Perussia, B., Starr, S., Abraham, S., Fanning, V., and Trinchieri, G. (1983) Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fc receptor functions. I. Characterization of the lymphocyte subset reactive with B73.1. J. Immunol., 130: 2133–2141. 16. Schlossman, S., Bloumsell, L., Gilks, W., et al. (1995) Leucocyte Typing V: White cell differentiation antigens. Oxford University Press, New York. 17. Wolf, S. F., Temple, P. A., Kobayashi, M., Young, D., Dicig, M., Lowe, l. Dzialo, R., Fitz, l., Ferenz, C., Hewick, R. M., Keleher, K., Herrmann, S. H., Clark, S. C., Azzoni, L., Chan, S. H., Trinchieri, G., and Perussia, B. (1991) Cloning of cDNA for Natural Killer cell Stimulatory Factor, a heterodimeric cytokine with multiple biologic effects on T and Natural Killer cells. J. Immunol., 146:3074–3081. 18. Griffin, J. D., Hercend, T., Beveridge, R., and Schlossman, S. F. (1983) Characterization of an antigen expressed by human natural killer cells. J. Immunol., 130: 2947–2951.

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19. Feickert, H. -J., Pietsch, T., Hadam, M. R., Mildenberger, H., and Riehm, H. (1989) Monoclonal antibody T-199 directed against human medulloblastoma: Characterization of a new antigenic system expressed on neuroectodermal tumors and natural killer cells. Cancer Res., 49:4338–4343. 20. Valiante, N. M., and Trinchieri, G. (1993) Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes. J. Exp. Med., 178:1397–1406. 21. Sivori, S., Vitale, M., Morelli, L., Sanseverino, L., Augugliaro, R., Bottino, C.,

Moretta, L., and Moretta, L. (1997) p46, a novel natural killer cell-specific surface molecule that mediates cell activation. J. Exp. Med., 186:1129–1136. 22. Vitale, M., Bottino, C., Sivori, S., Sanseverino, L., Castriconi, R., Marcenaro, E., Augugliaro, R., Moretta, L., and Moretta, A. (1998) NKp44, a novel triggering surface molecule specifically expressed by activated natural killer cells, is involved in non-major histocompatibility complexrestricted tumor cell lysis. J. Exp. Med., 187: 2065–2072.[tam]

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II. Antibodies to Mouse and Rat Natural Killer Cell Receptors The listed sources of antibodies are original authors, company, or hybridoma bank resource, ATCC (www.atcc.org).

Anti-Mouse Ly49 Mouse Ly49 receptors are expressed variably on subsets of NK cells and a small subset of T cells. Numerous separate genes have been identified in different mouse strains with distinct MHC class I binding specificities as designated below. It should be noted that the data summarized below have been obtained from direct binding studies, in vitro functional assays, or in vivo depletion studies. The results from these three types of assays do not always correspond. Individual references should be consulted to determine how a given specificity was defined. Antibodies to some isoforms have not been reported, and the cross-reactivities of most antibodies should be appreciated when typing Ly49 expression on NK cell clones.

Clone

Specificity

Species/Isotype

Source/Reference

A1

Ly49A (Dd , Dk , inhibitory) (specific for B6 but not BALB allele)

Mouse IgG2a

BD Pharmingen (1–4)

JR9-318

Ly49A (Dd , Dk , inhibitory)

Mouse IgG1

BD Pharmingen (5)

YE1/48

Ly49A (Dd , Dk , inhibitory)

Rat IgG2c

Biolegend (6, 7)

d

k

12A8

Ly49A (D , D , inhibitory) Ly49D (Dd, Ld , DSp2 , activating)

Rat IgG2a

BD Pharmingen, eBiosciences (8, 9)

4D11

Ly49A, weak (Dd , Dk , inhibitory) Ly49G2 (Dd, inhibitory)

Rat IgG2a

BD Pharmingen, ATCC, eBiosciences (10, 11)

4LO3311

Ly49C (Kb, Dd , Kd , inhibitory)

Mouse IgG3

S. Lemieux (12, 13)

5E6

Ly49C (Kb , Dd , Kd , inhibitory) Ly49I (Kb , H-2d , inhibitory)

Mouse IgG2a

BD Pharmingen (12, 14)

4E5

Ly49D (Dd, activating)

Rat IgG2a

BD Pharmingen, eBiosciences (15, 16)

YLI-90

Ly49I (Kb , H-2d , inhibitory)

Mouse IgG1

BD Pharmingen, eBiosciences

Ld ,

DSp2 ,

Anti-Mouse NKR-P1C (NK1.1, CD161, KLRB1C) NKR-P1C is found on most murine NK cells and a subset of T cells (including NKT cells) only in distinct strains of mice. NKR-P1C is expressed in the following strains of mice: C57BL, FVB/N, NZB, SJL, C57BR, and C57L (not in A, AKR, BALB/c, CBA/J, C3H, C58, DBA/1, DBA/2, or 129 strains).

Clone

Specificity

Species/Isotype

Source/Reference

PK136

Mouse NKR-P1C

Mouse IgG2a

ATCC, BD Pharmingen, Cedarlane, AbD Serotec, Southern Biotechnology Associates, Accurate, Invitrogen, eBiosciences, Santa Cruz Biotechnology, Biolegend (17, 18)

3H2788

Mouse NKR-P1C

Mouse IgG2a

Santa Cruz Biotechnology

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Appendix

Anti-Rat NKR-P1A (CD161) High-level expression of NKR-P1A is found on all rat NK cells and low-level expression is seen on most rat neutrophils, a subset of T cells, and reportedly on activated monocytes and a subset of dendritic cells.

Clone

Specificity

Species/Isotype

Source/Reference

3.2.3 10/78

Rat NKR-P1A Rat NKR-P1A

Mouse IgG1 Mouse IgG1

Thermo Scientific (19) Cedarlane, BD Pharmingen, Invitrogen, Accurate, AbD Serotec, Biolegend (20)

Anti-Mouse CD16 (Fcγ RIII) The transmembrane form of CD16 is expressed on mouse NK cells, macrophages, neutrophils, myeloid precursors, and a subset of thymocytes. As opposed to humans, no glycosylphosphatidylinositol (GPI)-linked form has been identified in mouse. It is important to note that the available antibodies also bind CD32 (Fc␥RII) on B cells and myeloid cells.

Clone

Specificity

Species/Isotype

Source/Reference

2.4G2

Mouse CD16/CD32

Rat IgG2b

BD Pharmingen, ATCC (21–23)

FCR4G8

Mouse CD16/CD32

Rat IgG2b

AbD Serotec

Mouse NKG2D (KLRK1)

Mouse NKG2D (KLRK1, blocking antibody)

Mouse NKG2D (KLRK1)

Mouse NKp46 (NCR1)

Mouse NKp46 (NCR1)

Mouse 2B4 (CD244)

Mouse CD49b (VLA-2), pan NK cell marker

All mouse and rat NK cells, some activated

1D11

CX5

C7

259018

29A1.4

2B4

DX5

Asialo GM1

Macrophages and some CD8+ T cells

Mouse NKG2D (KLRK1)

Mouse NKG2D (KLRK1)

Mouse NKG2A/C/E

14F-2

Mouse NKG2A (KLRC1)

16A11

20d5

191004

Rat IgG2a

Mouse CD94

18d3

Polyclonal rabbit IgG

Rat IgM

Mouse IgG2b

Rat IgG2a

Rat IgG2a

Armenian hamster IgG

Rat IgG1

Rat IgG

Rat IgG2b

Rat IgG2b

Rat IgG2a

Mouse IgG2b

Species/Isotype

Miscellaneous Anti-Mouse NK Cell Antibodies Clone Specificity

Note: As opposed to human, N-CAM is not expressed on mouse or rat NK cells.

Anti-CD56 (N-CAM)

AbD Serotec, Abcam, Novus

Accurate, Cedarlane, Wako

BD Pharmingen, Invitrogen, AbD Serotec, eBiosciences, Biolegend

BD Pharmingen, eBiosciences (17)

