Dendritic Cells in Clinics

M. Onji, S.M.F. Akbar Dendritic Cells in Clinics 2nd Edition M. Onji, S.M.F. Akbar Dendritic Cells in Clinics 2nd E...

Author: M. Onji | S.M.F. Akbar

31 downloads 942 Views 1MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!

Report copyright / DMCA form

DOWNLOAD PDF

M. Onji, S.M.F. Akbar

Dendritic Cells in Clinics 2nd Edition

M. Onji, S.M.F. Akbar

Dendritic Cells in Clinics 2nd Edition

Morikazu Onji, M.D. PH.D. Professor, Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan Sk. Md. Fazle Akbar, MBBS, PH.D. Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan

ISBN 978-4-431-79465-3

Springer Tokyo Berlin Heidelberg New York

Library of Congress Control Number: 2008928563

© Morikazu Onji and Sk. Md. Fazle Akbar 2008 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, speciﬁcally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microﬁlms or in other ways, and storage in data banks. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a speciﬁc statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publisher can give no guarantee for information about drug dosage and application thereof contained in this book. In every individual case the respective user must check its accuracy by consulting other pharmaceutical literature. Springer is a part of Springer Science+Business Media springer.com Printed in Japan Typesetting: SNP Best-set Typesetter Ltd., Hong Kong Printing and binding: Kato Bunmeisha, Japan Printed on acid-free paper

In Commemoration of The 44th Annual Meeting of the Japan Society of Hepatology 2008 Matsuyama, Japan

Foreword

It now has been clearly recognized that dendritic cells play a crucial role in the initiation and modulation of immune responses against infectious diseases and cancers; they are involved in both innate and acquired immunity. Since the identiﬁcation of myeloid dendritic cells by Steinman and Cohn in 1973, a huge volume of ﬁndings on dendritic cells has been obtained. It is known that there are subsets of dendritic cells with different phenotypes and functions, and the phenotypes and functions of individual dendritic cells have been studied extensively and been clariﬁed. On the basis of this cumulative research, dendritic cells are thought to be a possible potent adjuvant for both prophylactic and therapeutic vaccine against infectious diseases and cancers. Five years ago, Professor Onji and his colleagues, Drs. Akbar and Horiike, published the ﬁrst edition of Dendritic Cells in Clinics. The book was astonishing to me as the authors beautifully summarized a huge amount of information on dendritic cells and immunology for any reader to understand. Five years have passed since the publication of the ﬁrst edition, and during that time the advances in the ﬁeld have been tremendous. I know quite well that Professor Onji and Dr. Akbar, both talented physician–scientists, have worked very hard in this ﬁeld and have sought to apply an understanding of dendritic cells to the prevention and treatment of liver diseases. Thus it is timely for them to publish the second edition of the book at this moment. Thanks to Professor Onji and Dr. Akbar, we now have available in this book the latest knowledge on dendritic cells and its possible application in clinics. As Professor Onji’s colleague in the study of liver immunology and as a longstanding friend, it is my great honor to write this foreword. Michio Imawari, M.D. Professor and Chairman Department of Medicine Chief, Division of Gastroenterology Showa University School of Medicine Tokyo

VII

Preface

Dramatic changes have taken place in politics and economics in Japan during the last 5 years, and comparable changes have occurred in medical service delivery systems and education. New clinical training curricula following graduation from medical schools have brought revolutionary changes in medical education and the clinical research system. Although there has been some stagnancy in the nature of ongoing medical research in Japan, I believe that opportunities for great future developments are being provided. Recent successes in the development of stem cells and their possible applications in clinics inspire researchers in all ﬁelds. The ﬁrst edition of this book was published in 2004, when many clinicians had begun showing an interest in dendritic cells. It was one of the ﬁrst books about dendritic cells that described their relevance in clinical medicine. After its publication, we received many enthusiastic responses and also many criticisms. During the past 5 years, great advances have taken place in basic research and the clinical usefulness of dendritic cells. When the ﬁrst edition was published, the plasmacytoid dendritic cell was an important topic in the clinical ﬁeld, and the role of dendritic cells beyond that of T cell priming began to unfold. During the last few years, type-1 interferonproducing dendritic cells have provided more support to the idea that dendritic cells are essential to innate immunity. Discovery of different pattern receptors, including Toll-like receptors on dendritic cells, has ﬁrmly established the position of these cells in regulating innate immunity. Concurrently, it has been revealed that immune tolerance plays a critical role in controlling the immune system in living organisms and that critical events in this cascade are controlled by dendritic cells. Researchers have shown that regulatory dendritic cells prepared in vitro induce antigen-speciﬁc immune tolerance. Knowledge of regulatory dendritic cells deﬁnitely contributes to an understanding of pathogenesis and of the development of new therapy against immune-associated diseases. Thus, dendritic cells, the mighty immunocytes, induce both innate and adaptive immunity as well as regulating immune responses and immune tolerance. These advances in dendritic cell biology led to the revision of the ﬁrst edition of Dendritic Cells in Clinics, with an eye toward clarifying the implications of dendritic cells in the pathogenesis and treatment of human diseases. We have been working with dendritic cells for more than two decades, but our major focus is on liver immunology. We therefore feel a natural responsibility to respond to queries about dendritic cells in clinics during the 44th Annual Meeting of the Japan Society of Hepatology in June of 2008, to be organized by me and other members of our department at IX

X

Preface

Matsuyama, Japan. In this context, I hope that the newly published 2nd edition of Dendritic Cells in Clinics will provide some answers to questions about dendritic cells for use in humans. We would like to express my gratitude to my colleagues in our department and to the members of our Yellow Orchard Club. Morikazu Onji Sk. Md. Fazle Akbar January 2008

Contents

Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VII IX

1. History of Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

From Discovery to Clinical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

2. General Features of Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5

Dendritic Cells in Clinical Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Outline of DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Deﬁnition of DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Origin and Development of DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection of DC-Like Cells in the Intrauterine Life of Mouse and Man . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Origin of DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of DC Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dendritic Cells in Different Organs and Tissues . . . . . . . . . . . . . . . . . . . . . . . DCs in Lymphoid Organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DC in the Nonlymphoid Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Implications of Phenotypes and Subsets of DCs . . . . . . . . . . . . . . . Function of Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DCs in Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DC in Adaptive Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antigen Presentation and T-Cell Activation . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Implications of DC Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks: Implications of This Chapter in the Clinic . . . . . . . Recommended Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5 8 11 15

3. Interactions Between Dendritic Cells and Infectious Agents . . . . . . . . . . . .

41

Outline of Dendritic Cell in Microbial Infection . . . . . . . . . . . . . . . . . . . . . . Consequences of Interactions Between DC and Virus . . . . . . . . . . . . . . . . . Interactions Between Dendritic Cells and Bacteria . . . . . . . . . . . . . . . . . . . . Recommended Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

41 49 67 71

17 17 20 24 24 27 31 31 31 32 36 36 37 39

XI

XII

Contents

4. Dendritic Cells and Allergy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

73

Nature of Immune Responses and Allergic Diseases . . . . . . . . . . . . . . . . . . . Pathogenesis of Allergic Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dendritic Cells in Allergy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Allergen-Speciﬁc Immune Responses and Memory Lymphocyte Formation by DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Th2 Polarization Capacities of Both Myeloid and Plasmacytoid DCs . . . . Dendritic Cells and Drug Allergy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dendritic Cells in Animal Models of Allergy . . . . . . . . . . . . . . . . . . . . . . . . . Removal of Airway DCs from Sensitized Mice Eliminates Asthmatic Features Induced by Antigen Aerosol . . . . . . . . . . . . . . . . . . . Dendritic Cells in Human Allergy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Allergic Asthma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Atopic Dermatitis (AD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Allergic Rhinitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment of Allergic Conditions by DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recommended Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

73 74 75

5. Dendritic Cells and Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

87

General Consideration of Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dendritic Cells in Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Goal of DC Research in Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . . DCs in the Pathogenesis of Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . Putative Role of DCs in Induction of Autoimmunity . . . . . . . . . . . . . . . . . . DCs at the Tissues in Autoimmune Conditions . . . . . . . . . . . . . . . . . . . . . . . Peripheral Blood DCs in Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . IFN-α and Plasmacytoid DCs in Autoimmune Diseases . . . . . . . . . . . . . . . . DC-Based Therapy for Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DC-Based Therapy in Animal Models of Human Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Possible Use of DCs for Treatment of Human Autoimmune Diseases . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recommended Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

87 88 91 92 95 95 97 98 98 99 101 103 103

6. Dendritic Cells in Tumor Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

105

Basic Principles of Tumor Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dendritic Cells and Tumor Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Localization and Characterization of DCs in Tumors . . . . . . . . . . . . . . . . . . Immune Therapy of Tumors Using Dendritic Cells . . . . . . . . . . . . . . . . . . . DC-Based Therapy for Human Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recommended Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

105 107 121 126 128 139

7. Dendritic Cells in Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

141

Present Status of Organ Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acute and Chronic Rejection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

141 146

76 78 79 80 80 81 82 83 84 84 86

Contents

XIII

Roles of DCs in Minimizing Rejection of Transplants: Guidelines of DC-Based Therapy for Survival of Allografts . . . . . . . . . . . Controlling DC Functions as a Therapeutic Approach . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recommended Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

147 148 152 153

8. Dendritic Cell-based Immune Therapy: Concept, Design, Present Limitations, and Future Projections . . . . . . . . . . . . . . . . . . . . . . . . . .

155

Dendritic Cell (DC)-Based Therapy as Immune Therapy in Clinical Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of Immunity and Their Putative Roles in Host Defense . . . . . . . . . . Nonantigen-Speciﬁc Immune Therapy in Clinical Medicine . . . . . . . . . . . . Antigen-Speciﬁc Immune Therapy for Treatment of Patients with Different Pathological Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Strategy of Antigen-Speciﬁc Immune Therapy . . . . . . . . . . . . . . . . . . . . . . . . Limitations of Immune Therapy by Vaccines or Antigens in Patients with Cancer and Chronic Viral Infections and the Concept of DC-Based Immune Therapy . . . . . . . . . . . . . . . . . . . . . . . . Immunomodulatory and Therapeutic Efﬁcacy of Antigen-Pulsed DCs in Animal Models of Cancer and Chronic Viral Infection . . . . . . . . Limited Therapeutic Efﬁcacy of Antigen-Pulsed DCs in Patients with Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Different Immunomodulatory and Therapeutic Efﬁcacy of Antigen-Pulsed DCs in Animal Models of Human Diseases and Patients with Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Points of Attention for Production of Immunogenic Antigen-Pulsed DCs to Have Better Immunomodulatory Capacities in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Why There Is Concern If Antigen-Pulsed DCs with Different Levels of Maturation and Activation Are Present Among Bulk Populations of Antigen-Pulsed DCs . . . . . . . . . . . . . . . . . . . . . . . . . . Is There a Need to Check Chemokines on Antigen-Pulsed DCs? . . . . . . . . The Ratio of Mature DCs in Bulk Populations of DCs: Is It Important? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Checking of DCs for Their Capacity to Process and Present Antigens . . . . Choosing Antigens for Pulsing DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assessment of Immunogenicity of Antigen-Pulsed DCs . . . . . . . . . . . . . . . . Role of Innate Immunity During Induction of Antigen-Speciﬁc Adaptive Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recommended Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

155 156 158 159 159

160 161 162

162

164

165 165 165 166 167 167 167 169 169 171 173 179 187

1. History of Dendritic Cells

From Discovery to Clinical Applications In 1868, a medical student, Paul Langerhans, discovered a population of dendritically shaped cells in the suprabasal region of the epidermis. These cells now bear his name. The reactivity of these cells to gold salt made him believe that the cells represented sensory nerve endings. However, today we know that Langerhans cells (LCs) are dendritic leukocytes, which perform a variety of activities. In particular, they function as professional antigen-presenting cells (APCs). During the 1960s, a consensus developed among immunologists that three types of cells, that is, T lymphocytes, B lymphocytes, and macrophages, collaborate during the induction of antigen-speciﬁc immune response. The role of B lymphocytes in the production of antibodies was already known, and it was also clear that T lymphocytes played a dominant role in the induction of cell-mediated immunity. Nevertheless, there were several black holes regarding the mechanism of T-cell-mediated cellular immune responses. Two factors were mainly responsible for this state of affairs. First, it was known that T lymphocytes are devoid of receptors for microbial agents or tumor cells, so it was unclear how T lymphocytes could interact with these agents without having such receptors. Second, T lymphocytes are not equipped with an apparatus for capturing and internalization of microbes or their antigens, which is essential for the induction of antigen-speciﬁc immunity. Thus, it was speculated that, in addition to T lymphocytes, there should be one or more specialized types of cells in the immune system that would be capable of capturing, internalizing, and processing of microbes or their antigens in situ. Traditionally, cells with these capacities are regarded as APCs. The responsibility of uptake of microbes or their antigens was on the shoulder of macrophages, and these were the primary cells recognized as APCs until 1973, when Ralph Steinman and Zanvil Cohn introduced a new type of cell, the dendritic cell (DC), which functioned as an APC in immunology. They isolated and characterized this novel immunocyte from mouse spleen. DCs were larger than classical lymphocytes, had rough surfaces, and lacked some of the characteristic features of macrophages. Phase-contrast microscopy revealed many dendritic processes on their surfaces. Spleen DCs were not shown at this time to express surface markers of lymphocyte and monocyte lineages, but the technical limitations of the early 1970s should be borne in mind. Spleen DCs were motile and expressed very high levels of major histocompatibility (MHC) class I and II antigens. Functional analyses revealed that 1

2

1. History of Dendritic Cells

spleen DCs were highly potent stimulators of autologous and allogeneic T lymphocytes in vitro. DCs adhered on plastic surfaces initially, but became nonadherent after overnight culture. This unique property of spleen DC, along with their shape, motility, absence of phagocytic apparatus, and potent allostimulatory capacity, gave Steinman the scientiﬁc and logical basis to constitute a novel cell population. In time, new surface markers of DCs were identiﬁed and new functions of DCs were revealed. The potent antigen-capturing apparatus of DCs was also evident. Gradually, DCs gained recognition as APCs. DCs were localized, enriched, and isolated from other lymphoid and nonlymphoid tissues, including blood, lymph, thymus, and bone marrow. The functions of DCs from these tissues and organs were also characterized. During the 10 years or so from the discovery of spleen DCs in 1973 until the mid1980s, DC studies were dominated by immunologists. Few clinicians were interested in DCs because the concept of APCs was not clear in the physiological and pathological context. It was even unknown whether APCs had any role in the pathogenesis of immune-mediated diseases, persistent infections, and malignancies. Although lymphocytes occupied the central position of different immunological events, few experimental data were present regarding direct roles of lymphocytes in pathological conditions. The development of immune therapy in pathological conditions was limited to activating lymphocytes in vivo by immunomodulatory agents or the administration of lymphocytes activated in vitro. Needless to say, DC-based therapy was not even dreamed of by clinicians. Beginning in the mid-1980s, some important developments in this ﬁeld drew the attention of clinicians to APCs and DCs. Using immunohistochemical methods, some investigators detected DCs or DC-like cells at the site of pathological lesions in some human diseases. Furthermore, the prognosis of some diseases was related to the frequencies of inﬁltrated DCs in the pathological tissues; the more DCs that were present in the tissues, the better was the prognosis. By this time, a consensus was developed among immunologists regarding the roles of APCs during the induction of antigenspeciﬁc immune responses. The role of DCs in the pathogenesis of diseases and the potential scope of DC-based therapies for treating pathological conditions started to become evident about this time. It became evident that many pathological conditions might relate to the defective functioning of APCs and DCs. Researchers started to ﬁnd some answers to questions that had long been unanswerable by conventional wisdom. Clinicians started to consider why some patients suffering from chronic microbial infectious were unable to mount an effective immune response against the microbes or microbe-encoded antigens in vivo, even though there were abundant amounts of microbial agents and their antigens, nor was an immune response to microbe-encoded antigens seen in vitro. Interestingly, these patients did not show any features of a generalized immune deﬁciency, indicating that there might be a very speciﬁc defect in the processing of microbial agents or their antigens in these patients. Historically, this problem has been attributed to defective induction, production, and functioning of microbe- or microbial antigen-speciﬁc lymphocytes. However, DC researchers showed that the production of antigen-speciﬁc lymphocytes was dependent mostly on the recognition, processing, and presentation of antigens to lymphocytes by DCs. Clinicians predicted that the defective APC function of DCs might account for the impaired formation of antigen-speciﬁc lymphocytes in patients with persistent microbial infections. The

From Discovery to Clinical Applications

3

next step was comparatively easier; improvement of the functioning of DCs in vivo in these patients might have therapeutic beneﬁts. When impaired functions of DCs were a matter of concern for the clinicians dealing with persistent microbial infections, the opposite was true for transplant specialists. The induction of immune response to alloantigens is related to the rejection of transplanted organs. As DCs are the most potent stimulator of allogeneic lymphocytes, a role for DCs in the context of transplant acceptance or rejection was predicted. It was postulated that if the immunomodulatory capacity of DCs could be downregulated or if tolerogenic DCs could be propagated in vivo, the chances of acceptance of transplanted organs might be increased. Clinicians working with allergy and autoimmunity also found that DCs might have important roles in these conditions. At this time, the role of DCs as a regulator of immune response became evident. DCs were capable of polarization of T lymphocytes toward either T helper (Th) 1 or Th2 types of immune response based on the nature of antigens, the antigen dose, the duration of the ligation with T cells, and the availability of cytokines in the tissue microenvironment. Clinicians dealing with allergic diseases suspected that DCs might have a role in Th2 polarization in the context of immunity against allergens. The scope of therapy targeting DCs in allergic conditions was expanded. One of the main functions of DCs in the physiological condition is to induce immunogenic tolerance to self-antigens and harmless entities. Moreover, DCs play potent roles in inducing and controlling central and peripheral tolerance. Impaired functions of DCs in genetically susceptible hosts might induce and perpetuate autoimmunity. Many clinicians became interested in regulating the functions of DCs in vivo to handle autoimmune diseases. For a long time, it has been known that tumors somehow avoid the normal immune surveillance system, leading to their uncontrolled growth. It remained unknown, however, why tumors were able to escape from this surveillance. DCs should bear the primary responsibility for the recognition of tumors, and they would also be expected to induce innate and adaptive immunity against tumors. The uncontrolled growth of tumors, therefore, indicates some impairment in the functioning of DCs in tumorbearing patients. Several investigators have been able to show impaired functioning of DCs in almost all types of human and animal tumors. New information was accumulating regarding the nature of the defect in the DCs of tumor-bearing hosts. During the 1990s, several tumor-associated antigens (TAAs) were also discovered, and TAA-pulsed DCs were used for the treatment of tumors. The most important breakthrough regarding DCs was the development of techniques to enrich DCs from human peripheral blood. Various studies had reported the enrichment of DCs from human blood by several methodologies; however, the real breakthrough in this area came when abundant DCs could be produced by culturing an adherent population of human peripheral blood with a cocktail of cytokines. This effort initiated a series of functional studies using blood DCs from patients with different pathological conditions. From the discovery of DCs in 1973 up to the late 1990s, the role of DCs was evaluated mainly in the context of induction of adaptive immune responses. In 1999, some reports showed that DCs are not only related to adaptive immunity; a subset of DC precursors are potent producers of type 1 interferon (IFN). These DC precursors are

4

1. History of Dendritic Cells

called natural IFN-producing cells (NIPC). In addition to their potent capacity to produce type 1 IFN, these DCs were able to induce both Th1 and Th2 types of immune responses, depending on the nature of the stimulation and the tissue microenvironments. As type 1 IFN was widely used as an antiviral agent and for the treatment of malignancies, clinicians naturally became interested in ﬁnding ways to manipulate the functioning of NIPCs in vivo. During the 1990s, clinicians started to develop insights into the interactions between DCs and microbes, TAAs, autoantigens, allergens, and transplant antigens. Immunologists progressively developed better techniques for isolation of DCs from various tissues, including human blood. Different investigators published their data regarding the induction and production of various cytokines, chemokines, and immune modulators including type 1 IFN by different subsets of DCs. Researchers of various disciplines started to work together, and by the late 1990s, DCs began to move from the laboratory bench to the bedside. In addition to the DCs already described, there are some other cells with a dendritic morphology in the nervous system, but those are neither APC nor related to immune responses. Initially, DCs were mainly characterized in different pathological conditions to develop insights about roles of DCs in the pathogenesis of different diseases. From the early 1990s, DC-based therapy was started in animal models of human diseases, and DCs were ﬁrst used for human immune therapy in 1996. Now, DC-based therapy is mainly used for the treatment of patients with cancer. Recently, some investigators have used DCs for treatment of chronic viral infections and to overcome immune nonresponses for prophylactic purposes. Moreover, there is another population of cells with dendritic morphology related to immune responses, although these are not professional APCs of the immune system. These are called follicular dendritic cells (FDCs) because they were ﬁrst detected in the primary B-cell follicles of secondary lymphoid organs. FDCs share some morphological feature of DCs, but are quite different from antigen-presenting DCs, as already described. FDCs can retain antigen–antibody complexes on their surfaces for a long period but they cannot perform all the functions of professional APCs. Although the existence of these cells has been known since 1950, they were not characterized until 1962 and not named until 1978. During the 1980s to the early 1990s, these cells were isolated and functionally evaluated. The chronological history of immunology shows what we came to know about antibodies, “A,” and their protective capacities two centuries ago. In the last century, we developed tremendous insights regarding the phenotypes, functions, and mechanism of action of T and B lymphocytes, “B.” During the middle of the 20th century, many cytokines, “C,” which are able to regulate different immune reactions, entered the immunological arena. In the ﬁnal quarter of the last century, we achieved insights into APCs and DCs, “D.” Thus, it seems that the evolution of immunology progressed in alphabetical order: A, B, C, and D. Now, A, B, C, and D are meeting on the laboratory bench and at bedsides. Each discovery and innovation altered our understanding of the previous concepts tremendously. At this moment, we are unsure about “E” and its likely impact on its immediate predecessor D, the dendritic cells.

2. General Features of Dendritic Cells

Dendritic Cells in Clinical Medicine The book entitled Dendritic Cells in Clinics is intended to provide insights about dendritic cells (DC) to clinicians who are handling patients with cancers, chronic diseases, autoimmune diseases, and allergic manifestations. Much information has been accumulated regarding DC biology as a consequence of extensive work by basic immunologists and clinical researchers. The knowledge about DCs should be disseminated to clinicians in an amicable way so that these results can be applied in clinics for the beneﬁt of the patients. Various aspects of DC biology are progressing very rapidly. In this chapter, comprehensive outlines of DC have been provided so that clinicians can develop insights about the origin, the subsets, and the functions of DCs, which will help to develop roles of DCs in disease pathogenesis. Finally, DCs may be used for treating patients with different pathological conditions. In this context, we are interested in highlighting different features of human DCs. However, several important aspects of human DCs are yet to be explored. Accordingly, both murine DCs and human DCs are discussed in this chapter. Entry of DC in Clinics Different immunocytes have important roles in the pathogenesis of various diseases. In addition, cells that possess diagnostic, prognostic, and therapeutic utilities are also important in the clinic. As shown in Fig. 1, different cells in the peripheral blood have diagnostic and prognostic importance (Fig. 1A). However, most of these cells are not used for therapeutic purposes. On the other hand, DCs are used for therapeutic purposes in different diseases, although little is known about the diagnostic and prognostic importance of DCs in different pathological conditions (Fig. 1B). In addition, DCs have recently been used to overcome nonresponsiveness to prophylactic vaccine in humans. Diagnostic Importance of DCs There is a lack of consensus about the functions of DCs in different pathological conditions. Some investigators have reported that the functions of DCs are downregulated in some diseases, whereas others have shown that these are exacerbated in other diseases. The scenario becomes more complex because both impaired and 5

6

2. General Features of Dendritic Cells

(A) BLOOD CELLS

Clinical importance

Neutrophil

Diagnostic Prognostic Diagnostic Prognostic Diagnostic Prognostic Diagnostic Prognostic

Eosinophil Basophil Lymphocytes

(B) Apparent lack of diagnostic and prognostic implications of dendritic cells (DCs): 1. Blood DCs: Only available for clinical evaluation. Blood DCs are also few in number and there are different subtypes with contrasting functions. 2. Tissue DCs: Difficult to use for evaluation and clinical analyses. 3. Variations of DC number and function among individuals.

Therapeutic [limited] Monocytes Dendritic cells

Diagnostic Prognostic Therapeutic At Present: [cancer, viral diseases] In future: [autoimmune diseases, allergies, transplantation] Prophylactic As natural adjuvant

Why DCs bear therapeutic utility in clinics: 1. DCs are inducers of immunity and a few DCs can prime many immunocytes in vivo. 2. DC can induce both innate and adaptive immunity. 3. DCs are capable of inducing immune responses and immune tolerances. 4. As DCs are inducers of immunity or tolerance , the responses may be infinite. 5. DCs are safe for human usage.

Fig. 1. The clinical signiﬁcance of dendritic cells (DC) is different from other immunocytes in the blood. Different immunocytes, except DC, usually show diagnostic and prognostic importance (A). On the other hand, DCs exhibit potent therapeutic utility in the clinic (B)

unaltered functions of DCs have been reported from the same pathological conditions. Although there are several publications about altered functions of DCs in different pathological conditions, the real implications of DC in the diagnosis of different diseases are still elusive. Different investigators have used different types of DCs (peripheral blood DCs, cultured monocyte-derived DCs, or tissue-derived DCs) for functional analyses. Again, some investigators have checked the antigen-nonspeciﬁc functions of DCs whereas others have analyzed antigen-speciﬁc functions of DCs. In some diseases, cytokine production of DCs has been assessed, whereas the expressions of surface antigens have been elucidated by others. DCs from different stages of pathological conditions have been assessed in different diseases. In most cases, the functions of DCs have been elucidated as pilot studies. There are few investigations regarding the kinetics of DC function in normal healthy individuals. It seems that further controlled trials are required to develop insights whether DCs are involved in (1) initiation, (2) progression, and (3) complications of different pathological processes in human. To develop proper insights about these, representative DC populations should be used for functional analyses. Also, some speciﬁc functions of DCs should be checked in patients with different diseases. At present, it seems that the diagnostic importance of bulk populations of human DCs in different diseases or different stages of diseases is not so inspiring. However, studies about all major subtypes of DCs at different points of pathological processes may unveil some critical information that may help to establish the diagnostic importance of DCs in pathological conditions.

Dendritic Cells in Clinical Medicine

7

Prognostic Importance of DCs The functions of DCs have been analyzed in some pathological conditions to evaluate the prognostic importance of DCs. For example, the functions of DCs have been assessed in patients with acute resolved hepatitis C and chronic hepatitis C. The functions of DCs are diminished in patients with chronic hepatitis C, compared to patients with acute resolved hepatitis C. In these studies, DC functions were assessed in one group of patients with chronic hepatitis C and another group of patients with acute resolved hepatitis C. In most studies, the patients are from different geographical regions. These studies are not comparable regarding study design, study population, and methodologies. In fact, there is a paucity of information regarding DC function in the same population at different stages of a pathological process. Some studies indicate that the functions of DCs are upregulated by therapy in some diseases. However, little is known regarding age-related and sex-related variations of DC functions in normal individuals. For these reasons, the prognostic importance of DCs in different pathological conditions has not been properly established. Therapeutic Implications of DCs More studies are needed to establish the diagnostic and prognostic importance of DCs in different pathological conditions. Also, several clinical trials should be conducted to assess if DCs bear diagnostic or prognostic implications. Although the diagnostic and prognostic importance of DCs has not been established in different pathological conditions, DCs are now used for treating patients with different conditions. DCs are key regulators of innate immunity and play an active role during development of adaptive immunity in vivo. They produce various cytokines and instruct other immunocytes to do the same. Immature DCs can recognize, capture, and process antigens. Subsequently, DCs are activated and undergo maturation and activation. These DCs present antigens for induction of either immune responses or immune tolerances. Very few DCs can modulate immune responses. Several clinical trials have also shown that DCs are safe for human use. Thus, the therapeutic utility of DCs has been accepted by immunologists and clinicians, although critical discussion is continuing regarding the strategies of DC-based therapies in different pathological conditions. The prospect of DC-based therapy is bright because this therapy has been used in different types of cancers during the past decade. The safety of DC-based therapy has been shown by almost all investigators. Initially, DC-based therapies were performed as pilot studies only. At present, several clinical trials are going on to optimize DCbased therapies against cancers. Recently, DC-based therapy has been started in patients with chronic viral infections. Although there are numerous studies about the safety and efﬁcacy of DC-based therapies in animal models of allergic diseases and autoimmune diseases, DC-based therapies are yet to be carried out in patients with these diseases. Considering the importance of DC-based therapy in different pathological conditions, we discuss the scope and limitation of DC-based therapy in a new chapter of this book. There are several challenges to optimize DC-based therapies. The most notable one is about deﬁnition of DC and clariﬁcation of entity of DC. Basic immunologists are aware of diverse immunological features of DCs. However, it is not easy for clinicians

8

2. General Features of Dendritic Cells

to update different developments of DC biology, which is growing at an impressive speed. One of the main purposes of this chapter is to provide an outline of DC on the basis of present scientiﬁc knowledge.

Outline of DCs Entity of DC The current model of antigen presentation places DCs at the center of immunity. On the one hand, these cells are capable of inducing both innate immunity and adaptive immunity. On the other hand, DCs also acquire “self ”-products and constitutively present them in a tolerogenic fashion. This phenomenon is presently believed to contribute to the maintenance of self-tolerance. Several studies indicate that induction of immunogenic tolerance may be the main function of DCs in vivo. However, it is still unclear whether there are speciﬁed populations of immunogenic DCs and tolerogenic DC in vivo. Some recent studies have reported a tolerogenic population of DC in mice. However, the features and characteristics of tolerogenic DCs is unclear in humans in vivo. The present scientiﬁc understanding dictates that the nature of invading pathogens and tissue microenvironments are important to determine whether antigen presentation by DC would result in immune responses or immune tolerances. Immune responses are induced when DCs undergo maturation and activation after encountering microbial agents, cancer cells, transformed cells, allergens, or autoantigens. In contrast, immune tolerances are induced if maturation and activation signals are not properly provided by DCs. Antigen presentation by DCs in the absence of inﬂammatory mucosal milieu may also induce immune tolerance. Immune tolerances act at the interface between DCs and T lymphocytes and contribute to avoid the expansion of autoreactive T-cell clones. Although the tissue microenvironment and the maturation status of DCs are regarded as having major roles in immune responses and immunogenic tolerances, the cellular and molecular mechanisms may not be so simple. For example, in inﬂammatory tissue microenvironments, several antigens (microbial) and autoantigens may reside. However, immune responses are usually induced against microbial agents and rarely against autoantigens. On the other hand, although an inﬂammatory microenvironment prevails in many patients with cancers and chronic infections, DCs do not undergo maturation in these circumstances, and adequate levels of cancer-speciﬁc and microbial agent-speciﬁc immunity are not induced. These factors are a matter of great interest in the clinic. In fact, proper understandings about critical cellular and molecular mechanisms that trigger immune responses and immune tolerances by DCs are essential for designing clinically effective DC-based therapy. DCs were ﬁrst isolated from murine spleen in 1973. The most rapidly evolving aspect of DC biology is the magniﬁcent diversity of phenotypes that have been described from in vitro and in vivo studies. Also, DCs with different functional capacities have been detected. However, it is difﬁcult to provide proper deﬁnition of DC. We ﬁrst describe how DCs have been deﬁned until now and the various limitations of these approaches. Next, we provide a working deﬁnition of DCs. Initially, DCs were deﬁned as a trace population of immunocytes that did not express a marker of T lymphocytes, B lymphocytes, and macrophages. DCs were also

Outline of DCs

9

regarded as the most potent antigen-presenting cells (APCs). During the ﬁrst two decades after the discovery of DCs, thousands of reports have shown that DCs are professional APCs and that their main function is to induce immune responses. In the meantime, major progress has been made regarding isolation techniques of DCs, assessment of surface antigens by ﬂow cytometry, and functional characterization of DCs. The fundamental properties of DCs have not been changed; however, several new features of DCs have been identiﬁed. Based on these developments, a thorough discussion about the features and functions of DCs is now needed. DCs have either been enriched from or isolated from most tissues of the body. These cells are present in both lymphoid and nonlymphoid organs. DCs have also been isolated from human and murine peripheral blood. Different types of DCs have also been enriched from different organs and tissues. There is a need to make a distinction between the terms “isolation of DCs” and “enrichment of DCs.” The term isolation of DCs may be used when DCs are collected from tissues or blood without prolonged culture in vitro. On the other hand, the term enrichment of DC is suitable when DCs are ﬁnally available for characterization after culture in vitro. It is extremely important to assess whether isolated population of DCs or enriched population of DCs are characterized or used for DC-based therapy. For example, DCs can be isolated from fresh peripheral blood by cell-sorting techniques within a few hours. There may be some minor changes in the phenotypes and functions of DC during isolation. However, when DCs are enriched from different cellular populations by culturing in vitro in presence of different cytokines, drastic changes are detected between original cell populations and the ﬁnal cell populations. For example, when monocytes are cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 5–7 days, monocyte-derived DCs are generated. The phenotypes and functions of monocytes are altered during cultures. Although the resulting cells are called monocyte-derived DCs, it may be difﬁcult to get a natural counterpart of monocyte-derived DCs in vivo, especially in the steady state. The problem is further accentuated because different types of DCs have been described in the literature based on their phenotypes and functions: these include DC progenitors, DC precursors, immature DCs, mature DCs, myeloid DCs, lymphoid DCs, and plasmacytoid DCs. These DCs have been detected in cultures, and most of their counterparts are yet to be characterized in situ, especially in humans. Although DCs are derived from bone marrow from hematopoietic stem cells, little is known about the characteristic features of DC progenitors and DC precursors. DC progenitors exhibit immature phenotype and functions even after culture in vitro. The typical example of DC progenitors is liver DC progenitors. When nonparenchymal cells of the liver are cultured with GM-CSF for 7 days, a group of highly immature DC-like cells are detected in culture. These cells are usually resistant to maturation, even in presence of proinﬂammatory cytokines. Characteristic features of DC precursors have not been deﬁned for most DC subsets. Some of the DC precursors, such as monocytes, are highly differentiated, whereas the phenotypes of other DC precursors are yet to be fully described. Immature DCs are the sentinels of the immune system. They are mainly present in peripheral tissues, and are mainly responsible for recognition and capturing of antigens or transformed cells or tumor cells or apoptotic cells. They express low levels of costimulatory molecules. Maturating and migratory DCs represent DCs that have engulfed and processed antigens. They also show an

10

2. General Features of Dendritic Cells

alteration in chemokine receptor expression. Mature DCs are usually formed in the lymphoid tissues during their interaction with lymphocytes; however, these are also detected in the nonlymphoid tissues, especially in diseased organs. Tolerogenic DCs induce immune tolerance. They express lower levels of costimulatory molecules and produce antiinﬂammatory cytokines. Phenotypes of murine regulatory/tolerogenic DCs have been described in the literature, but almost nothing is known regarding regulatory and tolerogenic DCs in humans in situ. For practical purposes, there should be a clear distinction between immature DCs and regulatory DCs. Generally, it is assumed that antigen presentation by immature DCs in the steady state causes immune tolerance. However, it is still elusive whether there are different types of immature DCs or all immature DCs also possess the functional capacities of regulatory DCs. It may not be true that immature DCs in noninﬂammatory microenvironments are basically tolerogenic in nature. This concept may be supported if the cellular and molecular mechanisms underlying the induction of tolerance are analyzed. Commonsense indicates that immature and regulatory DCs must migrate to lymphoid tissues for induction of immune tolerance. If these DCs are phenotypically and functionally immature and reside in a tolerogenic microenvironment, then questions remain about their migratory capacities to lymphoid tissues for induction of immunogenic tolerance. Also, it is important to assess how these DCs interact with clonally selected lymphocytes at lymphoid organs. Little is known about the nature of exhausted DCs. Killer DCs express Fas ligand (Fas L) and might kill target cells. However, the capacity of killer DCs to destroy target cells in vivo has not been well documented. Recently, a new subset of DCs that express phenotypes of DCs and natural killer (NK) cells has been described. These DCs produce abundant amounts of interferon (IFN)-gamma and are called IFN-producing killer DCs (IKDCs). They also possess antigen-processing and -presenting capacities. IKDCs may be an important DC population for DC-based therapy because they can induce both innate and adaptive immunities. The progress of DC biology is extremely rapid. New subsets of DCs with different novel functions are emerging from extensive investigations by basic researchers. Investigators are also trying to develop more insights about their functions. These approaches are extremely necessary for use of DCs in clinics, especially for performing DC-based immune therapy. If proper insights are not developed about phenotypes, tissue localization, and functions of different subsets of DCs, development of effective regimens of DC-based immune therapy is not only difﬁcult but may be counterproductive. These factors are probably complicating the ongoing regimen of DC-based immune therapy in clinics. At present, the purposes of DC-based immune therapy are to induce antigen-speciﬁc immunity in patients with cancers and chronic infections. As both immunogenic and tolerogenic DCs have been detected, DC-based therapy may be counterproductive if proper DCs are not used for therapy. It seems that both types of DCs may be present in bulk population of DCs. In the near future, DCs will be used to treat patients with autoimmune diseases to induce antigen-speciﬁc immune tolerance. In addition to therapeutic purposes, DC-based vaccines have already been used to overcome nonresponse to prophylactic vaccine. In one pilot studies, we have used a DC-based prophylactic vaccine that induced protective antibodies in human vaccine nonresponders. In this context, clinicians should develop

Deﬁnition of DCs

11

clear insights so that the most suitable population of DCs may be used for therapy or prophylaxis in humans.

Deﬁnition of DCs DCs are a highly heterogeneous population of immunocytes with diverse phenotypes and functions. It is difﬁcult to deﬁne DCs on the basis of morphology, phenotypes, and functions because DCs exhibit widespread plasticity both in vivo and in vitro. Here, we ﬁrst describe the limitations to deﬁne DC. Next, a working deﬁnition of DC is provided. DCs can neither be deﬁned by morphology nor from their phenotypes nor by evaluating their functions. The causes underlying the difﬁculties of deﬁnition of DCs are shown in Table 1. Limitation of Deﬁnition of DCs by Dendritic Morphology The name “DC” implies that these cells should have dendritic processes. In fact, dendritic processes were detected in mice spleen DCs that were isolated by Ralph Steinmann in 1973 using density centrifugation and adherence on plastic surfaces in vitro. However, recent developments in imaging and other techniques indicate that DCs from many tissues and organs do not show such processes, especially in the steady state. Dendritic-like processes are not found in DCs in many organs in vivo. Table 1. Deﬁnition of dendritic cells A. Limitations of deﬁning dendritic cells Dendritic processes

DC surface antigens

DC-speciﬁc functions

B. Working deﬁnition of dendritic cells Combination of different features Expression molecules Migratory capacities Functional capacities

Expressed by some, but not all, DCs. Prominent in culture. However, may not be expressed in some DC subtypes, especially in situ. Lack of DC-speciﬁc antigen. DC-related antigens are expressed by other immunocytes in situ or by contaminated immunocytes in cultures. Functions of DCs can be performed by other immunocytes, although the magnitude may be different. This is difﬁcult to assess in situ.

MHC class II antigens, pattern recognition receptors. Migratory in nature. Professional antigen-presenting cells. Sense pathogen-associated molecular pattern. Antigen-processing capacities. Undergo self-maturation. Migration to lymphoid tissues. Activation and deactivation of lymphocytes. Inducers and producers of cytokines and other immunomodulators.

Dendritic cells (DC) cannot be deﬁned by a single speciﬁc characteristic (A). A working deﬁnition of DCs can be provided by combination of various phenotypic and functional features of DCs (B)

12

2. General Features of Dendritic Cells

Also, dendritic processes are not so prominent when DCs are isolated by cell-sorting techniques from different tissues. However, when these cells are cultured in presence of serum and other stimulatory agents, dendritic processes are detected in different types of DCs. Even if dendritic processes are detected on some DCs in culture, it is difﬁcult to deﬁne the nature of DCs by the presence or absence of dendritic processes. Several types of DCs, such as DC progenitors, DC precursors, immature DCs, maturating DCs, mature DCs, tolerogenic DCs, exhausted DCs, and killer DCs are present in the same organs or tissues at the same time. However, dendritic processes may be detected in some DCs only. In the other hand, many other cells such as those of monocyte and macrophage lineages exhibit dendritic-like processes. Limitation of Deﬁning DCs by Surface Markers When a cell cannot be deﬁned by morphology, the alternative way to identify that cell in different tissues and organs is by immunohistochemistry or ﬂow cytometry. To identify cells by these techniques, there should be some cell-speciﬁc markers that are expressed on those cells but not in adjacent cells or in contaminated cell populations. DCs were originally known to be negative for lineage markers (negative for markers of cells of myeloid and lymphoid lineages). However, recent investigations show that antigens of both myeloid and lymphoid lineages are constitutively expressed on different subsets of DCs. Moreover, several markers of myeloid and lymphoid cells could also be detected on DCs in vitro depending on the culture conditions. Although DCs express several antigens of APCs (MHC class II, CD1, CD80, CD86, CD40, and CD54) on their surface, none of these is DC speciﬁc. Many of these markers are detected on cells that are localized adjacent to DCs in vivo and in cells which contaminate DCs in cultures. Recently, many other receptors have been described in DCs, but none of these markers is DC speciﬁc. If there is any DC-speciﬁc marker, we have yet to detect that in DCs. The scenario becomes more complex when different types of antigens are expressed on different subsets of DCs. Some DCs express CD4 whereas others express CD8. Finally, the expression of surface antigens on DCs is dependent on stages of activation and maturation of DCs. Also, different surface antigens are detected on DCs at different stages of their life cycle. Limitations of Deﬁning DCs by Functional Capacities DCs are well known for their versatile functions. Different types of DCs exhibit different types of functions. Immature DCs are extremely efﬁcient to capture antigen, whereas the mature DCs usually present antigenic epitopes to T cells. Plasmacytoid DCs produce large amounts of type 1 IFN, but they are not efﬁcient APCs. Different types of DCs also produce different types of immune modulators including cytokines and chemokines. However, none of these functions is DC speciﬁc. Recently, it has been shown that regulatory/tolerogenic DCs induce immunogenic tolerance. Thus, it is becoming difﬁcult to deﬁne DCs from their functions.

Deﬁnition of DCs

13

Causes Underlying Confusion in Deﬁning DCs DCs can be deﬁned neither by their dendritic morphology nor by the expression of DC-speciﬁc surface antigens. Also, the functions of DCs are highly heterogeneous. We can get some answers to these queries if we look at the historical events relating to the discovery of DCs. DCs were ﬁrst isolated from murine spleen by overnight culture of spleen cells. Spleen is a secondary lymphoid organ, and considerable numbers of spleen DCs may have encountered microbial agents, their antigens, or other cellular sources of antigens. Some spleen DCs might have migrated from the peripheral tissues. These DCs are not in antigen-capturing mode; rather, they are mostly assigned to interact with T lymphocytes in the spleen. Most of the DCs in the spleen naturally exhibit a comparatively mature phenotype. Moreover, initially spleen DCs were isolated by overnight culture of spleen cells, which provided a further opportunity to undergo maturation of spleen DCs. Thus, dendritic processes were seen in mature DCs, as described by Ralph Steinman. The mature phenotype of spleen DCs was also reﬂected by their functional capacities. Very few spleen DCs were able to stimulate allogeneic T cells in an allogeneic mixed leukocyte reaction (MLR). Bulk populations of spleen DCs were also potent inducers and producers of various inﬂammatory cytokines and immune mediators. However, when fresh spleen DCs were enriched by cell-sorting techniques, comparatively less mature DCs were isolated. The T-cell stimulatory capacities of freshly isolated DCs are comparatively lower than those of cultured DCs. In the meantime, DCs were isolated from various tissues using cell-sorting techniques. Also, DC progenitors and DC precursors were isolated. The phenotypes of DCs are dependent on the microenvironment of the tissues. Although both spleen and thymus are lymphoid organs, DCs in the thymus perform a very specialized job. Accordingly, the phenotypes of thymic DCs are different from those of spleen DCs and other lymphoid organ-derived DCs. The phenotypes of DCs in nonlymphoid tissues are different from those of the lymphoid tissues. A working and tentative life history of DC is shown in Fig. 2. DCs are originated in the bone marrow. They migrate via the blood to nonlymphoid tissues. Naturally, most of these DCs bear an immature phenotype because these DCs are intended to capture microbes or their antigens or apoptotic cells or cell debris. In the steady state, antigen uptake by these DCs leads to immune tolerance, and this is possibly the main role of DCs in the physiological condition. However, it is not clear how immune tolerance can be induced without cells migrating to lymphoid tissues. It is accepted that when there is a danger signal or inﬂammatory stimuli at the peripheral tissues, some of these DCs capture antigen and undergo partial maturation. These maturating DCs may leave the nonlymphoid tissue and migrate to the lymphoid tissues. Based on these realities, the phenotypes of DCs in the nonlymphoid tissues are diverse depending on the nature of the organ. It is evident that DCs with different phenotypes and functions, such as DC progenitors, DC precursors, immature DCs, maturating DCs, migrating DCs, and mature DCs could be localized in most of the lymphoid and nonlymphoid tissues. However, it is not clear whether there is an organ-speciﬁc DC. Some insights about this could be found if tissue-speciﬁc DC progenitors can be detected in some organs. To have real insights about development of DCs, DC progenitors should be detected ﬁrst. However, this was not possible, for historical reasons. The phenotypes of DCs

2. General Features of Dendritic Cells

BONE MARROW

DC PROGENITORS OF DIFFERENT NATURE DC PRECURSORS OF DIFFERENT NATURE IMMATURE DC WITH DIFFERENT SUBTYPES

NONLYMPHOID TISSUES

MIGRATING DCs FROM NONLYMPHOID TISSUES

LYMPHOID TISSUES

DC PROGENITORS DC PRECURSORS

PERIPHERAL BLOOD

14

IMMATURE DC MATURING DC ANTIGEN-LOADED DC MATURE DC PLASMACYTOID DC REGULATORY DC LYMPHATICS IMMATURE DC MATURE DC ANTIGEN-LOADED DC REGULATORY DC PLASMACYTOID DC

Fig. 2. Life cycle of the dendritic cell (DC). DC progenitors and DC precursors are detected originating in the bone marrow. Then, they migrate to peripheral tissues via the blood. After interacting with different agents at the tissues, dendritic cells move to lymphoid tissues to interact with other immunocytes. Thus, different subtypes of DCs and DCs with different levels of activation and maturation can be detected in most tissues of the body. More mature DCs are by the larger font

could not be checked methodologically. It was well known that DCs including spleen DCs are bone marrow derived and are originated from hematopoietic progenitor cells of the bone marrow. However, bone marrow-derived DCs were isolated more than one decade after the isolation of spleen DCs. More interestingly, only now are we acquiring some insights about DC progenitors and precursors, more than two decades after the discovery of their mature counterpart (spleen DCs). Working Deﬁnition of DC We have mentioned that it is difﬁcult to deﬁne DCs properly on the basis of established criteria of deﬁnition of different types of immunocytes. However, a working deﬁnition of DCs on the basis of contemporary developments in science is required. It may take several decades to develop proper insights and appropriate criteria to deﬁne DCs. However, DCs are now used for treating patients with different pathological conditions. The clinicians should understand what DCs are. DCs are a member of

Origin and Development of DCs

15

the common leukocyte family. No DC-speciﬁc marker has been described so far. Accordingly, DCs are deﬁned based on a combination of parameters that include morphology, phenotype, cytokine secretion, immunomodulatory capacity, expression of chemokine and chemokine receptors, and migration in response to chemotactic stimuli. Human DCs are characterized by the surface expression of major histocompatibility complex (MHC) class II molecules. Initially, DCs were regarded as lineage negative, but that is no longer valid. The DC phenotype varies, depending on the stages of maturation and differentiation. CD1a is preferentially expressed on human immature DCs, whereas CD83 is typically upregulated in response to activation stimuli. DCs do not express a T-cell marker such as CD3, but some DCs express other T-cell differentiation markers such as CD4 and CD8. Murine DCs, but not human DCs, express some B-cell markers. Monocytes and macrophages share many common markers with DCs. These cells may also convert into DCs both in vivo and in vitro. DCs also express adhesion molecules, including CD11a, CD11c, CD50, CD54, and CD58, as well as the costimulatory molecules CD80, CD86, and CD40. Importantly, in response to activation stimuli, DCs express CCR7. DCs are also characterized by potent immunostimulatory capacity, which can be detected in MLR and by the ability to prime antigen-speciﬁc lymphocytes, both in vitro and in vivo. These functional properties are enhanced upon exposure to activating stimuli. Finally, DC immunogenicity is largely determined by the capacity to secrete cytokines such as tumor necrosis factor-alpha (TNF-α), IL-6, IL-12, IL-15, and IL-18, which contribute to activate lymphocytes and prime the subsequent immune responses. The only characteristic that is unique for DCs is their capacity to induce proliferation and polarization of naïve T cells. However, the presence of regulatory DCs may change this paradigm. All other functions of DCs can be performed by other cells, although DCs perform these functions most efﬁciently. A working deﬁnition of DCs might be given from the phenotypic and functional characteristics (see Table 1). DCs are a trace population of immunocytes with properties such as (1) expression of dendritic processes and some markers suggesting of their phylogeny and phenotype, (2) being capable of capturing, recognition, and processing of antigens, microbial agents, tumor cells and apoptotic cells, (3) having migratory capacity, and (4) possessing efﬁcient ability to interact with T lymphocytes. DCs may induce immune responses or immune tolerances depending on the nature of the DCs, tissue microenvironment, and extent of activation and maturation signals. Many new features of DCs have recently been discovered, and several old characteristic features of DCs may be invalid in the course of time.

Origin and Development of DCs The DC system is widely distributed throughout the body, and comprises Langerhans’ cell (LC) in epithelial locations, dermal DC in the dermis, thymic DC in the thymus, veiled cells in lymph, circulating DC in the blood, interdigitating cells in lymphoid organs, and interstitial DC in connective tissue of nonlymphoid organs. Various nomenclatures have been applied to deﬁne DCs in different tissues and to enumerate their characteristics and functions. For practical purposes, DCs can be divided into

16

2. General Features of Dendritic Cells

two main types: plasmacytoid DCs (pDCs) and conventional DCs (cDCs). Some investigators also list LC as a different type of DC, but they may be enlisted as cDCs. cDCs can also be divided into different subsets according to their tissue localization. In addition, some DCs are not found in the steady state but develop after infection or inﬂammation Monocyte-derived DCs are a typical example of this subset of DCs. pDCs are also regarded as type 1 IFN-producing cells, and they produce high levels of type 1 IFN in response to microbial stimulation. In clinical practice, different nomenclatures are applied to deﬁne different types of DCs. In addition to cDCs and pDCs, DC progenitors, DC precursors, immature DCs, maturating DCs, mature DCs, immunogenic DCs, regulatory DCs, tolerogenic DCs, interstitial DCs, and myeloid DCs are commonly described in the literature (Table 2 [A]). As there are many types of DCs in vivo, it should be clariﬁed whether there is a committed precursor for each type or whether all DCs develop from one common precursor. Basic scientists have been working to solve these issues. We provide next a simple description about the origin and life history of DCs. Initially, DCs were presumed to be derived from cells of myeloid lineage. Subsequently, data were cited that supported that DCs can be derived from cells of lymphoid linage. However, recent ﬁndings have demonstrated that DCs can develop from both myeloid and lymphoid-committed progenitors. Recent studies have shown that the common feature of the progenitors capable of developing into DCs is the surface expression of fms-like tyrosine kinase receptor 3 ligand (Flt3) receptor (Table 2 [B]). Also, various transcription factors and cytokines differentially regulate the development of different populations of DCs.

Table 2. Nomenclature and origin of dendritic cells (A) Nomenclature of dendritic cell (DC)

Conventional DC (cDC) Plasmacytoid DC (pDC) Langerhans’ cells (LC) DC progenitors, DC precursors Immature DC, maturating DC, mature DC Interstitial DC, lymphoid DC Immunogenic DC, tolerogenic DC, Regulatory DC

(B) Origin of dendritic cell (DC)

Common myeloid progenitors Common lymphoid progenitors Flt+ hematopoietic stem cells

(C) Appearance of dendritic cell in humans 4–6 weeks 11–14 weeks 14–17 weeks 16 weeks 23 weeks

Human yolk sac and mesenchyme Thymus and lymph nodes Nonlymphoid tissues except brain Spleen Skin and tonsils

The dendritic cell (DC) is called by different names on the basis of phenotype, function, and functional capabilities (A). DCs are originated from hematopoietic cells (B). In humans, DCs are detected in intrauterine life (C)

Origin of DCs

17

Detection of DC-Like Cells in the Intrauterine Life of Mouse and Man Genesis of Murine DCs Both CD11c+ cDCs and CD45RA+ pDCs are detected in small numbers in mouse thymus as early as embryonic day 17. CD4+CD8+ thymocytes are also detected in the thymus at this time, and a relationship between selection of thymocytes and development of DCs coincides. After that, the numbers of DCs are increased along with time. cDCs and pDCs are present in the spleen of day 1 newborn mice. In addition to spleen, DCs can be detected in low frequencies in other lymphoid tissues of neonatal mouse. Low numbers of DCs of may be related to the relative incompetence of immune responses to microbial infection of these mice. At about 5 weeks of age, the DC system is almost completed in mice. The composition of DC populations in the spleen of young mice and that of adult mice is different. Studies have shown that CD4+CD8− DCs are more frequent in neonatal mice spleen (50%–60%) compared to adult mice spleen (20%–30%). On the other hand, the ratio of CD4−CD8− DCs is more frequent in adult mice spleen. The cDCs of young mice are less efﬁcient than their adult counterparts in IL-12p70 and IFN-γ production and in antigen presentation. Detailed data about frequencies of pDCs of neonatal mice are not available, but pDCs of both neonatal and adult mice are capable of producing almost comparable levels of type 1 IFN. Thus, the neonatal DC system may not be fully developed in mice, and innate immunity may be the dominant form of immune response. Genesis of Human DCs The life history of human DCs is shown in Table 2 [C]. In human yolk sac and mesenchyma, MHC class II-expressing DC-like cells are detected as early as 4–6 weeks of gestation; this is earlier than formation of the liver, bone marrow, and thymus. MHC class II-expressing DC-like cells are also detected in the thymic medulla and paracortical area of mesentery lymph nodes at 11–14 weeks. DC-like cells are also detected at nonlymphoid tissues, except brain, at 12 weeks. These cells can be found in bone marrow at 14–17 weeks. DC-like cells are also seen in the T zone of spleen at 16 weeks, and in fetal skin and tonsillar crypts at 23 weeks of gestation. Human pDCs can be detected in fetal thymus and liver. Similar to murine DCs, human cord blood DCs have a limited ability to induce proliferation of T cells in response to phytohemagglutinin or concanavalin A.

Origin of DCs Logic That Supports Development of DCs from Myeloid Precursors DCs were originally believed to be of myeloid origin, based on certain similarities to monocytes/macrophages in terms of morphology, phenotype, endocytic potentials, and enzymatic activities. Studies have demonstrated that macrophages, granulocytes, and DCs can be developed from mouse blood and bone marrow proliferating MHC

18

2. General Features of Dendritic Cells

class-II-negative myeloid precursors in the presence of GM-CSF. DCs generated under these conditions possess homing capacities to T-cell regions of lymph nodes. They also possess DC-like function and are capable of stimulating allogeneic T cells. In the human, the CD34+ bone marrow-derived precursor differentiate into a bipotential precursor population and produce mature DC when cultured in the presence of GM-CSF and TNF-α or macrophages when cultured in the presence of colonystimulating factor (M-CSF). Peripheral blood monocytes differentiate into DC without proliferation and under various experimental conditions, which also provides an additional support to substantiate that DCs can be developed from cells of myeloid origin. Direct evidence for a myeloid origin of DC came from studies demonstrating that the transplantation of mouse bone marrow common myeloid progenitors (CMPs) into irradiated recipients led to the reconstitution of the cDCs and pDCs in the spleen and thymus. In fact, the Flt3+ fraction of CMPs that is able to produce all cDC and pDC subsets is found in the mouse spleen and thymus, and they have a dominant role in DC differentiation (Table 2 [B]). Concept of DC Development from Lymphoid Precursors Although several studies support that DCs can be developed from myeloid precursor populations, thymic cDCs and subpopulations of cDC in mouse spleen and lymph nodes express markers associated with lymphoid cells (CD4, CD8, CD2, and CD25). These factors indicate that DCs may have a lymphoid origin. Studies have shown that hematopoietic reconstitution studies with the earliest intrathymic lymphoid-restricted CD4loc-kit+Sca-1+Sca-2+ low CD4+ precursors resulted in development of predominantly the CD8+ DCs as well as T lymphocytes, B lymphocytes, and NK cells. However, only limited development of CD8− DCs and myeloid cells was seen in mouse spleen. Subsequent studies of the mouse bone marrow common lymphoid progenitor (CLP) also demonstrate the potential of these progenitors to differentiate into DCs both in vitro and in vivo. It was initially assumed that CLP mainly produce the CD8+ DC population (socalled lymphoid DCs); however, the CLP are capable of producing all splenic and thymic DC subsets. Moreover, both CD8+ and CD8− DCs were developed by reconstruction with bone marrow cells in the spleen. Recent studies on mouse bone marrow CLP provided direct evidence for the lymphoid origin of some DCs. The bone marrow CLP can generate all DC populations identiﬁed in lymphoid tissues, although most of these DC were CD8+. These studies have shown that both CMPs and CLPs are capable of producing DC or DC-like cells in vivo and vitro. It seems that CLP are more potent in DC production on a per cell basis than CMPs. However, if one considers the numbers of CLPs and CMPs, the CLP are less numerous than CMP in normal mouse bone marrow. Therefore, the overall contribution of CLP- and CMP-derived DCs may be similar in mouse lymphoid organs. The functional capabilities of DCs derived from different hematopoietic precursors are yet to be investigated. In humans, DCs can also be prepared from CLPs and also cells of myeloid origins. Human progenitor cells with lineage restrictions similar to those of the CLP in mouse have also been described and were shown to generate DCs in vitro. However, it is not clear if these precursors can generate all human DC subsets. Taken together, it is now

Origin of DCs

19

evident that DCs can be generated from both myeloid and lymphoid precursors. In fact, there may be some committed cells among myeloid and lymphoid precursors that play a cardinal role during differentiation of DCs. Flt3+ Cells as Precursor of Various Forms of DC Both CMPs and CLPs of the bone marrow can generate all the DC populations, and this suggests plasticity in developmental potentials of these early precursors. It also suggests that the CMPs and CLPs that can give rise to DCs may share some common features. Flt3L has been shown to act as a growth factor for hematopoietic progenitors and it can promote the expansion of both cDCs and pDCs in vivo and in vitro. However, the cells responding to Flt3L treatment and subsequently giving rise to cDCs and pDCs had not been fully characterized. Recent studies further examined the different mouse bone marrow hematopoietic precursor populations for the surface expression of Flt3 (the receptor for Flt3L) and tested them for early DC and pDC precursor activity. It was demonstrated that most DC precursor activity was within the bone marrow hematopoietic precursors expressing Flt3. The majority of mouse bone marrow CLP express high levels of Flt3, and these are the most efﬁcient precursors of both cDCs and pDCs. In contrast, only a small proportion of the CMP express Flt3, but the precursor activity for both cDCs and pDCs is within this minor Flt3+ CMP fraction. However, granulocyte and macrophage precursors and pro-B cells do not express Flt3 and have no cDC or pDC precursor activity. These ﬁndings indicate that the early precursors for all DC subtypes and for pDCs may be within the bone marrow Flt3+ precursor populations, regardless of their lymphoid or myeloid lineage orientation (see Table 2 [B]). This ﬁnding also emphasis the role of Flt3 signaling is for the development of both DCs and pDCs. However, it is not clear whether other subpopulations among Flt3+ cells are committed to develop some speciﬁc types of DCs. Development of DC from the Immediate Precursors A precursor that is at a developmental stage just before the formation of a phenotypically identiﬁable DC is termed an immediate DC precursor. The intermediate precursors have passed through their different pathways and then convert to DCs, if proper cytokines and culture conditions are provided. Two populations of DC precursors in mouse blood have been described. The CD45RA−CD11c+CD11b+ population represents an immature cDC that acquires the morphology of mature cDCs in the presence of TNF-α. They are also capable of stimulating T cells and producing IL-12 in response to microbial stimuli. The CD45RA+CD11cloCD11b− population represents the pDCs. These cells mature into cDC-like cells only in the presence of stimuli such as CpG DNA and GM-CSF, weakly stimulate T cells, and produce large quantities of type I IFN. Monocytes as Precursors of Human DC and Their Importance in the Clinic As described in this section, DCs can be developed from different precursor populations as well as from an early precursor population by culture with different cytokines

20

2. General Features of Dendritic Cells

or growth factors. However, most of these cell populations are not available for DC preparation in humans, especially for clinical usage. In addition, these precursors cannot be isolated in such a form that can be used for administration to humans. Human DCs are usually prepared from monocytes. Monocytes are cultured with different cytokines, and enriched populations of monocyte-derived DCs are produced.

