Advances in Immunology Volume 84

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ADVANCES IN

Immunology VOLUME 84

This Page Intentionally Left Blank

ADVANCES IN

Immunology EDITED BY FREDERICK W. ALT The Howard Hughes Medical Institute Childrens Hospital, Boston, Massachusetts

ASSOCIATE EDITORS FRANK AUSTIN Division of Rheumatology, Immunology & Allergy Harvard Medical School, Boston, Massachusetts

TADAMITSU KISHIMOTO Institute of Molecular & Cell Biology Osaka University Medical School, Osaka, Japan

FRITZ MELCHERS Department of Cell Biology University of Basel, Basel, Switzerland

JONATHAN UHR Department of MIcrobiology & Internal Medicine University of Texas, Dallas, Texas

EMIL R. UNANUE Department of Pathology & Immunology Washington University, St. Louis, Missouri

VOLUME 84

Elsevier Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK

This book is printed on acid-free paper. Copyright ß 2004, Elsevier Inc. All Rights Reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (www.copyright.com), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2004 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-2776/2004 $35.00 Permissions may be sought directly from Elsevier’s Science & Technology Right Department in Oxford, UK: phone: (þ44) 1865 843830, fax: (þ44) 1865 853333, E-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting ‘‘Customer Support’’ and then ‘‘Obtaining Permissions.’’ For all information on all Academic Press publications visit our Web site at www.books.elsevier.com ISBN: 0-12-022484-4 PRINTED IN THE UNITED STATES OF AMERICA 04 05 06 07 08 9 8 7 6 5 4 3 2 1

CONTENTS

Contributors

ix

Interactions Between NK Cells and B Lymphocytes Dorothy Yuan I. II. III. IV. V. VI.

Introduction B Lymphocytes NK Cells NK–B-Cell Interactions In Vitro In Vivo NK–B Cell Interactions The Role of NK–B-Cell Interactions in Autoimmune Diseases VII. NK–B-Cell Interactions during Infections VIII. Conclusions References

1 1 6 10 19 26 28 31 31

Multitasking of Helix-Loop-Helix Proteins in Lymphopoiesis Xiao-Hong Sun I. II. III. IV. V. VI.

Introduction HLH Proteins and B-Cell Development HLH Proteins and T-Cell Development HLH Protein and Notch Signaling HLH Proteins and Leukemia Conclusions References

v

43 54 58 63 64 67 67

vi

CONTENTS

Customized Antigens for Desensitizing Allergic Patients Fa´tima Ferreira, Michael Wallner, and Josef Thalhamer I. Introduction II. Allergy Diagnosis and Immunotherapy: From Extracts to Allergen Arrays and Engineered Vaccines III. Novel Antigen Preparations to Improve SIT IV. Customized Allergen DNA Vaccines V. Short Overview of Alternative Treatments and Supporting Adjuvants for SIT VI. Summary and Conclusions References

79 80 83 103 113 115 116

Immune Response Against Dying Tumor Cells Laurence Zitvogel, Noelia Casares, Marie O. PE´quignot, Nathalie Chaput, Matthew L. Albert, and Guido Kroemer I. II. III. IV.

Introduction Minimal Elements of the Antitumor Immune Response Subroutines of Cell Death: Apoptosis and Necrosis In Vivo Immunogenicity of Apoptotic Versus Necrotic Tumor Cells V. Apoptosis: A Tolerogenic Type of Tumor Cell Death? VI. Strategies to Enhance the Immunogenicity of Dying Tumor Cells VII. Concluding Remarks References

131 133 140 150 156 159 164 166

HMGB1 in the Immunology of Sepsis (Not Septic Shock) and Arthritis Christopher J. Czura, Huan Yang, Carol Ann Amella, and Kevin J. Tracey I. Introduction II. Modern Clinical Syndromes: Septic Shock is not Severe Sepsis III. TNF is a Mediator of Septic Shock Syndrome, but Not Severe Sepsis Syndrome IV. HMGB1 and Anti-HMGB1 in Severe Sepsis, Not Septic Shock V. Biochemistry and Molecular Biology of HMGB1 VI. HMGB1 Signaling via RAGE and TLR2

181 183 184 185 188 191

CONTENTS

VII. Cytokine Activities of HMGB1 VIII. HMGB1 in Arthritis IX. Perspective and Future References

vii 192 194 195 196

Selection of the T-Cell Repertoire: Receptor-Controlled Checkpoints in T-Cell Development Harald von Boehmer I. II. III. IV.

Thymic Selection: A Historical Perspective Early T-Cell Development Late T-Cell Development The Role of Thymic Selection in Recessive and Dominant Mechanisms of Self-Tolerance V. Concluding Remarks References

201 207 216 222 227 228

The Pathogenesis of Diabetes in the NOD Mouse Michelle Solomon and Nora Sarvetnick I. II. III. IV.

Introduction Origin of the NOD Mouse Pathogenesis of Diabetes in the NOD Mouse Leukocytes Involved in the Pathogenesis of Diabetes in the NOD Mouse V. Other Autoimmune Diseases Associated with the NOD Mouse VI. Breakthough Moments in the Study of the Pathogenesis of Diabetes in the NOD Mouse VII. Concluding Remarks References Index Contents of Recent Volumes

239 239 240 241 244 245 254 255 265 277


CONTRIBUTORS

Numbers in parenthesis indicated the pages on which the authors’ contributions begin.

Matthew L. Albert (131), Institut Pasteur, Paris F-75724, France Carol Ann Amella (181), Laboratory of Biomedical Science, Manhasset, New York 11030 Harald von Boehmer (201), Harvard Medical School, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 Noelia Casares (131), Institut Gustave Roussy, Villejuif F-94805, France Nathalie Chaput (131), Institut Gustave Roussy, Villejuif F-94805, France Christopher J. Czura (181), Laboratory of Biomedical Science, North Shore-LIJ Research Institute, Manhasset, New York 11030 Fa´tima Ferreira (79), University of Salzburg, Salzburg A-5020, Austria Guido Kroemer (131), Institut Gustave Roussy, Villejuif F-94805, France Marie O. Pe´quignot (131), Institut Gustave Roussy, Villejuif F-94805, France Nora Sarvetnick (239), Department of Immunology, The Scripps Research Institute, La Jolla, California 92037 Michelle Solomon (239), Department of Immunology, The Scripps Research Institute, La Jolla, California 92037 Xiao-Hong Sun (43), Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104 Jevin J. Tracey (181), Laboratory of Biomedical Science, Manhasset, New York 11030 Josef Thalhamer (79), University of Salzburg, Salzburg A-5020, Austria Michael Wallner (79), University of Salzburg, Salzburg A-5020, Austria Dorothy Yuan (1), Laboratory of Molecular Pathology, Department of Pathology, UT Southwestern Medical Center, Dallas, Texas 75390 Laurence Zitvogel (131), Institut Gustave Roussy, Villejuif F-94805, France Huan Yang (181), Laboratory of Biomedical Science, Manhasset, New York 11030 ix

advances in immunology, vol. 84

Interactions Between NK Cells and B Lymphocytes DOROTHY YUAN Laboratory of Molecular Pathology, Department of Pathology, UT Southwestern Medical Center, Dallas, Texas 75390

I. Introduction

Innate immunity is evolutionarily older than antigen-specific immunity and is crucial for its effector function. Natural Killer (NK) cells constitute one of the key components of the innate immune system in that they can be rapidly activated without the need for expansion of antigen-specific clones. They were initially perceived as serving as a first line of defense against tumors or virus-infected cells because of their ability to kill these targets; however, there is increasing awareness of their role in modulation of the immune system via their ability to produce a number of cytokines as well as via nonlytic interaction with target cells. B lymphocytes, by contrast, are important constituents of the specific immune system because of the presence of clonally distributed antigen receptors and the persistence of the progeny of specific clones that can continue to produce the relevant antibodies. An understanding of the interactions between these two cell types would provide important insights into the influence of the rapid but transient innate response to pathogens on the specific immune response that takes longer to initiate but is long lasting. In addition, detailed dissection of the interactions between these cell types and their regulatory loops may provide further clues for a number of pathogenic conditions that are attributed to immune system dysfunction. A critical analysis of the experiments performed to investigate the interaction between NK and B cells requires some understanding of their characteristics. Thus, in this chapter, the functional capabilities of each cell type is first described briefly followed by a review of the evidence for interactions between these two cells types provided by in vitro and in vivo experiments. Finally, the available data regarding the possible role of these interactions in the development of autoimmunity and in microbial infections are summarized. This exercise should serve to reconcile some of the apparently inconsistent data and point to the direction for further experimental approaches. II. B Lymphocytes

B lymphocytes are distinguished from other cell types by the presence of specific antigen receptors [BCR] on each cell. The BCR has two interrelated functions. The first is to initiate signal cascades that result in the transcription 1 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2776/04 $35.00

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of a variety of genes associated with B-cell activation, leading eventually to secretion of antibodies specific for the initiating antigen. The second is the uptake and targeting of antigen to the major histocompatibility complex (MHC) class II antigen processing and presentation pathway. In comparison to other antigen-presenting cells (APCs) the pathway is particularly important for the activation of T cells under conditions of low antigen concentration. A. T-Cell–Dependent Pathways of B-Cell Activation Signaling through the BCR alone is insufficient for full activation of B cells. Response to most antigens requires additional signals from antigen-specific helper T cells. These antigens are categorized as ‘‘T dependent’’ (TD). Other antigens can stimulate antibody production in the absence of MHC class II– restricted T-cell help and are classified as ‘‘T-cell–independent’’ (TI) antigens. The primary response to a TD antigen is initiated by the uptake of antigen by either nonspecific APCs or by B cells with specific receptors for the antigen. The APCs process antigen and migrate to the T zones of secondary lymphoid tissues. Cognate interaction between primed T cells and B cells first takes place in the outer T zone of secondary lymphoid organs (Liu et al., 1991). As a result of this interaction, antigen-specific B cells start to proliferate and differentiate both in follicles and in extrafollicular foci. This priming process usually takes 2–4 days in vivo, a period that is shortened in secondary antibody responses (Liu et al., 1991). Extrafollicular B blasts do not mutate their immunoglobulin V-region genes (Jacob and Kelsoe, 1992; McHeyzer-Williams et al., 1992) and they differentiate in situ into short-lived cells that produce low-affinity antibodies (Ho et al., 1986). In mice, this extrafollicular proliferation and differentiation occurs in the red pulp of the spleen adjacent to the T zone (Jacob et al., 1991) and in the medullary cord in lymph nodes (Kosco et al., 1989). B-cell proliferation in the follicles gives rise to germinal centers where the B blasts activate an immunoregion–directed hypermutation mechanism (MacLennan and Gray, 1986; McHeyzer-Williams et al., 1993). These cells are then subjected to a selection process, with the selected cells giving rise to long-lived antibody-producing cells (Tew et al., 1992) or memory cells (Luther et al., 1997). In addition to the genetic alteration of B cells by somatic hypermutation, class switch recombination can occur both in the germinal center and outside the follicles (Toellner et al., 1996). Recently a putative RNA editing enzyme activation-induced deaminase (AID) has been shown to regulate both class switch recombination and somatic hypermutation in mice and humans (Honjo et al., 2002). Class switch recombination replaces the immunoglobulin CH gene to be expressed from Cm to Cg, Ce, or Ca resulting in switching of immunoglobulin isotype from immunoglobulin M (IgM) to either immunoglobulin G (IgG), immunoglobulin E (IgE), or immunoglobulin A (IgA), respectively, without changing the antigenic specificity. The functional characteristics that

NK–B-CELL INTERACTIONS

3

distinguish each isotype determine the manner in which captured antigens are eliminated or the location where the antibody is delivered and accumulated. The activation of naive T cells requires two signals provided by APCs. The first signal is delivered through the T-cell receptor (TCR) upon engagement of MHC molecules loaded with the appropriate peptide. The second signal involves cross-linking of CD28 and other receptors on the T cell by costimulatory molecules expressed by APCs (Lenschow et al., 1996; Mondino et al., 1996). Many of the peptides recognized by CD4þ T cells in the context of MHC class II molecules derive from protein antigens internalized and degraded in the endocytic compartment of APCs (Cresswell, 1994; Germain, 1994). It has generally been assumed that the degradation of proteins and conversion to MHC class II–bound peptides occur constitutively, allowing presentation of any antigen that gains access to the endosomes of APCs. These might include selfproteins and antigens from infectious organisms. Although APCs are unable to discriminate between peptide sources, B-cell antigen presentation differs from that by other APCs in a number of ways. First, the presence of antigen-specific receptors allows them to process low concentrations of antigen that is perceived as nonself. Second, the stimuli that activate costimulatory molecules on B cells may differ from those of other APCs. In particular, dendritic cells that have been activated by and encounter with microbial antigens in the periphery and subsequently migrate to the lymphoid organs (Banchereau and Steinman, 1998) are able to present antigens more efficiently than APCs located in situ (Manickasingham and Reis e Sousa, 2000). In addition, proximity with T-cell ligands and perhaps other cell types together with their products may also differentially alter the extent of expression of costimulatory molecules on each cell type. Thus, signals that activate various APCs may differ depending on the site and type of antigen encounter. Whereas it is difficult to determine the relative role played by each type of APC for each antigenic response, it is clear that antigen-pulsed dendritic cells are powerful APCs that can significantly alter immune responses. However, a number of studies have also shown that antigen presentation is compromised in animals depleted of B cells (Ron and Sprent, 1987) or genetically engineered for B-cell deficiency (Linton et al., 2000; Macaulay et al., 1998; Rivera et al., 2001). B. T-Independent Pathways of B-Cell Activation In contrast to TD antigens, stimulation of B cells via TI antigens does not require antigen presentation by class II molecules. TI antigens are classified into two classes. TI-1 antigens require the mitogenic activity of some moieties, such as lipopolysaccharide (LPS) present on gram-negative bacteria, for effective stimulation of B cells. The receptor for LPS consists of several components including the toll-like receptor (TLR) TLR4 (or TLR2 in human cells) with an associated molecule MD-2, as well as RP105, the expression of which

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requires another extracellular molecule MD-1. Mutations of either the TLR4 or the RP105 gene result in LPS hyporesponsiveness (Nagai et al., 2002). Because TLR4 and RP105 are expressed on B cells and macrophages (Miyake et al., 1995), bacterial LPS can activate B cells directly and can induce macrophage cytokine production that would indirectly affect B cell responses. Antigens expressed by some viruses, such as the hepatitis B core antigen (Fehr et al., 1998), may have similar activity for TLRs expressed on B cells. TI-2 antigens are characterized by the repetitive polysaccharides present on encapsulated bacteria and the polysaccharide epitopes on the cell wall of nonencapsulated bacteria such as those present on bacteria such as Streptococcus pneumoniae and Haemophilus influenzae. These multiple repeating antigenic epitopes, which are also present on some viruses (Bachmann and Zinkernagel, 1996; Fehr et al., 1998) can mediate extensive cross-linking of antigen receptor and thereby activate B cells without the necessity for class II– mediated antigen presentation to elicit T-cell help. A significant fraction of TI-2 immune responses are mediated by marginal zone (MZ) B cells. Thus, mice deficient in the tyrosine kinase Pyk-2 and lack splenic MZ B cells have defective antibody responses to the TI-2 antigen TNP-Ficoll (Guinamard et al., 2000). Anatomically, the MZ lies between the white and red pulp of the spleen and is composed of B cells, macrophages, and dendritic cells (DCs) (Fagarasan and Honjo, 2000). The proximity of MZ B cells to the marginal sinuses allows them to be among the first cell population to be encountered by bloodborne antigens. Because Pyk-2 has been linked to migration and adhesion processes, this defect is likely partly responsible for the loss of MZ B cells in Pyk2–deficient mice. Interestingly, the resident macrophage of the MZ and metallophillic macrophages were present and correctly localized in the mice, suggesting that deficient response to TI-2 antigens is not due to reduced abilities of these macrophages to help in the response, as has been previously suggested (Sinha et al., 1987). Using a transgenic model consisting of B cells expressing BCR specific for TI-2 antigens, Balazs et al. (2002) demonstrated that a bloodborne circulating CD11clo DC population captures and transports bacterial antigens to the spleen to activate, clonally expand, and differentiate MZ B cells into plasmablasts. It is noteworthy however, that despite the depressed IgG3 and IgG2a responses to TNP-Ficoll in the Pyk-2–deficient mice, the relatively low IgG1 response was not compromised, suggesting that in addition to MZ B cells, other types of B cells can respond to TI-2 antigens. For example, B1 B cells, characterized mainly by their expression of CD5, have also been shown to have a preferential ability to respond to TI-2 antigens (Martin et al., 2001). Locally, peritoneal macrophages play a similar role to DCs for B1 B-cell plasmablast differentiation. Using an in vitro TI-2 model antigen consisting of monoclonal anti-IgD antibodies coupled to a high-molecular-weight dextran polysaccharide carrier


5

molecule (ad-dex), Vos et al. (2000) have shed significant light on the nature of the B-cell response to TI-2 antigens. They analyzed the signaling pathways resulting from the unique nature of B-cell activation by TI-2 antigens due to their capacity for multivalent binding of the BCR, as well as the second signals that are required for full activation. These second signals can be derived from microbial products and a number of cytokines, including IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, interferon-g (IFN-g), and granulocyte–macrophage colonystimulating factor (GM-CSF). Clearly these cytokines can originate from a number of cell types. Despite the usefulness of the ad-dex model, there are some differences that are not easily reconciled with in vivo findings. Mainly, in vitro ad-dex is much less efficient in stimulation of MZ than germinal center B cells (Vos et al., 2000). A possible difference may involve the ability of TI-2 antigens to activate the C0 system in vivo (Fagarasan and Honjo, 2000) resulting in the association of the C0 receptor, CD21, and CD19 to the BCR, thus lowering the threshold of activation. Both C3 and complement receptor (CR1/ 2)–deficient mice were unable to localize TI-2 antigens to MZ B cells, although localization of the MZ macrophages was unaffected. A dependence on complement has been documented also for TI-2 antigens expressed by viruses (Ochsenbein et al., 2000). In addition to the factors that affect homing of pathogens displaying TI-2 antigens to the MZ, there is strong evidence that a receptor on B cells, transmembrane activator interactor (TACI) (Mackay et al., 2003) is important for survival of MZ B cells (Balazs et al., 2002). TACI-deficient mice display normal to slightly elevated TD B-cell responses, but responses to TI-2 antigens are largely absent (Rivera et al., 2001; von Bulow et al., 2001). The tumor necrosis factor (TNF) homolog, B-cell activating factor (BAFF; also known as BlyS, TALL-1, THANK, or zTNF4) has been identified as a TACI ligand. BAFF is expressed on macrophages, monocytes, and DCs and is known to modulate Bcell development, survival, and activation (Mackay et al., 2003). Therefore, it is not surprising that other closely related TNF family members are important for the survivial of other B-cell subpopulations. Thus, BAFF is closely related to APRIL, a proliferation-inducing ligand that can bind to TACI and another member of this family, B-cell maturation antigen (BCMA), which is also constitutively expressed on mature B cells. Studies indicate that interactions between BCMA and BAFF are important, not for the initiation, but for the integrity of the germinal center environment (Vora et al., 2003). Only a cursory description of the wealth of information that is available regarding B-cell differentiation is included in this chapter; however, it should serve the purpose of showing that some understanding of the site of B-cell activation and the role of B cells in antigen presentation is crucial for the dissection of how NK cells can affect antigen-specific B-cell responses in vivo. In resting animals, NK cells generally reside in the red pulp of the spleen

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(Andrews et al., 2001) and are present in very low numbers in lymph nodes (Cavanaugh et al., 2003). However, after virus infection they have been shown to home to the MZ (Andrews et al., 2001; Salazar-Mather et al., 1996) where they have the potential to directly interact with B cells. Furthermore, because B-cell switch recombination in response to both TI and TD antigen can occur ouside the follicules, they can be targeted by NK cells at these sites as well. III. NK Cells

A. NK Cell Receptors NK cells are an integral component of innate immunity, both in the production of cytokines that can stimulate other cells of the immune system and in the direct destruction of infected or transformed cells. The actions of NK cells are modulated by the integration of inhibitory and activating signals sent by cell surface receptors upon binding to ligands. Inhibitory receptors on NK cells have specificity for epitopes on various MHC class I molecules and can discriminate target cells either by lack of identity to self or by reduced expression of these ligands on allogeneic or infected cells. In addition to class I molecules, activating receptors recognize additional ligands that are expressed on some tumors, stressed, or infected cells. MHC class I–specific NK cell receptors comprise two very different families of molecules, one made of immunoglobulin superfamily domains and the other resembling C-type lectins. Presently 14 killer immunoglobulin-like receptor (KIR) genes in humans have been identified. These KIRs differ from each other by the number of immunoglobulin domains and by the length of their cytoplasmic tails. The long tails contain immunoreceptor tyrosine-based inhibition motifs (ITIMs), which recruit the phosphatase SHP-1 upon receptor engagement and induce inhibitory signals. The KIRs with short cytoplasmic tails lack ITIMs and send activating signals to NK cells by association with the adaptor signaling molecule DAP12 via a charged amino acid in the transmembrane region. Rodents lack KIRs and instead use structurally distinct Ly49 C-type lectin receptors to bind MHC class I molecules on target cells. Members of the Ly49 gene family (Ly49a–w) are located in the NK cell gene complex (NKC) on mouse chromosome 6 or rat chromosome 4. The Ly49 family also includes both inhibitory and activating receptors. Both types of receptors can have specificity for mouse MHC class I antigens (McQueen and Parham, 2002). Within an individual organism, NK cells express different combinations of KIR or Ly49 molecules. Each cell must express at least one inhibitory receptor with specificity for self MHC class I, and the signal from the inhibitory receptor generally dominates the activating receptor. The dominance of the inhibitory receptor signal can explain many of the aspects of the phenomenon of ‘‘hybrid resistance’’ (Bennett, 1987). This refers to the situation where an


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(A B) F1 host, in defiance of conventional transplantation laws, rejects A or B grafts of parental origin (A and B referring to MHC genotypes). The missing-self hypothesis (Karre, 2002) proposes that NK cells could sense the presence of a complete set of MHC class I molecules in autologous grafts, which would prevent rejection. Conversely, the absence of some MHC class I genes in a parental to F1 graft would lead to rejection by NK cells. The differential expression levels of inhibitory versus activating receptors may also come into play (George et al., 1999). In addition, some non-MHC ligands also play an important role in overcoming these inhibitory signals. NKG2D receptors are expressed on most NK cells and on certain T-cell subsets (Vivier et al., 2002). Murine NKG2D recognizes retinoic acid early transcript (Rae-1) and antigen 60 (H60) molecules that are only distantly related to MHC class I (Diefenbach et al., 2000) and are induced by stress. NK cells can reject tumor cells expressing Rae-1 in the presence of a normal complement of conventional MHC class I (Cerwenka et al., 2000; Diefenbach et al., 2000). Human NKG2D was found to bind the polymorphic stress-inducible MICA and B (Bauer et al., 1999) and the human cytomegalovirus gpUL16-binding proteins ULBP1, 2, and 3 (Cosman et al., 2001). MICA and B molecules are normally expressed on only some restricted tissues, but can be induced on other cells by malignant transformation or virus infection (Groh et al., 1999). One explanation for the maintenance of tolerance despite the presence of these activating receptors may be based on the differential time course of appearance of the ligands during ontogeny. For example, H60 and Rae-1 in mice are generally expressed early in embryonic life (Cerwenka et al., 2000) before NK cells are even generated. However, by the time NKG2D-expressing NK cells appear, the ligands are no longer expressed, unless a pathological situation arises, such as infection or tumor transformation. This explanation is, however, more difficult to reconcile with the regulation of signaling from other activating receptors that recognize ligands that are not developmentally regulated (Lanier, 2000). The ligand for CD16 (Lanier et al., 1988) is the Fc portion of IgG and serves the important function of mediating antibody-dependent cellular cytotoxicity (ADCC) by NK cells. The ligands for CD244 (Garni-Wagner et al., 1993) and CD2 (Siliciano et al., 1985), expressed on both NK and subsets of T cells, are CD48 and CD58, which are expressed on a number of cell types. Although the ligand for NKR-P1 (Karlhofer and Yokoyama, 1991) is unknown, the receptor is expressed on all NK cells from a number of mouse strains. Except for CD244, these receptors function by association with adapter molecules, which contain ITAM motifs. These include CD3z and FcRIg, both present on NK cells. The activating receptor CD244 uses a signaling pathway involving the cytoplasmic tyrosine phosphatase SHP-2 and the Src homology 2 (SH2)–containing intracellular adaptor protein SAP (Tangye

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et al., 1999). Other co-receptors expressed on T cells that have ligands expressed by B cells and other APCs have also been shown to be present on NK cells. Transfectants expressing either CD86 or CD40 can be lysed by NK cells in vitro, suggesting that activation of either CD28 or CD40L can overcome the presence of inhibitory receptors; however, how these receptors are triggered in vivo is not clear because the transfected cells are also killed in CD28 / or CD40L / mice, respectively (Martin-Fontecha et al., 1999). Clearly the presence of more than one activating receptor on NK cells indicates that additive or synergistic interactions between multiple receptors may be necessary to stimulate cytotoxicity and cytokine production. There is mounting evidence supporting this idea (Martin-Fontecha and Carbone, 2001). There are three additional activating receptors expressed only on NK cells that are non–MHC class I specific (NKp46, NKp44, and NKp30 [Pende et al., 1999; Pessino et al., 1998; Vitale et al., 1998]). They recognize unknown determinants expressed by tumor cells and are activated via binding to different adaptor molecules, including CD3z, FcRe, FcRIg, and DAP12. NKp46 and NKp30 are conserved in mice (Biassoni et al., 1999). NKp44 and NKp46 have been shown to be specifically activated by hemagglutinins encoded by influenza- and Sendai-virus (Mandelboim et al., 2001). In contrast, some nonclassical MHC class I molecules are recognized by NK inhibitory receptors that appear earlier in differentiation than the Ly49 receptors. These receptors may also function to maintain tolerance. Mouse CD94– NKG2A, C, and E molecules interact with complexes consisting of nonclassical MHC molecule Qa-1b bound to a peptide derived from the leader sequence of a classical MHC class I protein (Salcedo et al., 1998). In humans, CD94– NKG2A and C receptors function in a similar manner by interacting with the complexes consisting of the nonclassical Human Leukocyte Antigen E (HLA-E) molecule (Braud et al., 1998; Lee et al., 1998). The Ly49D activating receptor of mice recognizes H2-Dd but also has the ability to recognize diverse ligands such as xenogeneic MHC-encoded ligands on rat lymphoblasts (Nakamura et al., 1999) and Chinese hamster ovary cells (Idris et al., 1999; Nakamura et al., 1999). Ly49H has been shown to have specificity for a murine cytomegalovirus (MCMV)-encoded MHC class I–like protein, m157 (Arase et al., 2002; Smith et al., 2002) and is directly responsible for clearance of experimental MCMV infection (Daniels et al., 2001; Lee et al., 2003). So far Ly49H has not been found to have specificity for a conventional class I epitope. Another MHC class I–like molecule CD1d is recognized by a subset of T lymphocytes in both mice and humans that coexpress the NK receptor NK1.1/NKRP1A (CD161). NK T cells are a heterogeneous subset of T lymphocytes, which display a CD4þ or CD4 CD8 double-negative phenotype, and coexpress a semi-invariant TCR, encoded in mice and humans by Va14-Ja281 and Va24-JaQ rearrangements, respectively (Bendelac et al.,


9

1997; Kronenberg and Gapin, 2002). Although natural antigens presented by CD1d to NK T cells are still unknown, a-galactosylceramide (a-GalCer), a glycosphingolipid isolated from marine sponges, specifically binds CD1d and activates NK T cells (Brossay et al., 1998; Kawano et al., 1997). The major characteristic of NK T cells that dictates their function is their ability to promptly secrete large amounts of diverse cytokines upon TCR engagement, leading in turn to a plethora of reported effects on the immune system, which may be attributed to their ability to activate NK (Carnaud et al., 1999) and B cells (Galli et al., 2003; Schofield et al., 1999). In most studies NK T cells are regulated via activation of the TCR and the role of NK receptors on these cells has not been extensively explored. A recent report, however, provides evidence that NK complex-dictated genes regulate the ability of NK T cells to determine the differential susceptibility of C57BL6 and BALB/c mice to the P. berghei cerebral malaria syndrome (Hansen et al., 2003). B. Regulation of NK Cell–Cytokine Secretion Although NK cell activation by a number of ligands generally results in the induction of both cytoxicity and cytokine secretion, there are examples that suggest that these two functions can be controlled by different biochemical pathways. B lymphocytes, at certain activation states, can induce NK cells to secrete IFN-g but are not killed by the activated NK cells (Michael et al., 1989). A number of B-cell tumors can also increase IFN-g secretion by NK cells and some of these are not killed by NK cells (Yuan et al., 1995). Further experiments show that the elevation of NK cell IFN-g messenger RNA (mRNA) abundance by B cells or a B-cell tumor results from signals that induce stabilization of preexisting IFN-g mRNA (Cheung et al., 1999; Wilder and Yuan, 1995). Thus, the mechanism differs significantly from induction of increased transcription of the gene that results from activation of NK cells by cytokines such as IFN-a/b or IL-12 (Chan et al., 1992; Cheung et al., 1999; Hodge et al., 2002). Interestingly, ligands that can stimulate other receptors such as CD28 and FcgRIII also increase mRNA levels by the mechanism of stabilization of the message and these effects synergize with those of IL-12 (Cheung et al., 1999). IFN-g mRNA contains several putative regulatory AUUUA pentameric repeats in its 30 UTR. These elements, which are important regulators of mRNA stability (Chen and Shyu, 1995), can be targeted by proteins that either enhance degradation (AUF1 [Laroia and Schneider, 2002]) or inhibit degradation (HuR [Gallouzi et al., 2000]). Thus, rapid changes in mRNA abundance is regulated by the relative levels of these proteins, which are determined by nuclear–cytoplasmic distribution (Atasoy et al., 1998). The signaling pathways involved in activation of the nuclear–cytoplasmic shuttling is not known, although findings dissociating NK cell cytotoxicity from cytokine secretion indicate that distinct pathways do indeed regulate the two effector

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functions. After stimulation of the human NK cell line YT by antibodies against CD244, an activating receptor, the resultant cytotoxicity is dependent on Erk1 and Erk2 and p38, whereas IFN-g production is p38 dependent but is Erk1/2 independent (Chuang et al., 2001). Further evidence is provided by the finding that Vav1 is involved in the induction of IFN-g production but not in cytotoxicity (Colucci et al., 2001). Finally, freshly isolated CD56hi human NK cells proliferate in response to IL-2 or IL-15 and are potent producers of both type 1 (IFN-g) and type 2 (IL-5 and IL-13) cytokines compared with the CD56lo NK cell subset, which does not readily proliferate, produces low levels of cytokines, and yet shows enhanced natural cytotoxicity (Cooper et al., 2001; Lanier et al., 1986). There is also evidence in cultured human NK cells for the existence of non-overlapping subsets of NK cells that can be differentiated by their ability to produce type 1, type 2, or type 0 cytokines (Loza and Perussia, 2001; Loza et al., 2002). There is no evidence for functionally distinct subsets of NK cells in mice with selective cytokine production. However, bulk cultures of mouse splenic NK cells produce different cytokines when propagated in the presence of different cytokines. IL-2–propagated NK cells produce low levels of IFN-g and IL-13 (Hoshino et al., 1999; Lauwerys et al., 2000). The levels of these cytokines, as well as others, such as IL-10, can be further elevated by the inclusion of IL-12 or IL-18 in the culture. Even IL-4 can enhance IFN-g production (Jay et al., 2003). However, whether the elevation of cytokine production reflects the general induction of increased NK cell activity in vitro is not clear because IL-12/IL-18 NK cells also exhibit greater cytotoxic activity (Lauwerys et al., 2000). Interestingly, although IL-15 is a potent growth factor for NK cells, it does not enhance cytokine production by NK cells (Lauwerys et al., 2000). Of all the possible cytokines that can be produced by NK cells, IFN-g may play the most prominent role in the regulation of in vivo responses.

IV. NK–B-Cell Interactions In Vitro

A. NK Cell Activation of B Lymphocytes in the Absence of Specific B Cell Activators The most direct way of demonstrating that NK and B cells can interact is to isolate purified populations of each cell type, coculture them, and determine the effect of these interactions on parameters of cellular activation such as induction of activation markers, proliferation, cytokine production, and in the case of B lymphocytes, immunoglobulin secretion and isotype switching. Freshly isolated B cells from peripheral lymphoid organs consist of a mixture of resting and partially activated lymphocytes. The more homogeneous resting


11

B cells can be isolated from the heterogeneous collection of activated B cells by fractionation on Percoll density gradients. These cells are not actively dividing, express low levels of class II molecules, and costimulatory ligand such as CD80, CD86, and class II molecules, as well as other activation markers such as CD69. Whereas most of them are double positive for IgM and IgD, the population can also contain a low number of memory cells that express IgG. The lowdensity B cells separated by the same Percoll gradient are much more heterogeneous in terms of surface marker expression, but in general they express higher levels of CD69 and CD86 and exhibit some spontaneous proliferation when placed in culture. The percentage of high-versus low-density B cells present in the mouse spleen varies greatly depending on the strain, the age, and the cleanliness of the housing conditions. The low-density B cells most likely represent cells that have been arrested at various stages of differentiation. Thus, although markers are available to distinguish subpopulations of B cells such as B1 versus B2 B cells and germinal center versus MZ B cells, these subpopulations may also be segregated into different fractions by density (Yuan and Dang, unpublished observations, 1999). The low-density B cells can respond more rapidly to polyclonal activators such as LPS and anti-BCR reagents (Snapper et al., 1993; Weiss et al., 1989). Their ability to present antigen also differs dramatically from that of resting B cells (Evans et al., 2000; Snyder et al., 2002). Therefore, to analyze the effects of NK cells on B-cell differentiation, we should examine each population separately. Thus, resting B cells can be shown to respond to NK cells in a number of ways. First, freshly isolated highly purified NK cells both from SCID mice that are devoid of B or T cells and from mice with a deletion of the IFN-g gene (IFN-g / ) can upregulate the activation marker CD69 and costimulatory markers such as CD86 (Fig. 1) after coculture for only a few hours. The interaction does not require MHC histocompatibility. Freshly isolated and IL-2–propagated NK cells can also induce a number of mRNAs associated with B-cell activation (Yuan and Gao, unpublished observations, 2002), such as lymphoid-specific interferon regulatory factor (LSIRF) [Mittrucker et al., 1997] and Blimp-1 (Calame, 2001). Of particular interest is the contact-dependent induction of expression of germline transcripts for IgG2a (Ig2a) together with mRNA for AID in resting B cells that are further depleted of IgG-expressing populations (Gao et al., 2001). Despite the induction of these transcripts necessary for switch recombination, B cells activated in this manner do not proceed to the synthesis of mRNA for switched transcripts encoding functional IgG2a heavy chains, indicating that additional factors are needed for the completion of switch recombination. Obvious factors that may be missing include IL-2 or IFN-g. However, addition of neither of these cytokines could drive further differentiation. In the same system, however, low-density B cells can be induced by NK cells to augment their production of IgG (Mikhael et al.,

12

DOROTHY YUAN

Fig 1 Induction of CD86 expression on B lymphocytes by natural killer (NK) cells. Small resting B lymphocytes were incubated with NK cells purified from (A) CB17.SCID or (B) GKO spleen cells in the presence or absence of interferon-g (IFN-g) (40 U/ml) or IL-4 (10 U/ml) as indicated, at the NK-to-B ratio of 0.25:1. In addition, B cells were incubated with mesenteric lymph node cells of a CB17.SCID mouse. After overnight culture, cells were analyzed by fluorescence-activated cell sorter for the presence of CD86 on CD19-positive cells.

