Immune Receptors (Methods in Molecular Biology, v748)

Methods

in

Molecular Biology™

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

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Immune Receptors Methods and Protocols

Edited by

Jonathan P. Rast Department of Immunology and Department of Medical Biophysics, University of Toronto and Sunnybrook Research Institute, Toronto, ON, Canada

James W.D. Booth Department of Immunology, University of Toronto and Sunnybrook Research Institute, Toronto, ON, Canada

Editors Jonathan P. Rast, Ph.D. Department of Immunology and Department of Medical Biophysics University of Toronto and Sunnybrook Research Institute, Toronto, ON Canada [email protected]

James W.D. Booth, Ph.D. Department of Immunology University of Toronto and Sunnybrook Research Institute, Toronto, ON Canada [email protected]

ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-138-3 e-ISBN 978-1-61779-139-0 DOI 10.1007/978-1-61779-139-0 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011930855 © Springer Science+Business Media, LLC 2011 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 Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface Immunology has made significant progress in the past decade, driven forward by rapidly advancing technology and a renewed interest in the vast realm of innate immunity. As the understanding of immune mechanisms matures, so too does the perception of the biological purpose to which these systems are directed; immunity not only provides a check on pathogenesis, but also serves as a primary regulator of all forms of microbial symbiosis, the necessity, complexity, and ubiquity of which is becoming increasingly apparent. The receptors that mediate these functions are at the front lines of both the protective and regulative roles of the immune system, and the techniques used to characterize these proteins are the subject of this volume. In the strictest sense, immune receptors are those proteins that form the link between the immune system and the outside world. These molecules either make direct contact with nonself or are evolutionarily tuned to respond indirectly to the presence of microbes by their sensitivity to correlative cellular disturbances. The repertoire of many of these recognition proteins can be viewed as an evolutionary snapshot of an ever-changing and unstable process in which pathogenic microbes are constantly breaking the receptor – nonself linkage in order to evade detection. Other immune receptors may form more evolutionarily stable associations with conserved microbial targets (e.g., interactions between microbial pattern molecules and TLRs). Links to commensal microbes are possibly a primary force in maintaining and containing these interactions. The most dynamic versions of nonself receptors are those of the adaptive immune system (e.g., T cell receptors and immunoglobulins) which diversify on the time-scale of the individual. Immune receptors mediate biological decisions with acute and dire consequences. Activation can elicit a cascade of cytotoxic events that require tight control. An immune response may be the “lesser of two evils” when appropriately activated or catastrophic when inappropriately launched. As such, immune decisions are the end results of complex processes of signal integration. This integration takes place both on the level of multireceptor complexes positioned at the initiation of signal generation and though coalescence of inputs at control points that are further downstream. The logic of signal integration lends specificity and flexibility to immunity that is only beginning to be understood. The contributions to this volume address a variety of experimental approaches to the characterization of immune receptors and the cell biology that mediates their functions. These include imaging techniques that aim to understand receptor localization and trafficking, techniques to measure receptor–ligand interactions, techniques to identify novel ligands, methods for the analysis of downstream signaling, as well as strategies for comprehensive genomic and proteomic characterization of immune receptors. Some of these techniques are specific for particular receptor subjects while others are broadly applicable to entire categories of proteins. The intent of the volume is that each of these technical descriptions and protocols will be useful both to investigators who are interested in carrying out these procedures and to those who seek a deeper understanding of the bench science that lies behind the immunology literature.

Toronto, ON, Canada

Jonathan P. Rast James W.D Booth v

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Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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  1 Screening for Ligands of C-Type Lectin-Like Receptors . . . . . . . . . . . . . . . . . . . . Elwira Pyz˙ and Gordon D. Brown   2 Yeast Surface Display of Lamprey Variable Lymphocyte Receptors . . . . . . . . . . . . Gang Xu, Satoshi Tasumi, and Zeev Pancer   3 Identification of Scavenger Receptor Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . Claudine Neyen, Annette Pluddemann, and Siamon Gordon   4 Construction, Expression, and Purification of Chimeric Protein Reagents Based on Immunoglobulin Fc Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . John P. Cannon, Marci O’Driscoll, and Gary W. Litman   5 Innate Immune Receptors for Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andrea Stutz, Damien Bertheloot, and Eicke Latz   6 Analysis of Receptor–Ligand Interactions by Surface Plasmon Resonance . . . . . . . Kimiko Kuroki and Katsumi Maenaka   7 Cell-Based Reporter Assay to Analyze Activation of Nod1 and Nod2 . . . . . . . . . . Birte Zurek, Harald Bielig, and Thomas A. Kufer   8 Determining FceRI Diffusional Dynamics via Single Quantum Dot Tracking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diane S. Lidke, Shalini T. Low-Nam, Patrick J. Cutler, and Keith A. Lidke   9 Ratiometric Analysis of Subcellular Recruitment of Fc Receptors During Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Patricia Mero and James W.D. Booth 10 Assessment of the Recycling of the Membrane-Bound Chemokine, CX3CL1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sajedabanu Patel, Ilya Mukovozov, and Lisa A. Robinson 11 Measuring Immune Receptor Mobility by Fluorescence Recovery After Photobleaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kristen Silver and Rene E. Harrison 12 Probing the Plasma Membrane Structure of Immune Cells Through the Analysis of Membrane Sheets by Electron Microscopy . . . . . . . . . . . . . . . . . . Björn F. Lillemeier and Mark M. Davis 13 Rapamycin-Based Inducible Translocation Systems for Studying Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michal Bohdanowicz and Gregory D. Fairn 14 Micropatterned Ligand Arrays to Study Spatial Regulation in Fc Receptor Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alexis J. Torres, David Holowka, and Barbara A. Baird



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15 CELLISA: Reporter Cell-Based Immunization and Screening of Hybridomas Specific for Cell Surface Antigens . . . . . . . . . . . . . . . . . . . . . . . . . Peter Chen, Aruz Mesci, and James R. Carlyle 16 Use of Colloidal Silica-Beads for the Isolation of Cell-Surface Proteins for Mass Spectrometry-Based Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yunee Kim, Sarah Elschenbroich, Parveen Sharma, Lusia Sepiashvili, Anthony O. Gramolini, and Thomas Kislinger 17 Transfection-Based Genomic Readout for Identifying Rare Transcriptional Splice Variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Larry J. Dishaw, M. Gail Mueller, Robert N. Haire, and Gary W. Litman 18 Characterizing Somatic Hypermutation and Gene Conversion in the Chicken DT40 Cell System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nagarama Kothapalli and Sebastian D. Fugmann 19 Characterizing Immune Receptors from New Genome Sequences . . . . . . . . . . . . Katherine M. Buckley and Jonathan P. Rast

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Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299

Contributors Barbara A. Baird  •  Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA Damien Bertheloot  •  Institute of Innate Immunity, Biomedical Center, University Hospitals, University of Bonn, Bonn, Germany Harald Bielig  •  Molecular Innate Immunobiology Group, Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany Michal Bohdanowicz  •  Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada James W.D. Booth  •  Department of Immunology, University of Toronto and Sunnybrook Research Institute, Toronto, ON, Canada Gordon D. Brown  •  Division of Immunology, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa; Section of Immunity and Infection, Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK Katherine M. Buckley  •  Department of Immunology and Department of Medical Biophysics, University of Toronto and Sunnybrook Research Institute, Toronto, ON, Canada John P. Cannon  •  Department of Pediatrics, University of South Florida College of Medicine, University of South Florida and All Children’s Hospital Children’s Research Institute, St. Petersburg, FL, USA James R. Carlyle  •  Department of Immunology, University of Toronto and Sunnybrook Research Institute, Toronto, ON, Canada Peter Chen  •  Department of Immunology, University of Toronto and Sunnybrook Research Institute, Toronto, ON, Canada Patrick J. Cutler  •  Department of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM, USA Mark M. Davis  •  Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA Larry J. Dishaw  •  Department of Pediatrics, University of South Florida College of Medicine, University of South Florida and All Children’s Hospital Children’s Research Institute, St. Petersburg, FL, USA; H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA Sarah Elschenbroich  •  Ontario Cancer Institute, University Health Network, Toronto, ON, Canada; Department of Chemistry and Pharmacy, Friedrich-Alexander University, Erlangen, Germany Gregory D. Fairn  •  Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada

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Sebastian D. Fugmann  •  Laboratory of Cellular and Molecular Biology, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA Siamon Gordon  •  Department of Biochemistry, Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Anthony O. Gramolini  •  Department of Physiology and Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada Robert N. Haire  •  Department of Pediatrics, University of South Florida College of Medicine and Children’s Research Institute, St. Petersburg, FL, USA Rene E. Harrison  •  Departments of Biological Sciences and Cell and Systems Biology, University of Toronto Scarborough, Toronto, ON, Canada David Holowka  •  Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA Yunee Kim  •  Institute of Medical Science, University of Toronto, Toronto, ON, Canada Thomas Kislinger  •  Ontario Cancer Institute and Campbell Family Cancer Research Institute and Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada Nagarama Kothapalli  •  Laboratory of Cellular and Molecular Biology, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA Thomas A. Kufer  •  Molecular Innate Immunobiology Group, Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany Kimiko Kuroki  •  Laboratory of Biomolecular Science, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Eicke Latz  •  Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA, USA; Institute of Innate Immunity, Biomedical Center, University Hospitals, University of Bonn, Bonn, Germany Diane S. Lidke  •  Department of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM, USA Keith A. Lidke  •  Department of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM, USA Björn F. Lillemeier  •  Howard Hughes Medical Institute and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA; Nomis Center for Immunobiology and Microbial Pathogenesis, The Salk Institute for Biological Studies, La Jolla, CA, USA Gary W. Litman  •  Department of Pediatrics, University of South Florida College to Medicine, University of South Florida and All Children’s Hospital Children’s Research Institute, St. Petersburg, FL, USA; H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA Shalini T. Low-Nam  •  Department of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM, USA Katsumi Maenaka  •  Laboratory of Biomolecular Science, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Patricia Mero  •  Department of Immunology, University of Toronto and Sunnybrook Research Institute, Toronto, ON, Canada

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Aruz Mesci  •  Department of Immunology, University of Toronto and Sunnybrook Research Institute, Toronto, ON, Canada M. Gail Mueller  •  Department of Molecular Genetics, All Children’s Hospital, St. Petersburg, FL, USA Ilya Mukovozov  •  Program in Cell Biology, The Hospital for Sick Children Research Institute, Toronto, ON, Canada Claudine Neyen  •  Department of Biochemistry, Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Marci O’Driscoll  •  Department of Molecular Genetics, All Children’s Hospital, St. Petersburg, FL, USA Zeev Pancer  •  Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD, USA Sajedabanu Patel  •  Program in Cell Biology, The Hospital for Sick Children Research Institute, Toronto, ON, Canada Annette Pluddemann  •  Department of Biochemistry, Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Elwira Pyz˙  •  Division of Immunology, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa; Department of Immunology, Institute for Cell Biology, Tübingen, Germany Jonathan P. Rast  •  Department of Immunology and Department of Medical Biophysics, University of Toronto and Sunnybrook Research Institute, Toronto, ON, Canada Lisa A. Robinson  •  Program in Cell Biology, The Hospital for Sick Children Research Institute, Toronto, ON, Canada Lusia Sepiashvili  •  Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada Parveen Sharma  •  Department of Physiology, University of Toronto, Toronto, ON, Canada Kristen Silver  •  Departments of Biological Sciences and Cell and Systems Biology, University of Toronto Scarborough, Toronto, ON, Canada Andrea Stutz  •  Institute of Innate Immunity, Biomedical Center, University Hospitals, University of Bonn, Bonn, Germany Satoshi Tasumi  •  Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD, USA; Fisheries Laboratory, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Shizuoka, Japan Alexis J. Torres  •  Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA Gang Xu  •  Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD, USA Birte Zurek  •  Molecular Innate Immunobiology Group, Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany

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Chapter 1 Screening for Ligands of C-Type Lectin-Like Receptors Elwira Pyz˙ and Gordon D. Brown Abstract In order to execute their immune functions, leukocytes interact with a broad range of cell types through cell surface receptors, such as those of the immunoglobulin and C-type lectin families, or indirectly through soluble factors. The characterization of activating and inhibitory counterparts of NK cell receptors on myeloid cells, as well as the identification of their physiological ligands, has provided new insights into the underlying mechanisms of immunity and homeostasis. Here, we describe methodology that can be employed to screen for endogenous ligands of type-II C-type lectin-like receptors using reporter cells and Fc fusion proteins. Key words: C-type lectin, Myeloid cell, BWZ.36 reporter cells, Fc fusion protein, Endogenous ligand

Abbreviations CRD CTLD CTLR MICL NKC NKCL PAMPs PRRs X-Gal

Carbohydrate recognition domain C-Type lectin-like domain C-Type lectin-like receptor Myeloid inhibitory C-type lectin Natural killer complex NK-like C-type lectin receptor Pathogen-associated molecule patterns Pattern recognition receptors 5-Bromo-4-chloro-3-indolyl-beta-d-galactopyranoside

1. Introduction C-type lectin-like receptors (CTLRs), often referred to as NK-like C-type lectin receptors (NKCL), are type II surface cell receptors that contain C-type lectin-like domains (CTLDs) and are expressed

Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_1, © Springer Science+Business Media, LLC 2011

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by many cells, including those of myeloid origin (1–3). The genes for myeloid expressed CTLRs are clustered within the natural killer complex (NKC) and encode molecules that share a common structure, comprising an extracellular carbohydrate recognition domain (CRD), a stalk region, a transmembrane region, and a cytoplasmic tail that may or may not contain a signalling consensus motif (4, 5). The receptors of group V CTLRs have been characterized in detail with regard to structure, distribution, and function (2, 3, 6, 7). They recognise a broad range of exogenous and endogenous molecules and are able to mediate the activation or inhibition of the function of a variety of cell types (2, 7). Some members of this family are non-opsonic pattern recognition receptors (PRRs) that bind to pathogen-associated molecule patterns (PAMPs) and serve to bridge innate and adaptive immune systems. Many of these molecules have been proposed to recognize endogenous ligands, and play a role in homeostasis, but the nature of these ligand(s) remains unidentified. Here, we present approaches to screen for endogenous ligands of CTLRs using BWZ.36 reporter cells and Fc fusion proteins. Such approaches aim to extend our understanding of the physiological functions of receptors encoded in the NKC.

2. Materials 2.1. Animals and Cell Culture

1. BALB/C and C57BL/6 mice were maintained under specific pathogen-free conditions and were used at 6–10  weeks of age. 2. Phoenix-ecotropic retrovial packaging cells, used to produce virus particles for transduction of BWZ.36 cells, were provided by Dr. G. Nolan. (www.stanford.edu/group/nolan/ retroviral_systems/retsys.html). 3. BWZ.36 cells containing an NFAT-LacZ construct were a gift from W. Yokoyama (Washington, USA). 4. Complete RPMI medium: RPMI medium (Gibco) supplemented with 10% heat-inactivated FCS, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM l-glutamine. 5. Complete DMEM medium: DMEM medium (Gibco) supplemented with 10% heat-inactivated FCS, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM l-glutamine. 6. Fugene 6 transfection reagent (Roche). 7. Selection antibiotics: hygromycin and puromycin (InvivoGen, USA).

1  Screening for Ligands of C-Type Lectin-Like Receptors

2.2. Cell Isolation and Flow Cytofluorometry

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1. TAC buffer: 20 mM Tris–HCl pH 7.2, 0.82% NH4Cl. 2. FACS Block: 5% heat-inactivated rabbit serum, 0.5% BSA, 2 mM NaN3, and 5 mM EDTA in PBS. 3. FACS Wash: 0.5% BSA, 2 mM NaN3, and 5 mM EDTA in PBS. 4. Antibodies: purified and biotinylated monoclonal anti-MICL (8), monoclonal anti-Dectin-1 (9) and appropriate isotype controls. 5. Secondary antibody: donkey anti-rat IgG-PE (Jackson Laboratories). 6. Streptavidin-PE and Streptavidin-APC (BD Biosciences).

2.3. Stimulation of Reporter Cells and In Situ b-Galactosidase Assay

(Protocol modified from that of W.L. Stanford; http://www. cmhd.ca/protocols/genetrap_pdf/Lac%20Z%20Staining.pdf) 1. Coating buffer: 0.1 M sodium carbonate and pH 9.5. 2. Sheep anti-mouse IgG (Jackson Laboratories). 3. Fix solution: 0.2% glutaraldehyde, 5 mM EGTA (from 0.5 M stock pH 7.3), 2 mM MgCl2 in 0.1 M sodium phosphate buffer, and pH 8.0 (see Note 1). 4. Wash buffer: 2  mM MgCl2, 0.02% Nonidet-P40 in 0.1  M sodium phosphate buffer, pH 8.0. 5. X-Gal stain: 1  mg/ml X-Gal (5-bromo-4-chloro-3-indolylbeta-d-galactopyranoside (FERMENTAS Life Sciences) powder dissolved in dimethyl formamide to 25 mg/ml), 5 mM potassium ferricyanide, and 5 mM potassium ferrocyanide in wash buffer (see Note 2).

2.4. Plasmid and Primers for Cloning of Chimeric Receptors and Fc Fusion Proteins

1. pFB-Neo vector (Stratagene). 2. pSecTag2 vector (Invitrogen). 3. pSecTag2-Fcmut vector provided by Dr. P. Taylor (University of Cardiff). 4. pMXs-IP cloning vector obtained from Prof. T. Kitamura (University of Tokyo). 5. Restriction enzymes, compatible buffers, and T4 ligase (FERMENTAS Life Sciences). 6. Advantage-HP-PCR Kit (BD Biosciences). 7. Gel Extraction and Miniprep DNA Purification Kits (Promega or QIAGEN).

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2.4.1. Cloning of Chimeric Receptors Used for Transduction of BWZ.36 Reporter Cells

Primers used to generate the CD3z-XhoI/XbaI: mCD3z-XhoIFor: 5¢-G TC TCG AGC CAC CAT GTT CAG CAG GAG TGC AG-3¢ and mCD3z-XbaI-Rev: 5¢-CGA TTC TAG AGT AGG CTT CTG CCA TCT TGT C-3¢. To clone MICL-SpeI/NotI, Dectin-1-SpeI/NotI as well as Clec2-SpeI/NotI fragments the following primers were used: mMICL-SpeI-For: 5¢-GAT ACT AGT CAT TCA CAG CAA AAA ACA GTC-3¢, mMICL-NotIRev: 5¢-GCG CGG CCG CGT AGC TAC CTG CTA TCC TCT GG-3¢, mBGR-SpeI-For: 5¢-CCA ACT AGT CCT TGG AGG CCC ATT GCA GTG G-3¢, mBGR-NotI-Rev: 5¢-TTT GCG GCC GCT TAC AGT TCC TTC TCA CAG AT-3¢, mClec2SpeI-For: 5¢-CTG CCA CTA GTT GGT GGC GTG TGA TGG C-3¢, mClec2-NotI-Rev: 5¢-CCA TGC GGC CGC ATT AAA GCA GTT GGT C-3¢. Primer used for sequencing: PSI: 5¢-CAC GTG AAG GCT GCC GAC C-3¢. See Fig.  1 for a schematic overview of the generation of the reporter construct.

2.4.2. Cloning of Fc-MICL Fusion Protein

Primers used to clone the complete MICL ORF into the pFBNeo vector:: mMICL-EcoRI-Fow: 5¢- GGG AGA ATT CCA CCA TGT CTG AAG AAA TTG TT-3¢ and mMICL-NotI-Rev: 5¢-GCG CGG CCG CGT AGC TAC CTG CTA TCC TCT GG-3¢. Primers used for the amplification of the mouse MICL CRD needed for cloning of FcMICL fusion protein into the pSecTag2 vector: mMICL(Fc)-KpnI-Fow: 5¢-ATA CAG GTA CCG CAA CAG AAA TGA TAA AAT CGA AT-3¢ and mMICL(Fc)-EcoRI-Rev: 5¢-CCG AGG AAT TCC CTG CTA TCC TCT GGG AG-3¢.

2.5. Biochemical Approaches

1. Nonidet-P40 lysis buffer: 25 mM Tris–HCl pH 8.0, 140 mM NaCl, 1.1% Nonidet-P40, and 4  mM EDTA supplemented with protease inhibitor cocktail (Roche Applied Sciences). 2. 2× reducing loading buffer: 0.25  M Tris–HCl pH 6.8, 4% SDS, 20% glycerol, 10% b-mercaptoethanol, and Bromophenol Blue. 3. SDS running buffer: 25  mM Tris–HCl base, 192  mM glycine, 0.1% SDS, and pH 8.3. 4. Transfer buffer: 25 mM Tris–HCl base, 192 mM glycine, and 20% methanol. 5. TBS ×10 (10× concentrated TBS): 24.23  g Trizma HCl, 80.06 g NaCl in 1 L water, and pH 7.6. 6. Add 0.1% Tween 20 to 1× TBS to obtain TBST. 7. Blocking buffer: 5% BSA in TBST or 10% non-fat milk ­powder in TBST. 8. Ponceau Stain (stock): 2% Ponceau S, and 5% Acetic Acid.

1  Screening for Ligands of C-Type Lectin-Like Receptors

mouse spleen cDNA or plasmid containing CD3ζ DNA

pFB-NeoMICL

pMXs-IP MCS XhoI

PCR digestion with XhoI and XbaI

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NotI digestion with XhoI and NotI

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pMXsCD3ζ/MICL XhoI

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Fig. 1. Cartoon representation of the cloning strategy used to generate chimeric CD3z /MICL receptor. (Details are in text).

3. Methods 3.1. Cell Culture Conditions

BWZ.36 and Phoenix-ecotropic retroviral packaging cells were cultured at 37°C with 5% CO2 in a H2O-saturated atmosphere in RPMI medium or DMEM complete medium, respectively.

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Transduced BWZ.36 cells that were maintained in medium also contain b-mercaptoethanol (100 mM), hygromycin (400 mg/ml), and puromycin (4 mg/ml) to select for the relevant gene of interest. Adherent Phoenix-eco, 293T, NIH-3T3, and CHO-K1 cells were detached from tissue culture plastic by incubation with 10 mM EDTA in PBS. 3.2. Isolation of Mouse Primary Cells (See Note 3)

1. Dissect the animals. Aseptically remove various mouse organs (spleen, thymus, heart, kidney, brain, testis, ovary plus uterus, liver, lung) and place them in cold PBS. 2. Disrupt organs by passing through a plastic 70-mm sieve placed on a tissue culture dish using a syringe plunger (see Note 4). 3. Transfer cell suspension, separately for each organ, into the 15 ml falcon tube and incubate for 10 min on ice to allow tissue debris to settle at the bottom. 4. Transfer the supernatant containing cells into a new tube and centrifuge for 10 min at 300 × g. 5. Discard supernatants and lyse erythrocytes by resuspending the cell pellet in TAC buffer. 6. After 10 min incubation at RT centrifuge samples for 5 min at 500 × g. 7. Wash cells twice with Complete RPMI medium (see Note 5).

3.3. Immunofluorescence and Flow Cytometry (See Note 6)

1. Block unspecific binding or binding to Fc receptors by incubating 106 mouse primary cells or cell lines in the presence of 2.4G2 (at 5  mg/ml) monoclonal antibody diluted in FACS Block, in volume of 50 ml for 30 min (see Note 7). 2. Stain cells with primary antibody of interest – add 50 ml of double concentrated antibody mix or appropriate isotype control (final concentration 10  mg/ml) to the samples and incubate at 4°C for 1 h. 3. Spin down the plates at 350 × g and 4°C for 3 min, remove antibodies, and wash cell pellet twice with FACS wash. 4. Stain cells for 30–40 min with fluorochrome-conjugated secondary antibody or Streptavidin-fluorochrome if using biotinylated antibodies, as recommended by the manufacturers. 5. Spin down the plates at 350 × g and 4°C, remove antibodies, and wash cell pellet three times with FACS wash. 6. Fix cells with 1% formaldehyde and analyze by flow cytometry (see Note 8).

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Given that access to human samples is limited, and because many members of group V C-type lectins are orphan receptors without described natural ligands, the analysis of these receptors in the mouse system is an advantage. Screening using BWZ.36 reporter cells, a system that was previously successfully used to identify ligands for many receptors (10–13), provides an attractive tool to look for ligands for myeloid cell C-type lectins. BWZ.36 cells containing an NFAT-lacZ construct were transduced with chimeric­ receptors comprising the extracellular and transmembrane domains of the relevant mouse C-type lectin-like molecule and the cytoplasmic tail of the CD3z chain (Fig.  2a). Since BWZ.36 cells express an NFAT-lacZ reporter vector, triggering of the CRD of the chimeric receptors with specific monoclonal antibody or ligand should induce the b-galactosidase gene that

3.4. Reporter Cell System

BWZ.36-CD3ζ/MICL

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BWZ.36-CD3ζ/Dectin-1 2A11

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Fig. 2. The functionality and specificity of cloned BWZ.36 reporter cells. (a) Cartoon representation of the chimeric CRD/ CD3z receptor and signal transduction pathway in BWZ.36 cells. CRD represents any C-type lectin-like receptor carbohydrate recognition domain. (b) Flow cytometric analysis of Dectin-1 and MICL on reporter cells. The anti-Dectin1 mAb (2A11) stains BWZ.36-CD3z/Dectin-1 cells, whereas the anti-MICL mAb (206) has specificity for BWZ.36-CD3z/MICL cells. Negative controls (cells stained with isotype control) are indicated by grey histograms. (c) IL-2 production from BWZ.36-CD3z/MICL reporter cells after 20 h stimulation with immobilized 206 mAb or PMA and ionomycin (PMA/Iono) as a positive control. (d) X-Gal staining of cells stimulated with immobilized anti-Dectin-1 and anti-MICL antibodies, or zymosan, a ligand of Dectin-1 (50 mg/ml). (Reproduced in part from ref. 8 with permission from European Journal of Immunology).

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can subsequently be measured quantitatively. Similarly, BWZ.36 cells make high levels of IL-2 upon activation that can be used as an alternative readout. 3.4.1. Generation of Plasmids

Plasmids used for gene transduction were generated by three way ligation of the following DNA fragments: pMXs-IP-XhoI/NotI, CD3z-XhoI/XbaI, and the CRD of the relevant C-type lectinSpeI/NotI (Fig. 1). 1. Digest the pMXs-IP cloning vector with XhoI and NotI restriction enzymes and isolate required pMXs-IP-XhoI/ NotI fragment by gel purification. 2. Amplify the CD3z-XhoI/XbaI fragment from mouse cDNA with mCD3z-XhoI-For and mCD3z-XbaI-Rev primers, digest the PCR product with XhoI and XbaI restriction enzymes and gel purify. 3. Generation of the CDR of relevant C-type lectin-SpeI/NotI fragments. Cloning of the CD3z/MICL chimaera is given below and in Fig. 1 as an example. Amplify the MICL-SpeI/NotI fragment from MICL cDNA containing plasmid, or mouse cDNA, using the mMICL-SpeI-For and mMICL-NotI-Rev primers. Digest PCR product with SpeI and NotI restriction enzymes and gel purify. 4. Perform the three way ligation: combine together in one ligation reaction three cloning DNA fragments: pMXs-IP-XhoI/ NotI, CD3z-XhoI/XbaI, and MICL-SpeI/NotI. 5. Transform competent E. coli bacteria. Screen for positive clones by colony PCR, and purify plasmid DNA. Sequence cloned constructs using plasmid-specific PSI primer.

3.4.2. Expression of Chimeric Receptors on the Surface of BWZ.36 Cells

To generate stable reporter cell lines, BWZ.36 cells that contain an NFAT-lacZ construct are retrovirally transduced with chimeric receptors comprising the extracellular and transmembrane domain of the C-type lectin of interest and the cytoplasmic tail of CD3z chain (Fig.  2a). To this end, transfect 293T Phoenix-ecotropic cells with DNA of pMXs-IP-CD3z/MICL, pMXs-IP-CD3z/ Dectin-1, or pMXs-IP-CD3z/Clec2 construct as described by the Nolan Lab (www.stanford.edu/group/nolan/retroviral_ systems/retsys.html). 1. A day before planned transfection, plate  1–2 × 106 of 293T Phoenix-ecotropic cells per well on six-well plate and place in incubator overnight. 2. The next day, transfect plated cells with 1 mg of DNA of interest using Fugene-6 transfection reagent according to manufacturer’s protocol and place in incubator overnight.

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3. Transfer plates to an incubator at 32°C and incubate overnight. 4. Collect culture supernatants containing virion particles, filter through a 0.45-mm filter, and add 8 mg/ml polybrene (final concentration). 5. Incubate BWZ.36 reporter cells (1 × 105  cells on 24-well plate) with filtered 293T Phoenix supernatants for 1–1.5  h at 37°C. 6. Centrifuge plates for 1.5 h at 37°C and 800 × g. 7. When centrifugation is finished, place the plates in the incubator overnight. 8. Exchange medium containing virus particles for fresh medium with selection antibiotics. We use hygromycin at 400 mg/ml and puromycin at 4 mg/ml. 9. After a selection time of 2–3 weeks, the presence of appropriate chimeric receptor on the cell surface can be confirmed by flow cytometry with antibodies specific for the extracellular region of each construct (Fig. 2b). 3.4.3. Testing of Functionality and Specificity of Reporter Cells with C-Type Lectin-Specific Monoclonal Abs

To test whether the generated reporter system is functional, BWZ.36 reporter cells (here, for example, BWZ.36-CD3z/ MICL and BWZ.36-CD3z/Dectin-1 cells) are stimulated with plate-bound C-type lectin-specific (anti-MICL and anti-Dectin-1) monoclonal antibodies. Alternatively, defined ligands of the relevant C-type lectin can be used as additional positive controls, e.g. zymosan that effectively stimulates Dectin-1 expressing cells. 1. Two days before planned experiments coat 24-well plates with 50 mg/ml of sheep anti-mouse IgG (which cross-reacts with rat IgG) (Jackson Laboratories) diluted in coating buffer. Incubate plates overnight at 4°C. 2. Next day, wash plates three times with PBS. Aspirate PBS and coat wells with required specific monoclonal antibody in coating­ buffer. Incubate plates overnight at 4°C (see Note 9). 3. Aspirate antibody solution and wash the wells with PBS three times. 4. Aspirate PBS and plate 3 × 105 of reporter cells per well (see Note 10). 5. Stimulate cells overnight at 37°C. 6. Spin plates down, collect and freeze down the culture supernatants for the IL-2 ELISA and perform b-gal assay for the cells remaining in the wells (see Subheadings  3.4.5 and 3.4.6). An example of results produced is shown in Fig. 2c, d.

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3.4.4. Screening for Ligands

These assays are performed once the reporter cell system has been established and the functionality and specificity of the assay have been proven. 1. Set up the coculture experiment with relevant BWZ.36 transductants and potential ligands. Because several C-type lectins recognize diverse ligands that may be regulated by pathological conditions, various stimuli, including a broad panel of different cell types of mouse and human origin, living cells, or components of pathogen cell walls, as well as mouse primary cells isolated from infected animals, can be used in the screening. In addition, the response of reporter cells to chemically modified (EDTA-, EGTA-, trypsin-pretreated or deglycosylated or necrotic) ligand positive cells might be analyzed, to dissect the possible involvement of these posttraslational modifications (see Note 11). For the assay performed on 24-well plates, mix 3 × 105 reporter cells with 3 × 105 cells of particular cell line or 1–2 × 106 of mouse primary cells. To check whether there are dose-dependent effects on BWZ.36 cells, it is usually recommended to perform a titration of ligand-expressing cells versus a fixed cell number of reporter cells, e.g. the titration of mouse primary cells between 0.5 and 5 × 106 cells per well. In the case when biologically active molecules are used, perform the titration of these stimuli as well. To prove the specificity of the ligand–receptor interaction, Fc fusion protein of a particular CTLR (at 10  mg/ml, see Subheading 3.5) can be included in the coculture as a competitor, to specifically inhibit the activation of reporter cells expressing the corresponding CTLR. 2. After overnight coculture at 37°C supernatants are frozen down at −20°C or are directly tested for IL-2 by ELISA, whereas the cells are stained using in situ b-gal staining protocol (see Subheadings 3.4.5 and 3.4.6).

3.4.5. Lac Z Staining (In Situ b-Galactosidase Assay)

(Modified from W.L. Stanford; http://www.cmhd.ca/protocols/ genetrap_pdf/Lac%20Z%20Staining.pdf) Because the BWZ.36 cells express an NFAT-lacZ construct, triggering of the chimeric receptor should induce b-galactosidase that can be subsequently measured quantitatively. In practice, after 20 h coculture, substrate X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) is added to the cells, and the b-Gal assay is performed. If the specific stimulation of reporter cells occurred, then the cells should turn blue (see Note 12). 1. Rinse cells twice with phosphate buffered saline (PBS). 2. Add fix solution and fix the cells for 30 min.

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3. Aspirate fix solution and wash cells with wash buffer three times for 5 min each time. 4. Add X-Gal stain to cells. Wrap plates in aluminium foil to prevent light exposure and incubate at 37°C (see Note 13). 5. Remove stain, rinse cells once with PBS or wash. Add fresh wash buffer and store samples at 4°C (see Note 14). The activation of reporter cells can be quantified by the detection of IL-2 in the culture supernatants. To measure the amount of IL-2 secreted by activated reporter cells, any commercially available IL-2 ELISA Kit (e.g. BD Pharmingen) can be used. An example of results produced is shown in Fig. 2c and Fig. 3.

3.4.6. IL-2 ELISA

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Fig. 3. mMICL recognizes an endogenous ligand. (a) IL-2 production from BWZ.36 reporter cells after coculture with single-cell suspensions isolated from various murine organs, as indicated. For such organs like bone marrow, thymus, heart, spleen, and kidney the difference between MICL and Dectin-1 reporter cells with regard to cytokine secretion was statistically significant. (b) Inhibition of IL-2 production by the inclusion of soluble Fc-MICL protein. The BWZ.36-CD3z / MICL reporter cells incubated with bone marrow, thymus, heart, spleen, and kidney cells in the presence of Fc-MICL protein secreted less IL-2 than these cells cocultured with ligand positive cells only. In the case of bone marrow, spleen, and kidney the inhibition was statistically significant. The data are presented as mean ± SEM of data pooled from three (a) and two (b) independent experiments. P values of t-test, *p < 0.05, **p < 0.01. (Reproduced from ref. 8 with permission from European Journal of Immunology).

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3.5. Looking for Ligands Using Fc Fusion Protein

Ligand–receptor interaction can also be explored using flow cytometric approaches and biochemically using purified recombinant Fc proteins.

3.5.1. Cloning of C-Type Lectin-Fc Fusion Protein

As an example, we describe the construction of the Fc-MICL fusion protein. A cartoon representation of the final recombinant protein is shown in Fig. 4. 1. Amplify the CRD of mouse MICL using MICL DNAcontaining plasmid as a template and specific primers with required restriction sites: mMICL(Fc)-KpnI-Fow and mMICL(Fc)-EcoRI-Rev. 2. Digest the obtained PCR product with restriction enzymes and isolate DNA by gel purification. 3. Clone KpnI-MICL-CDR-EcoRI fragment into the KpnI/ EcoRI sites of the pSecTag2-Fcmut vector upstream of the IgG “Fcmut” segment (14) (see Note 15). Fusion proteins of other C-type lectin-like molecules can similarly be generated using the same approach. Since the Fc is a type I protein, whereas CTLR are often type II proteins, the chimaera comprises a “head-to-head” orientation, which may sometimes lead to misfolding. However, we have successfully used this method to express MICL, Dectin-1, Clec2, Clec9A, and other fusion partners.

3.5.2. Expression of Fusion Fc Protein

1. Transfect 293T cells with DNA of Fc-MICL construct using, for instance, Fugene-6 transfection reagent and protocol (Roche). 2. Two days after transfection, culture transfected cells in the selection medium containing zeocin (at 250–400  mg/ml; Invivogen, San Diego). 3. After around 3–4  weeks selection, bulk up the cell culture, pool the culture supernatants and purify the Fc-MICL through a protein A sepharose column (see Note 16). Purified and dialyzed Fc proteins are often used for the immunization of animals, in order to produce C-type lectinspecific monoclonal antibodies. However, they are also very useful in immunoflow cytometry, immunohistochemistry, and biochemical approaches.

3.5.3. The Use of Fc Fusion Proteins in Flow Cytometry and in In Vitro Stimulation Assays

The Fc-C-type lectin fusion proteins can be used in flow cytometry analysis to identify endogenous or exogenous CTLD ligands expressed on the surface of cells or pathogens. Given the specificity of C-type lectin CRD for its ligand, the detection of Fc-protein positive cells should indicate the presence of cells expressing ligand for particular CTLR (an example using Fc-MICL is shown in Fig.  4). The ligand–receptor interaction, between CRD of

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Fig. 4. The use of Fc fusion proteins in flow cytometry and in in vitro stimulation assay: MICL as an example. (a) Schematic representation of the structure of the MICL-Fcmut fusion protein. Flow cytometric analysis of single-cell suspensions from various murine tissues, as indicated, and stained with Fc-MICL (dark line, lower panels) or Fc-Dectin-1 (grey line, upper panels). Secondarily, only staining is indicated by grey-filled histograms. Among tested mouse tissues, heart, lung, liver, kidney, and uterus, to some extent bone marrow and spleen appear to contain MICL-Fc positive cells, cells that might express MICL ligand(s). The data are representative of three independent experiments. (b) Inhibition of the binding of biotinylated Fc-MICL protein to mouse heart and liver cells upon the addition of purified Fc-MICL. In the case of heart, the inhibition was statistically significant. The data are presented as mean ± SEM of data pooled from three independent experiments. (Reproduced in part from ref. 8 with permission from European Journal of Immunology).

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tested lectin and ligand positive cells are detected using fluorochrome-labelled secondary Abs specific for Fc portion of Fc-fusion protein. Using this approach, we have detected ligands for MICL, Dectin-1, Clec2, and Clec9A. 1. Prepare cell homogenates from various mouse organs as described above (Subheading 3.2). 2. Stain the cells with Fc C-type lectin in the same way as with an antibody for 1 h on ice. Follow the cytofluorometry general protocol (Subheading 3.3; Note 8) (see Note 17). 3. After two step washing, stain cells with PE-labelled anti-human secondary antibody for 30–40  min (Jackson Laboratories). 4. Wash cells three times with FACS wash. Cells were analyzed by gating on populations based on SSC and FSC (see Note 18). Examples of ligands that were detected on cells from a variety of organs using Fc-MICL are shown in Fig. 4. Furthermore, the addition of unlabelled purified Fc protein prior to the staining with the corresponding biotinylated Fc fusion protein should inhibit the staining, and will help to demonstrate the specificity of the receptor–ligand interaction (Fig. 4b). Similarly, Fc fusion proteins should be able to block the interaction between receptor and its ligand(s) in reporter cell assays (Fig. 3b and Subheading 3.4.4). Further examples are presented below (Fig. 5a, b). 3.5.4. The Use of Fc Fusion Proteins in Biochemical Approaches

To confirm data obtained from flow cytometry analysis, it is possible to immunoprecipitate C-type lectin ligands from cells that appeared to be positive in the staining. “Far” Western Blotting of immunoprecipitates or whole cell lysates with Fc fusion protein allows the characterization of C-type lectin ligands with regard to their molecular size and glycosylation pattern while mass spectrometry analysis can define the identity and the nature of ligand. To isolate the protein single cell suspensions prepared from different mouse organs (Subheading 3.2) are lysed with lysis buffer. Alternatively, lysates from the intact tissues can be prepared following the protocol below. 1. Dissect the tissue of interest. Work quickly on ice to prevent degradation by proteases. 2. Weigh the tissues and place them in the Eppendorf or 15 ml falcon tube. For a ~5 mg piece of tissue, use 300 ml of lysis buffer and immediately homogenize with an electric homogenizer. Rinse the homogenizer blade twice with 300  ml of fresh cold lysis buffer (see Note 19).

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Fig. 5. The use of Fc fusion proteins in in vitro stimulation assay and in biochemical approaches: Clec2 as an example. (a) X-Gal staining of BWZ.36 cells expressing the various constructs (indicated on the left) after 20 h coculture with NIH3T3 and CHO-K1 cell lines. Only Clec2 reporter cells recognize a ligand present on NIH-3T3 and CHO-K1 cells. This recognition is specific because the addition of soluble Fc-Clec2 protein to the culture decreases the activation of the BWZ.36-CD3z/Clec2 reporter cells. (b) The use of soluble Fc-Clec2 protein to block the activation of Clec2 reporter cells cocultured with ligand bearing mouse lung cells. The data are representative of three (a) and two (b) independent experiments. (c) Western blotting of cell lysates prepared from different mouse cells and tissues probed with Fc-Clec2 fusion protein. Lysates prepared from lung, kidney, and uterus were positive when probed with Fc-Clec2 protein implying that Clec2 ligand is expressed in these organs. Given the molecular mass of the positive bands, it is highly likely that these correspond to podoplanin, a previously described ligand of Clec2 (17).

3. To complete the cell lysis rotate samples for 2 h at 4°C. 4. Centrifuge the samples for 20 min at 13,400 × g at 4°C. 5. Gently transfer the supernatants into new Eppendorf tubes placed on ice. 6. Determine the protein concentration (e.g. using a Bradford or a BCA assay (Pierce)) (see Note 20). 7. Separate proteins according to their molecular weight by SDS-PAGE. To denature the proteins, add 2× reducing loading buffer and boil the samples for 5  min at 100°C. Load samples (20–40  mg of total protein) and molecular weight marker onto gel. 8. Perform transfer of proteins onto the nitrocellulose membrane according to the manufacturer’s instructions. 9. To check the efficiency of the transfer, and comparable loading of samples, rinse the membranes with dH2O and stain

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them for 5 min with Ponceau stain. Wash away excess stain with dH2O. 10. To prevent non-specific background binding of the primary or secondary antibodies, incubate the membranes in the presence of the blocking buffer 5% BSA or 10% non-fat milk powder in PBS overnight with shaking. 11. Rinse the membrane using TBST and probe with 10 mg/ml of Fc fusion protein diluted in the blocking buffer. Incubate for 1–2 h at room temperature with shaking. 12. Wash membranes 4 × 15 min with TBST. 13. Add detection antibody, e.g. donkey anti-human IgG HRP (Jackson Laboratories) diluted 1:10,000 in TBST. Incubate the membranes for 1 h at RT. 14. Wash the membranes 4 × 15 min as described in step 5. 15. Develop the membranes using commercially available ECL detection kit (Amersham) and recommended autoradiography film. As an example, we show probing transferred cell lysates with Fc-Clec2 (Fig. 5c). 3.6. Discussion

Here, we describe how to screen for endogenous ligands of typeII CTLRs using BWZ.36 reporter cells and IgG-Fc fusion proteins. For some members of this receptor family, the screening for ligands may be complicated by a variety of factors, including low affinity of receptor–ligand interactions, or low-level expression of the ligand itself. Additionally, ligands may be expressed only by restricted cell types, during a particular time window or may be up- or downregulated upon stress or pathological conditions. All of these aspects should be taken into consideration when screening for ligands of C-type lectins. Nevertheless, the use of BWZ.36 reporter cells provides a highly sensitive assay which, when complimented with IgG-Fc fusion protein methodology, offers attractive tools to look for novel receptor–ligand interactions. In the long term, such studies provide more information on the function of CTLRs and their contribution to immunity, homeostasis, and disease.

4. Notes 1. To be prepared fresh each time. 2. This stain can be stored at 4°C in the dark but for no longer than 1 week. 3. Animals used in experiments are sacrificed under humane ethically approved conditions (e.g. through anoxia with CO2)

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and all procedures to obtain single cell suspension should be carried out under sterile conditions. 4. For bone marrow cells, cut the ends off the bones and flush out bone marrow with PBS using a syringe and needle. Leukocytes from murine peripheral blood, thioglycollateelicited neutrophils and peritoneal macrophages, as well as bone marrow-derived dendritic cells can be prepared according to previously described protocols (15, 16). 5. Mouse primary cells isolated in this way can be used in immunoflow cytometry, in in vitro functional assays or they can be lysed with NP-40 lysis buffer and analyzed biochemically. 6. Staining is performed on 96-well V-bottom bacterial plastic plates on ice. Primary antibodies are tested at 10 mg/ml while commercially available primary and secondary antibodies are used alongside matching isotype controls at the concentration recommended by the manufacturers. 7. 2.4G2 is a rat monoclonal antibody and cannot be used when anti-rat staining is performed. 8. Staining with Fc fusion proteins (e.g. Fc-MICL) can be performed as normal FACS staining, in which cells are labelled with 10  mg/ml of Fc proteins followed by secondary PE-labelled donkey-anti human IgG. 9. The concentration of antibody used depends on the receptor and should be optimized prior to the experiment. For the CTLRs we studied, we found that monoclonal antibodies at 10  mg/ml give appropriate responses. The same concentration of relevant isotype control antibody should be used as a negative control. 10. As an internal positive control for stimulation stimulate reporter cells with 40  ng/ml PMA (stock 0.1  mg/ml in DMSO) and 1.5 mg/ml ionomycin (stock 0.5 mg/ml). 11. The stimulations with mouse primary cells, cell lines, or biochemical molecules can be performed on normal 24- or 6-well plates. When adherent cell lines are used in the stimulation, it is recommended to plate them the evening before the planned experiment. 12. The staining is performed in the same plates that were used for stimulation, always at room temperature. 13. The blue colouration may sometimes appear within 1 h, but the plates are usually left for overnight incubation at 37°C. 14. Alternatively, 60% glycerol (in PBS) can be used to replace the wash buffer for long-term storage. 15. This segment contains mutations within human IgG1 that prevent the binding of the fusion protein to Fc receptors.

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16. Protein A is used due to relatively selective and strong binding to human IgG1 compared to bovine IgG in the medium. 17. For some mouse organs and tissues (gut, stomach, kidney), it is difficult to count the exact cell number required for the staining. In those cases, total suspension of cells are analyzed. 18. Fluorescent dyes, such as 7-AAD, can also be used to identify live versus dead cells. 19. Volumes of lysis buffer used need to be determined in relation to the amount of tissue present. The optimal concentration of prepared protein extract should be 1–5 mg/ml. 20. At this step, samples can be frozen down at −20°C or −80°C for later use.

Acknowledgments This work was supported by the Wellcome Trust and South African National Research Foundation. References 1. Sobanov, Y., Bernreiter, A., Derdak, S., Mechtcheriakova, D., Schweighofer, B., Duchler, M., Kalthoff, F., and Hofer, E. (2001) A novel cluster of lectin-like receptor genes expressed in monocytic, dendritic and endothelial cells maps close to the NK receptor genes in the human NK gene complex, Eur J Immunol 31, 3493–3503. 2. Pyz, E., Marshall, A. S., Gordon, S., and Brown, G. D. (2006) C-type lectin-like receptors on myeloid cells, Ann Med 38, 242–251. 3. Huysamen, C., and Brown, G. D. (2009) The fungal pattern recognition receptor, Dectin-1, and the associated cluster of C-type lectinlike receptors, FEMS Microbiol Lett 290, 121–128. 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. Drickamer, K., and Fadden, A. J. (2002) Genomic analysis of C-type lectins, Biochem Soc Symp 69, 59–72. 6. Kerrigan, A. M., Dennehy, K. M., Mourao-Sa, D., Faro-Trindade, I., Willment, J. A., Taylor, P. R., Eble, J. A., Reis e Sousa, C., and Brown, G. D. (2009) CLEC-2 is a phagocytic activation receptor

expressed on murine peripheral blood neutrophils, J Immunol 182, 4150–4157. 7. Robinson, M. J., Sancho, D., Slack, E. C., LeibundGut-Landmann, S., and Reis e Sousa, C. (2006) Myeloid C-type lectins in innate immunity, Nat Immunol 7, 1258–1265. 8. Pyz, E., Huysamen, C., Marshall, A. S., Gordon, S., Taylor, P. R., and Brown, G. D. (2008) Characterisation of murine MICL (CLEC12A) and evidence for an endogenous ligand, Eur J Immunol 38, 1157–1163. 9. Brown, G. D., Taylor, P. R., Reid, D. M., Willment, J. A., Williams, D. L., MartinezPomares, L., Wong, S. Y., and Gordon, S. (2002) Dectin-1 is a major beta-glucan receptor on macrophages, J Exp Med 196, 407–412. 10. Smith, H. R., Heusel, J. W., Mehta, I. K., Kim, S., Dorner, B. G., Naidenko, O. V., Iizuka, K., Furukawa, H., Beckman, D. L., Pingel, J. T., Scalzo, A. A., Fremont, D. H., and Yokoyama, W. M. (2002) Recognition of a virus-encoded ligand by a natural killer cell activation receptor, Proc Natl Acad Sci USA 99, 8826–8831. 11. Iizuka, K., Naidenko, O. V., Plougastel, B. F., Fremont, D. H., and Yokoyama, W. M. (2003)

1  Screening for Ligands of C-Type Lectin-Like Receptors Genetically linked C-type lectin-related ligands for the NKRP1 family of natural killer cell receptors, Nat Immunol 4, 801–807. 12. Carlyle, J. R., Jamieson, A. M., Gasser, S., Clingan, C. S., Arase, H., and Raulet, D. H. (2004) Missing self-recognition of Ocil/Clr-b by inhibitory NKR-P1 natural killer cell receptors, Proc Natl Acad Sci USA 101, 3527–3532. 13. Rosen, D. B., Bettadapura, J., Alsharifi, M., Mathew, P. A., Warren, H. S., and Lanier, L. L. (2005) Cutting edge: lectin-like transcript-1 is a ligand for the inhibitory human NKR-P1A receptor, J Immunol 175, 7796–7799. 14. Graham, L. M., Tsoni, S. V., Willment, J. A., Williams, D. L., Taylor, P. R., Gordon, S., Dennehy, K., and Brown, G. D. (2006) Soluble Dectin-1 as a tool to detect beta-glucans, J Immunol Methods 314, 164–169.

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15. Taylor, P. R., Brown, G. D., Geldhof, A. B., Martinez-Pomares, L., and Gordon, S. (2003) Pattern recognition receptors and differentiation antigens define murine myeloid cell heterogeneity ex vivo, Eur J Immunol 33, 2090–2097. 16. Lutz, M. B., Kukutsch, N., Ogilvie, A. L., Rossner, S., Koch, F., Romani, N., and Schuler, G. (1999) An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow, J Immunol Methods 223, 77–92. 17. Suzuki-Inoue, K., Kato, Y., Inoue, O., Kaneko, M. K., Mishima, K., Yatomi, Y., Yamazaki, Y., Narimatsu, H., and Ozaki, Y. (2007) Involvement of the snake toxin receptor CLEC-2, in podoplanin-mediated platelet activation, by cancer cells, J Biol Chem 282, 25993–26001.

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Chapter 2 Yeast Surface Display of Lamprey Variable Lymphocyte Receptors Gang Xu, Satoshi Tasumi, and Zeev Pancer Abstract The variable lymphocyte receptors (VLRs) of lamprey and hagfish comprise leucine-rich repeat modules, instead of the immunoglobulin-like domain building blocks of antibodies and T-cell receptors in jawed vertebrates. Both types of vertebrate-rearranging antigen receptors are similarly diverse, with repertoires that can potentially exceed 1014 unique receptors. In order to characterize antigen-binding properties of the VLRs, we developed a high-throughput yeast surface display platform for the isolation of monoclonal VLRs. We have isolated VLRs that specifically bind hen egg lysozyme, b-galactosidase, cholera toxin subunit B, R-phycoerythrin, and the blood group trisaccharides A and B, with binding affinities in the mid-nanomolar to mid-picomolar range. VLRs may, thus, be excellent single-chain alternatives to Ig-based antibodies for biotechnology applications. Key words: Lamprey, Variable lymphocyte receptors, Recombinant antibodies, Yeast surface display

1. Introduction The variable lymphocyte receptors (VLRs) of jawless fish, such as lamprey and hagfish, are the only known rearranging antigen receptors that are built from leucine-rich repeats (LRRs) instead of the immunoglobulin (Ig) superfamily domains that are building blocks of the B- and T-cell receptors of jawed vertebrates from shark to man (1–3). Members of the LRR-containing protein superfamily serve as cardinal microbial recognition molecules in the innate immune systems of plants and animals, for instance the LRR-containing plant Disease Resistance genes, Toll and Toll-like receptors, and the cytoplasmic nucleotide-binding site (NBS)-LRR proteins (4). These innate microbial recognition

Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_2, © Springer Science+Business Media, LLC 2011

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molecules have diverged to serve specific functions over very long evolutionary periods, like nearly all other genes in plant and ­animal genomes. In sharp contrast, in vertebrate lymphocytes, the rearranging antigen receptors are combinatorially assembled from hundreds of gene fragments, resulting in repertoires that can potentially exceed 1014 unique receptors (5, 6). Experimental data indicates that Ig-based antibodies can specifically bind virtually all types of antigens with high affinity. Little is known, however, about the antigen-binding properties of VLRs. Antigen recognition by VLRs from immunized lamprey has been shown for spore coats of anthrax (Bacillus anthracis) and their BclA glycoprotein component, for the human blood group trisaccharide antigens, and for hen egg lysozyme (HEL) (5, 7). In order to isolate and characterize VLR binders of specific antigens, we developed a yeast surface display (YSD) platform for the VLRs. Thus far, we have isolated clones that bind HEL, Escherichia coli b-galactosidase, cholera toxin subunit B, R-phycoerythrin (RPE), and the blood group trisaccharides A and B, with binding affinities in the mid-nanomolar to midpicomolar range comparable to high-affinity IgG antibodies with KDs in the low-nanomolar range (8). These monoclonal VLRs were isolated from libraries originating from immunized lamprey, as well as from nonimmunized animals, indicating that for most antigens there is no need for immunization in order to isolate specific ligand-binding clones (7). VLRs may, thus, be excellent single-chain alternatives to Ig-based antibodies for biotechnology applications, since both of these antigen receptors were optimized over hundreds of millions of years of evolution (9). The VLR diversity regions consist of sets of LRR modules, each with a highly variable sequence, as shown in Fig. 1. At both ends of the diversity region, there are capping modules, the N-terminal LRR (LRRNT), and the C-terminal LRR (LRRCT),

Fig. 1. A stick model of a lamprey mature VLRB. The VLR comprises a set of highly diverse LRR modules capped by disulfide-bonded N-terminal LRR (LRRNT, 24–32 amino acids) and C-terminal LRR (LRRCT, 45–62 amino acids). The 25-residue LRR1 is followed by one to ten 24-residue LRRVs and then a 16-residue truncated LRR, the connecting peptide (CP). The invariant portions of VLRBs include an N-terminal secretory signal peptide (SP) and an 81-residue C terminus that contains a threonine/proline-rich stalk (33 amino acids) and a glycosyl phosphatidylinositol (GPI) membrane anchor motif, which tethers the VLR to the lymphocyte surface. Seven cysteines in the 22-residue hydrophobic C-terminal domain may participate in VLR oligomerization.

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which are stabilized by two sets of intramodular disulfide bonds (10, 11). These disulfide bonds are essential for proper folding and stability of the VLR structure. Expression of recombinant VLRs, therefore, requires a eukaryotic host, such as the yeast Saccharomyces cerevisiae that possesses an efficient oxidative protein-folding machinery and secretory pathway, and is amenable to high-throughput screens (12, 13). We also noted that for optimal antigen binding, the VLRs require free N-termini. We, therefore, developed a YSD vector based on C-terminal fusion of the VLRs to the yeast surface-anchored flocculation protein Flo1p, as shown in Fig. 2a, b.

Fig. 2. (a) Yeast surface display of VLRs fused to the C-terminus of the Flo1p anchor. The hemagglutinin (HA)-tag serves for VLR detection via Alexa 488 labeled antibodies. Biotinylated ligands are detected via R-phycoerythrin conjugated to streptavidin (SA-PE). (b) The pYSD2 vector for VLR yeast surface display. The VLRs are expressed under the tightly regulated GAL1 promoter, fused between the authentic Flo1p leader and the yeast Flo1p C-terminus, which includes the surface-anchoring domain. The vector replicates in bacteria and yeast (ColE1, CEN6/ARS4) selected by kanamycin/ geneticin resistance. (c) VLRs are cloned directionally between two unique Sfi I sites. Protein detection and purification tags and the annealing sites for primers are indicated (YSD.F, YSD.R). (d) The homologous recombination cassette consists of two 49-bp direct repeats separated by a linker with a Pme I restriction site for plasmid linearization. HR.F, HR.R are primers for rolling-circle amplification across the plasmid.

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2. Materials 2.1. Construction of VLR YSD Library

1. The pYSD2 vector is available upon request following MTA. 2. Primers for PCR amplification of VLRA and VLRB (see Note 1). The primers carry overhangs with two unique Sfi I sites (underlined). VLRA.F 5-aaaaaaggccaccggggccAAAACGTGT GAAACGGTC; VLRA.R 5-aaaaaaggccccagaggccccCTCCAC GAATGGGCACT; VLRB.F aaaaaaggccaccggggccGCATGTC CCTCGCAGTGT; VLRB.R aaaaaaggccccagaggccccTGGGC ATTTCGAGGGGCT. 3. QIAquick PCR purification kit (QIAGEN). 4. TempliPhi 100 amplification kit (GE Healthcare). 5. Primers for the homologous recombination cassette: HR.F 5-AAACGGAATTAACCCTCCACT, HR.R 5-AAACCGG CGTAGAGGATGCA. 6. dNTPs, 25 mM each (Roche). 7. Yeast inorganic pyrophosphatase, phi29 DNA polymerase, Bovine serum albumin (BSA), and PmeI restriction enzyme (all from New England Biolabs).

2.2. Yeast Transformation

1. Yeast strain BJ5464 (ATCC 208288). 2. Bacto Peptone, Bacto Yeast Extract, and Bacto Agar (BD Biosciences). 3. Salmon sperm carrier DNA, MB-grade (Roche). 4. YPD Plus (Zymo Research). 5. Geneticin (G-418 Sulfate, American Bioanalytical). 6. 1 L YPD medium: 20 g Bacto Peptone and 10 g Bacto Yeast Extract. For YPD plates, add 18 g Bacto Agar. Add water to 950 mL and autoclave. Allow to cool to 55°C and add 50 mL of filter-sterilized 40% glucose. When needed, add in YPD medium, G-418 to 100 mg/mL and for plates, add G-418 to 300 mg/mL. Store at room temperature for up to 1 month or at 4°C for up to 6 months. 7. 1 L YPD medium (pH 4.5): YPD, including 10.4 g sodium citrate and 7.4 g citric acid monohydrate. 8. 1  L 2× YPD medium: 40  g Bacto Peptone and 20  g Bacto Yeast Extract. Add water to 900 mL and autoclave. Allow to cool to 55°C and add 100 mL of filter-sterilized 40% glucose. 9. Transformation buffer 1: 0.1  M lithium acetate (LiAc), 10 mM Tris–HCl, pH 7.5, 1 mM EDTA. Prepare fresh from the stock of 1 M LiAc (10.2 g of lithium acetate dihydrate in 100 mL water. Autoclave and store at room temperature).

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10. Transformation buffer 2: 40% PEG, 0.1  M LiAc, 10  mM Tris–HCl, pH 7.5, 1  mM EDTA. Prepare fresh from the stocks of 1 M LiAc and 50% PEG (50 g of PEG 3350 in water in 100 mL volume. Stir to dissolve at 70°C on a heating plate. Autoclave and store at room temperature securely capped to prevent evaporation. See Note 2). 2.3. Isolation of Antigen-Specific Monoclonal VLRs

1. 1 L YPG medium: 20 g Bacto Peptone and 10 g Bacto Yeast Extract. Add water to 933 mL and autoclave. Allow to cool to 55°C and add 67 mL of filter-sterilized 30% galactose. 2. Penicillin, 5,000 units per mL and Streptomycin, 5,000 mg per mL (Invitrogen). 3. MidiMACS with LS column (Miltenyi). Can separate 1 × 109 labeled yeast cells from a total of 1 × 1010 cells. 4. MiniMACS with MS column (Miltenyi). Can separate 5 × 107 labeled yeast cells from a total of 5 × 108 cells. 5. MACS buffer: PBS with 0.5% BSA, 2  mM EDTA, 0.1% Tween 20. Filter-sterilize and store at 4°C for up to 6 months. 6. FACS buffer: PBS with 0.5% BSA, 2 mM EDTA. Filter sterilize and store at 4°C for up to 6 months. 7. Anti-biotin microbeads (Miltenyi). 8. Rat anti-HA (high-affinity clone 3F10, Roche) or rat antiFLAG (Stratagene). 9. Streptavidin R-phycoerythrin (SA-PE) (Invitrogen). 10. Alexa Fluor 488 labeled donkey anti-rat IgG (Invitrogen).

3. Methods The pYSD2 vector consists of the Flo1p leader peptide connected via a short linker to the cloning sites region, which consists of two unique SfiI sites for directional cloning, followed by a set of tags for protein detection and purification, and then the Flo1p stalk and C-terminal anchor, as shown in Fig. 2b, c. The shuttle vector can propagate both in E. coli and yeast, selected for kanamycin or geneticin resistance. To characterize the natural VLR repertoire, we developed a procedure for efficient library construction that circumvents recombination among the VLR inserts during yeast transformation. In S. cerevisiae, linear fragments of double-stranded DNA can efficiently recombine based on homology regions spanning 30–50 bp, and even several bases of identity suffice to initiate recombination events. Thus, co-transformation of yeast with a gapped vector and

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molar excess of PCR amplicons of library inserts, which include overhangs homologous to the gap-flanking regions in the vector, result in highly efficient recombination between the vector and amplicons, as well as between related amplicons, reshuffling the genes in the library (14). Library construction by means of gap repair in vivo is about 100-fold more efficient than transformation with an equivalent aliquot of a plasmid library. However, this method produces low-quality VLR libraries perhaps due to the presence of multiple potential recombination sites in VLRs, which result in disrupted open reading frames. To take advantage of the high efficiency of yeast transformation with linear DNA, we created a cassette for intra-plasmid homologous recombination in the pYSD2 vector, consisting of two 49-bp direct repeats separated by an 8-bp PmeI restriction enzyme site, as shown in Fig. 2d. After ligation of inserts into the vector SfiI sites, the library is amplified via rolling-circle amplification. The amplified circular library is then linearized by PmeI digest and used to transform yeast. The vector is then recircularized in  vivo by homologous recombination between the two direct repeats in the pYSD2 plasmid. 3.1. Construction of VLR YSD Library

1. Amplify the diversity regions of lamprey VLRs from lymphocyte cDNA or genomic DNA (see Notes 1 and 3). 2. Digest 500  ng of the pYSD2 vector with SfiI restriction enzyme and gel purify the digested plasmid. Digest also 300 ng of the amplicon of VLR diversity regions with SfiI and column purify with QIAquick PCR. Set a ligation in 10 mL volume using 50 ng of the digested vector and 30 ng of the VLRA insert or 25  ng of the VLRB insert (molar ratio of about 5:1 insert to vector). Ligate overnight at 16°C. 3. Use 2 mL of the ligated library for rolling-circle amplification in 10 mL reaction of TempliPhi. Incubate for 4 h at 30°C. 4. Add 100 pmol of each of the primers HR.F and HR.R, and then in a PCR cycler, heat for 2 min at 95°C and chill to 4°C. Increase the volume of the reaction to 400 mL (can be split into two tubes of 200  mL) adding dNTPs to 1  mM (16  mL of 25 mM stock), 8 mL BSA (10 mg/mL), 4 mL pyrophosphatase (100 units/mL), 40 mL of the 10× buffer, and 8 mL of phi29 DNA polymerase (10 units/mL). Incubate 16 h at 30°C, and then add 100 pmol of the primers HR.F and HR.R and 6 mL of PmeI restriction enzyme (10 units/mL). Incubate at 30°C for 3 h, and then at 37°C for 1 h. Finally, heat inactivate the enzymes at 65°C for 20 min. Purify the amplified DNA using two columns of QIAquick PCR. Reapply the flow through to increase the yield, and elute each sample using 100 mL of the kit elution buffer heated to 70°C. Typical yields are 10–15 mg of the linearized library ready for transformation.

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1. Inoculate a yeast colony from a freshly streaked plate (see Note 4) into 20 mL YPD medium and grow overnight shaking at 250–300 RPM at 30°C (or longer at room temperature, 20–22°C). 2. Determine the culture cell density using a spectrophotometer. Dilute a sample 1:10 in water (10 mL culture in 90 mL water) and prepare a blank similarly (10 mL YPD medium in 90 mL water). For optimal results, only use a culture that has reached an OD546 between 2 and 4. 3. Dilute 30 OD units of the yeast culture into 200 mL of 2× YPD prewarmed to 30°C (OD546 of 0.15). 4. Grow the cells at 30°C to an OD546 of 0.6 (3–5 h; see Note 5). 5. Prepare Salmon sperm carrier DNA (10  mg/mL stock): Thaw the DNA on ice just before transformation. Aliquot 100  mL DNA in a tube and boil for 5  min at 100°C. Immediately place the DNA tube in an ice/water bath for 5 min. Repeat the boiling and quenching once more and keep the DNA on ice. 6. Spin the 200 mL culture at 700 × g for 5 min. 7. Resuspend the pellet in 120 mL of sterile water by vortexing. Pipet up and down if necessary. 8. Spin again at 700 × g for 5 min. 9. Decant supernatant and resuspend the pellet in 4  mL of Transformation buffer 1. 10. Spin at 700 × g for 5 min. 11. Decant supernatant and spin briefly again to remove all residual fluid. Resuspend the pellet in 2.4 mL of Transformation buffer 1. 12. Set a 50-mL tube on ice and add 10–15 mg of the linearized library DNA. 13. Add 80 mL of the denatured carrier DNA. 14. Add the yeast cells from step 11 and vortex to mix. 15. Add 10 mL of Transformation buffer 2 and vortex for 1 min to thoroughly mix the components. 16. Incubate in a heat block at 30°C for 15  min, and then for 30 min at 30°C shaking at 100 RPM. 17. Remove the tube from the shaker and add 640 mL DMSO. Immediately mix by gently swirling the tube (at this stage, the cells are becoming fragile). 18. Heat-shock in a heat block at 42°C for 5 min, and then for 20 min in an incubator shaking gently at 50 RPM. 19. Pellet the cells at 700 × g for 5 min, decant supernatant, and spin briefly again. Remove all residual fluid.

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20. Resuspend the pellet in 10 mL YPD Plus by gently pipetting up and down a 10-mL pipette (takes about 5 min to reach a single-cell suspension). 21. Allow the cells to recover for 2  h at 30°C shaking at 150–200 RPM. 22. Pellet the cells at 700 × g for 5 min. 23. Resuspend the cells in 10 mL of YPD (pH 4.5) supplemented with 100 mg/mL G-418 (citrate buffer at pH 4.5 inhibits the growth of contaminating bacteria, which may be resistant to G-418). 24. Check the titer of the library by plating aliquots on YPD agar plates supplemented with 300 mg/mL G-418. Typical yields are 5–50 × 106 individual clones. 25. Transfer all the transformed cells to a 2-L baffled flask containing 400 mL YPD (pH 4.5) supplemented with 100 mg/mL G-418. Measure the OD546 at the start of culture. 26. Culture for 2 days at 30°C shaking at 250–300 RPM. After that, measure again the OD546 to calculate the actual growth of the library. Then, based on the original titer of the library, passage an aliquot representing at least tenfold of the calculated library size in a 2-L baffled flask containing 400  mL YPD (pH 4.5) supplemented with 100 mg/mL G-418. For strain BJ5464, 1 unit of OD546 represents 3 × 107 cells. Repeat the passage once more (during the second and third passages, culture saturation should take less than a day). 27. The library can be stored for up to 1  month at 4°C. After that, passage an aliquot representing at least tenfold of the library size. 28. For long-term storage of the library, prepare frozen aliquots. Culture an aliquot representing at least tenfold of the library size in 100 mL YPD (pH 4.5) supplemented with 100 mg/mL G-418 at 30°C for 3  days (freezing the cells in stationary phase enhances their survival). 29. Measure the OD546 of the culture to estimate cell number. 30. Spin the culture for 10  min at 3,000 × g and decant supernatant. 31. Resuspend the pellet in YPD, 100 mg/mL G-418, at a final volume of 2.6 mL. 32. Prepare three 2-mL cryogenic tubes. To each tube, add 150 mL of sterile glycerol and 850 mL of the cell suspension (each tube should contain about 109 cells). 33. Chill the cells gradually to −80°C. First, place the tubes in a Styrofoam box at −20°C. After 24 h, transfer the box with the tubes to a −80°C freezer.

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34. To initiate culture from a frozen aliquot, thaw the cells at room temperature, transfer into 100 mL YPD, 100 mg/mL G-418, and culture at 30°C. Passage the library two to three times in order to dilute the dead cells. 3.3. Isolation of Antigen-Specific Monoclonal VLRs

1. In the morning, start a culture with an aliquot representing at least tenfold of the library size. Inoculate the culture at an OD546 between 0.05 and 0.1 in YPD (pH 4.5), 100 mg/mL G-418. Incubate at 30°C with shaking at 250 RPM. The best results are obtained with cultures expanded to an OD546 between 1 and 3. If the culture grew beyond OD546 of 3, dilute with fresh medium to an OD546 of 0.5 and culture at 30°C for about 2  h (doubling time is about 1.5  h at 30°C) to reach OD546 of 1, and then proceed to induction of the library. 2. Inoculate the starter cells into prewarmed YPG supplemented with 100 mg/mL G-418 at an OD546 of 0.05. Culture overnight at 30°C shaking at 250  RPM. The best results are obtained for induced cultures that reached an OD546 between 1 and 2. Library passages and induction can also be done at room temperature (20–22°C), with culture periods of the starter and induction extended to 16–24  h (in YPD, yeast grows nearly twice as fast as in YPG). 3. Spin 1 × 1010 induced cells in a centrifuge at 2,500 × g for 5 min and decant supernatant. 4. At this point, cells are prepared for magnetic separation (see Note 6). Wash the cell pellet with 50 mL of MACS buffer at room temperature, vortex to resuspend. 5. Repeat for a total of three washes. Resuspend the cell pellet in 5-mL MACS buffer. 6. Add biotinylated antigen to a final concentration of 0.5–1 mM (up to ten antigens may be used simultaneously). Rotate for 60 min at room temperature, followed by 10 min incubation on ice. 7. Pellet the cells in a refrigerated centrifuge at 4°C for 5 min at 2,500 × g and decant supernatant. 8. Wash the cell pellet with 50  mL of ice-cold MACS buffer. Repeat for a total of three washes. 9. Resuspend the cell pellet in 5 mL of ice-cold MACS buffer and add 100  ml anti-biotin Microbeads (up to 200  ml antibiotin Microbeads may be used for maximal enrichment). Rotate the tube for 30 min at 4°C. 10. Pretreat an LS column, loaded onto the magnet, by flowing 3 mL of ice-cold MACS buffer. 11. Pellet the cells at 4°C for 5  min at 2,500 × g and decant supernatant.

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12. Resuspend the cell pellet in 50 mL of ice-cold MACS buffer. Vortex to break any cell aggregates. 13. Immediately load 7 mL of the cell suspension onto the column on magnet. After the flow has stopped, briefly remove the column from magnet in order to release captured unlabeled cells, and immediately place it back on the magnet. Add 1 mL of ice-cold MACS buffer and let flow through. 14. Repeat until all cells have been loaded. 15. Wash the column with 3 mL of ice-cold MACS buffer. Make sure the upper loading chamber is washed of all the cells. To elute, remove the column from the magnet and place in a culture tube. Add 7 mL of YPD (pH 4.5) supplemented with 100  mg/mL G-418 and 1:100 dilution of Pen-Strep (to inhibit contaminating bacterial growth), and use the plunger to push the eluted cell suspension into the tube. Check the titer of eluted cells by plating aliquots. 16. Expand the eluted cell population, and passage an aliquot representing at least tenfold the size of the enriched cell population. Set a culture for induction of the enriched cell population for further enrichment of antigen binders via fluorescence-activated cell sorting (FACS, see Note 7). 17. Pellet the induced cells in a microfuge at full speed (16,000– 21,000 × g) for 1 min and carefully aspirate the supernatant. 18. Wash the cells with 1-mL MACS buffer at room temperature. Vortex to resuspend the pellet. 19. Repeat for a total of three washes and resuspend the cells in MACS buffer (see Note 8). 20. Label the cells with 1:1,000 dilution in MACS buffer of rat anti-HA (100 mg/mL stock) and with the biotinylated antigen at the desired concentration. 21. Rotate the cells at room temperature for 25  min, and then incubate on ice for 5 min. 22. Pellet the cells at full speed for 30 s at 4°C and wash with 1-mL ice-cold MACS buffer. Repeat for a total of three washes. 23. Label the yeast cells with a 1:200 dilution in ice-cold MACS buffer, of Alexa Fluor 488 labeled donkey anti-rat IgG and of SA-PE. 24. Incubate the cells on ice for 15–20 min shielded from light. Mix the tubes once or twice during the incubation. 25. Spin the cells at full speed at 4°C and aspirate supernatant, and then wash with 1 mL ice-cold FACS buffer for a total of three washes (FACS buffer used here, instead of the Tween-20 containing MACS buffer, to prevent distortion in fluid dynamics in the flow cytometer). Spin the cells for the last time, and decant

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Fig. 3. Isolation of HEL-binding VLRB clones from a YSD library of 1 × 107 clones. Each dot-plot represents 10,000 events. (a) The unsorted library is stained with 500 nM HEL, with no visible antigen binders. (b) After one round of enrichment with anti-biotin magnetic microbeads (MACS), the small population of double-positive cells in the gate was sorted. (c) The output of the first fluorescence-activating cell sorting (FACS) separation shows an enriched population of true binders, (d) which were further enriched during the subsequent sort. (e) Representative individual clones resulting from this screen.

and keep the pellets on ice. Immediately prior to sorting, resuspend the cells in ice-cold FACS buffer at a concentration that is appropriate for your cytometer. Set a gate to sort and collect the population of double-positive cells (see Note 9). 26. Successful enrichment of antigen-binding clones will result in at least three- to fivefold increase in the population of doublepositive cells after the second round of MACS, and after each subsequent round of FACS. An example of the process is shown in Fig. 3.

4. Notes 1. A set of primers devoid of overhangs may perform better for the first round of amplification of VLRs from cDNA or genomic DNA. Use these primers first, and then switch to the set listed in Subheading  2.1, item 2: VLRANT.F 5-AAAACGTGTGAAACGGTCACAG; VLRACT.R 5-CT CCACGAATGGGCACTCATA; VLRBNT.F 5-GCATGTC CCTCGCAGTGTTC; and VLRBCT.R 5-TGGGCATTTC GAGGGGCTAG. 2. It is essential to store the 50% PEG solution securely capped to prevent evaporation, which over time will increase the PEG concentration and affect the efficiency of transformation. 3. PCR amplification of VLRAs yields substantial amounts of amplicons also from the nonassembled germline VLRA gene, which are invariant and include stop codons in all three

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frames. The germline amplicons are shorter than the amplicons of mature VLRAs and, in the intervening sequence, include a unique AviII restriction site (TGCGCA). To eliminate the germline amplicons, amplify VLRA amplicons for the minimal number of PCR cycles required to see a product in an agarose gel, digest the PCR product with AviII, and then gel purify the remaining longer band, which corresponds to the mature VLRA amplicons. These can be amplified again to obtain sufficient amount of DNA. 4. A colony from a freshly streaked plate yields the highest transformation efficiency. 5. Optimal transformation efficiencies are achieved only if the majority of cells have undergone two cell divisions. Adjust the inoculum in 2× YPD accurately to an OD546 of 0.15 and proceed to the next step only once the culture has reached an OD546 of 0.6. This may take 3–5 h or longer. 6. Using a flow cytometer (FACS), it is impractical in most cases to enrich antigen-binding clones from a primary library of 1 × 106 clones or larger, since the fraction of positive clones is usually below 0.01%. One or two rounds of enrichment with magnetic beads (MACS) should increase this fraction to a size that is practical for FACS. We describe here one round of MidiMACS enrichment. To use the MiniMACS, adjust the volumes proportionally. 7. Several cell samples are required every time the FACS is turned on to set the parameters for the cytometer and for color compensation: (1) sample of unstained cells; (2) sample of VLR surface display level (rat anti-HA followed by Alexa Fluor 488 labeled donkey anti-rat IgG); and (3) sample of antigen-binding level (a known control clone can be used, stained with biotinylated antigen followed by SA-PE). For any new antigen, it is recommended to stain uninduced cells with the biotinylated antigen followed by SA-PE to detect nonspecific biding on the surface of yeast. Since binders of the secondary reagents can also be enriched, it is important to frequently stain induced cell population with Alexa Fluor 488-labled donkey anti-rat IgG, and separately with SA-PE, which should stain <0.01% of the cells. In the case of high background staining, the secondary reagents may be replaced with alternatives. 8. Typical labeling volume is 1  mL for up to 1 × 109  cells and 50–100 mL for 1–5 × 106 cells. It is recommended to maintain at least tenfold molar excess of antigen over the yeast-displayed receptors to prevent depletion of the antigen. Assuming 2.5– 10 × 103 receptors per yeast cell, the receptor concentration for 106 cells in 100 mL is about 0.17 nM (104 × 106), and the lowest antigen concentration is, therefore, 1.7  nM. For lower antigen

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concentrations, increase the labeling volume proportionally. For the stage of labeling with secondary reagents, use 0.5 mL volume for 1 × 109 cells and 50–100 ml for 1–5 × 106 cells. 9. Yeast cells grow poorly in liquid culture at concentrations of less than 104  cells per  mL. When sorting small numbers of cells, use plates instead of liquid culture to recover the cells.

Acknowledgments The authors would like to thank Dr. K. Dane Wittrup and Ms. S. Annie Gai from Massachusetts Institute of Technology for introducing us to the fascinating world of yeast surface display (YSD). This work was supported by NSF grant MCB-0614672, NIH grant AI083892, and Intercenter Collaboration Grant, UMBI. References 1. Pancer, Z., Amemiya, C. T., Ehrhardt, G. R., Ceitlin, J., Gartland, G. L., and Cooper, M. D. (2004) Somatic diversification of variable lymphocyte receptors in the agnathan sea lamprey Nature 430, 174–80. 2. Pancer, Z., Saha, N. R., Kasamatsu, J., Suzuki, T., Amemiya, C. T., Kasahara, M., and Cooper, M. D. (2005) Variable lymphocyte receptors in hagfish Proc Nat Acad Sci USA 102, 9224–9. 3. Litman, G. W., Dishaw, L. J., Cannon, J. P., Haire, R. N., and Rast, J. P. (2007) Alternative mechanisms of immune receptor diversity Curr Opin Immunol 19, 526–34. 4. Pancer, Z., and Cooper, M. D. (2006) The evolution of adaptive immunity Annu Rev Immunol 24, 497–518. 5. Alder, M. N., Rogozin, I. B., Iyer, L. M., Glazko, G. V., Cooper, M. D., and Pancer, Z. (2005) Diversity and function of adaptive immune receptors in a jawless vertebrate Science 310, 1970–3. 6. Rogozin, I. B., Iyer, L. M., Liang, L., Glazko, G. V., Liston, V. G., Pavlov, Y. I., Aravind, L., and Pancer, Z. (2007) Evolution and diversification of lamprey antigen receptors: evidence for involvement of an AID-APOBEC family cytosine deaminase Nat Immunol 8, 647–56. 7. Tasumi, S., Velikovsky, C. A., Xu, G., Gai, S. A. Wittrup, K. D., Flajnik, M. F., Mariuzza, R. A., and Pancer, Z. (2009) High-affinity lamprey VLRA and VLRB monoclonal antibodies Proc Natl Acad Sci USA 106, 12891–96.