Regulation of DC Development There are two major phases of the life cycle of a DC. One is the development of DCs from committed progenitors and precursors. It has been described that DCs can be developed from both CMPs and CLPs. The other is the differentiation, maturation, and activation of DCs. Different genes and proteins are regulated during these processes (Table 3, Fig. 3). Signaling During DC Development Different signals are required for the development of DCs from their precursors. Studies have shown the roles of different genes in this context. Zinc ﬁnger transcriptional regulator Ikaros play an essential role in DC development. All DCs subsets are ablated if there is a dominant-negative mutation in the Ikaros gene in the mouse. On the other hand, a null mutation in the Ikaros gene showed an absence of CD8− cDC and pDC and residual CD8+ cDC development. RelB, a member of the NF-kB (Rel) family, also affects development of DCs. Disruption of the RelB gene results in the defective development of cDCs, especially the splenic CD4+CD8− DC subset. However, the development of CD4−CD8+ and CD4−CD8− DCs is not substantially affected, indicating their functional speciﬁcity. The three interferon regulatory factors (IRF), namely, IRF-2, IRF-4, and IRF-8, also play important roles in the development of different DC populations. Mice lacking a functional IRF-2 or IRF-4 show impaired development of CD4+CD8− cDC. IRF-4-deﬁcient mice also show a defect in pDC subsets in the spleen. In contrast,

Table 3. Effect of different genes during development of dendritic cells Genes Effect Ikaros RelB IRF-2, IRF-4 IRF-4 IRF-8 Id2 Overexpression of Id2 Spi-B PU1 STAT3

Mutation of Ikaros gene cause ablation of DC subsets Disruption of ReLB gene results in impaired DC development Deﬁciency causes defective development of cDCs IRF-4-deﬁcient mice show distorted development of pDC Regulate development and function of all DC subsets Essential for development of cDCs and LC Inhibit pDC development Regulate pDC development Required for development of cDCs and pDCs Regulate development of DC caused by Flt3 stimulation

Different genes cause both positive and negative regulation of development of dendritic cells

Regulation of DC Development

(A)

Downregulation of genes during DC differentiation

21

1. Genes for CD14, CD163, C5a, CD88 2. Genes for cell adhesion and motility: galectin-2, CD11a/LFA-1, ninjurin-1, macmarcks, syndecan, CD44E, presenillin-1 3. Chemokines of IL-8 superfamily 4. Proinflammatory cytokines: TNF-alpha, TNF-alpha receptor, IL-6 5. Interferon regulatory factor (IRF)-7

Downregulation

(B) of proteins during DC differentiation

(C)

Upregulation of genes during DC differentiation

Upregulation of

(D) protein during DC

1. Calreticulin: required for presentation of antigen to CTL

1. MHC class II, CD1a, CD1b, CD1c, CD36, CD59, CD843, CD86, CCR7 2. Genes of cell motility, autotaxin-1, sematophoric-E 3. Antiinflammatory protein: cyclophillin C, TSG-6 4. Oesteopontin, Mac-2 binding protein 5. Interferon-regulatory factor (IRF)-4

1. Members of fatty acid-binding proteins: FABP4, FABP5, ACBP 2. Heat shock protein (hsp)-73, 27: related to antigen uptake

differentiation Fig. 3. Signal transduction during differentiation of dendritic cells (DC). Several genes and proteins are regulated during differentiation of DCs. Both downregulation (A and B) and upregulation (C and D) of genes and proteins are seen during DC differentiation

IRF-8 plays crucial roles in the development and function of CD4−CD8+ cDCs, pDCs, and LCs. Id2, a member of the inhibitory helix-loop-helix (HLH) transcription factor family, is upregulated during DC development and is required for the development of CD4− CD8+ cDCs and LCs. In contrast, overexpression of Id2 in hematopoietic stem cells inhibited the development of pDCs, indicating that Id2 acts as an inhibitor of pDC development. The ETS transcription factor Spi-B is expressed by pDCs, but not by cDCs. The knockdown of Spi-B mRNA expression in human hematopoietic progenitors led to the defective development of pDCs. The transcription factor PU.1, another member of the ETS family that interacts with Spi-B, IRF-4, and IRF-8, is also required for the development of both cDCs and pDCs, as shown by the fact that impaired development of cDCs from the hematopoietic progenitors in the embryo or in neonatal PU.1-deﬁcient mice and defective development of both cDCs and pDCs in the adult mice with induced deletion of PU.1 have been observed. The transcription factor STAT3 is required for Flt3L-dependent steady-state DC development. Deletion of STAT3 in hematopoietic cells abolished the effects of Flt3L on DC development; this also led to a profound deﬁciency in the DC compartment in lymphoid tissues. In contrast, deletion of STAT3 did not affect DC development in vitro in the presence of GM-CSF, indicating that STAT3 is not required for GM-CSFdependent inﬂammatory DC differentiation.

22

2. General Features of Dendritic Cells

Signaling During DC Differentiation Signaling pathways for DC differentiation are different from those of DC development. Recent studies by oligonucleotide array and proteomics analyses have shown that many novel genes and proteins have roles in DC differentiation, maturation, and function (see Fig. 3). RNA transcript levels for different genes expressed during DC differentiation have been determined. Monocytes of day 1 (CD14. monocytes), immature DCs (7 days of GM-CSF/IL-4 treatment), and mature DCs (14 days of GM-CSF/IL-4 plus TNF-α treatment) have been analyzed to develop insights about gene modulation during DC differentiation. Transcripts for approximately 40% of the 6300 unique genes were detected in all the cell populations tested. The expression levels of several genes differed during DC differentiation and maturation, by 2.5 fold or greater. The regulated genes were mostly involved in cell adhesion, motility, growth control, regulation of the immune response, antigen presentation, transcription, and signal transduction. During DC differentiation, the transcript levels of the genes for CD14, CD163, and C5a anaphylatoxin receptor (CD88) were strongly downregulated. Also, expression of galectin 2, CD11a/LFA-1 alpha, ninjurin 1, macmarcks, syndecan 2, CD44E, and presenilin 1 was downregulated. Expression of genes encoding proinﬂammatory cytokines and their receptors, such as prointerleukin-1, TNF-α, CD163, C5a anaphylatoxin receptor, IL-6 receptor, and TNF receptor were downregulated. Moreover, the expression of a set of chemokines belonging to the IL-8 superfamily such as CTAPIII, MIP2-, MIP2-, ENA78, PF4, and IL-8 was downregulated. Expression of IRF-7A, TAL2, NAP-2, EGF-response factor 2, CtBP, IEX-1, SAP49, HRH1, I-B alpha, Fyb, Net, and cyclophilin F genes was decreased. Of these genes, IRF-4 and IRF-7 are of particular interest. IRF-7 is traditionally involved in the activation of type 1 IFN and differentiation of monocytes into macrophages. The decrease in IRF-7 during DC differentiation versus increased expression in macrophage differentiation indicates a critical role for IRF-7 at the crossroad between macrophage and DC differentiation from monocytes. On the other hand, expression of cell-surface proteins MHC class II, CD1a, CD1b, CD1c, CD36, CD59, CD83, CD86, and CCR7 were upregulated. Expression of secreted proteins involved in cell motility, autotaxin-t, and semaphorin E was upregulated. The expression of genes encoding antiinﬂammatory proteins such as cyclophilin C was upregulated. Osteopontin, a key cytokine involved in T-lymphocyte activation, was upregulated. The maturation of DCs was accompanied by the upregulation of Mac-2binding protein, which stimulates the NK cell activities and induces the secretion of IL-2. Upregulation of transforming growth factor (TGF)-β was also observed during DC maturation. Expression of the IRF-4, C/EBPa, mrg1, PPARg, TRIP7, SLA, Rap1GAP, cAMP-dependent protein kinase, IP3 protein kinase B, cyclophilin C, and cyclins A1, D2, G2, and H genes, was increased during maturation. The decrease in expression of integrins and cell adhesion molecules and increase in expression of genes involved in cell motility might have an effect on the enhanced migration properties of DCs compared to their precursors. To explore the signaling by protein expression, total proteins were extracted and the protein spots whose intensities changed by 2.5 fold or greater during DC differ-

Regulation of DC Development

23

entiation or maturation from monocytes were analyzed. The proteins identiﬁed were members of speciﬁc families including chaperones, Ca2+. -binding proteins, fatty acidbinding proteins (FABP), and structural proteins. Expression of three members of the FABP family, FABP4, FABP5, and Acyl-CoA-binding protein was highly increased after 7 days of culture of monocytes. Heat shock protein (hsp)73, hsp27, and calreticulin were also regulated during DC differentiation. hsp70 is related to antigen uptake. Upregulation of hsp73 protein during DC differentiation was seen. In contrast to hsp27 and hsp73, the cognate chaperone protein calreticulin was downregulated during DC differentiation owing to posttranslational modiﬁcation. Calreticulin was mostly downregulated during DC maturation. Calreticulin is required for the presentation of antigenic peptides to CTLs at the cell surface. In addition, it has been recently reported that calreticulin elicits tumor- and peptide-speciﬁc immunity. Calreticulin displays in vivo peptide-binding activity and can elicit CTL responses against bound peptides. Role of Cytokines During DC Development In addition to the transcription factors that differentially regulate the development of distinct DC subsets via different signaling pathways, several cytokines have been shown to differentially promote the growth and differentiation of different DC subsets. Flt3L is a crucial factor in both human and mouse for promoting the development of both cDC and pDC in vivo and in vitro. A role of both GM-CSF and Flt3L has been shown in the development of cDC. However, only Flt3L is critical for the development of pDCs. GM-CSF promotes cDC development mediated by Flt3L at the expense of pDC development in vivo and in vitro. This pathway is regulated by STAT5. GM-CSF requires STAT5 to suppress Flt3L-driven pDC development from the lin−Flt3+ bone marrow progenitor population. STAT5 activation by GM-CSF rapidly attenuates the expression of critical pDC-related genes in lin−Flt3+ bone marrow progenitor cells cultured in Flt3L. GM-CSF therefore controls pDC development through a STAT5dependent pathway that impinges upon the pDC transcriptional network, inﬂuencing the production of DC subsets from the progenitor compartment. The cytokine TGF-β1 plays a critical role in LC development, as demonstrated by the fact that the in vitro generation of LCs from CD34+ progenitors can be greatly enhanced by TGF-β1. LCs is not detected in TGF-β1-deﬁcient mice, but bone marrow cells from TGF-β1-deﬁcient mice could give rise to LC after transfer into lethally irradiated recipients. Thus, it may be suggested that TGF-β1 exerts its effect through regulating the expression of its downstream transcription factor Id2, which is required for the normal development of LCs. Clinical Importance of Understanding Signal Transduction During DC Development and DC Differentiation Now, DCs are prepared from human peripheral blood by culturing with cytokines. A group of DC-like cells are obtained, and these cells are now used as DCs for treatment of patients with cancers and chronic diseases. Monocyte-derived DCs may not be the best DC population for therapeutic usage. Moreover, there are many types of DCs

24

2. General Features of Dendritic Cells

among monocyte-derived DC populations. Some of them may be tolerogenic DCs. Understanding the signal pathway for DC development will allow production of larger numbers of DCs with similar phenotypes and functions, and this will also allow development of abundant amounts of DCs in humans. Production of DCs from CLPs or CMPs by using Flt3L will allow obtaining comparatively homogeneous populations of DCs. As it is now becoming evident that several genes and proteins are upregulated during DC differentiation and maturation, manipulation of DCs by genetic approaches may allow development of proper types of DCs for clinical use.

Dendritic Cells in Different Organs and Tissues DCs originate in the bone marrow and are localized in different tissues (both lymphoid tissues and nonlymphoid tissues). Different types of DCs such as DC progenitors, early DC precursors, DC precursors, immature DCs, and mature DCs can be detected in both lymphoid and nonlymphoid tissues. Although DCs from bone marrow migrate to different tissues, DCs from nonlymphoid tissues may also migrate to lymphoid tissues. In the mouse, almost all dendritic cells express CD11c. In addition, there are different subtypes of DCs that express certain other surface antigens. The functions of different subtypes of DCs are also different. In human, some DCs do not express CD11c.

DCs in Lymphoid Organs Different DC populations in lymphoid organs are shown in Table 4. Thymic DCs The thymus is a primary lymphoid tissue where T-cell differentiation and selection occur and lead to the generation of naive CD4+ and CD8+ T cells, naturally occurring

Table 4. Phenotypes of dendritic cells in lymphoid tissue (A) Thymic dendritic cells General Conventional DC Plasmacytoid DC

MHC class II+, CD11c+, DEC-205+, CD8+, CD11bdull/−, CD86+, CD40+ CD11chighCD45RA−: CD8+Sirp-α-, and CD8−/loSirp-α+ CD11cintCD45RA+

(B) Spleen dendritic cells Conventional DC Plasmacytoid DC Interferon-producing killer DC

CD4-CD8+, CD4-CD8−, CD4+CD8− CD11cintCD45RA+B220+ CD11c+,NK1.1+B220+

(C) Lymph node dendritic cells Conventional DC Plasmacytoid DC

CD4-CD8+, CD4−CD8−, CD4+CD8−, CD8loCD205int CD8lowCD205hi CD11cintCD45RA+B220+

DCs in Lymphoid Organs

25

CD4+CD25+ regulatory T cells, and some of the double-negative invariant T-cell subsets, such as NKT cells or mucosa-associated invariant T cells. Mouse thymus contains both a pDC population (CD11cintCD45RA+ pDCs) and cDC subsets (CD11chiCD45RA− cDC). The cDCs can be further divided on the basis of expression of CD8 and the signal regulatory protein-α (Sirp-α) expression, as CD8+Sirp-α− and CD8−/loSirp-α+ cDC subsets. The major CD8+Sirp-α− subset is generated within the thymus from the earliest intrathymic progenitors, whereas the minor CD8−/loSirp-α+ cDC subset is originated from the peripheral migratory DCs. Dendritic processes are not usually seen on freshly isolated thymic DCs. Expression of MHC class I+, MHC class II+, CD11c+, DEC-205+, CD8+, CD11bdull/−, CD86+, and CD40+ have also been reported in thymic DCs. BP-1, a glutamyl aminopeptidase, is expressed on thymic DCs. Some thymic DCs express FasL, Thy 1.1, and Thy 1.2. The human thymus also contains pDCs and two subsets of mature CD11c+ cDC: CD11b−CD45ROlo DCs lack myeloid markers and produce IL-12. A minority of CD11b+CD45ROhi DCs express many myeloid markers, and they produce very low levels of IL-12. A third population of thymic DC has also been described that express CD11c− and CD123+ and do not produce IL-12. It is postulated that thymic DCs mostly presents self-antigen, rather than foreign antigen. Thymic cDCs play important roles in the induction of central tolerance through the process of negative selection as well as the generation of the naturally occurring CD4+CD25+ regulatory T cells. DC Populations in the Spleen Spleen DCs are usually detected by the expression of CD11c. On the basis of expression of CD4 and CD8, three cDC subsets have been identiﬁed in the spleen: these include CD4−CD8+, CD4−CD8−, and CD4+CD8−. Expression of CD205 is detected on the CD4−CD8+ cDCs subset. In contrast, the CD4−CD8− and CD4+CD8− cDCs do not express CD205. Different types of DCs in the spleen also distributed in different parts of the spleen. The CD8−CD205− cDCs are located in the marginal zone, whereas the CD8+CD205+ cDCs are in T-cell areas. However, DCs in the spleen are capable of migrating from one area to another area of the spleen. Marginal-zone DC can rapidly migrate into the T-cell-rich area after lipopolysaccharide stimulation. Some studies have reported that some splenic cDC can be generated in situ by the intrasplenic immediate cDC precursors. In addition to the cDCs, pDCs are also found in mouse spleen. They are deﬁned as CD11cintCD45RA+B220+. Similar to the blood pDC, the freshly isolated splenic pDC do not have the phenotypic and functional features of the antigen-presenting cDC. However, these DCs can act as cDCs in morphology during microbial infections. They represent the major cell type that produces large amounts of type 1 IFN. The most likely origin of pDC in the spleen is the peripheral blood, because cells with the characteristics of pDC can be found in mouse blood. It is unlikely that intrasplenic DC precursors differentiate into pDC. A newly identiﬁed DC lineage, namely IKDC, has also been found in mouse spleen. IKDCs have a molecular expression proﬁle of NK cells and DCs with a phenotype CD11c+NK1.1+B220+. Upon activation by various stimuli, IKDCs can produce large

26

2. General Features of Dendritic Cells

amounts of IFN-γ, kill typical NK target cells, and display some APC activity. Details of IKDC are not known at this point; however, it suggested that IKDC may arise from a unique differentiation pathway that diverges early from those responsible for NK, pDC, T, and B cells. The studies of splenic DC populations in human have been hampered by the limited tissue sources. Human splenic DCs have been located in marginal-zone, T-cell, and B-cell areas of the spleen. DC Populations in Lymph Nodes Different types of DCs are detected in mouse lymph nodes. CD8loCD205int and CD8loCD205hi cDC are detected in almost all lymph nodes. Subcutaneous lymph nodes contain a higher percentage of the CD8loCD205hi LC-like cells than the mesenteric lymph nodes. In addition to cDCs, a population of CD11c+ DCs expressing B220 and Gr-1 antigens has been detected from the spleen and the lymph nodes. A recent study has shown the existence of CD11clow, CD11b−, CD45RAhigh DCs in the spleen. These DCs may represent the pDCs or natural NIPC of the human peripheral blood, and these cells produce abundant amounts of type 1 IFN in vitro in response to viral infection. Although human pDCs are CD11c−, mouse pDCs are CD11c+. DCs in Human Tonsil In the human, different subsets of DCs have been characterized in the tonsils. Interdigitating DCs expressing CD11c, CD40, CD86, and CD83 are detected in the T-cell-rich areas of the tonsils. These DCs also express variable levels of CD83 and thus they can be divided into other subtypes. CD4+, CD3−, and CD11c− DCs are present at the T-cell-rich areas and around the high endothelial venules of tonsils. The precursors of these cells are also found in the blood. Cells with this phenotype are known as plasmacytoid T cells or pDCs. CD4+, CD11c+, and CD3− DCs can be found in the germinal center of the tonsil, and these are known as germinal center DC (GCDC). GCDC express receptors such as CD16, CD32, and CD64. Recently, an extensive classiﬁcation of tonsillar DCs both in situ and from isolate tonsillar cells was done. Five subsets of DCs in the tonsils were found based on the expression of HLA DR, CD11c, and CD123: (1) HLA DRmod, CD11c−, CD123+, (2) HLA DRhigh, CD11c−, CRMF44high, (3) HLA DRmod, CD11c−, CD13−, CRMF44−, (4) HLA DRmod, CD11c−, CD123−, and (5) HLA DRmod, CD11c+ germinal center DC. However, the functional implications of these subsets and their compartmentalization in the tonsils have not been deﬁned. Very little is known about the phenotype of murine tonsil DCs. DCs in Peyer’s Patch (PP) Peyer’s patch (PP) is the main lymphoid organ where antigens are available to the immune cells of the gut. Antigens may be transported to PP by M cells, a specialized

DC in the Nonlymphoid Tissues

27

cell of the gut. DCs have been described in two sites in the PP. One is in the T-cell areas of the PP and the other is the subepithelial area underlying the dome. DCs in the gut in general express CD11c. Subdome DCs are negative for DEC-205, but T-cell area DCs of the PP express DEC-205. Subdome DC express typical marker of myeloid DCs, CD11b, whereas T-cell area DC express CD8, a marker of lymphoid DC. A third population of DC, CD11c+ but negative for CD11b and DEC-205, are also found in the gut. Little is known regarding the DCs in PP in human. DCs in Cryptopatches In addition to PP, there is another lymphoid structure in the gut. These are solitary lymphoid structures, and it is presumed that they may represent sites for extrathymic T-cell differentiation. These are called cryptopatches in the mouse. Cryptopatch-like structures have also been detected in the rat. A structure resembling cryptopatches has been also characterized from human small intestine. MHC class II-positive DCs have been detected in the cryptopatches from human small intestine along with memory T cells and a variable B-cell component.

DC in the Nonlymphoid Tissues DCs have been enriched or isolated from most of the tissues of the body. Nonlymphoid tissues contain different types of DCs with diverse phenotypes and functions. In some tissues, DCs were localized immunohistochemically, whereas in others, DCs were propagated in vitro by culturing some progenitors or precursor populations of DCs in presence of various cytokines. It is well known that when DC progenitors and precursors are cultured in the presence of cytokines, the phenotypes and functions of DCs are altered. DCs in the Skin LCs resides mainly within the stratiﬁed squamous epithelia and represent 2%–4% of the epithelial cells. In the epidermis, LCs are located at the suprabasal position and remain attached to neighboring keratinocytes via a E-cadherin- and Ca2+-dependent mechanism. The density of LC varies between the locations. One study showed that there were about 200 LCs/mm2 in the palm, whereas about 930 LCs/mm2 are found in the epidermis of face and neck. Human LC express CD1a and CD1c. Recently, langerin, a 40-kDa molecule, has been detected in LCs. LCs can also be characterized by the presence of Birbeck granules. Langerin colocalizes with Birbeck granules. The precise functions of langerin and Birbeck granules have not properly elucidated. Langerin might be an antigencapturing apparatus that channels the antigen to Birbeck granules. Resident LCs display nonspeciﬁc esterase and ATPase activity. They also express Fc-IgG receptors type II (FCγRII, CD32), Fc-IgE receptor type I (FCγR1), and C3bi receptors (CD11bCD18). Langerin is also detected at the membrane of LCs. The phenotype of murine LCs is dependent on their level of maturation or activation. Resting LCs could be identiﬁed by their expression of CD11c, langerin, E-cadherin, and low costimulatory

28

2. General Features of Dendritic Cells

molecules. On the other hand, activated LC expresses very high levels of activation and maturation markers. In addition to LCs, there is another population of DCs in the skin. Human CD34+ hematopoietic progenitors differentiate into CLPs and CMPs. CMPs appear to differentiate into CLA+ (cutaneous lymphocyte antigen) and CLA− populations, which subsequently differentiate into CD11c+CD1a+ and CD11c+CD1a− DC, respectively. In contrast, CD11c+CD1a+ DCs migrate into the skin epidermis and become LCs, CD11c+CD1a− DCs migrate into the skin dermis and other tissues and become interstitial DCs. TGF-β plays a critical role in LC development, as demonstrated by the fact that the in vitro generation of LCs from CD34+ progenitors can be greatly enhanced by TGF-β and that TGF-β-deﬁcient mice lack LCs. Recent studies further demonstrated that Flt3+ CMPs as well as monocytes could all give rise to LCs in vivo. In mice, two populations of dermal DCs (DDC) have been described, CD11b+ and CD11b−. DDCs express most of the features of mature LCs, but they express intracytoplasmic transglutaminase clotting factor V111a (FXVIIIa), lower levels of CD1a and CD1c than LCs, and they express CD1b. DDCs can be distinguish from LCs by the absence of E-cadherin or langerin. DCs in Parenchymal Tissue DCs have been detected in most nonlymphoid tissues. In the lung, DCs are found in the bronchial mucosa either above or below the basement membrane or deep in the lamina propria of the bronchia. DCs are also seen at the bronchial-associated lymphoid tissue in the mucosa below the epithelium. Many DCs are found in the perivascular lymphoid tissue and the pleura. Lung DCs are immature in nature and very similar to freshly isolated blood DCs. CD11c is expressed on both DCs and monocytes of the lung, and thus it becomes difﬁcult to distinguish between these two related immunocytes in the lung. Lung DCs express high levels of MHC class II, and lymphocyte function associated antigen −3 and low levels of CD14. Lung DCs, similar to other immature DCs, express very low levels of CD86, CD80, and CD40. Based on the data of immunohistochemical, immunoﬂuorescence, and morphological studies, it is apparent that there are abundant numbers of DCs in the gut. The majority of DCs lie in the lamina propia (LP) underlying the epithelia. However, recent evidence suggests that DCs may extend their processes above the basement membrane. DCs in the gut following infection with Salmonella typhimurium are able to send dendritic processes outside the gut epithelium. DCs are able to open the tight junction of the epithelial cells and can directly sample the microbes in the gut lumen. As DCs contain tight-junction proteins such as occludin, claudin 1, and zonula occludens 1, the integrity of the epithelial barrier is preserved even after the dendritic processes come out as a periscope. Phenotypic studies with human gut DCs have not been adequately done. Immunohistochemical study has shown that both immature and mature DCs can be detected from the gut in human. Liver harbors different types of DCs with different levels of maturation, which include highly immature DC progenitors, CD11c+ DC that acts like DC of other nonlymphoid organs, and a mature population of DCs that express CD83 in human. Liver DC progenitors can be propagated by culturing a progenitor population in the hepatic

DC in the Nonlymphoid Tissues

29

nonparenchymal cells (NPC) with GM-CSF for 7 days. An immature population of DC progenitors that do not undergo maturation and activation in the presence of proinﬂammatory cytokines, such as IFN-γ and TNF-α, was enriched for functional study. However, these putative DC progenitors underwent maturation in the presence of intracellular matrix. It is elusive whether liver DC progenitors arose from a committed precursor of DCs in the liver or from the other hematopoietic stem cells. These cells have never been localized in vivo. CD11c+ DC was also located in the liver, mainly at the portal and along the central vein. Few CD11c+ DCs are seen in the hepatic parenchyma. These DCs express all phenotypic features of DCs and are potent stimulator of allogeneic T lymphocytes. Treatment of mice with GM-CSF induces a population of DEC-205+ DCs in the liver. Investigators have documented various DC subsets on the basis of expression of various antigens on their surface: (1) CD11c+, B220+, CD4+, (2) CD11c+, B220+, CD4−, (3) CD11c+, B220−, CD11b+, and (4) CD11c+, B220−, CD11b−. The subsets 1 and 2 resemble PDC, but these are Gr-1−. In another study, in which liver DCs were propagated byFlt-3L, two subsets of liver DCs were detected based on the mutually exclusive expression of CD11b and CD8. Very few studies have been undertaken regarding the phenotype of DCs in the human liver, especially in physiological liver. Flow cytometric study has revealed that liver-derived DCs express CD11c and CD123, but contamination from blood could not be completely eliminated in those studies. Mature DCs expressing CD83 have been detected in the liver, especially from the liver in pathological conditions. DC Populations in Blood Mouse blood contains two populations of DC precursors. Cells with the surface phenotype CD11c+CD11b+CD45RA− closely resemble the human immature CD11c+ precursor DCs and rapidly transform into CD8+ cDCs after stimulation with TNF-α. A second population of cells with the surface phenotype CD11cloCD11b−CD45RAhi that closely resembles human pDCs by morphology and function has also been detected in the blood. On stimulation with CpG DNA, these cells make large amounts of type 1 IFN and rapidly develop into DCs that bear CD8. Subsets of Peripheral Blood DC (PBDC) Based on the Expression of CD11c Precursor populations of DCs that are originated from CD34+ cells have been identiﬁed and characterized from human peripheral blood. Two main precursors of DCs in the peripheral blood were detected based on their expression of CD11c and CD123. One of them is CD11C+, CD123−/low and the other is CD11c−, CD123+. Both these subsets are HLA DR+ and negative for CD13. CD11c+ DC precursors undergo maturation in the presence of GM-CSF in vitro. They exhibit a myeloid appearance and are regarded as the myeloid DC. On the other hand, CD11c− DC precursors undergo maturation in presence of IL-3 and possess a lymphoid appearance; these cells are regarded as plasmacytoid DC. They express only low levels of adhesion and costimulatory molecules such as CD80, CD86, and CD40, suggesting that these cells are relatively immature. CD83 is not also expressed on

30

2. General Features of Dendritic Cells

DC precursors in the peripheral blood. However, when these DC precursors are cultured in vitro with inﬂammatory stimuli, they express high levels of adhesion and costimulatory molecules depending on the culture conditions. Although these DC precursors become DCs during in vitro cultures, there is no deﬁnitive proof that such an event takes place in vivo, especially in the peripheral blood. However, these precursor populations of DCs might migrate to the tissues in response to inﬂammatory or danger signals. Their differentiation in the tissues has not been studied in detail. However, the tissue microenvironment would inﬂuence further differentiations. Subset of DCs Based on the Expression of CD1a Although CD1a is known to be expressed on human LC, peripheral blood DC can be divided into three subsets based on the expression of CD11c and CD1a. Two of these subsets belong to CD11c+ DCs of the myeloid lineage: CD11c+, CD1a+, and CD11c+, CD1a−. Both CD11c+ fractions displayed myeloid markers such as CD13 and CD33 and possess the receptor for GM-CSF. In in vitro culture, CD11c+, CD1a+ DC acquired the morphology of LC and expressed langerin and E-cadherin along with production of typical Birbeck granules. However, CD11c+ CD1a− did not develop any feature of LC under any culture condition. The third subset of DCs in the blood is negative for both CD11c and CD1a and possibly belongs to CD11c− pDCs. Subsets of PBDC Based on the Expression of Blood Dendritic Cell Antigen (BDCA) Antigen that is speciﬁcally expressed on all types of DCs is lacking. However, some investigators have developed antibody that reacts with some antigens on peripheral blood DCs. Panels of antibodies that identify antigen on different PBDC subsets have been termed blood dendritic cell antigen (BDCA). There are four BDCAs and, of these four, three BDCAs react with different types of DC in the peripheral blood. BDCA-2 and BDCA-4 are detected on precursors of pDC (CD11c−, CD123+ subset of PBDC), whereas BDCA-3 binds to precursors of myeloid DC of the peripheral blood (CD11c+, CD123− subset). Phenotypes of Cultured Blood DCs Different populations of DCs can be derived by culturing DC precursors identiﬁed by their expression of CD11c, CD123, CD1, and ILTs. However, DCs are usually produced by culturing monocytes or an adherent population of peripheral blood mononuclear cells (PBMC) with GM-CSF and IL-4 for 7 days for clinical use. These cultured DCs express HLA DR and CD11c with low-to-moderate levels of costimulatory antigens such as CD86 and CD80, and only very low levels of CD83. These DCs are immature in nature but could be induced to maturation by a further culture with some inﬂammatory cytokines. A population of monocyte-derived DCs expresses DC-speciﬁc ICAM-3 grabbing nonintegrin (DC-SIGN). DC-SIGN has been reported to bind with human immunodeﬁciency virus (HIV)-1, but its implication for monocyte-derived DCs is yet to be established. DC-SIGN is not expressed on pDCs.

DCs in Innate Immunity

31

Clinical Implications of Phenotypes and Subsets of DCs DCs are highly heterogeneous population of immunocytes. They are distributed in almost all tissues of the body including the blood. These cells are now characterized to assess their roles in the pathogenesis of diseases. Also, DCs are used for induction of immunity in patients with cancers and chronic infections. We have just discussed that there are different types of DCs. The functions of different types of DCs are also highly heterogeneous. Some DCs are highly efﬁcient to induce immune responses, whereas others may induce immune tolerances. Some DCs are capable of recognition, capture, and processing of antigens or transformed cells, whereas others are highly potent to produce cytokines. In this context, proper insights are needed about populations of DCs. At present, little is known about diagnostic and therapeutic implications of DCs in various pathological conditions, which may be because blood DCs have been mostly characterized in different diseases. If it is possible to check the nature and function of tissue-derived DCs in different diseases, we may develop proper insights about variability of DC functions in different diseases, and this is also true in the context of DC-based therapy. Bulk populations of DCs are used for DC-based therapy. It is likely that DC-based therapy should be done with some particular subtype of DCs. When the target is to induce immune responses, DC-based therapy with bulk populations of DCs containing both immunogenic and tolerogenic DCs may not be an appropriate scientiﬁc approach.

Function of Dendritic Cells Historically, DCs are regarded as professional APCs. It has been shown that DCs are initiators of adaptive immunity. Subsequently, it was evident that DCs are capable of inducing both innate and adaptive immunity. Also, it was found that DCs induces both immune tolerance and immune responses. In the clinical setting, there is immense interest concerning DC functions. As regulatory of innate and adaptive immunity, the functions of DCs in vivo may be related to pathogenesis of different diseases, especially that of cancers, chronic infections, autoimmune diseases, allergic manifestations, and transplant rejection. On the other hand, the functional capacities of DCs in vitro have important implications for development of DC-based therapies for different diseases. In this context, the functions of DCs in the tissues may be dominant roles in the pathogenesis of diseases. On the other hand, blood-derived DCs are important for immune therapy. An outline of different functions of DCs is shown in Fig. 4.

DCs in Innate Immunity It has been assumed that DCs are professional APC and play a cardinal role during induction of adaptive immunity. However, the role of DC in innate immunity is becoming clear. DCs are localized in almost all tissues of the body. They are also capable of migrating to tissues of microbial infections very quickly. In some cases, mobilization of DCs has been seen before mobilization of other cells of innate immu-

32

2. General Features of Dendritic Cells

(A) DENDRITIC CELL IN INNATE IMMUNITY

1. Production of cytokines 2. Production of type 1 interferon 3. Interactions of DC and other immunocytes

(B) DENDRITIC CELL IN ADAPTIVE IMMUNITY Nature of function

Mediated by

Interaction with pattern recognition receptors

Toll-like receptors (TLRs) C-type lectine receptors (CLRs) and Pathogen-associated molecular pattern [PAMP]

Capture and internalization of antigens

Phagocytosis, endocytosis, macropinocytosis

Formation of MHC−antigen complex

MHC class I and MHC class II antigens

Maturation, activation, deactivation

PAMP−PRR interactions, cytokines, tissue environments

Migration

Chemokines, chemokine receptors

Antigen presentation and T-cell activation or deactivation

DC−T-cell interaction Cytokines in lymphoid tissue Nature of DCs and nature of antigens

Fig. 4. Multivariate functions of dendritic cells (DC). The DC is essential for both innate and adaptive immunity. Because of their variable functional abilities, DCs regulate the nature of immune responses

nity. DCs have specialized apparatuses to recognize different substances. DCs are also able to produce different cytokines, including type 1 IFN. They are able to instruct other cells of innate immunity to produce cytokines, chemokines, and other immune mediators. DCs at different stages of differentiation can regulate effectors of innate immunity such as NK cells and NKT cells. Both direct cell–cell interactions and indirect cytokine-mediated interactions have been implicated. Precursors of CD11c− DC can activate NK cells by releasing IFN-α, thereby inducing increased antiviral and antitumor immunity. DC at a later stage of differentiation may regulate the activity of NK cells and NKT cells through the release of cytokines such as IL-12, IL-15, and IL-18. Both murine and human NKT cells produce high amounts of IFN-γ or IL-4 and may thus determine the type of immune response. On recognition of alpha-glactocyloceramide/CD1d complex, NKT cells release IFN-γ. However, DC subsets differentially regulate NKT cytokine proﬁles, with monocyte-derived DC producing IFN-α release whereas plasmacytoid DC polarize NK T cells to IL-4 production.

DC in Adaptive Immunity Antigen Recognition The most critical single event that determines the subsequent cascades of antigen presentation is the recognition of antigens or antigen-bearing microbes or cells by

DC in Adaptive Immunity

33

DCs. Although DCs express different types of receptors, pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and C-type lectine receptors (CLRs) are important for antigen recognition. TLR include 10 (TLR1–10) and 12 (TLR1–9 and 11–13) family members in human subjects and mice, respectively. TLR usually recognize evolutionary conserved microbial molecules categorized into lipid, protein, and nucleic acids, according to their constituents. Ligation of TLRs with pathogenassociated molecular pattern (PAMP) leads to the activation of DC and immediately results in the production of proinﬂammatory cytokines, including IL-6 and TNF-α. These cytokines contribute to the induction of local innate immune responses. Thus, antigen recognition is related to both innate immunity and adaptive immunity. TLR regulate gene expression in DC by means of the activation of several transcription factors, including NF-κβ, mitogen-activated protein kinases (MAPK), and IRF. The CLR family includes DC-SIGN, BDCA-2, dendritic cell immunoactivating receptor (DCAR). These receptors recognize various saccharides, such as d-mannose, l-fucose, and N-acetylglucosamine, on the pathogen surface, and their major function is to internalize antigens for further processing and presentation by DC. It is not clear how regulatory DCs recognize antigens, and further study is needed to address this issue. It may be possible that tolerogenic DCs may also use PRRs for antigen recognition, but the nature of immune responses may be determined by other tissue-derived factors. In addition, DCs can also recognize various self- and altered self-entities. The antigen recognition function of DCs is an exciting area for clinical research. Proper immune responses are not seen in patients with cancers and chronic viral infections. On the contrary, exacerbated immunity is detected in patients with autoimmune diseases and allergic disorders. It is possible that impaired or exacerbated immunity in different diseases may be detected downstream of the antigen presentation capacity of DCs, that is, at the levels of antigen recognition stage. Antigen Capture After recognition of microbes or their antigens, the next important function of DCs is to capture and internalize these. Immature DCs can efﬁciently internalize a diverse array of antigens for processing and loading onto MHC molecules because of their high endocytic activities. Antigen capture by immature DC is mediated by distinct mechanisms. Macropinocytosis. This is a cytoskeleton-dependent ﬂuid-phase type of endocytosis mediated by membrane rufﬂing and the formation of large vesicles (1–3 μm). In DCs, macropinocytosis is constitutive, and enables a single a cell to take up a large volume of ﬂuid (half of a cell volume per hour). Fcg and Fce Receptor-Mediated Antigen Capture. Initially, it was described that DCs were devoid of Fc receptors; however, now several types of Fc receptors have been detected on various populations of DCs. Fc receptors allow the speciﬁc uptake of opsonized antigen. Fresh blood DCs express both CD32 and CD64, but not CD16. Similarly, human LC also expresses CD32, CD64. Human epidermal DCs, but not other DCs, express FcεR1. DCs are capable of internalization of various antigens by different receptors.

34

2. General Features of Dendritic Cells

Antigen Capture by Mannose Receptor and DEC-205. DCs express a variety of CLRs, which contain multiple carbohydrate-binding domains and are involved in the internalization of a variety of glycoproteins. One of the major differences between Fc receptor and mannose receptor lies in the fact that Fc receptors are usually degraded with their cargo; however, the mannose receptor releases its ligand at endosomal pH and is recycled. This mechanism provides supremacy of mannose receptors to uptake and accumulates many ligands by small numbers of receptors. Engulfment of Apoptotic Bodies by DCs. One of the major functions of DCs in steady state or in physiological condition is to induce immune tolerance. Again, crosspriming by DCs requires the uptake of apoptotic bodies. Thus, internalization of apoptotic bodies by DCs appears to be a highly efﬁcient machinery regarding the maintenance of homeostasis. DCs preferentially use the vitronectin receptor and the CD36/thrombospondin receptor for engulﬁng the apoptotic bodies. Engulfment of apoptotic bodies induces increases intracellular free Ca2+ concentration. The engulfment of antigens from necrotic bodies would depend on the nature of the necrotic tissues. If the antigens were free, those would be taken as nominal antigens However, if those are opsonized, the FC receptor might play a role in antigen capture. MHC Class II Loading Following internalization of antigens or microbes or apoptotic bodies, DCs must present these antigens to T cells. To do so, DC fulﬁlls the other requirements for antigen presentation by synthesizing and expressing high levels of class II MHC antigens. The intracellular class II MHC is found in both the late endocytic compartment and in an early endosomal compartment in DCs. In DCs, the majority of the intracellular class II MHC is found in late endocytic structures with numerous internal membrane vesicles and sheets. The major compartment contains newly synthesized MHC class II molecules that are targeted to this structure by an invariant chain. It also contains HLA DM molecules. In early DC, class II MHC molecules are localized to lysosomal compartments; however, in intermediate DC, class II MHC molecules are accumulated in distinctive nonlysosomal vesicles. In mature DCs, MHC–peptide complexes are present on the surface of DCs for long periods. This process allows a prolonged interaction between DCs and rare antigen-speciﬁc T lymphocytes. After the emergence of regulatory/tolerogenic DCs, the antigen presentation by DCs needs to be reevaluated. It is not clear whether similar pathways of antigen loading are utilized by immunogenic DCs and tolerogenic DCs. MHC Class I Loading MHC class I loading is critical for the activation of CD8+ T cells. DCs can capture exogenous antigen for presentation on MHC class I molecules, which ensures an efﬁcient generation of cytotoxic T lymphocyte (CTL). For class I loading, two pathways are required: (1) unconventional post-Golgi loading of MHC class I, and (2) involvement of a classical transporter associated antigen processing (TAP) loading

DC in Adaptive Immunity

35

mechanism. The peptides for class I MHC on DCs can be derived from nonreplicating microbes, soluble proteins, and apoptotic cells. The existence of MHC class I loading of regulatory DCs deserves evaluation. Activation and Maturation of DCs When microbes or their antigens are internalized by DCs and programmed to MHC class II or class I molecules, ultimately the antigen–MHC complex is expressed on the surface of the DCs. Several molecules including CD40, TNF-R, and IL-1R activate DCs and trigger their transition from immature antigen-capturing DCs to mature antigenpresenting DCs. DC maturation is a continuous process initiated in the periphery upon antigen encounter and/or inﬂammatory cytokines and completed during DC–Tcell interaction. Numerous factors are related to DC maturation, including (1) pathogen-related molecules such as lipopolysaccharides (LPS), bacterial DNA, and double-stranded RNA, (2) the relative presence of Proinﬂammatory and antiinﬂammatory signals in the local microenvironment including TNF-α, IL-1, IL-6, IL-10, TGF-β, and prostaglandins, and (3) signals that are given by T cells. Under the inﬂuence of these factors, several coordinated events are required for maturation of DCs: (1) loss of endocytic and phagocyte receptor; (2) upregulation of costimulatory molecules CD40, CD58, CD80, and CD86; (3) change in morphology; (4) shift in lysosomal compartments with downregulation of CD68 and upregulation of DC-lysosome-associated membrane protein (DC-LAMP); and (5) change in MHC class II compartments. The scenario that has been described here leads to activation of DCs and induction of immune responses. Evidence is still elusive about the deactivation of DCs or altered type of activation of regulatory DCs, when immune tolerance occurs as a result of antigen presentation by regulatory or tolerogenic DCs. Migration of DCs Several studies have shown that DC leaves the nonlymphoid organs through the afferent lymph. Pathogen products LPS and IL-1 and TNF-α, all mediators of DC maturation, also favor the migration of DC. Several chemokines are also involved in coordinated migration of DCs to lymphoid tissue. After antigen uptake, inﬂammatory stimuli turn off the response of immature DCs to macrophage inﬂammatory protein (MIP)-3α through either receptor downregulation or receptor desensitization. Upon maturation, DCs upregulate a chemokine receptor, CCR7, and acquire responsiveness to MIP-3β. Mature DCs entering the draining lymph node will be driven into the paracortical area in response to the production of MIP-3β and secondary lymphoid tissue chemokine (SLC). Also, mature can produce MIP-3β and SLC. Because two chemokines can attract mature DCs and naïve T cells, they are likely to play a key role in helping Ag-bearing DCs to encounter speciﬁc T cells. Upon encounter with T cells, which can take place not only in the secondary lymphoid organs but also in the site of tissue injury, DCs receive additional signals from CD40L that induce the release of chemokines such as IL-8, fractalkine, and macrophage-derived chemokines that attract lymphocytes.

36

2. General Features of Dendritic Cells

LCs in both human and mice migrate in response to proinﬂammatory cytokines Mobilization of LC by these cytokines might be inhibited by IL-4. TGF-β. that is required for LC development in vivo and in vitro prevents maturation of LC in response to IL-1, TNF-α, and LPS, but not to CD40 ligation.

Antigen Presentation and T-Cell Activation Experimental data are available regarding the presentation of antigen to T cells by DCs. Soluble antigen-pulsed DC elicits potent Ag-speciﬁc immune responses, and it has been demonstrated that DC–T-helper cell interaction in the periarteiolar lymphoid sheath by immunohistochemistry. In the presence of soluble antigens, T-helper cells primed by DCs can interact with B cells and may stimulate antigen-speciﬁc B lymphocytes for antibody production. DCs are equally important in priming naive T cells. In vitro, DCs can stimulate proliferation of allogeneic T cells directly in the absence of T-cell help. They can also generate antigen-speciﬁc CTL from naive T cells. Strong CTL responses can be induced in vivo by injection of mice with antigenbearing DCs, including allogeneic DCs, peptide-pulsed DCs, protein-loaded DCs, DCs transfected with DNA, DCs expressing virally encoded antigens, and DC pulsed with RNA. Although DCs can induce CTLs, they also require T-cell help. In the traditional model of CTL activation, CD4 and CD8 cells are thought to recognize antigens on the same DCs. However, conditioned DCs become a temporal bridge between T-helper cells and killer T cells. Conditioned DCs are formed by interacting with T-helper cells ﬁrst. These DCs having information from T-helper cells polarize the CTLs to become killer cells. Mature DCs appear to be essential to maintain survival of naïve CD4 T cells and immune T-cell memory. The mechanism of antigen presentation by regulatory DCs and immature DCs is not clear. Also, it is not known how antigen-speciﬁc immune tolerances are induced as a consequence of interactions between DCs and T cells.

Clinical Implications of DC Functions DCs perform several major functions in vivo. In the steady state, DCs scan the tissues and recognize autoantigens and apoptotic cells. When DCs recognize these elements in the tissues, they are capable of inducing innate immunity. The relationship between innate immunity and induction of immune tolerance is not clear. After processing, DCs process self-antigens and harmless entities in such a manner that immune tolerance is induced. Although cellular and molecular mechanisms underlying immune tolerances by DCs are not completely understood, DC plays a major role in the maintenance of homeostasis. On the other hand, in the presence of microbes, tumor cells, apoptotic cells, necrotic bodies, allergens, or alloantigens, DCs are activated and stimulated and induce antigen-speciﬁc immunity. As the roles of DCs during induction of immune tolerances and immune responses have been described, DCs have been analyzed in different pathological conditions to develop insights about pathogenesis of different diseases. Also, adoptively transferred DCs can modulate immunity of the hosts; DCs are also used to treat patients

Concluding Remarks: Implications of This Chapter in the Clinic

37

with cancer and other pathological conditions. Usually, bulk populations of DCs are used for treatment of different diseases in humans. As it is becoming clear that DCs regulate innate immunity, adaptive immunity, immune responses, and immune tolerances, proper types of DCs should be chosen to for treating patients. In this context, it is also required to assess different functional parameters of DCs in a case-by-case manner. This process may be difﬁcult, but it is essential for using DCs in clinical medicine.

Concluding Remarks: Implications of This Chapter in the Clinic In this chapter, we have mainly discussed (1) the clinical importance of DCs, (2) difﬁculty in deﬁning DCs, (3) a working deﬁnition of DC, (4) origins of DC including DC progenitors and DC precursors, (5) signals required for development and differentiation of DCs, (6) different types of tissue DCs, and (7) functions of different population of DCs. Because of their extreme plasticity and the limited knowledge about DCs, many important aspects of DC biology are largely unknown or incompletely understood. In this context, the main purpose of this chapter is to provide comprehensive understanding about DC to clinicians so that a proper regimen of DC-based therapies can be developed for human usage. Another aim is to develop insights about involvement of DCs in different pathological conditions. In this regard, the following points may be reemphasized. Present Status of DCs in Clinics. At present, the diagnostic and therapeutic importance of DCs in patients with different pathological conditions seems to be limited. However, this is mainly the result of limited understanding about human DCs and their subtypes. If more insights are developed about different subsets of DCs and their functions, the diagnostic and prognostic implications of DCs may be exposed. Also, clinicians should develop a proper protocol for studying the diagnostic and therapeutic importance of DCs. In spite of having limited diagnostic and therapeutic importance in patients with different pathological conditions, DC-based therapy seems to be a potentially important therapeutic approach for treating patients with cancers, microbial infections, autoimmunity, and allergic manifestations. Also, DCs may be used for preventing rejection of transplants. However, a careful protocol is required for using DCs in the clinical setting. These points are discussed in more detail in later chapters. Working Deﬁnition of DCs. DCs cannot be deﬁned from morphology, phenotype, and functions. In fact, there is no DC-speciﬁc marker or DC-speciﬁc function, which is true for many immunocytes. DC should be deﬁned from combinations of various characteristics such as dendritic morphology, expression of MHC class II and costimulatory molecules, production of cytokines (both proinﬂammatory and anti-inﬂammatory), and capacity to induce both immune responses and immunogenic tolerances. DCs are professional APCs and are capable of activating or deactivating the immune system. The migratory capacity of DCs represents a unique function of DCs. After capturing antigens, these cells undergo maturation and migration.

38

2. General Features of Dendritic Cells

Origin of DCs. DCs are bone marrow-derived leukocytes and may be developed form both CMPs and CLPs. The expressions of myeloid or lymphoid markers are not strictly related to their origin from CMPs or CLPs. Present studies indicate that Flt+ cells in the bone marrow populations may be the committed precursor of DCs; however, more studies are required to assess the presence of other committed precursors of DCs. DCs are detected in both mice and human in intrauterine life, and DCs are detected before the differentiation of some vital organs. The numbers of DCs increases with time after birth. Development and differentiation of DCs are under strict control of different transcriptional factors and cytokines. Phenotypes of DCs. Different types of surface markers are detected on DCs. In general, CD11c is a common marker of DCs in mice; however, CD11c− DCs are detected in humans. The phenotypes of DCs may be changed in culture under the inﬂuence of cytokines. In addition, there is extreme heterogeneity among some phenotypes. Some of the DCs express some antigens or transcription factors. The clinical implications of these subtypes are not also well understood. The phenotypes of regulatory DCs are not still clear. At present, we are probably using both immunogenic and tolerogenic DCs for therapy in the clinical setting, which may be one of the causes underlying the limited efﬁcacy of DC-based therapy. Again, this may be counterproductive in many pathological conditions. Functions of DCs. DCs can induce innate immunity as well as adaptive immunity. Although initially it was assumed that antigen presentation by DC causes immune responses, it is now clear that DCs are also able to induce immunogenic tolerances. To induce immune responses or immune tolerances, DCs must perform a series of activities. It is not well understood how immune responses and immune tolerances are induced by DCs. It is usually mentioned that the nature of DCs, agents, and tissue microenvironment determine the nature of immunity. However, further study is needed to clarify all types of DC functions and the ultimate outcome of immunity. DC-Based Therapy. DCs are used for treating patients with cancers. Although these cells can be used for treatment of other pathological conditions, clinical trials of DCbased therapy against chronic microbial infections, allergic diseases, and autoimmune diseases have not been started yet. Monocyte-derived DCs mainly are used for treatment of patients with cancers. The purpose of DC-based therapy in patients with cancers is to induce cancer-speciﬁc immunity in these patients. It is usually assumed that antigen-loaded monocyte-derived DCs would induce immune responses. However, the bulk population of these DCs may also contain tolerogenic DCs. Depletion of tolerogenic subsets of DCs from monocyte-derived DCs may allow development of more potent immunogenic DCs. In murine models, bone marrowderived DCs or spleen DCs are mainly used for therapy in animal models of human diseases. Some markers of regulatory DCs have recently been detected in mice. It may be possible to develop a better therapeutic regimen by depleting tolerogenic or regulatory DCs from the bulk population of DCs that are used for treatment. On the other hand, committed populations of regulatory DCs and tolerogenic DCs may be used to induce immune tolerances in subjects with autoimmunity or in the context of transplantation.

Recommended Readings

39

Recommended Readings Ardavin C (2003) Origin, precursors and differentiation of mouse dendritic cells. Nat Rev Immunol 3:582–590 Bell D, Young JW, Banchereau J (1999) Dendritic cells. Adv Immunol 73:255–323 Blom B, Spits H (2006) Development of human lymphoid cells. Annu Rev Immunol 24:287–320 Bonasio R, von Andrian UH (2006) Generation, migration and function of circulating dendritic cells. Curr Opin Immunol 18:503–511 Liu K, Waskow C, Liu X, Yao K, Hoh J, Nussenzweig M (2007) Origin of dendritic cells in peripheral lymphoid organs of mice. Nat Immunol 8:578–583 Manz M, Traver D, Akashi K, et al (2001) Dendritic cells development from common myeloid progenitors. Ann N Y Acad Sci 938:167–173 Niess JH, Reinecker HC (2006) Dendritic cells: the commanders-in-chief of mucosal immune defenses. Curr Opin Gastroenterol 22:354–360 Sato K, Fujita S (2007) Dendritic cells: nature and classiﬁcation. Allergol Int 56:183–191 Schuurhuis DH, Fu N, Ossendorp F, Melief CJ (2006) Ins and outs of dendritic cells. Int Arch Allergy Immunol 140:53–72 Shortman K (2000) Dendritic cells: multiple subtypes, multiple origins, multiple functions. Immunol Cell Biol 78:161–165 Shortman K, Caux C (1997) Dendritic cell development: multiple pathways to nature’s adjuvants. Stem Cells 15:409–419 Shortman K, Naik SH (2007) Steady-state and inﬂammatory dendritic-cell development. Nat Rev Immunol 7:19–30 Steinman RM (2003) Some interfaces of dendritic cell biology. APMIS 111:675–697 Steinman RM, Hemmi H (2006) Dendritic cells: translating innate to adaptive immunity. Curr Top Microbiol Immunol 311:17–58 Tacken PJ, Torensma R, Figdor CG (2006) Targeting antigens to dendritic cells in vivo. Immunobiology 211:599–608 Wu L, Dakic A (2004) Development of dendritic cell system. Cell Mol Immunol 1:112–118 Wu L, Liu Y-J (2007) Development of dendritic-cell lineages. Immunity 26:741–750

3 Interactions Between Dendritic Cells and Infectious Agents

Outline of Dendritic Cell in Microbial Infection Infection, Immunity, and Host Defense The body is constantly exposed to a remarkable variety of infectious agents, such as viruses, bacteria, fungi, and parasites. The individual pathogens differ with regard to their genetic organization, the tissue of localization and replication, the way of transmission, the mode of replication, the mechanism of causing disease, and the type of host response they elicit. To prevent entry of microbial agents in our body, epithelial surfaces act as a physical barrier. These epithelia comprise the skin and the mucosal linings of the gastrointestinal, respiratory, and urogenital tract. They also secrete antimicrobial substances, including lysozyme, defensins, and various other chemical and biological modulators. However, infectious agents enter the body of the hosts by means of a breach in epithelial surfaces or directly into the circulation. One of the main characteristics of the evolutionary process is to develop highly sophisticated defense mechanisms for survival of human beings and other living organisms in hostile environments. Only when pathogens come in contact with the epithelial barrier or cross the barrier or enter into circulation does the immune system initiate effector mechanisms to eliminate the infectious agent. The immune system is composed of cells and molecules that have specialized roles for defending against infection. The ﬁrst phase of host defense against invading microorganisms is dependent on the mechanisms of innate immunity. It is an evolutionarily ancient and universal form of host protection. Innate immune recognition is based on a limited number of highly conserved receptors that detect common microbial components. It allows the immune system to discriminate between non-self and self elements, and also between dangerous and nondangerous entities. This mechanism represents the major checkpoint in the decision to respond or not to respond to a particular antigen. The innate immune system is composed of different cells such as neutrophils, eosinophils, natural killer (NK) cells, macrophages, and dendritic cells (DCs). Also, soluble factors such as complement components, cytokines, and chemokines play important roles in innate immunity. In addition, the epithelial cells themselves have a role in innate immunity. Cells of the innate immune system possess pattern-recognition receptors (PRRs) that recognize pathogenassociated molecular patterns (PAMPs) from viruses, bacteria, fungi, and protozoa. In addition, these cells may also express other receptors that can allow them to recognize the microbial agents. 41

42

3. Interactions Between Dendritic Cells and Infectious Agents

The cells of the innate immune system are capable of directly killing microbial agents or may limit their activity by different mechanisms. In addition, microbial agents can be destroyed by chemical and biological mediators produced by cells of innate immunity. In some cases, however, microorganisms overcome these early lines of defense and the initiation of an adaptive immune response is required to ﬁght the infection. Adaptive immune responses are mediated by the generation of antigenspeciﬁc T and B cells. Antigen-primed T cells induce clonal expansion and differentiate into effector T cells that produce various cytokines or elicit cytolysis to eliminate target cells. B cells secrete immunoglobulins, which are responsible for eliminating extracellular microorganisms. Not only is the adaptive immune response associated with the activation of distinctive effector cells, B and T lymphocytes, but one of the special properties of adaptive immune system is to generate microbial agent-speciﬁc memory cells that prevent subsequent infections with the same pathogen. Taken together, the innate and adaptive immune systems form an integrated host defense machinery to confer optimal protection. Innate responses are generated at the periphery of sites of microbial penetration. On the other hand, the adaptive immune responses are generated at secondary lymphoid tissues, such as lymph nodes and the spleen. However, early cellular events of adaptive immunity begin at the tissues of infections or localization of microbial agents. There are some fundamental differences between innate and adaptive immunity regarding the purpose of immune responses, duration of action, natures of cells, and ﬁnal outcomes of these two types of immune system. Indeed, both these immune responses are interdependent. DCs in Host Defense and Immunity DC was ﬁrst discovered in 1973 in the lymphoid tissues as an antigen-presenting cell (APC). Early characterization of DCs revealed that DCs are capable of recognition, processing, and presentation of antigens for induction of antigen-speciﬁc adaptive immune responses. They were regarded as professional APC because their lymphocyte-priming capacities were signiﬁcantly higher than those of other APCs. In the course of time, it became evident that DCs are distributed in almost all tissues of the body. Also, it was found that DCs are not only inducers of immune responses but are capable of inducing immunogenic tolerance. In the meantime, the roles of DCs in innate immunity were disclosed. DCs maintain crosstalk with cells of innate immunity. The functions of DCs were impaired in animal models of chronic infections and also in patients with chronic infectious diseases. Subsequently, the role of DCs in the treatment of different animal models of chronic infections became evident. Different types of DCs with diverse phenotypes and functions have been detected in situ and in cultures. Now, it appears that DCs are players in innate immunity and champions of adaptive immunity. DCs in Innate Immunity Against Microbial Agents DCs are regarded as sentinels of the immune system, and they are continually scanning the hosts for presence of microbial agents, transformed cells, apoptotic cells, and various other substances in different tissues. Interaction between DCs and microbes is shown in Table 1. DC recognizes a limited but highly conserved set of molecular structures produced by pathogens, named PAMP. PAMPs are recognized through a

Outline of Dendritic Cell in Microbial Infection

43

Table 1. Interactions between dendritic cells and microbial agents Nature of interactions

Outcome of interactions

A. PAMPs of microbes/PRRs of DCs

Innate immune responses Recognition of microbes Production of cytokines and other factors Activation or deactivation Induction of adaptive immunity

B. Capture of microbial agents by DCs

Phagocytosis, endocytosis, micropinocytosis Crosstalk between innate and adaptive immunity

C. Microbial agents within DCs

Processing of microbial agents Activation or deactivation of DCs DCs in maturation and migratory modes

D. DCs with microbial antigens Final outcome of interactions between DCs and microbial agents

Immune responses or immune tolerances Acute and self-limiting infection Chronic and persistence infection

PAMP, pathogen-associated molecular pattern; PRR, pattern-recognition receptors

number of germline-encoded receptors of DCs, called PRRs. DC expresses members of the two most important families of PRRs, Toll-like receptors (TLRs) and C-type lectin receptors (CLRs). TLRs include 10 (TLR 1–10) and 12 (TLR 1–9 and 11–13) family members in human subjects and mice, respectively. TLR usually recognize evolutionarily conserved microbial molecules categorized into lipid, protein, and nucleic acids. In addition, engagement of TLRs with PRRs possesses immune adjuvant function; this also leads to activation of DCs. Different types of proinﬂammatory cytokines are produced by means of these interactions. Local innate immune responses may be induced on the basis of the nature of cytokines. TLR engagement causes activation of several transcription factors, such as NF-κβ and interferon regulatory factors (IRF), which may cause induction of several genes associated with the immune response. TLR signaling also induces the maturation of DCs, thus leading to increased immunogenicity and T-cell priming. It is not clearly known whether TLR signaling can induce antiinﬂammatory mediators and immune tolerances. The CLR family mainly includes DC-SIGN and blood dendritic cell antigen-2 (BDCA-2). These receptors recognize various saccharides, such as d-mannose, lfucose, and N-acetylglucosamine, on the pathogen surface. Their major function is to internalize antigens for further processing and presentation by DC. Internalization of various microbial agents by CLRs causes upregulation of MHC and costimulatory molecules on DCs. Also, proinﬂammatory cytokines are produced that determine the type of ensuing immune response. The production of antiinﬂammatory cytokines resulting from capture of microbes is a matter for further investigation. In addition to PAMPs, there may be a pathway of PAMP-independent innate activation of DC. It has been proposed that DC activation can be materialized by the recognition of endogenous host-derived molecules released by cells undergoing necrosis. Such endogenous “danger” signals may act as ligands for PRRs. Similarly, hyaluran degradation products, ﬁbronectin A, ﬁbrinogen, uric acid, heat shock proteins, and β-defensins can engage with putative endogenous TLR ligands of DCs. In addition, some signals that mimic PAMPs such as ATP and bradykinins may induce production

44

3. Interactions Between Dendritic Cells and Infectious Agents

of interleukin (IL)-12. Also, there is a TLR-independent pathway of DC activation by microbes. Microbe-infected tissues can activate DCs without a direct TLR engagement of DCs. For example, keratinocytes exposed to double-stranded RNA products produce cytokines. Also, cytokines produced by other cells of the immune systems may activate DCs; in turn, DCs activate the other cells of innate immunity. A type of DCs, plasmacytoid DC (pDC), produces abundant amounts of type 1 interferon (IFN) in response to stimulation with microbial agents or their products. Type 1 IFN possesses an important role in innate immunity and blocks replication of different viruses. Taken together, DCs express a highly sophisticated group of receptors. When these receptors are engaged with different pattern molecules of microbial agents, DCs may activate innate immunity. In addition, alteration of tissue death may cause induction of innate immunity through activity of DCs. Other cells of the innate immune system also act in association with DCs to induce innate immunity. Early inﬂux of DCs in different tissues after localization of microbes also indicates the activity of DCs in innate immunity. DCs in Adaptive Immunity Against Microbial Infections DCs are widely distributed in almost all tissues of the body. The frequencies of DCs may be lower compared with that of other immunocytes in some tissues. However, the DCs represent critical regulators of immunity because of their unique functional capacities. Moreover, when microbial agents are present in some tissues, DCs rapidly localize in that tissue. DCs act as cells of innate immunity through PRR-dependent or PRR-independent pathways, as described in Table 1. The activities of DCs in adaptive immunity are initiated after recognition of pathogens (see Table 1). In this step, DCs internalize microbial agents or their antigens by various receptors. Although PRRs on DCs are essential for sensing microbial agents, all types of PRRs are not required for antigen internalization. DC employs various types of manipulation to internalize microbial agents from different tissues, including phagocytosis, endocytosis, micropinocytosis, and receptor-mediated endocytosis. Scavenger receptors and CLRs are specialized in internalization of pathogens for subsequent processing and presentation. After internalization of microbial agents, DCs cleave these into antigenic peptides. The antigenic peptides of microbial agents are expressed with self-major histocompatibility complex (MHC) of DCs; this results in the acquisition of a migratory phenotype that allows them to exit the tissue and home to T-cell-rich areas of the secondary lymphatics. Maturation of DC is generally associated with a major switch from endocytotic to antigen processing and presentation activity, characterized by downregulation of PRRs, activation of antigenprocessing pathways, and, last, displaying and presenting loaded major MHC class II molecules and costimulatory molecules at their surface. Activation of MHC class I-restricted CD8+ T cells by DCs relies on viral replication, or cross-priming that may be initiated after uptake of cell-bound or cell-free antigens by speciﬁc receptors including Fcγ receptors and scavenger receptor A. DCs can receive antigen from living cells by cross-priming. Recognition and internalization of antigens by DCs is followed by antigen processing. Antigen may be processed in an immunogenic manner or a tolerogenic manner.