1991). This responsiveness of B cells that have been activated in vivo was also found in studies using activated B cells from trypanosome-infected mice (De Arruda Hinds et al., 2001). Thus, in vivo ligation by antigen is likely to be the missing signal required for productive switch recombination. However, the use of in vivo–activated B cells, some of which may have already undergone switch recombination, complicates the analysis of possible mechanisms. Although IL-2–propagated NK cells produce higher amounts of IFN-g than freshly isolated NK cells, the basal level of production is highly variable. This variability may account for the differences in abundance of IFN-g exhibited by different long-term clones of NK cells (Vos et al., 1998). Similarly, the level of production of other cytokines such as tumor necrosis factor-a (TNF-a), IL-13, and GM-CSF is also variable. When IL-2–propagated NK cells are cocultured with resting B lymphocytes in the absence of additional ligands, low levels of proliferation and IgM secretion can be induced (Yuan et al., 1992). These levels can be significantly increased by the addition of IL-5, although the extent of B-cell differentiation in terms of both proliferation and IgM secretion remains at best only 10% of that which can be induced by the polyclonal activator LPS. These results suggest that only a minor subset of B cells are responsive. In contrast to the induction of B-cell activation markers, however, the induction of IgM secretion by IL-2–propagated NK cells can be replaced by their culture supernatants. The nature of the active factor in the supernatants is not clear, although obvious candidates such as IFN-g, TNF-a, or GM-CSF have been discounted (Yuan et al., 1992). Moreover, further B-cell differentiation by switching to other isotypes was not induced.


13

Numerous studies have also examined the effect of NK cells on human B cells. The best comparison with the murine system is that of Gray and Horwitz (1995) who separated the resting population from in vivo preactivated cells in peripheral blood lymphocytes. The use of resting B cells as a target clarified previous experiments in which assays were performed with populations containing a heterogeneous mixture of B lymphocytes (Becker et al., 1990; Vyakarnam et al., 1985). They showed that these resting cells can be induced by autologous NK cells by becoming ‘‘primed’’ for immunoglobulin production via a cell-contact–dependent interaction that involves CD11a and CD54, but immunoglobulin secretion requires further activation by IL-2– activated NK cells and is mediated by cytokines. Under these conditions significant amounts of IgM and IgG that are comparable to these induced by pokeweed mitogen (PWM) can be induced. The production of IgG can be eliminated by the depletion of IgG-positive cells, indicating that although NK cells may be able to activate memory cells, they do not induce class switching. Interestingly, the factor secreted by NK cells is neither IFN-g nor a number of plausible cytokines that were tested. Similar experiments reported by Blanca et al. (2001) arrived at very different conclusions. First, although they also reported induction of IgG secretion by autologous NK cells, the levels of antibodies shown to be produced were some 10-fold lower than that induced by PWM. Second, they showed that the NK–B-cell interaction is mediated by a CD40–CD40L interaction, but a contact period of only 5 minutes was sufficient. Third, removal of IgG-positive cells did not affect the response, although removal of cells bearing CD5 and/or CD27 completely eliminated the secretion of immunoglobulin. The low levels of immunoglobulin synthesis induced in these cultures suggest that the interaction between CD40 and CD40L may be relevant only for this select population of B cells. Moreover, such low levels of immunoglobulin induced may reflect the ability of NK cells to promote selective survival of B-lymphocyte subpopulations. In this aspect it is also relevant to note that the induction of germline transcripts in mouse B cells has not been shown to require CD40–CD40L ligand–receptor interaction (Gao et al., 2001). In conclusion, the nature of molecules used in the NK–B-cell interaction is still not well defined. Whether other cell surface ligands in addition to adhesion molecules such as leukocyte function–associated antigen 1 (LFA-1) promote cell interactions is not known. Human invariant NKT cell clones can also induce syngeneic B cells to proliferate and to produce antibodies in vitro (Galli et al., 2003). The induction is effective both in the presence and in the absence of a-GalCer, the ligand for the TCR present on the cells, but is dependent on CD1d, suggesting that they may recognize a yet unidentified ligand expressed by B cells. However, in these studies the responding B cells consisted of CD19þ peripheral blood lymphocytes that may contain a significant fraction of in vivo–preactivated

14

DOROTHY YUAN

cells. Furthermore, the cytokine contribution of the activation from the in vitro–propagated NK cells was not evaluated. B. NK–Cell Activation of B Lymphocytes in the Presence of B-Cell Activators In contrast to induction of resting B cells in the absence of other B-cell activators, cross-linking of the BCR with anti-IgD conjugated to dextran (anti-ddex) makes them much more responsive to propagated NK cells (Snapper et al., 1993). Anti-d-dex has been used to simulate a TI-2 antigen in vitro. Activation of high-density B cells with this reagent without additional cytokines results in only B-cell proliferation. However, in the presence of propagated NK cells, B cells can be induced to produce significant amounts of IgM, although further differentiation by switching to downstream isotypes has not been reported. This effect is also mediated by a soluble factor. In this case IFN-g appears to be the dominant active cytokine and the extent of IgM secretion in the system is correlated with the amount of IFN-g produced by each inducing clone of NK cells (Vos et al., 1998). Because the addition of a number of cytokines, including IFN-g, can also induce IgM secretion, the significance of the role of IL-2– propagated NK cells in this system is not clear. However, the observation that depletion of NK cells that co-purify with B cells completely eliminates initiation of IgM production suggests that additional factors produced by NK cells may also play a role because only low levels of IFN-g are produced by freshly isolated NK cells. It is also interesting to note that although anti-d-dex has been shown to simulate TI-2 antigens in many aspects, removal of NK cells in vivo does not affect the response to TI-2 antigens in many aspects, removal of NK cells in vivo does not affect the response to TI-2 antigens (Koh and Yuan, 2000; Sungjin et al., 2000). Thus, the in vivo response of antigen-specific B cells may differ somewhat from polyclonal stimulation by anti-d-dex. Human cells previously stimulated with Staphylococcus aureus Cowan (SAC) were also shown to respond to NK cell clones (Becker et al., 1990). In this case direct cell contact, which may involve LFA-1, as well as soluble factors including IFN-g and TNF-a, were shown to be necessary. However, whether resting or previously activated B cells were responding is not clear because they were not separately analyzed. In contrast to the stimulation of immunoglobulin secretion, some clones of NK cells suppressed the antibody response to SRBC in vitro (Nabel et al., 1982). Similarly, suppression was shown to be exerted by in vivo–activated NK cells (Alexopoulou et al., 2001) or activated APCs (Abruzzo and Rowley, 1983). These results could be attributed to the inhibitory effect of NK cell–produced IFN-g on the SRBC response (Reynolds et al., 1987). A similar effect was demonstrated by the increase in anti-SRBC responses when NK cells were depleted from in vitro cultures (Robles and Pollack, 1989), suggesting that endogenous NK cells that


15

are not overtly stimulated may also have a suppressive effect. The differential effect of NK cells on the response of TI-2 versus TD antigen activation of B cells in vitro is interesting. Possibly different responding B cells are involved, but the presence of Tcells in the TD cultures also complicates interpretation of the data. The effect of NK cells on B cells that have already been induced to secrete immunoglobulin by other polyclonal activators yielded different results. In the murine system the incubation of LPS-stimulated B cells with NK cells resulted in a reduction of proliferation because of the secretion of IFN-g by activated NK cells (Michael et al., 1989). As a result of the decrease in proliferation, total immunoglobulin secretion, but not the amount of immunoglobulin produced per cell was reduced. Both the proliferative and the secretory response of human B cells activated by PWM has also been shown to be suppressed by NK cells (Arai et al., 1983; Katz et al., 1989; Tilden et al., 1983). In this case, other cytokines produced by NK cells, in particular TGF-b, can also suppress antibody responses via activation of CD8þ suppressor cells (Gray et al., 1994, 1998; Katz et al., 1989). Table I summarizes the essential findings regarding the activity of NK cells on B-cell immunoglobulin production in vitro. Induction of B-cell immunoglobulin secretion requires signals that can initiate B-cell proliferation and elevate transcription of the IgH and IgL chain genes as well as additional splicing factors to switch immunoglobulin production from the membrane to the secretory forms. The details of the mechanisms underlying these changes have not yet been completely elucidated. Thus, in the absence of additional signals, NK cells or cytokine, on their own, cannot initiate IgM secretion. Similarly, in human systems, with the exception of one study, NK cells cannot induce immunoglobulin secretion of resting B cells. The activation requirements of memory B cells may be different, but NK cells, even IL-2–activated cells, have not been shown to activate murine memory B cells, although human B cells can respond. In contrast, in vivo–activated B cells can be activated by NK cells to secrete IgM and other isotypes. The in vivo activation can also be mimicked by agents that can appropriate cross-linking the BCR or by other polyclonal activators. Thus, studies documenting the activity of NK cells on B-cell populations that are not adequately characterized are difficult to interpret. C. Activation of NK Cells by B Cells At certain differentiation stages, B cells can also exert an effect on NK cells. Michael et al. (1989) found that NK cells can be induced by low-density, in vivo–preactivated, but not resting B cells to increase their secretion of IFN-g. Interestingly, the level of induction was not increased by LPS stimulation of the B cells. In fact, even when resting B cells were stimulated with LPS, they did not become more effective inducers of IFN-g production (Mikhael et al., 1991) and the level of induction by low-density B cells was not increased

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DOROTHY YUAN

TABLE I Ability of NK Cells to Stimulate B Cell Subpopulations NK cells activation status

B-cell activation status

Additional known signals

Effect on Ig synthesis

Mediator

1

IL-2 propagated

Murine Resting

None

Ig2a, AID mRNA

2

IL-2 propagated

Murine Resting

IL-5

IgM

3

IL-2 propagated IL-2 propagated IL-2 propagated

Murine Resting Murine Resting Murine in vivo preactivated

Anti-d-dex

IgM

Cell contact, Not CD40CD40L Unknown cytokine Not IFNg, TNF-a IFN-g

LPS

IgM, IgG2a

IFN-g

None

IgM, IgG2a

6

NK clones

Srbc

7

Non cultured

Murine unfractionated Human

Suppression IgM, IgG Prime B cells

IFN-g and other cytokines Not tested

8

IL-2 activated IL-2 activated Non cultured

4 5

9 10

11

NK clones

12

NK T-cell clones

Resting Human Resting Human Resting IgGþ Human resting CD5þ and /or CD27þ Human unfractionated Human unfractionated

None

None

IgM

None

IgG

None

IgM, IgG, IgA

SAC

IgM, IgG

aGalCer

IgG

References (Gao et al., 2001)

(Yuan et al., 1992)

(Snapper et al., 1993) (Mikhael et al., 1991) (Yuan et al., 1992)

CD11a, CD54

(Nabel et al., 1982) (Gray and Horwitz, 1995)

Cytokines, not IFN-g Cytokines, not IFN-g CD40CD40L

(Gray and Horwitz, 1995) (Gray and Horwitz, 1995) (Becker et al., 1990)

CD11a and IFN-g, TNF-a Cell contact

(Blanca et al., 2001) (Galli et al., 2003)

by the addition of the mitogen, that would have activated adherent cells. Thus, induction of NK cells is not likely to be mediated indirectly by contaminating non–B cells in the culture. This difference in regulation may have significant consequences in terms of the cellular/cytokine amplification networks and is further discussed in Section V. Activated human B cells can also increase NK cell cytokine production (Becker et al., 1990; Wyatt and Dawson, 1991). Despite the activated status


17

of both human and murine B cells, they are not readily killed by NK cells, although they exhibit cell surface determinants that allow them to act as cold target inhibitors for the killing (Michael et al., 1989). Direct contact is necessary for the induction of NK cells in both systems, but further analysis is needed to determine whether the reported need for LFA-1–intercellular adhesion molecule (ICAM) interactions found for human cells (Becker et al., 1990) is the only requirement, because on their own, antibodies to these molecules did not inhibit NK–B-cell interactions in the murine system (Koh and Yuan, unpublished observations, 1996). It is clear, however, that the interacting receptors must be able to overcome the effect of the inhibitory receptors on NK cells during this interaction with syngeneic B cells. Although ligation of CD28 on NK cells in conjunction with FcgRIII can overcome the inhibition (Cheung et al., 1999), neither B7.1 nor B7.2 co-receptors expressed on B cells are involved in the activation, because CTLA4-immunoglobulin does not block the interaction (Koh, 1997). A recent publication claims that ligation of the activating receptor Ly49D on NK cells in the presence of IL-12 can also overcome inhibitory receptors (Ortaldo and Young, 2003) as evidenced by the induction of IFN-g secretion; however, because whole antibodies were used in the study, coligation of FcgRIII may participate in the activation, especially when stimulation with anti-Ly49D on its own induced low but clearly detectable levels of both IFN-g mRNA and protein. If the anti-Ly49D antibodies function in a similar manner as anti-CD28, the activity could be the result of synergy between a transcriptional increase of the IFN-g gene induced by IL-12 in combination with a stabilization of mRNA induced by the coligation of the Ly49D receptor with FcgRIII (Cheung et al., 1999). The syngeneic induction by activated B cells of NK cell cytokine secretion but not cytotoxicity differs significantly from the syngeneic induction of NK cells by DCs. One hypothesis is that whether DCs activate or are killed by NK cells depends on the ratio of the abundance of the two interacting cell types (Zitvogel, 2002) in that at high DC/NK cell ratios, they activate NK cells in both cytokine and cytotoxic functions. But at low DC/NK cell ratios, they are killed. Repeated attempts to document NK lysis of syngeneic B cells have not been successful, although it is clear that under the same conditions a subpopulation of LPS-activated B lymphocytes can be killed (Nabel et al., 1982). One difference between the two inducing populations may be the necessity for DCs to be propagated in culture to obtain sufficient numbers during which they may acquire additional ligands not expressed in vivo, whereas B-cell populations can be directly isolated from the animal. Second, the ability of activated DCs to secrete cytokines that can activate NK cells is much greater than that of B lymphocytes (Harris et al., 2000). The target molecule on NK cells that is triggered by activated B lymphocytes or DCs is not known. Recently, NKG2D on NK cells has been shown to be a

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DOROTHY YUAN

major receptor that allows NK cells to discriminate between normal versus abnormal (infected, activated, or transformed) cells (Diefenbach et al., 2002). Moreover, it is interesting that this receptor can be associated with different signaling molecules such as DAP12 that possess ITAMs in their cytoplasmic domains or with DAP10, which lacks a discernable ITAM and contains instead a YxxM motif, found also in the cytoplasmic domain of the CD28 and inducible costimulator (ICOS) molecules, where it is thought to impart a costimulatory signal. Therefore, the possibility exists that cells expressing different ligands may induce in NK cells either an activation or a costimulatory response via the NKG2D receptor, resulting in cytotoxicity and/or cytokine production (Diefenbach et al., 2002). Because ligands for NKG2 are upregulated in LPS-stimulated B cells (Diefenbach et al., 2000), this may be the triggering molecule for the induction of NK cytotoxicity against LPS-activated B cells, although blocking experiments are needed to confirm this hypothesis. However, Rae-1, on its own, is unlikely to be responsible for the induction of NK cell IFN-g secretion by B cells because both resting and activated B cells can be induced by LPS to elevate the abundance of mRNA for this family of ligands (Jennings and Yuan, unpublished observations, 2003); however, LPS-stimulated resting B cells are not effective inducers of IFN-g secretion. In conclusion, the in vitro studies described so far indicate that NK cells can exert various effects on both murine and human B-cell differentiation, although the extent of the effect may depend on the differentiation stage of the B cells used as targets. Thus, highly purified B cells that are devoid of memory cells may be able to respond to only a limited extent to direct stimulation by NK cells in the absence of additional B-cell stimulatory signals or cytokines produced by NK cells. However, as is discussed in a later section, by ‘‘priming’’ B cells in functions other than immunoglobulin secretion, even the limited differentiation may have important consequences. In contrast, when B cells are first activated by B-cell–specific reagents, NK cells can exert additional effects that are predominantly mediated by cytokines produced by NK cells. Because activated or memory B cells usually exhibit some basal level of immunoglobulin secretion, the influence of NK cells on increasing these levels may sometimes be difficult to dissociate from a survival-promoting effect of NK cells on B cells. In contrast to the induction of resting B cells by NK cells, activated B cells have been shown in both mouse and human systems to enhance NK cell cytokine production. It should be noted that the in vivo–activated B cells that are not further propagated in culture differ significantly from LPSstimulated B cells in their ability to activate NK cells. For both aspects of NK–B-cell productive interactions documented from the in vitro experiments, the nature of the ligand–receptor pairs involved is still largely unknown and should provide impetus for further study.


19

V. In Vivo NK–B Cell Interactions

Despite the body of convincing data showing in vitro interactions between NK and B cells, it is important to establish that such interactions are relevant in vivo. However, unlike the in vitro situation, productive cellular interactions in vivo take place within a complex microenvironment in which the movements of antigens, APCs, B cells, and T cells are governed by anatomical constraints. The most direct method to measure NK cell activation of B-cell activity in vivo is to evaluate its effect on TI responses in order to reduce the contributory role of T cells. This was first documented in experiments showing that injection of an NK cell clone together with B cells into irradiated animals resulted in the reduction of antibody response to the antigen TNP-Ficoll (Nabel et al., 1982). Though not tested directly on the antigen-activated cells, the reduction was attributed to cytotoxic activity of the NK cells. However, the effect may have been due to high levels of IFN-g secreted by the cloned NK cells, which reduced the proliferative response (Michael et al., 1989). An alternative approach to address this question is to test the effect of stimulation of NK cells in vivo on B-cell responses to a TI antigen. Wilder et al. (1996) showed that the injection of poly(I:C) before challenge with TNP-LPS resulted in specific increases in the IgG2a response and the increase requires the presence of NK cells. Similarly the IgG2a response to a TI-2 antigen, TNPFicoll, was also increased by the injection of Poly (I:C). Poly(I:C) is a synthetic analog of double-stranded RNA produced by RNA viruses, and there is considerable evidence that virus infection activates a cytokine loop that is initiated by production of IFN-a/b and IL-12, which can induce IFN-g production by NK cells. The production of IFN-g, in turn, further activates macrophages (Biron and Gazzinelli, 1995; Trinchieri and Gerosa, 1996). There is also much evidence that IFN-g enhances B-cell switching to IgG2a in the presence of polyclonal stimulators both in vitro (Snapper and Paul, 1987) and in vivo (Finkelman et al., 1988). Therefore, the effect on the antibody response is most likely due to the increased levels of IFN-g production, acting directly on antigen-activated B cells. However, unlike insights obtained from the induction of switch recombination to IgG1 by IL-4, the molecular mechanism by which IFN-g induces the switch to IgG2a has not been elucidated. Furthermore, other sources of stimulation may also participate in the induction of switch recombination to IgG2a upon activation of B cells in an antigenspecific manner. There are, for example, low but significant levels of IgG2a responses to TD antigens in both IFN-g / and IFN-gR / mice (Huang et al., 1993). IgG2a antibodies can be produced after some infections of IFN-g / mice (Markine-Goriaynoff et al., 2000). Moreover, it is interesting that although injection of IL-12 augments antibody responses (Buchanan et al.,

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DOROTHY YUAN

1998), injection of anti-IL-12 was found to completely abrogate IgG2a responses to TNP-LPS (Koh and Yuan, 1997). However, IL-12 on its own does not induce switch recombination in vitro (Metzger et al., 1997). Therefore, factors other than IFN-g may also induce switch recombination to IgG2a. Because cells containing recently switched transcripts can be detected outside of B-cell follicles (Toellner et al., 1996), it is conceivable that NK cells can play a contact-dependent role in addition to their ability to secrete IFN-g. Indeed the in vitro induction of germline transcripts for g2a and AID mRNA in the absence of IFN-g (Gao et al., 2001) by NK cells suggests that contact with NK cells may prime B cells to switch to IgG2a given appropriate further stimulation. The assessment of the effect of NK cells on TD immune responses is more difficult because of the involvement of T cells and APCs. In an attempt to reduce the complexity, Wilder et al. (1996) first primed mice with carrier only, then allowed them to rest, before injecting them with poly(I:C) followed by challenge intraperitoneally (IP) with the hapten-carrier conjugate. Thus, a primary B-cell response is assayed in the context of optimal T-cell help. Such a protocol resulted in increases in the IgG2a and IgG1 responses, although in this case only the increase in IgG2a was found to be dependent on the presence of NK cells. Thus, not only did the cytokine circuit induced by poly(I:C) activate NK cells, but other cell types may have been activated as well. Upon subcutaneous injection, poly(I:C) has been shown to activate DCs via stimulation of IFN-a/b–producing cells. Simultaneous injection of antigen results in enhancement of both IgG1 and IgG2a responses (Le Bon et al., 2001). Moreover, others have shown that IFN-a/b produced as a result of poly(I:C) stimulation can activate B cells directly in vitro in the presence of BCR ligation (Braun et al., 2002). In addition to an adjuvant, NK cells can also be activated by other means. Some evidence for B-cell stimulation of NK cells in vivo can be gleaned from the observation that a B-cell tumor, BCL1-C11, which can induce NK cell IFN-g production in vitro without being killed, can initiate a cascade in vivo whereby sufficient IFN-g is produced to alter the isotype distribution of antibodies induced by an antigen injected at the same time (Koh and Yuan, 1997). Although activation of NK cells in vivo clearly has an effect, either directly or indirectly, on B-cell antibody production, it is also important to determine whether NK cells play a role in antibody production without explicit stimulation. This question can be approached by depletion of NK cells before antigenic challenge. Early in vivo depletion experiments reported by Roble and Pollack (1986, 1989) suggested that NK cells exhibit negative regulatory effects on the IgG response of B cells; however, the readout of the effect was based on subsequent elicitation of the response in vitro. Direct in vivo responses were not measured. In none of the numerous subsequent studies involving


21

measurement of in vivo responses after depletion of NK cells was enhancement of antibody production reported, although other inconsistencies were found. In some cases exhaustive depletion accompanied by careful verification of the depletion failed to show changes in the response to different antigens (Wang et al., 1998; Wilder et al., 1996). On the other hand, using similar depletion strategies, others have uncovered significant effects on isotype switching (Korsgren, 1999; Shi et al., 2000). In most of these experiments T-cell priming was not first established, so effects on antibody responses could have been due to either deficiencies in generation of helper cells or the B-cell response itself. One problem associated with transient depletion experiments is that the removal of NK cells may be incomplete or variable. This problem can be overcome if genetically modified mice with a specific deletion of NK cells can be constructed. A number of such mouse strains exhibit defects in NK cell development and/or function, but these are usually accompanied by B- or T-cell defects as well (Ikawa et al., 2001; Kennedy et al., 2000; Wang et al., 1994). A model of NK-only deficiency was created by reconstituting the human CD3e transgenic animal, found to be deficient in both T and NK cells, with T-cell precursors to create a mouse deficient only in NK cells. However, the cytokine constitution of these mice may be disturbed (Wang et al., 1996). Furthermore, NKT cells are also decreased in these mice (Satoskar et al., 1999b). The Ly49A transgenic mouse (Sungjin et al., 2000) is deficient in only peripheral NK cells. The reason for the deficiency is not known, but the expression of transgenic Ly49 on cell types other than NK cells may differentially affect immune responses. A summary of the results from all of the systems that have analyzed the effect of NK depletion on B-cell antibody responses to defined antigens is presented in Table II. In view of the pronounced effect of poly(I:C) injection on antibody responses, it is also possible that inclusion of adjuvant during the immunization with specific antigens may influence the effect of NK cells. Therefore, Table II also indicates whether adjuvant was used during immunization. Comparison of these results provides some insight into the possible mechanism of the NK effect. First, it can be noted that in the absence of adjuvant, depletion of NK cells did not affect any of the IgG or IgM (not shown) responses to TI or TD antigens. Second, with the exception of one study, inclusion of adjuvant during injection of antigens, including a TI antigen, resulted in reduction of at least one of the IgG subclasses in the absence of NK cells. Although IgG2a is the subclass most frequently affected, other immunoglobulin classes, including a typical Th2-regulated response IgE, was also reduced. Interestingly, despite the association of IFN-g with switching to IgG2a, studies by Satoskar et al. (1999a) showed that in the human CD3e NK deficiency model, neither IFN-g nor IL-4 levels were affected after immunization. Therefore, the effect of NK cells may not be solely attributed to cytokines.

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DOROTHY YUAN

TABLE II Effect of Depletion by Anti-NK1.1 on Antigen-Specific IgG Responses IgG subclassa

1 2 3 4 5 6 7 8 9 10 11

Antigen

Adjuvant

IgG1

IgG2a

TNP-KLHb TNP-OVAb TNP-LPS OVA TNP-Ficoll KLHc OVAa DNP-Ficollc PyVc

None None None Alum None CFA CFA CFA None

Same Same

Same Same Same Same Same Reduced Reduced Reduced Reduced

OVAd AchR

Alum CFA

Same Same Same

Same

Reduced Same

IgG2b

IgG3

References

Same Same Same

Wilder et al., 1996 Wilder et al., 1996 Wilder et al., 1996 Wang et al., 1998 Sungjin et al., 2000 Satoskar et al., 1999 Satoskar et al., 1999 Buchanan et al., 1998 Szomolanyi-Tsuda et al., 2001 Korsgren et al., 1999 Shi et al., 2000

Same

Same

Reduced

a For each response reported, the comparative difference in responses between control and anti-NK1.1– treated animals is designated. b Animals were primed with carrier in RIBI but challenged with antigen in PBS. c Human CD3e mice were used. d IgE responses were also decreased.

In our laboratory we have adopted another method for developing a mouse strain that is deficient in only NK cells. The strategy is to construct a transgenic mouse that constitutively produces an anti-NK1.1 antibody identical to that produced by the PK136 hybridoma, which has a proven ability to delete NK cells specifically in vivo. Using such transgenic mice, we found that sufficient anti-NK1.1 antibodies are produced in the mice to kill all newly generated NK cells as soon as B cells are activated via endogenous or injected antigens. Because the PK136 transgenes do not code for the antigen receptor of the B cell in which it resides, activation occurs only as a result of stimulation via the endogenous BCR. Plasma cells generated after immunization with TI antigens (Garcia de Vinuesa et al., 1999; Koh and Yuan, 2000) and after virus infection (Slifka et al., 1998) have been shown to have long half-lives. Therefore, regardless of the initial stimulus, plasma cells producing the transgenic antibodies persist in the animal and maintain the absence of NK cells. It is also possible to use the bone marrow cells from these mice to create mixed radiation chimeras of the PK136 transgenic strain with cells from other strains. Figure 2A shows that for at least as long as 6 months, individual chimeric animals are uniformly depleted of NK cells to the same extent as intact mice that have recently been injected with anti-NK1.1 antibodies. Using three independent groups of animals, we examined the effect of depletion of NK


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Fig 2 Effect of natural killer (NK) cell depletion on the response to pneumococcal vaccine. (A) Peripheral blood lymphocytes from representative chimeric animals generated from C57BL /6 bone marrow cells or combined with an equal number of PK136 transgenic bone marrow cells were stained with anti-NK1.1 and anti-DX5 antibodies 4.5 months after transfer (background values varied between 0.2% and 0.4%). (B) Groups of chimeric animals generated in the same manner were immunized with Pneumovax in phosphate buffered saline (PBS) or in the adjuvant RIBI. Fourteen to twenty-four days later, serum samples collected were measured by isotypespecific enzyme-linked immunosorbent assay analysis against the same antigen. The mean of the responses in each group on day 14 is shown along with the standard error of the mean. Asterisks indicate p values .05 for T tests (assuming unequal variance) testing for the probability that the mean values between groups of animals are identical.

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cells on responses to a TI-2, antigen pneumococcal vaccine. Figure 2B confirms findings of previous experiments with TI-2 antigens, that the absence of NK cells does not greatly affect the response to antigens injected in PBS. However, when antigen is introduced in adjuvant, a robust IgG1 response can be generated in addition to IgG2a and IgG3 in chimeras generated with cells from intact mice. Significantly, the IgG1 response is much reduced when NK cells are chronically depleted. Surprisingly, the IgG2a response is not as affected, but the IgG3 response may also be reduced; however, the greater variability in the response of control animals diminished the statistical difference. Because NK T cells are not affected by the presence of the PK136 transgenes, as would be expected from previous assessment of the effect of transient injections of this antibody (Asea and Stein-Streilein, 1998; Smyth et al., 2001), the effect on IgG1 expression must be mediated by NK cells. These results show that NK cell elaboration of IFN-g may not be the only pathway by which B-cell responses can be affected. Other cytokines, such as IL-13, a Th2 cytokine, may be preferentially made under these conditions. Alternatively, the bloodborne DCs specialized in capturing TI-2 antigens (Balazs et al., 2001) may be influenced by NK cells in the presence of adjuvant. Although the mechanism of how DCs alter Th1 versus Th2 polarity is not clear, there is evidence that NK cells may play a role (Geldhof et al., 2002). Figure 3 illustrates the plethora of pathways that could be involved upon challenge with antigen in the presence of adjuvant or poly(I:C) with emphasis on how NK cells could be involved in the regulation of B-cell function. For simplicity, only pathways that could be occurring in the spleen upon introduction of bloodborne antigens are shown. Thus, in vivo NK–B-cell interactions can occur via several routes. (1) Upon activation by poly(I:C) or adjuvant, cytokines such as IFN-a/b, IL-12, produced by macrophages and/or DCs can activate NK (Biron et al., 1999) and B cells and APCs (Braun et al., 2002). IFN-g and TNF-a produced by activated NK cells can in turn further activate macrophages, thereby setting up a cytokine circuit that can affect B-cell responses indirectly. (2) B cells activated by TI antigens may be able to stimulate NK cells to produce IFN-g via direct cell contact and further amplify the circuit. In the absence of this amplification effect initiated by adjuvant, the level of IFN-g may be inadequate for significant alteration of the immune response. (3) Activated NK cells can also stimulate resting B cells to produce g2a germline transcripts in preparation for switch recombination to IgG2a. If B cells are then appropriately stimulated by either TI or TD antigens, they would be directed to preferential IgG2a production that is further enhanced by IFN-g. (4) Activated NK cells and/or IFN-g produced by NK cells can also enhance the ability of B cells and other APCs in their ability to present TD antigens to T cells. Whether preferential antigen presentation by B cells versus other accessory cells can differentially alter the polarity of Th1 versus Th2 stimulation is not clear.


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Fig 3 Possible interaction pathways in the spleen upon intraperitoneal injection of antigen in the presence of adjuvant or poly(I:C).

Even in the absence of IFN-g, NK cells may also be able to exert an effect on B-cell responses. First, stimulation by adjuvant could directly activate B cells so that they can stimulate NK cells. Second, resident activated accessory cells could also stimulate NK cells. In this case IL-13 production by NK cells may become more relevant, resulting in the activation of Th2 cells. Additionally, direct interactions between NK and B cells as well as unknown cytokines could replace IFN-g to stimulate B-cell IgG2a production. In addition, the preferential pathways initiated by antigen under the influence of different types of adjuvants may alter the relative contribution of cytokine circuits versus direct cell–cell interactions and could influence the manner in which NK cells affect B-cell responses. Thus, considering the multiple pathways by which B-cell immunoglobulin production can be regulated, the variable effects of NK cell depletion obtained in different experimental systems (Table II) is not surprising.

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It should be noted that with subcutaneous routes of immunization, DCs activated by either poly(I:C) or adjuvant may play a greater role because of their ability to capture antigen in the periphery for activation of T cells residing in the peripheral lymph nodes (Le Bon et al., 2001). Moreover, the role of NK cells may be less prominent because of their low abundance at these sites (Cavanaugh et al., 2003). Many questions remain regarding the interaction of NK and B cells in vivo. The readout of immunoglobulin secretion does not permit the analysis of whether the adjuvant effect is at the level of antigen presentation, of selective switch recombination, or of cytokine amplification of the response during the terminal phase. Additionally, factors that affect homeostasis of the participating cells may also play an important role. For example, the importance of BAFF, APRIL, and their counterreceptors in the development and maintenance of mature B cells in vivo (McHeyzer-Williams, 2003) as well as cytokines that are important in the maintenance of NK cell homeostasis (Koka et al., 2003) may all play important roles in the consideration of the effects of NK cells on B-cell activities. Another question concerns the significance of immunoglobulin isotype skewing directed by NK cells. In general IgG2a has been shown to be more effective mediators of ADCC, although the affinity of the antibodies may also play a significant role (Anasetti et al., 1987; d’Uscio et al., 1991; Kipps et al., 1985; Koh and Yuan, 2000; Steplewski et al., 1991). Furthermore, an intriguing finding made by Faquim-Mauro et al. (1999) raises the possibility that under the influence of NK cells, the properties of some antibodies produced may differ even if they belong to the same subclass. Thus, IgG1 antibodies produced in the absence of IL-4 are deficient in their ability to mediate an anaphylactic response. VI. The Role of NK–B-Cell Interactions in Autoimmune Diseases

Pathogenic antibody production is believed to be responsible for symptoms associated with a number of autoimmune syndromes. The underlying mechanism responsible for the activation of the autoimmune B cells is not evident in many cases. The finding that administration of antigen in the presence of adjuvant has profound effects on NK-mediated functions has relevance when one considers that a number of experimental models used to study autoimmunity rely on the injection of the inducing antigen in the presence of strong adjuvants, such as complete Freund’s adjuvant (CFA) (Damotte et al., 2003; Matsumoto et al., 1998; Shi et al., 2000; Zhang et al., 1997). For example, whereas removal of NK cells during experimental induction of the symptoms of myasthenia gravis by the injection of acetylcholine receptor in adjuvant results in amelioration of pathogenic antibodies (Shi et al., 2000), whether NK cells would play a similar role during the natural course of the disease is not clear. On the other hand, the adjuvant effect on NK cells may reflect, in an exaggerated manner, events


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occurring during an inflammatory response that can trigger endogenous nonspecific immune responses sometimes labeled the ‘‘danger signal’’ (Gallucci and Matzinger, 2001) and could lead to autoimmunity, given the appropriate genetic background and environmental conditions. One experimental system that does not involve the introduction of adjuvant is the induction of autoantibody production in (C57/BL/6 DBA/2) F1 hybrids upon injection of spleen cells from parental DBA/2 mice. In this case the presence of NK cells appeared to exert a protective role and removal of NK cells exasperated the autoimmune symptoms (Harada et al., 1995). Furthermore, infusion of IL-2–activated NK cells had a protective effect. Although they showed that the NK cells were not cytotoxic against the autoimmune B cells, possible cytokine effects were not investigated. Therefore, further studies aimed at unraveling the mechanism of protection may be informative for understanding the role of NK cells in spontaneous autoimmunity. Similar suppressive effects were found for NK cells in studies using a mouse strain predisposed to autoantibody production. The initiation of autoantibody production in C57BL/6.lpr mice that spontaneously develop autoantibodies was accelerated by depletion of NK cells (Takeda and Dennert, 1993). Conversely, injection of a semipurified population of NK cells delayed the onset. The interpretation of these studies is, however, confounded by the dysregulation of apoptosis resulting from the lpr gene defect, which affects multiple cell types. Similarly a role for NK cells has also been suggested by the evaluation of NK cell cytotoxic activity in MRL lpr/lpr mice (Nilsson and Carlsten, 1996; Magilavy et al., 1987). Disease symptoms have also been associated with the presence of NK cells or NK cell activity in NZB NZW mice (Gordon et al., 1989). Here also, the multiple defects in this strain make it difficult to identify a unique role for NK–B-cell interactions. More definitive answers may be derived from the utilization of congenic strains containing restricted loci derived from the NZM2410 strain on a C57BL/6 background (Morel et al., 2000). Some of these strains contain genes that are responsible for defects that are uniquely manifested by either B or T cells. Another particularly interesting strain B6.Sle.1 contains a cluster of functionally related genes that are responsible for spontaneous upregulation of activation markers on both B and T cells that correlate with the development of autoantibodies. Interestingly, however, radiation chimera studies show that presence of both cell types is not necessary for development of the symptoms (Mohan et al., 1997). Thus, it should be interesting to devise experimental strategies to analyze the possible role of NK cells in the modulation of disease progression. NKT cells have also been investigated as a possible candidate for the regulation of autoimmunity. Development of pathogenic symptoms attributed to T-cell dysfunction can be modulated by the activation of NKT cells in a number of autoimmune models (Mieza et al., 1996; Pal et al., 2001). Depending on the

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disease and time of activation of the cells relative to the disease process, NKT cells can either exacerbate or reduce disease symptoms. The mechanism is most likely due to modulation of the Th1 versus Th2 polarity through their ability to secrete cytokines such as IL-4, IL-10, transforming growth factor-a, or IFN-g (Kronenberg and Gapin, 2002). Furthermore, the secretion of these cytokines may be developmentally regulated (Benlagha et al., 2002). Interestingly, however, the development of autoantibodies has not been associated with the presence or absence of NKT cells (Luo et al., 2002). In conclusion, the rapid increase in the understanding of NK receptors and function provides excellent tools to reassess the role of NK cells in the regulation of many autoimmune diseases, especially those involving B-cell dysfunction. VII. NK–B-Cell Interactions during Infections