eBiosciences

R&D Systems

Biolegend, BioXCell, eBiosciences

Invitrogen, eBiosciences, Biolegend

Accurate, eBiosciences, Biolegend

Santa Cruz Biotechnology

R&D Systems

AbD Serotec, Accurate, eBiosciences

Santa Cruz Biotechnology, eBiosciences

AbD Serotec, Accurate, eBiosciences, Southern Biotechnology, Biolegend

Source/Reference

Appendix 527

528

Appendix

References 1. Nagasawa, R., J. Gross, O. Kanagawa, K. Townsend, L.L. Lanier, J. Chiller, and J.P. Allison (1987) Identification of a novel T cell surface disulfide-bonded dimer distinct from the ␣/␤ antigen receptor. J. Immunol., 138, 815–824. 2. Held, W., J. Roland, and D.H. Raulet (1995) Allelic exclusion of Ly49 family genes encoding class I-MHC-specific receptors on NK cells. Nature, 376, 355–358. 3. Karlhofer, F.M., R.K. Ribaudo, and W.M. Yokoyama (1992) MHC class I alloantigen specificity of Ly-49+ IL-2 activated natural killer cells. Nature, 358, 66–70. 4. Karlhofer, F.M., R. Hunziker, A. Reichlin, D.H. Margulies, and W.M. Yokoyama (1994) Host MHC class I molecules modulate in vivo expression of a NK cell receptor. J. Immunol., 153, 2407–2416. 5. Roland, J., and P.A. Cazenave (1992) Ly-49 antigen defines an alpha beta TCR population in i-IEL with an extrathymic maturation. Int. Immunol., 4, 699–706. 6. Brennan, J., G. Mahon, D.L. Mager, W.A. Jefferies, and F. Takei (1996) Recognition of class I major histocompatibility complex molecules by Ly49: specificities and domain interactions. J. Exp. Med., 183, 1553–1559. 7. Brennan, J., D. Mager, W. Jefferies, and F. Takei (1994) Expression of different members of the Ly-49 gene family defines distinct natural killer cell subsets and cell adhesion properties. J. Exp. Med., 180, 2287–2295. 8. Mason, L.H., S.K. Anderson, W.M. Yokoyama, H.R. Smith, P.R. Winkler, and J.R. Ortaldo (1996) The Ly-49D receptor activates murine natural killer cells. J. Exp. Med., 184, 2119–2128. 9. Raziuddin, A., A.L. Longo, L. Mason, J.R. Ortaldo, M. Bennett, and W.J. Murphy (1998) Differential effects of the rejection of bone marrow allografts by the depletion of activating versus inhibiting Ly-49 natural killer cell subsets. J. Immunol., 160, 87–94. 10. Salcedo, M., A.D. Diehl, A.M. Olsson, J. Sundback, K.L. Van, K. Karre, and H.G. Ljunggren (1997) Altered expression of Ly49 inhibitory receptors on natural killer. J. Immunol., 158, 3174–3180. 11. Mason, L.H., J.R. Ortaldo, H.A. Young, V. Kumar, M. Bennett, and S.K. Anderson (1995) Cloning and functional characteristics of murine LGL-1: a member of the Ly-49 gene family (Ly-49G2). J. Exp. Med., 182, 293–303.

12. Brennan, J., S. Lemieux, J.D. Freeman, D.L. Mager, and F. Takei (1996) Heterogeneity among Ly-49C natural killer (NK) cells: Characterization of highly related receptors with differing functions and expression patterns. J. Exp. Med., 184, 2085–2090. 13. Gosslin, P., Y. Lusignana, J. Brennan, F. Takei, and S. Lemieux (1997) The NK2.1 receptor is encoded by Ly49C and its expression is regulated by MHC class I alleles. Int. Immunol., 9, 533–540. 14. Stoneman, E.R., M. Bennett, J. An, K.A. Chesnut, E.K. Wakeland, J.B. Scheerer, M.J. Siciliano, V. Kumar, and P.A. Mathew (1995) Cloning and characterization of 5E6 (Ly49C), a receptor molecule expressed on a subset of murine natural killer cells. J. Exp. Med., 182, 305–313. 15. Mason, L.H., Willette-Brown, J., Anderson, S.K., Gosselin, P., Shores, E.W., Love, P.E., Ortaldo, J.R., and McVicar, D.W. (1998) Characterization of an associated 16-kDa tyrosine phosphoprotein required for Ly49D signal transduction. J. Immunol., 160, 4148–4152. 16. Ortaldo, J.R., R. Winkler-Pickett, A.T. Mason, and L.H. Mason (1998) The Ly49 family: Regulation of cytotoxicity and cytokine production in murine CD3+ cells. J. Immunol., 160, 1158–1165. 17. Sentman, C.L., J. Hackett, Jr., T.A. Moore, M.M. Tutt, M. Bennett, and V. Kumar (1989) Pan natural killer cell antibodies and their relationships to the NK1.1 antigen. Hybridoma, 8, 605–614. 18. Koo, G.C., and J.R. Peppard (1984) Establishment of monoclonal anti-Nk-1.1 antibody. Hybridoma, 3, 301–303. 19. Chambers, W.H., N.L. Vujanovic, A.B. DeLeo, M.W. Olszowy, R.B. Herberman, and J.C. Hiserodt (1989) Monoclonal antibody to a triggering structure expressed on rat natural killer cells and adherent lymphokineactivated killer cells. J. Exp. Med., 169, 1373–1389. 20. Kraus, E., D. Lambracht, K. Wonigeit, and T. H¨unig (1996) Negative regulation of rat natural killer cell activity by major histocompatibility complex class I recognition. Eur. J. Immunol., 26, 2582–2586. 21. Unkeless, J.C. (1979) Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J. Exp. Med. 150, 580–596.

Appendix 22. Kurlander, R.J., D.M. Ellison, and J. Hall, (1984) The blockade of Fc receptor-mediated clearance of immune complexes in vivo by a monoclonal antibody (2.4G2) directed against Fc receptors on murine leukocytes. J. Immunol., 133, 855–862.

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23. Perussia, B., M.M. Tutt, W.Q. Qiu, W.A. Kuziel, P.W. Tucker, G. Trinchieri, M. Bennett, J.V. Ravetch, and V. Kumar (1989) Murine natural killer cells express functional Fc␥ receptor II encoded by the Fc␥R␣ gene. J. Exp. Med., 170, 73–86.

530

Appendix

III. Transformed Natural Killer Cell Lines Human

KHYG-1 – IL-2-dependent NK-like cell line that is highly cytolytic, produces cytokines, lacks CD16 expression, and exhibits high spontaneous granule release, which may be due to constitutively polarized granules. This cell line expresses KIR3DL1 and KIR3DL2 on subsets of cells. Source: Health Science Research Resources Bank, Japan Health Sciences Foundation (www.jhsf.or.jp) Yagita, M., Huang, C.L., Umehara, H., Matsuo, Y., Tabata, R., Miyake, M., Konaka, Y., and Takatsuki, K. (2000) A novel natural killer cell line (KHYG-1) from a patient with aggressive natural killer cell leukemia carrying a p53 mutation. Leukemia. 14, 922–930. Suck, G., Branch, D.R., Smyth, M.J., Miller, R.G., Vergidis, J., Fahim, S., and Keating, A. (2005) KHYG-1, a model for the study of enhanced natural killer cell cytotoxicity. Exp. Hematol. 33, 1160–1171. Suck, G., Branch, D.R., Aravena, P., Mathieson, M., Helke, S., and Keating, A. (2006) Constitutively polarized granules prime KHYG-1 NK cells. Int. Immunol. 18, 1347–1354. NKL – An IL-2-dependent neoplastic NK-like cell line that is weakly cytolytic. Expresses ILT2, which is an inhibitory receptor that binds HLA-A, -B, and –G. Source: original authors Robertson, M. J., Cochran, K. J., Cameron, C., Le, J. M., Tantravahi, R., and Ritz, J. (1996) Characterization of a cell line, NKL, derived from an aggressive human natural killer cell leukemia. Exp. Hematol., 24, 406–415. NK3.3 – IL-2-dependent NK-like cell line that exhibits natural killing activity. Source: original authors Kornbluth, J., Spear, B., Raab, S.S., and Wilson, D.B. (1985) Evidence for the role of class I and class II HLA antigens in the lytic function of a cloned line of natural killer cells. J. Immunol., 134, 728–735. Kornbluth, J., Flomenberg, N., and Dupont, B. (1982) Cell surface phenotype of a cloned line of human natural killer cells. J. Immunol.,129, 2831–2837. NK-92 – An IL-2-dependent human NK-like cell line that is highly cytolytic, produces cytokines, and lacks CD16 expression. Source: ATCC (www.atcc.org) and DSMZ (www.dsmz.de) Gong, J. H., Maki, G., and Klingemann, H. G. (1994) Characterization of a human cell line (NK-92) with phenotypical

Appendix

531

and functional characteristics of natural killer cells. Leukemia, 8, 652–658. Maki, G., Klingemann, H. -G., Martinson, J. A., and Tam, Y. K. (2001) Factors regulating the cytotoxic activity of the human natural killer cell line, NK-92. J. Hematol. Stem Cell Res., 10, 369–383. YT – An IL-2-independent human NK-like cell line that mediates weak cytotoxicity through CD28 expression. It has come to our attention that many variants of this line exist and some exhibit natural cytotoxicity toward targets that are not normally killed by NK cells, while others do not express typical NK cell markers. One should characterize the line to determine its phenotype before use in functional and biochemical studies. Source: DSMZ (www.dsmz.de) Yodoi, J., Teshigawara, K., Nikaido, T., Fukui, K., Noma, T., Honjo, T., Takigawa, M., Sasaki, M., Minato, N., and Tsudo, M. (1985) TCGF (IL 2)-receptor inducing factor(s). I. Regulation of IL 2 receptor on a natural killer-like cell line (YT cells). J. Immunol., 134, 1623–1630. Azuma, M., Cayabyab, M., Buck, D., Phillips, J.H., and Lanier, L.L. (1992) Involvement of CD28 in MHC-unrestricted cytotoxicity mediated by a human natural killer leukemia cell line. J. Immunol.,149, 1115–1123. Rat

RNK-16 – An IL-2-independent spontaneous leukemic cell line from F344 rats which exhibits NK cell characteristics. The original line is IL-2 dependent, but many subclones currently available are IL-2 independent. Ward, J. M., and Reynolds, C. W. (1983) Large granular lymphocyte leukemia. A heterogeneous lymphocytic leukemia in F344 rats. Am. J. Pathol., 111, 1–10. Source: original authors.