Outline of Dendritic Cell in Microbial Infection

45

In the context of microbial agents, antigens are supposed to induce immune responses. However, tolerogenic or regulatory DCs may also their functions in this context. After antigen processing, DCs migrate to the lymphatics to present antigenic peptides to lymphocytes. At the secondary lymphoid tissues, DCs interact with clonally selected T cells, and T-cell scanning is facilitated by morphological features of the maturing DC. DC as Linkage Between Innate and Adaptive Immunity During Microbial Infections It is known that the innate immunity is mediated by stromal cells present in body lining tissues, such as epithelial cells, ﬁbroblasts, and endothelial cells, as well as by more specialized migratory immune cells such as NK cells, granulocytes, macrophages, and DC. Also, various soluble factors, such as complement components, cytokines, and chemokines are important effector mediators of innate immunity. On the other hand, lymphocytes are regarded as the main cells of adaptive immunity. Innate immune responses are generated at the periphery of sites of microbial penetration, whereas adaptive immune responses are generated at secondary lymphoid tissues, such as lymph nodes and the spleen. However, it is important to develop insights about the transmission of the signals of innate immunity at the tissue to immunocytes at the lymphoid organs, where adaptive immunity is induced. Also, it is important to have insights into how innate immunity instructs the adaptive immune system about the nature of pathogens. Most of these activities are performed by antigen-presenting DCs. DCs are not only able to stimulate naïve T cells with microbial antigens but also orchestrate protective immune responses; this is accomplished by the expression of variable sets of T-cell costimulatory and polarizing molecules that promote the development of an appropriate cell-mediated immune response. Innate stimulation of DCs can trigger their differentiation into immunogenic APC capable of priming and sustaining the expansion of naïve T cells. In addition, DCs direct T-cell effector differentiation, which is responsible for ensuring that the speciﬁcity of the innate immune system, which distinguishes between many classes of potential pathogens, is translated into an equally speciﬁc class of adaptive immune response. After the discovery of DCs, the many cellular and molecular events that underlie the linkage between innate and adaptive immunity became more clear. DCs are probably the most potent cells of the immune system as they are endowed with capacities to regulate both innate immunity and adaptive immunity. Different cytokines and immune modulators may act as a bridge between innate and adaptive immunity. Regarding DCs, different subtypes of conventional DCs (cDCs) have been deﬁned that differ in phenotype, localization, and immune function. The picture has been enlarged by the phenotypic and functional identiﬁcation of pDCs. Although their functional distinction in response to PAMP recognition and antigen capture and uptake is not absolute, cDCs and pDCs may have evolved to preferentially controlling bacterial and/or viral infections, respectively. This condition may be indicated by their distinct repertoire of PRRs and transcription factors. In fact, cDCs are major producers of inﬂammatory cytokines such as IL-12, TNF-α, IL-6, and IL-1α/β, and pDCs produce abundant amounts of type I IFN. pDCs and cDCs share a common program for chemokine induction after virus encounter, which allows for a coordinated attraction of the different immune effectors

46

3. Interactions Between Dendritic Cells and Infectious Agents

in response to viral infection. These actions of cDCs and pDCs allow DCs to act as a bridge between innate and adaptive immunity. It should be mentioned that the main function of DC-expressed PRRs may not be to activate DCs to become immunogenic APCs but, rather, to convey information about the nature of the insult, thereby allowing DCs to direct an appropriate class of immune response. Thus, innate immune responses do not only cause activation of DCs but also instruct about their differentiations. In addition, DCs also have crosstalk with NK cells. The functions of DCs are highly impaired in NK-depleted mice. On the other hand, when NK cells are activated in situ, DCs also undergo maturation. Also, activated DCs cause increased function of NK cells, which may not be speciﬁc for NK cells only; other cells of the innate immunity are linked with DCs in a similar manner. Taken together, DCs act as a bridge between innate and adaptive immunity. Moreover, ligation of PRRs on DCs and PAMPs of microbial agents not only activates innate functions of DCs but also instructs about the nature of adaptive immune responses. DC and Immune Tolerance in the Context of Microbial Infections DCs are considered as inducer of immune responses. However, if immune responses proceed in an uncoordinated manner, tissue injury may be inﬂicted in the hosts and the hosts may suffer from autoimmune diseases. Exacerbated and autoimmune conditions are checked by induction of tolerogenic immunity. In addition, if immune responses are induced in response to the thousands and millions of agents present in the environment, the host will become sick. One type of DCs (regulatory or to lerogenic DC), along with regulatory T cells, and different immune regulatory cytokines play a role to maintain homoeostasis. Interestingly, regulatory DCs also regulate the production and activities of regulatory T cells and antiinﬂammatory cytokines. The concept, characterization, and functions of regulatory DCs are discussed in a separate chapter of this book. We only discuss these DCs in the context of microbial infections. Induction of immunogenic tolerance is the primary function of DCs in steady-state conditions. Immature DCs and regulatory DCs play major roles in this context. However, after microbial infections, there is tissue inﬂammation, and maturation and activation of DCs occur. In this context, DCs are supposed to become unable to induce immunogenic tolerance. However, highly regulated mechanisms determine the extent of innate and adaptive immunity after microbial infections. The role of regulatory DCs has not been properly evaluated in microbial infections. However, we encounter different immune-mediated fallacies in clinics. The following points should have a relationship to DC-mediated immune tolerance against microbial agents. 1. Pathogenesis of chronic infection is still poorly understood. Although DCs can induce both innate and adaptive immunity, it is elusive why several pathogens establish a chronic infection in immune-competent hosts. These pathogens are never cleared from the hosts. Although virus-derived factors and host-related factors account for this, little is known about DCs in these situations. This gap in our under-

Outline of Dendritic Cell in Microbial Infection

47

standing regarding the pathogenesis of chronic viral infection can be explained by analyses of functions of regulatory DCs in these pathological conditions. Indirect evidence has shown that the frequencies of regulatory T cells, induced by regulatory DCs, are increased in patients with chronic viral infections. 2. Some patients exhibit an exacerbated response to microbial infections, whereas others do not show any features of disease after infection. Many patients develop progressive chronic infections and also their complications. The role of regulatory DCs may play a role in the determination of the clinical course of microbe-infected diseases. 3. The efﬁcacy of antimicrobial agents is highly heterogeneous even in patients with a similar load of microbial agents and similar tissue damage. The role of regulatory DCs needs to be examined in these cases. 4. The present era is a time of vaccine development against various microbial agents. Some potent vaccines are available against some microbial agents, although it seems that it may not be possible to develop a prophylactic vaccine against many microbial agents; this may be a matter of technical limitation. In addition, the responses to vaccines are highly heterogeneous among individuals. The regulatory DCs may determine the responses to prophylactic vaccines. Vaccine-induced induction of regulatory DCs and other immune regulatory cells may have a role during nonresponsiveness to prophylactic vaccine. Elucidation of Nature of Maturation of DCs for Development of DC-Based Therapy Against Chronic Microbial Infections The points that have been discussed here indicate that after entrance of microbial agents, DCs along with other cells of the innate immune system induce innate immune responses. On one hand, there will be production of various cytokines and thus an inﬂammatory mucosal milieu may prevail in the tissue of localization of microbial agents. The nature and extent of innate immunity may be capable of destroying the invading pathogens or control their growth. Subsequently, DCs would internalize, process, and present microbial agents for induction of adaptive immunity. The effector lymphocytes are supposed to destroy microbe-infected cells, and they have dominant roles during establishment of immunological memory. If the DC can perform its function and if other immunocytes of the host are capable of cooperating properly with DCs, the foregoing scenario can materialize. However, we experience different scenarios after microbial infection. Some patients do not exhibit visible innate and adaptive immunity and continue to harbor the viruses without any apparent tissue damage. Other patients harbor both microbial agents and also exhibit features of tissue damage. In some cases, there is an exacerbated immune response resulting in fulminant and life-threatening diseases caused by microbial infections. The clinical courses of diseases caused by infections with microbial agents are extremely diverse. Most of the cellular and molecular mechanisms underlying the progression and natural course of most diseases are unknown. DCs interact with PAMPs, and these encounters usually cause activation of DCs; DCs also receive instruction about successive immune responses. If this instruction is purposeful, the extent of successive innate and adaptive immunity may be fruitful. Patients infected with microbial agents should eradicate or eliminate the microbial

48

3. Interactions Between Dendritic Cells and Infectious Agents

agents after an acute episode of innate immunity. Also, the patients should develop antigen-speciﬁc memory lymphocytes to block reinfection with the same microbial agents. However, microbial infections are not controlled in many patients. Also, there are either no or impaired memory lymphocytes in patients with chronic infections. Several studies have indicated that both microbe-derived and host-related factors are related to the pathogenesis of chronic microbial infection. However, the real mechanism has not been explored, nor is effective treatment available to treat these subjects. From accumulating evidence, it may be suggested that the nature of DC–microbe interactions may have a dominant role in the pathogenesis of chronic viral infections and during development of complications. As shown in Fig. 1, DCs can be matured or activated in the peripheral tissues mainly by two major pathways: one is ligation of PRRs of DCs and PAMPs of microbial agents, and the other is by cytokines induced by other cells of innate immunity and also adaptive immunity. In vitro study has revealed that maturation of DCs without instructions from microbial agents may result in ineffective adaptive immune responses. It is possible that if DCs are activated nonspeciﬁcally by cytokines before getting instructions from PAMPs, the activated DCs may not be able to perform the different functions that have been shown in Table 1. This limitation may be related to the pathogenesis of chronic and persistent micro-

Maturation of DCs and nature of immunity Maturation of DCs by interactions with microbes or their antigens

Maturation of DCs from interaction between TLRs of DCs and PAMPs of microbes:

Instruction received from PAMPs about nature of microbes and successive immune responses

Maturation of DCs caused by interaction between CLRs of DCs and PAMPs of microbes:

Immune responses will depend on the nature of endocytosedmaterials and subsequent signals

Maturation of DCs by cytokines or environmental factors

Prior to engagement with PAMPs: 1.May lead to microbe nonspecific immunity 2.Impaired control of microbes

After engagement with PAMPs: 1.Microbe-specific immunity 2.Control of microbes

Fig. 1. The nature of maturation of dendritic cells (DCs) may inﬂuence subsequent immune responses. When dendritic cells undergo maturation as a consequence of uptake and processing of antigens, antigen-speciﬁc immunity can be induced. In contrast, maturation of DCs by inﬂammatory cytokines without involvement of antigen may downregulate antigen-speciﬁc immunity. TLR, Toll-like receptors; PAMP, pathogen-associated molecular pattern; CLR, C-type lectin receptors

Consequences of Interactions Between DC and Virus

49

bial infections. It seems that activated DCs are not major determinants of successive immune responses. However, the nature of activation and maturation of DCs seems to be important in the context of clinical courses of microbial infections (see Fig. 1). In this chapter, we provide an outline about the roles of DCs in the pathogenesis of some microbial infections. Finally, we explore how DC-based therapy can be developed against microbial infections.

Consequences of Interactions Between DC and Virus General Principle The role of DC during viral infection is not only complex but also confusing. In some viral infections, DCs act as professional APCs. However, it is not clear whether all steps of antigen presentation proceed in a coordinated manner. There are few studies in which the different steps of antigen presentations have been properly analyzed. Experimental data show that DCs cannot act as professional APCs in many viral infections. Again, it is elusive whether all steps of antigen presentation are disturbed or if some speciﬁc steps of antigen presentation are distorted in these cases. As shown in Table 2, in some viral infections, DCs may be infected by viruses whereas viruses are captured by DCs in other cases. Similarly, processing of viruses by DCs also varies considerably. In most cases, DC–virus interaction is evaluated by the T-cell stimulatory capacities of DCs. However, these approaches also have some inherent limitations. The consequences of DC–virus interactions in the pathogenesis of persistent infections with virus represent major topics of discussion. Several studies have shown that viruses are localized in DCs in different pathological conditions. It may appear that this is part of internalization of the virus by the DCs. However, the subsequent events of antigen presentation by DCs after internal-

Table 2. Viral persistency and involvement of dendritic cells A. Recognition of virus

Phagocytosis of apoptotic cells containing viruses by DCs Defective recognition of virus by DC Impaired innate immunity resulting from DC–virus interaction

B. Localization of virus in DC

Viruses establish infection in DC Virus is captured by DCs Virus captured by DCs may infect other immunocytes

C. Processing of virus

Normal loading of viral antigen to MHC complex Impaired processing of virus

D. Maturation and migration of antigen-loaded DC

Defective maturation of DCs in viral-infected tissue Maturation of DCs by cytokines but not by PAMPs Impaired migration resulting from lower expression of chemokine and chemokine receptor

E. T-cell stimulation

Impaired T-cell stimulation capacity Unaltered T-cell stimulatory capacity

50

3. Interactions Between Dendritic Cells and Infectious Agents

ization of virus have really been evaluated. Although the virus should be cleaved and processed in DCs, replication of virus is detected in DCs. To develop insights about these cellular and molecular events regarding DC–virus interactions, we discuss DCs in acute viral infection caused by two viruses, the inﬂuenza virus and the measles virus. These two viruses were selected for providing insights about DC–virus interactions because one of them causes activation of DCs whereas the other suppresses DC functions. Many people are infected with these viruses each year around the world. The public health burdens from these viruses are tremendous in both developed countries and developing nations. Moreover, the impact of these viruses on DC can be also assessed in vitro. We also discuss DCs in two chronic viral infections, human immunodeﬁciency virus (HIV) and human hepatitis viruses. HIV possesses cytopathic properties in the infected hosts. However, hepatitis viruses are not usually cytopathic. Infection with HIV and hepatitis viruses constitutes a major global public health problem. The diagnostic and prognostic importance of DCs in these infections is yet to be explored, but immune therapy using DC has been applied in animal models of these viral infections for the past two decades. Also, several in vitro studies have shown that immune therapy may be effective in patients infected with these viruses. Interaction of DCs with Viruses of Acute Infection (Inﬂuenza Virus and Measles Virus) During immune response to acute viral infection, a self-limiting tissue injury is inﬂicted. The incubation period from entry of virus to development of symptoms provides insights about the extent of innate and adaptive immunity during acute viral infections. The role of DCs in acute viral infection may be limited to innate immunity during initial stages of the disease if the incubation period is short. However, adaptive immunity, induced by DCs, also allows development of virus-speciﬁc memory lymphocytes and future protection from infection from the same virus. In some cases of acute viral infection, the infection takes an aggravated course and severe and fulminant diseases are seen. The role of DCs in the pathogenesis and clinical course of fulminant types of infection is not well understood. A wave of cytokine storm is believed to induce massive tissue damages in these patients. However, the contribution of DC in cytokine storm is elusive, but, not unexpected. DCs produce different cytokines and also induce other immunocytes to produce large amounts of proinﬂammatory cytokines and immune mediators. It is an open question why the exacerbated immune responses are not controlled at some point of severe and fulminant tissue damages as a result of certain viral infections. Study about regulatory DCs may unveil some mysteries in this respect. The functions of DCs have been investigated in inﬂuenza-infected subjects. Also, DCs have been transfected in vitro by inﬂuenza virus, and the phenotypes and functions of DCs have been characterized. Inﬂuenza virus is detected in DCs, and viral proteins are expressed on DCs. However, the replication of inﬂuenza virus is not so pronounced in DCs. Encounter of DCs with inﬂuenza virus causes maturation of DCs, as shown by the expression of DC-LAMP and CD83. Expression of MHC class I and MxA protein is also seen in inﬂuenza-infected DCs. These facts indicate that activation of DCs by inﬂuenza virus may be related to its self-limiting course of disease

Consequences of Interactions Between DC and Virus

51

activity. In addition to direct DC–inﬂuenza virus interaction, experimental study of cross-priming has been reported in inﬂuenza virus infection. Apoptosis occurred in monocytes infected by inﬂuenza virus. Immature DC phagocytoses this apoptotic product and induces inﬂuenza virus-speciﬁc, MHC class I restricted CD8+ CTL. As in other situations, the phagocytosis is observed in 80% of macrophages, 50% of immature DCs, and in less than 10% of mature DCs. However, the activity for CTL by cross-priming is most potent in immature DC and none in macrophages. Thus, DCs use unique pathways for the phagocytosis, processing, and presentation of antigen derived from apoptotic cells on MHC class I complex. CD36 might be important in this cross-priming system. In general, cDC has a potential for Th1 polarization, and pDCs produce type 1 IFN and polarize to Th2. However, in the presence of inﬂuenza virus and CD40L in vitro, pDCs may also polarize to Th1, with upregulation of major histocompatibility complex proteins and costimulatory molecules. pDCs probably participate in antiviral and proinﬂammatory responses, rather than only Th2 polarization and tolerance induction. These scenarios indicate that some DCs may be infected and killed by inﬂuenza virus, but the remaining DCs induce innate and adaptive immunity during inﬂuenza virus infections; this leads to protection from future infection by the same virus. However, it is still elusive whether any particular type of DC is susceptible to infection by inﬂuenza virus. The major limitations of these studies lie in the fact that bulk populations of DCs are used for the study. The effect of inﬂuenza on different subtypes may provide further information about DC–inﬂuenza virus interaction. In contrast to inﬂuenza virus, measles virus causes downregulation of DC functions. Immune suppression is also detected in measles-infected persons. However, it remains to be elucidated whether decreased functions of DCs are related to the immune suppressive state in measles-infected patients. The roles of other immunocytes during measles virus infection need to be explored. DC also processes and presents measles viruses for induction of measles-speciﬁc adaptive immunity, which may provide protection from future measles infection. Although studies are continuing about interactions between DCs and some viruses that cause acute infection, the clinical implications of these studies are still not clear. It is unlikely that DCs would show diagnostic and prognostic importance in acute viral infections. Also, DC-based immune therapy does not seem to be a treatment option against acute viral infection at this time. However, studies about interactions between DCs with inﬂuenza virus and measles virus provide broad insights about different cellular and molecular mechanisms in the context of DC/virus interactions. Chronic Viral Infection and DCs As we have discussed in the previous chapter, the roles of DCs have primarily been analyzed in chronic viral infections to evaluate if DCs bear diagnostic or prognostic importance. Finally, strategies have been developed to treat these patients using DCs. Usually viruses that cause chronic infection are noncytopathic; however, certain low cytopathic virus also causes chronic infections. In this section, we discuss HIV, which is a cytopathic virus and causes chronic infection. Also, we discuss two hepatitis viruses, hepatitis B virus (HBV) and hepatitis

52

3. Interactions Between Dendritic Cells and Infectious Agents

C virus (HCV). These viruses are noncytopathic in nature and cause a variety of infections, such as acute, fulminant, and chronic infections. DC–HIV Interactions, Clinical Consequences, and Opportunity for Development of DC-Based Therapy About 65 million people worldwide have been infected by HIV since the onset of the epidemic in the late 1970s, and 25 million have died so far from acquired immunodeﬁciency syndrome (AIDS). At present, about 40 million are still living with HIV, with an incidence of 4.1 million acquiring the virus and 2.8 million dying of AIDS every year. The HIV-1 penetrates the body through the sexual mucosa, blood, and the oropharyngeal and/or digestive mucosa. Heterosexual transmission is presently the most frequent contamination modality in Africa. An important characteristic of this virus is that its main target cell is one of the cells governing the immune system, the CD4+ cells. During the ﬁrst phase of primary infection, HIV infects a small proportion of CD4 cells with a memory phenotype. The antibodies targeted against the virus are not capable of neutralizing it, and the control of viral replication in CD4 cells relies therefore on the activity of cytotoxic T lymphocytes (CTLs). As DCs are essential for CD8-speciﬁc T-cell immune responses, interaction between HIV and DC is a topic in clinical immunology. HIV infection of DCs results in interference of the antigen-presenting function of DCs; this may be a key aspect in viral pathogenesis, and contributes to viral evasion of immunity. All types of DCs are susceptible to HIV infection, including cDCs, pDCs, and Langerhans cells (LCs). However, the susceptibility of DC progenitors and different precursor populations of DCs to HIV is not clear. Different DC populations express low levels of HIV receptor, CD4, and the coreceptors CC-chemokine receptor 5 (CCR5), CXC-chemokine receptor 4 (CXCR4), CCR3, CCR8, and CCR9. Among these, CCR5 and CXCR4 are the major HIV coreceptors. HIV can be divided into two types based on the requirements of coreceptors required for infection of DCs. R5 viruses (previously termed macrophage-tropic or nonsyncytium-inducing viruses) use CCR5 for entry into DCs, whereas X4 virus (previously termed T-cell line tropic or syncytium-inducing viruses) utilizes CXCR4 for infecting DCs. In general, immature DCs express higher levels of CCR5 and little or no surface CXCR4, whereas mature DCs tend to express less CCR5 and higher levels of CXCR4. cDCs are more susceptible to R5 HIV infection than pDCs from the same donor. Nevertheless, R5 HIV strains infect cDCs much more efﬁciently than X4 HIV strains, probably because they express higher levels of CCR5 than CXCR4. In addition to expressing receptors and coreceptors for HIV, DCs have a unique membrane transport pathway that facilitates the uptake of pathogens. DCs constitutively take up extracellular ﬂuid by macropinocytosis and engulf antigens and whole pathogens by endocytosis and phagocytosis by Fc receptors and C-type lectins, such as DC-SIGN. Four types of C-type lectin have been described that may be related to binding with HIV: DC-SIGN, langerin (CD207), mannose receptor (CD206), and an unidentiﬁed trypsin-resistant C-type lectin. However, it seems that no single C-type lectin is fully responsible for HIV binding on all DC subsets.

Consequences of Interactions Between DC and Virus

53

The Clinical Implication of Infection Versus Capture of HIV by DCs HIV can enter DCs by infection or can be attached in infectious form by capture. Two models are now presented regarding this. Transmission of virus from LC to T cells is dependent on CCR5-mediated infection of LC. Mannan (capable of blocking HIV envelope interactions with C-type lectins), as well as anti-DC-SIGN antibody, had no effect on LC-mediated transmission of virus to T cells. Furthermore, DC-SIGN might not be involved in sexual transmission of HIV in the genital tract because it is not expressed by LC within the vaginal epithelium and epidermis. Thus, it is likely that CCR5-mediated infection of LCs, and not DC-SIGN-mediated capture of HIV, is the major pathway involved in sexual transmission of HIV. On the other hand, DC-SIGN seems to be related to capture of HIV by DCs. DC-SIGN-mediated capture of HIV by DCs could play a role in the early events of sexual transmission by trapping small amounts of virus produced by LC and amplifying viral loads by migrating to lymph nodes and infecting T cells. Indeed, regardless of whether DCs are infected with HIV or HIV is captured by DCs, these actions render DCs infectious. Speciﬁcally, both HIV-infected DCs and DCs with captured HIV transmit robust infection to cocultured CD4+ T cells. Thus, DC–T-cell interactions, critical in the generation of immune responses, also provide rich microenvironments for ampliﬁcation of HIV replication. In general, the levels of HIV replication in DC-T-cell cocultures correlate directly with the degree of T-cell activation in vitro. In this setting, we can speculate that DC-SIGN-mediated infection of T cells is the major means by which DC promote infection of T cells, given that the absolute number of HIV-infected DC is relatively low in chronically infected individuals. Furthermore, not only does DC-SIGN promote infection of T cells that interact with DCs, this molecule also facilitates close interaction of HIV gp120 with CD4 and HIV coreceptors on the surface of DCs, thus enhancing infection of DCs as well. In summary, in contrast to sexual transmission of HIV that is mediated by CD4 and CCR5, DC-SIGN-mediated capture of HIV, with subsequent DC-mediated infection of T cells during immune responses, might contribute greatly to overall viral replication in chronic HIV disease. Transfer of Captured HIV by DCs Cells expressing DC-SIGN and the monocytic marker CD14 are able to increase HIV trans-infection of T cells. Similarly, DC-SIGN-expressing cells that were isolated from the rectal mucosa and that seemed to have an immature phenotype could efﬁciently bind and transfer HIV to CD4+ T cells. These DC-SIGN+ cells comprise only 1%–5% of total mucosal mononuclear cells, but they were shown to contribute more than 90% of virus binding. Binding of HIV is mainly mediated by DC-SIGN, as DC-SIGNspeciﬁc antibodies could block virus binding when more physiological amounts of virus were inoculated. HIV that is internalized after DC-SIGN binding retained infectivity and could be transmitted to target CD4+ T cells. DC-SIGN mediates rapid internalization of intact HIV into a low-pH, nonlysosomal compartment. The precise intracellular trafﬁcking and localization of internalized HIV within DCs remain to be elucidated.

54

3. Interactions Between Dendritic Cells and Infectious Agents

DC-SIGN-mediated HIV transmission is cell type dependent and requires cell–cell contact. Functional Implication of HIV–DC Interactions The functions of DCs are downregulated in HIV infection. Compared with DCs from healthy donors, DCs derived from the peripheral blood of HIV-infected individuals at different stages of infection have signiﬁcantly reduced efﬁciency in stimulating allogeneic T cells. The expression of costimulatory antigens such as CD80 and CD86 are downregulated in DC-SIGN+ DCs in acute HIV infection. Also, HIV-infected monocyte-derived DCs are resistant to maturation in culture and may stimulate production of the regulatory cytokine IL-10 by T cells. These facts indicate that infection of DCs by HIV causes an immunosuppressive state. The results suggest that productive HIV infection of DCs undermines the direct induction of T-cell-mediated immunity. On the other hand, some studies have indicated that monocytes derived from HIVinfected individuals can efﬁciently induce CD8+ CTL responses to various antigens. In addition, some researchers have shown that there are no functional defects in cytokine production following stimulation of HIV-infected myeloid DCs and pDCs. Thus, the functional implications of HIV infection of DCs are diverse and confusing. In summary, HIV establishes chronic infection in hosts and DCs are implicated in this process. Infection and localization of HIV has been found in DCs in situ and also in culture. CD4 antigen and also different receptors and co receptors are responsible for localization of HIV in DCs. Infection of DCs by HIV causes downregulation of the antigen-presenting function of DCs, which may hamper immune surveillance and allow establishing HIV infection in infected persons. Although there are some conﬂicting data about the role of HIV on DC function, that may represent differences in experimental conditions. Taken together, it seems that HIV infection compromises the functional capacities of DCs, but DCs with efﬁcient antigenpresenting capacities are also present in these subjects. Thus, it may be possible to develop a DC-based vaccine for induction of HIV-speciﬁc CD8+ T cells using DCs from HIV patients. Interactions Between DC and Hepatitis Viruses Although there are several types of hepatitis viruses, two of them cause chronic infection, HBV and HCV. Hepatitis A virus and hepatitis E virus cause self-limiting liver disease, and infected patients develop protective antibodies. Both HBV and HCV are noncytopathic viruses. Interaction Between HBV and DCs HBV is a member of the hepadna family and can cause acute as well as chronic infection. When HBV is not eliminated from or effectively controlled in HBV-infected persons within 6 months after initial infection, these persons are regarded as chronic HBV carriers. There are about 350 to 400 million chronic HBV carriers in the world.

Consequences of Interactions Between DC and Virus

55

The clinical course of chronic HBV carriers is highly variable. Some chronic HBV carriers never develop any features of liver injury; however, they harbor the virus for their whole life. On the other hand, considerable numbers of chronic HBV carriers develop progressive liver damage and complications such as liver cirrhosis and liver cancers. Different types of antiviral drugs are used to treat patients with chronic HBV infection, and many new antiviral drugs are emerging. In most cases, antiviral drugs reduce the levels of replication of the HBV, but the response is usually transient. In addition, antiviral drugs are recommended for patients with chronic HBV infection with features of ongoing immune responses. Thus, only about 10% of patients with total chronic HBV infection are recommended for treatment with antiviral drugs. Antiviral drugs cannot eradicate the HBV from chronic HBV carriers. Only the virus replication and liver damage can be controlled in about 20%–30% of chronic HBV carriers treated by antiviral drugs. Recent studies have revealed that use of antiviral drugs in chronic HBV carriers is also related to development of mutant strains of the HBV and also breakthrough hepatitis. In this context, there is a need to develop proper immune therapy against chronic HBV infection. Studies have revealed that HBV-speciﬁc immune responses possess antiviral capacities without causing damage to host tissues. These realities have initiated a study about DC–HBV interaction to develop an immune therapy, especially DC-based therapy, against chronic HBV infection. A major problem of research with HBV is the lack of a proper animal model of HBV. In addition, HBV viron cannot be efﬁciently propagated and grown in a culture system for in vitro study. Because of these factors, HBV transgenic mice (HBV-Tg) have been prepared by injecting the full genome of the HBV and are widely used to develop insights about DC–HBV interactions. Different lines of HBV-Tg have been prepared. Some of these mice express HBV DNA, or Dane particles, all HBV-related antigens. On the other hand, others express some speciﬁc HBV-related antigens. Characterization of DCs in HBV-Tg, a Murine Model of HBV Carrier State We used a line of HBV-Tg that expressed HBV DNA in the sera and the liver. Also, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen were detected in the sera. In spite of having all HBV-related mRNAs and proteins, these mice did not show any evidence of hepatitis. The absence of hepatitis in HBV-Tg provided an added beneﬁt to understand the interaction between DCs and HBV because factors related to hepatitis did not inﬂuence the functions of DCs of this HBV-Tg. Although abundant amounts of HBsAg were detected in HBV-Tg, HBsAg-speciﬁc humoral and cellular immune responses were not detected in HBV-Tg. It was ﬁrst assumed that this was the result of immunogenic tolerance to HBsAg, as we found very speciﬁc defects of antigen presentation in HBV-Tg. A series of coculture experiments in vitro showed that the functional impairment of spleen DCs was responsible for the defective immune response of HBV-Tg to HBsAg. The impaired APC function of spleen DCs from HBV-Tg was attributable to their low expression of MHC class II antigens and CD86. Spleen DCs from HBV-Tg were also poor inducers and producers

56

3. Interactions Between Dendritic Cells and Infectious Agents

of proinﬂammatory cytokines such as interleukin (IL)-12, tumor necrosis factoralpha (TNF-α), and interferon-gamma (IFN-γ). Different types of DCs have been isolated from liver of normal as well as HBV-Tg to assess the functional capacities. DC progenitors, a highly immature form of DC, have shown immature phenotype and low T-cell stimulatory capacity compared to liver DC progenitors from control mice. In an interesting study, we also found that antigen-nonspeciﬁc maturation of liver DCs did not induce antigen-speciﬁc immunity in successive challenge with antigens. This result provided some insights about difference in activation of DCs from PAMPs stimulation and cytokine stimulation. DCs have also been isolated from liver of HBV-Tg. Recently, we also showed that liver DCs from HBV-Tg that harbor HBV in situ had decreased T-cell stimulatory capacities compared with liver DCs from control mice (Fig. 2). In the meantime, methodology was developed to enrich DCs from a precursor population from human peripheral blood. We and others enriched DCs from patents with chronic hepatitis B (CHB). Using polymerase chain reaction (PCR) and the reverse transcriptase-PCR in situ hybridization method, we showed that 30%–40% of DCs from CHB patients harbored HBV DNA and HBV RNA. Also, the T-cell stimulatory capacities of DCs from CHB patients were signiﬁcantly lower than those of normal controls and patients with nonviral chronic hepatitis. DCs from these patients also produced and induced signiﬁcantly lower levels of IL-12 in DC–T-cell cultures compared to controls. Different independent laboratories, using enriched DCs from different ethnic groups, also reported impaired function of DCs from CHB patients. Further evidence was documented regarding a contribution of DCs in the pathogenesis of CHB when a disproportionate distribution of pDCs and myeloid DCs was reported in these patients. They reported increased frequencies of plasmacytoid DCs, decreased numbers of myeloid DCs, and functional impairment of myeloid DCs from CHB patients. Increased polarization of CHB patients to a Th2 polarization by plasmacytoid DCs may be related to the inability of CHB patients to clear HBV.

Levels of blastogenesis (CPM)

Liver DCs [Normal Mouse] Liver DCs [HBV-Tg]

8×103 6×103

N=5

4×103 P < 0.05 2×103

HBsAg-specific T cells Liver DCs

2×105 1×104

2×105 5×104

Fig. 2. Signiﬁcantly low capacities of liver dendritic cells from hepatitis B virus (HBV) transgenic mice to induce proliferation of hepatitis B surface antigen (HBsAg)-speciﬁc lymphocytes

Consequences of Interactions Between DC and Virus

57

DC in Chronic HCV Infection Because of the absence of a proper animal model of HCV carrier state that expresses the full genome of HCV, we studied DC functions by transfecting spleen DCs from normal C57BL/6 mice with adenovirus vector containing the gene of HCV. Spleen DCs expressing HCV gene products showed impaired allostimulatory function and low IL12 production, indicating that HCV would also affect the function of DCs. Further study by different groups also revealed that the expression of HCV-related proteins led to impaired transmission of intracellular peptide. Moreover, the expression of MHC class I and class II antigens was distorted by transfection of DCs with HCV. Also, the functions of DCs transfected with the HCV transcript were downregulated in culture. In patients with chronic hepatitis C (CHC), the function of DCs has been studied by various groups, including ours. In most of these studies, DCs were enriched from the peripheral blood mononuclear cells by culturing with a cocktail of cytokines. The allostimulatory capacity of DCs was signiﬁcantly decreased in patients with CHC compared to controls. DCs from CHC patients produced decreased amounts of IL-12. DCs also supported the replication of HCV, as evidenced by the presence of a minus strand of HCV RNA in DCs. DCs from CHC patients could not adequately activate NK cells in response to IFN-γ stimulation, probably because of decreased expression of MICA/B on DCs. As a consequence of the recent development of cell-sorting systems and understanding about different surface antigens of circulating DCs, different investigators have shown that circulating DCs from patients with CHC harbor HCV and show impaired function. However, some reports could not detect any notable defects of DC function in patients with CHC. The controversy about DC function in CHC patients may be attributable to differences in methodology and study protocol. We found that there was a lack of study about analyses of DC functions in the same patients at different time points. In this regard, we checked DC function in ten patients harboring HCV RNA. After antiviral treatment, these patients became negative for HCV RNA and DC functions were reassessed again. As shown in Fig. 3, the capacities of DCs to produce IL-12 (Fig. 3A) and stimulation of T cells (Fig. 3B) were signiﬁcantly increased in CHC patients when they became negative for HCV RNA. Although the effect of antiviral drugs on DC function cannot be completely disregarded for increased DC function, this study indicates that HCV RNA may directly interfere with functions of DCs in CHC patients. pDCs, which are recognized in the peripheral blood from their lineage negativity, CD11c negativity, and expression of CD4 and CD123 represents a precursor population of DCs. They lack potent antigen-capturing apparatus but can directly migrate to lymphoid tissues by means of their expression of L-selectin. pDCs are able to produce abundant amounts of type 1 IFN in response to some viruses. Studies have shown that pDCs from patients with CHC produce decreased amounts of IFN compared to control subjects. Altered Function and Phenotype of DCs in the Liver of Patients with Hepatitis Several studies and information are now accumulating regarding the spleen DC in HBV-Tg and blood DC and DC precursors in patients with CHB. Also, there are

3. Interactions Between Dendritic Cells and Infectious Agents

Interleukin-12 (pg/ml)

58

200

(A)

*

100

0 DC from HCV RNA (+) subjects

DC from HCV RNA (-) subjects

(B) Counts per minute (CPM)

DC from HCV RNA (+) subjects DC from HCV RNA )(-) subjects

60000 40000 20000

`Patient 1

2

3

4

5

6

7

8

9

10

Fig. 3. Comparative assessment of functional capacities of blood dendritic cells of same patients in HCV RNA (+) and HCV RNA (−) state. Loss of HCV RNA caused increased interleukin-12 (A) and increased T-cell stimulatory capacity of blood dendritic cells (B)

several studies about blood DCs from patients with CHC. However, these DCs are not directly related to the APC function of DCs. Rather, liver resident DCs are related to antigen-processing and -presenting functions of DCs. To develop insights about DCs in the liver, various DC-related antigens have been used. CD83, a marker of mature DC, has been used to detect the localization of mature DCs in the liver from patients with CHB and CHC. CD83-positive DCs were detected in the liver specimens from patients with CHB and CHC, but, it was not possible to relate the frequencies of CD83+ DCs and clinical course of chronic hepatitis. Isolating DCs from liver specimens collected during surgery, we checked the proportions of plasmacytoid and myeloid DCs among the liver inﬁltrating DCs. The numbers of pDCs were about threefold those in the blood, indicating that these cells may be localized preferentially in the liver in chronic hepatitis. What We Have Learned from Investigations Regarding DC–Virus Interactions in Acute and Chronic Viral Infection To understand DC–virus interaction, phenotypes and functions of DCs have been studied in both acute and chronic viral infections. Studies have been done in the

Consequences of Interactions Between DC and Virus

59

mouse by transfecting those with the virus. Also, investigations have been done in transgenic animals of different chronic viral infections. Finally, phenotypes and functions of DCs have been assessed in patients with viral infections. Understanding of DC–virus interactions has provided some insights about the basic mechanisms of host defense. Study of DCs in acute viral infection has shown that the functions of DCs are upregulated in some virus infections whereas these are downregulated in others. There are several underlying factors for this. The nature of the virus may have direct impact in these experiments. The experimental design may also be relevant in these circumstances. Different types of functions of DCs have been evaluated, and different types of DCs have also been used. However, the critical factor is whether upregulation or downregulation of DC functions in acute viral infection had any relationship to the ultimate clinical outcome. At least, a direct relationship between DC function and the clinical course of acute virus infection has not been established. Inﬂuenza virus activates DCs, whereas measles virus downregulates DC functions, but infections with both these viruses are self-limiting. However, acute infection is followed by (1) immune suppression, (2) severe tissue inﬂammation and life-threatening disease, and (3) different levels of protection after recovery. It seems that DCs have an important role in these aspects. Also, DC–virus interaction during acute viral infection may shed light regarding development of prophylactic vaccines against different viruses. However, the experiments about DC in acute infection should be designed more precisely to get some important information in the clinics. The functions of DCs should be evaluated during the incubation period before apparent disease is seen. Moreover, DC function should be assessed during different points after features of diseases are detected. Another important point is about the subtypes of DCs. Proper information about DC–virus interactions can be accumulated by checking tissue DCs during acute viral infection. When it is not possible to check tissue DCs in human, circulating DCs should be assessed for functional capacities. Antigen-speciﬁc functions should be evaluated. Evaluation of nonantigen-speciﬁc functions provides little information about the role of DCs in microbial infections. Development of treatment for acute viral infection by DCs has not surfaced. However, such possibilities cannot be completely ignored in future, especially in severe cases. It is to be noted that some acute viral infections progress to chronic and persistent infections. DC-based immune intervention at the acute phase of infection may block progression to chronic disease. We have discussed interactions between DCs and two types of viruses that cause chronic infection. There are many reasons to study DC–virus interactions in chronic viral infections. On one hand, these studies provide insights about the role of immune responses in the pathogenesis of chronic virus infection. On another, it will become clear whether there is any option for immune therapy in chronic viral infections. Although infection with these viruses ultimately causes chronic infection, initially these viruses cause an acute infection. The cellular and molecular events at the initial phase of DC–virus interaction may be important for progression to chronic viral infection. Thus, there may be several patterns of viral infections. In some virus infections, all patients develop chronic and persistent infection. In others, some patients develop acute infection whereas others progress to chronic infection. The natural course of viral infection may be determined during initial interactions between the

60

3. Interactions Between Dendritic Cells and Infectious Agents

virus and the hosts. In fact, the functions of DCs have not been analyzed in these situations. The functions of DCs have been explored after chronic infection has been established in viral-infected persons. Impaired phenotypes and functions of DCs have been detected in both HIV infection and HBV and HCV infections. However, recent studies are also pointing to data contradicting the ﬁndings of these studies. Also, there is a lack of consensus about the mechanisms of dysfunction of DCs during chronic infections. Implications of DC–Virus Interactions During Development of Immune Therapy It is interesting to note that impaired functions of DCs are found in both acute as well as chronic viral infection. However, in acute viral infection, the virus is controlled and the patients recover after a self-limiting infection. On the other hand, during chronic viral infections, the virus is not controlled and tissue damage and complications develop. The authors assume that study about DC–virus interactions and development of DC-based therapy are two separate issues. Traditionally, it is said that DC-based therapy is needed in patients with chronic viral infections because the functions of DCs are impaired in these subjects. In fact, DC-based therapy is not intended to activate DCs in patients with chronic viral infections. The aim of DC-based therapy is to induce protective and surveillance types of antigen-speciﬁc immunity in these subjects. Engineering Immune Therapy Against Chronic Viral infection by DCs In all types of chronic viral infection, antiviral immunity is not adequate, and this is true for HIV-infected as well as for HBV- and HCV-infected persons. As we mentioned in the initial part of this chapter, immune surveillance against the virus is an important factor for control of virus replication. If proper immune responses are induced and maintained after initial infection, most of the virus may be effectively controlled. There may be some exceptions to this rule, especially in the context of low cytopathic viruses. Also, different virus-related factors may also have a dominant role in this context. The logic for introducing DC-based immune therapy is shown in Table 3. The main purpose of immune therapy against chronic viral carriers is to induce antiviral immune responses in these subjects because they harbor viruses, but antiviral immunity is not induced. Again, polyclonal immunomodulators fail to induce antiviral immunity in these subjects. Also, treatment of these patients by vaccine or viral antigens has not resulted in induction of adequate levels of antiviral immunity. On the other hand, the protective effect of antiviral immunity is clear in chronic viralinfected persons. Some of these patients spontaneously control the viruses, and this is accompanied by antiviral immunity. On the other hand, some of these patients respond to antiviral agents, and these patients also acquire restoration of antiviral immunity. The main purpose of DC-based immune therapy in chronic viral infections is to induce antigen-speciﬁc immune responses in the host without causing damages to host tissues. The principle of DC-based immune therapy against chronic viral infections is similar to other DC-based immune therapy. DC-based immune therapy is now usually performed in patients with cancer. However, DC-based immune therapy against

Consequences of Interactions Between DC and Virus

61

Table 3. Concept of dendritic cell-based immune therapy against chronic viral infections Immunological status of chronic viral carriers

• Presence of virus and viral particles in chronic viral-infected subjects • Absence of adequate levels of antiviral immunity • Spontaneous recovery is associated with restoration of antiviral immunity • Antiviral therapy is related to restoration of antiviral immunity • Antiviral immunity is impaired but usually not immunocompromised • Functions of DCs and other immunocytes are distorted

Type of immune therapy required

• Virus-speciﬁc immunity • Nature of antiviral immunity: depends on nature of virus • Destruction of virus-infected cells versus control of virus replication

Relevance of DC-based immune therapy

• Polyclonal immune modulators have shown limited and ﬁnite action • Mere administration of antigen usually fails to induce antiviral immunity because of long-lasting tolerogenic state • Functions of endogenous DCs are defective in most cases and thus proper APC activities of these DCs are not expected

chronic viral infections should have some characteristic features. The strategy of DCbased therapy in chronic viral infection is shown in Table 4. 1. Impaired function of DCs in chronic viral carriers is not a prerequisite for DC-based therapy. DC-based therapy is not done to activate endogenous DCs, but to induce antiviral immunity. Even if the functions of DCs are normal or exacerbated, antiviral immunity is distorted in many patients with chronic viral infections. DC-based immune intervention can be applied in these circumstances. Recent studies have shown that the functions of DCs are not impaired in chronic HCV infection. However, this does not diminish the need of DC-based immune intervention in chronic HCV infection. 2. Immune therapy against cancer is carried out to destroy all cancer cells. Also, anticancer immunity (surveillance and protective) is intended so that recurrence of the cancers can be blocked. The purpose of DC-based immune therapy against chronic viral infections is basically different. In low-cytopathic virus, there is a need to destroy virus-infected cells. However, in noncytopathic virus infection, the virus should be destroyed mainly by a noncytopathic mechanism. Noncytopathic destruction of HBV has already been proven. Accordingly, DC-based therapy should be designed on the basis of the particular viral infection. 3. The proper antigen should be selected for loading DCs in vitro to prepare antigen-pulsed DCs. Viruses have several antigens. Some of them may be immunogenic whereas others may be tolerogenic. Immunogenic antigens should be used for preparing antigen-pulsed DCs. When the nature of the antigens is not clear, it is better to perform an in vitro study to assess the antigen nature.

62

3. Interactions Between Dendritic Cells and Infectious Agents

Table 4. Strategy of dendritic cell-based immune therapy against chronic viral infections Purpose DC function of viral carriers: Target ﬁxation

Nature of antigen

In vitro characterization of antigen-pulsed DCs Route of immunization Duration of immunization Assessment of efﬁcacy

Maintenance of antiviral immunity: No practical importance May be decreased, increased, or unaltered Cytolytic destruction of virus-infected cells Noncytopathic destruction of virus-infected cells Control of virus Antigen causing protective immunity Human grade materials Virus-infected cells, viral lysates: should be avoided Conﬁrmation of immunogenicity (surface antigen and cytokine production) Capacity to activate antigen-speciﬁc T cells in vitro Based on pilot studies On the basis of data of pilot studies Induction of antigen-speciﬁc immunity in vivo Control of virus and disease activity

4. Only one type of DCs is now used for treatment of humans. However, monocytederived DCs may not be the best candidate for induction of antigen-speciﬁc immunity in vivo. A study should be done to obtain the proper types of DCs for immune therapy in chronic viral infections. 5. The major challenge is to assess the functional capacities of antigen-pulsed DCs in vitro before their administration to patients with chronic viral infections. The functional capacities of antigen-pulsed DCs should be checked before their administration to patients with chronic infection in all cases. 6. Evaluation of therapeutic efﬁcacy should be done from two aspects. The immunomodulation capacities of DC-based therapies should be evaluated. Next, the therapeutic efﬁcacy should be assessed. DC-Based Therapy Against HIV Infection Vigorous HIV-1-speciﬁc CD4+ Th1 cell responses are associated with control of viremia and long-term nonprogression in HIV-infected individuals. This approach has been supported by highly active antiretroviral therapy (HAART) in HIV patients, which has shown that antiviral therapy was associated with enhanced HIV-1-speciﬁc CD4+ Th1 cell responses. However, use of antiviral drugs for prolonged period leads to declines in HIV-1-speciﬁc CD4+ Th1 cells and CD8+ CTL. This ﬁnding indicates that there should be some mechanism to maintain antiviral immune responses in HIV-infected persons. In vitro, DCs transduced with HIV proteins, such as Nef or Gag, or simply pulsed with heat-inactivated viruses or HIV RNA, generate HIV-speciﬁc T cells. As well, DCs can process extracellular HIV antigens for presentation by MHC class I molecules in the absence of viral replication; this process, however, requires adequate HIV–surface receptor interactions and fusion of viral and cellular membranes. This “cross-presentation” pathway may have a crucial role in the activation of CD8+ CTLs during viral infections. Monocyte-derived DCs or CD34+ cells that

Consequences of Interactions Between DC and Virus

63

can be expanded and differentiated in DCs can be also be loaded with HIV proteins and used for treatment. Recently, data of clinical trials have been reported in which human DCs have been loaded with HIV-related antigens and used in patients with AIDs. In a recent study, HIV-1-loaded viable monocyte-derived DCs have been administered to HIV patients three times at an interval of 2 weeks, and these patients were followed for 1 year without antiviral therapy. Plasma viral load levels were decreased by 80% (median) during the ﬁrst 112 days following immunization. Prolonged suppression of viral load of more than 90% was seen in eight individuals for at least 1 year. Also, the DCimmunized persons developed HIV-1-speciﬁc CD4+ T cells and CD8+ effector cells. Numerous strategies are currently being evaluated to develop therapeutic vaccines for patients with HIV infections. In vivo targeting of resident DCs may be the best means to establish the best protective primary immune responses. In particular, DNA-coated particles injected into skin via gene guns and topical protein patches represent vaccine strategies aimed at exploiting the immune-stimulating potential of LCs within the skin. DC-Based Therapy Against Chronic HBV Infection Promising data have been published about the possible usage of DC-based vaccine for treatment of CHB and CHC patients, but only one or two studies have really been conducted in clinics. The utility of DC-based therapy in chronic HBV infection has come from a study in which DC-targeting therapy showed excellent data in preclinical studies. Vaccine therapy, which is aimed at induction of antigen-speciﬁc immunity, has been shown to act via activation and maturation of DCs, which has been shown in both HBV-Tg and patients with CHB. Also, a combination of antiviral and vaccine therapy caused activation of endogenous DCs. HBsAg-pulsed DCs have shown potent immunomodulatory capacities in HBV-Tg. As shown in Fig. 4, two administrations of HBsAg-pulsed DCs induced HBsAg-speciﬁc humoral (Fig. 4A) as well as cellular immune responses (Fig. 4B) in almost all HBV-Tg. Recently, HBsAg-pulsed DCs have been used in patients with CHB. In preliminary studies, it has been shown that DC-based therapies are safe, but their antiviral capacity is yet to be explored. Development of a More Potent Regimen of DC-Based Therapy for CHB Patients The following points should be considered to develop more potent regimens of DC-based therapy against chronic HBV infection. 1. DCs may be generated without using IL-4, which can be produced by culturing monocytes with GM-CSF and other immunomodulators. 2. In addition to HBsAg, hepatitis B core antigen (HBcAg) should be used to load DCs with HBV-related antigens. In future, polymerase antigen can also be used. 3. Fundamental study should be done regarding the method of preparation of antigen-pulsed DCs.

3. Interactions Between Dendritic Cells and Infectious Agents

64

(A) (B)

HBsAg-SPECIFIC HUMORAL IMMUNITY SERUM HBsAg

HBsAgpulsed DCs

HBsAg-SPECIFIC CELLULAR IMMUNITY SERUM anti-HBs

300

600

200

400

Stimulation Index

800

10

Anti-HBs [mIU/ml]

400

HBsAg [ng/ml]

1000

N=5

5

1

100

200

ONLY HBsAg

0

2

4

6

UNPULSED DCs + HBsAg

HBsAgPULSED DCs

8 [Weeks]

Fig. 4. Potent antigen-speciﬁc immune modulation of antigen-pulsed DCs. Administration of hepatitis B surface antigen (HBsAg)-speciﬁc DCs for two times induced both HBsAg-speciﬁc humoral (A) and cellular (B) immunity in HBV transgenic mice

4. The numbers of DCs should be carefully ascertained. 5. DC-based therapy should be done as part of combination therapy, usually after or with antiviral treatment. DC-Based Therapeutic Approaches in Chronic HCV Infection HCV is not eliminated from patients with chronic HCV infection because of impaired HCV-speciﬁc adaptive immunity. This ﬁnding led to the assumption that induction of adaptive immunity may be a therapeutic approach for treating patients with chronic HCV infection. Although various studies in animal models of HCV indicate that DC-based immune therapy may be effective in chronic HCV carriers, no clinical trials have been conducted to treat these patients by DC-based vaccine. DC-Based Approaches for Improved Efﬁcacy of Prophylactic Vaccines Development of prophylactic vaccine is a challenge of our time. In addition, the efﬁcacy of prophylactic vaccines needs to be augmented in some cases. DC-based intervention can be effective in both situations. We have recently shown that HBsAgpulsed DCs are capable of inducing antibody to HBsAg (anti-HBs), the protective antibody against HBV, in HB vaccine nonresponders. These vaccine nonresponders could not develop anti-HBs when immunized with HB vaccine several times. However, anti-HBs were detected after a single injection of vaccine-pulsed DCs in HB vaccine nonresponders (Fig. 5).

Consequences of Interactions Between DC and Virus

65

NONRESPONDER-1 Before NONRESPONDER-2

After

NONRESPONDER-3 NONRESPONDER-4 NONRESPONDER-5 NONRESPONDER-6

10

20

30

40

500

550

ANTI-HBs (mIU/ml)

Fig. 5. Induction of antibody to hepatitis B surface antigen (anti-HBs) caused by administration of HBsAg-pulsed DCs in nonresponders to hepatitis B vaccines. HBsAg-pulsed DCs were administered to six vaccine nonresponders who never developed detectable levels of anti-HBs because of immunization with vaccine containing HBsAg. A single administration of HBsAgpulsed DCs induced anti-HBs in vaccine nonresponders

Limitations of Ongoing Regimen of DC-Based Therapeutic Approaches Against Chronic Viral Infection and Their Solution Although DC-based therapy may be a prospective immune therapeutic strategy, its application in clinics is not easy. At present, only a few studies have been published about DC-based therapy against chronic viral infections in humans. In addition, these are pilot studies. Present insights indicate that the following points should be considered for development of more potent DC-based therapies against chronic viral infections. 1. There should be a clear, if not complete, idea about the pathogenesis of chronic viral infections before immune therapy is considered for some chronic viral infections. 2. Understanding about the nature and character of various virus-related antigens and selection of appropriate antigens for production of antigen-pulsed DCs is needed. 3. Optimization of a clinical protocol to enrich DCs in human consumable form is required. 4. Extensive in vitro studies will optimize the protocol of preparation of immunogenic antigen-pulsed DCs. 5. Checking of the immunogenicity of antigen-pulsed DCs in vitro before administration to patients with chronic viral infections is urgent. 6. A dose-escalation study is needed. 7. Clinical study should be conducted in normal volunteers and then in patients with chronic viral infections. 8. The safety of antigen-pulsed DCs must be assessed.

66

3. Interactions Between Dendritic Cells and Infectious Agents

Table 5. Limitation of translation research regarding dendritic cell-based therapy against chronic viral infection Nature of study

Factors for difﬁculties in translation study

Translation study from animal model to patients

1. MODEL: animal model of chronic infection differs signiﬁcantly from patients with chronic viral infections. 2. NATURE AND TYPES OF DCS: Spleen and bone marrow DCs are used in animal. Only human blood DCs can be used in human. 3. AMOUNTS OF DCS: As per weight basis, 1000 times more DCs are used in mice. 4. EVALUATION OF EFFICACY: Immunogenicity is mainly evaluated.

Translation studies from cancer patients to patients with chronic viral infections

1. Concept of therapy: DC-based therapy has mainly, but not all, been done in patients with cancer at their terminal state. Chronic viral carriers are apparently healthy and immune competent. 2. DC-based therapy is done to destroy cancer-infected cells, but the purpose of therapy is different in chronic viral infections. 3. Little is known about cancer antigen, and several crude products are used for pulsing. 4. The functional capacities of DCs are rarely examined.

Historically, DC-based therapy has been done in animal models of human diseases ﬁrst. Then, this was done in cancer patients. The therapeutic efﬁcacy of DC-based therapy in cancers is not satisfactory. However, several clinical trials are now proceeding, and better regimens of therapeutic approaches are being developed. In the meantime, DC-based therapy has been done in animal models of chronic viral infections. The outcome of these animal studies shows that DC-based therapy may be a potent therapeutic approach for patients with chronic viral infections. However, there are several difﬁculties in translating the outcome of an animal study to patients with chronic viral infections (Table 5). Optimism About DC-Based Therapy in Chronic Viral Infections: DC-based therapy has just been started in patients with chronic viral infections. Animal studies have shown that the basic principle of DC-based therapy is scientiﬁcally valid, and it may be effective. DC-based therapy may be effective in patients with chronic viral infections (Table 6). The therapeutic efﬁcacy of DC-based therapy may be even better in chronic viral infections than in patients with cancers. Major improvements of application strategy are possible. More viral-related antigens can be used. Numbers of DCs can be isolated by advanced technique and administered. As the safety of antigen-pulsed DCs have been shown in patients with chronic viral infections, better efﬁcacy is expected if clinical trials are done with modiﬁed protocols.

Interactions Between Dendritic Cells and Bacteria

67

Table 6. Optimism about dendritic cell-based therapies in patients with chronic viral infections A. Most of the patients with chronic virus infections are immunocompetent, whereas most patients with cancers are immunocompromised. B. In most viral infections, the nature of different antigens including protective antigens is known. However, little is known about tumor-associated antigens. C. The functional capacities of dendritic cells of patients with chronic infections are comparatively better than those of patients with cancers. D. More potent antigen-pulsed dendritic cells can be produced by culturing dendritic cells of chronic viral-infected persons with known viral antigens. E. For treatment of chronic viral infections, induction of noncytopathic effecter lymphocytes may have therapeutic efﬁcacy. However, cytopathic T lymphocytes are required for destruction of cancer cells.

