CD137 Pathway: Immunology and Diseases

CD137 Pathway: Immunology and Diseases

CD137 Pathway: Immunology and Diseases Edited by

LIEPING CHEN John Hopkins University Baltimore, MD, USA

Editor: Lieping Chen Department of Dermatology and Oncology Johns Hopkins University School of Medicine 600 N. Wolf Street, Jefferson 1-121 Baltimore, MD 21205 [email protected]

Library of Congress Control Number: 2006933637 ISBN 10: 0-387-31322-2 ISBN 13: 978-0-387-31322-1 Printed on acid-free paper.  C 2006 Springer Science+Business Media, LLC. All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, Inc., 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.

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Preface It is still hard to believe that manipulation of a single protein on the cell surface or an interaction of two or more proteins, which at times is collectively referred to as a “pathway,” could have such a profound effect on our immune system. At present, a number of such proteins or pathways have been identified. These observations could only be interpreted in a way that, although tens of thousand of proteins are required for a perfectly healthy immune system, many of these pathways may work in either interconnected or linear fashion. Therefore, the combined understanding of each pathway, their interactions with other pathways, and the functional consequence, is a cornerstone for our interpretation of pathological basis of diseases and future treatments. It is important to stay abreast on the pace of progress, which I refer to as periodic summary of incremental and breakthrough discoveries in each pathway by the experts and the leader in the field. The CD137 Pathway: Immunology and Diseases represents such an effort and this is the first attempt to summarize our understanding of the CD137 pathway. The following chapters cover the majority of active areas of research in this pathway and also provide an essential resource to both the nonexperts and experts in the field. I would like to thank all of the authors for their contributions with this work and my administrative assistant, Jennifer Osborne, for her patience and assistance with editing. I would also like to thank The Johns Hopkins Medical Institutions and The National Institutes of Health for their financial support. Lieping Chen

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Contents 1. Genes, Transcripts and Proteins of CD137 Receptor and Ligand

1

Dass S. Vinay and Byoung S. Kwon Overview .............................................................................. Discovery, Alternative Names, and Structure of CD137 ........................... The CD137 Gene ..................................................................... The CD137 Protein ................................................................... CD137L ............................................................................... 5.1. The Discovery of CD137L ...................................................... 5.2. CD137L Structure and Expression ............................................. 6. CD137L Gene ......................................................................... 7. CD137L Protein ...................................................................... 7.1. Regulation of RNA and Protein Expression ................................... 7.2. CD137-CD137L in Health and Disease ........................................ 8. Future Directions ..................................................................... Acknowledgments .................................................................... References .............................................................................

1 3 4 5 6 6 6 7 7 8 9 11 11 11

2. CD137 Signal Transduction

15

1. 2. 3. 4. 5.

Hyeon-Woo Lee and Byoung S. Kwon 1. 2. 3. 4.

Background ........................................................................... CD137 (4-1BB) As an Immune Stimulator ......................................... CD137 Signal Transduction ......................................................... Concluding Remarks ................................................................. References .............................................................................

3. Significance of Reverse Signal Transduction for the Biology of the CD137 Receptor/Ligand System

15 16 17 22 23

29

Herbert Schwarz 1. Biology of Reverse Signaling Through CD137 Ligand ............................ 1.1. CD137 Ligand Activities on Monocytes and Macrophages .................. 1.2. CD137 Ligand Activities on Dendritic Cells .................................. 1.3. CD137 Ligand Activities on B Cells ........................................... 1.4. CD137 Ligand Activities on Bone Marrow Cells ............................. 1.5. CD137 Ligand Activities on T Cells ........................................... 1.6. CD137 Ligand Activities on Non-hematopoietic Cells .......................

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30 30 34 34 35 36 37

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Contents

1.7. CD137 Ligand Signal Transduction Pathway ................................. 1.8. Regulation of CD137 Ligand Signaling ....................................... 1.9. Influence of Soluble CD137 and Soluble CD137 Ligand on CD137 Ligand Signaling ....................................................... 2. Bidirectional Signaling in Other Receptor/Ligand Systems ....................... 3. Concluding Remarks ................................................................. References .............................................................................

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4. CD137 Signal in the Regulation of Innate Immunity

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39 40 41 42

Lieping Chen 1. 2. 3. 4. 5. 6.

Introduction ........................................................................... NK Cells .............................................................................. Macrophages/Monocyte .............................................................. Dendritic Cells ........................................................................ Granulocytes .......................................................................... Summary .............................................................................. References .............................................................................

5. Regulation of T Cell-Dependent Humoral Immunity Through CD137 (4-1BB) Mediated Signals

47 47 49 50 51 52 52

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Robert S. Mittler, Liguo Niu, Becker Hewes, and Juergen Foell 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction ........................................................................... T and B Cell Activation and Costimulation ......................................... CD137 Expression and T Cell Costimulation ...................................... Anti-CD137-Mediated Suppression of Humoral Immunity ....................... Anti-CD137 Induced Suppression of Autoantibodies .............................. DC Function, CD137 Expression and Signaling ................................... Anti-tumor Immunity and B Cells ................................................... Anti-CD137 mAbs Disrupt Hematopoiesis in Mice. .............................. Concluding Remarks ................................................................. References .............................................................................

6. CD137 in the Regulation of T Cell Response to Antigen

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Yuwen Zhu and Lieping Chen 1. CD137 in Na¨ıve T Cell Costimulation .............................................. 1.1. Effects of CD137 Engagement on CD8+ T Cells ............................. 1.2. Effects of CD137 on CD8+ T Cell Priming, Division and Survival ......... 1.3. Effects of CD137 on CD4+ T Cells: Positive or Negative? ................... 2. CD137 and Regulatory T Cells (Treg) .............................................. 3. CD137 and T Cell Anergy ........................................................... 4. CD137 and Memory T Cell (Tm) Response ........................................ 5. Conclusions and Perspectives ........................................................ References .............................................................................

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Contents

7. Autoimmune Diseases

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Yonglian Sun and Yang-Xin Fu 1. Introduction ........................................................................... 2. Role of CD137/CD137L Interaction in the Pathogenesis of Autoimmune Diseases ............................................................. 2.1. Lack of CD137/CD137L Interaction Prevents Autoimmune Diseases ...... 2.2. Soluble CD137 and Autoimmune Diseases ................................... 3. Treatment of Autoimmune Disease with Agonistic Anti-CD137 ................. 3.1. Experimental Autoimmune Encephalomyelitis (EAE) ........................ 3.2. Experimental Autoimmune Uveitis (EAU) .................................... 3.3. Systemic Lupus Erythematosus (SLE) ......................................... 3.4. Collagen-induced Arthritis (CIA) .............................................. 3.5. Chronic Graft-Versus-Host Disease (cGVHD) ................................ 4. Mechanisms Involved in CD137 Agonist-mediated Inhibition of Autoimmune Diseases ............................................................. 4.1. Apoptosis of T Lymphocytes ................................................... 4.2. Regulatory T Cells and IFN-γ .................................................. 4.3. Helper T Cell Anergy ........................................................... 4.4. B Cell Apoptosis ................................................................ 5. Summary .............................................................................. References .............................................................................

8. CD137/CD137 Ligand in Tumor and Viral Immunotherapy

97 98 98 99 100 101 102 103 105 105 106 106 107 108 109 109 110

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˜ Ignacio Melero, Oihana Murillo, Inigo Tirapu, Eduardo Huarte, Ainhoa Arina, Laura Arribillaga, and Juan Jose´ Lasarte 1. CD137 (4-1BB) and CD137 Ligand (4-1BB-Ligand) Meet Tumor Immunology ........................................................................... 1.1. The Arrival of Agonistic Anti-CD137 Monoclonal Antibodies .............. 1.2. A Comparison with Anti-CTLA-4 Monoclonal Antibodies .................. 1.3. Transfection of CD137 Ligand into Malignant Cells ......................... 2. Developments and Improvements on CD137/CD137 Ligand Therapeutic Strategies .............................................................................. 2.1. Immunization: CD137 Breaks Ignorance and Tolerance ..................... 2.2. CD137 as an Adjuvant for Adoptive T Cell Therapy and Bone Marrow Transplantation .................................................................. 2.3. CD137 Acting in Synergy with Cytokines and Other Costimulatory Molecules ........................................................................ 3. CD137/CD137 Ligand in the Antiviral Immune Response and in Viral Vaccination ............................................................................ 4. Reflections on the Mechanism(s) of Action ......................................... 5. Using Preclinical Information for Clinical Development of Immunotherapy .... References .............................................................................

Index ......................................................................................

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Contributors Ainhoa Arina Centro de Investigaci´on M´edica Aplicada y Cl´ınica Universitaria Universidad de Navarra Pamplona, Spain

Ignacio Melero Centro de Investigaci´on M´edica Aplicada y Cl´ınica Universitaria Universidad de Navarra Pamplona, Spain ˜ Inigo Tirapu Centro de Investigaci´on M´edica Aplicada y Cl´ınica Universitaria Universidad de Navarra Pamplona, Spain

Becker Hewes Department of Pediatric Hematology and Oncology Emory University School of Medicine 954 Gatewood Road Atlanta, GA 30329

Juan Jose´ Lasarte Centro de Investigaci´on M´edica Aplicada y Cl´ınica Universitaria Universidad de Navarra Pamplona, Spain

Byoung S. Kwon Department of Ophthalmology LSU Eye Center, Louisiana State University Health Sciences Center New Orleans, LA USA

Juergen Foell Division of Pediatrics, Hematology, Oncology, and Immunology Martin Luther University Halle-Wittenberg, 06097 Halle, Germany

Dass S. Vinay Department of Ophthalmology LSU Eye Center, Louisiana State University Health Sciences Center New Orleans, LA USA

Laura Arribillaga Centro de Investigaci´on M´edica Aplicada y Cl´ınica Universitaria Universidad de Navarra Pamplona, Spain

Eduardo Huarte Centro de Investigaci´on M´edica Aplicada y Cl´ınica Universitaria Universidad de Navarra Pamplona, Spain

Lieping Chen Department of Dermatology and Oncology Johns Hopkins University School of Medicine 600 N. Wolf Street, Jefferson 1-121 Baltimore, MD 21205

Herbert Schwarz Department of Physiology, National University of Singapore 2 Medical Drive, MD 9 Singapore 117597

Liguo Niu Emory Vaccine Center, Emory University School of Medicine 954 Gatewood Road Atlanta, GA 30329

Hyeon-Woo Lee Department of Pharmacology School of Dentistry Kyung Hee University Seoul 130-701, Korea

Oihana Murillo Centro de Investigaci´on M´edica Aplicada y Cl´ınica Universitaria Universidad de Navarra Pamplona, Spain

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xii Robert S. Mittler Department of Surgery, Emory Vaccine Center Emory University School of Medicine 954 Gatewood Road Atlanta, GA 30329 Yang-Xin Fu The Department of Pathology and Committee in Immunology The University of Chicago Chicago, Illinois, USA

Contributors Yonglian Sun The Department of Pathology and Committee in Immunology The University of Chicago Chicago, Illinois, USA Yuwen Zhu Department of Dermatology and Oncology Johns Hopkins University School of Medicine Baltimore, MD USA

1 Genes, Transcripts and Proteins of CD137 Receptor and Ligand Dass S. Vinay and Byoung S. Kwon

CD137 and CD137L belong to the tumor necrosis factor (TNF) superfamily, a group of cysteine-rich cell surface molecules. With a few exceptions, both CD137 and its ligand, CD137L, are activation induced. CD137 activates CD8+ T cells more strongly than CD4+ T cells, and is a potent inducer of IFN-γ. Stimulation through CD137L also relays activation signals to B cells and monocytes. These signals elicit activation of NF-κB via the TRAF-NIK pathway and lead to the induction of a plethora of immune modulators that accentuate the ongoing immune reaction. CD137 and CD137L-deficient mice develop normally, have normal numbers of T and B cells and only demonstrate modest immune malfunction. However, in vivo administration of agonistic anti-CD137 mAb protects strongly against a variety of autoimmune and non-autoimmune diseases. The basis of this protection is unclear; however, it seems to involve an indoleamine dioxygenase (IDO)-dependent process in which pathogenic T cells are killed/suppressed by “regulatory CD11c+ CD8+ T cells.” In this review, the origins and functional features of CD137 and CD137L are discussed.

1. Overview Co-stimulation, an integral component of immune regulation, is required for progressive T cell activation. T cell activation without co-stimulation induces anergy in which subsequent stimulation inhibits T cell responsiveness (Schwartz, 1990). Since the description of the two-signal model for T cell activation by Bretscher and Cohn (1970), understanding of the activation requirements of T cells has progressed rapidly and attained further prominence with the emergence of the CD28-B7 pathway. Several immunological ideas have since been refined, and a clearer picture of the events is slowly emerging. Dass S. Vinay • Department of Ophthalmology, LSU Eye Center, Louisiana State University Health Sciences Center, New Orleans, LA, USA Byoung S. Kwon • Department of Ophthalmology, LSU Eye Center, Louisiana State University Health Sciences Center, New Orleans, LA, USA and Immunomodulation Research Center and Department of Biological Sciences, University of Ulsan, Ulsan, Korea 1 CD137 Pathway: Immunology and Diseases. Edited by Lieping Chen, Springer, New York, 2006

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T cell

CD27 CD40 CD30

CD137 CD134 GITR CD70 CD154 CD30L CD137L CD134L GITRL

APC

Figure 1.1. Members of the stimulatory TNFR/TNF protein families. Although the patterns of expression of the various ligand–receptor pairs are represented in a generalized format, the actual expression patterns vary in individual cases, as some of the receptors and ligands (CD137/CD137L) can also be expressed on the same cell (see text). In most cases, the expression of CD137 or CD137L on the same type of cell was found to be functional. Unless mentioned, expression of the members of TNFR/TNF families shown is activation-induced (see text). The individual members of this superfamily are known to transmit either co-stimulatory or apoptotic signals.