8. Marks, J. D., and Bradbury, A. (2004) Selection of human antibodies from phage display libraries Methods Mol Biol 248, 161–76. 9. Binz, H. K., Amstutz, P., and Plückthun, A. (2005) Engineering novel binding proteins from nonimmunoglobulin domains Nat Biotechnol 23, 1257–68. 10. Kim, H. M., Oh, S. C., Lim, K. J., Kasamatsu, J., Heo, J. Y., Park, B. S., Lee, H., Yoo, O. J., Kasahara, M., and Lee, J. O. (2007) Structural diversity of the hagfish variable lymphocyte receptors J Biol Chem 282, 6726–32. 11. Velikovsky, C. A., Deng, L., Tasumi, S., Iyer, M. L., Kerzic, M. C., Aravind, L., Pancer, Z., and Mariuzza, R. A. (2009) Structure of a lamprey variable lymphocyte receptor in complex with a protein antigen Nat Struc Mol Biol 16, 725–30. 12. Chao, G., Lau, W. L., Hackel, B. J., Sazinsky, S. L., Lippow, S. M., and Wittrup, K. D. (2006) Isolating and engineering human antibodies using yeast surface display Nat Protoc 1, 755–68. 13. Gai, S. A., and Wittrup, K. D. (2007) Yeast surface display for protein engineering and characterization Curr Opin Struct Biol 17, 467–473. 14. Swers, J. S., Kellogg, B. A., and Wittrup, K. D. (2004) Shuffled antibody libraries created by in  vivo homologous recombination and yeast surface display Nucleic Acids Res 32, e36.

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Chapter 3 Identification of Scavenger Receptor Ligands Claudine Neyen, Annette Pluddemann, and Siamon Gordon Abstract Scavenger receptors (SRs) are structurally diverse but functionally related innate immune receptors involved in defence and clearance mechanisms. Their broad specificity relies on evolutionarily conserved pattern recognition domains which interact with a variety of microbial, apoptotic and modified self ligands. Studies of immune functions of SR-expressing cells require the identification of interaction partners for SRs. We have developed an ELISA-based method which allows for large-scale high-throughput screening of complex mixtures. The assay successfully identified bacterial and plasma ligands for macrophage scavenger receptor A and can be adapted to screen for novel exogenous or endogenous ligands for any immune receptor of interest. Key words: Macrophage, Innate immunity, Scavenger receptor, Ligand, Receptor, Interaction, Screening, ELISA, Far Western Blot

1. Introduction Scavenger Receptors (SRs) belong to a multifunctional family of structurally diverse innate immune receptors, and have been termed molecular flypaper for their low affinity and broad specificity-binding properties (1). The eight distinct classes defined to date play essential roles in immune defence and host homeostasis through recognition of a variety of microbial ligands, apoptotic cell epitopes, and modified or aged self molecules (2, 3). Conventional assays for the identification of scavenger receptor ligands use whole-cell approaches, based on the competition for binding and/or uptake of known ligands by a new candidate or on direct association of the new candidate ligand with receptorexpressing cells. While these functional tests are ultimately needed for validation of a novel ligand, they are somewhat cumbersome

Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_3, © Springer Science+Business Media, LLC 2011

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to deploy on large numbers of ligand fractions. A novel solidphase assay for the identification of ligands for macrophage scavenger receptor A (SR-A) has been described recently and includes a cell-free detection system, where potential ligands coated on ELISA plates are detected with post-nuclear cell lysates from receptor-positive and receptor-negative cells, followed by receptor-specific antibodies (4). This ELISA-based method lends itself to large-scale, high-throughput ligand screens, as it depends on cell lysate rather than living cells and can be carried out in microtitre plates. This, in turn, allows for extensive ligand fractionation prior to the assay, and has facilitated the identification of ligands even from such complex mixtures as bacterial lysates or human plasma (5, 6). Furthermore, the ligand can be of any molecular species, i.e. nucleic acid, protein, lipid, or carbohydrate, as long as it can be immobilized on ELISA plates. For protein ligands, coupling of the ELISA-based assay to an immune receptor Far Western blot allows specific detection of ligand-containing bands on SDS-Polyacrylamide Gel Electrophoresis (PAGE) gels and subsequent identification by mass spectrometry. The present method details a specific application of ligand identification for macrophage SR-A, but can easily be adapted to any other receptor of interest.

2. Materials 2.1. Cell Culture and Preparation of Cell Lysates 2.1.1. Cell Culture of Primary Cells

1. Phosphate-buffered saline (PBS) (Gibco, Invitrogen, UK): 1 mM KH2PO4, 3 mM Na2HPO47H2O, 155 mM NaCl and pH 7.4. 2. For bone marrow-derived macrophages (BMDMf): RPMI 1640 culture medium (Gibco, Invitrogen, UK) supplemented with 50  IU/ml penicillin G, 50  mg/ml streptomycin and 2 mM l-glutamine (PSG), 10% (v/v) foetal calf serum (FCS) and 15% (v/v) L-cell conditioned medium (R10LCM15). LCM is obtained by growing L929 fibroblasts in DMEM, 10% FCS and 2 mM l-glutamine for 10 days (see Note 1). 3. For human monocyte-derived macrophages (MDMf): RPMI 1640 culture medium supplemented with PSG and 5% (v/v) heat-inactivated (56°C for 30 min) autologous human serum for isolation (R5); X-VIVO 10 culture medium (Bio-Whittaker, Reading, UK) supplemented with 2% (v/v) heat-inactivated autologous human serum for growth (X-VIVO2). 4. BMDMf and MDMf are strongly adherent and, therefore, are best grown on 15-cm diameter bacteriological plastic (BP) dishes (Greiner, Gloucester, UK) to facilitate lifting of cells.

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5. To lift cells, use either 0.05% trypsin and 10 mM EDTA in PBS (trypsin-EDTA) or 4 mg/ml lidocaine-HCl and 10 mM EDTA in PBS (lidocaine-EDTA). 2.1.2. Cell Culture of Cell Lines

1. For CHO cells stably transfected with SR-A (CHO-SR-A): MAC (Mevalonate, acetylated LDL and Compactin) selection medium: HAM’s F12 culture medium supplemented with PSG, 3% (v/v) lipoprotein-deficient FCS, 250  mM mevalonate, 40  mM compactin/mevastatin and 3  mg/ml acLDL (7); PBS as above. 2. Cell lines are grown in 175-cm2 tissue culture (TC) flasks. 3. To lift and passage cells, use trypsin-EDTA or lidocaineEDTA as for primary cells.

2.1.3. Cell Lysis

1. NP-40 protein lysis buffer: 150 mM NaCl, 10 mM EDTA, 10  mM NaN3, 10  mM Tris–HCl, pH 8.0, 1  mM phenyl­ methylsulphonyl fluoride (PMSF), 5 mM iodoacetamide and 1% (v/v) Nonidet P-40 (NP-40). Store at 4°C. EDTA-free protease inhibitor cocktail tablets (one tablet per 25 ml lysis buffer) (Roche, UK) are added immediately prior to use, and the remaining buffer-containing protease inhibitor cocktail can be frozen at −20°C. 2. Cell lifters (Greiner).

2.2. ELISA for Immune Receptor Ligands

1. 96-well EIA/RIA high-binding polystyrene plates (Costar, Corning, New York, USA). 2. Coating buffer: PBS (Gibco, Invitrogen, UK) or any nondetergent saline buffer at physiological pH. 3. Control ligands (for SR-A): Acetylated LDL (acLDL, positive control) (Molecular Probes, Eugene, OR, USA) is stored at 4°C for up to 6 months; bovine serum albumin (BSA, negative control) and maleylated BSA (malBSA, positive control), prepared by reacting maleic anhydride and BSA as described in (8), are stored as aliquots at −20°C (see Note 2). 4. Blocking and assay buffer: 10 mg/ml LPS-free BSA (Sigma, Poole, UK) and 5 mM EDTA (see Note 3) in PBS, stored at 4°C for up to a month. 5. Wash buffer: PBS containing 0.1% (v/v) Tween-20 (PBS-T). 6. Post-nuclear cell lysates with and without receptor (e.g. SR-A) (originating either from wild type and receptor knockout animals or from untransfected and receptor-transfected cell lines) are diluted to a concentration of 50–100 mg/ml cell protein in assay buffer (see Note 4). 7. Primary antibodies: 2F8, a rat anti-murine SR-A monoclonal antibody (IgG2b) (AbD Serotec, Oxford, UK), or C6,

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a mouse anti-human SR-A monoclonal antibody (IgG1) (a gift from T. Kodama, (9)), and isotype-matched control antibodies are diluted in assay buffer at 10 mg/ml (see Note 5). 8. Secondary antibody: Horseradish peroxidase (HRP)-coupled anti-rat or anti-mouse antibodies (0.8 mg/ml) (Jackson laboratories) are diluted 1:1,000 in assay buffer. 9. TMB colour reagent (BD Biosciences, Pharmingen) stored at 4°C, stop solution 8 M H2SO4 and absorbance plate reader (450 nm wavelength). 2.3. Far Western Blotting for Immune Receptor Ligands 2.3.1. SDS-Polyacrylamide Gel Electrophoresis

1. BioRad Mini Gel apparatus (or equivalent), glass plates, and combs. 2. Separating buffer (4×): 1.5  M Tris–HCl, pH 8.8, stored at RT. 3. Stacking buffer (4×): 0.5 M Tris–HCl, pH 6.8, stored at RT. 4. 10% (w/v) SDS stock solution in water, stored at RT. 5. 30% acrylamide/bisacrylamide solution (37.5:1) (Merck Chemicals, UK), stored at 4°C. 6. N,N,N,N ¢-tetramethyl-ethylenediamine (TEMED) (SigmaAldrich, UK), stored at RT. 7. Ammonium persulfate (AMPS), prepared fresh 10% (w/v) solution in H2O, or stored at 4°C for up to 1 month. 8. Running buffer (1×): 25 mM Tris–HCl, 90 mM glycine, 0.1% (w/v) SDS, stored at RT. 9. Prestained molecular weight marker: Rainbow marker (GE Healthcare, UK), stored at −20°C. 10. Loading buffer (2×): 200 mM Tris–HCl, pH 6.8, 8 M urea, 2% (w/v) SDS, 1  mM EDTA, 0.25% (w/v) bromophenol blue, stored at RT.

2.3.2. Transfer

1. BioRad Mini Trans-Blot electrophoretic transfer cell (or equivalent). 2. Transfer buffer (1×): 25 mM Tris–HCl, 90 mM glycine, 20% (v/v) methanol, stored at RT, cooled during use in the transfer apparatus. 3. Supported nitrocellulose membrane (HybondC Extra, Amersham, GE Healthcare, UK) and 3MM Chr chromato­ graphy paper (Whatman, Maidstone, UK). 4. Optional: Ponceau staining solution, Pierce, UK.

2.3.3. Far Western Blot

1. Wash buffer: PBS-0.1% (v/v) Tween-20 (PBS-T). 2. Blocking and assay buffer: 4% (w/v) non-fat dry milk in PBS-T with 5 mM EDTA, prepared fresh.

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3. Post-nuclear cell lysate with and without SR-A as for ELISA, diluted to 0.6 mg/ml in assay buffer (see Note 4). 4. Primary and secondary antibodies as for ELISA, diluted in assay buffer to 10 mg/ml (primary Ab) and 1:2,000 (secondary Ab). 5. ECL reagents (Amersham, GE Healthcare, UK), Kodak film, film cassette. 6. Western blot stripping reagent (Restore Western Blot) (Pierce, Thermo Fisher Scientific, UK).

3. Methods 3.1. Cell Culture and Preparation of Cell Lysates 3.1.1. Cell Culture of Primary Cells

3.1.2. Cell Culture of Cell Lines

Methods for the isolation and maturation of murine BMDMf and human MDMf have been detailed elsewhere (10, 11). Briefly, isolate murine bone marrow from the femora and tibiae of wild type and receptor knockout mice and plate the equivalent of 2 bones per 15-cm diameter BP dish in 20 ml R10LCM15 medium. After 3 days of culture, feed cells with 15 ml R10LCM15, and after 6 days, replace medium with 20 ml R10LCM15 and replate any non-adherent cells to a new dish. At this stage, the culture should consist of differentiated BMDMf, identifiable by their spread morphology. Expression of various immune receptors may vary over time and with maturation or activation state in primary cells. It may be useful to quantify receptor expression in cell lysates by Western Blot. For human MDMf, isolate mononuclear cells by FicollHypaque density sedimentation, remove platelets by repeated washes in PBS and purify adherent monocytes via a 90  min incubation on gelatine-coated TC dishes in R5 medium, followed by repeated washes with warm PBS. After 24  h, lift adherent monocytes with lidocaine-EDTA and replate on 15-cm diameter BP dishes in X-VIVO2. Cells are ready to use after 5–7 days in culture. It may be of interest to prepare receptor-containing cell lysate from transfected cell lines rather than primary cultures, firstly because this allows the generation of large quantities of cell lysates without the need for laboratory animals and secondly because non-macrophage cell lines are likely to lack most immune receptors resulting in less non-specific background binding. For SR-A, the use of transfected cell line-derived lysates in Far Western blots considerably reduced background compared to BMDMf lysates. Culture cells to confluence in 175-cm2 TC flasks, maximizing receptor expression by targeted selective pressure where possible. For example, stably transfected CHO-SR-A cells are driven to high levels of SR-A expression by MAC selection medium, where

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serum is replaced with lipoprotein-deficient serum and the ­cholesterol necessary for cell proliferation is added in the form of SR-A-restricted acetylated LDL, leading to preferential expansion of SR-AHi cells. 3.1.3. Cell Lysis

1. Wash cells in 15-cm Petri dishes or 175-cm2 tissue culture flasks 1× with 10–15 ml ice-cold PBS. 2. Lift adherent cells by incubating 15–30  min with 3–5  ml lidocaine-EDTA on ice (see Note 6). 3. Detach cells by vigorous pipetting and by using a cell scraper and transfer loose cells to a 50-ml Falcon tube. Collect a maximum number of cells from the flask or dish by thoroughly rinsing with PBS (see Note 7). 4. Pellet cells by centrifugation for 5 min at (300×g) and discard supernatant. 5. Wash cells 2× by resuspending in 50 ml ice-cold PBS. This will remove the traces of lidocaine-EDTA. 6. Resuspend cell pellet in lysis buffer (1 ml per 107 cells) (see Note 8) by pipetting up and down until the cells are lysed (the cell suspension will turn transparent). Transfer into 1.5ml Eppendorf tubes and incubate 10–15  min on ice, with intermittent vortexing. 7. Remove nuclei and cellular debris by centrifugation of cell lysates for 5 min at maximal speed (14,100 × g) in a tabletop centrifuge. 8. Carefully remove clear supernatant and transfer to a new tube. 9. Measure protein concentration of the post-nuclear lysate, and store aliquots at −20°C, for up to 12 months.

3.2. ELISA for Immune Receptor Ligands

The ELISA detection method for novel immune receptor ligands is based on the following principle: the presence of candidate ligands coated on ELISA plates (see Note 9) is assessed in parallel with lysates from receptor-positive and receptor-negative cells. Bound receptor is detected with anti-receptor antibody followed by HRP-coupled secondary antibody (Fig. 1). Candidate ligands promoting receptor-specific binding are identified by comparing the wells detected with receptor-positive lysate to those detected with receptor-negative lysate (Fig. 2). An additional control can be inserted by replacing the anti-receptor antibody with an isotype control. Normalization of assay results to an internal control permits inter-assay comparability (see Note 10). The method described below is valid when either primary cells (WT and receptor knockout control) or cell lines (transfected with receptor and untransfected control) are used as the source of immune receptor. When using human MDMf, where no

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Fig. 1. Ligand identification ELISA: Controls. (a) Schematic ELISA. The minimal requirements for controlled ligand detection are conditions in wells 1–4 (positive/negative ligand control, positive/negative receptor control); wells 5–6 can be included to optimize the elimination of false positives (antibody control). (b) Experimental illustration of the schematic ELISA in (a). Each data point represents the average of triplicate wells in one distinct experiment. For all experiments, malBSA (positive ligand) and BSA (negative ligand) were coated at 125–500 ng/well and detected with either WT (receptor-positive) or SR-A−/− (receptor-negative) BMDMf lysate (62.5 mg/ml), followed by monoclonal rat anti-murine SR-A antibody or isotypematched control (10 mg/ml). One-way ANOVA with Tukey’s multiple comparisons test shows that well 1 is significantly different from all other conditions (P < 0.001), demonstrating the test’s power to reliably identify true ligands. All other means do not differ significantly, with the exception of well 2 compared to well 4, indicating that the non-specific background from receptor-positive lysates is significantly higher (P < 0.05) than that from receptor-negative lysates and that there is a marginal risk of identifying false positives. (c) ELISA documenting receptor-specific binding to various polyanions, including protein (AcLDL, positive control; BSA, negative control), polysaccharide (fucoidan, dextran sulphate, positive controls; chondroitin sulphate, negative control) and polynucleotide (poly I, positive control; poly C, negative control) ligands, coated at 10 mg/ml and detected as described in (b). Bars represent average + SD of quintuplicate repeats.

receptor-deficient control is available, the isotype control antibody must be included to provide a negative control. 1. The day before the ELISA, coat 96-well high-bind ELISA plates with 50  ml of control ligands at 10  mg/ml in PBS (acLDL or malBSA as positive control and BSA as negative control, in the case of SR-A) and 50 ml of candidate ligandcontaining samples (if concentration is known, coat at 10 mg/ml in PBS). Every sample should be coated at least in duplicates

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Fig. 2. ELISA ligand screen. The described ELISA was applied to fractions of delipidated human plasma eluted from an anion exchange column. Graph A shows traces obtained after detecting with receptor-positive lysate and anti-receptor antibody (filled circle), receptor-negative lysate and anti-receptor antibody (open circle) or receptor-positive lysate and isotype control antibody (triangle). In early eluting fractions (up to 30), all three traces overlap, probably indicating a component in these fractions which interacts with the assay components independently of the receptor. Conversely, traces in fractions 80–100 differ, indicating the presence of receptor-specific ligand. In Graph B, visualization of true positive candidate ligands has been enhanced by subtracting the trace from receptor-negative detection from the receptorpositive detection, yielding a receptor-specific signal trace. Due to the residual risk of identifying false positives described in Fig. 1b, it is likely that only candidate ligands in fractions 80–100 will prove true in further experiments, but candidates in fractions up to 30 cannot immediately be discarded.

to allow detection with receptor-positive and receptor-deficient cell lysates, followed by anti-receptor antibody. If enough materials are available, coat a third replicate for detection with receptor-positive lysate followed by isotype control antibody, to further limit the risk of false positives. Wrap plates in moist tissue paper and cellophane to avoid evaporation and incubate at 4°C overnight. Alternatively, coating can also be done on the day for 1 h at 37°C. 2. The next day, wash plates three times by immersing in PBS-T and vigorously inverting over a sink. After the last wash, tap plates face-down on tissue paper to absorb the remaining wash buffer. A plate washer can be used where available.

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3. All subsequent steps are done at room temperature. 4. Block wells by adding 150 ml blocking buffer and incubating for 2 h. During the blocking step, prepare the reagents for all subsequent steps and store at 4°C until needed. (a) Cell lysates: 50  ml per well, at 62.5  mg/ml cell protein (see Note 4) in blocking buffer. Total volume required = 50 ml × (no. of control ligands + no. of samples to test), prepare once with lysate from receptor-positive cells and once with lysate from receptor-deficient cells. If including an isotype control, double the volume of solution containing receptor-positive cell lysate (see Note 11). (b) Primary antibody (anti-receptor): 50  ml per well at 10 mg/ml in blocking buffer. Volume required = 50 ml × (no. of control ligands + no. of samples) × 2. If including an isotype control (also at 10 mg/ml), the volume required for isotype control antibody is 50  ml × (no. of control ligands + no. of samples). (c) HRP-coupled secondary antibody: 50 ml per well, diluted 1:1,000 in blocking buffer. Volume required = 50 ml × (no. of control ligands + no. of samples) × 2 or, if including an isotype control, 50  ml × (no. of control ligands + no. of samples) × 3. 5. Wash plates three times as described in step 2. 6. Add 50  ml receptor-positive cell lysates to one replicate of control and sample wells and 50  ml receptor-deficient cell lysates to the second replicate, and incubate for 2 h. If including an isotype control, you will have coated a third replicate of controls and samples; add 50 ml receptor-positive cell lysates to this replicate. 7. Wash plates five times as described in step 2. 8. Add 50 ml anti-receptor antibody to all wells and incubate for 1 h. If including an isotype control antibody at this step, add isotype control antibody to the extra replicate incubated with receptor-positive cell lysate at step 5. 9. Wash plates seven times as described in step 2. 10. Add 50 ml HRP-coupled secondary antibody to all wells and incubate for 45  min to 1  h. During this step, prepare adequate volume of TMB reagent: 50  ml × (no. of control ligands + no. of samples) × 2 (or × 3 if an isotype control antibody has been included) and allow warming to RT. Make sure an equal volume of stop solution is ready to use. 11. Wash plates nine times as described in step 2. 12. Add 50 ml TMB colour reagent to all wells, and wait for blue colour to develop (usually within 5–10 min). Once the positive

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control wells have reached an adequate colour intensity (deep turquoise colour), stop the reaction by adding 50 ml stop solution to all wells (wells turn yellow). Remove air ­bubbles (e.g. with a needle or pipette tip) and measure the absorbance at 450 nm in an absorbance plate reader. Avoid delays between the end of the assay and the read-out, since not all colour reagents remain stable even at 4°C and in the dark. 3.3. Far Western Blotting for Immune Receptor Ligands

While the ELISA-based immune receptor ligand screen facilitates isolation of candidate ligands from a large pool of molecules, such as human plasma or bacterial lysate, the immune receptor Far Western Blot is a preferable tool for the final identification of protein candidate ligands from semi-purified mixtures, since it includes an inherent fractionation step (SDS-PAGE) and can single out individual ligand-containing bands. Ideally, the immune receptor

Fig. 3. Far Western blot. (a) 5 mg/lane of BSA (− ligand) or malBSA (+ ligand) were separated by 8% SDS-PAGE, transferred to blotting membrane and detected by Far Western blot, with either receptor-positive lysate or receptor-negative lysate followed by anti-receptor antibody. Monomeric malBSA runs at 67 kDa; the higher MW bands contain multimeric aggregates. (b) 10  mg/lane of ligand-containing samples (total plasma lipoproteins (LP); lipoprotein-deficient plasma (LPDP)) were fractionated on a 6–20% SDS-PAGE gel, transferred to blotting membrane and detected by Far Western blot, with receptor-positive lysate and anti-receptor antibody, receptor-negative lysate and anti-receptor antibody or receptor-positive lysate and an isotype-matched control antibody. Circled bands appear only in the presence of receptor and anti-receptor antibody, and therefore correspond to candidate ligands. Other bands represent non-specific interaction with antibody as they appear in the isotype-matched control blot. (c) Sequential ion exchange fractions from human plasma were separated by 12% SDS-PAGE, transferred to membrane and detected by Far Western blot, with receptorpositive lysate and anti-receptor antibody, receptor-negative lysate and anti-receptor antibody or receptor-positive lysate and an isotype-matched control antibody. Several candidate ligands are identified above 50 kDa in fractions 1–4 and below 25 kDa in fractions 4–12.

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ELISA and the Far Western Blot should be coupled to identify protein candidate ligands from complex mixtures (Fig. 3). The same detection reagents as for ELISA apply to the Far Western Blot: cell lysates with and without receptor, anti-receptor antibody and HRP-coupled secondary antibody. To reduce the probability of false positives, an isotype control antibody for the anti-receptor antibody can be included, and is mandatory if using cell lysate from human MDMf, where no receptor-deficient control is available (see Note 12). 3.3.1. SDS-Polyacrylamide Gel Electrophoresis

These instructions assume the use of a BioRad Mini Gel apparatus; however, any other apparatus is also appropriate and volumes should be adjusted accordingly. When casting gels, it is essential to use clean glass plates and combs and minimize protein contamination (e.g. keratin) to optimize the conditions for subsequent mass spectrometry. 1. Prepare a gel of appropriate percentage using the component table below. (If no hypothesis or information is available on the projected size of the potential ligand of interest, a gradient gel (4–12% or 6–20%) is recommended). For optimal polymerization (if planning subsequent mass spectrometry), cast gels at 37°C.

6%

4% stacking gel

(per 1.5 mm gel)

20%

15% 12% 10% 8%

H2O (ml)

0.55

1.88 2.68 3.2

3.75 4.28 1.22

Tris–HCl (ml)

2

2

2

2

2

2

0.5

SDS 10% (ml)

80

80

80

80

80

80

20

30% Acryl V Bis (ml) 5.33

4

3.2

2.67 2.13 1.6

0.25

10% AMPS (ml)

40

40

40

40

40

40

20

TEMED (ml)

4

4

4

4

4

4

4

Total (ml)

8

8

8

8

8

8

2

2. While the gel is polymerizing, prepare samples. Detection of ligands by the Receptor Far Western Blot is optimal for blots with at least 5 mg protein per band; keep this in mind when running complex mixtures containing low levels of candidate ligand(s). Ligands should be in a low salt buffer to avoid smearing of gels. Depending on the thickness of the gel and the comb used, wells will take between 15 and 45 ml sample. Mix equal volumes of 2× loading buffer and sample, and heat at 95°C for 10 min. You will need to run at least two duplicates of the gel, one for protein staining (Coomassie Blue or

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other mass spectrometry-compatible stain) and one for transfer to blotting membrane. If you prefer to run Far Westerns with and without receptor simultaneously (recommended, see Note 12) rather than stripping and detecting consecutively, you will need two gels (or three if including an isotype control) for transfer. 3. Load gel and run at 160 V (constant voltage) for 50–60 min or until dye front has run out of the gel. 3.3.2. Transfer

These instructions assume the use of a BioRad Mini Trans-Blot electrophoretic transfer cell, but any other transfer apparatus is also appropriate. 1. While gels are running, prepare the following (for 1 gel transfer): One gel holder cassette, two fibre pads, four Whatman paper rectangles (9 × 7 cm for Mini gels), blotting membrane (8 × 6  cm for Mini gels) and tweezers. Wet fibre pads and blotting membrane by floating in transfer buffer. 2. Assemble the blotting cassette inside a container with transfer buffer by stacking one fibre pad, two Whatman papers, the gel, the blotting membrane, two Whatman papers and the second fibre pad on top of each other, submerged in transfer buffer. Gently press out any air bubbles from between the layers and close the cassette. Insert cassette(s) into transfer cell, add cooling block and fill to top with transfer buffer. Run at 120 mA (constant current) for 1.5–2 h (larger proteins take longer to transfer). (Optional: verify transfer by staining gel in Ponceau solution. This is recommended when using multiple replica membranes for simultaneous detection rather than a single membrane for sequential detection with stripping, in order to reliably compare banding patterns from different blots).

3.3.3. Far Western Blot

Apart from overnight blocking, all steps are carried out at RT on a horizontal rocker. Use a container fitted to the membrane size to minimize the amount of reagent necessary at each step. While rocking, the membrane should always be covered with liquid. For a mini gel-sized blot, 5  ml is a reasonable volume of lysate or antibody solution (see Note 13). 1. Block the membrane overnight at 4°C or for 1  h at RT in blocking buffer. 2. Wash 3× for 5 min in PBS-T. 3. Incubate for 2 h with cell lysates in blocking buffer. 4. Wash 3× for 5 min in PBS-T. 5. Incubate for 1 h with anti-receptor antibody or isotype control antibody in blocking buffer. 6. Wash 3× for 5 min in PBS-T.

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7. Incubate for 1 h with secondary antibody in blocking buffer. 8. Wash 3× for 5 min in PBS-T. While washing, prepare ECL reagent. 9. Add 1 ml ECL reagent to an 8 × 6 cm membrane, incubate for up to 5 min, lift the membrane, and then blot off excess ECL reagent. Fold the membrane between cellophane layers and press out the remaining ECL reagent by pressing with a paper towel. Place into X-ray film cassette. 10. Take film cassette with blot and Kodak film to dark room and develop at several exposure times. Comparison of Far Western blots developed with and without receptor should make it possible to single out ligand-containing bands on the corresponding SDS-PAGE gels. Cut out bands of interest and proceed to mass spectrometric identification of candidate ligands (see Note 14).

4. Notes 1. LCM provides the growth and differentiation factor CSF-1 which drives SR-A expression (12); for reproducibility, conditioned medium can be replaced with recombinant CSF-1 (500 U/ml or 0.22 nM) (13). 2. The following non-protein control ligand pairs can be used for SR-A ligand assays: nucleotides: polyinosinic acid (poly I, positive control) and polycytidylic acid (poly C, negative control), both from Sigma, UK; sulphated polysaccharides: fucoidan (derived from Fucus vesiculosus, positive control) from Sigma, UK, dextran sulphate (MW ~500,000 Da, positive control) and chondroitin sulphate (negative control) from Pharmacia Biotech, Uppsala, Sweden. Coat at 10 mg/ml as described for protein ligands. 3. Since SR-A is a divalent metal ion-independent receptor, EDTA increases stringency and reduces background. It may be necessary to omit EDTA if screening for ligands of a divalent metal ion-dependent receptor. 4. Adjust cell lysate concentration according to the availability of receptor. SR-A expression is increased in BMDMf by the presence of CSF-1 in the culture medium or by selective pressure through MAC medium in CHO-hSRA cells. It may also be necessary to survey receptor expression fluctuation over time to determine the best time point to lyse the cells; SR-A levels decrease rapidly in BMDMf after day 9 (post-isolation) of culture. Ideally, verify the receptor presence by Western

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Blot using the same antibody that will be used in ELISA and Far Western Blot. 5. Anti-receptor antibodies must not be blocking or recognizing the receptor-binding site, i.e. not promoting receptor– ligand dissociation, and must be able to recognize the receptor in ligand-bound form. Some receptors may bind their ligands only in a restricted pH range; SR-A, for example, is an endocytic receptor which releases its cargo in the endosomal compartment (pH below 5), therefore a low pH of the assay buffer may result in poor or no ligand binding. 6. Unless the immune receptor is trypsin-resistant, avoid cell contact with trypsin-EDTA before cell lysis. This also applies to passaging cell lines – if using trypsin-EDTA, allow sufficient generation time between cell passaging and cell lysis to avoid loss of surface receptor. 7. Lifting and pelleting of easily detached cells avoid cell loss from washes, allow lysis in a minimal volume, and subsequently result in highly concentrated lysates. However, strongly adherent cells can be lysed in the flask to prevent the loss of materials due to incomplete collection of cell lawns. In this case, wash cells 5× with 10–15  ml ice-cold PBS in the dish or flask, add 1 ml ice-cold lysis buffer per 107 cells to the dish or flask, incubate on ice for 10 min, scrape off cells with a cell lifter, and then carefully collect all lysate into 1.5-ml Eppendorf tubes. Proceed with clearing the lysates as described. 8. The number of cells recovered from a 175-cm2 TC flask or a 15-cm BP dish depends on the size of cells, as well as the strain or cell line. For BMDMf cultures from C57BL/6  J mice, 2 × 107 to 6 × 107 cells can be expected per plate. 9. Immobilization of ligands on ELISA plates may induce conformational changes. Since scavenger receptors are in part defined by their ability to bind modified self molecules, candidate ligands identified through this ELISA method should be considered with a note of caution, as they may have formed during immobilization. It is, therefore, crucial to confirm candidates by functional tests other than the ELISA or Far Western blot used for their identification. 10. Inter-assay comparability is achieved by normalizing samples to the internal positive control as follows: SR - A - specific binding (% ) =

-/(WTsample - SRA sample ) -/(WTpositive control - SRA positive control )

´ 100

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11. The ELISA can be modified to test competition between known and novel receptor ligands. In this case, incubate lysates for step 6 with a 40-fold molar excess of known ligands at RT while wells are blocking. Proceed as described. 12. The same blot can either be produced in replicates (to ­circumvent intrinsic variability between blots due to selfcasting of gels, use pre-cast gels) or repeatedly stripped and reprobed; in the latter case, start with negative controls (isotype and/or receptor-deficient lysate) and then strip and reprobe with cell lysate containing the receptor. This order of detection avoids “bleed-through” on control blots from strong receptor-reactive bands that were incompletely stripped. Replicate membranes have the advantage of carrying identical amounts of proteins (assuming reproducible loading of gels) which is not guaranteed with repeatedly stripped membranes. However, stripping and redetection of a single membrane may facilitate ligand identification from complex banding patterns, as films obtained from the same membrane can be superimposed. 13. To further minimize cell lysate and antibody solutions, enclose membranes in 15- or 50-ml Falcon tubes, protein side facing towards the tube contents, and incubate on a rolling stirrer. Watch out for leakage. 14. Once a candidate ligand has been identified, a variety of functional assays can be used to confirm receptor–ligand interaction in a whole cell setting. For example, wild type and receptor-knockout BMDMf can be compared for their ability to bind, endocytose, and degrade fluorescently or radioactively labelled ligands. References 1. Krieger, M., Acton, S., Ashkenas, J., Pearson, A., Penman, M., and Resnick, D. (1993) Molecular flypaper, host defense, and atherosclerosis. Structure, binding properties, and functions of macrophage scavenger receptors, J Biol Chem 268, 4569–4572. 2. Plüddemann, A., Mukhopadhyay, S., and Gordon, S. (2006) The interaction of macrophage receptors with bacterial ligands, Expert Rev Mol Med 8, 1–25. 3. Plüddemann, A., Neyen, C., and Gordon, S. (2007) Macrophage scavenger receptors and host-derived ligands, Methods 43, 207–217. 4. Plüddemann, A., Neyen, C., Gordon, S., and Peiser, L. (2008) A sensitive solid-phase assay for identification of class A macrophage scavenger receptor ligands using cell lysate, J Immunol Methods 329, 167–175.

5. Peiser, L., Makepeace, K., Pluddemann, A., Savino, S., Wright, J. C., Pizza, M., Rappuoli, R., Moxon, E. R., and Gordon, S. (2006) Identification of Neisseria meningitidis nonlipopolysaccharide ligands for class A macrophage scavenger receptor by using a novel assay, Infect Immun 74, 5191–5199. 6. Neyen, C., Plüddemann, A., Roversi, P., Thomas, B., Cai, L., van der Westhuyzen, D., Sim, R. B., and Gordon, S. (2009) Macrophage scavenger receptor A mediates adhesion to apolipoproteins A-I and E, Biochem 48, 11858–11871. 7. Gough, P. J., Greaves, D. R., Suzuki, H., Hakkinen, T., Hiltunen, M. O., Turunen, M., Herttuala, S. Y., Kodama, T., and Gordon, S. (1999) Analysis of macrophage scavenger receptor (SR-A) expression in human aortic

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atherosclerotic lesions, Arterioscler Thromb Vasc Biol 19, 461–471. 8. Butler, P. J., Harris, J. I., Hartley, B. S., and Lebeman, R. (1969) The use of maleic anhydride for the reversible blocking of amino groups in polypeptide chains, Biochem J 112, 679–689. 9. Tomokiyo, R., Jinnouchi, K., Honda, M., Wada, Y., Hanada, N., Hiraoka, T., Suzuki, H., Kodama, T., Takahashi, K., and Takeya, M. (2002) Production, characterization, and interspecies reactivities of monoclonal antibodies against human class A macrophage scavenger receptors, Atherosclerosis 161, 123–132.

10. Davies, J. Q., and Gordon, S. (2005) Isolation and culture of human macrophages, Methods Mol Biol 290, 105–116. 11. Davies, J. Q., and Gordon, S. (2005) Isolation and culture of murine macrophages, Methods Mol Biol 290, 91–103. 12. de Villiers, W. J., Fraser, I. P., and Gordon, S. (1994) Cytokine and growth factor regulation of macrophage scavenger receptor expression and function, Immunol Lett 43, 73–79. 13. Stanley, E. R. (1997) Murine bone marrowderived macrophages, Methods Mol Biol 75, 301–304.

Chapter 4 Construction, Expression, and Purification of Chimeric Protein Reagents Based on Immunoglobulin Fc Regions John P. Cannon, Marci O’Driscoll, and Gary W. Litman Abstract Recombinant fusion proteins incorporating experimental protein domains fused to immunoglobulin Fc regions have become widely utilized in studies of protein–ligand interactions. The advantages of these systems include an inherent increase in avidity provided by the multimerization of Fc regions, combined with robust detection methods based on numerous commercially available secondary reagents directed against the Fc tag. We describe a set of methods for subcloning, expression, and purification of chimeric protein reagents containing a protein domain (or domains) of interest fused to a C-terminal moiety derived from the Fc region of either IgG or IgM. Key words: Chimeric protein, Immunoglobulin, Fc, IgG, IgM, In vivo biotinylation, Soluble receptor, Avidity, Binding

1. Introduction Recombinant protein reagents in which a protein domain of interest has been fused to the Fc region of immunoglobulin (Ig) (typically IgG) have been used in diverse applications that require detection of binding of a receptor (or other) domain to target ligands (1–5). The IgG Fc moiety confers an intrinsic dimerization and bivalent binding function to the protein (with even higher valency provided by IgM Fc regions), providing an increase in avidity of binding; such an increase can be invaluable in the study of interactions of relatively low affinity. In addition, the widespread commercial availability of diverse, labeled secondary serological reagents capable of detecting the Fc region enhances experimental utility of such reagents.

Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_4, © Springer Science+Business Media, LLC 2011

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Plasmid DNA constructs

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Fig. 1. Schematic diagram of in vivo biotinylation of Fc-chimeric proteins.

We have generated a series of expression vectors that allow the rapid subcloning of cDNA fragments encoding protein domains of interest to the Fc regions of either human IgG1 or mouse IgM. Several of these vectors also encode a C-terminal target sequence for Escherichia coli biotin ligase (the product of the BirA gene); when included, this sequence allows the sitespecific addition of a biotin tag to a defined lysine residue near the C-terminus of the recombinant Fc fragment (Fig. 1) (6–8). The protocols below describe the subcloning and transfection of plasmid DNA vectors encoding Fc fusions. In addition, procedures for harvesting of cell culture supernatants, determination of protein yields and pull-down assays using in  vivo biotinylated Fc reagents are described.

2. Materials 2.1. Subcloning of cDNA Fragments Encoding Receptor Ectodomains into an Expression Vector

1. Expression vector plasmid DNA preparation(s) (either constructed locally or provided from external sources). 2. Sf i I restriction enzyme, restriction enzyme buffer, and acetylated bovine serum albumin (BSA) (New England Biolabs). 3. Polymerase chain reaction (PCR) reagents, including PCR buffer, deoxynucleotides, and Taq polymerase. (Any of several high-fidelity thermostable polymerases also can be used to reduce PCR-induced mutations during synthesis). 4. Chroma Spin 400 spin columns (Clontech, Mountain View, CA).

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5. Agarose gel electrophoresis reagents. 6. Costar Spin-X centrifuge tube filters (Corning Life Sciences, Corning, NY). 7. T4 DNA ligase and buffer. 8. Transformation-competent, recombination-deficient E. coli cells (e.g., TOP10, DH10B, and XL-1Blue); TOP10 (Invitrogen) are described in this protocol. 9. SOC bacterial growth medium (Invitrogen). 10. LB plates/broth containing 50 mg/ml ampicillin. 11. Qiagen plasmid miniprep/midiprep (Qiagen). 2.2. Culture and Transfection of HEK293T

1. HEK293T Cell culture medium: RPMI supplemented with 10% fetal bovine serum (FBS, HyClone), 2 mM GlutaMAX (Invitrogen) or l-glutamine (Cellgro, Mediatech), and 1 mM sodium pyruvate (Cellgro). 2. Opti-MEM I Reduced Serum Medium (Invitrogen). 3. Tissue culture incubator and necessary accessories, including cell culture plates and flasks. 4. 0.25% trypsin/1  mM ethylenediamine tetraacetic acid (EDTA) solution (Cellgro). 5. Lipofectamine 2000 (Invitrogen). 6. 100  mg/ml NaOH.

2.3. Harvesting, Purification, and Concentration of Fc Chimeric Proteins

d-biotin

(Sigma-Aldrich). Dissolved in 1  N

1. Sodium azide (Sigma-Aldrich). Prepare as stock solution of 4% in water; dilute to a final concentration of 0.02% as preservative. 2. rProteinA Sepharose Fast Flow beads (GE Healthcare). 3. Dulbecco’s phosphate-buffered saline (PBS), purchased as 10× concentrate (Cellgro). 4. 2 M Tris(hydroxymethyl)aminomethane (Tris) pH 9.0. 5. 100 mM glycine pH 2.5. 6. Amicon Ultra Centrifugal Filter Devices (Millipore).

2.4. Measuring Expression Levels by Dot Blot or Western Blot

1. Transfer membranes [either supported nitrocellulose (Nitropure, Micron Separations, Inc.) or polyvinylidene fluoride (PVDF, Immobilon P, Millipore)]. (Nitrocellulose may be used for either dot blot or Western blot; PVDF should be used only for Western blot). 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel casting plates and running apparatus. 3. 2 M Tris pH 9.

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4. 1 M Tris pH 6.8. 5. 40% acrylamide/bis-acrylamide solution (37.5:1, Electrophoresis Grade). Unpolymerized acrylamide is a neurotoxin; care should be taken to avoid inhalation, ingestion, or skin contact. 6. 20% (w/v) sodium dodecyl sulfate (SDS). 7. 10% (w/v) ammonium persulfate (APS); store in aliquots at −20°C. 8. N,N,N ¢,N¢-tetramethylethylenediamine (TEMED). 9. Water-saturated 1-butanol (n-butanol). Prepare by shaking equal volumes of water and 1-butanol together in a glass bottle and allowing phases to separate. Use the top (organic) phase. Store at room temperature. 10. SDS-PAGE running buffer: 25  mM Tris, 112  mM glycine, and 0.1% (w/v) SDS. Do not adjust pH. Store at room temperature. 11. Prestained molecular weight markers (such as Precision Plus Protein Dual Color Standards, Bio-Rad). 12. 4× SDS-PAGE loading buffer: 180  mM Tris pH 6.8, 40% glycerol, 4% SDS, 200  mM dithiothreitol (DTT), and bromophenol blue to color (only a few crystals are necessary per 10 ml of 4× stock). 13. Western Transfer Buffer: 20  mM Tris, 15  mM glycine, 1% SDS, and 20% (v/v) methanol. Do not adjust pH. Store at room temperature. 14. Genie gel blotting system and accessories (Idea Scientific Company) or equivalent apparatus. 15. Methanol (for PVDF membranes). 16. Tris-buffered saline (TBS): 140 mM NaCl, 27 mM KCl, and 250 mM Tris pH 7.4. This solution can be prepared as a 10× stock and diluted in water as needed. 17. TBS containing 0.1% Tween-20 (TBST). 18. 10% casein solution (Western Blocking Reagent, Roche). Diluted to 1% in TBS to make blocking buffer. 19. Horseradish peroxidase- or alkaline phosphatase-conjugated goat anti-human IgG, Fcg fragment-specific (Jackson Immunoresearch). 20. Horseradish peroxidase- or alkaline phosphatase-conjugated streptavidin (Jackson Immunoresearch). 21. DSB-X biotin goat anti-mouse IgM (Invitrogen). 22. Enhanced chemiluminescent (ECL) reagents (ECL Western Blotting Substrate, Pierce). 23. Alkaline phosphatase detection reagents.

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1. Dynabeads MyOne Streptavidin T1 magnetic beads (Dynal/ Invitrogen). 2. TBST. 3. Dynal magnetic particle concentrator (Dynal/Invitrogen). 4. Experimental solution representing the source of candidate interaction partners for an Fc chimeric protein. 5. 2× SDS-PAGE loading buffer (described above in Subheading 2.4).

3. Methods In the procedures below, cDNA fragments encoding selected domains are amplified by PCR such that each end bears an Sf i I site (see Note 1) compatible for insertion into a vector that encodes an Ig Fc region (IgG or IgM) and can include a C-terminal recognition site for E. coli biotin ligase (“Avitag”, http:// www. avidity.com; see also (9)). Constructs are transfected into human embryonic kidney (HEK293T) cells for expression. To produce in vivo biotinylation of the Fc reagents, Fc expression constructs are cotransfected with a second expression vector encoding a secreted form of E. coli biotin ligase, which enzymatically biotinylates the Avitag peptide at a single, defined lysine residue located six amino acids from the C-terminus. Site-specific, in vivo biotinylation proceeds very efficiently; in addition, in contrast to nonspecific chemical biotinylation (which we have encountered as problematic), potential interference with binding activity due to biotinylation of critical amino acids within the recombinant Ig domain is not a significant concern. Sf i I sites have been integrated into the vector because of the rare recognition site of Sf i I (5¢-GGCCNNNN^NGGCC) and the resulting capacity to produce two different, incompatible DNA ends after enzymatic cleavage by varying the two intervening sequences within the recognition sites (e.g., SfiIA and SfiIB; see Fig. 2). Expressed constructs can interact with either protein A or streptavidin/avidin for use in purification or affinity assays. Nonbiotinylated forms can be obtained by omitting transfection of the separate BirA gene, in which case anti-Ig secondary reagents or protein A products are used to detect or purify the chimeric forms. IgM Fc regions form pentamers (and hexamers) of individual dimeric Fc molecules, thus increasing the avidity over dimeric IgG-chimeric reagents, and thereby potentially facilitating the detection of low-affinity interactions. In the vectors used in our laboratory, the IgM Fc retains its hinge region, increasing the steric flexibility of the N-terminal regions. We have found that IgM chimeras multimerize successfully whether or not an Ig

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SfiIA

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GGC CAT TAT GGC C (TCGAGTCA) GGC CGC GGT GGC C

CMV b

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Fig. 2. Diagram of sample Fc chimera expression constructs and secreted biotin ligase expression vector. Reading frame of Sf i  I sites is noted. (a) IgG Fc expression construct with vector-supplied start codon and signal peptide; (b) IgG Fc expression construct without integrated start codon or signal peptide; (c) IgM Fc expression construct with vector-supplied start codon and signal peptide; (d) IgG Fc expression construct without integrated start codon or signal peptide; and (e) E. coli biotin ligase (BirA) expression construct. CMV human cytomegalovirus immediate–early promoter, SP mouse CD150 signal peptide, and IgG human immunoglobulin G1 Fc; pA bovine growth hormone polyadenylation signal, IgM mouse immunoglobulin M Fc, and SPk mouse Ig k-chain start codon and signal peptide.

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joining (J) chain cDNA has been cotransfected (unpublished observation); as a result, we do not typically contransfect J-chain constructs in experiments using IgM Fc reagents. 3.1. Subcloning of cDNA Fragments Encoding Receptor Ectodomains into an Expression Vector

1. Choose an appropriate vector into which a cDNA fragment encoding the ectodomain of interest is to be subcloned. Our laboratory has constructed a series of pcDNA3-based vectors (Invitrogen) that encode either IgG or IgM Fc regions as C-terminal tags and either include or do not include a vectorencoded signal peptide (Fig. 2). 2. Amplify the ectodomain-encoding cDNA by PCR from an appropriate template using oligonucleotide primers that contain Sf i I sites that match those of the selected vector. (Sf i I sites should be chosen to match the sequence and frame of the vectors shown in Fig. 2.) PCR is performed using standard protocols (as recommended by the thermostable polymerase distributor) using 1 ml of template (either plasmid DNA or cDNA) and 1 ml each of a 20 mM stock of each primer in a 50  ml reaction. Typically, 30 cycles of amplification are performed. 3. Cleave the PCR products with Sf i I restriction endonuclease. The PCR product does not need to be purified provided that the PCR reaction does not constitute over 50% of the Sf i I restriction enzyme reaction mixture. Typically, 15  ml of the PCR reaction is cut with Sf i I in a total volume of 30 ml using NEBuffer 2 supplemented with 100 mg/ml acetylated BSA. The reaction is incubated at 50°C for 1 h; heat inactivation of SfiI at 65°C for 30 min is optional, but not necessary. 4. Simultaneously, prepare an Sf i I-cleaved vector plasmid by digesting 5  mg of vector DNA with Sf i I and purifying the cleaved DNA by Chroma Spin 400 spin column, according to the manufacturer’s instructions, as summarized below: (a) Place the column into a 2-ml collecting tube, place the assembled column/tube into a 15-ml conical centrifuge tube, and pack the column by centrifuging at 700 × g for 5 min. (b) Remove and discard the collecting tube and load the column with the Sf i I reaction dropwise into the center of the top of the packed column. (c) Place the column in a new collecting tube and centrifuge at 700 × g for 5 min. (d) Discard the column; place cap on collecting tube, which contains the purified vector DNA, and store at either 4°C or −20°C. 5. Separate the Sf i I-cleaved ectodomain PCR amplicons by agarose gel electrophoresis and excise bands of the expected size

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using a scalpel or razor blade; remove the DNA from the agarose by loading the excised band into a Spin-X centrifuge tube filter and centrifuging at 20,000 × g for 5 min. 6. Combine insert and vector DNA fragments using T4 DNA ligase (according to standard methods) in a 10  ml reaction, incubated at room temperature for 1 h to overnight: (a) 1 ml purified Sf i I-linearized vector. (b) 6 ml Spin-X purified insert. (c) 2 ml 5× T4 ligase buffer. (d) 1 ml T4 ligase. 7. Transform a recombination-deficient E. coli host strain (e.g., chemically competent TOP10) with the ligation: (a) Thaw one 50-ml vial of chemically competent cells on ice. In many cases, the vial may be subdivided to extend the use of the cells when transforming multiple ligations on the same day (e.g., 2 × 25 ml for two ligations). (b) Add 2  ml of the ligation reaction to the cells and mix gently by finger tapping. (c) Incubate on ice for 10–30 min. (d) Heat-shock the cells for 30 s at 42°C without shaking. (e) Add 250 ml of prewarmed SOC medium. (An incubation on ice after heat shock is not necessary). (f) (Optional ) Incubate at 37°C for 1  h at 225  rpm in a shaking incubator. (g) Spread the entire transformation on an LB plate containing 50 mg/ml ampicillin. (h) Invert the selective plate(s) and incubate at 37°C overnight. 8. Screen the resulting colonies by direct colony PCR using vector-specific primers (T7: 5¢-ATTAATACGACTCACTATA GGG; BGHpA-REV: 5¢-AGGGGCAAACAACAGATGGC TG) and scoring for appropriately sized diagnostic amplicons (equal to the size of insert + approximately 1 kb that is derived from vector sequences). 9. Inoculate overnight cultures of positive colonies into LB medium containing 50 mg/ml ampicillin. 10. Purify plasmid DNA from the overnight cultures using Qiagen Miniprep Kits or a similar product; the DNA preparation can be used directly for transfection of HEK293T cells (see Subheading  3.2) and can also be sequenced to verify the integrity of the insert (see Note 2).

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3.2. Culture and Transfection of HEK293T 3.2.1. Cell Culture

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1. HEK293T cells are cultured at 37°C in a cell culture incubator containing 5% CO2, using RPMI medium supplemented with 10% FBS, sodium pyruvate, and GlutaMAX; use of antibiotics, such as penicillin/streptomycin, is optional (see Notes 3 and 4). 2. Cells are dislodged for passage by streaming medium (treatment with trypsin is generally not necessary, but can be performed if cells are particularly strongly attached) and reseeded at approximately 1:10 for passage every 3–4  days (see Note 5).

3.2.2. Transfection (See Note 6)

1. Plate cells in appropriately sized vessels and allow to expand until 90–100% confluent. (Typically, individual wells of a sixwell plate are sufficient for pilot transfections; T-75 or 10-cm tissue culture vessels/plates are used for scale-up). 2. Our laboratory uses Lipofectamine 2000 (Invitrogen) for all transfections; lipid:DNA complexes are prepared in OptiMEM I medium according to the manufacturer’s instructions (see Note 7). For one well of a six-well plate (volumes for other vessel sizes are indicated in the manufacturer’s instructions): (a) At least 1  h before transfection, change the growth medium of the six-well plate of HEK293T cells to 2 ml per well of prewarmed Opti-MEM I containing GlutaMAX (do not supplement with serum or antibiotics). (b) Dilute 4 mg DNA in 250 ml of Opti-MEM I in a 1.5-ml polypropylene tube; mix gently by inversion or pipetting up and down. (c) Mix the stock vial of Lipofectamine 2000 by gentle inversion and dilute 10 ml into 250 ml Opti-MEM I. Incubate for 5 min at room temperature. (Do not incubate longer than 25 min). (d) After the 5 min incubation, combine the two dilutions, mix by pipetting, and incubate for 20 min at room temperature to form lipid:DNA transfection complexes. Complexes are stable for 6 h at room temperature. (e) After the 20 min incubation, add the entire volume to a single well of a six-well plate containing cells to be transfected. Swirl to disperse complexes. (f  ) Incubate cells for 18–72 h prior to testing for expression. Complexes do not need to be removed after transfection.

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3. For in vivo biotinylation applications, the above transfection protocol is modified: (a) Replace 10% of the transfected DNA with BirA plasmid (resulting in a ratio of 9:1 Fc:BirA expression vector while maintaining the same total amount of transfected DNA, i.e., 4 mg for one well of a six-well plate). (b) Transfection and harvesting media may be supplemented with d-biotin at a concentration of 100  mg/ml (see Note 8). 3.3. Harvesting, Concentration, and Purification of Fc Chimeric Proteins 3.3.1. Harvesting of Cell Culture Supernatant Containing Recombinant Fc Chimeric Proteins

1. Harvesting of cell culture supernatants is typically performed from transfected T-75 flasks or 10-cm tissue culture plates. 2. Three days after transfection, remove the cell culture supernatant from the T-75 flask and replace with 20  ml of fresh Opti-MEM I supplemented with GlutaMAX (15  ml for a 10-cm plate). 3. Centrifuge the supernatant at 300 × g to remove debris. 4. Store the supernatant at 4°C; 0.02% sodium azide may be added as a preservative. 5. Continue incubating the freshly fed cells for a further 3 days and repeat steps 2–4 above; harvested supernatants may be pooled (see Note 9). 6. At least three successive harvests may be performed on a single transfected T-75 flask or 10-cm plate. After the third harvest, cell viability tends to decrease and cultures are typically discarded.

3.3.2. Purification of IgG Chimeras by Protein A Affinity

1. Protein A affinity purification is typically performed on 50 ml of harvested cell culture supernatant obtained as described above (see Note 10). 2. Using a pipette tip with 2 mm of its end removed by razor blade or scalpel, add 400 ml of rProteinA Sepharose Fast Flow slurry (containing approximately 200 ml of beads) per 50 ml supernatant to be purified to 10 ml PBS in a 50-ml conical centrifuge tube. Mix gently. 3. Centrifuge at 300 × g for 5 min to pellet washed beads. 4. Remove PBS and replace with 50 ml of cleared cell culture supernatant obtained as described above. Mix gently. 5. Rock gently for 1 h at room temperature. 6. Centrifuge at 300 × g for 5 min; remove supernatant with 10or 25-ml pipette. 7. Wash beads by resuspending in 20  ml PBS, mixing gently, centrifuging at 300 × g for 5 min, and removing PBS. 8. Repeat step 7 twice (for a total of three PBS washes).

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9. Elute the bound protein by resuspending the beads in 500 ml 100 mM glycine pH 2.5. Mix gently and transfer to a 1.5-ml Eppendorf microcentrifuge tube. 10. Incubate for 1  min at room temperature with gentle rocking. 11. Centrifuge at 300 × g for 1 min to pellet beads. 12. Remove glycine solution (containing eluted Fc proteins) and transfer to a fresh tube containing 25 ml of 2 M Tris pH 9.0. 13. Resuspend the beads in a new 500-ml aliquot of 100 mM glycine pH 2.5 and repeat steps 10–12. Each successive elution can be pooled with the previous ones; if pooling, add 25 ml of 2  M Tris pH 9.0 to the pooling tube before each 500  ml elution. 14. Continue elution for a total of four cycles (resulting in a total of 2 ml eluted protein). 15. Concentrate as described below. Store at 4°C in 0.02% sodium azide (see Note 11). 3.3.3. Concentration

1. Amicon Ultra centrifugal filter devices with a 10,000  Da molecular weight cutoff (MWCO) are used. Higher MWCO filters may be used for exceptionally large fusion proteins. Centrifugation in swinging bucket, as opposed to fixed, rotors is recommended. 2. Add up to 4 mL of sample to an Amicon Ultra filter unit. 3. Place cap on unit and centrifuge at (up to) 4,000 × g until approximately one-tenth of the original volume remains in the upper chamber of the unit (approximately 10–30  min depending on initial protein concentration of the solution; centrifuging at slower speeds will increase this time). 4. Add 4 ml of PBS or TBS to the remaining concentrate, mix gently, and repeat step 3. 5. Continue steps 3 and 4 until the original solution and three buffer changes have been spun through the device. 6. Recover the concentrated protein solution using a pipette inserted into the bottom of the filter. Pipette up and down to homogenize the solution and withdraw using a side-to-side motion to maximize recovery. 7. Rinse the filter with a further 100 ml of buffer (without centrifugation) and add the rinse to the concentrated protein. 8. Store at 4°C; 0.02% sodium azide may be added as a preservative.

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1. Remove 100 ml of medium; centrifuge at 300 × g for 1 min to pellet debris.

3.4. Checking Expression Levels by Dot Blot or Western Blot

2. Spot 5 ml of cleared supernatant onto a supported nitrocellulose filter; allow to air-dry for 10 min.

3.4.1. Preparation of Dot Blot

3. Immerse the filter in TBST and proceed to Immunodetection of dot blots or Western blots (below).

3.4.2. Preparation of Western Blot

1. Our laboratory uses a Hoeffer Mighty Small II minigel system for routine analysis of recombinant proteins. 2. Clean plates for casting a polyacrylamide gel by rinsing in water (cleaning with a detergent, such as Alconox, is optional). Rinse in 100% ethanol. 3. Prepare a 1.5-mm thick, 10% polyacrylamide gel by mixing 3 ml 2 M Tris pH 9, 3.75 ml 40% acrylamide:bis-acrylamide solution, 8 ml deionized water, 75 ml 20% SDS, 60 ml 10% APS, and 10  ml TEMED. Pour the gel, leaving space for a stacking gel, and overlay with water-saturated 1-butanol. The gel typically polymerizes within 45 min. 4. After polymerization, pour off the 1-butanol and rinse the top of the gel with water. 5. Pour 5% stacking gel by mixing 1.25  ml 1  M Tris pH 6.8, 1.25  ml 40% acrylamide:bis-acrylamide solution, 7.31  ml deionized water, 50 ml SDS, 80 ml APS, and 10 ml TEMED. Fill the gel mold to the top and insert the comb to form wells. The stacking gel typically polymerizes within 15–20  min. After the stacking gel has polymerized, remove the comb. 6. Mount the gel in the running unit, fill with SDS-PAGE running buffer, and rinse the wells by pipetting the running buffer up and down in each. 7. Load one lane of the polyacrylamide gel with 3–5  mL of prestained molecular weight markers. 8. For each experimental sample, mix 21  mL of cell culture supernatant or Protein A eluate with 7  mL 4× SDS-PAGE loading buffer (with or without added DTT), incubate at 95°C for 5  min, and pulse in a microcentrifuge. Load the entire sample into the well. 9. Connect the system to a power supply and electrophorese at 120  V until the bromophenol tracking dye has migrated approximately three-fourth of the way to the bottom of the gel (about 45 min). 10. Transfer the separated proteins to a membrane (either supported nitrocellulose or PVDF; note that PVDF membranes must be rinsed in 100% methanol and then Western transfer buffer to ensure proper saturation) by electrophoresis. Our laboratory uses a Genie system from Idea Scientific Company.

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Assemble the transfer according to the manufacturer’s instructions as summarized below: (a) In the transfer tray, stack in the following order: cathode, bubble guard, Scotch-Brite panel(s), and GB002 gel blot paper (Schleicher and Schuell). (b) Add Western transfer buffer to the transfer tray until approximately three-fourth full. (c) Continue stacking in the transfer tray by adding in the following order: SDS-PAGE gel, transfer membrane, GB002 gel blot paper, Scotch-Brite panel(s), bubble guard, and anode. (d) Insert assembled transfer tray into transfer tank, turn upright, connect to a 12  V power supply, and transfer proteins for 1 h at 12 V. (e) Disconnect and disassemble the transfer. For nitrocellulose membranes, remove and air-dry for 30  min. For PVDF membranes, rinse in 100% methanol and air-dry for 10 min. Mark the positions of wells and markers as needed with a pencil. 11. For nitrocellulose membranes, rehydrate by immersion in TBS. For PVDF membranes, immerse in 100% methanol and transfer to TBS to rehydrate evenly. Proceed to Immuno­ detection of dot blots or Western blots. 3.4.3. Immunodetection of Dot Blots or Western Blots

1. Block filters in TBS containing 1% casein (Roche). 2. Discard blocking solution and replace with either horseradish peroxidase-conjugated anti-human IgG antibody or horseradish peroxidase-conjugated streptavidin at a dilution of 1:5,000 in TBST containing 0.1% casein. Alkaline phosphatase-conjugated reagents can also be used. (For detection of IgM chimeras: Replace with DSB-X biotin goat anti-mouse IgM at a dilution of 1:1,000 in TBST containing 0.1% casein). 3. Incubate at room temperature for 1  h or at 4°C overnight with gentle rocking. 4. Remove the antibody (or streptavidin) solution. (These solutions can be reused multiple times if stored at 4°C in 0.02% sodium azide). 5. For detection of IgM chimeras: Replace the anti-mouse IgM antibody solution with horseradish peroxidase-conjugated streptavidin diluted at 1:1,000 in TBST containing 0.1% casein and incubate with rocking at room temperature for 30 min (see Note 12). 6. Wash filter(s) by rocking in TBST 3 × 10  min at room temperature.

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a

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Fig. 3. Sample Western blot analysis of immunoglobulin G- (IgG-) and IgM Fc chimeric protein expression. (a) Expression and covalent (disulfide-linked) multimerization of IgG Fc (left ) and IgM Fc (right ) chimeric protein constructs of catfish novel immune-type receptor 11 (NITR11). Reduction of disulfide bonds by treatment with dithiothreitol (DTT) before loading is indicated by “+” or “−”. Approximate molecular sizes in kilodaltons (kDa) are indicated. (Multimerization of IgG Fc chimeric constructs beyond simple dimers is conferred by inherent dimerization properties of NITR11; note that IgM Fc chimeric constructs are uniformly multimerized to very high molecular weight structures.) (b) Assessment of biotinylation of IgG Fc chimeras of NITR11. Transfection of Fc or E. coli biotin ligase (BirA) expression constructs in each sample is indicated at top by “+” or “−”. Detection using either anti-human IgG1 Fc antibody or streptavidin is indicated at right.

7. Wash filter(s) once for 5 min in TBS (without Tween-20) at room temperature. 8. Develop signal from the filters by using enhanced chemiluminescence (for HRP conjugates) or incubation in alkaline phosphatase detection reagent, such as Western Blue (Promega). 9. An example of detection of Fc fusions using anti-IgG, antiIgM, and streptavidin is provided in Fig. 3. 3.5. Biotin/ Streptavidin-Based Pull Down of Chimeric Reagents Using Dynal Magnetic Beads

1. Our laboratory uses Dynabeads MyOne Streptavidin T1 magnetic beads for streptavidin affinity isolation of biotinylated Fc chimeric reagents in complex with candidate ligands in solution. Pull-down experiments are performed according to the manufacturer’s instructions (see Note 13).

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2. Resuspend beads in their storage vial by rotation, inversion, or vortexing. 3. Add 20 ml of bead suspension to 1 ml of TBST, mix gently, and mount on a separation magnet until beads form a pellet (approximately 1–2  min). Remove TBS wash while tube is mounted on the magnet. 4. Remove the tube from the magnet. 5. Resuspend the beads in a solution containing candidate ligands or interaction partners for the domain represented by the Fc chimeric protein. 6. Add Fc chimeric protein to the suspended beads (optimal amount of Fc to be added must be determined by titration). 7. Rock suspension for 1 h at room temperature or overnight at 4°C. 8. Mount suspension on separation magnet and allow beads to form a pellet. Remove overlying solution, which may be saved for other experimental assays. 9. To wash, resuspend beads completely in 500–1,000 ml TBST, mount on separation magnet until beads pellet, and remove the TBST wash. 10. Repeat step 14 for a total of three to five washes (three washes are generally sufficient). 11. Sample can be eluted in 2× SDS-PAGE loading buffer for analysis by Coomassie stain, Western blot, or other appropriate assay. 12. Pull-down conditions may need to be optimized using different ratios of Fc chimeras to lysates, different amounts of magnetic beads, and different buffers with and without detergents.

4. Notes 1. Sfi  I was chosen as a cloning site due to its relative rarity (Sf i I has an 8 bp recognition sequence) and its ability to produce asymmetric cloning ends using a single recognition site (5¢-GGCCNNNN^NGGCC); other vectors/enzymes certainly could be used depending on the particular application. PCR oligonucleotide primers are generally designed to contain two additional bases upstream of the Sf iI site to ensure efficient cleavage by the restriction enzyme. 2. Endotoxin in plasmid preparations is generally not an issue unless its presence somehow might affect downstream applications or experiments.

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3. GlutaMAX (a commercially available l-alanyl-l-glutamine dipeptide supplement) is very useful to avoid issues of instability of l-glutamine upon storage at 4°C. 4. Other cell lines that encode the SV40 T antigen, such as COS7, as well as HEK293T should work in these applications, but we have not tested these lines directly. 5. HEK293T cells respond poorly to temperature “shocking” with cold cell culture medium and may detach spontaneously from the cell culture plate. For this reason, it is important that all media used in culture be prewarmed to 37°C before use. 6. We produce all of our recombinant proteins from transiently transfected HEK293T populations, rather than populations selected for integration of the vector using drug resistance (i.e., G418). HEK293T cells encode the SV40 T antigen, which allows episomal amplification of the transfected plasmid (which itself contains the SV40 origin of replication); as a result, expression levels are typically relatively high. 7. For reasons that are unknown, expression of proteins in OPTI-MEM appears to produce better yields when compared to the expression in serum-containing medium. Expression in serum-free medium also allows the use of Fc chimeric reagents in raw supernatants without complications from the presence of serum proteins. 8. We have verified that biotinylated Fc chimeras are compatible with surface plasmon resonance applications using either antiFc or streptavidin-based capture of the Fc chimera as immobilized ligand. 9. In our hands, cell culture supernatants often work as well as protein A-purified preparations in interaction assays (presuming sufficient affinity/avidity of the specific interaction and adequate concentration of the Fc chimera in unpurified supernatant). Nonpurified reagents can be useful in certain circumstances, as potential cofactors for function (e.g., metal ions or small molecules) may be depleted upon Protein A purification. 10. rProtein G (or a combination of Protein A and Protein G) can be equally effective in the purification of human IgG1 Fc-chimeric proteins. Protein A and Protein G exhibit differential affinity for the IgG Fc regions of antibodies from various mammalian species; therefore, care should be taken to verify compatibility for purification if expressing chimeras based on Fc regions other than that of human IgG1 (described here). 11. Yields of recombinant Fc chimeric proteins range from 10 mg to 1 mg per 100 ml of harvested cell culture supernatant.

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12. Either anti-Ig or streptavidin reagents can be used to detect the presence of biotinylated Fc chimeras in a sample; however, if the chimeric protein is not biotinylated, only anti-Ig reagents should be used. It should be noted that the detection method described in Subheading 3.4 for IgM chimeras is based on biotinylated anti-IgM reagents that were purchased by our laboratory for other applications. These reagents are useful for the detection of IgM Fc fusions, but are not suitable for the assessment of in vivo biotinylation of IgM Fc chimeras. It is likely that several different types of nonbiotinylated anti-mouse IgM antibodies and secondary reagents (e.g., from Jackson Immunoresearch) can be employed for this purpose; however, we have not currently tested such reagents directly and have chosen to present our current working protocol. 13. It is essential to remove free biotin from the culture medium by concentration (see Subheading 3.3) before conjugation to streptavidin beads, as free biotin in the medium will compete for binding to the beads and dramatically reduce or even eliminate conjugation. References 1. Lenschow, D., Zeng, Y., Thistlethwaite, J., Montag, A., Brady, W., Gibson, M., Linsley, P., and Bluestone, J. (1992) Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4lg. Science 257, 789–792. 2. Linsley, P., Wallace, P., Johnson, J., Gibson, M., Greene, J., Ledbetter, J., Singh, C., and Tepper, M. (1992) Immunosuppression in vivo by a soluble form of the CTLA-4T cell activation molecule. Science 257, 792–5. 3. Cerwenka, A., Bakker, A., McClanahan, T., Wagner, J., Wu, J., Phillips, J., and Lanier, L. (2000) Retinoic acid early inducible genes define a ligand family for the activating NKG2D receptor in mice. Immunity 12, 721–7. 4. Hamerman, J., Jarjoura, J., Humphrey, M., Nakamura, M., Seaman, W., and Lanier, L. (2006) Cutting edge: inhibition of TLR and  FcR responses in macrophages by triggering receptor expressed on myeloid cells (TREM)-2 and DAP12. J Immunol 177, 2051–5.

5. Cannon, J., Haire, R., Magis, A., Eason, D., Winfrey, K., Hernandez Prada, J., Bailey, K., Jakoncic, J., Litman, G., and Ostrov, D. (2008) A bony fish immunological receptor of the NITR multigene family mediates allogeneic recognition. Immunity 29, 228–237. 6. Parrott, M. and Barry, M. (2000) Metabolic biotinylation of recombinant proteins in mammalian cells and in mice. Mol Ther 1, 96–104. 7. Parrott, M. and Barry, M. (2001) Metabolic biotinylation of secreted and cell surface proteins from mammalian cells. Biochem Biophys Res Commun 281, 993–1000. 8. Parrott, M., Adams, K., Mercier, G., Mok, H., Campos, S., and Barry, M. (2003) Metabolically biotinylated adenovirus for cell targeting, ligand screening, and vector purification. Mol Ther 8, 688–700. 9. Beckett, D., Kovaleva, E., and Schatz, P. J. (1999) A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci. 8, 921–9.

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Chapter 5 Innate Immune Receptors for Nucleic Acids Andrea Stutz, Damien Bertheloot, and Eicke Latz Abstract The innate immune system has evolved to detect microbes and sterile tissue damage with the help of a series of signaling receptors. One key strategy is to detect infectious microbes or host cell damage by recognizing nucleic acids that are modified or appear in compartment normally devoid of nucleic acids. Here, we describe two methods that allow studying the molecular interaction between various nucleic acid recognizing signaling receptors with their ligands. A ligand pull-down assay can be used to show a known interaction between a ligand and its receptor or the method can be utilized as a discovery approach to identify an unknown receptor to a given ligand. An AlphaScreen experiment can be set up to assess the ligand binding affinity to a given receptor. Key words: Innate immunity, PYHIN, AIM2, TLRs, RLHs, RIG-I, AlphaScreen, Ligand pull down, Nucleic acid recognition

1. Introduction Life is ultimately based on passing along genetic information encoded in nucleic acids. A multitude of proteins are involved in storage, compartmentalization, and organization of nucleic acids, and a large number of proteins function in the translation of the genetic code into proteins. Infectious organisms, such as bacteria, viruses, or funguses, also depend on nucleic acids for information storage. The detection of foreign nucleic acids by the immune system has evolved as an important strategy to detect pathogens, and many different signaling receptors have evolved that detect the presence of foreign derived nucleic acids. These receptors vary in their ligand specificity, location, and cell-type distribution. It is becoming increasingly clear that the same receptors that function in the detection of nucleic acids from infectious organisms

Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_5, © Springer Science+Business Media, LLC 2011

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can also be involved in the detection of self nucleic acids that appear in certain disease states (1). For example, abundant cell damage can lead to the release of nucleic acids that can acutely trigger immune activation. Furthermore, a chronic imbalance of appearance of nucleic acids and their degradation can lead to chronic inflammation in susceptible individuals (2, 3). Nucleic acid recognizing receptors can broadly be categorized into transmembrane-signaling receptors (e.g., Toll-like receptor (TLR) family members) and cytosolic signaling receptors (e.g., RIG-I like helicases and PYHIN proteins). The outcome of receptor activation can either be a transcriptional activation of target genes or the induction of proteolytic cascades leading to the processing of procytokines and cell death (2, 4). Most mammalian cells express immune receptors that function in cell-autonomous anti-infective responses. RIG-I and MDA5 are members of the RLH receptor family and both can sense RNA ligands (5). Recently, it was discovered that RIG-I additionally detects the presence of cytosolic dsDNA in some cells via indirect means. Certain forms of dsDNAs are transcribed by polymerase III (also called Pol III) into RNA intermediates, which are recognized by RIG-I (6, 7). Immune cells are equipped with the broadest range of nucleic acid sensing signaling receptors. Among these, the bestcharacterized class is the TLR family, whose members are transmembrane receptors located in endosomal or lysosomal compartments or on the cell surface (4). These receptors function in the detection of nucleic acids derived from extracellular pathogens or released by host cells under stress conditions into the extracellular environment. Of note, the nucleic acid recognizing (TLR3, TLR7–9) appear to be activated in endosomal or lysosomal compartments, and it appears that the recognition of nucleic acids in these compartments is linked to the unique microenvironment. Indeed, proteolytic maturation of endosomal TLRs is linked to their activation, a fact that could be interpreted as a safeguard mechanism of endosomal TLR activation (8–10). In addition to the RLH family, the cytosol harbors the signaling receptor AIM2, a member of the PYHIN protein family. The PYHIN family proteins contain one or more DNA-interacting HIN200 domains and a single PYD (also called pyrin domain). Upon recognition of cytosolic dsDNA, AIM2 assembles the so-called inflammasome together with the adapter protein apoptosis-related speck-like protein (ASC) (11–13). AIM2 inflammasome assembly is followed by the activation of procaspase-1, which in turn activates the procytokines of the IL-1b cytokine family (14). It is expected that the cytosol contains additional, yet to be defined, nucleic acid recognizing signaling receptors. While an abundance of functional data have been acquired showing that the described receptors induce an antiviral or

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proinflammatory response toward different nucleic acids, the interaction process of ligands with their receptors has not been well characterized in most cases. In this chapter, we describe experimental protocols that can be utilized to characterize the interaction between signaling receptors and their nucleic acid ligands. In addition, the methods shown here can be used as a discovery approach to identify so far unknown receptors for nucleic acids. The discovery of nucleic acid recognizing immunesignaling receptors and the precise mechanisms of their molecular interaction with nucleic acids have led to novel drug targets. Undoubtedly, the pharmacologic interference with nucleic acid recognition evolves into a viable strategy for the treatment of a variety of infectious and inflammatory diseases.

2. Materials 2.1. Ligand Pull-Down Assay

1. 1 × 107 cells expressing the receptor of interest (see Note 1).

2.1.1. Cell Lysis

3. Lysis buffer: 137  mM NaCl, 20  mM Tris–Hcl (pH 7.4), 1 mM EDTA, 0.5% Triton X-100. Before usage, supplement the lysis buffer with 60  mM n-octylglucoside and protease inhibitors. For example, add leupeptin and aprotinin to a final concentration of 10 mg/ml and add phenylmethanesulfonylfluoride (PMSF) to a final concentration of 1 mM (see Note 2).

2. Ice-cold 1× DPBS (Gibco).

4. 6× SDS sample buffer: 47% (v/v) glycerol, 12% sodium dodecyl sulfate (SDS), 0.06% bromphenol blue, 600  mM Tris–HCl pH 6.8, 0.6 M dithiothreitol in water (see Note 3). Store in single-use aliquots at −20°C. 2.1.2. Pull Down

1. Biotinylated nucleic acid of your choice (see Note 4). 2. Streptavidin-coated agarose beads (Pierce). 3. Ice-cold lysis buffer (see Subheading 2.1.1). 4. 2× SDS sample buffer: Dilute 6× SDS sample buffer (see Subheading 2.1.1) 1:3 in water.

2.1.3. SDS-Polyacrylamide Gel Electrophoresis

1. 4× stacking gel buffer: 0.5 M Tris–HCl, pH 6.8, 0.4% SDS. Store up to 1 month at 4°C. 2. 4× separating gel buffer: 1.5 M Tris–HCl, pH 8.8, 0.4% SDS. Store up to 1 month at 4°C. 3. 10% ammonium persulfate (APS) in water. Freeze aliquots at –20°C. For short-term storage (up to 5 days), store at 4°C.

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4. N,N,N,N ¢-Tetramethyl-ethylenediamine (TEMED) and Acrylamide/Bis solution (29:1, 3.3% C) (Bio-Rad). Be careful, as unpolymerized acrylamide is a potent neurotoxin. 5. 2-Propanol. 6. 10× SDS running buffer: 0.25 M Tris base, 1.92 M glycine, 1% SDS. Store at 4°C. Dilute 100  ml to 1× with 900  ml water. 7. Molecular weight marker (PageRuler Plus Prestained Ladder, Fermentas or similar). 2.1.4. Immunoblotting

1. Transfer buffer: 20× stock (200 mM Tris, 2 M glycine) can be stored at room temperature for more than a month. From 20× stock, prepare 1  L of 1× transfer buffer: 50  ml 20× Transfer buffer, 100 ml ethanol, and 850 ml water. 2. Whatman 3 MM paper (Whatman). 3. Nitrocellulose membrane (Hybond ECL, 0.22 mm pore size, GE Healthcare). 4. Tris-buffered saline with Tween-20 (TBST): 10× stock: 1 M Tris–HCl, pH 7.5, 9% NaCl, 1% (v/v) Tween 20. Dilute 100 ml to 1× with 900 ml water. 5. Blocking buffer: 5% (w/v) powdered, nonfat milk in TBST. 6. Primary antibody for detecting your protein of interest, secondary horseradish peroxidase-coupled antibody specific for the host species of the primary antibody. 7. Hyperfilm ECL, ECL Plus Western Blotting Detection Reagent (Amersham Biosciences).

2.2. AlphaScreen 2.2.1. General Protocol

1. 50  mM (N-morpholino) ethanesulfonic acid, pH 6.5, 100 mM NaCl, 0.1% (vol/vol) BSA free of DNAse, protease and RNAse (Equitech), and 0.01% (vol/vol) Tween 20 (see Note 5). 2. FPLC-purified protein dialyzed into stock solution for storage: 20 mM Tris pH8.0, 100 mM NaCl, 1 mM EDTA, 50% (vol/vol) glycerol, 5  mM DTT, 0.05% (vol/vol) CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate) (see Note 6). TLR9–Fc stock solution is prepared on ice and stored in −80°C. For the assay, the protein stock solution is diluted into assay buffer to a final concentration of 50 nM. 3. Dilute the stock of donor and acceptor beads (stock = 5 mg/ml) 1:50 with assay buffer. Analyze for emitted fluorescence with an AlphaScreen-enabled multilabel reader (e.g., Envision; Perkin Elmer).

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3. Methods Here, we describe biochemical methods that can be utilized to identify the binding partners of immune-stimulatory nucleic acids and to characterize the relative affinity of a given nucleic acid to a purified receptor. The described methods have been utilized to characterize the binding of DNA or RNA to members of the TLR, RLH, or PYHIN protein families (13, 15–20). These methods can be adapted to other ligand/receptor interactions, and this methodology is not limited to the characterization of binding of nucleic acids to immune-signaling receptors. It is important to realize that binding of a nucleic acid to its respective receptor does not necessarily correlate to the activation of the receptor by the ligand. It is well established that certain nucleic acids can block the activation of a receptor in a competitive fashion. The mode of receptor activation may involve binding and ligand-specific induced conformational changes (18). 3.1. Ligand Pull-Down Assay

A ligand pull-down assay can be used to test whether a specific nucleic acid is bound by a receptor. It can be used to assess the interaction with a known ligand. In addition, this method can be utilized as a discovery approach aiming to identify an unknown interaction partner for a given immune stimulatory nucleic acid. To conduct a ligand pull-down assay, cell lysates containing your receptor of interest are needed. A biotinylated ligand could be added to the cell lysate and subsequently binds to the receptor of interest. Alternatively, the cells can be stimulated with a biotinylated ligand and are subsequently lysed (after the stimulation; the changes necessary to the protocol are found in Note 7). After the time necessary for the interaction to occur, the biotinylated ligand and its binding partners are precipitated by the use of streptavidincoated beads, which specifically bind the biotinylated ligand. After thorough wash steps, the interactions of target proteins with ligands are destroyed by heat denaturation or by specific digestion of the DNA. Proteins are then separated by denaturing polyacrylamide gel electrophoresis (PAGE) and visualized by immunoblotting to confirm the interaction with the biotinylated ligand. Alternatively, if pull-down experiments with larger preparations are performed, the proteins can also be detected by silver or coomassie-based staining methods. The ligand associated proteins can be identified by mass spectrometry analysis. The changes necessary for the mass spectrometry analysis can be found in Note 8. It is important to note that the ligand pull-down methodology cannot distinguish between direct interaction of the detected protein and an indirect interaction (i.e., when another protein directly interacts not only with the ligands, but also with the detected protein).