532

Appendix

IV. Natural Killer Cell Target Lines Human

771.221 – MHC class I-deficient human EBV-transformed B lymphoblastoid line. These cells do not express endogenous HLA-A, HLA-B, or HLA-C class I antigens due to gamma rayinduced mutations in the HLA complex. This line (and HLA transfectants) is commonly used to measure spontaneous (natural) cytotoxicity by human NK cells. Shimizu, Y., and DeMars, R. (1989) Production of human cells expressing individual transferred HLA-A,-B,-C genes using an HLA-A,-B,-C null human cell line. J. Immunol., 142, 3320–3328. Shimizu, Y., Geraghty, D.E., Koller, B.H., Orr, H.T., and DeMars, R. (1988) Transfer and expression of three cloned human non-HLA-A, B, C class I major histocompatibility complex genes in mutant lymphoblastoid cells. Proc. Natl. Acad. Sci. USA, 85, 227–231. Source: R. DeMars and a variety of labs that have generated HLA-A, B, C transfectants. K562 – A human chronic myelogenous leukemia cell line. Classical target cell for spontaneous (granule exocytosis-mediated, Ca2+ dependent) cytotoxicity. Lozzio, C.B., and Lozzio, B.B. (1975) Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood, 45, 321–334. Ortaldo, J.R., Oldham, R.K., Cannon, G.C., and Herberman, R.B. (1977) Specificity of natural cytotoxic reactivity of normal human lymphocytes against a myeloid leukemia cell line. J. Natl. Cancer Inst. (Bethesda), 59, 77–83. Source: ATCC. C1R – MHC class I-deficient ␥-irradiated variant of the human EBV-transformed Licr.Lon.Hmy2 B cell line. The cells express HLA-Cw4 and a low level of a mutant form of HLA-B35, designated B∗ 3503. This line (and HLA transfectants) is commonly used to measure spontaneous (natural) cytotoxicity by human NK cells. Storkus, W.J., Howell, D.N., Salter, R.D., Dawson, J.R., and Cresswell, P. (1987) NK susceptibility varies inversely with target cell class I HLA antigen expression. J. Immunol., 138, 1657–1659. Zemmour, J., Little, A.M., Schendel, D.J., and Parham, P.J. (1992) The HLA-A,B “negative” mutant cell line C1R expresses a novel HLA-B35 allele, which also has a point

Appendix

533

mutation in the translation initiation codon. J. Immunol., 148, 1941–1948. Source: ATCC. Daudi – Human Burkitt’s B-cell lymphoma line. This line expresses the NKG2D ligand, ULBP1. Klein, E., Klein, G., Nadkarni, J.S., Nadkarni, J.J., Wigzell, H., and Clifford, P. (1968) Surface IgM-kappa specificity on a Burkitt lymphoma cell in vivo and in derived culture lines. Cancer Res., 28, 1300–1310. Source: ATCC. THP-1 – An acute monocytic leukemia cell line that expresses both Fc␥RI and Fc␥RII, as well as the complement (C3b) receptor. The cells grow in suspension but are somewhat “sticky.” It can be used in redirected ADCC cytotoxicity assays. Importantly, this cell line is also insensitive to spontaneous (natural) cytotoxicity by IL-2-activated NK cells and clones. Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., and Tada, K. (1980) Establishment and characterization of a human acute monocytic leukemia cell line (THP1). Int. J. Cancer, 26: 171–176. Source: ATCC. RDMC – A human rhabdomyosarcoma, which is likely to be the same as RD. This cell line is adherent and can be used for redirected ADCC cytotoxicity assays using very low E:T ratios (i.e., also sensitive to spontaneous cytotoxicity). McAllister, R.M., Melnyk, J., Finkelstein, J.Z., Adams, E.C., Jr., and Gardner, M.B. (1969) Cultivation in vitro of cells derived from a human rhabdomyosarcoma. Cancer, 24: 520–526. Source: ATCC. Jurkat – A human CD3+ /CD4+ Fas (CD95)+ T-lymphoid cell line sensitive to spontaneous cytotoxicity by NK cells, both granule exocytosis- and Fas (CD95) mediated. Gillis, S., and Watson, J. (1980) Biochemical and biological characterization of lymphocyte regulatory molecules. V. Identification of an interleukin 2-producing human leukemia T cell line. J. Exp. Med. 152:1709–1719. Source: ATCC. Mouse

YAC-1 – Mouse lymphoma induced by Moloney leukemia virus (MLV) in A/Sn mouse. Classical target cell for spontaneous cytotoxicity. Evidence by Petersson et al. suggests that low MHC Class I expression by YAC-1 grown in vitro is due to high constitutive IL-10 production by the cell line. YAC-1 expresses the NKG2D ligand, RAE-1, and engagement of this receptor contributes significantly to NK cell-mediated cytotoxicity. Cikes, M., Friberg, S., Jr., and Klein, G. (1973) Progressive loss of H-2 antigens with concomitant increase of cell-surface

534

Appendix

antigen(s) determined by Moloney leukemia virus in cultured murine lymphomas. J. Natl. Cancer Inst. (Bethesda), 50, 347–362. Kiessling, R., Klein, E., and Wigzell, H. (1975) “Natural” killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Eur. J. Immunol.,5, 112–117. Petersson, M., Charo, J., Salazar-Onfray, F., Noffz, G., Mohaupt, M., Qin, Z., Klein, G., Blankenstein, T., and Kiessling, R. (1998) Constitutive IL-10 Production Accounts for the High NK Sensitivity, Low MHC Class I Expression, and Poor Transporter Associated with Antigen Processing (TAP)-1/2 Function in the Prototype NK Target YAC-1. J. Immunol., 161, 2099–2105. Source: ATCC. P815 – DBA/2 murine mastocytoma has been used as a target cell for cytotoxic assays. Clone P815-X2 is Fc␥RII/III negative, while clone P815␥ is Fc␥RII/III positive. P815␥ can be used in redirected cytotoxicity assays, provided low E:T ratios are used. It is also sensitive to spontaneous (natural) killing by IL-2-activated human NK cells. This cell line expresses H-2d . Lundak, R.L., and Raidt, D.J. (1973) Cellular immune response against tumor cells. I. In vitro immunization of allogeneic and syngeneic mouse spleen cell suspensions against DBA mastocytoma cells. Cell. Immunol., 9, 60–66. Plaut, M., Lichtenstein, L.M., Gillespie, E., and Henney, C.S. (1973) Studies on the mechanism of lymphocyte-mediated cytolysis. IV. Specificity of the histamine receptor on T cells. J. Immunol., 111, 389–394. Ralph, P., and Nakoinz, I. (1977) Antibody-dependent killing of erythrocyte and tumor targets by macrophage-related cell lines: enhancement by PPD and LPS. J. Immunol., 119, 950–954. Source: ATCC. RMA-S – A mutant of the murine RMA lymphoma line which has a defect in the TAP-2 transporter, resulting in the expression of only 5–10% of the wild-type H-2Db , Kb , and ␤2-microglobulin molecules. The RMA line expresses higher levels of these MHC class I molecules. Ohlen, C., Bastin, J., Ljunggren, H.-G., Foster, L., Wolpert, E., Klein, G., Townsend, A.R., and Karre, K. (1990) Resistance to H-2-restricted but not to allo-H2-specific graft and cytotoxic T lymphocyte responses in lymphoma mutant. J. Immunol. 145, 52–58. Franksson, L., George, E., Powis, S., Butcher, G., Howard, J., and Karre, K. (1993) Tumorigenicity conferred

Appendix

535

to lymphoma mutant by major histocompatibility complex-encoded transporter gene. J. Exp. Med. 177, 201–205. L1210 – A murine lymphocytic leukemia of DBA/2 origin that grows in suspension. It can be used, like P815, for redirected ADCC by human NK cells. Moore, G.E., Sandberg, A.A., and Ulrich, K. (1967) Suspension cell culture and in vivo and in vitro chromosome constitution of mouse leukemia L1210. J. Natl. Cancer Inst., 36: 405–421. Source: ATCC. L4 – Mouse T lymphoma induced in a C57BL/6 N mouse by 9,10-dimethyl-1,2-benzanthracene. This cell line expresses H-2b . It is important to note that this line also expresses Ly49A. Herberman, R.B. (1972) Serological analysis of cell surface antigens of tumors induced by murine leukemia virus. J. Natl. Cancer Inst. (Bethesda), 48, 265–271. Shevach, E.M., Stobo, J.D., and Green, I. (1972) Immunoglobulin and theta-bearing murine leukemias and lymphomas. J. Immunol., 108, 1146–1151. Source: ATCC. Rat

YB2/0 – Rat myeloma clone derived from the hybrid myeloma YB2/3HL as selected for the absence of Ig secretion. Kilmartin, J.V., Wright, B., and Milstein, C. (1982) Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J. Cell Biol., 93, 576–582. Source: ATCC.

536

Appendix

V. Killer Cell Immunoglobulin-Like Receptor (KIR) Nomenclaturea,b Alternative names (and closely related sequences)c

Receptor

HLA Specificity

Mass (kDa) CD designation

KIR2DL1

Cw2, Cw4, Cw5, Cw6

58

CD158a

NKAT1, cl-47-11, KAR-K6e, p58.1

KIR2DL2

Cw1, Cw3, Cw7, Cw8

58

CD158b1

NKAT6, cl-43

KIR2DL3

Cw1, Cw3, Cw7, Cw8

58

CD158b2

NKAT2, cl-6, KIR-023 GB, KAR-K7e, p58.2

KIR2DL4

Soluble G

49

CD158d

KIR-103AS, KIR-103LP, 15.212, NK3.3#27

KIR2DL5

Unknown

60

CD158f

KIR3DL1

Bw4

70

CD158e1

NKAT3, NKB1, AMB11, cl11, cl-2

KIR3DL2

A3, A11

70/140

CD158k

cl-5, AMC5, NKAT4, cl-1.1, 171c, 8-11c

KIR3DL3

Unknown

65

CD158z

KIR3DL7, KIR44, KIRC1

KIR2DS1

Cw2, Cw4, Cw5, Cw6

50

CD158h

EB6act1, EB6act2, p50.1

KIR2DS2

Cw1, Cw3, Cw7, Cw8

50

CD158j

NKAT5, cl-49, GL183act1, p50.2

KIR2DS3

Unknown

50

KIR2DS4

Cw3

50

CD158i

NKAT8, cl-39, cl-17, KARK1e, KKA3, p50.3

KIR2DS5

Unknown

50

CD158g

NKAT9

KIR3DS1

Unknown

60

CD158e2

NKAT10, KIR-123FM, C97.12#5, KIR-G1

cl-42,

NKAT7, 59C/K3

a KIR was originally adopted as an acronym for “killer cell inhibitory receptors, but subsequent studies confirmed that truncated forms of this receptor family were not inhibitory in function. In some reports, these truncated receptors have also been termed “KAR” for killer cell activating receptors. To avoid renaming the receptors entirely, the acronym KIR was adopted to denote killer cell immunoglobulin-like receptors by several investigators in the field. This nomenclature has become a standard. The nomenclature for individual receptors within the KIR family have been designated as “2D” or “3D” according to their number of extracellular immunoglobulin-like domains, which is followed by the letter “L” or “S” for long or short (truncated) cytoplasmic domain, respectively, and a definitive number for that specific receptor within each subgroup. b It should be noted that the diversity of KIR sequences may be much more complex than that presented in this table, due to numerous minor sequence polymorphisms that have been identified in cDNAs from individuals within the population. For an excellent alignment of many of these sequences, see www.ebi.ac.uk/ipd/kir/ c Alternative names are those originally designated by different investigators that separately cloned the receptors.