CD137 and CD137L are an important receptor–ligand pair that belong to the tumor necrosis factor (TNF) superfamily (Vinay and Kwon, 1998; Figure 1.1). This family includes proteins that have cytoplasmic death domains and can induce apoptosis, as well as others with no apparent homology in their cytoplasmic tails. The latter group of receptors is involved in gene activation and anti-apoptotic signaling. Signal transduction by members of this family occurs through TNF receptor-associated factors (TRAFs) which counteract apoptosis via inhibition of apoptosis proteins (IAPs) and/or nuclear factor kappa B (NF-κB) (Croft, 2003). CD137 exists as both a 30-kDa monomer and a 55-kDa homodimer (Pollok et al., 1993). It is inducible (Kwon and Weisman, 1989; Pollok et al., 1993) and is primarily expressed on activated CD4+ and CD8+ T cells, activated dendritic cells (Pollok et al., 1993) and activated NK and NKT cells (Melero et al., 1998). It is constitutively expressed on primary human monocytes, blood vessel endothelial cells, and human follicular dendritic, and CD4+ CD25+ regulatory T cells (Broll et al., 2001; Kienzel and von Kempis, 2000; Lindsted et al., 2003; McHugh et al., 2002). CD137 binds CD137 ligand (CD137L), a member of the TNF superfamily, and exists as a disulfide-linked homodimer (Goodwin et al., 1993). It is expressed

Genes, Transcripts and Proteins of CD137 Receptor and Ligand

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on mature dendritic cells (DeBenedette et al., 1997) as well as on activated B cells and macrophages (Pollok et al., 1994). In recent years, and especially after the generation of CD137 and CD137L knockout mice (DeBenedette et al., 1999; Kwon et al., 2002), this receptor–ligand pair has provided valuable insights into T cell immunity. Activation via CD137 generates unique biological signals. Although CD137 signaling preferentially promotes the proliferation and survival of CD8+ T cells (Shuford et al., 1997; Takahashi et al., 1999), it also supports IL-2 production by CD4+ T cells (Gramaglia et al., 2000), and prevents activation-induced cell death (Hurtado et al., 1997). CD137 has been to shown to transmit signals for potent and CD28-independent immune responses (Halstead et al., 2002; Saoulli et al., 1998). In spite of isolated reports that CD137 ligation supports Th2 responses (Chu et al., 1997), most workers believe that it amplifies Th1 responses (Kim et al., 1998). In vivo administration of agonistic anti-CD137 mAb eradicates established tumors (Melero et al., 1997), prevents the formation of autoimmune lesions (Foell et al., 2003; Seo et al., 2004; Sun et al., 2002), inhibits graft versus host disease (Kim et al., 2004), and reduces T-dependent B cells responses affecting humoral immunity (Mittler et al., 1999). Although there are few studies that relate CD137L and immune function, the available data suggests that CD137L is expressed on activated macrophages, and DC and B cells (Diehl et al., 2002; Futugawa et al., 2002; Laderach et al., 2003; Summers et al., 2001). Signaling through CD137L either by anti-CD137L mAb or by CD137 Fc/anti-Fc has been shown to promote B cell proliferation in the context of anti-μ (Pollok et al., 1994), and cytokine/chemokine production by monocytes and dendritic cells (Langstein et al., 1998; Wilcox et al., 2002). CD137-deficient mice develop normally and are viable and fertile; they make normal humoral responses to vesicular stomatis virus, exhibit moderately reduced anti-KLH IgG2a and IgG3 isotype responses, display diminished virus-specific cytokine production and CTL activity; and have increased turnover of myeloid precursor cells in the peripheral blood, bone marrow, and spleen (Kwon et al., 2002). Recent work in our laboratory demonstrates that CD137-null mice have suboptimal NK/NKT cells and associated functions, higher levels of LPS-induced septic shock and diminished IL-4-dependent Th2 immune responses (Vinay et al., 2004). CD137L knockout mice also develop normally and have roughly normal numbers of T cells but have impaired ability to generate CTL responses to influenza virus (DeBenedette et al., 1999).

2. Discovery, Alternative Names, and Structure of CD137 CD137 was first identified in screens for receptors on mouse concanavalin A-activated helper and cytotoxic T cell lines (Kwon and Weissman, 1989). CD137 (also called 4-1BB) was originally named “induced by lymphocyte activation” (ILA) in humans, and 4-1BB in the mouse (Pollok et al., 1993; Schwarz et al., 1993). It is a 30-kD glycoprotein and exists both as a monomer and a 55-kD

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dimer on the T cell surface. The entire gene spans approximately 13 kb of mouse chromosome 4.

3. The CD137 Gene Accession numbers: human (AL009183, AY438976) and mouse (U02567) The CD137 cDNA was initially isolated from activated murine T cells by a modified differential screening procedure (Kwon and Weissman, 1989). Its deduced amino acid sequence and its transcript expression profile indicated that it was an inducible T cell surface molecule (Kwon and Weissman, 1989; Pollok et al., 1993). CD137 was categorized as an early activation gene because the protein synthesis inhibitor, cyclohexamide, blocked formation of its transcripts (Kwon et al., 1987). The nucleotide sequence of CD137 contains a single long open-reading frame, starting with the ATG codon at positions 1–3. This reading frame encodes a polypeptide of 256 amino acids with a Mr of 27,587. The assigned ATG is preceded by an in-frame termination codon TGA (nucleotide residues -5 contains to 4) with eight out of nine residues similar to the consensus sequence (CCRC-CATGG, where “R” represents guanosine or adenine). The codon for the carboxy terminal leucine is followed by the translation termination codon TGA (nucleotide residues 769–771). The CD137 transcript contains an unusually long 3 -untranslated sequence that does not extend as far as the poly(A)+ tail. A potential polyadenylation signal is located at nucleotides 1158–1163. This signal may sometimes be functional because CD137 transcripts are of at least two different sizes. CD137 (Figure 1.2A and 1.2B) (accession number: U02567) is made up of eight exons and seven introns, in which there are two exons for the 5 UTR and eight for the coding region. Two kinds of UTR sequences were detected in the DNA sequence and found to be separated by an intron of ∼2.5 kb in length. The cysteine-rich extracellular domain is constructed from six exons, but most of the putative functional domains are encoded by separate exons. The signal sequence, transmembrane region, and serine, threonine, proline (STP)-rich region immediately proximal to the transmembrane domain are located in separate exons. The cytoplasmic domain that contains the p56lck -binding site is located in the last exon of the gene. Exon/intron boundaries were assigned by comparing the CD137 cDNA sequence with the genomic sequence. No TATA box-related elements were found in the flanking sequence of the type I 5 UTR. Instead, there were very good matches to the consensus TPA-responsive element (AP-1) at nt −18 to −10, and of the NF-κB-binding sequence at nt −49 to −39. Upstream of these elements, this region contains a potential ets-binding site at nt −169 to −162, an activator protein 2 (AP-2)-binding site at nt −498 to −494, and an SP-1 binding site at nt −522 to −516. The 5 flanking region of the type II 5 UTR contains a TATA-related element at nt −28 to −23. Two potential ets-binding sites appear at nt −15 to −8 and −139 to −132, two potential AP-2-binding sites at nt −89 to −82 and −331, and a very good match to an AP-1-binding site at nt −311 to −302.

Genes, Transcripts and Proteins of CD137 Receptor and Ligand

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Figure 1.2. Genomic organization of the human (A) and murine (B) CD137 receptors. Exons are shown as boxes; untranslated regions (UTR) are represented by black shading, and protein coding regions by no shading; they are labeled with Roman numerals. Lengths of introns and exons are shown in Arabic numerals (base pairs).

4. CD137 Protein Accession numbers: human (BC006196. L12964, U03397) and mouse (AK019885, BC028507, J04492, NM011612) The deduced sequence of the first 23 amino acids of CD137 cDNA has characteristics of the signal peptide of secretory and membrane-associated proteins (Blobel and Dobberstein, 1975) and fits the −1, −3 rule (von Heijne, 1983). The nucleotide sequence of murine CD137 has a single open reading frame which encodes a deduced polypeptide of 256 amino acids with a calculated mass of 27,587 Da. The first 23 amino acids appear to constitute a signal peptide, although the presence of lysine at −4 and glutamic acid at −5 is somewhat unusual. Two potential asparagine-linked glycosylation signals are located at positions 128 and 138. Thus the protein backbone of processed CD137 would have a mass of 25,167 Da. Its residues are arranged with spacing reminiscent of that seen in several

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groups of proteins including zinc finger DNA-binding proteins, the epidermal growth factor receptor, Drosophila Notch, and certain translation factors. The predicted protein contained an unusually large number (23) of cysteines in a region with four potential TNFR motifs, of which the first is partial and the third different from those of the TNFR. Following this ligand-binding domain is a stretch of amino acids (residues 140–185), in which almost 30% of the amino acids are serines and threonines, and potential sites of O-linked glycosylation reminiscent of those seen, for example, in the low-density lipoprotein receptor. Amino acids 186–211 constitute the hydrophobic transmembrane domain followed by a stop-transfer sequence containing several basic residues. This region may serve as a membrane-spanning anchor domain. The C-terminal part of the cytoplasmic domain contains two short runs of three and four acidic residues, respectively, and a sequence of five glycines followed by a tyrosine. The extracellular domain contains four potential C6 (CXn CXX CXn CXnC) motifs, of which the first is partial, and the third distinct from those of the nerve growth factor receptor and the TNF receptor. The amino acids flanking certain of the cysteine groups also resemble sequences found in other proteins. For example, if leucine is isomerized to isoleucine, there is an exact match to seven of the eight amino acids of a putative zinc finger structure in the yeast Elf-2β protein. These residues may represent metal binding sites but their role can not be accurately predicted. The human homologue of CD137 (huCD137) contains 255 amino acids with two potential N-linked glycosylation sites, and the molecular weight of its protein backbone is calculated to be 27 kD (Kwon et al., 1989). HuCD137 has features, such as a signal sequence and transmembrane domain, indicating that it is a receptor protein. It has 60% amino acid identity to mouse CD137. In the cytoplasmic domain, five regions are conserved between mouse and human, indicating that they may be important for CD137 function.

5. CD137L 5.1. The Discovery of CD137L The ligand for murine CD137 (CD137L) was originally identified by constructing soluble forms of the putative CD137 (encompassing amino acids 24–176; Goodwin et al., 1993; Kwon and Weissman, 1989). To ensure high-affinity binding of the soluble CD137 receptor to its putative ligand, and to aid purification of the soluble receptor, soluble CD137 was fused to the Fc portion of human IgG1 (Goodwin et al., 1993). Progress in CD137L biology has not gained as much momentum as that of its counterpart, CD137. A few isolated reports have suggested a role for CD137L in immune regulation (DeBenedette et al., 1999; Langstein et al., 1998; Pollok et al., 1994; Wilcox et al., 2002).

5.2. CD137L Structure and Expression CD137L is a 34 kD type II membrane glycoprotein with a carboxy-terminal extracellular domain and its gene is on chromosome 17 in the mouse (Goodwin

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et al., 1993). Although human CD137 ligand (huCD137L) is present in both T and B cells of the peripheral blood, the ligand is preferentially expressed in primary B cells and B cell lines. Daudi, a B cell lymphoma, is one of the B cell lines with the highest number of ligand molecules (Zhou et al., 1995). Scatchard analysis gave a kd of 1.4 × 10−9 M and number of ligand molecules per Daudi cell of 4.2 × 103 . Primary B cells when stimulated with pokeweed mitogen showed enhanced ligand expression. On the other hand, the ligand for murine CD137 is present at low levels on T cell lines (non-activated and anti-CD3-activated), pre-B cell lines and a number of immature macrophage cell lines. Also, CD137 AP (a fusion protein consisting of the extracellular domain of CD137 fused to human placental alkaline phosphatase) exhibited no binding to a glial tumor cell line, HeLa cells, or COS cells. On the other hand, anti-IgM-activated primary B cells showed higher binding of CD137 AP than anti-CD3-activated primary T cells. Scatchard analysis indicated that the A20 B lymphoma cells had 3680 binding sites per cell with a kd of 1.86 nM, and Western analysis showed that CD137L has a molecular mass of approximately 18–25 kD. Chalupny et al. (1992) reported that a fusion of the extracellular domain of CD137 with the Fc portion of human IgG1 bound to various extracellular matrix proteins. However, we and others have identified a high affinity ligand (CD137L) (Pollok et al., 1994; Goodwin et al., 1993) and cloned it (Goodwin et al., 1993). Consistent with its original assignment to the TNFR family, CD137L was found to be homologous to members of the emerging family of type II transmembrane glycoproteins that are counter-receptors to members of the TNFR superfamily (Armitage, 1994; Smith et al., 1994).

6. CD137L Gene Accession number: human (NM00381) and mouse (NM009494) The human CD137L gene (accession number: NM003811) (Figure 1.3A) is made up of three exons for the coding region, and two introns in which there is one exon for the 5 UTR. The mouse CD137L gene (accession number: NM009404) (Figure 1.3B), on the other hand, is composed of three exons and two introns.