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Every ligand pull-down experiment should include appropriate controls both at the level of the ligand (e.g., noninteracting nucleic acid, etc.) or at the level of protein (e.g., noninteracting TLR). 3.1.1. Cell Lysis

1. Wash the cells of interest twice with 10 ml ice-cold PBS to remove the remaining medium. 2. Aspirate the supernatant and take up the pellet in 2 ml of lysis buffer. Pipet gently up and down to ensure even lysis. Incubate cells for 10 min on ice (see Note 9). 3. Remove the nuclei by a centrifugation step (4°C) at 1,000 × g for 10 min, discard the pellet, and save the supernatant for the subsequent pull down. 4. Before proceeding to the pull down, save a fraction of the cell lysate to check for the overall expression of your receptor: Remove 100 ml of the cell lysate and add 20 ml of 6× SDS sample buffer. Boil the samples for 5 min at 95°C. After cooling to room temperature, the samples can be separated by SDS-PAGE.

3.1.2. Pull Down

1. Take 50 ml of a 50% slurry of streptavidin-coated beads per 1 ml of lysate (scale up or down as desired) in a separate tube (see Note 10) and wash them twice in 1 ml lysis buffer. To pellet the beads, a short spin for 5 s in a conventional microcentrifuge is usually sufficient. Keep the beads on ice and perform all centrifugation steps at 4°C. After washing, add the lysate to the beads. Rotate the tube for 1 h at 4°C. This socalled preclearing step reduces the amount of proteins that nonspecifically interact with the beads. 2. Spin down the beads, discard the pellet, and save the supernatant. 3. Divide the supernatant into fractions for the different pull downs. Make at least two fractions: one for the pull down with the biotinylated test ligand and one without any ligand (or a noninteracting ligand) as specificity control. 4. Add 1–5 mM final concentration of the biotinylated nucleic acid to a lysate fraction and rotate the tubes for 4 h at 4°C. 5. About 5 min before the end of the incubation time, take 50 ml of streptavidin-coated beads per 1  ml of lysate (scale up or down as desired) in a separate tube and wash them twice in 1 ml lysis buffer. 6. Mix the streptavidin-coated beads and lysates containing your oligonucleotides and rotate for 1 h at 4°C. 7. Pellet the beads by centrifugation and wash the beads at least four times with 1 ml of lysis buffer. To avoid drying of the beads, leave a small volume of lysis buffer on top of the beads between the wash steps.

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8. Add 2× SDS sample buffer (equal volume of the beads) and boil the samples for 5 min at 95°C. Spin down briefly to collect all the sample volume. After cooling to room temperature, the samples can be separated by SDS-PAGE. 3.1.3. SDS-Polyacrylamide Gel Electrophoresis

1. Clean the glass plates and spacers (1.5 mm) for two gels with water and ethanol and assemble them. 2. Prepare two 10% (see Note 11) separating gels (10 × 10 cm minigel): Mix 8.2 ml of water, 5 ml of 4× separating gel buffer, and 6.6  ml of the acrylamide/bis solution. Degas the solution by sonification in a water bath sonifier for 30 s. Add 200  ml of 10% APS and 8  ml of TEMED. Pour the gels between the glass plates, leaving space for the stacking gel (there should be 1 cm of stacking gel between the wells of the comb and the separating gel). Overlay the gels with 2-propanol. The gels should be polymerized within 30 min. 3. Remove the 2-propanol. Clean two 10-well combs with water and ethanol. 4. Prepare the 5% stacking gel: Mix 4.6 ml of water, 2 ml of 4× stacking gel buffer, and 1.4 ml of the acrylamide/bis solution. Degas the solution as above. Add 80 ml of 10% APS and 8 ml of TEMED. Pour the gel between the glass plates and insert the combs. The gels should be polymerized within 20 min. 5. Assemble the gels in the running apparatus and fill the upper and lower chambers with 1× running buffer. Remove the combs and wash the wells with running buffer using a thin pipet tip/syringe to remove gel fragments. 6. Load one gel with your pull-down samples: Load 50  ml of each sample in a well; load one well with 5 ml of the molecular weight marker. Load each sample and the marker twice so you can cut the membrane afterward and blot for your receptor of choice and an unrelated control receptor. 7. Load the other gel with the cell lysates to check for receptor expression. 8. Run the gels at 80 V until the dye front has left the stacking gel, and then increase the voltage to 100 V. Run the gels until the dye front is just about to run off the gel (if looking for large proteins, you can let the dye run off the gel).

3.1.4. Immunoblotting

1. This describes the general assembly of one wet blot sandwich. Cut a piece of membrane just a little larger than your gel and 4 Whatman paper pieces just larger than the membrane. For two gels, you need two membranes and eight Whatman papers. 2. Fill a tray (large enough to fit the two foams next to each other) with transfer buffer.

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3. Assemble a wet blot sandwich in the tray in the following order: foam – two pieces of Whatman paper – polyacrylamide gel – membrane – two pieces of Whatman paper – foam. Remove air bubbles by rolling a serological pipet over the sandwich. Repeat for the other gel. Assemble the sandwich into the transfer cassette and place it in the transfer tank (fill the transfer tank with transfer buffer) so that the membrane is between the gel and the anode (+). 4. Cool the transfer tank by placing it on ice or by water circulation. 5. Transfer at 80 V for 2 h. 6. Remove the membranes from the sandwich and place them separately in a 50-ml conical tube each. All following incubations are carried out on a rotating device or a roller mixer. 7. Block with 10  ml blocking buffer for 1  h at room temperature. 8. Cut the membrane with the pull-down samples so that you have two membrane pieces, each with all the samples and the protein ladder. 9. All following steps are described for one membrane piece. Treat the other pieces the same way with the respective antibodies. 10. Prepare 5 ml of an appropriate dilution of your primary antibody in blocking buffer (or 3% BSA in TBST) (see Note 12). Incubate the blot with the antibody solution either for 1 h at room temperature or overnight at 4°C. 11. Wash the blot with 15 ml of TBST for 10 min. Repeat twice. 12. Prepare 5  ml of an appropriate dilution of your secondary antibody in blocking buffer (or 3% BSA in TBST) (see Note 12). Incubate the blot with the antibody solution for 1 h at room temperature. 13. Wash the blot with 15 ml of TBST for 10 min. Repeat twice. 14. During the final wash, 1 ml of each part of the ECL reagent are mixed and incubated for 1 min. The membrane is blotted dry using a paper towel and incubated in the ECL reagent for 1 min. The membrane is again blotted dry and placed between two sheets of transparent plastic foil and into an X-ray cassette. In the dark, a film is exposed for a suitable amount of time. A typical result can be seen in Fig.  1, in which TLR9 was pulled down with CpG in a dose-dependent manner. 3.2. AlphaScreen Assay

The ligand pull-down procedure is limited by the amounts of proteins expressed per cell and by the fact that direct interaction cannot be readily distinguished from indirect interaction by one or more proteins that interact with the protein of interest and the

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Fig. 1. HEK cells expressing a chimeric protein consisting of the N-terminal domain of human TLR9 fused to the IgG2a Fc domain of a mouse Ab (TLR9-Fc) were lysed and incubated with phosphorothioate-modified biotinylated CpG-containing oligonucleotides (24-mer is the 2006 motif, the shorter versions are respective 3-prime truncations thereof). The biotinylated DNA was pulled down using a neutravidin-coated agarose or TLR9-Fc was precipitated with protein A-coated agarose (PAS) as a control. The proteins associated with the beads were separated by SDS-PAGE, and membranes were probed using an HRP-conjugated anti-mouse Ab.

ligand. The following assay describes a method that utilizes purified protein and a biotinylated ligand and can be used to assess the ligand-binding affinity to a given receptor. This assay can be scaled down to the assessment of binding in very small volumes (e.g., 5 ml in 1,536-well plates). The following method describes a homogeneous assay in which interactions between the nucleic acid ligands (or any other ligands) and the purified receptor are assessed in wells of microtiter plates. The assay is termed Amplified Luminescent Proximity Homogeneous Assay (Alpha), and since it is easily converted into a high content screening assay, it is called AlphaScreen. As its name indicates, this method measures the proximity of two molecules taking advantage of a luminescence-amplified detection procedure. In brief, two kinds of beads that are bound to the ligand or receptor, respectively, are used in this assay (Fig.  2). Donor beads contain the photosensitizer phthalocyanine, which upon illumination with 680  nm converts ambient oxygen to singlet oxygen with a single excited electron. This singlet oxygen can diffuse approximately over 200 nm in solution during its 4 ms half-life. When the acceptor bead is within 200 nm from a donor bead (i.e., when receptor/ligand interaction occurs), the energy is transferred from the singlet oxygen to thioxene derivatives contained into the acceptor bead. Once excited, the acceptor bead produces a light signal at 520–620 nm. If there is no acceptor bead within 200 nm from the donor bead, the singlet oxygen falls back to its unexcited state and no signal is produced. PerkinElmer provides a large variety of beads that leaves a great flexibility in designing experiments. The choice of beads depends on the experiment. Since a larger variety of anti-tag antibodies is available for the acceptor beads than for the donor beads, we recommend the usage of streptavidin-conjugated donor beads to bind to the biotinylated ligands. We have made good experiences with protein A-conjugated acceptor beads that bind specifically to the Fc domain of our fusion proteins. However, depending on

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chemical energy transfer by oxygen singlet

Emission 520-620 nm

biotinDNA Receptor

Donor bead Streptavidin

Acceptor bead

Fig. 2. Schematic of the AlphaScreen assay system. The donor beads are illuminated with a 680-nm emitting laser, which produces a singlet oxygen. If an acceptor bead is in the vicinity of 200 nm, the singlet oxygen leads to the generation of fluorescence in the range of 520–620 nm.

the tagging strategy of the protein expression, a variety of conjugated acceptor beads is available. For example, beads covered with anti-tag antibodies (such as anti-Flag, anti-myc, anti species IgG, nickel, etc.) can be purchased. The AlphaScreen beads can also be directly coupled to the test proteins using standard reductive amination protocols. The small size of the beads (250 nm in diameter) prevents settling of the beads during the assay and allows for easy liquid handling. The AlphaScreen assays are performed in white opaque plates in 96-, 384-, or 1,536-well formats. An AlphaScreen enabled microplate reader is required for the readout of the assay. These readers are available from various providers, including PerkinElmer, BMG LabTech, Beckman-Coulter, and BioTek. Appropriate controls should be added to each assay. For example, when binding of a nucleic acid to a receptor is tested, an alternative ligand could be used. In addition, it is also advisable to use a different receptor molecule in order to account for nonspecific interaction. Most AlphaScreen kits also contain a positive control that could be used to test for the influence of the buffer system used on the performance of the assay. 3.2.1. General Protocol

1. Prepare 1× assay buffer. 2. Prepare a gradient dilution of biotinylated oligonucleotide in assay buffer. The necessary volume depends on the well density used. The description here is an example of an assay in 384-well plates. A dilution from 100 to 0.1 nM usually covers the dynamic range of the assay. However, it is crucial to take into account the so-called hook effect, which depends on the bead capacity (see Note 13). 3. Pipet 7.5  ml of a 50  nM protein solution (e.g., TLR9-FC) into wells of 384-well plates (Proxiplate; Perkin-Elmer) and

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add 7.5 ml of increasing concentrations of biotinylated ligand (from step 2). Include controls, where beads are added without ligand or protein, respectively. Cover wells with sealer and incubate for 45 min at room temperature. 4. During incubation, prepare donor and acceptor beads stock solutions. Dilute the 5 mg/ml kit solutions to 100 µg/ml in assay buffer. 5. Add to each well 5 µl acceptor beads and 5 µl donor beads (final concentration 20 µg/ml each). Cover wells with sealer and incubate in the dark for 60 minutes (see note 14). 6. Analyze the plate with an AlphaScreen enabled multilabel reader (excitation with 680-nm laser source).

4. Notes 1. The number of cells needed depends on many factors, including the expression rate of your receptor of interest and the application. If pull-down experiments are planned for the identification of proteins using mass spectroscopy, at least tenfold more cells are required. 2. The water used for all buffers needs to be of Milli-Q quality. Dissolve aprotinin and leupeptin in water (10 mg/ml), and freeze the stocks in ready-to-use aliquots at −20°C for up to 6 months. For the PMSF stock solution (200 mM), we use anhydrous, highly pure ethanol as solvent and store the stock at 4°C for up to 6 months. The protease inhibitors need to be added to the lysis buffer directly before usage because of their short half-life in aqueous solutions, even on ice. Alternatively, cocktails of protease inhibitors that are available from various companies can be used. 3. Be careful when handling SDS as it is an irritant. Weigh SDS under a fume hood or wear a protective mask to avoid inhalation of the fine crystals. 4. Triethylene glycol (TEG)-biotinylated oligonucleotides are preferable, as the TEG spacer allows for a more unhindered binding of the biotin to the streptavidin, and therefore for a higher recovery of the tagged oligonucleotides from the lysates. Longer DNA strands can be labeled using biotin 3’ End DNA labeling (Pierce). Long RNA strands can be labeled using a photoactivatable biotin-labeling approach (e.g., EZ-Link Psoralen-PEG3-Biotin, Pierce). 5. The choice of buffer is very important. One should optimize pH, buffering capacity, and salt concentration for each interaction to be measured. For example, if the protein/ligand

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interaction is expected to proceed in the endosomal or lysosomal compartment (as is the case for nucleic acid recognizing TLRs), the buffers for the assay should be at a lysosomal pH of about 5. The buffer shown in this protocol was ­optimized for the assessment of binding of the N-terminal domain of TLR9 to CpG-DNA. The generic buffer for the AlphaScreen is different and can be prepared from the reagents provided by the PerkinElmer: 10× assay buffer: 1 M Tris, 0.1% Tween20, pH 8.0. Prepare fresh prior to assay 1× assay buffer: add 450 ml 10× assay buffer to 4.5 ml Milli-Q water and add 5 mg BSA (0.1%, w/v). When nonspecific binding is observed, a variety of detergents (0.1% (v/v) or less CHAPS, Tween-20 or Triton X-100) and BSA (up to 0.1% (w/v)) can be used Alternate blocking reagents, such as low molecular weight dextran or gelatin, can also be used. 6. Avoid using sodium azide as a microbicide, as it is a potent scavenger of the singlet oxygen that is used as a messenger of the signal. Proclin 300 (Sigma) can be used for all buffers as a preservative. 7. Alternatively to the cell lysis protocol, one can also stimulate cells with biotinylated nucleic acids. The binding process occurs in a more physiological environment than in the lysate. To stimulate your cells of interest before lysis, add the biotinylated nucleic acid stimulus to the cells in the fashion that would normally stimulate the cells with the respective nucleic acid (time of incubation, use of transfection reagents, etc.). Also include an unstimulated control without biotinylated ligand. Harvest the cells (using trypsin or a scraper), wash twice with PBS, and lyse the cells using lysis buffer. Then proceed with step 5 of the pull-down section. Since one cannot do a preclearing step with the streptavidin-coated beads, a control without biotinylated ligand is of special importance in this variation of the protocol. 8. Changes to the protocol for doing mass spectrometry: Lyse 1 × 108  cells with lysis buffer and use all the lysates for pull downs. Note to include a control for unspecific binding. Conduct the pull down as indicated, and add 50 ml of 2× SDS sample buffer to the beads. Take great care to prevent contamination of your samples and gel with other proteins. Wear gloves at all times. Pour only one gel and load the complete sample into one well, and do not run two lanes of each sample as stated in the above protocol. Subsequently, stain the gel with Colloidal Coomassie (e.g., GelCode Blue Safe Protein Stain, Pierce). To do so, wash the gel with 50 ml of ultrapure water for 15 min, and then discard the water. Repeat twice. Stain with 50  ml Coomassie solution (for 15  min to 1  h,

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depending on the amount of protein you loaded). Destain the gel with 50 ml of water for 1 h (or overnight, if needed). Protein bands should be visible in blue. Identify the unique bands that are obtained in your sample of interest (and not in the control samples). Cut out the bands of interest and ­prepare them for mass spectrometry by in-gel digestion (e.g., In-gel tryptic digestion kit, Pierce) and purify the peptides (e.g., using Pierce C-18 columns). 9. This time is usually sufficient to lyse the cell lines investigated in our laboratory. However, when you use this protocol for the first time, check a sample of your cells after the lysis with a light microscope. The cells should be destroyed and the nuclei should still be intact. The time for lysis may have to be increased if the cells are not destroyed. Take great care to not destroy the nuclei as they contain many proteins interacting with nucleic acids. These proteins might bind to your ligands unspecifically, increasing the background and interfering with the binding of your receptor of interest. 10. When pipeting agarose beads, cut off the top of the pipet tip or use large-bore pipet tips. This facilitates the handling. 11. This recipe is for a 10% gel which should be suitable for most receptors. However, if your receptor is smaller than 40 kDa or bigger than 150 kDa, you should consider pouring a gel with a higher or lower percentage, respectively. 12. An appropriate dilution of your antibody must be found empirically (as a guideline, refer to the dilution range in the manufacturer’s instructions). 13. The so-called hook effect can be observed in bimolecular detection systems which involve saturable reagents, such as beads or streptavidin. In these assays, the signal curves obtained by dilution series of the ligand or the receptor are generally bell-shaped. The curves are characterized by an initial concentration- and affinity-dependent signal increase followed by a signal plateau (called hook point) and a concentration-dependent titratable signal decrease (i.e., the hook effect). Above the hook point, the excess of target molecules oversaturates donor or acceptor beads leading to an interaction of the binding partners (e.g., nucleic acid and protein) without binding to both reporter beads. This oversaturation inhibits the interaction of donor/acceptor beads, and thereby decreases the signal in an affinity-independent manner. To avoid the hook effect, do not use a too high concentration of analytes in AlphaScreen assays and carefully titrate the reactants. It is important to accurately assess the protein concentration of the receptor protein before addition of carrier protein (BSA) and to carefully titrate the receptor protein.

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Various concentrations of protein (for example, 100  nM, 50 nM, 25 nM, and 12.5 nM) should be tested to determine the best signal-to-noise ratio. 14. The donor beads are light-sensitive, and regular laboratory illumination is detrimental for the performance of the assay. It is imperative to handle the donor beads under subdued light conditions (below 100  Lux). Alternatively, the room could be fitted with green filtered lighting (Recommended filter: Roscolux Chroma Green #389 from Rosco; fluorescent tube sleeves, Cat. #: 4812-389; or filter roll, Cat. #: R389. Rosco Labs: http:// www.rosco.com). References 1. Rock, K.L., Latz, E., Ontiveros, F. & Kono, H. The sterile inflammatory response. Annu Rev Immunol 28, 321–342 (2010). 2. Hornung, V. & Latz, E. Intracellular DNA recognition. Nat Rev Immunol 10, 123–130 (2010). 3. Pisetsky, D.S. The role of innate immunity in the induction of autoimmunity. Autoimmunity reviews 8, 69–72 (2008). 4. Takeda, K., Kaisho, T. & Akira, S. Toll-like receptors. Annu Rev Immunol 21, 335–376 (2003). 5. Takeuchi, O. & Akira, S. MDA5/RIG-I and virus recognition. Curr Opin Immunol 20, 17–22 (2008). 6. Ablasser, A., et al. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nat Immunol 10, 1065–1072 (2009). 7. Chiu, Y.H., Macmillan, J.B. & Chen, Z.J. RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell 138, 576–591 (2009). 8. Ewald, S.E., et al. The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor. Nature 456, 658–662 (2008). 9. Park, B., et al. Proteolytic cleavage in an endolysosomal compartment is required for activation of Toll-like receptor 9. Nat Immunol 9, 1407–1414 (2008). 10. Sepulveda, F.E., et al. Critical role for asparagine endopeptidase in endocytic Toll-like receptor signaling in dendritic cells. Immunity 31, 737–748 (2009). 11. Burckstummer, T., et al. An orthogonal proteomic-genomic screen identifies AIM2 as a

cytoplasmic DNA sensor for the inflammasome. Nat Immunol 10, 266–272 (2009). 12. Fernandes-Alnemri, T., Yu, J.W., Datta, P., Wu, J. & Alnemri, E.S. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature 458, 509–513 (2009). 13. Hornung, V., et al. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458, 514– 518 (2009). 14. Latz, E. The inflammasomes: mechanisms of activation and function. Curr Opin Immunol 22, 28–33 (2010). 15. Alagkiozidis, I., et al. Increased immunogenicity of surviving tumor cells enables cooperation between liposomal doxorubicin and IL-18. J Transl Med 7, 104 (2009). 16. Haas, T., et  al. The DNA sugar backbone 2¢ deoxyribose determines toll-like receptor 9 activation. Immunity 28, 315–323 (2008). 17. Schlee, M., et  al. Recognition of 5¢ triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus. Immunity 31, 25–34 (2009). 18. Latz, E., et  al. Ligand-induced conformational changes allosterically activate Toll-like receptor 9. Nat Immunol 8, 772–779 (2007). 19. Tian, J., et al. Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE. Nat Immunol 8, 487–496 (2007). 20. Latz, E., et al. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol 5, 190–198 (2004).

Chapter 6 Analysis of Receptor–Ligand Interactions by Surface Plasmon Resonance Kimiko Kuroki and Katsumi Maenaka Abstract Many immunological responses are often regulated by cell surface receptors in cell–cell recognition events. Such immune receptors on the cell surface typically exhibit low-affinity and fast-kinetic ligand interactions (e.g., Kd in the mM range, koff = 10−2 to 20 s−1). Real-time surface plasmon resonance (SPR) detection systems are generally useful for determining these binding parameters. However, several technical points should be considered because the determination of low-affinity binding and fast kinetics is often rather difficult. Here, we introduce a general procedure for SPR experiments and, moreover, show typical examples for ligand binding of immune cell surface receptors, including experimentally useful tips. We also show how to determine the thermodynamic characteristics using the nonlinear van’t Hoff and Arrhenius analyses. These affinity, kinetic, and thermodynamic parameters of immune–receptor binding are important for understanding immunological events as well as developing drugs and vaccines. Key words: Surface plasmon resonance, Affinity, Kinetics, Thermodynamics, Leukocyte immunoglobulin-like receptor, Human leukocyte antigen

1. Introduction Surface plasmon resonance (SPR) technology measures the mass concentration of biomolecules on sensor chips. Among the several SPR-based systems, the BIAcore (GE Healthcare) series are the most popular systems for monitoring the association and dissociation between ligands immobilized on the sensor chip and analytes flowed over the ligands in real time. The Biacore is useful for analyses with various nonlabeled biomolecules: proteins, peptides, nucleotides, carbohydrates, lipids, cells, viruses, etc. These biomolecules should be immobilized on appropriate sensor chips. If the regeneration step is optimized, then a sensor chip can be used more than 100 times. However, many immune cell surface Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_6, © Springer Science+Business Media, LLC 2011

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receptors show low-affinity ligand binding with a fast dissociation rate, and thus do not require the regeneration step. In order to understand receptor–ligand interactions, SPR is valuable because it can provide information not only about the specificity and affinity of binding, but also kinetic and thermodynamic information by monitoring in real time. This information is valuable for drug discovery and development. Thermodynamic investigations of the quality of antibodies used for medical treatments are often required for the Food and Drug Administration (FDA) approval. The recent trend has promoted an easy and simple strategy for determining such parameters by using linear and nonlinear van’t Hoff analyses, which simply require the acquisition of affinity parameters at several different temperatures to determine the enthalpy, entropy, and heat capacity of ligand binding. While previous reports pointed out some discrepancy between the van’t Hoff and calorimetric analyses, the feasibility of the van’t Hoff analysis was ensured by recently accumulated evidence showing that these thermodynamic values are essentially in good agreement with those directly determined by isothermal titration calorimetry (ITC). Therefore, many researchers use SPR analysis to determine the thermodynamic parameters, and actually the most advanced T-100 model of the BIAcore system has an automatic routine procedure for determining the affinities at different temperatures and calculating the thermodynamic parameters using van’t Hoff analyses.

2. Materials Several types of SPR equipment are available, including BIAcore (GE Healthcare), ProteOn XPR36 Technology (Bio-Rad Laboratories), and MultiSPRinter (Toyobo Corp.). While the latter two systems are generally more suitable for high-throughput measurements, the BIAcore is the most popular laboratory-based system. Therefore, here we mainly focus on the BIAcore system. The materials for SPR analyses using BIAcore systems are available from GE Healthcare. 2.1. Instruments

1. BIACORE instrument system GE Healthcare provides multiple systems according to the study design (e.g., basic research, drug discovery, manufacturing, and quality control) (http:// www.biacore.com). See Table 1. 2. Controlling PC. 3. BIACORE Control Software. 4. BIAevaluation Software. 5. Microcentrifuge.

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Table 1 Biacore systems (November 2009) Biacore X100

Biacore T100

Biacore 3000

Kinetic analysis (ka, kd)

Best

Best

Excellent

Single-cycle kinetics (Note 6)

Plus package

Yes

–

Affinity analysis (KD)

Best

Best

Best

Concentration analysis

Excellent (plus package)

Excellent

Good

Thermodynamic analysis

Good (plus package)

Excellent

Good

Sample recovery for MS

–

Excellent

Best

Analysis of low-molecular-weight interaction

Good

Best

Good

Detection spots

2

4

4

Dynamic range

70,000 RU

70,000 RU

70,000 RU

Analysis temperature (°C)

25 (4–40 plus package)

4–45

4–40

Cooled sample storage

–

Best

Excellent

Automated data evaluation

Excellent

Excellent

Good

Additionally, the Biacore A100 is suitable for high-throughput drug discovery screening, and the Biacore C is ideal for rapid and reliable protein quantification. The Biacore Flexchip for array-based parallel kinetic profiling is now available

2.2. Ligand Immobilization

2.2.1. Amine Coupling: Immobilization of Amine Groups (Lysine and Unblocked N-Termini)

Among the coupling methods, amine coupling is commonly used. GE Healthcare provides several other coupling kits: Thiol Coupling Kit, GST Kit for Fusion Capture, Mouse Antibody Capture Kit, and Human Antibody Capture Kit. 1. Sensor Chip CM5. 2. 100 mM N-hydroxysuccinimide (NHS) (see footnote 1). 3. 400 mM N-ethyl-N¢-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (see footnote 1). 4. 1 M Ethanolamine hydrochloride (pH 8.5) (see footnote 1). 5. Ligand protein diluted by appropriate acetate buffer.

2.2.2. Intrinsic Ligand Thiol Coupling: Immobilization of a ligand possessing a surface-exposed free cysteine or disulfide

Ligands need to be reduced under nondenaturing conditions to generate the free cysteine. 1. Sensor Chip CM5. 2. 100 mM NHS (see footnote 2). 3. 400 mM EDC (see footnote 2). These are contained in the Amine Coupling kit available from GE Healthcare.

1 

 These are contained in the Thiol Coupling kit available from GE Healthcare.

2

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4. 1 M Ethanolamine hydrochloride (pH 8.5) (see footnote 2). 5. 120  mM 2-(2-Pyridinyldithio)-ethaneamine hydrochloride (PDEA) (see footnote 2). 6. 150 mM Sodium borate buffer (pH 8.5) (see footnote 2). 7. 50 mM l-Cysteine (see footnote 2). 8. Ligand protein diluted by appropriate acetate buffer. 2.2.3. Surface Thiol Coupling: Immobilization of Amine or Carboxyl Groups

Reactive disulfides need to be introduced using PDEA. 1. Sensor Chip CM5. 2. 100 mM NHS (see footnote 2). 3. 400 mM EDC (see footnote 2). 4. 1 M Ethanolamine hydrochloride (pH 8.5) (see footnote 2). 5. 120 mM PDEA (see footnote 2). 6. 100 mM MES pH 5.0 (see footnote 2). 7. 40 mM Cystamine dihydrochloride (see footnote 2). 8. 150 mM Sodium borate buffer (pH 8.5) (see footnote 2). 9. 100 mM dithioerythritol (DTE) (see footnote 2). 10. 50 mM l-Cysteine (see footnote 2). 11. 1 M Sodium chloride (pH 4.0) (see footnote 2). 12. Ligand protein diluted by appropriate acetate buffer.

2.2.4. Aldehyde Coupling: Immobilization of Aldehyde Groups

Aldehydes need to be created by oxidizing cis-diols with periodate. 1. Sensor Chip CM5. 2. 100 mM NHS (see footnote 1). 3. 400 mM EDC (see footnote 1). 4. 1 M Ethanolamine hydrochloride (pH 8.5) (see footnote 1). 5. 5 mM Hydrazine monohydrate or 5 mM carbohydrazide. 6. 100 mM Sodium cyanoborohydride. 7. 100 mM Sodium acetate (pH 4.0, 5.5). 8. Sodium metaperiodate.

2.3. Running Buffers

The following buffers available from GE Healthcare are sterilefiltered and degassed, and thus are recommended as running buffers. Other buffers should be degassed and filtered through a 0.22-mm filter to remove particles. The buffers should include 0.005% Surfactant P20 to minimize the nonspecific adsorption of proteins.

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1. HBS-EP: 0.01  M HEPES pH 7.4, 0.15  M NaCl, 3  mM EDTA, 0.005% Surfactant P20. 2. HBS-P: 0.01  M HEPES pH 7.4, 0.15  M NaCl, 0.005% Surfactant P20. 3. HBS-N: 0.01 M HEPES pH 7.4, 0.15 M NaCl. 2.4. Regeneration Buffers

See Table 2 for some commonly used regeneration buffers.

Table 2 Some commonly used regeneration buffers NaCl (<1 M) 10 mM Gly–HCl (pH 1.5–3.0) Hydrochloric acid (pH 1.3, <100 mM) Phosphoric acid (<100 mM) Formic acid (<20%) 10 mM Gly–NaOH (pH 9–12) NaOH (<50 mM) Ethanolamine (<100 mM) Ethanolamine–HCl (<1 M) EDTA (0.35 M) (in case, where the interaction is dependent on divalent cations) Surfactant P20 (5%) Triton X-100 (5%) SDS (<0.5%) Octylglycoside (<40 mM) Acetonitrile (pH 7.5, <20%) Acetonitrile in sodium hydroxide (20%/100 mM, pH 10.4) DMSO (<8%) Ethylene glycol in HBS buffer (<50%) Ethanol (<20%) Formamide (<40%) Guanidine–HCl (<5 M) Urea (<8 M) Note: When thiol coupling of ligands is used, it is important to note that disulfide bonds are unstable at pH >9.5

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3. Methods 3.1. Preparing the System

1. Turn on the Biacore system and the personal computer. 2. Start the Biacore Control Software. 3. Set the temperature (4–40°C for Biacore 2000, 3000, X100, and A100, and 4–45°C for Biacore T100). A temperature change of 5°C takes about 60 min. Since small air bubbles are often formed at >30°C, complete degassing of the running buffer is important. Biacore models X100, A100, T100, and Flexchip have an in-line degasser. 4. Prepare an appropriate running buffer. 5. Dock the appropriate sensor chip (see Table  3). The carboxymethylated dextran matrix is useful for: (a) Making a hydrophilic environment on the surface of the sensor chip. (b) Dramatically reducing nonspecific binding of samples. (c) Increasing the flexibility of the immobilized ligands as if they were in solution. (d) Easily immobilizing multiple ligands to create a threedimensional structure on the chip. 6. Run Prime to equilibrate the flow system with running buffer. Place the running buffer and waste bottle at the appropriate locations before priming.

Table 3 Sensor Chips Sensor Chip CM5

Carboxymethylated dextran matrix, most widely used

Sensor Chip CM4

Low density of carboxymethylated dextran matrix to minimize nonspecific ligand immobilization

Sensor Chip CM3

Short carboxymethylated dextran matrix to minimize steric hindrance of ligands, but the degree of carboxylation is the same as with CM5

Sensor Chip C1

Carboxymethylated without dextran matrix, suitable for work with particles, such as cells and viruses

Sensor Chip NTA

NTA precoupled dextran matrix CM5 to immobilize His-tagged ligands

Sensor Chip SA

Streptavidin precoupled dextran matrix CM3 to immobilize biotinylated ligands

Sensor Chip L1

Dextran derivatized with hydrophobic compounds to immobilize liposomes

Sensor Chip HPA

Flat hydrophobic surface to immobilize lipid monolayers

Sensor Chip Au

Unmodified gold surface to design original surface chemistry

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7. Set the Rack. Specialized racks and vials for each Biacore machine are available. 8. Check the condition of the Sensor Chip. View the dips by selecting View Dips. A normal dip shows a symmetric curve with sufficient depth (Fig. 1). On the other hand, an abnormal dip shows a shallow or jagged curve. 3.2. Ligand Immobilization (Amine Coupling)

The molecules that are immobilized on the sensor chip are referred to as “Ligands.” Ligand immobilization is performed by direct covalent coupling or indirect capture coupling. The direct coupling methods can be used for almost fully purified proteins, but have some disadvantages: (1) heterogeneity of immobilized ligands and (2) inactivation of immobilized ligands due to blocking of the binding region by immobilization. On the other hand, the indirect coupling can be used only when a suitable binding site or a tag is available for the immobilized molecule. However, it has some advantages as compared to direct coupling: (1) inactivation of ligands does not normally happen, (2) a crude sample can be used, and (3) the orientation of the immobilized ligand is homogeneous. Particularly for receptors, this immobilization can replicate the cell surface orientation. Among the coupling methods, a commonly used method is amine coupling, which we discuss here (Fig.  2). At first, 70000

Reflectance

60000 50000 40000 30000 20000 10000 0

10

20

30

40

50

60

Pixels Fig. 1. Analysis of dips. Both the solid and dotted lines show normal dips.

Fig. 2. Diagram of amine coupling method.

K. Kuroki and K. Maenaka

carboxymethyl groups are activated by NHS, and highly reactive succinimide esters reacting with amines are created. Following the coupling of ligands, a high concentration of ethanolamine blocks the remaining activated carboxymethyl groups. References for other coupling methods are available in Biacore manuals or at the Web site (http:// www.biacore.com). There are several Inject methods in the Control Software, and it is important to check the sample consumption volume to be prepared. (a) INJECT: Generally used inject method, Injected volume + 30 ml necessary. (b) QUICKINJECT:  Low sample consumption method, Injected volume + 10 ml necessary. We usually use this Inject method, except for kinetic analysis. (c) KINJECT:  Appropriate for kinetic analysis because of the low dispersion for samples sensitive to dilution with running buffer. Control the dissociation time after the sample is injected. Injected volume + 40 ml necessary. COINJECT (sequential injections of different samples), BIGINJECT (Large volume injection), and MANUAL INJECT are also available if necessary. 1. Run the sensorgram (10 ml/min) and wait until the baseline becomes stable. If the baseline is difficult to stabilize, increase the flow rate for a while. 2. Perform preconcentration to concentrate the ligand on the dextran matrix of the sensor chip by electrostatic attraction. Inject a small amount of ligand diluted with preconcentration buffer (acetate buffers at pH 4.0–5.5 are available from GE Healthcare) on the nonactivated sensor chip, and select an appropriate pH at which the ligands will become wellconcentrated on the chip. In the case shown in Fig.  3, the 22000

pH4.5

20000 Response (RU)

90

18000

pH5.0

pH4.0

16000 14000 12000 1500

2000 Time (s)

Fig. 3. Sensorgram of preconcentration of a ligand protein.

2500

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ligand is most concentrated at pH 4.5. Generally, the highest pH that shows a sufficient response of immobilization should be normally selected for protein stability (see Note 1). 3. Mix the NHS and EDC in a 1:1 ratio gently (if necessary, degas by centrifugation), and immediately inject 100 ml of the mixture to activate the surface of the CM5 chip (10 ml/min). The next coupling step should be performed as soon as possible because the activated NHS–esters easily break down. 4. Inject the ligands (5–10 ml/min) diluted into the preconcentration buffer until the immobilization level reaches the target value. At least one flow cell should be used for a control ligand to record the nonspecific background response. 5. Inject 100 ml of ethanolamine hydrochloride (10 ml/min) to block the excess reactive surface. 6. Wash the chip with regeneration buffer (e.g., 10 mM glycine– HCl pH 2.0) if the proper regeneration method has already been determined. The regeneration step is useful to reuse the chip surface, but carefully confirm whether the ligand activity is unchanged and how many times regeneration can be performed. Ideally, regeneration should fulfill the following conditions: (1) maintaining the ligand activity, (2) completely dissociating the analytes, and (3) retaining the ligands on the sensor chip. In the case of typical fast ligand binding of immune cell surface receptors, such as MHC class I-LILRs (koff = 2.1 − 5.0 s−1) (Table 4), the dissociation is quite fast and the sensorgram quickly returns to the baseline. In such cases, the regeneration step is not necessary. Some commonly used regeneration buffers are listed in Table 2. When we analyze receptor–ligand interactions by SPR, we generally use biotinylated recombinant proteins as ligands to achieve a homogeneous molecular orientation on the chip surface and to avoid blocking the binding sites by coupling. The biotinylated ligands are immobilized onto the Sensor Chip SA or the SA-coupled CM5 Chip. For this method, we usually prepare the ectodomain of the ligand protein with the biotin ligase (birA) recognition sequence (GSLHHILDAQKMVWNHR) at the C-terminal end (for type I membrane proteins). Purified proteins with the birA recognition sequence are biotinylated by mixing the substrate samples, Biomix-A, and Biomix-B (8:1:1, respectively), and adding 1.0 mg of the birA enzyme (AVIDITY, LLC). Typically, for every 10 nmol of substrate at 40 mM, 2.5 mg of birA enzyme is recommended to complete the biotinylation in 30–40 min at 30°C. The biotinylated proteins are isolated from the free biotin by gel filtration chromatography or by dialysis. The purified biotinylated ligands are immobilized onto the SA-coupled CM5 chips. The amount of ligands needed for immobilization is modest (5–10 mg).

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Table 4 Examples of kinetic parameters of receptor–ligand interactions kon (× 105M−1/s)

koff (s−1)

Kd (mM)

References

LILRB1

HLA-G1

9.2

2.1

2.4

(4)

LILRB1

HLA-B35

5.0

3.7

7.4

(4)

LILRB1

HLA-Cw4

6.3

5.0

8.1

(4)

LILRB1

UL18

1.4

0.0028

0.0021

(5)

KIR2DL3

HLA-Cw7/DS11

2.1

1.1

5.2

(6)

CD8aa

MHC class I

≥1.0

≥18

~200

(7)

PILRa

CD99

3.9

1.2

3.0

(8)

CD22

CD45

≥1.5

≥18

117

(9)

CD80

CTLA-4

9.4

0.43

0.46

(10)

CD80

CD28

6.6

1.6

2.4

(10)

FcgRIIa, IIb, III

hFc1

3.8–4.4

0.31–0.69

0.72–1.9

(11)

TCR

MHC/peptide

0.009–0.2

0.01–0.1

1–90

(12, 13)

E-selectin

ESL-1

0.48

2.7

56

(14)

L-selectin

GlyCAM-1

>1

>10

108

(15)

P-selectin

PSGL-1

44

1.4

0.32

(16)

3.3. Preparing Analyte Samples

1. Purified analyte samples should be completely bufferexchanged into the running buffer. If this step is not accomplished, a bulk effect obscuring rapid binding events could occur (see Note 2). The concentration of analyte protein is dependent on the Kd value, and the required volume is dependent on the analysis method. 2. Before injection, analyte samples should be degassed by centrifugation at room temperature.

3.4. Analysis of Protein Interactions 3.4.1. Equilibrium-Binding Analysis

An equilibrium-binding analysis is performed by multiple sequential injections of an analyte at different concentrations. The typical receptor–ligand interaction is quite weak (Kd in the mM range) and shows fast association and dissociation (Table 4). In this case, the Kd value can be measured directly by an equilibrium-binding analysis. Here, we describe LILRB2 (22 kDa)–HLA-G (45 kDa) binding as an example. As for high-affinity interactions, an equilibrium-binding analysis is not suitable due to the very slow dissociation rates. Furthermore, if the binding is very strong and

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93

the bound analyte is difficult to dissociate from the ligands, then a regeneration step should be included after the injection step. Experiment

1. Dock a new CM5 chip, equilibrate the chip by running Prime with HBS–EP buffer, and immobilize SA diluted with 10 mM sodium acetate pH 5.0 buffer by amine coupling (>10,000 RU) (see Note 3). 2. Immobilize the ligands, including biotinylated BSA as a control protein on SA-immobilized Fc1 (2,000  RU) and C-terminal biotinylated HLA-G on SA-immobilized Fc2 (2,000 RU). Biotinylated HLA-G is prepared by a refolding method and purified as described previously (1, 2). Since the Biacore 3000 system has four flow cells, up to three kinds of ligands can be immobilized, in addition to a control ligand. 3. Prepare the analyte LILRB2 protein by the refolding method (3). Purified LILRB2, concentrated to 36.6 mM in HBS–EP buffer, is serially diluted twofold with HBS–EP buffer (0.07– 36.6  mM). Centrifuge all samples at max speed (e.g., 16,000 × g) in a microcentrifuge for 5 min at RT. The sample concentration needs to be sufficiently higher than the expected Kd. If there is no information available about the Kd, then about 50 mM will be sufficient to calculate the Kd of most of immune receptor–ligand interactions. 4. Inject 5 ml of the analyte in flow cells Fc1–Fc2 sequentially (10 ml/min) by Quickinject, from low to high concentration samples. Since the binding affinity between HLA-G and LILRB2 is weak and displays fast association/dissociation, it can be considered that there is no difference in the concentration in each flow cell. The time to the next injection should be estimated from the koff.

Data Analysis

Raw data from an equilibrium-binding analysis are shown in Fig. 4. Usually, we use a program to record the response (RU) 10  s before injection as a Baseline and 20  s after injection as a Response. 1. For each serially diluted analyte sample, the actual binding response is calculated from [Response]–[Baseline]. 2. The binding response (RU) at each concentration is calculated by subtracting the response measured in the control flow cell (Fc1: BSA) from the response in the sample flow cell (Fc2: HLA-G). This value is applied to the simple 1:1 Langmuir-binding model (A + B « AB). The Langmuir model is the most commonly used model to calculate binding affinity. This model is applied to the simple situation of an interaction between two samples (A, B). It is hypothesized that both the analyte and ligand are homogeneous, and that the analyte is monovalent.

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K. Kuroki and K. Maenaka Analyte concentration 34500

Response (RU)

34000 33500 33000 32500 32000 0

200

400

600

800

1000

Time (s) BSA (Control)

HLA-G

Fig. 4. Sensorgram of equilibrium-binding analysis. The gray line is BSA (Fc1), and the black line is LILRB2 (Fc2). Ten serially diluted LILRB2 samples are injected from low to high concentrations. 800 700 Response (RU)

600 500 400

LILRB2 vs HLA-G Kd=3.1µM

300 200 100 0 0

5

10

15

20

25

30

35

40

LILRB2 (µM)

Fig. 5. The affinity of the LILRB2–HLA-G interaction. The responses are plotted against the concentrations of injected LILRB2 protein. The solid line represents direct nonlinear fit of the 1:1 Langmuir-binding isoform to the data. The Kd value is determined as 3.1 mM.

3. Plot the analyte concentration on the X-axis and the Response (RU) on the Y-axis, as shown in Fig.  5. Affinity constants (Kd) are derived by nonlinear curve fitting of the standard Langmuir-binding isotherm:

y = Rmax · x / (K d + x )

Rmax : maximum response units.

4. The affinity constant (Kd) is also derived by Scatchard analysis, as shown in Fig. 6.

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6  Analysis of Receptor–Ligand Interactions by Surface Plasmon Resonance 300

LILRB2 vs HLA-G Kd=3.1µM

Bound (RU)

250 200 150 100 50 0

0

200

400

600

800

Bound/Free

Fig. 6. The Scatchard plot of the LILRB2–HLA-G interaction. The solid line is the linear fit. The Kd value is determined as 3.1 mM.

3.4.2. Kinetic Analysis



SPR analysis is also used to analyze high-resolution kinetic parameters. From a sensorgram, such as that in Fig.  7, kinetic parameters (ka, kd) are calculated. Here, we describe LILRB1 (22 kDa)–HLA-G (45 kDa) binding as an example. If the binding is quite strong and the bound analyte is difficult to dissociate from the ligands, then the regeneration step should be added after the injection, as shown in Fig. 7. From the association phase, the association rate constant (ka, M/s) between the ligand and the analyte is calculated, and the dissociation rate constant (kd, 1/s) is calculated from the dissociation phase. To avoid mass transport limitations and rebinding of analytes to the immobilized ligand before leaving the sensor surface, an excess of ligands should not be immobilized (see Note 4). In the beginning, multiple immobilization levels of ligand should be assessed. GE Healthcare recommends the level of immobilization as described below (s means the number of ligand-binding sites): Min (RU ) = 200 ´ 1 / s ´ (Ligand Mw / Analyte Mw ) Max (RU ) = 1, 000 ´ 1 / s ´ (Ligand Mw / Analyte Mw ) Mw: molecular weight.



In the case of LILRB1 (22,000 Da) and HLA-G (45,000 Da),

Min (RU ) = 200 ´ 1 / 1 ´ (45, 000 / 22, 000) = 409 RU

Max (RU ) = 1, 000 ´ 1 / 1 ´ (45, 000 / 22, 000 ) = 2, 045 RU.

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K. Kuroki and K. Maenaka association phase

dissociation phase

regeneration phase

Response (RU)

15500

15000

14500

14000 0

100

200

300

400

500

Time (s)

Fig. 7. Typical sensorgram of a binding analysis. During the association phase, the ligand binding is reflected by an increasing Response. At the end point of the analyte injection, the running buffer is flowed over the ligand and the dissociation phase begins. In order to start the new cycle of injection, the Response should return to the baseline level. In this case, the ligand is regenerated by the injection of regeneration buffer.

Experiment

1. Dock the CM5 chip, equilibrate the chip by running Prime with HBS–EP buffer, and immobilize SA diluted with 10 mM sodium acetate pH 5.0 buffer by amine coupling (>10,000 RU). 2. Immobilize the ligands, biotinylated BSA as a control protein on SA-immobilized Fc1 (800 RU) and biotinylated HLA-G on SA-immobilized Fc2 (800 RU). If it is possible, immobilize the ligand at different levels using three flow cells (Fc2–Fc4). 3. Prepare the analyte LILRB1 protein by the refolding method (3). Purified LILRB1 was concentrated to 3.0 mM in HBS–EP buffer and serially diluted twofold with HBS–EP buffer (0.19–3.0  mM). Centrifuge all samples at max speed (e.g., 16,000×g) in a microcentrifuge for 5 min at RT. In the kinetic analysis, Kinject (Injected volume + 40 ml) should be used in order to avoid sample dilution with running buffer. Furthermore, the analyte sample should be flowed over each flow cell independently, not sequentially, because the highest resolution detection is suitable for fast kinetics (see the following paragraph). 4. Inject 5 ml of the analyte in flow cells Fc1–Fc2 sequentially (50  ml/min) by Kinject, from low to high concentration ­samples. A higher flow rate should be used than that for an  equilibrium-binding analysis in order to minimize mass

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transport effects. Data collection at the maximum resolution (Data collection rate = high (see footnote 3)) and only one flow cell detection mode are recommended for measurements of very fast kinetics, such as cell surface receptor–ligand interactions. The durations of the association and dissociation phases should be determined in a preliminary experiment. Data Analysis (BIAevaluation Software)

Generally, the association of Analyte A and Ligand B results in the formation of complex AB. ka ¾¾ ® AB, A + B ¬¾¾ kd



where ka is association rate constant (M−1/s) and kd is dissociation rate constant (1/s). When the response achieves equilibrium, the rate of the concentration changes, which is dependent on the concentrations of A, B, and AB, and becomes zero.

d [AB] / dt = ka [A ][B] - kd [AB]



kd / ka = [A ][B] / [AB] = K d (dissociation constant, M ),

(1)

Ka (association constant, 1/M) = 1/Kd. A smaller Kd means higher affinity. In the Biacore systems, Analyte A always flows over Ligand B at the same concentration. Therefore, [A] = [A]0, [B] = [B]0 − [AB].

d [AB] / dt = ka [A ][B] - kd [AB] = ka [A ]0 ([B]0 - [AB])- kd [AB]. Next, relate the parameters in Eq. (1) to the SPR parameters. The concentration of complex AB ([AB]) is the response R (RU), [B]0 is the maximum response Rmax (RU), and the concentration of Analyte A [A]0 is C. dR / dt = kaC (Rmax - R ) - kd R

= kaCRmax - (kaC + kd )R



(kaC + kd ):



(2)

pseudo reaction rate constant.

Data collection rate: The data collection rate determines the number of points per second during the sensorgram. In Biacore 2000/3000, Low (0.1  point/s), Medium (1.0  point/s), and High (2.0  points/s) can be selected. The default setting (Medium) is adequate for most analyses (e.g., equilibrium-binding analysis).

3 

98

K. Kuroki and K. Maenaka 700 600 500 400 300 200

400 300 200 100

0

0 20

BSA

500

100

0

LILRB1

600

BSA

Response (RU)

Response (RU)

700

LILRB1

40

Time (s)

0

20

40

Time (s)

Fig. 8. Sensorgram of a kinetic analysis between HLA-G and LILRB1 at a single concentration. The solid line is the sensorgram of LILRB1, and the dotted line is the sensorgram of BSA after adjustment of the X- and Y-axes. Sensorgrams before subtraction of the control (left ) and after subtraction (right ) are shown.

The change rate of R follows a pseudo-first-order reaction. Here, the calculated pseudo (kaC + kd) is dependent on C; therefore, the ka can be calculated by plotting (kaC + kd) against the known C. By using the BIA evaluation software supplied by GE Healthcare, nonlinear fitting of the sensorgram by a nonlinear least-squares analysis can be performed, as described below. 1. Start the BIAevaluation software. 2. Open the result file, and display the sensorgram curves of the same analyte concentration of control (BSA) and HLA-G (Fc1 and Fc2, respectively). 3. Subtract the response in the control flow cell (Fc1: BSA) from the response in the sample flow cell (Fc2: HLA-G). It is important to adjust the baseline (Y-axis) just before the injection point and injection/dissociation point (X-axis) before subtracting. The subtraction of the control response from the sample response is performed for each analyte concentration response curve (Fig. 8). 4. Display the response plot drawn by (Fc2–Fc1) all dilution series samples (0.19, 0.38, 0.75, 1.5, and 3.0 mM of LILRB1). 5. Perform the fitting using the appropriate fitting model. In the case of the LILRB1–HLA-G interaction, the global fitting analysis using a 1:1 Langmuir-binding model was simultaneously performed with the raw data for the association and dissociation phases at different concentrations of LILRB1 (Fig. 9). As shown in Fig. 9, the small variation in the residuals (see footnote 4) means that the model is suitable for this analysis.  Residual plots: Plots of the difference between the experimental data and fitted data of each curve.

4

6  Analysis of Receptor–Ligand Interactions by Surface Plasmon Resonance

99

Fig. 9. The global fitting kinetic analyses of LILRB1 against HLA-G (4). The square dots are actual sensorgram data and the solid lines are nonlinear fits. The lower panel shows the residual errors from the fits. Kinetic parameters are also shown.

Several simultaneous ka/kd fitting models are available in the BIAevaluation software. (a) 1:1 Langmuir-binding model: Interaction between ligand A and analyte B. The simplest model, which is equivalent to the Langmuir isotherm for adsorption to a surface.

A + B « AB (b) 1:1 binding with drifting baseline: Sensorgram showing a linearly drifting baseline. Before using this model, try to eliminate the drift by subtracting the control sensorgram.



A + B « AB (c) 1:1 binding with mass transfer: Interaction showing mass transfer limitations. Before using this model, try to increase the flow rate or immobilize ligands at a lower level.



A + B « AB (d) Bivalent analyte: Interaction of a bivalent or dimeric analyte.



A + B « AB AB + B « AB2



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K. Kuroki and K. Maenaka

(e) Heterogeneous analyte–competition reactions: Two kinds of analytes competitively interact with the ligand.

A 1 + B « A 1B



A 2 + B « A 2B (f) Heterogeneous ligand–parallel reaction: Interactions between an analyte and a ligand possessing two binding sites with different binding affinities.



A + B1 « AB1



A + B2 « AB2 (g) Two-state reactions (conformation change): After formation of the AB complex, the complex changes its conformation.



A + B « AB « ABx Additionally, separate ka/kd models that fit ka and kd separately and describe only the association or dissociation phase of the sensorgram can be generated by the BIAevaluation software. However, simultaneous ka/kd fitting is preferable, if possible.

3.4.3. Thermodynamic Analysis

Experiment

The Biacore system can strictly control the temperature of flow cells. By measuring the change in the binding affinity at different temperatures, it is possible to estimate the thermodynamic parameters using van’t Hoff analysis, as described below. Here, we describe LILRB1 (22  kDa)–HLA-G (45  kDa) binding as an example. 1. Dock the CM5 chip, equilibrate the chip by running Prime with HBS–EP buffer, and immobilize SA (10  mM sodium acetate pH 5.0 buffer) by amine coupling (>10,000 RU). 2. Immobilize the ligands: Biotinylated BSA as a control protein on SA-immobilized Fc1 (2,000 RU) and C-terminal biotinylated HLA-G on SA-immobilized Fc2 (2,000 RU). 3. Set the temperature (e.g., 10°C, 15°C, 20°C, 25°C, and 30°C) and wait until the measured temperature is stable. 4. Prepare the analyte LILRB1 protein (50  mM) in HBS–EP buffer and serially dilute it twofold with HBS–EP buffer (0.2–50  mM). Centrifuge all samples at max speed (e.g., 16,000×g) in a microcentrifuge for 5 min at RT. 5. Inject 5 ml of the analyte Fc1 → Fc2 sequentially (10 ml/min) by Quickinject from low to high concentration samples.

Data Analysis

By measuring the change in the binding affinity at different temperatures, the van’t Hoff enthalpy can be calculated using the following equations.

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6  Analysis of Receptor–Ligand Interactions by Surface Plasmon Resonance

The change of Gibbs energy are DG / T = DH / T - DS



d (DG / T ) / dT = dDH / TdT - DH / T 2 - dDS / dT . (3) Here,



dDH = DC p dT and



dDS = DC p dT / T is applied to Eq. (3). d(DG / T ) / dT = DC p / T - DH / T 2 - DC p / T





= -DH / T 2 .



(4)

The standard state Gibbs energy change upon binding was obtained from DG = RT ln K d , where Kd is the dissociation constant and R is the gas constant. Therefore,



d ln K d / d (1 / T ) = -DH / R. The enthalpy (DH) can be calculated by plotting ln Kd against 1/T. On the other hand, the DG values of each data set are plotted against the temperatures, and are fitted with the nonlinear van’t Hoff equation DG = DH − TDS + DCp(T − 298.15) − DCpTln(T/298.15), where DH and DS are the binding enthalpy and entropy at 298.15  K, respectively, and DCp is the heat capacity, which is assumed to be temperature-independent. 1. The Kds are determined at different temperatures by an equilibrium-binding analysis (see Subheading 3.4.1). 2. The Gibbs energy (DG) at each temperature point is obtained from the Kd.



DG = RT ln K d . DG is plotted against the temperatures and is fitted with the nonlinear van’t Hoff equation (Fig. 10). DH, DS, and DCp are obtained simultaneously from this fitting (Table  5). These data are in good agreement with the values obtained from an ITC analysis (DG = −7.0  kcal/mol, DH = 2.4  kcal/mol, −TDS = −9.4 kcal/mol, DCp = −0.22 kcal/mol K) (4).

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Fig. 10. The plots of temperature dependence of DG (4). DG values at five temperature points were obtained from equilibrium-binding analyses. Filled triangles represent the plot fitted with the nonlinear van’t Hoff equation of the association between LILRB1 and HLA-G. Filled circles and squares are the associations with other tested HLA-class I molecules.

Table 5 Examples of the thermodynamic parameters of the receptor–ligand interactions DG DH −TDS DCp (kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol∙K) References

Analyte

Ligand

LILRB1

HLA-G1

−7.5

1.9

−9.4

−0.22

(4)

LILRB1

HLA-B35

−6.6

0.6

−7.2

−0.10

(4)

LILRB1

HLA-Cw4

−6.8

−0.2

−6.6

−0.16

(4)

KIR2DL3

HLA-Cw7/DS11

−7.2

−4.1

−3.1

−0.1

(6)

NKG2D

RaeI

−8.6

−5.2

−3.4

(17)

NKG2D

H60

−10.5

−23.6

13.1

(17)

PILRa

CD99

3.9

1.2

3.0

(8)

CD22

CD45

−5.1

−10.1

5.0

−4.4 to −6.4

−1.9 to −3.3

−0.08

(9)

−0.22 to −0.43

(11)

FcgRIIa, IIb hFc1

−7.9 to −8.3

FcgRIII

hFc1

−8.0

−15.4

7.4

−0.7

(11)

TCR

MHC/peptide

−7.1

−14.6

7.1

−0.62

(12, 18–22)

E-selectin

ESL-1

−5.7

−0.9

−4.8

(14)

DG, DH, −TDS, and DCp represent binding changes in standard state Gibbs energy, enthalpy, entropy, and heat capacity, respectively, calculated with the nonlinear van’t Hoff equation

6  Analysis of Receptor–Ligand Interactions by Surface Plasmon Resonance 3.4.4. Activation Energy Analysis

Experiment

103

Binding rate constants generally increase with temperature. The extent of this increase is a measure of the amount of thermal energy required to cross an energy barrier preventing the association or dissociation, and is referred to as the activation energy of association or dissociation (Eaon or Eaoff). By measuring the change in the kinetic parameters at different temperatures, the activation energies can be calculated. Here, we describe LILRB1 (22 kDa)– HLA-G (45 kDa) binding as an example. 1. Dock the CM5 chip, equilibrate the chip by running Prime with HBS-EP buffer, and immobilize SA (10  mM sodium acetate pH 5.0 buffer) by amine coupling (>10,000 RU). 2. Immobilize the ligands: Biotinylated BSA as a control protein on SA-immobilized Fc1 (800 RU) and biotinylated HLA-G on SA-immobilized Fc2 (800 RU). 3. Set the temperature (e.g., 10°C, 15°C, 20°C, 25°C, and 30°C) and wait until the measured temperature is stable. 4. Prepare the analyte LILRB1 (3.0 mM) in HBS–EP buffer and serially dilute it twofold with HBS–EP buffer (0.19–3.0 mM). Centrifuge all samples at max speed (e.g., 16,000×g) in a microcentrifuge for 5 min at RT. 5. Inject 5 ml of the analyte Fc1 → Fc2 sequentially (50 ml/min) by Kinject, from low to high concentration samples.

Data Analysis



The activation energy of association or dissociation (Eaon or Eaoff) was obtained using the Arrhenius equation. By assuming Ea is constant over the temperature range analyzed, ln k = ln A - E a / RT , where k is the kon or koff, A is a constant known as a preexponential factor, and R is the gas constant. The reaction enthalpy can be calculated from the relationship,



DH = E aon - E aoff . 1. The ka or kd is determined at different temperatures by kinetic analysis (see Subheading 3.4.2). 2. Eaon and Eaoff are obtained from Arrhenius plots (Fig. 11) (4). In the case of association between LILRB1 and HLA-G, the small temperature dependency resulted in low Eaon and Eaoff values (Eaon = 5.4 kcal/mol, Eaoff = 2.2 kcal/mol, DH = 3.2 kcal/ mol).

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Fig. 11. Temperature dependencies of kon (left ) and koff (right ) (4). Arrhenius plots of the natural logarithm on and off rates for LILRB1 binding derived over a range of temperatures (10–30°C). Filled circles and squares are associations with other tested HLA-class I molecules.

3.4.5. Shutdown

The shutdown methods differ, according to how long the system will not be in use (see Note 5): (a) <3  days until the next experiment using the same sensor chip The Standby procedure is useful to keep the instrument with the used sensor chip filled with the running buffer. The Standby procedure keeps the Biacore running at a slow flow rate (5 ml/min) for max 96 h. Make sure that sufficient running buffer is available. (b) 3–5 days To shut down the system for up to 5 days, run Prime with water followed by run Close with water. (c) >5 days Run shutdown method with 70% ethanol.

4. Notes 1. Ligands that are difficult to preconcentrate (low-weight molecules and peptides) should be diluted with 10  mM borate buffer (pH 8.5) because the response between NHS and amine is most efficient at pH 8.5. 2. Bulk effect: Bulk effects are due to differences in the refractive indexes of the running buffer and the sample solution. 3. In Biacore systems, one RU represents the binding of about 1 pg/mm2.

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4. Mass transport limitation and rebinding: The situation in which a massive amount of ligand causes two important problems in the kinetic analysis by SPR. The first one is the mass transport limitation, in which the rate at which the analyte binds the ligand can exceed the rate at which it is delivered to the surface. The reaction rate is limited by the physical transport of the analyte. When kon and koff are faster than the diffusion rate of the analyte, the depletion of the analyte close to the matrix occurs during the association phase, and then the binding is controlled by diffusion and not by interaction. As a result, an apparent kon is calculated that is slower than the actual kon. The effect of the high flow rate on mass transfer is not very large. On the other hand, the density of the immobilized ligand is quite important. Minimizing the ligand density is the most effective way to reduce mass transfer limitations. The next potential problem is the rebinding of analytes to the ligand before they leave the sensor surface. Consequently, the apparent koff is slower than the actual koff. This can also be avoided by decreasing the level of immobilized ligands. 5. Storage of sensor chips: Used sensor chips can be stored under dry or wet (with buffer in a 50-mL tube) conditions. Stable protein-, peptide-, or DNA-immobilized sensor chips can be stored under dry conditions. 6. Single-cycle kinetics (23): As shown in Table 1, Biacore X100 and T100 are capable of performing single-cycle kinetics. Since single-cycle kinetics enables kinetic analysis without the regeneration of a sensor chip, analyte samples are injected one after the other in the same cycle. This method is suitable for molecular interactions for which the kinetic parameters were previously difficult to determine due to the inability to regenerate ligands. Furthermore, it reduces the time required to develop the assay conditions. The Biacore software recommends five sample concentrations and a duplication cycle for this analysis to ensure a robust evaluation. References 1. Garboczi D.N., Hung D.T. and Wiley D.C. (1992) HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc Natl Acad Sci USA, 89, 3429–3433. 2. Reid S.W., Smith K.J., Jakobsen B.K., O’Callaghan C.A., Reyburn H., Harlos K., et al. (1996) Production and crystallization of MHC class I B allele single peptide complexes. FEBS Lett, 383, 119–123.

3. Shiroishi M., Tsumoto K., Amano K., Shirakihara Y., Colonna M., Braud V.M., et al. (2003) Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G. Proc Natl Acad Sci USA, 100, 8856–8861. 4. Shiroishi M., Kuroki K., Tsumoto K., Yokota A., Sasaki T., Amano K., et  al. (2006) Entropically driven MHC class I recognition by human inhibitory receptor leukocyte Ig-like

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receptor B1 (LILRB1/ILT2/CD85j). J Mol Biol, 355, 237–248. 5. Chapman T.L., Heikeman A.P. and Bjorkman P.J. (1999) The inhibitory receptor LIR-1 uses a common binding interaction to recognize class I MHC molecules and the viral homolog UL18. Immunity, 11, 603–613. 6. Maenaka K., Juji T., Nakayama T., Wyer J.R., Gao G.F., Maenaka T., et al. (1999) Killer cell immunoglobulin receptors and T cell receptors bind peptide-major histocompatibility complex class I with distinct thermodynamic and kinetic properties. J Biol Chem, 274, 28329–28334. 7. Wyer J.R., Willcox B.E., Gao G.F., Gerth U.C., Davis S.J., Bell J.I., et al. (1999) T cell receptor and coreceptor CD8 alpha bind peptide-MHC independently and with distinct kinetics. Immunity, 10, 219–225. 8. Tabata S., Kuroki K., Wang J., Kajikawa M., Shiratori I., Kohda D., et al. (2008) Biophysical characterization of O-glycosylated CD99 recognition by paired Ig-like type 2 receptors. J Biol Chem, 283, 8893–8901. 9. Bakker T.R., Piperi C., Davies E.A. and Merwe P.A. (2002) Comparison of CD22 binding to native CD45 and synthetic oligosaccharide. Eur J Immunol, 32, 1924–1932. 10. van der Merwe P.A., Bodian D.L., Daenke S., Linsley P. and Davis S.J. (1997) CD80 (B7-1) binds both CD28 and CTLA-4 with a low affinity and very fast kinetics. J Exp Med, 185, 393–403. 11. Maenaka K., van der Merwe P.A., Stuart D.I., Jones E.Y. and Sondermann P. (2001) The human low affinity Fcgamma receptors IIa, IIb, and III bind IgG with fast kinetics and distinct thermodynamic properties. J Biol Chem, 276, 44898–44904. 12. Willcox B.E., Gao G.F., Wyer J.R., Ladbury J.E., Bell J.I., Jakobsen B.K., et al. (1999) TCR binding to peptide-MHC stabilizes a flexible recognition interface. Immunity, 10, 357–365. 13. Ding Y.H., Baker B.M., Garboczi D.N., Biddison W.E. and Wiley D.C. (1999) Four A6-TCR/peptide/HLA-A2 structures that generate very different T cell signals are nearly identical. Immunity, 11, 45–56. 14. Wild M.K., Huang M.C., Schulze-Horsel U., van der Merwe P.A. and Vestweber D. (2001)

Affinity, kinetics, and thermodynamics of E-selectin binding to E-selectin ligand-1. J Biol Chem, 276, 31602–31612. 15. Nicholson M.W., Barclay A.N., Singer M.S., Rosen S.D. and van der Merwe P.A. (1998) Affinity and kinetic analysis of L-selectin (CD62L) binding to glycosylation-dependent cell-adhesion molecule-1. J Biol Chem, 273, 763–770. 16. Mehta P., Cummings R.D. and McEver R.P. (1998) Affinity and kinetic analysis of P-selectin binding to P-selectin glycoprotein ligand-1. J Biol Chem, 273, 32506–32513. 17. O’Callaghan C.A., Cerwenka A., Willcox B.E., Lanier L.L. and Bjorkman P.J. (2001) Molecular competition for NKG2D: H60 and RAE1 compete unequally for NKG2D with dominance of H60. Immunity, 15, 201–211. 18. Boniface J.J., Reich Z., Lyons D.S. and Davis M.M. (1999) Thermodynamics of T cell receptor binding to peptide-MHC: evidence for a general mechanism of molecular scanning. Proc Natl Acad Sci USA, 96, 11446–11451. 19. Anikeeva N., Lebedeva T., Krogsgaard M., Tetin S.Y., Martinez-Hackert E., Kalams S.A., et  al. (2003) Distinct molecular mechanisms account for the specificity of two different T-cell receptors. Biochemistry, 42, 4709–4716. 20. Garcia K.C., Radu C.G., Ho J., Ober R.J. and Ward E.S. (2001) Kinetics and thermodynamics of T cell receptor-autoantigen interactions in murine experimental autoimmune encephalomyelitis. Proc Natl Acad Sci USA, 98, 6818–6823. 21. Lee J.K., Stewart-Jones G., Dong T., Harlos K., Di Gleria K., Dorrell L., et al. (2004) T cell cross-reactivity and conformational changes during TCR engagement. J Exp Med, 200, 1455–1466. 22. Davis-Harrison R.L., Armstrong K.M. and Baker B.M. (2005) Two different T cell receptors use different thermodynamic strategies to recognize the same peptide/MHC ligand. J Mol Biol, 346, 533–550. 23. Karlsson R., Katsamba P.S., Nordin H., Pol E. and Myszka D.G. (2006) Analyzing a kinetic titration series using affinity biosensors. Anal Biochem, 349, 136–147.

Chapter 7 Cell-Based Reporter Assay to Analyze Activation of Nod1 and Nod2 Birte Zurek, Harald Bielig, and Thomas A. Kufer Abstract Nod1 and Nod2 are pattern recognition receptors of the mammalian innate immune system. They respond to bacterial peptidoglycan fragments and are implicated in host defense against a variety of ­different bacterial pathogens. Recent studies furthermore support additional functions of these proteins in the control of adaptive immune responses and intestinal homeostasis. Activation of Nod1 and Nod2 by their cognate elicitors triggers inflammatory responses driven by the activation of NF-kB and MAPK pathways. In this chapter, we describe a quick and reliable cell-based assay using a luciferase reporter to measure Nod1- and Nod2-mediated NF-kB activation. The described protocol was successfully applied to analyze the influences of overexpressed proteins and siRNA-mediated knock-down to provide new insights into the regulation of Nod1/2-specific signaling pathways. Furthermore, this method is well suited for downscaling to high-throughput screening applications. Key words: CARD4, CARD15, NLR, Bacteria, MDP, Tri-DAP, Luciferase assay, HTS

1. Introduction Early immune responses in vertebrates are triggered by the recognition of invariant motifs from invading microbes (microbeassociated molecular patterns, MAMPs) through specialized pattern recognition receptors (PRRs). Cells express a variety of PRRs, such as the membrane-bound toll-like receptors (TLRs), C-type lectins (CTLs), or the cytoplasmic RIG-I-like helicases (RLHs) (1). Another important type of cytoplasmic PRRs are the proteins of the nucleotide-binding domain, leucine-rich repeat-containing family (NLRs) (1, 2). Among the currently best-studied members of the NLR family are Nod1 and Nod2, which recognize different bacterial peptidoglycan motifs. The minimal structure required to

Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_7, © Springer Science+Business Media, LLC 2011

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activate Nod1 is the tripeptide l-Ala-g-d-Glu-meso-diaminopimelic acid (Tri-DAP) (3, 4), which is present in many Gram− as well as in some Gram+ bacteria. Nod2 recognizes muramyl dipeptide (MDP), a motif common for all peptidoglycans (5, 6). Nod1 and Nod2 display a tripartite domain architecture: The N-terminus contains caspase activation and recruitment domain(s) (CARDs), followed by a central NACHT domain which mediates activation and oligomerization of the protein (7, 8). The C-terminus consists of leucine-rich repeats (LRRs) mediating pathogen recognition (9, 10). Recognition of their cognate elicitors triggers downstream signaling by homo-oligomerization of Nod1/2 and recruitment of the serine–threonine kinase RIPK2 (also called RIP2, RICK, or CARDIAK) by homophilic CARD–CARD interactions. Thereby, RIPK2 gets activated by ubiquitination and, in turn, provides the link to TAK1 which activates the IKK complex (reviewed in (11)). This results in degradation of the NF-kB inhibitor IkBa, subsequent translocation of NF-kB (RelA) into the nucleus, and transcription of NF-kB-dependent proinflammatory genes (Fig. 1a). In addition, TAK1 activates MAPK pathways downstream of Nod1/2 (12–14). The activation of NF-kB and MAPK pathways induces an inflammatory response that supports the clearance of invading bacterial pathogens and controls the onset of an adaptive immune response (15, 16). Additionally, Nod1-mediated responses are involved in tissue homeostasis and the development of lymphoid structures in the gut (17). Here, we describe an adaptation of the classical luciferase reporter gene assay (18) to analyze Nod1 and Nod2 activation in human cell lines. In brief, the assay relies on the activation of Nod1 or Nod2 by the commercially available synthetic elicitors Tri-DAP or MDP, respectively, which leads to the nuclear translocation of NF-kB (p50/p65). P50/p65 binds to its response element on a luciferase reporter plasmid and luciferase gets transcribed. Luciferase accumulates in the cell and can be measured by the detection of chemiluminescence upon addition of firefly luciferin, as chemiluminescence shows a linear correlation to the amount of luciferase (19). Detection of a constitutively expressed reporter, here b-galactosidase (20), allows to adjust for variations in the transfection efficiency and to monitor cell-death, eventually induced by the experimental settings. Primarily, HEK293T cells are used, as they are easy to transfect and thus yield a robust readout. Furthermore, they offer advantages for the analysis of PAMP-mediated responses, as they do not express TLR2, 4, and 8 and are thus inert to stimulation with other classical bacterial PAMPs (21). In particular, they do not respond to contaminations with LPS, which is a major problem for the interpretation of PAMP effects in other systems.

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An NF-kB-driven luciferase reporter is used, as it turned out to be very robustly activated by Nod1 and Nod2. However, other reporter systems, such as IL-8, can also be successfully used. Different ways to normalize these assays have been reported (18). We have chosen a b-galactosidase reporter. This and the use of home-made luciferin make our protocol particularly cost-effective, and thus also applicable to laboratories with limited budget and for high-throughput screening (HTS) applications. Indeed, the provided protocol can efficiently be scaled down to 384-well plate applications without major changes and allows siRNA screening with very robust readouts (Bielig et al., unpublished data).

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By the use of differential readouts, the specificity for the Nod1/2 pathway can be determined. To this end, in our protocol, we use stimulation with TNF or Pam3CSK4 to activate the TNFR1 or TLR2 pathway, respectively (Fig. 1a, (22, 23)). When activations of Nod1/2 mutants are analyzed, results obtained with the described assay can be substantiated by the analysis of subcellular localization of overexpressed Nod1/2 constructs in human epithelial cells, such as HeLa and Caco2, where only signaling-active mutants of Nod1/2 show a pronounced membrane association (24–26, 34). Cell-based luciferase reporter assays for Nod1/2 have successfully been used to analyze the influences of chemical substances (27) and proteins, either by overexpression or by siRNA-mediated knock-down (24, 28–30, 35). The method described here provides a quick and reliable protocol that was successfully used for analyzing the effect of proteins on Nod2-mediated signaling (31).

2. Materials 2.1. Cell Culture

1. Cells: HEK293T cells were obtained from the ATCC (#CRL11268). 2. Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated (30 min, 56°C) fetal bovine serum (FBS) and Penicillin–Streptomycin (100  IU/ml and 100 mg/ml, respectively). 3. Phosphate-Buffered Saline (PBS). 4. Trypsin/EDTA solution (0.5%/0.2% in 10× PBS). 5. Transfection reagents: FuGene6 or X-tremeGENE9 (Roche) to transfect plasmid DNA, Hiperfect (Qiagen) for siRNA transfection. 6. TNF-a (Invivogen) is dissolved in sterile endotoxin-free water at 100 mg/ml and stored in aliquots at −20°C. Working solution is prepared by 1:100 dilution in endotoxin-free water. Freeze-thaw cycles should be avoided. 7. Tri-DAP (Invivogen) is dissolved in sterile endotoxin-free water at 1 mg/ml (2.56 mM) and stored in aliquots at −20°C. Working solution is prepared by 1:100 dilution in endotoxin-free water. 8. MDP (Invivogen) is dissolved in sterile endotoxin-free water at 1 mM (0.49 mg/ml) and stored in aliquots at −20°C. The aliquots should be stored at 4°C after thawing. Working solution is prepared by 1:500 dilution in endotoxin-free water. 9. Tissue culture test plates (96 F, TPP). 10. Nontransparent 96-well reading plates (F96 MicroWell plates, Nunc, #436111).

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1. pcDNA3 (Invitrogen). 2. Nod1 (such as pUNO-hNOD1, Invivogen). 3. Nod2 (such as pUNO-hNOD2a, Invivogen). 4. TLR2 (such as pUNO-hTLR2, Invivogen). 5. NF-kB reporter ((32) or any commercial, such as PathDetect® pNF-kB-Luc Cis-Reporter Plasmid, Stratagene, #219078). 6. b-Galactosidase expression plasmid (such as pCMVb Vector, Clontech). 7. RelA siRNA: 5¢-aag atc aat ggc tac aca gga-3¢ (Qiagen). 8. CTRL siRNA: AllStars negative control siRNA (Qiagen).

2.3. NF-kB Readout

1. Lysis buffer: 25  mM Tris–HCl pH 8.0, 8  mM MgCl2, 1% Triton, 15% glycerol. Triton should be added after autoclaving. Stable for several months at 4°C. Add 1 mM DTT freshly before use. 2. Reading buffer: To the lysis buffer described above, freshly add 1 mM DTT, 0.77 mg/ml d-luciferin (dissolved in 50 mM Tris–HCl pH 8.0 at 0.27 mg/ml, aliquots stored at −20°C; Sigma, #L-6882), and 1.33 mM ATP (dissolved in water at 100 mM, aliquots stored at −20°C; Sigma). 3. ONPG dilution buffer: 60 mM Na2HPO4, 40 mM NaH2PO4, 10  mM KCl, 1  mM MgSO4, pH  7.0. Sterile-filtered and stored at RT. 4. ONPG stock solution: 4  mg/ml 2-nitrophenyl betad -galactopyranoside (ONPG, Fluka) dissolved in ONPG dilution buffer. Solution is light sensitive and should not be used if colored. Stable for several weeks at 4°C. 5. ONPG-developing solution: Dilute ONPG stock solution 1:4 in ONPG dilution buffer.