Interactions Between Dendritic Cells and Bacteria Similar to interactions between viruses and DCs, the interaction between DC and bacteria is a regular event in the life cycle of living organisms. DCs are widely distributed in the body and present in almost all types of epithelial barriers, in parenchymal tissues, and in the blood, allowing different bacteria to encounter DCs. Direct recognition of bacteria and bacterial products is mediated by PRRs of DCs that recognize conserved microbial structures, so-called PAMPs. Although several receptors recognize microbial structures, the TLRs are the main receptors that recognize bacteria and induce immune responses. TLR recognize conserved bacteriaassociated molecules such as lipopolysaccharides, ﬂagellin, double-stranded RNA, and bacterial DNA containing CpG motifs. Engagement of the TLR induces nuclear translocation of the pro-inﬂammatory transcription factor NFκB. PRRs other than TLRs of DCs are also capable of recognizing and internalizing bacteria; these include lectin receptors such as the mannose receptor and scavenger receptors. In addition, Fc and complement receptors are used to internalize bacterial products bound to antibodies or complement. In vitro studies have shown that immature DC can internalize bacteria such as Salmonella typhimurium in a process that requires active cytoskeletal rearrangement. Splenic DC associate with Salmonella, Mycobacterium bovis, and Bacillus CalmetteGuérin within a few hours after intravenous infection. Immunoﬂuorescence microscopy has shown that Salmonella are contained within splenic DC isolated from infected mice. The full complexity of the interaction between DC and microbes is only now becoming clear. Exposure of DC to some bacterial antigens promotes the generation of a Th2 response, whereas exposure to bacteria containing lipopolysaccharides favors a Th1 response. In human studies using monocyte-derived DCs cultured with Vibrio cholerae, Th2 responses are favored, whereas Bordetella pertussis toxin or the viral mimic poly (I : C) generated Th1 responses. Taken together, interactions between DCs and bacteria are also heterogeneous and dependent on the nature of bacteria and also that of the DCs. Although antigen presentation of bacteria by DCs and other APCs is supposed to induce protective antibacterial immunity, this does not always happen, and many

68

3. Interactions Between Dendritic Cells and Infectious Agents

bacteria establish a chronic and persistent infection in the hosts by abrogating immune surveillance mechanisms of the hosts. DCs and other APCs also have a critical role in this regard. In this chapter, we do not describe the outcome of interactions between different bacteria and different subsets of DCs. In fact, there is a paucity of information regarding this outcome for most bacteria. Considering the public health importance of Mycobacterium tuberculosis, we discuss DC–M. tuberculosis interactions and their consequence. Various estimates indicate that one-third of the world’s population is infected with M. tuberculosis, but infection does not usually lead to active disease. In fact, in most cases of primary tuberculosis (TB), the individual is asymptomatic and noninfectious. This clinical latency often extends for the lifetime of the individual. Acute, active TB is seen in a small percentage of tuberculin-positive, latently infected individuals, probably as a result of the lack of initiation/maintenance of an appropriate immune response. Reactivation of latent infections can occur in response to perturbations of the immune response, thus ensuing active TB. However, in many cases of active TB, an obvious immunodeﬁciency is not found. M. tuberculosis is localized in both macrophages and DCs. As a result of infection with M. tuberculosis, macrophages undergo maturation. Also, some macrophages may remain in inactivated forms. When macrophages are activated by IFN-γ or TNFα, almost all M. tuberculosis resident within the phagosome of macrophages are killed. Infection of DC with M. tuberculosis leads to increased production of IL-12, TNF-α, IL-1, and IL-6. These cytokines play major roles in protective antimycobacterial immune responses. As noted earlier, IL-12 secreted by DC can potentiate IFN-γ and TNF-α secretion by T cells, and this in turn may serve to enhance the antimicrobial activity of macrophages to destroy invading bacilli. In addition to the production of proinﬂammatory cytokines, mycobacterial infection of DC is also associated with the secretion of IL-10, which may inhibit the cellular response to mycobacteria through the downregulation of IL-12 secretion. This inhibition may serve to limit the extent of DC and macrophage activation and thus regulate the potentially damaging immune response that occurs in tissues in vivo. Similar to nonactivated macrophages, DC may provide an environment within which M. tuberculosis can survive and replicate. However, DCs allow low levels of replication of M. tuberculosis. The low levels of turnover of M. tuberculosis within DC are not enough to kill the host cell. However, slow replication of M. tuberculosis in DCs is reﬂected by constant availability of antigens for presentation to T cells that will therefore potentiate the immune response. DC-SIGN is a type II transmembrane protein that belongs to the C-type lectin family, and it is expressed by DC derived from monocytes cultured with GM-CSF and IL-4, dermal DC, and interstitial DC in the lungs, intestine, rectum, cervix, and placenta, as well as in lymph nodes. The interaction of M. tuberculosis with DC-SIGN has been reported as one of the major examples of how a pathogen can subvert the function of DC through the interaction with a single receptor. In fact, it has been proposed that an indirect sign of immunosuppression caused by M. tuberculosis through DC-SIGN binding and signaling is represented by the increased secretion of the immunosuppressive cytokine IL-10 associated with a decrease secretion of IL-12.

Interactions Between Dendritic Cells and Bacteria

69

The cytokines for activation of macrophages may be induced by DCs. However, activation of DCs by cytokines does not lead to killing of bacteria. Following stimulation with IFN-γ, DC are able to control the replication of M. tuberculosis, but they are not killed by the DC. Instead, they appear to reside in vacuoles separated from the normal recycling pathway. DC may therefore be a reservoir for M. tuberculosis in vivo, particularly within lymph nodes to which they have migrated following the initial response to M. tuberculosis infection. The implication of these observations is that uptake of bacteria by the different APCs would inﬂuence the outcome of infection. Thus, uptake by DC could result in the transport of bacteria to lymph nodes. Survival and/or replication of mycobacteria within the DC is likely to induce T-cell activation but may also lead eventually to granuloma formation and persistence of infection, which would potentially contribute to disease pathogenesis. In contrast, uptake by macrophages may lead to lower T-cell-based immune responses and a failure to control infection. Alternatively, the mycobacteria may be killed, leading to disease resolution. There is no attempt, at least at present, to treat patients with chronic bacterial infections by DC-based therapy. One of the main underlying factors is related to availability of different potent antibacterial agents. Moreover, the role of immune responses during bacterial infections is not also evident. However, the incidence of antibiotic-resistant bacteria is rising and represents a major public health problem. Further study will unveil whether immune therapy will be a practical therapeutic option for bacteria-infected patients. Interactions Between Dendritic Cells and Parasites Dendritic cells are localized in almost all tissues of the body and encounter different types of endogenous and exogenous agents. Parasites are present in all living organisms, including human beings. DCs express different types of receptors that can recognize different types of agents. The expression of PAMPs in parasites has not been well characterized, and little is known about immune responses against parasites and their clinical implications. However, studies have shown DC harbors different types of parasites. These parasites enter directly into DCs or enter DCs after infecting other cells of the blood. Malaria The malaria parasite (Plasmodium falciparum) invades the human body by a mosquito. The parasites are mainly replicated in the liver and then released to blood. In the circulation, these parasites infect red blood corpuscles (RBCs). RBCs infected with malarial parasites bind to vascular epithelial cells, platelets, uninfected RBCs, macrophages, and myeloid DCs. P. falciparum malaria is characterized by the poor induction of long-lasting protective immune response. Infected RBCs bind to DCs via CD36, CD51 on DCs. DCs harboring malarial sites are resistant to maturation. Also, they are poor activators of T cells. Malarial parasiteinfected DCs also show low production of IL-12 and enhanced production of IL-10. Taken together, intact malaria-infected RBCs adhere to DCs, inhibit the maturation of DC, and subsequently reduce their capacity to stimulate T cells.

70

3. Interactions Between Dendritic Cells and Infectious Agents

The percentage of HLA-DR+ peripheral blood DCs was signiﬁcantly reduced in children with malaria. These data suggest that a proportion of peripheral blood DCs may be functionally impaired by the low expression of HLA-DR on their surface. Leishmania Leishmaniasis, a vector-borne parasitic disease, is transmitted during a sandy blood meal as the parasite is delivered into the dermis. It infects macrophages and DCs. DC-SIGN expressed on DC functions as a receptor for Leishmania amastigotes. The blocking monoclonal antibody dramatically reduces internalization of Leishmania amastigotes by immature DCs. DCs take up Leishmania major and, after this internalization, undergo changes in surface phenotype suggesting maturation. Infected DCs produce IL-12, which is important to induce the Th1 type immune response. This IL-12 production depends on interaction between CD40 and CD40 ligand. The activation of Th1 followed by IL-12 production by DCs is important to eliminate Leishmania. However, in Leishmania amazonensis infection in easy infectable mice (BALB/c mice), DC produce IL-4 rather than IL-12 and induce a Th2 type response. These data suggest that Leishmania amazonensis amastigotes may condition DCs of a susceptible host to a state that favors activation of pathogenic CD4+ T cells. DCs from mice with chronic Leishmania donovani infection fail to migrate from the marginal zone to the periarteriolar region of the spleen. Defective localization was attributed to TNF-α-dependent, IL-10-mediated inhibition of CCR7 expression. The defective DC migration plays a major role in the pathogenesis of leishmaniasis, and the immunosuppression is mediated, at least in part, through the spatial segregation of DCs and T cells. Toxoplasma Toxoplasma gondii is an obligate intracellular parasite. It infects a wide variety of nucleated cells of intermediate hosts. IFN-γ activation inhibits the replication of Toxoplasma gondii in DCs. DCs may act as effector cells in an early defense mechanism. Induction of chemokines by invading Toxoplasma gondii is important not only for the recruitment of DCs but also for the determination of their subsequent immunological function. For activation of DC, TLR is suggested to have a role. The effect of TLR-4, TLR-2, and MyD88 in Toxoplasma gondii-infected wild-type mice and deﬁcient mice have been checked. MyD88-deﬁcient mice died, whereas TLR-4- and TLR-2-deﬁcient mice survived after intraperitoneal infection of Toxoplasma gondii. In MyD88-deﬁcient mice, high levels of Toxoplasma gondii load were observed in the brain, liver, and other organs. Thus, studies have indicated that interaction between parasites and DCs leads to different types of immune responses. In some cases, the immune responses are diminished, and in others, response is exacerbated. However, detailed study about DC–parasite interactions has not been conducted. Also, little is known regarding the possible use of DCs in the context of parasite infection in humans in clinics.

Recommended Readings

71

Recommended Readings Andrieu JM, Lu W (2007) A dendritic cell-based vaccine for treating HIV infection: background and preliminary results. J Intern Med 261:123–131 Kawai T, Akira S. Antiviral signaling through pattern recognition receptors (2007) J Biochem (Tokyo) 141:137–145 Kaiserlian D, Dubois B (2001) Dendritic cells and viral immunity; friends or foes. Semin Immunol 13:303–310 Kis Z, Pallinger E, Endresz V, Burian K, Jelinek I, Gonczol E, Valyi-Nagy I (2004) The interactions between human dendritic cells and microbes: possible clinical applications of dendritic cells. Inﬂamm Res 53:413–423 Lu W, Arraes LC, Ferreira WT, Andrieu JM (2004) Therapeutic dendritic-cell vaccine for chronic HIV-1 infection. Nat Med 10:1359–1365 Nina Bhardwaj (1997) Interactions of viruses with dendritic cells: a double-edged sword. J Exp Med 186:795–796 Malmgaard L (2005) Dendritic cells, toll-like receptors, and T-cell responses: lessons from viral infections in vivo. Viral Immunol 18:584–594 Pasare C, Medzhitov R (2004) Toll-like receptors: linking innate and adaptive immunity. Microbes Infect 6:1382–1387 Piguet V, Steinman RM (2007) The interaction of HIV with dendritic cells: outcomes and pathways. Trends Immunol 28:503–510 Pohl C, Shishkova J, Schneider-Schaulies S (2007) Viruses and dendritic cells: enemy mine. Cell Microbiol 9:279–289 Pollara G, Kwan A, Newton PJ, Handley ME, Chain BM, Katz DR (2005) Dendritic cells in viral pathogenesis: protective or defective? Int J Exp Pathol 86:187–204 Pulendran B, Palucka K, Banchereau J (2001) Sensing pathogens and tuning immune responses. Science 293:253–256 Schneider-Schaules S, Meulen VT (2002) Triggering of an interference with immune activation: interactions of measles virus with monocytes and dendritic cells. Viral Immunol 15:417–428 Sewel AK, Price DA (2001) Dendritic cells and transmission of HIV-1. Trends Immunol 22:173–175 Smits HH, de Jong EC, Wierenga EA, Kapsenberg ML (2005) Different faces of regulatory DCs in homeostasis and immunity. Trends Immunol 26:123–129 van Kooyk Y, Engering A, Lekkerkerker AN, Ludwig IS, Geijtenbeek TB (2004) Pathogens use carbohydrates to escape immunity induced by dendritic cells. Curr Opin Immunol 16:488–493 Virgin HW (2005) Immune regulation of viral infection and vice versa. Immunol Res 32:293–315 Wu L, Kewal, Ramani VN (2006) Dendritic-cell interactions with HIV: infection and viral dissemination. Nat Rev Immunol 6:859–868 Yewdell JW, Hill AB (2002) Viral interference with antigen presentation. Nat Immunol 3:1019–1025

4. Dendritic Cells and Allergy

Nature of Immune Responses and Allergic Diseases Two distinct and highly sophisticated defense mechanisms have been developed in vertebrates for survival in hostile environments. One of them is the innate immune system, which is aimed to react rapidly (from within minutes to a few hours) and in a simple way to attack pathogens or dangerous elements in the body. Cells of the innate immune systems are capable of directly killing the target cells, or they may produce different types of cytokines and immune modulators to handle the pathogens. Innate immunity provides many essential immunological signals for induction of adaptive or acquired immunity. The acquired immune system has been evolved to provide a more adaptive and highly speciﬁc defense response to microbial agents, antigens, transformed cells, tumor cells, and substances, which are non-self, are dangerous to the host, and to which innate immunity is induced. Another fundamental property of the normal immune repertoire is to possess immunocytes that can block uncontrolled immune responses. The adaptive immune system also possesses the unique ability to induce tolerance of self-structures and possibly to nondanger elements. Various cells with tolerogenic properties and different cytokines take part during tolerance induction. There is plasticity among the cells of the immune system. It is interesting that a particular type of cell can induce both immune responses and immune tolerances, based on the nature of the invading agents, nature of participating cells, time of exposure of antigens, and tissue microenvironment. To maintain the normal homeostasis of the body, the affector and regulatory wings of different cells of the immune system interact in a coordinated way so that the magnitude and nature of the effector immune response become purposeful and functional in the context of a particular stimulus. In spite of the highly regulated immune system that vertebrates have, some susceptible individuals respond in an inappropriate manner to certain stimuli. These agents may come from the environment, foods, drugs, and unknown sources, and are generally regarded as allergens. Development of allergic manifestations following exposure to allergens is dependent on the personal characteristics of the individuals, including their genetic background. Allergic diseases are increasing in prevalence and constitute a major source of disability throughout the developed world. Although allergic manifestations are also substantial in the developing countries of the world, most of these remain unreported. 73

74

4. Dendritic Cells and Allergy

Any type of substance may cause an allergic reaction in a particular individual. Many people of the world exhibit allergic manifestations to different types of environmental factors (dusts, debris, dead and living insects) and also to different types of foods and drugs. The purpose of this chapter is to provide insights about the role of dendritic cells (DCs), the most antigen-presenting cells (APCs), in allergy. Recent studies have revealed that DCs may have a role in the pathogenesis of certain allergic manifestations, such as atopic (a term usually used interchangeably with allergy) asthma of the lung, atopic dermatitis (allergic eczema) in the skin, perennial and seasonal rhinitis (hay fever) in the upper airways, and allergic conjunctivitis in the eye. Allergic diseases show moderate to severe type of clinical manifestations, but at its most acute form and in some individuals, the allergic response manifests itself as the life-threatening systemic condition of anaphylaxis.

Pathogenesis of Allergic Reactions The allergic reactions may be divided into early-phase immediate type 1 hypersensitivity response and late-phase response. Patients with allergic diseases exhibit hypersensitive responses to different allergens and also tissue inﬂammations. Immunoglobulin E (IgE) plays a critical role in disease pathogenesis, especially in the pathogenesis of hypersensitive responses. Elevated levels of allergen-speciﬁc IgE are detected in allergic patients as a consequence of exposure with allergens. In individuals who are previously sensitized to an allergen, allergen-speciﬁc IgE antibodies bind to high-afﬁnity IgE receptors on the surface of different cells, mainly mast cells, in target tissues. These reactions are regarded as an early-phase response that causes degranulation and release of different mediators such as histamine, tryptase, and prostaglandins. Also, increased vascular permeability, local edema, and itching are detected in these patients. The immediate type 1 hypersensitivity response is frequently followed by a late-phase reaction. During late-phase response, eosinophil and CD4+ T lymphocytes inﬁltrate to the site of allergic responses. The late-phase response usually follows an early-phase response; however, an early-phase response is not essential for the late-phase response in all circumstances. Even in the absence of IgE sensitization and an early-phase response, allergen-speciﬁc T cells may be formed and elicit a direct late-phase response. Allergen-speciﬁc T-lymphocyte-mediated immune responses play a key role during delayed type IV cell-mediated hypersensitive response. The development and progression of allergic processes is dependent on tissue microenvironments that harbor the allergens. Even in the absence of recent allergen provocation, chronic inﬂammation is seen in the target organs of patients with allergic diseases. Eosinophils and CD4+ T lymphocytes are mainly responsible for tissue damage in allergic patients. Activated eosinophils contribute to disease chronicity by damaging epithelial cells and epidermal barriers and thus making allergic patients more vulnerable to environmental antigens. Although eosinophils have been regarded as the major cells that induce tissue assault during allergy, recent studies have indicated that allergen-speciﬁc CD4+ T lymphocytes are mainly responsible for tissue assaults of chronic allergic manifestations. The allergen-speciﬁc CD4+ T lymphocytes are able to maintain the chronic inﬂammation in allergic individuals even in the absence of fresh allergen.

Dendritic Cells in Allergy

75

Some characteristics features of inﬁltrating CD4+ cells in allergic diseases have been assessed. First, cloning experiments have revealed that these CD4+ T cells are skewed to the Th2 phenotypes. In humans, Th2 lymphocytes produce cytokines such as interleukin (IL)-4, IL-5, and IL-13, and these cytokines are responsible for much of the pathophysiology of allergic diseases. The involvement of a soluble mediator such as IL-4 has provided important insights about the pathogenesis of allergic diseases. IL-4 is not only produced by Th2 cells but also promotes differentiation of Th2 cells. Thus, IL-4 has a role in the initiation and also progression of allergic manifestations. Also, IL-4 can induce switching human B-cell immunoglobulin isotype production to IgE. Furthermore, IL-4, along with IL-13, acts as a growth factor for mast cell and leads to upregulation of the expression of vascular cell adhesion molecules on endothelial cells. Taken together, allergic manifestations are seen in genetically susceptible individuals in response to allergens. Many types of agents may act as allergens in susceptible individuals. Although more insight is needed regarding the pathogenesis of allergic diseases, it seems that histamine and other unknown allergy-related mediators are released from mast cells and play a dominant role in the imitation of allergic processes. However, the progression of allergic diseases may largely be inﬂuenced by immunocytes, such as allergen-speciﬁc Th2 cells. Also, cytokines produced by Th2 cells, such as IL-4, play an important role during progression of the allergic process.

Dendritic Cells in Allergy Roles of DCs in allergy are predicted by certain characteristic features of DCs, which include (1) the expression of the alpha chain of high-afﬁnity IgE receptor (FcεR1) on DCs, (2) the capacity of DCs to induce allergen-speciﬁc T and B lymphocytes and memory lymphocytes, and (3) the ability of DCs to induce Th2 polarization (Fig. 1). Implication of FcεR1 in Human DCs in Allergy Production of IgE is a common feature of most allergic diseases. A relationship between DCs and human allergic manifestations can be predicted because human DCs express FcεR1. FcεR1 is a member of the antigen receptor superfamily and is a tetrameric complex comprising two chains. The role of FcεR1 seems to be most relevant in humans because FcεR1 is conﬁned to the surface of the mast cells in rodents. However, in humans, the mRNA of FcεR1 is detected in the Langerhans’ cells (LCs) and circulating DCs. The exact role of FcεR1 in DCs has not been properly elucidated. However, DCs are professional APCs and are endowed with capacities to recognize, capture, process, and present antigens and allergens. FcεR1 may have a dominant role during capture of allergens. DCs utilize phagocytosis, ﬂuid-phase pinocytosis, and cell-surface receptor endocytosis for capture of antigens. Of these, cell-surface receptor-mediated endocytosis represents the most efﬁcient and speciﬁc pathway of antigen capture. As human DCs express FcεR1, this allows DCs to react with allergens by binding large amounts of IgE molecules with various speciﬁcities. The IgE–FcεR1 complexes would allow the capture of large allergens. Thus, huge amounts of allergens may be engulfed in DCs. In the absence of such

76

4. Dendritic Cells and Allergy

Involvement of DCs during allergic manifestations 1. 2.

Expression of FceR1 on human DCs

3. 4.

1.

Induction of allergenspecific memory T lymphocytes

TH2 polarization by DCs

2.

Increased IgE in allergic patients. FceR1 of DCs and IgE of allergic patients for FCeR1/IgE complex IgE/FCeR1 can engulf large amounts of allergens. DCs expressing FCeR1 are capable of induction of TH2 immunity

DCs are abundant at the site of entry of allergens, such as skin and nasal mucosa. DCs are capable of inducing allergenspecific T cells, B cells and memory lymphocytes.

DC/allergen encounters favor a T helper 2 type of polarization: 1. Low amounts of allergens 2. Low affinity of allergens 3. Prolonged TCR engagement 4. Cytokines 5. Thymic stromal lymphopoietin

Fig. 1. Dendritic cells (DCs) may be involved with initiation and progression of allergic manifestations. TCR, T-cell receptor

antigen-engulﬁng mechanisms, it may difﬁcult for DCs to endocytose huge amounts of allergens by the usual pathways. Aggregation of FcεR1 on DCs is followed by its internalization via receptor-mediated endocytosis. Internalization of large amounts of allergens allows DCs to induce the Th2 immune responses that are required for allergic manifestations.

Allergen-Speciﬁc Immune Responses and Memory Lymphocyte Formation by DCs After the entry of allergens, they are localized at certain tissues and induce the production of histamine and other substances from mast cells and eosinophils. These substances induce manifestations of allergic diseases. The tissue inﬂammation and damage may also be induced by early hypersensitive reactions. In the next phases, allergen-speciﬁc lymphocytes are formed. DCs are essential during the formation of these lymphocytes at the lymphoid tissues. The sites affected by allergic diseases typically lie at the interface between the body and the external environment. Huge numbers of DCs are available at these sites of the body. DCs are equipped with different antigen recognition apparatuses, which can recognizes microbes, transformed cells (apoptotic or tumor-like cells), and antigens by the pattern-recognition receptors (PRRs) of DCs. Apart from receptor-mediated recognition of microbial agents, antigens, or transformed cells, DCs can sense the alterations of the mucosal milieu. However, it is unclear whether DCs use any special types of recognition molecules to sense the

Allergen-Speciﬁc Immune Responses and Memory Lymphocyte Formation by DCs

77

presence of allergens. In the context of allergy, DCs may capture and internalize these agents by highly specialized systems such as macropinocytosis, receptor-mediated endocytosis, and phagocytosis. The tissue microenvironment of the primary hypersensitive state can facilitate the antigen recognition and capture of allergens by DCs. DCs process the allergens in their endosomal compartments; however, the outcome of allergen processing by DC is largely inﬂuenced by the nature of the allergens. The local tissue microenvironment and presensitization with allergens will be decisive factors in this context. However, these points are yet to be addressed in experimental systems. To induce an allergen-speciﬁc immune response, DCs would cleave the antigens into antigenic peptides, which would be expressed on their surface along with self-major histocompatibility complex (self-MHC) molecules. In the next phase, DCs expressing the antigenic epitopes of the allergen along with self-MHC molecules migrate to secondary lymphoid tissues for the induction of allergen-speciﬁc immune responses. This scenario of antigen presentation by DCs is a common feature of DCs. Different subtypes of DCs participate during adaptive immune responses. The role of DCs during antigen presentation by various subsets of DCs has not been well dissected. However, experimental evidence supports the proposed scenario of presentation of allergen in allergic diseases. In the skin, LC have been shown to uptake, process, and transport allergens to the local draining lymph nodes where they present the allergens to naïve T cells, indicating a dominant role of DCs in the primary allergen-speciﬁc immune response. Allergen-speciﬁc B cells are also produced as a result of presentation of allergens by DCs. Once allergen-speciﬁc helper T cells are formed in the lymphoid tissues, they interact with clonally selected B cells at the immunological synapse of lymphoid tissues to produce allergen-speciﬁc B cells. In successive steps, mobilization of allergen-speciﬁc CD4+ T lymphocytes and B cells would be under the control of sets of adhesion molecules and chemotactic gradients. When these cells are localized in the tissues where allergens are deposited, these cells perform their effector function, and also some cells can undergo a memory state. In this context, there are some differences between microbe-speciﬁc memorytype lymphocytes and allergen-speciﬁc memory lymphocytes. In the case of allergic diseases, allergens enter the body on a routine basis. However, that may not be the case in most microbial infections. Thus, allergen-speciﬁc memory lymphocytes may be persistently activated by allergens. Also, there is a chance that, as a consequence of constant exposure to allergens, the allergen-speciﬁc lymphocytes may exhibit hypo-responsiveness. It is questionable whether DCs are essential for the activation of allergen-speciﬁc memory T cells or whether other APCs can perform this action. B cells and macrophages and many other cells including epithelial cells possess APC functions. Theoretically, all types of antigen-presenting cells are able to present allergens to allergen-speciﬁc T cells and B cells; however, only DCs are endowed with all types of functions of antigen presentation. Other cells may be described as compromised APCs. For example, B cells are rare at the sites of entry and localization of allergens. Thus, their role is limited in the initiation and propagation of allergic manifestations. Epithelial cells can express MHC class II molecules, but they do not show CD40 and molecules involved in T-cell activation. Macrophages can maintain sustained T-cell responses; however, alveolar macrophages and interstitial macrophages that are important for handling of allergens usually fail to support T-cell proliferation in

78

4. Dendritic Cells and Allergy

an antigen-speciﬁc manner, although they can support cytokine production. In contrast, macrophages may downregulate the allergic manifestation by producing nitric oxide (NO), because NO can inhibit many DC functions and some macrophages such as pulmonary macrophages suppress T-cell function. Thus, it appears that presentation of allergens by DCs may be essential for the stimulation of allergen-speciﬁc lymphocytes and for the maintenance of chronic allergic conditions. Experimental evidence provides support for this because depletion of DCs from antigen-sensitized mice failed to develop eosinophilic inﬁltration after rechallenge with the original antigen. However, experimental evidence regarding the role of DCs in allergy is scarce. Because of ethical concerns, such studies cannot be conducted in humans. However, a role of DCs in human allergy has been shown. CD1a+ DCs isolated from bronchoalveolar lavage activate antigen-speciﬁc Th2 lymphocytes. Moreover, DCs are also abundant at the interface of body surfaces that are susceptible to meet with allergens. The intraepithelial DCs are highly sensitive to the action of inﬂammatory stimuli. Allergens and other stimuli induce expression of chemokines and might aid to the mobilization of DCs.

Th2 Polarization Capacities of Both Myeloid and Plasmacytoid DCs It is generally assumed that myeloid DCs support Th1 polarization whereas plasmacytoid DCs cause Th2 polarization. However, this not a rule, and both myeloid and plasmacytoid DCs can induce Th2 polarization in the context of allergic manifestations. Most of the DCs at the vicinity of the entry and localization of allergens are conventional myeloid DCs. Accordingly, it is of utmost importance to assess the mechanism of Th2 polarization by myeloid DCs in the context of allergy. Low Dose of Antigen. The dose of antigen is an important factor regarding the nature of Th polarization by DCs. Large amounts of antigens usually induce Th1 polarization whereas low doses of antigens cause Th2 polarization. In the context of allergy, the amount of environmental allergens is usually low, and thus it is logical to assume that a low dose of allergen might induce Th2 polarization. However, this idea cannot be applied to food allergens, when the dose of the antigens is usually very high. Low Afﬁnity of Antigen. Low afﬁnity of antigens induce Th2 polarization, but very little information is available regarding the comparative afﬁnity of allergens and other antigens that induce Th1 or Th2 types of immune responses. Prolonged T-Cell Receptor (TCR) Engagement. The duration of engagement of allergens with the TCR is an important factor for T-helper polarization. Prolonged TCR engagement causes a Th2 polarization. A low dose of antigen with low afﬁnity may favor a prolonged engagement with TCR. However, this phenomenon needs to be veriﬁed in the context of allergens. Role of Soluble Factors in Th2 Polarization. IL-4 is the main mediator of the Th2 response in humans. During primary allergic sensitization, NK cells may be a source of IL-4. In established allergic inﬂammation, mast cells, eosinophils, and Th2 cells can produce IL-4. IL-4 in the tissue microenvironment may act on DCs to induce Th2

Dendritic Cells and Drug Allergy

79

polarization. Among DC-derived cytokines, IL-6 may induce Th2 differentiation. In addition, IL-10 by inducing IL-4 may also potentiate a Th2 polarization. Moreover, DC-derived chemokines such as monocyte chemotactic protein (MCP)-1 could induce Th2 polarization in vitro. IL-12 is another important cytokine that mediates the polarization of T cells. Initially, it was assumed that IL-12 induces Th1 polarization, but now experimental data have indicated that the amount of IL-12 in the tissue microenvironment determines the nature of Th polarization. In allergic diseases, prostaglandins and IL-10 may downregulate the production of IL-12 and may favor a Th2 polarization. In addition, histamine, type 1 (IFN), and MCP 1 to -4 may also promote Th2 polarization. Thus, myeloid DCs can induce Th2 polarization in response to allergens. In addition to myeloid DCs, plasmacytoid DCs are well known for their capacity to induce Th2 polarization. Plasmacytoid DCs are negative for CD11c, but express CD4 and CD123. Plasmacytoid DCs produce huge amounts of type 1 interferon and induce T lymphocytes to differentiate into the Th2 phenotype under certain conditions. These cells induce Th2 polarization if treated in vitro with CD40L and IL-3. Recent studies have shown that plasmacytoid DCs are endowed with antigen capture and processing activity. The role of plasmacytoid DCs in allergic conditions has not been well explored. IL-3 may be derived from mast cells, which induces the maturation of plasmacytoid DCs. As mast cells are detected near allergic tissues, IL-3 derived from the mast cells may be related to the maturation of plasmacytoid DCs and polarization of T cells to the Th2 phenotype in allergic conditions. In addition to the aforementioned factors, recent studies have shown that the ability of DCs to induce Th2 responses appears to be dictated by the type of signals that the DC received at an immature stage. Thymic stromal lymphopoietin (TSLP), a epithelial cell-derived IL-7-like cytokine, regulates DCs to induce Th2 polarization. This effect has been specially seen in patients with atopic diseases. The human TSLP gene is localized in chromosome 5q22, which is close to the gene cluster encoding for all the Th2-related cytokines: IL-4, IL-5, IL-9, and IL-13. TSLP is mainly produced by epithelial cells in both mice and human beings. TSLP receptor R mRNA has been detected in myeloid DCs (mDCs) and plasmacytoid DCs. Thus, Th2 types of lymphocytes may be produced by interaction between TSLP and the TSLP receptor.

Dendritic Cells and Drug Allergy Little is known about the interaction between DCs with different drugs and their metabolites. β-Lactam is the most common agent involved in allergic reactions to drugs; it can form hapten–carrier conjugates with proteins that can be processed and presented by antigen-presenting cells to naive T lymphocytes. In this respect, a role of DC in drug-induced allergic reactions is predicted. In vitro studies have shown that lymphocytes from patients with drug-induced allergic manifestations exhibit speciﬁc lymphocyte proliferation after culture with the causative drug. Also, in vivo monitoring indicates an increase of CD4 activated cells, CD8 activated cells, or both in tissue and in peripheral blood, parallel to the evolution and the clinical manifestations of the disease. However, little is known about the initial steps mediated by DCs during drug-induced allergic manifestations. The hapten by itself is able to produce a maturation of DCs that induces speciﬁc T-cell responses. In addition, studies have shown

80

4. Dendritic Cells and Allergy

that DCs from patients with hypersensitivity reaction to amoxicillin showed phenotypic and functional maturation of DCs in vitro with amoxicillin. However, DCs from control subjects did not show such maturation as a result of culture with amoxicillin. Further study regarding interaction between dendritic cells and drug metabolites would unveil the role of DCs during drug-induced allergic manifestations.

Dendritic Cells in Animal Models of Allergy To develop insights about the role of DCs in allergic manifestations, investigators have checked DCs in animal models of allergy. Inhalation of ovalbumin (OVA) by rodents resulted in airway inﬂammation. Inﬁltration of eosinophils is detected at the site of inﬂammation. Also, DCs are localized at the sites of inﬂammation, and these DCs seem to capture inhaled ovalbumin. In the mouse model of experimental asthma, huge numbers of myeloid DCs are detected in the airway mucosa and bronchoalveolar lavage ﬂuid. These DCs may have migrated from bone marrow. Interestingly, the DCs of OVA-challenged mice had a mature phenotype, in clear contrast to DCs of naïve animals that show immature DCs. Other investigators have shown that airway DCs form clusters with primed T cells in the airway mucosa, leading to local maturation of DC function. Also, a subset of long-lived CD11b+ CD11c+ F4/80+ cells have been detected in the lungs of mice with airway inﬂammation, and this long-lived population had a prolonged capacity for presenting antigen to Th2 cells ex vivo. During antigen challenge of primed mice, there is increased migration of airway DCs to the mesenteric lymph nodes. The reason for this is unclear at present, but could involve the restimulation of resting central memory T cells, inducing their proliferation and differentiation to effector T cells that go back to the effector site and control allergic inﬂammation. Moreover, even during secondary immune responses to inhaled allergen, there might be activation of naïve antigen-speciﬁc T cells. Recent studies have shown that administration of OVA-pulsed myeloid DCs to the airways of naïve mice and rats induces sensitization to OVA, leading to a vigorous Th2 response and eosinophilic airway inﬂammation, goblet cell hyperplasia, and bronchial hyperreactivity after rechallenge of the airways with OVA aerosol. OVApulsed DCs are also capable of inducing the full asthmatic phenotype when injected intratracheally in primed mice, in the absence of antigen aerosol. In addition, eosinophilic airway inﬂammation could also be induced by repetitive injection of DCs not exposed to allergens. This ﬁnding indicates that intrinsic asthma is caused by DCs presenting some form of self-antigen, although the nature and properties of self-antigens have not been elucidated.

Removal of Airway DCs from Sensitized Mice Eliminates Asthmatic Features Induced by Antigen Aerosol A role of DCs in allergic airway inﬂammation is also predicted because if DCs are depleted from mice, then these mice do not show features of allergic diseases. Investigators have shown that CD11c-diphtheria toxin receptor transgenic mice, in which

Dendritic Cells in Human Allergy

81

airway DCs can be deleted conditionally through airway administration of diphtheria toxin, without affecting systemic DCs. Selective removal of airway CD11c+ cells completely eliminated airway eosinophilia, goblet cell hyperplasia, and bronchial hyperreactivity to metacholine concomitant with decreased Th2 effector generation. If we critically analyze these cellular events, some important information about the role of DCs in allergic conditions is exposed. Primary exposure of OVA via the respiratory route is a tolerogenic event, especially if OVA is presented by immature DCs and if there is no inﬂammatory microenvironment. However, when endogenous airway DCs are activated by transgenic expression of GM-CSF or by concomitant viral infection with inﬂuenza virus, tolerance is avoided and eosinophilic airway inﬂammation is ensured. From these models, it appears that DCs are responsible for sensitization to inhaled allergens, but, there is a need of danger signals for initiation and progression of allergic manifestations. The danger signals may come from eosinophils. More importantly, DCs are also attracted from the bone marrow and the bloodstream into sites of eosinophilic airway inﬂammation during the secondary immune response to ovalbumin. Moreover, DCs ensure the maintenance of eosinophilic airway inﬂammation and IgE synthesis in sensitized mice.

Dendritic Cells in Human Allergy Several studies in animal models of allergic diseases have shown that similar events may also happen in humans (Table 1). Human DCs may also be implicated in Th2 sensitization to common allergens in genetically predisposed individuals. To trace the basic mechanism of primary sensitization, we need to look at the immunological events during the neonatal period and early infancy. In these periods, DCs are functionally immature, and this period may be regarded as the “the peak period of sensitization” to allergens; this may explain the increased occurrence of allergic diseases in Western countries with improved socioeconomic status, which favors exposure to a low dose of allergens. Presentation of low levels of allergens to human DCs may cause decreased production of IL-12. These DCs would induce polarization to the Th2 type of immune response. Moreover, prolonged TCR ligation, which may be a common event in case of low dose of allergen is supposed to favor a Th2 polarization. Although these conceptions could not be well supported by human studies, at least one study favors this idea. Induction of novel Th2-dependent sensitization to bovine serum albumin was seen after repeated injection of DCs when bovine serum albumin was used in autologous DC preparation; this led to antibovine albumin IgE and anaphyTable 1. Dendritic cells (DCs) in human allergies 1. Increased localization of CD1a+ DCs in the lamina propria and epithelium of asthmatic patients. 2. Increased numbers of DCs in allergic rhinitis and atopic dermatitis 3. Th2 polarization of DCs in allergic condition. 4. Intervention studies by steroid reduce DC and improve allergic manifestations. 5. In patients with atopic dermatitis, the expression of IgE receptor is upregulated. 6. The frequencies of plasmacytoid DCs are increased in blood and decreased in skin in atopic dermatitis.

82

4. Dendritic Cells and Allergy

lactic reactions in human. To explore the role of DCs in human allergic diseases, we now discuss three common human allergic diseases.

Allergic Asthma Asthma is a chronic inﬂammatory disorder of the airways, characterized by the clinicopathological symptoms of intermittent and reversible airway obstruction, enhanced mucous production, chronic eosinophilic inﬂammation, and bronchial smooth muscle cell hypertrophy. These pathological changes cause impaired epithelial repair and airway remodeling. Patients with asthma suffer from both acute and chronic phases of the diseases. These two phases differ regarding the nature of the pathological lesions. In the acute phase, histamine is released from the mast cells of the airway. In the chronic phase, the inﬂammatory inﬁltrates in the airway mucosa and the alteration of mucosal milieu inﬂuence the pathological processes, which ultimately lead to permanent injury of the airway mucosa. In allergic asthma, IL-4 and IL-13 induce the expression of cell adhesion molecules on inﬂamed endothelium and epithelial production of chemokines, leading to the recruitment of inﬂammatory cells, stimulating the production of IgE by B cells and causing bronchial hyperreactivity. In asthma, both elevated IgE and Th2-type immune response are detected. It is already noted that the high rate of asthma in Western populations may be related to their higher level of hygienic standards. Low amounts of allergen and presence of fewer pathogens in the environment may endow the immune system to a Th2 response. Patients with asthma produce less IL-12 from peripheral blood mononuclear cells. Some common allergens, such as house dust mites and pollen allergens, contain substances that clearly polarize DC function toward Th2 induction. In this situation, myeloid DCs induce a Th2 type of immune responses. IL-23, a cytokine, produced by DCs also induces Th2 polarization; this is an IL-12-independent phenomenon. In patients with allergic asthma, CD1a+ DCs accumulate in the lamina propria and epithelium of steroid-naïve asthmatic patients, and that allergen challenge increases the number of myeloid DCs even further. Intervention studies with inhaled steroids in patients with asthma or allergic rhinitis have shown a correlation between the reduction in number and function of airway DCs and clinical efﬁcacy. The next important question is how the allergens gain access to the airway epithelium, which may be attained by increased transepithelial permeability in asthma. In fact, the bronchial epithelium becomes increasingly permeable to macromolecules after allergen deposition. In addition, allergen exposure induces asthmatic epithelial cells to express GM-CSF, which attracts DCs to the site of antigen contact. Studies have now shown that a putative MHC class II complex bearing DC precursors is accumulated in the airway epithelium after exposure to allergens, which reaches its maximum within 1 h after antigen exposure. After encountering allergens, DCs change their shape, and active DC surveillance within the epithelium is ampliﬁed and consequently results in an increase in the trafﬁc of these cells from the epithelium to the lymph nodes. For ampliﬁcation of an asthmatic attack, rechallenge with the allergens would lead to recruitment of new DCs from monocytes or other blood precursors. These monocyte-derived DCs from allergic asthma patients show phenotypic

Atopic Dermatitis

83

differences in the expression of HLA-DR, CD 11b, and the high-afﬁnity receptor for IgE, and even an upregulation of CD86, and develop into more potent accessory cells than those from normal subjects. Although airway DCs are critical in priming the immune system to inhaled allergens, other APC subsets may play a crucial role in the secondary immune response to “known” allergens. In this way, they may contribute to the chronicity of asthma.

Atopic Dermatitis (AD) AD represents a chronic, relapsing inﬂammatory skin disease with characteristic clinical features. Genetic background; environmental exposures such as food allergens, aeroallergens, microbial antigens, or stress; and distinct immunological predispositions all contribute to the development of recurrent, itchy eczematous skin lesions in some patients. It is assumed that allergens from the environment elicit or aggravate eczematous skin lesions; however, the exact pathophysiological pathway underlying this phenomenon is unclear. The expression of FcεR1 on LC and dermal DCs in the skin supports a role for DCs in the pathogenesis of AD. This idea is strongly supported by the observation that the presence of FcεR1-expressing LC bearing IgE molecules is a prerequisite to provoke eczematous lesions by application of aeroallergens on the skin of AD patients. However, in contrast to other allergic lesions, where a Th2 polarization is thought to be related to pathogenesis in allergic diseases, both CD4+ and CD8+ T cells play a role in the pathogenesis of AD. In patients with atopy, FcεRI is strongly upregulated on DC, including epidermal LC. In addition, FcεRI, in contrast to effector cells of anaphylaxis, on APCs is differentially regulated in individuals who are atopic and nonatopic. The FcεRIγ chain, which stabilizes surface expression of the FcεRI complex, is present in low amounts in professional APCs from nonatopic individuals, limiting the surface expression of the FcεRI complex and their IgE-binding capacity. In contrast, DCs of individuals who are atopic express substantial amounts of FcεRI, and the IgE–FcεRI binding stabilizes and increases the surface expression of this receptor. This concept also explains, at least in part, why FcεRI expression on myeloid DCs and plasmacytoid DCs in the peripheral blood and on distinct DC subtypes in the skin of patients with atopic dermatitis correlates with their serum IgE level. In addition, CD1a+ LCs express large amounts of costimulatory molecules such as CD86. This capacity of LCs to capture antigens by IgE receptors allows them to drive the activation of T cells. However, it is elusive why the LCs favor a Th2 polarization. Peripheral blood DCs from AD patients produce high levels of IL-10 and prostaglandin E2, which may favor Th2 polarization. After IgE-mediated binding and internalization, two types of cellular responses may happen. The antigen-bearing LC migrate to the peripheral lymph nodes and present the processed allergens efﬁciently to T cells, and antigen presentation after FcεRI binding is associated with Th2-type immune responses characterized by interleukin IL-4-, IL-5-, and IL-13-producing T cells. Next, the activated LC present allergen-derived peptides locally to transiting antigen-speciﬁc T cells and, thereby, induce a classic T-cell-mediated secondary immune response.

84

4. Dendritic Cells and Allergy

In addition to the foregoing descriptive mechanisms of a role of DC in AD, interaction between DCs and TSLP may also cause predominant Th2 immune responses in these cases. DC expressing TSLPR induce a unique type of Th2 polarization that is characterized by induction of different Th2 cytokines and tumor necrosis factor-α, but little or no IL-10. Several investigations are now progressing regarding activation of DCs by TSLP in atopic dermatitis. Using atopy patch test, it has been shown that high numbers of DCs invade the epidermis. The phenotypes of LC and other DC populations are also altered in patients with atopic dermatitis.

Allergic Rhinitis The role and function of APC in allergic respiratory disease still remain unclear. Relatively high numbers of both CD1a+ and HLA-DR-expressing DC are found in the columnar respiratory epithelium and the lamina propria of the nasal mucosa of patients suffering from grass pollen allergy. Some DCs of the respiratory epithelium contain Birbeck granules, a feature that classiﬁes them as LC. Whether the latter represent LC at a different maturation stage or DC of a different origin remains to be clariﬁed. The number of airway DCs is highest in the upper airways (600 ± 800/mm2) and decreases rapidly further down the respiratory tree, suggesting that higher numbers are necessary in the upper airways to cope with the increased antigen exposure. Indeed, it has been demonstrated in patients after allergen provocation testing that the number of DCs increases after antigen exposure. At the beginning of the provocation period, CD1a+ DC were observed in the subepithelial layer and around vessels, redistributing to the epithelium. In the second week of provocation, these cells were found throughout the whole depth of the epithelium. The pivotal role of airway DC for antigen processing was further demonstrated by their rapid steady-state turnover rate with a half-life of only 2 days. This ﬁnding strongly contrasts with the situation encountered in keratinized epithelia such as the normal human skin, where the corresponding DC populations, for example, LCs, are replaced with a half-life of 15 days or longer. The interaction of nasal DCs with other cell types such as mast cells that can be identiﬁed in the nasal mucosa remains to be elucidated. In addition to these, there are some other allergic conditions in which roles of DCs have not been explored. Drug-induced allergy represents one such condition. During this allergy, patients exhibit allergic manifestations after drug intake. Several drugs or their metabolites may be handled by DCs, but the cellular and molecular events regarding these have not been studied. Drug metabolites might alter the mucosal milieu and thus allow DCs to play a role in the induction of allergic manifestations.

Treatment of Allergic Conditions by DCs Different factors indicate that DCs are involved either in initiation or progression of allergic diseases. Accordingly, manipulation of DC functions in allergic patients may have therapeutic implications (Table 2). DC-based therapy in allergic conditions may be evaluated from two perspectives: (1) use of therapeutic agents that would target

Treatment of Allergic Conditions by DCs

85

Table 2. Dendritic cell-based therapeutic approaches in different allergic conditions 1. 2. 3. 4.

Alteration of Th2 type of immunity to Th1 type by in vitro manipulated DCs. Selective depletion of DCs in some allergic diseases. Use of DCs expressing inhibitory antigens. Use of allergen-speciﬁc regulatory DCs.

DC function in vivo, and (2) use of DCs that would directly alter the immune status of allergic patients in vivo. In view of the localization of DCs at the interface of tissues such as skin and nasal or lung mucosa, it is comparatively easy to target DCs for therapy. In the skin, ultraviolet radiation alters profoundly the biology of DC and is routinely used in the treatment of chronic inﬂammatory skin diseases. Similarly, glucocorticoids strongly affect the immunomodulatory capacities of DCs. In some cases, use of antiallergic drugs suppresses DC functions, although some DC-related antigens are unregulated on DCs. More recently, it has been shown that a new generation of immunosuppressive macrolides, that is, tacrolimus and ascomycin, which, in contrast to cyclosporin A, can be used topically, suppress the expression of costimulatory molecules, inhibit the appearance of distinct DC in inﬂammatory tissue reactions, and decrease the stimulatory activity of DC in vitro, as well as in vivo, after local application. Finally, local application of molecules interfering with the binding of IgE to its receptor or compounds inhibiting deﬁned activation mechanisms initiated by FcεRI-expressing DCs in situ could represent valuable alternatives in the future management of atopic conditions. In addition to various agents that affect DC function, DCs by themselves have the potential to be used as therapeutic agent. Recent progress made in understanding of DC and the techniques developed for their generation in vitro have led to an immunological revolution and opened new therapeutic options. In vitro manipulated DCs are now used for treatment of patients with cancers and chronic infectious diseases, but DC-based therapy is yet to be applied in patients with allergic diseases. However, a strategy of DC-based therapy is provided here that has been used in mice but has yet to be used in humans. These experimental approaches provide ideas about future treatment using DCs in patients with allergic diseases. Th2 immune responses are seen in patients with allergic diseases. In addition, these patients exhibit exacerbated immunity to allergens. Thus, the aim of therapy may be to alter the nature of immune response from the Th2 to Th1 type of immunity. Another therapeutic approach may be to downregulate allergen-speciﬁc immunity without compromising the immune response capacity of the hosts. Th2 types of immune responses of allergic patients may be altered to the Th1 type of immune response by using Th1-inducing immunomodulators. IL-12 may be used for this purpose. However, this approach has not given a good outcome in animal models of asthma. Adoptively transferred IL-12 overexpressing DCs could not induce a counterregulatory antigen-speciﬁc Th1 population in mice. Also, occurrence of eosinophilic airway inﬂammation upon rechallenge of the mice with relevant allergen could not be prevented. Thus, it is elusive whether this type of DC-targeting immune therapy can have any practical usage in patients with allergic diseases.

86

4. Dendritic Cells and Allergy

The Th2 type of immunity in allergic patients may be altered by conditional depletion of CD11c+ DCs from the airways of mice, which completely suppresses Th2mediated effector responses and cardinal features of asthma. In addition, induction of immune tolerance by regulatory DCs or induction of regulatory T cells in patients with allergic manifestations may have therapeutic efﬁcacy. Studies of production of regulatory DCs are in progress, as we discuss in another chapter of this book. If allergen-speciﬁc regulatory DCs can be prepared in vitro, a new way of immune therapy of allergic conditions by DCs can begin.

Recommended Readings Aiba S (2007) Dendritic cells: importance in allergy. Allergol Int 56:201–208 Akdis M, Blaser K, Akdis CA (2005) T-helper cell type-2 regulation in allergic disease. Eur Respir J 26:1119–1137 Duez C, Gosset P, Tonnel AB (2006) Dendritic cells and toll-like receptors in allergy and asthma. Eur J Dermatol 16:12–16 Lambrecht BN, van Rijt LS (2006) Infections and asthma pathogenesis: a critical role for dendritic cells? Novartis Found Symp 279:187–200 Novak N (2006) Targeting dendritic cells in allergen immunotherapy. Immunol Allergy Clin N Am 26:307–319 Novak N, Bieber T (2005) The role of dendritic cell subtypes in the pathophysiology of atopic dermatitis. J Am Acad Dermatol 53(2 suppl 2):S171–S176 Ong PY, Leung DY (2006) Immune dysregulation in atopic dermatitis. Curr Allergy Asthma Rep 6:384–389 van Rijt LS, Lambrecht BN (2005) Dendritic cells in asthma: a function beyond sensitization. Clin Exp Allergy 35:1125–1134

5. Dendritic Cells and Autoimmunity

General Consideration of Autoimmunity Autoimmune responses can arise because the repertoire of both T- and B-cell receptors, which allows recognition of pathogens, may contain receptors recognizing self-components. Ideally, autoreactive lymphocytes should be destroyed in the thymus during negative selection and induction of autoimmunity should be controlled. However, a great number of self-reactive lymphocytes escape thymic negative selection processes and form a peripheral pool of potentially autoimmune diseasemediating lymphocytes. On the other hand, self-tissues and -cells are routinely destroyed in our body and thus self-antigens are available in situ. If self-reactive lymphocytes are not completely destroyed in thymus by negative selection, autoantigens may activate these lymphocytes, and the feature of autoimmunity is developed. Thus, the autoimmune process is a normal phenomenon of living organisms. To block the activities of these autoreactive lymphocytes and minimize clinically apparent autoimmune diseases, another mechanism is active in situ. A population of tolerogenic immunocytes (T-regulatory cells) is present in our body. When these lymphocytes also fail to block progression of an autoimmune process, the pathological consequences of autoimmunity become manifest. It is therefore important to clarify the mechanisms leading to the initial activation of self-reactive lymphocytes that induce and sustain the autoimmune response. Also, insights are required regarding the causes underlying the inability of tissue-derived dendritic cells (DCs) to induce immune tolerance to self-antigens. In addition, another population of DCs, called regulatory DCs, should downregulate the autoimmune processes. Thus, development of autoimmune diseases depends on impairment of several mechanisms that are responsible for maintaining homoeostasis in situ. Multiple factors may trigger the development of autoimmune processes. However, genetic, environmental, and immunological factors are the main components that determine the susceptibility to autoimmune diseases. The genetic map of the hosts may inﬂuence the general immunoreactivity. Particularly, polymorphism in genes inﬂuencing lymphocyte homeostasis may increase the susceptibility to autoimmune disease. However, the onset of autoimmune tissue injury or a disease ﬂare is often triggered by factors such as hormones, drugs, and microbial infections. A close association has been detected between type I diabetes mellitus, lupus erythematosus, myocarditis, rheumatoid arthritis, and multiple sclerosis with microbial infections. 87

88

5. Dendritic Cells and Autoimmunity

Patients with chronic forms of autoimmunity may experience symptomatic disease ﬂares following infections. Microbial agents are recognized by the pattern-recognition receptors (PRRs). PRRs are expressed by different types of cells, but their expression on antigen-presenting cells (APCs) is related to induction of antimicrobial immune responses. The pathogen-associated molecular patterns (PAMPs) of microbial agents can stimulate PRRs. Infection with microbial agents activate different cells of innate and adaptive immunity. Infection-associated inﬂammation causes the release of cytokines and chemokines, which attract antiviral effector cells and lymphocytes of other speciﬁcities. In fact, an inﬂammatory microenvironment is established at the site of localization of microbial agents. Several factors may occur as a result ofthe persistence of the inﬂammatory mucosal milieu after infection and other causes. Self-antigens may be available in larger quantities as a result of the destruction of autologous cells by infection. Self-reactive lymphocytes may be activated in this situation, and this may lead to induction of autoimmune process or exacerbate preexisting autoimmune diseases in susceptible subjects. A second mechanism that may lead to the initiation of autoimmunity in the course of an infection is the activation of T or B cells via antigenic determinants shared between the pathogen and the host; this has been termed molecular mimicry. A role of molecular mimicry has have been described for human autoimmune diseases such as diabetes mellitus, multiple sclerosis, primary biliary cirrhosis, and Guillain–Barré syndrome. The frequencies and functions of regulatory T cells that usually inhibit autoimmune processes may be altered, thus allowing development of autoimmune diseases in some patients.

Dendritic Cells in Autoimmunity DCs in Maintaining Immune Tolerance and Protecting Against Development of Autoimmunity The exact mechanism underlying the development of autoimmune diseases is not clear. In fact, little is know about autoantigens in most autoimmune diseases. In addition, a latent condition of autoimmunity can be detected in many persons, but clinically apparent autoimmune diseases are not seen in these subjects. Several cells of the immune system and other factors (both immune-related and nonimmune-related) may have dominant roles in regulating the process of autoimmunity. In this chapter, we mainly discuss the roles of the DC during autoimmune diseases. As described in Table 1, DC is one of the main players that maintain tolerance at the tissue levels. Under normal conditions, the activities of DCs prevent induction or exacerbation of autoimmune diseases. DCs suppress the process of autoimmunity. Immature DCs are widely distributed in almost all tissues of the body. On the other hand, normal death and destruction of different cells is a common event in the life cycle of a living organism. Immature DCs in the tissues are capable of sampling debris of different cells of the body. DCs are the decision makers about induction of immune tolerance or immune responses. Under normal conditions, immature DCs induce immune tolerance, not immune responses; this may be a vital mechanism that controls the development of autoimmune diseases.

Dendritic Cells in Autoimmunity

89

Table 1. Dendritic cells (DCs) in the maintenance of homeostasis 1. DCs have roles during induction of tolerance in the thymus. 2. DCs scan different tissues and recognize, internalize, and process autoantigens and apoptotic cells. Presentations of these antigens by immature DCs induce immunogenic tolerance. 3. Regulatory DCs are capable of inducing immune tolerance. 4. DCs induce regulatory T cells in vivo and thus control the expansion and function of autoreactive T cells in the periphery. 5. DCs induce and produce different immunoregulatory cytokines and agents that restrict the activities of pathogenic T cells.

In addition to immature DCs, there is another population of DCs, called regulatory DCs. The phenotypes of these cells have recently been characterized in mice. These DCs seem to be committed immunocytes for induction of immune tolerance. However, more studies are needed to develop better insights about their phenotypes and functional plasticity. It is still elusive whether regulatory DCs among immature DCs maintain immune tolerances or if both these DCs are capable of inducing immune tolerances. Also, studies are needed to explore whether regulatory DCs can induce immune tolerances in the presence of an inﬂammatory mucosal milieu. In addition to direct action of immature DC and regulatory DCs to induce immune tolerance, DCs can also act indirectly for maintenance of the tolerogenic state. A population of T cells, regulatory T cells, is responsible for maintenance of immune tolerances. DCs also regulate the functional capacities of regulatory T cells. Also, DCs have some critical role during the production of regulatory T cells. In addition to production of various proinﬂammatory cytokines, DCs also produce different antiinﬂammatory cytokines. These cytokines may have a role in the maintenance of immune tolerance. Taken together, it seems that DCs prevent induction of autoimmune diseases, either by inducing immune tolerances or by instructing other immunocytes to do so. Role of DCs During Induction and Progression of Autoimmune Diseases DCs play major roles during maintaining immune tolerance in the steady state. Also, DCs block unwanted immune responses so that autoimmune diseases can be prevented even in inﬂammatory conditions. These facts indicate that impaired or improper functions of DC may also be related to the development of autoimmune diseases. Induction of immune tolerances and immune responses by DCs is determined by various factors at the tissue levels. It is true that immature DCs that induce immune tolerance can also induce immune responses. Irrespective of the nature of autoimmune diseases, several factors have a profound effect on the pathogenesis of autoimmune diseases, including (1) the proinﬂammatory mucosal milieu, (2) the nature of DCs that encounter self-antigens, (3) the nature of the self-antigens, and (4) the frequencies and functions of autoreactive lymphocytes. In this context, a role of DCs is predicted during induction and progression of autoimmune diseases, because DCs as regulator of immune responses and immune tolerances are capable of

90

5. Dendritic Cells and Autoimmunity

Table 2. Breakdown of homeostasis and role of DC during autoimmunity Presence of microbial agents with pathogen-associated molecular patterns (PAMPs) may alter DCs from tolerogenic mode to immunogenic mode. Availability of huge amounts of self-antigens from tissue destruction may be accompanied by proinﬂammatory cytokines and immunomediators. Inﬂammatory mucosal milieu caused by viral infections or drugs favor immune responses by DCs. Inﬂammatory condition downregulating the activity of regulatory T cells. Encounter between ignored antigens and DCs in lymphoid tissues. Changes in frequencies and functions of regulatory DCs. Increased activity of plasmacytoid DCs. Presentation of antigens by DCs for a prolonged period of time. Cytokine imbalance altering antigen presentation capacities of DCs.

modulating different factors related to induction and progression of autoimmune diseases (Table 2). DC is one of the key players of innate immunity and produces various cytokines by stimulation from various factors. The nature of cytokines that are produced by DCs by interactions with self-antigens or apoptotic cells in vivo has not been characterized. It is possible that under certain conditions and in genetically susceptible hosts, proinﬂammatory cytokines may be produced by immature DCs even because of encounter with self-products, which may distort the mucosal balance and provide a favorable condition for induction of autoreactive lymphocytes. In addition, some DCs, such as plasmacytoid DCs, are potent producer of type I interferon (IFN). Type 1 IFN is implicated in various autoimmune diseases. Thus, production of high levels of proinﬂammatory cytokines by DCs may disrupt the inﬂammatory balance of different tissues; this may cause formation of autoreactive T cells, which can induce destruction of various tissues. Different types of pathogens frequently enter the body via peripheral tissues, and DCs recognize those through PRRs. After recognition, DCs process those at their endosomal compartment. Then, DCs expressing the antigenic peptides migrate to lymphoid tissues and activate clonally selected lymphocytes to produce T-helper cells, cytotoxic T lymphocytes (CTLs), and antigen-speciﬁc B cells. The CTLs then come back to tissue with microbial infections and destroy the microbe-infected cells. Taken together, after microbial infections, DCs undergo maturation, and there may be tissue destruction and inﬂammation depending on the nature of the DC–microbe interactions. If both microbial agents and self-tissues are processed and presented by tissue DCs, autoreactive T cells may be produced, and these cells may cause tissue destruction in susceptible hosts. This event may happen at any time in the life cycle of the hosts, but autoimmune diseases are not so common. The balance between autoimmune and antiautoimmune phenomena may have a dominant role to determine whether autoimmune diseases occur. T cells that have not been negatively selected in the thymus and are of sufﬁciently high avidity possess the capacity to elicit autoimmune reactions. These autoreactive T cells are usually directed against immunologically ignored antigens, which are not present in the thymus or in peripheral lymphoid tissues such as lymph nodes or

Goal of DC Research in Autoimmune Diseases

91

spleen. Autoreactive T cells recognizing immunologically ignored antigens may be activated if their cognate antigen reaches lymphoid organs and is appropriately presented by DCs. In addition, antigen should be present in local lymphoid organs for a minimal time period (probably 4–6 days) to obtain optimal initial activation of T cells. In an inﬂammatory microenvironment, DCs can take up autoantigens and allow their prolonged presentation at the lymphoid tissues, which may cause induction of autoimmune diseases. The evidence is compelling that the aforementioned negative regulatory mechanisms, if they exist, are easily overruled, and that autoimmunity can be induced when immunologically ignored autoantigens are presented in secondary lymphoid organs above a minimal concentration for a sufﬁcient length of time. Also, DCs play a most critical role for recognizing the antigens and processing those and presenting them to lymphoid tissues. It has been suggested that DCs may also process exogenous self-antigens via MHC class I for presentation to CTLs, leading to induction of autoimmune diseases via a pathway called cross-presentation. Exogenous loading of MHC class I molecules on DCs can be achieved using soluble or cell-associated proteins and can be further enhanced by proteins from immune complexes with antibodies. In presence of tissue inﬂammation, autoreactive T cells may be stimulated and induce tissue damage. Also, activated DCs may downregulate the functional capacities of regulatory T cells. Finally, when the inﬂammatory mucosal milieu prevails, the production of regulatory DCs from immature DCs may be downregulated.