Appendix

537

VI. Inhibitory KIR Ligands Each inhibitory KIR recognizes a subset of HLA-A, -B, or -C molecules as ligands. KIR2DL1 recognizes group 1 HLA-C (possessing Ser at position 77 and Asn at position 80), while KIR2DL2 and KIR2DL3 recognize group 2 HLA-C (Asn at position 77 and Lys at position 80). KIR3DL1 engages with HLA-Bw4 alleles (Thr or Ile at position 80) and some HLA-A alleles that share the Bw4 characteristics but does not recognize HLA-Bw6 alleles (Asn at position 80). KIR3DL2 binds to HLA-A3 and HLA-A11 alleles. The following table lists the alleles within the HLA-B and HLA-C subgroups and some amino acid (AA) variations at position 80 for HLA-B. This table was generously provided by Dr. Mary Carrington (NCI-Frederick, Frederick, MD).

Bw4 (KIR3DL1)

AA80

Bw6

AA80

GROUP 1 HLA-C (KIR2DL2/KIR2DL3)

GROUP 2 HLA-C (KIR2DL1)

B0727

T

B0702

N

0102

0202

B0802

T

B0703

N

0103

0203

B0803

I

B0704

N

0104

0204

B1301

T

B0705

N

0105

0205

B1302

T

B0706

N

0302

0307

B1303

T

B0707

N

0303

0401

B1304

T

B0708

N

0304

0402

B1306

T

B0709

N

0305

0403

B1308

T

B0710

N

0306

0404

B1513

I

B0711

N

0308

0405

B1516

I

B0712

N

0309

0406

B1517

I

B0713

N

0311

0407

B1523

I

B0714

N

0312

0408

B1524

I

B0715

N

0313

0409

B1536

T

B0716

N

0314

0501

B1543

T

B0717

N

0701

0502

B1567

I

B0718

N

0702

0503

B1809

T

B0719

N

0703

0504

B2701

T

B0720

N

0704

0602

B2702

I

B0721

N

0705

0603

B2703

T

B0722

N

0706

0604

B2704

T

B0723

N

0708

0605

B2705

T

B0724

N

0710

0606

B2706

T

B0725

N

0711

0607

B2707

T

B0726

N

0712

0707

B2709

T

B0728

N

0713

0709

B2710

T

B0801

N

0714

1204

B2711

T

B0804

N

0715

1205

B2713

T

B0805

N

0801

1502

538

Appendix

(continued) Bw4 (KIR3DL1)

AA80

Bw6

AA80

GROUP 1 HLA-C (KIR2DL2/KIR2DL3)

GROUP 2 HLA-C (KIR2DL1)

B2714

T

B0806

N

0802

1503

B2715

T

B0807

N

0803

1504

B2716

T

B0808

N

0804

1505

B2717

T

B0809

N

0805

1506

B2719

T

B0810

N

0806

1508

B2720

T

B0811

N

0807

1509

B2721

T

B0812

N

0808

1510

B2722

T

B0813

N

0809

1511

B2723

T

B0814

N

1202

1602

B2724

T

B1309

N

1203

1701

B2725

T

B1401

N

1206

1702

B3701

T

B1402

N

1208

1703

B3702

T

B1403

N

1301

1801

B3703

T

B1404

N

1402

1802

B3704

T

B1405

N

1403

B3801

I

B1406

N

1405

B3802

T

B1501

N

1507

B3803

T

B1502

N

1601

B3804

T

B1503

N

1604

B3805

I

B1504

N

B3806

I

B1505

N

B3807

I

B1506

N

B3808

T

B1507

N

B4013

I

B1508

N

B4019

I

B1509

N

B4402

T

B1510

N

B4403

T

B1511

N

B4404

T

B1512

N

B4405

T

B1514

N

B4406

I

B1515

N

B4407

T

B1518

N

B4408

T

B1519

N

B4410

T

B1520

N

B4411

T

B1521

N

B4412

T

B1522

N

B4413

T

B1525

N

B4414

T

B1526

N

Appendix

539

(continued) Bw4 (KIR3DL1)

AA80

Bw6

AA80

B4415

T

B1527

N

B4416

T

B1528

N

B4417

T

B1529

N

B4418

I

B1530

N

B4420

T

B1531

N

B4421

T

B1532

N

B4422

T

B1533

N

B4423

T

B1534

N

B4424

T

B1535

N

B4425

I

B1537

N

B4426

T

B1538

N

B4427

T

B1539

N

B4428

T

B1540

N

B4429

T

B1542

N

B4430

T

B1544

N

B4701

T

B1545

N

B4704

T

B1546

N

B4901

I

B1547

N

B4902

T

B1548

N

B4903

I

B1549

N

B5101

I

B1550

N

B5102

I

B1551

N

B5103

I

B1552

N

B5104

I

B1553

N

B5105

I

B1554

N

B5106

I

B1555

N

B5107

I

B1556

N

B5108

I

B1557

N

B5109

I

B1558

N

B5110

I

B1559

N

B5111

I

B1560

N

B5112

I

B1561

N

B5113

I

B1562

N

B5114

I

B1563

N

B5115

I

B1564

N

B5116

I

B1565

N

B5117

I

B1566

N

GROUP 1 HLA-C (KIR2DL2/KIR2DL3)

GROUP 2 HLA-C (KIR2DL1)

540

Appendix

(continued) Bw4 (KIR3DL1)

AA80

Bw6

AA80

B5118

I

B1568

N

B5119

I

B1569

N

B5120

I

B1801

N

B5121

I

B1802

N

B5122

I

B1803

N

B5123

I

B1804

N

B5124

I

B1805

N

B5126

I

B1806

N

B5127

I

B1807

N

B5201

I

B1808

N

B5202

I

B1810

N

B5203

I

B1811

N

B5301

I

B1812

N

B5302

I

B1813

N

B5303

T

B1814

N

B5304

I

B1815

N

B5305

I

B2708

N

B5306

I

B2712

N

B5307

I

B2718

N

B5308

I

B3501

N

B5309

T

B3502

N

B5607

T

B3503

N

B5701

I

B3504

N

B5702

I

B3505

N

B5703

I

B3506

N

B5704

I

B3507

N

B5705

I

B3508

N

B5706

I

B3509

N

B5707

I

B3510

N

B5708

I

B3511

N

B5709

I

B3512

N

B5801

I

B3513

N

B5802

I

B3514

N

B5804

I

B3515

N

B5805

I

B3516

N

B5806

I

B3517

N

B5901

I

B3518

N

GROUP 1 HLA-C (KIR2DL2/KIR2DL3)

GROUP 2 HLA-C (KIR2DL1)

Appendix

(continued) Bw4 (KIR3DL1)

AA80

Bw6

AA80

A2301

I

B3519

N

A2302

I

B3520

N

A2303

I

B3521

N

A2304

I

B3522

N

A2305

I

B3523

N

A2306

I

B3524

N

A2307

I

B3525

N

A2308

I

B3526

N

A2402

I

B3527

N

A2403

I

B3528

N

A2405

I

B3529

N

A2406

I

B3530

N

A2407

I

B3531

N

A2408

I

B3532

N

A2409

I

B3533

N

A2410

I

B3534

N

A2411

I

B3535

N

A2413

I

B3536

N

A2414

I

B3537

N

A2415

I

B3538

N

A2416

I

B3539

N

A2417

I

B3705

N

A2418

I

B3901

N

A2420

I

B3902

N

A2421

I

B3903

N

A2422

I

B3904

N

A2423

I

B3905

N

A2424

I

B3906

N

A2425

I

B3907

N

A2426

I

B3908

N

A2427

I

B3909

N

A2429

I

B3910

N

A2430

I

B3911

N

A2431

I

B3912

N

A2432

I

B3913

N

A2433

I

B3914

N

A2501

I

B3915

N

GROUP 1 HLA-C (KIR2DL2/KIR2DL3)

GROUP 2 HLA-C (KIR2DL1)

541

542

Appendix

(continued) Bw4 (KIR3DL1)

AA80

Bw6

AA80

A2502

I

B3916

N

A2503

I

B3917

N

A2504

I

B3918

N

A3201

I

B3919

N

A3202

I

B3920

N

A3203

I

B3922

N

A3204

I

B3923

N

A3205

I

B3924

N

A3206

I

B3925

N

A3207

I

B3926

N

B4001

N

B4002

N

B4003

N

B4004

N

B4005

N

B4006

N

B4007

N

B4008

N

B4009

N

B4010

N

B4011

N

B4012

N

B4014

N

B4015

N

B4016

N

B4018

N

B4020

N

B4021

N

B4023

N

B4024

N

B4025

N

B4026

N

B4027

N

B4028

N

B4029

N

B4030

N

B4031

N

GROUP 1 HLA-C (KIR2DL2/KIR2DL3)