The participation of NK cells in the amplification of the cytokine circuit was first described in studies of microbial infections (Tripp et al., 1993). It is now apparent that activation of the innate immune system in response to infection can often be attributed to activation of members of the TLR family (Akira et al., 2001; Medzhitov and Janeway, 2002) expressed on many cells of the innate immune system and on B cells. These pattern recognition receptors can be triggered by gram-positive and gram-negative bacteria, as well as double-stranded RNA and hypomethylated CpG present in bacterial DNA. Despite the prominent role of NK cells as a member of the innate immune system, there is no evidence for the existence of TLRs on NK cells, although they can be easily activated by cytokines induced as a result of TLR engagement on other cell types. TLR3 has been shown to respond to double-stranded RNA (Alexopoulou et al., 2001), resulting in the production of type I interferons. Although increased IFN-a/b production is a hallmark of many viral infections (Biron, 1998), the mechanism by which RNA viruses engage TLR3 is not well understood (Lopez et al., 2003). Thus, whereas IFN-a/b clearly has an immunoregulatory role in the resolution of viral infections, the mechanism is still a subject of intense research (Nguyen et al., 2002). IFN-a/b production leading to activation of NK cell IFN-g production is an effective means of amplifying the cytokine cirucuit, but on the other hand, in some viral infections this cytokine inhibits another NK cell–activating cytokine IL-12 (Biron, 2001). In any case, many viral infections clearly result in enhanced NK cell expansion and function (Biron et al., 1999; Tay et al., 1998). Virus-induced alterations of MHC class I antigen expression using a number of strategies (Brutkiewicz and Welsh, 1995) can also elevate cytotoxicity of NK cells. In addition to resistance to MCMV (Arase et al., 2002; Smith et al., 2002), response to influenza and Sendai viruses (Mandelboim et al., 2001) may also be directly attributed to the expression of specific NK receptors. In a subset of infections, increased IFN-g


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levels contribute to the reduction of viral burden via mechanisms such as induction of nitric oxide synthase (Reiss and Komatsu, 1998). Moreover, IFN-g elaborated by NK cells early in the response is also most likely responsible for the increased level of IgG2a production often associated with a number of viral infections (Coutelier et al., 1987). Although synthesis of IgG2a antibodies is usually dependent on T-cell help, many viruses can express the appropriate repetitive antigenic determinants necessary for the elicitation of TI responses. Thus, both polio and polyoma virus have been shown to be TI-2 antigens in that they are poor immunogens in mice with defects in Bruton’s tyrosine kinase, which is required specifically for B-cell responses to TI-2 antigens (Pinschewer et al., 1999; SzomolanyiTsuda et al., 2001). Interestingly, although NK cells appear to be not required for a protective response to this virus, they are necessary for the generation of IgG2a antibodies (Szomolanyi-Tsuda et al., 2001), which may play a significant role in the reduction of viral burden. Furthermore, B-cell expression of the IFN-g receptor is required for increased IgG2a synthesis. Therefore, NK cells may function by amplification of the cytokine circuit to provide cytokine help, or possibly NK cell production of IFN-g is increased by B cells activated by viral infection. Because Rae-1 expression is upregulated by LPS stimulation of B cells, another possibility is that B cells activated by viral antigens can stimulate NK cells via the NKG2 receptor. The antibody response to a number of other viruses has also been shown to not require CD4 T-cell help (Szomolanyi-Tsuda and Welsh, 1998), suggesting that NK cells may provide an important source of second signal for TI responses. Because IgG2a may be more effective for mediating ADCC (Koh and Yuan, 2000), increased levels of this subclass of antibodies may provide a mechanism for controlling viral replication (Mochizuki et al., 1990) or maintaining viral latency (Kim et al., 2002). Antibodies also play a central role in the resolution of a number of bacterial infections. Other than the well-recognized effector functions of antibodies including agglutination, opsonization, and enhancement of complement activation, an unusual antibody-mediated function has been uncovered. Endocytosis of relevant antibodies by activated macrophages may allow them to retain the pathogen within an intracellular location inside a phagocyte (Edelson and Unanue, 2001). However, whether such antibodies are generated during the natural course of the infection is not known. Early studies have implicated NK cells and the cytokine loop in the early phases of the response against infection by the intracellular pathogen Listeria monocytogenes (Unanue, 1997). Amplification of the cytokine loop by many bacterial pathogens can occur upon capture of the pathogen in multiple ways, including phagocytosis, endocytosis, or via members of the TLR family. Thus, for a number of pathogenic bacterial infections, including Salmonella typhimurium (Ramarathinam et al., 1993) and Chlamydia trachomatis (Tseng and

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Rank, 1998), NK cells play a dominant role in the early production of IFN-g necessary for host defense. In other bacterial infections, however, NK cells may not be required (Brucella abortis [Fernandes et al., 1995]). In fact, in another study using an NK-deficient model, T-cell production of IFN-g during early infection by L. monocytogenes was found to be sufficient for activating the innate immune system (Andersson et al., 1998), although the general disruption of the lymphoid system in this mouse (Cao et al., 1995) may compromise the conclusion somewhat. Therefore, the importance of cytokine secretion by NK cells early in the response to bacteria may be determined by additional factors such as the time course of the infection and the rapidity of T-cell recognition of the processed antigen. A number of parasites have also been shown to activate the MyD88 pathway used by TLRs (Scanga et al., 2002). The activation of the cytokine circuit in this manner would explain the findings of NK involvement in some parasitic diseases. Early studies of Toxoplasma gondii infections (Denkers et al., 1993; Scharton-Kersten et al., 1996) showed that NK cells are the effectors of IFNg–dependent immunity. Not only are IL-1b–and IL-12–activated NK cells induced to secrete IFN-g, but they also express higher levels of CD28 (Hunter et al., 1997). The possibility that this upregulation is involved in NK–B-cell interactions is intriguing, especially in view of the finding that an intact B-cell response is also important for resistance to the parasite (Kang et al., 2000). Another mechanism for the resistance involving B cells may be attributed to the production of specific antibodies needed for ADCC mediated by NK cells against infected targets (Dannemann et al., 1989). NK cells are also the major source of IFN-g, which drives the differentiation of protective T-cell immunity in leishmaniasis (Scott, 1998; Scott and Hunter, 2002). The early activation of NK cells is probably also mediated by the activation of the TLRs on macrophages (Hawn et al., 2002). Although IgG2a is the predominant antibody produced as a consequence of the IFN-g production, antibodies probably play a subsidiary role to T cells in the manifestations or control of this disease (Brown and Reiner, 1999). On the other hand, antibodies play a prominent role in infections with Trypanosoma cruzi, the causative agent of Chagas’ disease, which also results in augmentation of NK cell activity (Brodskyn et al., 1996). Polyclonal B-cell activation is detected during infection with increases in circulating levels of both IgM and IgG2a (d’Imperio Lima et al., 1986; Spinella et al., 1992). Because NK cells can also augment the secretion of IgG2a in vitro from B cells obtained from infected animals even in the absence of further activation (De Arruda Hinds et al., 2001), the increase in polyclonal immunoglobulin in vivo may be a consequence of NK activation. NK cell depletion does, however, result in increased susceptibility to the infection (Cardillo et al., 2002). Moreover, even in the absence of IFN-g, IgG2a production is not reduced (Markine-Goriaynoff et al., 2000), suggesting


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that NK cells may also play an essential role in the initiation of the protective response (Paiva et al., 2002) by direct interaction with B cells. It is interesting that in some parasitic diseases such as Schistosoma mansoni, in which protective immunity is of the Th2 type (Stadecker, 1999), depletion of NK cells also induces alterations of regulatory mechanisms affecting disease manifestations (Asseman et al., 1996). It will be of interest to determine whether similar TLRs are activated by the pathogen. VIII. Conclusions

Through studies of the extent of NK cell participation during the early phases of infection by a number of organisms, NK cells have long been well established as being an important element of innate immunity that can boost immune resistance before the development of specific immunity. More recently, they have also been shown to be important in shaping the nature of the B- and T-cell responses. Because these studies are often complicated by the inflammatory processes arising from the particular infection, the specific mechanisms for how NK cells function have been more difficult to pinpoint. In this chapter, we scrutinized in vitro studies that show interactions between NK and B cells and compared these results with in vivo experiments that used defined antigens. Although some clues now emerge regarding the multiple pathways by which NK cells can potentially affect B-cell response, details regarding the relative importance of each of these pathways are still quite incomplete. Hopefully, however, this effort can point out further directions that need to be taken. The increased understanding of the functional relationship between NK and B cells may help resolve some outstanding problems in understanding the development of autoimmunity and aid in the design of appropriate vaccine strategies.

Acknowledgments I am grateful to the past and present members of my laboratory for their contributions and to Dr. Vandana Parikh and Dr. Michael Bennett for review of the chapter.

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Ramarathinam, L., Niesel, D. W., and Klimpel, G. R. (1993). Salmonella typhimurium induces IFN-g production in murine splenocytes. Role of natural killer cells and macrophages. J. Immunol. 150, 3973–3981. Reiss, C. S., and Komatsu, T. (1998). Does nitric oxide play a critical role in viral infections? J. Virol. 72, 4547–4551. Reynolds, D. S., Boom, W. H., and Abbas, A. K. (1987). Inhibition of B lymphocyte activation by interferon-gamma. J. Immunol. 139, 767–773. Rivera, A., Chen, C. C., Ron, N., Dougherty, J. P., and Ron, Y. (2001). Role of B cells as antigenpresenting cells in vivo revisited: Antigen-specific B cells are essential for Tcell expansion in lymph nodes and for systemic Tcell responses to low antigen concentrations. Int. Immunol. 13, 1583–1593. Robles, C. P., and Pollack, S. B. (1989). Asialo-GMþ1 natural killer cells directly suppress antibody-producing B cells. Nat. Immun. Cell. Growth. Regul. 8, 209–222. Ron, Y., and Sprent, J. (1987). T cell priming in vivo: A major role for B cells in presenting antigen to T cells in lymph nodes. J. Immunol. 138, 2848–2856. Salazar-Mather, T. P., Ishikawa, R., and Biron, C. A. (1996). NK cell trafficking and cytokine expression in splenic compartments after IFN induction and viral infection. J. Immunol. 157, 3054–3064. Salcedo, M., Bousso, P., Ljunggren, H. G., Kourilsky, P., and Abastado, J. P. (1998). The Qa-1b molecule binds to a large subpopulation of murine NK cells. Eur. J. Immunol. 28, 4356–4361. Satoskar, A. R., Stamm, L. M., Zhang, X., Okano, M., David, J. R., Terhorst, C., and Wang, B. (1999a). NK cell–deficient mice develop a Th1-like response but fail to mount an efficient antigen-specific IgG2a antibody response. J. Immunol. 163, 5298–5302. Satoskar, A. R., Stamm, L. M., Zhang, X., Satoskar, A. A., Okano, M., Terhorst, C., David, J. R., and Wang, B. (1999b). Mice lacking NK cells develop an efficient Th1 response and control cutaneous Leishmania major infection. J. Immunol. 162, 6747–6754. Scanga, C. A., Aliberti, J., Jankovic, D., Tilloy, F., Bennouna, S., Denkers, E. Y., Medzhitov, R., and Sher, A. (2002). Cutting edge: MyD88 is required for resistance to Toxoplasma gondii infection and regulates parasite-induced IL-12 production by dendritic cells. J. Immunol. 168, 5997–6001. Scharton-Kersten, T., Wynn, T., Denkers, E., Bala, S., Grunvald, E., Hieny, S., Gazzinelli, R., and Sher, A. (1996). In the absence of endogenous IFN-g, mice develop unimpaired IL-12 responses to Toxoplasma gondii while failing to control acute infection. J. Immunol. 157, 4045. Schofield, L., McConville, M. J., Hansen, D., Campbell, A. S., Fraser-Reid, B., Grusby, M. J., and Tachado, S. D. (1999). CD1d-restricted immunoglobulin G formation to GPI-anchored antigens mediated by NKT cells. Science 283, 225–229. Scott, P. (1998). Differentiation, regulation, and death of T helper cell subsets during infection with Leishmania major. Immunol. Res. 17, 229–238. Scott, P., and Hunter, C. A. (2002). Dendritic cells and immunity to leishmaniasis and toxoplasmosis. Curr. Opin. Immunol. 14, 466–470. Shi, F.-D., Wang, H.-B., Li, H., Hong, S., Taniguchi, M., Link, H., Kaer, L. V., and Ljunggren, H.-G. (2000). Natural killer cells determine the outcome of B cell–mediated autoimmunity. Nat. Immunol. 1, 245–251. Siliciano, R. F., Pratt, J. C., Schmidt, R. E., Ritz, J., and Reinherz, E. L. (1985). Activation of cytolytic T lymphocyte and natural killer cell function through the T11 sheep erythrocyte binding protein. Nature 317, 428–430. Sinha, A. A., Guidos, C., Lee, K. C., and Diener, E. (1987). Functions of accessory cells in B cell responses to thymus-independent antigens. J. Immunol. 138, 4143–4149. Slifka, M. K., Antia, R., Whitmire, J. K., and Ahmed, R. (1998). Humoral immunity due to longlived plasma cells. Immunity 8, 363–372. Smith, H. R., Heusel, J. W., Mehta, I. K., Kim, S., Dorner, B. G., Naidenko, O. V., Iizuka, K., Furukawa, H., Beckman, D. L., Pingel, J. T., et al. (2002). Recognition of a virus-encoded ligand by a natural killer cell activation receptor. Proc. Natl. Acad. Sci. USA 99, 8826–8831.


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Multitasking of Helix-Loop-Helix Proteins in Lymphopoiesis XIAO-HONG SUN Immunobiology and Cancer Program, Oklahoma Medical Research Foundation Oklahoma City, Oklahoma 73104

I. Introduction

The basic helix-loop-helix (bHLH) family of transcription factors plays a pivotal role in cellular differentiation, influencing diverse systems such as skeletal and cardiac muscle, pancreas, neuronal cells, adipocytes, chondrocytes, erythrocytes, and lymphocytes (Massari and Murre, 2000). This family consists of several dozen members, which are expressed either ubiquitously or tissue specifically. This chapter focuses on bHLH proteins involved in lymphoid development and tumorigenesis. Functions and properties of the bHLH proteins are first introduced, which will facilitate the understanding of their roles in the development and function of the lymphoid system. A. Helix-Loop-Helix Proteins in Lymphocytes Unlike the skeletal muscle system, in which a cell culture model can more or less mimic muscle differentiation, much of the investigation concerning the biological role of bHLH proteins in lymphoid cells relies on studies using transgenic or knockout mice (Engel and Murre, 2001). In lymphoid cells, the predominant bHLH proteins include products of the E2A, HEB, and E2-2 genes (Henthorn et al., 1990; Hu et al., 1992; Murre et al., 1989). The E2A gene produces two products, E12 and E47, which arise from alternatively spliced transcripts with different exons coding for the bHLH domains (Murre et al., 1989; Sun and Baltimore, 1991). E12, E47, HEB, and E2-2, collectively called E proteins, form homodimers or heterodimers to regulate transcription. As shown in (Fig. 1) these E proteins are highly homologous in the bHLH DNA binding and dimerization domain and in the two transcriptional activation domains, AD1 and AD2 (Aronheim et al., 1993; Massari et al., 1996; Quong et al., 1993). Additionly, these proteins contain three motifs with remarkable sequence homology, which we term E-protein homology domains, EHDs 1–3 (Nie et al., 2003). The structural conservation of the EHDs suggests a functional significance. However, the contribution of these domains to E-protein function has not been fully examined except that deletion of the EHD1 and EHD2 reduces the transcriptional activity of E47. Further, EHD3 is important for the degradation of E2A proteins (Nie et al., 2003). The fact that E proteins share extensive similarities in these various domains emphasizes 43 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2776/04 $35.00

Fig 1 Sequence alignment of E proteins. Domains with extensive sequence homologies are as labeled. The sequence alignments of the domains are shown below the diagram of a generic E protein in boxes with the same colors as those used for names. Sequences in red with yellow backgrounds are identical residues, whereas those in blue are similar residues. The number in front of each sequence for each domain indicates the position of the first residue in the context of the full-length protein. E12 and E47 only differ in the basic-helix-loop-helix (bHLH) domain. AD, activation domain; EHD, E-protein homology domain. (See Color Insert.)

MULTITASKING OF HLH PROTEINS IN LYMPHOPOIESIS

45

their functional redundancy. For example, Zhuang et al. (1998) has demonstrated that HEB can replace the function of E2A in B-cell development when inserted in the E2A locus. This indicates that the availability of all E proteins in a given cell must be taken into account when studying the role of E proteins in lymphocyte development and function. The issue of functional redundancy is perhaps best illustrated in B- and T-cell development of E2A-deficient mice. Because the E2A gene is expressed at a much higher level than HEB and E2-2 in the B lineage, E2A disruption is sufficient to cause a complete block of B-cell differentiation (Bain et al., 1994; Zhuang et al., 1994). In contrast, E2A and HEB are both expressed in the T lineage, and consequently, mutating either of the genes only partially impairs development (Bain et al., 1997a; Zhuang et al., 1996). The function of all E proteins can be inhibited by their dominant negative inhibitors, the Id proteins (Benezra et al., 1990; Christy et al., 1991; Riechmann et al., 1994; Sun et al., 1991). Four Id proteins (Id 1–4) have been identified. They are very similar in their HLH domains but share minimal homology in other regions except for a short stretch in the N-terminus. This structural characteristic suggests two possibilities, that HLH is the only functional domain to be conserved in all Id proteins or the remainder regions confer specificity to each Id protein. The affinities of Id1 to Id3 to E2A, E2-2, and HEB (also known as E2B) were compared in vitro and in vivo, but no significant differences were detected (Langlands et al., 1997; Sun et al., 1991). Furthermore, overexpression of either Id1 or Id3 appears to have similar inhibitory effects on lymphocyte development (Heemskerk et al., 1997; Jaleco et al., 1999; Kim et al., 1999; Sun, 1994). However, these studies do not rule out the possibility that Id proteins are posttranslationally modified in a different fashion to provide specificities for each protein. For example, Id2 and Id3 proteins are phosphorylated in vitro by cyclin A /E–cyclin-dependent kinase 2 (Cdk2) complexes at the Ser5 residue present in both proteins (Deed et al., 1997; Hara et al., 1997). Although this modification attenuates the inhibitory activity of both Id2 and Id3 on E12 homodimers, it surprisingly enhances the ability of Id3 to inhibit E12/myoD heterodimers. Furthermore, Id2 phosphorylation at the late G1 and S phases by cyclin A /Cdk2 correlates with the appearance of E2A DNA binding complexes in TIG3 human diploid fibroblasts. In contrast, expression of Id2 in the absence of cyclin A at the early G1 phase leads to inhibition of the DNA binding activity of constitutively expressed E2A proteins. Therefore, an alanine substitution mutation of Id3 at the Cdk2 phosphorylation site diminishes the ability of Id3 to promote the G1 to S-phase transition in rat embryonic fibroblasts. These results suggest a posttranslational regulation of Id function during cell cycle progression. However, whether Id function is differentially regulated during cell differentiation remains to be determined. In addition, Id1–Id3 proteins

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XIAO-HONG SUN

can also be phosphorylated by the cyclic adenosine monophosphate (cAMP)– dependent protein kinase and/or protein kinase C in vitro, but the functional consequences of the phosphorylation are not clear (Nagata et al., 1995). Another group of bHLH proteins includes the Tal1/SCL, Tal2, and lyl1 proteins (Begley et al., 1989; Chen et al., 1990; Cheng et al., 1993; Mellentin et al., 1989; Xia et al., 1991). They form heterodimers with E proteins and bind to DNA with a slightly different sequence preference compared to the E-protein homodimers (CAGATG over CAGGTG) (Hsu et al., 1994a; Sun and Baltimore, 1991). Tal proteins do not bind to Id proteins but compete with them to bind to E proteins (Hsu et al., 1994b; Sun et al., 1991). Although Tal/E protein heterodimers can bind to DNA, their transcriptional activity of E-box reporter genes is only 10% that of E-protein homodimers (Hsu et al., 1994b; Park and Sun, 1998). Accordingly, Tal proteins are considered another group of E-protein inhibitors. However, there are also reports that Tal /E protein heterodimers can cooperate in activating transcription with other transcription factors such as GATA1 and Lmo2, in the context of artificial or natural promoters where bind sites for each of the factors are present (Lam and Bresnick, 1998; Ono et al., 1998; Wadman et al., 1997). The Tal proteins are normally not expressed in the lymphoid lineages except during T-cell leukemogenesis caused by chromosomal translocation or interstitial deletion of the Tal1/SCL gene (Baer, 1993; Robb and Begley, 1997). In these situations, they appear to act as inhibitors of E proteins. The hairy/enhancer of the split (HES) family of bHLH proteins, including HES1 and HES5, represents the fourth group of bHLH proteins expressed in lymphoid cells (Kaneta et al., 2000; Tomita et al., 1999). HES proteins can form homodimers that bind to N-boxes (CACNAG) and recruit the transcriptional co-repressor, Groucho (Kageyama and Ohtsuka, 1999; Ohsako et al., 1994; Paroush et al., 1994). Because the basic regions of HES proteins contain a proline residue that disrupts the DNA binding helix, heterodimers between HES and E proteins are incapable of binding to DNA (Sasai et al., 1992). Therefore, they also serve as inhibitors for E proteins. The fact that HES1 is a downstream target of Notch signaling pathways provides a link between bHLH protein function and Notch signals ( Jarriault et al., 1995). Taken together, the net effect of bHLH proteins on lymphoid differentiation depends on the availability of all the E proteins and various inhibitors. B. Transcriptional Activation by E Proteins The DNA binding sequences of E proteins, E-boxes, have been found in the regulatory regions of many genes. Table I lists some of such genes related to the development and function of lymphocytes. These genes are considered the targets of E proteins because they satisfy at least one of the following criteria: (1) E-boxes are shown to be important for transcription of a target


47

TABLE I E2A Target Genes Gene

Aa Bb Cc

Dd

Note

Immunoglobulin m þ heavy chain

þ

Immunoglobulin k light chain Immunoglobulin l light chain Immunoglobulin e germline mRNA B29 (Igb) mb-1 (Iga)

þ

þ

þ

þ

þ

þ

l5

þ

þ

Vpre-B

þ

þ

þþ E2A and EBF act synergistically

EBF

þ

þ

þþ

RAG1 and RAG2

þ

þ

TdT AID CD4 pre-Ta

þ þ þ

CD21

þ

cdk inhibitor (p21) þ PAC-1 þ

þ þ þ

þ

þ

Cooperate with Ets Choi et al., 1996; Greenbaum and proteins Zhuang, 2002; Lenardo et al., 1987 þþ Greenbaum and Zhuang, 2002; Lenardo et al., 1987 þþ Greenbaum and Zhuang, 2002; Rudin and Storb, 1992 Sugai et al., 2003 þ

þ þþ E2A and EBF act synergistically þþ E2A and EBF act synergistically

þ

þ

þ

þ

þ

þ

In B cells but not T cells

E-box–mediated repression þ

þ

References

Greenbaum and Zhuang, 2002 Greenbaum and Zhuang, 2002; Sigvardsson et al., 2002 Greenbaum and Zhuang, 2002; Sigvardsson, 2000; Sigvardsson et al., 1997 Gisler and Sigvardsson, 2002; Greenbaum and Zhuang, 2002; Sigvardsson et al., 1997 Greenbaum and Zhuang, 2002; Smith et al., 2002 Hsu et al., 2003; Schlissel et al., 1991 Choi et al., 1996; Greenbaum and Zhuang, 2002 Sayegh et al., 2003 Sawada and Littman, 1993 Reizis and Leder, 2001; Takeuchi et al., 2001 Ulgiati et al., 2002 Prabhu et al., 1997 Grumont et al., 1996

a

E-box found to be important for the regulation of gene expression. The promoter or enhancer linked reporter gene activated by E proteins. Endogenous gene activated by ectopic expression of E proteins or inactivated by Id inhibitors. d E protein bound to the locus by chromatin immunoprecipitation. b c

gene, (2) a reporter gene constructed using the promoter or enhancer from a target gene can be activated by E proteins, (3) ectopic expression of E proteins or the inhibitors of E proteins can activate or repress the transcription of a target gene, or (4) E proteins are found to be bound to the regulatory regions of a target gene by chromatin immunoprecipitation assays. A number

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of genes involved in pre–B-cell receptor signaling such as Igm, mb-1, l5, VpreB, and B29 are well qualified to be the target genes of E proteins. E2A activates the transcription of these genes in cooperation with the EBF transcription factor, which itself is regulated by E2A. Genes crucial for the rearrangement of immunoglobulin (Ig) genes including RAG1, RAG2, and TdT are also controlled by E2A. Although finding these target genes helps explain why E proteins are important for B-cell development, lack of expression of these genes cannot account for the phenotype of E2A- or EBF-deficient mice. B-cell development arrests at a much earlier stage in these animals than in mice lacking any of the target genes (Gong and Nussenzweig, 1996; Kitamura et al., 1991, 1992; Mombaerts et al., 1992; Shinkai et al., 1992). By the same token, T-cell development is also blocked at the earliest stage (DN1) when E-protein function is completely inhibited by Id1 (Kim et al., 1999, 2002), whereas lack of pre-Ta expression impairs development at later stages (Fehling et al., 1995; Xu et al., 1996). These discrepancies suggest that additional target genes need to be identified to fully understand the role of E proteins in B- and T-cell development. Most likely, E proteins also control expression of genes whose expression is not necessarily restricted to the lymphoid lineage but are crucial for lymphocyte survival or proliferation. The p21 Cdk inhibitor and PAC1 dual-specificity phosphatase belong to this category, but many more remain to be found. The E2A proteins are exceptionally potent transcription activators. Two transcription activation domains, AD1 and AD2, were identified in these proteins through the GAL4 fusion assay. In these experiments, parts of the E2A protein were fused to the DNA binding domain of GAL4 and transcriptional activities were measured using a GAL4 reporter gene (Aronheim et al., 1993; Massari et al., 1996; Quong et al., 1993). Point mutations in AD1 or AD2 have been shown to completely abolish the activity when assayed separately as GAL4 fusion proteins (Massari et al., 1996; Quong et al., 1993). These mutations were then tested in the context of the full-length E47 protein with a multimerized E-box–driven reporter gene. Mutations in AD1 and AD2 each diminished about 50% of E47 transcriptional activity and combinating these mutations decreased the activity by about 80% (Nie et al., 2003). AD1 has been shown to bind to the SAGA complex, which contains subunits with histone acetyltransferase (HAT) activities and participates in chromosomal remodeling (Massari et al., 1999). AD2 interacts with p300, which also possesses HAT activities and acts as a coactivator for many transcription factors (Eckner et al., 1996; Qiu et al., 1998). It is through these interactions with transcription coactivators that E2A proteins exert their powerful effects on transcription of their target genes. In addition, interaction between the HAT and E2A proteins causes the acetylation of E2A proteins themselves, which enhances their transcriptional activity (Bradney et al., 2003).


49

However, the function of AD1 or AD2 appears to depend on the formation of E2A homodimers. Because the GAL4 DNA binding domain forms homodimers, the AD1- or AD2-GAL4 fusion proteins also have two copies of each AD. In contrast, the heterodimers between E2A and Tal1/SCL proteins are transcriptionally inactive because of the lack of a second copy of AD1 and AD2 (Park and Sun, 1998). Once the N-terminus of E2A including AD1 and AD2 is attached to Tal1, heterodimers between this chimera and E2A become potent transcription activators. This raises the possibility that E2A proteins can be used to inhibit transcription when they form heterodimers with other bHLH proteins such as Tal1, Twist, and ABF, which are transcriptionally inactive or repressive (Huang and Brandt, 2000; Massari et al., 1998; Spicer et al., 1996). Although heterodimers between E2A and the myogenic and neurogenic bHLH proteins such as MyoD and MASH activate transcription, whether AD1 and AD2 in E2A play any positive roles is not clear (Skerjanc et al., 1996; Weintraub et al., 1991). C. Transcriptional Regulation of Id Gene Expression The E proteins are thought to be ubiquitously expressed, albeit with some degree of variation at the protein level in different cell types and developmental stages (Herblot et al., 2002). In contrast, expression of the Id genes is highly dynamic during the differentiation of a variety of cell types. Thus, the function of E proteins can be regulated by controlling Id gene expression. The expression patterns of the four Id genes in hematopoietic and lymphoid tissues have not been systematically determined; therefore, the accumulated information, as summarized in Table II, appears fragmented and should be interpreted with caution (see references within). Expression of Id1–Id3 but not Id4 is found in lymphocytes and their precursors. During early lymphopoiesis in mouse bone marrow, Id1 is expressed in stem cells and early lymphoid progenitor cells (c-kithiSca-1 hiCD27þRAG1þ). Id1 expression declines as the cells become prolymphocytes or common lymphoid progenitors, although the expression continues in myeloid progenitors (Igarashi et al., 2002). Similarly, in human fetal hematopoiesis, Id1 is expressed at a high level in the most primitive progenitors (CD34þCD38 CD10 ) and its expression decreases as the cells differentiate along the B lineage. These observations suggest that downregulation of the Id1 gene is necessary for B-cell development to take place. However, as Id1 expression is being shut off, other Id genes are turned on in the same populations of cells, for example, Id3 in human fetal liver (Blom et al., 1999). This raises the question of whether downregulation of Id1 is meaningful if Id3 can replace the function of Id1. Likewise, Id2 is constitutively expressed in the thymus, which begs the question of whether the transient expression of Id3 after pre-T-cell receptor (pre-TCR) and T-cell receptor (TCR) signaling makes a difference. Data from analyses of Id3-deficient mice suggest that the

TABLE II Expression Patterns of Id Genes Organ Fetal liver

Id1

Id2

Id3

Human: Stem cells (CD34þ CD38 CD10 ) (Blom et al., 1999)

Mouse: Low in C-kitHi Sca1þ high in Ckitlo Sca1þ (Garrett and Kincade, 2003, personal communication)

Human: CD34þ CD38 CD10 < CD34þ CD38þ CD10 < CD34þ CD38þ CD10þ (Blom et al., 1999) Mouse: Low in progenitors, high in mature B cells (Garrett and Kincade, 2003, personal communication)

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Mouse: Widely expressed except in mature B cells (Perry and Sun, 2003, unpublished data; Yokota et al., 2003) Bone marrow

Human: CD34þ CD38 CD10 > CD34þ CD38þ CD10 > pro & pre-B < imimmature & mature B cells (Garrett and Kincade, 2003, personal communication) Mouse: Stem cells (Lin c-kithi Sca-1þ); Early lymphoid progenitors (Lin CD27þ c-kithi Sca-1hi RAG1þ); Myeloid progenitors (Lin c-kithi/lo0 ca1hi/lo RAG1 ) (Igarashi et al., 2002)

Human: CD34þ CD38 CD10 < CD34þ CD38þ CD10þ and pro to mature B cells (Garrett and Kincade, 2003, personal communication)

Thymus

Periphery

Mouse: (Perry CD4 CD8 CD44þCD25 and Sun, 2003, unpublished data) CD4/CD8 DP and CD8 SP (Yucel et al., 2003) Mouse: Very low levels (Sun, 1997, unpublished data)

Mouse: Widely expressed (Yucel et al., 2003)

Mouse: Transiently after pre-TCR or TCR signaling (Bain et al., 2001; Engel et al., 2001)

Human: Peripheral blood (Ishiguro et al., 1995)

Mouse: Splenic immature and mature B cells; downregulated during plasma cell differentiation (Pan et al., 1999; Shaffer et al., 2002).