Goal of DC Research in Autoimmune Diseases Autoimmune diseases play an increasing role in public health, especially in light of increasing aged population. Compared to male, females are affected more by autoimmune diseases. Although there are very few reports about the role of hormones on the activation and maturation of DCs, circumstantial evidence indicates a role of hormones, especially of female hormone, in the initiation or perpetuation of autoimmune diseases. A high dose of prolactin led to the maturation of DCs. Thyroid hormones also induce IL-12 from DCs, which explains the high serum level of IL-12 in the hyperthyroid state. The treatment of DCs with nonsteroidal antiestrogens, including tamoxifen, is able to inhibit the terminal maturation of DCs. These studies indicate that DCs may have some contribution to the pathogenesis of autoimmune diseases. However, the data of these studies should be carefully explained. Activation and maturation of different cells of the immune system are seen in autoimmune as well as other diseases. Studies about activation and deactivation of DCs would bear minimum importance if the strategies of these studies are not well explored. There should be more insights about types of DCs, and experimental conditions should be optimized. Again, it is important to describe what types of functions of DCs were evaluated. In general, upregulation of surface antigens on DCs or increased allostimulatory capacity of DCs is regarded as a sign of activation of DCs. This sign would have very little practical implication in the context of autoimmunity if antigen-speciﬁc functions of DCs were not evaluated. Autoimmune diseases show two peaks regarding age distribution, during the twenties and in the ﬁfties. Systemic lupus erythematosus (SLE) and HLA DR3+

92

5. Dendritic Cells and Autoimmunity

autoimmune hepatitis have peak onset in the twenties, and DR4+ autoimmune hepatitis and primary biliary cirrhosis (PBC) usually occur during the ﬁfties. In general, the frequencies and functions of DCs are downregulated with advancing age. However, little is known about whether the functions of both immunogenic DCs and tolerogenic DCs are downregulated along with age. On the other hand, along with the advancement of age, DCs and circulatory T cells would have more chances to encounter more autoantigens in vivo. More cells also undergo apoptosis along with age. DCs would have more chances to engulf more autoantigens in these circumstances. The changes of number and functions of DCs have been studied in a number of human autoimmune diseases as well as in animal models of autoimmune diseases. There are striking similarities in the behavior of DCs in affected organs, although the pathogenesis of various autoimmune diseases differs. DCs are accumulated in diseased tissues in autoimmune disease. DCs with markers of activation and maturation are usually detected in these conditions. DCs in affected tissues express MHC class II and costimulatory molecules including CD80 and CD86. The ﬁnal goal of DC research in autoimmunity is the development of therapy for these diseases. Also, it may be possible to block the initiation of autoimmune diseases in susceptible hosts before they become clinically apparent. Indirect evidence indicates that downregulation of DC functions may block progression of autoimmune diseases. The immunosuppressive drugs azathioprine, mizoribine, and cyclosporine are used for treatment of autoimmune diseases, and these drugs also downregulate DC functions. Also, drugs such as glucocorticoids and vitamin D that are used for treating autoimmune diseases also decrease the maturation and differentiation of DCs. However, these drugs have multiple functions, and drugs that would speciﬁcally affect the functions of DC are yet to appear. Treatment of autoimmune diseases by DCs may also be possible. It may be feasible to use tolerogenic DCs as therapeutic agent in autoimmune diseases. However, development of autoantigen-speciﬁc tolerogenic DCs is a major challenge for DC-based therapy against autoimmune diseases in humans.

DCs in the Pathogenesis of Autoimmune Diseases General Features The exact role of DCs in the pathogenesis of autoimmune diseases is not completely clear, but these cells are involved in the initiation and progression of autoimmune processes. As we discussed, exacerbated functions of some types of DCs may cause autoimmunity. For example, if the functions of immunogenic DCs are increased, that may lead to autoimmunity. On the other hand, impaired functions of tolerogenic DCs may cause autoimmune diseases. In Table 3, we have shown some data suggesting that DC activity may be associated with development of autoimmune diseases in animal models of human diseases and also in patients with autoimmune diseases. Animal models of spontaneous autoimmune disease have shown that DCs are among the ﬁrst cells to inﬁltrate the target tissue and are capable of presenting autoantigens to T cells in local draining lymph

DCs in the Pathogenesis of Autoimmune Diseases

93

Table 3. Dendritic cells in autoimmune diseases (A) Dendritic cells in animal model of autoimmune diseases • Thyroglobulin-pulsed DCs induce autoimmune diseases in genetically susceptible individuals. • Activated DCs are detected at the initial phase of experimental autoimmune gastritis. • Abnormal phenotype and functions of DCs in murine type-1 diabetes mellitus show skewing to a Th1 proinﬂammatory pattern. • Myelin basic protein-pulsed DCs induces autoimmune encephalitis. • Activated DCs in animal model of primary biliary cirrhosis. (B) Dendritic cells in autoimmune diseases • Increased DCs in synovial ﬂuids in patients with rheumatoid arthritis. • Impaired functions of DCs in multiple sclerosis, Sjogrens syndrome, and thyroiditis. • Increased numbers of DCs in juvenile chronic arthritis, psoriasis, encephalitis, diabetes, Sjogrens syndrome. • Increased IFN-α-producing DCs in systemic lupus erythematosus. • Increased activated DCs in colonic mucosa of ulcerative colitis. • Impaired functions of DCs in multiple sclerosis, Sjogrens syndrome, thyroiditis, rheumatoid arthritis.

nodes. We have reported the maturation and activation of DCs in animal models of autoimmune gastritis. It has also been shown that DCs expressing endogenous selfpeptides or pulsed ex vivo with immunogenic self-peptides can induce severe autoimmune disease. Abnormal phenotypes and functions of DCs have been reported in murine type 1 insulin-dependent diabetes that may skew the immune response toward pathogenic Th1 types. Also, defective numbers of regulatory T cells, including CD4+CD25+ T cells have been reported in nonobese diabetic mice, the murine model for type 1 diabetes. Defective numbers and impaired functions of regulatory T cells may compromise the functions of DCs and convert the tissue microenvironment from a tolerogenic to an immunogenic state, which may facilitate induction of autoimmune diseases. DCs were engineered to constitutively express a region of lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) that contains a dominant cytotoxic Tcell epitope. These DCs caused autoimmune diabetes when injected into mice that expressed LCMV-GP exclusively in pancreatic cells. This induction of autoimmunity was accompanied by the de novo formation of lymphoid tissue. DC pulsed in vitro with a peptide derived from myelin basic protein (MBP) also caused autoimmune diseases. Injection of the DC into transgenic mice that expressed T-cell receptors speciﬁc for the MBP peptide induced encephalitis. In these systems, DCs were modiﬁed to present antigens that are known to cause disease. In murine systemic lupus erythematosus models, increased numbers of DCs have been reported. DC can induce and maintain autoimmunity in a model system: DCs have also been characterized in human autoimmune diseases. An early inﬁltration of differentiated DCs into the synovial tissue before the exacerbation of disease has been observed in patients with rheumatoid arthritis. Increased numbers of DC have also been observed in the affected joints of rheumatoid arthritis patients. Synovial ﬂuid of patients with rheumatoid arthritis have shown to contain more DC precursors and myeloid DC growth factors, such as GM-CSF, TNF-α and IL-6-secreting DCs, than in healthy subjects. Impaired

94

5. Dendritic Cells and Autoimmunity

functions of DCs have been reported in multiple sclerosis, Sjögren’s syndrome, thyroiditis, and PBC. A type of DCs, plasmacytoid DCs, which produce abundant amounts of type 1 IFN, exhibit abnormal functions in patients with SLE. DCs are present in high numbers in the serum and synovial ﬂuid of patients with rheumatoid or juvenile chronic arthritis, psoriasis, diabetes, thyroiditis, and Sjögren’s syndrome, and high levels of circulating DC secreting proinﬂammatory cytokines are associated with multiple sclerosis. Thus, microenvironments that induce migration, accumulation, and maturation of DCs might be important for initiation of autoimmunity and onset of autoimmune diseases. However, a conclusion about involvement of DCs in autoimmune diseases should be developed carefully because mature DCs are found in various pathological conditions. It may be possible that, in addition to mature DCs, the tissue microenvironment of the hosts plays a critical role in the development of autoimmune diseases. In a study we found that DCs from patients with PBC produced increased amounts of nitric oxide compared to DCs from control subjects and patents with other inﬂammatory diseases of the liver (Fig. 1A). This effect was also related to reduced T-cellstimulating capacities of DCs of PBC patients (Fig. 1B). Thus, it is evident that administration of activated and matured DCs or DCs loaded with antigens can induce autoimmunity. Also, activated DCs have been localized in tissues from subjects with autoimmune diseases. However, the mechanisms of induction of autoimmunity by DCs remain elusive.

[B]

[A] 100%

*

¶ 800%

75%

600%

50%

400%

25%

200% 100% Controls

PBC

Allostimulatory capacity of DCs

Controls

PBC

NO production by DCs

Fig. 1. Increased nitric oxide (NO) production and low T-cell stimulatory capacity of monocytederived dendritic cells from patients with primary biliary cirrhosis (PBC). Dendritic cells from patients with PBC produced signiﬁcantly higher levels of nitric oxide (A) and also had low allostimulatory capacity (B). The T-cell stimulatory capacity and nitric oxide production of DCs from control subjects have been regarded as 100%

DCs at the Tissues in Autoimmune Conditions

95

Putative Role of DCs in Induction of Autoimmunity DCs have been detected in murine models of autoimmune diseases. DCs with different levels of maturation and activation have also been detected in the peripheral blood and in the tissues from patients with autoimmune diseases. Based on this information, it is usually assumed that DCs have a role in the initiation of autoimmune diseases. However, altered functions of DCs in patients with autoimmune diseases may reﬂect alteration of DC functions in a chronic pathological condition. Characterization of DCs in patients with autoimmune diseases provides very little information regarding the role of DCs in the initiation of autoimmune diseases. The role of DCs during induction of autoimmune process is difﬁcult to study in humans because this process usually starts long before the features of disease are seen. It may be possible to evaluate the role of DCs in autoimmunity in human by assessing DCs in drug-induced and virus-induced autoimmune diseases, but this is difﬁcult to study in other autoimmune diseases. Here, we describe studies in animal models of human diseases that provide some ideas about the involvement of DC during initiation of autoimmune diseases. Neonatal Balb/c mice thymectomized on day 3 after birth develop autoimmune gastritis that is progressive in nature. The lesion is characterized by inﬁltration of mononuclear cells in the gastric tissues. CD11c+ DCs have been localized from these mice as early as 3 weeks after thymectomy, which population increases with time. The DCs are usually clustered with CD4+ T cells. It is still unknown whether these DCs migrate from some other sources to the stomach, or whether DC progenitors or DC precursors at the stomach became CD11c+ DCs after thymectomy. The mobilization of DCs to the tissues may be an important step in the initiation of immune-mediated inﬂammation of that organ. The expression of chemokines in diseased organs and chemokine receptors on DCs would become important for understanding how early migration of DCs can be seen in these conditions. The presence of DCs indicates an involvement of DCs in the autoimmune process in murine models of experimental autoimmunity, but the real clinical relevance of these ﬁndings is far from clear.

DCs at the Tissues in Autoimmune Conditions DCs, activated DCs, and matured DCs have been localized in the tissues in animal models of autoimmune diseases and in patients with autoimmune diseases. CD11c+ DCs are detected at the portal areas in senescent female C57BL/6 mice that spontaneously develop PBC-like liver diseases (Fig. 2). DCs are frequently observed close to the bile duct epithelia. Some of these DCs express the phenotype of matured and activated DCs. Although DCs enriched from peripheral blood of PBC patients had impaired allostimulatory function, the CD11c+ DCs in the liver from the animal model of PBC had a phenotype of activated DCs, which may indicate that the phenotype and function of blood-derived DCs and tissue DCs may be different in pathological conditions. In conformity with the data of the animal model of PBC, mature DCs expressing CD83 have also been shown in the liver tissues from patients with PBC (Fig. 3).

96

5. Dendritic Cells and Autoimmunity

[A]

[B]

Fig. 2. Localization of dendritic cells in murine model of PBC. The murine model of PBC was established in senescent mice. CD11c+ DCs were seen at the portal areas and also at the parenchyma of the liver (A). Electron microscopy revealed the localization of CD11c on dendritic processes (B)

Fig. 3. CD83+ mature dendritic cells in the liver tissues of patients with PBC

Mature DCs expressing CD83 have been localized in the inﬂamed colonic mucosa from patients with ulcerative colitis. DCs expressing both CD83 and macrophage migration inhibitor factor were also located at the colonic mucosa from patient with ulcerative colitis. Although the real implication of this ﬁnding is not exactly known, macrophage migration inhibitory factor produced by DCs may help to mobilize macrophages at the site of tissue injury in patients with ulcerative colitis. A collaboration of DCs and macrophages may be involved in the pathogenesis of this disease. DCs

Peripheral Blood DCs in Autoimmune Diseases

97

expressing DC-SIGN, IL-12, and IL-18 are increased at the colonic mucosa of patients with Crohn’s disease. DCs have also been localized in target organs form patients with SLE, rheumatoid arthritis, autoimmune hepatitis, and other autoimmune diseases.

Peripheral Blood DCs in Autoimmune Diseases The frequencies and functions of DCs have been studied in various autoimmune diseases in human. DCs are enriched from precursor populations in the blood; however, it is now possible to isolate DC precursors directly from the blood. The functions of DCs, enriched by culturing monocytes or an adherent population of peripheral blood mononuclear cells, have been evaluated in PBC. The capacity of monocyte-derived DCs to stimulate allogeneic T cells was signiﬁcantly decreased compared to control subjects. Increased production of NO by DCs from PBC patients was responsible for this. Mature DCs in the liver tissues also expressed inducible nitric acid synthase, indicating their capacity to produce NO in the liver tissue. In addition to functional studies about peripheral blood DCs, DCs can also be used to detect autoantigen-speciﬁc T lymphocytes from patients with PBC. Patients with PBC are usually characterized by the presence of antibody to pyruvate dehydrogenase complex (PDC) in the sera and PDC-speciﬁc T cells in the liver. However, most of the patients with PBC do not show peripheral blood T-cell response to PDC when conventional lymphoproliferative assays are done using PDC. However, using PDC-pulsed monocyte-derived DCs, PDC-speciﬁc T cells were detected from the peripheral blood from most of the patients with PBC, even when anti-PDC antibody could not be detected by conventional methods. This study provides deﬁnitive evidence regarding the existence of autoantigen-speciﬁc lymphocytes in PBC patients. In autoimmune hepatitis, the frequency and nature of DC precursors were evaluated by ﬂow cytometry. The frequencies of myeloid DC precursors and plasmacytoid DC precursors were not different between patients and controls. However, the expression of HLA DR on myeloid DC precursors from patients with autoimmune hepatitis was decreased, but not on PBC. However, the expression levels of HLA DR and CD123 on precursors of plasmacytoid DC were signiﬁcantly decreased in patients with PBC and autoimmune hepatitis compared with healthy subjects. As plasmacytoid DCs play a role in the induction of immunogenic tolerance and Th2 polarization, defective expression of surface antigens on these DCs may underlie a switching of immune response to Th1 type in these autoimmune diseases. On the other hand, a completely different picture was seen in ulcerative colitis, another autoimmune disease of the colon. When precursor populations of DCs from the peripheral blood were cultured to enrich DCs from these patients, the numbers of DCs expressing CD83 antigen were signiﬁcantly higher than the controls. The T-cell-stimulatory capacity of DCs was signiﬁcantly higher in ulcerative colitis patients compared to controls. In human autoimmune diseases, understanding the nature and function of DCs would provide valuable information to follow up the patient, to have insight about

98

5. Dendritic Cells and Autoimmunity

the progression of disease, and to judge the efﬁcacy of therapy. However, this might not help with understanding the mechanism of the diseases. The mirror images of number and function levels of immune cells between peripheral blood and diseased organ tissue are experienced frequently. Thus, the gold standard would be to evaluate DCs in the affected tissues; however, more DC-speciﬁc markers would be required to study this in full detail.

IFN-a and Plasmacytoid DCs in Autoimmune Diseases A role of IFN-α is predictable in the pathogenesis of autoimmunity, although the prime function of this cytokine is to act during the induction of innate immunity. IFN-α is produced in and around the tissues of microbial invasion, and this cytokine can destroy the microbes directly. A role of microbial infections has been predicted in the induction of autoimmunity, and IFN-α can be involved in this process. Moreover, IFN-α can provide the requisite danger or alarm signals for the induction of adaptive immune responses. It has long been known that type 1 IFN are produced by leukocytes or ﬁbroblasts; however, little was known regarding the nature of IFN-producing cells. However, only recently it has been become evident that plasmacytoid DCs, a type of precursor of population of circulating DCs, are the most potent producers of IFN-α. These DCs are termed natural interferon-producing cells. Increased levels of IFN-α have been detected in the sera from patients with SLE. Plasmacytoid DCs produce huge amounts of IFN-α from culture with sera from SLE patients; possibly the DNA–anti-DNA complexes in SLE sera provide an activation signal for IFN-α production. Moreover, immune complexes in SLE sera may trigger other myeloid DCs or other cells to produce IFN-α. Plasmacytoid DCs that produce IFN-α have also been detected in the skin from cutaneous SLE. In addition to SLE, a role of IFN-α in autoimmunity can be postulated from the fact that IFN-α induces the exacerbation or acute onset of latent or remission stage of autoimmune liver diseases. Plasmacytoid DCs expressing CD123 and CD68 have recently been located in the liver tissues from patients with PBC.

DC-Based Therapy for Autoimmunity Circumstantial evidence has shown that DCs may induce autoimmunity because these cells regulate the nature and magnitude of immune responses. DCs are critical regulators of homeostasis. However, because of alteration of the tissue microenvironment, DCs may lose their capacity to block induction of autoimmune processes. Again, self-antigens may be presented by immunogenic types of DCs, thus increasing the risk of autoimmune diseases. Detection of activated and matured DCs in tissues and blood of patients with autoimmune diseases is not a speciﬁc ﬁnding that can be related to the role of DCs in the pathogenesis of autoimmune diseases. The authors have encountered activated DCs in various pathological conditions. However, the presence of DCs with altered functional capacities provides indirect support about the role of DCs in the autoimmune process.

DC-Based Therapy in Animal Models of Human Autoimmune Diseases

99

Table 4. Dendritic cell-based therapy against autoimmunity (A) Strategy of DC-based therapy against autoimmunity 1. Induction of tolerogenic environment in subjects with autoimmunity by in vitro manipulated DCs 2. The DCs should be antigen-speciﬁc with regulatory functions. 3. Administered DCs should be able to induce anti-inﬂammatory cytokines 4. Tolerogenic DCs should be capable of induction and maintenance of regulatory T cells. (B) DC-based therapy in animal models of autoimmunity 1. DCs from pancreatic lymph nodes downregulate diabetes of NOD mice. 2. IFN-γ-treated spleen DCs decrease diabetes in NOD mice. 3. Bone marrow-derived DCs pulsed with myelin basic protein (MBP) reduce experimental allergic encephalomyelitis (EAE). 4. TGF-β-treated DCs downregulate experimental autoimmune myasthenia gravis. 5. Downregulation of GVDH reaction by regulatory DCs.

Autoimmune diseases constitute a formidable burden in the context of therapy in many patients. The drugs that are used for autoimmune diseases control the process, but are endowed with severe side effects. In this context, developments of alterative therapeutic approaches are needed to combat autoimmune diseases in human. It has been suggested that DCs may be related to induction and pathogenesis of autoimmune diseases, and DC-based therapy against various diseases has been started. This effort has opened a ﬁeld to assess if there is any opportunity to use DC for treatment of autoimmune diseases. However, better understanding about the role of DCs in human autoimmune diseases will be needed to initiate DC-based therapy against autoimmunity. One of the major problems about immune therapy of autoimmune diseases is that in most cases the etiological agents of the autoimmune diseases are not known. Moreover, there may be several autoantigens in any speciﬁc autoimmune diseases, but probably only one or two autoantigens have been described. At present, patients with autoimmune diseases are treated by various agents that downregulate the functions of DCs in vivo. Corticosteroid is a representative drug of this class. In addition to corticosteroid, other immunosuppressive drugs also reduce the functional capacities of DCs. However, these drugs also decrease the activities of other immunocytes. DC-based therapy has been used in the murine model of autoimmunity for more than one decade. In most cases, it has been found that administration of in vitro manipulated DCs has downregulated the autoimmune processes in mice. A short account of the strategy of DC-based therapy in autoimmune diseases has been given in Table 4.

DC-Based Therapy in Animal Models of Human Autoimmune Diseases Injection of DCs isolated from draining lymph nodes of the pancreas protects NOD mice from disease development of type 1 diabetes mellitus. Also, it has been shown that murine bone marrow DCs are more effective in preventing diabetes in NOD

100

5. Dendritic Cells and Autoimmunity

mice than immature DCs. We have shown that IFN-γ-treated DCs can downregulate the pathogenesis of diabetes in NOD mice. It has also been shown CD40L blockade in the rat insulin promoter lymphochoriomeningitis virus model of autoimmune diabetes was found to completely prevent disease and that the protection could be transferred by a cell population that had features of both DC and NK cells. The therapeutic efﬁcacy of DC-based interventions in animal models of diabetes mellitus is related to induction of regulatory T cells and production of antiinﬂammatory cytokines. In experimental allergic encephalomyelitis (EAE), tolerance can be induced against acute EAE in Lewis rats by bone marrow-derived DC pulsed with encephalitogenic myelin basic protein (MBP) peptide. This therapeutic effect is associated with immature increased production of NO by DCs. NO by DCs causes high levels of IL-10 and low levels of IL-12, increased IFN-γ expression, and gradually reduced proliferative capacity and apoptosis of CD4+ T cells. In mice, repetitive injection of wild-type DC matured with TNF-α and pulsed with autoantigenic peptide can induce peptide-speciﬁc, IL-10-producing CD4+ T cells in vivo and prevent EAE. To treat animal models of DC experimental autoimmune myasthenia gravis (EAMG), DCs have been treated with TGF-β and administered subcutaneously to Lewis rat. In addition to these approaches, genetically engineered DCs have also been used for therapeutic purposes in autoimmune diseases of mice. The purpose is to produce DCs that induce antiinﬂammatory cytokines or the Th2 type of immune responses. The potential of genetically engineered DC for therapy of chronic systemic autoimmune disease has recently been demonstrated in a murine model of collagen-induced arthritis (CIA). A single systemic injection of IL-4-transduced DC to mice with established CIA reduced the severity of the disease, and disease was completely ameliorated within 1 week in at least 50% of the animals. Retrovirally transduced IL-4 DC also reduced the incidence and severity of CIA and suppressed established Th1 responses and associated humoral responses. These effects were achieved despite only transient persistence of the injected DC in the spleen. IL-4-transduced T cells or ﬁbroblasts failed to alter the course of the disease, indicating that the therapeutic effect was restricted to DC. A recent study demonstrated that DC engineered to express FasL was also effective in the prevention of CIA. DC engineered to express IL-4 can prevent autoimmune diabetes in NOD mice with already advanced insulitis. Recently, we administered parietal cell antigen-pulsed DCs in an animal model of autoimmune gastritis. As shown in Fig. 4, the levels of gastritis and the destruction of parietal cells were reduced by administration of antigen-pulsed DCs (Fig. 4). When these experimental data are considered, it becomes evident that immunogenic tolerance can be induced by DCs, or antigen-loaded DCs, or genetically transformed DCs in various animal models of human diseases. Recently, regulatory DCs have been prepared by culturing DCs with various agents. Antigen-speciﬁc regulatory DCs have also been prepared, and their immunomodulatory capacities have been assessed in various animal models. However, the speciﬁcity of antigen-speciﬁc regulatory DCs should be assessed more elaborately.

Possible Use of DCs for Treatment of Human Autoimmune Diseases

[A]

101

[B]

Fig. 4. Increased levels of gastritis and destruction of parietal cells in murine model of autoimmune gastritis (A). Administration of parietal cell-pulsed putative regulatory DCs caused reduced levels of gastritis and parietal cell destruction in mice with autoimmune gastritis (B)

Possible Use of DCs for Treatment of Human Autoimmune Diseases The potential roles of DCs in downregulating the autoimmune process have been conﬁrmed in animal models of human autoimmune diseases, but DC-based clinical trials have not yet been initiated in patients with autoimmunity. Several points must be well addressed before translation of the concept of DC-based therapy for autoimmune diseases in humans (Table 5). Some of these points are discussed next. Improper Understanding About Autoantigens in Human Autoimmune Diseases Major problems about immune therapy in autoimmune diseases lie in the fact that most of the autoantigens in human autoimmune diseases are not well characterized. Only some autoantigens are known in some autoimmune diseases. In this context, it is extremely difﬁcult to prepare antigen-pulsed tolerogenic DCs for human usage. When the nature of autoantigens is known, then it will be clear whether tolerogenic autoantigen-speciﬁc DCs may be produced or not. Antigen-Pulsed DCs Can Also Induce Autoimmunity Although DC have been used effectively in several models of autoimmune disease as a means to prevent or treat disease, it is important to realize that under some circumstances DC can induce autoimmunity, as has been shown in models of EAE, diabetes, and autoimmune thyroiditis. The ﬁnding is perhaps not surprising in view of the efﬁciency of these cells as inducers of the immune response. These studies highlight

102

5. Dendritic Cells and Autoimmunity

Table 5. Factors related to DC-based therapy for human autoimmune diseases • Most of the autoantigens are not characterized in human autoimmune diseases. It may not be possible to develop DC-based therapy against human autoimmune diseases until more autoantigens, especially target autoantigens, are discovered. • There may be several autoantigens in certain autoimmune diseases, and clear idea about these are necessary. • The phenotypes of regulatory DCs should be established. • Tolerogenic DCs in vitro may loose immune tolerance capacity after administration in vivo. • Tolerogenic DCs should be activated because they must migrate in vivo. • On the other hand, migration may alter tolerogenic DC to immunogenic DCs.

the requirement for a thorough understanding of DC biology and function before exploiting these cells as therapeutic agents. Also, antigen-pulsed DCs are used for treating patients with cancers and viral infections with an intention to induce anticancer and antiviral immune responses. Tolerogenic Antigen-Speciﬁc DCs May Contain Immunogenic Antigen-Pulsed DCs At present, DCs are cultured with autoantigen and different cytokines and immune regulator to prepare tolerogenic antigen-pulsed DCs for use in animal models of autoimmune diseases. Immunogenic antigen-pulsed DCs are also prepared in a similar manner. However, different types of immune modulators and cytokines are used for preparing immunogenic and tolerogenic antigen-pulsed DCs. The phenotypes and functions of bulk populations of tolerogenic antigen-pulsed DCs are evaluated before administration to animal models of autoimmunity. The expression of mean levels of surface antigens and production of mean levels of cytokines are decreased in tolerogenic DCs that are used for treatment of autoimmune diseases. However, many DCs within the bulk population of DCs may be immunogenic in nature. The Nature of Tolerogenic DCs May Be Altered In Vivo Tolerogenic antigen-pulsed DCs are immunosuppressive in vitro, mainly because they produce various antiinﬂammatory cytokines and downregulate proliferation of antigen-speciﬁc lymphocytes in vitro. However, when these DCs are injected into humans, these may be activated and become immunogenic in situ. Moreover, a proinﬂammatory mucosal milieu prevails in patients with autoimmunity. It is still elusive whether tolerogenic DCs can maintain their tolerogenic nature even in vitro. Detection of Regulatory DCs in Humans It will be extremely helpful if a population of regulatory DCs is detected in the human. This type of DCs has been characterized in mice.

Recommended Readings

103

Concluding Remarks Study about the immune pathogenesis of various autoimmune diseases indicates that DCs may have a dominant role in initiation and perpetuation of autoimmunity and autoimmune diseases. The identiﬁcation of autoantigens in these pathological conditions would be important to reafﬁrm the role of DCs in autoimmune diseases. The progress in molecular biology about the human genome contributed to the role of sensitive gene single nucleotide polymorphism in autoimmune diseases. Fusion between immunological and genomic research may contribute to rapid progress in the research of autoimmune diseases. Many possibilities for DC-based immunotherapy are available for clinical experiments. The prospect of immunotherapy using antigen-pulsed DCs, tolerogenic DCs, and antigen-speciﬁc immunosuppression via DCs is exciting. However, it is extremely important to determine the strategy of intervention for treating human autoimmune diseases.

Recommended Readings Feili-Hariri M, Flores RR, Vasquez AC, Morel PA (2006) Dendritic cell immunotherapy for autoimmune diabetes. Immunol Res 36:167–173 Hardin JA (2005) Dendritic cells: potential triggers of autoimmunity and targets for therapy. Ann Rheum Dis 64(suppl) 4:86–90 Kuwana M (2002) Induction of anergic and regulatory T cell by plasmacytoid dendritic cells and other dendritic cell subsets. Hum Immunol 63:1156–1163 Ludewig B, Krebs P, Junt T, et al (2003) Dendritic cell homeostasis in the regulation of self-reactivity. Curr Pharm Design 9:221–231 Manuel SL, Rahman S, Wigdahl B, Khan ZK, Jain P (2007) Dendritic cells in autoimmune diseases and neuroinﬂammatory disorders. Front Biosci 12:4315–4335 Mehling A, Beissert S (2003) Dendritic cells under investigation in autoimmune disesases. Crit Rev Biochem Mol Biol 38:1–21 Penna G, Giarratana N, Amuchastegui S, Mariani R, Daniel KC, Adorini L (2005) Manipulating dendritic cells to induce regulatory T cells. Microbes Infect 7:1033–1039 Tarner IH, Fathman CG (2006) Does our current understanding of the molecular basis of immune tolerance predict new therapies for autoimmune disease? Nat Clin Pract Rheumatol 2:491–499 Thomson AG, Thomas R (2002) Induction of immune tolerance by dendritic cells: implications for preventative and therapeutic immunotherapy of autoimmune disease. Immunol Cell Biol 80:509–519 Xiao BG, Huang YM, Link H (2006) Tolerogenic dendritic cells: the ins and outs of outcome. J Immunother 29:465–471

6. Dendritic Cells in Tumor Immunology

Basic Principles of Tumor Development General Features Tumorigenesis, a multistage process, is regulated by mechanisms such as genetic factors of the host, various environmental factors, and different host-derived factors. Cancer development represents an outcome of the process of tumorigenesis. In this chapter, we use the terms tumorigenesis and carcinogenesis to indicate the process of cancer development. A cancer mass may become clinically visible in a genetically susceptible host under the inﬂuence of different procarcinogenic environmental factors. A series of mutations and/or epigenetic changes is required to drive the transformation of a normal cell into a malignant tumor cell. When a cell is exposed to chemical, physical, or microbial carcinogens, initiation of carcinogenesis usually begins with DNA damage. The next phase is dependent on repair of damaged DNA. If not repaired, DNA damage could produce genetic mutations. The majority of these DNA alterations do not lead to cancer risk because these alterations represent an essential part of the normal life cycle. However, the damage of critical genes could be lethal, and sometimes the mutations create a growth advantage for a cell because of increased proliferation or reduced cell death. The most relevant in this regard are mutations activating proto-oncogenes and inactivating tumor suppressor genes. In the next phase, clonal expansion of an altered cell results in the formation of preneoplastic cells producing nodules, polyps, or papillomas. Finally, the progression stage is characterized by the transformation of preneoplastic cells into malignant tumor invading surrounding tissues and forming metastases. Angiogenesis is also essential for growth of cancer. Some investigators have shown that tumor cannot develop beyond 1–2 mm in diameter without induction of angiogenesis. To invade the surrounding tissues, epithelial tumor cells have to lose some adhesion molecules that keep them attached to each other. Also, the cancer should produce enzymes able to dissolve the elements of the basement membrane. It is not possible to determine when tumorigenesis of a cell begins in vivo. Accordingly, the duration between start of tumorigenesis and detection of clinically visible tumor cannot be conﬁrmed in humans. Circumstantial evidence indicates the time between the start of tumorigenesis and development of clinically visible cancer is highly variable. In some cases, long-stage processes of tumorigenesis proceed before a cancer becomes clinically visible. However, other cancers can grow very rapidly. 105

106

6. Dendritic Cells in Tumor Immunology

Most of the tumors are antigenic, even if they may not be completely immunogenic. Many tumor cells express molecularly deﬁned antigens that are recognized by cytotoxic and/or helper T cells. Moreover, manipulation of the immune system can lead to complete tumor eradication. In spite of these characteristics of tumor cells, the normal immune system is rarely a signiﬁcant barrier to tumor growth and development. In most circumstances, tumor develops in the presence of an apparently competent host immune system. If these factors are considered, it becomes apparent that DNA damage is not properly repaired in some cancer-developing hosts. In addition, the antioncogenic factors cannot block tumorigenesis in some patients. Angiogenesis probably helps further the growth of cancers. Finally, immune mechanisms may fail to restrict the development of tumorigenesis. Immune Surveillance Mechanism in Cancers Tumorigenesis is a normal event in the life history of a living organism. In some individuals, clinically detectable cancers are never seen. On the other hand, cancer grows unrestrictedly in some persons. The factors that control development of transformed cells to clinically visible cancers may be numerous, but a dominant one is immune surveillance. The concept of immune surveillance was ﬁrst proposed by Ehrlich in the early 20th century. It has been proposed that eradication of nascent transformed cells may be achieved before they are clinically detected. In the mid-20th century, Burnet suggested that immune surveillance mechanisms are responsible for detecting and eliminating tumor cells and that these represent a central mechanism by which tumor development is kept in check. They have postulated that the immune system constantly surveys the newly developing tumors and, so long as it is effective, prevents the development of neoplastic disease. It was assumed that clinically evident tumors represent exceptions that had slipped through the immunological net. The growth of arising tumor-like cells is controlled by the immune system in the majority of cases, and this may happen hundreds or thousands of times during our lifetime. However, sometimes the immune surveillance mechanism is evaded and malignant tumors are detected. The increased incidence of tumors in immunosuppressed recipients following organ transplantation, and in patients with inborn or acquired defects of the immune system, supports this concept. These ideas were supported by experimental results showing strong immunemediated rejection of transplanted tumors in mice. However, the role of cancer immune surveillance is not applicable in all situations. In fact, immune surveillance mechanisms prevent the outgrowth of tumor cells induced by potential oncogenic viruses, but the action of immune surveillance against chemically induced tumors is not common. Evidence also contradicts a clear role of immune surveillance during tumorigenesis. Although immune surveillance is important for preventing cancer development, immunosuppressed patients are not also at an increased risk of developing cancers. The most frequent epithelial malignancies such as breast, lung, colon, or prostate cancers are not common in immunosuppressed individuals. Intriguingly, the occurrence of some epithelial cancers is even reduced in human immunodeﬁciency virusinfected patients. With recent improvements of the viral detection system, it is

Dendritic Cells and Tumor Immunity

107

Table 1. Immune surveillance system against tumors (A) Need of immune surveillance against tumors 1. Tumor spreads similar to a massive infection. 2. Reappearance of tumors, either at the original site or in another location, is common. 3. All types of antitumor therapy can destroy or ablate only part of the tumors, but rarely can block recurrence of tumor. 4. Cells at the initial state of tumorigenesis can neither be detected nor destroyed by available therapeutic strategies. (B) Role of immune surveillance mechanism on tumorigenesis 1. Immune surveillance mechanism would patrol the body to detection of transformed cells or tumor cells. 2. The cells and mediators of immune surveillance system are capable of mobilizing at the site of tumor cells. 3. After recognizing tumor cells, immunocytes can activate cells of innate immunity. 4. Subsequently, antitumor adaptive immunity is induced. 5. Cells of the immune surveillance system provide environments for activity of other effector immunocytes so that development of tumor can be controlled.

becoming clear that tumors that develop in patients with defects in the immune function are mostly associated with viral infections. It seems that the real meaning of immune surveillance is yet to be well understood in the context of various cancers. In spite of evidence against the entity of immune surveillance, the concept of immune surveillance has been revived recently by observations that spontaneous as well as chemically induced tumors develop more frequently in genetically immunocompromised (knock-out) animals. Thus, newer insights are developing regarding the role of immune surveillance in different types of cancers. This chapter of this book is not intended to provide a detailed analysis about the controversies of immune surveillance in cancers. As in any dynamic branch of science, it will take more time to speciﬁcally deﬁne the actual role of immune surveillance in cancer. Immune surveillance may not be a universal phenomenon for all types of cancers. However, circumstantial evidence indicates that immune surveillance is an integral part of control of tumorigenesis in humans (Table 1).

Dendritic Cells and Tumor Immunity General Features and Dendritic Cells (DC) in the Context of Cancer Immunity We have already described the phenotypes, tissue distribution, physiological behavior, and functions of DC in other chapters of this book. However, here we discuss some relevant features of DCs presented in each chapter of this book so that readers can follow the role of DCs in the context of that particular chapter. DCs are bone marrow-derived antigen-presenting cells (APC) and are widely distributed in the body. They have been detected from most tissues in humans and mice. Different types of DCs are present in vivo, and they express different types of surface antigens. DCs or DC-like cells are found as DC progenitors, DC precursors, immature DCs, mature DCs, killer DCs, exhausted DCs, immunogenic DCs, and tolerogenic

108

6. Dendritic Cells in Tumor Immunology

DCs. Different forms of DCs are present in different tissues and at different compartments of the same tissues. They are also highly heterogeneous in the context of their levels of maturation and functions. Some DCs are key players of innate immunity and are capable of inducing killing of transformed cells and cancer cells. They can also induce different cytokines and instruct other cells of the innate system to produce immune mediators to handle cancer cells. DCs are also regulators of adaptive immunity. The immature tissue-derived DCs are usually in antigen-capturing mode, and recognize, capture, and internalize tumor cells or their antigens. After capture, tumor cells and tumor-associated antigens (TAAs) are processed in the endosomal compartment of DCs. If an inﬂammatory environment or danger signals are present in the microenvironment of DCs, DCs may undergo maturation and migrate to lymphoid tissues. In the lymphoid tissues, DCs present antigen to T cells and also B cells at the immunological synapses. Experimental data have shown that presentation of TAA by DCs may induce TAA-speciﬁc immunity or TAA-speciﬁc immune tolerance. When the ﬁrst edition of this book was written, little was known about the role of DCs in the induction of regulatory T cells and immunogenic tolerance. Now, several studies have shown that the nature, doses, and afﬁnity of antigens, tissue microenvironments, and various immune-related signals critically regulate whether antigen presentation by DCs leads to induction of immune responses or immunogenic tolerance. In addition, regulatory DCs have been detected in mice, although their phenotypes are yet to be described in humans. Generally, it is assumed that in presence of proinﬂammatory cytokines or danger signals, antigen presentation by DCs causes immunity, whereas in presence of antiinﬂammatory cytokines or in the absence of danger signals, immune tolerance is induced. However, the immunological events are not so simple, and induction of immunity or tolerance is tightly regulated at different stages of signal transduction. These factors are highly important in the context of development of DC-based immune therapy against cancer, which is discussed in a later section of this chapter. Plasmacytoid DC (pDC), a subtype of DCs, have a dominant role during immune responses against transformed cells, precancerous cells, or cancers cells. pDCs produce abundant amounts of type 1 interferon (IFN) and possess a direct role in innate immunity. Normally, pDCs favor a T-helper 2 type of polarization. However, in the presence of microbial agents or danger signals, they can induce Th1 polarization. Recruitment of DCs at the site of transformed cells may be another crucial factor for tumor immune surveillance. DCs express various types of receptors to recognize the presence of danger signals. Among them, pattern-recognition receptors (PRRs) are most important. Different types of PRRs are expressed on different subsets of human and murine DCs. Also, DCs can activate macrophages. Several studies have shown that there is an intimate crosstalk between DC and natural killer (NK) cells. DCs as Scanners of Tumor Cells In Vivo DCs act as sentinels of the immune system. In this regard, these cells posses immune scan properties. Tumorigenesis can be compared with chronic infections. The tumorigenesis begins with the transformation of normal cells to tumor-like cells. In the course of time, the tumor-like cells develop progressively. Although different genetic

Dendritic Cells and Tumor Immunity

109

factors and environmental factors are capable of inducing tumorigenesis, the process should be controlled by cells of immune system. As shown in Table 2, DCs represent one of the best scanners in vivo in the context of tumorigenesis. DCs are tissue resident cells, and they are highly mobile. They are also capable of recognizing microbes, their antigens, and abnormal cells (dead cells, altered cells, transformed cells, and tumor cells). They produce various cytokines and can activate cells of innate immunity. They are also capable of internalizing transformed cells or cancer cells to induce antigen-speciﬁc immune responses. DCs in vivo should act as the sentinels of the immune system and scan whether any cells are undergoing malignant transformation. The recognition of transformed cells or tumor cells is vital for subsequent antitumor immunity. It is true that different factors are related to the development of clinically apparent tumors, but impaired function of DCs or defective interactions between DCs and tumor cells may give rise to clinical cancers. A clinically visible cancer indicates that the immune scanning properties of DCs failed to work properly as a result of some factors (summarized in Table 3). With time, tumor cells grow unrestrictedly, and they may produce numbers of mediators that can downregulate the scanning capacity of DCs; this allows further development of tumors. The scanning property of DCs is not only important during tumorigenesis but also during immune therapy of tumors. Generally, tumor masses are ablated by surgery

Table 2. Dendritic cell as scanner of tumor cells 1. Dendritic cells (DCs) are widely distributed in almost all tissues of the body. Thus, they are capable of scanning the body for presence of tumors. 2. Capable of mobilization at the tissue of tumor formation. DCs can sense the presence of tumor and migrate to tumor tissues. 3. DCs can sense the presence of altered or transformed cells due to presence of specialized receptors of DCs. 4. DCs produce innate cytokines by themselves and activate cells of innate immunity. Thus, the growth of tumors may be blocked by mobilization of DCs at the site of tumor growth. 5. Recognition of tumor cells by DCs may initiate cascades of immune responses, and ﬁnally tumor-speciﬁc T cells and B cells would be formed. 6. DCs are also capable of inducing tumor-speciﬁc memory T cells for control of recurrence of tumor.

Table 3. Apparent failure of scanning functions of dendritic cell during tumorigenesis 1. Defective functions of dendritic cells before development of tumor caused by precancerous diseases. The etiological agents of tumors may interfere with functions of dendritic cells. 2. The process of tumorigenesis leads to impaired functions of dendritic cells. 3. Tumor-associated antigens may undergo mutations: dendritic cells become incapable of recognizing mutated tumor antigens. 4. A single tumor may have several antigens; some of them remained masked and others may not be recognized by dendritic cells. 5. Engulfment of tumor cells may cause apoptosis and other functional defects of dendritic cells: successive steps of immune responses may be distorted. 6. Impaired functions of dendritic cells in tumor microenvironments.

110

6. Dendritic Cells in Tumor Immunology

or destroyed by different types of antitumor approaches. However, complete removal of the tumor mass is not achieved in most cases, which leads to regeneration of the tumor. If DCs of the tumor-bearing hosts possess efﬁcient scanning functions, it may be possible to block progressive development of cancers after traditional anticancer therapy. Recurrence of cancer can also be blocked. Putative Roles of DCs in Tumor Immune Surveillance: A Complex Issue in Immunity As immune scanners, DCs can recognize transformed cells or precancerous cells at their early stage. As professional APCs, DCs can induce innate and adaptive immunity and may block further progression of tumorigenesis. In addition, some DCs expressing FasL are able to kill target cells in vitro. These DCs have been termed “killer DCs.” However, their capacity to destroy the target cells or tumor cells has not been directly evaluated in vivo. Recently, another population of DCs has been characterized in mice. These DCs are called interferon-producing killer DC (IKDC). They can kill target cells and are also capable of antigen presentations. The functional implications of killer DCs and IKDCs have not been studied in humans; however, experimental evidence supports dominant roles of these cells during immune surveillance. If tumor cells escape killing by killer DCs and IKDCs, DCs can induce anticancer adaptive immunity after recognizing and internalizing transformed cells. If DCs can recognize the transformed cells and induce potent antitumor immune responses, growth of tumors may be controlled. However, little is known regarding the role of DCs during the early phase of tumorigenesis. The major problem is related to the absence of a precancerous state of different types of tumors. Accordingly, the role of DCs in immune surveillance could not be well characterized in different models of cancers. The role of DCs in tumor immune surveillance should be cautiously explained. Analyzing the functions of DCs in patients with cancers, it is usually postulated that the immune surveillance capacities of DCs are distorted. However, this is an oversimpliﬁcation of a very complex issue. Indeed, impaired functions of DCs in patients with established cancers do not represent the activities of DCs during initial phases of cancers because the functions of most immunocytes are impaired in patients with established cancers. In addition, the allostimulatory capacities of DCs are evaluated as representative functions of DCs. However, the allostimulatory capacities of DCs, although representing nonspeciﬁc immune-activating capacities of DCs, are not representative of their functions in cancer immune surveillance. The allostimulatory capacities of DCs do not represent the antigen-recognizing, -capturing, and -processing capacities of DCs. Moreover, DCs with increased allostimulatory capacities are not able to induce antigen-speciﬁc activation of T cells in all circumstances. It may not be possible to use TAA for evaluation of the functions of DCs from tumor-bearing hosts to assess the real roles of DCs during cancer immune surveillance. In general, inﬁltration of DCs in tumor nodules, especially that of mature DCs, is considered as a good prognostic marker. If mature DCs are present in tumor nodules, it is usually considered that DCs are either helping killing of tumor cells or also inducing antitumor immunity. Indeed, evidence supporting this concept is also accumulating because increased localization of DCs in tumors has been found to be related to

Dendritic Cells and Tumor Immunity

111

a better prognosis. However, this concept has not been accepted, and many investigators have shown that mature DCs can be present in tumor tissues with no protective effect. The discussion that we have presented so far provides an impression that DCs possess tumor-scanning properties, produce inﬂammatory cytokines, induce various cytokines from other cells of the innate immune system, and are capable of recognition, internalization, processing, and presentation of antigens for induction of adaptive immunity. Taken together, it appears that DCs are a committed population with anticancer activities. However, DCs represent a highly heterogeneous type of cells with different phenotypes and functions. Thus, even if the main function of DCs is to play an anticancer role in vivo, these cells may also have carcinogenic properties under certain conditions. These properties should be discussed in detail because the present regimen of DC-based therapy is not yielding proper anticancer effects. To develop better therapeutic regimes using DCs, all types of functional capacities of DCs should be addressed and conﬁrmed. In this context, we also discuss putative procarcinogenic roles of DCs. Putative Roles of DCs in Tumorigenesis/Carcinogenesis It is generally accepted that DCs play key roles during cancer immune surveillance; however, DCs may also have some activities during tumorigenesis (Table 4). During the initial phages of tumorigenesis, transformed cells or precancerous cells come in contact with immature DCs. At this phase, proinﬂammatory cytokines or danger signals may be absent during the encounter of tumor products and DCs. There is a strong possibility that presentation of TAA or precancerous cells by immature DCs would lead to production of regulatory T cells and immune tolerance, which may allow cancer cells to evade immune surveillance. Once immune tolerance is developed to TAA-associated antigens, it is difﬁcult to induce anticancer immunity during successive challenge with same TAAs. Thus, the production of regulatory DC and regulatory T cells as a result of interactions of tumor products and DCs may hinder immune surveillance properties of DCs. The strategies of DC-based therapy should be modiﬁed if these events can be conﬁrmed in patients with cancers. Several cancers develop from a precancerous state. The precancerous tissue usually suffers from chronic inﬂammatory changes. However, the inﬂammatory changes are not uniform over the entire tissue. Inﬂammatory mucosal milieu of different tissues may induce nonspeciﬁc maturation of tissue-derived DCs. Nonantigen-speciﬁc

Table 4. Putative role of dendritic cells in the progression of tumors 1. Recognition of tumor cells by pattern recognition receptors of dendritic cells may not provide adequate dangers signals for immune responses. 2. Tumor cells may interact with regulatory dendritic cells. 3. Dendritic cell–tumor cell interactions may lead to production of regulatory T cells. 4. Production of reactive oxidative species and other immune modulators by activated dendritic cells may act as procarcinogenic factors. 5. Other immunocytes, activated by dendritic cells, may produce procarcinogenic factors. 6. Promotion of angiogenesis by dendritic cell-derived factors.

112

6. Dendritic Cells in Tumor Immunology

maturation of DCs is fundamentally different from TAA-speciﬁc maturation of DCs. In the case of TAA-speciﬁc maturations, DCs become matured after recognition, internalization, and processing of TAAs. However, in the case of nonspeciﬁc maturation, DCs are matured by proinﬂammatory cytokines in tissue microenvironments; this may be a common feature in subjects with chronic tissue inﬂammations. Once DCs are matured in a nonspeciﬁc manner, activated DCs may produce mutagenic reactive oxygen species (ROS) or nitric oxide. These substances may cause DNA damage and neoplastic transformation. Production of ROS and nitric oxide is not a special matter during the life cycle of a host. Different phagocytic cells may produce ROS by to engulfment of different tissues. However, professional phagocytic cells have evolved perfect mechanisms of limiting the extracellular spill of ROS. The tumorigenic effects of DCs results from the fact that these are professional APCs, but not professional phagocytic cells, and the phagocytic apparatus of DCs is not so perfect. Phagocytosis is only part of the DC function and is not a main function of DCs. Although more studies are required in this context, defective phagocytosis of different types of tissues by activated DCs may be related to increased risk of carcinogenesis. DCs are producers of different cytokines and also induce several cytokines from different types of immunocytes including NK cells, NKT cells, macrophages, and activated T cells. One of the functions of these cytokines is to induce increased cell turnover rate, impaired differentiation, and disorganized apoptosis, which may cause errors during DNA synthesis. Some of the putative roles of cytokines have been shown in malignancy in mice. Prolonged secretion of IL-15 enhances the formation of lymphomas in mice. TNF-α is essential for the elicitation of skin carcinomas by chemical promoters, IL-6 is necessary for the development of carcinogen-induced lymphoma, and macrophage migration inhibitory factor (MIF) antagonizes the function of p53. It is too early to indicate that cytokine produced by DCs may have tumorigenic properties, because many proinﬂammatory cytokines have also been used for treatment of experimental tumors in mice. However, the roles of these cytokines in the initial stage of tumorigenesis may determine the whole process of cancer development. It is commonly believed that immunocytes inﬁltrating the cancer tissue may have an anticancer effect because these cells can destroy cancer tissues. Although these cells are usually believed to be anticancer, there is no evidence to substantiate these claims. The still unanswered question is whether these cells are involved in promoting progression of cancers or in the destruction of tumors. The protective role of inﬂammatory cells in the development of tumors has been demonstrated in a series of studies. However, the intensity of the leukocyte inﬁltrate still remains of unproved prognostic signiﬁcance. Although macrophages and neutrophils are capable of phagocytosis and antibody-dependent cellular cytotoxicity toward tumor cells and can secrete a number of cytokines, there are an equal number of studies that implicate the role of these cells in the neoplastic progression. In addition to these effects, DCs can compromise immune surveillance mechanism against tumors by activating macrophages, neutrophils, and eosinophils because these cells have shown both anticancer as well as protumorogenic effects. Moreover, transformed cells or the early stage of tumor nodules can acquire more aggressive growth by producing chemokines that attract inﬂammatory cells. The

Dendritic Cells and Tumor Immunity

113

“tumor-friendly” mediators released by these cells may act back and stimulate tumor cell proliferation or inﬂuence angiogenesis, invasion, and metastasis (numerous chemokines). Moreover, several mediators (PGE2, TGF-β, VEGF) are capable of preventing the development of speciﬁc immune response against tumors. Taken together, it seems that the process of tumorigenesis may be aggravated by DCs under certain conditions. In one hand, DCs are essential for tumor immune surveillance. On the other hand, DCs can induce tumorigenesis. The situation becomes more complex because there are several types of DCs. Also, DCs with different maturational states are present in vivo. More studies are required regarding these factors, especially in the context of development of DC-based immune therapy against tumors. Impaired Innate Functions of DCs During Tumorigenesis Initially, it was assumed that DCs are regulators of adaptive immunity because most of their functions were related to induction of antigen-speciﬁc immunity. It is now evident that DCs also can act as cells of innate immunity. They can produce different cytokines including type 1 IFN. Moreover, they are capable of directly killing target cells. In addition, DCs can activate other cells of innate immunity. The functions of DCs are impaired in subjects with tumors; however, it is not still clear whether DC functions are impaired at the early stage of tumorigenesis or distorted at a later state. In vitro studies have shown that cytokine production by DCs is downregulated in cultures containing tumor cells or cancer cell lines. Thus, evidence indicates that tumor cells may compromise the innate functions of DCs. However, little is known about the effects of tumor cells on DCs at the initial state of tumorigenesis in vivo. Theoretically, transformed cells may not be recognized by DCs because of impaired interactions between pathogen-associated molecular patterns (PAMPs) of transformed cells and PPRs of DCs. In fact, the expression of PAMPs by transformed cells is elusive. Again, transformed cells or precancerous cells can directly diminish the innate immune functions of DCs. Also, DCs in the initial tumor microenvironment may not be able to activate other cells of innate immunity. Little is known about the effector functions of killer DCs and IKDCs during tumorigenesis. Tumor cells may produce various types of antiinﬂammatory cytokines and immune mediators. These substances may downregulate the innate functions of DCs. An account of tumor cell and DC interactions and their consequences is shown in Table 5. Defective Capacities of DCs to Induce Adaptive Immunity During Tumorigenesis The process of tumorigenesis can be prevented at an initial stage if the tumor cells or transformed cells are recognized by host DCs. Recognition of tumor cells by DCs would cause antigen processing and activation of DCs, which is supposed to follow induction of adaptive immune responses against the tumor cells; this may kill tumor cells by effector lymphocytes. Also, an immune surveillance mechanism may be induced that can block further progression of the process of tumorigenesis. To block the process of tumorigenesis, the DCs should perform several activities in a coordinated manner. However, tumors develop and clinically visible tumors are detected, which indicates that DCs are incapable of inducing adequate levels of tumor-speciﬁc

114

6. Dendritic Cells in Tumor Immunology

Table 5. Interaction between dendritic cells and tumor cells and development of clinically visible tumors (A) 1. 2. 3. 4.

Impaired innate immunity Decreased production of innate cytokines by DC. Decreased activation of other cells of innate immunity. Diminished expressions of pattern recognition receptors by DCs. Impaired interactions between pathogen-associated molecular patterns and pattern recognition receptors in tumor microenvironment.

(B) Impaired antitumor adaptive immunity A. Deceased mobilization of DCs and their progenitors from bone marrow 1. Diminished production of DC progenitors and DC precursors at bone marrow. 2. Impaired expressions of chemokine and chemokine receptors in cancer patients. 3. Effect of tumor cells in the bone marrow and tissues. B. Diminished recognition of tumor cells by DCs 1. Tumor cells with decreased pathogen-associated molecular patterns (PAMPs) or PAMP-like molecules. 2. Distorted expression of pattern-recognition receptors (PPRs) on DCs. 3. Tumor microenvironment. C. Impaired internalization and processing of tumor cells 1. Presence of tumor cells may affect phagocytosis, endocytosis, and pinocytosis of DCs. 2. Loading of tumor cells to MHC class I and class II compartments downregulated. 3. Tumor cells may induce apoptosis of DCs. D. Diminished migration of DC to lymphoid tissues 1. Impaired expression of chemokines and chemokine receptors. E. Diminished T-cell stimulation 1. Decreased migration of DCs from tumor tissues to lymphoid organs. 2. Inability of DCs to activate T cells. 3. Alteration of lymphoid microenvironments. 4. The nature of TAA may not support antitumor immunity.

immunity in situ. We discuss next possible mechanisms underlying ineffective antitumor immunity following DC–tumor cell interactions (see Table 5). Defective Mobilization of DCs in the Tumor Microenvironment To accomplish adequate levels of antitumor immunity, different immunocytes, especially DCs, should mobilize at the site of tumorigenesis. Mobilization of sufﬁcient numbers of different types of DCs is dependent on various factors: (1) development of DC progenitors and DC precursors in the bone marrow, and (2) migration of DC precursors from the bone marrow to the blood and then from the blood to the tissues. In addition, some DC precursors in tissues may also differentiate into immature DCs based on the local microenvironment. Mobilization of DC progenitors and DC precursors from the bone marrow and blood to the tissue of localization of tumor-like cells would require the expression of chemokines and chemokine receptors at the tissues of localization of tumor-like cells and DCs. Also, DCs migrate to tissues in response to the presence of inﬂammatory factors in the tissues. When tumorigenesis proceeds in tissues, the expression of chemokine receptor may be diminished. Moreover, changes in the local tissue microenvironment may affect the mobilization of DCs from

Dendritic Cells and Tumor Immunity

115

blood and bone marrow at the site of tumorigenesis. For example, high levels of IL-6 in local tissue may convert DC progenitors and DC precursors toward a macrophage lineage, whereas GM-CSF would ensure transition to immature DCs. If the tumor cells or transformed cells do not provide danger signals, DCs may not be mobilized at the tissue; this might also occur if the amounts of TAAs are very small or TAAs remain sequestered in the tumor-like cells. Inadequate production of cytokines or expression of other relevant proteins in tissues harboring the tumor cells may also result in defective DC mobilization. Defective expression of chemokines in tissues and chemokine receptors on DCs may disrupt the normal mobilization of DCs in response to tumor cells. Even the local microenvironment might be distorted in these tissues in such a way that the signals from the local tissue might be disrupted, which may happen in the cirrhotic liver and in the lungs of heavy smokers. Thus, in spite of having an adequate pool of DC precursors in the bone marrow and blood, these DC precursors might not be able to mobilize at the tissues during tumorigenesis. The mobilization capacity of DCs would be further worsened if the function of DCs, or the expression of chemokines or their receptors, were already impaired by a prolonged pathological process in the premalignant state. Thus, tumor-like hepatocytes in patients with liver cirrhosis or gastric epithelial cells in patients with gastritis or inﬂamed lung in heavy smokers might not be able to induce proper signals for DC mobilization. The alteration of tissue architecture in these premalignant states may also obstruct DC mobilization by blocking migration of DC precursors through vessels and by inducing an abnormal expression of chemokines. Many of these events regarding early mobilization of DCs in tumor tissues could not be shown by experimental evidence. However, these are major areas of research interest because clinical application of DCs for therapy and their success is dependent on better understanding of these factors. This scenario is not only true for the induction of immune response to so-called putative premalignant cells or tumor-like cells but is equally valid in the context of developing tumors. When a tumor attains a considerable size, huge numbers of DCs should be mobilized at the site of the developing tumor. Although we are discussing mobilization of DCs at the site of tumors, mobilization of other immunocytes including TAA-speciﬁc cytotoxic T lymphocytes (CTLs) is essential for the eradication of tumor. Taken together, the following points are important in the context of improper mobilization of DCs and DC-related immunocytes at the site of tumor cells. 1. The rate of hematopoiesis, especially production of DC precursors, may be altered during the process of tumorigenesis. This is especially important if cancer cells are present in the bone marrow. The presence of metastatic cancer cells will complicate the situation. 2. There may be an inherent limitation of tumor cells to provide adequate danger signals for induction of innate as well as adaptive immunity. The expression of PAMPs or PAMP-like molecules in tumor cells has not been well characterized. It is also tempting to asses if virus-related tumor cells express virus-related PAMPs or if these tumor cells lose these PAMPs during tumorigenesis. For example, hepatocytes infected with hepatitis viruses express different types of PAMPs. The expressions of PAMPs by malignant hepatocytes have not been assessed. However, indirect evidence indicates that the expression of PAMPs may be downregulated in cancer cells, because

116

6. Dendritic Cells in Tumor Immunology

many cell-surface antigens including MHC antigens are highly downregulated in many patients with tumors. However, it is not clear whether these factors affect antitumor adaptive immune responses. 3. The intensity and the magnitude of expression of chemokines and chemokine receptors on blood vessels, tissues, DC precursors, and immature DCs may not be adequate for induction of antitumor adaptive immune responses. 4. The proinﬂammatory cytokines and immune modulators may not be available at the site of localization of the initial state of tumorigenesis. Also, various antiinﬂammatory cytokines may not allow DCs to be immunogenic at these circumstances. 5. Developing tumors may have a capsule, and these capsules may block migration of DCs to the site of tumorigenesis. For example, in cirrhotic liver and in capsulated HCC, the capsules may form a formidable barrier for the entry of DCs. 6. Migration of regulatory DCs may also hinder adaptive antitumor immune responses. Taken together, the normal body homeostasis and anticancer immunity are maintained by a highly regulated migration of DCs or their progenitors or precursors to tissue of tumorigenesis. However, this may be impaired by multiple factors, such as the direct effect of tumorigenesis or alteration of tissue microenvironments during tumorigenesis. Mobilization of DCs is also important in the context of DC-based therapy against cancers. Defective Recognition and Internalization of Tumor Cells by DCs Impaired mobilization of DCs and other immunocytes at the site of tumorigenesis may compromise the adaptive antitumor immunity of the tumor-bearing hosts. In addition, interactions of DCs with tumor cells are also important determinants of tumor-speciﬁc adaptive immunity. At the initial phase of tumorigenesis, DCs get signals from the tissue harboring transformed cells or premalignant or malignant cells to mobilize at the affected organs. After mobilization, tumor cells should be recognized by DCs and it is one of the most important steps in immune surveillance. The recognition of tumor cells by DCs should be mediated by interactions between receptors of DCs and ligands of tumor cells. Different PRRs of DCs may act in this respect. Although interactions of PRRs or DCs and PAMPs of microbial agents have been analyzed, little is known about the method of recognition of tumor cells by DCs. Impaired expression of various recognition molecules by tumor cells or defective expression of recognition receptors by DCs will compromise tumor cell–DC interactions. The role played by the tumor microenvironment is also important in this context. After recognition of tumor cells or TAAs, DCs should internalize those for induction of tumor-speciﬁc adaptive immunity. DCs use phagocytosis, endocytosis, and pinocytosis for capturing antigens. The levels of expression of these receptors on DCs have not been well evaluated in tissues harboring tumor-like cells. It is indeed extremely difﬁcult because the expression of these receptors is extremely heterogeneous among different subsets of DCs. Also, the expression of antigen-capturing apparatus is different among tissues. The nature and functions of the antigen internalization apparatus of DCs in tumor-bearing tissue may be impaired. It is also

Dendritic Cells and Tumor Immunity

117

unknown if defective engulfment of transformed cells by DCs underlies the defective antigen-speciﬁc immune response in these situations. Although there is little information regarding the behavior of DCs in the microenvironment of transformed cells, the tumor microenvironment is usually hostile for proper functioning of DC. Tumors produce a variety of cytokines and mediators that may compromise the antigen-capturing capacities of DCs. In addition, the antigen-capturing capacities of DCs may be variable in the context of tumor cells, TAAs, transformed cells, and others. The viability of DCs and functional capacities of DCs after capturing tumor antigens are also important in the context of cellular events that happen subsequent to antigen capture. Engulfment of tumor cells may induce apoptosis of DCs. Finally, even after engulfment of tumor cells or TAAs, DCs may just ignore these cells because of a lack of danger signals. The following points regarding recognition and capture of tumor cells or transformed cells by DCs might be relevant in the context of tumor immunity. 1. The precise mechanism of capture of tumor cells and TAAs by DCs is largely unknown. 2. Also, expression levels of different PRRs on DCs are not known. 3. Expression of PAMPs or similar antigen recognition molecules by tumor cells is important in this context. 4. There are differences between capture of normal antigens and tumor antigens. 5. The existence and nature of danger signals in cancer microenvironments. If these are different from normal tissues, there is a need to characterize the factors. 6. Survival of DCs after capturing of tumor cells. 7. The roles of tumor-derived factors on antigen capture capacity of DCs. 8. It is important to develop insights if PRRs of DCs are involved in this process; identiﬁcation of ligands for PPRs in tumor-like cells and in malignant cells is important, as these may be highly variable depending on the nature and genesis of the transformed cells. For example, if microbial infections underlie the pathogenesis of tumor cells, relevant PPRs on DCs might be involved during recognition of antigen. Processing of Tumor Cells or TAAs by DCs TAA-speciﬁc immune response depends on the processing of tumor cells or TAAs by DCs. DCs are special APCs because they bear an extraordinary capacity to process internalized antigens. At least, studies in vitro have supported these concepts. However, many of these events have not been explored in situ and thus confusion prevails about the cellular events related to antigen presentation by tumor cells. It has been well known for a long time that both DCs and macrophages are able to capture and internalize tumor antigens or antigens, but induction of a primary immune response is mediated by DCs, not by macrophages. The main factor underlying this depends on the intracellular migration and processing of engulfed tumor cells or TAAs. Macrophages possess strong phagocytic receptors, but they are almost incapable of transmitting the internalized antigens into MHC class I or class II compartments. On the other hand, DCs can load the internalized antigens on the

118

6. Dendritic Cells in Tumor Immunology

endosomal compartment, which induces the formation of MHC class II compartment (MIIC). The migration of these MIIC to the surface of the DCs would allow the DCs to express the Ag along with self-MHC. One of the factors that compromise antitumor immunity may be related to improper delivery of antigens to the MIIC compartment. Also, the migration of the MIIC complex containing tumor-derived antigens to the surface of the DCs is also critically important. To have insights about the relationship between antigen processing by DCs and the induction of antitumor immunity by DCs, the following points are relevant. 1. 2. 3. 4. 5.