GROUP 2 HLA-C (KIR2DL1)

Appendix

543

(continued) Bw4 (KIR3DL1)

AA80

Bw6

AA80

B4032

N

B4033

N

B4034

N

B4035

N

B4036

N

B4037

N

B4038

N

B4039

N

B4040

N

B4042

N

B4101

N

B4102

N

B4103

N

B4104

N

B4105

K

B4106

N

B4201

N

B4202

N

B4204

N

B4409

N

B4501

N

B4502

N

B4503

N

B4504

N

B4505

N

B4601

N

B4602

N

B4702

N

B4801

N

B4802

N

B4803

N

B4804

N

B4805

N

B4806

N

B4807

N

B5001

N

B5002

N

GROUP 1 HLA-C (KIR2DL2/KIR2DL3)

GROUP 2 HLA-C (KIR2DL1)

544

Appendix

(continued) Bw4 (KIR3DL1)

AA80

Bw6

AA80

B5004

N

B5401

N

B5402

N

B5501

N

B5502

N

B5503

N

B5504

N

B5505

N

B5507

N

B5508

N

B5509

N

B5510

N

B5511

N

B5601

N

B5602

N

B5603

N

B5604

N

B5605

N

B5606

N

B6701

N

B6702

N

B7301

N

B7801

N

B7802

N

B7803

N

B7804

N

B7805

N

B8101

N

B8201

N

B8202

N

B8301

N

GROUP 1 HLA-C (KIR2DL2/KIR2DL3)

GROUP 2 HLA-C (KIR2DL1)

INDEX

2B4 (CD244) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67, 522, 527 721.221 cells (target cell line, human) . . . . . . . . . . . . . . . . . 92 293FT cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214, 218 3’-RACE (rapid amplification of cDNA ends) . . . . 383, 389 5’-RACE (rapid amplification of cDNA ends) . . . 378, 382, 384–385 3T3 cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .215, 293 293T cells (or HEK-293T or HEK293) . . . . 219, 235–239, 243, 275–282, 295–296

A A9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414–415 Acid stripping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320–322 Actin . . . . . . . . . . . . . . . . . . . . . . . . . 71, 73, 131, 133, 139–142 Activation marker . . . . . . . . . . . . . . . . . . . . 115–116, 430–431 Adenovirus (adenoviral) . . . . . . . . . . . . . . . 201, 206, 210, 293 Adhesion . . . . . 68–69, 89–90, 220, 317–319, 322, 339, 522 Adoptive transfer (of lymphocytes) . . . . . 114–115, 420–421 Altered self . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 Amphotropic retrovirus . . . . . . . . . . . . . . . . . . . . . . . . 129, 200 Analysis of variance (ANOVA) . . . . . . . . . . . . . . . . . . . . . . 403 Antibody-dependent cellular cytotoxicity (ADCC). . . . .68, 78, 309, 533, 535 Antibody titration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 Antisense transcripts . . . . . . . . . . . . . . . . . . . . . . . . . . . 381–383 Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149, 245, 394 Automated magnetic cell sorter (AutoMACS) . . . 244, 251, 254, 270

B Backcrossing mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Bacterial artificial chromosome (BAC). . 108, 110, 268, 502 Bactrim (Trimethoprim-Sulfoxaxole) . . . . . . . . . . . . . . . . . . 53 B cells . . . . . . . . . . . . . . . . . . 3–7, 30, 101, 137, 326, 458, 526 Bicistronic expression vector . . . . . . . . . . . . . . . . . . . . . . . . 200 Bi-directional promoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 Blasticidin . . 71, 84, 145, 180, 183, 191, 194, 219, 287, 293 Blastocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 β2m (β2 microglobulin) . . . . . . . . . . . . . . . . . . . . . . . . . 74, 534 Bone marrow (BM) . . . . . . . . . . . . . . . . . . . . . . 52, 54, 98, 103 Bone marrow chimeras (bone marrow chimeric mice) . . 115 Bone marrow-derived DC . . . . . . . . . . . . . . . . . . . . . . 103–104 Bone marrow-derived macrophages . . . . . . . . . 103–104, 249 Boolean analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345, 359 BOSC cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265–266 Brefeldin A . . . . . . . . . . . 41, 43, 47, 100, 116, 162, 168–169, 339, 349, 418, 437 Bromodeoxyuridine (BrdU). . . . . . . . . . . . . . . . . . . . .418–419 BSC-1 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414, 416–417 BSL2 (biological safety level 2) . . . . 111–112, 200, 205, 230 BSL3 (biological safety level 3) . . . . . . . . . . . . . . . . . 414, 435 BW cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252, 260–261, 271 BW zeta assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252, 258–262

C C1498 (NKT-like cell line, mouse) . . . . . 314, 316–320, 322 Calcein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Calcium signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149–157 CD3ζ (CD3zeta) . . . . . . . . . . . . 258–260, 286, 288–289, 292 CD7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 CD11b (Mac-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 CD11c . . . . . . . . . . . . . 99, 101, 108–110, 114–115, 121–122 CD16 (FcγRIII) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526 CD25 (IL-2Rα) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430 CD27 . . . . . . . . . . . . . . . 28–29, 33–34, 36–37, 419, 477, 507 CD28 . . . . . . . . . . . . . . . . . . . . . . . . . . . 68, 138, 178, 275, 531 CD34 . . . . . . . . . . . . . . . . . 1–3, 8–10, 14, 16–19, 20–25, 244 CD40 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103, 106–107, 111 CD45. . . . . . . . . . . .1–2, 53, 57, 61–62, 64–65, 99, 101, 420 CD48 . . . . . . . . . . . . . . . 68, 84, 178, 181–182, 187–190, 195 CD56bright , CD56high . . . . . . . . 17, 27, 344, 431, 448, 467 CD56dim , CD56low . . . . . . . . . . . 17, 27, 34, 343–345, 351, 357–360, 431, 456 CD56 (NCAM) . . . . . . . . . . . . . . . . . . . . . . . . . . . 72, 337, 354 CD63 (LAMP-3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 CD69 . . . . . . . . 102, 115–116, 430–431, 433, 438, 440–441 CD94 . . . . . . . . . . . . . . . . . . . 2–3, 9–10, 14, 16, 68, 199, 419, 430–431, 520, 527 CD94/NKG2 receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431 CD107a (LAMP-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 CD107b (LAMP-2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 CD117 (c-kit) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506 CD122 (IL-2Rβ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506 CD127 (IL-7Rα) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506–507 CD161 (NKR-P1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521 cDNA library . . . . . . . . . . . . . . . 286–287, 289–292, 294–295 CellQuest software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 CellTracker . . . . . . . . . . . . . . . . . . . 72, 79–81, 85–86, 93, 327 CFSE, Carboxyfluorescein diacetate succinimidyl ester . . . . . . . . . . . . . 139, 315, 318–319, 326–329, 331–332, 413, 420, 424, 426 Chemokines . . . . . . . . . 28, 89, 209, 335–336, 339, 360, 365 Chimera chimera founder mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 chimeric receptor. . . . . . . . . . . . . . . . . . . . . . . . . . .286, 292 Chloramphenicol acetyltransferase (CAT) reporter . . . . 378 Chlorophenol red galactoside (CPRG) . . . . . . . . . . 288–289 Chloroquine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235, 238, 442 Chorionic villi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448 Chromium-51 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Chromium release assay . . . . . . . . . . . . . . . . . . . . . . . . . 94, 119 Cis interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 Collagenase . . . . . . 29–31, 36, 101, 105, 450, 453, 459, 511 Colocalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141–144, 147 Commensal bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506 Compensation for flow cytometry . . . . . . . . . . . . . . . 350, 358 Congenic mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53, 61, 99

545

NATURAL KILLER CELL PROTOCOLS

546 Index

Conjugation (conjugates) . . . . . . . . . . . . . . 78–80, 89, 90–94, 130–131, 139, 146, 318, 322, 350, 355 COS-7 cells . . . . . . . . . . . . . . . . . . . . . . . . . 251, 255, 256–258 CpG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103, 111 Cre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107–110, 268 Cryptopatches (CP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506 Crystal violet solution . . . . . . . . . . . . . . . . . . . . . 215, 413, 417 C-type lectin . . . . . . . . . . . . . . . . . . . . . . . . . . 40, 299, 313, 394 Cytomegalovirus (CMV) . . . . 193, 244, 300, 393, 411, 430 Cytotoxicity assay (killing assay) spontaneous release . . . . . . . . . . . . . 81–82, 119, 423, 470 total release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

D DAP12 . . . . . . . . . . . 177–178, 394, 398, 400, 405, 407, 520 DAPI . . . . . . . . 142–143, 496–497, 501, 509, 512–513, 515 Dead cell exclusion markers . . . . . . . . . . . . . . . . . . . . 350, 354 Decidua . . . . . . . . . . 448–449, 452–453, 458, 466–467, 469, 491–493, 501 Decidualization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467, 498 Decidual stromal cells . . . . . . . . . . . . . . . . . 449, 454, 456, 460 Degranulation . . . . . . 69, 72, 78, 86, 89, 336–337, 339–340, 343–345, 346–349 Dendritic cells (DC) . . . . . . . . 4, 28, 98, 103, 106, 108–114, 121–122, 335, 429, 506, 526 DERL7 (NK-like and T-like cell line, human) . . . 211–212, 216–217, 220 Diphtheria toxin (DT) . . . . . . . 98, 101, 107–111, 113, 115, 121–122, 383, 389, 489 Diphtheria toxin receptor (DTR) . . . . . . . . 98–99, 107–110, 114–115, 122 DNAM-1 (CD226) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67–68 DNA quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 Dolichos biflorus (DBA) lectin . . . . . . . . . . . . . . . . . . . . . . . . 469 Drosophila melanogaster . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 DX5 (CD49b) . . . 37, 42, 48, 102, 106, 151, 164–165, 244, 419, 477, 496–497, 501–502, 527