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Mouse: Splenic IgD immature B cells and down-regulated in IgDþ mature B cells, Splenic NK cells and B cells, CD4þ CD3 IL-7Raþ cells of embryonic intestine (Fukuyama et al., 2002; Hacker et al., 2003; Sugai et al., 2003; Yokota et al., 1999)

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transient expression of Id3 is important (Rivera et al., 2000). Perhaps, the precise timing when each Id gene is expressed and the posttranslational modification of the Id proteins contribute to an optimal level of Id function in a given cell. Mechanisms involved in transcriptional regulation of the Id1, Id2, and Id3 genes have been investigated. The Id1 gene is expressed in a series of interlenkin-3 (IL-3)–dependent hematopoietic or lymphopoietic progenitor cell lines and its expression is stimulated by IL-3 (Mui et al., 1996; Quesenberry et al., 1996; Sun et al., 1991; Wilson et al., 1991; Xu et al., 2003). Therefore, many of the studies were carried out using these IL-3– dependent cell lines as model systems. An enhancer, called the Pro-B enhancer (PBE) has been found approximately 3 kb downstream of the Id1 gene, which is largely responsible for Id1 expression (Saisanit and Sun, 1995). Within the enhancer, three C/EBP binding sites were found to be absolutely essential for enhancer activity (Saisanit and Sun, 1997; Xu et al., 2003). STAT5 binding sites are also present in the enhancer, through which IL-3 stimulates Id1 gene expression by a novel mechanism involving protein deacetylation. Upon binding of IL-3 to its receptor, STAT5 is activated, enters the nucleus, and binds to the STAT5 sites in the PBE. STAT5 recruits histone deacetylases, which deacetylate C/EBPb bound nearby. The transcriptionally active form of C/ EBPb dimers can then bind DNA with high affinities and activate transcription (Xu et al., 2003). Consistently, Id1 gene expression is downregulated by treatment of the IL-3–dependent Ba/F3 cells with an HDAC inhibitor, trichostatin A (Rascle et al., 2003; Xu et al., 2003). The connection between the JAK-STAT pathway and Id1 expression would be helpful in understanding the regulation of Id1 expression during hematopoiesis where cytokines and growth factors play critical roles. In myoblasts, several Egr1 sites in the promoter region have been shown to be important for Id1 expression (Tournay and Benezra, 1996). These sites couId also be crucial for Id1 expression upon serum stimulation because the Egr1 transcription factor is a downstream effector of serum stimulation. Furthermore, Id1 has been shown to be a direct target of bone morphogenic proteins (BMPs). Activation of Smad transcription factors by BMP-mediated signaling allows them to bind to the promoter of Id1 gene and stimulate transcription (Katagiri et al., 2002; Korchynskyi and ten Dijke, 2002; Lopez-Rovira et al., 2002). BMP is also implicated in the activation of Id2 and Id3 gene expression (Hollnagel et al., 1999). Transcription of the Id2 genes is often influenced by oncogenic proteins. The promoter of the Id2 gene contains multiple Ets and myc binding sites. The Ets sites mediate transcriptional activation by Ewing sarcoma (EWS)– related proteins such as the EWS-FL1 protein, which is a fusion between the EWS gene product and an Ets transcription factor (Fukuma et al., 2003; Nishimori et al., 2002). Similarly, the myc sites are thought to be responsible


53

for Id2 overexpression in neuroblastoma cells, in which the N-myc gene is amplified (Lasorella et al., 2002). The Id3 gene was originally identified because of its response to serum stimulation (Bain et al., 2001; Christy et al., 1991). During pre-TCR and TCR signaling by anti-CD3 treatment of double-negative or double-positive thymocytes, activation of the ras-Erk MAP kinase pathway leads to activation of Egr1 transcription, which is followed by Id3 transcription (Bain et al., 2001; Engel et al., 2001). Overexpression of Egr1 by itself stimulates Id3 transcription. Conversely, Egr1-deficient mice have reduced Id3 expression after treatment with anti-CD3 antibodies (Bettini et al., 2002). These findings suggest that Id3 expression is tightly linked to the ras-Erk signaling pathway, which is influenced by a variety of developmental or growth stimuli. D. Control of Cell Cycle and Growth by bHLH Proteins In NIH3T3 fibroblasts, overexpression of E47 or introduction of antisense oligonucleotides against Id1 to Id3 arrests cell cycle at the G1 to S-phase transition (Barone et al., 1994; Hara et al., 1994; Peverali et al., 1994). Conversely, overexpression of Id1 accelerates cell proliferation (Prabhu et al., 1997). Similarly, Id1 delays the senescence of human primary fibroblasts and keratinocytes, whereas loss of Id1 leads to presenescence (Alani et al., 2001; Hara et al., 1996; Nickoloff et al., 2000; Tang et al., 2002). The mechanisms by which the HLH proteins influence the cell cycle may be partially explained by the altered expression of cyclin-dependent kinase inhibitors (CKIs). E47 activates the transcription of the p21 CKI gene through multiple E-boxes present in its promoter (Prabhu et al., 1997). Alternatively, Id1 reduces expression of the p16 CKI by blocking the binding of Ets proteins to the promoter (Ohtani et al., 2001). E-boxes are also present in the promoter of the p16 CKI gene and are thought to mediate transcriptional activation (Alani et al., 2001). However, overexpression of E47 alone is insufficient to alter p16 transcription (Prabhu and Sun, 1997, unpublished data). It is likely that more bHLH protein–regulated genes will be found to influence cell cycle control in a cell-type–specific manner. In addition, Id2, but not Id1 or Id3, has also been shown to bind the retinoblastoma protein (Rb) and its relatives such as p107 and p130 (Lasorella et al., 1996, 2000; Iavarone et al., 1994). Interaction between Id2 and the active hypophosphorylated form of Rb inhibits the function of Rb and sequesters Id2. The interaction has also been demonstrated genetically by an increased survival rate of Rb / Id2 / mice compared to Rb / mice, which die at embryonic day 14.5 of massive proliferation, poor differentiation, and apoptosis in nervous and hematopoietic systems (Lasorella et al., 2000). Overexpression of Id2 overrides p16-mediated growth arrest of human glioma cells (Lasorella et al., 1996). The Id2 gene is thought to be a direct target of

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N-myc, which is overexpressed in neuroblastoma cells (Lasorella et al., 2000, 2002). Analysis of primary neuroblastoma specimens initially showed correlations of N-myc amplification with Id2 expression (Lasorella et al., 2002), as well as Id2 expression with poor prognosis of the disease. However, reports with similar analyses challenge these findings (Sato et al., 2003; Vandesompele et al., 2003; Wang et al., 2003). The ability of HLH proteins to influence cell cycle progression may have a direct impact on their function in regulating cell differentiation and tumor formation. Although loss of E2A proteins resulting from somatic mutation has not been found in human tumors, Id gene overexpression is shown in various malignancies. Id1, Id2, or Id3 is overexpressed in prostate, breast, ovarian, esophageal, nasopharyngeal, medullary thyroid, astrocytic, and pancreatic cancers, melanomas, and in lymphocytic and myeloid leukemia (Hu et al., 2001; Maruyama et al., 1999; Ouyang et al., 2002; Schindl et al., 2003; Singh et al., 2002; Takai et al., 2001; Vandeputte et al., 2002; Wang et al., 2002; Wilson et al., 2001). Because Id gene expression is stimulated during cell cycle progression, Id overexpression observed in cancer cells may be due to rapid proliferation of cancer cells. This can be used as an indicator of the aggressiveness of the tumors and their prognosis (Kebebew et al., 2000; Schindl et al., 2001; Schoppmann et al., 2003). Alternatively, Id overexpression may cooperate with other oncogenic factors to promote malignant growth such as that seen in transgenic mice expressing Id in T cells and intestinal epithelial cells (Kim et al., 1999; Morrow et al., 1999; Wice and Gordon, 1998). II. HLH Proteins and B-Cell Development

A. Early B-Cell Development The E2A gene was cloned based on the ability of its products to bind to the mE2 and kE2 sites in the intronic enhancers of the immunoglobulin m heavy and k light chain genes (Murre et al., 1989). However, its importance for B-cell development was not demonstrated until E2A-deficient mice were created (Bain et al., 1994; Zhuang et al., 1994). These mice are completely devoid of B cells in the bone marrow and peripheral lymphoid organs. This result would have been unanticipated knowing that two other genes, E2–2 and HEB, share extensive structural and functional similarities with E2A (Henthorn et al., 1990; Hu et al., 1992). Analyses of the E2–2– and HEB-deficient mice suggest that E2–2 and HEB are expressed at much lower levels than E2A because lack of E2–2 or HEB expression causes much less impairment of B-cell development than E2A deficiency (Zhuang et al., 1996). Nevertheless, trans-heterozygotes between E2A and E2–2 or HEB display more severe defects in B-cell differentiation compared to heterozygous mutants of each gene, suggesting that the functions of the three genes are similar, and a combined dose of these


55

E proteins determines the outcome of B-cell development. By the same logic, this combined dose of E proteins can also be regulated by the level of their inhibitors such as Id1. Accordingly, expression of the Id1 gene under the control of a B-cell–specific promoter is sufficient to block B-cell development in transgenic mice similarly to E2A deficiency, but the degree of blockage depends on the level of Id1 expression in the transgenic mice (Sun, 1994). The inhibitory effect of Id3 on B-cell development has also been shown in human B-cell cultures (Jaleco et al., 1999). Likewise, expression of another inhibitor of E2A, SCL/TAL1, also partially inhibits B-cell development (Herblot et al., 2002). Loss of E-protein function results in arrest of B-cell development at a very early stage without any discernable effect on the differentiation of myeloid lineages. In E2A-deficient mice, this arrest is mapped at the fraction A stage (Hardy nomenclature) (Bain et al., 1997b; Hardy et al., 1991). These mice have reduced numbers of fraction A cells and lack B cells at later stages (Bain et al., 1997b). However, because fraction A includes a mixture of pre-pro–B cells, natural killer (NK) cells, and uncommitted progenitors (Tudor et al., 2000), it is not clear how early the arrest occurs. The developmental block in Id1 transgenic mice is at the early pro-B–cell stage (Kincade nomenclature) (Kincade et al., 2002), but this may be determined by the timing of transgene expression (Medina, Kincade, and Sun, 2000, unpublished data). Additionally, transplantation experiments have shown that the developmental defect is intrinsic to B cells rather than the microenvironment in which they reside (Zhuang et al., 1996). B-cell–specific gene expression and immunoglobulin gene rearrangement are dramatically reduced in the bone marrow of Eprotein–deficient and Id1 transgenic mice relative to wild-type mice (Bain et al., 1994; Sun, 1994; Zhuang et al., 1994). However, these results do not necessarily indicate that E proteins are responsible for the expression or rearrangement of these genes, but reflect that bone marrow samples without E-protein function are depleted of B cells. The early developmental arrest, which results in very few progenitor B cells in the bone marrow available for study, has made further biochemical investigation into the underlying molecular mechanisms very difficult. Consequently, little is known about how Eprotein deficiency leads to such an early developmental arrest or what are the cellular events that take place before the arrest. For example, whether the apparent block is due to the death of B cells during the course of maturation or the inability of development to proceed is unclear. To address these inadequacies, genetic approaches have been taken to probe the molecular mechanisms of B-cell deficiency resulting from loss of E-protein functions. Several crosses have been made with a number of genetically manipulated mouse models. Crossing Id1 transgenic mice with transgenic mice expressing either a productively rearranged Ig heavy chain or both

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functional heavy and light chains did not rescue the B-cell defect in Id1 transgenic mice (Vladimirova and Sun, 1996, unpublished data). This suggests that the B-cell deficiency observed in Id1 transgenic mice is not primarily due to lack of Ig gene rearrangement or expression. Several genes encoding proteins involved in forming pre-B–cell receptors (pre-BCRs) are regulated by E2A, as discussed in Section IB. However, pre-BCR deficiency arrests development at the transition from fraction B to fraction C stages, which is much later than the arrest caused by E2A deficiency (Gong and Nussenzweig, 1996; Kitamura et al., 1991, 1992; Mombaerts et al., 1992; Shinkai et al., 1992). Thus, although E proteins may be involved in the formation of pre-BCR, they must activate additional genes to drive the differentiation of early B-cell progenitors. Also, the Bcl2 transgene, expressed under the control of the promoter and enhancer of the Ig heavy chain gene (Strasser et al., 1991), cannot rescue B-cell development in Id1 transgenic mice (Sun, 1996, unpublished data). The functional interaction between E proteins and two other transcription factors prominent in B-cell development, EBF and Pax5, has been investigated. Disruption of the EBF gene blocked B-cell development at a slightly later stage, that is, the transition from fraction A to fraction B, than mutation of the E2A gene (Lin and Grosschedl, 1995). The arrest caused by Pax5 ablation is even later than EBF deficiency (Urbanek et al., 1994). Separate data have suggested that EBF is a target gene of E proteins, whereas Pax5 is activated by EBF (Greenbaum and Zhuang, 2002; O’Riordan and Grosschedl, 1999; Smith et al., 2002). Therefore, it has been tempting to propose a linear relationship among the three transcription factors, with E2A leading the pathway, followed by EBF, and then Pax5. To test this relationship, we generated transgenic mice expressing the EBF or Pax5 complementary DNA (cDNA) driven by the mb-1 promoter and Ig heavy chain enhancer. These transgenic mice were crossed with Id1 transgenic mice, in which the Id1 gene is expressed from the same promoter. EBF or Pax5 could partially rescue the B-cell deficiency in the Id1 transgenic mice by facilitating differentiation from the B220þIgM to B220þIgMþ stage (Vladimirona and Sun, 1996, unpublished data). However, total numbers of B cells in the Id1 transgenic bone marrow did not increase as a result of EBF or Pax5 expression. It is likely that EBF and Pax5 can partially substitute for E2A because of overlapping functions shared by these transcription factors, but they are not able to provide the unique activities of E2A needed at earlier stages of development. Likewise, comparison of the phenotype of E2Aþ/ EBFþ/ mice with either E2Aþ/ or EBFþ/ mice reveals a synergistic effect of the two proteins on B-cell development at stages beyond fraction A (O’Riordan and Grosschedl, 1999). In the E2Aþ/ EBFþ/ mice, B-cell development cannot reach the fraction C stage and differentiation to the fraction B stage is impaired. However, these blocks are less complete and later than those in either E2A /


57

or EBF / mice. Careful examination of gene expression in the trans-heterozygous mice shows decreased expression of a number of genes encoding proteins involved in pre-BCR formation, as well as EBF and Pax5 transcription factors. These findings are consistent with various reports that these genes are direct targets of E2A and/or EBF (Table I and references within). Therefore, the phenotype of the E2Aþ/ EBFþ/ mice may be attributed to a combined effect because of low levels of these gene products. Furthermore, the ability of E2A and EBF to influence Ig gene rearrangement may also contribute to the B-cell defects when half doses of E2A and EBF are present (Goebel et al., 2001; Romanow et al., 2000). As mice age, their B-cell development slows down (Sherwood et al., 2000). In particular, their pre-B–cell populations diminish compared to young mice. This is accompanied by low levels of l5 and VpreB surrogate light chains expressed in pre-B cells of the aged mice (Sherwood et al., 1998). Interestingly, the level of E2A proteins in IL-7 expanded pro/pre-B cells or Lipopolysaccharide (LPS)-treated splenic B cells also decreases in old mice, which may account for the reduced expression of l5 and VpreB genes (Frasca et al., 2003; Sherwood et al., 2000). However, mechanisms underlying the down regulation of E2A proteins during aging remain unknown. It is not clear whether aged B cells are unable to respond to the stimulation by IL-7 or LPS to express E2A or whether E2A proteins are destabilized. B. Late Stages of B-cell Development When mature B cells enter the periphery, they undergo further differentiation in an antigen-dependent manner. The differentiation processes include class-switch recombination (CSR) and somatic hypermutation (SHM), which generates memory B cells and high-affinity antibody–secreting plasma cells. E proteins have been implicated in both processes. E2A is expressed abundantly in the dark zone of germinal centers, where isotype switching takes place, and is upregulated by stimuli for B-cell activation (Goldfarb et al., 1996; Quong et al., 1999). CSR replaces the IgM constant region with the constant regions of other isotypes through their 50 switch regions. Different stimuli induce the recombination at different switch regions in vitro. Expression of the E-protein inhibitors, Id1 or Id3, in B-cell lines or primary activated B cells impairs spontaneous CSR or cytokine-induced CSR, suggesting that E proteins play an important role in class switching (Goldfarb et al., 1996; Quong et al., 1999). Conversely, IgE CSR is enhanced by disruption of the Id2 gene, indicating that Id2 normally inhibits CSR (Sugai et al., 2003). Interestingly, although E2A is known to activate transcription through E-boxes present in the promoters of germline transcripts originating from the switch regions (Ma et al., 1997), the inhibition of CSR by Id proteins is not accompanied by reduced levels of these transcripts (Goldfarb et al., 1996; Quong et al.,

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1999). Recently, the Aicda gene encoding an activation-induced deaminase (AID) has been found to be a direct target of the E2A gene (Sayegh et al., 2003). The finding that AID is a crucial enzyme for initiating CSR and SHM (Honjo et al., 2002) helps explain how E proteins enhance CSR in B cells. E-boxes are also found to enhance SHM of an Igk transgene when placed between the V and J segments (Michael et al., 2003). These E-boxes can bind to the E47 protein as determined using electrophoretic mobility shift assays in vitro. However, the two E-boxes do not increase levels of the Ck transcript. The involvement of E2A proteins in SHM of the transgene also remains to be investigated. Id2 is present at high levels in IgMþIgD immature B cells and their expression is downregulated during differentiation to the IgMloIgDþ mature B-cell stage (Becker-Herman et al., 2002). Lack of Id2 increases the population of IgDþCD23þ mature B cells. Similarly, Id3 is also expressed in mature B cells and becomes downregulated upon plasma cell differentiation (Pan et al., 1999; Shaffer et al., 2002). Id3-deficient B cells have a greater tendency to differentiate and class switch when induced in vitro, but they proliferate poorly in response to BCR cross-linking (Pan et al., 1999). This suggests that Id3 is normally important for clonal expansion of activated B cells. III. HLH Proteins and T-Cell Development

A. Developmental Defects in E-Protein–Deficient Mice In contrast to B-cell development, disruption of the E2A gene causes moderate impairment of T-cell differentiation in the thymus (Bain et al., 1994; Zhuang et al., 1994). E2A-deficient mice have reduced numbers of CD4 /CD8 double-positive (DP) cells and increased numbers of double-negative (DN) cells, particularly the cells at the DN1 stage (Bain et al., 1997a; Zhuang et al., 1996). T-cell development is less affected by E2A deficiency than B-cell development because the E2A functional homolog, HEB, is expressed in the thymus. In fact, E2A and HEB proteins are thought to form heterodimers in T cells (Bain et al., 1997a). HEB appears to play a larger role in T-cell development than E2A does (Barndt et al., 1999). Although the phenotype is variable in HEB / mice, a significant reduction in thymic cellularity is consistently observed in 3-week-old mice (70–95% reduction) and E18.5 fetuses (30–80% reduction). In addition to an increased percentage of DN cells and a reduced number of DP cells, a dramatic increase in the immature CD8 single-positive population is found in HEB-deficient mice. However, when both E2A and HEB are inhibited by expression of inhibitors such as Id1, Id2, Tal1, or a dominant-negative mutant of HEB, dramatic blockage of T-cell development is observed (Barndt et al., 1999; Kim et al., 1999, 2002; Morrow et al., 1999). Id1 and the p22 form of Tal1/SCL are


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expressed using the lck proximal promoter, which produces Id1 or Tal1 transcripts beginning at the DN1 stage (Kim and Sun, 1999, unpublished data). These transgenic mice display severe impairment of T-cell development in a dose-dependent manner. The thymuses of Id1 homozygous transgenic mice or Id1 and Tal1 trans-heterozygous transgenic mice contain predominantly DN1 cells (Kim et al., 1999, 2002). Because expression of both Id1 and Tal1 in the trans-heterozygous transgenic mice has a synergistic effect on inhibition of T-cell development, these inhibitors likely act through binding to their common targets, E proteins. In contrast, heterozygous Id1 or Tal1 transgenic mice have some thymocytes that develop beyond the DN stage, even though dramatically reduced numbers of total thymocytes are also observed. These transgenic mice also feature an increased proportion of CD8þ cells, consisting of immature and mature single positive cells and dramatically decreased numbers of DP cells. The phenotype of these heterozygous transgenic mice is similar to mice deficient of either E2A or HEB, which have partial activities of E proteins. Expression of the Id2 cDNA in transgenic mice results in a marked increase in the number of the immature single positive cells in addition to higher numbers of DN cells and lower numbers of DP cells (Morrow et al., 1999). Furthermore, mutations of three amino acids in the basic region of HEB abolish DNA binding activities of both HEB homodimers and HEB/E2A heterodimers. These mutations, collectively called bm, have been introduced into the HEB gene through gene targeting. Consequently, T-cell development is completely blocked at the transition from the DN to the DP stage (Barndt et al., 2000). The precise point of development arrest is thought to be at the DN2 or DN3 stage, when HEB begins to be expressed (Zhuang, 2003, personal communication). The HEBbm/bm mice have more severe defects than HEB / mice because of the dominant negative effect of HEBbm. Taken together, the phenotypes of these transgenic and knock-in mice, though somewhat variable depending on the gene expressed and the timing of expression, underscore the similarities of E-protein functions in B and T cells. E proteins are as essential for T-cell development as they are for B-cell development. The difference largely resides in the E proteins they express; that is, B cells contain primarily E2A proteins and T cells use both E2A and HEB. B. Apoptosis of the Developing Thymocytes in Id1 Transgenic Mice A striking characteristic of the Id1 and Tal1 transgenic thymus is the presence of a massive amount of apoptotic cells (Kim et al., 1999, 2002). This was initially observed by light scatter analysis, which revealed 40–60% of thymocytes outside of the viable cell gate. Fragmentation of DNA was also

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detected in the apoptotic cells. This has been confirmed by TUNEL analysis (Kim et al., 2002), as well as by deoxyuridine Triphosphate (dUTP) labeling and annexing V-staining assays (Qi and Sun, 2004, submitted). Based on the scatter plots shown in Barndt et al. (2000), it is likely that the HEBbm/bm mice display a similar phenotype. To determine the nature of the apoptotic cells, TCR gene rearrangements in the apoptotic cells have been carefully examined (Kim et al., 1999). Apoptotic cells from heterozygous Id1 transgenic mice were purified by centrifugation on a Ficoll cushion. DNA from these cells was then electrophoresed on agarose gels and the fragmented DNA was excised and purified. This procedure ensured that the isolated DNA was indeed from apoptotic cells. Surprisingly, rearrangement at the TCR b, a, and d loci in the apoptotic cells was found to be as efficient as in transgenic or wild-type viable thymocytes (Kim et al., 1999). Furthermore, sequence analysis of the rearranged segments revealed that the apoptotic cells had as many productive TCR b and a rearrangement events as wild-type thymocytes (Qi and Sun, 2004, submitted). These results thus suggest that the apoptotic thymocytes in the heterozygous transgenic mice have functional pre-TCRs or TCRs and the death of developing thymocytes is not due to lack of signaling from these receptors. Consistently, expression of functional TCR or constitutively active lck cannot rescue the T-cell defect seen in Id1 transgenic mice (Qi and Sun, 2004, submitted; Sun, 2002, unpublished data). The TCR transgenes also failed to improve T-cell development in the HEBbm/bm mice (Barndt et al., 2000). Additional attempts have been made to rescue T-cell development in Id1 transgenic mice by manipulation of gene expression, but they have been largely unsuccessful. The apoptosis and developmental defect (particularly the reduced thymic cellularity) of the Id1 transgenic mice cannot be alleviated by expression of the Bcl-2 transgene, mutation in the FAS or FAS ligand gene, disruption of the p53 gene, or elimination of the IL-2 or IL-7 signaling pathways (Sun, 2001, unpublished data). Similarly, Bcl-2 could not rescue E2A-deficient mice and lack of IL-7 signaling further reduces the number of thymocytes (Kee et al., 2002). Therefore, the death of developing thymocytes remains a mystery, but the role of E proteins in pre-TCR and TCR signaling may shed some light on the apoptosis of Id1 transgenic thymocytes as discussed in the following section. C. E Proteins and Pre-TCR or TCR Signaling The connection between E2A proteins and pre-TCR signaling was discovered through genetic crosses with RAG-deficient mice. Mutation of the E47 exon in the E2A gene or expression of Id1 enables RAG-deficient thymocytes to differentiate to the DP stage (Engel et al., 2001; Kim et al., 2002). This suggests that downregulation of E2A function is one of the steps necessary for


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T cells to progress to the DP stages. Indeed, artificial activation of pre-TCRs by treating RAG-deficient mice with the anti-CD3 antibody reduces the DNA binding activity of E proteins (Engel et al., 2001; Kim et al., 2002), which could be due to induction of Id3 expression by pre-TCR/MAP kinase pathways (Bain et al., 2001; Engel et al., 2001). The level of E2A proteins also decreases with the same treatment (Nie and Sun, 2003, unpublished data). These observations led to the postulation that E proteins create a checkpoint for pre-TCR signaling so that when the signal is strong enough to diminish the overall function of E protein, T-cell development can proceed. However, removal of E proteins is not the only factor needed for development because these RAGdeficient cells, though able to differentiate to the DP stage, do not proliferate as vigorously as wild-type cells. The RAG2 / E47 / mice have slightly more thymocytes than RAG2 / mice but less than E47 / mice (Engel et al., 2001). The number of thymocytes in the RAG1 / Id1tg mice does not increase (Kim et al., 2002). Therefore, the proliferative effect of pre-TCR signaling cannot be substituted by downregulation of E-protein function. The role of E proteins in TCR signaling has been illustrated through examination of the phenotypes of E47- or Id3-deficient mice crossed with TCR transgenic mice. E47 deficiency favors positive selection of either MHC class I–restricted H-Y TCR or class II–restricted AND TCR (Bain et al., 1999). Conversely, disruption of the Id3 gene inhibits negative selection so that DP cells survive in the Id3 / H-Y TCR but not Id3þ/ H-Y-TCR transgenic male mice (Rivera et al., 2000). Id3 mutation also inhibits positive selection and consequently fewer CD4 SP cells are found in Id3 / AND-TCR transgenic mice. Together, it appears that the function of E proteins is to dampen the signals from TCRs either directly by influencing the signaling pathway or indirectly by augmenting the cellular responses to the signal. Consistent with this notion, the Id1 transgenic CD4þ thymocytes, where E-protein function is more completely abolished, display hyperresponsiveness to artificial stimulation through TCR. Wild-type SP thymocytes normally proliferate upon treatment with antibodies against CD3 and CD28, which activate both TCR and the costimulatory receptor. The proliferation of Id1 transgenic CD4þ cells is independent of costimulation by anti-CD28 and is suppressed by an inhibitor of nuclear factor-kB (NF-kB) activation (Qi and Sun, 2004, submitted). Furthermore, when Id1 transgenic mice are crossed with the H-Y or AND-TCR transgenic mice under conditions for positive or negative selection, only DN cells remain in the thymus, even though either heterozygous Id1 or TCR single transgenic mice have 80% of their thymocytes at stages beyond DN (Qi and Sun, 2004, submitted). This phenotype appears to be different from that observed with E47 / mice discussed earlier. However, this may be due to a quantitative difference in the level of E-protein function. With reduced E-protein function, positive selection is enhanced.

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Without E-protein function, signals for positive selection or any signals from the transgenic TCR become so amplified that negative selection or apoptosis occurs. This could explain why massive apoptosis takes place in an Id1 transgenic thymus with the natural TCR repertoire. D. E Proteins and NF-kB Activation To understand the mechanism by which E proteins influence pre-TCR or TCR signaling, the activities of transcription factors typically downstream of these signaling pathways have been examined (Kim et al., 2002). Among AP-1, NFAT, and NF-kB transcription factors, the DNA binding activity of NF-kB is dramatically increased in thymocytes from Id1 transgenic mice and from mice expressing the p22 form of Tal1. A similar observation is also obtained in transgenic mice producing the p42 form of Tal1 (O’Neil et al., 2003). The NF-kB complex primarily consists of homodimers or heterodimers of the c-rel protein (Kim et al., 2002), which is thought to be the protein responsible for constitutive NF-kB activity in lymphocytes. The activation of NF-kB correlates with and increased level of phospho-IkB and IkB kinase (IKK) activity (Kim et al., 2002; O’Neil et al., 2003). However, what mediates the activation of IKK is not known. The significance of these biochemical changes in the thymus was revealed by examination of the phenotypes of Id1 transgenic mice, in which NF-kB activity is altered (Kim et al., 2002). When Id1 transgenic mice were crossed with transgenic mice expressing a constitutively active form of IKKb, which causes activation of NF-kB, T-cell development was further impaired. Conversely, T-cell development was partially rescued with expression of a degradation-resistant form of IkB, which constitutively inhibits NF-kB. Our recent data show that disruption of the c-rel gene has a similar effect (Xiong and Sun, 2003, unpublished data). Therefore, these results suggest that superactivation of NF-kB contributes, at least in part, to the T-cell defect observed in Id1 transgenic mice. This is in agreement with the finding that superactivation of NF-kB results in the differentiation of RAG-deficient cells to the DP stage and the enhancement of positive selection (Voll et al., 2000). NF-kB superactivation can have many ramifications. It may augment preTCR or TCR signaling beyond the tolerable range and cause apoptosis of the thymocytes. Activation of NF-kB could also increase the production of various cytokines that may be harmful for thymocytes. For example, the tumornecrosis factor-a (TNF-a) gene is under the control of NF-kB, whereas TNF-a itself is also a potent activator of NF-kB. We have found that TNF-a and interferon-g (IFN-g) are indeed overproduced in the Id1 transgenic thymus (Cochrane, Yang, and Sun, 2003, unpublished data). Disruption of the gene encoding TNF-a or TNF receptor 1 rescues T-cell development in Id1 transgenic mice to a similar extent as the IkB inhibitor (Xiong and Sun, 2003,


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unpublished data). In contrast, mutation of the IFN-g receptor had no effect on T-cell development in Id1 transgenic mice (Yang and Sun, 2003, unpublished data). However, the causal relationship between NF-kB activation and cytokine production, or vice versa, remains to be determined. IV. HLH Protein and Notch Signaling

The Notch signaling pathway plays crucial roles in lymphocyte development. It is thought to be involved in lineage fate decisions including the B versus T, ab versus gd, and the CD4þ versus CD8þ choices (Allman et al., 2002). However, how Notch executes these decisions is largely unknown. Because E proteins are intimately involved in lymphocyte development, it would be convenient for Notch to use E proteins as downstream effectors. Indeed, the link between Notch signaling and E proteins was found by Ordentlich et al. (1998), who showed that constitutively activated Notch receptors inhibit the transcription activity of E47. Upon further examination, Nie et al. (2003) have found that Notch signaling induces a ubiquitin-mediated and proteasome-dependent degradation of E2A proteins, which explains the apparent reduction of E47 transcriptional activity in the presence of activated Notch. Furthermore, E2A ubiquitination depends on its phosphorylation by Erk kinases and can therefore be regulated by signals controlling Erk activity in a cell type or developmental stage–specific manner. For example, Notch induces E2A degradation in B cells where sustained levels of activated Erk exist but only cause E2A degradation in T cells during transient activation of Erk. We, therefore, formulated a hypothesis with respect to how Notch might choose the T-over B-cell fate in the thymus. If multipotent progenitors arriving in the thymus begin to differentiate along the B lineage, Notch signals would cause E2A degradation in the presence of high levels of Erk in B cells and differentiation would be aborted. On the other hand, if they become T cells, which tightly regulate their Erk activity, E2A proteins would be stable and available for promoting their differentiation. This hypothesis helps explain why the Notch1-deficient thymus permits B-cell development but awaits vigorous proof from genetic and biochemical studies. Notch-induced E2A degradation may also occur during T-cell development because the level of E2A proteins decreases progressively at DP and SP stages. These stages follow pre-TCR and TCR signaling, respectively, which results in Erk activation. Downregulation of E2A levels may be important for the differentiation to DP and SP stages. Another group of bHLH proteins involved in Notch signaling is the HES protein family. HES1 is the best-known target gene of Notch signaling (Jarriault et al., 1995). Upon ligand binding to Notch receptors, their intracellular domains translocate into the nucleus and form complexes with RBP-Jk/

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CBF1, which then bind to the promoter of HES1 and activate HES1 transcription (Tamura et al., 1995). Although the phenotypes of Notch1- and RBP-Jk–deficient mice are similar (Han et al., 2002; MacDonald et al., 2001; Radtke et al., 1999), they are different from that of the HES1-deficient mice (Tomita et al., 1999). Because mutation of HES1 causes embryonic lethality, lymphopoiesis in the mutant mice has been studied by examination of lymphotes generated after transplanting fetal liver cells into RAG-deficient recipients. HES1-deficient cells fail to expand at the CD44þ CD25þ (DN2) stage, even though the small number of cells present is able to undergo TCR rearrangement and differentiation. Unlike Notch1 or RBP-Jk deficiency, HES1-deficient cells do not differentiate into B cells. It is not clear how HES1 functions to facilitate the expansion of developing T cells, but it is known that HES1 can bind to N-boxes as homodimers and repress transcription (Kageyama and Ohtsuka, 1999). HES1 also forms heterodimers with E proteins, which is thought to inhibit their function (Sasai et al., 1992). Curiously, this phenotype of reduced thymic cellularity is also seen in Id1 and Tal1 transgenic mice or HEB-dominant negative mutant mice, as well as in E2A- or HEB-deficient mice, albeit to a lesser extent (see Section III.A). Although apoptosis of the developing T cells contributes to the loss of thymocytes, the inability to proliferate at the DN2 stage could explain the extremely small number of DN2 cells detected in these mice. If E proteins and HES1 share similar functions in the proliferation of thymocytes, a new mechanism must be invoked by which E protein and HES1 heterodimers act together either as transcriptional activators or as transcriptional repressors, a possibility worth testing. V. HLH Proteins and Leukemia

Disruption of the E2A gene or inhibition of E-protein function by Id or Tal1 proteins in mice leads to the development of T-cell lymphomas in the thymus at the age of 3–6 months (Bain et al., 1997a; Kelliher et al., 1996; Kim et al., 1999; Morrow et al., 1999; Yan et al., 1997). These data establish that E proteins act as tumor suppressors and are consistent with the findings that E2A overexpression arrests cell cycle progressions and that Id1 overexpression is found in numerous types of cancers (see Section I.D). The aberrant expression of the Tal1 gene is found in 70% of the human childhood lymphoblastic leukemias (Baer, 1993). Tal1 expression inhibits the function of E proteins and enables the proliferation of T cells (Park and Sun, 1998; Park et al., 1999). The surface marker phenotypes of the E-protein–deficient lymphomas vary from tumor to tumor, but most of them express either CD4 or CD8. This suggests that either tumors arise during the course of differentiation or tumor cells continue to differentiate after transformation. It is, however, remarkable that


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E-protein–deficient mice are initially impaired in T-cell development and have varying degrees of reduced thymic cellularity. Over the 3- to 6-month period before the typical onset of T-cell lymphoma, additional mutations must have accumulated in the surviving thymocytes to transform them into cancer cells. The mechanism by which loss of E-protein function leads to T-cell lymphoma is poorly understood. Although E2A is shown to regulate the expression of cyclin-dependent kinase inhibitor, p21CIP, this cannot be the entire reason because disruption of the p21 gene is not lymphomagenic (Deng et al., 1995). The c-myc gene is amplified in E2A-deficient tumors, but it is not clear whether this is the primary cause for the tumor or is secondary to the uncontrolled growth of tumor cells (Bain et al., 1997a). E47 has also been shown to activate the expression of a number of cell cycle regulators in lymphoid cell lines, but this has not been verified in primary lymphocytes (Zhao et al., 2001). However, as discussed in the previous section, E proteins appear to negatively influence pre-TCR or TCR signaling, whereas inhibition of E-protein activities allows a functional amplification of signals. In fact, Id1 transgenic thymocytes undergo costimulation-independent proliferation in vitro. Therefore, reduced levels of E-protein activity perhaps potentiate the cells to proliferate. Normally, the default outcome for this higher tendency of proliferation is apoptosis of the thymocytes. However, if the cells manage to survive as a result of additional genetic mutations or epigenetic changes in gene expression, cells can undergo cancerous growth. If this is the case, it will be interesting to determine whether antiapoptotic genes are overexpressed in the tumor cells. This hypothesis would help explain the apparently contradicting phenomenon in these E-protein–deficient mice, that is, they are lymphopenic at early ages but develop lymphoma later. Interestingly, expression of a constitutively active form of Notch1 or Notch3 also causes T-cell lymphoma, though more rapidly than E-protein–deficient mice (Bellavia et al., 2000; Pear et al., 1996; Pui et al., 1999). Notch signaling may cause alterations in multiple cellular processes, but Notch-induced degradation of E proteins could be one of the downstream events involved in lymphomagenesis. Notch requires an intact pre-TCR signaling pathway to induce lymphoma, but E-protein deficiency does not (Bellavia et al., 2002; Engel and Murre, 2002). However, this does not mean that they act through different mechanisms. Because Notch causes E2A degradation only in the presence of active Erk, pre-TCR signaling may result in Erk activation, thereby allowing Notch to induce E2A ubiquitination and degradation. This mechanism would also be applicable to nonlymphoid tissues where both constitutive Notch signaling and loss of E-protein function are found to be involved in tumorigenesis, such as in the formation of breast cancer (Callahan and Raafat, 2001; Lin et al., 2000).

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Fig 2 Diverse functions of E proteins in B- and T-cell development. B- or T-cell development is depicted with a block colored with a gradient of green or yellow, symbolizing the maturity of the cells. Key events during the differentiation are marked on the top. The positive effects are pointed out with arrows and the negative effects are shown with bars. The multiple steps of pre–T-cell receptor (TCR) and TCR signaling are illustrated with gray arrows with associated cellular events labeled. The outcomes of the signaling events are indicated with single letter-coded circles, as indicated at the bottom of the figure. Because Id3 has a positive role in B-cell receptor (BCR)– dependent proliferation, E proteins are inferred to negatively influence the proliferation as shown with a broken line. X is placed upstream of IKK to mean that unknown factors are involved in IKK activation in Id1 transgenic mice. Id, Tal1, and Notch signaling are three groups of factors that inhibit the activities of E proteins and thus have opposite effects on the differentiation processes compared to E proteins. (See Color Insert.)


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VI. Conclusions

It is now well established that E proteins play multiple roles during lymphocyte development, partly by acting as ‘‘yin’’ and ‘‘yang’’ in antigen receptor signaling as illustrated in (Fig. 2). On one hand, E proteins are instrumental in the production of premature and mature antigen receptors, which are crucial driving forces of lymphocyte differentiation. On the other hand, E proteins are also responsible for gauging the signaling strength of TCRs to ensure the survival of developing T lymphocytes. Whether E proteins play similar roles in BCR signaling remains to be determined. Furthermore, although the roles of E proteins in later stages of lymphocyte development are better understood, the molecular and cellular events occurring at the earliest developmental block in both E-protein–deficient B and T cells are elusive. The quest for understanding these early events will be aided by further information about the differentiation processes of early lymphoid progenitors. Key findings may also come from the identification of E-protein target genes, which influence the overall well-being of developing cells but whose expression is not necessarily restricted to the lymphoid system. One outstanding question is how E-protein deficiency leads to the dramatic reduction in the numbers of B and T cells even at very early stages. The connection between Notch signaling pathways and E-protein function has also added another layer of complexity but will shed light on the operation of the overall differentiation program controlled by these regulators.

Acknowledgments I wish to thank Drs. Paul Kincade and Scott Perry for critical reading of the manuscript and members of my laboratory for discussion and providing unpublished data. I also acknowledge Dr. F. W. Kincade and Y. Zhuang for communicating unpublished data.

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Xia, Y., Brown, L., Yang, C. Y. C., Tsan, J. T., Siciliano, M. J., Espinosa, R., Le Beau, M. M., and Baer, R. (1991). TAL2, a helix-loop-helix gene activated by the t(7;9)(q34;q32) translocation in human T cell leukemia. Proc. Natl. Acad. Sci. USA 88, 11416–11420. Xu, M., Nie, L., Kim, S. H., and Sun, X. H. (2003). STAT5-induced Id-1 transcription involves recruitment of HDAC1 and deacetylation of C/EBPbeta. EMBO J. 22, 893–904. Xu, Y., Davidson, L., Alt, F. W., and Baltimore, D. (1996). Function of the pre-T-cell receptor alpha chain in T-cell development and allelic exclusion at the T-cell receptor beta locus. Proc. Natl. Acad. Sci. USA 93, 2169–2173. Yan, W., Young, A. Z., Soares, V. C., Kelley, R., Benezra, R., and Zhuang, Y. (1997). High incidence of T-cell tumors in E2A-null mice and E2A/Id1 double-knockout mice. Mol. Cell. Biol. 17, 7317–7327. Yokota, Y., Mansouri, A., Mori, S., Sugawara, S., Adachi, S., Nishikawa, S., and Gruss, P. (1999). Development of peripheral lymphoid organs and natural killer cells depends on the helix-loophelix inhibitor Id2. Nature 397, 702–706. Yokota, T., Kouro, T., Hirose, J., Igarashi, H., Garrett, K. P., Gregory, S. C., Sakaguchi, N., Owen, J. J. T., and Kincade, P. W. (2003). Unique properties of fetal lymphoid progenitors identified according to RAG1 gene expression. Immunity 19, 365–375. Yucel, R., Karsunky, H., Klein-Hitpass, L., and Moroy, T. (2003). The transcriptional repressor Gfi1 affects development of early, uncommitted c-Kitþ T cell progenitors and CD4/CD8 lineage decision in the thymus. J. Exp. Med. 197, 831–844. Zhao, F., Vilardi, A., Neely, R. J., and Choi, J. K. (2001). Promotion of cell cycle progression by basic helix-loop-helix E2A. Mol. Cell. Biol. 21, 6346–6357. Zhuang, Y., Barndt, R. J., Pan, L., Kelley, R., and Dai, M. (1998). Functional replacement of the mouse E2A gene with a human HEB cDNA. Mol. Cell. Biol. 18, 3340–3349. Zhuang, Y., Cheng, P., and Weintraub, H. (1996). B-lymphocyte development is regulated by the combined dosage of three basic helix-loop-helix genes, E2A, E2-2, and HEB. Mol. Cell. Biol. 16, 2898–2905. Zhuang, Y., Soriano, P., and Weintraub, H. (1994). The helix-loop-helix gene E2A is required for B cell differentiation. Cell 79, 875–884.