Intracellular digestion of tumor cells within DCs. Migration of TAAs to endosomal compartments of DCs. Formation of MIIC in the DCs. Mobilization of MIIC to the surface of DCs. Role of tumor-related cytokines and modulators in these events.

Defective Migration of DCs from Tumor Microenvironment to Lymphoid Tissue If DC precursors are mobilized properly in the tumor-harboring tissues, if they can capture and process tumor-like cells or TAAs efﬁciently, and if TAAs are expressed on the surface of DCs along with self-MHC antigens, these antigen-bearing DCs will move to lymphoid tissues to interact with clonally selected lymphocytes. Normally, uptake of antigens and successful processing of these antigens through endosomal compartments lead to activation of DCs, which is manifested by upregulation of costimulatory molecules such as CD86, CD80, and CD40. At the same time, the activity of the antigen-capturing apparatus of DCs and the expression of CC chemokine receptor (CCR)6 is downregulated. The expression of CCR7 is unregulated. Thus, antigen-capturing DCs turn into antigen-presenting DCs. It is possible that DCs expressing TAAs may not migrate to lymphoid tissue efﬁciently, but conclusive evidence regarding this is lacking. These pathways of DC activation have not been explored in vivo. It is true that DCs with defective maturation and activation status are detected in tumor tissues. However, it does not indicate that DCs expressing TAAs had defective migration. It is possible that tumor-derived factors may downregulate expression of chemokine and chemokine receptors. In addition, antiinﬂammatory cytokines may reduce migration of DCs. The following points may be relevant to migration of antigen-loaded DCs in tumor microenvironments. 1. Because of improper mobilization of DCs in tumor tissue, decreased recognition and internalization of tumor antigens by DCs, and diminished processing of tumor antigens by DCs, DCs may not enter into the antigen-presenting mode from the antigen-capturing mode in tumor microenvironments. 2. This alteration may result from the presence of various cytokines such as IL-10, vascular endothelial growth factor, and IL-6. These cytokines and modulators can inﬂuence the function of DCs at different levels of Ag presentation. Defective Presentation of TAAs by DCs in the Lymphoid Tissues Little is known about interactions between antigen-expressing DCs and T cells at lymphoid tissues. Some data about this are available from in vitro studies, but

Dendritic Cells and Tumor Immunity

119

conﬁrmation of these ﬁndings in vivo is difﬁcult. Normally, DCs interact with several T cells for prolonged periods of time, and thus many T cells can be primed by few DCs. However, the interactions between DCs and T cells in the context of cancer immunology are not known. The phenotype and maturation status of DCs in secondary lymphoid organs in patients with tumor should be explored to provide insight about this. However, this is a very difﬁcult task, because it is extremely difﬁcult to locate TAA-speciﬁc DCs in situ. Exploration of interactions between TAA-pulsed DCs and T cells is more difﬁcult. Again, similar studies have not been done in patients with early stages of tumor development. Thus, it is uncertain whether the lack of activated DCs in cancer tissues is a generalized defect of advanced cancer patients or whether this reﬂects some immunoregulatory defects of these patients to TAA. The following points should be considered regarding Ag presentation in the lymph nodes in the context of tumor immunology. 1. Enumeration of TAA-speciﬁc DCs by immunohistochemistry from lymphoid tissue. 2. Evaluation of frequencies of TAA-speciﬁc DCs in lymphoid tissues by ﬂow cytometry. 3. If TAAs are not well characterized, functional assays may be carried out to assess if DCs in the lymphoid tissues can activate lymphocytes in cultures. 4. Cytokine production capacities of DCs in culture also provide indirect evidence of T-cell stimulatory capacities of DCs. 5. Evaluation of frequencies of activated T cells and activated DCs in secondary lymphoid organs, especially during the early stage of cancers and in the precancerous state, is needed. Mature DC in Tumor Tissues Generally, the role of DCs should come to an end after interacting with lymphocytes and presenting TAAs to T lymphocytes in immunological synapses in the lymphoid tissues. However, presentation of tumor antigens may induce both immune responses and immune tolerances. Because of this, we discuss more about DCs in tumor immunology. After interacting with lymphocytes, DCs may undergo apoptosis as fully differentiated and mature immunocytes. However, mature DCs are detected in tumor tissues. It is not still clear whether these DCs have any role in antitumor immunity. First, the mechanisms of maturation of DCs at tumor tissues are not clear. However, this is clinically important because the absence or low frequencies of mature DCs have been reported in the tissues of most cancer patients (Table 6). DCs with markers of maturation and activation are detected in the tissues. Moreover, some studies have shown that the prognosis of cancer may be dependent on the frequencies of mature DCs, although recent studies have also cited data countering this. We focus on the following points about the role of mature DCs in cancer tissue. 1. DCs in the cancer tissues may undergo maturation at the local tissue by several means. Immature DCs may be matured by proinﬂammatory cytokines in the cancer tissue in an antigen-nonspeciﬁc manner. Many cancer tissues express high levels of TNF-α. This cytokine can induce maturation of immature DCs. The clinical signiﬁcance of these mature DCs would be minimal in the context of antitumor immune

120

6. Dendritic Cells in Tumor Immunology

Table 6. Clinical relevance of tumor-inﬁltrating dendritic cells (A) Localization and implications of dendritic cells in tumors 1. Less in tumor Tumors of oral, head, neck, oropharynx, salivary gland, arsenic skin, basal cells, cervix, hepatoma, melanoma, skin. 2. DCs with immature phenotype in cancers Tumors of thyroid gland, breast. 3. Inﬁltration of DCs: related to better prognosis Endometrial, larynx, nasopharyngeal, Oral and tongue, cervical, esophageal, gastric, lung, prostate. 4. Unrelated to prognosis: Oral squamous, bronchoalveolar. (B) Approach to develop insights about role of tumor-inﬁltrating dendritic cells 1. Checking of mature and immature DCs, also DC-like cells in tumor tissues: this may provide insights about regulatory DCs in cancer tissues. 2. Characterization of different subtypes of DC in tumor tissues: may provide idea if any speciﬁc types of DCs are affected in particular cancers. 3. Isolation of DCs from tumor tissues and evaluation of DC-related functions. 4. Characterization of tumor-associated antigen (TAA)-speciﬁc functions of tumor-inﬁltrating DCs.

responses, but they may provide an inﬂammatory mucosal milieu and thus help to establish immunogenic microenvironments. In addition, these DCs can produce different proinﬂammatory cytokines and downregulate the effects of antiinﬂammatory cytokines, which may be related to clinical observation of increased mature DCs and better prognosis of cancers. 2. After processing tumor antigens, some DCs expressing tumor antigens may not migrate to lymphoid tissues because of impaired expression of chemokines and chemokine receptors. These antigen-bearing DCs may express markers of maturation. However, they are not supposed to have any dominant role in antitumor immunity because they have not interacted with clonally selected lymphocytes at the lymphoid tissues. 3. Mature DCs may have come from the lymphoid tissues after presenting tumor antigens to T lymphocytes in the immunological synapses. In the lymphoid tissues, DCs and lymphocytes make a very strong contact by the effect of several adhesion molecules. When activated lymphocytes leave the lymph nodes and migrate to peripheral tissues, DCs may also come to the peripheral tissues with these lymphocytes. Whatever may be the mechanisms of localization of mature DCs in tumor tissues, the presence of these DCs has clinical signiﬁcance. These DCs can be located in tumor tissues by immunohistochemistry. However, it would be premature to explain their functional implications at this moment. Initially, it was reported that mature DCs at tumor tissues are related to better prognostic importance; however, recent studies also show that immature DCs in cancer tissues may relate to better prognosis of tumors. The functions of mature DCs in tumor tissues have not been well explored. Mature DCs are not related to recognition, capture, processing, and presentation of antigens,

Localization and Characterization of DCs in Tumors

121

meaning that these DCs are not designed to act as APCs. Although it is difﬁcult to precisely deﬁne their roles, these DCs may be important for performing some effector function of immune responses. The activated CTL and other activated T cells in the cancer microenvironment must survive to perform their functions, and several cytokines are required for this. Mature DCs may be essential for the survival and functioning of the CTLs in cancer tissues by secreting a wide variety of cytokines. The existence of mature DCs in cancer tissue is a reality, but exploration of the following points will provide insights about their real implication in tumor immunity. 1. The mechanisms of accumulation of mature DCs in tumor tissues are unclear. It is important to assess if they are matured in situ or they have migrated from other tissues including lymphoid tissues. 2. Very few mature DCs are seen in peripheral blood from both controls and cancer patients. It is important to ascertain the source of mature DCs in the peripheral blood. 3. The expression of chemokine and chemokine receptors at cancer tissues should be evaluated to assess the mechanisms of localization at cancer tissues. 4. The relationship between mature DCs with NK cells and CTLs should be assessed. 5. The functional capacities of mature DCs should be evaluated. 6. Mature DCs are usually deﬁned by expression of CD83 antigens, but other markers of maturation should be checked in these DCs. 7. T-cell polarization capacities of mature DCs in vitro should be analyzed. Concluding Remarks An effective antitumor immune response is needed for control of tumors and to establish an effective scanning system against recurrence of cancers. However, this is dependent on interaction between DCs and tumor cells. We have discussed various factors that may be relevant to improper antitumor immune responses in cancer patients. It is important to induce both innate immunity and tumor-speciﬁc adaptive immunity by DC-based therapy. However, understanding about DC–tumor cell interactions is vital to develop such therapeutic maneuvers. Although the production of regulatory DCs is still not clear in tumor microenvironments, several studies have shown that increased regulatory T cells are related to diminished immune surveillance capacities of cancer patients. The activities of regulatory DCs and production of regulatory T cells should be analyzed in cancer patients.

Localization and Characterization of DCs in Tumors DCs are localized in almost all peripheral tissues and lymphoid organs. In normal conditions, tissue DCs are responsible for recognition of tumor cells and their antigens. Subsequently, DCs perform varieties of functions, such as antigen internalization, antigen processing, and antigen presentation. In physiological conditions, immune responses or immune tolerances are induced by antigen presentation by DCs. The ultimate outcome of the nature of immunity is dependent on various

122

6. Dendritic Cells in Tumor Immunology

factors, and we have discussed these in detail during the description of general features of DCs. In tumor patients, the functions of DCs may be altered in tumor microenvironments. Thus, the scanning properties of DCs cannot act properly, and progressive growth of tumors is seen. Different types of DCs are detected in patients with cancers. The real implications of these DCs are not clear. These DCs may perform different types of functions and may have antitumor activity. Again, these DCs may be responsible for aggravation of tumor. Investigations are now ongoing about the implications of tumor-inﬁltrating DCs. DCs in Human Tumors Although immunohistochemical study represents the best method for identifying DCs in situ, it is impossible to detect all types of DCs in tissue sections by this approach. DCs are highly heterogeneous regarding their expression of surface antigens, and there is no DC-speciﬁc surface antigen. Accordingly, studies about DCs in cancer tissues have checked only certain types of DCs. The functional signiﬁcance of these DCs has not been evaluated, and it is elusive whether they have any role during initiation, progression, and complications of cancers. The limited clinical implications of localization of DCs in tumor tissues are evident from the low speciﬁcity of different DC-related antigens. The most commonly applied markers for DCs in human tissues identify the HLA class II molecules and the associated invariant chains, expressed in high density on the cell surface of DCs. The S100 families of intracytoplasmic calcium-binding proteins have traditionally been used to demonstrate dendritic populations such as Langerhans’ cells and interdigitating DCs in the lymph nodes. The speciﬁcity of staining is low because many other types of cells such as lymphoid cells, nerve, fat, carbohydrates, and melanocytes also express S100 protein. CD68 antigen has been found to be expressed on some DCs, but this is basically a marker of macrophages. CD68 is also expressed on developing myeloid cells and on various other tumor cells. Some of the immune accessory molecules, such as CD40, CD80, and CD86 and the adhesion molecules CD11c, ICAM-1, and ICAM-3 have also been detected in cancer tissues, but many non-DC cells also express these antigens. Newer molecules of interest for the study of DCs in situ include CD83 and CMRF-44 and CMRF-56. CD83 is unique for monocyte-derived cultured blood DCs. DCs have been detected in the tumor tissues from several malignancies, such as basal carcinoma of the skin, breast cancer, nasopharyngeal cancers, oropharyngeal cancers, laryngeal cancers, oral cancers, cancers of salivary glands, cancers of head and neck, bronchoalveolar carcinoma, cervical carcinoma, endometrial carcinoma, gastric cancer, hepatocellular carcinoma, Hodgkin disease, lung cancer, melanoma, prostate cancer, and thyroid cancer (see Table 6). Many studies have shown that the numbers of DCs are fewer in tumor tissue compared to noncancerous tissues and normal tissues of the same organs. For example, in patients with hepatocellular carcinoma, mature DCs expressing CD83 are not detected in cancer nodules. However, mature DCs expressing CD83 are detected in the adjacent cirrhotic nodules (Fig. 1). Immature DCs have mainly been localized in the tumor tissues in some malignancies. In several tumors, a relationship has been shown between the frequencies of DCs and the prognosis of tumors. However, most of the recent studies have shown that there

Localization and Characterization of DCs in Tumors

[A]

123

[B]

Fig. 1. Absence of CD83+ mature DC at hepatocellular carcinoma nodules [A]. Localization of mature DC at adjacent cirrhotic nodule [B]

may not be a relationship between the prognosis of tumors with the frequencies and nature of inﬁltrating DCs. Studies about the localization of DCs in cancer patients represent pioneer investigations about the DC–tumor relationship, and one of the causes of introducing DCbased therapy in cancers was initially validated because there were few DCs in cancer tissues. Also, the presence of DCs with impaired functional capacities showed the need of DC-based therapy against cancer. Now, several limitations regarding localization of DCs in cancer tissues have surfaced. None of these studies represents a randomized controlled study, and there is no control population in most studies. The tumor tissues were also collected from patients with different stages of tumors. Different types of monoclonal antibodies were used to deﬁne DCs. The criteria for determining prognosis are not also well characterized. Even with all these methodological and technical limitations, some ideas are developing about the tissue-inﬁltrating DCs in cancers. Our main intention is to apply different information about DCs for diagnostic, prognostic, and therapeutic purposes in patients with cancers. In this context, the diagnostic importance of DCs in cancer patients is not so bright. Also, the prognostic importance of DCs is controversial, although some studies have shown that DCs may have prognostic implications in cancer patients. DCs may have a role in the pathogenesis of cancers, but present approaches of detection of DCs in cancer tissues are unlikely to provide critical information about this. The purpose of detecting DCs in cancer tissue is still elusive. To demonstrate roles of DCs during initiation of tumorigenesis, DCs should be checked either at the precancerous state or during the early phase of cancers. Moreover, there are various subsets of DCs in situ with different types of functional capabilities. Some DCs induce immunity whereas others cause

124

6. Dendritic Cells in Tumor Immunology

tolerance. Although increased frequencies of DCs have been shown to be related to better prognosis, it is necessary to evaluate what types of DCs have been localized, whether immunogenic or tolerogenic. The authors assume that study about regulatory DCs may provide new information about the role of DCs in cancers. Low expressions of HLA DR and CD83 have been found in DCs in cancer tissues. Considering the role of DC in immunity, the absence of CD83+ cells has been regarded as few mature DCs occurring in cancers. These data can also be interpreted in another manner. The absence of CD83+ DCs may be related to the presence of abundant amounts of DCs that have not matured in cancer tissues. These DCs may represent regulatory DCs. Studies are required to solve these points. The functions of tumor-inﬁltrating DCs should be studied more elaborately. The functional study should not only include evaluation of nonspeciﬁc functions of DCs but also the antigen-handling capacity of DCs. It is true that these types of studies are extremely hard to conduct in human malignancies, but these limitations must be overcome to bring DCs from the bench to the bedside. Implication of Tumor-Inﬁltrated DCs in Tumor Tissues Both immature and mature DCs have been localized in cancer tissues from patients with different types of cancers. In some patients, mature DCs were few in tumor nodules but more numerous in adjacent tissues. In others, no deﬁnite pattern was detected about distribution of mature DCs. However, these studies provide no direct evidences about the role of DCs during initial states of tumorigenesis. Rather, they reﬂect the conditions of DCs from prolonged coexistence of DCs in cancer microenvironments. DCs in the tumor tissues can pick up TAAs and may shuttle these to lymph nodes for the induction of TAA-speciﬁc immunity. Although this seems to be too simplistic, experimental evidence either supporting or contradicting this concept is lacking. Indeed, the presence of increased numbers of immature DCs makes the cancer tissues a suitable site for capture of TAAs. However, it is not clear whether antigen presentation by these DCs would lead to immune responses or immune tolerance. If these DCs capture TAAs, the following points are important regarding the function of cancer-inﬁltrated DCs in the tumor tissues. 1. The mobilization of cancer-inﬁltrating DCs in cancer tissues and nature of signals required for their mobilization. 2. The expression pattern of PRRs on these DCs. Also, it is important to assess how these DCs recognize tumor cells. The expression of PAMP-like agents on tumor tissues, especially on early tumor-like cells, is not known. 3. Expression of TAAs by tumor-inﬁltrating DCs. Also, information is needed about different types of TAAs. 4. The functional capacities of antigen-capturing apparatus such as the capacity to capture TAAs by macropinocytosis, phagocytosis, and receptor-mediated endocytosis of tumor-inﬁltrating DCs need to be deﬁned. 5. The migratory capacities of tumor-inﬁltrating DCs should be analyzed. 6. The capacity of tumor-inﬁltrating DCs to undergo maturation and activation following uptake and processing of tumor antigen or tumor cells. 7. The ability of tumor-inﬁltrating DCs to stimulate T cells.

Localization and Characterization of DCs in Tumors

125

Cancer-inﬁltrating DCs may have a functional capacity similar to tissue-derived immature DCs. However, if they lose some of the fundamental features of tissue DCs, then it is likely that tumor antigens in the tissues will not be handled by tumorinﬁltrating DCs. For example, tumor-inﬁltrating DCs in some tumors may have potent capacities to capture TAAs but may be unable to undergo maturation in the cancer microenvironment. However, in some other cancers, the DCs may not be able to recognize TAAs, but may be able to undergo maturation in the cancer microenvironment. If the function of cancer-inﬁltrating DCs can be studied in precancerous state, more insights will be developed regarding the role of DCs in the initiation of tumorigenesis. This insight is also important for the development of DC-based therapy in cancer. DCs with immature phenotype have been detected in tumor tissues. The functional implications of these DCs have been shown. However, the presence of mature DCs in the vicinity of cancer nodules and the absence of mature DCs in cancer tissues provide indirect evidence that the cancer microenvironment may be hostile for the induction of maturation of DCs in situ. In addition, some in vitro studies have documented that cancer-derived factors block the maturation of DCs. Our present understanding indicates that mature DCs are formed at the lymphoid tissues, not at the peripheral tissues. Although mature DCs have been detected in the peripheral tissues from patients with cancers, the functions of these DCs have not been studied in detail. It is unknown whether these DCs have already captured TAAs and have undergone maturation. Also, it is unsure whether there is an antigen-independent pathway of maturation of DCs by the inﬂuence of local cytokines. It is not impossible that mature DCs in the tissues represent a defective population of DCs that have captured antigens, but were unable to move to lymph nodes as a result of ineffective expression of chemokines or their receptors. Whatever the mechanism of formation of mature DCs in cancer tissue might be, it is important to know their functions. If these DCs have not matured in an antigen-dependent manner, they may be a source of several cytokines in vivo. TAA-speciﬁc CTLs and other lymphocytes migrate to the cancer tissues, although these are formed in the lymphoid tissues. These activated lymphocytes are assigned to kill TAA-expressing tumor cells. However, these lymphocytes may also undergo apoptosis if certain cytokines are not present in the cancer microenvironment. DCs matured in vitro are a unique source of various cytokines and mediators. Mature DCs in the cancer nodules may have such a role for the survival and function of activated lymphocytes. A lack of mature DCs may thus make the activated lymphocytes more prone to be killed or to undergo apoptosis in vivo. Peripheral Blood DCs in Patients with Tumors Although tissue DCs have been characterized in patients with several tumors, it is not possible to collect tissue DCs from tumor patients for serial studies. Again, it is not possible to study the phenotypes, functions, and subtypes of tumor-inﬁltrating DCs, especially in human tumors. Moreover, it is not feasible to study the prognosis of DC-based therapy by analyzing tumor-inﬁltrating DCs because it is impossible to collect several tissue samples from cancer patients. One of the ways to overcome these problems is to use blood-derived DCs for functional studies. However, it is necessary to show whether there is any relationship between phenotypes and functions of

126

6. Dendritic Cells in Tumor Immunology

tumor-inﬁltrating DCs and blood DCs in tumors. To address this issue, investigators have enriched DCs from peripheral blood and tumor tissues from patients with tumors. DCs from different cancer patients have shown impaired allostimulatory capacity, defective maturation, and lower expression of costimulatory molecules such as CD86 and decreased capacity to produce IL-12, a key cytokine required for antitumor immunity. However, these studies have not checked the TAA-speciﬁc functional capacities of DCs. Blood-derived DCs are also important for DC-based therapy, which is discussed in more detail in this chapter and also in other chapters of this book.

Immune Therapy of Tumors Using Dendritic Cells Basic Principles and Design of DC-Based Therapy for Tumors Patients with clinically visible tumors harbor the tumor cells for a long time, varying from some months to many years. In spite of harboring tumor cells, the patients do not show effective antitumor immune responses. The tumors also are not destroyed and controlled. Patients with cancers are treated by traditional approaches, such as ablation of tumor, administration of anticancer drugs, or radiotherapy, but there are several limitations of these therapeutic approaches. In some cases, the traditional therapies can not be employed because of the patient’s condition, side effects of anticancer therapies, or localization of tumors in inoperable areas of the body. In others, the efﬁcacy of antitumor therapy is not satisfactory. Finally, the recurrence of tumors cannot be controlled by traditional anticancer therapy. Investigations for many decades indicate that naturally occurring defense systems should be established in cancer patients so that growth of visible cancer can be blocked and recurrence of cancer can be controlled (Table 7). Although immune therapy may represent such a therapeutic regimen, designing immune therapy for treatment of human cancer is needed. First, it is necessary to assess what type of immune interventional strategy would be useful for speciﬁc

Table 7. Scientiﬁc and logical aspects of dendritic cell-based immune therapy in tumor-bearing hosts 1. Antitumor immunity is impaired in cancer patients. 2. Data show that removal of tumor by ongoing therapies results in transient restoration of antitumor immunity. 3. Present antitumor therapies are incapable of complete destruction of cancer tissues and blocking cancer recurrence. 3. Functions of DCs are impaired in most patients with cancers. 4. Antigen-pulsed DCs can restore antigen-speciﬁc immunity in various animal models of human diseases. 5. Antigen-pulsed DCs can induce antigen-speciﬁc cytotoxic T cells (CTLs). 6. CTLs can kill cancer cells and establish anticancer immune surveillance mechanism. 7. Administration of antigen-pulsed DCs is safe for cancer patients. 8. Immune response can be induced against tolerogenic antigens by administration of antigen-pulsed DCs.

Immune Therapy of Tumors Using Dendritic Cells

127

cancers. We have discussed the guidelines and strategy of immune therapy including DC-based therapy in a separate chapter of this book. Here, we provide some outlines for DC-based therapy in cancer patients. There are several types of immunity that can be induced. However, immune therapy that induces cancer cell destruction, provides protective immunity, and induces long-term surveillance immune responses against cancers is required for cancer patients. Why DC-Based Therapy Is Considered for Treatment of Tumor As DCs are critical regulators of antigen-speciﬁc immunity in vivo, DC-based therapy has been considered for treating patients with cancers. DCs are professional APCs and capable of recognition, internalization, and processing of antigen from all types of human tissues. Antigen-bearing DCs are also capable of interacting with clonally selected lymphocytes. Finally, they can either activate lymphocytes or deactivate those for induction of immune responses or immune tolerances. The main purpose of DC-based therapy in cancer patients is not to perform effector functions but to act as inducers of cancer antigen-speciﬁc immunity. It is expected that if DC-based therapy can induce antitumor immunity in cancer-bearing hosts, it may be possible to generate CTLs that will destroy cancer cells. The action is also supposed to be inﬁnite. Also, if the surveillance type of immunity can be induced by administering DCs, that can block the recurrence of cancers. Is There a Need to Have Defective Function of DCs for DC-Based Immune Therapy? To validate DC-based immune therapy against cancers, initially it was mentioned that the functions of DCs in the peripheral blood of cancer patients were impaired. Next, DCs with impaired phenotypes and functions were detected in cancer tissues from cancer patients. Recent insights about DC and antigen-speciﬁc immune therapy indicate that impaired functions of DCs are not required for DC-based therapy in tumor patients. The functions of DCs may be affected by various means in tumor-bearing hosts. Most importantly, the DC functions that are evaluated provide very limited insights about the role of DCs in antitumor immunity. DCs of patients with tumors are detected immunohistochemically, but the subtypes of DCs are not detected, and the functional capacities of these DCs are not clear. Moreover, TAA-speciﬁc DCs are neither detected nor are their functions assessed. Even in most patients with tumors, the roles of different TAAs of a particular tumor are not understood. Alterations of functions of DCs may just reﬂect the role of the tumor microenvironment on immunocytes. Taken together, there is no scientiﬁc logic that the functions of DCs should be impaired for doing DC-based therapy. On the other hand, DC-based therapy is carried out to induce anticancer immunity capable of destroying tumor cells and also scanning the body against tumors. When a clinically visible tumor is seen, irrespective of the DC function of tumor patients, DC-based therapy can take place in these patients. Another notable idea is that DC-based therapy can induce antitumor immunity. The purpose is not to activate endogenous DCs of tumor patients. However, further study may be required to address various aspects of this issue.

128

6. Dendritic Cells in Tumor Immunology

Does It Make Sense to Use DC-Based Therapy When Anticancer Immunity Was Not Induced by Endogenous DCs of Cancer-Bearing Hosts? Tumor is clinically detectable when the immune surveillance system of the hosts fails to block the growth of cancers. As the sentinel and police of immune system, this also indicates a failure of DC activity in cancer patients. Thus, it is natural to ask if there is any beneﬁcial effect of DC-based therapy in cancer patients when endogenous DCs of cancer-bearing hosts could not block tumorigenesis. Experimental data indicate that antigen-pulsed DCs can restore immune responses when antigen-speciﬁc immune responses are not induced by endogenous DCs in the presence of abundant amounts of antigens in situ, as has been shown not only in different models of cancers but also in various infections diseases. Various factors may be related to this. To induce antigen-speciﬁc immunity, DCs must recognize, internalize, process, and present antigens. When antigen-pulsed DCs are prepared, antigens are processed by DCs in culture in vitro, and antigen-pulsed DCs express antigenic peptides. Thus, mobilization, recognition, capture, and processing of antigens are accomplished in vitro. These DCs can directly activate T cells in vivo. The efﬁcacy of antigen-pulsed DCs has been shown in different models in which antigen-pulsed DCs induced antigen-speciﬁc immunity, although this was not induced by antigen or traditional vaccines.

DC-Based Therapy for Human Tumors After several preclinical trials with DC-based therapy in animal models of cancers, DC-based therapy was ﬁrst used in human cancer in 1996. Now, several clinical trials are ongoing in which DC-based therapy is used for patients with different cancers. The general regimen of DC-based therapy against cancer has been compiled in Table 8. Although DC-based therapy has inspired considerable optimism, the therapeutic efﬁcacy of present regimens of DC-based therapy is not satisfactory. In this context, we describe the following factors related to developing more effective regimens of DC-based therapy.

Table 8. Ongoing regimens of dendritic cell-based immune therapy against tumor patients 1. DC precursors are isolated from peripheral blood of patients with cancers and cultured with cytokines to produce DCs. 2. DCs are loaded with cancer products such as antigens, epitopes, cancer tissues, RNA, fusion products, and exosomes. 3. Additional antigens are used to load DCs in some cases. 4. The immunological features of tumor-pulsed DCs are either characterized or partially analyzed or not characterized before administration to patients. 5. Antigen-speciﬁc immunomodulatory capacities of tumor-pulsed DCs are evaluated in only some cases in vitro before administration to cancer patients. 6. Different amounts of antigen-loaded DCs are administered for different duration in cancer patients. 7. The immune responses of cancer patients from administration of cancer antigen-pulsed DCs are either evaluated or no further analyses are done. 8. The therapeutic efﬁcacy of tumor-pulsed DCs is assessed by different means.

DC-Based Therapy for Human Tumors

1. 2. 3. 4. 5. 6. 7.

129

Types and sources of DCs. Tumor products, TAAs: method of loading of DCs with tumor antigens. Dose, frequency, and route of administration of DCs. Regulation of migration of antigen-pulsed DCs. DC–T-cell interaction at lymphoid tissues. Induction of regulatory T cells and tolerogenic T cells. Regulatory DCs and their possible roles.

Types and Sources of DCs There are few options regarding DCs that can be used for DC-based therapy against cancer in humans. Monocyte-derived DCs that are enriched from human peripheral blood have mainly been used for DC-based therapy. These cells offer the advantages of high yield, purity, and feasibility, especially when leukapheresis is used to collect the starting monocytes. These DCs can also be successfully cryopreserved, even after loading with antigens. DCs derived from the CD34+ hematopoietic progenitor cell (HPC) have also been used for DC-based therapy. Two subsets of DCs are found in these populations: Langerhans’ cells or epidermal DCs and monocyte-derived or interstitial DCs. These two DC subsets differ in markers and some functions. Langerhans’ cells are ineffective at stimulating B-cell development directly, a function that may require the expression of B-cell-activating factor belonging to the TNF family (BAFF) on monocyte-derived DCs. CD34-derived DCs may have improved efﬁcacy at eliciting CTLs. Plasmacytoid DC, a subset of DC, is now available. However, these DCs are not usually used for DC-based therapy. These cells can be collected from the peripheral blood: lineage−, CD4+, HLA DR+, CD123+, and CD11c−. They produce abundant amounts of type 1 IFN and usually induce Th2 cytokines. However, they can also induce Th1 polarization in the presence of viral infection. Recently, another population of DCs have been detected in mice. These regulatory DCs induce immune tolerance. The phenotypes of murine regulatory DCs have been described, but nothing is known about regulatory DCs in human. However, immature DCs can induce immune tolerance if activating and maturation signals are not properly provided. Bulk populations of monocyte-derived DCs may contain different types of DCs, including regulatory DCs. Thus, further studies are required to ﬁnd the best DC population for loading with antigens. Combinations of different subtypes of DCs may give better therapeutic potentials. However, the best DC populations in clinics are yet to be developed. Tumor Products and TAAs: Method of Loading One of the critical determinants of DC-based immune therapy against cancer is the method of preparation of cancer antigen-loaded DCs. This is a very critical area of developing DC-based therapy because little is known about most cancer antigens. Because of the unavailability of proper cancer antigens, DCs are cultured with different types of tumor products. It is expected that DC will accomplish at least three or four functions in culture, including recognition of antigens in culture, internalization of antigens, and processing of antigens. Although our main intention is to provide an

130

6. Dendritic Cells in Tumor Immunology

outline about tumor-related products for human usage, some tumor products that are used in mice are also discussed. DCs can be loaded with cancer antigens by various means, and we describe these methodologies in detail. Transfecting DC with Tumor-Derived RNA Transfection of DCs with RNA derived from tumor tissues can lead to the expression of wide varieties of tumor peptides on DCs. The RNA can be used directly or after ampliﬁcation from small amounts of starting material. This method is useful when tumor antigens are not well characterized. Also, DCs may be loaded with different types of tumor-related antigens, if these are transfected with tumor-derived RNA. The use of RNA for loading DCs may be highly important because only a few tumor cells in a biopsy can be useful. These RNA approaches have the potential to elicit immune responses to proteins uniquely expressed in a patient’s tumor. At the same time, this approach may be counterproductive because some of the proteins produced from the transfected total RNA may have immune inhibitory capacity in vivo. DCs transected with RNA may become regulatory DCs. However, this problem can be overcome if RNA-pulsed DCs are challenged to activate antigen-speciﬁc lymphocytes. However, this is not usually done in clinics. In addition, the nature of the antigens is usually unknown in tumor-derived RNA. Transfecting Using Viral Vectors Adenoviral vectors encoding the gene of target proteins can be efﬁciently transfected to immature DCs. These transfected DCs may undergo maturation with standard stimuli to carry out normal functions such as IL-12 production, antigen presentation to CD8+ T cells, and antitumor immunity in mice. Investigators have used Poxvirus vectors for transfecting DCs. However, the infection is abortive, with only early viral gene products being expressed. When this is applied to immature DC, infection is followed by overt cytotoxicity. Nevertheless, efﬁcient presentation of the recombinant gene is observed in culture, possibly through cross-presentation of dying infected DCs by other noninfected DCs. Several other virus vectors such as retroviruses, lentiviruses, and inﬂuenza virus can be used to transfect DCs. In addition to use of viral vectors for transfecting DCs in vitro, these can also be used to target DCs in vivo. The HIV-1 envelope may target DCs via a lectin called DC-SIGN. The lymphocytic choriomeningitis virus targets mouse DCs and the Dengue virus targets human LCs and monocyte-derived DCs. These approaches need to be optimized for loading DCs with tumor antigens for human usage. Culture of DCs with Protein Antigens DCs are equipped with several antigen internalization machineries. Also, DCs may use different types of uptake apparatuses for internalizing antigens. In phagocytosis, several different receptors contribute to uptake of cells. Adsorptive uptake is mediated via clathrin-coated vesicles, such as mannose receptor-mediated uptake of certain glycoproteins. Uptake by ﬂuid-phase pinocytosis and phagocytosis usually ceases upon full maturation of DCs, but adsorptive pinocytosis via clathrin-coated vesicles

DC-Based Therapy for Human Tumors

131

can persist. In addition to these methods, DCs can uptake antigens by receptors. One DC receptor, DEC-205, greatly enhances the efﬁciency of MHC class II-mediated presentation after the adsorptive uptake step. DEC-205 is unique because it does not recycle through peripheral endosomes, as is typical for most adsorptive receptors. Instead, the cytosolic domain is able to move the receptor more deeply into the cell through MHC II late endosomes or lysosomes. This unusual trafﬁc pattern is associated with a 10- to 100-fold increase in presentation of ligands relative to the classical macrophage mannose receptor, which recycles through peripheral endosomes. Several pathways target the MHC class I compartments of DCs, which is remarkable because in most cells MHC I molecules are loaded from newly synthesized proteins. Heat shock proteins (hsp), particularly hsp70, can deliver peptides to MHC class I in vivo. Heat shock proteins are therefore being considered as adjuvant for peptide delivery to DCs and even for modifying other aspects of DC function such as maturation. Loading of DCs with Dead or Dying Tumor Cells or Cell Lines This pathway allows DCs to phagocytose tumor cells or their fragments. It is now evident that tumor-associated peptides also can be presented from just one or two dead cells per DC. Both apoptotic and necrotic types of cell death can be followed by cross-presentation. In some cases, necrotic cells also cause maturation of DCs. However, there are some limitations to using tumor cells as the source of antigen. On one hand, dead cells loaded with self-peptides can lead to autoimmunity. On the other hand, presentations of dying cells by DCs may induce tolerance to self-peptides. The major potential with the use of tumor cells as the source of antigen is to allow DCs to be charged with a wide array of tumor peptides and on both MHC class I and class II molecules. Fusion Between DCs and Tumor Cells Another strategy to deliver several tumor antigens to DCs is to fuse the two cell types. Fused mixtures of tumor cells and DCs enhance resistance to mouse mammary tumors, human ovarian cancer, hepatocellular carcinoma, and advanced renal cancer. Although these results are encouraging, more studies are needed to optimize the use of fusion products for the treatment of patients with cancers. Exosomes Exosomes are small, membrane-bound vesicles both released from tumor cells and presented by DCs. These can also be released by DCs that have processed tumor cells. Exosomes contain MHC class I and II products, costimulatory molecules such as CD86, and other cell-derived products such as heat shock proteins. However, it may not be feasible to obtain large amounts of tumor cells from most patients for this approach. However, exosomes could be developed into a standard form of tumor antigen, which would be captured and presented by DCs in vivo or by ex vivo-derived DCs. If various methods of preparation of antigens-pulsed DCs are critically analyzed, it become evident that two important factors determine the nature and quality of

132

6. Dendritic Cells in Tumor Immunology

tumor antigen-pulsed DCs. The ﬁrst factor is the source of the antigens. There is little information about tumor antigens, except in some diseases. In addition, there is little information about the immunogenicity of different types of tumor antigens. Next, tumor antigens are susceptible to undergo mutation. Thus, it is extremely difﬁcult to select proper tumor products for preparing tumor antigen-pulsed DCs. If DCs can be loaded with different tumor antigens, it may be possible to have better immunomodulatory capacity and therapeutic efﬁcacy of DC-based immune therapy against cancers. In one study in mice, we induced destruction of tumors by administration of ethanol. Subsequently, immature DCs were administered directly to ethanol-treated tumors. By this approach, the sizes of the tumors were decreased compared to control groups that received only DCs (Fig. 2). The next problem is the method of loading of tumor antigens. As described here, there are many methods that can be used for preparing antigen-pulsed DCs, but there are few options in clinics. In the hospitals, clinicians culture DCs with tumor products that have been described here. Then, DCs are retrieved from the cultures and it is assumed that antigen-pulsed immunogenic DCs have been prepared, which should not be a proper approach for DC-based therapy in clinics. The nature and immunogenicity of antigen-pulsed DCs should be checked. The best way is to challenge these DCs to stimulate antigen-speciﬁc lymphocytes and to analyze their pattern of production of cytokines. However, this may not be possible in some cases because the nature of the tumor antigen is not known. It is a matter of open debate whether we should use tumor antigen-pulsed DCs when the nature of the tumor antigen is not known. Culture of DCs with crude tumor products may give rise to regulatory DCs, and they can induce regulatory T cells, which may have extremely bad consequences for cancer patients. It is almost certain it will be difﬁcult to get good therapeutic efﬁcacy of tumor antigen-pulsed DCs until our understanding about tumor antigens becomes more

200

p < 0.0005

Tumor size (mm in diameter)

p < 0.0005

180 160 140

P>0.05

120 100 80 60

0

3 Only DC

0

3

(Weeks)

Ethanol plus DC

Fig. 2. Maturation of dendritic cells (DCs) and anticancer immunity in a murine model of colon cancer. Immature bone marrow DCs were injected 48 h after administration of 100% ethanol in a murine model of colon cancer, developed at the skin of mice. The size of cancers signiﬁcantly reduced in all mice injected with ethanol and DCs, but not in mice injected with only DCs

DC-Based Therapy for Human Tumors

133

expanded. This issue will be more important when patients with early stages of cancer are treated by antigen-pulsed DCs. Dose, Frequency, and Route of Administration of DCs The dose of antigen-pulsed DCs is important in the context of DC-based antitumor immunotherapy. There is a paucity of information regarding the effect of DC dose on subsequent antitumor immune response in vivo. Studies in vitro have shown that a low dose of DCs, similar to a low dose of antigen, can polarize toward Th2. However, the dose of injected DCs should not be too large because that may hinder the DCs in gaining access to the lymphatics. The dose of DCs should be optimized on the basis of overall treatment protocol. The frequency of injection of DC is also an important factor. After injection, DCs systematically induce a series of cellular events, the ﬁnal product being the Ag-speciﬁc CTL and CD4+ T cells. These CTLs are located in the lymphoid tissues and would eventually migrate to the tissue of localization of tumors. A rapid injection schedule may cause death of DCs by the CTLs, because these injected DCs would express tumor antigens; this makes them an easy target for destruction by Ag-speciﬁc CTLs. The immunogenicity of DCs after different routes of administration in humans also needs to be compared. Some data suggest that the subcutaneous and intradermal routes lead to greater nodal migration than the intravenous route. The ﬁrst two injection protocols also induce improved Th1 polarization. Recently, DCs have been directly injected to lymph nodes with an intention to direct interaction with T cells. However, the antitumor effect of intranodal administration of DC in clinical trials has not been evaluated. A new approach is to directly inject DCs into tumor, which may facilitate the afferent limb of immunity in vivo. A portion of the tumor can be necrotized and, subsequently, immature DCs may be injected after 1 or 2 days. The principle is to pulse immature DC in vivo with all available TAAs and to provide certain maturation signals, which may come from the necrotizing tissues. Migration of Injected DCs During Immunotherapy After administration, antigen-pulsed DCs are supposed to migrate to the draining lymph node. The longevity of these DCs in lymphoid tissues is important for the efﬁcacy of DC-based therapy. In mice, treatment of DCs with CD40L enhances their emigration from the tissues to the lymph nodes. However, only a very small number of the injected DCs can be traced in the draining lymph node (10% or less). The expression of CCR7 on DCs is required for effective migration. One of the major limitations of ongoing regimen of DC-based therapy is that the migration pattern of antigen-loaded DCs has not been checked in different patients with cancers. Activation of T Cells by DCs The evaluation of therapeutic potential of DC-based therapy is another important aspect. However, the criteria for determining complete responders and partial responders would depend on the study population and the nature of the tumor.

134

6. Dendritic Cells in Tumor Immunology

Factors Related to Presentation of Tumor Antigen-Pulsed DCs at Lymphatics There is a paucity of information regarding antigen-pulsed DCs and T-cell interaction in vivo. Most of the insights have been developed from in vitro studies. The tissue microenvironments of lymph nodes play a critical role in this regard that is poorly understood; this is usually evaluated from tumor-speciﬁc immune responses. The problem lies in the fact that in most cases tumor-speciﬁc immune responses cannot be evaluated because information about tumor antigens is limited. Factors Related to Effector Functions of Lymphocytes Induced by Antigen-Pulsed DCs The primary purpose of using DC-based immune therapy is to induce tumor-speciﬁc immunity in cancer-bearing hosts. However, the ﬁnal target is to evaluate if the tumor antigen-speciﬁc immune effector cells were functionally potent to kill cancer cells and induce anticancer immune surveillance. The ultimate efﬁcacy of these can be judged by evaluating the anticancer effect of DC-based vaccine in cancer patients. However, this is not achieved in most patients with cancers after immunization with DC-based vaccine. For the time being, the capacity of effector T cells to kill tumor cells in vitro may provide some insights about the therapeutic efﬁcacy of DC-based vaccines. DC-Based Immune Therapy in Human Tumors DC-based immune therapy is applied for various human cancers at present. Hsu et al. reported about DC-based immune therapy in humans in 1996. During the past decade, DC-based immune therapy has been conducted in different types of cancers. The initial DC-based vaccine therapy was designed to demonstrate feasibility, that is, a lack of acute toxicity and responses to MHC class I-restricted tumor peptides. Also, most studies were accomplished in patients with advanced cancers. Studies with a longer follow-up or with revaccination strategies are underway to test the potential of antigen-bearing DCs to stabilize metastatic disease and even induce complete remissions. Melanoma Melanoma is the best-studied tumor from an immunological perspective. In contrast to other tumors where little is known regarding TAAs, several melanoma-related antigens have been deﬁned. Some of these antigens are also expressed on normal tissues; therefore, the possibility of tolerance to these antigens exists. Several DC-based studies have been carried out in patients with stage IV melanoma. When DCs were pulsed with melanoma peptides, including MAGE-3, tyrosinase, gp100, and Melan A/MART1, immune response to these peptides was detectable after administration of melanoma antigen-pulsed DCs, indicating that tolerance to these antigens is partial. However, the therapeutic efﬁcacy of these approaches is yet to be clariﬁed. It seems that their capacity to expand T cells and to kill melanoma targets is still inconclusive, although some patients have been beneﬁted from DC-based therapy. In one clinical trial in which 18 stage IV melanoma patients were injected

DC-Based Therapy for Human Tumors

135

subcutaneously with CD34-derived DCs pulsed with tumor peptides, injectioninduced antigen-speciﬁc T cells were detected in some patients. The preliminary data suggest a correlation between immune responses and early clinical outcome. Not only do clinical trials in melanoma patients exhibit a role of DC-based therapy in these patients, but also melanoma may be used as a model disease condition to optimize requirements for more robust immune responses and to achieve further clinical efﬁcacy. Prostate Cancer In most of the published studies, tissue-speciﬁc self-antigens such as prostate-speciﬁc antigen (PSA) have been targeted, using either blood DCs or monocyte-derived DCs. The level of serum PSA, which reﬂects the tumor burden, was reduced in about 25% of patients. In two studies, injection of blood-derived DCs pulsed with Ag fusion protein led to more than 50% reduction in serum PSA in some patients. In these studies, the evidence for immunogenicity of DCs was based on antigen-speciﬁc proliferative responses after vaccination. Prostate cancer offers two key challenges for research in DC therapy: the availability of serum markers to follow the tumor cell burden, and deﬁned prostate-restricted antigens and carcinoma lines to monitor the T-cell immune response. Lymphoma and Myeloma The target selected in published studies for both these tumors is a tumor-speciﬁc antigen, the idiotype of the monoclonal Ig expressed as a surface receptor or secreted by the tumor. In some studies, patients also received soluble antigen, which complicated the evaluation of the response. Occasional tumor regressions were observed in some patients with lymphoma. Both low-grade, non-Hodgkin’s lymphoma and myeloma respond to, but are not cured by, standard chemotherapy and may therefore be amenable to vaccination in the setting of minimal residual disease. Virus-Associated Malignancies In virus-associated malignancies, virus-derived antigens that are foreign to the immune system serve as attractive targets for immune therapy. However, one major problem would be in chronic viral infection, when the viral antigens behave as selfantigens. Examples of such viruses and associated malignancies include hepatitis B virus/hepatitis C virus and hepatocellular carcinoma (HCC), human papillomavirus, and cervical cancer, EBV, and lymphoma or nasopharyngeal cancer. Among these, HCC is one of the major public health problems. Patients chronically infected with hepatitis B virus or hepatitis C virus develops progressive liver diseases such as chronic hepatitis and liver cirrhosis. In the course of time, considerable numbers of these patients develop HCC. Because of recent improvements in imaging techniques, it is now possible to detect HCC nodules at an early stage. Also, early detection of HCC nodules is possible in developed countries because of improved follow-up of the patients. Technical developments now make it possible to ablate HCC nodules almost completely. However, recurrence of HCC nodules is a common problem. To address

136

6. Dendritic Cells in Tumor Immunology

these problems, DC-based therapy has been started in HCC patents. The data of preliminary clinical trials show promise. However, major improvements are needed regarding antigens, the method of loading DCs with antigens, and the proper design of DC-based therapy for obtaining proper therapeutic responses. Renal Cancer DCs have been loaded with crude lysates of autologous tumors for treating patients with renal cancer. In another study, fusion products of allogeneic DCs and patientderived renal cancer cells were used. Other Tumors Immunotherapy directed against other tumors falls into two broad categories: one is to target deﬁned antigens such as overexpressed self-antigens (Her2/neu, CEA, muc-1) and mutant oncoproteins (bcr-abl, p53, mutant ras), and another is to use crude antigenic preparations derived from autologous tumor lysates, peptides eluted with acid from cells, tumor-derived RNA, or heat shock proteins. Injection of tumor lysate-pulsed DCs led to marked tumor regression in a child with metastatic ﬁbrosarcoma. Development of a Better Regimen of DC-Based Therapies in Patients with Cancer Similar to most new therapeutic options, DC-based therapy was ﬁrst used in animal models of human cancers. Subsequently, DC-based therapy has been applied in patients with cancers, and one decade has passed after the ﬁrst use of DC-based therapy in patients with lymphoma. Several clinical trials are now ongoing with DCbased vaccine for treatment of cancer patients around the world. Most of the studies are pilot studies. These studies will deﬁnitely provide important insights about the scope and limitations of DC-based therapy in human cancer. In the meantime, randomized controlled trials of DC-based therapy in human cancer will be done. These studies will unveil the real prospects of DC-based immune intervention in the near future. It is time to discuss different points for development of better regimens of DC-based therapies against human cancers. Before this, there is a need to recognize various limitations of the ongoing regimens of DC-based therapy (see Fig. 3). Patient Selection. DC-based therapy has mostly been done in patients with advanced cancers. These patients are usually immunocompromised because they have been suffering from their cancer for a long period of time. The DCs of these patients exhibit impaired functional capacities. Also, the functions of other immunocytes are downregulated in these patients. It is not still clear whether DCs of these patients can efﬁciently be loaded with tumor-derived antigens in vitro. However, it is natural that new types of therapies are usually tested in these types of patients. As the safety of DC-based therapy has been conﬁrmed in these patients, now DC-based therapy should be done in patients at early stage of malignancy, which is not only important

DC-Based Therapy for Human Tumors

Nature of limitation

Cause

Possible solution

Patient selection

Patient in terminal stages Patients in early stage Immunomodulatory capacity low

Types of DCs

Bulk population of DCs may contain both immunogenic and tolerogenic DCs

Use of immunogenic DCs

Types of tumor antigens

Immunogenicity of tumor products is largely unknown

Use of immunogenic tumor antigens only

Nature of antigen-pulsed DCs May be immunogenic or tolerogenic

Checking before administration

Amounts, route, and duration Determine nature of immune of immunization responses

Preclinical studies are needed

Clinical assessment

No fixed criteria

137

Protocol can be improved if antigen-pulsed DCs are immunogenic, even if a therapeutic response is not achieved

Fig. 3. Present limitations of dendritic cell-based therapy against tumors. The underlying cause is shown. An outline has been given regarding to achieve better clinical outcome of dendritic cell-based theraphy against tumors

for showing the efﬁcacy of DC-based therapy but has tremendous importance in clinics. The real potential of DC-based therapy in cancer patients should be clariﬁed. Otherwise, this evolving therapy will not be practiced by clinicians, as has happened in many immune therapies. Also, DC-based therapy should be carried out in precancerous patients with cancers to provide more information about the effect of DCs in blocking recurrence of cancers. Types of DCs. As it is becoming clear that there may be both immunogenic and tolerogenic DCs, DC-based therapy should be administered with immunogenic DCs only. The existence of tolerogenic DCs in a monocyte-derived DC population has not been evaluated. Studies should be done to clarify these points. Cancer Antigens. In most cancers, little is known about TAAs. There may be several TAAs in a single cancer. Preclinical studies should be done to assess immunogenic TAAs because many TAAs may not be protective for the hosts. Also, studies should be done to determine universal TAAs in different cancers. The immunogenicity of crude tumor products and tumor-related materials should be evaluated before loading DCs with those materials. Pulsing of TAAs or Cancer Products. Culture of DCs and tumor products is not committed to produce immunogenic tumor antigen-pulsed DCs, which may induce production of tolerogenic tumor antigen-pulsed DCs. Addition of maturating factors will not ensure production of immunogenic antigen-pulsed DCs because tolerogenic DCs may also be produced in presence of maturating signals such as

138

6. Dendritic Cells in Tumor Immunology

IL-10. Accordingly, tumor antigen-pulsed DCs should be characterized before clinical trials. Dose, Duration, and Route of Administration of DCs. This is an ongoing challenge to develop DC-based vaccines for cancer immune therapy, which can only be addressed if more clinical trials are done with antigen-pulsed DCs in different types of cancers. Induction of Antigen-Speciﬁc Regulatory T Cells by DC-Based Vaccine in Cancers. This process may be a very complex challenge in every ﬁeld of immune therapy. The methods of production of immunogenic DCs and tolerogenic DCs in vitro are not much different. In vivo, there is strict regulation during induction of immunity and tolerance. The picture has become complex as the concept of regulatory DCs has been exposed. DCs that are used to load with antigen in vitro may contain tolerogenic DCs. It is not possible to deplete tolerogenic DCs from a bulk population of DCs. However, TAA-pulsed DCs should be carefully checked to assess their potential to induce tolerogenic T cells in vitro. DC-Based Therapy as a Multidisciplinary Approach for Treatment of Cancer The therapy of cancer is a major challenge of all branches of medicine and science. Conventional therapeutic approaches such as surgery, radiotherapy, and chemotherapy are widely used to treat these patients: however, these therapeutic approaches are mostly unable to destroy residual cancer and block recurrence of cancers. In many cases, the cancer is located in an inaccessible area and traditional therapeutic approaches cannot be applied. When a visible cancer is detected, it implies that the immune surveillance system of the hosts failed to block the process of carcinogenesis. Thus, immune therapy that can establish an immune surveillance system may be a therapeutic option for treating cancer patients. After using different immunomodulators, antigen-pulsed DCs now seem to be an effective therapeutic approach for these patients. It is unlikely, however, that the present regimen of DC-based vaccines will be an independent therapeutic approach in patients with cancers. The present regimen of tumor antigen-pulsed DCs is not so effective in patients with cancers for several reasons: (1) the protocol of production of immunogenic tumor antigen-pulsed DCs has not been optimized, mainly because adequate amounts of tumor antigens are not available; (2) difﬁculty in loading antigen on DCs because of the impaired functional capacities of DCs of cancer-bearing hosts; and (3) impaired activation of different immunocytes by tumor antigen-pulsed DCs caused by the high tumor load in patients with cancer. Based on these realities, we have provided a proposal of using DC-based therapy as part of a multidisciplinary therapeutic approach in cancer patients (Fig. 4). DC-based therapy can be done with surgery, radiotherapy, or chemotherapy. It may be possible to obtain abundant amounts of tumor products by surgery for optimization of the protocol of production of tumor antigen-pulsed DCs. In addition, we can acquire more and functionally potent DCs from cancer patients after conventional anticancer treatments. Finally, surgery, radiotherapy, and chemotherapy usually

Recommended Readings 1. 2. 3.

Antigen-pulsed DCs after surgery and radiotherapy

Antigen-pulsed DCs after chemotherapy

4. 5.

139

Availability of larger amounts of tumor antigens. Characterization of immunogenic and tolerogenic tumor antigens. Decreased tumor loads favor induction of immunity caused by administration of antigen-pulsed DCs. Increased amounts of DCs may be enriched from cancer patients. The activity of endogenous DCs may be upregulated because of a low cancer mass after surgery

1. Use of chemotherapeutic agents may have both positive effects and negative effects. 2. Chemotherapeutic agents may further downregulate DC function in vivo. 3. The functions of DCs may be activated by chemotherapy. 4. The therapeutic strategy should be designed in a case-by-case manner.

Fig. 4. Multidisciplinary therapeutic approach including dendritic cell-based therapy may be a better immune therapeutic approach against cancer. Traditional tumor therapeutic approach would reduce tumor burden, which may facilitate the immunomodulatory and therapeutic efﬁcacy of dendritic cell-based therapy in these patients

reduce cancer burden, and this may be related to the better therapeutic potential of tumor antigen-pulsed DCs in cancer patients. However, some conventional therapeutic approaches induce immune suppression in cancer-bearing hosts. Accordingly, a multidisciplinary approach using tumor antigen-pulsed DCs may not be useful after all types of conventional therapy in cancer patients. There is a need to select the treatment regimen on a case-by-case basis for combination therapy in cancer patients. Cancer is a chronic as well as a complex disease. There are no established criteria of prognosis of cancer patients. Increased survival and regression of tumor may be good prognostic markers; however, considering the difﬁculty of treatment of cancer, prolonged survival and improved quality of life may be also be used as markers of prognosis. DCs are now used as an attractive target for therapeutic manipulation of the immune system in cancer patients to increase otherwise insufﬁcient antitumor immune responses of these patients. It appears that tumor antigen-pulsed DCs may be a potent therapeutic vaccine against cancer if applied as part of the multidisciplinary therapeutic approaches against cancer.