E Ecotropic retrovirus . . . . . . . . . . . . . . . . . . . . . . . 129, 200, 266 Ectromelia virus (ECTV) footpad infection . . . . . . . . . . . . . . . . . . . . . . 412, 417–418 preparation of stocks . . . . . . . . . . . . . . . . . . . . . . . 415–416 purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416 quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416–417 resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417 Effector cell . . . 78–80, 82, 91, 94, 119–120, 130, 178–179, 181, 185, 187, 190, 195, 261–262, 271, 306–308, 339, 422, 340–343, 470, 505, 533–534 Enhanced green fluorescent protein (EGFP or GFP) . . 25, 93, 108, 200, 204, 218–219, 235, 239, 242, 300, 304–306, 308 EL4 (target cells, mouse) . . . . . 178–183, 185–190, 193–195 Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 Endometrial (endometrium) . . . . . . . . . . . 467, 469, 498–499 Endothelial cells . . . . . . . . . . . . . . . . . . . . 4, 12, 448, 454, 486 Enhanced yellow fluorescent protein (EYFP or YFP) . 217, 314, 318–320, 322 env (envelope) . . . . . . . . . . . . . . 201, 210, 230, 234–235, 296 Enzyme-linked immunosorbent assay (ELISA) . . 179–181, 184–187, 194, 239, 271, 456 Erythrocyte (red blood cell; RBC) lysis . . . . . . . . . . 433, 436

ES (embryonic stem) cells . . . . . . . . . . . . . . . . . . . . . . 267–269 Estradiol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451, 456 Europium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . 345, 347, 532–533 Expression cloning . . . . . . . . . . 285–287, 291–292, 294, 405 Extravillous trophoblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448

F False positive/negative amplification . . . . . . . . 229–230, 373 Fas ligand (CD178) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 Fcγ receptor blockade (2.4G2 antibody), Fc receptor blocking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 Fc . . . 3, 7, 29, 32, 42, 44, 68, 101, 116–118, 163, 166, 263, 275–282, 347, 431, 509, 533–534 Fc receptors. . . . . . . . .32, 116–118, 166, 169, 263, 340, 420 Feeder cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253–255 Femur . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30–31, 103 Fetus . . . . . . . . . . . . . . . . . . . . . . . . . . . 466–467, 479, 491, 497 Fibronectin . . . . . 18, 64, 220, 452, 457, 459, 461–462, 476, 494–495 Ficoll (Ficoll-Paque), Histopaque . . . . . . . . . . . . . . 2, 28, 30, 250, 253 Fixation of cells formaldehyde . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42, 354 glutaraldehyde . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471 methanol . . . . . . . . . . . . . . . . . . . . . . . . . 161, 167, 172–173 paraformaldehyde . . . . . . . . . . . . . 90, 173, 337, 413, 478 Flow cytometry cell sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160, 242 FACS (fluorescence-activated cell sorting) . . . . . . 10, 33, 41, 44, 63, 77, 167, 169, 421 flow cytometer . . . . . . . . . . . . . 10, 33, 36, 44, 48, 80, 83, 90, 119, 315, 339, 343, 421, 496, 514 recombinant receptor. . . . . . . . . . . . . . . . . . . . . . . . . . . .281 FlowJo software . . . . . . . . . . . . . . . . . . . . . . 54, 151, 153, 338, 344, 355, 496 Flt3-ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Fluorescent proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 137–138, 145, 217–219 Forward genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394 F(ab’)2 fragment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302 Framework genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374 F344 rat strain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

G gag . . . . . . . . . . . . 53, 55, 201, 210, 214, 219, 287, 289, 291, 293–294, 296, 405 Gene targeting. . . . . . . . . . . . . . . . . . . . . . . . . . . .114, 267–269 Gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 Genetic background (mice) . . . . . . . . . . . . . . . . . . . . . . . . . 269 Geneticin (G418, neomycin) . . . . . . . . . . . 71, 213, 252, 301 Genomic DNA . . . . . . . . . . . . . . . . . 211, 268, 356, 381–383, 385–386, 402–403 Germ line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Germline chimeric mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Giemsa stain . . . . . . . . . . . . . . . . . . . . . . . . . 432, 434, 442–443 Glycoprotein. . . . . . . . . . . . . . . . 276, 281–282, 314, 467, 469 Glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275–282 Granule polarization . . . . . . 69, 72, 78, 80–81, 86, 140–143 Granulocyte-macrophage colony stimulating factor (GM-CSF) . . . . . . . . . . . . . . . . . . . . 101, 104, 448 Granzyme . . . . . . . . . . . . . 102, 346, 348, 412, 419–420, 507 Gut associated lymphoid tissue (GALT). . . . . . . . . . . . . .506

NATURAL KILLER CELL PROTOCOLS 547 Index H H2 (histocompatibility 2 locus, mouse MHC class I) . . 394 H60 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99, 117, 119 HeLa cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 Hemacytometer or hemocytometer or Haemocytometer . . . . . . . . 1, 5, 7, 16–17, 19, 21, 23, 25, 75, 278, 307, 328, 435, 436 Hematopoietic progenitor (or precursor) cells (HPC). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1, 16 Hematoxylin and eosin (H&E) staining . . . . . . . . . 470, 475, 486–487 Heparin . . . . . . 250, 253, 336, 340, 346, 373, 419, 434, 471, 478–479 Hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439, 449, 452 Heterotypic interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 Histology. . . . . . . . . . . . . . . . . . . 471, 473–474, 479, 481–482 HLA (human leukocyte antigen locus, human MHC) . . . . . . . 68, 356, 362, 367, 448, 457, 461, 532, 537–544 Hofbauer cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457 Homologous recombination . . . . . . . . . . . . . . . . . . . . 268, 366 Homotypic interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 Human immunodeficiency virus (HIV) . . . . . . . . . 210, 234, 236, 239, 244, 315, 439, 452 Hydrocortisone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17, 20 Hygromycin . . . . . . . . . . . . . . . . . . . . 71, 84–85, 200, 217–218

I ICAM-1 (CD54) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 ICAM-2 (CD102) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 ICAM-3 (CD50) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Ig (immunoglobulin) fusion (or fusion Ig) . . . . . . . . 256, 271 IL-2 . . . . . . . . . . . 90, 98, 101, 178, 181, 184–190, 202, 211, 216, 229, 236, 240, 251, 261, 418, 506, 534 IL-2 secretion assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260–261 IL-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16, 19, 24 IL-7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16, 18–19, 24, 62, 236 IL-12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101, 216, 430, 437 IL-15 . . . . . . . . . . . . 16–17, 19, 98, 114, 122, 236, 241, 327, 461, 476, 507 IL-18 . . . . . . . . . . . . . . . . . . . . . . . . . . . 430, 432, 437, 439–440 IL-22 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507–508, 515 Image quantitation . . . . . . . . . . . . . . . 128, 133–134, 139, 144 Immunofluorescence (immunofluorescent) . . . 3, 9, 44, 115, 118, 121 Immunological synapse . . . . . . . . . . . . . . . . . . . . 127–147, 149 Immunophenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . 335, 353 Immunoreceptor tyrosine-based activating motif (ITAM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178, 394 Immunoreceptor tyrosine-based inhibitory motif (ITIM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40, 394 Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 Indo-1 AM dye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 Indomethacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Induced self . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39, 366 Inhibitory synapse . . . . . . . . . . . . . . . . . . . . . . . . . . 78, 139–143 Insect cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Interferon (IFN)-α . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101, 163, 430 (IFN)-β . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101, 159, 170 (IFN)-γ . . . 27, 42, 44–46, 48, 160–161, 164, 167–169, 173, 211, 342, 351, 419, 437, 515 type 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 Internal ribosomal entry site (IRES) . . . . . 54–55, 108, 110, 145, 200–201, 204, 287, 293

Intracellular cytokine (interferon-γ) staining . . . . . . . . . . . 39 Ionomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41, 43 IRBC (Plasmodium falciparum schizont infected red blood cells) . . . . . . . . . . . . . . . . . . . . . . 430–437, 441–444 Irradiation gamma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 lethal . . . . . . . . . . . . . . . . . . . . 52, 59, 60–62, 64, 109, 114 Isolated lymphoid follicles (ILF). . . . . . . . . . . . . . . . . . . . . 506 Isotype control . . . . . . 18, 23, 111, 118, 132, 164–166, 171, 186, 495

J Jurkat cell line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

K KHYG-1 (NK-like cell line, human) . . . . . . . . . . . . 207, 226 Killer cell immunoglobulin (Ig)-like receptors (KIR) . . 313, 366, 377–391, 519, 536 KIR genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . 356, 365–374 KIR Haplotype, Group A or Group B . . . . . . 354, 357, 361 KIR locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 KIR promoter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 KLRG1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116, 506–507 Knockdown . . . . . . . . 17, 209, 217–218, 220, 223–224, 229, 246, 346 Knock-in mouse . . . . . . . . . . . . . . . . . . . . . . . . . . 107, 110, 267 Knockout (KO) mouse . . . . . . . . . . . . . . . . . . . . . . . . . 250, 269 Kozak initiation sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 K562 (target cell line, human) . . . . . . . . . . . . . . 129, 337, 347