7. CD137L Protein Accession numbers: human (AI357267, BM790119, U03398, NM003811) and mouse (NM009404) CD137L is a 34 kD glycoprotein with probable involvement of the N-linked sites, and possibly also the three putative O-linked sites (Goodwin et al., 1993). Under reducing conditions, CD137L has an apparent MW of ∼97 kD suggesting that it is a disulfide-linked homodimer. The C terminal 200 residues of full-length recombinant CD137L appear to contain glycosylation sites and at least one interchain disulfide bond capable of generating homodimers. This region has the lowest degree (14–16%) of sequence identity with various other family members. Based on sequence data, CD137L has a tertiary structure very similar to that of TNF and LT-α, which is consistent with its being oligomeric. The region between strands D

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Vinay S. Dass and Byoung S. Kwon

Figure 1.3. Genomic organization of human (A) and murine (B) CD137 ligands. Exons are shown as boxes; untranslated regions (UTR) are represented by black shading, and protein coding regions by no shading; they are labeled with Roman numerals. Lengths of introns and exons are shown in Arabic numerals (base pairs).

and I is not well conserved and can only be loosely aligned using secondary structure prediction techniques. There are two conserved residues, Leu164 and Trp166, in the loop between B-strands B and B . These residues immediately precede a segment of the loop, residues Gln169 through Ala172, which is important for the activity of TNF (Goh and Porter, 1990). The hydrophobic side-chains of Leu164 and Trp166 are buried and appear to anchor this important loop in the structure. The C-terminal residues of TNF and LT-α together form β-strand I, an integral part of the tertiary fold. There are seven extra C-terminal residues in CD137L; these probably form a flexible tail that does not exist in other family members. This putative tail is reminiscent of the N-terminal tails of soluble TNF and LT-α (9 and 25 residues, respectively) that modulate the biological activities of these cytokines (Goh and Porter, 1990). Cysteine placement varies across the ligand family. Of the three cysteines in the extracellular domain of CD137L, the second and third, both in loops at the same end of the b-sandwich, are well positioned to form a disulfide bond. The first cysteine (Cys137), nine residues N-terminal to the homologous region, is thus probably the cysteine involved in the homodimer link.

7.1. Regulation of RNA and Protein Expression Barring a few cases, the expression of CD137 and CD137L is activationinduced. CD137 is not detected (<3%) on resting T cells and T cell lines. However, when the T cells are stimulated with a variety of agonists (plate-bound anti-CD3, ConA, PHA, IL-2, IL-4, anti-CD28, PMA, and ionomycin alone or in various combination), in the presence of APCs, it is stably upregulated (Garni-Wagner et al., 1996; Kwon et al., 1987, 1989; Pollok et al., 1993). Using T cell clones, Kim et al. (2003) have shown that CD137 mRNA can be detected within 3 h of stimulation, and CD137 is surface-expressed by 12 h post-stimulation reaching maximal levels by 24 h. Induction is inhibited by cyclosporin A (Kwon et al.,

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1989) and cyclohexamide (Kwon et al., 1987), and actinomycin D halved CD137 transcript levels in activated lymphocytes within 30 min, pointing to a relatively short half-life (Schwarz et al., 1995). The fact that CD137 appears late during activation has led to the suggestion that it may be localized to memory T cells (Garni-Wagner et al., 1996). Interestingly, CD137 is also upregulated by DNA damaging agents such as anti-cancer drugs, or γ-irradiation, in human PBMC (Kim et al., 2002). Using 5 -deletion constructs of the CD137 promoter in luciferase reporter assays, Kim et al. (2003) demonstrated that the transcriptional elements mediating CD137 upregulation are located between ∼0.9 and ∼1.1 kb of the translational start site. Further characterization of these sites by electrophoretic mobility shift assays and site-directed mutagenesis revealed that nuclear factor κB (NF-κB) and activating protein-1 (AP-1) are involved. Also, MEK and c-Jun N-terminal kinase-1 are required for activation-dependent CD137 upregulation. CD137L, on the other hand, is highly expressed on mature B and macrophage cell lines as well as on anti-μ-activated B cells and dendritic cells (Pollok et al., 1994). Like CD137, CD137L is inducible in T cells (Goodwin et al., 1993).

7.2. CD137-CD137L in Health and Disease The importance of CD137-CD137L interactions has been underscored in several experimental systems (Vinay and Kwon, 1998). Expression of CD137/CD137L has been reported in various clinical disorders. Yamada-Okabe et al. (2003) demonstrated that thyroid hormone induced CD137 expression along with caspases. Also, expression of CD137 was detected in the inflammatory tissue in Crohn’s disease (Maerten et al., 2004). Interestingly, soluble forms of CD137 and CD137L were found in serum samples collected from humans with rheumatoid arthritis, and the levels of circulating CD137 and its ligand closely mirrored disease severity (Jung et al., 2004). Serum levels of soluble CD137L are also reported in patients with hematological malignancies (Salih et al., 2001). Furthermore, expression of CD137L was localized to the aortic tissue of subjects with Takayasu’s arthritis (Seko et al., 2004). Likewise, Wan et al. (2004) have demonstrated expression of CD137 in both hepatocellular carcinoma and non-tumorous regions of the liver. A similar finding of CD137 expression at tumor sites was reported by Zhang et al. (2003). In addition, the peripheral blood of liver-transplant patients not only contained CD137 but its expression was correlated with clinical severity. Furthermore, both CD137 and its ligand have been detected in human neurons, astrocytes, and microglia, as well as peripheral blood samples from chronic heart failure patients (Yndestad et al., 2002) while patients with acute myocarditis and dilated cardiomyopathy had high levels of CD137L (Seko et al., 2002). Lim et al. (2002) have demonstrated CD137-like molecules in the islet-infiltrating mononuclear cells and gray matter of the brain, but the significance of such expression is unclear. In addition, CD137L expression in the hearts of mice with acute myocarditis was shown to be caused by Coxsackie Virus B3 (Seko et al., 2001). Interestingly, Salih et al. (2001) detected constitutive CD137L expression on carcinoma cells where it appeared to be functional as the cells were able to prime T cells and induce cytokine production in co-culture assays. Taken together,

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Cytokine Secretion (IL-2, IL-6, IFN-γ, TGFβ etc)

(

Proliferation CD8αβ+) CD4+

T cell TCR CD137 Anti-CD137

Anti-CD137 Anti-μ

IgM MHC (B cell)

CD137 (DC, Mø)

APC

Cytokines/Chemokines/Antigens Up-regulated B cell Proliferation (IL-8, IL-12, IFN-γ, ICAM etc)

Figure 1.4. Consequences of CD137/CD137L signaling in immune competent cells. Co-stimulatory signals transmitted by cell-surface-derived determinants are mandatory for successful T cell activation. CD137/CD137L is an important receptor/ligand system concerned with immune regulation. Ligation of T cells either by anti-CD137 or CD137L-bearing transfectants, or soluble CD137, in the context of suboptimal doses of anti-TCR (anti-CD3) mAbs, provides strong co-stimulation leading to cell proliferation, cytokine induction, and production of immune effectors. The available evidence suggests that CD137 signaling is biased to CD8+ T cell as opposed to CD4+ T cell activation. Although CD137 has been considered a predominantly T cell antigen, other cell types (monocytes, dendritic cells etc.) also bear functional CD137 molecules. Ligation of CD137 on monocytes or dendritic cells by either anti-CD137 or soluble CD137 results in the secretion of characteristic immune modulators that further potentiate CD137/CD137L signaling. Bidirectional signals are known to exist in the case of CD137/CD137L. Thus, cross-linking of CD137L on B lymphocytes, together with ligation of the B cell receptor by anti-μ, leads to robust B cell proliferation.

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these data clearly highlight the important role of the CD137-CD137L pathway in health and disease.

8. Future Directions It is becoming clear that the CD137/CD137L interaction is unique and distinct from any other pathway with co-stimulatory properties (Figure 1.4). Given the effect of anti-CD137 mAb in protecting against disease, a major challenge is to streamline and design effective treatments for various autoimmune and nonautoimmune disorders. The fact that a novel regulatory CD11c+ CD8+ cell population expanded by anti-CD137 mAb can block the development of autoimmune diseases provides an additional interest to CD137 signaling. Our laboratory, which was the first to describe this novel cell type, is actively pursuing the question whether these cells can replace anti-CD137 mAb therapy in combating various immune disorders. Our unpublished results thus far look promising and demonstrate that adoptively transferred CD11c+ CD8+ (anti-CD137 mAb-expanded) cells are on par with anti-CD137 mAb therapy in treating inflammatory bowel disease and halting the spread of herpes simplex virus by selectively targeting autoreactive T cells.

Acknowledgments This work was supported by a US Public Service Health Grant RO1EY013325 (to BSK) as well as a Departmental Core Grant (P30EY002377). It was also supported by KRF-201-E00008 and KRF-2005-084-E00001, SRC funds to the Immunomodulation Research Center, University of Ulsan (Ulsan, Korea), and the International Cooperation Research Program from KOSEF and the Korean Ministry of Science and Technology.

References Armitage, R.J. (1994). Tumor necrosis factor receptor superfamily members and their ligands. Curr. Opin. Immunol., 6, 407–413. Blobel, G., and Dobberstein, B. (1975). Transfer to proteins across membranes. II. Reconstitutuion of functional rough microsomes from heterologous components. J. Cell. Biol., 67, 852–862. Bretcher, P., and Cohn, M. (1970). A theory of self-non-self discrimination. Science, 169, 1042–1049. Broll, K., Richter, G., Pauly, S., Hofstaedter, F., and Schwarz, H. (2001). CD137 expression in tumor vessel walls. High correlation with malignant tumors. Am. J. Clin. Pathol., 115, 543–549. Chalupny, N.J., Peach, R., Hollenbaug, D., Ledbetter, J.A., Farr, A.G., and Aruffo, A. (1992). T cell activation molecule 4-1BB binds to extracellular matrix proteins. Proc. Natl. Acad. Sci., U.S.A., 89, 10360–10364. Chu, N.R., DeBenedette, M.A., Stiernholm, B.J., Barber, B.H., and Watts, T.H. (1997). Role of IL-12 and 4-1BBL in cytokine production by CD28+ and CD28− T cells. J. Immunol., 158, 3081– 3089.

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Croft, M. (2003). Co-stimulatory members of TNFR family: keys to effective T cell immunity. Nat. Rev. Immunol., 3, 609–620. DeBenedette, M.A., Shahinian, A., Mak, T.W., and Watts T.H. (1997). Co-stimulation of CD28− T lymphocytes by 4-1BB ligand. J. Immunol., 158, 551–559. DeBenedette, M.A., Wen, T., Bachmann, M.F., Ohashi, P.M., Barber, B.H., Stocking, K.L., Peschon, J.J., and Watts, T.H. (1999). Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to influenza virus. J. Immunol., 163, 4833–4841. Diehl, L., van Mierlo, G.J., den Boer, A.T., van der Voort, E., Fransen, M., van Bostelen, L., Krimpenfort, P., Melief, C.J., Mittler, R., Toes, R.E., and Offringa, R. (2002). In vivo triggering through 4-1BB enables Th-independent priming of CTL in the presence of an intact CD28 costimulatory pathway. J. Immunol., 168, 3755–3762. Foell, J., Strahotin, S., O’Neil, S.P., McClausland, M.M., Suwyn, C., Haber, M., Chander, P.N., Bapat, A.S., Yan, X.J., Chiorazzi, N., Hoffmann, M.K., and Mittler, R.S. (2003). CD137 costimulatory T cell receptor engagement reverses acute disease in lupus-prone NZB × NZW F1 mice. J. Clin. Invest., 111, 1505–1518. Futugawa, T., Akiba, H., Kodama, T., Takeda, K., Hosoda, Y., Yagita, H., and Okukura, K. (2002). Expression and function of 4-1BB and 4-1BBL on murine dendritic cells. Int. Immunol., 14, 275–276. Garni-Wagner, B.A., Lee, Z.H., Kim, Y.J., Wilde, C.E., Kang, C.Y., and Kwon, B.S. (1996). 4-1BB is expressed on CD45 RAhi ROhi translational T cells in humans. Cell. Immunol., 169, 91–98. Goh, C.R., and Porter, A.G. (1990). Structural and functional domains in human tumor necrosis factors. Protein Eng., 4, 385–389. Goodwin, R.G., Din, W.S., Davis-Smith, T., Anderson, D.M., Gimpel, S.D., Sato, T.A., Maliszewski, C.R., Brannan, C.I., Copeland, N.G., Jenkins, N.A., Farrah, T., Armitage, R.J., Fanslow, W.C., and Smith, C.A. (1993). Molecular cloning of a ligand for the inducible T cell gene 4-1BB: A member of an emerging family of cytokines with homology to tumor necrosis factor. Eur. J. Immunol., 23, 2631–2641. Gramaglia, I., Cooper, D., Miner, K.T., Kwon, B.S., and Croft, M. (2000). Co-stimulation of antigenspecific CD4 T cells by 4-1BB ligand. Eur. J. Immunol., 30, 392–402. Halstead, E.S., Mueller, Y.M., Altman, J.D., and Katsikis, P.D. (2002). In vivo stimulation of CD137 broadens primary antiviral CD8+ T cells responses. Nat. Immunol., 3, 536–541. Hurtado, J.C., Kim, Y.J., and Kwon, B.S. (1997). Signals through 4-1BB are costimulatory to previously activated splenic T cells and inhibit activation-induced cell death. J. Immunol., 158, 2600– 2609. Jung, H.W., Choi, S.W., Choi, J.I., and Kwon, B.S. (2004). Serum concentrations of soluble 4-1BB and 4-1BB ligand correlated with the disease severity in rheumatoid arthritis. Exp. Mol. Med., 36, 13–22. Kienzel, G., and von Kempis, J. (2000). CD137 (ILA/4-1BB), expressed by primary human monocytes, induces monocyte activation and apoptosis of B lymphocytes. Int. Immunol., 12, 73–82. Kim, Y.J., Kim, S.H., Mantel, P., and Kwon, B.S. (1998). Human 4-1BB regulates CD28 co-stimulation to promote Th1 cells responses. Eur. J. Immunol., 28, 881–889. Kim, K.M., Kim, H.W., Kim, J.O., Baek, K.M., Kim, J.G., and Kang, C.Y. (2002). Induction of 41BB (CD137) expression by DNA damaging agents in human T lymphocytes. Immunology, 107, 472–479. Kim, J.O., Kim, H.W., Baek, K.M., and Kang, C.Y. (2003). NF-kB and AP-1 regulate activationdependent CD137 (4-1BB) expression in T cells. FEBS Lett., 541, 163–170. Kim, J., Choi, W.S., La, S., Suh, J.H., Kim, B.S., Cho, H.R., Kwon, B.S., and Kwon, B. (2004). Stimulation with 4-1BB (CD137) inhibits chronic graft-versus-host disease by inducing activationinduced cell death of donor CD4+ T cells. Blood, 105, 2206–2213. Kwon, B.S., Kim, C.S., Prystowski, M.B., Lancki, D.W., Sabath, D.E., Pan, J.L., and Weissman, S.M. (1987). Isolation and initial characterization of multiple species of T lymphocyte subset cDNA clones. Proc. Natl. Acad. Sci. U.S.A., 84, 2896–2900. Kwon, B.S., and Weissman, S.M. (1989). cDNA sequences of two inducible T cell genes. Proc. Natl. Acad. Sci. U.S.A., 86, 1963–1967.