3. Methods The method described here is well suited for the specific analysis of signaling events involving the PRRs Nod1 and Nod2, as it is quick, cost-effective, and yields highly reproducible results. It uses HEK293T cells that do express low levels of endogenous Nod1 and Nod2 (4). However, they only respond marginally to stimulation with Nod1/2 agonists, thus Nod1/2 responsiveness is boosted by overexpression. At this step, it is essential to carefully titrate the amount of plasmid used for an optimal readout (Fig.  1c, d), as overexpression of Nod1/2 leads to autoactivation. Briefly, HEK293T cells are seeded in 96-well plates and are transfected with Nod1/2 expression plasmids and a reporter

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system consisting of an NF-kB reporter plasmid and a constitutive ß-galactosidase plasmid. Cells are subsequently stimulated with the cognate elicitors and incubated overnight. To measure NF-kB activation, cells are lysed with a detergent-containing buffer, and luciferase activity is determined in a luminometer. Part of the lysate is preserved and mixed with a solution containing ONPG to determine ß-galactosidase activity. This is used as an indicator for transfection efficiency and cell-death and serves to normalize the readout (Fig. 1b). The method is suited to be combined with different experimental approaches: 1. To analyze the effects of individual, overexpressed proteins on Nod pathways. Here, the protein of interest is coexpressed with Nod1 or Nod2 and the reporter system (Subheading 3.1). 2. To test the influence of chemical substances (inhibitors) on Nod signaling. To this end, the substance is added to the cells after transfection (Subheading 3.1). 3. To determine the effects of siRNA-mediated knock-down of single genes on Nod signaling. To this end, cells are transfected with siRNA 48–72  h prior to transfection of the reporter system (Subheading 3.2). For HTS applications, it is recommended to adapt the protocol to smaller plates to increase effectiveness. Importantly, it is possible to test whether an observed effect is specific for Nod-mediated NF-kB activation rather than affecting the components of the canonical NF-kB pathway or by inducing other NF-kB-activating pathways. For this purpose, a differential readout is performed by activating NF-kB independently of Nod1/2, for example, by the TLR2 or TNF pathway (Fig. 1a). These pathways also lead to the activation of NF-kB and merge with the Nod1/2 pathway at the level of TAK1, allowing the discrimination of specific effects acting in the Nod1/2 pathway upstream of TAK1 (Fig. 1a). To test TLR2-mediated NF-kB activation, HEK293T cells are complemented by transfection with a TLR2 expression plasmid and the reporter system and are subsequently stimulated with Pam3CSK4 , as they do not express endogenous TLR2 (21). To test NF-kB activation via the TNF pathway, only the reporter plasmids are transfected as HEK293T cells express endogenous TNFR1 and can be stimulated with TNF. 3.1. Nod1/2 NF-kBLuciferase Assay 3.1.1. Transfection (Day 1)

1. Human HEK293T cells are maintained in 10-cm dishes until they reach about 80% confluence. The medium is removed and cells are washed once with 1× PBS (see Note 1). To detach the cells, they are incubated for 5  min with 2  ml trypsin/ EDTA at 37°C. Subsequently, 8 ml DMEM (supplemented

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with FCS and PenStrep) is added, and cells are counted using a Neubauer counting chamber or another appropriate technique. 2. The cell number is adjusted with DMEM (supplemented with FCS and PenStrep) to 3 × 105 cells/ml. Subsequently, 100 ml of the cell suspension is added to each well of a 96-well cell culture plate using a multichannel pipette (3 × 104  cells per well). Cells are incubated at 37°C in 5% CO2 for 1 h while the transfection mixes are prepared. 3. Transfection mixes are prepared at least for triplicate assays. A 3 ml total volume of DNA mix adjusted with H2O is used per well. Plasmids to be added per well: 8.6 ng b-gal plasmid, 13 ng NF-kB reporter gene plasmid (see Notes 2 and 3), and 0.5 ng Nod1 or 0.1 ng Nod2 plasmid (see Notes 4 and 5). The DNA mixes are adjusted with pcDNA3.1 to a total amount of 51 ng DNA per well. 17 ml DMEM (without supplements) is added per well to obtain a final volume of 20 ml per well. When preparing the master mixes, 10% more volume than needed should be added. For controls, refer to Notes 6 and 7. OPTIONAL: Expression plasmid encoding for a candidate protein to test is added to the transfection mix. 4. 0.17 ml Fugene6 or X-tremeGENE9 (see Note 8) per well is added to the transfection mix from step 3 and mixed by tipping or brief vortexing of the tube and incubated for 20 min at RT. Each sample should be prepared in triplicates and always be analyzed with and without elicitor stimulation. 5. During the incubation time, elicitor dilutions (see Note 9) are prepared (see Subheading 2.1). 6. Transfection and stimulation of the cells: 20 ml of the transfection mix from step 4 is carefully added to each well, and then the cells are stimulated with 500 nM Tri-DAP (see Note 10) (when transfected with Nod1) or 50  nM MDP (when transfected with Nod2). Cells are incubated at 37°C in 5% CO2 for approximately 16 h. 3.1.2. Readout (Day 2)

1. The medium is discarded using a multichannel pipette. Then, 100 ml cold lysis buffer is added to each well (see Note 11) and the plate is incubated for 5 min on an orbital shaker. The shaking should be rough enough to support lysis of the cells as seen by the formation of a clear lysate with the appearance of precipitated material. 2. 50 ml of each lysate is transferred to a nontransparent 96-well reading plate for luciferase activity measurement. 3. The remaining 50 ml of the lysates are left in the incubation plate, and 100  ml ONPG-developing solution is added per

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well. The plate is incubated at 37°C for about 45 min until the samples appear yellow. Then, absorption is measured in a photometer at 405 nm (620 nm reference). 4. In the meantime, luciferase activities of the lysates are measured in the reading-plate using a commercial luminometer with dispenser. The luminometer is programed to dispense 100 ml reading buffer per well and to start the luminescence measurement instantly thereafter. Alternatively, it is possible to add the reading buffer manually with a multichannel pipette prior to the readout. Setting details for the luminometer cannot be provided as they depend on the type of machine used (see Note 12). 5. Data are collected from the luminometer and the photometer, and normalized relative light units (nRLU) are calculated by dividing the luciferase by ß-galactosidase values. From these values, the mean and standard deviation (SD) for each sample is calculated from the triplicates. This is done using a software package that offers spreadsheet and statistics functionality. 3.2. Nod1/2 siRNA NF-kB-Luciferase Assay 3.2.1. Transfection of siRNA (Day 1)

1. Human HEK293T cells are maintained as described in step 1 in Subheading 3.1.1. 2. The cell solution is adjusted with DMEM (supplemented with FCS and PenStrep) to a concentration of 4 × 104 cells/ml. 100 ml per well are seeded in a 96-well cell-culture test plates using a multichannel pipette, resulting in a final number of 4 × 103 cells per well (see Note 13). Cells are incubated at 37°C in 5% CO2 for 1 h while the siRNA transfection mixes are prepared. 3. Preparation of the transfection mixes: For one well, the total volume of 20  ml transfection mix is used, adjusted with DMEM (without supplements). 20  nM siRNA and 0.8  ml HiPerfect are added and mixed by inverting or vortexing the tube. After 10 min incubation at RT, the solution is carefully added to the cells and incubated at 37°C in 5% CO2.

3.2.2. Change of Growth Medium (Day 2)

The growth medium is changed after ~20 h: The medium is carefully aspirated and replaced by 100  ml fresh DMEM (supplemented with FCS and PenStrep). It is recommended not to aspirate the medium completely to avoid detaching of the cells.

3.2.3. Transfection of Reporter System (Day 3–4)

Dependent on depletion characteristics of the siRNA, cells are transfected after 48 or 72  h following siRNA transfection (Subheading 3.2.1) as described in Subheading 3.1.1 steps 3–5; however, 21.5 ng b-gal plasmid instead of 8.6 ng is used to facilitate b-gal detection.

3.2.4. Readout (Day 4–5)

The readout is performed as described in Subheading 3.1.2 (see Note 14).

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4. Notes 1. HEK293T cells do not adhere very strongly. Be very careful when aspirating growth medium and while washing the cells. Use cells with low passage numbers, as HEK293T cells tend to get less active at passage numbers higher than 8. 2. Dilute the plasmid stocks with sterile H2O to a working concentration of 100 ng/ml and keep on ice. They are stable for several months and should be stored at −20°C. It is recommended to use endotoxin-free water. 3. It is also possible to assay Nod1/2-mediated activation of other pathways rather than NF-kB by the use of appropriate luciferase reporter constructs (33). 4. Prepare fresh dilutions for the Nod1 and Nod2 plasmids at a concentration of 1 ng/ml which should be discarded after use. If more than 5 ng/well is tested, prepare 10 ng/ml dilutions. 5. Nod proteins show autoactivation when overexpressed which masks elicitor-mediated activation. Therefore, it is important to determine the amount of plasmid to be transfected for the particular laboratory settings (cell line, media, incubator, etc.). It is highly recommended to optimize the ratio of specifically activated protein (activated with Tri-DAP or MDP) to autoactivated protein. In our hands, optimal amounts for Nod1 and Nod2 are 0.5 ng and 0.1 ng per well, respectively (Fig. 1c, d). It is further advisable to use untagged proteins as epitope fusions might influence Nod1/2 activity. 6. To assure a low basal NF-kB activation level and assay functionality, controls are mandatory for each experiment. To this end, a transfection mix containing only the reporter system (b-gal, NF-kB reporter, and pcDNA3.1 plasmids) is prepared and cells are stimulated with10 ng/ml TNF or left untreated. 7. To test whether an observed effect is indeed specific for Nodmediated signaling, a differential readout by activating NF-kB by Nod1/2-independent pathways needs to be performed. For example, TLR2 and TNFR signaling can be used (but other suited pathways also can be included here). For this purpose, cells are either transfected with 10 ng TLR2 expression plasmid and NF-kB-luciferase/ß-galactosidase reporter plasmids or the reporter plasmids alone (see Note 6). These cells are subsequently stimulated with 0.5 mg/ml Pam3CSK4 or 10 ng/ml TNF, respectively (Fig. 2b, c). 8. When using several different master mixes, it is more efficient to prepare a quantity of medium with FuGene6, which is then readily added to each master mix. Make sure that the solution is well mixed before adding it to the DNA mixes.

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Fig. 2. Examples of the assay results when combined with siRNA-mediated knock-down (Subheading 3.2). (a) Timeline of the experiment (for details, see Subheading 3.2). (b and c) Cells were transfected with 20 nM RelA, Rip2, or CTRL siRNA. After 48 h of incubation, cells were transfected with 0.5 ng Nod1, an NF-kB-luciferase reporter, and a ß-galactosidase plasmid and subsequently stimulated with 500 nM Tri-DAP (b) or 10 ng/ml TNF (c). Cells were lysed after 16 h incubation, and luciferase and ß-galactosidase activities were determined. Data are represented as mean nRLU + SD (n = 3). (d) Visualization of the siRNA knock-down efficiency by Western blot (see Note 14). Cells were treated the same way as for the reporter gene assay. Lysates were separated on a 10% SDS-polyacrylamide gel and blotted onto a nitrocellulose membrane. Proteins were detected using an RelA-specific antibody. Detection with a GAPDH-specific antibody served as loading control. (e) Reproducibility of the siRNA Nod1 activity assay. An assay after Subheading 3.2 was performed as in (b). The graph shows the combined values of three independent 96-well plates with 20 reactions each (n = 60). Empty: no siRNA transfected, mock: cells treated with HiPerfect only, CTRL: cells transfected with CTRL siRNA, and RelA: cells transfected with RelA-specific siRNA. Boxes represent 75 and 25% percentile with the median presented as line, whiskers represent minimum and maximum values.

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9. MDP and, especially, TNF do not tolerate many freeze-and-thaw cycles. Make sure that you use small aliquots. The diluted working solutions can be stored at 4°C for about 1 week. 10. There are different Tri-DAP solutions available. Be aware that some are sold as racemats (like the one used here), where only one enantiomer is recognized by Nod1, thus the bioactive concentration might differ. 11. When handling 96-well plates, it is most efficient to use multichannel pipettes. Furthermore, for accurate measurements, it is indispensable to guarantee bubble-free liquid handling. An easy and efficient solution to bypass this problem is to use automatic multichannel pipettes. 12. As the photomultipliers of different luminometers have different sensitivities, it is recommended to measure the kinetic of the reaction to find the proper integration time. The luciferase signal reaches a peak after a few seconds, and then slightly decreases and reaches a plateau, where it remains stable for at least 15 min. Therefore, it is also possible to manually add the reading buffer to each well before reading the plate in the luminometer. 13. The cell number in siRNA assays depends on the total incubation time during the experiment. 4 × 103 cells per well should be used for 48 h siRNA knock-down, whereas for 72 h knockdown a lower cell number (3 × 103 cells per well) is seeded. 14. Effective siRNA-mediated knock-down of the target gene can be monitored by performing western blot analysis of additional samples (pool at least three wells) lysed in SDSPAGE buffer (Fig. 2d).

Acknowledgments We thank Dr. Katja Lautz and Andreas Neerincx for critical reading of the manuscript. The authors acknowledge support by the Deutsche Forschungsgemeinschaft (DFG) grant SFB670-NG01. References 1. Akira, S., Uematsu, S., and Takeuchi, O. (2006) Pathogen recognition and innate immunity. Cell 124, 783–801. 2. Fritz, J. H., Ferrero, R. L., Philpott, D. J., and Girardin, S. E. (2006) Nod-like proteins in immunity, inflammation and disease. Nat Immunol 7, 1250–7. 3. Chamaillard, M., Hashimoto, M., Horie, Y., Masumoto, J., Qiu, S., Saab, L., Ogura, Y., Kawasaki, A., Fukase, K., Kusumoto, S.,

Valvano, M. A., Foster, S. J., Mak, T. W., Nunez, G., and Inohara, N. (2003) An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat Immunol 4, 702–7. 4. Girardin, S. E., Boneca, I. G., Carneiro, L. A., Antignac, A., Jehanno, M., Viala, J., Tedin, K., Taha, M. K., Labigne, A., Zahringer, U., Coyle, A. J., DiStefano, P. S., Bertin, J., Sansonetti, P. J., and Philpott, D. J. (2003)

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Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 300, 1584–7. 5. Girardin, S. E., Boneca, I. G., Viala, J., Chamaillard, M., Labigne, A., Thomas, G., Philpott, D. J., and Sansonetti, P. J. (2003) Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem 278, 8869–72. 6. Inohara, N., Ogura, Y., Fontalba, A., Gutierrez, O., Pons, F., Crespo, J., Fukase, K., Inamura, S., Kusumoto, S., Hashimoto, M., Foster, S. J., Moran, A. P., Fernandez-Luna, J. L., and Nunez, G. (2003) Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn’s disease. J Biol Chem 278, 5509–12. 7. Inohara, Chamaillard, McDonald, C., and Nunez, G. (2005) NOD-LRR proteins: role in host-microbial interactions and inflammatory disease. Annu Rev Biochem 74, 355–83. 8. Lukasik, E., and Takken, F. L. (2009) STANDing strong, resistance proteins instigators of plant defence. Curr Opin Plant Biol 12, 427–36. 9. Inohara, N., Koseki, T., del Peso, L., Hu, Y., Yee, C., Chen, S., Carrio, R., Merino, J., Liu, D., Ni, J., and Nunez, G. (1999) Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB. J Biol Chem 274, 14560–7. 10. Tanabe, T., Chamaillard, M., Ogura, Y., Zhu, L., Qiu, S., Masumoto, J., Ghosh, P., Moran, A., Predergast, M. M., Tromp, G., Williams, C. J., Inohara, N., and Nunez, G. (2004) Regulatory regions and critical residues of NOD2 involved in muramyl dipeptide recognition. Embo J 23, 1587–97. 11. Kufer, T. A. (2008) Signal transduction pathways used by NLR-type innate immune receptors. Mol Biosyst 4, 380–6. 12. Girardin, S. E., Tournebize, R., Mavris, M., Page, A. L., Li, X., Stark, G. R., Bertin, J., DiStefano, P. S., Yaniv, M., Sansonetti, P. J., and Philpott, D. J. (2001) CARD4/Nod1 mediates NF-kappaB and JNK activation by invasive Shigella flexneri. EMBO Rep 2, 736–42. 13. Opitz, B., Puschel, A., Beermann, W., Hocke, A. C., Forster, S., Schmeck, B., van Laak, V., Chakraborty, T., Suttorp, N., and Hippenstiel, S. (2006) Listeria monocytogenes activated p38 MAPK and induced IL-8 secretion in a nucleotide-binding oligomerization domain 1-dependent manner in endothelial cells. J Immunol 176, 484–90. 14. Hsu, Y. M., Zhang, Y., You, Y., Wang, D., Li, H., Duramad, O., Qin, X. F., Dong, C., and Lin, X. (2007) The adaptor protein CARD9 is

required for innate immune responses to intracellular pathogens. Nat Immunol 8, 198–205. 15. Fritz, J. H., Le Bourhis, L., Sellge, G., Magalhaes, J. G., Fsihi, H., Kufer, T. A., Collins, C., Viala, J., Ferrero, R. L., Girardin, S. E., and Philpott, D. J. (2007) Nod1mediated innate immune recognition of peptidoglycan contributes to the onset of adaptive immunity. Immunity 26, 445–59. 16. Magalhaes, J. G., Fritz, J. H., Le Bourhis, L., Sellge, G., Travassos, L. H., Selvanantham, T., Girardin, S. E., Gommerman, J. L., and Philpott, D. J. (2008) Nod2-dependent Th2 polarization of antigen-specific immunity. J Immunol 181, 7925–35. 17. Bouskra, D., Brezillon, C., Berard, M., Werts, C., Varona, R., Boneca, I. G., and Eberl, G. (2008) Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis. Nature 456, 507–10. 18. Kain, S. R., and Ganguly, S. (2001) Overview of genetic reporter systems. Curr Protoc Mol Biol Chapter 9, Unit9 6. 19. White, E. H., Rapaport, E., Seliger, H. H., and Hopkins, T. A. (1971) The Chemi- and Bioluminescence of Firefly Luciferin: An Efficient Chemical Production of Electronically Excited States. Bioorganic Chemistry 1, 92–122. 20. Miller, J. H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 21. Kurt-Jones, E. A., Sandor, F., Ortiz, Y., Bowen, G. N., Counter, S. L., Wang, T. C., and Finberg, R. W. (2004) Use of murine embryonic fibroblasts to define Toll-like receptor activation and specificity. J Endotoxin Res 10, 419–24. 22. Chen, G., and Goeddel, D. V. (2002) TNFR1 signaling: a beautiful pathway. Science 296, 1634–5. 23. Kawai, T., and Akira, S. (2007) Signaling to NF-kappaB by Toll-like receptors. Trends Mol Med 13, 460–9. 24. McDonald, C., Chen, F. F., Ollendorff, V., Ogura, Y., Marchetto, S., Lecine, P., Borg, J. P., and Nunez, G. (2005) A role for Erbin in the regulation of Nod2-dependent NF-kappaB signaling. J Biol Chem 280, 40301–9. 25. Barnich, N., Aguirre, J. E., Reinecker, H. C., Xavier, R., and Podolsky, D. K. (2005) Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor{kappa}B activation in muramyl dipeptide recognition. J Cell Biol 170, 21–6. 26. Kufer, T. A., Kremmer, E., Adam, A. C., Philpott, D. J., and Sansonetti, P. J. (2008) The

7  Cell-Based Reporter Assay to Analyze Activation of Nod1 and Nod2 pattern-recognition molecule Nod1 is localized at the plasma membrane at sites of bacterial interaction. Cell Microbiol 10, 477–86. 27. Huang, S., Zhao, L., Kim, K., Lee, D. S., and Hwang, D. H. (2008) Inhibition of Nod2 signaling and target gene expression by curcumin. Mol Pharmacol 74, 274–81. 28. Barnich, N., Hisamatsu, T., Aguirre, J. E., Xavier, R., Reinecker, H. C., and Podolsky, D. K. (2005) GRIM-19 interacts with nucleotide oligomerization domain 2 and serves as downstream effector of anti-bacterial function in intestinal epithelial cells. J Biol Chem 280, 19021–6. 29. Kufer, T. A., Kremmer, E., Banks, D. J., and Philpott, D. J. (2006) Role for erbin in bacterial activation of Nod2. Infect Immun 74, 3115–24. 30. Yamamoto-Furusho, J. K., Barnich, N., Xavier, R., Hisamatsu, T., and Podolsky, D. K. (2006) Centaurin beta1 down-regulates nucleotidebinding oligomerization domains 1- and 2-dependent NF-kappaB activation. J Biol Chem 281, 36060–70.

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31. Bielig, H., Zurek, B., Kutsch, A., Menning, M., Philpott, D. J., Sansonetti, P. J., and Kufer, T. A. (2009) A function for AAMP in Nod2mediated NF-kappaB activation. Mol Immunol 46, 2647–54. 32. Munoz, E., Courtois, G., Veschambre, P., Jalinot, P., and Israel, A. (1994) Tax induces nuclear translocation of NF-kappa B through dissociation of cytoplasmic complexes containing p105 or p100 but does not induce degradation of I kappa B alpha/MAD3. J Virol 68, 8035–44. 33. Werts, C., le Bourhis, L., Liu, J., Magalhaes, J. G., Carneiro, L. A., Fritz, J. H., Stockinger, S., Balloy, V., Chignard, M., Decker, T., Philpott, D. J., Ma, X., and Girardin, S. E. (2007) Nod1 and Nod2 induce CCL5/ RANTES through the NF-kappaB pathway. Eur J Immunol 37, 2499–508. 34. Zurek et al. (2011) Innate Immun, PMID: 21310790. 35. Bielig et al. (2010) ChemMedChem 5(12), 2065–71. PMID:20973121.

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Chapter 8 Determining FceRI Diffusional Dynamics via Single Quantum Dot Tracking Diane S. Lidke, Shalini T. Low-Nam, Patrick J. Cutler, and Keith A. Lidke Abstract Single-particle tracking (SPT) using fluorescent quantum dots (QDs) provides high-resolution spatial– temporal information on receptor dynamics that cannot be obtained through traditional biochemical techniques. In particular, the high brightness and photostability of QDs make them ideal probes for SPT on living cells. We have shown that QD-labeled IgE can be used to characterize the dynamics of the highaffinity IgE Receptor. Here, we describe protocols for (1) coupling QDs to IgE, (2) tracking individual QD-bound receptors, and (3) analyzing one- and two-color tracking data. Key words: FceRI, High-affinity IgE receptor, Single-particle tracking, Quantum dots

1. Introduction Many key events in the immune response are controlled by the multichain immune recognition receptor family, which includes the T- and B-cell receptors and the high-affinity IgE Receptor (FceRI) of mast cells and basophils (1). Cross-linking of these receptors initiates Src kinase family-mediated phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs). In the case of FceRI, cross-linking by multivalent antigen triggers a complex signaling pathway that ultimately leads to degranulation and release of key mediators of allergic inflammation. Antigen binding is associated with changes in receptor dynamics and topography, suggesting an important role for receptor motion in signaling.

Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_8, © Springer Science+Business Media, LLC 2011

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Single-particle tracking (SPT) is a powerful tool for studying the subtleties of protein behaviors and interactions. By measuring the motion of individual proteins, SPT can reveal details of protein dynamics at the molecular level. The use of bright and photostable Quantum Dots (QDs) for SPT has extended the capabilities of this technique by allowing for longer tracking times and facilitating multicolor measurements (2–5). Simultaneous multicolor tracking allows for direct comparison of protein dynamics when different species are labeled with distinct QDs and can provide information about protein–protein interactions. We have previously shown that QD-labeled IgE can bind FceRI without activating the receptor and can be used to track receptor dynamics in the resting and activated state (2, 6). Here, we describe methods for generation of QD-IgE and collection and analysis of SPT data.

2. Materials 1. Phosphate-buffered saline (PBS), pH 7.4 (Gibco 10010-023, Carlsbad, CA). 2. 6-((6-((biotinoyl)amino)hexanoyl)amino)hexanoic acid, succinimidyl ester (Biotin-XX-SE, Invitrogen B-1606, Carlsbad, CA). 3. Streptavidin–conjugated QDs (SAvQD) (Molecular Probes, Eugene, OR). 4. Hanks’ balanced salt solution (HBSS): HBSS (Invitrogen 14065-056) supplemented with 10 mM HEPES, 0.05% BSA, 5.5 mM glucose, 0.7 mM MgSO4, 0.16 mM CaCl2, 0.13% NaHCO3. 5. LabTek eight-well coverslip chambers (Nunc 155411, Rochester, NY). 6. Minimum Essential Medium (MEM, Gibco 11095, Carlsbad, CA) supplemented with 10% Fetal Bovine Serum (MEM/FBS). 7. IgE as prepared in (7) and stored in Borate-Buffered Saline (BBS), pH 8.3. 8. FluoReporter® Biotin Quantitation Assay (Molecular Probes F30751, Carlsbad, CA). 9. Two-color image splitter, such as Cairn Optosplit (Faversham, UK) or Optical Insights Dual View (Santa Fe, NM). 10. Appropriate emission filters and dichroics for QDs of interest (Chroma, Rockingham, VT or Semrock, Rochester, NY). 11. Rat Basophilic Leukemia (RBL-2H3) cells (ATCC CRL2256).

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3. Methods 3.1. Generating QD-IgE

3.1.1. IgE Biotinylation

This protocol describes methods for IgE biotinylation and conjugation to commercial SAvQDs (see Note 1). Monovalent IgE is prepared through labeling of reactive primary amine groups with biotin-XX-SE. We have found that incubation of a 4:1 biotin-XXSE:IgE reaction stoichiometry for 15 min at room temperature consistently results in <1 biotin:IgE ratio. 1. Add 200 ml of 10 mM IgE stock solution to a 2-ml reaction tube that contains a micromagnetic stir bar. If the IgE is in pH 7 buffer, add 20 ml of 1 M NaHCO3 solution to raise the pH to > 8. 2. Make a 4 mg/ml (7 mM) solution of biotin-XX-SE in DMSO. DMSO stocks of Biotin-XX-SE can be stored at −20°C for up to six months. Biotin-XX-SE can be dissolved in aqueous buffer, but should be made immediately prior to the conjugation process, as solutions of these compounds will gradually hydrolyze in water. 3. While gently stirring the IgE, add 1.15 ml (final concentration of 40 mM) of the biotin-XX-SE solution to the reaction tube containing the IgE. 4. Allow the reaction to stir for 15 min at room temperature. 5. Prepare PD SpinTrap G-25 (GE Healthcare, 28-9180-04) spin columns at room temperature as directed. Ensure that the resin does not dry out. We have found that separation performed at room temperature increases recovery from the column. (a) Resuspend resin by vortexing. (b) Remove storage buffer by centrifugation for 1  min at 800 × g. (c) Wash column with 300 ml of appropriate buffer (1× PBS) and spin for 1  min at 800 × g; dispose of flow through and replace column in collection tube; repeat these washes five times. (d) Apply 70–130 ml of sample to the center of the column in a fresh collection tube. (e) Elute by centrifugation for 2  min at 800 × g; the final conjugate is in the collection tube. 6. Transfer the contents of the collection tube to a 0.5-ml centrifuge tube. Store at 4°C. 7. Determine IgE concentration by spectrophotometric absorbance measurement: C = A280 /eL; where A280 is the absorbance at 280  nm, e is the extinction coefficient for IgE (203,000 M−1 cm−1), and L is the cuvette path length (typically 1 cm).

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3.1.2. IgE:Biotin Quantitation

A kit is available to quantify the degree of IgE biotinylation (FluoReporter® Biotin Quantitation Assay) using a 96-well format fluorescence reporter assay (see Note 2). The FluoReporter® system uses a fluorescent avidin reagent that is bound to quenching ligands. Biotin displaces the quencher, resulting in a fluorescent signal that is proportional to the concentration of biotin added. 1. Optional: The IgE may be proteolytically cleaved to expose the biotin molecules completely. If this step is included, use a biotinylated IgG that is included as a control. We do not typically include this digestion step in our experiment. 2. Using a volume of 50 mL per well, serially dilute the biotinylated lysine standard to cover the range of 0–80 pmol standard; repeat in triplicate. 3. Aliquot 50 mL of two-fold serially diluted biotinylated IgE to get concentrations within the sensitivity range of the assay. 4. Add the Biotective Green reagent to initiate the reaction; incubate for 5 min, covered, at room temperature, rocking constantly. 5. Immediately after the incubation, read fluorescence with 485 nm excitation and 530 nm emission using a fluorescent plate reader. 6. Biotinylated lysine data is fit to a quadratic equation to generate a standard curve from which the concentration of biotin in IgE samples can be determined. 7. The ratio of biotin concentration to that of IgE is the degree of labeling (DOL).

3.1.3. QD-IgE Conjugation

1. Dilute SAvQDs to 40 nM in 100 ml of PBS + 1% BSA. 2. Dilute Biotin-IgE to 40 nM in 100 ml of PBS + 1% BSA. 3. Add Biotin-IgE to the SAvQD and mix several times with a micropipette. 4. Incubate for at least 30 min at 4°C with gentle agitation. The result is a 20 nM stock of QD-IgE that can be stored at 4°C for up to 2 weeks (see Note 3).

3.2. Cell Labeling and Microscopy 3.2.1. Labeling Cells

1. RBL-2H3 cells are plated in 8-well chambers for 1–2 days prior to the experiment in MEM/FBS. An initial density of 50,000 cells/well or 25,000 cells/well results in an appropriate single-cell density after 1 or 2 days, respectively. 2. Wash cells two to three times with 200 ml HBSS. 3. Label cells by adding 200 ml of 50–200 pM QD655-IgE in HBBS and incubating for 10–15  min at room temperature (or 10 min at 37°C in cell culture incubator). (a) For two-color QD tracking, label cells by simultaneously adding 50 pM of each color QD-IgE in HBSS.

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4. Wash cells at least four times with 200 ml HBSS to remove unbound QDs. 5. Optional: Add this step after QD-IgE labeling in cases, where saturating nonfluorescent IgE (dark IgE) is required (i.e., for cross-linking studies). Add 200 ml of 0.5 mg/ml dark IgE in HBSS to each chamber and incubate for 10–15 min at RT (or 10  min at 37°C in incubator). For long-term incubation, serum-free MEM can be used instead of HBSS. Wash cells two to three times with 200 ml HBSS to remove any excess dark IgE. 6. Add 200 ml of HBSS to each well and maintain in this buffer for imaging. 3.2.2. Single-Particle Tracking

1. Place eight-well chamber containing QD-IgE primed cells on the microscope stage and allow time for temperature equilibration. We use an inverted wide-field microscope (Olympus IX71) equipped with an objective heater (Bioptechs) to maintain temperature at 34–36°C. Excitation (see Note 4) is from a mercury lamp (436/10 nm BP filter), and emission is collected by an electron-multiplying CCD camera (emCCD, Andor iXon). For two-color imaging, an image splitter that projects two-color channels simultaneously on the emCCD is used with appropriate QD emission filters. For example, we use an OptoSplit II (Carin Research) equipped with a 600 nm dichroic and 655/40  nm and 585/20 BP emission filters (Chroma). 2. Locate a single cell and focus on the apical membrane. A 60× water, 1.2 NA objective is recommended to avoid aberrations induced by oil/water index of refraction mismatch. 3. Begin acquisition of time series. Data is typically collected at 20–33 frames per second. Longer exposure times give better signal-to-noise ratio, but at the expense of temporal resolution.

3.3. Analysis 3.3.1. Single QD Tracking

A 2D Gaussian is used to represent the microscope point spread function (PSF), with sPSF values measured for each color channel using immobilized fluorophores on a coverslip. See Fig.  1 for more PSF details. Localization accuracy is limited primarily by the microscope PSF, number of photons collected from the fluorophore, and the background count rate (8). For image processing, we use and recommend MATLAB (The MathWorks, Natick, MA) in combination with the freely available DIPimage toolbox (Delft University of Technology, http://www.diplib.org/). 1. Convert CCD image data to photon counts (9). (a) Subtract the camera offset pixel-wise for each image frame. The camera offset image is the mean image of ~1,000 frames taken with no light on the CCD.

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Fig. 1. Single QD-IgE-FceRI Tracking. (a) Images from one frame of two-color QD-IgE tracking on the surface of RBL cells showing each channel (QD655 on the left, QD585 on the right) as projected by the Optosplit. Inset on the lower left shows the PSF fits of a single QD655. These images were acquired with excitation intensity of 4.7 W/cm2 (436/10 BP), resulting in average photon counts of 314 and 172 for QD655 and QD585, respectively. The localization error is then 24 nm for QD655 and 36 nm for QD585. (b) Selected trajectories of FceRI labeled with QD655-IgE (white) or QD585-IgE (black ) overlaid on the transmission image.

(b) Divide the offset corrected images by the CCD gain. The gain can be found as the slope of the variance vs. intensity plot generated from a stack of ~20 images of an identical object. The variance and intensity are calculated along the third dimension. Slightly out-of-focus fluorescent beads mounted on a coverslip make a good, photostable test object. Gain calculation is implemented as DIPimage function “cal_readnoise”. 2. Find coordinates of QDs. (a) Identify the areas of interest. Initial segmentation of each image is performed by subtracting a Gaussian filtered image with filter kernel sk = 2*sPSF from a filtered image with sk = sPSF. A threshold of the standard deviation across the resulting image is used to identify candidate-fit regions. (b) Maximum Likelihood Estimate of QD position, emission rate, and background count rate (8). A box of ~6* sPSF pixels centered around the center of mass of each contiguous area found in part (a) is used as a fitting region for a single-molecule fit. (c) Reject objects that are not single molecules. Objects that do not pass a shape test or do not pass an emission rate threshold are removed from further analysis. 3. Build trajectories from QD coordinates (2). (a) Build short, continuous trajectories. Starting with the first frame, all coordinates found in the subsequent frame are compared to each set of coordinates from the current frame. If they pass a threshold probability given by P (r,Dt) = exp[−r2/(4DDt)]), where P is typically 0.01, D is an estimated diffusion constant, and r is the distance

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between coordinates, they are connected to build a ­trajectory. If more than one particle passes this threshold, the coordinate with the highest P value is selected. (b) Connect short trajectories. The x,y, and t coordinates at the beginning and end of each short trajectory are used to repeatedly connect short trajectories into final trajectories. Using the same equation as above, the threshold probability for this step is typically 10−4. We also give an upper limit for the distance and time interval between coordinates permitted for two short trajectories to be joined into a longer trajectory. 3.3.2. Calculation of Diffusion Coefficients

Once trajectories have been obtained from the time series, diffusion coefficients (D) can be extracted. Here, we outline the generation of mean square displacement (MSD) plots, which can then be fit to models of diffusion (see also (2)). The best fit model reveals the type of motion (i.e., free, restricted, directed diffusion, or immobile) and diffusion coefficients for each molecule (Fig. 2a and b). 1. For each trajectory, generate an MSD plot (see Fig. 2). 2. Fit to the appropriate model to obtain diffusion coefficient. Free: MSD = offset + 4DDt; Restricted: MSD = offset + (L2/3)*(1 − exp(−Dt/t); t = L2/12D; Directed diffusion: MSD = offset + 4DDt + v2Dt2, where offset is the y-axis offset and related to the localization accuracy, Dt is time interval, L is the length of one side of the area that the molecule is restricted in, and v is transport velocity. The statistical error increases with large time intervals; therefore, no more than the first 25% of the MSD plot should be considered for fitting. 3. To compare aggregate data, generate a Cumulative Probability Analysis (CPA) plot (see Fig. 2c). Due to the spread of values for SPT data, diffusion coefficients are better represented by a CPA than by an average value (10). Comparison of CPA plots for different conditions immediately reveals differences in diffusional dynamics (Fig. 2c). A CPA plot can be generated from fits to the above models. To remove the influence of restricted diffusion from the calculation of D, fitting the free diffusion model to the first few points of the MSD is often used. The number of points used can vary; we use D1-3 while others report D2-4 or D1-5.

3.3.3. Two-Channel Overlay

To properly analyze two-color single-QD tracking data (i.e., differentiating between dimers and colocalized QDs), optimal interchannel registration is required. Using software to directly overlay data from each channel by just an image shift will result in suboptimal

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Fig. 2. Analysis of QD-IgE-FceRI Single-Particle Trajectories. (a) Example trajectories showing the four modes of motion that are observed for FceRI (Free, Restricted, Directed, Immobile).

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interchannel colocalization since an image splitter may introduce nonuniform chromatic aberrations into the separate images projected onto the detector. 1. Collect calibration image(s) (see Note 5). A calibration image should consist of single-point sources spanning the field of view. Fiducial calibration data is optimal due to its well-distributed sampling of the entire field of view and the ability to optimize the localization accuracy of the point source(s) (11) (see Fig. 3). It is important to obtain a test and training image for calibration in order to define the localization accuracy achieved using an independent test set.

Fig. 3. Fiducial Calibration Data Set. (a) Fiducial bead data shown for each channel using a single broad spectrum bead (200 nm TetraSpeck Bead from Invitrogen, T7283) imaged by stepping the stage (5 mm) to sample the entire emCCD. (b) Analysis of the calibration image shows the imaged positions for each channel, the linearly shifted channel 2 data, and the transformed channel 2 data. This transformation vector is then used to transform the channel 2 SPT data to allow overlay with channel 1 trajectories.

Fig. 2. (continued)  (b) An MSD plot for a QD-IgE-FceRI trajectory (gray circles) shows that free diffusion (i.e., linear fit, solid black line) best represents the data. Also shown are the shapes of curve obtained from restricted diffusion, immobile molecules, and directed diffusion. The inset highlights the differences between the fits. (c) CPA plot comparing free (dashed line) and cross-linked (solid line ) FceRI motion. The leftward shift of diffusion coefficients seen for the solid line indicates a decreased diffusion rate upon cross-linking with 1 mg/ml DNP-BSA. The number of trajectories (N) and median diffusion coefficients (D) are reported.

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2. Localize calibration points in each channel using a 2D Gaussian PSF fit model. The width of the microscope PSF is proportional to wavelength, and therefore the PSF for each channel is different. Since localization accuracy is essential for optimal colocalization, identifying the sigma for the 2D Gaussian PSF model for each channel is recommended. 3. Train and test the calibration model. (a) Identify a proper calibration model for your instrumental setup. We use a polynomial (specifically a + bx + cy + dxy, where a,b,c,and d are coefficients and x and y are spatial coordinates) to describe the relationship between positions in each channel. Other calibration models are also available (11). (b) Use a training calibration image to build the model (find coefficients), and test the calibration image to estimate the prediction accuracy of the calibration. See Fig. 3 for more details on channel transformation. If calibration images are acquired before and after data acquisition, then train the model with the preacquisition calibration and test it with the postacquisition calibration data. This will give insights into the instrument stability during the acquisition of experimental data. 4. Apply calibration model to QD data. The coordinates obtained from SPT of raw data are corrected to correspond with the reference channel using the calibration model. Trajectories should always be obtained from unshifted images. 3.3.4. Correlated Motion Analysis

Pair-wise comparisons of two-color QD-IgE trajectories permit further description of receptor behaviors, namely, the presence of interactions whose lifetimes are longer than the imaging acquisition time. This analysis shows the degree of correlated motion by determining parameters for each candidate pair in sequential frames. The trajectories of receptors during an interaction will be coordinated which is seen as a decrease in the uncorrelated jump distance parameter (see Fig. 4). 1. Determine initial separation between two trajectories; if this is less than a specified cutoff (i.e., 500 nm), continue to analyze the candidate pair. 2. Uncorrelated jump distance is determined for all candidate pairs at each time step using: Di = |J1i–J1i(J1i∙J2i)/(|J1i||J2i|)|, where J1i = r1i+1–r1i and J2i = r2i+1−r2i and ri is the position of a QD at time i. The uncorrelated jump distance parameters are averaged and binned in 50  nm separation intervals for plotting.

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Fig. 4. Correlated Motion Analysis of Simulated Single-Particle Trajectories. (a) A schematic of two-color QD tracking shows single molecules that are initially separated, but in frame 3, form a dimer and continue to move in a coordinated manner, reflecting the interaction between the receptors. (b and c) Simulated two-particle trajectories of receptors demonstrating uncorrelated and correlated motion, respectively. Particle trajectories are plotted in three dimensions using coordinates and time frame. (d) Global analysis of many simulated trajectories demonstrates the trends seen in the cases of correlated (black) and uncorrelated (gray ) motion. In the former case, as particles come within the interaction distance and move together, the uncorrelated jump distance parameter markedly decreases. Simulation conditions included 10 particles for each QD color, 5,000 frames, diffusion coefficient = 0.07 mm2/s, and a 25 nm interaction distance. Note that for FceRI, we found uncorrelated motion (2).

4. Notes 1. We recommend the SAvQDs from Invitrogen that are coated with polyethylene glycol (PEG) 2000 as this reduces nonspecific binding compared to non-PEG-coated QDs. These QDs contain 3–5 SAv molecules allowing for the IgE:QD stoichiometry to be varied, though we have shown that higher ratios than 1:1 IgE:QD can lead to FceRI activation (2). 2. Labeling conditions can be optimized by first using a fluorescent (rather than biotin) derivative of succinimidyl ester such that the conjugation chemistry is the same and the dye-toprotein ratio can be easily determined by spectrophotometric absorption measurements. 3. QD-IgE should be diluted to appropriate concentrations in HBSS just before use since buffers containing divalent cations can cause SAvQD aggregation over time. 4. While QDs are highly photostable, illumination with intense light can increase QD blinking and even lead to blue-shift and photodegradation of the QD emission. Therefore, optimal

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illumination power (typically controlled through neutral density filters) that maximizes QD signal but does not lead to blue-shifting should be determined for each system. 5. We have observed sporadic instrumental drift when using an image splitter; therefore, it is important to investigate stability when performing experiments in which nanometer overlay accuracy is important. We have found it good practice to obtain a calibration image both before and after data acquisition to ensure instrument stability. 6. In SPT, the parameters for single-molecule thresholds and trajectory connection probability thresholds should be optimized on a subset of data and checked manually before proceeding with an automated analysis of larger data sets.

Acknowledgments This work was supported by NIH P50GM085273, ACS-IRG #192, and the Human Frontier Science Program. References 1. Sigalov, A.B. (2004) Multichain immune recognition receptor signaling: different players, same game? Trends Immunol 25, 583–89 2. Andrews , N.L., Lidke, K.A., Pfeiffer, J.R, Burns, A.R, Wilson, B.S., Oliver, J.M.,and Lidke, D.S. (2008) Actin restricts FceRI diffusion and facilitates antigen-induced receptor immobilization. Nat Cell Biol 10, 955–63 3. Dahan, M., Lévi, S., Luccardini, C., Rostaing, P., Riveau, B., Triller, A. (2003) Diffusion dynamics of glycine receptors revealed by ­single-quantum dot tracking. Science 302, 442–45 4. Lidke, D.S., Lidke, K.A., Reiger, B., Jovin, T.M., and Arndt-Jovin D.J. (2005) Reaching out for signals: filopodia sense EGF and respond by directed retrograde transport of activated receptors. J Cell Biol 170, 619–26 5. Roullier, V., Clarke, S., You, C., Pinaud, F., Gouzer, G.G., Schaible, D., Marchi-Artzner, V,, Piehler, J., and Dahan, M. (2009) Highaffinity labeling and tracking of individual histidine-tagged proteins in live cells using Ni2+ tris-nitrilotriacetic acid quantum dot conjugates. Nano Lett 9, 1228–34 6. Andrews, N.L., Pfeiffer, J.R., Martinez, A.M., Haaland, D.M., Davis, R.W., Kawakami, T.,

Oliver, J.M., Wilson, B.S., Lidke, D.S. (2009) Small, mobile FceRI aggregates are signaling competent. Immunity 31, 469–79 7. Liu, F.T., Bohn, J.W., Ferry, E.L., Yamamoto, H., Molinaro, C.A., Sherman, L.A., Klinman, N.R., and Katz, D.H. (1980) Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization. J Immunol 124, 2728–37 8. Smith, C.S. et al (Submitted) Fast, single-molecule localization that achieves theoretically minimum uncertainty. 9. Lidke, K.A., Rieger, B., Lidke, D.S., and Jovin, T. M. (2005) The role of photon statistics in fluorescence anisotropy imaging. IEEE Transactions on Image Processing 14, 1237–45 10. Ehrensperger, M.V., Hanus, C., Vannier, C., Triller, A., and Dahan, M. (2007) Multiple association states between glycine receptors and gephyrin identified by SPT analysis. Biophysical Journal 92, 3706–18 11. Churchman, L.S., Okten, Z., Rock, R.S., Dawson, J.F., and Spudich, J.A. (2005) Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. Proc Natl Acad Sci U S A 102, 1419–23

Chapter 9 Ratiometric Analysis of Subcellular Recruitment of Fc Receptors During Phagocytosis Patricia Mero and James W.D. Booth Abstract Numerous immune receptors have the ability to mediate phagocytosis of large particles by triggering dynamic local rearrangement of the cytoskeleton and cell membrane. Different receptors can be differentially recruited to sites of particle binding, which in turn can have important functional consequences with respect to engulfment and downstream signaling. Using Fcg receptor-mediated phagocytosis of IgG-coated particles as a model, we describe a method for analyzing nascent phagocytic cups and quantifying relative receptor levels at sites of phagocytosis. This technique is based on a ratiometric analysis of subcellular localization of exogenously expressed receptors carrying different fluorescent protein tags. This approach could be applied more generally to the analysis of surface membrane protein localization in the context of any dynamic cellular process. Key words: Phagocytosis, Fc receptors, Fluorescence microscopy

1. Introduction Phagocytosis is the process by which immune cells engulf large particles (>0.5  mm). It can be triggered by the engagement by particles of a number of different phagocytic receptors, among which the FcgR that mediate phagocytosis of IgG-coated particles have been most extensively studied (1). Phagocytosis involves a rapid and dynamic remodeling of both cytoplasmic and membrane proteins at the sites of particle binding (2). Membrane proteins (in particular, the receptors that directly mediate binding of opsonized particles) can become enriched at such sites relative to their concentration in the plasma membrane as a whole. Some receptors may become relatively more enriched than others, depending on factors such as their binding affinities for the particle.

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This can have important functional consequences, particularly in the case of activating and inhibitory Fcg receptors, which have opposing effects on signaling for phagocytosis (3, 4). There are several challenges associated with observing recruitment of membrane proteins to phagocytic cups. One is simply that of “catching” the appropriate stage of a highly dynamic process by live cell microscopy. A second is quantitating local changes in membrane protein concentration by image analysis. One simple and commonly used approach for such quantitation is line scanning, e.g., drawing a line that crosses the cell membrane both at the site of particle binding (the phagocytic cup) and elsewhere, and comparing the heights of the fluorescence intensity peaks where the line crosses each membrane. A drawback of this approach, however, is that it is very sensitive to local fluctuations in fluorescence along the plasma membrane, depending on the exact points through which the line is drawn. Ratiometric approaches have proven useful in quantitating the recruitment of cytoplasmic proteins to phagocytic cups (5); here, we describe a ratiometric approach we have developed for comparing the local concentrations of two surface membrane proteins during phagocytosis. We sought to measure the relative recruitment to sites of phagocytosis of IgG-coated particles of two different membrane proteins: the activating receptor FcgRIIA and the inhibitory receptor FcgRIIB, which support and oppose phagocytosis, respectively. Of note, expression of transfected activating FcgR is sufficient to confer the ability to perform phagocytosis of IgGcoated beads on otherwise nonphagocytic cells, such as fibroblasts (6). In this chapter, we describe approaches to follow phagocytosis using such “engineered phagocytes” expressing FcgR carrying distinguishable fluorescent protein tags. First, we address strategies for observing early phagocytic events by live cell microscopy. We then describe the analysis of resulting image data to quantitate the relative enrichment of two different FcgRs at sites of phagocytosis. Our approach relies on measuring the ratio of signal from the two different fluorophores on a pixel-by-pixel basis. This allows sampling of the entire region of the phagocytic cup or plasma membrane to obtain a robust measure of relative enrichment of the two receptors based on changes in this local ratio. We have used this approach in studies of FcgR (3), but note that the same ratiometric approach could be used for the analysis of recruitment of other phagocytic receptors during phagocytosis of appropriate particles. More broadly, it could be used for the analysis of any cellular process that involves dynamic subcellular relocalization of surface membrane proteins, either to compare the relative localization of two functionally important proteins as in the example discussed or to study local enrichment of individual membrane proteins by using a second, evenly distributed plasma membrane protein as a reference.

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2. Materials 2.1. Transfection and Cell Culture

1. ts20 Chinese hamster fibroblasts or similar cell line (see Note 1). 2. Lipofectamine 2000 (Invitrogen). 3. Optimem (Invitrogen). 4. Purified transfection-grade DNA for expressing receptors with appropriate distinguishable fluorescent tags, e.g., FcgRIIA-YFP and FcgRIIB-CFP (see Note 2). 5. 6-well or 35-mm tissue culture plates. 6. 25-mm round #1 cover glass (Fisher). 7. Alpha-Minimal Essential Media. 8. Fetal Bovine Serum (FBS).

2.2. Phagocytic Particle Preparation

1. 3-mm diameter polystyrene beads (Polysciences Inc. 17134-15). 2. Purified bulk human IgG (Sigma I-4506). 3. Phosphate-buffered saline (PBS). 4. Thermomixer capable of maintaining 37°C and 1,050  rpm (Eppendorf 21516-170).

2.3. Microscopy

1. Confocal microscope (inverted). 2. Attofluor cell chamber for live imaging (Molecular Probes A-7816). 3. Heated stage assembly suitable for holding cell chamber. 4. Phenol red-free HEPES-buffered RPMI (HPMI) (Wisent, Quebec).

2.4. Image Quantitation

1. Zeiss LSM 510 or similar software, with capacity to add and obtain ratios of separate channels and to export processed files in TIFF format. 2. Microsoft Excel. 3. ImageJ freeware (http://rsb.info.nih.gov/ij/) (see Note 3).

3. Methods 3.1. Transfection

1. ts20 cells grown in AMEM with 10% FBS at 34°C and 5% CO2 should be split 1 day before transfection into 6-well or 35-mm tissue culture plates, with each well/dish containing one 25-mm round cover glass. Cells should be plated so that the confluence on the day of transfection is no more than 40–50% in order to achieve ~70% confluence 1 day posttransfection. If using a cell

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line that is particularly sensitive to transfection reagent, cells can be plated at a higher density, but ensure that the confluence posttransfection is no more than 70% (see Note 4). 2. For each well to be transfected, add 100 ml of Optimem into each of two 1.5-ml Eppendorf tubes (see Note 5). To one tube, add 5 ml of Lipofectamine 2000 (pipetting directly into the Optimem and making sure not to touch the sides of the tube with the transfection reagent). To the second tube, add 0.5  mg of each DNA construct to be transfected (e.g., FcgRIIA-YFP and FcgRIIB-CFP) for a total of 1 mg of DNA. Incubate for 5 min at room temperature. 3. Mix the contents of the two tubes by adding the solution containing the DNA to the tube containing Lipofectamine 2000 and pipetting up and down three to five times or flicking the tube to mix. Incubate at room temperature for 15–20 min. 4. Add the mixture (total of 200  ml) to cells (can be added directly to media with serum) and swirl gently to mix. 5. Incubate cells for 16–24 h. 3.2. Phagocytic Particle Preparation

1. Polystyrene beads are sold as a 2.5% w/v suspension; mix well by inverting the tube several times prior to each use (see Note 6). 2. To opsonize beads, add 70  ml of bead suspension into a 1.5-ml Eppendorf tube. Wash beads by adding 1 ml of 1× PBS and mixing well. Spin beads for 15 s at no more than 5,000 g and aspirate supernatant. Some beads will smear up the side of the tube rather than pelleting – this is normal and the smear can be rinsed off the side of the tube in the next step. 3. Resuspend the washed beads in 157.5 ml of 1× PBS, and add 17.5 ml of human IgG (50 mg/ml stock in PBS). Mix well. 4. Place Eppendorf tube in thermomixer and mix at 37°C, 1,050 rpm, for 1 h. 5. To remove free IgG, spin down beads for 15  s at no more than 5,000 g and aspirate supernatant. To wash, add 1 ml of 1× PBS, mix well, and repeat the spin step. Wash two more times with PBS and finally resuspend in 150 ml of PBS. 6. Particle preparation can be scaled up or down to suit the scale of the experiment. Approximately 10–15  ml of beads are required per condition to be imaged.

3.3. Microscopy

1. Warm up the confocal microscope and prepare settings before the first coverslip is prepared for imaging so that it is ready to be used immediately once beads are added to the cells. 2. Once the phagocytic particles are prepared, transfer one coverslip of transfected cells to an Attofluor cell chamber. Add

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phenol red-free HPMI (warmed to room temperature) to the cell chamber to completely cover the cells (~400 ml), and add 10 ml of bead suspension to the media (see Note 7). The bead suspension should be well mixed prior to addition and can be distributed in the cell chamber by gentle swirling. The chamber can be covered at this point with the lid of a 35-mm tissue culture dish to prevent evaporation. It takes approximately 15–20 min for beads to settle completely; however, depending on the activity of the receptor, phagocytosis can proceed very quickly once beads have started to land on the cell layer; hence, it may be necessary to begin imaging in as little as 10 min post bead addition. 3. Place the cell chamber onto the heated stage assembly that has been prewarmed to 34°C (or 37°C for cell lines that are normally grown at 37°C). Once on the heated stage, the chamber must be covered with a lid (as above) or with a heated stage cover in order to prevent evaporation. 4. Examine the cells through the microscope eyepiece using epifluorescence at a wavelength that makes the transfected cells visible (see Note 8). The polystyrene beads have a soft glow under most wavelengths of light, and hence are also visible as dark orbs with dimly glowing outer shells. Look around the coverslip for transfected cells that are well spread, have distinct cell membranes, and are sufficiently separate from other transfected cells as to make quantitation of membrane fluorescence unambiguous. It is not necessary to find cells that are completely isolated, but cells that have at least one edge of their plasma membrane unattached to other cells result in better quality images. The best phagocytic cups for imaging are those that form along a free edge of plasma membrane, rather than particles that land on top of a cell. Once the particles have started to settle on to the monolayer, it will be possible to identify cells that have particles bound to their membranes. These particles will appear less mobile than their free counterparts, and it will be possible to observe them moving into the cells (see Note 9). 5. Once an appropriate cell is found, switch to viewing it by laser scanning through the confocal interface. 6. To capture a good-quality image of the phagocytic cup, it is necessary to make sure that the acquired fluorescence signals of both tagged receptors are adequate. To this end, scan each channel individually using fast XY and make the appropriate adjustments to the amplifier gain. The intensity of each fluorophore should be sufficient to be seen easily above background, but no pixels should be offscale. The levels for each channel need to be adjusted for each cell that is imaged to account for variations in expression levels between transient transfectants.

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7. Acquire two-channel fluorescence images using multitrack settings. Simultaneous acquisition of a brightfield channel is useful to confirm the position of beads. 8. Depending on the goal of the experiment, follow the progression of phagocytosis by time-lapse imaging or repeat steps 3–6, acquiring images of phagocytic cups on other cells on the same coverslip. 3.4. Quantitation

1. Open an image file and create a new image that is the average of the two fluorescence channels using the formula (channel 1 + channel 2) (in LSM 510, this is done by selecting 2 Process → ADD) and save this file as a TIFF (see Note 10). 2. Using the original confocal file, create a ratio image using the channel 1 + 1 formula *60 (using Process → RATIO) and save channel 2 + 1 this file as a TIFF (see Note 11). 3. Open the average and ratio images in ImageJ. Select the average image and convert it to 8-bit (if not 8-bit already) and threshold the image (Ctrl + Shift + T), setting the mode to black and white. Set the bottom limit to 0, and adjust the top limit until the phagosome and plasma membrane of the cell of interest are white and everything else is black (see Note 12). 4. In the ImageJ console, select Process → Image Calculator and choose AND to perform a logical AND operation on the ratio and average images; create the resulting masked image file in a new window. Crop the image to include only the cell(s) of interest and save the file (for future reference). 5. Using the freehand selection tool, draw a region of interest (ROI) around the phagosome in the masked image and create a histogram of values in the ROI for the region by selecting Analyze → Histogram from the ImageJ console. Capturing extra black space is not a problem, as these masked regions with zero intensity drop out of the subsequent analysis. 6. Open an Excel spreadsheet and paste the histogram values starting at row 5. This will create a spreadsheet with 2 columns: column 1 will be numbers 0–255 (the bit depth of an 8-bit image, defining 256 levels of opacity) and column 2 will be integers indicating how many pixels fall into each level. Once this is done, create a third column using a formula that will multiply all the scaled ratio values from column 1 with each corresponding pixel number value from column 2 (e.g., cell C5 = A5*B5) and apply the formula to all 256 rows. 7. Select an unused cell and define its contents by the formula cell = SUM(C6:C256)/SUM(B6:B256), where C = column 3

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and B = column 2 as outlined above; this will give you the average value of pixels of the ratio image in the phagocytic cup, excluding the masked pixels (which have a value of zero). 8. Return to the cropped, masked image in ImageJ and use the freehand selection tool to draw a new ROI around the plasma membrane. It does not matter how much black space is included in the ROI, but avoid areas of cell–cell contact, phagosomes, and any internal fluorescence (e.g., endosomal compartments) that are not masked by thresholding. Create a histogram for the ROI and copy and paste the histogram values into Excel as in steps 5 and 6. Once you have calculated the average pixel intensities for the phagocytic cup and plasma membrane, determine the enrichment factor by dividing the average pixel intensity of the phagosome by that of the plasma membrane (see Fig. 1 for a schematic overview of the quantitation operation).

Fig. 1. Schematic overview of enrichment quantitation method.

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4. Notes 1. Ideally, the cell line should be adherent (to glass), flat and well spread, easily transfectable, and should not express endogenous Fc receptors. Cells that form single-cell layers are the most suitable, as cells that tend to clump or grow on top of each other are difficult to image. 2. Any two distinguishable fluorescent proteins could be used; however, if fluorophores are selected between which fluorescence resonance energy transfer (FRET) can occur (e.g., CFP and YFP), there may be some skewing of apparent local intensity ratios, since occurrence of FRET between receptors will increase the apparent local concentration of the acceptor fluorophore and decrease that of the donor. To avoid this, fluorophores with more widely separated spectra (e.g., CFP and mCherry) could be employed. 3. We describe a quantitation approach using several commonly available or free software programs; a similar strategy could, however, be implemented in a consolidated manner in a single image analysis package. 4. The confluence of cells on the day of imaging is crucial to obtaining a satisfactory result. If the cells are too sparse, it will be difficult to catch an adequate number of phagocytic events. On the other hand, if the cells are overconfluent, imaging becomes difficult as cells tend to grow on top of one another, often with the result that many of the transfectants end up buried in an inaccessible layer. The ts20 cells that are used in this protocol are not sensitive to transfection reagent (i.e., will not be killed off), and hence it is appropriate to seed them low (~40% confluent) on the day prior to transfection. If using a cell line that is known to be adversely affected by transfection reagent, seeding at a higher confluence may be necessary. 5. It is recommended that Optimem be used as the diluent for Lipofectamine 2000. While other serum-free media can be used instead, it has been our experience that using Optimem significantly increases transfection efficiency. 6. Particles should be prepared fresh on the day of the experiment. 7. Phenol red-free media is essential at this step, as phenol red is itself fluorescent and interferes with imaging. It is also recommended that the media be no warmer than room temperature at this point, as cooler temperature prevents any particles that settle from being immediately phagocytosed. 8. The use of fast XY scanning in the confocal software to scan the coverslip is not recommended. The particles are far less visible

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under the confocal scanning than under epifluorescent light, and hence phagocytic events are nearly impossible to detect until they are at a significantly advanced stage. If the goal is to catch phagocytic cups as they form rather than catching fully formed phagosomes, scanning by eye using epifluorescent light is preferred. An alternative strategy is to use the fast XY function to scan the coverslip before particles have started to settle to identify transfected cells that are good candidates for imaging, and then wait for particles to land in that area. We do not recommend this approach, as it is impossible to predict when/ if a particle will settle onto any given cell and it is possible that the majority of transfectants will have phagocytosed numerous particles during the wait. Once cells have become filled with particles, they are no longer suitable for imaging, so a coverslip may become unusable while waiting for a well-positioned phagocytic event to occur on a specific cell. 9. For active phagocytic receptors (e.g., FcgRIIA), particle uptake is very rapid and can be perceived even when observing the cells through the eyepiece. As a general rule for active receptors, if a particle appears to be bound to a cell, it should be completely engulfed in a time frame of 5–10 min. If a particle appears to be bound but has not formed a cup within 2–3 min, it is unlikely that the particle will be engulfed and moving on to another event is recommended. It should be noted that when inhibitory receptors are present or with lowaffinity activating receptors, cup formation can be weak and full engulfment may take much longer or may not happen at all. In these cases, catching cups is more difficult and may require several coverslips of cells in order to acquire a sufficient number of useful images. 10. When obtaining the ratio of two different channels on a pixel-by-pixel basis, any pixels where there is no fluorescence signal will give meaningless ratio values (background noise divided by background noise). To exclude these pixels, the ratio image is analyzed after applying a mask to consider only pixels that had fluorescence (determined using the image that is the average of both fluorescence channels) above a threshold value. 11. The multiplier (60) in the ratio formula is used to spread the pixel ratio values over the full 256 bit depth (most pixel ratio values will be <4). The multiplier should be reduced if pixels within the cell membrane in the resulting ratio image are offscale; for most images, a multiplier between 20 and 60 works well. 12. In the case where the fluorescence in the phagosome is substantially higher than that in the plasma membrane (due to active receptor recruitment), different threshold values may

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need to be used to cleanly select the plasma membrane and phagosome. In general, we find that calculated enrichment factors do not vary substantially based on the choice of threshold values used. References 1. Greenberg, S. and Grinstein, S. (2002) Phagocytosis and innate immunity. Curr Opin Immunol 14, 136–145. 2. Swanson, J. A. (2008) Shaping cups into phagosomes and macropinosomes. Nat Rev Mol Cell Biol. 9, 639–649. 3. Syam, S., Mero, P., Pham, T., McIntosh, C. A., Bruhns, P., and Booth, J. W. (2010) Differential recruitment of activating and inhibitory Fc gamma RII during phagocytosis. J Immunol 184, 2966–2973.

4. Nimmerjahn, F. and Ravetch, J. V. (2008) Fcgamma receptors as regulators of immune responses. Nat Rev Immunol 8, 34–47. 5. Henry, R. M., Hoppe, A. D., Joshi, N., and Swanson, J. A. (2004) The uniformity of phagosome maturation in macrophages. J Cell Biol 164, 185–194. 6. Indik, Z., Kelly, C., Chien, P., Levinson, A. I., and Schreiber, A. D. (1991) Human Fc gamma RII, in the absence of other Fc gamma receptors, mediates a phagocytic signal. J Clin Invest 88, 1766–1771.

Chapter 10 Assessment of the Recycling of the Membrane-Bound Chemokine, CX3CL1 Sajedabanu Patel, Ilya Mukovozov, and Lisa A. Robinson Abstract Fractalkine (CX3CL1) is a membrane-anchored chemokine whose N-terminus contains a unique CX3C motif that is cleaved and released. The membrane-bound form functions as an adhesion molecule and the secreted form as a chemotactic factor. Like other chemokines, CX3CL1 is regulated at the levels of transcription and translation. Recent evidence points to additional functional regulation by cellular trafficking owing to the unique transmembrane structure. CX3CL1 is the only chemokine known to undergo constitutive internalization. To understand mechanisms governing the regulation and processing of such membrane-bound proteins, it is vital to study their subcellular distribution and transport. The methods outlined in this chapter describe (1) transfection of mammalian cells with plasmids encoding the expression of green fluorescent protein-tagged CX3CL1; (2) immunofluorescence antibody labeling as well as fluorescence recovery after photobleaching to study internalization of CX3CL1 by endocytosis; and (3) acid-stripping assays to study the recycling of internalized CX3CL1 back to the plasma membrane. Together, these methods allow for the examination of subcellular distribution and traffic of recycling membrane proteins. Key words: Chemokine, Fractalkine, FRAP, Endocytosis, Exocytosis, Antibody labeling, Acid stripping

1. Introduction Chemokines are a class of proinflammatory cytokines that possess the ability to attract and activate leukocytes. They serve as cues that direct migration of leukocytes to an injured or inflamed site and that facilitate adhesion of leukocytes to endothelium. The leukocyte adhesion cascade involves capture, rolling, tethering, and firm adhesion of leukocytes, mediated by selectins and integrins. These events culminate in diapedesis of leukocyte

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across the endothelium and their migration into the injured tissue. Chemokines play a key role as mediators of immune and inflammatory responses (1, 2). Among more than 50 chemokines identified so far, fractalkine (CX3CL1) is the sole member of the CX3C family and has unique structural and functional features. Unlike most other chemokines, CX3CL1 exists in two forms: a soluble chemotactic glycoprotein and a membrane-anchored protein with adhesive properties (3, 4). Like other chemokines, CX3CL1 biosynthesis is regulated at both transcriptional and translational levels (4–8). The unique transmembrane domain suggests additional mechanisms that may govern its regulation. To understand these regulatory mechanisms, it is vital to first investigate the subcellular distribution and trafficking of the protein. In this chapter, we aim to describe the basic methods that we have successfully used to study the localization and recycling of CX3CL1 (9). The methods outlined allow similar examination of recycling of other membrane-bound proteins. A number of methods have been developed to study subcellular trafficking of membrane proteins. The choice of method depends primarily on the question that needs to be addressed, cost, and the reagents available. To track the movement of proteins from the cell surface to intracellular compartments biochemically, radiolabeling of the protein of interest can be used (10). Alternatively, immunofluorescent labeling of the protein using specific antibodies can be performed, a method that we have adopted to study CX3CL1 distribution and trafficking (9). This is a simple, yet effective way to determine if the membrane protein of interest undergoes internalization and to gain insight into the subcellular localization of the protein. However, cross-linking by antibodies can alter the native traffic of the protein being studied. Consequently, such immunolocalization studies with antibody labeling need to be performed with Fab fragments as probes. Where these antibody Fab fragments are not available, another approach based on fluorescence recovery after photobleaching (FRAP) can be used to study the internalization process. FRAP is a technique used to monitor the mobility and dynamics of fluorescent proteins in live cells. Here, protein molecules within a small region are irreversibly photobleached by high-intensity laser excitation. Subsequent movement of unbleached fluorescent molecules into the bleached area is then monitored over time using a low-intensity laser (11, 12). Because anti-CX3CL1 Fab fragments were not readily available, we used FRAP to study internalization of the chemokine. Replenishment of the fluorescence of green fluorescent protein (GFP)-tagged CX3CL1 in the entire juxtanuclear compartment was monitored following its photobleaching. To further examine if an internalized protein is recycled back to the plasma membrane, acid stripping assays can be performed. Here, cells expressing the protein of interest are

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labeled with specific antibody at 37°C to allow internalization of the antibody bound to its target antigen. Cells are then incubated in an acidic solution to remove antibody bound to CX3CL1 at the cell surface. This is followed by quantification of the reappearance of the protein of interest on the plasma membrane by immunofluorescence. Here, we outline the basic methods we used to study CX3CL1 subcellular distribution and trafficking in cells. These include immunofluorescence labeling of CX3CL1-expressing mammalian cells, FRAP to study the endocytosis of the chemokine, and acid stripping to examine its exocytosis.

2. Materials 2.1. Reagents and Antibodies

1. ECV-304 cells (American Type Culture Collection, Manassas VA). 2. Plasmid-encoding protein of interest with GFP tag (in our case, the fusion protein is CX3CL1-GFP). 3. ECV-304 cells stably expressing CX3CL1 (ECV-CX3CL1) (9). 4. Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS). 5. Medium 199 (Invitrogen) containing 10% fetal calf serum and 500 mg/ml G418 (Invitrogen). 6. FBS. 7. Phosphate-buffered saline (PBS; Invitrogen) (See Note 1). 8. HEPES solution (Invitrogen). 9. 16% paraformaldehyde solution (Fisher Scientific, Rockford, IL), methanol-free (see Note 2). 10. Permeabilization solution: 0.1% (v/v) Triton X-100 in PBS. 11. Blocking solution: 5% donkey serum (DS) in PBS. 12. Antibody dilution buffer: 3% (w/v) donkey serum in PBS. 13. Primary antibodies: Goat anti-CX3CL1 (stock concentration = 0.1 mg/ml, dilute 1:40 to give working concentration of 2.5 mg/ml) (R & D Systems, Inc., Minneapolis, MN). 14. Secondary antibodies: Cy3-conjugated anti-goat IgG (stock concentration = 1.5  mg/ml, dilute 1:500 to give working concentration of 3  mg/ml) (Jackson Immunoresearch Laboratories, Bar Harbor, ME). 15. DAKO fluorescent-mounting Carpinteria, CA).

medium

(DAKO

Corp,

16. Live-cell imaging buffer: Phenol red-free DMEM supplemented with 10% (v/v) FBS and 25 mM HEPES.

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17. LipoD293TM DNA in vitro transfection reagent (SignaGen® Laboratories). 18. Acid wash solution: 150  mM NaCl, 50  mM glycine, 0.1% BSA, pH 2.5. 19. Recovery buffer: 150  mM NaCl, 20  mM HEPES, 1  mM CaCl2, 1 mM MgCl2, 5 mM KCl, pH 7.4. 2.2. Equipment and Laboratory Supplies

1. Attofluor ® cell chamber (Molecular Probes). 2. 25-mm round glass coverslips (Fisher Scientific). 3. Microscope coverslips (25 × 40 × 0.15  mm) (VWR) (see Note 3). 4. Six-well tissue culture plates (Becton Dickinson and Company). 5. Incubator at 37°C (with 5% CO2). 6. Tissue culture flasks (25 cm2, 75 cm2) for adherent cell line. 7. Confocal laser scanning microscope capable of selective photobleaching (e.g., Zeiss LSM510).

3. Methods 3.1. Transfection of ECV-304 Cells with CX3CL1-GFP Expression Plasmid

3.1.1. Day 1

A detailed protocol is outlined for transiently transfecting ECV304 cells with plasmid-encoding CX3CL1-GFP fusion protein using LipoD293TM DNA in vitro transfection reagent (see Note 4). Cells are seeded on 25-mm coverslips on Day 1, transfected with the plasmid on Day 2, and imaged on Day 3. 1. ECV-304-CX3CL1-GFP cells are grown in DMEM supplemented with 5% FBS in 25- or 75-cm2 tissue culture flasks. 2. Seed ~1 × 106 ECV-304-CX3CL1-GFP cells onto 25-mm coverslips in a six-well tissue culture plate so that they are 90–95% confluent the following day (see Note 5).

3.1.2. Day 2

1. Replenish the wells to be transfected with fresh 1  ml of DMEM media supplemented with 5% FBS 30–60 min before transfection. 2. For each well, dilute 2 mg of plasmid DNA (see Note 6) with 100 ml serum-free DMEM into sterile eppendorf tubes. Mix by tapping the tube and spin down briefly. 3. For each well, dilute 6 ml of LipoD293™ reagent into 100 ml of serum-free DMEM. Mix by tapping the tube and spin down briefly. 4. Add 100 ml of the diluted LipoD293™ reagent immediately to 100  ml of the diluted DNA solution all at once to yield

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200 ml of the LipoD293™/DNA complex. The diluent volume used for DNA and the transfection reagent depends on the surface area of each dish. See manufacturer’s instructions for dishes of other sizes. 5. Mix the solution immediately and spin down the tubes briefly. 6. Incubate the LipoD293™/DNA mix for 15  min at room temperature (see Note 7). 7. Following incubation, add the 200  ml LipoD293™/ DNA mixture drop-wise onto the medium in each well and gently swirl the plate to mix. 8. Incubate plate in an incubator at 37°C (with 5% CO2) for 12–18 h to allow expression of the protein (see Note 8). 3.1.3. Day 3

1. Remove LipoD293™/DNA complex-containing medium and replace with fresh DMEM media with 5% FBS 12–18 h posttransfection. 2. Confirm GFP expression by fluorescence microscopy (see Note 9).

3.2. Endocytosis: Antibody Uptake Method

1. Grow ECV-CX3CL1 cells to confluence on glass coverslips in a six-well plate. 2. To label membrane-associated CX3CL1, incubate cells with primary anti-CX3CL1 antibody (2.5 mg/ml) at 37°C or 4°C for 1–2 h (see Note 10). 3. Wash the cells three times with PBS, 2 ml/well. 4. Fix the cells using 4% paraformaldehyde (see Note 11) for 20 min at room temperature and perform three washes with PBS, 2 ml/well. 5. Permeabilize cells using 0.1% Triton, and block with 5% donkey serum in PBS at room temperature for 1 h (see Note 12). 6. To visualize antibody-labeled CX3CL1 that has undergone internalization, incubate cells with a fluorophore-conjugated secondary antibody (e.g., Cy3-conjugated anti-goat IgG) for 1 h on ice (Fig. 1). 7. Perform three washes with PBS, 2 ml/well, and mount the coverslips onto glass slides using DAKO fluorescent-mounting medium (see Note 13). 8. Capture images using an epifluorescent microscope (e.g., Leica DMIRE2 microscope) and image acquisition software (e.g., OpenLab or VolocityTM). 9. Measure endomembrane immunofluorescence intensity using image analysis software (e.g., VolocityTM imaging software) (see Note 14).

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a

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c CX3CL1 anti-CX3CL1 Ab fluorophoreconjugated secondary Ab

Fig. 1. Antibody Uptake Protocol. (a) Cells are incubated with primary antibody for 1 h at 37°C. Antibody-labeled CX3CL1 is internalized by endocytosis. (b) Cells are fixed and permeabilized, and nonspecific binding is blocked. (c) Cells are incubated with a fluorophore-conjugated secondary Ab. The ratio of intracellular to total fluorescence intensity is measured by microscopy.

3.3. Endocytosis: Fluorescence Recovery After Photobleaching

3.3.1. Preparation of Samples

We utilized this technique to complement the immunofluorescence labeling with bivalent anti-CX3CL1 antibodies since cellular trafficking could potentially be altered by antibody clustering. The information in this section intends to provide general guidelines applicable for FRAP experiments, and assumes that the experimenter is acquainted with the use of a confocal laser scanning microscope capable of selective photobleaching (see Note 15). 1. ECV-304 cells transfected with CX3CL1-GFP (See Subheading  3.1) cells are grown on 25-mm coverslips in a six-well plate in DMEM with 5% FBS. 2. Aspirate medium from the well and carefully transfer the 25-mm circular coverslip from the six-well plate onto the Attofluor®cell chamber. 3. Gently add live-cell imaging buffer to the chamber and mount on a heated stage maintained at 37°C.

3.3.2. FRAP Procedure

1. Start and warm up the microscope. Identify and focus the desired cell (Fig. 2). 2. Selecting regions of interest (ROIs): A circular region that encompasses the entire juxtanuclear region of ECV- CX3CL1GFP is selected for photobleaching. At least two other ROIs with the same diameter are also selected: one of them in the plasma membrane of the cell (to serve as a control, unbleached reference) and the other in an area outside the cell (background reference) (see Note 16). 3. Selecting bleaching conditions: Set parameters to acquire at least five pre-bleached images (to provide a reference point for fluorescence recovery), and to acquire images at 45 s time intervals for 15 min (see Note 17). 4. Image and data acquisition: A 30-mW argon laser set at 25% intensity is used to irreversibly photobleach the entire juxtanuclear region of ECV-CX3CL1-GFP. The 488-nm laser line

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c CX3CL1-GFP

Fig. 2. Fluorescence recovery after photobleaching (FRAP) protocol. (a) Juxtanuclear compartment of CX3CL1-GFP cells is photobleached. (b) Juxtanuclear fluorescence intensity is allowed to recover. (c) Fluorescence intensity of juxtanuclear compartment is measured.

of the Zeiss LSM 510 confocal microscope at full power is used for photobleaching, and 22% intensity used for image acquisition (see Note 18). 3.3.3. FRAP Quantification

1. Collect the mean fluorescence intensity for all the ROIs from the time series of images, and import the readings to an appropriate statistical package (see Note 19). 2. Plot a graph between the percent recovery of fluorescence intensity versus time for ECV-CX3CL1-GFP after performing the background correction and normalizing the data (see Note 20). (a) Background correction: The mean background fluorescence (background measurement obtained from an area outside the cell) should be subtracted from the mean fluorescence intensity of ROIs for each time point. (b) Normalization: The fluorescence intensity of the average pre-bleach intensity is normalized to 1 to allow for comparison between different experiments. Furthermore, the data is also normalized to the unbleached region to account for any unwanted photobleaching that may occur during image acquisition. The normalized data obtained can then be averaged for different cells to perform the standard deviation or standard error calculations.

3.4. Exocytosis: Acid-Stripping Assay

1. Incubate cells with anti-CX3CL1 antibody (2.5  mg/ml) for 1  h at 37°C (5% CO2) to allow internalization of labeled CX3CL1 from the plasma membrane. 2. To remove membrane-associated CX3CL1, perform two washes with acidic buffer solution on ice, 2 ml/well, for 30 s (Fig. 3). 3. Perform a wash with recovery buffer, 2 ml/well, for 3 min at room temperature. 4. Allow membrane-associated CX3CL1 to recover for various time points (see Note 21).

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a

b

c

d CX3CL1 anti-CX3CL1 Ab fluorophoreconjugated secondary Ab

Fig. 3. Acid-Stripping Protocol. (a) Cells are incubated with primary antibody for 1 h at 37°C. (b) To remove membraneassociated anti-CX3CL1 Ab, acid washes are performed. (c) Membrane-associated CX3CL1 is allowed to recover for various time points. (d) Cells are fixed and incubated with a fluorophore-conjugated secondary Ab. Cell surface fluorescence intensity is measured.

5. Wash the cells three times with PBS, 2 ml/well. 6. Fix the cells with 4% PFA for 20 min at room temperature. 7. Wash the cells three times with PBS, and incubate with Cy3conjugated secondary antibody diluted in PBS containing 3% donkey serum. 8. Perform three washes with cold PBS and mount the coverslips using DAKO fluorescent-mounting medium (see Note 13). 9. Capture images using an epifluorescent microscope (e.g., Leica DMIRE2 microscope) and image acquisition software (e.g., OpenLab or VolocityTM). 10. Image acquisition settings (e.g., exposure time, gain, contrast, etc.) must be identical to allow the assessment of recovery of fluorescence over time. Measure cell surface immunofluorescence intensity using image analysis software (e.g., VolocityTM imaging software).