L LacZ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 LAK (lymphokine-activated killer) cells . . . . . . . . . 239–243 Lamina propria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505–544 Lamina propria lymphocytes (LPL) . . . . . . . . 506, 508, 510, 513–515 Langerhans cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Langerin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108, 110 Lavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326–331 Lentivirus . . . . . . . . . 210, 218–220, 223, 233–246, 291, 293 Leukocyte receptor complex (LRC) . . . . 366, 520–521, 527 LFA-1 (CD11a/CD18) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Ligand. . . . . . . . .54, 110–111, 290–292, 299–309, 313–322 Ligand capture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318 Listeria monocytogenes . . . . . . . . . . . . . . . . . . . . . . . . . . 103, 111 Liver, hepatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36, 105 LNK (NK-like cell line, mouse) . . . . . . . 211–212, 218, 220 Locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267–268, 393–409 Long-terminal repeat (LTR) elements . . 15, 17, 19, 23–24, 110, 200, 203, 224, 233, 440, 499 LOU rat strain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 LoxP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107, 108 LPS (lipopolysaccharide) . . . . . . . . . . . . . . 102, 111, 121, 290 Luciferase reporter firefly (Photinus pyralis) . . . . . . . . . . . . . . . . . . . . . . . . . . 378 Renilla . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380, 387–388 Lung . . . . . . . . . . . . . . . . . . . . . . . 30–32, 35–37, 101, 105, 327 Ly49 . . . . . . . . . . . . . 40, 44–49, 62, 178, 269, 299–309, 320, 377, 394–399, 402–403, 405 Lymph nodes . . . . . . . . . . . . . . . . . . . . 1–14, 36, 424, 496, 514 Lymphocyte differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Lymphocytic choriomeningitis virus (LCMV) . . . . . . . . . 99, 102–103, 112–114, 122, 162–163, 171

NATURAL KILLER CELL PROTOCOLS

548 Index

Lymphoid tissue inducer cells (LTi) . . . . 506–508, 512–515 Lympholyte-M . . . . . . . . . . . . . . . . . . . . . . . . . . . 101, 106, 492 Lymphoma . . . . . . . . . . . . . . 99, 211, 253, 258, 326, 332, 533 Lymphopoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Lymphoprep . . . . . . . 336, 340, 450, 452–455, 457, 459–460 Lytic granules . . . . . . . . . . . . . . . . 80, 131, 137–138, 140–143 Lytic synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131, 140–141

M m157 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300, 394 Macrophages . . . . . . . 98, 101, 103–104, 106–110, 429, 448, 454, 461, 521, 526–527 MACS columns/beads (magnetic cell sorting) . . . . . . . . 314 Major histocompatibility complex (MHC) class I (MHC-I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Malaria (Plasmodium parasites) . . . . . . . . . . . . . . . . . . 429–445 Mapping genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400 Marginal zone macrophages . . . . . . . . . . . . . . . . . . . . 108–109 Masking . . . . . . . . . . . . . . . . . . . . . 68, 314, 316–317, 321–322 Mast cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4, 521 Maternal-fetal interface . . . . . . . . . . . . . . . . . . . . . . . . 365, 466 MC57G cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412 MCMV (mouse cytomegalovirus) resistance . . . . . 393–409 M-CSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101, 104 Mean fluorescence intensity (MFI) . . . . . . . . . 139, 147, 290, 294, 317, 319–322 Medroxyprogesterone 17-acetate (MPA) . . . . . . . . . 451, 456 Melanoma . . . . . . . . . . . . . . . . . . . . . . . 98, 326–327, 329, 332 Menstrual cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456, 467 Mesenteric lymph nodes (MLN) . . . . . . . . . . . . . . . . . . . . 506 Mesometrial deciduas . . . . . . . . . . . . . . . . . . . . . . . . . . 466, 491 Metallophilic Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . 109 [35 S]-methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262 MHC class I-deficient mice (β2m−/− or Kb Db−/− ) . . . 46 Microsatellites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402 Microscopy confocal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128, 320 electron . . . . . . . . . . . . . . . . . . . . . . . . . . . 72, 471–472, 478 laser capture microscopy (LCM) . . . . . . . . 469, 474–475, 482–483 live cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 total internal reflection fluorescence (TIRF) microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 Microspot culture of uterine NK cells. . . 476–477, 494–495 Microtubular organizing center (MTOC) . . . 131, 140–143 Microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . 149, 420–421, 426 MIP-1β . . . . . . . . . . . . . . . . . . . . . . . . 336–339, 342, 345, 349 “Missing self ”. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 Moloney murine leukemia virus (MMLV) . . . . . . . . . . . 200, 287–289, 291 Monensin . . . . . . . . . . . . . . . . . . . . . . . . 47, 100, 116, 339, 348 Mouse embryonic fibroblasts (MEF) . . . 398, 400, 407, 410 Multi-color flow cytometry . . . . . . . 342, 350, 353–355, 361 Multiplex PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365–374 Multiplicity of infection (MOI) . . . 112, 216, 218, 240–242, 244, 287, 294, 407, 414 Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300–303, 308 Mycoplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440 Myeloid cells . . . . . . . . . . 4, 98, 102–103, 106, 107–110, 526

N Natural cytotoxicity receptors (NCR) . . . . . 67–68, 276, 282 Natural killer cell complex locus (NKC) . . . . . . . . . . . . . . 394

Negative selection . . . . . . . . . . 236, 240, 244, 254, 268, 340, 421, 461 NFAT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286, 400, 405, 407 Nick-A retroviral packaging cell line . . . . . . . . . . . . . . . . . 289 NIH 3T3 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 NK1.1 (NKR-P1C). . . . . . . . . . . . . . . . . . . . . . . . . . . . .41, 515 NK3.3 (NK-like cell line, human) . . . . . . . . . . 201, 207, 530 NK-92 (NK-like cell line, human) . . . . . . . . . 200, 207, 212, 220, 227 NK cell activation . . . . . . . . . . . 39, 67–86, 98–99, 115–116, 140, 314, 346, 349, 418, 522 alloreactivity . . . . . . . . . . . . . . . . . . . . . . 469, 497, 499, 532 antibody depletion/blockade . . . . . . . . . . . . . . . . . . . . . 412 cloning . . . . . . 16, 17, 206, 224, 235, 242, 285–286, 378 decidual . . . . . . . . . . . . . . . . . 448–451, 455–456, 461, 467 development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330, 378 differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236 education . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 endometrial (eNK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467 immature (iNK) . . . . . . . . . . . . . . . . . . . . . . . 314, 468, 486 immunological synapse . . . . . . . . . . . . . . . . . . . . . 127–147 licensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39–49 maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2, 16, 24, 394 mucosal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505, 512–514 polyclonal NK cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 precursor (pre-NK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 primary NK cells . . . . . . . . . . . 69, 94, 210, 233–246, 346 progenitor (pro-NK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467 purification of NK cells . . . . . . . . . . . . . . . . 1–14, 69, 200, 279–280, 416, 450–451, 459 self tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39, 41 uterine NK (uNK) . . . . . . . . . . . . . . . . . . . . . . . . . 497, 502 NKG2D . . . . . 41, 67–68, 99, 116, 397, 412, 419, 426, 507, 515, 521, 533 NKL (NK-like cell line, human). . . . . . . . . . . .129, 211, 346 NKp46 . . . . . . 42, 46, 48, 68, 116, 123, 254, 282, 506–509, 512–515, 527 Nkrp1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41, 286, 292, 295 NKT cells . . . . . . . . . . . . . . . . . . . . . . . . . 30, 37, 244, 419, 525 Non-allelic homologous recombination (NAHR) . . . . . . 366

O Orthopoxvirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411

P P815 cells (target cell line, mouse) . . . . . . . . . . . . . . . . . . . 263 PCR-SSP (sequence-specific priming) . . . . . . . . . . . 365–374 Percoll . . . . . . . . . . . . 29, 31–32, 36, 101, 413, 509, 511–512 Perforin . . . . . . . . . . 72, 78, 80–81, 117–119, 131, 209, 335, 348, 412, 467, 507 Pericentrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131, 140 Periodic Acid Schiff ’s (PAS) reagent . . . . . . . . 469–470, 500 Peripheral blood mononuclear cells (PBMC) . . 28, 30, 250, 253–254, 339–340, 344, 346, 356, 358, 430, 432, 434–435, 440, 444 Peritoneal exudate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 Permeabilization . . . . 42, 100, 129, 161, 167, 171–173, 342, 413, 509, 514 Peyer’s patches (PP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506 Phalloidin . . . . . . . . . . . . . . . . . . . . . . . 129, 131–132, 139–140 Phenotype . . . . . . . . 13–14, 24, 28, 210, 234, 254, 255, 267, 394–396, 400, 457, 461, 506, 531 Phoenix cells . . . . . . . . . . . . . . . . . . . . . . . . . 201, 203–204, 230