Customized Antigens for Desensitizing Allergic Patients ´ TIMA FERREIRA, MICHAEL WALLNER, AND JOSEF THALHAMER FA University of Salzburg, Department of Molecular Biology Salzburg, Austria

I. Introduction

The most effective allergy treatment is one that specifically and with maximum safety cures clinical symptoms caused by allergens in any given patient. In this context, allergen-specific immunotherapy (SIT) offers the best possibility of accomplishing this goal. The process is highly specific because the treatment is targeted at those allergens that the patient and the physician identified as the responsible ones (Frew, 2003). In its conventional form, SIT involves repeated administration of whole allergen extracts to patients displaying immunoglobulin E(IgE)-mediated allergic symptoms and inflammatory reactions caused by natural exposure to allergens. The drawback of this approach is that extracts are complex mixtures that are difficult to standardize. In addition, systemic administration of active allergens can cause severe IgEmediated side effects during the treatment and therapeutic effective doses often cannot be achieved because of nonstandardized extracts or side effects. For the best performance of this tailor-made type of treatment, the first crucial step is the exact identification of the molecules causing the allergic reaction, that is, a clear diagnosis establishing the association between the disease manifestation and the IgE-mediated immune reactions. This can be accomplished with a molecule-based diagnosis. Knowledge of the pattern of IgE reactivity at a molecular level allows the formulation of vaccines containing only the allergens to which the patient is allergic, consisting, for example, of cocktails of pure and standardized recombinant allergens instead of whole extracts. Moreover, the selected recombinant allergens can be modified to reduce the risk of IgE-mediated side effects. In this way, higher doses of allergen can be administered to allergic patients increasing the efficacy of the treatment without compromising safety. Genetic immunization is an attractive alternative for SIT using protein antigens. It has been shown that intramuscular or intradermal injection of plasmid DNA encoding clinically relevant allergens can induce long-lasting immune responses with a Th1 bias and promote the formation of interferon-g (IFN-g)–producing CD4þT cells. In addition, immunization with plasmid DNA encoding modified allergen genes would fulfill the requirements of efficacy and safety in SIT. 79 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2776/04 $35.00

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The increasing knowledge of the mechanisms operating in Th1 and Th2 types of immune responses opened various possibilities to modulate an allergic (Th2-type) response. The working hypothesis is that Th1-biased stimuli could prevent the development of an allergic response and might even convert established Th2 responses. Therefore, designed antigens in combination with immunomodulatory adjuvants form the basis for novel and optimized allergen vaccines. II. Allergy Diagnosis and Immunotherapy: From Extracts to Allergen Arrays and Engineered Vaccines

So far only allergen extracts are routinely applied for diagnostic and therapeutic purposes. These extracts are difficult to standardize regarding their allergen content; several allergens might be underrepresented because of degradation, whereas other nonallergenic components are present and there might be even contamination with allergens from other sources. Thus, extractbased diagnosis goes in the direction of identifying allergenic sources and not individual allergenic molecules, which does not allow the discrimination between co-sensitization and cross-sensitization in patients showing adverse reactions to more than one allergenic source (Pauli, 2000). The development of an allergic disease always requires contact with a sensitizing agent, which does not necessarily have to be the elicitor of the allergic symptoms. Allergen cross-reactivity occurs when IgE antibodies originally raised against one allergen bind or recognize a similar protein from another source (Aalberse et al., 2001). The interaction with such homologous proteins can then trigger allergic reactions or can be completely irrelevant for the patient. The inadequacy of diagnosis with extracts in cases of cross-reactivity is clearly demonstrated by a survey of mite-allergic Orthodox Jews. Because of strict adherence to kosher dietary laws, the consumption of shellfish is prohibited in this population. Fernandes et al. (2003) showed that IgE antibodies of these mite-allergic individuals cross-reacted with shrimp tropomysin Pen a 1, despite that direct contact with this aliment did not occur. When tested with single allergenic components, such tropomyosin-based cross-reactivity could be easily predicted. Furthermore, with allergen extracts there is always the risk of contamination as seen for mite allergens in animal hair dander extracts producing false-positive results on skin tests (van der Veen et al., 1996). The consequences of such incorrect diagnoses can be taken to the next level, considering cases of indirect sensitization of mite-allergics patients who developed positive radioallergosorbent test (RAST) signals for shrimp and snails after receiving specific immunotherapy for dust mite allergy (van Ree et al., 1996). Induction of new IgE specificities has also been reported for birch- and grass pollen-allergic individuals (Ball et al., 1999b; Moverare et al., 2002a).

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Sixty-five percent of patients treated with birch pollen rush immunotherapy developed low levels of IgE antibodies toward additional birch pollen allergens. Among these newly recognized allergens, the highly cross-reactive Bet v 2 and Bet v 4 proteins could represent a risk of clinical consequences for the patient. All these findings demonstrate the unpredictability and shortcomings of diagnosis and immunotherapy using allergen extracts. A. Molecule-Based Diagnosis: Clustering of IgE Reactivity Patterns A question that arises is how far the allergy diagnosis can be simplified as a result of cross-reactivity phenomena. A study by van Ree et al. (1999) demonstrated that a combination of birch, hazel, and alder pollen extracts compared to birch or alder extracts alone was equally efficient in diagnosis. Also, a combination of Bet v 1 and Bet v 2 allergens covered around 94% of the tree-pollen–positive sera used. The remaining few patients were suspected of having IgE against either other minor birch pollen allergens or cross-reactive carbohydrate determinants (CCDs) (van Ree et al., 1999). Even when using purified allergens, false-positive results may occur because of CCDs present on plant and invertebrate proteins. A sensitization toward CCDs usually occurs via pollen or insect stings and results in a wide pattern of cross-reactivity to various kinds of food. Anti–carbohydrate IgE antibodies seem to have poor biological activity, and until now their clinical relevance could not be proven. Nevertheless, anti–CCD IgE antibodies frequently produce positive reactions in in vitro tests. To minimize these false-positive results, the use of recombinant allergens seems obligatory. However, depending on their way of production (e.g., Escherichia coli, Pichia pastoris, insect cells, and tobacco plants), recombinant allergens might also show different forms of glycosylation. All allergens produced in prokaryotic cells are not glycosylated, but because of problems regarding protein folding, eukaryotic expression systems are increasingly being employed for the production of recombinant allergens. The glycosylation of these recombinant allergens does not often correlate with their naturally occurring counterparts. In this way, incorrectly glycosylated recombinant allergens could also react with anti– carbohydrate IgE antibodies and produce false-positive results. In terms of immunotherapy with recombinant allergens, the role of glycans for protein folding and their effects on the human immune system need to be further evaluated (Mari, 2002; Mari et al., 1999; van Ree, 2002). The precise knowledge of cross-reactivity patterns offers the possibility to define marker allergens indicative of certain linked allergic diseases and to develop strategies for the direct treatment of allergies (Kazemi-Shirazi et al., 2002). Because many allergens have become available as recombinants, few representative allergens covering most of the IgE epitopes could be selected

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for diagnostic purposes. If a patient mounts IgE antibodies against Bet v 1, that individual will likely develop symptoms upon contact with other allergenic sources harboring the corresponding cross-reactive allergens either in pollen or in foods (Ebner et al., 1996). This notion holds true for many cross-reactive allergens (Aalberse et al., 2001; Sicherer, 2001; Vieths et al., 2002). Therefore, such marker allergens could help redefine the current concept of allergy diagnosis, so they not only are useful for the diagnosis of existing allergies, but also might help predict potential sensitization to multiple allergenic sources. For timothy grass pollen allergy, a panel of allergens, either recombinant or natural, can substitute allergen extracts used in diagnosis. A limited number of allergen molecules were sufficient to diagnose sensitization to this allergenic source in 99% of all patients tested. Moreover, a sensitization profile could be determined, identifying and quantifying the amount of IgE bound to the particular allergens (Valenta et al., 1999a). A study comparing the IgE binding pattern of timothy grass pollen allergens in different populations had similar results (Laffer et al., 1996). A multipopulation study using molecule-based diagnosis was performed for the selected recombinant birch pollen allergens, rBet v 1, rBet v 2, and rBet v 4. The study revealed different population-dependent recognition patterns of these allergens among birch pollen–allergic patients (Moverare et al., 2002b). With classic diagnostic measures, such patterns could not be determined, which implies that birch pollen–allergic individuals not yet sensitized to Bet v 1 could, for instance, develop such a sensitization upon SIT treatment. Another survey among allergic individuals from the urban areas of Zimbabwe was performed using recombinant timothy grass pollen and dust mite allergens. The allergen-recognition patterns were compared with the ones obtained from patients in central Europe, and striking differences were observed. This profile could be explained by a primary sensitization to Bermuda grass and not to timothy grass pollen. The unusual recognition profile toward mite allergens might also be due to different sensitization with cross-reactive allergens compared to the European population. Interesting to remark is the existence of African patients reactive to birch pollen extracts because of a sensitization to profilin (Westritschnig et al., 2003). All these findings point out the necessity of molecule-based diagnosis for allergies. Developments in this direction have already been done with the introduction of allergy chips in a microarray format. In one study, 94 purified recombinant or natural allergens were spotted robotically in triplicates on preactivated glass slides and incubated with patients’ sera. The results were verified by nitrocellulose blots and skin prick tests, and most of the allergens spotted on the chips gave results comparable to RAST-based results. The advantages of such a system are obvious: Only some hundred picograms


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of protein is needed per slide, the system is predestined for high throughput, and allergen-bound IgE antibodies can be quantified (Hiller et al., 2002). With the array technology, it will be further possible to detect different immunoglobun classes bound to the immobilized allergens, which could be useful to analyze the course of an allergic disease over time, enabling a clinician to monitor allergy treatment (Harwanegg et al., 2003). For the establishment of molecule-based allergy diagnosis on a large scale, well-characterized allergens have to be available in large amounts, with low or no batch-to-batch variations. The use of recombinant allergens offers the best prospect for rational and accurate allergy diagnosis. Over the past few years, allergens from various allergenic sources including pollen, food, house dust mite, cockroach, animal dander, insect venom, latex, molds, and fungi have been cloned and characterized and are now available as recombinant proteins (Andersson and Lidholm, 2003; Arruda et al., 2001; Chapman and Wood, 2001; Kurup et al., 2002; Lorenz et al., 2001; Muller, 2002; Sussman et al., 2002; Thomas et al., 2002). Thus, molecule-based allergy diagnosis should be gaining importance rapidly. III. Novel Antigen Preparations to Improve SIT

Modification of allergens to improve conventional SIT should aim at the production of molecules with reduced IgE binding epitopes (hypoallergens) while preserving structural motifs necessary for T-cell recognition (T-cell epitopes) and for induction of IgG antibodies reactive with the natural allergen (blocking antibodies). The uptake of allergens by antigen-presenting cells (APCs) is mediated and facilitated by the interaction of the allergen with specific IgE (Maurer et al., 1995; van der Heijden et al., 1993) and leads to higher Th2 cytokines and IgE production (Akdis et al., 1998). Modified allergens lacking IgE binding (hypoallergens) might avoid these pathways and preferentially target APCs that use phagocytosis or pinocytosis for antigen uptake (e.g., monocytes, macrophages, and dendritic cells [DCs]). This, in turn, induces a balanced Th0- or Th1-like cytokine production by T cells and low IgE and high IgG production by B cells (Fig. 1). The presence of intact T-cell epitopes on hypoallergens would also target T cells, allowing administration of higher doses to induce tolerance of allergen-specific T cells and alteration of cytokine production toward a Th1-like pattern. In this way, vaccine preparations consisting of hypoallergens could replace natural extracts and increase the efficacy and safety of SIT. Other promising approaches include protein fusions and synthetic peptides. Synthetic peptides containing T-cell epitopes were shown in clinical studies to be safe and to induce allergenspecific T-cell hyporesponsiveness. (Fig. 2) gives an overview of designed allergens to be used in immunotherapy of allergic diseases.

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Fig 1 Possible advantages to the use of hypoallergens for specific immunotherapy. Low density of immunoglobulin E (IgE) epitopes would not trigger IgE-mediated side effects upon injection and bypass IgE-mediated allergen presentation, which leads to a Th2-biased immune response. (See Color Insert.)

A. Hypoallergens The idea of modifying allergens to decrease their allergenicity (IgE binding activity) while preserving their antigenicity (T-cell reactivity) is not new. In fact, earlier studies showed that chemical modification of allergens (e.g., polyethylene glycol [PEG], glutaraldehyde, formaldehyde, etc,) could be used to abrogate IgE binding. Some of these preparations are available in clinical practice (Akdis and Blaser, 2000). More recent developments include chemical modification of allergens by conjugation with synthetic oligonucleotides containing immunostimulatory sequences from bacterial DNA (allergenISS conjugates). Allergen-ISS conjugates are promising therapeutics. In clinical trials, an Amb a 1-ISS preparation was shown to be safe and effective for treating ragweed-allergic patients. Recombinant DNA techniques offer the unique possibility of arbitrarily altering the nucleotide sequence of a gene to produce novel ‘‘mutant’’ proteins or genetically engineered proteins displaying altered properties. This is particularly interesting for the production of allergen mutants with predefined properties regarding allergenicity and antigenicity. Akdis et al. (1998) showed that bee venom phospholipase A2 (PLA)


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Fig 2 Cartoon representation of customized allergens to improve specific immunotherapy. Examples of naturally occurring hypoallergens are scarce and cannot be widely explored. Modification of allergens can be accomplished by chemical means and by genetic engineering approaches. Synthetic peptides include mimotopes, B-cell epitope–derived nonanaphylactic peptides, and T-cell epitope–containing peptides. (See Color Insert.)

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preparations lacking the native conformation and antibody binding activity were exclusively presented by monocytes and induced a Th1-biased cytokine profile, leading to IgG4 production by B cells. In contrast, folded PLA with full antibody binding activity was processed and presented by B cells, stimulated Th2-like cytokines, and induced IgE antibodies. Thus, the three-dimensional structure of an antigen and its recognition by different APCs seem to be crucial aspects in the development of distinct patterns of T-cell cytokines. These findings give further support to the use of hypoallergens in SIT. 1. Chemically Modified Allergens a. Allergoids. The first attempts of chemically modify allergens and allergen extracts for use in SIT were preparations of aluminium hydroxide (alum)– precipitated aeroallergens or alum-precipitated pyridine extracts with the aim to retain allergens at the injection site. Other modifications such as emulsions of aqueous allergen extracts in mineral oil were also tested but were abandoned because of lack of proof of efficacy and concerns about noxious side effects. Further, urea-denatured, PEG conjugated, and poly d-glutamic acid:d-lysine– linked allergens were investigated for the potential to suppress IgE antibody production. However, clinical trials revealed that the specific IgE levels in these patients were not greatly diminished and specific IgG antibodies raised extensively (Akdis and Blaser, 2000; Middleton et al., 1993). In other approaches, oligomers were produced by chemical modification of allergens with glutaraldehyde or paraformaldehyde. These chemical modifications seem either to destroy or to mask structural B-cell epitopes, resulting in significantly reduced IgE binding activity but retained immunogenicity. These allergoids seem to be better tolerated by patients, because higher doses of antigen can be administered during SIT and consequently, fewer injections are necessary for successful treatment (Akdis and Blaser, 2000; Middleton et al., 1993). Another possibility of creating chemically modified allergens with reduced allergenic potential is carbamylation. Treating proteins with potassium cyanate at neutral pH levels leads to a transformation of the e-amino group of lysines into ureido groups. Thus, the native monomeric character of the allergens is preserved. The advantage is that monomeric allergoids can be administered to the organism not only subcutaneously, but also through a mucosal pathway. These allergoids can be produced either from allergenic extracts or from purified single allergens. In animal models, the allergoids induced IgG antibodies that recognized the native allergens (Mistrello et al., 1996). Commercially available allergoids are produced by chemical modification of allergen extracts with aldehydes and are available only for a limited panel of allergens. These allergoid preparations consist of bulky macromolecules with undefined structure and sequence. Even though the standardization of


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allergen extracts has improved, the batch-to-batch reproducibility still remains suboptimal. A better approach would probably rely on the use of recombinant allergens for the production of allergoids. b. Maleylated Allergens. Many proteins are known to become ligands for scavenger receptors (SRs) when chemically modified to enhance their negative charges (Abraham et al., 1995). SRs are members of the so-called patternrecognition receptors (PRRs), which are expressed by APCs and are involved in the recognition of foreign ligands during early phases of the immune response (Gordon, 2002). SRs bind to various polyanionic ligands (e.g., oxidized low-density lipoprotein, maleylated proteins, polyguanylic acid, and fucoidin) and deliver them into the endolysosomal pathway. The enhancement of negative charges in proteins can be accomplished by alteration of the e-amino group of lysine residues with acetic acid or maleic anhydrides. It has been shown that maleylation of protein antigens converts them into SR ligands and their delivery through SRs results in increased immunogenicity with a dominant Th1-type immune response (Abraham et al., 1995; Singh et al., 1998). Therefore, specific targeting of allergens to SR represents an attractive approach to modulate allergen-specific immune responses. Maleylation of whole Orchard grass pollen proteins was reported to decrease IgE binding activity tested by immunoblots and to reduce skin reactivity (Cirkovic et al., 1999). A maleylated form of shrimp tropomyosin, a major cross-reacting crustacean food allergen, displayed reduced allergenicity as compared to the native counterpart (Rajagopal et al., 2000). Analysis of the cytokine profiles after stimulation of splenocytes isolated from Balb/c mice immunized with maleylated tropomyosin showed a dominant Th1 immune response. Similar results were obtained by targeting a Der p 1 peptide to macrophages via SR (Bhatia et al., 2002). In addition, SR targeting of the Der p 1 peptide and elicitation of Th1-dominant responses was also accomplished by simple coadsorption of the peptide with maleylated poly-l-lysine on alum (Bhatia et al., 2002). The possibility of coadsorption would be particularly interesting for the formulation of multivaccines for immunotherapy of patients allergic to several allergens. c. Allergen-ISS Conjugates (AIC). One of the lessons from DNA immunization (see Section IV) was that certain immunostimulatory sequences (ISSs) of bacterial DNA can deviate the normally occurring Th2 response to allergens toward a nonallergic Th1 response. Taking advantage of this knowledge, oligodeoxynucleotides (ODNs) containing ISS molecules (ISS-ODN) were conjugated to allergens and led to the design of a novel type of modified allergen (allergen-ISS conjugate [AIC]) for use in SIT (Tighe et al., 2000). The immunostimulatory property of bacterial DNA was discovered nearly 20 years ago (Tokunaga et al., 1984) and showed that bacterial DNA differs from that of mammalians in the frequency of CpG dinucleotides, which are

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avoided in mammals (CpG suppression) and not methylated in bacteria. Studies with synthetic ODNs have revealed a hexameric consensus sequence for immunostimulatory CpG motifs consisting of a central CpG dinucleotide flanked by two purines at the 50 end and two pyrimidines at the 30 end (50 PuPuCGPyPy-30 ). It turned out that both base-sequence and backbone chemistry influence the immunostimulatory efficacy of individual CpG motifs, which also exhibit marked species specificity (Krieg, 2002). i. Recognition and Signal Transduction Induced by Immunostimulatory CpG DNA. The cellular events of activation of APCs by CpG DNA strikingly parallel those achieved with other microbial products. These substances are recognized by APCs via PRRs. PRRs were developed early in evolution and represent a mechanism of the innate immune system to signal ‘‘danger’’ based on structural surface characteristics that are shared by a variety of infectious agents but are absent on host cells. CpG-mediated intracellular signaling requires endocytosis of CpG DNA/toll-like receptor (TLR) complexes and acidification of endosomes. Signal transduction involves the myeloid differentiation factor MyD88, IRAK, and TRAF6, activation of IkB kinase, mitogenactivated protein kinases, the stress kinases N-terminal c-Jun kinases JNK1/2, and p38, and finally results in transcriptional activation of multiple genes involving ATF-2, AP-1 and nuclear factor-kB (NF-kB). Many immunoregulatory genes contain NF-kB–responsive elements in their promoter regions and inhibition of NF-kB abrogates CpG-mediated immune cell activation (Takeda et al., 2003). ii. Cellular Targets of CpG Action. Several immune cell species have been identified that respond to CpG DNA. These include B and T cells, natural killer (NK) cells, and APCs (dendritic cells and macrophages). The direct action on APCs is probably most important for the immunostimulatory effect of CpG DNA. These cells, when encountering CpG DNA, undergo dramatic physiological changes affecting both the immunoregulatory functions of the APC themselves and via secretion of cytokines the activation status of other immune cells. NK cells: Activated NK cells play an important role in the early phase of an immune reaction. These cells provide the majority of IFN-g before this cytokine is supplied in relevant concentrations by activated T cells. However, purified NK cells appear not to be activated directly by CpG DNA but are potently stimulated by type 1 interferons, IL-12, and tumor necrosis factor-a (TNF-a), which are secreted by macrophages and dendritic cells (DCs) in response to CpG DNA (Ballas et al., 1996). Macrophages: Macrophages are induced by CpG DNA to secrete a panel of cytokines, in particular type 1 interferons (IFN-a/b), IL-1b, TNF-a, IL-6, and IL-12. One of the features of interferons is to enhance the cytolytic activity of NK cells and in humans facilitate Th1 cell development by


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promoting the expression of IL-12 receptor on these cells. IL-12 is a key cytokine for the development of cell-mediated immunity and the development of Th1-type immune reactions in general. In addition to these effects, CpG DNA increases the expression of several surface molecules on macrophages, such as major histocompatibility complex class I (MHC-I), intercellular adhesion molecule 1 (ICAM-1), CD40, or CD80/86. These molecules are involved in antigen presentation and activation of lymphocytes. The alterations triggered by CpG oligonucleotides have been shown to persist for a prolonged time (Kobayashi et al., 1999). DCs: DCs are unique in their ability to activate naive T lymphocytes, and therefore, play a key role as APC for the induction of a primary immune response. Similar to macrophages, bone marrow–derived DCs respond directly to CpG DNA by secretion of TNF-a, IL-6, and IL-12 and with increased levels of CD40, CD86, and MHC-II on their surface (Sparwasser et al., 1998). CpG motifs have been found that induce IFN-g and IFN-a production in human cells, and functionally distinct types of CpG motifs have been designed to specifically address immunocompetent cells (Krieg, 2002). T cells: The Th1-biased danger signal inherent to CpG DNA and acting on the innate immune system also has a fundamental influence on the Th1/Th2 balance. IFN-g promotes Th1 cells by enhancing IL-12 production by macrophages and expression of IL-12 receptors on CD4þ T cells. IL-12, in turn, mediates the differentiation of Th1 cells by activation of STAT4. Simultaneously, IFN-g suppresses the development of Th2 cells. Although many processes that occur after activation with various microbial products are strikingly similar, CpG DNA may be superior over other adjuvants because of its ability to trigger the release of large amounts of IL-12 (more than lipopolysaccharide) [LPS] [Cowdery et al., 1999]). Thus, comparing an adjuvant’s potential to induce IL-12 versus TNF-a (and IL-1) might provide a means for evaluating its applicability. iii. Preclinical Studies with Allergen-ISS Conjugates. Soon after the discovery of the Th1-inducing capacity of ISS-containing CpG motifs, experiments with model allergens proved that systemic or mucosal application of CpG-ODN led to reduction of IL-5 and inhibition of airway hyperesponsiveness and eosinophilia of the airway, lung parenchyma, and blood. The effect on T-cell cytokine production was proven to be indirect via stimulating APCs and NK cells to generate IL-12 and interferons. A single dose of CpG application was as effective as multiple injections of corticosteroids, but only CpG treatment was able to induce allergen-specific IFN-g production and balance a Th2-type response (Broide et al., 1998; Spiegelberg et al., 1998). The effect of CpG motifs was also tested in murine models using CpG-ODN coadministered with antigen. Treatment of previously sensitized animals with CpG-ODN prevented allergen-induced airway inflammation (Kline

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et al., 1998). Using a protective approach with CpG-ODN coinjected with the major birch pollen allergen (Jahn-Schmid et al., 1999) and ragweed allergen (Sur et al., 1999), which was applied 48 hours before allergen challenge, confirmed the Th1-modulatory capacity of CpG motifs. The effects of CpGODN were sustained for several weeks and the reaction after boosting indicated a strong Th1-biased memory response. Although coinjection of allergens with CpG-ODN also induces Th1 responses, chemical coupling of the allergen to CpG-ODN proved superior, as demonstrated with the ragweed allergen Amb a 1 (Tighe et al., 2000). Synthetic phosphorothioate (but not phosphodiester)-ODN proved more effective concerning the desired Th1-inducing capacity (Jahn-Schmid et al., 1999; Parronchi et al., 1999). CpG-ODN conjugated to allergen can mask allergenic determinants, thus interfering with cross-linking of Fc IgE receptors on mast cells and basophils. Coupling the allergen to ISS-ODN also was superior to a mixture of CpGODN and allergen with respect to the immunogenicity profile. A possible explanation for this is that both the allergen and the CpG motifs are delivered together to the same APC for processing and induction of type 1 cytokine secretion. This proved to be the case since much less ISS-ODN in the form of an AIC was necessary to induce the same IgG2a antibody titer as compared to allergen CpG-ODN mixtures. AIC induced a Th1 response to the allergen, as shown by strong IgG2a and almost no IgE antibody production and antigenspecific IFN-g–secreting spleen cells (in contrast to control mice, which produced IgG1 and IgE antibodies and showed elevated levels of IL-5). Sensitization with allergen and treatment with AIC induced IgG2a antibodies without significantly increasing the IgE titer, and spleen cells from AICtreated mice showed significantly more IFN-g secretion than spleen cells from the control groups, both pointing to a therapeutic potential of AICs (Spiegelberg et al., 2002). The effects of AIC have also been tested in a mouse model of asthma (Shirota et al., 2000). Ovalbumin(OVA)-CpG-ODN conjugates were applied intratracheally and the effects on airway eosinophilia and airway hyperresponsiveness were studied. AIC reduced airway eosinophilia and hyperresponsiveness significantly more effectively than a mixture of OVA and CpG-ODN and the suppressive effect lasted for more than 2 months. In addition, the AIC induced a striking increase in IFN-g secretion by OVA activated lymph node cells and concomitantly decreased the IL-4 and IL-5 secretion, suggesting a switch from a Th2 to a Th1 anti-OVA T-cell response. These results were confirmed in a similar model system with the clinically relevant ragweed allergen Amb a 1 (Santeliz et al., 2002). Furthermore, CpG-ODN inhibited early and late phases of murine-allergic conjunctivitis after sensitization with short ragweed (Magone et al., 2000).


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Whereas the protective capacity of AIC seems very effective under various conditions, therapeutic protocols dealing with balancing ongoing Th2-type responses were not successful in all cases. Prevaccination with CpG-ODN coapplied with the mosquito salivary antigen Aed a 2 inhibited the development of IgE antibodies after sensitization. However, an established Th2-type response was not influenced by administration of CpG-ODN together with recombinant Aed a 2 (Peng et al., 2001). The Th1-modulating capacity of CpG-ODN coupled to or mixed with the allergen obviously also depends on the antigenic and/or immunogenic nature of the allergen and the Th2-stimulating protocol (Hochreiter et al., 2001), and in the case of covaccination of allergen together with CpG-ODN, prepriming with CpG motifs apparently is superior over simultaneous application (Kobayashi et al., 1999). Experiments focused on the mechanisms by which AIC stimulates Th1-type responses show enhanced binding of AIC to DC, compared to native allergen, thereby improving the phagocytosis of the allergen-CpG-ODN conjugate. The enhanced binding was not sequence specific because AIC containing a non–CpG-ODN also bound better to DC than allergen without coupled ISS-DNA. However, the induction of type 1 cytokine secretion by DC occurred only with allergenDNA conjugates that contained CpG-ODN (Shirota et al., 2001). Based on the findings that DCs, like T cells, can be subgrouped into type 1 and type 2 cytokine-secreting cells and that DCs from atopic patients secrete more type 2 cytokines than DCs from nonallergic humans (Caron et al., 2001; Uchida et al., 2001), it may be of interest to determine the secretion profile of DCs from atopic patients after immunotherapy in general and especially after treatment with AIC. iv. Clinical Trials with AIC. The preclinical data obtained in mice and in in vitro studies with human lymphocytes, both indicating the Th1-stimulating potency of CpG motifs (Bohle et al., 1999; Marshall et al., 2001; Tighe et al., 2000), led to the development of an early phase clinical trial with patients suffering from ragweed seasonal allergic rhinitis. AIC with an average of four ISS-ODN conjugated per molecule of Amb a 1 was tested for safety, tolerability, and immune response in comparison to a licensed ragweed allergen extract in a phase I trail (Creticos et al., 2000). This clinical study with a quantitative intradermal procedure indicated that more than 100-fold higher quantities of Amb a 1 in the form of AIC were necessary to induce the same wheal and flare skin test reaction as native ragweed allergen. Preliminary data from early phase I/II clinical trials showed that the Amb a 1–specific IgE response was similar among study subjects who received subcutaneous injections of AIC and those who received placebo injections (Creticos et al., 2001; Dieudonne´ et al., 2001). In contrast, IgG anti–Amb a 1 antibody titers after six weekly injections with AIC appeared comparable to titers measured in sera from individuals who received immunotherapy for more than 1 year with licensed ragweed allergen

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extracts. Additional phase II efficacy studies are underway to assess further the safety, immune response, and efficacy of AIC for immunotherapy of ragweed allergic rhinitis. Concluding, AIC seems to display the required features of modern vaccines for an improved SIT such as a high safety profile and strong immunogenicity. In addition, and in contrast to SIT with allergen extracts, the curative principle can be clearly attributed to the balancing of the allergic Th2-type response by counteracting with Th1-inducing stimuli. Whereas the mechanisms of conventional SIT are still poorly understood and controversially discussed, SIT with AIC uses an adjuvant agent with well-known and defined functional characteristics. However, it must be stated that despite encouraging preliminary data from initial clinical trials, most of the optimistic interpretations still originate from animal experiments. Moreover, most of these data come from experiments with model molecules such as ovalbumin or E. coli b-galactosidase. Both antigens are highly immunogenic and differ from clinically relevant allergens in many aspects. The main potential for harmful side effects of AIC, however, may result from the downstream events of CpG DNA, which are similar to those of other bacterial products, such as LPS. CpG DNA was able to induce septic shock symptoms, and it was found to synergize with LPS in the induction of TNF-a (Cowdery et al., 1996; Sparwasser et al., 1997). With respect to clinical trials, it must be emphasized that overwhelming Th1 immunity with forced application of CPG motifs or treatment with AIC holds the risk of potential promotion of autoimmunity (Segal et al., 1997). Profound knowledge of the immunogenic nature of the selected allergen, the proper choice of AIC dose, and vaccination protocols should help avoid these risks. 2. Naturally Occurring Hypoallergens Complementary DNA (cDNA) cloning of allergens showed that many major allergens are encoded by gene families. Sequence polymorphisms have been described for ragweed Amb a 1 (Griffith et al., 1991), hazel Cor a 1 (Breiteneder et al., 1993; Luttkopf et al., 2002), birch Bet v 1 (Swoboda et al., 1995), group 1 (Au et al., 2002; Chang et al., 1999) and group 5 grass allergens (Muller et al., 1998b; Wurtzen et al., 1999), apple Mal d 1 (Helsper et al., 2002; Son et al., 1999), celery Api g 1 (Hoffmann-Sommergruber et al., 2000), Parietaria Par j 1 (Duro et al., 1997), olive Ole e 1 (Gonzalez et al., 2002), group 1 and 2 dust mite allergens (Smith et al., 2001a, 2001b), latex Hev b 7 (Sowka et al., 1999), and cow dander Bos d 2 (Rautiainen et al., 2001). Despite extensive characterization of isoallergens at the DNA level, very little information is available about their relevance at the level of expressed protein. Analyses at the protein level were performed for Bet v 1 and Mal d 1 isoforms (Helsper et al., 2002;


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Swoboda et al., 1995). The possibility that isoallergen sequences result from cloning artifacts cannot be ruled out, especially those obtained by polymerase chain reaction (PCR) cloning. In fact, this was shown to be the case for one Mal d 1 isoform (Helsper et al., 2002). Because of sequence variations, isoallergens might have different antigenic and/or allergenic activities. Differences in T-cell reactivity of isoforms have been reported for Cor a 1 (Schenk et al., 1994), Bet v 1 (Ferreira et al., 1996), Phl p 5 (Muller et al., 1998b; Wurtzen et al., 1999), and Der p 2 (Hales et al., 2002). Investigation of the IgE binding activity of isoallergens led to the identification of naturally occurring Bet v 1 hypoallergens (Ferreira et al., 1996). Isoforms Bet v 1d, Bet v 1g, and Bet v 11 were found to be highly antigenic in T-cell proliferation assays and low in their allergenic activities in vitro (Ferreira et al., 1996) and in vivo (Arquint et al., 1999). It is possible that the low IgE binding activity of these isoforms resulted from incorrect folding during the recombinant production. However, the crystal structure of the hypoallergenic isoform Bet v 11 was found (Markovic-Housley et al., 2003) and has been shown not to be significantly different from the structure of the high IgE binding isoform Bet v 1a (Gajhede et al., 1996). Thus, the low IgE binding activity of certain isoforms is not due to problems in the recombinant production leading to unfolded proteins. Such well-characterized molecules would be excellent candidates for specific immunotherapy. However, naturally occurring hypoallergens have not been identified for other allergen families. Instead, genetic engineering has been widely used to generate hypoallergens. 3. Engineered Hypoallergens Genetic engineering involves the targeted modification of a protein to alter its function or properties in a predictable manner. This requires the complete understanding of the relationship between structure and function/properties for precise and effective manipulation. Genes can be altered by changing specific base pairs (mutated gene), by introducing a new piece of DNA into the existing DNA molecule (chimeric or hybrid gene), by fusions (fused genes), and by deletions (truncated gene or fragments). With the exception of the DNA shuffling approach, which bypasses the need to identify amino acid residues or motifs that are important to structure and function, engineering of allergens usually requires knowledge of B- and T-cell epitopes and in some cases, the three-dimensional structure of the allergen. No matter how allergen genes have been altered, putative hypoallergens must be subjected to a series of in vitro and in vivo evaluation procedures before being considered for therapeutic purposes (Fig. 3). Presently, the allergen databank (www.allergen. org) from the Allergen Nomenclature Subcommittee of the International

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Fig 3 Task tree for genetic engineering and evaluation of hypoallergenic molecules for specific immunotherapy. The DNA shuffling approach bypasses the need to identify B- and T-cell epitopes of allergens.