4. Notes 1. PBS must be sterile and endotoxin-free, as it is used for washing live cells. Use sterile pipettes and proper aseptic techniques. 2. Store in opaque container at 4°C. Prepare a 4% working solution before each experiment. Label all containers as carcinogenic. 3. Glass coverslips should be washed with HCl and autoclaved prior to use to remove debris from the surface. Briefly, wash with 70% ethanol for 30 min, rinse with H2O, wash with 1 M HCl for 30 min, rinse with autoclaved H2O, allow to air dry, and autoclave. 4. We found that LipoD293TM DNA in vitro transfection reagent yielded the best results for expression of CX3CL1-GFP cDNA

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plasmid in ECV-304 cells. ECV-304 is a bladder epithelial cancer cell line that possesses characteristics of endothelial cells (13, 14). Generally, the choice of transfection reagent and optimal conditions need to be empirically determined, and depend on the cells used as well as the size and purity of the DNA expression plasmid. 5. It is critical to have a cell confluency of 90–95% to achieve maximum transfection efficiency with LipoD293TM DNA in vitro transfection reagent. We notice a decrease in transfection efficiency at cell densities less than 80% confluent. If you notice that the cells are overconfluent the day after transfection, they can be split into separate wells after trypsinization and allowed to settle for 4–5 h. 6. Plasmid DNA can be prepared using commonly available maxi-prep kits (such as those made by QIAGEN), according to the manufacturer’s protocol, and should be resuspended in endotoxin-free Tris–EDTA (TE) buffer. The DNA stock concentration should be between 0.1 and 2 mg/ml. 7. Do not keep the LipoD293™/DNA complex longer than 20 min. 8. For certain cell types, particularly primary cells, the LipoD293™/DNA complex can be toxic. In this instance, remove the LipoD293™/DNA complex and replace with fresh DMEM media (with 5% FBS) 4–5 h after transfection. 9. We generally achieve 60–65% transfection efficiency with this protocol. Lower transfection efficiencies could arise if the cells have been in culture for a prolonged period of time or if the plasmid DNA is degraded. If low efficiencies are obtained, try the transfection with cells at a lower passage and use freshly isolated plasmid. 10. A fluorophore-conjugated membrane marker may be used to clearly label the plasma membrane 48 h prior to the performance of experiments. This will aid the analysis by clearly outlining the plasmalemma, and will help to discern the fluorescence intensity of CX3CL1 originating from the plasmalemma (15). 11. For up to 5 × 106 cells/ml, add 1 ml of 4% PFA. 12. Triton X-100 is a commonly used permeabilization agent for immunofluorescence staining, as it efficiently solvates cellular membranes without disturbing protein–protein interactions. Due to its viscosity, it can be prepared as a 20% stock solution in deionized water and stored at 4°C. Triton is usually used at concentrations ranging from 0.1 to 1% for permeabilization. 13. Do not aspirate the PBS from the final wash, as coverslips are easier to remove when immersed in PBS. Remove excess PBS

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from the coverslip by tilting and drying the edge on a kimwipe. Flip the coverslip so that the cells are facing down when mounting onto the glass slide. Use a single drop of mounting medium, and ensure that air bubbles are avoided by discarding the first drop from the bottle. It is helpful to lower one edge of the coverslip into the mounting medium, and then slowly lower the opposite edge, allowing the mounting medium to spread evenly under the coverslip. 14. The ratio of endomembrane fluorescence intensity to total fluorescence intensity is then used to quantify the amount of endocytosis that had occurred from the plasmalemma. Should the experimental conditions studied result in a difference in endocytosis, the underlying mechanism(s) can be investigated further by inhibiting individual endocytic pathways, such as clathrin- or caveolin-mediated endocytosis (15). 15. FRAP studies are based on the bleaching and recovery characteristics of the molecules under observation. These characteristics vary among different cell types and fluorophores used. Thus, optimization of experimental conditions is required. 16. Serial monitoring of a control ROI is performed to quantify the amount of unwanted bleaching caused by repeated image acquisition. Monitoring a region outside the cell provides a readout of the background fluorescence emitted by factors besides the object of interest (e.g., autoflourescence from medium, glass, reflected light). 17. The parameters that influence the bleaching process and require fine-tuning are laser power, zoom factor, and scan speeds. Increasing laser power not only allows for faster bleaching, but can also harm sensitive cells. Zooming in generally speeds up the bleaching process, and slower scan speed results in more energy radiation (longer pixel dwell time). 18. If considerable photobleaching is observed in the control area, the time interval between images can be increased and/ or laser power be decreased. Alternatively, the detector gain settings can also be increased. 19. The graphing and data analysis can be performed by most common statistical packages, such as Excel (Microsoft), Kaleidagraph (Synergy Software), or SPSS (SPSS Inc). The mean fluorescent intensities for the selected regions for each time point can be determined by most confocal operating software (e.g., Zeiss LSM, Leica LCS) or alternatively with other image processing software (e.g., the freeware ImageJ). 20. The background quantification can also be carried out with the confocal operating software (e.g., Zeiss LSM, Leica LCS) or with image processing software (e.g., the freeware ImageJ) that can perform time series calculations. Depending on the ­experiment and the question that needs to be addressed, data

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evaluation can be performed in several ways to yield meaningful results (11, 12). 21. The time frame used to allow for the recovery of plasmalemma CX3CL1 fluorescence is dependent on the experimental conditions used. Ideally, a time-course experiment should be performed to determine an optimal time to recovery of plasmalemma fluorescence. The fluorescence intensity of plasmalemma is an index of the amount of recycling that had occurred, and is compared across conditions to see if a given treatment significantly affects the rate of CX3CL1 recycling. References 1. Schall, T.J., Bacon, K.B. (1994) Chemokines, leukocyte trafficking, and inflammation Curr Opin Immunol 6, 865–873 2. Baggiolini, M. (1998) Chemokines and leukocyte traffic. Nature 392, 565–568 3. Bazan, J.F., Bacon, K., Hardiman, G., Wang, W., Soo, K., Rossi, D., Greaves, D.R., Zlotnick, A., and Schall, T. S. (1997) A new class of membrane-bound chemokine with a CX3C motif Nature 385, 640–644 4. Pan, Y., Lloyd, C., Zhou, H., Dolich, S., Deeds, J., Gonzalo, A., Vath, J., Gosselin, M., Ma, J., Dussault, B., Woolf, E., Alperin, G., Culpepper, J., Gutierrez-Ramos, J.C. and Gearing, D. (1997) Neurotactin, a membraneanchored chemokine upregulated in brain inflammation Nature 387, 611–617 5. Fong, A., Robinson, L., Steeber, D., Tedder, T., Yoshie, O., Imai, T., and Patel, D. (1998) Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion and activation under physiological flow J Exp Med 188, 1413–1419 6. Harrison, J., Jiang, Y., Wees, E., Salafranca, M., Liang, H., Feng, L., and Belardinelli, L. (1999) Inflammatory agents regulate in  vivo expression of fractalkine in endothelial cells of the rat heart J Leukocyte Biol 66, 937–944 7. Ludwig, A., Berkhout, T., Moores, K., Groot, P., and Chapman, G. (2002) Fractalkine Is Expressed by Smooth Muscle Cells in Response to IFN- g and TNF-a and Is Modulated by Metallo­proteinase Activity J Immunol 168, 604–612 8. Muehlhoefer, A., Saubermann, L., Gu, X., Luedtke-Heckenkamp, K., Xavier, R., Blumberg, R. S., Podolsky, D. K., MacDermott, R. P., and Reinecker, H. (2000) Fractalkine Is

an Epithelial and Endothelial Cell-Derived Chemoattractant for Intraepithelial Lymphocytes in the Small Intestinal Mucosa J Immunol 164, 3368–3376 9. Liu, G, Kulasingam, V., Alexander, R.T., Touret, N., Fong, A.M., Dhavalkumar, D.P., and Robinson, L.A. (2005) Recycling of the membrane-anchored chemokine, CX3CL1. J Biol Chem 280:19858–19866 10. Touret, N., Furuya, W., Forbes, J., Gros, P., and Grinstein, S. (2003) Dynamic Traffic through the Recycling Compartment Couples the Metal Transporter Nramp2 (DMT1) with the Transferrin Receptor. J Biol Chem 278: 25548–25557 11. Lippincott-Schwartz, J., Presley, J., Zaal, K., Hirschberg, K., Miller, C., and Ellenberg, J. (1999) Monitoring the dynamics and mobility of membrane proteins tagged with green fluorescent protein. Methods Cell Biol 58, 261–281 12. Reits, E., and Neefjes, J. (2001) From fixed to FRAP: measuring protein mobility and activity in living cells Nat Cell Biol 3, 145–147 13. Su, T., Cariappa, R., and Stanley, K. (1999) N-glycans are not a universal signal for apical sorting of secretory proteins FEBS Lett. 453, 391–394 14. Haller, C., Kiessling, F., and Kubler, W. (1997) Polarized expression of heterologous membrane proteins transfected in a human endothelial-derived cell line Eur J Cell Biol 75, 353–361 15. Huang, YW., Su, P., Liu, GY., Crow, MR., Chaukos, D., Yan, H., Robinson, LA. (2009). Constitutive Endocytosis of the Chemokine CX3CL1 Prevents Its Degradation by Cell Surface Metalloproteases. J Biol Chem 284: 29644–29653

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Chapter 11 Measuring Immune Receptor Mobility by Fluorescence Recovery After Photobleaching Kristen Silver and Rene E. Harrison Abstract The coordinated effort of cells in the immune system relies heavily on surface receptor interactions. Immune receptor mobility provides vital information on the function and responses of immune cells, and these measurements shed light on their interactions with other membrane, cytosolic, and extracellular matrix proteins. These measurements can be obtained using the fluorescence recovery after photobleaching (FRAP) technique in living cells. We describe here general approaches for FRAP using green fluorescent protein fusion proteins. Key words: FRAP, GFP, Confocal laser scanning microscopy, Region of interest, Immune receptors, Diffusion

1. Introduction The mobility of various cellular macromolecules has been viewed and recorded over the last 30 years by the application of fluorescence recovery/redistribution after photobleaching (FRAP), also referred to as fluorescence photobleaching recovery (FPR). Peters et al. first characterized this now relatively noninvasive and simple technique, where they followed the fluorescent dye fluorescein isothiocyanate (FITC) coupled to proteins and lipids and measured the lateral diffusion into the bleached zone of an erythrocyte (1). FRAP was also one of the techniques that advanced the fluid mosaic model of the plasma membrane (2). Diffusion can be calculated as motion in three dimensions, but for the purpose of this review of immune receptors, diffusion in two dimensions will be considered. FRAP has been used to image and calculate ­diffusion coefficients for a variety of green fluorescent protein

Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_11, © Springer Science+Business Media, LLC 2011

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(GFP)-tagged proteins in different cellular compartments, ­including the nucleoplasm (3), cytosol (4), mitochondria (5), Golgi (6), and endoplasmic reticulum (ER) (7). This protocol, therefore, can be applied to other macromolecules within the cell; however, some of the intracellular compartments, such as the Golgi and ER, require more elaborate mathematical modeling. In this chapter, the mobility of immune receptors that are involved in both Brownian diffusion and binding events within the plasma membrane is discussed and an FRAP protocol described using a two-dimensional diffusion model. The popularity of FRAP was enhanced with the advent of GFP technology and the development of confocal laser scanning microscopes (CLSMs) with FRAP capabilities. The CLSM enables precise diffusion measurements while offering high spatial resolution due to its ability to eliminate out-of-focus fluorescence. DIC images can also be taken concurrently on the CLSM to ensure that cells are viable during the imaging process (8). Currently, FRAP is the primary technique employed for determining the diffusional mobility of macromolecules in vivo and in near real time. FRAP is based on the principle that fluorescent molecules can be irreversibly photobleached after being subjected to constant laser excitation and emission. This repeated exposure to high-intensity illumination produces an irreversible covalent modification due to the interaction of fluorescent molecule with oxygen, resulting in a nonfluorescent protein (9). Subsequent monitoring of the movement of fluorescent proteins into the photobleached area can then provide information on receptor dynamics and interactions along the plasma membrane. 1.1. FRAP and Immune Receptors

FRAP has, in particular, been used in a number of studies to assess the mobility of various different immune receptors. Below is an overview with a few examples of the measurements taken from various immune receptors using FRAP. The first direct measurement of lateral diffusion of the T cell receptor (TCR) in live T lymphocytes was recorded by Favier et al. using FRAP (10). The results from these experiments indicated that TCRs are freely mobile on the cell surface of T cells and are not constrained by obstacles, such as the actin cytoskeleton. FcgR3B is a GPI-linked receptor involved in the degranulation of neutrophils. Based on suggestive evidence that this ­receptor physically interacts with complement receptor 3 (CR3) on the plasma membrane, FRAP was employed to provide physical­ evidence of receptor interactions. This study determined that the lateral diffusion of FcgR3B was constrained by CR3 because the diffusion coefficient was decreased when cells were transfected with both receptors compared to a transfectant that expressed only FcgR3B (11). FRAP was also used to disprove interactions between two receptors, FcgRI and CR3, which showed marked differences in lateral mobility in the plasma membrane, ruling out a constitutive association between these receptors (12; see Fig. 1).

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Fig. 1. FcgR and CD11b have different mobility in resting cells. Representative FRAP experiments in RAW264.7 cells transiently transfected with a chimeric GFP-labeled FcgRI receptor (a–c) or labeled with FITC anti-CD11b (d–f ). Images acquired before, immediately after, and 50 s following bleaching are shown. Areas that underwent photobleaching are shown by open arrows, and control regions left unbleached are shown by solid white arrows. Size bars = 5  mm. Representative recovery curves for FcgRI (open circles) and CD11b (solid triangles) are depicted in (g). The times for 50% recovery of fluorescence (t1/2) and fractional recovery were determined from two different bleached regions in 20 separate determinations and are tabulated in (h). Data are means ± S.E. *, p < 0.05; **, p < 0.001.(Reproduced from Ref. (13) with permission from Journal of Biological Chemistry).

Another Fc receptor, FcgR2a (CD32), displays different mobility states depending on the activation status of macrophages. FcgR2a showed reduced lateral diffusion in macrophages treated with phorbol myristate acetate (PMA) compared to cells not treated with PMA. This suggested that activation of protein kinase C creates interactions of Fcg2a with proteins via its cytoplasmic domain to reduce its lateral mobility (13). FRAP was used in a study by Triantafilou et al. to determine if more than one receptor is involved in the binding of lipopolysaccharide (LPS) by immune cells (14). The mobility of the LPS receptor CD14 was measured prior to and after stimulation with LPS. As has been done for many receptors, fluorescently labeled antibody Fab fragments were used to label CD14, negating the

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need for transfection of GFP chimeras, which can be difficult to introduce into immune cells. The researchers found similar diffusion coefficients for CD14 before and after stimulation with LPS indicating that the ligand does not affect the mobility of the receptor. Using FITC, the diffusion of LPS was also studied and surprisingly found to be immobile in many cells. The researchers showed, using FRAP, similar immobility of two heat-shock proteins, Hsp70 and 90, and implicated these proteins in immobilizing the bulk of LPS after a brief, dynamic association with CD14. Another study used FRAP to study the assembly of TLR signaling complexes in response to lipopeptides (15). 1.2. Future Directions of FRAP

With the growing ability to characterize immune receptors and other macromolecules, the use of FRAP becomes an increasingly useful approach to discern the function and properties of membrane proteins and their role in complex immune processes within living cells. Use of cotransfected cells or dual-color Fab antibody fragments makes it possible to track several proteins simultaneously within the cell. Moreover, FRAP technology is now being incorporated into total internal reflection fluorescence (TIRF) and multiphoton microscopes to more precisely visualize the plasma membrane and allow the use of photobleaching studies within tissues and organisms.

2. Materials 2.1. Cell Culture and Transfection

1. Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). 2. 25-mm round coverslips (#1.5) and microscope slides for analysis of fixed samples. 3. Transfection reagent of choice, e.g., FuGENE HD (Roche Diagnostics). 4. 16% Paraformaldehyde (PF) (Canemco): Prepare 4% PF in 1× PBS, store at 4°C. 5. Mounting media: Dako Mounting Media (DakoCytomation).

2.2. Setup: Laser Scanning Confocal Microscope and FRAP

1. HPMI: RPMI 1640 containing 25 mM HEPES (Wisent). 2. Phosphate-Buffered Saline (PBS): 8  g NaCl, 0.2  g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, dissolve in 800 mL of distilled H2O. Once prepared, autoclave and store at room temperature. 3. Attofluor cell chamber (Molecular Probes). 4. An inverted Zeiss LSM510 laser scanning confocal ­microscope using LSM510 Meta System program or equivalent microscope and software (e.g., Leica TCS SP2 CLSM) (see Note 1).

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3. Methods In FRAP, a small region of interest (ROI) is irreversibly ­photobleached by a high-intensity laser to create two mutually exclusive subpopulations. One population is the molecules in the photobleached ROI and the other population is the surrounding fluorescent molecules. Immediately thereafter, sequential images are taken with a relatively low-intensity laser to monitor the recovery of fluorescence into the ROI until equilibrium is attained and a plateau on the FRAP curve is reached. Several parameters can be determined using the raw FRAP data collected, such as kinetics and mobile fraction (% recovery). The diffusion time (tD), which is the amount of time required for half the molecules to diffuse back into the ROI, can be determined from the FRAP curve (16). The diffusion coefficient (rate of mobility) can be calculated from this diffusion time; see below in Data Analysis Subheading  3.5. As mentioned, fluorescently labeled Fab fragments specific for the extracellular portion of receptors can be used for the analysis of endogenous receptors by FRAP in living cells. With transfected receptors, enhanced green fluorescent protein (EGFP) chimeric proteins are generally the tool of choice for FRAP due to the ability of EGFP to not photobleach significantly when pulsed with a low-intensity laser, but to bleach quickly and irreversibly under high-intensity lasers while leaving the cell free from intracellular damage (17). EGFP is also not cytotoxic and can be imaged for a long time due to its photostability (17). Typically, argon lasers at 10–25 mW are used to excite EGFP at 488 nm (9). FRAP can also be applied to kinetic studies, where the interaction of a fluorescent ligand with a nonfluorescent binding partner is studied (8). 3.1. Transfecting Cells for FRAP

1. Cells are seeded into six-well tissue culture plates containing 25-mm round coverslips and grown in DMEM supplemented with 10% FBS in a humidified atmosphere with 5% CO2, until the cells reach 70–80% confluency (see Note 2). 2. In an Eppendorf tube, add 100 ml of serum-free DMEM and 3 ml of FuGENE HD and let stand for 5 min at room temperature. Add 2  mg of an expression vector for a chimeric fluorescent protein (EGFP or other variant) to the microcentrifuge tube for 20 min at room temperature. The transfection solution is then added (100  ml/well) to each well containing 1 mL of media with serum (see Notes 3 and 4). 3. Wait for 18–24  h to allow expression of the plasmid while cells are kept in a 37°C incubator with 5% CO2.

3.2. Fixed Cell FRAP Preparations

1. Prior to doing live cell FRAP, establish FRAP parameters (see Note 5). To do this, fix the transiently transfected cells in 4% PF in PBS for 10 min at room temperature.

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2. Wash out PF with PBS several times, and place PF waste into a hazardous waste container. 3. Place a drop of Dako mounting media onto a microscope slide, and place inverted coverslip on top (see Note 6 and 7). 4. Locate EGFP-transfected cell on CLSM microscope, and select and photobleach ROI (see Subheading 3.3 below on setting up FRAP parameters). 3.3. Laser Scanning Confocal Microscope Preparation for FRAP

Data Acquisition 1. These instructions assume the use of an inverted Zeiss LSM510 using the LSM510 software. Before preparing sample, turn on the microscope and set up the software to fit parameters of choice. Below are guidelines used for FRAP, and parameters may vary with sample and experimental design. 2. Wash the Attofluor cell chamber with 70% ethanol before inserting round coverslip and attaching lid. Add about 1 ml of HPMI media into the chamber (see Note 8). 3. Affix Attofluor cell chamber into the stage adaptor with screws (finger tight) on a flat surface to ensure that the coverslip is level and place into heated stage. 4. After adding oil to immersion objective, secure heated stage and carefully raise the objective until cells are visible. Locate a transfected cell. 5. Objective: 63× (can also use 40 or 100× objective). 6. Laser of choice: If using GFP or Cy2-conjugated antibody to visualize the protein of interest, use 488-nm argon ion laser (500–530 band pass filter) (see Note 9). 7. Pixel resolution: 512 × 512 pixel frame. 8. Under Scan control ³ mode: scan speed: set to the maximum: 10 = 786  ms; Under scan control ³ channel: Select pinhole: 2.98 airy units = around 284 mm Pinhole can be set between the values 1 and 2.98 airy units (the higher the pinhole number, the greater the depth of field during the recovery phase) (see Note 10). 9. Bit acquisition: Under pixel depth ³ data depth: Select 9 or 12 (12 gives a better dynamic range). 10. Track: If acquiring images for FRAP of a single fluorophore, then choose single track; if doing dual-color FRAP, use multitrack. 11. Number of Prebleach images “Scan number”: To judge the intensity of fluorescence prior to the bleach, at least 2–5 images should be taken; the preintensity fluorescence is taken as an average of the prebleach images. Under bleach ­control > bleach after number of scans: Select anywhere from 2 to 5 images. Make sure to save, store, check off, and apply this new setting.

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12. Shape of ROI: To quantify simple FRAP experiments, choose rectangles or circles. Instructions below relate to this model of FRAP. Other shapes or freehand forms require more complex mathematical models. 13. ROI size: The size of the ROI should not be greater than 10% of the size of the cell in view. If the ROI is larger, it may skew the fluorescence recovery because too much of fluorophore will be photobleached to measure accurate recovery and cellular photodamage may occur. To obtain ROI, use the drawing tool in software to form a circular or rectangular shape. ROIs are typically 2-mm spot diameter. You will need to choose two ROIs; one ROI will be the unbleached control. 14. Laser power: Under bleach control ³ excitation of bleach: (The ROI should be bleached by at least 50%; the laser power should be adjusted for this). Use full laser power excitation 100% with 100% transmission. Repetitive scanning of the cell surface/ROI after photobleaching is done with an attenuated laser beam. Recovery laser strength is set to 50% power and 3% transmission. 15. Bleaching time “Iterations”: This is the number of times the bleach will occur. This should be done as fast as possible to allow quick monitoring of recovery. It is usually set to ten iterations. Under bleach control ³ bleach parameter: iterations, amount of bleaching: set to 10 which will bleach the ROI within seconds. The duration of bleaching should be kept to the minimum time required for the area in question to be fully bleached; this can be optimized using fixed FRAP. 16. Duration of postbleaching acquisition “Cycle delay”: Under time series ³ stop series: number: 30 or as many images in total you would like to acquire (defines total # of images). You want to see a plateau reached in the recovery phase. Once recovery has reached completion, continue imaging for analysis purposes. Choose at least 30–50 time points during recovery. The more postbleach scans acquired the more information that can be extracted. Time intervals between images can vary. Under time series ³ cycle delay: msec time interval is usually used. However, if you are expecting fast recovery, you can have a time interval of 0 s (no cycle delay) between scans. You can adjust the cycle delay during imaging to change it to have progressively faster or slower time intervals. Make sure to save your format and apply (see Note 11). 17. Find cell and do fine focus with fast x/y : Scan area to find the cell of interest and focus, adjust detector gain and amplifier offset, and take single image; crop image in the window of the image you just acquired; press single again to get updated cropped image. At this point, you may want to readjust the detector gain, amplifier offset, and focus knob on the microscope.

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18. Under bleach control ³ bleach regions window: Use define region under bleach parameter, make circular or rectangular ROIs. Be sure to add to list and save. To ensure that the size of ROIs is consistent (e.g., 20 × 20), look under bleach control window. Uncheck control ROI at this time. 19. Start Bleach (Bleach B): In time series control, press Bleach B. Once the bleaching has started, press mean ROI, which gives a graph of bleaching intensity values. If you wish to change time intervals after bleaching has occurred, look on the graph to see when bleaching has ended, and press pause in time series window – cycle delay. At this point, you can change the time interval (see Note 12). 3.4. After Photobleaching

1. After photobleaching, you can see a gallery of all images or slice-through images in your bleached image acquired window. Press mean ROI; check off the ROI setting that you saved previously, in bleach regions window check back control, to see control values. 2. To view the measurements recorded, see “Show table”. A graph can be imaged, as well as the raw prebleached, bleached, and postbleached values. 3. After imaging is done, take a postbleached image and/or Z stack. 4. To save image with ROI drawn on the image, save using “contents of image”. 5. Save table as a text file. This table can be uploaded into MS Excel. 6. Save all images as TIF and LSM files. Images can be exported as TIF files (contents single: saves one image with circles and graph acquired. Export raw series: saves each single image acquired). Final publication images can then be generated using Adobe Photoshop and Illustrator.

3.5. Data Analysis

See Tables 1 and 2 for a sample data set and analysis. 1. In an excel spreadsheet (see Table 1), copy and paste the time column from your LSM data into column 2. In column 1, label which rows are pre- versus postbleached intensity values (see Note 13). 2. In column 3, copy and paste the photobleached raw intensity values from LSM data, and do the same for the unbleached control raw data values in column 4 (see Note 14). 3. Take the average of the prebleached values in both unbleached control and bleached treated groups. 4. To convert raw data values from LSM to show the percent change in fluorescence intensity for both unbleached control

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Table 1 Example FRAP data set and calculations to normalize data and corrected ratios in percentage Time (s)

Raw values

Raw values

% ROI1

% ROI2

Ratio

Ratio %

Prebleach 1

0

10,000

10,000

  99.90

100.50

0.994005994

  99.40

Prebleach 2

0.99

10,020

  9,900

100.10

  99.50

1.006054552

100.61

Postbleach 1

1.97

  4,500

  9,700

  44.96

  97.49

0.461136801

  46.11

Postbleach 2

2.96

  5,600

  9,600

  55.94

  96.48

0.57983683

  57.98

Postbleach 3

3.94

  5,800

  9,500

  57.94

  95.48

0.606866817

  60.69

Postbleach 4

5.58

  5,900

  9,450

  58.94

  94.97

0.620596335

  62.06

Postbleach 5

6.57

  6,000

  9,420

  59.94

  94.67

0.633124837

  63.31

10,010

  9,950

Average of prebleached

Table 2 Example of FRAP data set and calculation of percent recovery Time (s)

New time (s)

Ratio %

% Change

Total bleach

% Recovery

1.97

0

46.11

 0

53.89

 0

2.96

0.99

57.98

11.87

22.02

3.94

1.97

60.69

14.58

27.05

5.58

3.61

62.06

15.96

29.61

6.57

4.6

63.31

17.2

31.91

and bleached treated groups, and to set the prebleached values as 100%, take the values in columns 3 and 4 and divide by the average you calculated in step 3 and multiply by 100. Do this for all the values and place in column 5 for bleached spot (ROI1%) and column 6 for unbleached control spot (ROI2%). 5. To normalize the values to account for any nonspecific changes in intensity due to factors, such as focus drift or overall photobleaching during postbleach image acquisition, take the ratio of bleached % values in column 5 to unbleached control values in column 6, (column 5/column 6) = ratio, and enter in column 7. 6. In column 8, take the ratio values and express as a percentage (multiply all values in column 7 by 100).

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7. Open a new worksheet to continue the analysis (see Table 2). Column 1 is the recovery time (only postbleach time points); to do this, copy and paste time points excluding the prebleach time points from column 2 in worksheet 1. In column 2, set the first postbleach time point to 0, and calculate subsequent time points by subtracting the original first time point. For example, in column 1, if the time points are 1.97, 2.96, 3.94…, then in column 2, the first value is 0 (1.97–1.97), the second value is 0.99 (2.96–1.97), etc. 8. In column 3, copy and paste corrected ratio % values from column 8 in worksheet 1, excluding prebleach values. 9. To calculate the percentage change from the first postbleach value, in column 4, set the first value to 0 (0% change); for values following, take column 3 values and subtract the first postbleach value in column 3 from all values. 10. To calculate total bleach in column 5, subtract the first postbleach intensity value (the first value in column 3) from 100. 11. Lastly, to calculate percent recovery in column 6, take column 4 values, divide by the value in column 5, and multiply by 100 to give a percentage. 12. Plot recovery of the relative fluorescence intensity in the ROI as a function of time (18). Make a scatter graph recovery curve, with the x axis values being the column 2 time points in worksheet 2, and column 6 as % recovery values in worksheet 2 (see Fig. 1g as an example of a recovery curve). 13. t1/2, also known as the diffusion time, is the amount of time it takes for the ROI to reach 50% of its initial fluorescence intensity. This value can be determined from the diffusion recovery curve (see Fig.  1h for an example of the t1/2 of FcgRI and CD11b) by fitting the curve to an exponential equation using software, such as Microcal Origin 6.0, or by visual inspection; more complex fitting equations can be found in (17). 14. The immobile fraction is the difference between the initial and final fluorescence intensities and is defined as the amount of fluorescence that is retained in the ROI (9). The percentage of fluorescent molecules that are found outside of the ROI that are able to freely diffuse into the ROI (bleached zone) is reflected by the mobile fraction (18). The mobile fraction can be measured by the ratio of fluorescence that occurs in the ROI after full recovery (F∞) with the amount of fluorescence before bleaching (Fi) and right after photobleaching (Fo): R (mobile fraction) = (F∞ − Fo)/(Fi − Fo) (19) (see Note 14). 15. To calculate diffusion coefficient, use the formula: D = w2/4t1/2, where D = diffusion coefficient, w = the radius of the laser beam, and t1/2 = diffusion time (19).

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4. Notes 1. A wide-field or two-photon microscope can also utilize FRAP; however, confocal is the most commonly used system and is the microscope discussed in this protocol. 2. Use a cell line capable of being transiently or stably transfected with a GFP fusion protein. 3. Fab fragments of antibodies can also be used instead of chimeric proteins. For example, for antibody labeling with an FITCconjugated monoclonal antibody, label cells with 30  mg/ml antibody in PBS at room temperature for 10 min. Wash cells several times with PBS and view cells immediately. 4. Use FuGENE HD according to manufacturer’s instructions; other transfection reagents are also suitable, such as Lipo­ fectamine. 5. Fixed FRAP will allow you to (1) set the number of iterations required to bleach the ROI by at least 50%; (2) ensure that fluorescence does not reappear in the ROI after it has been photobleached, as effective photobleaching should be irreversible (allow visualization for at least a couple of minutes); and (3) ensure that the expressed protein is distributed normally within the cell. 6. Slides can be stored in a microscope slide box for several months, but should be viewed as soon as possible. 7. Ensure that no air bubbles are present in the Dako mounting media. Wait for at least 4 h for Dako to harden before imaging cells. 8. Buffering with HEPES maintains appropriate pH in the absence of CO2; media formulated without phenol red reduces background fluorescence. 9. Adjust laser choice according to fluorophore used (e.g., Cy3 or mCherry bleaching: use 543-nm laser). 10. You can set this parameter to the maximum (if cells are not adherent, use large pinhole to compensate for a drift in focus). 11. If a 37°C temperature-controlled microscope stage is unavailable, be sure to view samples within 1 h in room temperature conditions, especially when using antibody labeling. 12. Green bars on graph indicate start and end of bleach; blue bars indicate when the cycle delay interval has been changed. 13. As well, data can be imported into an MS Excel spreadsheet for normalization as described. Other software can also be used to analyze FRAP data, such as ImageJ.

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14. Postbleach values should be decreased by at least 50% from prebleached values. 15. Factors that may alter the mobile fraction include the interaction with other proteins, barriers within the membrane, and various microdomains found within the plasma membrane (13).

Acknowledgments This work was supported by a Canadian Institutes of Health Research (CIHR) grant, MOP-68992. R.E.H. is the recipient of a CIHR New Investigator Award and an Ontario Early Researcher Award. K.S. is a recipient of a Natural Science and Engineering Research Council doctoral scholarship. References 1. Peters, R., Peters, J., Tews, K.H. and Bahr, W. (1974) A microfluorimetric study of translational diffusion in erythrocyte membranes. Biochim Biophys Acta 3, 282–94. 2. Tocanne, J.F., Dupou-Cezanne, L., Lopez, A., and Tournier, J.F. (1989) Lipid lateral diffusion and membrane organization. FEBS Lett 257, 10–16. 3. Phair, R and Misteli, T. (2000) High mobility of proteins in the mammalian cell nucleus. Nature 404, 604–9. 4. Swaminathan, R., Hoang, C., and Verkman, A. (1997) Photochemical properties of green fluorescent protein GFP-S65T in solution and transfected CHO cells: analysis of cytoplasmic viscosity by GFP translational and rotational diffusion. Biophys J 72, 1900–07. 5. Partikian, A., Olveczky, B., Swaminathan, R., Li, Y., and Verkman, A. (1998) Rapid diffusion of green fluorescent protein in the mitochondrial matrix. J Cell Biol 140, 821–9. 6. Cole, N.B., Smith, C.L., Sciaky, N., Terasaki, M., Edidin, M and Lippincott-Schwartz, J. (1996) Diffusional mobility of golgi proteins in membranes of living cells. Science 273, 797–801. 7. Dayel, M.J., Hom, E.F., and Verkman, A.S. (1999) Diffusion of green fluorescent protein in the aqueous-phase lumen of the endoplasmic reticulum. Biophys J 76, 2843–51. 8. White, J and Stelzer, E. (1999) Photobleaching GFP reveals protein dynamics inside live cells. Trends Cell Biol 9, 61–65. 9. Lichtman, J.W. and Conchello, J.A. (2005) Fluorescence microscopy. Nat Methods 2, 910–9.

10. Favier, B., Burroughs, N.J., Wedderburn, L., and Valitutti, S. (2001) TCR dynamics on the surface of living T cells. Int Immunol 13, 1525–32. 11. Poo, H., Krauss, J.C., Mayo-Bond, L., Todd, R.F, and Petty, H.R. (1995) Interaction of Fcg receptor type 3B with complement receptor type 3 in fibroblast transfectants: evidence from lateral diffusion and resonance energy transfer studies. J Mol Biol 247, 597–603. 12. Jongstra-Bilen, J., Harrison, R, and Grinstein, S. (2003) Fcg-receptors Induce Mac-1 (CD11b/ CD18) Mobilization and Accumulation in the Phagocytic Cup for Optimal Phagocytosis. J Biol Chem 278, 45720–9. 13. Zhang F., Yang, B., Odin, J., Shen, Z., Lin, C., Unkeless, J., and Jacobson, K. (1995) Lateral mobility of FcgR2a is reduced by protein kinase C activation. FEBS Lett 376, 77–80. 14. Triantafilou, K., Triantafilou, M., Ladha, S., Mackie, A., Dedrick, R., Fernandez, N., and Cherry, R. (2001) Fluorescence recovery after photobleaching reveals that LPS rapidly transfers from CD14 to hsp70 and hsp90 on the cell membrane. J Cell Sci 114, 2535–45. 15. Manukyan, M., Triantafilou, K., Triantafilou, M., Mackie, A., Nilsen, N., Espevik, T., Wiesmuller, K., Ulmer, A., and Heine, H. (2005) Binding of lipopeptide to CD14 induces physical proximity of CD14, TLR2 and TLR1. Eur J Immunol 35, 911–21. 16. Sprague, B.L. and McNally, J.G. (2005) FRAP analysis of binding: proper and fitting. Trends Cell Biol 15, 84–91.

11  Measuring Immune Receptor Mobility by FRAP 17. Pucadyil, T and Chattopadhyay, A. (2006) Confocal fluorescence recovery after photobleaching of green fluorescent protein in solution. J Fluoresc 16, 87–94. 18. Axelrod, D., Koppel, D., Schlessinger, J., Elson, E., and Webb, W. (1976) Mobility

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­ easurent by analysis of fluorescenece m ­photobleaching recovery kinetics. Biophys J 16, 1055–69. 19. Reits, E., and Neefjes, J. (2001) From fixed to FRAP: measuring protein mobility and activity­ in living cells. Nat Cell Biol 3, 145–7.

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Chapter 12 Probing the Plasma Membrane Structure of Immune Cells Through the Analysis of Membrane Sheets by Electron Microscopy Björn F. Lillemeier and Mark M. Davis Abstract This chapter describes a method to generate plasma membrane sheets that are large enough to visualize the membrane architecture and perform quantitative analyses of protein distributions. This procedure places the sheets on electron microscopy grids, parallel to the imaging plane of the microscope, where they can be characterized by transmission electron microscopy. The basic principle of the technique is that cells are broken open (“ripped”) through mechanical forces applied by the separation of two opposing surfaces sandwiching the cell, with one of the surfaces coated onto an EM grid. The exposed inner membrane surfaces can then be visualized with electron dense stains and specific proteins can be detected with gold conjugated probes. Key words: Plasma membrane, Transmission electron microscopy, Protein distribution

1. Introduction The plasma membrane is the outer border of a cell and physically separates its interior from the surrounding environment. However, the plasma membrane is not an inert shell. Rather, it is utilized in many cellular processes, and therefore its composition and ­structure are of great interest to many researchers. One of the early attempts to describe the plasma membrane was the “fluid mosaic” model by Singer and Nicholson (1). In this model, the plasma membrane is a homogeneous two-dimensional lipid bilayer in which proteins can diffuse freely. Over the last decades, this view has been revised, as it became clear that diffusion in the plasma membrane is slower than expected, molecules are not

Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_12, © Springer Science+Business Media, LLC 2011

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evenly ­distributed over the cell surface, and molecules are confined to membrane domains and/or surface areas with dimensions of less than one micrometer. These findings led to more complex models, including the “lipid raft” (2), “picket fence” (3), and “protein island” models (4). However, the actual organization of the plasma membrane and its associated molecules remains controversial. Many biological processes in immune cells utilize and reorganize the plasma membrane. Most immune cells are activated via cell surface receptors, which in some cases is accompanied by the reorganizations of the plasma membrane, most dramatically seen in the formation of the immunological synapse (5). The systematic functions of immune cells often involve the plasma membrane, e.g., endocytosis, phagocytosis, and secretion. Here, we describe a method based on Sanan et al. (6) that has been successfully used in the analyses of the two-dimensional architecture of the plasma membrane in T cells (4, 7), B cells (8), mast cells (9, 10), and other cell types (6, 11, 12). It is based on the generation of plasma membrane sheets attached to EM grids (Fig.  1), by breaking (“ripping”) cells open through forces applied by separating two opposing surfaces sandwiching the cells of interest (Fig. 2). This procedure has been used with a variety of modifications, each optimized for the study of a particular question. In this chapter, the method is divided into four steps: (1) Surface choices and preparations, (2) Cell binding to primary surface, (3) Generation of plasma membrane sheets, and (4) Labeling of plasma membrane sheets. For each step, up to three possible protocols are shown, which can be “mixed and matched,” and further adapted, to suit any specific study aim.

Fig. 1. (a) Whole plasma membrane sheet from an activated T cell bound to an EM grid coated with stimulatory ligands (peptide-MHC II and co-stimulatory B7.1 molecules). (b) Magnified area of a plasma membrane sheet from a quiescent T cell bound to an EM grid coated with poly-l-lysine. Ectopically expressed myristoylated “non-raft” and myristoylated + palmitoylated “raft” markers are labeled with 5 and 10 nm gold particles, respectively.

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Fig. 2. Simplified three-step illustrations of the “ripping” procedures to obtain plasma membrane sheets. (For simplification, working surface, filter papers and acetate disks are not included in the illustration.) (a) Example for the generation of plasma membrane sheets from the adherent cell side attached to a coated EM grid (primary surface). A PVDF membrane is used as secondary surface. (b) Example for the generation of plasma membrane sheets from the solvent exposed cell side attached to a PLL-coated EM grid (secondary surface). A PLL-coated cover glass is used as primary surface.

2. Materials 2.1. Equipment

1. Carbon vacuum evaporator. 2. Electric glow discharger (e.g., Bench Top Turbo III from Denton Vacuum). 3. Film casting device. 4. Forceps (nonmagnetic) type 7 and N5. 5. Glass beakers (50 ml). 6. Glass plate (at least 10 × 10 cm). 7. Hydrophobic slide marker. 8. Incubator or slide warmer. 9. Rubber cork #3 or #5. 10. Vacuum tip connected to a liquid trap.

2.2. Consumables

1. Acetate disks (0.22 mm pore size; 25 mm diamerter). 2. Acetic acid. 3. Acetone ACS and EM grade. 4. Ashless filter paper. 5. Biotin-N-hydroxysuccinimide ester (NHS-biotin).

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6. Bovine serum albumin (BSA). 7. Coverslips (25 mm diameter). 8. Deionized water. 9. EM grids (hexagonal). 10. Fetal calf serum (FCS). 11. Formvar (1%) in ethylene dichloride. 12. Glass slides (75 × 25 mm). 13. Glutaraldehyde (4%). 14. Glycine. 15. Hank’s balanced saline solution (HBSS). 16. Methanol. 17. Osmium tetroxide. 18. ParafilmM®TM. 19. Paraformaldehyde (16%). 20. Petri dishes. 21. Phosphate-buffered saline (PBS). 22. Poly-l-lysine HBr (PLL) (150–300 kDa). 23. Polyvinylidene fluoride membranes (PVDF). 24. Sodium cacodylate. 25. Streptavidin. 26. Syringes + 0.2 mm filters. 27. Tannic acid. 28. Uranyl acetate. 2.3. Study-Specific Reagents

1. Biotinylated ligands and/or antibodies to adhere cells. 2. Tissue culture media. 3. Stimulatory antibodies/reagents. 4. Primary antibodies/probes (possibly gold conjugated) to detect molecules of interest. 5. Differently sized gold-conjugated secondary antibodies/ streptavidin.

2.4. Buffers

1. HEPES buffer (+Ca/Mg): 20 mM HEPES pH 7.4, 150 mM sodium chloride (+2.5  mM magnesium chloride, 0.5  mM calcium chloride). 2. Cytosol buffer: 20 mM HEPES pH 7.4, 140 mM potassium chloride, 10 mM sodium chloride, 2.5 mM magnesium chloride, 0.5  mM calcium chloride, 50  mM b-mercaptoethanol.

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3. Methods 3.1. Surface Choices and Preparations

3.1.1. Pretreatment of Glass Coverslips for Efficient Coating

The generation of plasma membrane sheets requires the simultaneous interaction of cells with two opposing surfaces (Fig.  2). Therefore, for each study suitable surfaces have to be found or developed. The “primary” surface is initially used to capture and adhere cells as efficiently as possible. In the case of adherent cells, this can be achieved through simply cultivating the cells on ­surfaces. Alternatively, immobilized ligands to surface receptors, or affinity reagents, like antibodies or streptavidin, can capture and adhere cells. The latter is generally faster and occurs with higher affinities. These primary surfaces can also be used to manipulate (activate, polarize, etc.) the cells of interest. The “secondary” surface is added so that the forces necessary to “rip” the cells can be applied, and in most cases, binds the cells nonspecifically, quickly, and only for a short period of time. In the methods described here, the secondary surfaces interact with the cells for 10–20 s, but up to 15 min have been reported (12). Depending on which surface is used to coat the EM grid, plasma membrane sheets originating from the adherent (Fig. 2a) or solvent exposed side (Fig. 2b) can be analyzed while attached to a surface. This section describes the preparation of glass surfaces (Subheading 3.1.1) or EM grids (Subheading 3.1.2), which can then be coated with two different methods (PLL, Subheading 3.1.3 and immobilized proteins, Subheading 3.1.4). Alternatively, polyvinylidene fluoride (PVDF) membranes (Subheading 3.1.5) can be used instead of the above. 1. Round glass coverslips are cleaned by sequentially dipping and gently agitating them in deionized water, 20% acetic acid, deionized water, acetone, and three times deionized water. 2. Place coverslips on top of ashless filter paper inside an autoclavable container, cover with aluminum foil, and dry autoclave for 20 min. 3. Just prior to coating with primary or secondary surfaces, electric glow discharge the top of coverslips for 90 s under air of 50–100 mTorr to negatively charge the surface.

3.1.2. Preparation of EM Grids for Efficient Coating

1. Wash (and if available sonicate) EM grids sequentially in deionized water, 20% acetic acid, three times deionized water, and two times EM grade acetone. Air-dry EM grids and protect from dust (see Notes 1 and 2). 2. Score formvar film (generated on a glass slide with a film ­casting device) with a razor blade, and slowly dip the slide ­perpendicularly into deionized water to float the film onto

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the water surface. Place multiple EM grids with their rough side down on top of the film. 3. Carefully place a clean piece of parafilm on top, sandwiching the EM grids between formvar and parafilm. This is best done by “rolling” the parafilm slowly onto the EM grids, starting on one side of the formvar film and without water running on the top of the parafilm. 4. Place the sandwich with the formvar side up on ashless filter paper, gently remove any trapped air with an ashless filter wedge, and air-dry. 5. Slightly carbon coat EM grids in a vacuum evaporator, and just before coating with the primary or secondary surfaces, glow discharge for 30 s under air of 50–100 mTorr to negatively charge the surface. 3.1.3. Poly-l-Lysine as Primary and/or Secondary Surfaces

PLL surfaces bind cells nonspecifically due to the ion bonds between the positively charged lysines and negative charges on the cell surface. 1. Incubate the glow discharged glass coverslips or EM grids with 0.2  mg/ml PLL in deionized water for 20–30  min at room temperature (RT). EM grids are incubated either by placing them on a glass slide with the parafilm side attached to the glass and placing liquid on top, or by removing them from their parafilm support and inverting them onto droplets (see Note 3). Up to four EM grids can be conveniently incubated on a single droplet. 2. Rinse coverslips or EM grids with deionized water, remove excess water by touching the edge of the EM grid with an ashless filter paper and air-dry them for at least 1 h. EM grids that have been removed from their parafilm support can be washed by inverting and floating them on water in a small Petri dish.

3.1.4. Immobilized Ligands and/or Antibodies as Primary Surfaces

These surfaces bind specific receptors or other molecules on the cell surface and can be used to activate the signaling pathway downstream of a specific receptor. In the case of T cells, specific major histocompatibility complex II molecules together with the co-stimulatory receptor B7.1 have been used successfully (4, 7). In this case, T cell signaling was initiated by the surfaces, and the T cells polarized toward the adhesion site. In different studies, other endogenous ligands or antibodies against surface molecules can be used in similar fashion. 1. Recombinantly expressed ligands or antibodies are purified, and chemically or enzymatically biotinylated. Using site-specific biotinylation on a single amino acid is preferred, as it will orientate the protein on the surface and prohibit functional loss due to high biotin incorporation.

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2. PLL is modified at 10 mg/ml with 50-fold excess of NHSbiotin in 50 mM sodium phosphate pH 7.8 for 2  h at RT. Nonsoluble NHS-biotin is removed by centrifugation. The biotinylated PLL is then filtered through a 0.2  mm cut-off syringe filter and dialyzed against deionized water. (Biotinylated PLL can be stored at -20°C). 3. Glow discharged coverslips or EM grids are incubated with 0.1 mg/ml biotinylated PLL for 20–30 min at RT. 4. Rinse coverslips or EM grids with deionized water, remove excess water by touching the edge of the surface with an ­ashless filter paper, and air-dry them for at least 1 h. 5. Block surfaces with HEPES buffer + 10% FCS or alternatively + 1% BSA for 1  h at RT in a humidity chamber, and wash three times with HEPES buffer. 6. Incubate surface with 20–50 mg/ml streptavidin in HEPES buffer + 0.2% BSA for 1 h at RT in a humidity chamber and wash three times with HEPES buffer. 7. Incubate with a combined concentration of biotinylated ligands and/or antibodies of approximately 400 pmol/ml in HEPES buffer + 0.2% BSA for 1 h at RT in a humidity ­chamber and wash three times with HEPES buffer. This step should be optimized (time and protein concentrations) for different ligands and antibodies. After steps 4, 5, 6, or 7, the surfaces can be stored for up to several days under HEPES buffer, with or without BSA, in a humidity­ box at 4°C. However, some recombinant ligands might not be stable under these conditions for an extended period of time. 3.1.5. Polyvinylidene Fluoride Membranes as Secondary Surface

PVDF membranes have high protein binding capacity and bind well to cells. Due to their flexibility, they are an easy material for the generation of membrane sheets. PVDF membranes do not allow TEM analysis of the plasma membrane attached to them, and are only suitable as secondary surface. 1. PVDF membranes are activated by soaking them in methanol for ~2 min. 2. Wash and equilibrate membranes in HEPES buffer + Ca/Mg for at least 5 min.

3.2. Cell Binding to Primary Surfaces

Plasma membrane sheets are bound to EM grids via the extra­ cellular leaflet and the cytosolic leaflet is exposed to solvent. Thus, they can only be sufficiently labeled with reagents that recognize the cytosolic portion of molecules. Proteins or other markers, fused to intracellular tags (HA-tag, Myc-tag, GFP, etc.), can be ectopically expressed in cells and detected in plasma membrane sheets with the appropriate antibodies. In studies that require the

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detection of extracellular molecules, gold-conjugated probes have to be used prior to the ripping procedure. Live cells can be labeled when bound to the primary surface if a study focuses on the solvent exposed cell side, or alternatively in suspension for either side. However, in this latter case, the label may inhibit binding to the primary surface. Multivalent probes can multimerize their targets and/or induce endocytosis in live cells, which will affect the results of the TEM analyses. In studies, where cells are activated by receptor cross-linking this can be a desired effect. Multimerization and activation can be prevented by fixation of the cells prior to labeling. However, the degree of fixation has to be optimized to avoid multimerization, but still allow binding to the surfaces and ripping of the cell. Here, three possible conditions that have been successfully used to bind cells to primary surfaces are described. 3.2.1. Short-Term Binding of Suspension Cells to Primary Poly-l-Lysine Surface

1. Grow up enough cells to cover 60–80% of the primary ­surfaces. If EM grids are the primary surface, bind them with the attached parafilm proximal to the glass slide and draw a border around them with a hydrophobic slide marker. For the appropriate number of cells, take the additional area ­surrounding the EM grid into account. 2. Wash the cells two times either in tissue culture media without serum or in HBSS. Resuspend the cells in enough media or buffer to cover the primary surface with an appropriate amount of liquid. 3. Apply the cells to the primary surface and place in a tissue culture incubator with humidity control, or on a slide warmer surrounded by moist tissues (to avoid drying of the samples) at 37°C for 30–60 min. The duration has to be optimized for different cell types. 4. Rinse and remove excess cells by sequentially dipping and then gently agitating the surface two times in HEPES buffer + Ca/Mg and continue with the ripping procedure.

3.2.2. Cell Activation and Binding to Immobilized Ligand Surfaces

This procedure is similar to the binding of cells to PLL surfaces (Subheading  3.2.1). However, some adjustments have to be considered. 1. The cell number has to be increased so that 60–80% of the primary surfaces are covered within the time used to activate cells with immobilized ligands. For example, to obtain the same number of T cell membrane sheets on surfaces coated with immobilized ligands in 5 min as on PLL coated surfaces in 30–60 min the T cell concentration has to be tripled. 2. Any necessary supplement for the activation and adhesion of the cells on immobilized ligand surfaces (Calcium, Magnesium, FCS, etc.) has to be added to the media or HBSS.

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3. Before the ripping procedure, protein in the media or buffer has to be removed to increase the binding efficiency to the secondary surface. 3.2.3. Cell Growth on Primary Surface and Labeling of Extracellular Receptors

1. Cells are grown on PLL surface (coverslips or EM grids) or directly on glass coverslips under the same conditions as used in normal cell culture condition. Coverslips can be submerged under media in tissue culture plates with wells ~1.5 times wider than the coverslip itself. Cells are grown on EM grids in a similar fashion as described for the binding to PLL surfaces (Subheading  3.2.1). Alternatively, EM grids can be bound to the bottom of a tissue culture plates or wells via the attached parafilm. 2. Continue with either a or b. (a) Cells that are activated with antibody-gold conjugates against cell surface receptors, and when multimerization is desired, are treated as established for other experimental procedures that use the same ­antibodies for receptor cross-linking of adherent cells (9, 10). (b) Multimerization of the cell surface receptor can be avoided by washing the cells three times in PBS + Ca/Mg, ­followed by fixation in the same buffer with 0.5% paraformaldehyde for 5 min at RT (9, 10). Cells are then labeled with the gold conjugated antibodies as described in step 2a. 3. Rinse and remove excess cells by sequentially dipping and gently agitating the surface two times in HEPES buffer + Ca/Mg and continue with the ripping procedure.

3.3. Generation of Plasma Membrane Sheets

Depending on what surface is used to coat the EM grid, the plasma membrane attached to either the primary surface ­(adherent cell side; Fig. 2a) or secondary surface (solvent exposed cell side; Fig. 2b) can be analyzed. In general, the primary surfaces yield larger, more intact and higher numbers of plasma membrane sheets. Therefore, if the study permits it, the EM grid is ideally coated with the primary surface. The ripping procedure can take place on ice or at RT. If cold conditions are required, place all beakers with buffers in and the ripping surface on compressed ice in a large insulated tray. Under any conditions, excessive amounts of drying that would destroy the membrane sheets or condensation that affects ­buffer concentrations should be avoided (see Note 4). Here, two procedures are described that were ­successfully used to generate plasma membrane sheets from the adherent (Subheading  3.3.1) and the solvent exposed side (Subheading 3.3.2) of T cells.

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3.3.1. Ripping Procedure for the Analysis of Adherent Cell Side (Fig. 2a; Steps 5–14 Are Also Used in Subheading 3.3.2)

1. Place an ashless filter paper on a clean glass surface and equilibrate it thoroughly with HEPES buffer + Ca/Mg. Place an acetate disk on top of filter paper and remove excess buffer with a vacuum tip. 2. Equilibrate EM grids (two can be easily ripped simultaneously) coated with primary surface and bound cells (Subheading 3.2) by dipping and gently agitating it in HEPES buffer + Ca/Mg (see Note 5). 3. Place EM grids with the cell side up on the acetate disk (Fig. 2a, left). Remove any excess liquid carefully with a vacuum tip without drying the surface. 4. Place a PVDF membrane or other secondary surface (Subheading 3.1) equilibrated with HEPES buffer + Ca/Mg and excess liquid removed (by touching the edge of the surface with an ashless filter paper) on top of EM grids (PLL surface can be placed dry on top of cells). Make sure no air is trapped between EM grids and secondary surfaces. 5. Place another acetate disk, previously equilibrated in HEPES buffer + Ca/Mg, and a rubber cork on top of the sandwich (Fig. 2a, middle or Fig. 2b middle for Subheading 3.3.2). 6. Apply vertical pressure to the sandwich for 5–20 s by firmly bearing down with the cork and at the same time remove excess buffer with a vacuum tip. This step requires practice and differs between cell and surface types. If not enough force is applied, the secondary surface does not bind the cells and no ripping occurs; and if too much force is applied, the membrane sheets become damaged. 7. Remove the rubber cork while keeping the rest of the sandwich in place with a pair of forceps. 8. Immerse the whole sandwich thoroughly in cytosol buffer. This will avoid drying of the specimen while the sandwich is disassembled. 9. Remove the top acetate disk with a pair of forceps while keeping the rest of the sandwich in place with another pair of forceps. 10. Lift the secondary (or primary for Subheading 3.3.2) surface with a pair of forceps while pushing down on the bottom acetated disk with another pair of forceps. The EM grids should stick to the surface. If EM grids remain on bottom acetate disk, immerse them quickly in cytosol buffer and continue with step 12. This can happen if too few cells are used or the secondary surface is not binding well enough to the cells. 11. Remove the EM grids vertically from the surface with a fine tipped pair of forceps. If the EM grid accidentally slides along the secondary (or primary for Subheading 3.3.2) surface, the

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cells might rip in a way that results in damaged plasma membrane sheets (Fig.  2a, right or Fig.  2b right for Subheading 3.3.2; see Note 6). 12. Quickly invert and float EM grids on cytosol buffer in a small Petri dish for 30 s to 1 min. This will remove cell debris from the ripping procedure. 13. Float EM grids on cytosol buffer + 4% paraformaldehyde + 0.25% glutaraldehyde for 20 min at RT. Depending on the labeling method, the concentrations of the fixatives and time of fixation have to be optimized. Some antibodies do not recognize their epitopes after stringent fixation (see Note 7). 14. Float EM grids for 10 min in PBS + 25 mM glycine in order to quench excess fixation reagents and continue with labeling of plasma membrane sheets. 3.3.2. Ripping Procedure for the Analysis of Solvent-Exposed Cell Side (Fig. 2b)

1. Place an ashless filter paper on a clean glass surface and equilibrate it thoroughly with HEPES buffer + Ca/Mg. Place an acetate disk on top of filter paper and remove excess buffer with a vacuum tip. 2. Equilibrate EM grid coated with the secondary surface (Subheading 3.1) with HEPES buffer + Ca/Mg and place it on top of the acetate disk with the coated side facing up (two can easily be ripped simultaneously, and PLL-coated EM grids can be placed dry). Remove excess buffer carefully with a vacuum tip. 3. Equilibrate primary surface with bound cells (Subheading 3.2) by dipping and gently agitating it in HEPES buffer + Ca/Mg (Fig. 2b, left; see Note 5). Remove excess liquid by touching the edge of the surface with an ashless filter paper. 4. Place secondary surface with the bound cells facing down on top of the EM grid and continue with steps 5–14 as described in Subheading 3.3.1.

3.4. Labeling of Plasma Membrane Sheets

Multiple membrane-associated molecules of interest can be labeled with different sized gold-conjugated detection reagents (Fig. 1b). However, more than two different sizes can make the identification of the gold species difficult during analyses. Probes specific to certain molecules can be directly conjugated to colloidal gold, thus no secondary label is required. 1. Rinse EM grids with the fixed plasma membrane sheets attached twice by floating on PBS. 2. Invert the EM grids onto droplets (50–100 ml) of the primary staining reagents in PBS + 0.2% BSA and incubate between 1h and overnight at RT in a humidity chamber. Antibodies have been successfully used in concentrations of 10–100 mg/ml;

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­ owever, this has to be optimized for every probe. If multiple h probes are used, they should be applied as a mixture to ­minimize effects due to steric inhibition of binding (see Note 7). 3. Rinse EM grids three times for 5 min at RT by floating them on PBS. 4. Invert EM grids onto droplets (50–100  ml) of mixtures of gold-conjugated secondary probes in PBS + 0.2% BSA for 1–3 h at RT in a humidity chamber. Gold reagents are mostly stored at concentrations of an OD520nm of ~1.0 and are used in 5- to 20-fold dilutions. 5. Rinse EM grids three times for 5 min at RT by floating them on PBS. 6. Postfix EM grids on a droplet of 2% glutaraldehyde in PBS for 10 min at RT in a humidity chamber. 7. Rinse EM grids sequentially in PBS + 25  mM glycine, PBS, and 100 mM cacodylate for 5 min at RT. 8. Incubate grids on droplets of 100  mM cacodylate + 1% osmium tetroxide for 10 min at RT in a humidity chamber under a chemical hood. Rinse one time on 100 mM cacodylate and twice on deionized water for 5 min each time at RT. 9. Incubate grids on droplets of 1% tannic acid (freshly filtered) for 10 min at RT in a humidity chamber and rinse three times on deionized water for 5 min at RT. 10. Incubated grids on droplets of 1% uranyl acetate (freshly filtered) for 10 min at RT in a humidity chamber and rinse for 30 s to 1 min on deionized water. 11. Remove excess liquid by touching the edge of the EM grid with an ashless filter paper, air-dry for ~15  min on ashless ­filter paper with the coated side facing up, and store at RT for analysis by TEM. The specimens are imaged using a transmission electron microscope (TEM), and the gold distribution in the resulting images can be analyzed for clustering, sizes of clusters, co-localization, and other parameters by a multitude of statistical methods (e.g., (13, 14)).

4. Notes 1. EM grids are best handled with nonmagnetic, fine tipped and curved type 7 forceps, and coverslips are most conveniently manipulated with reverse action type N5 forceps. 2. The choice of EM grids is crucial for the preparation of plasma membrane sheets. The EM grid should distribute the force

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during the attachment of the secondary surface equally onto the carbon-coated formvar sheet. Hexagonal nickel EM grids have wide metal bands forming the mesh, which is ideal for this procedure. EM grids made from wire cut the formvar/ carbon sheet and are not suited for this procedure. 3. Due to surface tension of liquid trapped between the two tines, EM grids easily become attached to the surface of forceps during their release. Therefore, when removing liquid by touching the edge of the EM grid with an ashless filter paper, touch the gap between the tines simultaneously, which will remove any liquid between them. When floating EM grids on liquid, make sure that the EM grid is in contact with liquid surface during the opening of the tines, which will ensure that it floats onto the liquid. 4. The ripping conditions can be optimized by TEM analyses of plasma membrane sheets labeled with the electron dense stains only or by the analyses of cells labeled with a fluorescent membrane marker (e.g., DiO, DiI, or DiD) on an inverted fluorescence microscope. For the latter, submerge the EM grid with the plasma membrane sheet side down in a microscopy chamber. 5. The efficiency of the ripping procedure can be increased for some cells, by incubating them in hypotonic buffer (25–75 mM salt) for a short period of time prior and during the ripping procedure. However, this potentially induces changes in the cell morphology and may activate stress-related responses. 6. If PVDF membranes are used as secondary surfaces, it is easier to pick the EM grids with forceps, when the membrane is bend between two fingers with the EM grid on the outside of the arch. If glass coverslips are used, place them on top of the EM grids with the edge of the EM grid slightly extended past the surface of the coverslip. This makes the removal of the EM grids from the coverslips easier. 7. Fixation and binding condition for the labeling can be ­optimized by preliminary experiments using fluorescenceactivated cell sorting (FACS). This enables many conditions to be examined quickly.

Acknowledgments The authors thank Dr. Bridget S. Wilson for advice on TEM and plasma membrane sheet preparation, and Dr. Fleur E. Tynan for comments on the manuscript.

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References 1. Singer SJ, Nicolson GL. (1972) The fluid mosaic model of the structure of cell membranes. Science 175 (23):720–31. 2. van Meer G, Simons K. (1982) Viruses budding from either the apical or the basolateral plasma membrane domain of MDCK cells have unique phospholipid compositions. Embo J 1 (7):847–52. 3. Sako Y, Kusumi A. (1994) Compartmentalized structure of the plasma membrane for receptor movements as revealed by a nanometerlevel motion analysis. J Cell Biol 125 (6):1251–64. 4. Lillemeier BF, Pfeiffer JR, Surviladze Z, Wilson BS, Davis MM. (2006) Plasma membraneassociated proteins are clustered into islands attached to the cytoskeleton. Proc Natl Acad Sci USA 103 (50):18992–7. 5. Monks CR, Freiberg BA, Kupfer H, Sciaky N, Kupfer A. (1998) Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395 (6697):82–6. 6. Sanan DA, Anderson RG. (1991) Simultaneous visualization of LDL receptor distribution and clathrin lattices on membranes torn from the upper surface of cultured cells. J Histochem Cytochem 39 (8):1017–24. 7. Lillemeier BF, Mortelmaier MA, Forstner MB, Huppa JB, Groves JT, Davis MM. (2010) TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation. Nat Immunol 11 (1):90–6.

8. Kim JH, Cramer L, Mueller H, Wilson B, Vilen BJ. (2005) Independent Trafficking of Ig-{alpha}/Ig-{beta} and {micro}-Heavy Chain Is Facilitated by Dissociation of the B Cell Antigen Receptor Complex. J Immunol 175 (1):147–54. 9. Wilson BS, Pfeiffer JR, Oliver JM. (2000) Observing FcepsilonRI signaling from the inside of the mast cell membrane. J Cell Biol 149 (5):1131–42. 10. Wilson BS, Pfeiffer JR, Surviladze Z, Gaudet EA, Oliver JM. (2001) High resolution mapping of mast cell membranes reveals primary and secondary domains of Fc(epsilon)RI and LAT. J Cell Biol 154 (3):645–58. 11. Prior IA, Muncke C, Parton RG, Hancock JF. (2003) Direct visualization of Ras proteins in spatially distinct cell surface microdomains. J Cell Biol 160 (2):165–70. 12. Morone N, Fujiwara T, Murase K, et al. (2006) Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography. J Cell Biol 174 (6):851–62. 13. Ripley BD. (1977) Modeling spatial patterns. J. R. Stat. Soc. B39:172–212. 14. Zhang J, Leiderman K, Pfeiffer JR, Wilson BS, Oliver JM, Steinberg SL. (2006) Characterizing the topography of membrane receptors and signaling molecules from spatial patterns obtained using nanometer-scale electron-dense probes and electron microscopy. Micron 37 (1):14–34.

Chapter 13 Rapamycin-Based Inducible Translocation Systems for Studying Phagocytosis Michal Bohdanowicz and Gregory D. Fairn Abstract Phagocytosis is an immune receptor-mediated process whereby cells engulf large particles. The process is dynamic and requires several localized factors acting in concert with and sequentially after the engagement of immune receptors to envelope the particle. Once the particle is internalized, the nascent ­phagosome undergoes a series of events leading to its maturation to the microbicidal phagolysosome. Investigating these dynamic and temporally controlled series of events in live cells requires noninvasive methods. The ability to rapidly recruit the proteins of interest to the sites of phagocytosis or to nascent phagosomes would help dissect the regulatory mechanisms involved during phagocytosis. Here, we describe a general approach to express in RAW264.7 murine macrophages, a genetically encoded rapamycin­-induced heterodimerization system. In the presence of rapamycin, tight association between FK506-binding protein (FKBP) and FKBP rapamycin-binding protein (FRB) is observed. Based on this principle, a synthetic system consisting of a targeting domain attached to FKBP can recruit a protein of interest fused to FRB upon the addition of rapamycin. Previously, this technique has been used to target lipid-modifying enzymes and small GTPases to the phagosome or plasma membrane. The recruitment of the FRB module can be monitored by fluorescent microscopy if a fluorescent protein is fused to the FRB sequence. While the focus of this chapter is on phagocytic events, this method can be employed to study any organelle of interest when the appropriate targeting sequence is used. Key words: Phagocytosis, Macrophage, Rapamycin and Heterodimerization

1. Introduction Phagocytosis is a complex process utilized by specialized cells, such as macrophages, neutrophils, and dendritic cells, to ingest target particles >0.5 mm (1). By clearing pathogens and apoptotic bodies, phagocytosis plays an essential role in development and immunity (2). Regardless of the nature of the particle, the process

Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_13, © Springer Science+Business Media, LLC 2011

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of phagocytosis consists of two basic steps: phagosome formation and phagosome maturation. During phagosome formation, the plasma membrane forms projections called pseudopods that crawl along the surface of a recognized particle. Once the pseudopods surround the particle, they fuse and generate an intracellular vacuole or phagosome. Phagosome formation is a highly orchestrated event, which remodels the cytoskeleton, evokes lipid signaling, and appropriates membrane from the exocytic/­ recycling compartments. The newly formed phagosome undergoes a series of fusion events with endosomes and lysosomes leading to its maturation. The resulting phagolysosome is more acidic than the nascent phagosome and has increased degradative and microbicidal capacities (3, 4). Phagosome formation is a receptor-mediated event, in which immune receptors recognize either intrinsic surface features of a particle or extrinsic molecules, such as immunoglobulins, coating the particle (5). Receptor engagement activates tyrosine kinases, which recruit adaptor molecules, activate small GTPases of the Rho family, trigger lipid signaling, and remodel filamentous actin (5). Actin dynamics are closely linked to the phospholipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5P)2), and to the small GTPases, Rac1 and Cdc42 (6). PtdIns(4,5)P2 is a constitutive component of the plasma membrane that becomes enriched at the base of the phagosomal cup. Later on, PtdIns(4,5)P2 also accumulates at the leading edge of the pseudopods, where it supports the actin polymerization that drives the extension of the pseudopods. Just prior to phagosome sealing, the PtdIns(4,5)P2 disappears from the base of the cup with a concomitant disappearance of polymerized actin (6, 7). Investigating phagocytosis or other dynamic processes has presented several obstacles to researchers. The cytoskeletal rearrangements, GTPase activation, and lipid metabolism during phagocytosis are transient in nature and localized to a small portion of the cell’s total plasma membrane. This hinders classical biochemical approaches involving fixation or subcellular fractionation from elucidating the underlying mechanism. Progress has been made in recent years with the advent of live cell imaging. To exploit this new technology, several plasmid-based biosensors have been developed to detect a variety of phospholipids via fluorescent microscopy. These biosensors are chimeric constructs consisting of a fluorescent protein, such as green fluorescent protein (GFP), fused with a specific lipid-binding domain (8). These lipid probes have allowed researchers to monitor lipid dynamics. Similar probes have been developed to visualize the polymerization of actin or the activation of Cdc42 and Rac1 (9, 10). Such a probe can describe the physical and temporal localization of the molecule of interest, but to understand how the visualized molecule is regulated, the probe is used in combination with functional

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assays, such as pharmacology, gene knockdown, or dominant negative mutants. These techniques can have various limitations, including nonspecific activity, slow onset of action, and compensation by nontarget proteins. For example, the phosphatidylinositol 4-phosphate 5-kinase family of enzymes are responsible for producing the majority of PtdIns(4,5)P2, and over-expression of any of these enzymes can inhibit phagocytosis (11). These results suggest that the increased (or untimely) production of PtdIns(4,5) P2 prevents proper actin dynamics and pseudopod extension. As a result, it has been difficult to examine the roles of different isoforms of the phosphatidylinositol 4-phosphate 5-kinases in phagocytosis. Recent results suggest that the phosphatidylinositol 4-phosphate 5-kinases are displaced from the forming phagosome and that inducible recruitment of the kinases can inhibit phagocytosis (12). This and other instances have highlighted the utility of having a controllable system to recruit/remove or activate/ inactivate proteins. Here, we describe a general method for controllable recruitment to aid in the dissection of temporally sensitive signaling events. The method was first developed to investigate the ability of constitutively active Rac1 and Cdc42 to induce phagocytosis in the absence of receptor signaling (13, 14). The method takes advantage of a cell-permeable compound, rapamycin, to induce protein dimerization. Rapamycin binds with high affinity to intracellular proteins of the FK506-binding protein (FKBP) family. One such protein, FKBP12, when bound to rapamycin interacts with the protein kinase FRAP. The region of FRAP binds to FKBP12-rapamycin through a 11-kDa domain referred to as FRB. Native Rac1 and Cdc42 are peripheral membrane proteins that are posttranslationally modified by the addition of lipid molecules to the C-terminal region. Replacement of this C-terminal region with the FRB domain generates a soluble protein that upon the addition of rapamycin interacts with FKBP12. The generation of chimeric FKBP12 proteins allows for the recruitment of the Cdc42-FRB to the desired location. Such a strategy has been used to target proteins to the plasma membrane via the N-terminal 11 amino acids of Lyn kinase (15), to the mitochondria by using the N-terminal region of Tom70 (16), and to the late Golgi via the Golgi localization domain of sialyl-transferase (17). The basis of this controllable system is the generation and expression of the constructs of interest. Transfection of the cells with plasmid DNA is essential for the process. Primary macrophage is difficult to transfect; therefore, we routinely use a murine macrophage cell line RAW264.7 when studying phagocytosis. Described below is a method for controllable recruitment and removal of proteins from a phagosome during the uptake of IgG-coated red blood cells (Fig. 1).

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Fig. 1. Rapamycin-induced heterodimerization system and examples of its application. (a) One protein containing an FKBP domain (FKBP12) and another protein containing an FRB domain (mTOR) combine to form a tripartite complex with the addition of rapamycin, a natural macrolide which has binding sites for both domains. The resulting complex is essentially irreversible. These domains can be fused with the existing probes/effectors and targeting sequences to create novel rapamycin heterodimerization systems. (b) Cells were transfected with plasmids encoding the Tom70-FKBP and FRBGFP-3E constructs. At time = 0, the FRB-GFP-3E is seen on both the plasma membrane and phagosome (arrow ).

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2. Materials 2.1. Cell Culture

1. RAW264.7 murine macrophage cell line (ATCC, Manassas, VA). 2. 1× Dulbecco’s modified Eagle’s medium (DMEM) (Wisent, Mississauga, ON, Canada), supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Wisent). 3. 1× solution RPMI 1640 buffered with 25 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) (Sigma-Aldrich). 4. Sterile 1× phosphate-buffered saline (PBS). 5. 0.05% trypsin solution containing 0.53  mM ­ethylenediaminetetra-acetic acid (EDTA) buffered with sodium bicarbonate (Wisent). 6. T-25 tissue culture flasks (Sarstedt). 7. Tissue culture plates with 6 or 12 wells (Becton Dickinson). 8. Round glass coverslips (18-mm diameter for 12-well plates or 25 mm for 6-well plates) (Fisher Scientific). 9. Humidified tissue culture incubator at 37°C with 5% CO2.

2.2. Transfection

1. Fugene HD transfection reagent (Roche). 2. Serum-free DMEM (Wisent).

2.3. Plasmid Isolation

1. Bacterial stock (typically E. coli) transformed with the plasmid of interest. 2. Sterile Luria-Bertani (LB) bacteriological medium: Tryptone 10 g/L, NaCl, 5 g/L, and yeast extract 5 g/L (BioShop). 3. Antibiotic stocks for plasmid selection (ampicillin 100 mg/mL, kanamycin 10  mg/mL, or chloramphenicol 34  mg/mL) (Sigma-Aldrich). 4. High-Speed plasmid Maxi kit (QIAGEN).

2.4. Phagocytosis Assays

1. Sheep red blood cells (MP Biomedicals, Solon, OH) supplied as a 10% suspension. 2. Rabbit anti-sheep red blood cell IgG (MP Biomedicals, Solon, OH) supplied at 40 mg/mL.

Fig. 1. (continued) Sixty seconds post addition of rapamycin, the FRB-GFP-3E is no longer on the plasma membrane or phagosome (arrow), but has been recruited to the mitochondria. (c) Cells were transfected with plasmids encoding Lyn11FRB and FKBP-YFP-PIP5K. At time = 0, the FKBP-YFP-PIP5K is largely cytosolic while 120 s after the addition of rapamycin, the FKBP-YFP-PIP5K is recruited to the phagosome and plasma membrane. (d) Phagocytic index of cells expressing FKBP-YFP-PIP5K and Lyn11-FRB. Rapamycin was added prior to the initiation of phagocytosis, and the recruitment of the FKBPYFP-PIP5K inhibits the internalization of IgG-opsonized sheep red blood cells.

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3. DyLight 488-, DyLight 594-, and DyLight 649-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). 4. Rapamycin (Sigma-Aldrich). 5. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich). 6. Fixation solution: 4% paraformaldehyde in PBS (made from 16% parafomaldehyde stock (Electron Microscopy Sciences). 2.5. Microscopy

1. A spinning-disc confocal microscope equipped with a 63× or 100× magnifying objective, a light source and filter set that is appropriate for the fluorescent protein of choice, and an objective heater. The system in our laboratory consists of a Zeiss Axiovert 200  M inverted microscope (Carl Zeiss) equipped with diode-pumped solid-state laser (Spectral Applied Research, Richmond Hill, ON, Canada) providing lines 440, 491, 561, 638, and 655  nm, a motorized X − Y stage (API, WA) and a piezo focus drive (Quorum Technologies, Guelph, ON, Canada). Images on this system are captured using a back-thinned EM-CCD camera (Hamamatsu) controlled by the software Volocity version 4.1.1 (Improvision Inc., Waltham, MA). 2. Live-cell imaging chamber for 18 or 25  mm coverslips (Invitrogen). 3. Digital temperature regulator with a heated P insert (PeCon, Germany). 4. Image analysis software, such as Volocity or ImageJ (http:// rsb.info.nih.gov/ij/).

3. Methods 3.1. Cell Culture

1. RAW264.7 macrophages are routinely cultured in 10 mL of DMEM supplemented with FBS (5% v/v) at 37°C and 5% CO2 in a T-25 tissue culture flask. Once the macrophage reach ~80–90% confluence, they are trypsinized and used to seed tissue culture plates (6- or 12-well) containing sterile glass coverslips (see Note 1).

3.2. Transfection of Cells

1. Prior to trypsinizing the macrophages, add to a 12-well tissue culture plate sterile 18  mm coverslips using sterile tweezers. Add 1 mL of DMEM + FBS to each well. If using a 6-well tissue culture plate, use 25 mm coverslip and 2 mL of medium. 2. Use RAW264.7 macrophages in a T-25 flask that have reached 80–90% confluency and wash once with 10 mL of sterile prewarmed PBS. Aspirate off the PBS gently.

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3. Add 1  mL of warmed (37°C) 0.05% trypsin/ 0.053  mM EDTA to the T-25 flask. Gently rock the flask to ensure that the trypsin solution is evenly over the cells. Incubate for 2–5 min while observing for detachment of the macrophage cells. 4. When the cells begin to detach from the bottom of the flask, use a sterile rubber scraper to detach the remaining cells. 5. Add 9 mL of warmed DMEM + FBS to the cell suspension. This medium contains a trypsin inhibitor and sufficient amounts of divalent cations to prevent further proteolysis. Gently pipette up and down to disperse any clumped cells. 6. To the 12-well plate containing coverslips (steps 1 and 2), add one drop of cell suspension from step 5. 7. Incubate the plate, now containing RAW cells seeded on coverslips, overnight in a tissue culture incubator at 37°C with 5% CO2. 8. Day 2 – Warm serum-free DMEM to room temperature. 9. Transfections are performed as described in the Fugene HD product literature (Roche). Typically, 2 mg of plasmid DNA and 6 mL of Fugene HD are used. This is sufficient to transfect 2 wells of the 12-well plate. When multiple plasmids are to be cotransfected, the combined amount of DNA should be 2–3 mg. For the rapamycin heterodimerization experiments, we routinely cotransfect with three plasmids, in which case the total plasmid DNA used is 3 mg (see Notes 2 and 3). 10. The Fugene HD–DNA is allowed to complex by incubating at room temperature for 15 min. 11. 500  mL of DMEM + 5% FBS is added to the DNA–Fugene complex. Half of this solution is added to each of two wells. 12. Cells are returned to the cell culture incubator and incubated overnight. There is no need to remove this specific transfection reagent. 3.3. Sheep Red Blood Cell Opsonization

1. This can be done 60–90  min before the initiation of the experiment. Draw 200 mL of red blood cells from the bottle using a 1-mL syringe with a 23½ gauge needle (see Note 4). 2. Spin at 6,000×g for15 s in a microfuge, aspirate supernatant, and resuspend by pipettting in 1 mL of PBS. 3. Spin at 6,000×g for15 s in a microfuge, aspirate supernatant, and resuspend by pipettting in 200 mL of PBS. 4. To the red blood cell suspension, add 4  mL of rabbit antisheep IgG. 5. Incubate for 1 h at 37°C on a rotator.

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6. The IgG-opsonized red blood cells are washed three times with PBS as before. Resuspend the cells in 200 mL of PBS. 7. 25  mL of the IgG-opsonized red blood cell suspension is added per well of a 12-well plate. 3.4. Phagocytosis and Image Acquisition

1. Turn on the spinning disc confocal microscope and light source. 2. Turn on the digital heater to warm the objective and the P insert. These must be allowed to equilibrate to 37°C prior to acquiring images (or movies) of cells undergoing phagocytosis. 3. Setup the experimental acquisition parameters for the experiment using the Volocity software. 4. Warm an aliquot of HPMI prior to beginning the experiment. 5. Using forceps, remove a coverslip from one of the wells containing transfected cells and transfer to a fresh plate. To this well, add 1 mL of cold (4°C) HPMI and keep the plate on ice. 6. Next, add 25 mL of IgG-opsonized red blood cell suspension to this well. To synchronize phagocytosis, the plate is transferred to a bench-top centrifuge fitted with a rotor for tissue culture plates and spun at 250 × g for 1 min. 7. Transfer the coverslip to a live-cell imaging chamber and add 500 mL of cold HPMI to the chamber. 8. Quickly transfer the live-cell imaging chamber to the microscope and focus on a region of interest. The focal plane should be set so that imaging is through the middle of the cell. If desired, Z-stacks can also be acquired; however, excess imaging can lead to photobleaching. 9. Once the cell or region of interest is in focus, the cold HPMI should be aspirated and fresh, warm HPMI should be added to the chamber. After the addition of the warmed medium, phagocytosis will begin almost immediately and image acquisition should commence. Images can be taken manually over time or an image acquisition protocol can be set up for a time-lapse series of images (see Note 5). 10. If the experiment involves phagosome formation, 1 mL of a 10 mM stock solution rapamycin can be added immediately to induce the recruitment of the chimeric protein of interest to the plasma membrane/forming phagosome. If the experiment involves studying an aspect of phagosome maturation, the rapamycin can be added 3 to 5  min post initiation of phagocytosis (see Notes 6–9).

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1. Warm an aliquot of HPMI to 37°C prior to beginning the experiment. 2. Aspirate the media from each of the wells of the 12-well plate. Replace with cold (4°C) HPMI. 3. Next, add 25 mL of IgG-opsonized red blood cell suspension to this well. To synchronize phagocytosis, the plate is transferred to a bench-top centrifuge fitted with a rotor for tissue culture plates and spun at 250 × g for 1 min. 4. Replace the cold HPMI with the prewarmed (37°C) HPMI. 5. Add 1 mL from a 10 mM stock of rapamycin or solvent control (DMSO) to each well. 6. Incubate at 37°C for 5–10 min. 7. Aspirate off the HPMI and add 1 mL of cold PBS. Keep the plate on ice. 8. The uninternalized IgG-opsonized red blood cells can be stained using 1 mL of DyLight 649-coupled donkey anti-rabbit IgG/well with incubation for 2 min at room temperature. 9. Wash each well twice with 1 mL of cold PBS and aspirate off the medium. 10. Add 1 mL of 4% formaldehyde to each well and incubate on ice for 30 min. 11. Aspirate formaldehyde solution and wash the cells with 1 mL of PBS supplemented with 0.1 M glycine. 12. If so desired, fixed cells can be permeabilized by adding 1 mL 0.1% triton X-100 in PBS and incubating at room temperature for 1 h. All red blood cells (internalized and external) can then be stained with either DyLight 488- or DyLight 594-conjugated donkey anti-rabbit IgG. 13. Coverslips can be transferred to a live-cell imaging chamber and imaged using the microscope. 14. In cases where the protein of interest is tagged with GFP, external beads can be labeled with the DyLight-594 and total beads with DyLight-649. 15. Count the total number of beads internalized (DyLight-649 positive, DyLight-594 negative) for between 100 and 500 cells.

4. Notes 1. Low-passage subconfluent RAW cells tend to transfect in higher numbers and with higher expression levels than high-passage RAW cells. This is especially important when trying to express multiple constructs, like the rapamycin heterodimerization

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system, because the transfection efficiency decreases as the number of constructs increases. 2. We try to transfect more of the targeting domain and less of the soluble domain because at high levels, the soluble domain may start to mislocalize before rapamycin is added and it may fully saturate the targeting domain once rapamycin is added, leading to an excess of unheterodimerized soluble domain. 3. The method relies on the generation of chimeric proteins. Due to steric or other geometric limitations, not all chimeric proteins generated remain functional. 4. An alternative to sheep red blood cells, polystyrene beads can be used as a target particle. One advantage is that they are available in a variety of sizes from 0.5 to 8.3 mm. When using polystyrene beads, it is important to use beads containing 2% DVB. Beads without DVB do not opsonize efficiently with IgG. 5. For time-lapse imaging of synchronized phagocytosis, we typically capture one frame every 5–15 s for 5–10 min. 6. The rapamycin system has a significant drawback in that it is only slightly reversible, thus only one experiment can be done per coverslip and a return of function/loss of function with dissociation of the rapamycin complex cannot be studied. 7. Ensure that the coverslip holder is washed well after every experiment. The heterodimerization system is sensitive to even minute levels of rapamycin, thus the coverslip holder should be washed in warm, soapy water, scrubbed with 70% ethanol, and rinsed with Millipore water in between every experiment. 8. The rate of the heterodimerization reaction depends on how much rapamycin is added. However, cells may bleb if too much is added or if it is not mixed in well. 9. Rapamycin inhibits FRB-containing proteins, like mTOR, and induces autophagy. These secondary effects can be controlled by using an empty rapamycin-binding domain as a negative control or they can be avoided by using the rapamycin analogue, AP21967 (ARIAD Pharmaceuticals), a heterodimerizing agent that selectively interacts with mutant FRB and not wild-type FRB.

Acknowledgments The authors would like to thank Dr. Sergio Grinstein (The Hospital for Sick Children) for guidance during the preparation of this manuscript. M.B. holds a Canadian Institutes of Health Research (CIHR) MD/PhD studentship and a McLaughlin fellowship. GDF holds a postdoctoral fellowship from CIHR.