NATURAL KILLER CELL PROTOCOLS 549 Index Phorbol ester (PMA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41, 43 Phospho-STAT (pSTAT) . . . . . . . . . 161–167, 169–171, 173 Phytohemagglutinin (PHA) . . . . . . . . . . . . . . . . . . . . 251, 253 PKH26. . . . . . . . . . . . . . . . . . . . . .72, 79, 85, 90–93, 327, 329 Placenta . . . . . . . . . . . 365, 448–449, 466, 469, 481, 492, 497 Plaque Assay . . . . . . . . . . . . . . . . . . . . . . . . . 102, 401, 416–417 Plaque forming units (PFU) . . . . . . . . . . 113, 163, 395, 402, 408, 412, 415, 417, 425 Plasmodium falciparum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429 PlatE cells . . . . . . . . . . 19, 53, 55–56, 64, 191, 292, 304, 357 Pluronic F-127 . . . . . . . . . . . . . . . . . . . . . . . . . . . 150–151, 156 Point mutation . . . . . . . . . . . . . . . . . . . . . . . . 52, 264, 303, 532 Poisson distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294 pol (viral polymerase) . . . . . . . . 55, 201, 210, 214, 235, 289, 293–294, 405 Polybrene (hexadimethrine bromide) . . . . . . . . 59, 129, 202, 235, 252 Polychromatic flow cytometry . . . . . . . . . . . . . . . . . . . . . . . 359 Poly-D-lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72, 80 Poly I:C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102, 111 Poly-L-lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129, 131–132 Polymerase chain reaction (PCR) . . . . . . . . 52, 55, 179, 182, 192–193, 216, 219, 229, 250, 258, 264, 379, 397, 400, 489–490, 495 Positive selection . . . . . . . . . . . 2, 7, 217–218, 244, 414, 421, 455, 461 Pregnancy. . . . . . . . . . . . . . . . . .365, 447–462, 466, 469, 478, 480, 492, 498 Priming . . . . . . . . . . . . . . . . . . . . . . 97–98, 106–107, 114–115, 122, 385 Proliferation . . . . . . . . . . . . . . . . . . . 16–17, 26, 149, 160, 241, 294, 418–421, 495 Protein purification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258 Puromycin . . . . . . . . . . . 53, 56, 84, 180, 191, 194, 202, 215, 219, 225, 228, 231, 238, 241, 246, 266, 293, 314, 405

Q qRT-PCR (quantitative real-time PCR), qPCR (quantitative PCR). . . . . . . . . . . . . .216, 219, 246 Quantitative imaging . . . . . . . . . . . . . . 90, 128, 133–134, 144 Quantum-dot (Qdot) nanoparticles . . . . . . . . 337–338, 342, 355–356, 361–362

R Radioresistant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109, 122 Rag2−/− Il2rg −/− mice . . . . . . . . . . 326–327, 329–330, 332 Rag2−/− mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326, 330 Recombinant glycoprotein . . . . . . . . . . . . . . . . . 276, 281–282 Recombinant receptor-Fc chimera (receptor-Ig fusion protein) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 Recombinase . . . . . . . . . . . . . . . . . . . . 107, 211, 267–268, 330 Red blood cells (RBC; erythrocytes) . . . . . . . . . . . . . . . . . 436 Redirected killing (reverse ADCC) . . . . . . . . . . . . . . . . . . 262 Reporter assay . . . . . . . . . . . . . . 285–286, 291, 380, 387–388, 390, 398, 408 Reporter cell . . . . . . . . . . . . . . . . 285–296, 395, 400, 407–408 Restriction f ragment length polymorphisms (RFLPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402–403 Retroviral transduction. . . . . . . . . . .17–18, 20–23, 179, 182, 191–192, 194, 199–208, 265–267 Retrovirus . . . . . . . . . . . 16, 53, 129, 200, 204, 207, 223–231, 265–266, 285, 292, 295, 398, 405

Retrovirus rescue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285, 293 Reverse genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 R837 (imiquimod) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102, 111 RMA-S . . . . . . . . . . . . . . . . . . . . . 99, 117, 119, 326, 329, 534 RNAi (RNA interference) . . . . . . . . . . . . 223–224, 241–242, 245–246 RNase protection assay (RPA) . . . . . . . . . . . . . . . . . . 383–384 RNK-16 (NK-like cell line, rat) (also RNK) . . . . . 300, 302, 305, 309, 531 RORγt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506–509, 512–515 Rosa26 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107–108 RPMI-8866 (target cell line, human) . . . . . . . . . . . . 250, 253 RT-PCR (real-time polymerase chain reaction) . . . . . . . 179, 182, 192–193, 229, 269, 378–379, 382, 489–490, 495

S S2 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67–86 Saponin . . . . . . . . . . . . . . . . . . . . . . . . . 42, 100, 117–119, 131, 337, 445 Schizont . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431–432, 434, 436, 442 Scintillation counter . . . . . . . . . . . . . . . . . . 252, 263, 307, 423 Secondary lymphoid tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Secretory lysosomes . . . . . . . . . . . . . . . . . . 335–336, 339, 347 Shipping of blood samples . . . . . . . . . . . . . . . . . . . . . . . . . . 346 Short hairpin RNA (shRNA) . . . . . 211, 217–218, 223–231, 233–246 Sialic acid binding immunoglobulin-like lectin (Siglec) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314, 320 Signal Transducer and Activation of Transcription (STAT) . . . . . . . . . . . . . . . . . . . 162, 164, 167, 171 Signal transduction . . . . . . . . . . . . . . . . . . . . . . . . . 89, 199, 249 Simple Sequence Repeats (SSRs) . . . . . . . . . . . . . . . . . . . . 402 SIN (self-inactivating) vector . . . . . . . . . . . . . . . 210, 235, 293 Small interfering RNA (siRNA) . . . . . . . . . . . 135, 145, 209, 224, 245, 346 Spectral overlap . . . . . . . . . . . . . . . . . . . . . . . . . . 342–343, 350, 357, 362 SPICE (Simplified Presentation of Incredibly Complex Evaluations) software . . . . . . . . . . . 338, 345, 355, 359–360 Spiral arteries . . . . . . . . . . . . . . . . . . . . . . . . 448–449, 469, 486 Spleen . . . 30–31, 35, 60, 104, 111, 113, 165, 172, 395, 403, 415, 418, 422, 496, 514, 534 Stem cell . . . . . . . 16–17, 24, 51–65, 236, 240–241, 336, 531 Stem cell factor (SCF) . . . . . . . . . . 54, 60, 62, 212, 220, 236, 240, 244, 461 ‘Stepdown’ thermal cycling . . . . . . . . . . . . . . . . . . . . . . . . . . 386 Streptavidin . . . . . . . . . . 42, 44, 47, 132, 150–154, 156, 166, 187, 261, 315, 319, 342, 500 Stroma, stromal cells . . . . 4, 16, 19, 24, 236, 448, 450–451, 453–454, 460, 467, 470

T TA cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55, 379 Target cells . . . . . . . . . 67, 69, 75, 78, 81, 130, 178, 184–186, 230, 250, 261–263, 288, 300, 337, 344, 366, 422, 522 T cells. . . . . . . . . .2, 30, 37, 69, 97, 117, 161, 163, 177, 242, 254, 331, 347, 454, 467, 506–508, 521, 525, 534 T cell antigen receptor (TCR) . . . . . . . . . . . 28, 69, 173, 178, 181, 184, 187, 193, 196, 290, 477, 521 Tetramer . . . . . . . . . . . . . . . . . . . . . . . . . 37, 156, 316–317, 321

NATURAL KILLER CELL PROTOCOLS

550 Index

Th17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506 TNF-α (tumor necrosis factor) . . . . . . . . . . . . 336–339, 342, 345, 349, 430 Tonsil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4, 10, 12 TRAIL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Transcriptional control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 Transduction . . . . . . 17–18, 20–23, 55, 61, 63–64, 135–137, 199–207, 216, 223, 229, 233, 235, 239–240, 242, 245, 265–267, 288, 406 Transfection calcium phosphate transfection . . . . . . . . . 70–71, 75–76, 84, 214 electroporation . . . . . . . . . . . . . . 224, 252, 258, 260, 301, 304, 314, 380, 388, 390 lipofection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 nucleofection . . . . . . . . . . . . . . . . . . . . . . . . . . 133–134, 210 stable . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69, 183, 260 transient transfection . . . . . . . . . . . 73–74, 178, 191, 276, 277–279, 288, 291, 405 Transgenic mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99, 108 Trans interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314 Trimester of pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . 448, 456 Trophoblast cells . . . . . . . . . . . . . . . . . 448, 451–453, 456–459 Trophozoites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436, 442 Trypsin . . . . . . . . . . . . . . . . . . . 17, 19, 53, 100, 182, 212, 237, 278, 399–400, 407, 451, 456, 461 Tubulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129, 131, 140 Tumor immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . 210, 347 Tumor necrosis factor (TNF)-α . . . . . . . . 27, 209, 336–339, 342, 349, 351, 430 Toll-like Receptors (TLR) . . . . . . . . 98, 106–107, 110–111, 121–122

U U6 promoter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235, 242, 245 ULBP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Umbilical cord blood (UCB) . . . . . . . . . . . . 16–18, 20, 22, 25 Uninfected red blood cells (URBC) . . . . . . . . . . . . . 436–437 Uterine stromal cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466 Uterus . . . . . . . . 448, 466, 479–480, 490–491, 497–498, 501

V Vaccinia virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 Vesicular stomatitis virus (VSV) . . . 210, 233–238, 242, 293 Viability tests 7AAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33, 156 propidium iodide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 trypan blue. . . . . . . . . . . . . . . . . . . . . . . . . . . .432, 476, 494 Villous trophoblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448, 457 Virus packaging cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 VSV-G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237–238, 242, 293

W Western blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . 60, 161, 229

X YAC-1 (target cell line, mouse) . . . . . . . . . . . . . . . . . . 41, 533 YB2/0 (target cell line, rat) . . . . . . . . . . . . 300, 304–306, 309 YT-Indy (NK-like cell line, human) . . . . . . . . . . . . . . . . . 378 YT (NK-like cell line, human) . . . . . . . . . . . . . 211, 378, 531 YTS-Eco (NK-like cell line, human) . . . . . . . . . . . . . . . . . 252 YTS (NK-like cell line, human) . . . . . . . . . 90, 94, 129, 200, 250, 252, 264, 346