Union of Immunological Societies (IUIS) contains a list with more than 400 allergens and almost 200 isoallergens. For most of these allergenic proteins originating from various sources, the complete cDNA sequences have been determined and in some cases, the three-dimensional structures. This large number of available sequences goes parallel with an impressive number of


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publications dealing with B- and T-cell epitopes of allergens. In the next sections, we discuss approaches for genetic engineering and provide examples of engineered allergens and their evaluation. Fragments and oligomeric hypoallergenic forms of Bet v 1 were the most extensively characterized preparations and are now in clinical trials. Unfortunately, no clinical evaluation of wild-type recombinant Bet v 1 in comparison with extracts and engineered hypoallergenic forms has been performed. a. Site-Directed Mutants. Knowledge of crucial amino acid residues or motifs involved in IgE recognition can be used to alter dominant epitopes by site-directed mutagenesis. Data showing that Bet v 1 and other closely related tree pollen allergens consist of a mixture of closely related isoforms displaying striking differences in their ability to bind IgE (Breiteneder et al., 1993; Ferreira et al., 1996) constitute the basis for engineering a full-length Bet v 1 hypoallergen (Ferreira et al., 1998). The patterns of amino acid substitutions in tree pollen isoallergens and their IgE binding activities were analyzed using a computer algorithm developed to predict functional residues in protein sequences. The amino acid residues occurring in positions 113, 57, 125, 112, 30, and 10 of Bet v 1a were substituted using in vitro site-directed mutagenesis by those present in the same positions of low IgE binding isoforms. In this way, a Bet v 1 mutant carrying six point mutations was produced, which displayed extremely low IgE binding activity for all patients tested. In vivo (skin prick) tests indicated that the potency of the six-point mutant to induce typical urticarial skin reactions in allergic individuals was dramatically reduced (100- to 1000-fold) compared to Bet v 1a. T-cell clones (TCCs) established from the peripheral blood of birch pollen–allergic patients and reactive with Bet v 1a were also activated by the six-point mutant. IgE recognition of a group of calcium-binding allergens found in various pollens is influenced by bound calcium. This led to the idea of disruption of the EF-hand calcium-binding domains for engineering hypoallergenic mutants. In this way, calcium-binding-deficient mutants of Bet v 4 (Engel et al., 1997) and Bra r 1 (Okada et al., 1998) with reduced IgE binding activities were generated. However, data concerning T-cell recognition of calcium-binding allergens and their engineered counterparts are not available. B- and T-cell epitope mapping and sequence comparison of group 5 allergens from different grasses provided the basic information for introducing point mutations in highly conserved sequence domains of Lol p 5. Hypoallergenic forms of Lol p 5 were produced containing all relevant T-cell epitopes (Swoboda et al., 2002). Likewise, hypoallergenic variants of latex Hev b 5 (Beezhold et al., 2001), apple Mal d 1 (Son et al., 1999), egg Gal d 1 (Mine et al., 2003), and peanut Ara h 1, Ara h 2, and Ara h 3 (Bannon et al., 2001) were also generated by site-directed mutagenesis. Modified peanut allergens retained the ability to stimulate T-cell proliferation using peripheral blood

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mononuclear cells (PBMC) from allergic patients. In addition, peanut hypoallergens have been tested in a murine model of peanut anaphylaxis (Li et al., 2003) and shown to be effective. Hypoallergenic variants of the major allergen of Par j 1 displaying altered conformation were constructed (Bonura et al., 2001). Par j 1 is a member of the nonspecific lipid transfer proteins (nsLTPs) with a characteristic a-a-a-a-b structure that is stabilized by four disulfide bonds (Colombo et al., 1998). Targeting these disulfide bonds by site-directed mutagenesis resulted in molecules with altered conformation and decreased IgE binding activity but also activated allergen-specific T cells. Disruption of the native conformation by targeting disulfide bonds could be an approach generally used for engineering allergenic nsLTPs, including food-derived members. Disulfide bonds stabilizing the antigenic structure of major allergens of house dust mites were also targeted by site-directed mutagenesis. Hypoallergenic variants of Der p 2 (Smith and Chapman, 1996), Der f2 (Takai et al., 1997), and Lep d 2 (Eriksson et al., 2001; Kronqvist et al., 2001) were produced and evaluated for their IgE-mediated reactions and cellular responses. One potential problem when targeting the conformation of allergens might be the solubility of the final product, because denatured or unfolded proteins tend to form aggregates. Protein preparations consisting of aggregates are not suitable candidates for vaccine development. b. Deletion Mutants. Hypoallergenic variants can be engineered by deleting DNA segments in the gene corresponding to IgE binding epitopes. This approach was successfully used for the timothy grass pollen allergen Phl p 5b (Schramm et al., 1999). Epitope mapping was performed using overlapping recombinant fragments and at least four continuous IgE binding epitopes were identified. Deletions avoiding identified T-cell epitopes were then performed within these IgE binding regions. Some of these deletion mutants showed reduced IgE binding properties, no histamine-releasing activity, reduced skin reactivity, and no significant changes in T-cell reactivity. A similar approach was used to engineer hypoallergens of the American cockroach Per a 1 allergen (Wu et al., 2002). Based on the results obtained by proteolytic fingerprinting, a deletion mutant of ryegrass Lol p 1 was produced, which displayed decreased IgE binding activity and did not trigger histamine release up to a concentration of 10 mg/ml (Tamborini et al., 1997). This mutant was not tested in T-cell proliferation assays and for skin reactivity. c. Fragments. The disruption of the three-dimensional structure by fragmentation could be a useful approach to reduce the anaphylactic potential of allergens. IgE epitopes can be formed by a stretch of amino acids in a row (continuous epitopes) or by nonadjacent sequence elements brought together by folding (discontinuous or conformation-dependent epitopes). IgE recognition of continuous epitopes might also depend on their conformation, which


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might only occur in the context of the folded allergen molecule. The threedimensional structure of Bet v 1 was disrupted by expressing in E. coli two fragments of the cDNA corresponding to amino acids 1–74 and 75–160 (Vrtala et al., 1997). The fragments exhibited random coil conformation and almost no allergenicity. Together, the fragments harbored all relevant T-cell epitopes. Skin reactivity and histamine release were greatly reduced when compared to the native intact Bet v 1 allergen (Pauli et al., 1999; van Hage-Hamsten et al., 1999). Further, immunization of mice and rabbits with Bet v 1 fragments induced IgG antibodies that inhibited binding of IgE from allergic patients to wild-type Bet v 1 (Vrtala et al., 2000). Clinical trials now show the efficacy of vaccines based on Bet v 1 fragments for immunotherapy of birch pollen–allergic patients. Nonanaphylactic fragments of the major house dust mite allergen Der f 2 were produced by C- and N-terminal deletions and were mixed after separate refolding of the denatured fragments (Takai et al., 1999). Fragments of the calciumbinding allergens Bet v 4 (Twardosz et al., 1997) and Aln g 4 (Hayek et al., 1998) and an N-terminal fragment of Lol p 1 from ryegrass (Tamborini et al., 1997) also showed decreased IgE binding activities. However, they were not investigated concerning T-cell reactivity and immunogenicity. d. Oligomers. Vrtala et al. (1999) constructed oligomeric forms of the major birch pollen allergen Bet v 1. Two or three copies of the full-length Bet v 1 molecule were linked by short spacers, expressed in E. coli, and purified to homogeneity. In vitro studies using patients sera showed comparable IgE binding for the rBet v 1 monomer, dimer, and trimer, whereas the trimer demonstrated profoundly reduced histamine release of patients’ basophils compared to the monomer. This reduced anaphylactic potential was verified in vivo by skin testing of birch pollen–allergic patients. When compared to the Bet v 1 monomer, the Bet v 1 trimer induced similar proliferation and cytokine production upon stimulation of Bet v 1–specific TCCs. In vivo studies in animal models showed the ability of the Bet v 1 trimer to induce IgG blocking antibodies, which inhibit binding of human IgEs to rBet v 1 (Pauli et al., 1999; van Hage-Hamsten et al., 1999; Vrtala et al., 2001). As for the Bet v 1 fragments, clinical trials are also underway with the Bet v 1 trimer. It will be interesting to compare the efficacy and safety of the different hypoallergenic preparations of Bet v 1. e. Chimeras. King et al. (2001) reported an approach to genetically modify allergens in which hybrids are prepared consisting of a small portion of the guest allergen of interest and a large portion of a homologous but weakly crossreacting host protein. The idea is that the homologous host protein serves as a scaffold to maintain the native structure of the guest allergen of interest to preserve conformational-dependent B-cell epitopes, but at a reduced density. The homologous allergens from yellow jacket venom Ves v 5 and from paper

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wasp Pol a 5 (59% sequence identity) show very limited cross-reactivity of antibodies from sensitized patients. Hybrids of these two molecules containing 10–49 residues of Ves v 5 showed a 100- to 3000-fold reduction in allergenicity determined by histamine release assays with basophils from yellow jacket– sensitized patients. Such an approach could be employed for several allergens with known homologs displaying low allergenic activity. Whether injections of such hybrids would induce novel IgE specificities remains to be established. A promising approach to generate allergen chimeras is DNA shuffling. When regions of a protein that are critical for structure and/or function are known, site-directed mutagenesis is an appropriate and well-established approach to create molecules with desired properties. Unfortunately the information required for such a rational approach in protein design cannot always be provided. Therefore, within the last decade a new DNA-based recombination technique termed DNA shuffling has gained prominence. DNA shuffling was demonstrated to be superior over alternative random mutagenesis techniques such as error-prone PCR or degenerate cassette mutagenesis (Patten et al., 1997; Punnonen, 2000). The ambition of this in vitro evolution process is to mimic and profoundly accelerate natural design processes and to guide them in a certain direction accomplished by selective screening. The DNA shuffling technique itself comprises the fragmentation of two or more related genes and their random reassembly, followed by an amplification step. Because of sequence similarities of the parental genes, the gene fragments can form areas of recombination during the ‘‘shuffling’’ process, which results in the generation of new full-length genes. Libraries created in this way consist of highly diverse sequences and can be subjected to a diversity of screening procedures for the selection of molecules fulfilling the desired requirements (Stemmer, 1994a, 1994b). This trend toward in vitro protein evolution is also reflected in the literature, where many publications describing superior molecules are becoming available. The DNA shuffling technique has been successfully applied to generate improved variants of enzymes, proteases, antibodies, viral gene therapy vehicles, and cytokines (Kurtzman et al., 2001; Leong et al., 2003; Raillard et al., 2001; Soong et al., 2000). Because of the enormous potential of this technique, procedures for optimal shuffling have been intensively investigated. Consequently, a large panel of DNA shuffling techniques have been developed using enzymebased approaches such as different endonucleolytic or restriction enzyme digestion protocols or primer-based methods (Kikuchi et al., 1999; Kurtzman et al., 2001). Following these techniques, parameters as the number of template sequences, the degree of sequence homology among the templates, and the number of crossovers per kilobase DNA can be adapted to particular needs.


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DNA shuffling seems to be an ideal strategy to be exploited for generating novel allergy therapeutics and improved tools for allergy diagnosis. In terms of allergy treatment, reduced IgE binding capacity and conserved T-cell– activating properties are two critical demands. Diminished IgE reactivity could be achieved by minimizing either the number of epitopes per shuffled molecule to zero or one that would avoid cross-linking of IgE receptors on effector cells. Further, the affinity of IgE antibodies toward these epitopes could be reduced. Nevertheless, the shuffled products should be able to influence the T-cell population of an allergic individual by either shifting the typical Th2 toward a Th1 response or inducing T-cell tolerance. Considering cross-reactive allergens, the generation of allergen chimeras associating T-cell epitopes from several allergens but at the same time with extremely reduced anaphylactic potential also becomes possible by applying DNA family shuffling. Chimeric proteins obtained by family shuffling could be beneficial not only for allergy treatment but also for diagnostics, especially when considering allergens with superior IgE binding activities (Crameri et al., 1998; Ferreira et al., 2002; Punnonen, 2000). In a preliminary experiment, a group of pollen allergens derived from birch, hazel, and alder pollen, all belonging to the highly cross-reactive Bet v 1 family, were selected for DNA family shuffling. The resulting chimeric allergens were shown to be composed of several epitopes derived from different parental genes. Interesting results were obtained regarding the IgE reactivity of the chimeric Bet v 1 molecules when tested with birch pollen–allergic patients’ sera. In these experiments, candidate molecules showing either elevated or diminished IgE binding activity compared to the parental proteins could be identified. The antigenicity of the chimeric Bet v 1 molecules was comparable to that of wild-type Bet v 1a, as determined by in vitro T-cell proliferation assays using T-cell lines from Bet v 1–sensitized mice and Bet v 1–specific T-cell lines derived from birch pollen–allergic individuals (Wallner et al., 2002; Ferreira, 2003, unpublished data). A. Synthetic Peptides In principle, synthetic peptides could be used in three approaches of SIT. Nonanaphylactic peptides derived from surface-exposed areas and comprising IgE binding epitopes of allergens could be used for immunization aiming at the induction of blocking IgG antibodies. The same idea is behind the concept of mimotopes: synthetic peptides mimicking IgE-binding epitopes of allergens. Some aspects could limit the use of peptides corresponding to B-cell epitopes. In general, peptides are not good antigens and induce poor antibody responses. The application of suitable carriers or multimerization could be used to improve their immunogenicity, although multimerization might also increase the risk of IgE cross-linking events. In addition, the opinion that

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blocking IgG antibodies has beneficial effects could be challenged, because many mast cells are present on the mucosa, and thus, IgE would encounter the allergen before it could be captured by IgG antibodies. Very promising is the approach using peptides containing T-cell epitopes. Clinical studies have shown their applicability and effectiveness in inducing T-cell hyporesponsiveness. 1. B-cell Epitope–Derived Peptides Fragmentation of allergenic molecules offers a possibility to convert these proteins into hypoallergenic derivatives. First, developments in this direction were done some decades ago when low-molecular-weight allergen fragments were produced by proteolytic digestion of allergen extracts. The idea was to destroy IgE epitopes and to create peptides unable to cross-link effector cell–bound IgE (Valenta et al., 1999b). In principle, one can differentiate between two types of B-cell epitopes, conformational and continuous epitopes. By epitope mapping and structural analysis, one could identify those areas responsible for IgE binding to an allergen. For the Phl p 1 allergen, five continuous IgE epitopes were identified by gene fragmentation. These epitopes represented only a portion of the Phl p 1 B cell epitopes. However, it was demonstrated that grass pollen–allergic patients mounted protective IgG antibodies against these fragments after receiving SIT immunotherapy with grass pollen extracts (Ball et al., 1999a). In another study, nonanaphylactic peptides based on identified B-cell epitopes of Phl p 1 were synthesized. All peptides lacked secondary or tertiary structure as determined by nuclear magnetic resonance (NMR) analysis. The low allergenicity of these peptides was demonstrated by enzyme-linked immunosorbent assay (ELISA), basophil histamine release, and skin prick tests. In animal models, the synthetic peptides were able to induce IgG antibodies that recognized intact Phl p 1 and inhibited binding of human IgE antibodies to the native allergen (Focke et al., 2001). 2. T-cell Epitope–Containing Peptides Peptide-based immunotherapy uses peptide fragments containing T-cell– reactive epitopes instead of complete allergen molecules. In principle, these peptides are unable to cross-link two IgE molecules necessary to activate mast cells but can induce allergen-specific T-cell hyporesponsiveness (Briner et al., 1993; Tarzi and Larche, 2003). Earlier studies with two relatively long peptides (27 amino acids) of the major cat allergen Fel d 1 showed some clinical efficacy for cat-allergic patients. However, the approach was associated with a high frequency of adverse reactions, both immediate and late-onset symptoms (Norman et al., 1996). The immediate reactions very likely resulted from cross-linking of IgE. Using shorter peptides to avoid IgE cross-linking, a number of studies were performed in which peptides were administered to


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allergic asthmatic subjects (Haselden et al., 1999; Oldfield et al., 2001, 2002). At higher peptide doses, MHC-restricted activation of allergen-specific T cells led to late asthmatic reactions. Because these peptides were not capable of inducing in vitro histamine release from basophils and acute bronchoconstriction, the observed reactions were assumed to be independent of IgE. Rechallenge with peptides was associated with a marked reduction of the late asthmatic reaction and in the in vitro proliferation/cytokine production of PBMC, suggesting the induction of a state of allergen-specific hyporesponsiveness, or tolerance. To evaluate the potential of allergen-derived peptides as a form of immunotherapy for cat allergy, a randomized, double-blind, placebo-controlled clinical trial was performed with a mixture of 12 peptides generated from the sequence of Fel d 1. The study showed statistically significant reductions in early and late-phase skin reactions to allergen and a reduction of Th1 and Th2 cytokines in PBMC after allergen stimulation. The reduction in the skin reactions to allergen was accompanied by an increase in PBMC-derived IL-10 production, suggesting a role of regulatory T cells in tolerance induced by peptides. Short peptides from phospholipase A2, the major allergen of bee venom, were also clinically evaluated (Muller et al., 1998a). Successfully treated patients showed an immunomodulation toward the whole allergen with specific T-cell anergy and a decrease in specific IgE/IgG4 ratio. More recently, a doubleblind, placebo-controlled phase I clinical trial in patients allergic to bee venom was conducted to evaluate long synthetic overlapping peptides (LSPs) derived from phospholipase A2 sequence (Fellrath et al., 2003). The results demonstrated that LSPs were safe and induced Th1-type immune deviation, allergen-specific IL-10 production, and T-cell hyporesponsiveness. Together, all these results indicate that allergen peptide immunotherapy might be an effective and safe alternative to conventional SIT. 3. Mimotopes Artificial peptide structures mimicking natural antibody binding epitopes of an allergen, the so called mimotopes, have been described for the major birch pollen allergen Bet v 1. These peptides were obtained from phage libraries, which displayed random peptides of defined length fused to phage surface proteins. In this way, phage ligands for the monoclonal mouse anti-Bet v 1 IgG BIP 1, termed Bet mim 1, and for a pool of polyclonal patients’ IgEs, termed Bet mim E, were selected (Jensen-jarolim et al., 1998). Because most B-cell epitopes are conformational, these two peptide mimotopes were modeled on the three-dimensional structure of Bet v 1. The mimotope Bet mim E was found to cover an area assembled by the amino acids 9–22 and 104–113, which are found in proximity on the surface of Bet v 1. This correlates with the data obtained by Ferreira et al. (1998) where they identified six amino acid positions crucial for IgE binding to Bet v 1. Three out of these six amino acids are

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within the area deduced for IgE binding by Bet mim E. A possible therapeutic use of such mimotopes could be the induction of IgG blocking antibodies in patients sensitized to a single allergen. This was shown by in vitro studies using sera from mice immunized with Bet v 1 IgE mimotopes attached to the surface of phages. These mice produced IgG antibodies, which could drastically reduce human IgE binding toward Bet v 1 (Ganglberger et al., 2000, 2001b). For the use of mimotope-based vaccines in humans, phages might not represent the ideal vector. Although phages are immunogenic vectors and the mimotopes presented on the phage surface remain in a stable conformation, phages could infect E. coli cells in the gut of the patient, which might have unforeseeable consequences. Further, hundreds of mimotopic peptides are presented on a single phage particle and the application of phages as immunogenic vectors might be associated with a high risk because of crosslinking of membrane-bound IgE on effector cells. Therefore, mimotopes bound to the surface of a streptococcal albumin-binding protein were tested. With this system, the mimotopes retain the same conformation as on the phage particles, protein also serves as an immunogenic vector, and the mimotopes are presented in a monovalent manner, which would reduce the risk of IgE cross-linking (Ganglberger et al., 2001a). Mimotopes have not only been identified for Bet v 1 but also for Bet v 2, Der p 1, and group 1 grass pollen allergens (Furmonaviciene et al., 1999; Leitner et al., 1998; Suphioglu et al., 2001). Approaches using mimotopic peptides will certainly help identify more structural B-cell epitopes on allergens. Also, therapeutic applications with these molecules would seem possible. A. Protein Fusions An alternative approach consisting of a fusion of two unrelated allergens from the same species was implemented in a study with the timothy grass pollen allergens Phl p 1, Phl p 2, Phl p 5, and Phl p 6 (Linhart et al., 2002). Dimeric fusion molecules (rP2-P6, rP6-P2, and rP5-P1) were constructed, which showed similar secondary structures as equimolar mixes of the individual allergens, as determined by far-UV CD. IgE inhibition experiments revealed that the fusions represent the full spectrum of B-cell epitopes and all three fusions induced strong proliferative response in PBMCs from grass pollen–allergic patients. It was also observed that the immunogenicity of these hybrids was altered compared to the parental allergens. Molecules showing low immunogenicity as monomers displayed increased potential of T-cell stimulation as fusion partners. These findings offer novel possibilities in terms of SIT. Because allergen extracts used for immunotherapy bear not only the risk of de novo sensitization toward new allergens, also the different allergens administered are not equally efficient in stimulating the human immune system. The latter seems true also for equimolar combinations of


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recombinant allergens. By fusing grass pollen allergens, it was possible to increase the immunogenicity of allergens displaying weak immunogenic activity, which seems ideal for combinatorial allergy vaccines. IV. Customized Allergen DNA Vaccines

A number of preclinical studies proved the enormous potential of DNA immunization for the treatment of allergy. The antiallergic effect of DNA vaccines can be clearly attributed to the recruitment of Th1 cells and the establishment of a balancing Th1-biased cytokine milieu, and because of their unique features, DNA vaccines enable both preventive and therapeutic applications. A major selling point for DNA vaccines is their high safety profile. The lack of viral antigens (like with viral vector vaccines) and serious side effects in animals and humans makes them attractive, especially when multiple immunizations are required. However, critics have been raising a concern of the hypothetical possibility that plasmid DNA could integrate into the genome of the transfected host cell with the risk of tumorigenesis, for example, through inactivation of tumor suppressor genes. Several studies have addressed this concern and have concluded that integration would occur at a rate that is several orders of magnitude below the rate of spontaneous mutations (Martin et al., 1999). A. Short Introduction into DNA Vaccines DNA vaccines, also called genetic vaccines, introduced a new era in vaccine research and development in the 1990s (Tang et al., 1992; Ulmer et al., 1993). The principles underlying this new generation of vaccines differ significantly from those of former generations of vaccines. Instead of an antigen itself, the genetic information for a particular protein is delivered. After injection of the gene of interest, host cells take up the DNA. The encoded gene is subsequently transcribed and translated and the newly synthesized protein is processed and presented to the immune system. DNA vaccines not only carry the genetic information for the antigen of interest but also deliver an adjuvant effect as a result of the presence of immunostimulatory CpG motifs within the bacterial backbone (see Section III). Considering the similarity of the cellular events to the endogenous pathways preceding antiviral immune responses, it was originally postulated that this new technology would be useful only for vaccination against viral infections (Ulmer et al., 1993). However, within a few years after the first reports of this new generation of vaccines, the number of publications describing the usefulness of DNA vaccines soared. They demonstrated their effectiveness in a wide variety of disease models including protective immunity against bacterial and

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parasitic infections and offered new perspectives for the treatment of cancer, autoimmunity, and allergic diseases (Donnelly et al., 1997; Gurunathan et al., 2000; Leitner and Thalhamer, 2003). Various strategies have been developed to modulate the immune response after DNA immunization, such as (1) modification of the vector by inserting or deleting various regulatory sequences and optimization of promoters, (2) codelivery of genes or adjuvant molecules with regulatory and/or stimulatory properties such as costimulatory molecules [e.g., B7-1, B7-2], adhesion molecules [ICAM-1, leukocyte function–associated antigen-3], or cytokines (e.g., granulocyte–macrophage colony-stimulating factor, IFN-g, IL-2), and (3) employing different methods and sites of vaccine delivery. All of these possibilities, either as single approaches or in combination, led the way for new approaches to vaccine development and basic immunological research (Gurunathan et al., 2000; Leitner et al., 2001). B. History and Development of Allergen DNA Vaccines Immunotherapeutic approaches for the treatment of allergic diseases are generally aimed at either inducing tolerance or redirecting the immune response toward a Th1-type reaction. For the latter strategy, DNA immunization has already yielded promising results. In the initial proof-of-principle experiment, a DNA vaccine encoding the model antigen b-galactosidase was employed (Raz et al., 1996). Intradermal DNA immunization of BALB/c mice elicited a typical Th1-biased humoral immune response with elevated IgG2a antibody levels. CD4þ T cells isolated from immunized animals secreted IFN-g, but no IL-4 or IL-5. In contrast, immunization with protein stimulated a predominant Th2-type response characterized by high IgG1 levels, IgE production, and IL-4 and IL-5 expression. Injection of the DNA vaccine before protein immunization prevented IgE formation, and moreover, DNA application after protein immunization elicited a therapeutic effect. In the same year, the protective antiallergic effect of DNA vaccines was demonstrated for the first time using a clinically relevant allergen, the dust mite protein Der p 5. This DNA construct prevented IgE synthesis in rats (Hsu et al., 1996). Furthermore, preimmunization with the DNA vaccine reduced histamine release in bronchoalveolar fluids and airway hyperresponsiveness after challenge with aerosolized allergen. Transfer experiments indicated an important role not only for CD4þ T cells but also a supportive role for CD8þ T cells in mediating antiallergic effects. A number of clinically relevant allergens have been tested in DNA vaccine studies. Several publications confirmed the protective and/or therapeutic potential of DNA vaccines with constructs encoding the allergens b-lactoglobulin (Adel-Patient et al., 2001), Der p 1 and 2 (Jacquet et al., 2003; Kwon et al., 2001; Wolfowicz et al., 2003), Der f 11 (Peng et al., 2002), Bet v 1 (Hartl et al.,


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1999a; Hochreiter et al., 2003), and Art v 1 (Bauer et al., 2003). Furthermore, DNA immunization has been shown to reduce anaphylaxis after oral gene delivery of chitosan-DNA nanoparticles encoding the dominant peanut allergen Ara h 2 (Roy et al., 1999). In sum, these publications demonstrate that simple DNA constructs encoding the wild-type allergen gene without further modifications fulfill the basic requirements and can be immunogenic and effective in the protection and treatment of allergies. However, there is still much room for improvement not only in terms of efficacy but also regarding the safety of such vaccines. These aspects are summarized in Fig. 4. C. Strategies to Increase Safety of Allergy DNA Vaccines A major drawback of SIT is the common occurrence of side effects, essentially anaphylactic reactions induced by cross-linking of preexisting IgE antibodies on mast cells. Moreover, the injection of high doses of antigen can lead to

Fig 4 Task tree for the construction of gene vaccines with optimized safety and immunogenicity profiles. GV, gene vaccine; MIDGE, minimalistic immunogenic defined expression vector.

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anaphylaxis during the course of treatment via stimulation of ‘‘therapy-induced’’ IgE antibodies. Both reasons for these unwanted side effects are prevented by general features of DNA vaccines. The amount of translated antigen after DNA immunization in the skin comes to at least three to five orders of magnitude lower than the amount of protein injected during a typical SIT protocol. Because of the Th1-biased immune response type of intradermal or intramuscular application of DNA vaccines, de novo synthesis of IgE antibodies is inhibited. Furthermore, the high purity of the DNA vaccine avoids the induction of new allergic reactions via cross-reacting impurities, a possibly drastically underrated problem of SIT with allergen extracts. Nevertheless, before entering clinical trials, several concerns must be addressed: (1) the danger of systemic immune responses with side effects resulting from widespread distribution of the DNA vaccine transcript in various host tissues after immunization (Slater et al., 1998), (2) induction of autoimmune responses resulting from excessive Th1 immune activation induced by DNA vaccination, and (3) anaphylactic side effects as described for SIT, which are caused by preexisting and/or newly generated IgE antibodies. To overcome these concerns, strictly allergen-specific vaccination protocols inducing mild Th1-type responses and the creation of hypoallergenic DNA vaccines are required. This necessitates the development of optimized DNA vaccines with respect to immunogenicity and safety. 1. DNA Vaccines Translating Hypoallergenic Allergen Derivatives To increase the safety of DNA vaccines for allergies, the translation of native allergenic determinants must be avoided to prevent anaphylactic responses. These responses are caused by cross-linking of preexisting or vaccine-induced IgE antibodies on mast cells. However, to guarantee the recruitment of allergen-specific Th1 cells, T-cell epitopes on the protein must not be destroyed. Using the major birch pollen allergen Bet v 1, we evaluated two approaches to meet these requirements: (1) cutting the allergen gene into overlapping fragments, which lack any antigenic determinant of the native allergen but display the original repertoire of T-cell epitopes, and (2) using artificial hypoallergenic derivatives. For this purpose, DNA vaccines coding for two hypoallergenic Bet v 1 fragments (Vrtala et al., 2000) and for an artificial hypoallergenic Bet v 1 mutant (Ferreira et al., 1998) have been constructed. Vaccination with both types of vaccine was protected in an antigen-specific manner from IgE production and IgE-mediated cell release after repeated sensitization with recombinant allergen. Compared to the wild-type DNA vaccine that stimulates a strong humoral response, the DNA vaccine encoding the hypoallergenic mutant of Bet v 1 triggered a reduced and transient


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antibody response against wild-type Bet v 1, thus indicating the altered allergenic and immunogenic properties of the mutant allergen. The vaccine encoding the two hypoallergenic fragments of Bet v 1 stimulated no antibody response against Bet v 1, confirming that the fragments do not display allergy-associated epitopes present on the entire wild-type Bet v 1 molecule. However, the fragment vaccine triggered the recruitment of Bet v 1–specific Th1 cells as indicated by IgG2a antibodies, IgE suppression, and antigenspecific IFN-g production after sensitization with the native allergen. A therapeutic approach with sensitization followed by DNA immunization with the fragment-DNA vaccines revealed a remarkable and sustained antigenspecific reduction of IgE and IgE-mediated release thus, confirming the excellent therapeutic capacity of this type of DNA vaccine (Hochreiter et al., 2003). 2. Ubiquitination: A Routine Strategy to Produce Hypoallergenic DNA Vaccines Knowledge-based approaches to reduce anaphylactic properties of proteins by fragmentation or deliberate mutation, as described earlier, are time consuming and their suitability must be tested on a case-by-case basis. Therefore, we used ubiquitination of DNA vaccines as an alternative to develop a routine approach for destroying IgE binding epitopes on allergens in order to avoid recognition by preexisting IgE antibodies. Simultaneously, any T-cell epitope of the allergen would be preserved. This approach takes advantage of the fact that ubiquitination leads to proteasomal degradation of translated gene products. Resulting peptides are then transported by the Tap1/Tap2 heterodimer into the endoplasmic reticulum (ER), where they associate with MHC-I molecules and b2-microglobulin, which are in turn delivered to the cell surface for recognition by CD8þT cells. By still not fully understood cross-priming mechanisms, peptide epitopes can gain access to the MHC-II presentation pathway and activate CD4þT cells (Bristol et al., 2000). Under normal conditions, only a small percentage of plasmid-encoded proteins become ubiquitinated after DNA immunization. Stably linking ubiquitin to the antigen of interest has been demonstrated to efficiently channel the hybrid protein into the polyubiquitination pathway. As a result, the immune response is shifted toward a Th1-type, and simultaneously, antibody reactions are abolished while cytotoxic T-cell responses are enhanced (Rodriguez et al., 1997). To take advantage of this mechanism, we constructed a DNA vaccine encoding a Bet v 1-ubiquitin fusion molecule. Immunization with this vector yielded no antibody production (confirming the expected degradation of the allergen) but resulted in a 100% suppression of IgE antibodies and IgE-mediated cell release in a protective experimental design. In a therapeutic setting, IgE production and IgE-mediated cell release

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after sensitization markedly reduced treatment with the DNA vaccine, thus clearly demonstrating the antiallergic efficacy of hypoallergenic ubiquitin-fusion constructs (Hartl et al., 2004; Leitner and Thalhamer, 2003). 3. Vaccine Dose Reduction with Self-Replicating Vaccines Independent from these approaches, which led to an optimized safety profile of allergy gene vaccines, the high amount of plasmid DNA necessary for intramuscular or intradermal needle injection remains a weak point of DNA vaccines for allergy treatment. Using a biolistic device (such as the gene gun) enables the induction of strong immune responses with 100-fold lower quantities of plasmid DNA compared to needle injection. Unfortunately, immunization with gene gun results in a serological Th2-biased immune response with the production of high amounts of IgG1 and even IgE antibodies (Feltquate et al., 1997; Hochreiter et al., 2001; Weiss et al., 2002). These facts make the biolistic devices useless or at least questionable for any application concerning protection and/or therapy of allergic diseases. The principle of self-replicating RNA offered a solution of the dose problem and led to the development of a novel type of genetic vaccines. The genome of alphaviruses encodes a replicase enzyme used by the virus to efficiently replicate the viral genome inside the host cell cytoplasm. In theory, host cells only need to pick up a single molecule of this self-replicating RNA and still produce sufficient amounts of antigen to induce an immune response. It has been previously shown that antigen production in cells transfected with selfreplicating RNA is rather independent of the amount of RNA or DNA used for transfection (Leitner et al., 2000). Similar to the infection of cells with alphaviruses, the transfection of mammalian cells with self-replicating RNA that is based on alphaviral replicase causes host cell apoptosis (Ying et al., 1999). This is believed to be a protective response of the host cell attempting to limit viral spread. Therefore, it is assumed that self-replicating RNA vaccines achieve their high effectiveness by safely mimicking a viral infection of the transfected host cells. This might trigger powerful antiviral host pathways that would enhance the immune response to the antigen encoded by the self-replicating RNA. RNA stability still remains a problem for these vaccines during in vitro production. To facilitate and simplify vaccine production and handling, as well as the storage requirements for RNA, the sequence of the self-replicating RNA molecule is reverse transcribed into DNA and cloned into a conventional DNA plasmid. Now, the self-replicating RNA is produced from this plasmid by transfected cells using a conventional promoter. DNA replicons have successfully been applied in tumor models (Leitner et al., 2003; Ying et al., 1999) with humoral and cellular immune reactions displaying Th1-biased profiles. The Th1-type response was not induced by CpG motifs within the plasmid DNA


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because the amount of plasmid DNA used for immunization with DNA replicons (10 ng to 1 mg plasmid DNA) is far less than the effective concentration of Th1-inducing CpG motifs. The Th1-biased danger signals obviously come from the initiation of the antiviral defense pathways of mammalian cells and from activation of APCs via ISSs displayed during apoptosis (Kibler et al., 1997; Restifo, 2000). Intradermal injection of nanogram quantities of a DNA replicon vaccine encoding b-galactosidase is sufficient to trigger a Th1-biased immune response as indicated by IgG2a antibody and IFN-g expression correlated with lacking IgE production and IL-5. Fluorescence activated cell sorting (FACS) analysis revealed both CD4þ and CD8þ as the IFN-g–producing cells. Moreover, two injections of DNA replicons were sufficient to protect against the induction of an allergic immune response after sensitization with recombinant b-galactosidase. Vaccination induced 100% inhibition of cross-linking IgE antibodies as detected with a sensitive rat basophil leukemia cell release assay. The protective efficacy of DNA replicons is not restricted to immunodominant antigens such as b-galactosidase but could also be demonstrated with the clinically highly relevant allergen Bet v 1 (Thalhamer, 2003, unpublished data). DNA replicons are the latest generation of DNA vaccines. Three aspects make them highly interesting candidates for effective and safety-optimized vaccine approaches in allergy: (1) The very small amounts of plasmid DNA necessary for the induction of antiallergic immune responses will increase the acceptance of DNA vaccination for allergy treatment, (2) the induction of apoptosis of cells transfected with the DNA replicon vaccine leads to ‘‘selfremoval’’ of the vaccine after triggering the immune response, and (3) DNA replicon vaccines gain their immunogenicity via viral danger signals and induction of antiviral immune response types. The latter aspect attracted attention after clinical trials with DNA vaccines indicated poor immunogenicity of the constructs in humans in several cases (Leitner, 2001). Differences in the responsiveness of mice and primates to plasmid DNA-related danger signals via TLRs are assumed to be the major reason (Bauer et al., 2001; Takeda et al., 2003). TLR-independent danger signals make DNA replicon vaccines promising candidates for highly immunogenic DNA vaccines in humans. D. Improvement of Immunogenicity and Tailor-Made Immune Responses Immunogenicity of DNA vaccines depends on various factors including the antigen expression level, the form of the antigen (intracellular, membrane bound, or secreted), and the immunostimulatory ‘‘danger’’ signals. Therefore, many attempts have been made to optimize these parameters by approches such as vector modification, improving costimulation, and

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co-delivery of immunomodulatory sequences and molecules. Here, some of those approaches that specifically apply to allergen DNA vaccines are discussed. 1. Harmonization of the Codon Usage An issue that would mainly affect antigens not normally expressed by mammalian cells (such as allergens) is the codon usage that can be addressed by recoding (also called harmonization of a protein). The expression level of antigens encoded on DNA vaccines is influenced predominantly by two parameters: the vector itself and the codon usage. With respect to the former, a panel of optimized vectors with sophisticated features have been developed and most of them are commercially available. In contrast, however, the aspect of codon usage has largely been ignored. Suboptimal codon usage after transfection of mammalian cells with heterologous genes (such as genes encoding plant allergens) can severely diminish protein expression. This problem was demonstrated with DNA vaccines encoding the major mite allergen precursor ProDer p 1 (Jacquet et al., 2003; Massaer et al., 2001) and the major mugwort allergen Art v 1 (Bauer et al., 2003). Constructs encoding the plant gene Art v 1 revealed no response or only marginal immune responses. Immunogenicity of the Art v 1 DNA vaccine could not be enhanced by well-established immune modulations such as the addition of a eukaryotic leader sequence, co-injection of a GM-CSF encoding plasmid as adjuvant or cationic liposome-mediated intradermal gene delivery (Gurunathan et al., 2000; Leitner et al., 2001). The subsequent analysis of the gene sequence for the allergen Art v 1 revealed that the codon usage frequencies are strikingly different from those prevalent in mammals (Nakamura et al., 2000). Therefore, we replaced each codon of Art v 1 with the codon displaying the highest frequency for the same amino acid in mammals (Bauer et al., 2003). This resulted in a synthetic (recoded) gene encoding the amino acid sequence of Art v 1 with the mammalian codon usage. The optimized Art v 1 gene has a high GC content (71%) and a distinct codon bias for guanosine and cytosine at the third codon position (86.9% G or C at the third codon position) compared to the wild-type Art v 1 gene (53% GC and only 35.5% G or C at the third codon position). Compared to the wild-type gene, protein expression with the recoded gene was increased about 180-fold and intradermal DNA immunization elicited strong immunogenicity. The type of immune response induced by the recoded DNA vaccine fulfilled the criteria necessary for allergen DNA vaccines, for example, a Th1-biased antibody profile with high IgG2a titers, no or only marginal IgE levels, and antigen-specific IFN-g expression. Despite its strong immunogenicity the recoded construct did not induce release-mediating IgE antibodies as measured by a rat basophil leukemia cell release assay (Bauer et al., 2003).