Recommended Readings Banchereau J, Palucka AK (2005) Dendritic cells as therapeutic vaccines against cancer. Nat Rev Immunol 5:296–306 Barratt-Boyes SM, Figdor CG (2004) Current issues in delivering DCs for immunotherapy. Cytotherapy 6:105–110 Cranmer LD, Trevor KT, Hersh EM (2004) Clinical applications of dendritic cell vaccination in the treatment of cancer. Cancer Immunol Immunother 53:275–306

140

6. Dendritic Cells in Tumor Immunology

Engleman EG, Fong L (2003) Induction of immunity to tumor-associated antigens following dendritic cell vaccination of cancer patients. Clin Immunol 106:10–15 Fricke I, Gabrilovich DI (2006) Dendritic cells and tumor microenvironment: a dangerous liaison. Immunol Invest 35:459–483 Gabrilovich D (2004) Mechanisms and functional signiﬁcance of tumour-induced dendritic-cell defects. Nat Rev Immunol 4:941–952 Kim R, Emi M, Tanabe K (2006) Functional roles of immature dendritic cells in impaired immunity of solid tumour and their targeted strategies for provoking tumour immunity. Clin Exp Immunol 146:189–196 Mocellin S, Mandruzzato S, Bronte V, Lise M, Nitti D (2004) Part I: Vaccines for solid tumours. Lancet Oncol 5:681–689 Mocellin S, Semenzato G, Mandruzzato S, Riccardo Rossi C (2004) Part II: Vaccines for haematological malignant disorders. Lancet Oncol 5:727–737 O’Neill DW, Adams S, Bhardwaj N (2004) Manipulating dendritic cell biology for the active immunotherapy of cancer. Blood 104:2235–2246 Tuyaerts S, Aerts JL, Corthals J, Neyns B, Heirman C, Breckpot K, Thielemans K, Bonehill A (2007) Current approaches in dendritic cell generation and future implications for cancer immunotherapy. Cancer Immunol Immunother 56:1513–1537 Osada T, Clay TM, Woo CY, Morse MA, Lyerly HK (2006) Dendritic cell-based immunotherapy. Int Rev Immunol 25:377–413 Schott M (2006) Immune surveillance by dendritic cells: potential implication for immunotherapy of endocrine cancers. Endocr Relat Cancer 13:779–795 Schuler G, Schuler-Thurner B, Steinman RM (2003) The use of dendritic cells in cancer immunotherapy. Curr Opin Immunol 15:138–147 Svane IM, Soot ML, Buus S, Johnsen HE (2003) Clinical application of dendritic cells in cancer vaccination therapy. APMIS 111:818–834 Soruri A, Zwirner J (2005) Dendritic cells: limited potential in immunotherapy. Int J Biochem Cell Biol 37:241–245 Steinman RM, Pope M (2002) Exploiting dendritic cells to improve vaccine therapy. J Clin Invest 109:1519–1526 Zhong H, Shurin MR, Han B (2007) Optimizing dendritic cell-based immunotherapy for cancer. Expert Rev Vaccines 6:333–345

7. Dendritic Cells in Transplantation

Present Status of Organ Transplantation The transplantation of organs has expanded greatly over the past ﬁve decades. Transplantation represents the ﬁnal therapeutic choice in patients with end-organ failure who can beneﬁt from kidney, liver, heart, and lung transplantation. Hematopoietic cell transplantation and solid organ transplantation are deﬁnitive therapies for several otherwise fatal conditions. There have been tremendous developments regarding surgical procedures, applications of anesthetics, and technical understanding about preservation of organs during the past two or three decades, which have brought about a silent revolution regarding organ transplantation. From the surgical point of view, the transplantation operation is usually successful; however, the lifelong management of the different issues of posttransplantation patients is complex and difﬁcult. The major problems following transplantation are the acute and chronic rejection of transplanted organs. Immunosuppressive agents are now widely used in most organ transplant recipients, leading to dramatically improved short-term (1–3 year) patient and graft survival rates, with 1-year graft survival approaching or exceeding 80% for many organ systems. However, despite these dramatic improvements in short-term graft survival, as well as a signiﬁcant reduction in acute rejection rates, little improvement has been made in long-term graft survival. It is now well known that both nonimmune and immunerelated factors are responsible for graft loss. Among nonimmune factors, ischemia reperfusion, infection, drug-speciﬁc toxicities, hypertension, and dyslipidemia are notable. However, among the immune-related factors, alloimmunity leading to chronic rejection plays a dominant role in rejection of most organs. Two approaches may allow solving these problems. The ﬁrst would be to generate artiﬁcial organs and to optimize their use in the clinical setting, which would deﬁnitely revolutionize organ transplantation. However, this does not seem to be possible in the near future. The next is to develop insights about the causes underlying rejection of allografts and to develop a remedy for organ rejection. Nature of Problems with Transplant Survival Posttransplant immune complications include graft rejection by the host and injury to the host mediated by the graft. The donor organs that are transplanted to allogeneic recipients contain several types of cells, including parenchyma cells, nonparenchymal 141

142

7. Dendritic Cells in Transplantation

cells (endothelial cells, ﬁbroblasts, vascular cells, and specialized cells of that organs), and different types of cells of the immune system. All types of cells from the donor may play some role during transplant rejections because they all have allogeneic major histocompatibility complex (MHC) antigens. However, the cells of the immune system play the most dominant role in this regard because some of the cells of the immune system express both MHC class II and MHC class I antigens. Moreover, the cells of the immune system are migratory and can undergo maturation and activation in the recipients, which provides them with additional ability to induce alloantigenspeciﬁc immune response in the recipients. The role of immunocytes in transplant rejection was evident when it was shown that depletion, deactivation, and suppression of leukocyte population in the donor organs improved the chances of allograft acceptance. To deplete leukocytes from the donor organs, researchers have used techniques such as culture in low-temperature and in hyperbaric oxygen conditions. In addition, some investigators have used an intermediate transplantation approach in which a donor organ was ﬁrst transplanted into an intermediate host under severe immunosuppressive conditions, which led to severe functional impairment of the leukocytes of the donor organs. This organ from the intermediate host was then retransplanted into another recipient. Depletion and functional impairment of leukocyte populations led to increased survival of allografts after transplantation. These factors indicate that donor immunocytes may have played a dominant role in rejection of transplanted organs. However, for a long time the nature of leukocytes that play the critical role in this context was elusive. Recently, it is becoming clear that antigen-presenting dendritic cells (DCs) are primarily responsible for this. DCs are a very versatile population of immunocytes with different phenotypes, migration patterns, and functions. Studies have revealed that both donor DCs and recipient DCs have important roles during rejection of transplants. Dendritic Cells in the Context of Organ Transplantation We have described the origin, phenotypes, tissue localization mechanism of maturation, and functions of DCs in another chapter of this book. Here, we provide a short description about DCs that is necessary for explaining their role transplant rejections (Table 1). DCs are mostly derived from hematopoietic precursors present in the bone marrow, and they move to both the lymphoid and nonlymphoid tissues through the blood. At peripheral tissues, DCs recognize and internalize antigens and migrate through afferent lymphatics to the T-cell areas of the lymph nodes. Migration of DCs occurs both in the steady state and in inﬂammatory conditions. In the steady state, DCs carry self-antigen and play a cardinal role for induction of immunogenic tolerance. A second way of tolerance induction by DCs may be by recirculation from lymph node to thymus. However, the existence of this pathway is yet to be conﬁrmed. When inﬂammatory conditions prevail, immune responses are induced by the processing and presentation of antigens by DCs. In addition to these recognized characters of DCs, studies have shown that DCs possess the capacity of self-renewal in nonlymphoid tissues. This capability is greatest in the skin, but it also present, although at a lower level, in other tissues such as spleen, lymph nodes, and dermis. However, renewing of DCs can be detected only in the steady state. In the inﬂammatory mucosal milieu, the self-renewing DC progenitors are eliminated and replaced by blood-borne monocyte-derived DC precursors.

Present Status of Organ Transplantation

143

Table 1. The role of dendritic cells during rejection of allografts Hematopoietic stem cells transplantation: Both donor DCs and recipient DCs induce alloantigen-speciﬁc immune responses. Solid organ transplantation: Donor DCs play transient and limited role in antigen presentation. Recipient DCs act as main antigen-presenting cells for rejection of donor allograft. Maturation signals provided for activation of DCs after transplantation: Initial maturational signals may be provided during surgery. Interaction of PRRs with alloantigen may facilitate migration of DC precursors from bone marrow and blood. DC and variation of organ rejections: The relative frequencies of different types of DCs in transplants and recipients may determine the extent of rejection. For example, liver DCs may contain a less immunogenic population.

The DCs that are responsible for antigen capture and presentation are regarded as conventional DCs (cDCs). Another type of DCs, plasmacytoid DCs (pDCs), are also detected in different tissues in both human and mouse. In the steady state, pDCs are generated in the bone marrow, circulate in the blood, enter lymph nodes through the high endothelial venules, and accumulate in the paracortical T-cell-rich area of lymph nodes. In this way, the migration pattern of pDCs closely resembles that of lymphocytes and is distinct from that of cDCs. Migration of pDCs into lymph nodes is greatly enhanced when lymph nodes drain at a site of inﬂammation. Very low frequencies of pDCs can be detected in nonlymphoid peripheral tissue such as the skin and the gut in the steady state, but they can be found in these tissues during inﬂammation. In the absence of inﬂammatory signals, antigen presentation by DCs causes immune tolerance. However, when DCs present antigens derived from a dangerous entity, antigen-speciﬁc immunity is induced. This mechanism is also applicable in the case of alloantigens, and presentation of these antigens by DCs would lead to immune responses. The role of DCs in transplant rejection is different between hematopoietic cell transplantation (HCT) and solid organ transplantation. In HCT, the major alloreactivity reaction is mediated by the graft immune system against host antigens. Accordingly, DCs of both donors and recipients play important roles in antigen presentation. On the other hand, with solid organ transplantation, the major alloreactivity reaction is mediated by the host immune system against the graft. Donor DCs play a transient and limited role in antigen presentation, whereas the majority of professional antigen presentation is performed by the host DCs. Recipient DCs acquire, process, and present donor alloantigen in the context of recipient MHC class II. Donor DCs are also called passenger leukocytes, and they may directly initiate and prime immune responses to alloantigens. Support for this idea has been generated from studies that have shown that depletion of donor DCs from transplant organs partially inhibits priming and prolongs graft survival. However, immunity is eventually generated, and tolerance is not achieved. These factors indicate a dominant role for indirect alloantigen presentation by recipient DCs to prime the immune response for subsequent rejection.

144

7. Dendritic Cells in Transplantation

In solid organ transplantation, secondary lymphoid organs play important roles during presentation of antigen to prime T cells to reject allografts. Investigations have unveiled that draining lymph nodes and secondary lymphoid organs are responsible for presenting antigen to T cells that are destined either to become proinﬂammatory T cells or to become anergic and tolerant. The choice between priming versus tolerization within secondary lymphoid organs is regulated by the presence or absence of other strong secondary signals, such as inﬂammatory cytokines. The immune responses that contribute to the rejection of allografts are comparable to the immune reaction that happens in allogeneic mixed lymphocyte reaction (AMLR) in vitro. During allogeneic MLR, DCs present alloantigens to allogeneic T cells and induce their proliferation. In addition to their strong stimulatory capacity in allogeneic MLR, DCs can also act as initiators, propagators, and regulators of immune responses. DCs can undergo activation by means of an alteration of the mucosal milieu of the tissue microenvironment. DCs are passenger leukocytes and can migrate to the lymphoid tissues for presenting alloantigen and other antigens for the induction of immune response. In the transplantation situation, the surgical intervention might represent potent stimuli for this activation, which can be responsible for maturation of DCs. These activated DCs would have the best chance to migrate to the lymphoid tissues of the recipients, where they can directly activate T cells. They can also release antigens from their MHC groove and thus may indirectly activate T cells. All these activities of the donor DCs would facilitate the rejection of allografts. On the other hand, DCs are not only committed to induce immune response, but they are potent inducers of immune tolerance. DCs do not represent a homogeneous cell population; rather, there are different subtypes of DCs in both human and mouse. Some of these DCs are committed inducers of immune tolerance. In addition, cytokines and immunomodulators can alter the nature of the DCs from the immunogenic to tolerogenic state. These studies indicate DC might be a foe in the context of transplantation because they induce immune response and facilitate allograft rejection. However, if donor DCs can be altered to a tolerogenic population, that would deﬁnitely increase the chance of acceptance of allograft. In addition, there are several ways and means to downregulate DC function in vivo. However, the use of DCs in a transplant situation would be dependent on the understanding of the nature of DCs and the ways of manipulating them. DC-Related Immunological Events During Allograft Rejection Different experimental evidence shows that DCs from both the donor and the recipient might constitute a formidable obstacle to allograft acceptance (Table 2). The cellular and molecular mechanism relating to allograft rejection and the activities of DCs in this context cannot be studied in detail in human transplantation. However, insights about these mechanisms have been developed from animal experiments with a transplantation model and by analyzing the immunological events in allogeneic MLR. During the initial phase of allogeneic MLR, discrete aggregates of DCs and T cells are formed. Allogeneic DCs can directly stimulate the puriﬁed CD4+ and CD8+ T lymphocytes in allogeneic MLR; this results in the growth and proliferation of T

Present Status of Organ Transplantation

145

Table 2. Pathways of rejection of transplant and involvement of dendritic cells Direct pathway of allorecognition: Alloreactive T cells ligate with foreign MHC antigens on donor DCs; this leads to migration of DCs to lymphoid organs and activation of recipient T cells, which may be the leading cause of acute rejection. Support for this hypothesis: Although liver transplants are easily accepted, if mice are treated by FLT3L and liver is transplanted, liver DCs are matured and liver transplantation is quickly rejected. Liver allograft is also rejected if treated with proinﬂammatory cytokines that cause DC maturation. Indirect pathway of allorecognition: DCs of donor represent a source of different antigens. Immature DCs of donors engulf apoptotic cells and necrotic materials. They migrate to lymphoid tissues, and eluted peptides will be captured and processed by the recipient’s DCs. The recipient T cells will be activated and induce rejection of transplants, which may be responsible for chronic rejection.

cells, which lead to the production of T blasts. Once T blasts are formed by DCs, other APCs such as macrophages and B cells can also induce vigorous proliferation of T blasts. After transplantation, donor DCs migrate to the lymphoid tissue of the recipients and induce alloantigen-speciﬁc activation of recipient T cells. The surgical procedure of transplantation might provide stimulatory and activation signals for migration of donor DCs to the recipient’s lymphoid organs. The donor allograft contains a variety of DC populations, such as DC progenitors, DC precursors, immature DCs, mature DCs, immunogenic DCs, and tolerogenic DCs. The capabilities of different populations of donor DCs to stimulate allogeneic T cells of the recipients have not been properly examined. However, the levels of maturation and activation of DCs are related to their allostimulatory capacities. It is important to assess how the levels of activation and maturation of DCs are altered by anesthesia and the surgical procedures. The inherent characteristic features of DCs of the organs are also important in this regard. Liver allografts are well accepted even against MHC mismatch. The liver harbors a considerable number of DC progenitors with very special phenotypes and functions. In contrast to immature DCs and DC precursors, liver DC progenitors do not undergo maturation in presence of known stimulatory signals such as tumor necrosis factor-alpha (TNF-α) and interferon (IFN)-α. This fact may underlie the excellent acceptance of liver allograft even under a MHC mismatch; however, the liver also contains immunogenic cDCs, and these DCs are capable of inducing immunity. Thus, the migration pattern of different subsets of DCs after transplantation may be important for survival of allografts. It is also important to assess how the migration of DCs in initiated after transplantation. The chemokines may play a role in this situation. However, a parenchyma factor may also be important. Increased expression of ligands for pattern-recognition receptors (PRRs) at the recipient parenchyma cells may also induce maturation and migration of DCs. Nevertheless, the expression of PRRs on donor DCs would be important in this regard.

146

7. Dendritic Cells in Transplantation

T cells can recognize the MHC alloantigen by two distinct pathways. One of them is called the direct pathway of allorecognition and the other is the indirect pathway of allorecognition. In the direct pathway of allorecognition, T-cell receptors of alloreactive T cells ligate with the foreign major histocompatibility complex (MHC) antigens on DCs. The migration of donor DCs to recipient lymphoid tissues, and subsequent activation of recipient T cells, represent the direct pathway of allorecognition. In the indirect pathway of allorecognition, T cells recognize allogeneic MHC molecules as peptides presented in the context of self-MHC antigens. This recognition system is somewhat similar to that of recognition of nominal antigens. DCs of the donor represent a source of various antigens. Immature DCs of the donors may have engulfed various apoptotic and necrotic bodies that may have undergone processing in the donor DCs. These DCs would migrate to the lymphoid tissues of the recipients. At the lymphoid tissues, MHC peptides might be eluted from the peptide-binding groves of the MHC molecules. These eluted MHC peptides would be captured by recipient DCs as nominal antigens. After processing the captured antigens, recipient DCs would activate alloantigen-speciﬁc T cells, which would lead to the activation and proliferation of T cells. The role of macrophages as a source of eluted MHC is controversial. Although macrophages possess high potentiality to capture apoptotic cells and other dying cells, they probably digest the antigens completely and thus cannot supply the antigen in peptide form to recipient T cells.

Acute and Chronic Rejection In general, it is thought acute rejection is the ultimate end product of direct allorecognition. As mentioned, donor DCs play critical roles in this regard. Donor DCs as passenger leukocytes are released or these DCs migrate from the allograft to the recipient tissues. The roles of various chemokines or signals during their migration from donor tissue have not been well explored, but these DCs would induce a powerful stimulus for the proliferation of allogeneic T cells of the recipients. In addition, the interaction between DCs and T cells would lead to Th1 polarization and production of various inﬂammatory cytokines. When T cells of the recipients are activated, they will ultimately cause damage of transplants. Traditionally, it is assumed that chronic rejection is the result of indirect allorecognition, as has been shown by the progressive form of allograft injury characterized primarily by persistent, but patchy, inﬂammation of the allograft, hyperplasia of arteries, interstitial ﬁbrosis, and destruction and atrophy of parenchymal elements and organ-associated lymphoid tissue. In contrast to acute rejection, chronic rejection develops over a longer period of time. Indirect allorecognition, which allows the recipient DCs to present donor MHC peptides to autologous T cells, may be one of the main factors underlying the development of chronic rejection. Chronic rejection may also develop in many cases from inadequately controlled acute rejection and in patients not compliant with immunosuppressive therapy. Inﬁltration of the allograft by recipient accessory cells, including macrophages and DCs, is a hallmark feature of chronic rejection. The inﬂammatory inﬁltrates in chronic rejection are often arranged into nodular aggregates, some of which contain a germinal center in secondary lymphoid follicles. The number of recipient DCs in chronically rejected organs directly correlates with the overall severity of inﬂammation.

Roles of DCs in Minimizing Rejection of Transplants

147

Roles of DCs in Minimizing Rejection of Transplants: Guidelines of DC-Based Therapy for Survival of Allografts Various studies indicate that DCs are one of the most important immunocytes that are directly or indirectly related to rejection of transplants. Also, other cells of the immune system are related to this process. DCs are critical immunocytes in transplantation acceptability because many of the primary events that ultimately lead to transplant rejection are initiated by DCs. In many cases, the primary pulsing of these immunocytes may be mediated by DCs. Survival of transplants is dependent on various techniques, some of which occur as a natural consequence of donor–recipient interactions, whereas others need to be induced for survival of transplants. Different mechanisms that are relevant to protection of allografts are described in this section so that the most appropriate one can be developed for protecting transplants in humans (Table 3). Chimerism and DCs Chimerism is the coexistence of cells from genetically different individuals. In the context of transplantation, it is detected that donor-derived passenger leukocytes, mainly DCs, can remain in the recipient for a long time. Chimerism may be divided into three categories: full chimerism, mixed chimerism, and microchimerism. In full chimerism, the entire recipient hematolymphoid system is replaced by that of the donor. In mixed chimerism, a functionally integrated immune system is composed of various proportions of hematolymphoid cells from the donor and recipient. Microchimerism is similar to mixed chimerism; however, trace populations of multilineage donor hematopoietic cells persist. The presence of chimerism is thought to be associated with graft acceptance and may even play a role in maintaining unresponsiveness to the graft. It is well accepted that tolerance achieved by hematopoietic chimerism is the ideal and most robust form of tolerance. A certain degree of chimerism can be achieved if the inoculum contains cells capable of self-renewal, for example, bone marrow cells. The critical factors that control different types of chimerism are not

Table 3. DC-based intervention for survival of allografts Deletion of T cells by DCs: 1. Antigen-presenting cell (APC)-induced activation induces cell death. 2. Blockade of costimulatory molecules on allogeneic DCs. 3. Immature DCs cultured with anti-CD40 induce increased apoptotic death of T cells. Tolerance induction of regulatory DCs: 1. Regulatory DCs that produce IL-10 in vitro may increase survival of transplant by their capacity to tolerize the immune system. 2. Regulatory DCs may induce tolerogenic regulatory T cells and protect transplant from rejection. 3. Deviation of immune responses to Th2 may help survival of transplants. Tolerance induction by targeting in DC of recipient: 1. Drugs such as salicylates and corticosteroids may block DC maturation in vivo.

148

7. Dendritic Cells in Transplantation

well understood. However, a relationship between development of chimerism and use of immunosuppressive drugs is seen. The robust tolerance associated with mixed chimerism is strictly dependent on the persistence of donor cells. Successful mixed chimerism can induce clonal deletion of self-reactive T cells in the thymus, which is mediated primarily by DCs. Donor DCs are able to migrate into the recipient thymic medulla and mediate negative selection of T cells potentially reactive against the allograft. Donor precursor cells can also mature into DCs and migrate into peripheral lymph nodes and into the interstitial spaces of organs where they can mediate immunogenic or tolerogenic reactions. The mixed chimerism approach to tolerance induction, which is successful in small experimental animals, cannot be seen in humans, and thus has not been clinically accepted.

Controlling DC Functions as a Therapeutic Approach Insights Developed about Roles of DCs from Liver Transplantation Control of Liver Allograft Rejection Caused by Virus Infection The therapeutic efﬁcacy of tolerance induction by DCs can be explained in the clinical situation from liver transplantation. Liver transplantation is unique that HLA matching is not necessary. However there is an absolute demand for HLA matching during the transplantation of most organs, bone marrow, kidney, heart, and intestine. Spontaneous tolerance is induced after liver transplantation in many cases without immunosuppressive drugs. Liver transplantation can protect other organ grafts from the same donor transplanted in conjunction with the liver. This acceptance may be lost by removal of donor leukocytes before liver transplant. A part of the immunological strange phenomena in liver transplantation would be explained by the characteristics of liver DCs. Liver DCs are inherently immature and a considerable proportion of these secrete IL-10. These DCs have the features of tolerogenic DCs. Thus, transplantation of liver may induce tolerance because of the high proportion of immature and tolerogenic DCs in the liver. The liver is a hematopoietic organ, and may have an advantage in being a continuous source of donor hematopoietic cells. Donor-derived immature DCs may promote donor-speciﬁc tolerance induction. Large numbers of donor leukocytes present in liver allograft cause overstimulation or abnormal early activation of recipient T cells that leads to their exhaustive proliferation and deletional tolerance. Alloreactive host T-cell apoptosis in experimental liver transplantation is associated with tolerance, whereas less apoptosis is seen with rejection. The donor may play a role in inducing apoptosis in host T cells via death ligand–receptor pathways. Neutralization of IL-12 produced by liver-resident DCs restores long-term allograft survival and enhances alloreactive T-cell apoptosis. This ﬁnding suggests that suppression/inhibition of donor DC function promotes tolerance induction. The immature state of normal liver-derived DCs may be important in inherent liver tolerogenicity. Administration of liver DC progenitors before transplantation has been shown to increase allograft survival.

Controlling DC Functions as a Therapeutic Approach

149

These investigations indicate that manipulation of functions of DCs may allow developing interventional strategy for protecting allografts. Although DC-based approaches are not used to protect allografts in humans at present, various studies in animal models of transplantation indicate that DC-based intervention may be a practical option in clinics in future. In this section of the book, we do not give a lengthy discussion about animal study data that indicate possibilities of DC-based interventional strategies for protecting transplant organs. Readers are encouraged to check the important reviews in this regard, listed in “Recommended Readings.” We mainly discuss the scope and limitations of DC-based therapy in the context of transplantation in humans. Strategy of DC-Based Immune Therapy to Protect Transplants Induction of Immune Tolerance by Manipulating DC–T-Cell Interactions The predominant role of the direct pathway provides a rational basis for the manipulation of donor-derived DCs to prevent donor-derived acute graft rejection and to promote tolerance induction, as has been supported by various experimental data. When DCs are stimulated, the chances of acceptance of allografts are decreased. Treatment of mice with fms-like tyrosine kinase 3 ligand (Flt-3L) causes increased numbers of DCs in the liver. Transplantation of Flt3-L-treated liver allograft would be quickly rejected, although normally these allografts are accepted without immunosuppressive therapy. Similarly, acute rejection of liver allograft is seen if the recipients are treated with IL-12 or IL-2. In fact, activation of DCs has taken place as a result of use of these immunomediators. These studies indicate that if a reverse attitude can be employed, it may be possible to protect transplants. Activated T cells can be deleted or minimized by use of antiinﬂammatory agents, blockade of costimulatory antigens, or downregulation of migratory capacities through blocking of chemokines. Many of these approaches are used in the clinical setting; however, the appropriate protocol is yet to be developed in humans. Tolerance induction may be achieved by inducing regulatory T cells and anergic T cells (see Table 3). Immature donor DCs may deliver signal 1 in the absence of signal 2, and this would eventually lead the production of regulatory T cells. These regulatory T cells produce IL-10 and transforming growth factor-beta (TGF-β), but low levels of IL-2 and no IL-4. The induction of regulatory T cells has been shown in both mice and humans. Intravenous injection of humans with monocyte-derived immature DC pulsed with inﬂuenza matrix protein would lead to the production of matrixspeciﬁc IL-10. In addition, constant stimulation of human cord blood T cells with immature DCs would lead to the production of IL-10-producing T-regulator cells, as has also been seen in the transplantation setting. Murine B-cell lineage-associated DCs derived from liver resident progenitors have been shown to induce regulatory T cells in vitro, which also led to prolongation of allograft survival in vivo. This approach has an additional beneﬁt because there is no need to alter DCs. However, the efﬁcacy of this approach is questionable because administered immature DCs may be activated after injection into recipients. Immune tolerance can be induced by immune deviation also. The major damage in the transplantation setting is made by T cells of Th1 phenotype. DCs are able to

150

7. Dendritic Cells in Transplantation

induce both Th1 and Th2 types of immune responses. If the immune response during transplantation can be skewed to the Th2 phenotype, that would lead to survival of transplanted organs. However, several studies must carried out to assess how immune deviation may be induced in vivo. Although deletion of T cells, induction of regulatory T cells, and deviation of immune response to Th2 phenotypes might induce immune tolerance, the timing of DC administration and allograft operation are critical factors for successful allograft acceptance. Bone marrow-derived DCs cultured with suboptimum doses of GM-CSF retained an immature phenotype. When these DCs were administered into the mouse 7 days before transplantation, they survived indeﬁnitely. However, when DCs were administered before 3, 14, or 28 days, this approach did not result in allograft survival. Blocking of maturation of DCs might also facilitate tolerance induction. DC maturation is mediated by nuclear translocation of NF-κβ. Various drugs such as salicylates and corticosteroids block the maturation of DCs through this pathway. Immature DCs can also be produced by targeting NF-κβ pathway by antisense oligonucleotides. Short oligonucleotides with consensus binding sequences to NF-κβ inhibit DC allostimulatory capacity by blocking NF-κβ translocation. DCs treated with NF-κβ ODN for up to 36 h and administration to fully allogeneic recipients as a single intravenous dose, 7 days before transplantation, would prolong allograft survival. DCs of both donors and recipients origin may act as a foe to cause acute as well as chronic rejection of allografts. Direct allorecognition by donor DCs induces acute rejection, whereas the indirect allorecognition by recipient DCs causes chronic rejection. As immune response to alloantigens induces the rejection of allografts, it might be possible to reverse the process by manipulating the function of DCs. The relative contribution of direct and indirect allorecognition has been examined in a murine model of skin graft rejection. In this model, during acute rejection, 90% of the responding T cells responded to directly presented donor MHC peptides, whereas less than 10% of T cells recognized allopeptides presented indirectly. Manipulating Donor DCs 1. Identifying the Migratory DCs of Donor and Modifying Their Nature. It is almost clear that acute rejection of transplants is mainly mediated by donor DCs and passenger leukocytes. However, it may not be true that all types of donor DCs take part in rejection of allografts. In fact, study is needed to assess the nature of DC subsets that mainly migrate from donor tissues to recipient lymph nodes. If it is found that one or more subsets of donor DCs preferentially migrate, then these DCs will be the target of therapy in the context of transplantation biology. Depletion of these DCs may save a transplant from rejection. 2. Preservation of Organs So That DCs Do Not Undergo Activation. It is generally presumed that activated DCs would migrate from transplants to donor lymph nodes. The activation signals may be provided during surgical maneuvers. However, this approach is unavoidable. However, the utmost care should be taken so that donor DCs are not activated during preservation of allografts. Addition of antiinﬂammatory agents during preservation of allografts may be useful.

Controlling DC Functions as a Therapeutic Approach

151

3. Use of Donor DCs for Induction of Immune Tolerance to Donor Antigens. Several methods may be employed to realize this. It is now well known that DCs can induce immunogenic tolerance, as has been convincingly proved in animal models of human diseases. However, studies about their efﬁcacy have not been conducted in humans. 4. Administration of Tolerogenic DCs of Donor Before Transplantation. Donor DCs can be enriched from peripheral blood mononuclear cells and cultured with different antiinﬂammatory cytokines and maturation signals to produce regulatory DCs. When these DCs are injected into transplant recipients, these persons may develop immune tolerance against donor MHC antigens. If transplantation is carried out subsequently, the donor allografts may not induce rejection of allografts. At least, acute rejection may be avoided. Another notable point of this approach is that donor DCs can be collected before transplantation; this is one type of presensitization. Manipulation of Recipient DCs Use of Regulatory DCs of Recipients The T cells of the recipients are activated and induce immunological rejection. Thus, downregulation of recipient T-cell activity may have therapeutic potential. In this regard, it is important to induce regulatory T cells in recipients so that these T cells may downregulate the activity of pathogenic T cells. Regulatory T cells may be induced by administration of regulatory DCs. The protocol for preparation of regulatory DCs has not been optimized in humans. However, on the basis of studies in mice, these DCs can be prepared. DC Subsets and Their Use in Induction of Tolerance Although DCs could be manipulated in the animal model of transplantation, this is not a simple and viable task in human transplantation. However, now different subsets of DCs with diverse functions are known. For example, plasmacytoid DCs can be isolated from human peripheral blood. These cells are not good stimulators in allogeneic MLR, and they induce the Th2 phenotype after CD40 ligation. G-CSFmobilized PDC can induce tolerance. The role of these DCs in prevention of hostversus-host diseases has been shown. Utility of DCs for Induction of Immunity in the Context of Transplantation In addition to induction of tolerance by DCs in the context of transplantation, DC-based intervention may be required for induction of immune responses. When transplants are derived from patients with chronic viral infection or transplantation is done in these patients, transplants may be rejected because of de novo activation of viruses after transplantation. This rejection can be blocked by inducing protective immunity against the viruses. However, it is extremely hard to induce antiviral immunity by immunization with traditional vaccines because patients are usually given immunosuppressive agents to block rejection of transplants. We used antigen-pulsed

152

7. Dendritic Cells in Transplantation

HBsAg-pulsed DCs Duration

0

2W

4W

8W

12W

16W

20W

Anti-HBs (mIU/ml)

FK506 2.0 mg/kg/day

N=8 500 375 250 125 0

2

4

8

12

16

20 W

Fig. 1. Hepatitis B virus (HBV) transgenic nice (HBV-Tg) were administered with FK-506, an immunosuppressive agent, once daily for 20 weeks (W). Hepatitis B surface antigen (HBsAg)-pulsed murine spleen DCs were administered twice at 2 and 4 weeks (W) after start of administration of FK-506. Antibody to HBsAg (anti-HBs) was detected in all HBV-Tg because of administration of HBsAg-pulsed DCs

DCs in a murine model of chronic hepatitis B virus (HBV) infection receiving immunosuppressive agents. Administration of HBV-related antigen-pulsed DCs induced protective antibody in immunosuppressive HBV transgenic mice when this could not be achieved by traditional vaccinations (Fig. 1).

Concluding Remarks It is evident that DCs play a dominant role during the rejection of allografts. Accumulated evidence also suggests that DCs are involved in the process of peripheral Tcell tolerance, which may increase the chance of allograft acceptance. Techniques have also been developed to isolate or enrich abundant amounts of DCs in vitro. The various DC subsets can also be produced in vitro. Advances in vectorology and gene therapy would allow us to produce tolerogenic DCs. All these experimental data strongly suggest that it is now feasible to start DC-based alloantigen-speciﬁc T-cell hyporesponsiveness as an approach to improve allograft acceptance. However, one of the main obstacles is that we have very little information regarding the mechanism of induction of peripheral T-cell tolerance in the normal steady state. Moreover, there are very few insights about the cellular and molecular events underlying the maintenance of this tolerance. The most important factor is not the mechanism of induction of tolerance after transplantation, but to have proper understanding about the induction and maintenance of tolerance to self- and non-self-antigens in the steady state.

Recommended Readings

153

Recommended Readings Benseler V, McCaughan GW, Schlitt HJ, Bishop GA, Bowen DG, Bertolino P (2007) The liver: a special case in transplantation tolerance. Semin Liver Dis 27:194–213 Martinez OM, Rosen HR (2005) Dendritic cells, tolerance and therapy of organ allograft rejection. Contrib Nephrol 146:105–120 McCurry KR, Colvin BL, Zahorchak AF, Thomson AW (2006) Regulatory dendritic cell therapy in organ transplantation. Transplant Int 19:525–538 Merad M, Collin M, Bromberg J (2007) Dendritic cell homeostasis and trafﬁcking in transplantation. Trends Immunol 28:353–359 Morelli AE, Thomson AW (2003) Dendritic cells: regulators of alloimmunity and opportunities for tolerance induction. Immunol Rev 196:125–146 Morelli AE, Thomson AW (2007) Tolerogenic dendritic cells and the quest for transplant tolerance. Nat Rev Immunol 7:610–621 Nouri-Shirazi M, Thomson AW (2006) Dendritic cells as promoters of transplant tolerance. Expert Opin Biol Ther 6:325–339 Raimondi G, Thomson AW (2005) Dendritic cells: tolerance and therapy of organ allograft rejection. Contrib Nephrol 146:105–120 Young JW, Merad M, Hart DN (2007) Dendritic cells in transplantation and immune-based therapies. Biol Blood Marrow Transplant 13:23–32

8. Dendritic Cell-based Immune Therapy: Concept, Design, Present Limitations, and Future Projections

Dendritic Cell (DC)-Based Therapy as Immune Therapy in Clinical Medicine Immune therapy seems to be a rational therapeutic choice for treating patients with immune-mediated anomalies. In some diseases, immune-related factors are primarily responsible for disease pathogenesis. Autoimmune diseases are a typical example. In addition, an immune-mediated phenomenon is related to rejection of organ transplants. Thus, immune therapy may be used for treating autoimmune diseases. Rejection of transplanted organs may be delayed or blocked by immune intervention strategies. In addition to a direct role of immunity in disease pathogenesis, the immune responses of the hosts play critical roles during the initiation, progression, and complications of many pathological conditions such as chronic microbial infections, cancers, and allergic diseases. Microbial agents are primary etiological factors of different microbial infections. However, the clinical course of the disease depends on the immune responses of the hosts to the microbial agents. If the host immune responses against the microbial agents are adequate and purposeful, the microbes are eradicated or controlled after an acute and self-limiting infection. On the other hand, chronic infections are established in some patients because of impaired immune responses of the hosts to invading pathogens. Genetic mutations in critical genes of the hosts, various environmental factors, and microbial agents are related to initiation of tumorigenesis. However, this is usually controlled by the host immune system, and rarely are clinically visible tumors detected. Progression of cancer indicates failure of host immune surveillance mechanisms. Thus, either exacerbated or decreased immune responses are related to the pathogenesis of various diseases. In this context, the concept of immune therapy has surfaced. Immune therapy may be broadly divided into two categories. Some immunomodulatory drugs have been used for a long time for treatment of different pathological conditions. Corticosteroid is a representative member of this group. Different immunosuppressive and immunomodulator drugs are commercially available and are used in clinics. A second type of immune therapy may be regarded as an evolving or emerging immune therapeutic approach. This type of immune therapy is designed by evaluating the role of immune responses in different pathological conditions. Most of these immune therapeutic approaches are started as pilot studies. 155

156

8. Dendritic Cell-based Immune Therapy

Cell-based immune therapy represents one of the most promising evolving and emerging immune therapeutic approaches in clinical medicine. Among cell-based therapies, dendritic cell (DC)-based immune therapy has emerged as a means of inducing antigen-speciﬁc immunity in patients with cancers. Studies in animal models of cancers revealed that DC-based immune therapy possesses potent immunomodulatory and therapeutic efﬁcacy against cancer. In patients with cancer, DC-based therapy has shown some therapeutic efﬁcacy, but more potent regimens of DC-based therapy are needed in the clinic. In this chapter of the book, we mainly discuss the concept, design, present limitations, and future projections of DC-based therapy in patients with cancer as well as those with chronic viral infections.

Types of Immunity and Their Putative Roles in Host Defense Different types of immune therapy have been applied to treat patients with different pathological conditions, but few of them could stand the test of time. There may be several underlying factors for lack of general acceptability of immune therapy, which seems to be more relevant in the context of evolving and emerging immune therapies. The ﬁnal purpose of immune therapy in different pathological conditions is to modulate host immunity so that it can cure the disease or control the progression of different diseases. Immune therapy is a generalized term; however, the purpose of immune therapy should be speciﬁc and clear when this is applied to treat patients with different pathological conditions. For example, the purpose of immune therapy for patients with cancers is different from that of patients with autoimmunity. Again, the aim of immune therapy for patients with chronic microbial infections is different from immune therapies of patients with cancers and autoimmunity. As shown in Fig. 1, different types of immune responses may be seen in vivo. Some of them may also be induced by different immune therapeutic approaches. There is lack of consensus about the nomenclature of these immune responses. Immune surveillance is an inherent property of living organisms, and this type of immunity is induced in the hosts when they are challenged with non-self agents or dangerous elements. Recent studies also indicate that hosts are usually protected from unwanted attack by autoantigens because of the presence of immune surveillance mechanisms. Immune surveillance against tumors is one of the common mechanisms that allow the host to block the growth of clinically visible tumors. Immune ignorance is seen in some patients after being infected with some microbial agents or after development of transformed cells or precancerous cells. In spite of harboring abundant amounts of harmful agents, these subjects do not exhibit adequate levels of immunity against these agents. It seems that these agents have been either completely or partially ignored by the host immune system. Protective immunity provides protection against various harmful agents, which can be induced by vaccines in normal individuals. Protective immunity can also be developed after development of acute inﬂammatory diseases following microbial infections, as is also detected in some patients with chronic viral infections spontaneously or after antiviral therapy. The nature of protective immunity in patients with tumors is not clearly deﬁned because of incomplete understanding

Types of Immunity and Their Putative Roles in Host Defense

157

TYPES OF IMMUNITY IMMUNE SURVEILLANCE

Immunity with scanning capacity

IMMUNE IGNORANCE

Inadequate response to pathogens

IMMUNE PROTECTION

Immunity that protects from environmental agents and autoimmunity

IMMUNE PATHOGENESIS

Immunity that causes tissue destruction

IMMUNE TOLERANCE

Immunity that protects host from exacerbated immunity

IMMUNE REGULATION

Immunity that balances between immune response and immune tolerance

Fig. 1. Different types of immunity in vivo. Immunity is a generalized term and does not reﬂect the real implications in clinics. There are several types of immunities in vivo. There is also a close relationship between different types of immunities

about various tumor-associated antigens (TAA). Protective immunity is usually antigen speciﬁc and protects the hosts from future invasion with the same type of agents. The nature and magnitude of protective immunity are important for their protective capacities. Development of protective immunity may be preceded by tissue damage. Tissue damage may cause eradication or reduction of infectious and harmful agents. Pathogenic immunity is a type of immune response that can induce damage of the host tissues. In some cases, this may be beneﬁcial for the hosts but in other cases it may have a detrimental effect. In acute viral infections, pathogenic immunity causes destruction of virus-infected cells and is followed by development of protective immunity. On the other hand, pathogenic immunity causes tissue damage in patients with chronic viral infections. Recent studies have unveiled that regulatory immunity is an inherent property of all hosts. The role of immune regulation in host defense is now under critical analyses. Studies in animal models have shown that regulatory immunity may be induced in vivo by different types of immune interventional approaches. Induction of immune tolerance against different agents is a daily event in the life cycle of living organisms, protecting them from unwanted immune responses and autoimmune diseases. However, immune tolerance to tumors and microbial agents

158

8. Dendritic Cell-based Immune Therapy

may be detrimental to the hosts because these agents cannot be eradicated from the hosts as a consequence of the immune tolerances. It appears that different types of immune responses are present in situ. At present, immune therapy aims to activate or deactivate host immunity. However, the precise goal of immune therapy should be clear during its design. Immune therapy is applied in patients with tumors to induce pathogenic, protective, and surveillance types of immunities. The cancer cells should be destroyed by immunocytes that would be induced during immune therapy, which should lead to protective immunity against cancer. On the other hand, the purpose of immune therapy against chronic vial infection is to induce protective immunity with minimum tissue damage. Immune therapy should be designed to reduce viral replication without or with minimum destruction of self-tissues. Immune therapy against autoimmune diseases is intended to induce tolerogenic and regulatory immunity. Taken together, the design of immune therapy should be different on the basis of nature of pathological conditions and the immune status of the hosts.

Nonantigen-Speciﬁc Immune Therapy in Clinical Medicine As shown in Fig. 1, there are different types of host immune responses. In the context of immune therapy, there is no universal protocol that would be effective in all types of pathological conditions. Different types of immune responses should be induced for therapeutic purposes in different diseases. Also, the strategy of immune therapy should be different on the basis of the immune status of the patients. It is extremely difﬁcult to induce speciﬁc types of immune responses in humans in vivo by immune therapeutic strategies. From the practical point of view, immune therapy may be divided into two main categories: (1) nonantigen-speciﬁc immune therapy, and (2) antigen-speciﬁc immune therapy. Nonantigen-speciﬁc immune therapeutic agents include different polyclonal immunomodulators such as cytokines, growth factors, and different types of immune stimulators. Also, immunosuppressor agents such as corticosteroid hormones and immunosuppressive drugs may be included in this group. These immunomodulatory agents are usually available as drugs on the market. Use of these commercially available drugs activates or deactivates host immunity. Generally speaking, these drugs induce an overall effect on host immunity. However, it is not clear what type of immunity would be activated or deactivated by nonantigen-speciﬁc agents. Different cytokines, such as interferon (IFN)-γ or interleukin (IL)-12, or antibodies (antitumor necrosis factor-alpha) or immunomodulators such as OK-432, or growth factors are now used for activation or deactivation of immune responses of patients with chronic viral infections, cancer, and autoimmune diseases. Some pilot studies have shown that antigen-nonspeciﬁc immunity has therapeutic potential in some diseases. However, the actions of nonantigen-speciﬁc immune therapeutic agents are usually ﬁnite, and the clinical effects cease once the therapy is discontinued. Moreover, these immunomodulators may not induce proper types of immunity required for therapeutic effects. However, antigen-nonspeciﬁc immune therapeutic agents are easy to apply and commercially available. Moreover, the indications and contraindications of these drugs have been well analyzed by commercial companies.

Strategy of Antigen-Speciﬁc Immune Therapy

159

Antigen-Speciﬁc Immune Therapy for Treatment of Patients with Different Pathological Conditions The concept of antigen-speciﬁc immune therapy originated after analyzing the immune responses of patients with cancer and chronic viral infections. The mechanisms underlying pathogenesis of cancers and chronic viral infections have not been completely explored, but host-related factors, viral factors, and environmental factors are related to the pathogenesis. These points have been discussed elaborately in other chapters of this book. As patients with cancer and chronic infections exhibit impaired antigen-speciﬁc immunity in spite of harboring abundant amounts of tumor-related antigen (TAA) and viral antigens, it is assumed that these patients may have developed immune tolerance to different viral antigens and cancer-related antigens. These patients are characterized by (1) impaired innate immunity after encountering microbes and tumor cells, (2) defective immune surveillance mechanisms, and (3) impaired priming of adaptive immunity. Although impaired immune responses are related to pathogenesis of cancers, these patients are usually treated by surgery, local ablations methods, radiotherapeutic approaches, chemotherapy, and other methods. The limitations of ongoing regimens of anticancer therapy are evident. Many patients with advanced cancer cannot be treated by surgery, and cancers in many organs cannot be properly approached. The efﬁcacy of radiotherapy and chemotherapy is not satisfactory. In addition, these therapeutic approaches compromise the quality of life. Cancer recurrences represent another major problem in cancer patients. Even after almost complete eradication of cancers, cancer recurrence is often detected either at the original site or at other, distant locations. The standard therapeutic approach for treating patients with chronic viral infections is to use antiviral drugs. However, these drugs are indicated in only a group of patients with chronic viral infections. In addition, these therapies are effective in fewer patients. Antiviral drugs cause reduction of replication of the viruses but are incapable of their complete eradication. Moreover, use of antiviral drugs causes different types of side effects, some of which may be life threatening. In this context, it is required to develop alternate and effective therapeutic approaches against different diseases. One interesting observation provides a scientiﬁc logic for immune therapy using antigen-speciﬁc immunomodulators in patients with cancers and chronic viral infections. Analyses of immune responses before and after traditional therapies have shown that patients who respond to different ongoing regimens of therapeutic approach usually exhibit restoration of antigen-speciﬁc immunity. However, these effects are not seen in nonresponders.

Strategy of Antigen-Speciﬁc Immune Therapy The traditional method of induction of antigen-speciﬁc immunity is to administer antigens in adjuvant or vaccines containing antigens. This procedure is usually effective to induce antigen-speciﬁc immunity in normal individuals and is widely used to induce protective immunity against several microbial agents by prophylactic vaccines. However, vaccine is not usually used as a therapeutic tool against chronic infections or cancers. It is assumed that patients with chronic viral infections and

160

8. Dendritic Cell-based Immune Therapy

cancers are tolerant to the virus and cancer-related antigens. Indeed, these patients harbor huge amounts of antigens in vivo, but fail to exhibit adequate levels of antigenspeciﬁc immunity. In this context, it is natural to ask why administration of antigens can induce antigen-speciﬁc immunity in these patients when immune responses could not be detected because of the persistent presence of circulating or tissue-localized antigens. The answer to these queries could not be provided for a long time, although it was speculated that administration of vaccine may have immunomodulatory and therapeutic effects in patients with chronic viral infections. The rationale of vaccine therapy became evident when investigators provided evidence about the mechanisms underlying impaired antigen-speciﬁc immune responses of patients with cancers and chronic viral infections. To induce adequate levels of antigen-speciﬁc immunity, antigens should be recognized and processed by the antigen-presenting cells (APCs) of the hosts. Then, the processed antigens should be presented to lymphocytes for induction of antigen-speciﬁc immunity. It seems that circulating tumor antigens of cancer patients or viral antigens of patients with chronic viral infections act as harmless entities. These antigens may not have been recognized and processed by the APCs of the hosts. Also, adequate levels of antigen-speciﬁc immunity may not be induced in these patients because of the immune escape mechanism of the viral-infected or tumor-bearing hosts. Finally, even if antigen-speciﬁc immunity is induced in these patients, it may not be strong enough to protect the hosts and to establish an immune surveillance system. However, when so-called tolerogenic antigens have repeatedly been administered along with adjuvant, antigen-speciﬁc immunity was induced in animal models of cancers and chronic viral infections. In addition to antigen-based vaccines, epitopebased vaccines and DNA-based vaccines have also been used to induce antigenspeciﬁc immunity. Although the underlying mechanisms are not completely understood, it seems that antigens in adjuvant induce dangerous signals for induction of antigen-speciﬁc immunity. In the next phase, different pilot studies have carried out in patients with cancers by vaccine therapy in which TAAs or products containing TAAs have been administered for induction of TAA-speciﬁc immunity and treatment of cancers. The ﬁnal therapeutic evaluation of vaccine therapy is difﬁcult to assess because only a few controlled trials have been conducted with vaccine therapy in cancer patients. In addition to cancer patients, vaccine therapy has also been given to patients with viral chronic infections. However, the clinical efﬁcacy is yet to be conﬁrmed by controlled trials. Although preliminary data have shown promising and inspiring therapeutic efﬁcacy of vaccine therapy, the therapeutic efﬁcacy of these approaches could not be conﬁrmed in controlled clinical trials.

Limitations of Immune Therapy by Vaccines or Antigens in Patients with Cancer and Chronic Viral Infections and the Concept of DC-Based Immune Therapy Immune therapy by antigen-based vaccines, peptides, antigenic epitopes, and DNAbased vaccines has been given mainly to patients with tumors; however, the therapeutic efﬁcacy of these approaches could not be conﬁrmed by randomized trials.

Immunomodulatory and Therapeutic Efﬁcacy of Antigen-Pulsed DCs

161

There are several causes underlying the limited efﬁcacy or nonefﬁcacy of the present regimens of antigen-speciﬁc immune therapy in these patients. Selection of proper antigens for vaccine therapy is crucial for the therapeutic efﬁcacy of antigen-based therapy. There may be several antigens of certain viruses, and there are many tumorrelated antigens in all types of tumors. Some of these antigens may induce protective immunity in vivo whereas others may not. If the purpose of immune therapy is to induce protective or surveillance types of immunity, and if the antigens of the vaccines are not able to do that, therapeutic efﬁcacy of vaccine therapy cannot be expected (see Fig. 1). In most cancers, little is known about TAAs. Moreover, the natures of immune responses induced by different TAAs have not been well evaluated. The same picture is applicable in chronic virus infection. At present, there is no standard protocol of vaccine therapy against chronic viral infections and cancers. The nature of antigens, amounts of vaccines, frequencies of administration of vaccines, and duration of vaccinations for treating patients with chronic viral infections and cancers have not been optimized. It is difﬁcult to obtain therapeutic effects if these variables are not optimized. In addition, there may be other factors causing low therapeutic efﬁcacy of vaccine therapy. Protein-based, DNA-based, or epitope-based vaccines are administered with the assumption that these vaccines would be recognized and processed by the endogenous APCs, especially DCs, of the patients. It is also expected that presentation of administered antigens by endogenous DCs would induce antigen-speciﬁc immunity in these patients. However, the functions of DCs are usually impaired in patients with cancers and chronic viral infections. Moreover, antigen presentation by DCs in vivo may induce both immune responses and immune tolerances. DCs of patients with cancers or chronic viral infections may not be able to perform proper processing and presentation of viral and cancer antigens for induction of desired types of immunity in vivo. These observations indicated that if DCs of patients with cancers and chronic viral infections can be activated in vitro in presence of antigens, then antigen-loaded activated DCs may induce potent immune modulation in vivo.

Immunomodulatory and Therapeutic Efﬁcacy of Antigen-Pulsed DCs in Animal Models of Cancer and Chronic Viral Infection Antigen-pulsed DCs have been extensively used in different animal models of human diseases, applied to induce immunity and treat animal models of tumors and chronic infectious diseases. Also, antigen-pulsed DCs have been used to deactivate immune responses and induce immune tolerances in animal models of autoimmune and allergic diseases. In animal models, either only antigen-pulsed DCs have been used or other immunomodulators have been used with antigen-pulsed DCs. The overall immunomodulatory capacities and therapeutic efﬁcacies of antigen-pulsed DCs are not only inspiring, but excellent. Administration of antigen-pulsed DCs has cured cancer, prolonged survival, and protected hosts from successive cancer challenge. In the context of viral infections, antigen-pulsed DCs drastically reduced viral replication and induced protective immunocytes and antibodies against different viral antigens. Antigen-pulsed DCs have also exhibited signiﬁcant capacities to block

162

8. Dendritic Cell-based Immune Therapy

autoimmunity in several animal models of human diseases. Also, transplants have been accepted as a result of induction of immune tolerance by antigen-pulsed DCs in different animal models.

Limited Therapeutic Efﬁcacy of Antigen-Pulsed DCs in Patients with Cancer Data from different clinical trials have shown that antigen-pulsed DCs are safe for cancer patients, but the therapeutic efﬁcacy of antigen-pulsed DCs is not so apparent. The immunomodulatory capacities of TAA-pulsed DCs have not been explored because of the limited understanding about tumor antigens and the nature of tumorspeciﬁc immune responses. The role of antigen-pulsed DCs in cancers has been elaborately discussed in the relevant chapter of this book (Chapter 6). Here, we discuss some points related to the limited therapeutic efﬁcacy of antigen-pulsed DCs in humans.

Different Immunomodulatory and Therapeutic Efﬁcacy of Antigen-Pulsed DCs in Animal Models of Human Diseases and Patients with Cancer More work is needed to clarify the mechanisms underlying the immunomodulatory and therapeutic efﬁcacy of antigen-pulsed DCs as it differs between animal models and patients. Some possible causes are shown in Fig. 2.

Ag-PULSED MURINE DC

Ag-PULSED HUMAN DC

SPLEEN, BONE MARROW, BLOOD, SYNGENEIC

ONLY BLOOD: AUTOLOGOUS

ANTIGENS

RECOMBINANT Ag OTHER SOURCES

SELECTED ANTIGENS

CULTURE CONDITIONS

FETAL CALF SERA

AMOUNTS OF DC

LARGE AMOUNTS

FEW

STATUS OF DC

POTENT FUNCTIONS

POOR FUNCTIONS

SOURCES OF DC

AUTOLOGOUS SERA

Fig. 2. Possible causes of high immunogenicity of antigen-pulsed DCs in murine system and low immunoregulatory and therapeutic potentials of human dendritic cells (DCs). Ag, antigen

Different Immunomodulatory and Therapeutic Efﬁcacy

163

Inherent Differences Between Animal Models of Human Diseases and Patients with Different Diseases Animal models of cancers are prepared by implanting cancer cells at the skin or in some part of the body. In some cases, carcinogenic agents are administered for development of different experimental cancers. Visible cancers are developed within days or weeks after implantation of cancer cells, which is fundamentally different from human cancer development. In most cases, human cancers develop as a consequence of genetic mutation, or the effects of different types of carcinogens, or following chronic inﬂammation. Usually, it takes several years for development of a clinically visible cancer in human. Accordingly, the immune status of cancer-bearing experimental animals and cancer patients is signiﬁcantly different. The nature of immune responses that would be induced by administration of antigen-pulsed DCs in animal models of cancers and human cancers is also different. Also, the functional capacities of effecter immunocytes that control growth of tumors differ between animal models of cancers and patients with cancers. Differences Regarding Therapeutic Protocol of Production of Antigen-Pulsed Murine and Human DCs 1. DCs are isolated from the bone marrow or spleen of mice and cultured with antigens for production of antigen-pulsed DCs. On the other hand, human DCs are isolated from precursor population of DCs in the peripheral blood. Murine antigenpulsed DCs, especially antigen-pulsed murine spleen DCs, are more immunogenic than human antigen-pulsed DCs, as has been found by comparing the functional capacities of antigen-pulsed murine and human DCs using the same antigens. However, if other antigens are used, different data may be accumulated. 2. For treating patients with cancers, DCs from the same patients should be used. On the other hand, DCs from healthy mice are used for preparing antigen-pulsed DCs for treating animal models of cancers. The functional capacities of DCs of diseased human and healthy mice are usually different. 3. Huge numbers of antigen-pulsed DCs are administered to mice with experimental cancers, whereas comparatively fewer numbers of antigen-pulsed DCs are injected into patients with cancers. 4. To prepare human antigen-pulsed DCs, autologous sera are used, whereas in mice fetal calf sera usually are used. Fetal calf sera induce the production of more immunogenic antigen-pulsed DCs. 5. The availability of antigens that can be used in human is a major limitation for DC-based therapy in patients with cancer. However, different types of antigens can be used for treatment of the mouse model of cancers and chronic infectious diseases. There have been few clinical trials with antigen-pulsed DCs in patients with chronic viral infections. Antigen-pulsed DCs have been used in patients with chronic hepatitis B. One of the antigens of hepatitis B virus (HBV) has been used to prepare antigenpulsed DCs. Antigen-pulsed DCs are safe for chronic HBV-infected patients, but therapeutic efﬁcacy is yet to be conﬁrmed by conducting more clinical trials.

164

8. Dendritic Cell-based Immune Therapy

Here, we have mentioned several factors for low therapeutic efﬁcacy of antigenpulsed DCs in patients with cancer, which may provide the impression that there is limited scope for using DC-based therapy in human. This is not true. It is natural that the efﬁcacy of immune therapy would differ between patients and animal models of the disease. New therapeutic approaches are developed after proper understanding of these limitations. In this context, it is necessary to pay attention to how more effective regimens of immune therapy may be developed by manipulating other variables. We have focused on different factors that can be manipulated to develop effective DC-based immune therapy against human diseases. We mainly concentrate on factors such as (1) production of human DCs, (2) methods of loading of DCs with antigens, and (3) immunization protocols of antigenpulsed DCs for obtaining a better therapeutic outcome of antigen-pulsed DCs in human diseases.

Points of Attention for Production of Immunogenic Antigen-Pulsed DCs to Have Better Immunomodulatory Capacities in Humans Most of the studies about antigen-pulsed DCs in human have been performed to treat patients with cancers. We discuss mainly the methods of improving ongoing protocols of preparation of antigen-pulsed DCs for humans on the basis of published studies. Minimizing the Effect of Contaminant Cells on the DC Preparation DCs are enriched in vitro from DC progenitors and DC precursors in the blood by culturing mononuclear cells of the peripheral blood with GM-CSF and IL-4; this gives DC populations that contain 70%–80% HLA DR+ DCs. However, the contaminant cell populations may have an impact on the immunomodulatory capacities of DCs. If the contaminant cells contain regulatory T cells, these may downregulate the functional capacities of DCs. On the other hand, if some activated T cells are present in DC populations, maturation of DCs can be induced during antigen pulsing. The nature of contaminant cells is not assessed during production of DCs but may be crucial for the immunomodulatory capacities of antigen-pulsed DCs in humans. Heterogeneous Maturation and Activation of Antigen-Pulsed DCs in Bulk Population of DCs To prepare antigen-pulsed DCs, human DCs are cultured with different types of antigens. The bulk population of DCs contains mainly immature DCs but may also contain progenitors and precursors of regulatory DCs. To prepare TAA-pulsed DCs, these DCs are cultured with tumor products either with or without maturation signals; thus, both TAA-pulsed immunogenic and TAA-speciﬁc tolerogenic DCs may be produced under this culture condition. The phenotypes of antigen-pulsed DCs are checked in some, but not in all, cases before administration to patients with cancers. Even if the mean ﬂuorescence intensity of HLA DR or CD86 is increased, it does not

The Ratio of Mature DCs in Bulk Populations of DCs: Is It Important?

165

indicate that all antigen-pulsed DCs are activated or matured. At present, no protocol has been presented that can guarantee the preparation of only mature and activated immunogenic antigen-speciﬁc DCs. However, new methodologies should be developed to realize this for better therapeutic efﬁcacy of antigen-pulsed DCs in human.

Why There Is Concern If Antigen-Pulsed DCs with Different Levels of Maturation and Activation Are Present Among Bulk Populations of Antigen-Pulsed DCs The nature of antigen-pulsed DCs has been little discussed until now in the context of DC-based therapy because little was known about regulatory DCs. However, the impact of regulatory DCs should be seriously considered for the therapeutic efﬁcacy of antigen-pulsed DCs. DC precursors, immature DCs and mature DCs are cultured to prepare antigen-pulsed DCs. The immature antigen-pulsed DCs may induce regulatory T cells after their administration into patients with cancers. These DCs may also induce antigen-speciﬁc tolerance in patients with cancers. These factors may be responsible for the low effectiveness or noneffectiveness of antigen-pulsed DCs for treatment of patients with cancers. In addition to concern about therapeutic efﬁcacy, administration of these DCs may be unethical in patients with cancer because these DCs may induce a state of immune tolerance for these patients.

Is There a Need to Check Chemokines on Antigen-Pulsed DCs? After administration, antigen-pulsed DCs should migrate to lymphoid tissue to induce antigen-speciﬁc immunity. If these DCs do not express CCR7 or other chemokines that facilitate migration of DCs, then it is unlikely that they will be able to move to lymphoid tissues efﬁciently. If they cannot migrate, the administered DCs might undergo apoptosis, and antigens can be captured and processed by regulatory DCs or immature DCs in the cancer microenvironment. The cancer microenvironment is usually hostile for immune responses because it contains various factors that downregulate immune responses.