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References 1. Swanson J.A. (2008) Shaping cups into ­phagosomes and macropinosomes. Nat Rev Mol Cell Biol 9,639–49. 2. Huynh K.K., Kay J.G., Stow J.L., and Grinstein, S. (2007) Fusion, fission, and secretion during phagocytosis. Physiology 22, 366–72. 3. Underhill D.M. (2005) Phagosome maturation: steady as she goes. Immunity 23, 343–4. 4. Kinchen J.M. and Ravichandran K.S. (2008) Phagosome maturation: going through the acid test. Nat Rev Mol Cell Biol 9,781–95. 5. Groves E., Dart A.E., Covarelli, V. and Caron, E. (2008) Molecular mechanisms of phagocytic uptake in mammalian cells. Cell Mol Life Sci 65,1957–76. 6. Yeung, T. and Grinstein, S. (2007) Lipid ­signaling and the modulation of surface charge during phagocytosis. Immunol Rev 219,17–36. 7. Botelho R.J., Teruel, M., Dierckman, R., et  al   (2000) Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis. J Cell Biol 151,1353–68. 8. Varnai, P. and Balla, T. (2007). Visualization and manipulation of phosphoinositide dynamics in live cells using engineered protein domains. Pflugers Arch 455, 69–82. 9. Kraynov, V.S., Chamberlain, C., Bokoch, G.M., Schwartz, M.A., Slabaugh, S., and Hahn, K.M. (2000) Localized Rac activation dynamics visualized in living cells. Science 290,333–7.

10. Riedl, J., Crevenna, A.H., Kessenbrock, K., et al (2008) Lifeact: a versatile marker to visualize F-actin. Nat Methods, 5, 605–7. 11. Scott, C.C., Dobson W., Botelho, R.J., et  al (2005) Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis. J Cell Biol 169, 139–49. 12. Fairn, G.D., Ogata, K., Botelho, R.J., et  al. (2009) An electrostatic switch displaces phosphatidylinositol phosphate kinases from the membrane during phagocytosis. J Cell Biol 187,701–14. 13. Castellano, F. and Chavrier, P. (2000) Inducible membrane recruitment of small GTP-binding proteins by rapamycin-based system in living cells. Methods Enzymol 325,285–95. 14. Castellano, F., Montcourrier, P., and Chavrier, P. (2000) Membrane recruitment of Rac1 triggers phagocytosis. J Cell Sci 113, 2955–61. 15. Inoue, T., Heo, W.D., Grimley, J.S., Wandless, T.J., and Meyer, T. (2005) An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways. Nat Methods 2, 415–8. 16. Silvius, J.R., Bhagatji, P., Leventis, R., and Terrone, D (2006). K-ras4B and prenylated proteins lacking “second signals” associate dynamically with cellular membranes. Mol Biol Cell 17, 192–202. 17. Pecot, M.Y. and Malhotra, V. (2006) The Golgi apparatus maintains its organization independent of the endoplasmic reticulum. Mol Biol Cell 17, 5372–80.

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Chapter 14 Micropatterned Ligand Arrays to Study Spatial Regulation in Fc Receptor Signaling Alexis J. Torres, David Holowka, and Barbara A. Baird Abstract Fc receptor signaling plays a fundamental role in immune responses. A plethora of Fc ­receptors (e.g., Fc gamma, Fc-alpha, and Fc-epsilon) are expressed on different immune cells, including natural killer cells, macrophages, mast cells, and neutrophils. Receptor clustering and activation by multivalent ligands or opsonized particles induce a signaling cascade that leads to targeted secretion of chemical mediators (i.e., histamine, cytokines, and chemokines) and phagocytosis, among other responses. Spatial targeting and compartmentalization are common mechanisms of regulation in Fc receptor signaling. However, the tools for studying these dynamic interactions have been limited. To overcome these limitations in our model system, microfabricated surfaces containing spatially defined ligands are used to cluster­ and activate IgE receptors (FceRI), involved in allergic responses by mast cells. Micron-scale control of cell activation allows investigation of spatially regulated mechanisms for intracellular signaling with ­fluorescence microscopy. This approach in conjunction with biochemical techniques has proven to be valuable for investigating immune receptor signaling. Key words: IgE receptors, Supported membranes, Microfabrication, Photolithography, Immune response, Mast cell, Fluorescence microscopy, Cytoskeleton

1. Introduction Cellular receptors for immunoglobulins (Fc receptors) are widely expressed on the surface of highly specialized cells that form part of the innate and adaptive immune system (1, 2). These cells are sensitized by the binding of antibodies produced in response to foreign molecules. Receptor activation elicits different cellular responses that depend mainly on the cell and receptor type but also on the presence of other co-stimulatory signals (3). Such responses include the release of preformed mediators, de novo protein synthesis, phospholipid metabolism, changes in cell morphology,

Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_14, © Springer Science+Business Media, LLC 2011

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endocytosis­, and phagocytosis. One important class of Fc receptor is the ­receptor for IgE (FceRI), which is responsible for the release of chemical mediators that cause inflammatory and allergic reactions (4), and is found primarily on mast cells and basophils. Cross-linking of IgE-FceRI by multivalent antigen (ligand) triggers intracellular signaling events, leading to multiple cellular responses. In the earliest signaling events, antigen-induced clustering of IgE-FceRI causes stable association with ordered lipid domains, cytoskeletal redistribution, and Lyn kinase phosphorylation of the FceRI b and g subunits (5). The latter initiates a series of signaling events that induces Ca2+ mobilization and other downstream signaling steps leading to degranulation and secretion of preformed mediators (6). Elucidation of signaling pathways involved in receptor signaling is crucial for the development of therapeutics and prevention of immune diseases. The current understanding of FceRI signaling has been mostly dependent upon biochemical and genetic characterization of protein interactions. Spatial orchestration of these signaling events is a critical aspect, but studies have been limited by these common approaches. We have recently established the use of microfabricated surfaces containing spatially defined ligands to investigate spatial regulation of cell signaling (7, 8). A recent review discussing the applications of micro and nanofabrication in receptor signaling and cell biology has been published elsewhere (9). Herein, we present a detailed description of an approach we developed to investigate the spatial regulation in FceRI signaling on RBL-2H3 cells by microfabricated surfaces using the polymer lift-off method and 2,4-dinitrophenyl (DNP) ligands. It should be noted that even though the procedure described here is specific for the IgE receptor system, the same principles are applicable to other receptor systems and can be adapted by selecting the appropriate ligands and surface functionalization schemes.

2. Materials 2.1. Microfabrication

1. Parylene C dimer (Speciality Coating System, Indianapolis, IN). 2. Parylene Deposition System PDS 2010 LABCOTER (Speciality Coating System, Indianapolis, IN). 3. Computer-Assisted Design (CAD) software. 4. Cleaning solution (piranha etch ) composed of a freshly ­prepared mixture of concentrated sulfuric acid and 30% (v/v) hydrogen peroxide solution (3:1 v/v) (see Note 1). 5. Silicon wafers containing a ~50–100 nm oxide layer or glass wafers (optical quality for microscopy, see Note 2).

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6. Positive photoresist (see Note 3). 7. Resist spinner and hot plates. 8. Chrome-coated mask. 9. Developing solutions for dissolving exposed photoresist. The choice of developing solution depends on the photoresist used. 10. Optical pattern generator for mask design (e.g., DWL66 Heidelberg Mask Writer). 11. g-line (435 nm) or i-line (365 nm) stepper. 12. Reactive Ion Etcher (e.g., Oxford PlasmaLab 80+). 2.2. RBL-2H3 Cell Culture

1. Minimum Essential Medium (MEM, Gibco, Invitrogen) supplemented with 20% (v/v) Fetal Bovine Serum (Atlanta Biologicals, GA) and 50  mg/mL Gentamicin (Gibco, Invitrogen). 2. Trypsin Solution (Gibco, Invitrogen).

2.3. Supported Lipid Bilayer Preparation

1. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0-18:1 PC) (POPC, Avanti Polar Lipids, Al). 2. 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6((2,4-dinitrophenyl)amino)caproyl) (DNP-Cap-PE, Avanti Polar Lipids, Al). 3. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Lissamine Rhodamine B Sulfonyl) (Lissamine Rhodamine PE, Avanti Polar Lipids, Al). Stock solutions of lipids are prepared in chloroform at a concentration ranging from 1 to 25 mg/mL. Store stock solutions in glass vials desiccated at −20°C. 4. Argon or nitrogen gas (Airgas Inc.). 5. Phosphate-Buffered Saline (PBS): 137  mM NaCl, 2.7  mM KCl, and 10 mM sodium phosphate, pH 7.4. 6. Acrodisc 25-mm syringe filter and 0.2 mm HT Tuffryn membrane (Pall Co., NY). 7. Plasma cleaner PDC-32G (115V) (Harrick Plasma, NY) connected to a vacuum pump with a minimum pumping speed of 1.4 m3/h and a maximum ultimate total pressure of 200 mTorr. 8. Parafilm M Laboratory Wrapping Film (Fisher Scientific).

2.4. Ligand Carrier Immobilization

1. (3-Mercaptopropyl) trimethoxysilane (MPTS) (SigmaAldrich) stored under argon in a desiccator at 4°C. 2. 4-Maleimidobutyric acid N-succinimidyl ester, GMBS (Sigma-Aldrich). Prepare a 1 M stock solution in dimethylformamide (DMF) and store at −20°C in small aliquots (­stable for weeks in DMF). 3. Toluene (Mallinckrodt Chemicals, NJ).

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4. 2,4-Dinitrophenylated bovine serum albumin (DNP-BSA) (Molecular Probes, Invitrogen) conjugated to a fluorophore (e.g., Alexa 568 or Cy3). 5. Phosphate buffer: 0.1  M Sodium Phosphate and 0.15  M NaCl, pH 7.3. 2.5. Transfection

1. Gene Pulser Xcell electroporator system (Bio-Rad Laboratories). 2. Electroporation buffer: 137 mM NaCl, 2.7 mM KCl, 1 MgCl2, 5.6 mM glucose, and 20 mM Hepes (pH 7.4). 3. DNA construct encoding for the green fluorescent protein (GFP)-fusion protein of interest.

2.6. Cell Activation

1. Monoclonal anti-2,4-dinitrophenyl IgE (Sigma-Aldrich). 2. Buffered Saline Solution (BSS): 135 mM NaCl, 5.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5.6 mM glucose, and 20 mM Hepes (pH 7.4) containing 1 mg/ml BSA. 3. 35 × 10-mm tissue culture dish. 4. Micropatterned substrate.

2.7. Fixation and Imaging

1. Formaldehyde 3.7%, freshly prepared in PBS from a 37% stock. 2. BSA. 3. Glass Bottom Culture Dishes (No. 1.5) (MatTek Co., MA).

3. Methods 3.1. Microfabrication

Due to the highly technical nature of the microfabrication ­process and variations in instrumentation between different facilities, specific details are not described. Photolithography instrumentation is expensive and not easily accessible outside dedicated clean room facilities; moreover, instrument models vary greatly between facilities, and some procedures need to be adapted and/or optimized depending on the available instrumentation. Many universities have access to clean room facilities and technical staff who train in the fabrication process. We suggest initial collaborations with groups with experience in microfabrication. The fabrication procedure described here is straightforward and can be easily followed by a person with basic knowledge in the field. Alternatively, some companies or facilities may be able to process custom orders. The fabrication of the patterned substrate involves a series of steps that are summarized in Fig. 1. Once the patterned parylene

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Fig. 1. Schematic diagram of patterned surface microfabrication and deposition of biological material (e.g., ligand).

wafers are prepared, they can be stored for several months (in a desiccator) for further use. Immobilization of the biomaterial is then performed in the laboratory. 1. Design the pattern with desired feature size and shape in a CAD software. The pattern size and spacing should be enough to include several features per biological cell and large enough (>500  nm) for efficient visualization using fluorescence microscopy. 2. Design Chrome mask: The mask is fabricated by transferring the CAD pattern using an optical pattern generator on a chrome (~80  nm layer)-evaporated glass plate (5″ by 5″) coated with photoresist. After light exposure and photoresist development, the chrome is removed from areas unprotected by photoresist by using acid etch. Once prepared, the same mask can be used indefinitely for the photolithographic

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­ reparation of the patterned wafers and can be stored for p years without significant loss of quality. 3. Clean the wafer with piranha solution. 4. Coat the wafers with a thin layer (~1 mm) of Parylene C polymer using the Parylene Coating System. The amount of parylene dimer loaded into the system and other instrument settings have to be optimized to achieve the desired layer thickness. 5. Spin-coat wafers with photoresist to get a uniform layer of ~1.6–2.0 mm and place wafers on a hot plate for ~0.5–2 min (soft-bake) to evaporate any excess solvent and promote adhesion of the resist to the wafer. Soft-bake conditions (baking time and temperature) depend on the choice of photoresist. 6. Expose the photoresist-coated wafer to electromagnetic radiation through the mask (prepared previously as depicted in steps 1 and 2) by using a 5× g-line (435 nm) or 10× i-line (365  nm) stepper. The optimal focus and exposure time should be determined experimentally. 7. Place the exposed wafers on a hot plate for ~0.5–2 min (postbake) to anneal the surface. Post-bake conditions depend on the choice of photoresist. 8. Dissolve the exposed photoresist (develop) using the appropriate solvent for the photoresist and characterize the photoresist film thickness. Some facilities have automatic instrumentation for wafer development, such as the Hama­ tech-Steag wafer processors. 9. Etch the wafer using Reactive Ion Etcher with oxygen as the reactive species (see Note 4). 10. Dissolve the remaining photoresist with acetone while spinning the wafer using a spin-coater. 11. Store the wafer for future use. 12. Before ligand immobilization on the surface, cut the silicon wafers into 8 × 8 mm pieces. These small pieces are used for cell experiments (see Note 5). When working with glass wafer, cut the wafer into 22 × 22 mm pieces by scratching the surface on the side opposite to the parylene layer using a diamondscribing tool with the help of a rubber ruler. Carefully detach the coverslip from a glass bottom culture dish (No. 1.5) and replace with patterned parylene glass piece by using an adhesive, such as Sylgard 182 silicone elastomer (Dow Corning), with the parylene layer facing the interior of the dish.

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Two different methods for immobilizing 2,4-dinitrophenyl (DNP) ligand to the patterned substrates are presented here. The first method requires supported lipid bilayers for ligand presentation while the other, alternative method involves covalent coupling of a ligand carrier to the surface (see Fig.  2). Both the immobilization schemes are equivalent in their ability to activate receptor clustering. However, we find that supported lipid bilayers provide some significant advantages. Supported lipid bilayers tend to be inert and more resistive to nonspecific protein absorption, in contrast to the SiO2 surface which is more prone to protein absorption and cell adhesion (10, 11). Our experimental system uses IgE antibodies specific to DNP molecules. Cells are sensitized with anti-DNP IgE antibodies, where they form tight complexes (high affinity, low dissociation) with FceRI receptor on the cell surface. IgE-FceRI clustering and activation by the immobilized ligands recruit a series of signaling proteins that, if labeled, can be visualized with fluorescence microscopy (Fig. 3). For other experimental systems, the choice of ligand and immobilization scheme may need to be adapted (see ref. 9). For example, biotin–streptavidin interaction can be used to tether biotinylated proteins on a surface modified with biotin groups attached to streptavidin (12). 1. Prepare a solution containing 89 mol% POPC, 10 mol% DNP-cap-PE, and 1 mol% lissamine rhodamine PE by mixing stock solutions in chloroform (see Note 6). The amount of lipids used should amount to a total lipid concentration of 1 mM after reconstitution in buffer (see step 3). Use a glass vial to mix the lipids.

Fig. 2. Micropatterned ligand array after polymer lift-off. (a) Array of immobilized fluorescent BSA visualized after partially peeling the parylene layer. Nonspecific bound material is removed with the polymer, leaving a surface with clearly defined features and low background fluorescence. (b) Cartoon showing possible ligand immobilization schemes. The ligand of interest (e.g., DNP) can be incorporated into a supported lipid bilayer (top) or covalently attached to the surface via a protein carrier, such as BSA (bottom ).

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Fig. 3. Cell activation with the patterned lipid bilayers containing DNP ligands. (a) Cartoon representing RBL mast cells interacting with the patterned ligand array. FceRI-IgE binding and clustering by the spatially restricted DNP molecules cause localized recruitment of signaling proteins, such as Lyn kinase (zoomed box). (b) Confocal images of RBL cells stimulated by ligand-containing lipid patterns at 37°C for ~30  min. Distinctive local accumulation of Lyn kinase (upper ) and actin (lower ) shown in green can be visualized under the clustered FceRI-IgE. Supported membranes (red ) are labeled by adding 1 mol% Lissamine Rhodamine PE to the lipid mixture. Scale bar corresponds to 20 mm.

2. Evaporate the solvent with a stream of argon or nitrogen gas to create thin lipid film in a glass vial. Lipid films may be stored under argon for weeks at −20°C in a desiccator. 3. Hydrate the film by adding ~2  mL of PBS for 5  min. The ­temperature of the buffer should be above the gel-liquid crystal­ transition temperature of the major lipid and should

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be ­maintained during the hydration period. The effects of buffer pH and ionic strength on supported lipid bilayer formation and stability are addressed in ref. 13. 4. Vortex for 30 s to create large multilamellar vesicles (LMVs). 5. Sonicate the samples with a probe sonicator with suspension at ~10°C higher than the transition temperature of major lipids until the suspension becomes clear, corresponding to unilamellar vesicles (between 5 and 10 min). Use water bath to control temperature and prevent overheating. 6. Filter the liposome suspension through a 0.2-mm filter (Acrodisc) to remove particles deposited during sonication. 7. Plasma clean the substrate (parylene-patterned surface) for ~20 s to make the surface hydrophilic. 8. Cut a piece of Parafilm M, place the patterned parylene substrate on the film, add lipid suspension, and incubate for ~10 min. The hydrophobicity of the parafilm causes the solution to remain on the substrate. 9. Fill four 100 × 50-mm crystallizing dishes with distilled deionized water. 10. Rinse the substrate vigorously in water iteratively in each of the crystallizing dishes. Use a small (35 × 10-mm or smaller) dish to transfer the substrate from each crystallizing dish to the other to avoid drying the surface, and hence destroying the bilayer. After the second rinse, mechanically peel off the parylene layer with tweezers. This yields supported lipid bilayers patterned on the substrate. Rinse once more with water (see Note 7). 11. Place the patterned substrate on the 35 × 10-mm culture dish and replace water with BSS. Keep the substrate hydrated at all times. 12. Experiment with cells should ideally be done same day as lipid preparation (see Note 8). 3.3. Covalent Ligand (Attached to Carrier Protein) Immobilization Using Silane Chemistry (Alternative Method)

1. Rinse patterned parylene substrate gently with acetone and dry with nitrogen gas in a glass dish. 2. Prepare a 2% (v/v) solution of MPTS in toluene. Aminosilanes and their fumes are irritant and highly corrosive. The reactions must be done carefully in a chemical hood. 3. Incubate patterned parylene substrate with the MPTS solution for 20–30  min in the hood at room temperature in a glass dish to functionalize the surface with thiol groups. Rinse surface with acetone, and dry using nitrogen or argon gas. When working with glass wafers, this reaction should be carried out before using adherents to attach the glass piece to the plastic dish (as described in Subheading 3.1, step 12).

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4. Rinse the surface with absolute ethanol, and add the 2 mM of the GMBS cross-linker solution in absolute ethanol for 1 h (see Note 9). 5. Rinse substrate with phosphate buffer. 6. Immediately incubate patterned parylene substrate with 25–100  mg/ml of protein (DNP-BSA) in phosphate buffer for 2 h (up to overnight). 7. Rinse with phosphate buffer to remove unbound proteins. 8. Mechanically peel off parylene under buffer to reveal the patterned protein and rinse the surface with buffer. 9. Block the surface with PBS containing 10 mg/mL BSA for 2 h (up to overnight) to minimize nonspecific binding. 3.4. RBL-2H3 Cell Culture

1. Grow adherent RBL-2H3 cells in media to sub-confluence in 25-cm2 flask in a 5% CO2 incubator. 2. Harvest cells using trypsin solution to detach the cells from the flask and resuspend in media or BSS at 0.5 × 106 cells/mL.

3.5. Cell Transfection

1. Harvest the cells and resuspend at 1 × 107 cells per ml in electroporation buffer. 2. Mix ~5–10 mg of plasmid DNA with 500 mL cell suspension. 3. Electroporate the cells by using an exponential pulse of 280 V and 950 mF in a 4-mm cuvette by using a Gene Pulser Xcell electroporator system. 4. Recover electroporated cells (avoid floating dead cell debris formed during electroporation), plate cells in medium at ~60% confluency, and incubate at 37°C for 24 h (see Note 10).

3.6. Cell Activation with Patterned Ligand

1. Sensitize the cells by adding IgE to a final concentration of ~0.5 mg/mL and incubate for 1 h keeping the cells in suspension at 37°C (overnight incubation of attached cells with IgE is also possible). Remove excess IgE by washing and resuspension in BSS containing 1 mg/mL BSA and resuspend the cells at a concentration of ~0.5 × 106 cells/mL. 2. Place patterned substrate on 35 × 10-mm tissue culture dish and add ~1 mL of cell suspension (see Note 11). 3. Incubate cells with the patterned substrate for 15–45 min at 37°C. It will take about 5 min for the cells to settle and begin attaching to the surface. For live cell imaging, skip steps 5–6 and immediately image cells using fluorescence microscope. 4. After the appropriate incubation time, rinse the cells with BSS to remove cells that are still in suspension. 5. Fix cells by adding 3.7% formaldehyde (in PBS) and incubate for 10 min. Remove fixative and quench reaction by adding PBS containing 10 mg/mL BSA and incubate for 5 min.

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6. Rinse cells with PBS. Fixed cells can be stored at 4°C in PBS with 0.01% (w/v) NaN3 to prevent microbial growth for weeks. 7. Image cells using fluorescence microscope. If using inverted fluorescence microscope and patterned silicon substrate, put silicon substrate upside down inside a glass bottom dish filled with PBS buffer to visualize fluorescence features (see Note 12).

4. Notes 1. Piranha solution is extremely corrosive and reactive, and handling and disposal should be done with extreme caution (always use a hood and protective equipment). Some facilities may have access to automated wafer cleaning instrumentation, such as the Hamatech Wafer Processor, that are useful for reducing exposure to dangerous chemicals. 2. Fabrication on glass wafer is desired when a transparent substrate is required. For example, Total Internal Reflection Fluorescence Microscopy (TIRFM) requires a transparent substrate for the propagation of an evanescent wave. Fabrication of glass wafers involves the same steps as those for silicon wafers, but can be more difficult because of the fragility and thickness of optical glass wafers, which are much thinner (0.16–0.19 mm) than standard silicon wafers (>0.4 mm). For confocal and epifluorescence imaging, silicon wafers are sufficient. In some confocal systems, scattered light from the silicon surface can be visualized as background noise. This background noise is small compared to the signal obtained from the labeled cells and can be improved by collecting fluorescence from wavelength far from the excitation wavelength. 3. Many different options are available for photoresist, and the choice mainly depends on the desired feature size and the available instrumentation (i.e., i-line or g-line stepper). We found that both SPR 220-3.0 (i-line or g-line) and SPR 955 cm-2.1 (i-line) (Rohm and Haas Electronic Materials LLC, MA) work well with our fabrication scheme. 4. The optimal time for oxygen etching should be optimized experimentally. Ideally, the majority of the ~1.6–2 mm photoresist layer should be consumed without exposing the parylene layer. This step is important because under-etching leaves residual parylene on the patterns and over-etching consumes the parylene layer. After etching, the remaining thickness of the palylene features should be confirmed with a profilometer. 5. The lattice structure of the silicon wafers is responsible for its brittle nature. This property allows easy cutting of wafers by

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slightly scratching a corner with a diamond-scribing tool and applying small pressure on both sides of the scratch. 6. The ligands are chosen to be specific for the experimental setting. For other receptors, the lipid composition may be changed and other lipids with modified head groups may also be used for ligand presentation. For example, avidin–biotin interactions are exploited by using biotinylated lipids. As an alternative to ligand presentation with supported lipid bilayers, an alternate method for covalently immobilizing ligands is presented in Subheading 3.3. 7. Once the supported lipid bilayer has formed, it is important to keep it hydrated. Exposure to air destroys the supported lipid bilayer (13). 8. Although it may be possible to use the supported membranes days after preparation, it is recommended to use them within a few hours of preparation. Supported membranes tend to expand within hours of preparation in a pH- and ionic strength-dependent manner (13), and this affects the quality of the patterned ligands. 9. After the MPTS covalently attaches to the silicon dioxide surface, its thiol group reacts specifically with the maleimide moiety on the GMBS cross-linker. This activates the surface with succinimide residues that can react with amine groups of an antibody or other protein to form stable amide bonds (14, 15). 10. Chemical transfection methods may also be used to introduce GFP-encoding DNAs into the cells. We chose to describe electroporation for its simplicity. 11. Although supported lipid bilayers are usually a better choice for preventing nonspecific interactions, one advantage of covalent immobilization of proteins is that the modified surfaces can be stored for weeks at 4°C without significant loss of functionality and short periods of drying do not affect the immobilized ligand. 12. In cases where the protein to be investigated is not available as GFP-fusion protein, immunofluorescence microscopy can be used to detect proteins. In such cases, fluorescent lipids cannot be used to visualize the patterned features because the detergent used to permeabilize cells (Triton X-100) partially dissolves the supported lipid bilayers. To overcome this technical limitation, fluorescently labeled anti-DNP IgE can be used to sensitize the cells, and hence visualize the clustered receptors (see ref. 11 for example). Covalently immobilized protein carriers are not affected by permeabilizing reagents.

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Acknowledgments This work was supported by the Nanobiotechnology Center (NSF: ECS9876771) and by NIH grants R01-AI18306 and R01-AI22449. References 1. Bruhns, P., Frémont, S., and Daëron, M. (2005) Regulation of allergy by Fc receptors. Curr Opin Immunol 17, 662–669. 2. Nimmerjahn, F., Ravetch, J. V., and Frederick, W. A. (2007) Fc-Receptors as Regulators of Immunity, Adv Immunol 96, 179–204. Academic Press. 3. Kraft, S., and Novak, N. (2006) Fc receptors as determinants of allergic reactions, Trends Immunol 27, 88–95. 4. Kinet, J.-P. (1999) THE HIGH-AFFINITY IgE RECEPTOR (FceRI): From Physiology to Pathology, Annu Rev Immunol 17, 931–972. 5. Holowka, D., Gosse, J. A., Hammond, A. T., Han, X., Sengupta, P., Smith, N. L., Wagenknecht-Wiesner, A., Wu, M., Young, R. M., and Baird, B. (2005) Lipid segregation and IgE receptor signaling: A decade of progress, Biochim Biophys Acta 1746, 252–259. 6. Rivera, J., Fierro, N. A., Olivera, A., and Suzuki, R. (2008) New Insights on Mast Cell Activation via the High Affinity Receptor for IgE, Adv Immunol 98, 85–120. Academic Press. 7. Orth, R. N., Wu, M., Holowka, D. A., Craighead, H. G., and Baird, B. A. (2003) Mast Cell Activation on Patterned Lipid Bilayers of Subcellular Dimensions, Langmuir 19, 1599–1605. 8. Wu, M., Holowka, D., Craighead, H. G., and Baird, B. (2004) Visualization of plasma membrane compartmentalization with patterned

lipid bilayers, Proc Natl Acad Sci USA 101, 13798–803. 9. Torres, A. J., Wu, M., Holowka, D., and Baird, B. (2008) Nanobiotechnology and Cell Biology: Micro- and Nanofabricated Surfaces to Investigate Receptor-Mediated Signaling, Annu Review Biophys 37, 265–288. 10. Kam, L., and Boxer, S. G. (2001) Cell adhesion to protein-micropatterned-supported lipid bilayer membranes, J Biomed Mater Res 55, 487–95. 11. Torres, A. J., Vasudevan, L., Holowka, D., and Baird, B. A. (2008) Focal adhesion proteins connect IgE receptors to the cytoskeleton as revealed by micropatterned ligand arrays, Proc Natl Acad Sci USA 105, 17238–44. 12. Doh, J., and Irvine, D. J. (2006) Immunological synapse arrays: Patterned protein surfaces that modulate immunological synapse structure formation in T cells, Proc Natl Acad Sci USA 103, 5700–05. 13. Cremer, P. S., and Boxer, S. G. (1999) Formation and Spreading of Lipid Bilayers on Planar Glass Supports, J Phys Chem B 103, 2554–59. 14. Shriver-Lake, L. C., Donner, B., Edelstein, R., Breslin, K., Bhatia, S. K., and Ligler, F. S. (1997) Antibody immobilization using heterobifunctional crosslinkers, Biosens Bioelectron 12, 1101–06. 15. Vijayendran, R. A., and Leckband, D. E. (2000) A Quantitative Assessment of Heterogeneity for Surface-Immobilized Proteins, Anal Chem 73, 471–480.

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Chapter 15 CELLISA: Reporter Cell-Based Immunization and Screening of Hybridomas Specific for Cell Surface Antigens Peter Chen, Aruz Mesci, and James R. Carlyle Abstract Monoclonal antibodies (mAbs) specific for cell surface antigens are an invaluable tool to study immune receptor expression and function. Here, we outline a generalized reporter cell-based approach to the generation and high-throughput screening of mAbs specific for cell surface antigens. Termed CELLISA, this technology hinges upon the capture of hybridoma supernatants in mAb arrays that facilitate ligation of an antigen of interest displayed on BWZ reporter cells in the form of a CD3z-fusion chimeric antigen receptor (zCAR); in turn, specific mAb-mediated cross-linking of zCAR on BWZ cells results in the production of b-galactosidase enzyme (b-gal), which can be assayed colorimetrically. Importantly, the BWZ reporter cells bearing the zCAR of interest may be used for immunization as well as screening. In addition, serial immunizations employing additional zCAR- or native antigen-bearing cell lines can be used to increase the frequency of the desired antigen-specific hybridomas. Finally, the use of a cohort of epitope-tagged zCAR (e.g., zCARFLAG) variants allows visualization of the cell surface antigen prior to immunization, and coimmunization using these variants can be used to enhance the immunogenicity of the target antigen. Employing the CELLISA strategy, we herein describe the generation of mAb directed against an uncharacterized natural killer cell receptor protein. Key words: Monoclonal antibody, Hybridoma technology, High-throughput screening, BWZ reporter cell assay, CD3z-fusion chimeric antigen receptor, b-Galactosidase (b-gal)

1. Introduction Monoclonal antibodies (mAbs) can be instrumental in studying protein expression and function, receptor signaling pathways, ligand specificities, protein–protein interactions, and a myriad of other cellular and molecular properties (1). Conventional immunization approaches often require a large amount of purified, nonnative

Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_15, © Springer Science+Business Media, LLC 2011

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(e.g., refolded) protein or the synthesis of several small peptide ­fragments of the antigen of interest. Such approaches frequently yield mAbs specific for improperly folded forms of the antigen of interest or mAbs with only weakly reactive or highly cross-reactive properties. In addition, subsequent hybridoma screening can be laborious, frequently involving sequential analysis of duplicate ­samples. For example, flow cytometric analysis of cells ectopically expressing an antigen usually involves parallel analysis of mAb reactivity toward a parental cell line for each individual hybridoma supernatant. This screening can involve costly secondary reagents, and the identification of mAbs with a desired specificity can be obscured by oligoclonal hybridoma outgrowths within single wells. On the other hand, whole cell-based immunization approaches avoid large-scale production and purification of the antigen of interest, and can be used to generate an enriched library of hybridomas and mAbs specific for native antigenic epitopes (1). Furthermore, when the immunogen is coupled to a reporter cell that generates endogenous enzyme (2, 3), hybridoma selection can be simplified into an efficient high-throughput screening (HTS) technology (1, 4–6). Here, we report an enzymatic reporter cell-based adaptation of hybridoma screening technology, termed CELLISA (1). This method involves central use of the BWZ.36 reporter cell line [hereafter, BWZ (3)] bearing a CD3z-fusion chimeric antigen receptor (zCAR) (1, 4–8). The cell surface zCAR serves as a target for the desired antibodies, both in  vivo during immunization, as well as in vitro during hybridoma screening, since the reporter cell produces b-galactosidase (b-gal) upon ligation of the antigenic epitopes presented by the mAb-immobilized zCAR (1). CELLISA offers several advantages: (1) large-scale production of the protein of interest is unnecessary; (2) antigens can be presented in their native form at the cell surface; (3) a single cell line can be used for immunizations as well as screening, since BWZ cells produce their own b-gal enzyme in response to zCAR ligation (thus, obviating the need for expensive secondary reagents); (4) HTS of hybridoma supernatant arrays can be achieved in a single day using an inexpensive colorimetric substrate; (5) duplication of effort is not required during primary HTS, although secondary validation of hybridoma specificities can be performed using the parental BWZ cell line; and (6) mAb of desired specificity can be uniquely identified among oligoclonal hybridoma populations, since only positive ligation events are visualized even in the presence of mixed mAb specificities. In addition, we recently modified our CELLISA immunization strategy in order to optimize the generation of mAbs of desired specificity and quality. In order to amplify the response to the antigen of interest, we took advantage of several previously observed immune phenomena. First, to encourage linked recognition (the promotion of T:B collaboration during an immune response

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by inclusion of both B and helper T cell epitopes into a single immunogen), we constructed an epitope-tagged variant of our original zCAR vector (zCARFLAG) (1). Here, the inclusion of a strongly immunogenic and highly charged FLAG epitope sequence (DYKDDDDK) serves to enhance the presentation of helper T cell epitopes in the linked antigen following the uptake by primary FLAG-specific B cells. Of course, the FLAG epitope offers several additional advantages, including the ability to detect surface expression of the tagged antigen, thus by proxy inferring similar surface expression of the native (nontagged) antigen of interest. Furthermore, the FLAG tag serves to encourage intramolecular epitope spreading (the directed expansion of antibody responses from a dominant primary epitope to secondary or cryptic epitopes on a linked protein sequence). This can enhance the overall immunogenicity or breadth of the antibody response to an otherwise weak target antigen, amongst a multitude of cellular antigens. Finally, to encourage immune focusing toward the target antigen itself (4), and thereby discourage the expansion of antibody responses toward nontarget cellular antigens, the same zCAR vectors are used to transduce distinct cell lines (for example, from different species). The use of different zCAR-bearing cell lines during the priming and boosting immunizations focuses the antibody response toward the shared antigen of interest, while at the same time promoting antibody class switching to IgG isotypes. Importantly, following hybridoma generation, supernatants are screened using the native (nontagged) zCAR-bearing BWZ reporter cells in order to visualize only desired specificities. Taken together, these flexible adaptations were used to derive novel mAbs specific for an uncharacterized and conserved (i.e., weakly immunogenic) NK cell receptor, mouse NKR-P1G (9, 10).

2. Materials 2.1. Preparation of Retroviral Vectors: Amplification, Gel Purification, and Cloning

1. Expand High-FidelityPLUS Polymerase kit (Roche Diagnostics, Mannheim, Germany). 2. PCR-grade dNTP: 100 mM stocks, dATP, dCTP, dGTP, and dTTP. 3. Primers (See Table 1). 4. Molecular biology grade water. 5. Preparative grade agarose. 6. TAE buffer (10×): 400  mM Tris base, 0.2  N acetic acid, 10 mM EDTA dissolved in water, and pH adjusted to 7.5. 7. 0.5% bromophenol blue and xylene cyanol solution for agarose gel loading, 10×, in 1× TAE buffer.

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Table 1 Primers used to amplify variants of the mouse NKR-P1G coding sequence Primer set

Primer sequence

P1GFL-5¢ P1GFL-3¢

ctcgaggccaccATGGATGCACCAGTGCTCTATG agcggccgcTCAGACGTGTTTCAGTGTCTTTTG

P1GEC-5¢ P1GEC-3¢

ctcgagCAAAAACCTCTAATAGAAAAATGCAG agcggccgcTCAGACGTGTTTCAGTGTCTTTTG

P1GECD-5¢ P1GECD-3¢

ctcgagCAAAAACCTCTAATAGAAAAATGCAG agcggccgcGACGTGTTTCAGTGTCTTTTGG

NKR-P1GFL, full-length coding sequence; NKR-P1GEC, ectodomain sequence; and NKR-P1GECD, stop-codon (TGA)-deleted ectodomain sequence. Italicized font, restriction enzyme sites; underlined font, Kozak consensus sequence.

8. DNA markers: 1 kb PLUS DNA Ladder (Invitrogen) reconstituted with TAE buffer (1×) and loading dye (1×) at 100 ng/mL. 9. Ethidium bromide solution, 10 mg/mL in 1× TAE buffer. 10. Rapid Gel Purification kit (Invitrogen). 11. pcDNA3.1/V5/HIS/TOPO kit (Invitrogen). 12. Rapid Plasmid Purification kit (Invitrogen). 13. Xho I and Not I restriction endonucleases. 14. LigaFast Rapid DNA Ligation System (Promega). 2.2. Generation of Cell Lines: Cell Lines, Cell Culture, Transfection, and Sorting

1. BWZ.36 mouse reporter cells (Dr. Nilabh Shastri, U.C. Berkeley, CA, USA) (2, 3). 2. P3-X63-Ag8.653.1 (P3) mouse myeloma cells (11) or Sp2/0 myeloma cells. 3. 293T human embryonic kidney cells. 4. YB2/0 rat myeloma cells. 5. Dulbecco’s Modified (DMEM-HG).

Eagle’s

Medium–High

Glucose

6. Fetal bovine serum (FBS). 7. Penicillin–streptomycin supplement, 10,000  U/mL and 10,000 mg/mL. 8. Gentamicin supplement, 50 mg/mL (Gibco). 9. 2-mercaptoethanol solution in D-phosphate-buffered saline (PBS), 55 mM (Gibco). 10. HEPES supplement, 1 M (Hyclone).

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11. Sodium pyruvate supplement, 100 mM (Hyclone). 12. Glutamax supplement, 100× (Gibco). 13. PBS (Hyclone). 14. EDTA. 15. Trypsin solution, 2.5% in PBS (Gibco). 16. Effectene Transfection Kit (Qiagen). 17. Polybrene (hexadimethrine bromide; Sigma Aldrich). 18. FACS buffer: Hank’s balanced salt solution (HBSS; Hyclone) with 0.5% bovine serum albumin (BSA, EMD Chemicals). 19. Flow cytometry buffer: FACS buffer plus 0.03% NaN3 (EMD Chemicals). 20. Primary antibody: Anti-FLAG-biotin (Sigma Aldrich). 21. Secondary reagent: Invitrogen).

Streptavidin-phycoerythrin

(SA-PE,

22. Propidium iodide solution, 50  mg/mL in PBS (Sigma Aldrich). 2.3. Hybridoma Generation and Screening: Immunization, Fusion, and CELLISA

1. 1-mL syringe with tuberculin slip tip (BD). 2. 25G-5/8” Precision Glide Needle (BD). 3. 40-mm cell strainer (BD Falcon). 4. Lympholyte-Mammal cell separation medium (Cedarlane Laboratories Ltd.). 5. Polyethylene glycol (PEG 1500), 50% solution. 6. Hypoxanthine aminopterin thymidine (HAT) supplement (50× HAT; Sigma Aldrich). 7. Hypoxanthine thymidine (HT) supplement (50× HT; Sigma Aldrich). 8. 2× freezing medium (20% DMSO, 80% FBS). 9. High-binding chemistry plates (Corning Life Sciences). 10. AffiniPure goat anti-rat IgG + IgM (H + L) polyclonal antiserum (Jackson Immunoresearch). 11. CPRG buffer: [90 mg/L chlorophenol-red-b-d-galactopyranoside (Roche Diagnostics), 9 mM MgCl2, 0.1% NP40] in PBS (Hyclone, Logan, UT). 12. Confirmatory antibodies: AffiniPure goat anti-rat IgG-PE [F(ab¢)2 fragment, Fcg-specific] and AffiniPure goat anti-rat IgM-PE [F(ab¢)2 fragment, Fcm-specific] (Jackson Immunoresearch). 13. PMA/ionomycin mixture, 100× in medium: 5 mg/mL phorbol12-myristate-13-acetate (PMA; Sigma Aldrich), 100 mM ionomycin (Sigma Aldrich).

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3. Methods 3.1. PCR Amplification of the Ectodomain of the Antigen of Interest

Here, we use NKR-P1G as an example, but other surface antigens are processed similarly. 1. A preexisting pCR2.1 plasmid clone encoding the full-length NKR-P1G molecule of C57BL/6 origin (manuscript in preparation) is used as the PCR template. 2. PCR reactions are set up using the three primer sets indicated in Table  1 under the following conditions: 0.4  mM each primer; 0.5  mM each dNTP; and 2.5  mM MgCl2 final. Amplification is performed as follows: initial denaturation, 95°C, 2  min; 25 amplification cycles (denaturation, 94°C, 30 s; annealing, 45°C, 30 s; and extension, 72°C, 1 min); final extension, 72°C, 5 min; and cooled to 4°C following amplification. The resulting PCR products are loaded in a 2% agarose gel and separated at a setting of 120 V, 400 mA for 10 min. 3. The bands are visualized via ethidium bromide staining (0.3 mg/mL final) using a Gel Doc 2000 gel-imaging system as shown in Fig. 1, and the bands corresponding to the products are excised and purified using the “Rapid Gel Purification Kit” according to the manufacturer’s instructions. 4. The TOPO cloning reaction is set up using the purified PCR product according to the manufacturer’s instructions (see Note 1). After 5 min, 1–2 mL of the reaction product is used to transform “One Shot Top 10” chemically competent bacteria included with the kit. Transformed cells are plated, colonies are selected and cultured, and then plasmid DNA is extracted using the “Rapid Plasmid Purification Kit” according to the manufacturer’s instructions. 5. In order to identify correct inserts, plasmids are cut using XhoI and NotI restriction enzymes at 5  units per mg of plasmid, and the reaction products are loaded in a 2% agarose gel. The bands corresponding to the inserts are imaged, excised, and purified as above. 6. The purified inserts are subcloned as follows: (1) the full-length (FL) NKR-P1G-coding sequence insert is cloned into the pMCIG retroviral vector (4); (2) the NKR-P1G ectodomain (EC) insert is cloned into the “Type II” zCAR retroviral vector (1); and (3) the stop-codon-deleted ectodomain (ECD) insert is cloned into the “Type II–FLAG” zCAR retroviral vector (see Note 2). Ligations are performed at a molar ratio of 4:1 insert:vector using the “LigaFast Rapid Ligation Kit” according to the manufacturer’s instructions. The reactions are allowed to proceed for 20  min at room temperature, and the reaction products are transformed using “One Shot Top 10” competent bacteria; plasmid isolation is carried out as described above.

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a P1GIC

P1GTM

NKR-P1GFL

P1GEC

X

N CD3

IC

PIB TM

P1GEC X

CD3

IC

PIB TM

P1GEC

1000 500 100

P1GFL

654

N Flag N

X

b

CD3 /P1G

P1GEC

465

CD3 /P1GFLAG

TGA

P1GEC

462

Fig. 1. PCR amplification and cloning of the NKR-P1G coding sequence variants into the native and CD3z-fusion chimeric antigen receptor (zCAR)-based retroviral vectors. (a) Schematic diagram of the three NKR-P1G variants. Rectangles indicate protein domains; bold text indicates NKR-P1G insert sequences; italics indicate sequences present in the pMCIG, zCAR, and zCARFLAG fusion vectors; Flag, sequence encoding the DYKDDDDK epitope tag; X, Xho I restriction site; N, Not I restriction site; and TGA, stop codon. (b) PCR products amplified for subcloning of the mouse NKR-P1G inserts. Full-length (P1GFL), extracellular domain (P1GEC), and stop-codon-deleted ectodomain (P1GECD) variants are shown with their corresponding sizes (in bp) below the bands. Standard bands from the 1 kb Plus DNA Ladder and their sizes are given on the left.

3.2. Generation of Cell Lines Expressing NKR-P1G

1. In general, BWZ.36 (3), YB2/0, and 293T cells are maintained in complete culture media (DMEM-HG plus 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, 100 mg/mL gentamicin, 110 mg/mL sodium pyruvate, 10 mM HEPES, 2 mM Glutamax, 55 mM 2-mercaptoethanol, and pH 7.4). The cells are passaged by 1/3 splits when approaching confluence, using 0.25% trypsin plus 2 mM EDTA in PBS (BWZ) or 2 mM EDTA in PBS alone (YB2/0 and 293T) to maintain healthy cultures. 2. One 100-mm dish of 293T cells in log phase growth and approaching confluence is passaged onto three new 100-mm dishes the evening prior to transfection (see Note 3). 3. The Effectene transfection protocol (as supplied by the manufacturer) is carried out using 0.5 mg of gag/pol plus 0.5 mg amphotropic VSV-G env packaging constructs (4), and 1.0 mg of one of the three proviral constructs are generated as above into each 100-mm dish of 293T cells in order to generate the retroviral supernatants.

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4. 18–24 h after transfection, the cell culture media is replaced with fresh complete media (10% FBS) (see Note 4). 5. The next day, at 48 h post-transfection, all cell supernatants are collected without disruption of the plated cells and centrifuged at 800 × g for 5¢ to remove cellular debris (see Note 5). 6. BWZ.36 and YB2/0 cells are resuspended at 1 × 105 cells/mL in DMEM (10% FBS). In two separate 6-well dishes, three wells are seeded with 2 × 105 BWZ.36 or YB2/0 cells to ­prepare for “spin-fection.” To each well, 2 mL of retroviral supernatant, 0.5 mL of FBS, 0.5 mL of fresh complete media (10% FBS), and 8 mg/mL of polybrene (from an 800 mg/mL stock in DMEM) are added. The plates are centrifuged at 1,000 × g for 90  min as close to 37°C as possible and then placed into the incubator (see Note 6). 7. 72 h after retroviral transduction, cells are washed and resuspended in 0.5 mL of FACS buffer, then sorted for high GFP expression. The cells are then washed and plated in 6-well dishes, then transferred to 100-mm dishes upon reaching confluence. Cell sorting is repeated for high-level GFP expression 1–2 more times, as required (see Note 7). 8. In order to visualize surface expression of the epitope-tagged zCAR on BWZ.CD3z/P1GFLAG and YB.CD3z/P1GFLAG transductants, flow cytometry is performed as follows: 106 cells are stained in 100  mL of flow cytometry buffer with a-FLAG-biotin primary antibody at a dilution of 1/200 for 30  min, followed by washing. The cells are then stained in 100 mL of buffer containing SA-PE at a dilution of 1/600 for 20 min, then washed again. Following the final wash, 100 mL of buffer containing 5 mg/mL propidium iodide is added to the cells, and the samples are analyzed using a FACSCalibur flow cytometer. As shown in Fig.  2, all sorted transductants express high levels of GFP, and FLAG epitope expression is

Fig. 2. Flow cytometric analysis of BWZ transductants of the NKR-P1G variants. 106 cells were stained with a-FLAG-biotin mAbs at a 1/200 dilution, followed by secondary streptavidin-APC at a 1/1,000 dilution. Cells were gated on according to the size and lack of propidium iodide to exclude dead cells and cellular debris.

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confirmed on BWZ.CD3z/P1GFLAG cells, indicating that the fusion receptor is expressed at the cell surface. 3.3. Immunization and Hybridoma Generation

1. For immunization using mouse receptor-bearing cell lines, a total of 6 × 106 cells are injected intraperitoneally (i.p.) into Lewis rats (usually, 2 × 106 cells are used to immunize mice). Minimally, this can be performed using the BWZ.zCAR cells (see Note 8); however, multiple zCAR- and native antigenbearing cells (mouse BWZ, rat YB2/0) can be used to elicit immune focusing (4). In this case, we use 106 of each cell line generated (BWZ.NKR-P1GFL, BWZ.CD3z/P1G, BWZ. CD3z/P1GFLAG, YB.NKR-P1GFL, YB.CD3z/P1G, and YB.CD3z/P1GFLAG), which are pooled into one 15-mL tube. The cells are washed three times with 5  mL of serum-free DMEM (0% FBS; see Note 9), and resuspended in 1-mL serum-free media for i.p. injection into a Lewis rat. 2. At approximately 3–4 week intervals after the initial injection, the second and third immunizations are carried out using the protocol described above. The first boost is performed with the same numbers and types of cells as the initial injection; however, in order to minimize antibody responses to endogenous BWZ cell antigens or to the FLAG epitope tag, 3 × 106 of each of the YB.NKR-P1GFL and YB.CD3z/P1G variants are used in the final boost (Fig. 3). 3. Three days after the final boost, the rat is euthanized using CO2, and the spleen is harvested. Splenocytes are isolated,

Time:

0 wk

4 wk

Immunization:

1st

2nd

Cells:

#Cells:

BWZ.NKR-P1GFL BWZ.CD3 /P1G BWZ.CD3 /P1GFLAG YB.NKR-P1GFL YB.CD3 /P1G YB.CD3 /P1GFLAG

1x106 each

7 wk 7 wk + 3d

3rd Harvest

BWZ.NKR-P1GFL YB.NKR-P1GFL BWZ.CD3 /P1G YB.CD3 /P1G BWZ.CD3 /P1GFLAG YB.NKR-P1GFL YB.CD3 /P1G YB.CD3 /P1GFLAG

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filtered through a 40-mm cell strainer, and separated by densitygradient centrifugation following underlay of the cell suspension with Lympholyte-Mammal cell separation medium, according to the manufacturer’s instructions (see Note 10). 4. Hybridoma fusion is carried out using standard protocols (1, 4). Cells are maintained at 37°C during the entire hybridoma fusion protocol, using a double-walled water bath. Briefly, splenocytes and P3 myeloma fusion partner cells are washed (500 × g, 5¢) three times in serum-free media. The splenocytes and P3 cells are mixed together at a 1:1 ratio during the final wash, and all medium is removed using a pipette tip. Fusion is performed by dropwise addition of 2 mL fresh PEG 1500 over a period of 1 min, with gentle mixing using the pipette tip and gentle agitation of the tube (light tapping). The cells are then gently mixed as above for 1 additional minute (total fusion, 2 mL PEG over 2 min). The PEG-fused cells are then washed by dropwise addition of 4 mL serum-free media over a period of 2 min with gentle mixing/agitation; this is followed by a more rapid dropwise addition of 14 mL serum-free media over a period of 3 min (total wash, 18 mL over 5 min). The fused cells are collected by centrifugation at 500 × g for 5¢, then the cell pellet is forcefully dispersed using 10 mL of complete hybridoma medium (20% FBS) ejected from a 10-mL pipette; this is repeated once (20 mL total). Cells are then resuspended to a final volume of complete hybridoma medium (20% FBS) required to distribute all the fused cells into 30 × 96-well flatbottom plates to a final volume of 100  mL per well. Fused cells are allowed to recover overnight in the incubator. 5. The next day, 100 mL of complete hybridoma medium (20% FBS) supplemented with 2× HAT is added to each well (total well volume, 200  mL in 1× HAT: 100  mM hypoxanthine, 0.4 mM aminopterin, 16 mM thymidine), and selection is allowed to carry on for 1 week. Afterward, 100 mL of complete 1× HT media is added to each well (total well volume, 300 mL, in 1× HT with diluted aminopterin), and hybridomas are allowed to grow further until significant growth is observed (~day 7–14; see Note 11). When rapidly growing hybridomas first emerge, 150 mL supernatant from each well is removed using a multichannel pipette and collected in a 96-well array (total, 30 plates; see Note 12); the supernatant volume is replaced with 150  mL of fresh 1× HT media added back to the growing hybridomas. Supernatant collection is repeated the next day (another 150 mL) to complete one array of hybridoma supernatants (total volume, 300 mL per well; stored at 4°C, frozen at −20°C, or used immediately for CELLISA). A duplicate supernatant array of ~200–300 mL is harvested over the next 1 or 2 days. If CELLISA screening is not carried out at this

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time, the hybridomas are spun down (500 × g, 3 min), residual media removed, and an equal volume of 2× freezing medium is added directly to the hybridomas; finally, the hybridoma plates are gently agitated or mixed, wrapped in stacks of ~10 plates using parafilm, then frozen at −80°C (optional). 3.4. CELLISA Screening

1. In order to test for the presence of specific (in this case, anti-NKR-P1G) antibodies in the hybridoma supernatants, CELLISA capture plates are generated by immobilizing goat anti-rat IgG/IgM capture antibody (10 mg/mL in 50–75 mL) on 96-well high-binding chemistry plates overnight (see Note 13). The CELLISA plates are washed five times with 200 mL of PBS, then 100 mL hybridoma supernatants are added (in an ordered 30-plate array), and the plates are incubated ­overnight. CELLISA plates are next washed five times with 200 mL of PBS, then ~5–10 × 104 BWZ.CD3z/P1G cells (in 200  mL of complete media plus 10% FBS) are added to each well. The reporter cells are then stimulated for 18–24 h. After the incubation period, the CELLISA plates are centrifuged to pellet the reporter cells, and the medium is discarded (see Note 14). Finally, 100  mL of CPRG buffer is added to each well, and colorimetric changes are monitored and quantitated after 3 h (and again overnight) by plotting the difference of the optical density (OD) values at 595 and 655 nm (see Note 15) (1, 4). As shown in Fig.  4, NKR-P1G specificities are detected in multiple wells. 2. In our example, one hybridoma, corresponding to plate 1, row H, column 8 (1H8; Note 12 regarding the “comet” effect observed in column 8, rows H-A) is selected for further subcloning (see Note 16). The resulting monoclonal population is denoted 1H8. 3. In order to confirm the specificity, the BWZ and YB transductants used in the immunizations, along with appropriate control cells, are characterized by flow cytometry. In the example, transient 293T transfectants of NKR-P1G are also analyzed in parallel. Here, cell staining is carried out as described above using a primary 1/10 dilution of 1H8 supernatant followed by a secondary 1/200 dilution of the goat anti-rat-IgG-PE or goat anti-rat-IgM-PE reagents. As shown in Fig. 5, all three NKR-P1G transductants are recognized by 1H8 mAb + anti-rat-IgG-PE; however, the untransduced parental cells, or cells expressing an alternate fusion receptor (BWZ.CD3z/P1F; a related molecule belonging to the NKR-P1 gene family (9, 10)), do not react with 1H8 mAb. In addition, the NKR-P1G transductants are not recognized by 1H8 + anti-rat-IgM-PE secondary reagent. These results show that 1H8 selectively recognizes the mouse NKR-P1G

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Well (Plate/Row/Column) Fig. 4. Primary CELLISA screen using BWZ.CD3z/P1G reporter cells. Primary screen of hybridoma supernatants in 96-well arrays using BWZ.CD3z/P1G reporter cells. Polyclonal goat anti-rat-IgG/M antibodies were plate-bound overnight, washed, used to capture hybridoma supernatants overnight, then 75 × 103 reporter cells were added per well and stimulated overnight. Plate numbers are indicated in the top right corners, rows are indicated by letters (A–H), and individual bars are shown for each column within a row (1–12; numbers not shown). OD595/655, difference of optical density values at 595–655 nm. Clone 1H8 was selected for subcloning and further analysis. Note the reverse “comet” effect for plate 1, rows H-A, column 8, as the 1H8 supernatant was inadvertently carried over in the direction of pipetting (in this case, from row H to row A, as one set of tips was used per plate, direction of pipetting noted); this effect usually indicates a strong mAb specificity and the sensitivity of the CELLISA method.

molecule expressed at the surface of the transductants and that 1H8 mAb is a rat IgG2ak isotype antibody. The staining of transient 293T transfectants with 1H8 mAb + anti-rat-IgGPE confirms this specificity (Fig. 5). 4. To further demonstrate functional specificity of the mAb, the reporter cell assay is repeated with captured supernatant as described above. In parallel, 100 mL of a-FLAG mAb is directly immobilized on a plate at a concentration of 30 mg/mL (1). Reporter cell stimulation using 0.5  mg/mL PMA and 10 mM ionomycin is included as a positive control (1, 4). As expected, in our example, 1H8 mAb-mediated stimulation is observed for BWZ.CD3z/P1G and BWZ.CD3z/P1GFLAG cell lines (but neither parental BWZ– cells, nor BWZ.NKRP1G transductants lacking the CD3z-fusion moiety) while a-FLAG mAb selectively activates only the epitope-tagged

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Fig.  5. Secondary flow cytometric analysis of 1H8 mAb specificity for NKR-P1G on various transductants and trans­ fectants. (a) Flow cytometric analysis of NKR-P1G expression at the cell surface on BWZ transductants using 1H8 mAb. Cells were stained with 1H8 supernatant at a 1/10 dilution, followed by washing and secondary goat anti-rat-IgG-PE at a 1/200 dilution. Shaded area, 1H8 staining; thin line, goat anti-rat-IgG-PE secondary reagent alone. RCN, relative cell number. (b) Flow cytometric analysis of NKR-P1G expression on YB2/0 transductants, stained and analyzed as in (a). (c) Flow cytometric analysis of NKR-P1G expression on 293T transfectants, stained as in (a); top plot, goat anti-rat-IgGPE secondary reagent alone versus IRES-GFP reporter expression; bottom plot, 1H8 mAb plus secondary goat anti-ratIgG-PE secondary reagent versus IRES-GFP reporter expression. (d) Flow cytometric analysis of NKR-P1G expression on BWZ.CD3z/P1G transductants using 1H8 mAb plus two distinct secondary reagents: goat anti-rat-IgG-PE [F(ab¢)2 fragment, Fcg-specific) (shaded area) versus goat anti-rat-IgM-PE [F(ab¢)2 fragment, Fcm-specific) (thin line). This indicates that 1H8 mAb is a rat IgG isotype mAb.

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Fig. 6. Secondary reporter cell analysis of 1H8 mAb specificity for NKR-P1G. Immobilized antibody-mediated stimulation of BWZ transductants using 1H8 mAb or a-FLAG mAb. In the left panel, goat anti-rat-IgG/M antibody (10 mg/mL) was immobilized on wells, and 10 mL of 1H8 supernatant was captured overnight. Following washing, 75 × 103 reporter cells were added to each well. OD595/655 indicates the difference of the optical density values at 595–655 nm; samples were analyzed in triplicate, and error bars indicate standard deviation. In the middle panel, purified mouse a-FLAG mAb (30 mg/mL) was immobilized on wells overnight without capture, and reporter cells were stimulated overnight as above. In the right panel, reporter cells were stimulated nonspecifically using PMA/ionomycin overnight as a positive control.

BWZ.CD3z/P1GFLAG transductants (Fig.  6). These results indicate that 1H8 mAb recognizes the native mouse NKRP1G molecule, independently of the FLAG epitope, on the surface of the reporter cells, and this approach can be generalized to other cell surface receptors.

4. Notes 1. The TOPO cloning reaction should be performed immediately after PCR to ensure optimal cloning of PCR products. To conserve reagents, the TOPO reaction can be scaled down to use only half the recommended amount of cloning vector (i.e., 0.5 mL) as long as the other reagents are also scaled down. 2. Minimally, the zCAR vector alone (here, the type II vector encoding CD3z/P1GEC) can be used for subsequent immunizations and CELLISA screening; however, the other two vectors are included here as controls to optimize immunization and illustrate surface antigen expression.

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3. For optimal transfection efficiency, it is important to ensure that the 293T cells are in a uniform monolayer of dispersed single cells, with minimum contact between neighboring cells. This can be achieved by forcefully dispersing the cells against the dish surface using a pipette when splitting. 4. The retrovirus produced in the manner presented here will be replication incompetent, but nonetheless amphotropic (4). Therefore, extended biosafety level 2 precautions should be taken to prevent exposure of the handler to the virus and the possibility of contamination of other samples. A waste container with bleach should be kept in the biosafety cabinet, where all pipettes that have come in contact with the virus are to be rinsed prior to disposal. Care should also be taken to properly decontaminate the biosafety cabinet following retrovirus handling, for instance wiping down and UV treating the cabinet, according to the manufacturer’s instructions. 5. Flow cytometric analysis of the virus-producing 293T tranfectants is recommended, in that the proportion of cells expressing GFP is a good indicator of transfection efficiency and quality of the retrovirus produced (³50% GFP+ cells is optimal). 6. During spin-fection (4), maintaining a temperature close to 37°C is crucial for ensuring membrane fluidity and efficient retroviral transduction of the cells. After the centrifugation step, it is possible to observe some amount of pelleted virus, which appears as a “fine dust” at the bottom of the wells. This observation can serve to indicate efficient retrovirus production. 7. Optionally, cells expressing FLAG-tagged variants can also be sorted using an a-FLAG antibody to select for cells expressing high levels of the fusion receptor at the cell surface. However, mAb-mediated cross-linking of the zCAR on BWZ cells can lead to activation-induced cell death in some instances, so high GFP reporter expression is usually sufficient. 8. Immunization can be performed using the BWZ.zCAR cells alone, if the antigen of interest is highly immunogenic (e.g., highly divergent). However, because the immunization step is independent of CELLISA screening, theoretically, any form of the antigen or any immunization protocol may be followed here (e.g., purified protein antigen, peptide antigen, or adjuvant-enhanced immunization); the resulting hybridomas may be screened by CELLISA using the BWZ.zCAR cells to ensure subsequent specificity for native cell surface protein. 9. It is crucial to use serum-free immunization buffer to minimize the host immune response to bovine serum proteins. 10. It is crucial to isolate as many splenocytes as possible to preserve all the specificities available from immunization. All of the tools used during the homogenization step should be rinsed with media into the cell strainer, and a plunger from a syringe

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can be used to disrupt all the tissues into a single cell suspension to ensure that cell loss is minimal during this process. Red blood cell lysis is sometimes used in lieu of Lympholyte separation; however, this can lead to some loss of viability of the splenocytes, so care should be taken. 11. When fusing mouse splenocytes with the mouse P3 fusion partner, rapidly growing hybridomas usually first emerge by day ~7–9; when fusing rat splenocytes with P3 cells, this usually takes somewhat longer, occurring by day ~9–11; and when fusing hamster splenocytes with P3 cells, outgrowth can take significantly longer, occurring by day ~11–14. Choosing a myeloma fusion partner closely related to the splenocyte species origin can ensure more efficient viable hybridoma fusion and outgrowth. 12. If fresh tips are not used for each well when using a 12-well multichannel pipette to harvest the original supernatants from the 96-well plates to transfer them into fresh empty plates (or preprepared CELLISA screening plates, see below), the direction of the handling should be noted and kept constant for all handling (e.g., row A-H or row H-A in the case of conserving tips by using one set of tips per hybridoma plate). This ensures that if small amounts of contaminating supernatants are carried over from well to well, the results can be interpreted correctly. Consistently, if a strong mAb is carried over during handling, a linear series of activated wells in a single column (a “comet” effect) can be observed during CELLISA development, where the degree of activation follows a titration series starting at the well containing the actual specificity. This phenomenon has been observed in this instance (Fig. 4) and with other antibodies made by our group (1, 4), and usually indicates strong mAb specificity in the initial supernatant well. 13. To conserve the capture reagent, the primary antibody volumes in the original CELLISA capture plates can be transferred to new plates prior to washing; this generates additional plates for further use, for instance in validation experiments. We have reused the primary capture antibody successfully up to five to seven times with minimal loss of sensitivity during validation and subcloning. 14. In order to minimize the contribution of the culture medium (phenol red) to the colorimetric background observed (uncleaved CPRG is yellow while enzymatic cleavage results in a red-purple color), the 96-well plate should be blotted on a piece of clean paper towel after the medium is “flicked out” or discarded; this ensures a minimal amount of residual media; optionally, the plates can be washed once using PBS. 15. There are a few points that should be kept in mind when interpreting results of the BWZ reporter cell assay. Firstly, if there are cell types present in the assay other than the BWZ

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cells themselves (e.g., macrophages), these cells can increase the background signal observed (4). Secondly, if a ligand recognized by the zCAR of interest is present on the BWZ cells themselves, a low level of “cis” or auto-activation can be observed, even in the absence of antibody stimulation (this depends on the levels of specific zCAR surface expression – usually, if cis receptor–ligand interactions occur, only low GFP/zCAR levels may be achieved in the initial sorts) (8). Nonetheless, antibody-mediated stimulation of BWZ.zCAR reporters should still be visible above this baseline activation (8). Thirdly, it is important to ensure that the plates used in the assay are free of contamination, since bacterial, yeast, or fungal b-gal activities can result in false-positive signals. 16. We usually subclone hybridomas by limiting the dilution into one 96-well plate each at 1 cell/well and 0.1 cell/well and repeating this three times to ensure: (1) that the hybridoma is truly monoclonal, having only 1 specificity; (2) that the hybridoma of interest is not lost due to outgrowth of other cells or clones; and (3) that high-level antibody secretion is maintained by the clones over time to ensure stable secretion when scaling up mAb production. These plates can be screened and validated by CELLISA using the “recycled” primary antibody. References 1. Mesci, A., and Carlyle, J. R. (2007) A rapid and efficient method for the generation and screening of monoclonal antibodies specific for cell surface antigens. J Immunol Methods 323, 78–87. 2. Karttunen, J., Sanderson, S., and Shastri, N. (1992) Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. Proc Natl Acad Sci USA 89, 6020–4. 3. Sanderson, S., and Shastri, N. (1994) Lacz Inducible, antigen/MHC-specific T-cell hybrids. Int Immunol 6, 369–76. 4. Carlyle, J. R., Jamieson, A. M., Gasser, S., Clingan, C. S., Arase, H., and Raulet, D. H. (2004) Missing self recognition of Ocil/Clr-b by inhibitory NKR-P1 natural killer cell receptors. Proc Natl Acad Sci USA 101, 3527–32. 5. Sancho, D., Mourao-Sa, D., Joffre, O. P., Schulz, O., Rogers, N. C., Pennington, D. J., Carlyle, J. R., and Reis e Sousa, C. (2008) Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin. J Clin Invest 118, 2098–110. 6. Voigt, S., Mesci, A., Ettinger, J., Fine, J. H., Chen, P., Chou, W., and Carlyle, J. R. (2007) Cytomegalovirus evasion of innate immunity by

subversion of the NKR-P1B:Clr-b missing-self axis. Immunity 26, 617–27. 7. Carlyle, J. R., Mesci, A., Ljutic, B., Belanger, S., Tai, L. H., Rousselle, E., Troke, A. D., Proteau, M. F., and Makrigiannis, A. P. (2006) Molecular and genetic basis for strain-dependent NK1.1 alloreactivity of mouse NK cells. J Immunol 176, 7511–24. 8. Tai, L. H., Goulet, M. L., Belanger, S., Troke, A. D., St-Laurent, A. G., Mesci, A., ToyamaSorimachi, N., Carlyle, J. R., and Makrigiannis, A. P. (2007) Recognition of H-2K(b) by Ly49Q suggests a role for class Ia MHC regulation of plasmacytoid dendritic cell function. Mol Immunol 44, 2638–46. 9. Carlyle, J. R., Mesci, A., Fine, J. H., Chen, P., Belanger, S., Tai, L. H., and Makrigiannis, A. P. (2008) Evolution of the Ly49 and Nkrp1 recognition systems. Semin Immunol 20, 321–30. 10. Mesci, A., Ljutic, B., Makrigiannis, A. P., and Carlyle, J. R. (2006) NKR-P1 biology: from prototype to missing self. Immunol Res 35, 13–26. 11. Kearney, J. F., Radbruch, A., Liesegang, B., and Rajewsky, K. (1979) A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J Immunol 123, 1548–50.

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Chapter 16 Use of Colloidal Silica-Beads for the Isolation of Cell-Surface Proteins for Mass Spectrometry-Based Proteomics Yunee Kim, Sarah Elschenbroich, Parveen Sharma, Lusia Sepiashvili, Anthony O. Gramolini, and Thomas Kislinger Abstract Chaney and Jacobson first introduced the colloidal silica-bead protocol for the coating of cellular plasma membranes in the early 1980s. Since then, this method has been successfully incorporated into a wide range of in vitro and in vivo applications for the isolation of cell-surface proteins. The principle is simple – cationic colloidal silica microbeads are introduced to a suspension or monolayer of cells in culture. Electrostatic interactions between the beads and the negatively charged plasma membrane, followed by cross-linking to the membrane with an anionic polymer, ensure attachment and maintain the native protein conformation. Cells are subsequently ruptured, and segregation of the resulting plasma membrane sheets from the remaining­ cell constituents is achieved by ultracentrifugation through density gradients. The resulting membrane-bead pellet is treated with various detergents or chaotropic agents (i.e., urea) to elute bound proteins. If proteomic profiling by mass spectrometry is desired, proteins are denatured, carbamidomethylated, and digested into peptides prior to chromatography. Key words: Colloidal silica, Plasma membrane isolation, Cell-surface proteins, Membrane proteomics

1. Introduction Cell-surface receptors represent an important class of membrane and membrane-associated proteins. Analysis of the membrane proteome is hampered by the heterogeneity and amphiphilic nature of these proteins. The low abundance of this class of proteins – only 2% of all cellular proteins belong to the plasma membrane (1) – further impedes their detection. Although immense scientific interest has propelled the invention and refinement of various methods tackling the aforementioned obstacles, coverage of the membrane proteome remains far from being exhaustive. Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_16, © Springer Science+Business Media, LLC 2011

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Proteomic principles for the isolation of cell-surface proteins encompass centrifugation (2–4) and extraction techniques (5, 6), affinity-based approaches (e.g., lectin- and antibody-based protocols) (7–11), as well as methods exploiting the chemical characteristics of the analytes. The latter have proven to be valuable tools for the mass spectrometry (MS)-based description of cell-surface proteomes and mainly comprise protocols for the chemical capture of glycoproteins, biotinylation, and silica-bead coating (12). The chemical capture of both glycoproteins and biotinylation relies on the chemical labeling of specific residues in targeted proteins, whereas silica-bead coating exploits the electrostatic forces between the target and reagent. With glycosylation being one of the most abundant posttranslational modifications, a comprehensive profile of cell-surface proteins can be established by the chemical capture of N- or both N-and O-glycosylated proteins using hydrazide chemistry (13, 14). Captured proteins can be subsequently enriched and analyzed. Similarly, selective covalent linking of biotinylation reagents to plasma membrane protein moieties, followed by affinity purification and MS detection, has proven to be a valuable method for cell-surface protein isolation, and biotinylation reagents can be tailored to meet the needs of different experimental conditions (15). In vivo applicability of biotinylation has been demonstrated, though extravasation of the reagent into surrounding tissues remains a caveat of this technique (16). The introduction of a chemical modification, which persists on the peptide and will thus be detected in downstream MS analysis, also adds confidence in the identification of bona fide cell-surface proteins. The high specificity conveyed by targeting selected residues (i.e., glycosylation site, residues targeted by the biotinylation reagent used), though, comes at the expense of tagging only surface proteins that contain these moieties. Important in vitro (3, 14, 17, 18) and in vivo studies (16, 19, 20) have applied all three protocols to the study of diverse scenarios (e.g., cancer, signaling pathways, and stem cell markers), thus validating their feasibility and contributing to our state of knowledge. A continued role of these approaches, possibly implementing improvements to increase efficiency, in future cell membrane research is anticipated. In this chapter, we focus on the isolation of cell-surface proteins using silica-bead coating. This method, successfully applied to the isolation of membrane proteins in their physiological state for nearly 30 years, is based upon the binding of the negatively charged membrane to a positively charged solid support. It thus represents a further development of earlier approaches exploiting this physical principle, like binding to polylysine or polyethylenecoated beads (21). In brief, the method relies on electrostatic attachment of the negatively charged cell-surface to cationic silica microbeads,

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Silica Bead Coating Silica bead particles PAA cross-linking PAA

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Fig. 1. Silica-beads are attached to the positively charged cell-surface. Subsequently, attached beads are cross-linked and the remaining free positive charges are blocked using polyacrylic acid (PAA). Cell lysis and ultracentrifugation yield purified, silica-bead-bound membrane proteins. These are eluted from the beads and can subsequently be prepared for mass spectrometric analysis.

followed by cross-linking of the beads by an anionic polymer (see Fig. 1). After cell rupture, the procedure yields large open sheets of high-density plasma membranes, which can be easily separated from cell debris and lysate by centrifugation. Furthermore, the rapid coating of surface proteins and subsequent cross-linking guarantee the preservation of native protein orientation in the lipid bilayer, prevent the formation of membrane vesicles upon cell lysis by stabilizing an open sheet structure, and shield the targeted proteins from chemical or enzymatic manipulation. Co-isolation of intracellular material is kept at a minimum by immediate blockage of silica-beads through cross-linking. Thus, in their first description of the technique, Chaney and Jacobson report a 15- to 17-fold enrichment of probed plasma membrane markers (21). Furthermore, the method is capable of distinction between different plasma membrane domains (e.g., the apical and basolateral sides of the plasma membrane) (22–24). An early study by Jacobson’s group reports the successful application of the method to selectively describe the topology of three distinct domains of HeLa cell plasma membranes (22). Similarly, the apical and basolateral plasma membranes of bovine aortic endothelial cells were characterized (24). An adapted ­version of the method allowed Sambuy and Rodriguez-Boulan to

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investigate the apical membrane of canine endothelial kidney cells closely on the microscopic level to further our understanding of epithelial cell-surface polarity (25). Stolz and coworkers used the silica-bead procedure to monitor changes in both extracellular matrix components and epithelial growth factor receptor occurring on the surface of sinusoidal endothelial liver cells in response to partial heparectomy in rats (26). The more recent coupling of the silica-bead method to powerful MS platforms has led to an enhanced detection of proteins in isolated fractions, thus making the approach an attractive technique in proteomics today. For instance, Rahbar and Fenselau integrated an adapted protocol successfully into their proteomics work-flow to characterize the plasma membrane of suspension and adherent cell cultures. In their study with human multiple myeloma (RPMI 8226) and human breast cancer cells (MXR MCF-7), a yield of 50% plasma membrane-annotated proteins was reported (23). They also exploited a similar strategy to quantify changes in cell-surface proteins between drug-susceptible and drug-resistant human breast cancer cell lines (MCF-7 and MXR MCF-7 cells) (27). Recently, Hör and colleagues combined the technique with quantitative proteomics to identify novel plasma membrane protein substrates for the ubiquitin ligase MARCH9 (28). Several studies have reported the feasibility of silica-bead coating in vivo, thus enabling the capture of cell-surface proteins in their native microenvironment, which is grossly modified under tissue culture conditions (20). The group of Schnitzer used a modified protocol to isolate endothelial cell-surface membrane proteins in vivo from different rat tissues by perfusing the vasculature of animals with a silica-bead solution (20, 29). Not only did their work facilitate proteomic mapping of the targeted cells, but it also revealed putative receptor targets that are accessible to biological agents from the vasculature, which could in turn have great implications, e.g., for antibody-based cancer therapy. Li et  al. recently adapted this strategy for the isolation of plasma membrane proteins from freshly isolated mouse hepatocytes and from rat liver sinusoidal endothelial cells (30, 31). Arjunan and colleagues have reported a conflicting observation regarding the silica-bead coating method for the isolation of cardiac microvascular surface proteins. Although the authors could convincingly demonstrate the adherence of the silica-beads to the luminal surface of the cardiac microvasculature, only a small number of proteins/plasma membrane proteins were identified. The authors argue that the rigid structure of the cardiac muscle tissue results in the loss of silica-beads during tissue homogenization (19). Although proteins annotated to other sub-cellular structures are often identified in any organellar proteomics study, and reasons for this have been intensely debated in the field for decades (32), the protocol has proven successful in our hands (33), even for the isolation of cardiac plasma membrane proteins

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(unpublished data). Interestingly, the silica-bead approach promises to be especially valuable for the characterization of endothelial cells in tissue types with more penetrable vascular systems (e.g., fenestrated endothelium), which in turn hampers alternative approaches, like biotinylation (31). With the advent of high-throughput mass spectrometry and sophisticated bioinformatics platforms, the field of cell-surface proteomics has rapidly evolved, allowing for comprehensive coverage of cell-surface proteomes, cell signaling pathway analyses, as well as the discovery of therapeutic targets. The silica-bead coating method of cell-surface protein isolation, in particular, has held a marked position in membrane proteomics and, in many cases, proven to be superior to other cell-surface isolation techniques. It is expected that the technique will continue to facilitate the expansion of our understanding of this important class of proteins.

2. Materials 2.1. Reagents

1. HPLC-grade solvents (water, methanol, acetonitrile, formic acid, trifluoroacetic acid (TFA), acetone, HCl). 2. MES (2-(N-morpholino)ethanesulfonic acid), NaCl, sucrose, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), urea, Triton® X-100, CaCl2 KCl, NH4HCO3, and Na2CO3 are of biotechnology grade. 3. Colloidal silica-beads (LUDOX® CL colloidal silica, suspension in water), polyacrylic acid (PAA), Nycodenz® (Histodenz™), and iodoacetamide (IAA) (Sigma-Aldrich Co). 4. Protease inhibitor (Complete Mini, EDTA-free) (Roche Applied Science). 5. Dithiothreitol (DTT). 6. PPS Silent® Surfactant (Protein Discovery, Inc). 7. MacroSpin™ Columns (The Nest Group, Inc). 8. Proteomics-grade trypsin.

2.2. Silica-Bead Coating of Plasma Membranes and Cell Rupture

1. MES-buffered saline (MBS): 25  mM MES, pH 6.5, and 150 mM NaCl. 2. Colloidal silica-bead solution: 1 or 10% for coating, dissolved in MBS. 3. PAA solution: 0.1%, dissolved in MBS. 4. Sucrose/HEPES solution: 250 mM sucrose, 25 mM HEPES, pH 7.4, 20 mM KCl, and 20 ml/ml protease inhibitor (see Note 1). 5. Glass dounce homogenizer. 6. Syringe and needle (for suspension cells, see Note 2).

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2.3. Density Gradient Centrifugation of Plasma Membrane Proteins

1. Density gradient medium: Nycodenz® gradient of 27.5–40.0% in sucrose/HEPES solution. 2. Ultracentrifuge capable of reaching 100,000 × g (our lab uses rotor # SW40 Ti, Beckman Coulter). 3. Ultracentrifuge tubes (Ultra-Clear Beckman Instruments, Inc).

2.4. Protein Elution/ Solubilization

Centrifuge

Tubes,

1. TX-100 Buffer: 1% Triton® X-100, 400  mM NaCl, and 25 mM HEPES. 2. Urea Buffer: 8 M urea, add 100 mM DTT for a final concentration of 2 mM (see Note 3). 3. PPS Silent® Surfactant solution: 0.2% PPS Silent® Surfactant in 50 mM NH4HCO3 and 40% methanol. 4. Wash Buffer: 0.025 M Na2CO3.

2.5. Protein Precipitation for TX-100 Buffer-Treated Samples

1. Acetone (prechilled to –20°C).

2.6. Preparation of Sample for MS

1. Resuspension Buffer: 8 M Urea, 100 mM Tris, and pH 8.5. Before use, add 100 mM DTT at a ratio of 1:50. 2. 100 mM IAA, freshly prepared. 3. 100 mM NH4HCO3, pH 8.5. 4. 100 mM CaCl2. 5. MS-grade trypsin. 6. 2.5% TFA. 7. Desalting column; (e.g., MacroSpin™ column kit). 8. Acetonitrile. 9. HPLC-grade water. 10. 0.1% TFA. 11. Elution Buffer: 70% acetonitrile, 0.1% TFA. 12. 5% acetonitrile, 0.1% formic acid buffer. 13. 200 mM HCl.

2.7. General Materials

1. Shaker or rotator. 2. Cell culture supplies (scraper, plates, microscope). 3. Vortex. 4. Bench-top centrifuge. 5. Microfuge tubes. 6. Incubator set at 37°C.