Genes, Transcripts and Proteins of CD137 Receptor and Ligand

13

Kwon, B.S., Kestler, D.P., Eshhar, Z., Oh, K., and Wakulchik, M. (1989). Expression characteristics of two potential T cell mediator genes. Cell. Immunol., 121, 414–422. Kwon, B.S., Hurtado, J.C., Lee, Z.H., Kwack, K.B., Seo, S.K., Choi, B.K., Koller, B.H., Wolisi, G., Broxmeyer, H.E., and Vinay, D.S. (2002). Immune responses in 4-1BB (CD137)-deficient mice. J. Immunol., 168, 5483–5490. Laderach, D., Wesa, A., and Galy, A. (2003). 4-1BB ligand is regulated on human dendritic cells and induces the production of IL-12. Cell. Immunol., 226, 37–44. Langstein, J., Michel, J., Fritsche, J., Kreutz, M., Anderson, R., and Schwarz, H. (1998). CD137 (ILA/4-1BB), a member of the TNF receptor family induces monocyte activation via bidirectional signaling. J. Immunol., 160, 2488–2494. Lim, H.Y., Kim, K.K., Zhou, F.C., Yoon, J.W., Hill, J.M., and Kwon, B.S. (2002). 4-1BB-like molecule is expressed in islet-infiltrating mononuclear cells and in the gray matter of the brain. Cell. Biol. Int., 26, 271–278. Lindstedt, M., Johansson-Lindbom, B., and Borrebaeck, C.A. (2003). Expression of CD137 (4-1BB) on human follicular dendritic cells. Scand. J. Immunol., 5, 305–310. Maerten, T., Geboes, K., De Hertogh, G., Shen, C., Cadot, P., Bullens, D.M., Van Assche, G., Penninckx, F., Rutgeerts, P., and Cueppens, J.L. (2004). Functional expression of 4-1BB (CD137) in the inflammatory tissue in Crohn’s disease. Clin. Immunol., 112, 239–246. McHugh, R.S., Matthew, J.W., Piccirillo, C.A., Young, D.A., Shevach, E.M., Collins, M., and Byrne, M.C. (2002). CD4+CD25+ immunoregulatory T cells: Gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity, 16, 311–323. Melero, I., Shuford, W.W., Newby, S.A., Aruffo, A., Ledbetter, J.A., Hellstrom, K.E., Mittler, R.S., and Chen, L. (1997). Monoclonal antibodies against 4-1BB T cell activation molecule eradicate established tumors. Nat. Med., 3, 682–685. Melero, I., Johnston, J.V., Shuford, W.W., Mittler, R.S., and Chen, L. (1998). NK1.1 cells express 4-1BB (CDw137) costimulatory molecule and are required for tumor immunity elicited by anti-4-1BB monoclonal antibodies. Cell. Immunol., 190, 167–172. Mittler, R.S., Bailey, T.S., Klussman, K., Trailsmith, M.D., and Hoffmann, M.K. (1999). Anti-4-1BB monoclonal antibodies abrogate T cell-dependent humoral immune responses in vivo through the induction of helper T cell anergy. J Exp Med., 190, 1535–1540. Pollok, K.E., Kim, Y.J., Zhou, Z., Hurtado, J., Kim, K.K., Pickard, R.T., and Kwon, B.S. (1993). Inducible T cell antigen 4-1BB. Analysis of expression and function. J Immunol., 150, 771–781. Pollok, K.E., Kim, Y.J., Hurtado, J.C., Zhou, Z., Kim, K.K., and Kwon, B.S. (1994). 4-1BB T cell antigen binds to mature B cells and macrophages and co-stimulates anti-μ-primed splenic B cells. Eur. J. Immunol., 24, 367–374. Salih, H.R., Schmetzer, H.M., Burke, C., Straling, G.C., Dunn, R., Pelka-Fleiscischer, R., Nuessler, V., and Kiener, P.A. (2001). Soluble CD137 (4-1BB) ligand is released following leukocyte activation and is found in sera of patients with hematological malignancies. J. Immunol., 167, 4059–4066. Saoulli, K., Lee, S.Y., Cannons, J.L., Yeh, W.C., Santana, A., Goldstein, M.D., Bangia, DeBenedette, M.A., Mak, T.W., Choi, Y., and Watts, T.H. (1998). J. Exp. Med., 187, 1849–1862. Schwartz, R.R. (1990). A cell culture model for T lymphocyte clonal anergy. Science, 248, 1349–1356. Schwarz, H., Tuckwell, J., and Lotz, M. (1993). A receptor induced by lymphocyte activation (ILA); a new member of the human nerve growth factor/tumor necrosis factor receptor family. Gene, 134, 295–298. Schwarz, H., Valbracht, J., Tuckwell, J., von Kempis, J., and Lotz, M. (1995). ILA, the human 4-1BB homologue, is inducible in lymphoid and other cell lineages. Blood, 85, 1043–1052. Seko, Y., Takahashi, N., Oshima, H., Shimozato, O., Akiba, H., Takeda, K., Kobata, T., Yagita, H., Okumura, K., Azuma, M., and Nagai, R. (2001). Expression of tumor necrosis factor (TNF) ligand superfamily costimulatory molecules CD30L, CD27L, OX40L, and 4-1BBL in murine hearts with acute myocarditis caused by coxsackievirus B3. J. Pathol., 195, 593–603. Seko, H., Ishiyama, S., Nishikawa, T., Kasajima, T., Hiroe, M., Suzuki, S., Ishiwata, S., Kawai, S., Tanaka, Y., Azuma, M., Kobata, T., Yagita, H., Okumura, K., and Nagai, R. (2002). Expression of tumor necrosis factor ligand superfamily costimulatory molecules CD27L, CD30L, OX40L, and 4-1BBL in the heart of patients with acute myocarditis and dilated cardiomyopathy. Cardiovasc. Pathol., 11, 166–170.

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Seko, Y., Sugishita, K., Sato, O., Takagi, A., Tada, Y., Matsuo, H., Yagita, H., Okumura, K., and Nagai, R. (2004). Expression of costimulatory molecules (4-1BBL and Fas) and major histocompatibility class I chain-related A (MICA) in aortic tissue with Takayasu’s arteritis. J. Vasc. Res., 41, 84–90. Seo, S.K., Choi, J.H., Kim, Y.H., Kang, W.J., Park, H.Y., Suh, J.H., Choi, B.K., Vinay, D.S., and Kwon, B.S. (2004). 4-1BB-mediated immunotherapy of rheumatoid arthritis. Nat. Med., 10, 1088–1094. Shuford, W.W., Klussman, K., Tritchler, D.D., Loo, D.T., Chalupny, J., Siadak, A.W., Brown, T.J., Emswiler, J., Raecho, H., Larsen, C.P., Pearson, T.C., Ledbetter, J.A., Aruffo, A., and Mittler, R.S. (1997). 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell response. J Exp Med., 186, 47–55. Smith, C.A., Farrah, T., and Goodwin, R.G. (1994). The TNF receptor superfamily of cellular and viral proteins; activation, co-stimulation, and death. Cell, 76, 959–962. Summers, K.L., Hock, B.D., McKenzie, J.L., and Hart, D.N. (2001). Phenotypic characterization of five dendritic cell subsets in human tonsils. Am. J. Pathol., 159, 285–295. Sun, Y., Lin, X., Chen, H.M., Wu, Q., Subudhi, S.K., Chen, L., and Fu, Y.X. (2002). Administration of agonistic anti-4-1BB monoclonal antibody leads to the amelioration of experimental autoimmune encephalomyelitis. J. Immunol., 168, 1457–1465. Takahashi, T., Mittler, R.S., and Vella, A.T. (1999). 4-1BB is a bona fide CD8 T cell survival signal. J Immunol., 162, 5037–5040. Vinay, D.S., and Kwon, B.S. (1998). Role of 4-1BB in immune responses. Sem. Immunol., 10, 481–489. Vinay, D.S., Choi, B.K., Bae, J.S., Kim, W.Y., Gebhardt, B.M., and Kwon, B.S. (2004). CD137-deficient mice have reduced NK/NKT cell numbers and function, are resistant to lipopolysaccharideinduced shock syndromes, and have lower IL-4 responses. J. Immunol., 173, 4218–4229. Von Heijne, G. (1983). Patterns of amino acids near signal-sequence cleavage sites. Eur. J. Biochem., 133, 17–21. Wan, Y.L., Zheng, S.S., Zhao, Z.C., Li, M.W., Jia, C.K., and Zhang, H. (2004). Expression of costimulator 4-1BB molecule in hepatocellular carcinoma and adjacent non-tumor liver tissue, and its possible role in tumor immunity. World J. Gastroenterol., 10, 195–199. Wilcox, R.A., Chapoval, A.I., Gorski, K.S., Otsuji, M., Shin, T., Flies, D.B., Tamada, K., Mittler, R.S., Tsuchiya, H., Pardoll, D.M., and Chen, L. (2002). Expression of functional CD137 receptor by dendritic cells. J. Immunol., 168, 4262–4267. Yamada-Okabe, T., Satoh, and Yamade-Okabe, H. (2003). Thyroid hormone induces the expression of 4-1BB and activation of caspases in thyroid hormone receptor-dependent manner. Eur. J. Biochem., 270, 3064–3073. Yndestad, A., Damas, J.K., Geir Eiken, H., Holm, T., Hauh, T., Simonsen, S., Froland, S.S., Gullestad, L., and Aukrust, P. (2002). Increased gene expression of tumor necrosis factor superfamily ligands in peripheral blood mononuclear cells during chronic heart failure. Cardiovasc. Res., 54, 175–182. Zhang, H., Merchant, M.S., Chua, K.S., Khanna, C., Helman, L.J., Telford, B., Ward, Y., Summers, J., Toretsky, J., Thomas, E.K., June, C.H., and Mackall, C.L. (2003). Tumor expression of 4-1BB ligand sustains tumor lytic T cells. Cancer Biol. Ther., 2, 579–586. Zhou, Z., Kim, S.H., Hurtado, J.C., Lee, Z., Kim, K.K., Pollok, K.E., and Kwon, B.S. (1995). Characterization of human homologue of 4-1BB and its ligand. Immunol. Lett., 45, 67–73.

2 CD137 Signal Transduction Hyeon-Woo Lee and Byoung S. Kwon

1. Background T lymphocytes have critical roles in clearing cells expressing foreign antigens. The proliferation of T cells and their differentiation into effector and memory cells confers T cell-mediated adaptive immunity (Abbas et al., 1997). To undertake these antigen-specific functions, T cells require two signals: ligation of the T cell receptor (TCR) by the MHC/peptide complex on the antigen presenting cell (APC), and cross-linking of co-stimulatory receptors on the T cell with corresponding ligands on the APC (Carreno and Collins, 2002; Chambers and Allison, 1999). In addition, cytokines synthesized and released by the APC can modulate T cell functions in a paracrine way (Beginat et al., 2003; Kanegane and Tosato 1996; Lantz et al., 2000; Lodolce et al., 1998; Nakajima et al., 1997; Schluns et al., 2000; Unutmaz et al., 1994; Unutmaz et al., 1995; Zhang et al., 1998). Although the exact mechanisms by which several co-stimulatory molecules can interact to stimulate T cells remain to be uncovered, co-stimulation appears to be an accurate process that subtly evokes T cell immunity (Rothstein and Sayegh, 2003; Samia and Mohamed, 2004; Van Parijs, and Abbas, 1998). Co-stimulatory signals determine whether antigen-priming T cells become fully activated or antigen-specifically inactivated (Samia and Mohamed, 2004). If the concentration of antigen or its affinity for TCR is low, co-stimulatory signals tend to enhance antigen-specific TCR signals and fully activate T cells by activating their own signaling pathways or enhancing those generated by the TCR (Gravestein et al., 1998; Kenneth and Thompson, 2002). Co-stimulation can promote both early antigen-priming T cell activation and late T cell differentiation to effector or memory cells (Michael, 2003). At the same time, negative co-stimulatory signals may prevent unnecessary activation of T cells and hence autoimmune responses (Rothstein and Sayegh, 2003; Samia and Mohamed, 2004). Many proteins have been identified as co-stimulatory molecules required for optimal activation of T lymphocytes. CD28/CTLA-4 –CD80 (B7-1)/CD86 (B7-2) is the best-studied co-stimulatory pathway (June et al., 1990; Lenschow et al., 1996; Mueller, 2000; Okkenhaung et al., 2001; Watts and DeBenedette, 1999). Hyeon-Woo Lee • Department of Pharmacology, School of Dentistry, Kyung Hee University, Seoul 130-701, Korea. Byoung S. Kwon • Immunomodulation Research Center, University of Ulsan, Ulsan 680-749, Korea and LSU Eye Center, 2020 Gravier Street New Orleans LA 70112. 15 CD137 Pathway: Immunology and Diseases. Edited by Lieping Chen, Springer, New York, 2006

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The B7 ligands on APCs, B7-1 and B7-2, bind to both an activating (CD28) and an inhibitory (CTLA-4) receptor on T cells. These co-stimulatory ligands and receptors are trans-membrane proteins belonging to the immunoglobulin superfamily. Although they are considered to play a critical role in the activation of T cells, others have been found to augment antigen-driven T cell responses. Thus, several members of the tumor necrosis factor receptor (TNFR)/ligand family including CD137/CD137L, OX40/OX40L, GITR/GITRL, CD27/CD27L, and CD30/CD30L also act as co-stimulatory receptors/ligands enhancing T cell responses following initial activation (Gravestein and Borst, 1998; Mueller et al., 1989; Watts and DeBenedette, 1999; Weinberg et al., 1998). Studies of CD137 (4-1BB) have shed light on its roles in autoimmunity, and in anti-viral and anti-cancer responses.