The Ratio of Mature DCs in Bulk Populations of DCs: Is It Important? For practical purpose, when 70%–80% DCs are immunogenic, it is assumed that these DCs are good candidates for therapy. Recently, different studies have indicated that DCs should be 80% pure for clinical use for patients with cancers. This is probably the most practical approach for using DCs clinically because there is no means to prepare a more puriﬁed population of DCs from human blood. However, the limitations should also be considered. If 20% of the DCs are not maturing and remain immature, these may induce immunogenic tolerance. Thus, more study is needed to

166

8. Dendritic Cell-based Immune Therapy

obtain a homogeneous population of antigen-speciﬁc mature DCs for human therapy.

Checking of DCs for Their Capacity to Process and Present Antigens

Stimulation Index

Autologous DCs are used for treatment of patients with cancer. Many of these patients have been suffering from their cancers for a long period of time. The functional capacities of DCs are usually highly impaired in these cases. Considerable numbers of patients with cancer also suffer from impaired nutritional status. There are few studies about the role of nutrients in the functional capacities of DCs. Recently, we investigated the antigen-processing and -presentation capabilities of DCs in mice with protein-energy malnutrition (PEM). This study provided insights about the role of nutrition on DC function. When the nutritional status of the host is compromised as a secondary effect of different environmental agents or pathological processes, the real implication of nutrition on DC function remains elusive. Our study revealed that DCs from mice with PEM were almost incapable of processing and presenting speciﬁc antigens both in vitro and in vivo (Fig. 3). As patients with cancer exhibit nutritional anomalies, their DCs may also be incompetent to process and present antigens, and this may be one of the main factors for the low immunomodulatory capability of antigen-pulsed DCs of cancer patients. At present, the functional capacities of DCs of cancer patients are not tested before their use in immune therapy, but this is necessary for better therapeutic efﬁcacy of antigen-pulsed DCs in human diseases.

6

*

4

2 1

HBsAg-specific memory lymphocytes

2×105

2×105

1×104

HBsAg-pulsed DCs (Control mice) HBsAg-pulsed DCs (PEM mice)

2×105

1×104

Fig. 3. Chronic undernutrition induces impaired functional capacities of dendritic cells (DCs). DCs from control nutrient mice and mice with protein-energy malnutrition (PEM) were cultured with hepatitis B surface antigen (HBsAg) to prepare HBsAg-pulsed DCs. HBsAg-pulsed DCs from control mice induced proliferation of HBsAg-speciﬁc lymphocytes, whereas HBsAgpulsed DCs from PEM mice were almost incapable of inducing antigen-speciﬁc proliferation of lymphocytes

Role of Innate Immunity During Induction of Antigen-Speciﬁc Adaptive Immunity

167

Choosing Antigens for Pulsing DCs To prepare antigen-pulsed DCs for treatment of cancers, DCs are cultured with different tumor-related products. We have provided a detailed account about this in the relevant chapter about DCs in tumorigenesis. It is well known that some cancerrelated products may be immunogenic whereas others may be tolerogenic. Only immunogenic antigens should be used for pulsing DCs with cancer antigens. Little is known about the nature of various cancer antigens, which should be always borne in mind for preparing immunogenic antigen-pulsed DCs for different pathological conditions.

Assessment of Immunogenicity of Antigen-Pulsed DCs Full characterization of antigen-pulsed DCs before their administration to patients is needed for better therapeutic effects of antigen-pulsed DCs. Mere examination of expression of HLA DR and CD86 may not be sufﬁcient. The cytokine production capacities of the antigen-pulsed DCs should be checked. Also, it should be assessed whether the antigen-pulsed DCs can induce proliferation of antigen-speciﬁc lymphocytes in vitro.

Role of Innate Immunity During Induction of Antigen-Speciﬁc Adaptive Immunity The purpose of immune therapy by antigen-pulsed DCs against cancers and chronic infectious diseases is to induce strong antigen-speciﬁc immunities in the patients. However, activities of the cells of innate immunities are essential for induction and maintenance of antigen-speciﬁc immunities. Under normal conditions, tumor cells or microbial agents induce innate immunity in the hosts, allowing destruction of the majority of harmful and dangerous agents. Next, innate immunity provides directions about the type of adaptive immunity, and long-lasting protective immunity is generated. In the case of immune therapy, there is a need to assess if antigen-pulsed DCs can induce both innate as well as adaptive immunity. In a recent study, we found conclusive data regarding the need of natural killer (NK) cells during induction of antigen-speciﬁc adaptive immunity. Mice with depleted NK cells could not exhibit adequate levels of antigen-speciﬁc humoral and cellular immune responses by means of administration of vaccines containing antigens, mainly because the vaccines could not induce innate immunity in the NKdepleted mice. However, when NK-depleted mice were immunized with activated antigen-pulsed spleen DCs that stimulated both innate and adaptive immunity, antigen-speciﬁc immunity was seen in all the NK-depleted mice (Fig. 4). A putative design of developing immune therapy using antigen-pulsed DCs is shown in Table 1.

8. Dendritic Cell-based Immune Therapy

168

Interleukin12p70

10

3000

7.5

pg/ml

4000

Tumor necrosis factor-α

5

2000

*

2.5

0 DC DC (Control) (NK-depleted )

1200 800

*

*

400

1000

0

Interferon-γ

0 DC DC (Control) (NK-depleted )

DC DC (Control) (NK-depleted )

Stimulation index

Anti-HBs (mIU/ml)

NK-depleted mice immunized with HBsAg NK-depleted mice immunized with HBsAg-pulsed DCs

[A] 200

* 0

N=5

N=5

30

[B]

10

*

0

N=5

N=5

Fig. 4. Innate immunity is essential for induction of antigen-speciﬁc adaptive immunity through cytokine production by dendritic cells (DCs). Depletion of natural killer (NK) cells resulted in signiﬁcantly lower levels of cytokine production by spleen DCs (upper panel). NKdepleted mice could not produce hepatitis B surface antigen (HBsAg)-speciﬁc humoral (A) and cellular immune responses (B) (lower panel) resulting from immunization with vaccine containing HBsAgs. However, NK-depleted mice showed HBsAg-speciﬁc immunity when administered with activated HBsAg-pulsed DCs that induced higher levels of various proinﬂamamtory cytokines from spleen cells (lower panel)

Table 1. Designing dendritic cell (DC)-based immune therapy for humans Purpose-based designing of interventional strategy: A. Treatment of tumor: Induction of cytopathic T cells capable of destroying tumor antigen-bearing tumor cells. B. Treatment of chronic virus infection: differs with the nature of the virus. In most cases antigen-pulsed DCs should control viral replication, mainly by a noncytopathic mechanism. C. Treatment of autoimmunity and protection of transplants: Induction of regulatory DCs and regulatory T cells by antigen-pulsed DCs. [There is no universal strategy or design of DC-based immune therapy] Optimization of immunization protocols Antigens: Should be selected on the basis of nature of immune responses. DCs pulsed with crude products may be counterproductive. Dose, duration, and amounts of DCs: Should be conﬁrmed by preclinical study in vitro. Nature of antigen-pulsed DCs: Dependent on the purpose of therapy. However, these DCs should have migratory capabilities. Antigen-pulsed DCs should also be able to induce antigen-speciﬁc immunity or tolerogenicity in vitro.

Recommended Readings

169

Concluding Remarks Several guidelines regarding preparation, pulsing, and use of antigen-pulsed DCs for treatment of cancers have been provided in the literature. However, the real situation about DC-based therapy in human is complex, for all types of emerging and evolving therapies. It appears that the clinical outcome of DC-based therapy in cancer is not satisfactory. However, cancer is a complex disease entity and there are few treatment options in these patients. Moreover, patients with advanced cancers have usually been treated by DC-based immune therapy. In addition to cancers, DC-based immune therapy has been started in patients with chronic viral infections. Some clinical trials have conﬁrmed the safety and immunomodulatory capacities of antigen-pulsed DCs in patients with chronic hepatitis B virus infection. We have used DCs to induce humoral immunity when that could not be induced by administration of prophylactic vaccines in normal individuals. Thus, the spectrum of using antigen-pulsed DCs is progressing rapidly from cancers to various pathological conditions including prophylactic purposes. These therapeutic approaches will also unmask the real potential of antigen-pulsed DCs in human. Although it has been shown that the present regimens of DC-based therapy are not so effective for treatment of patients with cancers, this potentially important therapeutic approach may have signiﬁcant therapeutic efﬁcacy in other pathological conditions. The purpose of immune therapy in cancer patients and in patients with other diseases is different. The main purpose of using antigen-pulsed DCs in cancer patients is to induce cytotoxic T lymphocytes (CTL). These CTLs are expected to kill tumor cells and block progression of tumor regeneration. Even if antigen-pulsed DCs are unable to induce adequate levels of tumor-speciﬁc CTLs in cancer patients, this does not reduce the chance of potent therapeutic efﬁcacy of antigen-pulsed DCs in other pathological conditions. There is a strong possibility that antigen-pulsed DCs will show more efﬁcacy in noncancerous patients compared to cancer patients. Most of noncancerous patients are immune competent and have increased numbers of DCs compared to cancer patients. Also, the functional capabilities of DCs are more potent in noncancerous patients than cancer patients. These points have been described so that more potent therapeutic regimens can be developed using DC against different diseases.

Recommended Readings Banchereau J, Palucka AK (2005) Dendritic cells as therapeutic vaccines against cancer. Nat Rev Immunol 5:296–306 Fazle Akbar SM, Abe M, Yoshida O, Murakami H, Onji M (2006) Dendritic cell-based therapy as a multidisciplinary approach to cancer treatment: present limitations and future scopes. Curr Med Chem 13:3113–3119 Figdor CG, De Vries IJ, Lesterhuis WJ, Melief CJ (2006) Dendritic cell immunotherapy: mapping the way. Nat Med 10:475–480 Mocellin S (2005) Cancer vaccines: the challenge of developing an ideal tumor killing system. Front Biosci 10:2285–2305

170

8. Dendritic Cell-based Immune Therapy

Slingluff CL Jr, Engelhard VH, Ferrone S (2006) Peptide and dendritic cell vaccines. Clin Cancer Res 12:2342s–2345s Steinman RM, Banchereau J (2007) Taking dendritic cells into medicine. Nature (Lond) 449:419–426 Tuyaerts S, Aerts JL, Corthals J, Neyns B, Heirman C, Breckpot K, Thielemans K, Bonehill A (2007) Current approaches in dendritic cell generation and future implications for cancer immunotherapy. Cancer Immunol Immunother 56:1513–1537

Authors

Morikazu Onji, M.D., Ph.D. Curriculum vitae Date of birth: Place of birth: March 1973 May 1973

February 13, 1948 Ehime, Japan Graduated from Kagoshima University School of Medicine Resident, The First Department of Internal Medicine, Okayama University Medical School June 1974 Medical Staff in Internal Medicine, Saiseikai Imabari Hospital April 1976 Clinical and Research Fellow, First Department of Biochemistry, Osaka City Medical Board April 1977 Assistant Professor, The Third Department of Internal Medicine, Ehime University School of Medicine November 1984 Ph.D. (Doctor of Medicine), Ehime University May 1985 Associate Professor (Lecturer), The Third Department of Internal Medicine, Ehime University School of Medicine. January 1986 Research Fellow, Royal Free Hospital, London University May 1994-present Professor and Chairman, The Third Department of Internal Medicine, Ehime University April 2000–2005 Head, Endoscopy Center, Ehime University Hospital April 2000–2005 Deputy Director, Ehime University Hospital Awards 1988 1988 1991 1992

Young Investigation Award from Japan Society of Hepatology Award from Kanae Foundation for Life and Socio-Medical Sxcience Investigator’s Award from Japan Gastroenterology Endoscopy Society Investigator’s Award from the Foundation for Viral Hepatitis Research.

Society Memberships American Association for the Study of Liver Diseases (AASLD) The European Association for the Study of Liver (EASL) International Association for the Study of Liver Diseases (IASL) Asia-Paciﬁc Association for the Study of the Liver (APASL) Japanese Society of Gastroenterology (Member of Council) Japan Society of Hepatology (Member of Council) Liver Cancer Study Group of Japan (Member of Council) Japanese Gastroenterological Endoscopy Society (Member of Council) Japanese Society of Allergology (Member of Council) 171

172

Authors

Japanese Society of Mucosal Immunology (Executive Board Member) Japan Diabetic Society (Member of Council) Japan Society of Metabolism and Clinical Nutrition (Executive Board Member)

Sk. Md. Fazle Akbar, MBBS, Ph.D. 1980 1985 1993 1993 1994 1996 1998 2007

Medical Graduate, Rajshahi Medical College, Bangladesh. Evaluator, Communicable Disease Control, D/G Health, Bangladesh. Ph.D. in Medical Science, Ehime University, Ehime, Japan. Research Assistant, Institute of Post Graduate Medicine & Research, Dhaka, Bangladesh. Post-Doctoral Fellow, Ehime University School of Medicine, Japan. Assistant Professor, Department of Hygiene, Ehime University School of Medicine, Japan. Assistant Professor, Third Department of Internal Medicine, Ehime University School of Medicine, Japan. Lecturer, Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine.

Society Memberships American Association for the Study of Liver Diseases (AASLD) The European Association for the Study of Liver (EASL) Asia-Paciﬁc Association for the Study of the Liver (APASL) Japanese Society of Gastroenterology Japan Society of Hepatology Japanese Society for Immunology

Bibliography

1986–1996 Akbar SMF, Onji M, Inaba K, Yamamura K-I, Ohta Y (1993) Low responsiveness of hepatitis B virus-transgenic mice in antibody response to T-cell dependent antigen: defect in antigen-presenting activity of dendritic cells. Immunology 78:468–475 Akbar SMF, Inaba K, Onji M (1996) Upregulation of MHC class II antigen on dendritic cells from hepatitis B virus transgenic mice by interferon-gamma: abrogation of immune response defect to a T-cell-dependent antigen. Immunology 87:519–527 Kanaoka M, Kumon I, Michitaka K, Onji M, Nadano S, Ohta Y (1986) An electron microscopic study of follicular hepatitis. J Clin Electron Microsc 19:559–560 Kumon I, Onji M, Nadano S, Kanaoka M, Ohta Y (1989) Detection of hepatitis B surface antigen in lymphoid follicle in patients with chronic hepatitis type B. Gastroenterol Jpn 24:738 Kumon I (1992) In site characterization of mononuclear cell phenotype in intrahepatic lymphoid follicle in patients with chronic viral hepatitis. Gastroenterol Jpn 27: 638–645 1997 Akbar SM, Kajino K, Tanimoto K, Kurose K, Masumoto T, Michitaka K, Horiike N, Onji M (1997) Placebo-controlled trial of vaccination with hepatitis B virus surface antigen in hepatitis B virus transgenic mice. J Hepatol 26:131–137 Akbar SM, Masumoto T, Onji M (1997) Role of lymphoid dendritic cells in persistent hepatitis B virus infection. Dendritic Cells 7:16–18 Kurose K, Akbar SM, Yamamoto K, Onji M (1997) Production of antibody to hepatitis B surface antigen (anti-HBs) by murine hepatitis B virus carriers: neonatal tolerance versus antigen presentation by dendritic cells. Immunology 92:494–500 Onji M, Kumon I, Nadano S, Akbar SMF, Michitaka K (1997) Existence of hepatitis B surface antigen in intrahepatic lymphoid follicles in patients with chronic viral hepatitis. Dendritic Cells 7:12–15 1998 Akbar SM, Onji M (1998) Hepatitis B virus (HBV) transgenic mice as an investigative tool to study immunopathology during HBV infection. Int J Exp Pathol 79:279–291 Hiasa Y, Horiike N, Akbar SM, Saito I, Miyamura T, Matsuura Y, Onji M (1998) Low stimulatory capacity of lymphoid dendritic cells expressing hepatitis C virus genes. Biochem Biophys Res Commun 249:90–95 Onji M, Shinomiya M, Masumoto T, Yamauchi Y, Yamamoto K, Hoiike N, Michitaka K, Akbar SM (1998) Dendritic cells in the liver of patients with primary biliary cirrhosis. Dendritic Cells 8:7–9 173

174

Bibliography

Shinomiya M, Masumoto T, Nadano S, Akbar SMF, Onji M (1998) Lymphoid dendritic cells in the liver of patients with primary biliary cirrhosis and its mouse model. Hepatol Res 1:84–94 Yamamoto K, Akbar SM, Masumoto T, Onji M (1998) Increased nitric oxide (NO) production by antigen-presenting dendritic cells is responsible for low allogeneic mixed leucocyte reaction (MLR) in primary biliary cirrhosis(PBC). Clin Exp Immunol 114:94–101 1999 Akbar SM, Kajino K, Tanimoto K, Yamamura K, Onji M, Hino O (1999) Unique features of dendritic cells in IFN-γ transgenic mice: Relevance to cancer development and therapeutic implications. Biochem Biophys Res Commun 259:294–299 Akbar SM, Abe M, Masumoto T, Horiike N, Onji M (1999) Mechanism of action of vaccine therapy in murine hepatitis B virus carriers: Vaccine-induced activation of antigen presenting dendritic cells. J Hepatol 30:755–764 Akbar SM, Onji M, Hino O (1999) Antigen-presenting dendritic cells in the pathogenesis and therapy of cancer. Recent research development in cancer. Research Signpost, Trivandram, India, pp 229–236 Akbar SM, Yamamoto K, Abe M, Ninomiya T, Tanimoto K, Masumoto T, Michitaka K, Horiike N, Onji M (1999) Potent synergistic effect of sho-saiko-to, a herbal medicine, during vaccine therapy in a murine model of hepatitis B virus carrier. Eur J Clin Invest 29:786–792 Akbar SM, Horiike N, Onji M (1999) Prognostic importance of antigen-presenting dendritic cells during vaccine therapy in a murine hepatitis B virus carrier. Immunology 96:68–108 Ninomiya T, Akbar SM, Masumoto T, Horiike N, Onji M (1999) Dendritic cells with immature phenotype and defective function in the peripheral blood from patients with hepatocellular carcinoma. J Hepatol 31:323–331 Onji M, Akbar SM, Hiasa Y, Masumoto T, Horiike N, Michitaka K (1999) Intrahepatic dendritic cells. Prog Hepatol 5:15–22 Onji M (1999) In what types of diseases does anti-carbonic anhydrase II antibody positivity have clinical signiﬁcance? J Gastroenterol 34:431–432 Shinomiya M, Akbar SM, Sinomiya H, Onji M (1999) Transfer of dendritic cells (DC) ex vivo stimulated with interferon-gamma (IFN-γ) down-modulates autoimmune diabetes in non-obese diabetic (NOD) mice. Clin Exp Immunol 117:38–43 Tanimoto K, Akbar SM, Michitaka K, Onji M (1999) Immunohistochemical localization of antigen presenting cells in liver from patients with primary biliary cirrhosis: highly restricted distribution of CD83-positive activated dendritic cells. Pathol Res Pract 195:157–162 Tanimoto K, Akbar SM, Nakatya M, Kobayashi F, Chen S, Masumoto T, Michitaka K, Horiike N, Onji M (1999) Immunohistochemical detection of CD83 antigen in liver tissue sections. Dendritic Cells 9:26–28 Tamaru M, Matsuura B, Onji M (1999) Dendritic cells produce interleukin-12 in hyperthyroid mice. Eur J Endocrinol 141:625–629 2000 Akbar SM, Onji M (2000) Immune therapies for chronic hepatitis B virus carrier: role of recently developed vaccine therapy and traditional Chinese herbal medicine (sho-saikoto). Cent Eur J Immunol 25:37–42 Chen S, Akbar SM, Tanimoto K, Ninomiya T, Iuchi H, Michitaka K, Horiike N, Onji M (2000) Absence of CD83-positive mature and activated dendritic cells at cancer nodules

Bibliography

175

from patients with hepatocellular carcinoma: relevance to hepatocarcinogenesis. Cancer Lett 148:49–57 Masumoto T, Yamamoto K, Abe M, Akbar SM, Onji M (2000) Role of dendritic cells in patients with primary biliary cirrhosis. In: Matano S, Tuttle RH, Ishida H, Goodman M (eds) Autoimmune liver disease: its recent advances. University of Tokyo, Tokyo, Japan Ninomiya T, Matsui H, Akbar SM, Murakami H, Onji M (2000) Localization and characterization of antigen-presenting dendritic cells in the gastric mucosa of murine and human autoimmune gastritis. Eur J Clin Invest 30:350–358 Okazaki A, Tanimoto K, Akbar SM, Ninomiya T, Kumagi T, Abe M, Hiasa Y, Masumoto T, Michitaka K, Horiike N, Onji M (2000) Expression of inducible nitric oxide synthase on antigen-presenting cells in patients with primary biliary cirrhosis. Dendritic Cells 10:23–24 Onji M, Akbar SM, Hiasa Y, Masumoto T, Horiike N, Michitaka K (2000) Intrahepatic dendritic cells. In: Progress in hepatology, vol 5. Liver and immunology. Elsevier, Amsterdam, pp 13–22 Shinomiya M, Nadano S, Shinomiya H, Onji M (2000) In situ characterization of dendritic cells occurring in the islets of nonobese diabetic mice during the development of insulitis. Pancreas 20:290–296 Tanimoto K, Akbar SM, Shudo M, Kobayashi F, Shintani I, Nakaya M, Kumagi T, Abe M, Hiasa Y, Masumoto T, Michitaka K, Horiike N, Onji M (2000) Immunohistochemical localization of CD83+ dendritic cells in patients with primary biliary cirrhosis. Dendritic Cells 10:19–21 2001 Abe M, Akbar SM, Horiike N, Onji M (2001) Induction of cytokine production and proliferation of memory lymphocytes by murine liver dendritic cell progenitors: role of these progenitors as immunogenic resident antigen-presenting cells in the liver. J Hepatol 34:67–71 Akbar SM, Horiike N, Onji M, Hino O (2001) Dendritic cells and chronic hepatitis viruscarriers. Intervirology 44:199–208 Akbar SM, Yamamoto K, Miyakawa H, Ninomiya T, Abe M, Hiasa Y, Masumoto T, Horiike N, Onji M (2001) Peripheral blood T-cell responses to pyruvate dehydrogenase complex in primary biliary cirrhosis: role of antigen-presenting dendritic cells. Eur J Clin Invest 31:639–646 Ikeda Y, Akbar SM, Matsui H, Onji M (2001) Characterization of antigen-presenting dendritic cells in the peripheral blood and colonic mucosa of patients with ulcerative colitis. Eur J Gastroenterol Hepatol 13:841–850 Kumagi T, Akbar SM, Horiike N, Onji M (2001) Increased serum levels of macrophagemigration inhibitory factor (MIF) at the sera in alcoholic liver diseases and their expression in liver tissues. Clin Biochem 34:189–193 Murakami H, Akbar SM, Matsui H, Onji M (2001) Macrophage migration inhibitory factor in the sera and at the colonic mucosa in patients with ulcerative colitis; clinical implications and pathogenic signiﬁcance. Eur J Clin Invest 31:337–343 Oka Y, Akbar SM, Horiike N, Joko K, Onji M (2001) Mechanism and therapeutic potential of DNA-based immunization against the envelope proteins of hepatitis B virus in normal and transgenic mice. Immunology 103:90–97 Onji M, Akbar SM, Horiike N (2001) Dendritic cell-based immunotherapy for hepatocellular carcinoma. J Gastroenterol 36:794–797 Tanimoto K, Akbar SM, Michitaka K, Horiike N, Onji M (2001) Antigen-presenting cells at the liver tissue in patients with chronic viral liver diseases: CD83-positive mature dendritic cells at the vicinity of focal and conﬂuent necrosis. Hepatol Res 21:117–125

176

Bibliography

2002 Abe M, Kajino K, Akbar SM, Yamamura K, Onji M, Hino O (2002) Loss of immunogenicity of liver dendritic cells from mouse with chronic hepatitis. Int J Mol Med 9:71–76 Akbar SM, Horiike N, Onji M (2002) Therapeutic vaccine in chronic hepatitis B virus [HBV]-carriers. In: Recent research development in immunology, vol 4. Research Signpost, Trivandrum, Kerala, India, pp 695–702 Hiasa Y, Akbar SM, Abe M, Michitaka K, Horiike N, Onji M (2002) Dendritic cell subtypes in autoimmune liver diseases; decreased expression of HLA DR and CD123 on type 2 dendritic cells. Hepatol Res 22:241–249 Horiike N, Akbar SM, Ninomiya T, Abe M, Michitaka K, Onji M (2002) Activation and maturation of antigen-presenting dendritic cells during vaccine therapy in patients with chronic hepatitis due to hepatitis B virus. Hepatol Res 23:38–47 Ikeda Y, Akbar SM, Matsui H, Onji M (2002) Antigen-presenting dendritic cells in ulcerative colitis. J Gastroenterol 37:53–55 Murakami H, Akbar SM, Matsui H, Horiike N, Onji M (2002) Macrophage migration inhibitory factor activates antigen-presenting dendritic cells and induces inﬂammatory cytokines in ulcerative colitis. Clin Exp Immunol 128:504–510 2003 Abe M, Akbar SM, Horiike N, Onji M (2003) Inability of liver dendritic cells from mouse with experimental hepatitis to process and present speciﬁc antigens. Hepatol Res 26:61–67 Abe M, Akbar SM, Horiike N, Onji M (2003) Defective antigen-presenting cell capacity of murine dendritic cells due to starvation. Nutrition 19:265–269 Abe M, Akbar SM, Hasebe A, Horiike N, Onji M (2003) Glycyrrhizin enhances interleukin10 production from liver dendritic cells in mice with hepatitis. J Gastroenterol 38:962–967 Arima S, Akbar SM, Michitaka K, Horiike N, Nuriya H, Kohara M, Onji M (2003) Impaired function of antigen-presenting dendritic cells in patients with chronic hepatitis B: localization of HBV DNA and HBV RNA in blood DC by in situ hybridization. Int J Mol Med 11:169–174 Ikeda Y, Akbar SM, Matsui H, Murakami H, Onji M (2003) Depletion and decreased function of antigen-presenting dendritic cells due to lymphocytapheresis in ulcerative colitis. Dis Colon Rectum 46:521–528 Kumagi T, Akbar SM, Horiike N, Onji M (2003) Increased survival and decreased tumor size due to intratumoral injection of ethanol followed by administration of immature dendritic cells. Int J Oncol 23:949–955 2004 Akbar SM, Murakami H, Horiike N, Onji M (2004) Dendritic cell-based therapies in the bench and the bed sides. Curr Drug Targets Inﬂamm Allergy 3:305–310 Akbar SM, Furukawa S, Horiike N, Onji M (2004) Vaccine therapy for hepatitis B virus carrier. Curr Drug Targets Infect Disord 4:93–101 Akbar SM, Furukawa S, Hasebe A, Horiike N, Michitaka K, Onji M (2004) Production and efﬁcacy of a dendritic cell-based therapeutic vaccine for murine chronic hepatitis B virus carrier. Int J Mol Med 14:295–299 Akbar SM, Furukawa S, Onji M, Muarta Y, Niya T, Kanno S, Murakami H, Horiike N (2004) Safety and efﬁcacy of hepatitis B surface antigen-pulsed dendritic cells in human volunteers. Hepatol Res 29:136–141

Bibliography

177

Furukawa S, Akbar SM, Hasebe A, Horiike, Onji M (2004) Production of hepatitis B surface antigen-pulsed dendritic cells from immunosuppressed murine hepatitis B virus carrier: evaluation of immunogenicity of antigen-pulsed dendritic cells in vivo. Immunobiology 209:551–557 Furukawa S, Akbar SM, Hasebe A, Horiike, N, Onji M (2004) Induction and maintenance of anti-HBs in immunosuppressed murine hepatitis B virus carrier by a novel vaccination approach: implication during liver transplantation in hepatitis B virus-infected subjects. J Gastroenterol 39:851–858 Hiasa Y, Takahashi H, Shimizu M, Nurita H, Tsukiyama-Kohara K, Tanaka T, Horiike N, Onji M, Kohara M (2004) Major histocompatibility complex class-I presentation impaired in transgenic mice expressing hepatitis C virus structural proteins during dendritic cell maturation. J Med Virol 74:253–261 Kobayashi Y, Murakami H, Akbar SM, Matsui H, Onji M (2004) A novel and effective approach of developing aggressive experimental autoimmune gastritis in neonatal thymectomized Balb/c mouse by polyinocynic:polycytidylic acid. Clin Exp Immunol 136:423–431 Murakami H, Akbar SM, Matsui K, Horiike N, Onji M (2004) Decreased interferon-alpha production and impaired T helper 1 polarization by dendritic cells from patients with chronic hepatitis C. Clin Exp Immunol 137:559–565 Tsubouchi E, Akbar SM, Murakami H, Horiike N, Onji M (2004) Isolation and functional analysis of circulating dendritic cells from hepatitis C virus (HCV) RNA-positive and HCV RNA-negative patients with chronic hepatitis C: role of antiviral therapy. Clin Exp Immunol 137:417–423 Tsubouchi E, Akbar SM, Horiike N, Onji M. Infection and dysfunction of circulating blood dendritic cells and their subsets in chronic hepatitis C virus infection. J Gastroenterol 39:754–762 2005 Akbar SM, Onji M (2005) Preventive and therapeutic approaches against hepatitis B virus by vaccine. In: Hepatitis B annual, vol 2, issue 1. Kalinga Gastroenterology Foundation. MedKnow Publication, Mumbai, India, pp 31–55 Akbar SM, Furukawa S, Nakanishi S, Abe M, Horiike N, Onji M (2005) Therapeutic efﬁcacy and decreased nitrite production by bezaﬁbrate in patients with primary biliary cirrhosis. J Gastroenterol 40:157–163 Hasebe A, Akbar SM, Furukawa S, Horiike N, Onji M (2005) Impaired functional capacities of liver dendritic cells from murine hepatitis B virus (HBV) carriers: relevance to low HBV-speciﬁc immune responses. Clin Exp Immunol 139:35–42 Hiraoka A, Horiike N, Akbar SM, Michitaka K, Matsuyama T, Onji M (2005) Soluble CD163 in patients with liver diseases; very high levels of soluble CD163 in patients with fulminant hepatic failure. J Gastroenterol 40:52–56 Hiraoka A, Horiike N, Akbar SM, Michitaka K, Matsuyama T, Onji M (2005) Expression of CD163 in the liver of patients with viral hepatitis. Pathol Res Pract 201:379–384 Horiike N, Akbar SM, Michitaka K, Joukou K, Yamamoto K, Kojima N, Hiasa Y, Abe, Onji M (2005) In vivo immunization by vaccine therapy following virus suppression by lamivudine: a novel approach for treating patients with chronic hepatitis B. J Clin Virol 32:156–161 Kumagi T, Akbar SM, Horiike N, Kurose K, Hirooka M, Hiraoka A, Hiasa Y, Michitaka K, Onji M (2005) Administration of dendritic cells in cancer nodules in hepatocellular carcinoma. Oncol Rep 14:969–973 Onji M, Akbar SM (2005) On dendritic cell-based therapy for cancers. J Zhejang Univ Sci B 6:1

178

Bibliography

2006 Akbar SM, Horiike N, Onji M (2006) Immune therapy including dendritic cell-based therapy in chronic hepatitis B virus infection. World J Gastroenterol 12:2876–2883 Akbar SM, Abe M, Yoshida M, Murakami H, Onji M (2006) Dendritic cell-based therapy as a multidisciplinary approach to cancer treatment: present limitation and future scopes. Curr Med Chem 13:3113–3119 Akbar SM, Murakami H, Horiike N, Onji M (2006) Rationale for designing of antigenspeciﬁc immune therapy including dendritic cell-based therapy in patients with chronic hepatitis B virus infection. Anti-infect Agents Med Chem 5:75–86 Akbar SM, Yoshida O, Horiike N, Onji M (2006) Immune therapy of cancers by antigenpresenting dendritic cells: can it stand the test of time. Curr Trends Immunol 7:97–103 Akbar SM, Yoshida O, Horiike N, Onji M (2006) Antiviral drugs for treatment of chronic hepatitis B virus infection: what is expected and what can be achieved in developing countries. Curr Top Virol 5:31–38 2007 Akbar SM, Furukawa S, Yoshida O, Hiasa Y, Horiike N, Onji M (2007) Induction of antiHBs in HB vaccine nonresponders in vivo by hepatitis B surface antigen-pulsed blood dendritic cells. J Hepatol 47:60 Akbar SM, Yoshida O, Abe M, Hiasa Y, Onji M (2007) Engineering immune therapy against hepatitis B virus: Hepatol Res 37(suppl):S351–S356 Akbar SM, Yoshida O, Onji M (2007) Revisiting the monotherapy with antiviral drugs in patients with chronic hepatitis B: ethical and scientiﬁc basis of combination therapy and their application in clinics. In: Willis AP (ed) Hepatitis B research advances. Nova, Hauppauge, NY, pp 9–20 Mashiba T, Udaka K, Hirachi Y, Hiasa Y, Miyakawa T, Satta Y, Osoda T, Kataoka S, Kohara M, Onji M (2007) Identiﬁcation of CTL epitopes in hepatitis C virus by a genome-wide computational scanning and a rational design of peptide vaccine. Immunogenetics 59:197–209 Niiya T, Akbar SM, Yoshida O, Miyake T, Matsuura B, Murakami H, Abe M, Hiasa Y, Onji M (2007) Impaired dendritic cell function resulting from chronic undernutrition disrupts the antigen-speciﬁc immune response in mice. J Nutr 137:671–675 2008 Torisu M, Murakami H, Akbar SM, Matsui H, Hiasa Y, Matsuura B, Onji M (2008) Protective role of interleukin-10-producing regulatory dendritic cells against murine autoimmune gastritis. J Gastroenterol 43:100–107

Yoshida O, Akbar SM, Miyake T, Abe M, Hiasa Y, Matsuura B, Onji M (2008) Impaired dendritic cell (DC) functions due to depletion of natural killer (NK) cells disrupt antigen-speciﬁc immune responses in mice: restoration of adaptive immunity in NK-depleted mice by antigen-pulsed DC 152:174–181

Glossary

• Anergy Failure to make an immune response following stimulation by potential antigens. • Antigen presentation The process by which certain cells in the body (antigen-presenting cells) express antigen on their cell surface in a form recognizable by lymphocytes. • APCs (antigen-presenting cells) A variety of cell types that carry antigen in a form for presentation to lymphocytes. Only dendritic cells (DCs) can present antigen to naïve lymphocytes. Presentation of antigens by APCs may cause immune responses or immune tolerances. • Antigen processing The conversion of an antigen within antigen-presenting cells in a form that can be recognized by lymphocytes. • Antigen recognition An inherent capacity of antigen-presenting cells that enables them to sense the presence of antigens or microbial agents in the mucosal milieu. • Apoptosis Programmed cell death, which involves nuclear fragmentation and condensation of cytoplasm, plasma membranes, and organelles into apoptotic bodies. • B220 Common leukocyte antigen that is expressed on different cells including some DCs. • BDCA (blood dendritic cell antigen) Antigen expressed on circulating peripheral blood DCs. Four BDCA antigens (1–4) have been reported. • Birbeck granule Rod-shaped or tennis racket-shaped trilaminar cytoplasmic structure in Langerhans’ cells. Birbeck granules are present in immature or semimatured Langerhans’ cells, but are reduced and lost at their maturation. • cDC (conventional DC) DCs were described as myeloid and plasmacytoid DCs on the basis of their development from common myeloid progenitors (CMP) and common lymphoid progenitors (CLP). It now evident that CMP and CLP can give rise to different types of DCs. Accordingly, DCs with antigen-processing and antigen-presentation capacities are regarded as conventional DCs. 179

180

Glossary

• CD1a Member of immunoglobulin supergene family. Expressed on a subset of blood DCs and Langerhans’ cells. • CD11b Also known as Mac-1. A subset of DCs expresses this marker. • CD11c Also known as leukocyte surface antigen p150/90. A member of the integrin family. It is expressed on most of the DC populations in mouse and human. • CD34 Expressed on hematopoietic progenitor cells. Its cellular function includes cell–cell adhesion. As the differentiation of hematopoietic progenitor cells progresses, CD34 is progressively lost. DCs can be directly obtained from cultures of CD34+ hematopoietic progenitor cells. • CD40 Expressed on DCs. Its ligation with CD40L induces the activation and maturation of DCs. • CD45RA Leukocyte common antigen, CD45, is a tyrosine phosphatase. It has several isoforms, and the CD45RA isoform is expressed on naïve T cells. • CD62L Mediates homing of immunocytes to high endothelial venules of peripheral lymphoid tissue and leukocytes rolling on activated endothelium at inﬂammatory sites. It is expressed on plasmacytoid DCs. • CD80 and CD86 Costimulatory molecules present on antigen-presenting cells. These molecules provide costimulation for T-cell activation via ligation with CD28 and suppression via ligation with CTLA-4. • CD83 A member of the immunoglobulin superfamily. Expressed on mature DCs from different sources. • CD95 (Fas) A molecule expressed on a variety of cells that acts as a target for ligation by FasL. This molecule works for apoptosis. • CD123 Interleukin-3 receptor. The expression of CD123 on plasmacytoid DCs, but not on myeloid DCs, has enabled isolation of plasmacytoid DCs from peripheral blood. • Central tolerance Tolerance of T cells or B cells induced during their development in the thymus. • Chimerism The coexistence of cells from genetically different subjects. • CLA (cutaneous leukocyte antigen) Expressed on Langerhans’ cells that colonize the skin. CLA is a coreceptor for E-selectin (CD62 E), which is expressed on the surface of activated endothelial cells. Expression of CLA allows the attachment and transendothelial migration of circulating cells to the dermis.

Glossary

181

• CLP (common lymphoid progenitor) Progenitors that give rise to cells of lymphoid lineages. Previously, it has been assumed that a subtype of DCs, lymphoid DCs, originate from CLP. However, recent study indicates that CPL may give rise to putative myeloid DCs. • CLR (C-type-lectin receptor) A type of PRR that recognize PAMPs. Unlike the TLR family, members of the CLR family mediate not only pathogen recognition, but also self-antigen and non-selfantigen uptake and interactions between cells. • CMP (common myeloid progenitor) Progenitors that give rise to cells of myeloid lineages. Previously, it has been assumed that a subtype of DCs, myeloid DCs, originate from CMP. However, recent study indicates that CMP may give rise to putative lymphoid DCs. • Costimulation The signals required for the activation of lymphocytes, in addition to the antigenspeciﬁc signal delivered via their antigen receptors. CD28 is an important costimulatory molecule for T cells and CD40 for B cells. • Conditioned DC Activation of DCs by T-helper cells (conditioning of DCs). These conditioned DCs can stimulate cytotoxic T cells without the presence of helper T cells. • Cryptopatch Cryptopatches are the solitary lymphoid structures found in murine and human gut. These were ﬁrst identiﬁed in gut-associated murine lymphoid tissues where the generation of IL-7-dependent lymphohematopoietic progenitors for T- and/or B-cell descendants may start to take place. Cryptopatches also contain DCs. • Danger signal The signals that induce an immune response. Danger signals may be provided by cytokines, inﬂammatory mucosal milieu, microbial agents, and other non-self entities. • DC progenitors Hematopoietic progenitor cells capable of producing DCs or DC precursors. These cells retain the capacity of proliferation. • DC precursors These cells are more committed to become DCs than DC progenitors. Some of the DC precursors are well differentiated; however, the others have not been well characterized. Monocytes are the most common DC precursors. • DC-SIGN (dendritic cell-speciﬁc ICAM-grabbing non-integrin, CD207) DC-SIGN is a mannose-speciﬁc C-type lectin expressed by DCs. DC-SIGN binds to ICAM-3 on T lymphocytes, therefore playing an important role in the activation of T lymphocytes. DC-SIGN acts as a coreceptor for HIV, and the virus may remain bound to DC-SIGN for protracted periods. • DEC-205 A 205-kDa mouse glycoprotein lectin that is recognized by the monoclonal antibody NLDC-145. This molecule is important for trapping of antigen by DCs. Abundant expression of DEC-205 is seen in mouse Langerhans’ cells and interdigitating dendritic cells. Very low levels of DEC-205 are expressed by B cells, activated macrophages, and epithelial cells of the intestine and bronchia.

182

Glossary

• Exhausted DCs DCs stimulated for a prolonged period become nonresponsive to those stimuli and are regarded as exhausted DCs. However, the exhausted DCs might be functional again if these are cultured in media free from the original stimuli. • FC receptor A type of phagocytic receptor. Antigens, microbes, and apoptotic cells may be internalized by cells having these receptors. There are different types of FC receptors. • Flt-3 ligand (fms-like tyrosine kinase receptor 3 ligand) Flt-3L stimulates the proliferation of stem and progenitor cells including DCs through binding to Flt-3 receptor, which is a type III receptor tyrosine kinase member of the platelet-derived growth factor (PDGF) family. Flt-3L induce proliferation of both myeloid and lymphoid DCs. • Germinal centers Areas of secondary lymphoid tissue in which B-cell differentiation and antibody class switching occur. • GCDC (germinal center dendritic cells) DCs presenting in both dark and light zones of germinal centers in human tonsils, spleen, and lymph nodes; associated with germinal center T cells. • GM-CSF (granulocyte-macrophage colony-stimulating factor) Required for differentiation of DCs from DC progenitors, DC precursors, and other cells of hematopoietic lineage. • HBV (hepatitis B virus), HCV (hepatitis C virus) HBV is a member of the hepadna virus family. It is a DNA virus. HCV is a member of the Flaviviridae family. Both HBV and HCV cause acute as well as chronic liver diseases including liver cirrhosis and hepatocellular carcinoma. • HEV (high endothelial venule) An area of venules from which lymphocytes and other cells would migrate into lymph nodes. • ICAM-1 (CD54), ICAM-2 (CD102), and ICAM-3 (CD50) (intercellular adhesion molecules) Cell-surface molecules found on a variety of leukocytes and nonhematogenous cells. • Immature DCs DCs expressing low levels of costimulatory molecules and with a potent antigencapturing capacity. • Innate immunity Immune response that is induced immediately after the entry of microbial agents or other agents. May be induced against tumor cells. Innate immunity is very quick, and different leukocytes act as effector cells of innate immunity. It also provides direction to adaptive immunity. • IKDC (interferon-producing killer dendritic cell) These cells express both markers of DC and natural killer cells. They produce IFN-γ and possess APC function. • Interleukin (IL) A group of mediators that regulate the growth and proliferation of different cells both positively and negatively. There are several interleukins.

Glossary

183

• IL-3 This cytokine induces the maturation of human peripheral blood plasmacytoid DCs in vitro. • IL-4 This cytokine is used to generate DCs from human monocytes. • IL-10 An immunoregulatory cytokine. It directly acts on mouse Th cells. In humans, it acts via APCs. Usually attributed to Th2 polarization. Tolerogenic DCs can be produced by culturing DCs with IL-10. • IL-12 Most of the DCs can produce IL-12 in response to various stimulations. It is a key cytokine for the development and maintenance of cellular immunity. It induces Th1 polarization. Important survival factor for CTL, Th1, and DCs. • IL-13 Can be used to generate DCs in spite of IL-4. Causes maturation of monocytederived DCs grown in GM-CSF and IL-4. • IL-15 Produced by DDCs, and mature monocyte and CD34+-derived DCs. IL-15 induces the proliferation of T cells and promotes the generation of CTLs. • IL-18 Also called IFN-γ-inducing factor. Acts synergistically with IL-12. Enhances production of IL-1, IFN-γ, GM-CSF, and IL-13. Increases in mature DCs. • IDC (interdigitating dendritic cells) DCs in the T-cell areas of peripheral lymphoid organs such as the spleen, lymph nodes, and Peyer’s patch. These cells express MHC II, invariant chain, high levels of self-antigens, accessory molecules such as CD40 and CD86, a multilectin receptor for antigen presentation called DEC-205, the integrin CD11c, several antigens within the endocytic system, and, in the human system, molecules termed S100b, CD83, and p55. • IFN-a (interferon-a) Also called type 1 IFN. IFN-α has potent antiviral potential. Plasmacytoid DCs produce abundant amounts of IFN-α in response to viral stimulation. • IRF (interferon regulatory factor) Members of the IRF family induce the sequential expression of type 1 IFN genes. Viruses, DNA motifs, and double-stranded RNA might activate different IRF molecules. The expression of IRF molecules is regulated during maturation and activation of DCs. • Killer DCs DCs expressing FasL. They are able to kill target cells. • Langerhans’ cells Antigen-presenting cells of the skin expressing Birbeck granules and langerin. • Langerin A type II Ca2+-dependent lectin displaying mannose-binding speciﬁcity. Langerin colocalizes with Birbeck granules. Transfection of langerin cDNA into ﬁbroblasts results in Birbeck granule formation.

184

Glossary

• Lineage Markers that are present on myeloid or lymphoid cells. In general, DCs are lineage negative, but some DCs express variable amounts of lineage markers. • Myeloid DCs Mouse DCs that are CD8− and CD11b+. In humans, CD11c+ DCs are regarded as myeloid DCs. Recently, the myeloid DCs are no longer regarded as a speciﬁc entity of DCs. These DCs are classiﬁed within conventional DCs. • NIPC (natural interferon-producing cell) Plasmacytoid DCs that are CD11c−, CD4+, CD123+. They produce abundant amounts of type 1 interferon after viral stimulation. • Nef, gag, gp 120 These are antigens related to HIV. • NK (natural killer) cells A group of cells with intrinsic ability to recognize and destroy some virally infected cells and tumor cells. • NKT cell Lymphocytes having markers of both T and NK cells. These cells express Vα24+ receptor. NKT cells can recognize alpha-galactosylceramide through binding with the CD1d molecule. • NF-kb (nuclear factor kb) A transcription factor that plays a central role in immunological processes by regulating genes involved in immune and inﬂammatory responses. • ODN (oligodeoxynucleotide) Activate immune cells including DCs through NF-κB signaling pathway. ODNs or decoy ODNs might regulate immune response both positively and negatively, respectively. • Oral tolerance Induction of immune tolerance by ingestion of food antigens via the oral route. • PALS (periarteriolar lymphoid sheath) Present in the lymphoid tissues. A particular type of DCs is localized in this compartment of the lymphoid tissues. • PAMP (pathogen-associated molecular pattern) PAMPs are speciﬁc motifs within microbial products, including intermediate products of infection and replication. These are recognized by specialized receptors of the host cell. • PRR (pattern recognition receptor) The PRRs represent a family of germline-encoded proteins that are essential to the recognition of PAMP by the innate immune system. • Peripheral tolerance A state of speciﬁc immunological unresponsiveness. • Peyer’s patches Collections of lymphoid cells in the wall of the gut that form a secondary lymphoid tissue. • pDC (plasmacytoid dendritic cell) Large lymphocyte having plasma cell-like appearance. These cells produce abundant amounts of type 1 interferon after viral stimulation. They can also act as professional antigen-presenting cells.

Glossary

185

• Professional APC Antigen-presenting cells being able to stimulate and induce proliferation of naïve T cells. • relB A subunit of the nuclear factor (NF)-κB transcription factor. It is expressed on different DC populations and implicated in DC differentiation and maturation. • Regulatory DC DCs that induce immune tolerance and immune regulation. Regulatory DCs are produced in vitro by culturing immature DCs with different cytokines. These have been shown in situ in mice, but, their phenotype is yet to be assessed in humans. • SLC (secondary lymphoid chemokine) This is a chemotactic cytokine and expressed highly within lymphoid organs. It binds to CCR7 and is a potent attractant for T cells and mature DCs. • SCF (stem cell factor) SCF is an essential hematopoietic progenitor cell growth factor with proliferative and antiapoptotic functions. • S-100 proteins Intracytoplasmic calcium-binding proteins, expressed on some DCs as well as on other lymphoid cells, nerves, chondrocytes, fat, and melanocytes. • TAA (tumor-associated antigen) Peptide antigen expressing in tumor cells, not in normal cells. TAA can induce TAA-speciﬁc immunity in some circumstances. • TAP (transporter associated with antigen processing) This pathway is required for the intracellular transport of antigens to MHC class I molecules. • TLR (Toll-like receptor) The TLRs are a type 1 transmembrane receptor that possess an extracellular leucine-rich repeat domain and cytoplasmic domain homologous with that of the interleukin 1 receptor (IL-1R) family. Different TLRs are expressed by different DC populations. • Thymic dendritic cell DCs in thymus are BP-1+, CD11c+, DEC-205+, M342+, and CD8+. Thymic DCs play the main role for central tolerance. • TNF (tumor necrosis factor) A cytokine released by activated macrophages and other cells that is structurally related to lymphotoxin released by activated T cells. • TNFR (TNF receptor) There are two TNFR, TNFRI (p55) and TNFRII (p75). Both can induce signal transduction. • TGF (transforming growth factors) A group of cytokines identiﬁed by their ability to promote ﬁbroblast growth, which is generally immunosuppressive. TGF-β promotes growth of LCs from monocytes. • Tolerogenic DCs DCs manipulated in vitro are shown to exploit the same mechanisms that normal DCs employ to induce/maintain peripheral and central tolerance under steady-

186

Glossary

state conditions. Tolerogenic DCs would be an invaluable tool for therapy of allograft rejection, autoimmune diseases, or allergies. • TSLP (thymic stromal lymphopoietin) IL-7-like cytokine, produced by different epithelial cells. TSLP activates DCs to polarize T cells and also for formation of memory lymphocytes.

Subject Index

a acquired immuno-diﬁciency syndrome (AIDS) 52 acute rejection 146, 150, 151 adaptive immune system 73 adaptive immunity 7 Ag-speciﬁc CTL 132 allergen 73, 77 allergen-speciﬁc immune responses 76 allergic asthma 82 allergic diseases 73, 76 allergic rhinitis 81, 84 allergy 80 allogeneic mixed lymphocyte reaction (AMLR) 144 allograft 142, 144, 152 angiogenesis 105 anticancer immune surveillance 133 antigen presentation 34, 36, 38, 49, 117 antigen recognition 32 antigen-loaded activated DCs 161 antigen-loaded DC 14 antigen-presenting cell 1, 42, 107, 160 antigen-pulsed DCs 61, 65, 102, 126, 131, 138, 161, 162, 163, 165, 167 antigen-speciﬁc immune responses 160 antigen-speciﬁc immune therapy 158, 159 antigen-speciﬁc immunity 60 antigen-speciﬁc lymphocytes 129

anti-HBs 64 antitumor immunity 109 antiviral immunity 60 APCs 2 Apoptotic Bodies 34 apoptotic cells 36 atopic dermatitis 81, 83 autoantigens 8, 36, 91, 101 autoimmune diseases 46, 89, 95, 98, 101 autoimmune gastritis 93 autoimmune hepatitis 92 autoimmunity 3, 87, 99 b B cells 77 B lymphocytes 1 bacteria 67 Birbeck granules 27, 84 blood DCs 31 blood dendritic cell antigen (BDCA) 30 blood-derived DCs 125 breakthrough hepatitis 55 c cancer 105, 110 cancer antigen 66 capture 7 CCR5 52 187

188

Subject Index

CD11c 24, 28, 57 CD11c+ DC 29 CD1a 15, 30 CD1a+DCs 78, 82 CD1a+LCs 83 CD4 15, 52 CD4+ T lymphocytes 77 CD40 26 CD68 antigen 122 CD8 15 CD8+ cytotoxic T lymphocytes (CTL) 62 CD83 15, 58, 95 CD86 26 cells 41 CHB 63 chemokine receptor 114 chemokines 45, 114 chimerism 147 chronic HBV carriers 54 chronic hepatitis B (CHB) 56 chronic hepatitis C (CHC) 57 chronic rejection 146 chronic viral infections 48 CLA+ (cutaneous lymphocyte antigen) 28 CMRF-44 122 CMRF-56 122 collagen-induced arthritis (CIA) 100 colony-stimulating factor (M-CSF) 18 common lymphoid progenitor (CLP) 18, 24 common myeloid progenitors (CMPs) 18, 24 complement components 45 concanavalin A 17 conditioned DCs 36 conventional DCs (cDCs) 16, 45, 143 corticosteroid 99 costimulatory molecules 118 CpG DNA 19, 29 CpG motifs 67 cross-presentation 91 cross-priming 44 crosstalk 42 cryptopatches 27 C-type lectine receptors (CLRs) 33

cultured monocyte-derived DCs 6 cytokine 6, 37, 45 cytotoxic T lymphocytes (CTLs) 90, 115 d danger signal 13 DC differentiation 22 DC mobilization 115 DC precursors 12, 114, 164 DC progenitors 12, 114, 164 DC-based immune therapy 61, 169 DC-based immunocytes 138 DC-based therapy 2, 4, 37, 65, 66, 85, 121, 127, 128, 136 DC-lysosome-associated membrane protein (DC-LAMP) 35 DC-related antigens 122 DC-SIGN 53, 68, 70, 96 DC-virus interaction 49, 59 DEC-205 34, 130 deﬁnition of DCs 8 dendritic cell (DC) 1, 2, 41, 65, 66, 67, 75, 81, 107, 123, 142, 156 diabetes 94 diagnostic 5 diagnostic and prognostic implications of DCs 37 diagnostic importance of DCs 123 differentiations 46 direct pathway of allorecognition 145 donor DCs 143 donor-derived DCs 149 DS-SIGN 52 e early-phase immediate type 1 hypersensitivity response 74 endocytosis 44, 116 enrichment of DCs 9 eosinophils 74 exhausted DCs 10 exosomes 131 experimental allergic encephalomyelitis 100

Subject Index

f fatty acid-binding protein (FABP) family 23 FcεR1 75, 83, 85 fms-like tyrosine kinase 3 ligand (Flt-3L) 19, 23, 149 fms-like tyrosine kinase receptor 3 ligand (Flt3) receptor 16 follicular dendritic cells (FDCs) 4 functions 11 fusion 131 g GM-CSF 18 graft survival 141 h HB vaccine 64 HBsAg-pulsed DCs 63 HBV transgenic mice (HBV-Tg) 55, 63 HCV RNA 58 heat shock protein (hsp) 23, 73, 130 helix-loop-helix (HLH) 21 hepatic nonparenchymal cells (NPC) 28, 29 hepatitis B surface antigen (HBsAg) 55 hepatitis B virus (HBV) 51, 54, 60, 152, 163 hepatitis C virus (HCV) 51, 57, 60, 61, 64 hepatocellular carcinoma (HCC) 122, 135 highly active antiretroviral therapy (HAART) 62 histamine 75, 82 HIV gp120 53 HIV-1 130 homoeostasis 46 host defense 41 human allergy 81 human cord blood DCs 17 human DCs 5 human hepatitis viruses 50

189

human immunodeﬁciency virus (HIV) 50, 62 hypo-responsiveness 77 i ICAM-3 grabbing nonintegrin (DC-SIGN) 30 IFN-α 32, 98 IFN-producing killer DCs (IKDCs) 10, 25, 110, 113 Ikaros 20 IL-12p70 17 IL-23 82 immature DCs 10, 12, 14 immune ignorance 156 immune response 3, 121, 126 immune suppression 139 immune surveillance 3, 106, 107, 113, 127, 156, 159 immune therapy 125, 155 immune tolerance 89, 111, 121, 126, 144, 149, 157 immunity 41 immunogenic DCs 8, 92, 137 immunogenic tolerance 3, 142, 165 immunoglobulin E (IgE) 74, 75, 82, 85 immunological memory 47 immunosuppressive 142 immunosuppressive drugs 92 immunosuppressive state 54 indirect pathway of allorecognition 145 infection 41 inﬂammatory stimuli 13 inﬂuenza virus 50, 51, 59 innate immune system 73 innate immunity 7, 31, 109, 159 interferon regulatory factors (IRF) 20 internalization of the virus 49 isolation of DCs 9 k killer DCs

10, 110, 113

190

Subject Index

l lamina propia (LP) 28 Langerhans cells (LC) 16, 23, 27, 53, 75, 83 langerin 27, 30 late-phase response 74 Leishmaniasis 70 liver 58, 95 liver cancers 55 liver cirrhosis 55 liver DC 56, 148 liver transplantation 148 L-selectin 57 lymphocytic choriomeningitis virus glycoprotein 93 lymphoid 9 lymphoma 135 m M. tuberculosis 68 macrophage migration inhibitory factor 96 macrophages 1, 77, 112 macropinocytosis 33, 124 major histocompatibility complex (MHC) 146 Mannan 53 maturating DCs 12 maturation 119 mature DC 10, 12, 14, 110, 120 measles virus 50, 59 melanoma 134 memory cells 42 memory lymphocytes 48 MHC class I 117 MHC class I complex 51 MHC class I molecules 62, 91 MHC class II 34 MHC class II antigens 11 microbial agents 8 microorganisms 42 micropinocytosis migration 133 MIIC 118 mixed leukocyte reaction (MLR) 15 molecular mimicry 88

monocyte 19, 22 monocyte-derived DCs 9, 23, 128 morphology 11 multidisciplinary approach 138, 139 murine DCs 5 mutagenic 112 mutant 55 MyD88 70 myelin basic protein (MBP) 93, 100 myeloid DCs 56, 97 myeloma 135 n naïve T cells 45 natural interferon-producing cells (NIPC) 4, 26 natural killer (NK) 32, 41 NF-kB (Rel) family 20 nitric oxide 78, 100, 112 NK cell 18, 25, 121, 167 NK cell activities 22 NK-depleted 46 NK-depleted mice 167 NKT 32 NOD mice 99 nonantigen-speciﬁc immune therapy 158 nonlymphoid 9 nonlymphoid tissues 13 o organ transplantation 141 organ-speciﬁc DC 13 origins of DC 37 ovalbumin 80 OVA-pulsed myeloid DCs 80 p PAMP-independent innate activation 43 parasites 69 pathogen-associated molecular pattern (PAMP) 33, 42, 69, 88, 113 pathogenic immunity 157

Subject Index

pattern-recognition receptors (PRRs) 33, 43, 67,145 PDC 97 peripheral blood DC (PBDC) 6, 29 peripheral blood mononuclear cells (PBMC) 30 Peyer’s Patch 26 phagocytosis 44, 116, 124 phenotypes 11, 31 Phenotypes of DCs 38 pinocytosis 116 plasmacytoid DC (pDC) 16, 44, 56, 79, 98, 108, 129, 143 Plasmodium falciparum 69 poly (I:C) 67 PPRs 88, 113 presentation 2 primary biliary cirrhosis (PBC) 92, 94, 95, 97 process 7 processing 2 professional APC 31, 49 prognostic 5 prognostic importance of DCs 123 prophylactic vaccines 47, 64, 159, 169 prostate cancer 134 protective immunity 156, 157, 161 protein-energy malnutrition (PEM) 166 psoriasis 94 PU.1 21 pyruvate dehydrogenase complex (PDC) 97 r reactive oxygen species (ROS) 112 receptor-mediated endocytosis 44, 124 recognize 7 regulator 3 regulatory DC 10, 14, 47, 87, 89, 100, 102, 129, 132, 165 regulatory immunity 157 regulatory T cells 47, 108, 111, 150, 151, 164 renal cancer 135 rheumatoid arthritis 93

191

s S100 122 scanners 109 secondary lymphoid tissue chemokine (SLC) 35 self-antigens 3, 87 “self ”-products 8 Sjögren’s syndrome 94 Skin 27 spleen dendritic cells 24 STAT3 21 subtypes 6 synovial tissue 93 systemic lupus erythematosus (SLE) 91, 98 t T lymphocytes 1 TAA-pulsed DCs 162 TAA-speciﬁc DCs 119, 127 TAA-speciﬁc immunity 124 T-cell activation 36 T-cell receptor 78 TGF-β 28 Th1 polarization 51 Th1 population 85 Th2 51, 81 Th2 polarization 78, 79 therapeutic 5 thymic DCs 24, 25 thymic stromal lymphopoietin (TSLP) 79, 84 thymus 13, 90 thyroiditis 94 tissue-derived DCs 6 TLR 33, 67, 70 tolerance 147 tolerogenic DC 8, 38, 92, 101, 137, 148, 152 tolerogenic immunity 46 Toxoplasma gondii 70 transfecting DCs 130 transplant recipients 141 transplantation 147, 151 T-regulatory cells 87 TSLP receptor 84, 79

192

Subject Index

tuberculosis (TB) 68 tumor 106 tumor antigen-pulsed DCs 139 tumor-associated antigens (TAAs) 3, 108, 111, 115, 117, 118, 125, 137, 160 tumor immunity 107 tumor necrosis factor (TNF)-α 18 tumorigenesis 105, 109, 116, 124, 155 tumor-inﬁltrating dendritic cells 120

type 1 diabetes mellitus 99 type 1 insulin-dependent diabetes 93 type 1 interferon (IFN) 4, 22, 90, 108 u ulcertative colitis 96 v vaccine therapy 160, 161