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2. Th1/Th2 Modulation of Allergy DNA Vaccines An objective of DNA vaccines against allergies is the induction of a Th1-type cellular immune response, thus suppressing or converting the Th2-type response that is responsible for the high IgE production. Therefore, approaches that drive a Th1 response are potentially useful, such as the additional delivery of immunostimulatory DNA sequences (CpG motifs), Th1-stimulating cytokines, or ubiquination of the allergen. However, it should be emphasized that the intention must not be to trigger overwhelming Th1 responses, and combinations of strong Th1 stimuli must also be seen under the light of a potential risk to induce autoaggressive reactions. The extent of the Th1-biased reaction can be modulated by additional delivery of immunostimulatory CpG sequences. Coinjection of CpG-ODNS along with the plasmid DNA vaccine represents a simple approach to use the Th1-stimulatory capacity of CpG motifs (Hartl et al., 1999a, 1999b). CpG motifs can also be added to the gene of interest by appending a CpGrich sequence, which codes for certain amino acids. We have attached the amino acid sequence Tyr-Asn-Asn-Val-Asn-Val-Asp-Val-Asp-Val to the carboxyterminus of the Bet v 1a gene. This appendage is encoded by a sequence containing four CpG motifs and does not disturb the correct folding of the protein. Both approaches, appending and coinjection of CpG sequences, increased the Th1 bias of the immune response with respect to humoral and cellular responses (Hartl et al., 1999a, 1999b). Th1-directed immunomodulation was also achieved in DNA fusion vaccines encoding the gene of the model allergen ovalbumin fused to the IL-12 gene or a sequence encoding nine amino acids of IL-1b (Maecker et al., 1997). In a publication, fusion of the ovalbumin gene with the IL-18 gene, a cytokine that can indirectly act as a Th1 stimulator, demonstrated both protective and therapeutic efficiency as measured by airway hyperreactivity in a murine asthma model (Maecker et al., 2001). Another approach used the Th1-inducing capacity of viral vector vaccines. A replication-deficient adenovirus vector expressing the model allergen b-galactosidase (b-gal) was evaluated for its suitability to protect from or to treat an allergic reaction. Similar to intramuscular or intradermal plasmid DNA immunization, this vaccine elicited a Th1 type of immune response with increased titers of IgG2a antibodies and high frequency of IFN-g– producing T cells. Furthermore, prevaccination abolished the production of IgE and modulated the Th2-biased response to a more Th1-orientated response after sensitization with b-gal protein, thus indicating its protective efficacy. However, similar to a DNA vaccine encoding bovine b-lactoglobulin (Adel-Patient et al., 2001), the adenovirus vector vaccine also displayed an impaired therapeutic efficacy (Sudowe et al., 2002).

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Depending on the immunization schedule, the strength of nonspecific Th1-mediating signals, such as the number of immunostimulatory CpG motifs within the backbone and/or gene of interest, can have different influences on the protective and therapeutic efficacy of DNA vaccines. This may also account for the reported antigen-independent antiallergic properties of DNA vaccines (Jilek et al., 2001). Within a narrow time window after DNA immunization, a strong systemic Th1 effect triggered by CpG motifs obviously can mediate a nonspecific suppression of Th2 responses. However, this antigen-independent suppressive effect is not long lasting. E. DNA Multivaccines The approaches described are not restricted to the application of DNA vaccines encoding a single allergen. Intradermal injection of a plasmid DNA mixture of the 17-kd tree-pollen–related family (Bet v 1, Mal d 1, Cor a 1, and Aln g 1) elicited immune responses comparable to those induced by immunization with the single constructs. Both humoral and cellular responses indicated a Th1biased reaction with increased IFN-g and decreased IL-5 expression and various degrees of cross-reactivity between the different allergens. This cross-reaction also proved to have cross-preventive capacity; for example, DNA prevaccination with Mal d 1 revealed a similar protective efficacy against the challenge with the allergen mixture as preimmunization with the whole mixture. Similar results were found with a DNA multivaccine encoding Bet v 1 and its related food allergens (Mal d 1, Dau c 1, and Api g 1) and with a vaccine mixture coding for the profilins Art v 4, Lyc e 1, and Cap a 2 (Thalhamer, 2003, unpublished data). Complex mixtures of DNA vaccines raise the problem that upon using the aliquots of plasmid doses of single vaccines, the necessary dose of DNA vaccine would be 10-fold for a multivaccine containing 10 compounds. We, therefore, tried to circumvent this problem by creating fusion vaccines containing several allergens arranged in a string-of-bead manner, either by using the respective entire genes (gene A – gene B – gene C) or by splitting genes into fragments (gene A1 – gene B1 – gene C1 – gene A2 – gene B2 – gene C2). The latter approach additionally intends to include the hypoallergenic aspect by partially destroying the native conformation of the allergens. Preliminary experiments demonstrated the feasibility of this approach in principle. The whole-gene fusion vaccine induced antibody responses equal to the multivaccine or the respective single-gene vaccine. Fusion of gene fragments reduced the antigenicity with respect to the IgG response, indicating the assumed loss of native B-cell epitopes. Furthermore, both attempts led to allergen-specific immune responses with the desired Th1 nature pointing to the maintenance of the desired T-cell stimulatory capacity (Thalhamer, 2003, unpublished data). Summing up, in the first decade of DNA vaccines, a huge number of publications demonstrated the usefulness of this novel vaccination approach.


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Amazingly, these very simple constructs not only showed efficacy against highly immunogenic pathogens, but also appeared to be practicable for difficult conditions such as cancer and allergies. One of the advantages of DNA vaccines proved to be the ‘‘clean’’ immune response that is induced, that is, the absence of side effects both in animals and apparently in humans based on the limited data available. However, in the case of poorly recognized antigens, it is necessary to put strong adjuvant-type signals back into the vaccine. Better understanding of the signals that trigger innate immune responses will allow us to design more targeted strategies. Ideally, such strategies only induce a desired innate immune response while avoiding unwanted responses such as the excessive production of inflammatory cytokines. Replicase-based DNA vaccines appear to trigger one of those innate pathways in the same way as certain viral infections, but without the problems associated with viral vectors. The triggering of PRRs (Takeda et al., 2003) and immunostimulatory apoptosis (Restifo, 2000), thus stimulating the innate immune system simultaneously with DNA vaccination, may give DNA vaccines the necessary ‘‘bite’’ to induce optimized protective and/or therapeutic immune response. V. Short Overview of Alternative Treatments and Supporting Adjuvants for SIT

The abnormal switch to IgE production against ubiquitous proteins is triggered by IL-4–producing Th2-type cells and leads to the establishment of specific IgE antibody responses, which are boosted by repeated allergen contact. These events can be influenced at different levels and a number of strategies have been developed that intend to suppress or mitigate allergic diseases. In terms of the progression of the ‘‘immunological disorder allergy,’’ the production of IgE antibodies represents a very late state of the course of the disease. Neutralization of circulating IgE antibodies prevents from binding to their high-affinity mast cell receptors and should help avoid the final release process. However, intervention at this level will be restricted to the treatment of symptoms. A number of publications, including clinical trials, demonstrated that anti-IgE antibodies display some beneficial effects with a high safety profile (D’Amato, 2003). Supporting application of anti-IgE antibodies together with standard therapy concepts may be very helpful to reduce the rescue medications and improve the quality of life. Blocking the IL-4–dependent actions of Th2 cells with antibodies against IL-4 represents another approach using antibodies as a therapeutic tool. In the development of allergy, IL-4 acts much closer to the initial triggering processes of allergic diseases than IgE. Nevertheless, from an immunological point of view, this approach will induce neither allergen-specific curing nor long-lasting balancing of allergic responses, and moreover, IL-13 can replace several

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functional activities of IL-4. In general, anti-IL-4 and IL-5 treatment has been disappointing and contradictory (Barnes, 2001). Approaches to intervening in IgE-mediated allergic diseases also include the inhibition of allergen-specific IgE synthesis via blocking the activation or preventing the synthesis of IL-4 and IL-13. For this purpose, IL-4 mutant proteins were developed that act as antagonists of both IL-4 and IL-13 activity. Mutation led to an altered IL-4, which retained its binding capacity to the IL-4 receptor but lost the proliferative activity, thus uncoupling binding and signal transduction (Grunewald et al., 1998). The mechanisms and consequences of inhibiting IL-4 and IL-13 synthesis still need to be elucidated. A 2001 study evaluated the therapeutic potential of inhaled recombinant human soluble IL4 receptor and found clinical benefits for patients with moderate asthma who require daily inhaled corticosteroids (Borish et al., 2001). Without any doubt, allergen-specific induction of tolerance would be the most elegant approach to protect from or to cure allergic diseases. In its optimal form, it would be free of any side effects (e.g., anaphylactic reactions driven by therapy-induced IgE production) and would lack the risk of strong Th1-biased approaches (e.g., overwhelming Th1 reactions leading to autoimmunity). Animal experiments and initial clinical trials indicated the potential of approaches such as local nasal immunotherapy (Giannarini and Maggi, 1998; Hufnagl et al., 2003). The role of IL-10 (and TGF-b) in the induction of antiallergic tolerance was confirmed by animal experiments (Zemann et al., 2003) and by clinical studies investigating the consequences of SIT on the immune status of patients (Francis et al., 2003; Jutel et al., 2003). Taken together, the data indicate that nasal or oral tolerance induction may be a potential approach for an alternative SIT. However, the results also indicate that the success of these attempts was restricted to the protective application with much less efficacy concerning a downregulation of already established Th2-type responses. With increasing insight into the mechanisms of Th1- and Th2-type immune responses, various possibilities arose to modulate an allergic Th2 immune response. Most of the attempts were based on the hypothesis that Th1-biased stimuli could prevent the development of an allergic response and even may exert the power to convert an established Th2-type response. Bacille Calmette– Gue´rin (BCG), as one of the candidates, fulfills the criteria of Th1 induction with an acceptable safety profile and a long clinical tradition. Animal experiments revealed that BCG, alone or in combination with allergen, induced antiallergic effects such as reduced IgG1 and IgE production, airway responsiveness, eosinophilic influx, and Th2 cytokines in bronchoalveolar lavage fluids (Herz et al., 1998; Hubeau et al., 2003). A 2002 clinical trial proved the therapeutic effect of BCG application in asthmatic patients (Choi and Koh, 2002). Another group of bacilli, the lactic acid bacteria (LAB), such as


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lactobacilli, also proved to be antiallergic adjuvant candidates. The possible role of specific LAB strains in the prevention of allergic diseases has become more evident (Bjorksten et al., 1999). A 2003 study confirmed the capacity of two LAB strains to prevent and modulate allergic immune responses (Repa et al., 2003). A practicable Th1 adjuvant potential was also demonstrated for heat-killed Listeria monocytogenes, an intracellular bacterium triggering strong signals to the innate immune system including Th1 danger signals, in a food allergy model (Li et al., 2003). LPS represents a ubiquitous molecule of our environment and a number of important immunoregulatory processes have coevolved with this molecule. With respect to allergy and asthma, there is still the question whether LPS is beneficial or disease promoting. Animal experiments and clinical trials with LPS or lipid A as adjuvants coadministered with allergens proved Th1-inducing capacity and partly improved the efficacy of SIT without triggering harmful side effects (Drachenberg et al., 2001; Ormstad et al., 2003). With the availability of cytokines as recombinant molecules or expression plasmids, it became obvious to use the Th1-inducing cytokines IFNg, IL-12, and IL-18 as adjuvants to counteract Th2-biased allergic responses. IFN-g causes side effects upon systemic use, but mucosal transfer of adenovirus-mediated IFN-g was tolerated and able to reduce Th2-mediated lung parameters in animal experiments (Behera et al., 2002). Indirect stimulation of IFN-g via IL-12 and/or IL-18 also induced Th2-inhibiting effects with partly contradictory results concerning the cooperation of the two cytokines (Kim et al., 1997; Lee et al., 1999), indicating that any application of IL-18 must be critically assessed. In combination with IL-12, this cytokine displays doubleedged features. Depending on the cytokine environment, it can trigger both inflammatory Th1-biased immune response types and allergic Th2-dominated ones (Wild et al., 2000). Recently, substances such as synthetic imidazoquinolines (Brugnolo et al., 2003), microbicides (Tsuji et al., 2003), and antimycotics were reported to display Th2-suppressive properties (Kanda et al., 2001) and may be added to the list of future adjuvants for supporting SIT. VI. Summary and Conclusions

Molecular cloning and recombinant production of allergens opened new possibilities for the increasing problem of allergies. The concept of moleculebased instead of allergen extract–based diagnosis was developed and is already being implemented in the field. Molecule-based diagnosis allows not only the precise identification of allergen recognition patterns of individual patients and the quantification of IgE levels to each allergen, but it might also help to predict potential sensitization to multiple allergenic sources resulting from the cross-reactivity phenomenon. Information about the sensitization profile of

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individual patients forms the basis for the development of customized forms of immunotherapy based on the use of recombinant and synthetic protein antigens. For this purpose, a variety of preparations are being developed for different allergies. Major goals are to increase safety by minimizing the risk of IgE-mediated side effects and to improve efficacy of specific immunotherapy by counterbalancing the ongoing Th2-biased allergic response. Synthetic peptides in question are mimotopes, such as artificial peptide structures mimicking IgE binding epitopes, B-cell epitope–derived peptides, and T-cell epitope–containing peptides. Recombinant-based approaches are mostly focused on genetic engineering of allergens to produce molecules with reduced allergenic activity and conserved antigenicity, such as hypoallergens. An alternative to genetic engineering is the chemical modification of pure allergens with immunostimulatory DNA sequences (allergen-ISS conjugates), which mask IgE epitopes and add a desirable Th1-inducing character to the allergen molecule. Several of these customized allergen preparations have been already evaluated for their safety in clinical provocation studies. So far, clinical trials showed the efficacy and safety of immunotherapy with T-cell epitope– containing peptides and with allergen-ISS conjugates for cat-allergic and ragweed pollen–allergic patients, respectively. In addition, two preparations consisting of hypoallergenic derivatives are being evaluated for immunotherapy of birch pollen–allergic patients. In parallel, several animal studies have now demonstrated the potential of genetic immunization for allergy treatment in the future. The antiallergic effect of DNA vaccines translating wildtype allergen genes or hypoallergenic derivatives is attributed to the recruitment of Th1 cells and the establishment of a balancing Th1-biased cytokine environment. Acknowledgments The work of the authors was supported by the Joint Research Project S88-B01 (S8802-B01, S8811B01, S8813-B01) and Project P16456-B05 of the ‘‘Fonds zur Fo¨rderung der Wissenschaftlichen Forschung, FWF’’, Austria.

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Immune Response Against Dying Tumor Cells LAURENCE ZITVOGEL,* NOELIA CASARES,* MARIE O. PE´QUIGNOT,* NATHALIE CHAPUT,* MATTHEW L. ALBERT,{ AND GUIDO KROEMER* *Institut Gustave Roussy Villejuif, France { Institut Pasteur Paris, France

I. Introduction

Despite a concerted effort on the part of scientists and clinicians, cancer remains one of the leading causes of death in industrialized countries. Furthermore, in spite of stunning progress in cancer cell biology, the clinical management of cancer is still largely based on surgical resection, local irradiation, and the instillation of toxic compounds (‘‘chemotherapy’’) that kill any kind of proliferating cell, though cancer cells somewhat more efficiently, at least at an early stage. Though effective (albeit toxic) in the short term, patients typically develop chemotherapy-resistant tumor cells after several rounds of darwinian selection, resulting in the eventual demise of the patient. Such escape mutants also arise in the setting of more ‘‘selective’’ compounds such as radioactive iodine, hormone receptor antagonists, or tyrosine kinase inhibitors. Based on this frustrating experience, therapies that bypass drug resistance and are less toxic to patients are urgently needed. Cancer immunotherapy offers one particularly exciting alternative therapeutic strategy. Severe immunodeficiencies increase the frequency of spontaneously arising tumors, both in humans and in mice. For example, mice that are deficient in lymphocytes and/or the interferon-g (IFN-g) system have a higher incidence of carcinogen-induced sarcoma, lymphoma, and spontaneous epithelial tumors than immunocompetent mice (Kaplan et al., 1998; Shankaran et al., 2001; Street et al., 2002). Similarly, perforin-deficient mice exhibit an abnormally high rate of spontaneous lymphoma and lung adenocarcinoma (Smyth et al., 2000), and neutralization of tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) by blocking antibodies promotes carcinogen-induced tumor development in mice (Takeda et al., 2002). The tumors arising in perforin-deficient or TRAIL-suppressed mice are readily rejected when transplanted into immunocompetent controls (Smyth et al., 2000; Takeda et al., 2002), thus confirming that their development is dictated by the absence of an immune response, rather than by cell autonomous phenomena. Immunocompromised patients having undergone allotransplantationassociated immunosuppression exhibit an increased incidence of virally 131 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2776/04 $35.00

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induced skin neoplasia and lymphoproliferative disease, as well as non–virusassociated epithelial cancers (Bleday et al., 1993; Nalesnik, 2002; Penn, 2000; Trofe et al., 2002; Wu and Orengo, 2002). Although these observations suggest the existence of an immunosurveillance system that participates in the suppression of tumor development, there are also strong arguments to assume that such an immunosurveillance has limited importance, at least once tumors have been established. If cycles of immune pressure and immune escape were operative during tumor development, we would expect—in a Gedankenexperiment—that phases of progressive tumor growth will be interrupted by one or more periods of contraction. Nonetheless, once clinically detectable, solid tumors generally grow and continue to grow, without evidence of significant drops or depressions. In addition, there is normally no clear-cut clinical or histological evidence of local inflammation in uninfected tumors, with the notable exception of melanoma (Mihm et al., 1996), renal carcinoma (Nakano et al., 2001), and ovarian carcinoma (Zhang et al., 2003), in which the presence of tumor-infiltrating lymphocytes (TILs) has a favorable prognostic impact. It can be argued that the generation of immunoresistant tumor cell variants is a side effect of the initial steps of carcinogenesis, as a result of genomic instability and dysregulation that characterizes the transformed genome (Khong and Restifo, 2002). In that case, the selection for immunoresistance would take place early and thus be clinically imperceptible. Immunoresistance would be acquired before tumors reach a cell mass of 109 cells or more, and only once the battle has already been lost, tumors would become clinically detectable and enter a phase of ever progressive growth. Although doubts can be shed on the importance of immunosurveillance in normal cancer development, it is commonly agreed that the immune system can be manipulated to respond to established and developing tumors, especially in experimental animals. Antitumor responses can be induced by vaccination, usually by providing a formulation of concentrated tumor antigens whose optimal presentation is ensured in vitro (by pulsing dendritic cells [DCs]) or in vivo by the application of suitable immunostimulatory agents (adjuvants, cytokines, or blockade of T-cell inactivation). Moreover, antitumor responses can be induced by passive transfer of tumor-specific cytotoxic T lymphocytes (CTLs) that have been selected and expanded in vitro. Promising results have been obtained in several clinical studies employing such strategies (Banchereau et al., 2001; Coulie et al., 2001; Dreno et al., 2002; Dudley and Rosenberg, 2003; Pardoll, 2002; Phan et al., 2003a; Ribas et al., 2003; Rosenberg, 2001). In this context, clear evidence in favor of tumor escape mechanisms has been obtained. For instance, melanomas from patients experiencing partial responses after T-cell–based immunotherapies reportedly lose b2-microglobulin (and hence, major histocompatibility complex [MHC]

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class I) expression or downmodulate the target antigen (Khong and Restifo, 2002; Restifo et al., 1996). The central dilemma of cancer immunotherapy resides in the striking contrast between the lack of spontaneous antitumor immune responses and the apparent possibility to induce active antitumor immune responses experimentally. How do cancer cells tolerize (or paralyze) the immune system or simply manage to be ignored? And how is it possible that anticancer therapy by irradiation or by drugs, ideally resulting in massive death of cancer cells, does not elicit an immune response in the patient? Irradiation and chemotherapy mostly induce a type of cell death, apoptosis, which is widely thought to be immunologically silent or even tolerogenic. Thus, paradoxically, the standard treatments that are used in the clinical management of both solid and diffuse tumors would suppress any possibility that the patient’s immune system eradicates those residual tumor cells that will ultimately cause relapse. This chapter critically examines this hypothetical scenario and raises the question how the modality of tumor cell death and/or the immune system can be manipulated so dying tumor cells become immunogenic. II. Minimal Elements of the Antitumor Immune Response

A. Specific Tumor Antigens Tumor antigens can be categorized in the following groups: (a) nonmutated shared antigens (e.g., MAGE, BAGE, RAGE, and NY-ESO), which are expressed in testes and in multiple tumor cells, (b) differentiation antigens (e.g., prostate-specific membrane antigen [PSMA] and prostate-specific antigen [PSA] in prostate carcinoma, Mart1/MelanA and tyrosinase present in many melanoma, and carcino embryonic antigen [CEA] present in a large percentage of colon cancers), which are tissue restricted and present in lineage-specific tumor cell, (c) mutated oncogenes and tumor suppressor genes (e.g., mutated ras, rearranged bcr/abl, mutated p53), which provide novel epitopes for immune recognition, (d) unique idiotypes (e.g., immunoglobulin antigensin myeloma and B-cell myeloma, T-cell receptor [TCR] expressed in CTCL), (e) oncovirus-derived epitopes (e.g., the human papillomavirus–encoded E6 and E7 proteins, Epstein–Barr virus [EBV]–associated antigens present in primary brain lymphoma), and (f) nonmutated oncofetal proteins such as CEA, a-fetoprotein, and survivin (Ribas et al., 2003). In experimental models, it is possible to elicit specific T-cell responses against such tumor antigens, and this can lead to tumor prophylaxis or eradication, at least in murine models. The existence of T cells specifically recognizing different tumor antigens and mediating antitumor responses in vitro or in vivo has been extensively documented. There are benefits and risks associated with targeting each group of tumor antigens. For example, in responses

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specific for differentiation antigens, tumor immunity may result in autoimmune responses. Breast or ovarian cancers that ectopically express CDR2 (normally confined to neurons and testis) can elicit an autoimmune response against CDR2, which then causes paraneoplastic cerebellar degeneration (Albert et al., 1998a). Similarly, in therapeutic trials of patients with melanoma, it has been reported that patients develop vitiligo (Mackensen et al., 2000; Yee et al., 2000). It is unclear whether antitumor immune responses specific for differentiation antigens can be dissociated from the autoimmune response to such targets. On the other hand, the development of interventions targeting mutated antigens has been more difficult because of the diversity of individual tumors and the polymorphism of the immune system. It is particularly interesting that apoptotic cell death can lead to unmasking (or destruction) of specific tumor antigens. Thus, different classes of proteases selectively activated during the death of apoptotic cells may expose hidden or cryptic antigens that were previously ignored by T cells (Rosen and Casciola-Rosen, 1999). For instance, hepatocellular carcinoma (HCC) is accompanied by an autoantibody response to nuclear proteins in 30% of patients. Several targets of the antinucleolar autoantibody response in HCC were identified, including nucleophosmin/B23, fibrillarin, and NOR-90 (Imai et al., 1992). Interestingly, anti-B23 autoantibodies are also found in patients with other tumors (e.g., breast cancer) (Brankin et al., 1998), indicating that this immune response may indeed be linked to novel features of the transformed cell. B23 and in particular a B23 variant expressed in tumors (that is transcribed starting on methionine residue 7) has been shown to be a substrate of granzyme-B, and the resulting cleavage product is recognized by autoantibodies (Ulanet et al., 2003). Novel tumor antigens can also be created by the cell surface expression of proteins that are normally secluded in the cytoplasm. For example, the prominent lymphoplasmacytic cell infiltrate characteristic of medullary carcinoma of the breast (MCB) contains an oligoclonal B cell population that recognizes bactin expressed on the surface of MCB cells, after apoptosis induction, perhaps as the result of an aberrant granzyme-B activation (Hansen et al., 2001). Thus, in particular circumstances, malignancy can lead to the generation of neo-autoantigens that are created, presumably as a result of cell death, by cleavage by granzyme-B (normally restricted in expression to CTLs, natural killer [NK] cells, NK T cells [NKTs], and gd T cells). In contrast, immunodominant epitopes may be destroyed as a result of apoptosis, subsequent to caspase activation. For example, injection of C57BL/6 mice either with apoptotic RMA lymphoma cells or with bone marrow-derived dendritic cells (BM-DCs) pulsed with apoptotic RMA cells mount a specific and protective CTL response; however, it is not directed against the immunodominant CTL epitope gag85–93 because this epitope is destroyed by active caspases (Castiglioni et al., 2002).

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B. Exosomes and Heat Shock Proteins as a Source of Multiple Tumor Antigens A novel source of undefined tumor antigens has been found in small vesicles secreted by hematopoietic and tumor cells. Such vesicles, which are called exosomes, originate from multivesicular late endosomes (MVBs) that are secreted upon fusion of the external limiting membrane of MVB with the plasma membrane. Exosomes were initially described as vesicles containing proteins discarded by reticulocytes during their transformation into red blood cells (Johnstone et al., 1987). Interest in exosomes was renewed after their description in antigen-presenting cells (APCs) (Raposo et al., 1996) and the observation that they can stimulate immune responses in vivo (Zitvogel et al., 1998). Importantly, exosomes contain MHC class I (and when produced by professional APCs, MHC class II) peptide complexes, heat shock proteins (HSPs), and cytosolic antigens that could account for their immunogenicity in vitro and in vivo (Thery et al., 2002b). MHC–peptide complexes contained in exosomes can indeed be transferred to DCs to induce primary T-cell responses leading to tumor eradication (Thery et al., 2002a). Exosomes may also transfer antigens from tumor cells to DCs. Like DCderived exosomes, exosomes produced by melanoma tumor cells contain MHC class I molecules. Interestingly, tumor cell–derived exosomes contain tumor antigens, such as MelanA/Mart1, in the case of melanoma cell tumors, or HER-2/neu for ovarian tumors. DCs expressing the appropriate MHC class I molecules can trigger effective T-cell activation in vitro, when they are fed with exosomes derived from tumor cells expressing the corresponding antigen but not the restriction element (the MHC class I recognized by the T-cell clone). Tumor cell–derived exosomes may, therefore, be used as a source of antigen for cross-presentation by DCs, and DCs loaded with tumor cell– derived exosomes induce tumor rejection (Wolfers et al., 2001). Surprisingly, this effect is not entirely tumor specific, because exosomes from some tumors can protect against allogeneic tumors, whereas irradiated tumor cells protect against tumor challenge in a strictly tumor-specific manner (Wolfers et al., 2001). The reason for this ‘‘cross-protection’’ remains unclear, but it could be due to an enrichment of shared tumor antigens in exosomes. Exosomes have been identified in vivo, first by Denzer et al. (2000a,b) by electron microscopy of follicular DCs and later by Andre´ et al. (2002) in tumoral biological fluids. Exosomes from ascites harvested from metastatic patients may constitute an abundant source of candidate tumor antigens (such as HER-2/neu) and are able to elicit antitumor T-cell responses in vitro when transferred to DCs (Andre´ et al., 2002). Exosomes derived from DCs or tumor cells do not induce DC maturation by themselves and thus are not immunogenic unless they are delivered with additional stimuli. Thus, the functional

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outcome of exosome-mediated stimulation depends on external factors, such as local inflammation, the route of injection (when exosomes are used for vaccination), and the cellular source of exosomes. According to one report (Karlsson et al., 2001), 40-nm vesicles produced by rat intestinal epithelial cells cultured in the presence of IFN-g (to induce MHC class II expression) and predigested ovalbumine induced antigen-specific tolerance. Similarly, vesicles purified from the serum of rats previously fed an ovalbumine diet also induced some tolerogenic effects to ovalbumine in delayed-type hypersensitivity (DTH) assays (Karlsson et al., 2001). The vesicles used in these studies, however, were not fully characterized, and whether they are genuine exosomes remains a matter of debate. Proteomic analyses of DC-derived exosomes revealed a particular abundance of molecules such as tetraspanins, CD11b, and lactadherin, which have a putative role in targeting APCs (Thery et al., 2001). However, the exact role of these proteins in the immunogenicity of exosomes remains elusive. Although several similarities exist between exosomes and apoptotic microvesicles, exosome production is not enhanced in apoptotic cells, suggesting that living and dying cells may produce different types of membrane vesicles (Thery et al., 2001). The differential immunogenicity of exosomes secreted from stressed/dying and nonstressed tumor cells awaits further studies. Interestingly, exosomes contain elevated levels of several HSPs, in particular of the HSP60, HSP70, and HSP90 families, depending from which cell type the exosomes have been derived (Skokos et al., 2003; Thery et al., 1999; Wubbolts et al., 2003). Molecular chaperones (such as HSP70, Grp94, but also HSP60, HSP90, and calreticulin) can serve as immune adjuvant for crosspriming with antigenic peptides (Srivastava et al., 1994). APCs internalize HSPs with bound peptides, presumably through receptor-mediated endocytosis (Singh-Jasuja et al., 2000), resulting in antigen presentation via MHC class I molecules (Castellino et al., 2000). For example, HSP70–peptide complexes (HSP70–PC) purified from tyrosinase-positive (but not from tyrosinasenegative) melanoma cells resulted in the specific DC-mediated activation of a class I–restricted tyrosine peptide-specific cytotoxic T-cell clone (Noessner et al., 2002). HSPs, thus, deliver chaperoned peptides from non-APCs, including tumor cells, to MHC molecules of APCs. CD91 is a common receptor for HSPs Gp96, HSP90, HSP70, and calreticulin, allowing for the uptake of complexes of peptides with HSPs by macrophages and DCs and presentation of these peptides through the classic endogenous pathway (Basu et al., 2001). In addition, the extracellular domain (ECD) of CD40 can reportedly bind to the aminoterminal adenosine triphosphatase (ATPase) domain of HSP70. This interaction would be found when HSP70 is in its adenosine diphosphate (ADP)–bound state (after ATP hydrolysis) and therefore tightly bound to its peptide substrate. Apparently, the

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CD40–HSP70 interaction stimulates DC cells via p38 (Becker et al., 2002). HSP60 stimulates DCs for maturation and release of tumor necrosis factor-a (TNF-a), IL-12, and IL-1b (but not IL-10) and stimulates several kinases (p38, JNK, and ERK) and IkB phosphorylation via toll-like receptor-4 (TLR4) (Flohe et al., 2003). HSP70 may also elicit cytokine secretion via TLR2 and TLR4 with their cofactor CD14 (Asea et al., 2002) and thus may stimulate DC maturation. Indeed, HSP70 causes MyD88 relocalization (a typical downstream effect of TLR ligation), and MyD88-deficient DCs do not respond to HSP70 with proinflammatory cytokine production (Vabulas et al., 2002). In addition, HSP70 expressed on the surface of tumor cells can activate the NK cell receptor CD94 (Gross et al., 2003a) and facilitate perforin-independent apoptosis by specific binding and uptake of granzyme-B (Gross et al., 2003b). The improved NK-, lymphokine activated killer cell (LAK)-, or CTL-mediated lysis of tumor cells then conceivable could lead to cross-presentation of antigen by DCs to prime adaptive T-cell immunity. Together, these data suggest that HSPs can enhance the antitumor immune response at three levels, by increasing ‘‘signal one,’’ through the delivery of antigenic peptides to the APC, by increasing ‘‘signal two,’’ that is, by enhancing DC activation and maturation, and possibly by providing a stimulus for ‘‘signal three,’’ for the production of proinflammatory cytokines. It has been suggested (but by no means proven) that HSPs would come into action in particular when tumor cells die from necrosis, in which case HSPs (and HSP–peptide complexes) would shed in the extracellular space and become available for binding to surface receptors of APCs. In contrast, HSPs would be confined within apoptotic cells, bodies, and blebs. However, there is some evidence that HSPs can increase the immunogenicity of apoptotic cells and that HSP-bound antigenic peptides are transferred within the phagosome or possibly the endoplasmic reticulum (ER) of the APC, postretrograde transport of internalized HSPs (Moroi et al., 2000). Alternatively, HSPs could serve as vehicles of tumor antigens associated with exosomes (Thery et al., 1999). This means that conditions (and in particular stress) leading to an increase in the expression of HSPs may favor immune responses against tumors (and perhaps against tumor-derived exosomes). C. Dendritic Cells: The Key Unlocking the Immune Response Although there is some evidence that tumor cells can directly prime naive T cells (Ochsenbein et al., 2001), it is commonly assumed that the tumor itself does not present tumor autoantigens to T cells efficiently. Rather, DCs are capable of capturing tumor-derived antigen for the generation of MHC–peptide complexes and the initiation of CD4þ and CD8þ T-cell immune responses. With respect to the activation of CD8þ T-cells or CTLs, the mechanism of

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antigen presentation is referred to as cross-presentation for the crossing of the classically defined MHC class I restriction for endogenously produced antigen (Fig. 1). That cross-presentation is a cardinal feature of antitumor immune responses has become clear first in a series of experiments showing that immunity to tumors that express influenzavirus nucleoproteins is mediated by indirect presentation of antigens from tumor cells by bone marrow–derived APCs (presumably DCs) (Huang et al., 1994). More recently, it was demonstrated in vitro and later in vivo that DCs are the only APC capable of antigen cross-presentation. These rare bone marrow–derived leukocytes possess high levels of MHC and costimulatory molecules, have the capacity to produce immunostimulatory cytokines (e.g., IL-12), and are capable of trafficking antigen from the periphery to the T-cell areas of lymph nodes (Banchereau and Steinman, 1998). DCs use a variety of surface receptors for the phagocytosis of apoptotic cells, including the avb5 integrin, which works in concert with CD36 (Albert et al., 1998b), FcR that engulf antibody-opsonized dying cells (Rovere et al., 1998a), and scavenger receptors (Peiser et al., 2002). Moreover, opsonization of apoptotic cells by the complement factor iC3b can facilitate their uptake by immature DCs (Verbovetski et al., 2002). With respect to internalization via

Fig 1 The minimal components of the antitumor immune response. When tumor cells enter the apoptotic pathway, they display ‘‘eat-me’’ signals that lead to specific interactions with antigenpresenting cells and in particular dendritic cells (DCs) (step 1). DCs take up antigen and present tumor-derived peptides bound to major histocompatibility complex (MHC) class I molecules while expressing costimulatory signals (exemplified by B7) on the surface. In doing so, DCs migrate to the draining lymph node (step 2), where they undergo cognate interactions with MHC class I–restricted naive CD8þ T cells (cytotoxic T lymphocyte [CTL] precursors) and MHC class II–restricted CD4þ T cells (which provide help to either the CTL precursor or the DC, step 4). The CD8þ T cells then exit from lymph nodes (step 5) into peripheral locations to exert their cytotoxic activity against the tumor (step 6).