2. CD137 (4-1BB) As an Immune Stimulator CD137 (4-1BB), a 30-kDa glycoprotein, was identified as an inducible protein following activation of mouse T cells and was originally named “induced by lymphocyte activation (ILA)” in humans (Kwon and Weissman, 1989; Schwarz et al., 1993). Although 4-1BB is known to be expressed in a variety of cells including activated T cells, natural killer (NK) cells, regulatory T cells, monocytes, neutrophils, eosinophils and dendritic cells (DCs), the main focus of studies of 41BB has been on its effects on T cells, especially on CD8+ T cell-mediated adaptive immunity (Brown et al., 1997). The immune response induced by anti-4-1BB monoclonal antibodies is mediated by both CD8+ and CD4+ T cells and is accompanied by a marked augmentation of tumor-selective cytolytic T-cell activity (Melero et al., 1997). Activation of DCs, via the production of cytokines (IL-6 and IL-12) and the expression of co-stimulatory molecules (CD80 and CD86), may also contribute to the positive effects of 4-1BB by modulating innate immunity (Futagawa et al., 2002; Kwon et al., 2002; Wilcox et al., 2002). The activation of DCs by NK cells and the subsequent development of anti-tumoral CTL responses facilitated by 4-1BB-activated DCs may account for the synergistic effects observed in combination therapy (Pan et al., 2004). Activated CD4+ CD25+ regulatory T cells express 4-1BB (Zheng et al., 2004), and cross-linking of 4-1BB inhibits the immunosuppressive effect of these cells (Choi et al., 2004). It is now clear that 4-1BB-mediated co-stimulatory signaling plays a role in enhancing T cell survival and expansion, promoting differentiation, increasing cytokine expression, promoting rejection of cardiac and skin allografts, eradicating established tumors, enhancing integrinmediated cell adhesion, and increasing T-cell cytolytic potential (Kwon et al., 2000; Melero et al., 1997; Shuford et al., 1997; Tan et al., 2000; Vinay and Kwon, 1998). In an experiment involving 4-1BBL−/− mice infected with influenza virus, the knockout mice had lower numbers of Db /NP366-374-specific CD8+ T cells late in the primary response (Bertram et al., 2002). It was shown that 4-1BB enhanced the survival of CD8+ T cells by increasing expression of the anti-apoptotic genes, bcl-XL and bfl-1, and stimulated cell cycle progression of the CD8+ T cells by amplifying the expression of cyclins D and E and down-regulating or degrading the cell cycle-dependent kinase inhibitor, p27kip1 (Lee et al., 2002, 2003a). These

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effects of 4-1BB are generated by specific 4-1BB-mediated signal transduction pathways, as discussed in the following section. Upon cross-linking of 4-1BB in CD8+ cells by anti-4-1BB Ab, the expression of a memory CD8+ cell marker (CD45RO) and CC chemokine receptor 6, as well as the content of granzyme B, increased (Kim et al., 2002), indicating that 4-1BB has a key role in the differentiation of memory CD8+ T cell. Interestingly, a recent study showed that in vivo cross-linking of 4-1BB suppressed autoimmune diseases such as collagen-induced rheumatoid arthritis (Seo et al., 2004). This effect is attributable to 4-1BB-evoked differentiation of CD8+ T cells to CD8+ CD11c+ T cells that secrete IFN-γ, which in turn acts on DCs or macrophages in a paracrine manner. IFN-γ-induced activation of APCs causes secretion of indolamine 2,3-dioxygenase (IDO), which kills adjacent antigen (collagen)-specific CD4+ T cells. The differentiation of CD8+ T cells to CD8+ CD11c+ T cells may be a general response to cross-linking of 4-1BB in vivo because CD8+ CD11c+ T cells are also produced by in vivo challenge with anti-4-1BB Ab in cancer models (Kwon, unpublished data). 4-1BB, therefore, is a double-edged sword: it is immunosuppressive in autoimmune diseases and immunostimulatory towards cancers or viral infections. 4-1BB-mediated differentiation to CD8+ CD11c+ T cells seems to be central to both actions, and the nature of the dominant immune response in each case may determine the outcome. In models of autoimmune disease where immune responses are mainly mediated by antigen-specific CD4+ T cells, 4-1BB activates CD8+ T cells and induces them to differentiate into CD8+ CD11c+ T cells, which deplete antigen-specific CD4+ T cells (Seo et al., 2004). In tumor models, however, CD8+ T cells predominate and attack the cancer cells; in this case, the activation and differentiation of CD8+ T cells in response to 4-1BB may be directly involved in eradicating tumor cells.

3. CD137 Signal Transduction Although 4-1BB is involved in modulating cellular responses in various types of cells, studies of its signaling pathway have been performed mainly in T lymphocyte. Cellular events in CD8+ T cells in response to cross-linking 4-1BB with 4-1BBL or anti-4-1BB Ab are decisive in modulating host immunity. 4-1BB is one of type I membrane receptors of TNFR superfamily (Vinay and Kwon, 1998). Signaling by the TNFR family is thought to be initiated by ligand-induced trimerization of the monomeric receptor (Chan et al., 2000). The extracellular domains of the TNFR contain characteristic cysteine-rich domains, through which receptors involve the trimerization by physical interaction with the trimeric ligand. Although it is not completely uncovered, it seems that TNF receptors on cell membrane exist as preassembled trimeric form in the absence of ligand, and ligand binding causes to change its conformation to facilitate an intracellular signaling pathway. One critical event common to signal transduction of the TNFR family is the association of TNFR with series of the TNF receptor associated factor (TRAF) family initiating their own signaling pathways. To date, TRAFs are composed of seven molecules and a few isoforms. TRAFs act as an adaptor molecule integrating molecules required for not only TNFR signals but also signals of innate immunity

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such as those of IL-1 receptor and the Toll-like receptors. As other TNFR family, 4-1BB uses TRAFs as adaptor molecules to propagate its own specific signal transductions. The cytoplasmic domain of 4-1BB is capable of associating specifically with TRAF1, TRAF2, and TRAF3 (Arch and Thompson, 1998; Jang et al., 1998; Saoulli et al., 1998). It possesses two consensus TRAF2-binding sequences, (P/S/A/T)X(Q/E)E (Ye et al., 1999). Study using point mutation analysis indicated that the C-terminal site (PEEE246−250 ) is more important than the N-terminal site (TTQE234−237 ), although it has not been clearly defined whether both sites are involved in the binding activity for TRAF2 (Jang et al., 1998). The former site is associated with TRAF1 and TRAF3, which suggests that all the three TRAFs compete to bind to that site. Therefore, it appears to be a regulatory mechanism by which 4-1BB-evoked biological responses are governed at this proximal step. Numbers of studies have shown that TRAF2 is required for 4-1BB-exerted activation of transcriptional factors such as NF-κB and AP-1 (Saoulli et al., 1998). Studies to date have shown that 4-1BB produces signals through TNF receptor-associated factor–NF-κB inducing kinase–NF-κB (TRAF–NIK–NF-κB) (Arch and Thompson, 1998; Jang et al., 1998) and TRAF–apoptosis signal-regulating kinase–p38 mitogen-activated protein kinase (TRAF–ASK–p38MAPK) or stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathways (Saoulli et al., 1998; Cannons et al., 2000). Studies of the 4-1BB signaling pathway promoting the survival and multiplication of CD8+ T lymphocytes have been reported (Figure 2.1). 4-1BB signaling pathway specific for the survival of CD8+ T lymphocytes was studied (Lee et al., 2002). Cross-linking 4-1BB up-regulated expression of the anti-apoptotic genes bcl-XL and bfl-1 and increased production of Bcl-XL protein. These effects appear to be responsible for 4-1BB-enhanced survival of primary CD8+ T lymphocytes. Although 4-1BB-mediated ERK1/2 and/or PI3-kinase signals enhanced proliferation of primary CD8+ T lymphocytes, these pathways were not involved in the 4-1BB-mediated increase in Bcl-XL expression. It is 4-1BBmediated NF-κB activation that provides CD8+ lymphocytes with prolonged survival via up-regulation of Bcl-XL and Bfl-1 expression. The critical importance of NF-κB activation by 4-1BB for Bcl-XL and Bfl-1 induction in our study is consistent with recent studies showing NF-κB-dependent up-regulation of Bcl-XL and Bfl-1 expression in other contexts (Grillot et al., 1995; Jones et al., 2000; Lee et al., 1999). For instance, CD28-mediated NF-κB activation is essential for BclXL induction and anti-apoptotic effects in primary human CD4+ T lymphocytes (Khoshnan et al., 2000). Similarly, NF-κB-mediated up-regulation of Bcl-XL and Bfl-1 is important for CD40 survival signaling in B lymphocytes (Lee et al., 1999). Although it has been shown that the PI-3 kinase/Akt pathway plays a key role in NF-κB activation (Burr et al., 2001; Ozes et al., 1999) and subsequent Bcl-XL expression (Brennan et al., 1997; Jones et al., 2000), this pathway is not involved in 4-1BB-mediated up-regulation of Bcl-XL and Bfl-1 in primary CD8+ T lymphocytes. LY294002, a PI-3 kinase blocker, abolished 4-1BB-mediated T cell proliferation to the same extent as did PDTC, an NF-κB blocker. However, LY294002 did not block 4-1BB-mediated up-regulation of Bcl-XL , whereas PDTC did. These data indicate that 4-1BB-induced PI3-kinase and NF-κB signals have separate

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Figure 2.1. 4-1BB signal transduction pathways in T lymphocytes. 4-1BB is expressed following TCR ligation with antigens, or CD3 ligation with anti-CD3 mAb. The 4-1BB is located in lipid raft domains where it apparently interacts with TRAF upon ligation with anti-4-1BB mAb. This causes re-location of the lipid rafts to the area of contact between T cell and APC, and subsequently reactivates TCR signaling pathways via as unknown mechanism(s). This involves recruitment of TCR signalosomes to the lipid rafts, which leads to increased activation of NF-κB, intracellular [Ca2+ ], and phosphorylation of ERK1/2. 4-1BB-mediated activation of NF-κB increases transcription of the anti-apoptotic genes, bcl-XL and bfl-1, which in turn enhances T cell survival. At the same time activation of ERK1/2 by 4-1BB stimulates transcription of cyclin D2. The increase in intracellular Ca2+ resulting from PLC-γ activation by 4-1BB then activates the Ca2+ /calmodulin-dependent phosphatase, calcineurin. The latter dephosphorylates the transcription factor NFAT, which is translocated to the nucleus where it increases transcription of IL-2 and IFN-γ. The IL-2 is secreted and binds to IL-2 receptors in an autocrine or paracrine fashion, and the IL-2/IL-2 receptor signaling pathway provides a mechanism that enhances translation of the cyclin D2 transcripts made in response to 4-1BB-mediated ERK1/2 activation. This stimulates multiplication of T cells.

physiological functions: only the NF-κB signal triggers Bcl-XL and, potentially, Bfl-1 expression. These results explain how engagement of the co-stimulatory molecule 4-1BB enhances survival of CD8+ T lymphocytes through NF-κB activation. This mechanism could account for 4-1BB-induced long-term survival of CD8+ T lymphocytes in vivo. Cross-linking 4-1BB also enhances cell cycle progression of primary CD8+ T lymphocytes in both IL-2-dependent and -independent ways (Lee et al., 2003a). Several molecules have been identified that play key roles in regulating the cell cycle in T cells (Botz et al., 1996; Leone et al., 1997; Sherr and Roberts, 1999). Studies of cyclin/cyclin-dependent kinase (cdk) holoenzymes and cdk inhibitors such as p27kip1 and p21cip1 have resulted in important advances in understanding the cell cycle of T cells (Poltak et al., 1994; Toyoshima and Hunter, 1994). CD28 costimulation enhances the clonal expansion of T cells via PI3K/Akt -pathwaymediated down-regulation of p27kip1 (Appleman et al., 2002). Mitogenic ligands