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3. Methods 3.1. Silica-Bead Coating of Plasma Membranes

For adherent cells (recommended volumes are for the confluent monolayer adherent cells growing on 100-mm diameter plates, see Note 4): 1. Discard cell culture media. 2. Place tissue culture plates containing the monolayer of cells on a flat bed of ice. 3. Gently wash the cells three times with 10 ml of ice-cold MBS. 4. Overlay with 1% ice-cold colloidal silica-bead solution. Incubate for 10 min on ice and adjust the plates accordingly to ensure complete coverage (see Note 5). Discard solution. 5. Wash the cells three times with 10 ml of ice-cold MBS. 6. Overlay with 10 ml of ice-cold 0.1% PAA solution. Incubate for 10 min on ice and adjust the plates accordingly to ensure complete coverage (see Note 6). Discard solution. 7. Add 2 ml sucrose/HEPES solution to the first plate, scrape the cells into the solution, then transfer cell suspension to the next plate, and harvest the cells into the same solution. After the harvesting of all plates, combine cell suspensions (see Note 7). For suspension cells: 1. Transfer the suspension cells from flask to centrifuge tubes and sediment the cells by centrifugation at 1,000 × g and 4°C for 5 min. 2. Wash the cell pellet three times with ice-cold MBS. 3. Prepare a 10% silica-bead solution and add the cells dropwise, using a syringe and needle or a pipette (see Note 2). Place on ice and rock gently for 10 min (see Note 8). 4. Transfer the cells to a centrifuge tube and sediment by centrifugation at 1,000 × g and 4°C for 5 min. 5. Wash the pellet three times with ice-cold MBS. 6. Prepare a 0.1% PAA solution and add cells drop-wise. Place on ice and rock gently for 10 min. 7. Transfer the cells to a centrifuge tube and sediment by centrifugation at 1,000 × g and 4°C for 5 min. 8. Wash the pellet three times with ice-cold MBS.

3.2. Cellular Rupture

1. Place the harvested cells in a centrifuge tube and centrifuge at 1,000 × g and 4°C for 5 min. 2. Remove the supernatant. If desired, save an aliquot of the supernatant for Western Blotting (see Note 8).

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3. Resuspend the cell pellet in 1 ml of sucrose/HEPES. Place in a glass dounce homogenizer and homogenize to rupture the cells (at least 5 strokes or until the solution becomes cloudy, see Note 9). 4. Take a small aliquot (50 ml) and check under a microscope to ensure the complete rupture of cells. If desired, save an aliquot of this fraction for Western Blotting (see Note 8). 3.3. Density Gradient Centrifugation of Plasma Membrane Proteins

1. Prepare a discontinuous Nycodenz® sucrose/HEPES gradient (as shown in Fig. 2 and Table 1, see Note 10). 2. Dilute sample with Nycodenz® and place it on top of the Nycodenz® sucrose/HEPES gradient (recommended: 25% Nycodenz®, see Note 11). 3. Gently add 1  ml of sucrose/HEPES solution to cover the sample. 4. Prepare an exact counterbalance to each tube. 5. Ultracentrifuge at 100,000 × g and 4°C for 1 h (see Note 12). 6. Once the membrane pellet has reached the bottom of the tube, discard the supernatant (see Note 13). 7. Resuspend the membrane pellet in 500  ml of 0.025  M Na2CO3. Vortex and shake or rotate for 30 min at 4°C. 8. Centrifuge on a bench-top centrifuge at 5,000 × g and 4°C for 20  min. Remove the supernatant and proceed to the Subheading 3.4 – Protein Elution/Solubilization.

Fig. 2. Careful layering of the Nycodenz® solutions (as prepared according to Table 1) in increasing density yields a discontinuous gradient for comprehensive purification of silica-bound membrane proteins.

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Table 1 Recipe for a 27.5–40% discontinuous Nycodenz® gradient preparation 100% Nycodenz® (ml)

Sucrose/HEPES solution (ml)

Final volume (ml)

Gradient (%)

1.375

3.625

5

27.5

1.5

3.5

5

30

1.75

3.25

5

35

2.0

3

5

40

3.4. Protein Elution/ Solubilization

For the elution/solubilization of membrane proteins, three alternative reagents may be used as follows (see Note 14): 1. TX-100 buffer: Resuspend the pellet in 200  ml of TX-100 Buffer. Transfer the suspension to a microfuge tube. Shake or rotate for 1 h at 4°C. Centrifuge on a bench-top centrifuge at 5,000 × g and 4°C for 20 min to bring the pellet back down. Collect the supernatant (if desired, take an aliquot of this membrane-enriched fraction for Western Blotting) and proceed to the Subheading  3.5 – Protein Precipitation for TX-100 Buffer-Treated Samples. 2. 8 M Urea with 2 mM DTT: Resuspend the pellet in 200 ml Urea Buffer. Incubate with shaking or rotation for 30 min at 37°C. Centrifuge on a bench-top centrifuge at 5,000 × g and 4°C for 20 min. Collect the supernatant (if desired, take an aliquot of this membrane-enriched fraction for Western Blotting) and proceed to the Subheading 3.6 – Preparation of Sample for MS step 2 (see Note 15). 3. PPS Silent® Surfactant solution: Resuspend the pellet in 100– 250  ml of PPS Silent® Surfactant solution. Gently pipette repeatedly (if desired, take an aliquot of this membraneenriched fraction for Western Blotting). Proceed to the Subheading 3.6 – Preparation of Sample for MS step 1.

3.5. Protein Precipitation for TX-100 Buffer-Treated Samples (See Note 16)

1. To the volume of recovered supernatant, add 5 times the volume of acetone (at –20°C). 2. Allow proteins to precipitate overnight at –20°C. 3. Bring down protein pellet by centrifugation at 5,000×g and 4°C for 20 min. 4. Resuspend the pellet in ice-cold acetone and centrifuge again. Repeat. 5. Discard acetone and dry pellet for 30 min at 37°C. 6. Proceed to the Subheading 3.6 – Preparation of Sample for MS step 1.

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3.6. Preparation of Sample for Mass Spectrometry

For approximately 30–200 mg of protein 1. For TX-100 Buffer or PPS Silent® Surfactant-treated samples, prepare Resuspension Buffer and add 50 ml to the recovered membrane-enriched fractions. Shake or rotate at 37°C for 30 min. 2. Add freshly prepared 100 mM IAA for a final concentration of 8 mM. Shake or rotate at 37°C for 30 min. 3. Dilute sample with 100  mM NH4HCO3. Add enough to bring the urea concentration from 8 to ~1.5 M. 4. Add 100 mM CaCl2 for a final concentration of 2 mM. 5. Add trypsin for enzyme:protein of 1:30–1:40. 6. Digest overnight at 37°C with shaking or rotation (see Note 17). 7. Stop the digestion: For samples solubilized in TX-100 Buffer or 8 M Urea with 2 mM DTT, stop the digestion by adding ~50 ml of 2.5% TFA and proceed to desalting. For samples solubilized in PPS Silent® Surfactant solution, stop the digestion by adding HCl to a final concentration of 200  mM. Incubate at 37°C for 45  min. Centrifuge sample at 5,000 × g at 4°C for 10  min (see Note 18). Collect the supernatant and proceed to desalting. Desalting (each centrifugation step is performed at ~200 × g, see Note 19). 8. Condition the MacroSpin™ column with 500  ml of acetonitrile. Centrifuge and discard flow-through in the collection tube. 9. Condition the column two times with 200 ml of HPLC-grade water. 10. Add sample to the column. Centrifuge and discard flowthrough from the collection tube. 11. Wash the column by centrifuging with 200 ml of 0.1% TFA. Repeat. 12. Into a new collection tube, elute the peptides by adding 200 ml of Elution Buffer to the column. Centrifuge. Add an additional 200  ml of Elution Buffer. Centrifuge and collect the recovered eluate (peptides). 13. Concentrate the peptides by drying at 45°C using a vacuum concentrator. 14. Resuspend the resulting peptide pellet in ~40  ml of 5% ­acetonitrile 0.1% formic acid buffer (see Note 20).

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4. Notes This protocol has delivered acceptable results in our hands using the procedure and materials described above. We strongly recommend that users complete several quality controls prior to using the isolated membrane fractions. For example, complete rupture of cells should be ensured under the microscope. Fractions obtained from Subheading 3, steps 2 and 4 should be probed by Western Blotting against known intracellular organelle and membrane protein markers (see Fig. 3). If necessary, adjust the protocol. Critical readjustment of conditions might also be necessary when applying the protocol to a new cell type. The following notes are provided as guides for the development and optimization of silica-bead coating protocols that are suited to individual experimental needs. 1. Protease inhibitor should be added to the sucrose/HEPES solution immediately before use. The purpose of the protease inhibitor is to block the enzymatic activity of endogenous proteases that will be deliberated during cell homogenization and might otherwise contribute to protein degradation.

Fig. 3. Purity of sample fractions obtained from the Subheading 3, Steps 2 and 4 can be probed by Western Blotting (Figure from Elschenbroich et  al. J Proteome Res. 2009; 10:4860–9).

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2. For suspension cells, ensure that the gauge of the needle is not so small that it would force premature rupture of the cells. If cells are large or easily perturbed, use a pipette and pipette tip instead. 3. Prepare a stock solution of 8 M urea, 100 mM Tris, pH 8.5 (can be kept at room temperature), and a separate stock solution of 0.1 M DTT (which must be frozen at –20°C). Before use, add 100 mM DTT at a ratio of 1:50 to arrive at a final concentration of 2 mM DTT. 4. During the whole procedure, ensure cooling of sample preparation to prevent degradation of proteins. 5. Make sure the cell layer is completely coated with silica-bead solution during the entire incubation step and readjust the plate position if necessary. Gentle movement of the plates after half of the incubation time ensures thorough silica-bead coverage of cells. During this incubation step, silica-beads tightly attach to negative charges on the cell-surface. After incubation, discard silica solution completely by soaking up the remaining liquid with a laboratory wipe (to this purpose, plates can be placed onto the wipe with part of the rim facing upside-down). It is important to remove all excess silica-beads to prevent them from reacting with the PAA cross-linker. 6. During this step, PAA links silica-beads to each other, thus not only completing the formation of pellicles, but also neutralizing all exposed positive charges on the nonattached surface of the beads, which might otherwise capture intracellular proteins after cell rupture. 7. The total volume of the solution should be kept to a minimum, thus enhancing the complete sedimentation of particles during centrifugation. This is especially important when applying the protocol for the first time or to new cell types. As stated several times, complete cell harvest should be ensured using a microscope. 8. Depending on the cell type used, intact cells or cellular debris form the pellet in this centrifugation step. Cells that are easily disrupted break open during scraping, thus releasing intracellular proteins into the buffer (in this case, the supernatant might be kept if these proteins are of interest). For more sturdy cell types (e.g., skeletal muscle cells), intact cells are collected through centrifugation. If cells rupture during this step and consequently release intracellular proteins into the supernatant, they might be saved (storage at –80°C). An aliquot of this fraction can be subjected to Western Blotting to probe for the presence of intracellular marker proteins. 9. Homogenization of cells should be complete while avoiding the extended exposure to the shear forces. It is, therefore,

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necessary to optimize the number of strokes for each cell type individually using a microscope to monitor the cell rupture. Furthermore, with sturdier cells, an additional lysis procedure may be required. This can be achieved by the addition of lysis buffer followed by alternative cellular disruption methods (e.g., nitrogen cavitation, sonication, etc.). 10. Prepare solutions as outlined in Table 1. To prepare a discontinuous Nycodenz® gradient, very carefully place solutions on top of each other, starting with the densest. Then, very slowly let the following solution slide down the wall of the centrifugation tube. This avoids mixing of solutions of different density. If the gradient is prepared correctly, clear boundaries will be visible between layers. 11. Dilute 100% Nycodenz® 1:2 with sucrose/HEPES-buffer and add to an equal amount of prepared sample (thus arriving at a final Nycodenz® concentration of 25% in the sample). Place the sample carefully on top of the gradient preparation. The sample must be the least dense of all gradient layers, so it can be assumed that only the silica-bead pellicles will migrate through the gradient during ultracentrifugation but not the sample solution as a whole. 12. The ultracentrifugation time and speed must be optimized according to the type of rotor to allow for sedimentation of the silica-coated membrane pellet. 13. The silica-bead pellicles should form a firm pellet after this step. If the pellet does not reach the bottom of the tube, transfer it to a microfuge tube with some of the gradient medium, and then spin at 14,000 × g and 4°C for 20  min. Then, add homogenization buffer to decrease the density of the gradient and spin again using the same conditions. This should allow for sedimentation of the pellet. 14. Three different reagents for the elution/solubilization of proteins are described here. Note that these reagents have produced acceptable results in our hands, but optimizations according to the unique conditions of each experiment are warranted, and thus other reagents may be substituted. 15. Samples treated with 8 M urea and 2 mM DTT in the ­elution/ solubilization step already have sufficient urea to denature proteins. Thus, Resuspension Buffer need not be added, and IAA can be added directly to this sample. 16. Triton® X-100 detergent may interfere with peptide detection and must, thus, be completely removed prior to MS analysis. This can be achieved by precipitation of the proteins and subsequent resolubilization into another buffer. 17. Do not exceed 18 h of digestion to avoid nonspecific cleavage of proteins.

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18. At low pH, the PPS molecule is cleaved and subsequently removed by centrifugation, thereby eliminating the interfering effects of the detergent on MS sensitivity. 19. Desalting is necessary to remove buffer salts, which interfere with MS analysis. 20. Store at 4°C if MS analysis is to be carried out within 1 week. Otherwise, store at −80°C until use. References 1. Kearney, P., Thibault, P. (1953) Bioinformatics meets proteomics – bridging the gap between mass spectrometry data analysis and cell biology. J Bioinform Comput Biol 1, 183–200 2. Brakke, M. K. (1953) Zonal separations by density-gradient centrifugation. Arch Biochem Biophys 45, 275–90. 3. Dormeyer, W., van Hoof, D., Braam, S. R., Heck, A. J., Mummery, C. L., and Krijgsveld, J. (2008) Plasma membrane proteomics of human embryonic stem cells and human embryonal carcinoma cells. J Proteome Res 7, 2936–51. 4. Lund, R., Leth-Larsen, R., Jensen, O. N., and Ditzel, H. J. (2009) Efficient isolation and quantitative proteomic analysis of cancer cell plasma membrane proteins for identification of metastasis-associated cell surface markers. J Proteome Res 8, 3078–90. 5. McCarthy, F. M., Burgess, S. C., van den Berg, B. H., Koter, M. D., and Pharr, G. T. (2005) Differential detergent fractionation for nonelectrophoretic eukaryote cell proteomics. J Proteome Res 4, 316–24. 6. McCarthy, F. M., Cooksey, A. M., and Burgess, S. C. (2009) Sequential detergent extraction prior to mass spectrometry analysis. Methods in molecular biology (Clifton, N.J) 528, 110–8. 7. Elortza, F., Nuhse, T. S., Foster, L. J., Stensballe, A., Peck, S. C., and Jensen, O. N. (2003) Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins. Mol Cell Proteomics 2, 1261–70. 8. Ghosh, D., Krokhin, O., Antonovici, M., Ens, W., Standing, K.G., Beavis, R.C., Wilkins, J.A. (2004) Lectin affinity as an approach to the proteomic analysis of membrane glycoproteins. J Proteome Res 4, 841–50. 9. Guo, L., Eisenman, J. R., Mahimkar, R. M., Peschon, J. J., Paxton, R. J., Black, R. A., and Johnson, R. S. (2002) A proteomic approach for the identification of cell-surface proteins shed by metalloproteases. Mol Cell Proteomics 1, 30–6.

10. Lawson, E. L., Clifton, J.G., Huang, F., Li, X., Hixson, D.C., Josic, D. (2006) Use of magnetic beads with immobilized monoclonal antibodies for isolation of highly pure plasma membranes. Electrophoresis 27, 2747–58. 11. Watarai, H., Hinohara, A., Nagafune, J., Nakayama, T., Taniguchi, M., and Yamaguchi, Y. (2005) Plasma membrane-focused proteomics: dramatic changes in surface expression during the maturation of human dendritic cells. Proteomics 5, 4001–11. 12. Elschenbroich, S., Kim, Y., Medin, J. A., and Kislinger, T. (2010) Isolation of cell surface proteins for mass spectrometry-based proteomics. Expert Rev Proteomics 7, 141–54. 13. McDonald, C. A., Yang, J. Y., Marathe, V., Yen, T. Y., and Macher, B. A. (2009) Combining results from lectin affinity chromatography and glycocapture approaches substantially improves the coverage of the glycoproteome. Mol Cell Proteomics 8, 287–301. 14. Wollscheid, B., Bausch-Fluck, D., Henderson, C., O’Brien, R., Bibel, M., Schiess, R., Aebersold, R., and Watts, J. D. (2009) Massspectrometric identification and relative quantification of N-linked cell surface glycoproteins. Nat Biotechnol 27, 378–86. 15. Elia, G. (2008) Biotinylation reagents for the study of cell surface proteins. Proteomics 8, 4012–24. 16. Rybak, J. N., Ettorre, A., Kaissling, B., Giavazzi, R., Neri, D., and Elia, G. (2005) In vivo protein biotinylation for identification of organ-specific antigens accessible from the vasculature. Nat Methods 2, 291–8. 17. Conn, E. M., Madsen, M. A., Cravatt, B. F., Ruf, W., Deryugina, E. I., and Quigley, J. P. (2008) Cell surface proteomics identifies molecules functionally linked to tumor cell intravasation. J Biol Chem 283, 26518–27. 18. Nunomura, K., Nagano, K., Itagaki, C., Taoka, M., Okamura, N., Yamauchi, Y., Sugano, S., Takahashi, N., Izumi, T., and Isobe, T. (2005) Cell surface labeling and mass spectrometry reveal diversity of cell surface markers and

16  Isolation of Cell-Surface Proteins for Mass Spectrometry s­ ignaling molecules expressed in undifferentiated mouse embryonic stem cells. Mol Cell Proteomics 4, 1968–76. 19. Arjunan, S., Reinartz, M., Emde, B., Zanger, K., and Schrader, J. (2009) Limitations of the colloidal silica method in mapping the endothelial plasma membrane proteome of the mouse heart. Cell Biochem Biophys 53, 135–43. 20. Durr, E., Yu, J., Krasinska, K. M., Carver, L. A., Yates, J. R., Testa, J. E., Oh, P., and Schnitzer, J. E. (2004) Direct proteomic mapping of the lung microvascular endothelial cell surface in  vivo and in cell culture. Nat Biotechnol 22, 985–92. 21. Chaney, L. K., and Jacobson, B. S. (1983) Coating cells with colloidal silica for high yield isolation of plasma membrane sheets and identification of transmembrane proteins. J Biol Chem 258, 10062–72. 22. Mason, P. W., and Jacobson, B. S. (1985) Isolation of the dorsal, ventral and intracellular domains of HeLa cell plasma membranes following adhesion to a gelatin substrate. Biochim Biophys Acta 821, 264–76. 23. Rahbar, A. M., and Fenselau, C. (2004) Integration of Jacobson’s pellicle method into proteomic strategies for plasma membrane proteins. J Proteome Res 3, 1267–77. 24. Stolz, D. B., and Jacobson, B. S. (1992) Examination of transcellular membrane protein polarity of bovine aortic endothelial cells in vitro using the cationic colloidal silica microbead membrane-isolation procedure. J Cell Sci 103, 39–51. 25. Sambuy, Y., and Rodriguez-Boulan, E. (1988) Isolation and characterization of the apical surface of polarized Madin-Darby canine kidney epithelial cells. Proc Natl Acad Sci U S A 85, 1529–33. 26. Stolz, D. B., Ross, M. A., Salem, H. M., Mars, W. M., Michalopoulos, G. K., and Enomoto, K. (1999) Cationic colloidal silica membrane perturbation as a means of examining changes at

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the sinusoidal surface during liver regeneration. Am J Pathol 155, 1487–98. 27. Rahbar, A. M., and Fenselau, C. (2005) Unbiased examination of changes in plasma membrane proteins in drug resistant cancer cells. J Proteome Res 4, 2148–53. 28. Hor, S., Ziv, T., Admon, A., and Lehner, P. J. (2009) Stable isotope labeling by amino acids in cell culture and differential plasma membrane proteome quantitation identify new substrates for the MARCH9 transmembrane E3 ligase. Mol Cell Proteomics 8, 1959–71. 29. Oh, P., Li, Y., Yu, J., Durr, E., Krasinska, K. M., Carver, L. A., Testa, J. E., and Schnitzer, J. E. (2004) Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy. Nature 429, 629–35. 30. Li, X., Jin, Q., Cao, J., Xie, C., Cao, R., Liu, Z., Xiong, J., Li, J., Yang, X., Chen, P., and Liang, S. (2009) Evaluation of two cell surface modification methods for proteomic analysis of plasma membrane from isolated mouse hepatocytes. Biochim Biophys Acta 1794, 32–41. 31. Li, X., Xie, C., Cao, J., He, Q., Cao, R., Lin, Y., Jin, Q., Chen, P., Wang, X., and Liang, S. (2009) An in vivo membrane density perturbation strategy for identification of liver sinusoidal­ surface proteome accessible from the vasculature. J Proteome Res 8, 123–32. 32. Bergeron, J. J., Au, C. E., Desjardins, M., McPherson, P. S., and Nilsson, T. (2010) Cell biology through proteomics--ad astra per alia porci. Trends Cell Biol 20, 337–45. 33. Elschenbroich, S., Ignatchenko, V., Sharma, P., Schmitt-Ulms, G., Gramolini, A. O., and Kislinger, T. (2009) Peptide separations by ­on-line MudPIT compared to isoelectric ­focusing in an off-gel format: application to a ­membrane-enriched fraction from C2C12 mouse skeletal muscle cells. J Proteome Res 8, 4860–69.

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Chapter 17 Transfection-Based Genomic Readout for Identifying Rare Transcriptional Splice Variants Larry J. Dishaw, M. Gail Mueller, Robert N. Haire, and Gary W. Litman Abstract Understanding the transcriptome, defined as the complete transcriptional component of the genome, is far more complex than originally considered. Even with the near fully resolved human and mouse genomes, for which extensive databases of transcribed sequence data (e.g., expressed sequence tags) are available, it is presently not possible to experimentally recover or computationally predict the full range of transcription products that derive from multiexon genes. Many genes are tightly regulated, which could include alternative processing of RNA, and lead to significant underrepresentation of many transcripts. A multitude of factors in addition to cell lineage- and developmental stage-specific expression as well as shortcomings in computational methods result in a less than complete understanding of transcriptional complexity. Here, we describe an approach to predict and evaluate a more complete repertoire of transcriptional products that derive from specific genetic loci with attention toward analysis of immune receptor genes. This approach is particularly useful in identifying gene products, including alternative splice forms, that originate from complex multigene families. Key words: Transcriptome, Alternative splicing, HEK293T, BAC/PAC expression

1. Introduction Accurate prediction and characterization of transcriptional repertoires, particularly those arising from multiexon genes or gene loci associated with complex immune receptor families, are of essential importance to functional biology. Conventional expressed sequence tag (EST) databases are an invaluable resource, but can be limited by developmentally regulated genes or (despite the efforts at normalization) remain biased toward relatively abundant

Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_17, © Springer Science+Business Media, LLC 2011

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predominant transcript

rare variant transcripts

Fig.  1. Illustration of a hypothetical case of alternative splicing. In many RT-PCR ­experiments, a single predominant transcript can be detected and characterized for multiexon genes. In some cases, alternatively spliced products can be detected. Other alternatively spliced products are generally predicted to be under strict genetic control or occur in tissue- and/or cell-specific environments such that their detection in ­standard RT-PCR or cDNA/EST libraries is difficult.

transcripts (see Fig.  1). Likewise, computational methods that predict gene products (1) can be biased toward predictions that follow typical transcriptional rules and are only as effective as the algorithm’s ability to recognize canonical splice sites. These algorithms can predict gene products inaccurately from complex loci that include repetitive DNA or coding regions from tightly clustered multigene families. Even though modern high-throughput methods for DNA sequence determination have diminished reliance on large insert DNA libraries in genomic assemblies, these resources are commercially available for commonly studied species or can be produced for other species as well as different individuals from the same species. Libraries remain a significant resource for a wide range of functional investigations. Large insert DNA libraries also afford a means to characterize the complete transcriptional potential of a specific locus. The method described here (see Fig.  2) provides a highly informative approach for evaluating alternative splicing. The approach is based on large insert genomic clones (e.g., bacterial artificial chromosomes, BACs), which have been selected on the basis of a genetic region of interest, either by sequence prediction or via library screening. DNA is purified from the large insert clone and transfected into human embryonic kidney (HEK)293T cells (or other empirically tested cell lines) without any modification of the genomic clone, and RACE RT-PCR is used to recover and characterize a range of transcription products.

17  Identifying Rare Transcriptional Splice Variants

large genomic inserts

genomic DNA

245

Screen library with oligo probes

Bacterial artificial chromosomes (BAC)

kb ladder

characterize variants

expected

TAcloning

alternate products

transfect BAC clones into HEK293T cells isolate total RNA

RT- PCR colony PCR

Fig. 2. Schematic illustration of the procedure for BAC/PAC transfection and RNA prediction. Library screen-mediated BAC or PAC clones are selected and transfected into HEK293T cells. Total RNA is recovered and BAC-specific transcription is detected by RT-PCR. Unpurified or unfractionated PCR products are cloned and screened by colony PCR for variation in insert size. Alternative splicing is characterized by sequencing variable-sized inserts.

2. Materials 2.1. BAC and PAC Library Screening

1. Commercial sources of BAC or PAC genomic libraries (available as filter arrays) and archived clones are available for a wide range of animal species and genetically defined animal strains (e.g., BACPAC Resources; Children’s Hospital Oakland Research Institute, Oakland, California, USA). For the materials used in this description, an amphioxus, Branchiostoma floridae, BAC library was purchased from BACPAC Resources. PAC libraries for amphioxus and zebrafish, Danio rerio, as well as clones were courtesy of Dr. C.T. Amemiya (see Note 1). 2. Probes/primers (20–40 nucleotides, avoiding repetitive sequence; oligonucleotides should be test hybridized when possible). 3. dCTP (alpha-32P) 3,000 Ci/mmol. 4. Terminal deoxynucleotidyl transferase (20,000  U/ml), 10× buffer, 2.5  mM CoCl2 (New England Biolabs), and deionized water (or molecular grade water).

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5. Prehybridization/hybridization buffer: 6× SSC (20× SSC: 3 M NaCl, 0.3 M Na Citrate), 5× Denhardts (40× Denhardts: 0.02% (w/v) Ficoll, 0.02% (w/v) polyvinylpyrolidone, 0.02% (w/v) BSA), and 0.5% (w/v) SDS, 0.05% (w/v) sodium pyrophosphate. 6. Wash buffer: 6× SSC, 0.5% (w/v) SDS. 7. Filter strip buffer: 1 mM EDTA, 0.1% (w/v) SDS. 2.2. Alternative Random Labeling Approach

1. dCTP (alpha-32P) 3,000 Ci/mmol. 2. Gel purification kit, such as column-based kits from Qiagen. 3. Random primers (random 7-mers, mixed at 1 mg/ml each). 4. 10× RLK buffer: 500 mM Tris–HCl (pH 6.8), 50 mM MgCl2, 100 mM beta-mercaptoethanol, 4 mg/ml BSA, 2 mM dATP, 2 mM dTTP, and 2 mM dGTP. 5. Klenow fragment of DNA polymerase I (5,000 U/ml). 6. Radioactive labeling stop solution: 20  mM NaCl, 20  mM Tris–HCl pH 7.5, 2  mM EDTA, 0.25% SDS, and 1  mM dCTP. 7. Prehybridization/hybridization buffer: SET buffer (0.6  M NaCl (5 M stock), 0.02 M EDTA (0.5 M stock, pH 8), 0.2 M Tris–HCl (2 M stock, pH 8), 0.5% SDS (20% w/v stock), and 0.1% Na pyrophosphate). 8. Blot wash buffer: 1× SSC, 0.05% Na pyrophosphate, 0.1% SDS. 9. Strip buffer: 1 mM EDTA, 0.1% SDS.

2.3. Isolation of the BAC and PAC Clones

1. LB medium supplemented with chloramphenicol (12.5  mg/ ml) for BAC clones or kanamycin (25 mg/ml) for PAC clones. 2. Maxi kit: Nucleobond BAC 100 (Macherey-Nagel, Cat No. 740–579).

2.4. Cell Culture

1. HEK293T cells. 2. RPMI media: RPMI 1640 media (Gibco-BRL), 10% (v/v) Fetal bovine serum, 2  mM glutaMAX (Invitrogen), and 1  mM sodium pyruvate (Cellgro). Antibiotic is not necessary.

2.5. Cell Culture Transfection

1. Transfection media: Opti-MEM1 (Gibco, Invitrogen).

2.6. Screening for Transcripts

1. Gene-specific oligonucleotides: PCR primers.

2. Transfection reagent: Lipofectamine 2000 (Invitrogen).

2. Reverse transcriptase: Superscript III and associated reagents (Invitrogen). 3. PCR reagents. 4. TOPO cloning kit (Invitrogen).

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3. Methods A single genomic locus is employed as a template to capture representative transcripts. The approach can be used independently or in conjunction with EST and cDNA library screening. Two different methods are employed to capture extended chromosomal regions: (1) a BAC spanning a region of interest is identified from a relational (contig) map and (2) specific BACs or PACs are identified by library screening using gene-specific probes (oligonucleotides or PCR products). If a genome sequence is available, oligonucleotides complementary to the ends of the locus can be used to verify that positively hybridizing genetic regions are complete or a related (crosshybridizing) member of a gene family is represented in the captured BAC insert. The transcriptional complexity of a locus is assessed by transfecting a BAC clone (see Note 2) into a eukaryotic tumor cell line (e.g., HEK293T), which is somewhat indiscriminate in its transcriptional readout, and the transcription products are recovered. This approach has been evaluated in our laboratory using previously sequenced and assembled BAC and PAC clones as well as unsequenced BAC clones (identified through screening). A variety of transcripts, some of which could not be predicted by conventional approaches, have been recovered. The method can provide a more comprehensive analysis of transcriptional repertoires to facilitate the characterization of unknown or incompletely characterized genetic regions, and thereby further illuminate the complexities of gene expression and regulation. 3.1. BAC and PAC Library Screening

1. Hybridization of labeled oligonucleotides to genomic library filters remains a significant technology for detecting and characterizing individual genetic loci (2). 2. Oligonucleotides, rather than PCR products, are easy to label, provide sufficient signal, exhibit very little if any background, and are very easy to strip from filters (see Note 3). 3. Choose appropriate oligonucleotides for library screening. We recommend dual screening with two or more oligonucleotide probes. The first oligonucleotide can be designed to target the coding region, and other oligonucleotides can be designed to flank both sides of the coding region. Implementing this type of process increases the recovery efficiency. 4. End labeling with a single radioactive nucleotide and the TdT enzyme is rapid, efficient, and highly sensitive. End label the 20–40 bp oligo as such: 30 ng oligo, 5 ml 10× NEB buffer 4, 5 ml 2.5 mM CoCl2, 3 ml 32P dCTP, 1 ml TdT, and deionized water to 50 ml total; incubate at 37°C for 30 min.

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5. Prehybridization is carried out for 1 h at 55°C (layer filters between nylon mesh); add probe, without purification (see Note 3). A small hybridization buffer volume is preferred and hybridize overnight at 55°C. 6. Wash filters at 55°C at least five times, 20 min each. Increase the wash volume in the last two washes; filters are transferred to large-volume glass bakeware. 7. Expose filters (wrapped in plastic wrap) to X-ray film overnight (to several days) at −80°C. Some positives may be evident overnight, but the background development assists in accurate reading of coordinates. 8. Do not allow filters to dry. Once hybridization results have been scored, probes should be stripped from filters. 9. Oligo probes can be effectively stripped by bringing strip buffer to a boil and pouring over the top of filters. Continue to soak filters in buffer for 15 min at room temperate; repeat. Verify that the probes have been adequately stripped by exposing the filter to X-ray film for several days. 10. In rare cases, oligo hybridization may be insufficient for library screening. We recommend an alternative screening approach using random-primed probes (see Note 2 and Subheading 3.2). 3.2. Random Label Kit-Based Probes

1. RT-PCR products were recovered from a 1% agarose gel. Approximately 25  ng template gel-purified DNA, 3  ml of random 7-mer mix, and water to 42 ml are mixed by pipetting. Heat at 95°C for 5 min and quickly place on ice. Add 5 ml of 10× RLK buffer, 3  ml of 32dCTP, and 1  ml of Klenow fragment. 2. Incubate at 37°C for 10 min. 3. The reaction is terminated by adding 50  ml of the Labeling Stop Solution. Estimate the specific activity of probe with a 1:20 dilution of the probe spotted onto a Whatman DE81 filter paper. Wash filter with phosphate buffer, deionized water, rinse in 95% ethanol, and dry by vacuum filtration. Detect the specific activity. A probe of at least 15,000 counts/min (after the 1:20 dilution) is usually optimal. 4. Place library filters in a closed, leak-proof, cylinder between nylon screens to separate the filters (or any suitable leakproof container) and add several milliliters of prehyb/hyb buffer. Incubate with rotation (or with agitation) for at least 1 h at 65°C. 5. Reduce the volume of prehyb/hyb buffer to a minimum required to adequately cover the filters and add the probe (melted at 95°C for 10 min in a small volume, <1 ml, of TE). Incubate overnight at 65°C with rotation (or agitation).

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6. Wash filters in several volumes of wash buffer at least five times for 20 min each at 52°C. For high-stringency washes, increase the temperature to 65°C and if necessary, use 0.1× SSC in wash solution. 7. Strip filters carefully with boiling strip buffer; see the method above. 3.3. Isolation of the BAC and PAC Clones

1. A single colony BAC or PAC clone from a fresh streak is grown in a starter culture and transferred to 150 ml of media (in appropriate antibiotic) and grown overnight at 37°C. 2. Isolated BAC or PAC DNA with a Maxi kit according to manufacturer’s instructions. 3. Carefully quantitate DNA; keep the DNA stock semi-sterile as this will be used to transfect into culture.

3.4. Cell Culture

1. Purified BAC or PAC DNA is transfected into HEK293T cell line (see Note 4), which was originally derived from embryonic kidney cells immortalized with adenovirus (3). Recent microarray studies of HEK cells have shown that although they were derived from embryonic kidney, they demonstrate many characteristics of neuronal progenitors (4), which likely explains their extensive transcriptional capacities. 2. Prior to transfection, HEK293T cells are grown until at least 90% confluent in RPMI media.

3.5. Cell Culture Transfection

1. BAC DNA is transfected into the cultures using Lipofectamine-2000 (Invitrogen) according to the manufacturer’s recommendations. Optimizations can be carried depending on the nature of the foreign DNA, but we have found that the efficiency of RNA synthesis from the transfected template DNA is enough for RT-PCR detection, if the transfected DNA is at least 1 mg DNA/cm diameter of culture plate (i.e., 10  mg of 50–150  kb BAC DNA/10-cm plate). 2. After 72 h, remove media and immediately lyse the cells for total RNA recovery using 1  ml of a single step guanidinebased RNA isolation reagent (see Note 5) per 10-cm plate. 3. DNase-treat the total RNA, if necessary (see Note 6).

3.6. Screening for Transcripts

1. Gene-specific oligonucleotides: At least one gene-specific oligonucleotide probe is required to identify transcripts. An upstream-sense oligonucleotide can be used to screen the transcripts via 3' RACE-type approaches with standard oligo-dT-primed cDNAs as template (5). 2. Random-primed double-stranded DNA probes (RLK, Random Label Kit-derived methods) are commonly used to hybridize DNA blots, primarily restriction enzyme digested

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blots or Southern blots, and can be used to hybridize genomic library filters. However, this method is only advisable if end-labeled oligonucleotides fail to provide sufficient signal. PCR product-derived dsDNA probes can yield considerable background. If the probe hybridizes to repetitive DNA, stripping of the blot can prove exceptionally difficult. 3. Reverse transcription is carried out using Superscript III using at least 5  mg of the total RNA (most of the RNA in this procedure originates from the HEK293T cells): 5 mg of total RNA, 1 ml of 10 mM dNTP mix, and 1 ml of 50 mM oligo(dT)20 are heated to 65°C for 5  min and rapidly chilled on ice; combine (20 ml total) 4 ml of the 5× first strand buffer, 1 ml of 0.1 M DTT, 1 ml of RNase-OUT, and 1 ml of Superscript III and gently mix by pipetting. Incubation is carried out at 50°C for 45 min. RT enzyme is inactivated by heating for 10 min at 70°C; dilute to 50 ml with molecular grade water and use 2 ml for PCR reaction screening. 4. PCR reactions are used to screen for gene-specific transcripts. We perform PCR reactions according to the standard protocols (as recommended by the manufacturer) using 2  ml of cDNA as template and 1  ml of 20  mM gene-specific sense primer and 1 ml of 20 mM Oligo-dt primer (see Note 7) in 50 ml of reaction volumes. 5. The results of the PCR screen are analyzed using 15 ml of the reaction in a 1.5% agarose gel. If PCR products are visible (see Note 8), 2 ml of PCR reactions are cloned directly (without any purification) into a TOPO cloning vector for sequence analysis (see Fig. 2). 6. Screen is conducted by colony PCR using TOPO-specific vector primers (see Note 9), and at least one representative colony for each size product is selected for sequence analysis. 7. Confirm that the method has generated at least a set of known or “predicted” transcripts. Examine sequences to identify “alternative” transcripts (see Note 10). 8. Alternative approaches are available to detect transcripts from “unknown” genetic regions (see Note 11).

4. Notes 1. BACPAC Resources are a leading commercial provider of BAC and PAC libraries. The amphioxus (6) and zebrafish (7) PAC libraries and individual clones utilized for this method have been provided courtesy of Dr. Amemiya (Benaroya Research Institute, Seattle, WA, USA).

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2. A variety of approaches have been developed to modify the existing BAC or PAC clones for optimizing gene expression and/or gene delivery and integration in functional transgenesis systems (8–13). In our case, we were looking for a more comprehensive method to rapidly identify transcripts from a given genomic locus, and not to generate transgenic lines or animals for functional experiments. These options, of course, are open to the user. 3. Typically, the specific activity is adequate for homologous screening; end-labeled oligonucleotides do not require column purification, which avoids additional radioactive waste. 4. We have used HEK293T cells extensively in BAC and PAC transcription studies because they are easily accessible, easy to maintain, transfect at high efficiency, and effectively transcribe foreign DNA. The introduced DNA should be “foreign” so that transcripts originating from the BAC clone can be distinguished from endogenous transcripts, i.e., human BACs should not be transcribed in HEK293T cells. In this case, an alternative immortalized line, such as the mouse NIH 3T3 line, can be employed. Neither BAC nor PAC plasmid vectors encode the SV40 origin of replication (to take advantage of the SV40 large T antigen that HEK293T cells incorporate, as opposed to HEK293 cells), and very little evidence suggests that either cell line would be a superior expresser of large insert genomic clones. Questions such as these need to be addressed on an individual basis. 5. For total RNA isolation, RNA-bee (Tel-Test, Inc) has worked well, but others, such as Trizol (Invitrogen) or Tri Reagent (Molecular Research Center), are also suitable. 6. Total RNA isolated with one-step extraction reagents typically does not have a significant amount of contamination from genomic DNA. Transfected cell lines, on the other hand, tend to have large amounts of foreign DNA that could prove problematic in RT-PCR if the genes of interest have short or no introns. If the coding regions transcribed are separated by several introns, we have found that contaminating transfected BAC DNA does not pose a problem and DNase treatment can be avoided. 7. Oligo(dT)20 can be used as an antisense primer in PCR. Sometimes, PCR products are poorly resolved owing to primer slippage along the poly-A tail during reverse transcription or during the actual PCR priming event. This can be offset by using an anchored poly-dT primer, such as oligo(dT)20VN, in both RT reactions and in PCR or using a modified anchored oligo-dT primer, which possesses a 5¢ adaptor sequence that functions as a nested priming site for subsequent reactions (see Zhang and Frohman (1997) for a modified version of the 3¢ RACE oligo-dT primer designated Qt) (5).

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8. Occasionally, one round of PCR is insufficient. Some loci do not transcribe as efficiently as others. In cases where no, or faint, PCR products result, a second round of (nested) PCR is recommended. Take 1 ml of a 1:20 dilution of the first-round PCR and conduct a second round of PCR with the same primers or, if possible, a nested sense primer. 9. Usually, T7 and T3 or M13F and M13R are used as priming sites for the TOPO TA cloning kit (Invitrogen). In some cases, screening can be based on a more specific approach, such as one gene-specific primer and one vector-anchored primer. 10. We have employed this method to transcribe BAC and PAC DNA for predicting mRNA transcripts from several different immune receptor genes in amphioxus, including VCBPs (14) and zebrafish NITR genes ((15), Dishaw et al. unpublished). In one notable example, an assembled amphioxus PAC clone 30b18, which encodes a full-length VCBP3 gene locus, was transfected under standard conditions. Sequence prediction had previously revealed a prototypic VCBP3 transcript and two additional, but noticeably divergent, chitin-binding domain(CBD)-encoding open reading frames (ORFs), which encoded putative splice sites (14). To determine how this locus is transcribed, PAC clone 30b18 was transfected into HEK293T. RT-PCR revealed a predicted, full-length VCBP3 transcript, several variant transcripts, and a single transcript which spliced into the second alternative CBD-encoding ORF (see Fig. 3). This hypothetical transcript is disrupted by a splice-induced frameshift and generates stop codons downstream of the immunoglobulin domains, thus rendering the CBD a 3¢ UTR region (with polyadenylation signals). We confirmed this transcript by designing a primer specific to this CBD. Twenty individual amphioxus were screened in an

L

V1 domain

V2 domain

Alternative CBDs

CBD

a ∗

b

∗

results in Ig-only VCBP3

b

a Typical VCBP3

Ig-only VCBP3

Fig. 3. Detection of alternative splicing using PAC transfection and RT-PCR from HEK293T cells. In this example, amphioxus PAC clone 30b18 was identified by hybridization with a VCBP3 probe. Transfection and expression in HEK293T cells revealed both typical (a) and alternative (b) splicing patterns. Splicing into an alternate downstream exon generates a frameshift, premature stop codons, and polyadenylation signals. This unusual product was confirmed in amphioxus using RT-PCR directed at the alternate downstream exon.

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assay by RT-PCR using this primer and our standard upstream VCBP3 V1domain primer. The alternatively spliced product was confirmed in a single amphioxus individual. The RT-PCR product was a closely related splice variant that utilized the second alternate CBD of the locus (in place of the “normal” CBD) and encoded the splice-induced frameshift identified previously. The downstream CBD-encoding ORF is out-offrame with the upstream Ig domains and appears to behave as an alternative termination exon and provides polyadenylation signals. This transcript may be limited to certain haplotypes or maintained under specific regulation and suggests a functional relevance to VCBP3 without a CBD. 11. The method described here is effective at predicting transcripts from known genetic regions. Although the detection of transcripts is not possible without some prior sequence knowledge from which to design specific oligonucleotide primers, a variety of approaches exist for gathering transcriptional information from unknown genetic regions. Locus-specific primers can be designed by attaining flanking genomic sequence data using a variety of inverse PCR or genome-walking approaches (16, 17). Anchored BAC or PAC fragments are utilized to capture unknown cDNAs (18, 19). Alternatively, “light sequencing” of BAC or PAC clones can be used. In this last example, the clones are digested or sheared to generate fragments of 1–3 kb in size and ligated into a sequencing vector (not T-overhang), and randomly sequenced. Sequencing in the neighborhood of 100 random clones can provide essential information about flanking genetic regions. References 1. Salzberg, S., and Yorke, J. (2005) Beware of mis-assembled genomes. Bioinformatics 21, 4320–1. 2. Amemiya, C. T., Ota, T., and Litman, G. W. (1996) in “Nonmammalian Genomic Analysis: A Practical Guide” (Birren, B., and Lai, E., Eds.), Academic Press, Inc., San Diego. 3. Graham, F., Smiley, J., Russell, W., and Nairn, R. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36, 59–74. 4. Shaw, G., Morse, S., Ararat, M., and Graham, F. (2002) Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK293T cells. FASEB J 16, 869–71. 5. Zhang, Y., and Frohman, M. (1997) Using rapid amplification of cDNA ends (RACE) to

obtain full-length cDNAs. Methods Mol Biol 69, 61–87. 6. Amemiya, C., Prohaska, S., Hill-Force, A., Cook, A., Wasserscheid, J., Ferrier, D., PascualAnaya, J., Garcia-Fernàndez, J., Dewar, K., and Stadler, P. (2008) The amphioxus Hox cluster: characterization, comparative genomics, and evolution. J Exp Zool B Mol Dev Evol 310, 465–77. 7. Amemiya, C., and Zon, L. (1999) Generation of a zebrafish P1 artificial chromosome library. Genomics 58, 211–3. 8. Kim, S., Horrigan, S., Altenhofen, J., Arbieva, Z., Hoffman, R., and Westbrook, C. (1998) Modification of bacterial artificial chromosome clones using Cre recombinase: introduction of selectable markers for expression in eukaryotic cells. Genome Res 8, 404–12.

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9. Heintz, N. (2001) BAC to the future: the use of bac transgenic mice for neuroscience research. Nat Rev Neurosci 2, 861–70. 10. Giraldo, P., and Montoliu, L. (2001) Size matters: use of YACs, BACs and PACs in transgenic animals. Transgenic Res 10, 83–103. 11. Al-Hasani, K., Simpfendorfer, K., Wardan, H., Vadolas, J., Zaibak, F., Villain, R., and Ioannou, P. (2003) Development of a novel bacterial artificial chromosome cloning system for functional studies. Plasmid 49, 184–7. 12. Wang, Z., Longacre, A., and Engler, P. (2004) Retrofitting BACs with a selectable marker for transfection. Methods Mol Biol 256, 69–76. 13. Sparwasser, T., and Eberl, G. (2007) BAC to immunology–bacterial artificial chromosomemediated transgenesis for targeting of immune cells. Immunology 121, 308–13. 14. Dishaw, L., Mueller, M., Gwatney, N., Cannon, J., Haire, R., Litman, R., Amemiya, C., Ota, T., Rowen, L., Glusman, G., and Litman, G. (2008) Genomic complexity of the variable region-containing chitin-binding proteins in amphioxus. BMC Genet 9, 78. 15. Yoder, J., Litman, R., Mueller, M., Desai, S., Dobrinski, K., Montgomery, J., Buzzeo, M.,

Ota, T., Amemiya, C., Trede, N., Wei, S., Djeu, J., Humphray, S., Jekosch, K., Hernandez Prada, J., Ostrov, D., and Litman, G. (2004) Resolution of the novel immune-type receptor gene cluster in zebrafish. Proc Natl Acad Sci U S A 101, 15706–11. 16. Tsaftaris, A., Pasentzis, K., and Argiriou, A. (2010) Rolling circle amplification of genomic templates for inverse PCR (RCA-GIP): a method for 5’- and 3’-genome walking without anchoring. Biotechnol Lett 32, 157–61. 17. Tsuchiya, T., Kameya, N., and Nakamura, I. (2009) Straight walk: a modified method of ligation-mediated genome walking for plant species with large genomes. Anal Biochem. 388, 150–60. 18. Parimoo, S., Patanjali, S., Shukla, H., Chaplin, D., and Weissman, S. (1991) cDNA selection: ­efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments. Proc Natl Acad Sci U S A 88, 9623–7. 19. Lovett, M., Kere, J., and Hinton, L. (1991) Direct selection: a method for the isolation of cDNAs encoded by large genomic regions. Proc Natl Acad Sci U S A 88, 9628–32.

Chapter 18 Characterizing Somatic Hypermutation and Gene Conversion in the Chicken DT40 Cell System Nagarama Kothapalli and Sebastian D. Fugmann Abstract The secondary immunoglobulin gene diversification processes, somatic hypermutation (SHM), immunoglobulin gene conversion (GCV), and class switch recombination, are important for efficient humoral immune responses. They require the action of activation-induced cytidine deaminase, an enzyme that deaminates cytosine in the context of single-stranded DNA. The chicken DT40 B-cell line is an important model system for exploring the mechanisms of SHM and GCV, as both processes occur constitutively without the need for stimulation. In addition, standard gene targeting strategies can be used for defined manipulations of the DT40 genome. Thus, these cells represent an excellent model of choice for genetic studies of SHM and GCV. Problems arising from defects in early B-cell development that are of concern when using genetically engineered mice are avoided in this system. Here, we describe how to perform gene targeting in DT40 cells and how to determine the effects of such modifications on SHM and GCV. Key words: DT40, Somatic hypermutation, Gene conversion, AID, Gene targeting, Immunoglobulin

1. Introduction The efficient recognition and clearance of pathogens by the humoral immune system relies on a highly diversified repertoire of immunoglobulin (Ig) molecules. Upon encountering an antigen, the Ig genes of mature B cells in the periphery undergo two related secondary diversification processes to improve antigen recognition: somatic hypermutation (SHM) and Ig gene conversion (GCV) (1). Both processes are initiated by activation-induced cytidine deaminase (AID), which converts C to U in the context of single-stranded DNA (2). The resulting G:U mismatches are replicated or repaired in an error-prone fashion, leading to single

Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_18, © Springer Science+Business Media, LLC 2011

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point mutations in the case of SHM and multiple nucleotide changes in the case of GCV, respectively. The resulting subtle changes in the antigen recognition domain of the Ig polypeptides lead ultimately to higher affinity antigen receptors. The chicken DT40 B cell line has become a standard tool to study the molecular mechanism of both SHM and GCV (3). This cell line constitutively undergoes AID-dependent diversification of the IgH and IgL genes and allows for defined modifications and deletions of individual gene loci by standard gene targeting. Over the years, a large body of evidence has emerged from individual (and combined) gene deletion with respect to the involvement of distinct DNA repair enzymes in SHM and GCV. More recently, the DT40 model system has been utilized to identify cis-acting elements controlling SHM and GCV (4, 5). Using a systematic deletion approach, we have identified a ­cis-acting regulatory DNA sequence within the IgL gene that ­targets these processes specifically to this locus. Here, we describe how to generate DT40 cell lines with a defined alteration of a gene locus by gene targeting and provide a protocol to analyze and quantify SHM and GCV processes in such cells. Gene targeting in embryonic stem (ES) cells is a standard technique to generate genetically modified mice with targeted modification (e.g., deletions) of distinct locations within their chromosomes (6). This method relies on spontaneous homologous recombination between the chromosome and a plasmid containing two regions of homology (matching two neighboring regions on the chromosome) that flank a selection cassette. The result of a successful recombination is the exchange of the intervening region on the chromosome with the drug selection cassette. Using a respective selection drug, the cells in which the desired recombination events have occurred can be grown selectively. While this method works with relative efficiency in ES cells, it works poorly in almost all other cell types and cell lines. The basis for this discrepancy is not well understood. One notable exception is the chicken DT40 B cell line, as its genome can be altered in the very same fashion with reasonable efficiencies (7). For the analysis of SHM and GCV in the DT40 B cell line, a specific subclone is frequently used, clone 18 (CL18) (8). This particular clone carries a premature stop codon in the recombined allele of the IgL gene that renders it surface IgM− (the other allele is in its unrearranged germline configuration). Specific GCV events revert this stop codon, turning this particular cell and all its progeny into a surface IgM+ phenotype. Cell surface expression of IgM (and the events altering its status) is detectable by fluorescence-activated cell sorting (FACS), allowing for a convenient fast method to assess the ability of respective cell clones to undergo GCV. As this method is relatively inaccurate, it is necessary to confirm the phenotype by DNA sequencing of the IgL gene

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locus. Importantly, this scores all GCV events (and SHM events), including those that do not lead to a reversion of the premature stop codon, and is useful in identifying mutation events when a particular DT40 subclone is only available as an IgM+ variant.

2. Materials 2.1. Generation of Targeting Constructs

1. Phusion DNA polymerase. 2. Zero Blunt TOPO PCR cloning kit for sequencing. 3. Restriction enzymes. 4. pLox selection cassette plasmids: These plasmids were generated by H. Arakawa and J. M. Buerstedde (9).

2.2. Culture of DT40 Cells

1. Complete DT40 culture medium: Roswell Park Memorial Institute (RPMI-1640) medium, with l-glutamine supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% chicken serum, Penicillin/Streptomycin, 2 mM l-glutamine, and 10 mM HEPES (pH 7.4). FBS is heat inactivated by incubation at 55°C for 15 min. 2. Freezing medium: Mix 9  mL of complete DT40 culture medium and 1  mL of dimethylsulfoxide, and sterile filter through 0.22 mm syringe filter.

2.3. Cell Transfection by Electroporation

1. Selection medium: Complete DT40 culture medium is supplemented with the appropriate amount of the respective selection drug (see Table 1). Prepare selection medium fresh before using it, as the selection drugs have limited stability when stored at 4°C. 2. Hemocytometer.

Table 1 Selection drugs for DT40 cell transfectants Selection drug

Stock solution

Final concentration in selection medium

Hygromycin B

50 mg/mL in water

2.0–2.5 mg/mL

Puromycin

10 mg/mL in water

0.5 mg/mL

Blasticidin

10 mg/mL in water

20–30 mg/mL

Mycophenolic acid (Ecogpt)

20 mg/mL in DMSO

15–20 mg/mL

Histidinol

50 mg/mL in water

1 mg/mL

G418 (neomycin)

50 mg/mL in water

2 mg/mL

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3. Bio-Rad Gene Pulser and cuvettes (0.4  cm gap) (Bio-Rad, Hercules, CA). 4. 1× PBS. 2.4. Genomic DNA Preparation

1. 5-mL tubes. 2. 1× PBS. 3. 2× DNA lysis buffer: 200  mM NaCl, 40  mM Tris–HCl (pH 8.0), 2 mM EDTA, and 1% SDS. 4. Phenol:Chloroform:Isoamyl alcohol (25:24:1, v/v), molecular biology grade. 5. Chloroform. 6. Ethanol, molecular grade. 7. RNase A stock solution (100 mg/mL). 8. Proteinase K stock solution: Dissolve lyophilized proteinase K in 10  mM Tris–HCl (pH 7.4), 20  mM CaCl2, and 50% glycerol at 10 mg/mL. 9. 7.5 M ammonium acetate solution: Dissolve 5.78 g in water and adjust volume to 10 mL. 10. 10 mM Tris–HCl solution (pH 8.0): Dilute 1 M Tris–HCl (pH 8.0) 1:100 in water.

2.5. Southern Blotting

1. 0.25 M HCl solution: Add 21 mL of 37% HCl to a final volume of 1 L of water. 2. Transfer buffer: 0.4 M NaOH and 1 mM EDTA. 3. GeneScreen Plus membrane (Perkin Elmer, Waltham, MA) (see Note 1). 4. Whatman 3 M filter paper. 5. Heat-denatured salmon sperm DNA. 6. Expresshyb hybridization solution (Clontech, Mountain view, CA). 7. NEBlot kit (New England Biolabs, Ipswich, MA). 8. Illustra MicroSpin G-50 columns (GE healthcare, Waukesha, WI). 9. 2× SSC solution: 300  mM NaCl, 30  mM sodium citrate (pH 7.0). 10. Wash solution 1: 2× SSC, 0.1% SDS. 11. Wash solution 2: 0.1× SSC, 0.1% SDS.

2.6. Removal of Selection Cassettes Using Cre Recombinase

1. Serum-free medium: Complete DT40 culture medium without FBS and chicken serum. 2. Cre deletion medium: Add purified recombinant His-TatNLS-Cre (HTNC) protein to 300 mL serum-free medium at

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a final concentration on 6 mM (make this fresh immediately before use). 3. Recombinant HTNC protein: Express and purify HTNC according to Peitz et al. (10). Protein can be stored at −20°C in buffer containing 50% glycerol. 2.7. SHM and GCV Analysis by FACS and DNA Sequencing

1. Anti-chicken IgM, Birmingham, AL).

PE-conjugated

(Southern

Biotech,

2. Staining solution: Dilute anti-chicken IgM 1:200 in 1× PBS. Make fresh as required. 3. 1× PBS. 4. FACS tubes: Falcon 2052. 5. Phusion High-Fidelity PCR kit (New England Biolabs). 6. Oligonucleotides for VJ amplification: CVLF1 (5¢-ccatggc ctgggctcctctcctcctg-3¢) and CLA2 (5¢-gacagcacttacctgga cagctg-3¢). 7. Zero Blunt TOPO PCR cloning kit for sequencing (Invitrogen). 8. PureLink 96 Plasmid Purification System (Invitrogen). 9. Oligonucleotide for DNA sequencing: CHVLSR (5¢-agcctg ccgccaagtccaag-3¢).

3. Methods The generation of DT40 cells with defined alterations of their genome and the subsequent analyses of their phenotypes with respect to SHM and GCV are based on a number of individual standard molecular biology techniques described in detail below. The general experimental scheme providing an overview of the individual steps to be taken is shown in Fig. 1. 3.1. Generation of Targeting Constructs

1. The left and right homology regions (also called arms) in the gene targeting vector flank the chromosomal fragment to be deleted. Design PCR primers to amplify the arms such that the size of the products is at least 1,500 bp (the longer the better, but remember that the final construct can get very large). Add suitable restriction sites to 5¢-end of the primers to facilitate the subsequent cloning step. Choose BamHI if possible for the ends between which the selection cassette will be inserted (see Notes 2 and 3). 2. Amplify the arms by PCR from genomic DNA of DT40 cells using Phusion polymerase. Clone the products into the pCR4 blunt TOPO vector (Invitrogen) (see Note 4) and confirm their identity by DNA sequencing. Excise the fragments with

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N. Kothapalli and S.D. Fugmann Generate the gene targeting plasmid (including a loxP site-flanked selection cassette) Transfect DT40 cells with linearized targeting plasmid by electroporation Genotype the drug-resistant clones by Sounthern blot analyses (Optional: check for the absence of random integration events) Perform Cre-mediated deletion of the selection cassette for two independent clones Confirm Cre-deletion of selection cassette by Sounthern blot analyses Culture 12 subclones of two independent Cre-deleted subclones for 4 weeks Perform FACS analysis on days 14, 21, and 28 On day 28, prepare genomic DNA from two subclones per genotype PCR amplify VJ region and clone it into pCR4 vector Sequence 48 plasmid minipreps from each cloned PCR fragment (i.e. 96 per genotype) Analyze sequences using ClustalW and BLASTN to identify GCV and SHM events Calculate mutation event frequencies (number of mutation events / total number of base pairs sequenced)

Fig.  1. Flowchart of the experimental strategy. All important steps on the way from ­generating the targeting plasmid to the final calculation of mutation event frequencies are shown. A conservative time frame for generating and analyzing one phenotype is about 6 months.

an appropriate restriction digest, and clone them either sequentially (or via a 3-way ligation in a single step) into a linearized pBluescript plasmid. 3. Excise the selection cassette(s) from the respective pLox plasmids using BamHI, and insert it between the two arms of the targeting vector. 4. Prior to transfection, linearize 25–30 mg of the plasmid DNA (per transfection) by restriction digest using an enzyme that cuts only the pBluescript backbone but not within/between the homology arms or the selection cassette. Purify the linearized DNA by phenol/chloroform extraction followed by ethanol precipitation, and dissolve the plasmid in 50 mL of 1× PBS. 3.2. Culture of DT40 Cells

1. DT40 cells (wild type and genetically modified) are cultured in complete medium at 41°C with 5% CO2. Split the cells frequently to maintain a cell density between 1×105 and

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2×106  cells/mL. All cell culture procedures are performed under sterile conditions in a biological safety cabinet. 2. Freezer stocks are made for long-term storage of cell clones. Spin down a total of 1–2×106 cells at 300 × g, 25° for 5 min. Resuspend the pellet in 1  mL of freezing medium, and transfer it into a cryogenic vial. Wrap the cryovial in bubble wrap and place it in a −80°C freezer to slowly cool the cell suspension to avoid ice crystal formation. For long-term storage, transfer the freezer stocks to a liquid nitrogen or −140°C freezer (see Note 5). 3.3. Cell Transfection by Electroporation

1. Count the cells using a hemocytometer, and spin down 1×107 cells per transfection at 300 × g, 25°C for 5 min. Resuspend the pellet in 10 mL of cold 1× PBS and centrifuge again at 300 × g, 25°C for 5 min. Aspirate the supernatant, and resuspend the cell pellet in 0.8  mL of 1× PBS containing 25–30  mg linearized plasmid DNA (i.e., targeting vector). Mix thoroughly by pipetting up and down gently. 2. Transfer the cell/DNA suspension into a prechilled electroporation cuvette (0.4 cm gap), and place it on ice for 10 min. Adjust the settings on the Bio-Rad Gene Pulser electroporator to: Voltage = 580  V, Capacitance = 25 mFD, and Resistance = ¥ W. Wipe the outside of the cuvette dry using a kimwipe, place it in the electroporator pod, and initiate the pulse (see Note 6). The time constant (t) of the pulse decay should be between 0.6 and 0.8 ms (see Note 7). Place the cuvette back on ice for 10 min immediately after the electroporation. 3. Transfer the cells from the cuvette to a T75 flask containing 20 mL of prewarmed medium using a 1 mL pipet (see Note 8), and let them recover overnight in an incubator at 41°C with 5% CO2. On the next day, spin down the transfected cells at 300 × g, 25°C for 5 min, and resuspend the pellet in 40 mL of the appropriate selection medium. Plate the entire amount of cells as 200 mL aliquots in two 96-well plates, and let them grow for up to 10 days. 4. Individual colonies of cell clones appear 5–7 days after plating. Visually inspect each well, and choose wells with single colonies for further analyses (see Note 9). Once a colony occupies about 15–25% of the surface of the well, transfer it to a single well of a 6-well plate containing 2 mL of complete medium (without the selection drug). Grow the cells until the density is high enough to make freezer stocks, and continue the culture to obtain enough cells for genomic DNA preparation.

3.4. Genomic DNA Preparation

1. The drug-resistant cell clones are the result of the desired targeted integration events (at the gene locus of interest), aberrant targeted integration events (in which the gene locus

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of interest was altered but not with the expected outcome), or random integration events (somewhere else in the genome). To determine the genotype of each clone, the first step is to prepare genomic DNA and to use this genetic material for subsequent Southern Blots (see Subheading 3.5). 2. Genomic DNA is prepared using the proteinase K/phenol method. Spin down 1–2×106 cells at 300 × g, 4°C for 5 min in a 5-mL tube (Falcon 2063). Discard the supernatant and completely resuspend the cell pellet in 500 mL 1× PBS. Add 500  mL DNA lysis buffer and lyse the cells by repeatedly inverting the tube (see Note 10). The cell lysate is highly viscous, and it is advisable to use wide-bore tips for all subsequent pipetting steps to avoid shearing of the high-molecular genomic DNA. 3. To eliminate RNA and protein contamination from the genomic DNA preparation, RNase A and proteinase K digestions are performed. These can be done either sequentially or in a single step. First, add 10 mL RNase A stock solution to the cell lysate, and incubate at 37°C for 1 h. Second, add 10 mL of proteinase K stock solution and incubate at 55°C for at least 1 h. Alternatively, add both enzymes at the same time and incubate the reaction at 55°C overnight. 4. Subsequently, a phenol/chloroform extraction is performed to remove peptides and lipids from the lysate. Add 1 mL of phenol/chloroform/isoamylalcohol (see Note 11) to the proteinase K digest, and mix the sample well by inverting the tubes multiple times until a homogenous emulsion forms (see Note 12). Centrifuge the sample at 4,500 × g, 4°C for 10 min to separate the aqueous and organic phases. The aqueous phase (top layer) contains the genomic DNA, the white interface residual proteins, and the organic phase (lower phase) lipids and other hydrophobic molecules. Carefully remove the aqueous phase with a wide-bore pipet (see Note 13), and transfer it to a fresh 5-mL tube (Falcon 2063) containing 1 mL of chloroform. Mix the sample thoroughly by repeated inversion of the tube, and spin at 4,500 × g, 4°C for 10 min. Again, remove the aqueous phase carefully, and transfer it to a fresh 5-mL tube (Falcon 2063). Add 100 mL of 7.5 M ammonium acetate solution and 2 mL of 100% ethanol to precipitate the genomic DNA (see Note 14). Mix the sample gently by inverting the tube and place it on ice for 30 min. Spin down the genomic DNA (see Note 15) at 4,500 × g, 4°C for 10 min. Wash the pellet with 70% ethanol, and briefly air dry it. Add 50 mL of 10 mM Tris–HCl (pH 8.0) to the DNA pellet, and let it dissolve overnight at 4°C. The resulting genomic DNA solution should be highly viscous. Measure the DNA concentration using a spectrophotometer (see Note 16). Genomic DNA samples are stored at 4°C (see Note 17).

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1. Southern Blots are used to determine the genotype of the drug-resistant cell clones. The restriction enzyme(s) and the probe should be chosen such that the expected band pattern of the targeted integrant clearly differs from that of the parental cell line (see Fig. 3a). The probe that is used for hybridization should not be part of the targeting construct itself, as this might identify “false-positive” clones that have a random integration of the targeting vector. 2. For southern blotting, complete digestion of genomic DNA is critical. Use 10 mg of genomic DNA and digest it overnight with the respective restriction enzyme(s) in a total volume of 50 mL. For complete digestion, use 3–5 units of restriction enzyme per mg of DNA (see Note 18). It is important to also digest a sample of the genomic DNA of the parental cell line (on occasion, the sequence databases are incorrect, and a comparison of the experimentally obtained band pattern from the parental and potential targeted integrants helps to circumvent such issues). 3. Pour a 0.8% agarose gel using 1× TBE (add 0.5 mg/mL final concentration of ethidium bromide to visualize the DNA and the size marker after the run). Load the entire digest of each sample per lane, and load one lane with an appropriate DNA size standard. Perform electrophoresis in 1× TBE at 35–40 V for 15–16  h (the time of electrophoresis and percentage of the gel can be varied depending on the expected sizes of the bands). Visualize the DNA digest pattern (it is a continuous streak rather than distinct bands) with UV light (254 nm) on a transilluminator, and take a picture of the entire gel using a gel documentation system (see Fig.  3b). It is important to include a ruler in the picture to be able to accurately determine the band sizes in the final Southern blot pattern. 4. The DNA is transferred onto a GeneScreen Plus membrane by capillary action using the alkaline transfer method. Place the gel into a plastic container large enough to hold 500 mL of buffer. Submerge the gel in 500 mL of 0.25 M HCl and shake gently for exactly 17  min. The acid breaks the DNA into smaller pieces and facilitates the transfer of larger fragments. Pour off the acid and rinse the gel twice briefly with distilled water to remove the remaining acid. Soak the gel for 40–45 min with gentle agitation in 500 mL of transfer buffer to denature the DNA fragments completely. Place the gel in a standard capillary transfer Southern Blot setup (see Fig. 2 and Note 19), and transfer overnight. 5. Disassemble the transfer setup, and mark the position and orientation of the wells and the side of the membrane that faced the gel on the membrane using a ballpoint pen (this is important as it is impossible to determine the position of the

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Weight

glass plate Whatman paper (3 sheets)

glass plate Whatman paper

stack of paper towels GeneScreen Plus transfer membrane agarose gel transfer buffer

Fig. 2. Southern blot transfer by capillary action. Schematic drawing of a standard setup for Southern blot transfer. All important elements are shown. The plastic wrap is essential to focus the capillary action to the gel and to prevent evaporation of the transfer buffer.

membrane relative to the gel later on). Rinse the membrane briefly with 2× SSC to neutralize the transfer buffer, and crosslink the DNA to the membrane using the auto-crosslink setting of a Stratalinker (Stratagene, Cedar Creek, TX) (see Note 20). Transfer the membrane to a hybridization bottle with the gel side of the membrane facing the inside of the tube (see Note 21). To prehybridize the membrane, add 10 mL of ExpressHyb solution and 100 mL of freshly heatdenatured salmon sperm DNA to the bottle, and rotate it in a hybridization oven at 62°C for 2–3 h. 6. Radiolabel the DNA probe specific for the DNA fragments of interest with 32P a-dCTP using the NEBlot kit according to the manufacturer’s protocol (see Note 22). The excess radionucleotides are removed using sephadex G-50 spin columns. The percentage of incorporation of radioactivity is determined using a liquid scintillation counter by measuring the radioactivity of 1-ml aliquots taken before and after the G-50 purification. The incorporation should be at least 30%. Denature the probe at 95°C for 5 min, and place it immediately on ice for 2 min. Add the probe to the hybridization bottle (see Note 23) and continue to rotate it at 62°C overnight. 7. Remove the unbound excess probe by washing the membrane twice with wash solution 1 at 62°C for 30  min and twice with wash solution 2 at 62°C for 30 min. The last wash should remove very little radioactivity. Remove the membrane from the hybridization bottle and soak up any excess liquid by blotting on a whatman filter paper. Wrap the membrane in saran wrap to prevent potential contamination of the phosphoimaging screen and cassette. Expose an erased phosphoimaging screen with the membrane for 2–3  h (or overnight, depending on the strength of the signal). Scan the phospoimaging screen using a phosphoimager (see Fig. 3b). Alternatively, X-ray film can be used and developed under appropriate conditions.

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a left arm A A

selection cassette

right arm targeting vector

puroR

A probe

A

A puroR

b

probe

ethidium bromide-stained gel M

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P

M

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Southern blot + +

P

*

5 kb 4 kb 3 kb 2 kb

Fig. 3. Gene targeting and genotyping by Southern blot. (a) Schematic representation of the gene targeting strategy to replace the endogenous chicken IgL enhancer (open oval ) and its flanking region with a SV40 enhancer (filled oval ). The targeting vector consists of a left arm, a right arm, and a puromycin-resistance cassette (puroR) flanked by two loxP sites (triangles ). The parental allele and the expected targeted allele are shown and the position of the AvrII sites (A) are marked. The position of the probe used for the Southern blot analyses is indicated. (b) Left panel : AvrII restriction digest of genomic DNA from ten puromycin-resistant clones (31–40) and the parental cell line (P); right panel: corresponding Southern blot after hybridization with the probe shown in (A). The sizes of the bands in the marker lane (M) are shown on the left. Two clones (+) show a targeted allele (arrow ), the non-targeted parental allele is marked with an asterisk (Note that the two alleles of the parental cell line have a different restriction pattern in this case).

8. Determine the size of the visible DNA fragments using the ruler of the gel picture as the scale. Typically, two bands are visible per lane, corresponding to the two alleles. For clones with a successful targeted integration event, only one of the bands should be in a different position compared to the pattern obtained for the parental cell line. Use such positive clones for further analyses. To rule out that such clone also carries an additional random integration of the transfected plasmid DNA, additional Southern blots with cassette probes should be performed.

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3.6. Removal of Selection Cassettes Using Cre Recombinase

1. After targeted integration of the gene targeting vector, the antibiotics resistance cassette used for the selection of clones still resides within the disrupted genomic locus. To avoid interference of the transcribed selection marker with the surrounding locus, the cassette is flanked by two loxP sites and can be removed using Cre recombinase. This ultimately leaves a single loxP site behind as the footprint of the genetic manipulation. In the procedure described below, we use a cell-permeable version of the Cre protein (10). 2. Spin down 3×105 cells at 300 × g, 25°C for 5 min. Remove the supernatant completely, and resuspend the cell pellet in 300 mL of Cre deletion medium. Incubate the cell suspension at 41°C, 5% CO2 for 1–2 h to allow the Cre recombinase to enter the cells and to excise the selection cassette. Stop the reaction by adding 6  mL of complete medium, dilute the cells 1:10,000, and plate them as 200-mL aliquots (=one cell per well) in a 96-well plate to obtain single-cell clones. Individual colonies start to appear in about 3–5 days (see Note 24). Transfer single-cell clones to individual wells in a 6-well plate (see Note 25), and grow to obtain sufficient cell numbers for making freezer stocks and to prepare genomic DNA. The removal of the selection cassette is tested for each clone by Southern blot analysis using a suitable restriction enzyme/probe combination.

3.7. SHM and GCV Analysis by FACS and DNA Sequencing

1. After obtaining cell clones with the desired genotype by gene targeting (and successful removal of the selection cassette, if required), the SHM and GCV phenotypes of such cells are determined over 4 weeks of continuous culture. It is important to culture the parental cell line and a nontargeted “random” integration cell clone in parallel as controls. 2. Ensure that the starting cultures of all cell lines are mostly surface IgM− by cell sorting or subcloning (see Note 26) for the FACS-based gene conversion assay. The determination of the mutation event frequency (i.e., both GCV and SHM) by DNA sequencing is independent of the surface phenotype of the starting population. 3. The cells are seeded out in 96-well plates by limiting the dilution to obtain single-cell subclones. After 1 week, 12 individual single-cell clones from each genotype are transferred into 6-well plates containing 2 mL media per well. The cultures of all single-cell clones are split daily (at a ratio of 1:4 to 1:6) to maintain a cell density between 1×105 and 2×106 cells per mL. 4. On days 14, 21, and 28, the surface IgM phenotype is determined by FACS analysis. Spin down 1 mL of the culture of each clone at 300 × g, 25°C for 5 min. Discard the supernatant,

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2 100 100

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Fig. 4. GCV analysis by FACS. (a) FACS plot of a single DM3.5 subclone after 28 days of culture. The percentage of cells within the IgM+ gate is shown. (b) Scatter plot of the percentage of IgM+ cells in a 4-week culture experiment of 12 ­subclones of DM3.5. Each diamond corresponds to an individual subclone, and the average at each time point is indicated by a bar. (Note: We use the DT40 DM3.5 clone as an example for this chapter. The first report of its phenotype can be found in ref. 4).

and wash the cell pellet with 1 mL ice-cold 1× PBS. Resuspend the cells in 200  mL of staining solution and incubate for 45 min on ice in the dark. Wash the cells once with 1 mL ice-cold 1× PBS, and resuspend in 500  mL 1× PBS. Perform flow cytometry of the samples using FACS Calibur (Becton Dickinson) or equivalent instrument. The PE-conjugated a-chicken IgM is detected in the FL2 channel (see Fig. 4). Always use an unstained cell sample and samples of stained IgM+ and IgM− cultures as controls to ensure the correct compensation settings. The frequency of IgM+ cells in each experimental sample is correlated to the frequency with which GCV occurs in this subclone. The data are typically represented as scatter plots with the average percentage of IgM+ cells of each genotype indicated (see Fig. 4). 5. On day 28, cells are harvested for sequence analysis. Prepare genomic DNA from two subclones of each genotype, and amplify the VJ region using primer pair CVLF1/CLA2 using phusion DNA polymerase (see Note 27). Purify the reaction products (approx. 850 bp in size) by agarose gel electrophoresis, followed by gel extraction. Clone the purified PCR products into pCR4 using the Zero Blunt TOPO PCR cloning Kit (Invitrogen). 6. Set up 48 plasmid minipreps per cloned PCR product (i.e., 96 plasmid minipreps of each genotype), and perform the purification using the PureLink 96 plasmid purification system (Invitrogen) according to manufacturer’s protocol. Submit the minipreps to DNA sequencing with primer CHVLSR.

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7. The sequence analysis to identify GCV and SHM events is performed using Web-based bioinformatics software. Align all sequences using ClustalW (http://www.ebi.ac.uk/clustalw) (11) (see Note 28). The dominant sequence represents the parental sequence (see Fig.  5). Verify all differences in the nucleotide sequences by cross-checking with the chromatogram. Determine the type of each mutation (GCV or SHM) using the NCBI BLASTN program (http://blast.ncbi.nlm. nih.gov) (12). Choose approximately 50 nucleotides of sequence that contain the respective sequence alterations (in the case of GCV, these clusters of individual mutations can stretch over tens of nucleotides) and compare them to the “nucleotide collection nr/nt” database with the parameters “Organism: Gallus gallus” and “Entrez query: pseudo.” Perfect matches to mutated nucleotides in the query sequence are considered GCV events (see Note 29). Mutations with no match are considered non-templated SHM events. Count the numbers of mutation events (multiple changes by one GCV event represent a single event). Identical mutation events occurring in multiple sequences are counted only once.

Fig.  5. GCV and SHM analysis by DNA sequencing. (a) ClustalW alignment of 12 sequences obtained from a single subclone of DM3.5. Mutations are underlined and highlighted in bold. (b) Results of BLAST alignment searches in the pseudoVL gene (YVL) database for matches with mutated sequences, A04 and G02. Perfect matches with YVL8 and YVL5 were found, respectively. This indicates that these two pseudogenes served as donors for the respective GCV events.

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Completely identical mutated sequences are excluded, as they may be the result of a PCR amplification of a single event. Determine the total number of bases analyzed by choosing a window starting about 50 bp from the sequencing primer binding site and ending with the last nucleotide of PCR primer CVLF1 (excluding the bases of low-quality sequences and identically mutated sequences). Calculate the mutation event frequency by dividing the number of mutation events by the number of bases analyzed. This number is a measure of the GCV/SHM phenotype of the respective genotype.

4. Notes 1. Any positively charged nylon hybridization transfer membrane will work. 2. Make sure that none of the restriction sites is found with the arm itself. Add additional 4 nt to the 5¢-end of the oligos if you want to digest and clone the PCR products directly into pBluescript. 3. A comprehensive review of the design and generation of gene-targeting vectors for DT40 cell has been published recently (13). 4. Any commercially available blunt PCR product cloning vector will suffice. 5. Extended storage at −80°C reduces the viability of the cells. 6. The electroporator apparatus itself does not need to reside in the sterile environment of a biological safety cabinet. 7. The optimal voltage setting varies from individual electroporator to individual electroporator. If the time constant is to high/low, increase/decrease the voltage accordingly. 8. A small amount of cell suspension will remain in the cuvette; it is not necessary to retrieve it, and attempts to recover it by repeated pipetting will reduce the cell viability (as the cells are very fragile at this moment). 9. It is important to check the plates for colonies starting at day 3 in order to identify “true” single-cell colonies. When the colonies get too large, it is impossible to determine whether they originated from a single-cell clone. 10. Do not vortex the lysate, as this will lead to shearing and fragmenting of the high-molecular-weight genomic DNA. 11. Avoid the aqueous layer of TE buffer that lies on top of the phenol:chloroform:isoamylalcohol mix in the stock bottle.

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12. Do not vortex the crude genomic DNA preparation. This will lead to shearing of the high-molecular-weight DNA. 13. Try to avoid the interphase and the organic phase. It is good enough if you recover 90–95% of the aqueous phase. If you still get some of the interphase and/or organic phase, you will be able to get rid of it during the chloroform extraction. 14. Alternatively, the genomic DNA can be precipitated by adding 100 mL of 3 M sodium acetate solution (pH 4.8) and 2.5 mL of 100% ethanol. 15. The genomic DNA precipitate looks like a white tangled string that can usually be seen by eye. 16. Use a Nanodrop (Thermo Fisher Scientific, Wilmington, DE) spectrophotometer to directly measure the concentration using 2 mL of the DNA solution. Alternatively, dilute an aliquot of the DNA in a larger volume of water sufficient for a traditional cuvette-based spectrophotometer. 17. Do not freeze the genomic DNA solutions, as this leads to fragmentation of the high-molecular-weight genomic DNA. 18. Some restriction enzymes are methylation sensitive and do not cut the genomic DNA if the cytosines are methylated. Also, some enzymes exhibit star activity, and prolonged digests should be avoided in such cases. 19. Presoak all whatman paper sheets and the membrane in transfer buffer, and avoid trapping air bubbles between the gel and the membrane. 20. The cross-linking is not essential as the DNA is already bound to the membrane by strong ionic bonds, but still it does not hurt. 21. Avoid trapping bubbles underneath the membrane, as this will lead to blank spots on the Southern blot. 22. Equivalent random hexamer/octamer-based labeling kits from other manufacturers work equally well. 23. Add the probe directly to the hybridization mix, and avoid pipetting it directly onto the membrane as this leads to dark spots and smudges on the Southern blot after exposure. 24. Mark wells containing single colonies early on to avoid mixed cell populations. 25. Twelve clones are usually sufficient to obtain a couple of Credeleted clones. 26. A culture with less than 5% IgM+ cells can be easily generated by seeding cells out by limiting the dilution to obtain singlecell clone. After 7–10 days, the surface IgM phenotype can be determined to identify mostly IgM− subclones that are suitable as a starting population for the SHM and GCV analysis.