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the avb5 integrin, the intracellular signaling pathway has been characterized; it recruits a CrkII–Dock180 molecular complex, which in turn activates Rac1 for cytoskeletal rearrangement and the formation of a phagocytic cup capable of enveloping apoptotic cells (Albert et al., 2000). Although which pathways are critically required remains unclear, it is evident that DCs are able to present internalized antigen derived from apoptotic cells to T cells (Albert et al., 1998c), through a pathway that likely involves capture of the apoptotic cell, proteolysis by cathepsin D in the acidic endosomal compartment, further cytosolic proteolysis by the proteasome, translocation into the lumen of the ER by specific transporters associated with antigen presentation (TAP), and their loading into newly synthesized MHC class I molecules. An alternative pathway for antigen presentation may involve phagosomes containing apoptotic cells fusing with the ER, thus generating a specialized mixed compartment for optimal antigen presentation and offering an explanation for the particular efficacy of DCs as a professional APC (Fonteneau et al., 2003b; Guermonprez et al., 2003; Houde et al., 2003). DCs are effective in presenting antigens taken up from dying tumor cells. Thus, for instance, DCs from HLA-A*0201 patients with paraneoplastic cerebellar degeneration phagocytose apoptotic tumor cells and induce autologous T cells to lyse cells expressing the tumor antigen CDR2 (Albert et al., 1998a). Monocyte-derived DCs loaded with dead melanoma cells (killed with betulinic acid, a bona fide apoptosis inducer) (Fulda and Debatin, 2000) can prime naive CD45RAþCD27þCD8þT cells against the four shared melanoma antigens: MAGE3, gp100, tyrosinase and Mart1 (Berard et al., 2000). Similarly, immature DCs loaded with apoptotic bodies from multiple myeloma cells (irradiated), then stimulated with TNF-a for maturation, stimulate significantly higher T-cell proliferation than DCs pulsed with freeze-thaw lysates (Hayashi et al., 2003). By contrast, monocytes or macrophages, though more efficient at phagocytosis, reportedly cannot stimulate resting CD8þ T cells to develop into CTLs or are by far less efficient in doing so as compared to DCs (Albert et al., 1998c; Yrlid and Wick, 2000). Although most studies focus on DC interactions with classic T cells, it is important to note that DCs may also engage and activate gd T cells, NK cells, and NKT cells. The interaction between DCs and T lymphocytes occurs by antigen presentation in a multimolecular complex called the immunological synapse, which contains an antigen epitope bound to MHC and flanked by receptor–ligand interactions from costimulatory and adhesion molecules, formed by transient cell membrane contacts between the interacting cells (van Der Merwe and Davis, 2002). A similar ‘‘synapse’’ may be the morphological substrate of the immunostimulatory interaction between DCs and NK cells (Borg et al., 2004).

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D. Effector Cells of the Antitumor Immune Response: T and NK Cells One of the principal functions of CD4þ T lymphocytes is to provide help to APCs (in particular to DCs) for the activation of CD8þ T cells and the maintenance of memory responses. This help is mediated in part by the interaction between CD40-L on the surface of the CD4þ T cell and the CD40 receptor on the surface of the APC (Grammer and Lipsky, 2000). Although tumor-specific CD4þ T cells with cytotoxic properties have been reported, CD8þ T cells are considered the principal killer cells of the adaptive immune response. When interacting with tumor cells bearing the peptide– MHC class I complex that their TCR can engage activated CD8þ T cells induce apoptosis via two mechanisms: the predominant Ca2þ perforin– granzyme-B pathway and the CD95–CD95 ligand (FAS–FAS-L) pathway. In the perforin–granzyme-B pathway, perforin facilitates the delivery of granzyme-B (and other granzymes) from the lumen of the CTL granule to the cytosol of the tumor cell (Barry and Bleackley, 2002). Granzyme-B, which is a cysteine protease, then directly sets off the caspase activation cascade (by direct proteolytic activation of proenzymes) and/or cleavage of the proapoptotic Bcl-2 family protein Bid, in turn triggering mitochondrial failure and indirectly activating effector caspases activation. In contrast, the interaction of the CD95 ligand (CD95L, on the surface of CTL) with CD95 (expressed on tumor cells) can trigger the recruitment of the so-called ‘‘death-inducing signaling complex’’ (DISC), a complex that causes the apical activation of caspase-8 and then stimulates the activation of other caspases, either directly (in a proteolytic cascade) or indirectly (via activation of Bid) (Scaffidi et al., 1998). In addition, infiltrating T cells may secrete cytokines (in particular IFN-g and TNF-a) that stimulate tumor cell death, either directly (by ligation of specific cytokine receptors on the tumor cell surface) or indirectly (by stimulating local inflammation and macrophage-mediated tumor cell lysis). NK cells serve as the first line of the innate immunological defense, recognizing and eliminating cells with aberrant or loss of MHC class I expression. Thus, for tumors with low to undetectable MHC molecule expression, NK cells may be the predominant immune effector cells, killing primarily through the perforin–granzyme-B pathway and perhaps by means of IFN-g secretion. Alternatively, the presence of still undefined surface receptors may result in the engagement of activation receptors of NK cells. III. Subroutines of Cell Death: Apoptosis and Necrosis

Tumor cells can die through a variety of different subroutines of cell death, depending on the death-inducing stimulus and on the particular conditions of the cell’s metabolic and signal transduction networks. ‘‘Programmed cell

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death’’ may be considered a type of cell death that involves an active metabolism (with sustained ATP levels, at least at the beginning of the process), whereas ‘‘accidental cell death’’ (frequently called necrosis) would involve cell death with low or minimal ATP levels. However, ‘‘programmed cell death’’ is by no means synonymous with ‘‘apoptosis’’ and can be divided into different subtypes. Thus, cells can die from apoptosis (Kerr et al., 1972) or from other types of cell death considered to be different from apoptosis, namely apoptosis-like programmed cell death (Leist and Jaattela, 2001), necrapoptosis (Jaeschke and Lemasters, 2003), aponecrosis (Formigl et al., 2000), oncosis (Majno and Joris, 1995), paraptosis (Sperandio et al., 2000), limoktonia (Xue et al., 2001), autoschizis (Jamison et al., 2002), autophagic cell death (Bursch, 2001), mitotic catastrophe (Roninson et al., 2001), or terminal senescence (Roninson, 2003), just to mention a few expressions used in the specialized literature. These different types of cell death are defined by morphological criteria, a fact that misguides into the illusion that each of these categories is biochemically and functionally uniform. For example, apoptosis, by far the most common subroutine of cell death, also nicknamed type 1 programmed cell death, is defined as a type of cell death in which chromatin loses its substructures (chromatinolysis) and condenses (pyknosis), nuclear fragments are generated (karyorrhexis), the cells shrink, cytoplasmic organelles manifest no major ultrastructural changes, the plasma membrane remains near to impermeable, blebbing of the plasma membrane occurs, and eventually the cells breaks up in apoptotic bodies (Kerr et al., 1972). In contrast, autophagic cell death, type 2 programmed cell death, is characterized by the accumulation of multiple autophagic vacuoles containing degenerating cytoplasmic organelles without (or before?) cells undergoing apoptosis (Boya, 2003; Lockshin, 2001). Mitotic castastrophe has been tentatively defined as a type of cell death resulting from abnormal mitosis, which usually ends in the formation of large cells with multiple micronuclei and uncondensed chromatin (Roninson et al., 2001). Oncosis (frequently also called necrosis) is characterized by swelling of the cell, vacuolization of mitochondria and the ER, and an early rupture of the plasma membrane (Majno and Joris, 1995). Though interesting starting points and certainly relevant as historical anecdotes, we argue that phenomenological definitions are not operative and not particularly helpful. In medical jargon, it would be analagous to describing death by a ruptured cerebral aneurysm as ‘‘a type of death in which the patient, apparently in good health, having made some physical effort, screams and then collapses, dying after a short period of convulsion’’ without mentioning the underlying cause of the disease. For the sake of this discussion and in hopes of offering molecular definition to morphological classifications of death, we

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briefly summarize the biochemical definition of the most prominent types of cell death. A. Biochemical Definition of Apoptosis Apoptosis is a highly organized mode of cell death that is employed to eliminate superfluous, aged, injured, or infected cells in diverse biological settings. Anticancer chemotherapy is also largely mediated by apoptosis (Debatin et al., 2002; Herr and Debatin, 2001). Moreover, when the immune system (and in particular cytotoxic T and NK cells) attacks cancer cells, cell death involves apoptosis (Barry and Bleackley, 2002). During apoptosis, cells are dismantled from within and display plasma membrane alterations that provoke their removal by phagocytic cells. Thus, apoptosis is largely interpreted as a regulated process that involves the controlled demise of cellular structures and removal of the resulting debris so collateral damage to surrounding tissue may be minimized. As apoptosis is induced by a plethora of different inducers (Kroemer, 1995; Kroemer et al., 1995; Penninger and Kroemer, 1998) yet demonstrates a rather stereotypical pattern of morphological changes, irrespective of the cell type and the initial trigger, several investigators have postulated the existence of a so-called ‘‘central executioner’’ (Martin and Green, 1995) or ‘‘death machine’’ (Chinnaiyan and Dixit, 1996). Activation of the hypothetical central executioner during the effector stage would allow the ‘‘decision to die,’’ streamlining the specific and sometimes tissue-specific apoptotic pathways into one common mechanism of death and clearance. At present, two separate yet mechanistically intertwined phenomena are acknowledged to play a pivotal role in the death–life decision, namely caspase activation and mitochondrial membrane permeabilization (MMP). Caspases, a family of aspartic acid–specific proteases, are widely thought to be the demolition experts, coordinating and executing the apoptotic process (Nicholson and Thornberry, 1997). Because of their potentially lethal nature, caspases are synthesized as inactive precursors (zymogens) that either require limited proteolysis at internal aspartic acid (Asp) residues to become fully active and/or have to interact with an allosteric activator. This strategy provides an important means of keeping caspase activities under control and reduces the possibility that cells will inadvertently enter apoptosis. Caspases have a rare substrate specificity for Asp and require limited processing at Asp residues to become fully activated. Thus, caspases either become activated through autoproteolysis or are activated by other caspases or noncaspase proteases with a similar specificity for Asp residues. The 13 known mammalian caspases are expressed as single-chain proenzymes composed of three domains: an N-terminal propeptide (or pro-domain), a large subunit, and a small subunit. The group of caspases involved in apoptosis can be divided into several functional subgroups. Initiator or apical caspases (caspases-2,-8,-9,-10) are

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Fig 2 A scheme of the apoptotic program with particular emphasis on caspase activation cascades. Different types of signals or damage elicit the activation of a range of distinct initiator caspases that in turn stimulate the activation of the effector caspases. Effector caspases then cleave essential cellular components, inactivate survival pathways, and stimulate other catabolic hydrolases, thus sealing the cell’s fate.

responsible for initiating apoptotic caspase-activation cascades (Fig. 2). They have long N-terminal prodomains that contain recognizable protein–protein interaction motifs (caspase recruitment domains [CARDs] or death effector domains [DEDs]) that are also found in molecules that promote caspase activation. The second subgroup, the downstream or effector caspases (caspase-3,-6,-7), are held responsible for the dismantling of the cell during apoptosis and have relatively short prodomains or no prodomain at all. Effector caspases (Fig. 2) orchestrate the direct dismantling of cellular structures, disruption of cellular metabolism, inactivation of cell death inhibitory proteins, and the activation of additional destructive enzymes such as the caspase-activated DNAse, the DNAse largely responsible for internucleosomal DNA fragmentation (Adams, 2003). Caspase-1 is the prototype of the four inflammatory caspases; the family includes caspase-1,-4,-5, and -13 (caspase11 and -12 are the mouse homologs) (Nicholson and Thornberry, 1997). Knockout mice lacking caspase-1,-11, and-12 have been generated and all exhibit rather selective defects in cell death (Joza et al., 2002). With respect

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to their role in inflammation, caspase-1 and-5 have been shown to associate with Pycard/ASC and NALP1, a pyrin domain–containing protein with homology to NOD proteins (Tschopp et al., 2003). Together, these proteins constitute the inflammasome—a large signal-induced multiprotein complex that results in the activation of proinflammatory caspases and the cleavage of IL-1b and IL-18 (Martinon et al., 2002). The link between secretion of proinflammatory cytokines, cell death, and protective immunity must be explored. Apical caspases are typically activated by the recruitment of several procaspase molecules into protein complexes by specific adaptor molecules, thereby facilitating their allosteric activation and/or the close proximity between several caspase zymogens and their auto-proteolytic maturation. It appears that each apical caspase can be activated in a specific fashion. For example, the CARD of caspase-9 can interact with the CARD of Apaf-1, which in turn is activated and oligomerized when it comes into contact with cytochrome c leaking out from mitochondria. Thus, loss of the integrity of the outer mitochondrial membrane (which normally sequesters cytochrome c) leads to the prompt activation of caspase-9, through the cytochrome c/Apaf-1/caspase9 activation complex (also called apoptosome) (Wang, 2002). In contrast, caspase-2 (which also possesses a CARD) is likely to be activated by another protein complex that forms in the nucleus after DNA damage (Read et al., 2002). Caspase-8 and-10 (which both possess DED) are activated upon homotypic interaction with the DED of FADD, which in turn is either activated upon its recruitment into the DISC of so-called ‘‘death receptors’’ (the prototype being CD95/Fas) (Krammer, 2000). This implies that different types of cellular damage, affecting, for instance, different organelles, trigger distinct initiator caspases (Ferri and Kroemer, 2001b). Although there has been a marked tendency in the field to consider caspases as the principal regulators and executors of apoptotic cell death, it has become clear that the point of no return of the cell death process is largely determined by MMP (Penninger and Kroemer, 2003; Perfettini and Kroemer, 2003; Zamzami and Kroemer, 2003). Thus, multiple signal transduction pathways including caspases and caspase cleavage products (e.g., the caspase-8 cleavage product of Bid, a proapoptotic Bcl-2 protein) act on mitochondria to induce MMP, which is locally regulated by antiapoptotic members of the Bcl-2 family (inhibitors of MMP) and proapoptotic Bcl-2 homologs (inducers of MMP) (Cheng et al., 2001; Kroemer, 1997). MMP results in progressive bioenergetic failure with a loss of the mitochondrial transmembrane potential (DCm), as well as in the release of potentially toxic proteins from mitochondria (Fig. 3). Such apoptogenic effectors include the caspase-9 activator cytochrome (Liu et al., 1996), meaning that there is a considerable degree of cross-talk between caspase activation and MMP, to the extent that MMP constitutes a near-to-obligatory link between apical caspase activation and the explosive lethal activation of

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Fig 3 A scheme of the apoptotic program with particular emphasis on mitochondrial membrane permeabilization (MMP) as a central mechanism of cell death. A number of proapoptotic signal transduction pathways (some of which involve the activation of apical caspases) lead to mitochondrial dysfunction with the consequent release of a plethora of caspase activators and caspase-independent death effectors. These effectors can stimulate caspase-dependent apoptosis (full-blown apoptosis), caspase-independent subapoptosis (with some but not all morphological features of apoptosis), and cell death with prominent features of necrosis, depending on the local concentration of the death effectors and their endogenous inhibitors.

downstream effector caspase. In addition, MMP can culminate in the release of a series of caspase-independent death effectors, one of which is apoptosisinducing factor (AIF), a flavoprotein that translocates into the nucleus where it causes large-scale DNA fragmentation (Susin et al., 1999). Depending on the relative abundance of these effectors (e.g., caspases, AIF) versus their cellular inhibitors, the cell death process may involve a larger contribution of either caspases or caspase-independent death effectors, with important effects on the biochemistry of cellular dismantling (Green and Kroemer, 1998). Indeed, it appears that under the seemingly homogenous cover of ‘‘apoptosis,’’ cell death phenomena involving a variable degree of caspase activation and/or MMP are hidden.

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As we discuss, the central point is that in distinguishing death pathways, we may uncover important mechanisms by which dying cells control immune responsiveness. One important counterpoint to this argument has been offered by Green and Evan (2002), who argue that cell death is likely the summation of multiple hits—that it may appear as if one particular pathway were mediating cell death—when in reality that particular pathway may merely be the final straw that breaks the ‘‘apoptotic camel’s back.’’ We agree that in wild-type cells, a complex integration of death pathways occurs but argue that in fact the use of distinct death pathways may influence immunity in diverse ways. B. Incomplete Apoptosis without Caspase Activation ‘‘Complete’’ apoptosis, as described earlier, can be defined as a process in which caspases are activated and MMP occurs. However, in many cases, cell death exhibiting most if not all morphological signs of apoptosis is not accompanied by full caspase activation and/or lacks some of the features of MMP. Frequently, this type of ‘‘incomplete apoptosis’’ (also called apoptosis-like programmed cell death [Jaattela and Tschopp, 2003]) lacks full-blown chromatin condensation and oligonucleosomal DNA fragmentation (Susin et al., 2000). In contrast, we are not aware of convincing data showing that cell death can occur without some features of mitochondrial dysfunction, suggesting that MMP does constitute an important point of no return of the death process (Kroemer and Reed, 2000; Kroemer et al., 1998). Here, we enumerate a few examples of incomplete apoptosis that may be relevant to this discussion. Cross-linking of a number of cell surface receptors with monoclonal antibodies (mAbs) can provoke rapid cell death without the morphological features of apoptosis. For example, rituximab, a chimeric mAb targets the pan-B-cell marker CD20 and has been successfully used for the treatment of relapsed or refractory CD20-positive follicular non-Hodgkin’s lymphoma (Smith, 2003). In vitro data suggest that it can cause phosphatidylserine exposure and MMP without caspase activation (Chan et al., 2003). Ligation of CD47 by its natural ligand thrombospondin (TSP), or cross-linking by CD47 antibodies triggers caspase-independent cell death in normal and chronic lymphocytic leukemic B cells, through a pathway that involves the proapoptotic Bcl-2 family protein BNIP3 to mitochondria, dissipation of the DCm yet no cytochrome c release (Lamy et al., 2003). Importantly, BNIP3 is also involved in other pathophysiologically relevant models of caspase-independent cell death, including myocardial infarction (Kubasiak et al., 2002). Other examples include use of anti-CD99 (Pettersen et al., 2001) and anti–MHC class II antibodies (Nagy and Mooney, 2003), which kill cells without caspase activation. Alternatively, genetic alterations in the cell may modify the death pathway used. MCF-7 breast cancer cells treated with the active form of vitamin D3 or the vitamin D analog EB 1089, which now enters phase III trials, succumb

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in vitro with an apoptosis-like morphology. However, such cells do not show evidence of active caspases, instead they die via a calpain-dependent pathway (Mathiasen et al., 2002). A second example concerns the treatment of BCRABL–positive leukemic cells with imatinib mesylate (STI-571, Gleevec), a highly specific tyrosine kinase inhibitor. Again, a caspase-independent cell death with DCm loss (albeit no release of cytochrome c or AIF) is observed (Okada et al., 2004). In contrast, treatment of gliomas with flavopiridol induced cell death without mitochondrial cytochrome c release (and hence, without caspase activation), yet AIF does translocate from mitochondria to the nucleus, presumably accounting for the execution phase of apoptosis (Alonso et al., 2003). Together, these examples illustrate that some mechanisms of cell death induction may induce apoptosis with nonclassic features (in particular, absent caspase activation). It is, however, an ongoing conundrum whether this kind of manipulation, if applied to the treatment of cancer, may modulate an antitumor immune response. In addition to apoptosis, a number of different types of cell death that are energy demanding and thus belong to the realm of ‘‘programmed cell death’’ have been defined in morphological terms. In the next paragraphs, we critically address the question whether ‘‘autophagic cell death,’’ ‘‘mitotic catastrophe,’’ and ‘‘premature senescence’’ do share biochemical features of apoptosis. C. Autophagic Cell Death During macroautophagy, cytoplasmic organelles or a portion of the cytosol is engulfed by a structure known as autophagosome, which is defined by a double membrane from the rest of the cell. Subsequently, the autophagosome fuses with lysosomes, and the inner membrane of the resulting autophagolysosome, as well as the sequestered materials, is digested by lysosomal enzymes. Autophagic cell death (also called type 2 cell death), as characterized by the appearance of abundant vacuoles in the cytoplasm, has been initially described as a prominent form of programmed cell death in development (Lockshin and Zakeri, 2002). In addition, autophagy-inducing stimuli acting on cancer cells include starvation, hypoxia, radiation, antiestrogens, and cytokines such as TNF-a, IFN-g, and the tumor suppressor protein DAP kinase (Ogier-Denis and Codogno, 2003). Other tumor-relevant inducers of morphological signs of autophagic cell death include arsenic (in malignant glioma) (Kanzawa et al., 2003), oncogenic Ras (Kitanaka et al., 2002), DAP kinase (Inbal et al., 2002), and lysosomotropic agents such as chloroquine, hydroxychloroquine (Boya et al., 2003b), norfloxacine, and cyprofloxacine (Boya et al., 2003a). HeLa cells stimulated with IFN-g have also been reported to manifest some features of autophagic cell death (Inbal et al., 2002). Currently, the biochemical mechanisms of type 2 cell death remain unknown. As such, the definition of this modality of death is strictly morphological. What is known is that the accumulation of autophagic vacuoles may be due

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to either a stimulation of autophagy (and hence, the formation of autophagosomes) or an inhibition of their turnover (by fusion with lysosomes and subsequent degradation). In addition, there is some evidence suggesting that autophagic cell death just constitutes an early morphological manifestation of cellular damage that will lead to apoptosis at later stages and hence involves caspase activation and/or induction of MMP (Boya et al., 2003a,b). D. Mitotic Catastrophe The expression ‘‘mitotic catastrophe’’ has been widely used to describe yet another form of death affecting mammalian cells occurring during or shortly after mitosis, without cellular shrinkage or apoptotic chromatin condensation (Roninson et al., 2001). However, most reports describing ‘‘mitotic catastrophe’’ actually show cells with some phenotypical characteristic of apoptosis (such as hypercondensed chromatin aggregates) (Heald et al., 1993), which were interpreted in the past ‘‘premature chromatin condensation’’ (Chakrabarti and Chakrabarti, 1987; Sperling and Ra, 1974). Thus, there is no consensus on the distinctive morphological appearance of mitotic catastrophe as far as the extent of chromatin condensation (which is the morphological hallmark of apoptosis) (Ferri and Kroemer, 2001a; Kerr et al., 1972) is concerned, and there is increasing evidence that death through mitotic catastrophe may result from nuclear caspase-2 activation and subsequent MMP and thus may indeed constitute a special case of apoptosis, at least in biochemical terms (Castedo et al., 2004a,b,c). E. Premature Senescence Premature senescence (also called cytostasis) is characterized by terminal cell cycle arrest and may be triggered by DNA-damaging or differentiationinducing chemotherapies (Schmitt, 2003). The principal effectors of premature senescence are p53, p21, and p16INK4a (Schmitt et al., 2002b; te Poele et al., 2002). The downstream determinants of apoptosis (specifically Bcl-2 in a murine transgenic lymphoma model) can profoundly influence the response of malignant cells to chemotherapy in vivo, inducing a shift from apoptosis to senescence-like growth arrest (Schmitt et al., 2002a). Similarly, in vitro the suppression of apoptosis by the pan-caspase inhibitor Q-VD-OPH can lead to a shift from apoptosis to senescence, as defined by the cells flatten down, becoming irreversibly arrested in G1, overexpress p21 and the senescenceassociated b-galactosidase (Rebbaa et al., 2003). Conversely, HCT116 colon carcinoma cells treated with SN38 (the active metabolite of irinotecan) demonstrate a senescent phenotype (which depends on p21 and p53 but not on Bax). When treated with anti-sense Bcl-XL oligonucleotides, such cells manifest a switch from senescence to apoptosis (Hayward et al., 2003). Senescenceassociated b-galactosidase induction has also been found in vivo, in human

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breast tumor specimens post-neoadjuvant chemotherapy (te Poele et al., 2002). It is not known whether senescent cells finally undergo apoptosis and it is also a complete enigma whether senescent (dormant?) tumor cells are simply ignored by the immune system or whether they may actually constitute an extension of the immunogical ‘‘self’’ and thus trigger tolerance. F. Necrosis Necrotic cell death is frequently viewed as a type of cellular demise that occurs beyond any kind of regulation, as a result of ‘‘accidental cell death,’’ without any of the hallmarks of apoptosis. Rather, necrosis would be accompanied by a cellular hydrops or ‘‘oncosis,’’ shortly before the cells burst with release of cytosol and organelle contents into the intercellular space. One interpretation of the phenomenon of necrosis is to assume that acute disruption of the cellular metabolism, for instance, by ATP depletion, would be incompatible with any kind of cellular regulation. However, at least in some instances, stimuli that normally would induce apoptosis can induce necrosis in vitro, if the cellular ATP production is blocked (Leist et al., 1997). In some examples, antiapoptotic proteins from the Bcl-2 family can still block necrotic cell death, indicating that there is some kind of biochemical overlap in the regulation of apoptosis and necrosis (Kroemer et al., 1998). Accordingly, intermediate phenotypes (such as ‘‘necro-apoptosis’’) have been observed (Jaeschke and Lemasters, 2003). The mouse fibrosarcoid L929 cell line can be manipulated to undergo typical necrosis (by addition of TNF-a) or apoptosis (by addition of agonist CD95 antibodies), and the mechanistic differences between apoptosis and necrosis have been investigated to some extent in this model (Beyaert et al., 2002). Thus, in L929 cells, necrosis is not accompanied by the release of mitochondrial intermembrane proteins and lacks signs of caspase activation but is associated with an increased ROS production by mitochondria (Denecker et al., 2001). Inhibition of caspases can shift an apoptotic response to a necrotic one (Hirsch et al., 1997; Vercammen et al., 1998), and vice versa, inhibition of HSP90 can shift the necrotic death subroutine to apoptosis (Vanden Berghe et al., 2003). These findings illustrate that the current definition of necrosis, in biochemical terms, is still arguable. However, we suggest that a strong delineation should be made between the two forms of cell death with ‘‘programmed cell death’’ involving an active process that consumes energy (or at least maintains cellular viability at the beginning of the process) versus necrosis, where no ATP is consumed during what is typically an explosive death. Though of questionable relevance for physiological questions, there are examples of anticancer treatments/interventions that can be expected to induce necrosis as the preponderant death modality. This applies to cauterization, electrocoagulation, cryosurgical procedures, local application of microwaves,

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embolization, and to some extent photodynamic therapy. Unfortunately, there are no studies, at least to our knowledge, whether this kind of intervention stimulates anticancer immune responses in patients. IV. In Vivo Immunogenicity of Apoptotic Versus Necrotic Tumor Cells

Given that apoptosis is a physiological phenomenon affecting several million cells per second (Thompson, 1995), it is tempting to assume that in immunological terms, apoptosis must be either silent or tolerogenic (Fig. 4). Similarly, if apoptosis is the preponderant type of cell death induced by anticancer chemotherapy, yet chemotherapy does not trigger any antitumor immune response, then it is tempting to expect that tumor cell apoptosis is immunologically ‘‘null’’ or negative (although it is also possible that chemotherapy is simply immunosuppressive). However, intramuscular DNA vaccines elicit improved CD4þ and CD8þ responses, when proapoptotic genes (caspase-2 or -3, CD95) are coinjected (Chattergoon et al., 2000; Sasaki et al., 2001), indicating that apoptosis by itself is not by definition tolerogenic. Nonetheless, numerous investigators have formulated theories, including the following apoptosis occurring in a fashion in which the dying cells are not ‘‘seen’’ by the immune system (e.g., as the result of rapid phagocytosis by neighboring cells and macrophages) (Kerr et al., 1972); apoptosis as intrinsically immunosuppressive (because of the production or elicitation of immunosuppressive cytokines) (Fadok et al., 1998); apoptosis as a trigger for antigen-specific immune tolerance (Sauter et al., 2000; Steinman et al., 2000) (perhaps because of the deletion of antigen-specific T cells (Albert et al., 2001; Kurts et al., 1997b); apoptotic cells as inducers of regulatory T cells (Ferguson et al., 2002); and/or that apoptosis would fail to emit the ‘‘danger signals’’ required to trigger an active immune response (Gallucci et al., 1999; Matzinger, 2002). In strict contrast, it is widely assumed that necrosis, the type of cell death that leads to ‘‘spilling of the cell’s content into the tissue’’ (Savill and Fadok, 2000), causes local inflammatory reactions, and provides ‘‘danger signals’’ that (Matzinger, 2002) must be highly immunogenic. DCs treated with freeze-thaw lysates from fibroblasts (but not apoptotic fibroblast cells generated by ceramide treatment) express B7.2, MHC class II, and CD40 (Gallucci et al., 1999; Sauter et al., 2000). This suggests that lysates contain ‘‘danger signals’’ endowed with the capacity of stimulating DCs (and by extension T cells). Indeed, necrotic cells (but not apoptotic cells) activate nuclear factor-kB (NF-kB) and induce expression of genes involved in inflammatory and tissue repair responses, in fibroblasts, macrophages, and DCs (Basu et al., 2000; Li et al., 2001), in a reaction that depends on the expression of TLR2 (Li et al., 2001). Such genes include the cytokine-induced neutrophil chemoattractant (KC) and macrophage inflammatory protein-2 (MIP-2),

Fig 4 Schematic illustration of the immune response initiated by dying tumor cells. (A) When dying cells are recognized by macrophages (or neighboring cells) at an early stage of apoptosis, corpses are cleared in a silent fashion, without any local response. (B) At a later stage of apoptosis, macrophages are stimulated to reduce the production of proinflammatory mediators and to increase that of anti-inflammatory cytokines. The net result

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metalloproteinase-3, and vascular endothelial growth factor (VEGF). Necrotic (but not apoptotic) cells release the chromatin protein high-mobility group box chromosomal protein-1 (HMGB1), and this protein can actually stimulate inflammation. This is possible because apoptosis leads to hypoacetylation of histone H4, which causes HMGB1 to bind to chromatin, where it is retained (Scaffidi et al., 2002). Altogether many studies have produced evidence that necrotic cells must be more immunostimulatory than apoptotic ones. If the aforementioned assumption—that apoptosis is the nonimmunogenic form of cell death—is true, then one corollary would include that necrotic tumor cells must be a more potent source of antigen for anticancer vaccination. This is not the case in most studies that have comparatively addressed the immunogenicity of apoptotic versus necrotic tumor cells (Table I). It can be argued that these studies possess several methodological problems rendering the results difficult to interpret. First, in most instances, ‘‘necrosis’’ is produced by repeated freezing and thawing, a process that is unlikely to mimic necrotic cell death (oncosis) induced by hypoxia or anticancer therapy in vivo. Moreover, in some instances, ‘‘necrotic cells’’ have been obtained by induction of apoptosis, followed by cellular lysis (e.g., freeze-thaw postinfluenza infection) (Fonteneau et al., 2003). Many experiments have been undertaken under the influence of the misguided view that the apoptotic death comes in a single variety. In experiments performed by immunologists, tumor cell apoptosis is mostly induced by ultraviolet B (UVB) induction—that is, with one particular death inducer that, different from metabolic or differentiating agents, induces apoptosis without HSP70 expression and thus fails to ‘‘stress’’ the cells well before antigen transfer to the immune system (Gregoire et al., 2003). Worse, in most if not all cases the ‘‘apoptotic’’ population generated by treatment of cancer cells in vivo is actually a mixture of cells that are in the process of apoptosis, of late-stage apoptotic cells, and of cells that have undergone secondary necrosis (i.e., lysis after apoptosis). Such cells are not (or rarely) seen in vivo (Castedo et al., 1995, 1996), because in situ dying cells are rapidly and efficiently cleared by professional and nonprofessional phagocytes (Surh and Sprent, 1994). of this effect is immune suppression. (C) If apoptotic cells interact with immature dendritic cells (iDCs), such cells can cause tolerance, either by transfer of the antigen to resident lymph node DCs, by eliciting immunosuppressive mechanisms, or by silencing (anergizing or deleting) tumor antigen–specific T cells. (D) If apoptotic cells interact with iDCs, which subsequently mature by stimulation of toll-like receptors (either by endogenous ligands or by pathogen-associated molecular patterns [PAMPs]) and/or receive stimuli via engagement with T-cell help (exemplified by CD40L), they become effective in cross-priming cytotoxic T lymphocyte precursors. (E) Necrotic cells (and in particular heat shock protein–expressing stressed cells) may stimulate themselves and iDCs to undergo maturation, thereby stimulating an active immune response. Consult the main text for a detailed discussion of the experimental evidence for or against these mechanisms.

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Attempts have been undertaken to purify apoptotic cells, for instance, by staining them with propidium iodide plus annexin-V-FITC followed by fluorescence-activated cell sorting (FACS) of the propidium iodide-negative, annexin-V-FITC–positive population (Kotera et al., 2001). However, this manipulation masks phosphatidylserine residues on the surface of apoptotic cells (because of binding of annexin-V-FITC), thereby interfering with the recognition of the apoptotic cell by phosphatidylserine receptors and possibly altering its in vivo fate. Moreover, it is reasonable to expect that even this population of cells is at a stage of apoptosis beyond that usually found in vivo because evidence suggests that phagocytosis may occur upstream of our earliest measures of cell death (Brown et al., 2002; Castedo et al., 1995, 1996; Kurosaka et al., 2003). Keeping these critiques in mind, it is still revealing to consider that in most experiments in which DCs were pulsed with apoptotic or necrotic tumor cells, followed by evaluation of their antitumor vaccination efficacy in vivo, DCs pulsed with apoptotic tumor cells turned out to be more potent activators of T-cell responses (Table I). The one exception is the observation that DCs pulsed with lysates form B16 melanoma cells or with FACS-purified apoptotic cells (annexin VþPI cells after UVB induction) were found equivalent in the induction of a protective antitumor (Kotera et al., 2001). In addition, in vivo induction of apoptosis in established hemagglutinin (HA) transfected AB1 mesotheliomas with gemcitabine caused cross-presentation of HA to CD8þ T cells, proliferation of HA-specific CD8þ T cells, in vivo CTL activity, and no signs of deletion or functional tolerance of HA-specific CD8þ T cells (Nowak et al., 2003a). Altogether these data are in apparent conflict with what might have been expected, namely that necrosis would be more immunostimulatory than apoptosis. To date, there are only a few convincing series of experiments suggesting that apoptotic cells are less immunogenic than necrotic debris. One example is the PROb rat colon cancer model system. A PROb clone (REGb) that failed to undergo normal apoptosis but spontaneously died from atypical cell death (without caspase activation and without mitochondrial cytochrome c release), different from the parental PROb line (Larmonier et al., 2002), tended to induce a protective antitumor immune response when inoculated subcutaneously (Bonnotte et al., 2000). Fluorescence-labeled REGb cells were found as apoptotic bodies within DCs of the T-cell area of the draining lymph node several days after injection. When these cells were transfected with Bcl-2, no such relocalization to the draining lymph node was observed and the priming of CD8þ T cells was impaired (Bonnotte et al., 2000), whereas tumor rejection was suppressed (Bonnotte et al., 1998). Similarly, a PROb clone manipulated to stably express an antisense cytochrome c complementary DNA (cDNA) underwent atypical apoptosis without caspase activation (Schmitt et al., 2004). In vivo

TABLE I Comparative Analysis of Necrotic and Apoptotic Tumor Cells as Elicitors of the Antitumor Immune Response Tumor cell type B16 melanoma

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E.G7 thymoma expressing ovalbumin SCCVII squamous carcinoma PROb rat colon carcinoma

Death inducers A: UVB+FACS purification of AnnV+PI cells N: lysate A: irradiation N: freeze thaw A: irradiation N: freeze thaw A: butyrate

CT26 colon carcinoma

N: lysate A: g-irradiation

RENCA renal carcinoma

N: freeze thaw A: g-irradiation N: freeze thaw

Observation

Conclusion

Reference

A ¼ Na

Kotera et al., 2001

A>N

Strome et al., 2002

DCs pulsed with A have higher anti-tumor activity than DCs pulsed with N.

A>N

Strome et al., 2002

Thioglucollate-induced peritoneal exudate cells pulsed with A elicit higher anti-tumor activity than cells pulsed with N.

A>N

Henry et al., 1999

DCs pulsed with A are more efficient anti-tumor vaccines.

A>N

Scheffer et al., 2003


A>N


DCs pulsed with A or N have similar antitumor activity in vivo. A induces more IL-12 p70 secretion than N in vitro. No difference in HSP70 content. DCs pulsed with A have higher anti-tumor activity than DCs pulsed with N.

B16 melanoma

RMT T-cell lymphoma

PROb rat colon carcinoma

PROb rat colon carcinoma

A: g-irradiation N: freeze thaw A: mitomycin plus serum depletion or UVB N: freeze thaw None

None


A>N


Subcutaneous of A (but not N) enhances the frequency of mice not developing tumors.

A>N

Ronchetti et al., 1999

A clone expressing antisense cytochrome c dies from atypical (nonapoptotic) cell death without caspase activation and is more immunogenic than the vector-only control line. A subclone undergoing atypical (nonapoptotic) cell death without caspase activation is more immunogenic than the wild-type control line.

A

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