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initiate up-regulation of cyclin D protein and inactivate the cyclin D/cdk4 or 6 complex, which then sequesters p27kip1 from the inactive cyclin E/cdk2/p27kip1 complex and generates an active cyclin D/cdk4 or 6/p27kip1 complex (Sherr and Roberts, 1999). This active form phosphorylates and inhibits the activity of the retinoblastoma tumor suppressor gene product (Rb) that binds to and blocks the activity of transcription factor E2F. Active E2F then increases transcription of genes, such as cyclin E required for S phase entry (Matakeyama et al., 1994; Resnitzky and Reed, 1995). Sequestration of p27kip1 from inactive cyclin E/cdk2/ p27kip1 onto cyclin D/cdk4 or 6 also results in increased levels of active cyclin E/cdk2, which further phosphorylates Rb and activates E2F. As the levels of cyclin E and cyclin E/cdk2 rise, p27ki p1 protein is phosphorylated and degraded via the ubiquitin-proteasome pathway (Montagnoli et al., 1999; Tsvetkov et al.,1999). Degradation of p27kip1 further increases the level of active cyclin E/cdk2 complexes. As a general rule, mitogenic stimuli promote the G1 /S phase transition by means of this positive autoregulatory feedback loop. 4-1BB engagement by anti-4-1BB increased expression of cyclin D2 and cyclin E, and down-regulated p27kip1 protein. These effects of 4-1BB were responsible for 4-1BB-enhanced cell cycle progression of primary CD8+ T lymphocytes. Stimulation by 4-1BB of ERK1/2 and LY294002-sensitive PI3K signal pathways independently increased transcription of the cyclin D2 gene. It seems that 4-1BB co-stimulation evokes the phosphorylation of ERK1/2, which then causes increased transcription of the cyclin D2 gene. Following this, the cyclin D2 mRNA is translated via a 4-1BB-evoked IL-2/wortmannin-sensitive PI3K/mTOR signal pathway. The increased cyclin D2 expression induced by TCR/CD3, together with 4-1BB co-stimulation, appears to be an initial event that triggers up-regulation of cyclin E expression and down-regulation of p27kip1 , thereby promoting the G1 /S phase transition. 4-1BB co-stimulation is known to enhance the secretion of IL-2, which acts in an autocrine or paracrine manner on the IL-2R on CD8+ T cells (Brennan et al., 1997; Moon and Nelson, 2001; Schluns et al., 2000). IL-2 promotes cell cycle progression through the PI3K/Akt/mTOR pathway, which phosphorylates and activates p70S6K , thereby eliciting translation of cyclin D (Gingras et al., 2001; Kuo et al., 1992). The cyclin D2 mRNA generated in response to TCR/CD3 plus 4-1BB is only in part translated via an IL-2/wortmannin-sensitive PI3K/mTOR pathway because neither anti-IL2 mAb, wortmannin, nor rapamycin completely blocked the increase in cyclin D2 protein. However, the increase was completely inhibited by LY294002 plus PD98059, apparently because transcription and translation of cyclin D were blocked. Whether or not some IL2R/PI3K/Akt/mTOR-independent translational machinery is involved in translating the remainder of the cyclin D2 mRNA remains to be established. LY294002 inhibits the stimulation of proliferation and cyclin D2 expression and the down-regulation of p27kip1 more effectively than PD98059, rapamycin, wortmannin, or anti-IL-2 mAb. It may, therefore, inhibit a pathway operated by TCR/CD3 plus 4-1BB that evokes up-regulation of cyclin D2 transcription and translation and does not depend on either MEK1/2-mediated transcription or IL-2/wortmannin-sensitive PI3K/mTOR-mediated translation. The mechanism(s) by which the LY294002-sensitive PI3K signal up-regulates cyclin D2 transcription and translation remains unclear. In addition, there is a difference

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in the effect of wortmannin and LY294002 and this needs further study. LY294002 and wortmannin are known to be potent inhibitors of the same isoforms of PI3K catalytic subunits, although LY294002 acts differently from wortmannin as it competes reversibly for the ATP-binding site (Adi et al., 2001; Vlahos et al., 1994). It is conceivable that in CD8+ T cells LY294002 acts on unknown isoforms of PI3K subunits that are insensitive to wortmannin. These isoforms could be responsible for up-regulation of cyclin D2 transcription and its translation. 4-1BB elicits up-regulation of cyclin D2 and cyclin E expression, and down-regulation of p27kip1 , mainly through ERK1/2 and PI3K. This mechanism could account for the 4-1BB-induced clonal expansion of CD8+ T lymphocytes following antigen challenge in vivo. As in CD8+ T lymphocytes, cross-linking of 4-1BB also enhances the survival and cell cycle progression of primary CD4+ T lymphocytes (Lee et al., 2003b). 4-1BB engagement by anti-4-1BB stimulates expression of the antiapoptotic genes bcl-X L and bcl-2 that are critical for survival. 4-1BB ligation also induces expression of cyclins D2 and E, and down-regulates p27kip1 protein. These effects of 4-1BB are responsible for the enhanced cell cycle progression of primary CD4+ T lymphocytes. 4-1BB-deficient CD4+ T cells had lowered responsiveness to Ova323−339 antigen in the 4-1BB knockout/DO11, 10 TCR inorganic mouse model, confirming that 4-1BB acts as a co-stimulator in the primary response to peptide antigen. These findings indicate that in CD4+ T lymphocytes, 4-1BB-mediated responses could occur via intracellular signaling pathways similar to those in CD8+ T lymphocytes. Recent work has presented several findings related to the early 4-1BB signaling pathways in CD8+ T cells (Nam et al., 2005). Cross-linking of 4-1BB in primary CD8+ T lymphocytes increased tyrosine phosphorylation of TCR pathway proteins. Since pre-treatment of CD8+ cells with PP2, which blocks Src tyrosine kinases such as Lck and Fyn (Salazar and Rozengurt, 1999), completely abolished this 4-1BB-evoked tyrosine phosphorylation, it was clearly the result of tyrosine phosphorylation of one of the early TCR pathway proteins by Lck. Cross-linking of 4-1BB on CD8+ T cells in p815-m4-1BBL transfectants caused the redistribution of lipid rafts to the area of contact of the T cells with the p815m4-1BBL cells. TCR pathway proteins such as Lck, pTyr, PKC-θ and SLP-76 were also redistributed to the lipid rafts, and other TCR pathway proteins such as PKC-θ and SLP-76 were translocated from the Brij 58-soluble to the insoluble fraction, indicating that these proteins were also recruited to the lipid rafts. Cross-linking of 4-1BB recruited 4-1BB itself to lipid rafts in the area of cell contact. TRAF2 was also recruited, as shown by confocal microscopy, and by its appearance in the Brij 58-insoluble fraction. Cross-linking of 4-1BB increased intracellular Ca2+ , apparently due to translocation of PLC-γ1 to the lipid rafts and its subsequent activation. Methyl-β-cyclodextrin, which disrupts raft formation (Marwali et al., 2003), blocked translocation of PLC-γ1 to the detergent-insoluble fraction, redistribution of lipid rafts and of Lck, and degradation of IκB-α, as a result of cross-linking of 4-1BB. Finally, PP2, a Src kinase inhibitor, or CsA, a calcineurin inhibitor, suppressed 4-1BB-mediated proliferation. CsA completely

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blocks 4-1BB-induced IL-2 and IFN-γ mRNA expression. Because we have observed similar 4-1BB effects in CD4+ T cells, these could be early molecular events underlying the role of 4-1BB in survival and cell cycle progression in both CD8+ and CD4+ T cells (Lee et al., 2002, 2003a, 2003b). It is noteworthy that 4-1BB, a TNFR family member, activates TCR signaling pathways. 4-1BB is known to evoke a variety of cellular responses via TNFR signaling pathways (Arch and Thompson, 1998; Cannons et al., 2000; Jang et al., 1998; Saoulli et al., 1998). It activates NF-κB, p38 MAPK, SAPK/JNK and ERK1/2 via TRAF/TRADD in T cells, and it is generally accepted that its cross-linking recruits or stimulates the TNFR “signalosome” by which 4-1BB activates downstream signaling. The mechanism(s) by which cross-linking of 41BB activates TCR signaling remains to be uncovered. There are several plausible mechanisms. First, since 4-1BB increases the adhesion of T cells to extracellular matrix proteins (Chalupny et al., 1992; Kim et al., 1999), its ligation could extend the duration of interaction between TCR and antigen/MHC as a result of increased adhesion of the T cells to the APCs. Second, recruitment of TRAF2 to the raft fraction could initiate tyrosine phosphorylation and activation of Lck via an interaction of TRAF2 with an unidentified protein kinase. Third, the binding of 4-1BB to TRAF2 upon 4-1BB engagement (Arch and Thompson, 1998) could redistribute lipid rafts, which might by default relocate Lck to the cell contact area. Lck in turn could be activated by the same route as that responsible for stimulation of the TCR by antigens or by anti-CD3 Ab. It has been reported that cross-linking CD40, another member of TNFR family, recruits CD40, TRAF2 and 3, and Lyn Src family kinase to lipid rafts and initiates tyrosine phophorylation of intracellular protein substrates. This promotes ERK1/2 and p38 MAPK activation, which leads to the production of various cytokines in dendritic cells (Vidalain et al., 2000). Recruitment of CD40, TRAFs and Lyn to lipid rafts following CD40 ligation may be a prerequisite for the effects of tyrosine phosphorylation of intracellular proteins on subsequent signaling pathways and cytokine production. Finally, 4-1BB-mediated activation of classical signal transduction pathways could modulate TCR signaling proteins and initiate or enhance TCR signaling cascades. It is believed that the findings that cross-linking of 4-1BB recruits 4-1BB and TRAF2 to lipid rafts and activates TCR signaling pathways accounts for how engagement of 4-1BB activates NF-κB and ERK1/2 and enhances the survival and expansion of T lymphocytes.

4. Concluding Remarks To date, roles of 4-1BB in immune system have been extensively studied and appreciated as a critical co-stimulatory molecule. It has also been shown to regulate functions of innate immunity. Elucidation of 4-1BB signal transduction can provide valuable information on underlying molecular mechanisms by which cross-linking of 4-1BB modulates immune responses. Since 4-1BB is a pivotal costimulatory molecule in both adaptive and innate immunity, its signal transduction

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enlightens us on precise intracellular events not only for specific 4-1BB-exerted cellular responses but also general, but critical, immune responses such as differentiation of memory T cells, which is apparently enhanced by cross-linking of 4-1BB in vivo. In the innate immune system, 4-1BB signal transduction has rarely been studied. Therefore, it is necessary to elucidate its signaling pathway in innate immune system so as to completely understand roles of 4-1BB in immune system. A thorough understanding of the cellular and molecular mechanisms underlying 4-1BB-induced immune responses promises to provide avenues for improved immunotherapy against various diseases including tumor, viral infection, and autoimmune diseases. This work was supported by a US Public Service Health Grant RO1EY013325 (to BSK) as well as a Departmental Core Grant (P30EY002377). It was also supported by the SRC funds to the Immunomodulation Research Center, University of Ulsan (Ulsan, Korea), Korea Research Foundation Grant (KRF-2004003-100349) and the International Cooperation Research Program from KOSEF and the Korean Ministry of Science and Technology.

References Abbas, A.K., Lichtman, A.H., and Pober, J.S. (1997). Cellular and Molecular Immunology. W.B. Saunders, Philadelphia, Pennsylvania. Adi, S., Wu, N.-Y., and Rosenthal, S.M. (2001). Growth factor-stimulated phosphorylation of Akt and p70S6K is differentially inhibited by LY294002 and wortmannin. Endocrinology, 142, 498–501. Appleman, L.J., van Puijenbroek, A.A. F.L., Shu, K.M., Nadler, L.M., and Boussiotis, V.A. (2002). CD28 costimulation mediates down-regulation of p27kip1 and cell cycle progression by activating of the PI3K/PKB signaling pathway in primary human T cells. J. Immunol., 168, 2729–2736. Arch, R.H., and Thompson, C.B. (1998). 4-1BB and Ox40 are members of a tumor necrosis factor (TNF)-nerve growth factor receptor subfamily that bind TNF receptor-associated factors and activate nuclear factor κB. Mol. Cell. Biol., 18, 558–565. Beginat, J., Sallusto, F., and Lanzavecchia, A. (2003). Cytokine-driven proliferation and differentiation of human na¨ıve, central memory and effector memory CD4+ T cells. Pathologie Biologie., 51, 64–66. Bertram, E.M., Lau, P., and Watts, T.W. (2002). Temporal segregation of 4-1BB versus CD28-mediated costimulation: 4-1BB ligand influences T cell numbers late in the primary response and regulates the size of the T cell memory response following influenza infection. J. Immunol., 168, 3777–3785. Botz, J., Zerfass-Thome, K., Spitzovsky, D., Delius, H., Vogt, B., Eilers, M., Hatzigeorgiou, A., and Jansen-Durr, P. (1996). Cell cycle regulation of the murine cyclin E gene depends on an E2F binding site in the promoter. Mol. Cell. Biol., 16, 3401–3409. Brennan, P., Babbage, J.W., Burgering, B.M.T., Groner, B., Reif, K., and Cantrell, D.A. (1997). Phosphatidylinositol 3-kinase couples the interleukin-2 receptor to the cell cycle regulator E2F. Immunity, 7, 679–689. Brown, T.J., Emswiler, J., Raecho, H., Larsen, C.P., Pearson, T.C., Ledbetter, J.A., Aruffo, A., and Mittler. R.S. (1997). 4-1BB costimulatory signals preferentially induce CD8 T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J. Exp. Med., 186, 47–55. Burr, J.S., Savage, N.D., Messah, G.E., Kimzey, S.L., Shaw, A.S., Arch, R.H., and Green, J.M. (2001). Cutting edge: Distinct motifs within CD28 regulate T cell proliferation and induction of Bcl-XL . J. Immunol., 166, 5331–5335. Cannons, J.L., Choi, Y., and Watts, T.H. (2000). Role of TNF receptor-associated factor 2 and p38 mitogen-activated protein kinase activation during 4-1BB-dependent immune response. J. Immunol., 165, 6193–6204.