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27. The PCR program may need to be optimized for the individual brand of PCR machine and the DNA polymerase used. A starting point is: 98°C for 2 min, followed by 35 cycles of 98°C for 20 s, 60°C for 20 s, and 72°C for 20 s. A final extension at 72°C for 5 min concludes the reaction. 28. Remove all low-quality sequences that are short or contain multiple Ns prior to performing the CLUSTALW alignments, as such sequences will create a seemingly large number of (false-positive) mutations. 29. Always check whether the neighboring mutations could also be a result of the very same GCV event.

Acknowledgments This work was supported entirely by the Intramural Research Program of the NIH, National Institute on Aging. References 1. Maizels, N. (2005) Immunoglobulin gene diversification. Annu. Rev. Genet. 39, 23–46. 2. Neuberger, M. S., Harris, R. S., Di Noia, J., and Petersen-Mahrt, S. K. (2003) Immunity through DNA deamination. Trends Biochem Sci 28, 305–12. 3. Arakawa, H., and Buerstedde, J. M. (2009) Activation-induced cytidine deaminase-­ mediated hypermutation in the DT40 cell line. Philos Trans R Soc Lond B Biol Sci 364, 639–44. 4. Kothapalli, N., Norton, D. D., and Fugmann, S. D. (2008) Cutting Edge: A cis-Acting DNA Element Targets AID-Mediated Sequence Diversification to the Chicken Ig Light Chain Gene Locus. J Immunol 180, 2019–23. 5. Blagodatski, A., Batrak, V., Schmidl, S., Schoetz, U., Caldwell, R. B., Arakawa, H., and Buerstedde, J. M. (2009) A cis-acting diversification activator both necessary and sufficient for AID-mediated hypermutation. PLoS Genet 5, e1000332. 6. Koller, B. H., and Smithies, O. (1992) Altering genes in animals by gene targeting. Annu Rev Immunol 10, 705–30. 7. Buerstedde, J. M., and Takeda, S. (1991) Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell 67, 179–88.

8. Buerstedde, J. M., Reynaud, C. A., Humphries, E. H., Olson, W., Ewert, D. L., and Weill, J. C. (1990) Light chain gene conversion continues at high rate in an ALV-induced cell line. EMBO J. 9, 921–7. 9. Arakawa, H., Lodygin, D., and Buerstedde, J. M. (2001) Mutant loxP vectors for selectable marker recycle and conditional knock-outs. BMC Biotechnol. 1, 7. 10. Peitz, M., Pfannkuche, K., Rajewsky, K., and Edenhofer, F. (2002) Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes. Proc Natl Acad Sci USA 99, 4489–94. 11. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., and Higgins, D. G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–8. 12. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local alignment search tool. J Mol Biol 215, 403–10. 13. Arakawa, H., and Buerstedde, J. M. (2006) Dt40 gene disruptions: a how-to for the design and the construction of targeting vectors. Subcell Biochem 40, 1–9.

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Chapter 19 Characterizing Immune Receptors from New Genome Sequences Katherine M. Buckley and Jonathan P. Rast Abstract Genome sequences are quickly being generated from a variety of organisms and provide researchers with an abundance of previously inaccessible information and an important source of insight into immune mechanisms. There are a variety of methods to accurately characterize genes from new genome sequences, but immune receptors pose special challenges for these techniques. Immune receptors, particularly those that directly recognize pathogens, often diverge rapidly among species and are commonly found in large, complex multigene families. Because of these characteristics, immune receptors tend to be overlooked or misannotated in large-scale genomic surveys. We describe here a computational strategy to characterize homologs of immune receptors and also to identify putative novel receptors from newly assembled genome sequences. The description of these protocols is aimed at a typical immunologist, and a substantial knowledge of bioinformatics is not expected. The approach is based on using low-stringency sequence searches to identify divergent homologs. For receptors with multiple domains, the intersection of low-stringency searches can be used to identify divergent receptor sequences with high confidence. For multigene families, these predictions can be refined using sequence conservation among gene family paralogs. This strategy has recently been useful in identifying novel expansions in immune receptors in a number of animal genomes and will likely continue to revolutionize our view of animal immunity as new genomes emerge. Key words: Genomics, Evolution, Toll-like receptors, Nod-like receptors

1. Introduction As sequencing technology improves, genomes from a wide variety of species are quickly being resolved. This provides biologists with a wealth of information that was previously inaccessible given the constraints of traditional, hybridization-based techniques in molecular biology. Because of these advances, annotation of genes from imperfect genome sequence is falling upon researchers with little experience in bioinformatics and molecular evolution. Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0_19, © Springer Science+Business Media, LLC 2011

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Characterizing immune receptors from a newly sequenced genome is a two-step process. The first step is to accurately predict the coding sequence and gene structure. Next, these gene models are used to identify homologs of genes from other organisms as well as those encoding novel receptors. Although the primary focus of this manuscript is to present techniques for the second of these objectives, we also highlight some of the limitations of gene prediction algorithms and describe protocols for analyzing genome sequences directly. In addition to the well-characterized human, mouse, and Drosophila genomes, immune gene repertoires have been characterized from a number of other organisms, including the tunicate (1), amphioxus (2), sea urchin (3), mosquito (4, 5), honeybee (6, 7), beetle (8), and two Cnidarian species (9, 10), with many more genome projects underway. These analyses have yielded important insights regarding the evolution of immunity. For example, gene families encoding pattern recognition receptors are notably expanded in the purple sea urchin (11, 12), amphioxus (13, 14), and polychaete (15). A large group of Nod-like receptors (NLRs) with unique domain architecture were identified in three fish species (16). Components of toll-like receptor (TLR) signaling and complement pathways have been found in Cnidarians, suggesting ancient origins for these proteins. These breakthroughs would not have been possible without accurate genome analyses, and further analysis of the immune gene repertoires in these and other emerging genomes will continue to reshape our understanding of immune evolution. Bioinformatic identification of genes encoding immune receptors is a challenging task. Unlike genes involved in many other cellular processes that tend to be conserved across a range of phylogenetic taxa, immune genes tend to evolve quickly, most likely as a result of heavy selective pressure from pathogens. Immune receptors are often encoded in large, multigene families that further complicate both genome sequence assembly and repertoire analysis. Special considerations and techniques are, therefore, necessary to characterize immune receptors. An overview of the strategy is shown in Fig. 1. Low-stringency searches can be used to uncover divergent sequences. These sequences can be used to query the genome and generate a larger set of candidate receptor homologs. Divergent, multidomain immune receptors can be identified using the intersection of low-stringency search techniques. For multigene families, coding sequence of putative homologs can be refined using sequence conservation among family members. This type of analysis will be tailored to the specific aims of the researcher, and, as such, we have left some details vague, but a key approach is working with a high number of false positives (see Note 1) and the emphasis on conserved characteristics of ­protein sequence and domain architecture. To accomplish this, we use

19  Characterizing Immune Receptors from New Genome Sequences

Genome Sequence

Translate genome EMBOSS

Generate gene models Gene prediction programs

Set of gene predictions

Domain predictions

Re-analyze the genome using candidates from each cluster

Homologous Test of homology Align domains and build trees Reciprocal BLAST Unique domain architecture

Novel immune receptors

Initial set of candidate immune receptors Multigene families

Cluster sequences CD-HIT, trees

Immune receptor homologs

Nonhomologous

Isolate candidate receptors using homolgs from related taxa BLAST, HMMER, EMBOSS

275

Refined set of candidate immune receptors

Cluster representatives

Low stringency set of candidate immune receptors

Singlecopy genes

Refine the immune receptors Concatenate and/or divide models Dot plots to refine coding sequence Differentiate alleles from loci Characterize protein sequences by identifying glycosylation sites, transmembrane domains, etc.

Fig. 1. Steps to characterizing immune receptors from a newly sequenced genome. The flowchart illustrates the overall approach for bioinformatic isolation of genes encoding immune receptors. Given a sequenced genome, gene models are produced with a variety of gene prediction algorithms or the genome is translated to supply amino acid sequence used to identify putative receptor domains. Depending on the sequence conservation, candidates can be isolated using lowstringency searches. If the receptors form a multigene family, they are clustered and reanalyzed with representatives from each cluster. A candidate immune receptor is refined by concatenating or separating faulty gene models. This data is then used to demonstrate homology to known receptors or to identify novel immune receptor sequences.

many tools and resources that are freely available (Table 1). The protocols include examples of programs that, although of limited scope, provide an idea of how these methods can be applied.

2. Materials 1. A genome assembly, genomic trace sequences, or gene models. These sequences can be downloaded from genome-sequencing center Web sites (see Note 2) and are typically in FASTA format (see Note 3). Genomic traces are the raw sequences used to assemble the genome and vary in length based on the sequencing method. A complete assembled genome consists of a set of contig or scaffold sequences. A contig is a region of sequence that has been continuously

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Table 1 Where to find commonly used bioinformatics software and sequencing centers Program

Description

HMMER

Predicts protein domains within http://www.hmmer. known sequences and isolates janelia.org proteins with given domains

PFAM

Database of domain profiles

http://www.pfam.sanger.ac.uk/ (48)

SMART

Identifies domains within protein sequences

http://www.smart.embl-heidel- (31) berg.de/

BLAST

Identifies similar sequences

http://www.ftp.ncbi.nlm.nih. gov/blast/executables/ blast+/LATEST

(26)

Genscan

Predicts gene models

http://www.genes.mit. edu/GENSCAN.html

(18)

EMBOSS

Suite of bioinformatics tools

http://www.emboss. sourceforge.net

(28)

YASS

Generates dot plots

http://www.bioinfo. lifl.fr/yass

(39)

Clustal

Sequence alignment tool

http://www.clustal.org

(32)

MEGA

Edits alignments and builds phylogenetic trees

http://www.megasoftware.net/ (33)

Bioedit

Sequence alignment editor

http://www.mbio.ncsu. edu/BioEdit/ bioedit.html

(35)

http://www.expasy. ch/tools

(49)

ExPASy Proteomics Server Online tools for modifying sequences and predicting protein characteristics

Web site

Ref. (27)

Center for Biological Sequence Analysis Technical University of Denmark

Online tools for modifying sequences and predicting protein characteristics

http://www.cbs.dtu. dk/services

(50)

Max Planck Institute Bioinformatics Toolkit

Online tools for modifying sequences and predicting protein characteristics

http://www.toolkit.tuebingen. mpg.de

(51)

CD-HIT

Sequence clustering

http://www.bioinformatics. org/cd-hit

(34)

AUGUSTUS

Gene model prediction

http://www.augustus. gobics.de

(22)

GenePalette

Scaffold annotation

http://www.genepalette.org

(36)

Open Bioinformatics Foundation

Source of Bioperl, Biopython, and Biojava scripts

http://www.open-bio.org

(continued)

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Table 1 (continued) Program

Description

Web site

NHGRI sequencing centers

Sequencing center

http://www.genome.gov/

Joint Genome Institute

Sequencing center

http://www.jgi.doe.gov/

Ensembl

Genome annotation

http://www.ensembl.org

Ref.

assembled using sequence similarity of the genomic traces. Scaffolds are built by piecing together contigs using pairs of end sequences from clones, and thus consist of both contig sequence and gaps. Many genome projects generate gene models, which are computational predictions of gene sequences. 2. Computer.  A Linux workstation or access to cloud computing (see Note 4). The analyses described here can be completed on a reasonably sized desktop computer. Although there are more sophisticated programs that can be run on high-power computers or clusters, biologists should not be limited by a lack of computing power. More processors and increased RAM expedite the programs, but, generally speaking, a standard desktop computer is sufficient for many forms of analyses. We find that a Linux workstation is the most amenable to this type of investigation (see Note 5). 3. Bioinformatics programs.  The bioinformatics programs described are available from sources listed in Table 1. All of the programs are freely available for academic users and are easy to install on a Linux computer (see Note 5). See program documentations for installation details. The programs described here represent a small sampling of the many available, and researchers are encouraged to find others that may better suit their needs. 4. Set of query sequences from other taxa.  Amino acid sequences are nearly always more useful in this type of analysis due to the high divergence of immune receptors. If available, a set of sequences from taxa at a variety of phylogenetic distances is preferred. 5. PFAM profiles for domains of interest.  PFAM profiles collect domain-type sequences across genes and species. Information can be extracted from these profiles that allow for searches that are far more powerful than those that can be achieved using single sequences. Profiles for common domains of immune receptors are listed in Table 2. A complete set can be downloaded from http://pfam.sanger.ac.uk/.

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Table 2 PFAM domains commonly found in immune receptors Accession

ID

Description

PF00047

ig

Immunoglobulin domain

PF07654

C1-set

Immunoglobulin C1-set domain

PF05790

C2-set

Immunoglobulin C2-set domain

PF07679

I-set

Immunoglobulin I-set domain

PF07686

V-set

Immunoglobulin V-set domain

PF00560

LRR_1

Leucine-rich repeat

PF07723

LRR_2

Leucine-rich repeat

PF07725

LRR_3

Leucine-rich repeat

PF01463

LRRCT

Leucine-rich repeat C-terminal domain

PF01462

LRRNT

Leucine-rich repeat N-terminal domain

PF00530

SRCR

Scavenger receptor cysteine-rich domain

PF01582

TIR

TIR domain

PF08357

SEFIR

SEFIR domain

PF00619

CARD

Caspase recruitment domain

PF00531

Death

Death domain

PF01335

DED

Death effector domain

PF02758

PAAD_DAPIN

PAAD/DAPIN/Pyrin domain

PF05729

NACHT

NACHT domain

PF00653

BIR

Inhibitor of Apoptosis domain

PF00270

DEAD

DEAD/DEAH box helicase

PF00059

Lectin_C

Lectin C-type domain

PF00147

Fibrinogen_C

Fibrinogen beta and gamma chains, C-terminal globular domain

3. Methods The first step in identifying homologs of immune receptors is to collect a set of coding sequences from the genome of interest. This can be done using gene prediction programs by extracting open reading frames or by translating raw genomic sequence. The state of the genome at the time of release will vary substantially with different projects, particularly as the genome-sequencing technology changes. For example, traditional Sanger, 454, and short

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sequence methods, such as SOLiD and Illumina, each offer a ­different set of challenges (see Note 6). Most genome projects generate a set of gene models that can be downloaded as a FASTA sequence file and translated (see the transeq tool in the EMBOSS suite; see Note 7). If a gene set is not available, one can be generated using gene prediction programs or by collecting open reading frames from the genomic sequence. In most cases, a combination of these methods is optimal, as both have their own advantages. Large gene families encoding immune receptors can be masked by algorithms that identify genomic repeats and omitted from the gene models entirely. An example of this is a subfamily of NLR genes in bony fishes (16). These genes were identified only after searching the genome sequence directly, rather than the predicted genes, because they were initially excluded from the gene models as repeats. The large gene families that are typical of immune receptors can also be difficult for genome assembly algorithms. Conversely, translating genomic sequence omits important information on splicing signals, which means that domains containing introns may be difficult to find. These two techniques complement each other and should be used together to generate a comprehensive set of potential coding sequences. 3.1. Assembling a Gene Catalogue 3.1.1. Translating the Genome to Find Open Reading Frames

Although most immune receptors are encoded by multiexon genes, individual domains are often encoded in single exons that can be identified by translating the genome. This avoids pitfalls of gene prediction programs and can be used on trace sequences in addition to assembled genomes (see Note 6). There are a number of ways to translate genomic sequence. The EMBOSS getorf tool extracts open reading frames from DNA sequences and is shown as an example below. 1. Install the EMBOSS package (Table 1; see Note 5). 2. Given the genome sequence in FASTA format (genome_ scaffolds.fas; see Note 3), run getorf with the following command: >getorf –sequence genome_scaffolds.fas –outseq genome_ORF.fas –minsize 200 Running getorf without arguments prompts the user for the name of the input and output files (see Note 8). The –sequence and –outseq arguments specify the paths and filenames of the input and output files, respectively. The –minsize argument specifies the minimum size of the open reading frames in nucleotides (default = 30). Increasing this value reduces the file size and eliminates noise, but discretion should be used to ensure that domains are not missed on the basis of size. If the domain of interest is typically encoded in a single exon, set the –minsize argument to slightly smaller than the size of

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the domain. The output of getorf is a FASTAformatted file containing the predicted open reading frames. By default, getorf finds open reading frames in all six frames. For a full list of arguments, use “getorf –h”. 3.1.2. Predicting Gene Models

A number of programs have been developed for the complex task of predicting open reading frames from eukaryotic genomes (17). We describe two of these slightly different programs here. Genscan predicts genes using characteristics of transcription, translation, and splicing signals, as well as the sequence composition of coding vs. noncoding regions (18). A Web server is available for predicting genes from single scaffolds (Table 1), and the program is available to download for local use. However, Genscan is designed to identify genes from single scaffolds, which is useful if a scaffold has been identified through other means, but, although it is possible to run on a whole genome, it is relatively difficult. A number of other gene-finding programs are available, such as FGENESH (19), mGENE (20), GeneMark (21), and AUGUSTUS (22). As an example, we have outlined how to use AUGUSTUS to predict gene models below. AUGUSTUS uses a hidden Markov model (HMM) method and can generate gene models from a mediumsized genome in a few hours. This program has been used to predict genes from the beetle (8), mosquito (23), and nematode (24, 25) genomes and can be trained for any taxa using ESTs. 1. Download and install AUGUSTUS (Table  1). Given a genome sequence (genome.fas), use the following: >augustus –-species=species genome.fas >genome_ AUGUSTUS.out The --species argument indicates which species-specific set of parameters will be used. AUGUSTUS includes parameter files for a number of taxa, including prokaryotes and plants, as well as a number of insects and vertebrates. Select a closely related species or, if possible, train AUGUSTUS using ESTs. AUGUSTUS lacks an argument to specify the location of the results file. Instead, use “>” followed by a path and file name, which is a UNIX command to direct the output to a file. 2. The output of AUGUSTUS is a gff-formatted file that lists the location of each exon on the scaffold followed by the protein sequence. Convert to FASTA format for further analysis using a PERL or Python script (see Note 9).

3.2. Isolating a Set of Candidate Genes

Due to the high sequence divergence of immune receptors, lowstringency search techniques are necessary to isolate an initial set of candidate homologs (Fig. 1). Three general approaches for this are described here. First, for closely related genes, Basic Local Alignment Search Tool (BLAST; (26)) is used to find homologs with similar sequence. Second, although sequences may have

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FREP

Ectodomain Cytoplasm TLR VLR

NLR C C C

SRCR Dectin

RLR C C D D D

C D

NITR

TCR

KIR

IL1R

BIR

CTLD

LRR

SRCR

ITIM

ITAM

Ig

D

DEATH

FBG

C

CARD

DEAD

PYRIN

TIR

NACHT

Fig. 2.  Domain architectures for common immune receptors. Although frequently divergent in primary sequence, immune receptors are characterized by conserved combinations of specific domains. These domains may also be shuffled in novel immune receptors. PFAM numbers for each of the domains are shown in Table 2. Abbreviations are as follows: BIR, Baculovirus inhibitor of apoptosis protein repeat; CARD, caspase recruitment domain; CTLD, C-type lectin domain; FBG, fibrinogen; Ig, immunoglobulin; IL1R, IL-1 receptor; ITAM, immunoreceptor tyrosine-based activating motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; LRR, leucine-rich repeat; KIR, killer inhibitory receptor; NITR, novel immunetype receptor; NLR, Nod-like receptor; RLR, Rig-I-like receptor; SRCR, scavenger receptor cysteine-rich; TCR, T cell receptor; TIR, toll/interleukin-1 receptor; TLR, toll-like receptor; VLR, variable lymphocyte receptor.

diverged, immune receptors are often characterized by conserved domain combinations (Fig. 2). The HMMER suite provides tools for bioinformatic identification of specific protein domains in a set of sequences (or a translated genome) and for characterization of protein domains within a particular sequence (27). Finally, in some cases, immune receptors can be identified through characteristic patterns of conserved residues using tools within the EMBOSS suite (28). The optimal approach will vary given the sequence conservation and characteristics of the receptors in question, and a combination of these methods is usually applied. 3.2.1. Identifying Homologs Using Sequence Similarity with BLAST

BLAST is a standard bioinformatics tool used to identify similar sequences (26). The BLAST algorithm finds short matches between pairs of sequences and then lengthens the region of similarity using other nearby short matches. Searches for immune receptors in divergent species almost always rely on amino acid similarity, as nucleotide sequence searches have little discriminatory power when applied to quickly evolving immune genes. Even using amino acid sequence, the utility of BLAST in identifying immune receptors may be limited, as it relies on regions of relatively high sequence similarity. However, if sequences from closely related taxa are available

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or if the sequences are under evolutionary constraints, BLAST is a good first step to identify homologs. The key is to reduce the search stringency and to analyze a large number of possible hits. BLAST is also good at identifying partial matches that can serve as a target region for the expansion by manual curation. 1. Download and install the BLAST toolkit (Table 1; see Note 5) 2. Build a sequence database using formatdb. Given a file containing translated sequences (gene_models.fas), use the following: >formatdb –i gene_models_nt.fas –o T –p T The –o argument indicates whether (T) or (F) formatdb parses the sequence identifiers and produces indices that allow sequence retrieval by identifier, which increases the speed of the searches (default = F). Use “The -o argument indicates T” whenever the database sequences are in FASTA format. This generates seven files with the same name as gene_models.fas, but different extensions (.phr, .pin, .psq, .pnd, .pni, .psd, and .psi; only the first three are created for databases formatted with “o F”). The –p argument (“protein”) specifies whether the database sequences are amino acids (T) or nucleotides (F). For a nucleotide sequence database, five files are created (file extensions:. nhr, .nin, .nsq, .nsd, and .nsi; only the first three files are created with “–o F”). 3. Make a file of the sequences to BLAST (query sequences) in FASTA format (see Note 3). Multiple query sequences can be analyzed simultaneously if they are in the same file. When possible, the query sequences should consist of homologs from a number of taxa at a variety of phylogenetic distances. 4. BLAST the query sequences against the custom database as follows: >blastall –p blastp –d gene_models.fas –i query_sequences.fas –o output_results.out –a 8 –e 100 –v 1000 –b 50 –m 8

Table 3 BLAST programs Database sequences Query sequences

Nucleotide Amino acid

Nucleotide blastn tblastx a tblastn

Amino acid blastx blastp

tblastx uses an amino acid intermediate, in that it searches a translated database with a translated query sequence. a

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A number of arguments are available within the BLAST toolkit; consult the documentation for details. The arguments that we manipulate most frequently are outlined below (see Note 8). ●●

●●

●●

●●

●●

●●

The –p argument indicates the BLAST program to be used, which depends on whether the query and database sequences are nucleotides or amino acids (Table 3). For immune receptors, the nucleotide sequence divergence is typically greater than the sensitivity of BLAST searches, and, as such, it is best to search with amino acid query sequences (tblastn or blastp) or against databases containing either amino acid (blastx) or translated nucleotide sequences (tblastx). The –d, -i, and -o arguments indicate the paths and filenames of the file used to generate the sequence database, the input file containing the query sequence(s), and the output file to store the results. The default for –o is stdout, meaning the results are displayed on screen. The –a argument specifies the number of processors on which to run the search (default = 1). When analyzing a large number of queries or using a large database, increasing this value to the maximum number of processors on the computer expedites the analysis. The –e argument sets the maximum Expectation value (E-value; default = 10) of hits that are reported. The E-value for a hit with a given score is the number of alignments with an equal or better score that is expected due to chance given the size of the database. Lower E-values are more significant. Increasing this value not only allows a greater number of hits, but also increases the number of nonspecific hits that are reported (see Note 1). The –v and –b arguments set the numbers of sequences for which to show one-line descriptions and alignments (defaults are –v = 500 and –b = 250). Adjusting these values customizes the results file. Increase –v such that hits with the maximum E-values are identified. If alignments are unnecessary, set –b 0 to reduce the file size. The –m argument specifies the format of the output file (values range from 0 to 11; default = 0). A complete description of the formats is found in the documentation, but, in addition to the default, the -m 8 format is particularly useful. This formats the results in a tabulated file that can be easily imported into a spreadsheet for further analysis.

5. When sequence similarity is weak, use Position-Specific Iterated (PSI)-BLAST (29, 30)), which is part of the BLAST toolkit. PSI-BLAST works similarly as blastp, except that rather than using a standard amino acid substitution matrix,

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it generates a custom substitution matrix through iterative BLAST searches. This method identifies sequence similarity by weighting residues that are important in the query sequence (as determined by conservation). In the first iteration, a single query sequence is searched against the database with a standard substitution matrix. Results from this iteration are then used to generate a matrix for scoring subsequent BLAST searches. The process is repeated until the searches converge (fail to find new sequences). As with most of these similarity searches, optimal results are usually obtained when single protein domains are used as the queries, rather than entire sequences. Run PSI-BLAST as below, given a file containing a single protein query sequence in FASTA format (input_ sequence.fas) and a formatted database of protein sequences (gene_models.fas). >psiblast –query input_sequence.fas –db gene_models.fas –out results_file.out –num_ iterations 10 –inclusion_ethresh 0.1 See the PSI-BLAST help files (>psiblast –help) for a description of the arguments not described below. ●●

●●

●●

The –query, -db, and –out arguments specify the paths and filenames of the file containing the query sequence, file used to generate the database, and the results file (default is stdout). The –num_iterations argument specifies how many iterative BLAST searches to be performed (default = 1). If the searches converge before this limit is reached, the program stops. The –inclusion_ethresh argument specifies the E-value below which sequences are included in the matrix for iterative BLAST searches (default = 0.002). Raising the value reduces the stringency of the searches.

6. Retrieve the sequences of interest. The specifics of this step vary substantially among genome projects. If possible, use a custom script (see Note 9). The Unix command grep is a command line text search tool that can also be manipulated to retrieve sequences. 3.2.2. Identifying Immune Receptors by Domain Architecture Using HMMER

A second method of receptor analysis is to find proteins with relevant domains or combinations of domains (Fig. 2). Identifying genes with unique domain architectures is sometimes a means to find homologs for distant or quickly evolving species, where primary sequence has diverged. HMMER is a powerful tool to identify protein domains using profile HMMs, which contain information about the frequencies of amino acids within protein domains (27), and is typically more sensitive than BLAST. The user’s guide for HMMER3 is very helpful, and, for all the functions listed below, help can be obtained with “-h”.

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1. Download and install HMMER3 (Table 1). 2. Collect the relevant domain alignments (Table 2; Fig. 2), for example, from PFAM (http://pfam.sanger.ac.uk/). Click on “Alignments” and download the full alignments by selecting the “Full” button next to “PFAM alignments” and clicking “Download”. Extract the compressed alignment files and rename informatively (i.e., domain.ann) 3. Build an HMM profile from the alignment file with the following: >hmmbuild domain.hmm domain.ann where domain.ann is the alignment file and domain.hmm is the path and filename of the HMM profile to be created. An HMM profile can be built from any sequence alignment in Stockholm or FASTA format using hmmbuild. The hmmbuild command can also generate multiple HMM profiles from an input file that contains more than one alignment. 4. Identify proteins that contain domain(s) of interest from a large database of sequences using hmmsearch. If the domain.hmm file contains multiple HMM profiles, hmmsearch searches for all of them. The following is an example for the usage of hmmsearch: >hmmsearch –o results.out ––noali –A alignment.out ––tblout results.tblout ––domtblout results.domtblout –E 100 –incE 100 domain. hmm sequence_database.fas A number of options are included in hmmsearch that allow for significant customization of both the search strategy and output format. The argument order is irrelevant, except for the last two (the HMM profile must be penultimate and the input sequence file last). ●●

●●

●●

●●

The –o argument specifies the path and filename of the output (default is stdout). The –A argument generates a file containing an alignment of the domains from the identified sequences with E-values better than the inclusion threshold (see below). The –tblout and –domtblout arguments specify the paths and filenames of tabulated files that summarize the results by target and domain, respectively, that can be imported into a spreadsheet for further analysis. HMMER output files are not only designed to be readable, but also contain a significant amount of information in various formats. Taking advantage of these summary output files to collect data is easier than parsing the complete output file for specific information. The --noali argument removes the alignments from the main output file. Although these alignments are

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useful to compare the potential hits to the HMM profile, excluding them reduces the file size. ●●

●●

The –E argument takes an integer and specifies the maximum E-value at which hits are reported. The default value is 10, which means that approximately 10 false positives are reported for each query. Increasing this value reduces the search stringency (see Note 10). The –incE argument specifies the E-value at which sequences are included in the domain alignment file (default = 0.01).

5. To identify domains within a specific sequence (or set of sequences), use hmmscan. Build a database of HMM profiles by collecting alignments of interest, concatenating into a single file, and using hmmbuild (myHMMs.hmm). A more comprehensive approach is to download the complete set of PFAM alignments (ftp://ftp.sanger.ac.uk/pub/databases/ Pfam/). Although the files are large, building the HMM profiles for this database takes about 2 h on a reasonably sized computer. Prepare the database of HMM profiles for hmmscan with hmmpress as below. >hmmpress myHMMs.hmm This creates four files with the same name as myHMMs, but with the file extensions – .h3m, .h3i, .h3f, and .h3p. Given a prepared HMM profile database, use hmmscan: >hmmscan –o results.out ––noali –A alignment. out ––tblout results.tblout ––domtblout results.domtblout –E 100 –incE 100 myHMMs. hmm query_sequences.fas The arguments for this command are the same as those for hmmsearch. 6. To get information quickly from a single sequence, the same analysis can be performed with the online tool, Simple Modular Architecture Research Tool (SMART; (31)). SMART uses HMM profiles to identify domains within protein sequences and contains a substantial amount of information about known domain architectures. Go to http://smart.embl-heidelberg. de/, paste the candidate protein sequence into the “Sequence” box, check the boxes next to “PFAM domains” and “Signal sequences”, and click “Sequence SMART”. 7. Identify and collect proteins with known domain architectures (Fig. 2) by analyzing the intersection of sets of proteins with single domains. Specifics of this vary depending on the receptor being analyzed, but this can be done either with a custom script or using Microsoft Excel (see Note 9). A good example of the power of this type of combinatorial search can

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be found in our searches for TLRs in the sea urchin genome sequence. Here, both leucine-rich repeats (LRRs) and TIR domains were highly divergent from those of mammals and insects, but a search for genes containing both the domain types (even with E values >100) returned almost exclusively genuine TLR genes (11). 3.2.3. Identifying Homologs Using Specific Residue Patterns with EMBOSS

For some immune receptors, only a few key residues will be ­conserved among taxa. Two tools within the EMBOSS package search for sequences with specific patterns. The first, fuzzpro, searches amino acid sequences, whereas the second, fuzztran, searches translated nucleotide sequences. The usage is the same; the only difference is the format of the input sequences. 1. Install EMBOSS (Table 1; see Note 5). 2. Align homologs from other taxa to identify a conserved amino acid pattern. Examples of these patterns may be characteristic residues, used in protein folding or interactions, or active sites for enzymes. Conservation over a wide phylogenetic range of organisms is optimal. 3. Given a set of candidate sequences in FASTA format in a single file (gene_models.fas), run either fuzztran or fuzzpro, depending on the format of the sequences. >fuzzpro Search for patterns in protein sequences Input protein sequence(s): gene_models.fas Search pattern: CX(10)(DE)X(8)C Output report: sequences_pattern.out In the above example, sections in bold are entered by the user; the program prompts for the rest. The output report contains the sequence name, the number of times the pattern was found, the position of the pattern, and the pattern sequence. The regular expression capacity of the search pattern allows for flexibility in the sequences that can be captured. A complete description of the pattern syntax can be found in the help file (fuzzpro –h). In brief, use IUPAC one-letter codes for amino acids, where X stands for any amino acid, sets of amino acids can be included (using brackets, e.g., (AG) stands for alanine or glycine) or excluded (using curly brackets, e.g., {AG} stands for any amino acid except alanine or glycine), and repetitive amino acids are specified such that A(3) will find AAA (spaces between conserved residues are specified as X(10), which will find any ten consecutive amino acids). The example shown identifies the sequences containing two cysteine residues, separated by any ten amino acids, an aspartate or glutamate, and any other eight residues.

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4. Isolate sequences containing the pattern of interest. Because this method provides the lowest stringency for identifying immune receptor homologs, other lines of evidences should be used to show homology. 3.3. Refining the Homolog Candidates

Given an initial set of candidate homologs, these sequences can be manually curated, refined, and used to iteratively search for additional sequences. Species-specific gene family expansions often generate paralogs that are more similar to each other than to homologs from other taxa. It is common for the initial searches to uncover outliers within a new genome that can then be used to identify further gene family members.

3.3.1. Clustering the Sequences to Iteratively Search the Genome

One disadvantage of the methods outlined above is that the lowstringency search criteria can produce a number of candidate homologs that need to be validated. Some domain types that are important in immune receptors can also be found in many other proteins such that searches for these domains isolate hundreds to thousands of candidate sequences. For example, our analysis of the sea urchin genome uncovered more than 1,500 immunoglobulin domains, of which only a fraction are of interest from an immunological perspective (11). One approach to sort through this large numbers of genes is to cluster similar domain sequences and analyze limited representatives from each closely related cluster. This “cluster and conquer” technique can significantly reduce the amount of work needed to manually curate the candidates. Domains should be clustered, rather than the complete receptor sequences, as domain reorganization and nonconserved sequences between domains may preclude robust analysis. Clustering can be done either by aligning and building trees with standard phylogenetic methods or using tools that identify sequence similarity without a priori alignments. Sequences can be aligned with ClustalX (32), and trees can be built using the neighbor-joining method within MEGA (33). Due to the high sequence divergence, alignments of domain sequences will usually contain many columns with gaps. To avoid eliminating all information from the sequences, pairwise gap deletion should be used rather than complete deletion. Pairwise deletion of gaps identifies groups of similar sequences, but cannot resolve the high order relationships among these groups, which is sufficient for this purpose. To avoid the complications of alignment sequences, the clustering program CD-HIT (34) can be used. 1. To run CD-HIT locally, download and install the program (Table 1). Given a FASTA file containing the sequences to cluster (candidate_sequences.fas), run CD-HIT as follows: >cd-hit –i candidate_sequences.fas –o candidate_clusters.out –c 0.8 The -i and -o arguments specify the paths and filenames of the file containing the input sequences and

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output file, respectively. The output of this analysis is a FASTA-formatted file containing representatives from each cluster and a file detailing the sequences in each cluster and their similarities. The -c argument sets the threshold at which sequences are clustered (default = 0.9). By decreasing this threshold, sequences with less similarity are clustered. See the documentation for a further description of the arguments (or use “>cd-hit –h”). 2. CD-HIT can also be run through a Web server (Table  1). Upload a FASTA file containing the sequences of interest, adjust the parameters as necessary, and submit the analysis. Output files for the Web version of CD-HIT can be downloaded or visualized in the Web browser, where it is possible to navigate the clusters by size or length and see a distribution of the number of sequences in each cluster. Results will also be sent via e-mail, which is useful for large analyses that require some time. 3. Select a representative from each cluster for further analysis and repeat the strategies above, particularly BLAST searches, to bootstrap other candidates from the genome. 3.3.2. Using Common Domain Architectures to Refine Gene Models

Gene prediction programs are prone to errors in either incorrectly concatenating multiple genes into single models (a common problem with tandemly arrayed immune receptors) or breaking single genes into separate models. Therefore, while these gene model prediction programs are valuable for identifying divergent immune receptors, it is important to analyze the genomic locations of the gene candidates. Novel architectures should be experimentally validated when possible. ESTs can provide evidence supporting novel structures. An alignment editor, such as Bioedit (35) or MEGA (33), or a program, such as GenePalette (36), which facilitates the annotation of large scaffolds using a graphic interface, is valuable for this type of analysis.

3.3.3. Using Sequence Conservation to Refine Gene Models

Another challenge for gene prediction algorithms is identifying short exons. This is of particular relevance for immune receptors, such as NLRs, in which individual LRRs are encoded in single, short exons (<75 nt; (37, 38)) that are commonly missed by gene prediction algorithms. One method for identifying coding sequences not included in gene models is to identify the regions of conservation among related members of a multigene family. Although intron and intergenic sequences quickly diverge, coding sequence is more likely to be conserved among paralogs. A dot plot is the easiest way to identify conserved regions of sequence (Fig.  3). Ideally, two sequences from the same taxa are used. Recently diverged sequences may be too conserved in noncoding regions to identify coding sequences, whereas other pairs may be too divergent (Fig. 3). The dot plot tool, YASS (39), can

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Fig. 3.  Using sequence conservation to identify coding sequence not included in the gene predictions. Dot plots are a convenient way to visualize conservation between long sequences. The dot plots shown here were generated with YASS (39). The optimal sequence divergence and analysis parameters are shown in (a). Dot plots can be generated using two related sequences and used to identify conserved segments that are not included in the gene model (indicated by the gray boxes ) to refine the coding sequences (black boxes ). Conserved sequence that was added to the gene is outlined on the dot plot with dotted lines. Examples of parameters that are too stringent (b) or too tolerant (c) are shown.

be downloaded or run through a Web server (Table 1) and allows flexibility in the parameters, as well as the ability to examine specific regions of the dot plot and quickly retrieve the sequences. 1. Go to the YASS Web server (http://bioinfo.lifl.fr/yass/yass.php). 2. Upload or paste two sequences that will be used to generate the dot plot. 3. Adjust the dot plot parameters such that sequence conservation is apparent but not masked by excess noise (see Fig. 3, for example). The parameters that will affect this include the scoring matrix and E-value threshold. See the YASS documentation for details. 4. Identify the regions of conservation near, but not included in the gene models. 5. Isolate the sequences in these conserved regions. 6. Translate the sequences to find putative coding regions. The ExPASy server (Table 1) has a convenient online translation tool.

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7. Validate the new gene models when possible. BLAST the new sequence against the nonredundant database at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) or a custom database of sequences as above to explore the similarity to receptors in other species. 3.3.4. Differentiating Alleles from Loci in Multigene Families

Distinguishing allelic gene copies from genes that derive from different loci, particularly for multigene families, is a difficult task for immune receptors for a number of reasons. Interallelic sequence diversity for these divergent genes may be greater than the differences between paralogs. This is especially true for outbred organisms, which represent a growing proportion of genomes being sequenced. These repeated, similar genes also present a challenge to genome assembly algorithms. Use the genomic traces to accurately estimate the number of alleles without introducing the potential biases of genome assembly errors (see Note 6).

3.3.5. Characterizing Immune Receptor Homologs

Potential homologs can also be analyzed by predicting protein characteristics, such as the presence of transmembrane domains, signal peptides, or potential glycosylation sites. Pattern recognition receptors can be secreted (e.g., collectins), transmembrane (e.g., TLRs), GPI-linked (e.g., lamprey VLRs), or cytosolic (e.g., NLRs or RLRs), which has important implications for their function (Fig. 2; (40)). TLRs are also heavily glycosylated (41). These aspects of protein structure can, therefore, provide important information for describing immune receptors. Online tools for these analyses are shown in Table 1. The TMHMM and PSORT algorithms predict transmembrane and secreted proteins, respectively (42). Glycosylation sites can be predicted with the programs, NetOGlyc and NetNGlyc (43). GPI anchors can be predicted with PredGPI (44). Many other aspects can also be analyzed using tools on these proteomic servers.

3.4. Demonstrating Homology

The standard method for proving sequence homology is to align sequences and build phylogenetic trees. The mosaic domain architectures of immune receptors may preclude robust tree building on complete sequences, although it may be possible to analyze domain sequences individually. Particular attention should be paid to the treatment of gaps, with the alignments carefully examined and curated by hand. Conclusions of homology should be drawn using appropriate out-group sequences to root the tree and measures of tree support, such as bootstrap or jackknife methods, Bayesian posterior probabilities, or maximum likelihood estimations. The reciprocal best hit method can also be used to tentatively identify homologous genes. Two genes from different genomes are deemed orthologous if, given comprehensive sets of genes from both genomes, the best BLAST hit for each gene is the other (45). In situations where sequences are too

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divergent to build reliable trees or when reciprocal BLAST searches fail, unique domain architecture can provide evidence for homology, although, for some genes, similar architecture may arise independently in distant phyla (46). Another important outcome of this type of analysis, however, is the identification of putative novel immune receptors. Receptors that contain immunerelated domains, but in unique organizations, or complex multigene families that are under strong diversifying selection are good candidates for genes with immune function and can be investigated more thoroughly.

4. Notes 1. Immune receptors present special, unique challenges at nearly all levels of genomic analysis when compared to many other genes. Their presence as multigene families and similarity among paralogs often complicate the accurate assembly and make it difficult to distinguish alleles from loci. Their pathogen response function drives the diversifying selection such that primary sequence similarity is quickly lost between related taxa. As such, the optimal strategy for identifying immune receptors is to collect a large set of candidate genes using low-stringency search strategies and then refine the set using other data. Fully annotating these genes requires a certain amount of patience and willingness to analyze false positives. 2. Most major sequencing centers maintain Web sites to store their sequencing data. Generally, the large genome files can be downloaded as compressed files from an ftp site. Extract the file to access its contents. A few examples of sequencing centers are shown in Table  1. Additionally, Ensembl maintains an online database for a number of genome sequences, as well as an easy-to-use genome browser (http://www. ensembl.org/index.html). 3. Sequences in FASTA format are represented with a one-line header that starts with a “>” and sequence name followed on the next line by the nucleotide or amino acid sequence in IUPAC one-letter designations. An example is shown below. >Sequence_001 VNLICSLCVYAYNVLTSRHADGKCWDDLHVQWPWKWWFTCGS MCHLACGLPIQKFCLHVHYQGVFYLRTRR >Sequence_002 APGTDMHCAADTFLAKKNQDLRRPEFRMRMERVHDNRG GLTLDVVGPFSTTQNPDKQVHAEAKGYYMFHIE

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4. As an alternative to a standard desktop computer, cloud computing offers a few advantages. Cloud computing is an Internet-based resource such that computers are shared by many users on large servers and processor space is allocated on demand. These virtual computers can be designed to exact specifications, including extremely powerful machines, and are relatively inexpensive to use. Cloud computing may be particularly attractive to immunologists who plan to look only at a specific genome for a specific gene family, where a powerful computer may be needed only for a short time. A number of cloud computing services are available, including Amazon’s Elastic Compute Cloud (http://aws.amazon.com/ec2/), IBM (http://www.ibm.com/ibm/cloud/), and Microsoft’s Windows Azure (http://www.microsoft.com/windowsazure/ windowsazure/). 5. For many biologists, the most intimidating and frustrating aspects of the protocols described here may be installing and running the bioinformatics programs. We are hesitant to recommend specific operating systems or software or to provide explicit installation instructions, given the rapid rate at which computers and software change. However, the easy-to-use, open-source, Linux-based operating system, Ubuntu (http:// www.ubuntu.com/), offers some advantages for users unaccustomed to a Linux environment. Installing and upgrading the operating system are straightforward, and the interface is similar to Windows or Mac. The most powerful aspect, however, is the sudo apt-get install command. In Ubuntu, sudo gives the operator root permission. The apt-get utility installs or upgrades software, for example, “>sudo aptget install blast” downloads and installs BLAST. EMBOSS, ClustalX, and HMMER are also available. See http://www.debian.org/distrib/packages for a full list of packages and more information. 6. Although the protocols outlined here are largely aimed at assembled genomes, many of these techniques can be performed with raw genomic trace sequences. Traces that are a few hundred nucleotides long can be used in BLAST searches or translated into open reading frames that can be analyzed for specific domains with HMMER or conserved sequences in EMBOSS. They also provide a valuable resource for verifying novel sequences within assembled genomes or, for multigene families, gene copy number. 7. The EMBOSS suite is a valuable set of bioinformatics tools. Many of these tools can be recreated using other programming languages, but EMBOSS provides an accessible starting point for most biologists. Only a few tools within the suite are mentioned here, but many more are available (for the full list, see http://emboss.sourceforge.net/apps/).

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8. The programs described here are largely run from the command line as follows: >program –argument1 value1 –argument2 value2 such that the program name is followed by a number of arguments. Arguments are used to specify information that the program needs, such as the location of an input file, or to customize specific parameters, such as setting the maximum number of hits. Argument names are usually indicated with a “-” and are followed by a value, which can either be a word or an integer. Some arguments are required, whereas others are optional; for the commands described here, the required arguments are shown in bold. If optional arguments are not specified, they are set to default values. Unless otherwise specified, arguments are case sensitive, and the order is irrelevant. Instructions on the program syntax or a full list of arguments can typically be obtained with either “>program –h” or “>program –help”. 9. The Open Bioinformatics Foundation (http://www.open-bio. org/) oversees the BioPerl, BioPython, and BioJava projects. Each of these open source projects produces modules for manipulating and analyzing biological data in their respective languages. They are a rich set of tools and can easily be used given some basic programming knowledge and are also supported by a wide community of open source programmers. 10. Although E-values are a good way to understand the significance of a score, strict adherence to a maximum E-value limit will sometimes eliminate true homologs. Some domains are more difficult to identify due to low conservation or short length. For example, LRRs are particularly hard to define, although other programs are available to identify LRRs (47). Short domains, such as DEATH domains, are also hard to find. Other evidence, such as neighboring domains, can be used to support domain predictions (i.e., adjacent CARD, NACHT, and LRR domains with suboptimal individual E-values most likely comprise an NLR-like gene). References 1. Dehal, P., Satou, Y., Campbell, R. K., Chapman, J., Degnan, B., De Tomaso, A., Davidson, B., Di Gregorio, A., Gelpke, M., Goodstein, D. M., Harafuji, N., Hastings, K. E., Ho, I., Hotta, K., Huang, W., Kawashima, T., Lemaire, P., Martinez, D., Meinertzhagen, I. A., Necula, S., Nonaka, M., Putnam, N., Rash, S., Saiga, H., Satake, M., Terry, A., Yamada, L., Wang, H. G., Awazu, S., Azumi, K., Boore, J., Branno, M., Chin-Bow, S., DeSantis, R., Doyle, S., Francino, P., Keys, D.

N., Haga, S., Hayashi, H., Hino, K., Imai, K. S., Inaba, K., Kano, S., Kobayashi, K., Kobayashi, M., Lee, B. I., Makabe, K. W., Manohar, C., Matassi, G., Medina, M., Mochizuki, Y., Mount, S., Morishita, T., Miura, S., Nakayama, A., Nishizaka, S., Nomoto, H., Ohta, F., Oishi, K., Rigoutsos, I., Sano, M., Sasaki, A., Sasakura, Y., Shoguchi, E., Shin-i, T., Spagnuolo, A., Stainier, D., Suzuki, M. M., Tassy, O., Takatori, N., Tokuoka, M., Yagi, K., Yoshizaki, F., Wada, S.,

19  Characterizing Immune Receptors from New Genome Sequences Zhang, C., Hyatt, P. D., Larimer, F., Detter, C., Doggett, N., Glavina, T., Hawkins, T., Richardson, P., Lucas, S., Kohara, Y., Levine, M., Satoh, N., and Rokhsar, D. S. (2002) The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins. Science 298, 2157–67. 2. Holland, L. Z., Albalat, R., Azumi, K., BenitoGutierrez, E., Blow, M. J., Bronner-Fraser, M., Brunet, F., Butts, T., Candiani, S., Dishaw, L. J., Ferrier, D. E., Garcia-Fernandez, J., Gibson-Brown, J. J., Gissi, C., Godzik, A., Hallbook, F., Hirose, D., Hosomichi, K., Ikuta, T., Inoko, H., Kasahara, M., Kasamatsu, J., Kawashima, T., Kimura, A., Kobayashi, M., Kozmik, Z., Kubokawa, K., Laudet, V., Litman, G. W., McHardy, A. C., Meulemans, D., Nonaka, M., Olinski, R. P., Pancer, Z., Pennacchio, L. A., Pestarino, M., Rast, J. P., Rigoutsos, I., Robinson-Rechavi, M., Roch, G., Saiga, H., Sasakura, Y., Satake, M., Satou, Y., Schubert, M., Sherwood, N., Shiina, T., Takatori, N., Tello, J., Vopalensky, P., Wada, S., Xu, A., Ye, Y., Yoshida, K., Yoshizaki, F., Yu, J. K., Zhang, Q., Zmasek, C. M., de Jong, P. J., Osoegawa, K., Putnam, N. H., Rokhsar, D. S., Satoh, N., and Holland, P. W. (2008) The amphioxus genome illuminates vertebrate origins and cephalochordate biology. Genome Res 18, 1100–11. 3. Sodergren, E., Weinstock, G. M., Davidson, E. H., Cameron, R. A., Gibbs, R. A., Angerer, R. C., Angerer, L. M., Arnone, M. I., Burgess, D. R., Burke, R. D., Coffman, J. A., Dean, M., Elphick, M. R., Ettensohn, C. A., Foltz, K. R., Hamdoun, A., Hynes, R. O., Klein, W. H., Marzluff, W., McClay, D. R., Morris, R. L., Mushegian, A., Rast, J. P., Smith, L. C., Thorndyke, M. C., Vacquier, V. D., Wessel, G. M., Wray, G., Zhang, L., Elsik, C. G., Ermolaeva, O., Hlavina, W., Hofmann, G., Kitts, P., Landrum, M. J., Mackey, A. J., Maglott, D., Panopoulou, G., Poustka, A. J., Pruitt, K., Sapojnikov, V., Song, X., Souvorov, A., Solovyev, V., Wei, Z., Whittaker, C. A., Worley, K., Durbin, K. J., Shen, Y., Fedrigo, O., Garfield, D., Haygood, R., Primus, A., Satija, R., Severson, T., Gonzalez-Garay, M. L., Jackson, A. R., Milosavljevic, A., Tong, M., Killian, C. E., Livingston, B. T., Wilt, F. H., Adams, N., Belle, R., Carbonneau, S., Cheung, R., Cormier, P., Cosson, B., Croce, J., Fernandez-Guerra, A., Geneviere, A. M., Goel, M., Kelkar, H., Morales, J., Mulner-Lorillon, O., Robertson, A. J., Goldstone, J. V., Cole, B., Epel, D., Gold, B., Hahn, M. E., HowardAshby, M., Scally, M., Stegeman, J. J., Allgood, E. L., Cool, J., Judkins, K. M., McCafferty, S. S., Musante, A. M., Obar, R. A., Rawson, A. P.,

295

Rossetti, B. J., Gibbons, I. R., Hoffman, M. P., Leone, A., Istrail, S., Materna, S. C., Samanta, M. P., Stolc, V., Tongprasit, W., et  al. (2006) The genome of the sea urchin Strongylocentrotus purpuratus. Science 314, 941–52. 4. Holt, R. A., Subramanian, G. M., Halpern, A., Sutton, G. G., Charlab, R., Nusskern, D. R., Wincker, P., Clark, A. G., Ribeiro, J. M., Wides, R., Salzberg, S. L., Loftus, B., Yandell, M., Majoros, W. H., Rusch, D. B., Lai, Z., Kraft, C. L., Abril, J. F., Anthouard, V., Arensburger, P., Atkinson, P. W., Baden, H., de Berardinis, V., Baldwin, D., Benes, V., Biedler, J., Blass, C., Bolanos, R., Boscus, D., Barnstead, M., Cai, S., Center, A., Chaturverdi, K., Christophides, G. K., Chrystal, M. A., Clamp, M., Cravchik, A., Curwen, V., Dana, A., Delcher, A., Dew, I., Evans, C. A., Flanigan, M., GrundschoberFreimoser, A., Friedli, L., Gu, Z., Guan, P., Guigo, R., Hillenmeyer, M. E., Hladun, S. L., Hogan, J. R., Hong, Y. S., Hoover, J., Jaillon, O., Ke, Z., Kodira, C., Kokoza, E., Koutsos, A., Letunic, I., Levitsky, A., Liang, Y., Lin, J. J., Lobo, N. F., Lopez, J. R., Malek, J. A., McIntosh, T. C., Meister, S., Miller, J., Mobarry, C., Mongin, E., Murphy, S. D., O’Brochta, D. A., Pfannkoch, C., Qi, R., Regier, M. A., Remington, K., Shao, H., Sharakhova, M. V., Sitter, C. D., Shetty, J., Smith, T. J., Strong, R., Sun, J., Thomasova, D., Ton, L. Q., Topalis, P., Tu, Z., Unger, M. F., Walenz, B., Wang, A., Wang, J., Wang, M., Wang, X., Woodford, K. J., Wortman, J. R., Wu, M., Yao, A., Zdobnov, E. M., Zhang, H., Zhao, Q., et  al. (2002) The genome sequence of the malaria mosquito Anopheles gambiae. Science 298, 129–49. 5. Christophides, G. K., Zdobnov, E., BarillasMury, C., Birney, E., Blandin, S., Blass, C., Brey, P. T., Collins, F. H., Danielli, A., Dimopoulos, G., Hetru, C., Hoa, N. T., Hoffmann, J. A., Kanzok, S. M., Letunic, I., Levashina, E. A., Loukeris, T. G., Lycett, G., Meister, S., Michel, K., Moita, L. F., Muller, H. M., Osta, M. A., Paskewitz, S. M., Reichhart, J. M., Rzhetsky, A., Troxler, L., Vernick, K. D., Vlachou, D., Volz, J., von Mering, C., Xu, J., Zheng, L., Bork, P., and Kafatos, F. C. (2002) Immunity-related genes and gene families in Anopheles gambiae. Science 298, 159–65. 6. Consortium, T. H. G. S. (2006) Insights into social insects from the genome of the honeybee Apis mellifera. Nature 443, 931–49. 7. Evans, J. D., Aronstein, K., Chen, Y. P., Hetru, C., Imler, J. L., Jiang, H., Kanost, M., Thompson, G. J., Zou, Z., and Hultmark, D. (2006) Immune pathways and defence mechanisms in honey bees Apis mellifera. Insect Mol Biol 15, 645–56.

296

K.M. Buckley and J.P. Rast

8. Richards, S., Gibbs, R. A., Weinstock, G. M., Brown, S. J., Denell, R., Beeman, R. W., Gibbs, R., Beeman, R. W., Brown, S. J., Bucher, G., Friedrich, M., Grimmelikhuijzen, C. J., Klingler, M., Lorenzen, M., Richards, S., Roth, S., Schroder, R., Tautz, D., Zdobnov, E. M., Muzny, D., Gibbs, R. A., Weinstock, G. M., Attaway, T., Bell, S., Buhay, C. J., Chandrabose, M. N., Chavez, D., Clerk-Blankenburg, K. P., Cree, A., Dao, M., Davis, C., Chacko, J., Dinh, H., Dugan-Rocha, S., Fowler, G., Garner, T. T., Garnes, J., Gnirke, A., Hawes, A., Hernandez, J., Hines, S., Holder, M., Hume, J., Jhangiani, S. N., Joshi, V., Khan, Z. M., Jackson, L., Kovar, C., Kowis, A., Lee, S., Lewis, L. R., Margolis, J., Morgan, M., Nazareth, L. V., Nguyen, N., Okwuonu, G., Parker, D., Richards, S., Ruiz, S. J., Santibanez, J., Savard, J., Scherer, S. E., Schneider, B., Sodergren, E., Tautz, D., Vattahil, S., Villasana, D., White, C. S., Wright, R., Park, Y., Beeman, R. W., Lord, J., Oppert, B., Lorenzen, M., Brown, S., Wang, L., Savard, J., Tautz, D., Richards, S., Weinstock, G., Gibbs, R. A., Liu, Y., Worley, K., Weinstock, G., Elsik, C. G., Reese, J. T., Elhaik, E., Landan, G., Graur, D., Arensburger, P., Atkinson, P., Beeman, R. W., Beidler, J., Brown, S. J., Demuth, J. P., Drury, D. W., Du, Y. Z., Fujiwara, H., Lorenzen, M., Maselli, V., et  al. (2008) The genome of the model beetle and pest Tribolium castaneum. Nature 452, 949–55. 9. Putnam, N. H., Srivastava, M., Hellsten, U., Dirks, B., Chapman, J., Salamov, A., Terry, A., Shapiro, H., Lindquist, E., Kapitonov, V. V., Jurka, J., Genikhovich, G., Grigoriev, I. V., Lucas, S. M., Steele, R. E., Finnerty, J. R., Technau, U., Martindale, M. Q., and Rokhsar, D. S. (2007) Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Science 317, 86–94. 10. Chapman, J. A., Kirkness, E. F., Simakov, O., Hampson, S. E., Mitros, T., Weinmaier, T., Rattei, T., Balasubramanian, P. G., Borman, J., Busam, D., Disbennett, K., Pfannkoch, C., Sumin, N., Sutton, G. G., Viswanathan, L. D., Walenz, B., Goodstein, D. M., Hellsten, U., Kawashima, T., Prochnik, S. E., Putnam, N. H., Shu, S., Blumberg, B., Dana, C. E., Gee, L., Kibler, D. F., Law, L., Lindgens, D., Martinez, D. E., Peng, J., Wigge, P. A., Bertulat, B., Guder, C., Nakamura, Y., Ozbek, S., Watanabe, H., Khalturin, K., Hemmrich, G., Franke, A., Augustin, R., Fraune, S., Hayakawa, E., Hayakawa, S., Hirose, M., Hwang, J. S., Ikeo, K., Nishimiya-Fujisawa, C., Ogura, A., Takahashi, T., Steinmetz, P. R., Zhang, X., Aufschnaiter, R., Eder, M. K., Gorny, A. K., Salvenmoser, W., Heimberg, A. M., Wheeler, B. M., Peterson, K. J., Bottger,

A., Tischler, P., Wolf, A., Gojobori, T., Remington, K. A., Strausberg, R. L., Venter, J. C., Technau, U., Hobmayer, B., Bosch, T. C., Holstein, T. W., Fujisawa, T., Bode, H. R., David, C. N., Rokhsar, D. S., and Steele, R. E. (2010) The dynamic genome of Hydra. Nature 464, 592–6. 11. Hibino, T., Loza-Coll, M., Messier, C., Majeske, A. J., Cohen, A. H., Terwilliger, D. P., Buckley, K. M., Brockton, V., Nair, S. V., Berney, K., Fugmann, S. D., Anderson, M. K., Pancer, Z., Cameron, R. A., Smith, L. C., and Rast, J. P. (2006) The immune gene repertoire encoded in the purple sea urchin genome. Dev Biol 300, 349–65. 12. Rast, J. P., Smith, L. C., Loza-Coll, M., Hibino, T., and Litman, G. W. (2006) Genomic insights into the immune system of the sea urchin. Science 314, 952–6. 13. Huang, S., Yuan, S., Guo, L., Yu, Y., Li, J., Wu, T., Liu, T., Yang, M., Wu, K., Liu, H., Ge, J., Yu, Y., Huang, H., Dong, M., Yu, C., Chen, S., and Xu, A. (2008) Genomic analysis of the immune gene repertoire of amphioxus reveals extraordinary innate complexity and diversity. Genome Res 18, 1112–26. 14. Zhang, Q., Zmasek, C. M., Dishaw, L. J., Mueller, M. G., Ye, Y., Litman, G. W., and Godzik, A. (2008) Novel genes dramatically alter regulatory network topology in amphioxus. Genome Biol 9, R123. 15. Davidson, C. R., Best, N. M., Francis, J. W., Cooper, E. L., and Wood, T. C. (2008) Tolllike receptor genes (TLRs) from Capitella capitata and Helobdella robusta (Annelida). Dev Comp Immunol 32, 608–12. 16. Stein, C., Caccamo, M., Laird, G., and Leptin, M. (2007) Conservation and divergence of gene families encoding components of innate immune response systems in zebrafish. Genome Biol 8, R251. 17. Picardi, E., and Pesole, G. (2010) Computational methods for ab initio and comparative gene finding. Methods Mol Biol 609, 269–84. 18. Burge, C., and Karlin, S. (1997) Prediction of complete gene structures in human genomic DNA. J Mol Biol 268, 78–94. 19. Salamov, A. A., and Solovyev, V. V. (2000) Ab initio gene finding in Drosophila genomic DNA. Genome Res 10, 516–22. 20. Schweikert, G., Zien, A., Zeller, G., Behr, J., Dieterich, C., Ong, C. S., Philips, P., De Bona, F., Hartmann, L., Bohlen, A., Kruger, N., Sonnenburg, S., and Ratsch, G. (2009) mGene: accurate SVM-based gene finding with an application to nematode genomes. Genome Res 19, 2133–43.

19  Characterizing Immune Receptors from New Genome Sequences 21. Borodovsky, M., Lomsadze, A., Ivanov, N., and Mills, R. (2003) Eukaryotic gene prediction using GeneMark.hmm. Curr Protoc Bioinformatics Chapter 4, Unit4 6. 22. Stanke, M., and Waack, S. (2003) Gene prediction with a hidden Markov model and a new intron submodel. Bioinformatics 19 Suppl 2, ii215–25. 23. Nene, V., Wortman, J. R., Lawson, D., Haas, B., Kodira, C., Tu, Z. J., Loftus, B., Xi, Z., Megy, K., Grabherr, M., Ren, Q., Zdobnov, E. M., Lobo, N. F., Campbell, K. S., Brown, S. E., Bonaldo, M. F., Zhu, J., Sinkins, S. P., Hogenkamp, D. G., Amedeo, P., Arensburger, P., Atkinson, P. W., Bidwell, S., Biedler, J., Birney, E., Bruggner, R. V., Costas, J., Coy, M. R., Crabtree, J., Crawford, M., Debruyn, B., Decaprio, D., Eiglmeier, K., Eisenstadt, E., El-Dorry, H., Gelbart, W. M., Gomes, S. L., Hammond, M., Hannick, L. I., Hogan, J. R., Holmes, M. H., Jaffe, D., Johnston, J. S., Kennedy, R. C., Koo, H., Kravitz, S., Kriventseva, E. V., Kulp, D., Labutti, K., Lee, E., Li, S., Lovin, D. D., Mao, C., Mauceli, E., Menck, C. F., Miller, J. R., Montgomery, P., Mori, A., Nascimento, A. L., Naveira, H. F., Nusbaum, C., O’Leary, S., Orvis, J., Pertea, M., Quesneville, H., Reidenbach, K. R., Rogers, Y. H., Roth, C. W., Schneider, J. R., Schatz, M., Shumway, M., Stanke, M., Stinson, E. O., Tubio, J. M., Vanzee, J. P., VerjovskiAlmeida, S., Werner, D., White, O., Wyder, S., Zeng, Q., Zhao, Q., Zhao, Y., Hill, C. A., Raikhel, A. S., Soares, M. B., Knudson, D. L., Lee, N. H., Galagan, J., Salzberg, S. L., Paulsen, I. T., Dimopoulos, G., Collins, F. H., Birren, B., Fraser-Liggett, C. M., and Severson, D. W. (2007) Genome sequence of Aedes aegypti, a major arbovirus vector. Science 316, 1718–23. 24. Ghedin, E., Wang, S., Spiro, D., Caler, E., Zhao, Q., Crabtree, J., Allen, J. E., Delcher, A. L., Guiliano, D. B., Miranda-Saavedra, D., Angiuoli, S. V., Creasy, T., Amedeo, P., Haas, B., El-Sayed, N. M., Wortman, J. R., Feldblyum, T., Tallon, L., Schatz, M., Shumway, M., Koo, H., Salzberg, S. L., Schobel, S., Pertea, M., Pop, M., White, O., Barton, G. J., Carlow, C. K., Crawford, M. J., Daub, J., Dimmic, M. W., Estes, C. F., Foster, J. M., Ganatra, M., Gregory, W. F., Johnson, N. M., Jin, J., Komuniecki, R., Korf, I., Kumar, S., Laney, S., Li, B. W., Li, W., Lindblom, T. H., Lustigman, S., Ma, D., Maina, C. V., Martin, D. M., McCarter, J. P., McReynolds, L., Mitreva, M., Nutman, T. B., Parkinson, J., Peregrin-Alvarez, J. M., Poole, C., Ren, Q., Saunders, L., Sluder, A. E., Smith, K., Stanke, M., Unnasch, T. R., Ware, J., Wei, A. D., Weil, G., Williams, D. J., Zhang, Y., Williams, S. A., Fraser-Liggett, C.,

297

Slatko, B., Blaxter, M. L., and Scott, A. L. (2007) Draft genome of the filarial nematode parasite Brugia malayi. Science 317, 1756–60. 25. Dieterich, C., Clifton, S. W., Schuster, L. N., Chinwalla, A., Delehaunty, K., Dinkelacker, I., Fulton, L., Fulton, R., Godfrey, J., Minx, P., Mitreva, M., Roeseler, W., Tian, H., Witte, H., Yang, S. P., Wilson, R. K., and Sommer, R. J. (2008) The Pristionchus pacificus genome provides a unique perspective on nematode lifestyle and parasitism. Nat Genet 40, 1193–8. 26. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–402. 27. Eddy, S. R. (1998) Profile hidden Markov models. Bioinformatics 14, 755–63. 28. Rice, P., Longden, I., and Bleasby, A. (2000) EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 16, 276–7. 29. Altschul, S. F., and Koonin, E. V. (1998) Iterated profile searches with PSI-BLAST--a tool for discovery in protein databases. Trends Biochem Sci 23, 444–7. 30. Schaffer, A. A., Aravind, L., Madden, T. L., Shavirin, S., Spouge, J. L., Wolf, Y. I., Koonin, E. V., and Altschul, S. F. (2001) Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements. Nucleic Acids Res 29, 2994–3005. 31. Letunic, I., Doerks, T., and Bork, P. (2009) SMART 6: recent updates and new developments. Nucleic Acids Res 37, D229–32. 32. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–82. 33. Kumar, S., Nei, M., Dudley, J., and Tamura, K. (2008) MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform 9, 299–306. 34. Huang, Y., Niu, B., Gao, Y., Fu, L., and Li, W. (2010) CD-HIT Suite: a web server for clustering and comparing biological sequences. Bioinformatics 26, 680–2. 35. Hall, T. A. (1999) BioEdit: a user friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nuc Acids Symp Ser 41, 95–98. 36. Rebeiz, M., and Posakony, J. W. (2004) GenePalette: a universal software tool for

298

K.M. Buckley and J.P. Rast

genome sequence visualization and analysis. Dev Biol 271, 431–8. 37. Istomin, A. Y., and Godzik, A. (2009) Understanding diversity of human innate immunity receptors: analysis of surface features of leucine-rich repeat domains in NLRs and TLRs. BMC Immunol 10, 48. 38. Martinon, F., and Tschopp, J. (2007) Inflammatory caspases and inflammasomes: master switches of inflammation. Cell Death Differ 14, 10–22. 39. Noe, L., and Kucherov, G. (2005) YASS: enhancing the sensitivity of DNA similarity search. Nucleic Acids Res 33, W540–3. 40. Iwasaki, A., and Medzhitov, R. (2010) Regulation of adaptive immunity by the innate immune system. Science 327, 291–5. 41. Botos, I., Liu, L., Wang, Y., Segal, D. M., and Davies, D. R. (2009) The toll-like receptor 3:dsRNA signaling complex. Biochim Biophys Acta 1789, 667–74. 42. Chen, Y., Yu, P., Luo, J., and Jiang, Y. (2003) Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. Mamm Genome 14, 859–65. 43. Hansen, J. E., Lund, O., Tolstrup, N., Gooley, A. A., Williams, K. L., and Brunak, S. (1998) NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility. Glycoconj J 15, 115–30. 44. Pierleoni, A., Martelli, P. L., and Casadio, R. (2008) PredGPI: a GPI-anchor predictor. BMC Bioinformatics 9, 392.

45. Moreno-Hagelsieb, G., and Latimer, K. (2008) Choosing BLAST options for better detection of orthologs as reciprocal best hits. Bioinformatics 24, 319–24. 46. Zhang, Q., Zmasek, C. M., and Godzik, A. (2010) Domain architecture evolution of pattern-recognition receptors. Immunogenetics 62, 263–72. 47. Offord, V., Coffey, T. J., and Werling, D. LRRfinder: A web application for the identification of leucine-rich repeats and an integrative Toll-like receptor database. Dev Comp Immunol. 48. Sonnhammer, E. L., Eddy, S. R., Birney, E., Bateman, A., and Durbin, R. (1998) Pfam: multiple sequence alignments and HMM-profiles of protein domains. Nucleic Acids Res 26, 320–2. 49. Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R. D., and Bairoch, A. (2003) ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31, 3784–8. 50. Hansen, J. E., Lund, O., Engelbrecht, J., Bohr, H., Nielsen, J. O., and Hansen, J. E. (1995) Prediction of O-glycosylation of mammalian proteins: specificity patterns of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase. Biochem J 308 ( Pt 3), 801–13. 51. Biegert, A., Mayer, C., Remmert, M., Soding, J., and Lupas, A. N. (2006) The MPI Bioinformatics Toolkit for protein sequence analysis. Nucleic Acids Res 34, W335–9.

Index A Acid stripping..........................................144, 145, 149–150 Activation-induced cytidine deaminase (AID)....... 255, 256 Affinity, determination..................................................... 92 AIM2...................................................................................70 AlphaScreen......................................................... 72, 76–81 Alternative splicing..........................................244, 245, 252 Amine coupling......................... 85, 89–91, 93, 96, 100, 103 Antibody labeling................................................... 144, 165 Association rate constant, determination.................... 95, 97 Avidity................................................................51, 55, 66, 91

B Bacterial artificial chromosome (BAC).................. 244–253 Beta-galactosidase.................................. 7, 10, 22, 108, 109, 112, 114–116, 210 Biacore..........................83, 85, 88–90, 93, 97, 100, 104, 105 Bioinformatics.................................................276, 277, 294 Biotin ligase............................................... 52, 55, 56, 64, 91 Biotin quantitation................................................. 122, 124 Biotinylation....................................... 52, 55, 59, 64, 67, 91, 123, 124, 174, 228, 231 BLAST........................... 268, 276, 280–284, 289, 291–293 BWZ cells...............................................210, 220, 223, 225

C Cdc42.........................................................................184, 185 CD3zfusion.............................................215, 217, 219, 220 Cell binding to surfaces...................................170, 175–177 Cell culture.2, 5–6, 12, 27, 36–37, 39–40, 53, 59, 60, 62, 66, 110, 113, 114, 124, 135, 158, 177, 187–189, 197, 204, 212–213, 216, 230, 232, 233, 246, 249, 261 Cell growth..................................................................... 177 CELLISA.............................................................. 209–225 Cell isolation...................................................................... 3 Cell lysis......................... 15, 37, 40, 48, 71, 74, 80, 224, 229 Cell permeabilization..................................................... 151 Cell signalling..................................................................... 2 Cell surface receptors..................... 1, 91, 170, 177, 222, 227 Cellular rupture...................................................... 233–234 Chemiluminescence....................................................... 108

Chemokine............................................................. 143–153 Chicken.................................................................. 255–271 Chimeric protein........................... 51–67, 77, 159, 190, 192 Class switch recombination............................................ 211 Cloning.............................3–5, 8, 12, 25, 65, 211–212, 214, 215, 219, 222, 246, 250, 252, 257, 259, 267, 269 Cloud computing........................................................... 293 Colloidal silica beads.............................................. 227–240 Colorimetric detection........................................... 219, 224 Computational methods................................................. 244 Confocal microscopy......................................135, 136, 148, 149, 156, 158, 160–162, 165, 188, 190 Contig .............................................................247, 275, 277 Correlated motion analysis..................................... 130, 131 Cre recombinase..............................................258–259, 266 C-type lectin.................................................1–18, 107, 281 Cytidine deaminase........................................................ 255 Cytoskeleton........................................................... 156, 184

D Density gradient centrifugation.......................218, 232, 234 Diffusion coefficients, calculation............127, 155, 159, 164 Dissociation rate constant, determination.................. 95, 97 DNA isolation BAC/PAC........................................................ 249–253 Genomic................................... 18, 26, 31, 82, 243–253, 258, 259, 261–263, 265–267, 269, 270, 275, 277–279, 289, 291–293 plasmid............................................................. 151, 187 Domain architecture.......................................108, 274, 281, 284–287, 289, 291, 292 Dot plot.....................................................31, 276, 289, 290 DT40 cells.......................................................257, 259–261

E Electron microscopy grids, preparation.................. 173–174 Electroporation........................ 198, 204, 206, 257–258, 261 ELISA . .................................................9–11, 36–45, 48, 49 EMBOSS................................ 276, 279, 281, 287–288, 293 Endocytosis......................145, 147–149, 152, 170, 176, 196 Epitope spreading........................................................... 211 Exocytosis........................................................145, 149–150

Jonathan P. Rast and James W.D. Booth (eds.), Immune Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 748, DOI 10.1007/978-1-61779-139-0, © Springer Science+Business Media, LLC 2011

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Immune Receptors 300  Index

  

F

L

Far western blotting...................................14, 38–39, 44–47 FceRI...............................................121–132, 196, 201–202 Fc fusion proteins............................................... 2–4, 12–17 Fixation...........................................................181, 188, 198 FKBP.................................................................12, 185, 186 FLAG tag............................................................... 211, 223 Flow cytometry...............................................6, 12–14, 213 Fluorescence-activating cell sorting (FACS)................. 3, 6, 14, 17, 25, 30–32, 181, 213, 216, 256, 259, 266–269 Fluorescence microscopy................................................ 205 Fluorescence recovery after photobleaching.......... 148–149, 155–166

Lamprey............................................................. 21–33, 291 Leucine-rich repeats (LRRs).......21, 22, 108, 278, 281, 287, 289, 294 Leukocyte immunoglobulin-like receptor (LILR)........... 83 Library construction................................................... 25, 26 Library screening.................................................... 245–248 Ligand arrays.......................................................... 195–206 Ligand immobilization......................85–86, 88–9, 200–203 Ligand pulldown assay......................................... 52, 71–76 Linux...................................................................... 277, 293 Lipid bilayer preparation................................................ 197 Live-cell imaging............................. 145, 148, 188, 190, 191 Luciferase................................................108–110, 112–117

G Gene conversion..................................................... 255–271 Gene prediction....................... 274, 275, 278, 279, 289, 290 Gene targeting................................. 256, 259, 265, 266, 269 Genome analysis..............................................274, 288, 292 Genomics.... 26, 31, 243–253, 258, 259, 261–263, 265–267, 269, 270, 275, 277–279, 289, 291–293 Green fluorescent protein................ 144, 155, 159, 184, 198

H HEK293T cells....................................................55, 58, 59, 66, 108, 110–112, 114, 115, 245, 246, 249–252 Heterodimerization.................................186, 189, 191, 192 Hidden Markov model....................................280, 284, 286 High throughput screening.............................109, 112, 210 HMMER........................................ 276, 281, 284–287, 293 Homologous recombination..................................23, 24, 26 Human leukocyte antigen (HLA).................................... 83 Hybridoma............................................................. 209–225

I IgE receptors.......................................................... 121, 196 Illumina sequencing....................................................... 279 ImageJ ..................................... 135, 138, 139, 152, 165, 188 Image quantitation......................................................... 135 Imaging, live cell...............145, 148, 184, 188, 190, 191, 204 Immunization..............................................12, 22, 209–225 Immunoblotting..............................................72, 73, 75–76 Immunodetection....................................................... 62–64 Immunofluorescence................................................. 6, 144, 145, 147, 148, 150, 151, 206 Immunoglobulin...................................................21, 51–67, 195, 252, 255, 278, 281, 288 Innate immunity.................................. 2, 21, 35, 69–82, 195 Isothermal titration calorimetry (ITC)..................... 84, 101

K Kinetic analysis.............................. 85, 90, 95–100, 103, 105

M Macrophages bone marrow-derived.................................................. 36 human monocyte-derived........................................... 36 RAW264.7................................................185, 187, 188 MACS enrichment........................................................... 32 MAP kinase................................................................... 108 Mass spectrometry................36, 45, 46, 73, 80, 81, 227–240 Mast cell..................................................121, 170, 196, 202 MATLAB...................................................................... 125 Membrane proteomics.................................................... 231 Microfabrication..................................................... 196–200 Monoclonal antibody.............................. 6, 7, 9, 17, 38, 165 Multigene families................... 244, 274, 275, 289, 291, 292 Muramyl dipeptide (MDP)............. 108–110, 113, 115, 117 Myeloid cell........................................................................ 7

N NF-kB.............................................................108, 111–116 NK receptor................................................................ 1, 211 Nod1....................................................................... 107–117 Nod2....................................................................... 107–117 Nod-like receptor.................... 107, 274, 279, 281, 291, 294 Nucleic acid receptors................................................. 69–82

O Opsonization.......................................................... 189–190

P Paralogs...................................................288, 289, 291, 292 Pattern recognition receptors (PRRs)..........2, 107, 111, 274 P1-derived artificial chromosome (PAC)............... 245–253 Peptidoglycan......................................................... 107, 108 PFAM domains...................................................... 278, 286 Phagocytic cup........................................134, 137–139, 141 Phagocytic index.................................................... 187, 191 Phagocytosis............................ 133–142, 170, 183–192, 196

Immune Receptors 301 Index     

Phagosome.......................138, 139, 141, 142, 184–187, 190 Phenol red............................... 135, 137, 140, 145, 165, 224 Phospholipid.......................................................... 184, 195 Photobleaching.........144, 146, 148–149, 152, 155–166, 190 Photolithography............................................................ 198 Plasma membrane, isolation........................................... 230 Plasma Membrane Sheets....... 170, 171, 173, 175, 177–181 Plasmid biosensor........................................................... 184 Polylysine coating........................................................... 228 Polymerase chain reaction (PCR).............................3, 8, 12, 24, 26, 31, 32, 52, 55, 57, 58, 65, 211, 214–215, 222, 244–253, 257, 259, 267, 269, 271 Polystyrene beads.............................................135–137, 192 Protein A..................................12, 18, 53, 55, 60–62, 66, 77 Protein expression................................................64, 78, 209 Protein G.......................................................................... 66 Protein solubilization...............................232, 234, 235, 239 Proteomics.............................................................. 227–240 PYHIN...................................................................... 70, 73

Q Quantum dots (QDs)............................................. 122–132

R RACE .............................................................244, 249, 251 Rak1................................................................................ 109 Rapamycin.............................................................. 183–192 Ratiometric analysis................................................ 133–142 RAW264.7 cells............................................................. 157 RBL–2H3 cells................................ 122, 124, 196, 197, 204 Recycling........................................................................ 143 Recombinant antibodies................................................. 174 Restriction digest.................................................... 260, 265 Retroviral transduction................................................... 216 Rig-I................................................................................. 70 Rig-I like helicase..................................................... 70, 107

S Scaffold............................................ 275–277, 279, 280, 289 Scavenger receptor.......................................35–49, 278, 281 SDS-PAGE............ 15, 44, 47, 53–55, 62, 63, 65, 74, 75, 77 Sea urchin........................................................274, 287, 288

SfiI cloning....................................................................... 65 Single particle tracking............ 122, 125, 127, 129, 130, 132 siRNA............................................. 109–112, 114, 116, 117 SOLiD sequencing......................................................... 279 Soluble receptor.............................................................. 185 Somatic hypermutation.......................................... 255–271 Southern Blotting............................................258, 262–265 Streptavidin..........3, 6, 23, 25, 54, 55, 63–67, 71, 73, 74, 77, 79–81, 88, 122, 172, 173, 175, 201, 213, 216 Supported membranes.....................................201–203, 206 Surface plasmon resonance................................. 66, 83–105

T TAK1..................................................................... 108, 112 Toll-like receptors (TLRs)........................................ 21, 68, 70, 73, 74, 80, 107, 158, 274, 281, 287, 291 Transcriptional splicing.......................................... 243–253 Transcriptome................................................................ 243 Transfection......2, 8, 12, 52, 53, 55, 58–60, 64, 80, 108, 110, 112–115, 135–136, 140, 146–147, 150, 151, 158, 165, 185, 187–189, 192, 198, 204, 206, 212–213, 215, 216, 223, 243–253, 257, 260, 261 Transmission electron microscopy (TEM).................... 175, 176, 180, 181 Tri-DAP..........................................108–110, 113, 115–117 Type II cell surface receptor............................................... 1

V Van’t Hoff analyses........................................................... 84 Variable lymphocyte receptor............................. 21–33, 281

W Western blot............................14, 15, 36, 38–39, 44–48, 53, 54, 62–64, 72, 116, 117, 233–235, 237, 238

Y Yeast surface display (YSD)........................................ 21–33 Yeast transformation........................................24–25, 27–29

Z Zebrafish.........................................................245, 250, 252