24

Hyeon-Woo Lee and Byoung S. Kwon

Carreno, B.M., and Collins, M. (2002). The B7 family of ligands and its receptors: New pathways for costimulation and inhibition of immune responses. Annu. Rev. Immunol., 20, 29–53. Chalupny, N.J., Peach, R., Hollenbaugh, D., Ledbetter, J.A., Farr, A.G., and Aaruffo, A. (1992). T-cell activation molecule 4-1BB binds to extracellular matrix proteins. Proc. Natl. Acad. Sci. USA., 89, 10360–10364. Chambers, C.A., and Allison, J.P. (1999). Costimulatory regulation of T cell function. Curr. Opin. Cell. Biol., 11, 203–210. Chan, F.K.-M., Siegel R.M., and Lenardo, M.J. (2000). Signaling by the TNF receptor superfamily and T cell homeostasis. Immunity, 13, 419–422. Choi, B.K., Bae, J.S., Choi, E.M., Kang, W.J., Sakaguchi, S., Vinay, D.S., and Kwon, B.S. (2004). 4-1BB-dependent inhibition of immunosuppression by activated CD4+CD25+ T cell. J. Leukoc Biol., 75, 785–791. Futagawa, T., Akiba, H., Kodama, T., Takeda, K., Hosoda, Y., and Yagita, H. (2002). Expression and function of 4-1BB and 4-1BB ligand on murine dendrine dendritic cells. Int. Immunol., 14, 275–286. Gingras, A.-C., Raught, B., and Sonenberg, N. (2001). Regulation of translation initiation by FRAP/mTOR. Genes Dev., 15, 807–826. Gravestein, L.A., Amsen, D., Boes, M., Calvo, C.R., Kruisbeek, A.M., and Borst, J. (1998). The TNF receptor family member CD27 signals to Jun N-terminal kinase via Traf-2. Eur. J. Immunol., 28, 2208–2216. Gravestein, L.A., and Borst, J. (1998). Tumor necrosis factor receptor family members in the immune system. Semin. Immunol., 10, 423–434. Grillot, D.A., Merino, R., and Nunez, G. (1995). Bcl-XL displays restricted distribution during T cell development and inhibits multiple forms of apoptosis but not clonal deletion in transgenic mice. J. Exp. Med., 182, 1973–1983. Jang, I.K., Lee, Z.H., Kim, Y.-J., and Kwon. B.S. (1998). Human 4-1BB (CD137) signals are mediated by TRAF2 and activate nuclear factor-κB. Biophys. Biochem. Res. Commun., 242, 613– 621. Jones, R., Parsons, G.M., Bonnard, M., Chan, V.S.F., Yeh, W.-C., Woodgett, J.R., and Ohashi, P.S. (2000). Protein kinase B regulates T lymphocyte survival, nuclear factor κB activation, and Bcl-XL levels in vivo. J. Exp. Med. 191, 1721–1734. June, C.H., Ledbetter, J.A., Linsley, P.S., and Thompson, C.B. (1990). Role of the CD28 receptor in T-cell activation. Immunol. Today, 11, 211–216. Kanegane, H., and Tosato, G. (1996). Activation of na¨ıve and memory T cells by interleukin-15. Blood, 88, 230–235. Kenneth, A. T., and Thompson, C. B. (2002). Activation and Inhibition of lymphocytes by costimulation. J. Clin. Invest., 109, 295–299. Khoshnan, A., Tindell, C., Laux, I., Bae, D., Bennett, B., and Nel, A.E. (2000). The NF-κB cascade is important in Bcl-XL expression and for the anti-apoptotic effects of the CD28 receptor in primary human CD4+ lymphocytes. J. Immunol., 165, 1743–1749. Kim, Y.J., Brutkiewicz, R.R., and Broxmeyer, H.E. (2002). Role of 4-1BB (CD137) in the functional activation of cord blood. Blood, 100, 3253 and 3260. Kim, Y.Z., Mantel, P.L., June, C.H., Kim, S.H., and Kwon, B.S. (1999). 4-1BB costimulation promotes human T cell adhesion to fibronectin. Cell. Immunol., 192, 13–21. Kuo, C.J., Chung, J., Fiorentino, D.F., Flanagan, W.M., Blenis, J., and Crabtree, G.R. (1992). Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase. Nature, 358, 70–73. Kwon, B., Lee, H.W., and Kwon, B.S. (2002). New insights into the role of 4-1BB in immune response: Beyond CD8+ T cells. Trend Immunol., 23, 378–380. Kwon, B., Moon, C.H., Seo, S.K., and Kwon, B.S. (2000). 4-1BB: Still in the midst of darkness. Mol. Cells, 30, 119–126. Kwon, B.S., and Weissman, S.M. (1989). cDNA sequences of two inducible T-cell genes. Proc. Natl Acad. Sci. USA., 86, 1963–1967. Lantz, O., Grandhean, I., Matzinger, P., and Di Santo, J.P. (2000). Gamma chain required for na¨ıve CD4+ T cell survival but not for antigen proliferation. Nat. Immunol., 1, 54–58.

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Lee, H.H., Dadgostar, H., Cheng, Q., Shu, J., and Cheng, G. (1999). NF-κB-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. Proc. Natl. Acad. Sci. USA., 96, 9136–9141. Lee, H.W., Nam, K.O., Park. S.J., and Kwon, B.S. (2003a). 4-1BB Enhances CD8+ T cell expansion by regulating cell cycle progression through changes in expression of cyclins D2 and E and cyclin-dependent kinase inhibitor p27kip1 . Eur. J. Immunol., 33, 2133–2141. Lee, H.W., Nam, K.O., Seo, S.K., Kim, Y.H., Kang, H., and Kwon, B.S. (2003b). 4-1BB cross-linking enhances the survival and cell cycle progression of CD4 T lymphocytes. Cellular Immunol., 223, 143–150. Lee, H.W., Park, S.J., Choi, B.K., Kim, H.H., Nam, K.O., and Kwon, B.S. (2002). 4-1BB promotes the survival of CD8+ T lymphocytes by increasing expression of Bcl-XL and Bfl-1. J. Immunol., 169, 4882–4888. Lenschow, D.J., Walunas, T.L., and Bluestone, J.A. (1996). CD28/B7 system of T cell costimulation. Annu. Rev. Immunol., 14, 233–258. Leone, G., DeGregori, J., Sears, R., Jakoi, L., and Nevins, H. (1997). Myc and Ras collaborate in inducing accumulation of active cyclin E/Cdk2 and E2F. Nature, 387, 422–426. Lodolce, J.P., Boone, D.L., Chai, S., Swain, R.E., Dassopoulos, T., Trettin, S., and Ma, A. (1998). IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity, 9, 669–676. Marwali, M.R., Rey-Ladino, J., Dreolini, L., Shaw, D., and Takei, F. (2003). Membrane cholesterol regulates LFA-1 function and lipid raft heterogeneity. Blood, 102, 215–222. Matakeyama, M., Brill, J.A., Fink, G.R., and Weinberg, R.A. (1994). Collaboration of G1 cyclins in the functional inactivation of the retinoblastoma protein. Genes Dev., 8, 1759–1771. Melero, I., Shuford, W.W., Newby, S.A., Aruffo, A., Ledbetter, J.A., Hellstrom, K.E., Mittler, R.S., and Chen, L. (1997). Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat. Med., 3, 682–685. Michael, C. (2003). Costimulation of T cells by OX40, 4-1BB, and CD27. Cytokine Growth Factor Rev., 14, 265–273. Montagnoli, A., Fiore, F., Eytan, E., Carrano, A.C., Draetta, G.F., Hershko, A., and Pagano, M., (1999). Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation. Genes Dev., 13, 1181–1189. Moon, J.J., and Nelson, B.H. (2001). Phosphatidylinositol 3-kinase potentiates, but does not trigger, T cell proliferation mediated by the IL-2 receptor. J. Immunol., 167, 2714–2723. Mueller, D.L. (2000). T cell: A proliferation of costimulatory molecules. Curr. Biol., 10, R227–R230. Mueller, D.L., Jenkins, M.K., and Schwarz, R.H. (1989). Clonal expansion versus functional clonal inactivation: a costimulatory signaling pathway determines the outcome of T cell antigen receptor occupancy. Annu. Rev. Immunol., 7, 445–480. Nakajima, H., Shores, E.W., Noguchi, M., and Leonard, W.J. (1997). The common cytokine receptor gamma chain plays an essential role in regulating lymphoid homeostasis. J. Exp. Med., 185, 189–195. Nam, K.O., Kang, H.S., Shin, M., Cho, K.H., Kwon, B., Kwon, B.S., Kim, S.J., and Lee, H.W. (2005). Cross-linking of 4-1BB activates TCR-signaling pathways in CD8+ T lymphocytes. J. Immunol., 174, 1898–1905. Okkenhaung, K., Wu, L., Garza, K.M., La Rose, J., Khoo, W., Odermatt, B., and Mak T.K. (2001). A point mutation in CD28 distinguishes proliferative signals from survival signals. Nat. Immunol., 2, 325–332. Ozes, O.N., Mayo, L.D., Gustin, J.A., Pfeffer, S.R., Pfeffer, L.M., and Donner, D.B. (1999). NF-κB activation by tumour necrosis factor requires the Akt serine-theonine kinase. Nature, 401, 82–85. Pan, P.Y., Gu, P., Li, Q., Xu, D., Weber, K., and Chen, S.H. (2004). Regulation of Dendritic Cell Function by NK Cell: Mechanisms Underlying the Synergism in the Combination Therapy of IL-12 and 4-1BB Activation. J. Immunol., 172, 4779–4789. Poltak, K., Kato, J.-Y., Solomon, M.J., Sherr, C.J., Massague, J., Roberts, J.M., and Koff, A. (1994). p27Kip1 , a cyclin-cdk inhibitor, links transforming growth factor-b and contact inhibition to cell cycle arrest. Genes Dev., 8, 9–22.

26

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Resnitzky, D., and Reed, S.I. (1995). Different roles for cyclins D1 and E in regulation of the G1-to-S transition. Mol. Cell. Biol., 15, 3463–3469. Rothstein, D.M., and Sayegh, M.H. (2003). T-cell costimulatory pathways in allograft rejection and tolerance. Immunol. Rev., 196, 85–108. Salazar, E.P, and Rozengurt, E. (1999). Bombesin and platelet-derived growth factor induce association of endogenous focal adhesion kinase with Src in intact Swiss 3T3 cells. J. Biol. Chem., 274, 28371– 28378. Samia, J.K., and Mohamed, H.S. (2004). The Roles of the new negative T cell costimulatory pathways in regulating autoimmunity. Immunity, 20, 529–538. Saoulli, K., Lee, S.Y., Cannons, J.L., Yeh W.C., Santana, A., Goldstein, M.D., Bangia, N., DeBenedette, M.A., Mak, T.W., Choi, Y., and Watts, T. H. (1998). CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand. J. Exp. Med., 187, 1849–1862. Schluns, K.S., Kieper, W.C., Jameson, S.C., and Lefrancois, L. (2000). Interleukin-7 mediates the homeostasis of na¨ıve and memory CD8 T cells in vivo. Nat Immunol., 1, 426–432. Schwarz, H., Tuckwell, J., and Lotz, M. (1993). A receptor-induced by lymphocyte activation (ILA): A new member of the human nerve growth factor/tumor necrosis factor receptor family. Gene, 134, 295–298. Sherr, C.J., and Roberts, J.M. (1999). CDK inhibitors: Positive and negative regulators of G1 -phase progression. Genes Dev., 13, 1501–1512. Seo, S.K., Choi, J.H., Kim, Y.H., Young, H., Kang, W.J., Suh, J.H., Choi, B. K., Vinay, B.S., and Kwon, B.S. (2004). 4-1BB-mediated immunotherapy of rheumatoid arthritis. Nat. Med., 10, 1088–1094. Shuford, W.W., Klussman, K., Tritchler, D.D., Loo, D.K., Chalupny, J., Siadak, A.W., Brown, T.J., Emswiler, J., Raecho, H., Larsen, C.P., Pearson, T.C., Ledbetter, J.A., Aruffo, A., and Mittler, R.S. (1997). 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J. Exp. Med., 186, 46–55. Tan, J.T., Ha, J., Cho, H.R., Tucker-Burden C., Hendrix, R.C., Mittler, R.S., Pearson, T.C., and Larsen, C.P. (2000). Analysis of expression and function of the costimulatory molecule 4-1BB in alloimmune responses. Transplantation, 70, 175–183. Toyoshima, H., and Hunter, T. (1994). p27, a novel inhibitor of G1 cyclin/Cdk protein kinase activity, is related to p21. Cell, 78, 67–74. Tsvetkov, L.M., Yeh, K.H., Lee, S.J., Sun, H., and Zhang, H. (1999). p27 (Kip1) ubiquitination and degradation is regulated by the SCF (Skp2) complex through phosphorylated Thr187 in p27. Curr. Biol., 9, 661–664. Unutmaz, D., Baldoni, F., and Abrignani, S. (1995). Human na¨ıve T Cells activated by cytokines differentiate into a split phenotype with functional features intermediate between na¨ıve and memory T cells. Int. Immunol., 7, 1417–1424. Unutmaz, D., Pileri, P., and Abrignani, S. (1994). Antigen-independent activation of na¨ıve and memory resting T cell by a cytokine combination. J. Exp. Med., 180, 1159–1164. Van Parijs, L., and Abbas, A.K. (1998). Homeostasis and self-tolerance in the immune system: Turning lymphocytes off. Science, 280, 243–248. Vidalain, P.O., Azocar, O., Servet-Delprat, C., Rabourdin-Combe, C., Gerlier, D., and Manie, S. (2000). CD40 signaling in human dendritic cells is initiated within membrane rafts. EMBO J., 19, 3304– 3313. Vinay, D.S., and Kwon, B.S. (1998). Role of 4-1BB in immune response. Semin. Immunol., 10, 481–489. Vlahos, C.J., Matter, W.F., Hui, K.Y., and Brown, R.F., (1994). A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem., 269, 5241–5248. Watts, T.H., and DeBenedette, M.A. (1999). T cell co-stimulatory molecules other than CD28. Curr. Opin. Immunol., 11, 286–293. Weinberg, A.D., Vella, A.T., and Croft, M. (1998). Ox-40: Life beyond the effector T cell stage. Semin. Immunol., 10, 471–480. Wilcox, R.A., Chapoval, A.I., Gorski, K.S., Otsuji, M., Shin, T., Flies, D.B., Tamada, K., Mittler, R.S., Tsuchiya, H., Pardoll, D.M., and Chen, L. (2002). Cutting edge: Expression of functional CD137 receptor by dendritic cells. J Immunol., 168, 4262–4267.

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Ye, H., Park, Y.C., Kreishman, M., Kieff, E., and Wu, H. (1999). The structure basis for the recognition of diverse receptor sequence by TRAF2. Mol. Cell, 4, 321–330. Zhang, X., Sun, S., Hwang, I., Tough, D.F., and Sprent, J. (1998). Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity, 8, 591–599. Zheng, G., Wang, B., and Chen, A. (2004). The 4-1BB Costimulation Augments the Proliferation of CD4+CD25+ Regulatory T Cell. J. Immunol., 173, 2428–2434.

3 Significance of Reverse Signal Transduction for the Biology of the CD137 Receptor/Ligand System Herbert Schwarz

CD137 is a member of the TNF receptor family and it has originally been identified as a potent T cell costimulatory molecule. Recently, it has become evident that CD137 signals can also inhibit T cell activity under certain conditions. The CD137 receptor/ligand system has the ability to signal bidirectionally. CD137 ligand is also expressed as a cell membrane protein and it can also transduce signals into the cells it is expressed on, referred to as reverse signaling. The signals through CD137 ligand are activating or costimulatory for antigen presenting cells (APC). Together with CD137, which can deliver costimulatory signals to T cells, the CD137 receptor/ligand pair can therefore form a potent proinflammatory system enhancing immune reactions by stimulating APC as well as T cells. CD137 ligand signals, however, negatively regulate T cell proliferation and survival, and it is possible that this activity of CD137 ligand participates in the termination of immune responses. The bidirectional signaling capacity allows the CD137 receptor/ligand system to mediate extensive crosstalk between immune cells and between immune and non-immune cells. CD137 (4-1BB, induced by lymphocyte activation, ILA) is a member of the tumor necrosis factor (TNF) receptor family (Kwon and Weissman, 1989; Schwarz et al., 1993). CD137 has originally been identified as a potent T cell costimulatory molecule and a promising target for immunotherapy of cancer (Melero et al., 1997). Recent evidence also demonstrates a role for CD137 signaling in T cells for inhibiting immune responses and autoimmune disease (Foell et al., 2003; 2004). The other chapters of this book and several recent reviews provide a comprehensive overview over the various activities of CD137 signaling on immune functions and their potential therapeutic applications (Al-Shamkhani, 2004; Croft, 2003; Kwon et al., 2000; Sica and Chen, 2000). Interaction of CD137 with CD137 ligand, however, not only initiates a signal into the CD137-expressing cell but also into the CD137 ligand-expressing Herbert Schwarz • Department of Physiology, National University of Singapore, 2 Medical Drive, MD 9, Singapore 117597 29 CD137 Pathway: Immunology and Diseases. Edited by Lieping Chen, Springer, New York, 2006

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Herbert Schwarz

monocyte

activated T cell

Costimulation

Activation

CD137 ligand

CD137

Figure 3.1. Schematic representation of bidirectional signaling mediated by the CD137 receptor/ligand system between a monocyte and an activated T cell. In this case both cells receive activating signals, through CD137 ligand and CD137, respectively.

cell. In other words, the CD137 receptor/ligand system can signal bidirectionally (Figure 3.1). Signal transduction through the ligand is referred to as reverse signaling. The CD137/CD137 ligand system shares the ability of bidirectional signal transduction and reverse signaling with many other members of the TNF receptor and TNF families (Arens et al., 2004; Eissner et al., 2004). Reverse signaling in the TNF family is possible because most of its members are expressed as membrane proteins with cytoplasmic domains (Gravestein and Borst, 1998; Lotz et al., 1996). The designation as ligands for these molecules is based on historical reasons but in functional terms they would more aptly be described as counter receptors. For the CD137 receptor/ligand system reverse signal transduction, that is signaling through CD137 ligand is an activity which has received far less attention than signal transduction through CD137 and its initiated biological activities. This chapter aims to summarize and review what is known about signal transduction through CD137 ligand and that side of the CD137/CD137 ligand biology (Table 3.1). In the following, the description of the effects of CD137 ligand signaling is organized according to the cell types involved.

1. Biology of Reverse Signaling Through CD137 Ligand 1.1. CD137 Ligand Activities on Monocytes and Macrophages CD137 ligand is expressed constitutively on peripheral monocytes and monocyte/macrophage cell lines (Table 3.2). The signal through CD137 ligand activates monocytes and the CD137 ligand signal alone is sufficient for activation. When CD137 ligand is crosslinked by recombinant CD137 protein or an anti-CD137 ligand antibody, it induces adherence of monocytes within a few hours. The adherent monocytes change their shape over the course of a week and adopt the three basic macrophage morphologies, which are round, elongated, and branched cells (Langstein and Schwarz, 1999). Concomitantly with adherence the CD137 ligand signaling induces the expression of proinflammatory cytokines (TNF, IL-6, IL-8, IL-12) and activation markers (ICAM-1), and it inhibits expression of anti-inflammatory cytokines (IL-10) and differentiation markers (FcγRIII), (Laderach et al., 2003; Langstein et al., 1998).

Significance of Reverse Signal Transduction for the Biology

31

Table 3.1. Activities of CD137 Ligand Signaling by Cell Type. (Trg.: CD137 ligand-transgenic; Def.: CD137 ligand-deficient; H: human; M: mouse; S: species.) Cell type

S

Activity

System

Reference

Monocytes Macrophages

H

Induction of TNF, IL-6, IL-8, IL-12, ICAM and adherence. Induction of M-CSF and prolongation of survival. Induction of proliferation and endomitosis. Inhibition of FcγRIII and IL-10 Amplification of cell numbers

in vitro

Langstein et al., 1998; Langstein and Schwarz, 1999; Langstein et al., 1999; Langstein et al., 2000; Laderach et al., 2003; Ju et al., 2003

M Dendritic cells

H

M B cells

H

M M T cells

H

M

Trg. mice

Zhu et al., 2001

Induction of IL-12, CD11c, CD86, CD137 ligand, MHC class II and cellular adherence Induction of IL-6, IL-12, CD80 and CD86

in vitro

Kim et al., 2002; Laderach et al., 2003

in vitro

Futagawa et al., 2002

Costimulation of proliferation and immunoglobulin secretion Costimulation of proliferation Elimination of peripheral B cells

in vitro

Pauly et al., 2002

in vitro Trg. mice

Pollok et al., 1994 Zhu et al., 2001

Inhibition of proliferation and induction of cell death by apoptosis Inhibition of proliferation

in vitro

Schwarz et al., 1996; Michel et al., 1999; Ju et al., 2003

Def. mice

Kwon et al., 2002

Bone marrow cells

M

Induction of proliferation. Inhibition of osteoclast differentiation

in vitro

Saito et al., 2004

Carcinoma cells

H

Induction of IL-8

in vitro

Salih et al., 2000

The degree of monocyte activation by CD137 ligand signaling is dosedependent. IL-8 release correlates with the concentration of recombinant CD137 protein (Langstein et al., 1998) and reducing the CD137 signal strength also delays adherence and morphological changes (unpublished observation). The process of monocytes adherence is at least in vitro associated with a cellular activation. However, activation induced by CD137 ligand signaling is not merely a consequence of the monocytes starting to adhere, since activation can also be induced through CD137 ligand when the cells are cultured under non-adherent conditions. However, crosslinking of CD137 ligand is essential for activation as soluble CD137 protein is inactive and has no effects on monocytes (Langstein et al., 1998). The CD137 ligand signal also induces expression of macrophage colonystimulating factor (M-CSF), an essential survival factor for monocytes and significantly prolongs survival of monocytes in vitro (Langstein and Schwarz, 1999a). Neutralizing anti-M-CSF antibodies block CD137 ligand-mediated cell survival

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Herbert Schwarz

Table 3.2. CD137 Ligand Protein Expression by Tissue. Listed are Only Reports Which Demonstrate CD137 Ligand Expression at the Protein Level. (Const.: constitutive; H: human; M: Mouse; S: species.) Cell type

S

Expression

Reference

T cells - Primary, resting - Jurkat, CEM - T cells clones - Primary - Primary - T cell clones - EL-4, WR-19L, BW5147 - L5178Y

H H H M M M M M

Negative Constitutive Inducible by anti-CD3 Negative Const., low levels, not inducible Const., low levels, not inducible Constitutive Negative

Salih et al., 2000 Palma et al., 2004 Alderson et al., 1994 Futagawa et al., 2002 Pollok et al., 1994 Pollok et al., 1994 Futagawa et al., 2002 Futagawa et al., 2002

B cells - Primary - Primary - Daudi, SKW6.4 - Raji, IM-9 - Primary - 2PK-3, A20 - K46J, M12

H H H H M M M

Zhou et al., 1995 Jung et al., 2004 Zhou et al., 1995 Palma et al., 2004 DeBenedette et al., 1997 Pollok et al., 1994 DeBenedette et al., 1995

- A20, BCL1-B20, 2-PK-3 - BAL17

M M

Induced by pock weed mitogen Constitutive Constitutive Constitutive Induced by CD40L and cAMP Constitutive Constitutive, enhanced by cAMP Constitutive Negative

Monocyte, Macrophage - Primary

H

Constitutive

H M

Constitutive Constitutive

Laderach et al., 2003; Ju et al., 2003; Jung et al., 2004 Ju et al., 2003 Pollok et al., 1994; Futagawa et al., 2002

H H

Constitutive on subpopulation Constitutive, enhanced by CD137 ligand Constitutive, enhanced by IL-1 and α-CD40 Const., enhanced by LPS, TNF/ PGE2, IFN-γ, α-CD40, dsRNA Const., enhanced by LPS/α-CD40 Constitutive

- HL60 - RAW264.7, J774.1, P388D1 Dendritic cells - Isolated from tonsil - Derived from umbilical cord blood progenitors - Deriv. fr. monocytes or hematop. progenitors - Derived from monocytes

H H

- Isolated from spleen

M

Sarcoma: Colon (Colo 205, HT29, HCT116), lung (LX 1, L2987), breast (SKBR), ovarian (A2780), prostate (PC3)

H

Futagawa et al., 2002 Futagawa et al., 2002

Summers et al., 2001 Kim et al., 2002 Laderach et al., 2003 Lee et al., 2003

Futagawa et al., 2002 Salih et al., 2000

Significance of Reverse Signal Transduction for the Biology

33

confirming that M-CSF is essential for monocyte survival and demonstrating that also CD137-induced monocyte survival is mediated by M-CSF. Contrary to what might have been expected based on the life-prolonging effects of the CD137 ligand signal on monocytes, it also enhanced the rate of apoptosis. Apoptosis as measured by the amount of fragmented DNA was several-fold increased throughout a 1 week culture period by CD137 ligand signaling. At the same time, the percentage of living cells was also increased (Langstein et al., 1999). Simultaneous induction of apoptosis, together with activation and proliferation is well known in lymphocytes as activation induced cell death (Green et al., 2003). However, this concept was not easily transferable to monocytes/macrophages as these cells have been assumed not to be able to proliferate. But when tested whether the CD137 ligand signal can induce proliferation it was indeed noticed that proliferation did occur. H3 -thymidine incorporation increased steadily with time upon CD137 ligand signaling and at its peak at day 8 was increased by 30-fold. Close to 10% of the cells incorporated Bromo-deoxyuridine within a 1 h labeling interval, demonstrating that a large proportion of the cells were active in replicating their DNA (Langstein et al., 1999). But while DNA was replicated and the cells grew in size, their numbers did not increase implying that endomitosis occurred. Indeed, many of the cells contained several nuclei (Langstein et al., 1999). Although induction of monocyte proliferation has also been described for M-CSF, the CD137 ligand signal is more potent in inducing proliferation and endomitosis in monocytes, indicating that it induces the release of additional factors besides M-CSF. These are also induced in monocytes upon CD137 ligand signaling and are released as conditioned supernatants from CD137-treated monocytes transfer the proliferative potential to na¨ıve monocytes (Langstein et al., 1999). This CD137 ligand-induced proliferation and endomitosis of monocytes overcompensates the loss of monocytes by CD137 ligand-induced apoptosis and allows the monocyte population to expand, not in numbers but in size. Though CD137 can activate monocytes, it is less potent than classical monocyte activators such as LPS (Langstein et al., 2000). However, the CD137 ligand signal synergizes with LPS in activating monocytes since it is able to further increase cytokine release after maximum stimulation by LPS has been reached. Also, the combination of LPS and the CD137 ligand signal, but not either alone can induce myc expression in monocytes. (Langstein et al., 1998; Langstein et al., 2000). It is in inducing proliferation that CD137 ligand exceeds the potency of other monocyte growth and activation factors and that may be its more important function in vivo. The activating effects of CD137 ligand on monocytes have also been documented in vivo. Transgenic mice overexpressing CD137 ligand on antigen presenting cells (APC) develop a threefold increased number of macrophages (Zhu et al., 2001). CD137 ligand-deficient mice on the other hand have an increased number of myeloid progenitor cells in the peripheral blood, bone marrow, and spleen (Kwon et al., 2002). It seems these cells cannot fully mature to monocytes, macrophages, and possibly dendritic cells due to the lack of the CD137 ligand signal. CD137 ligand provides potent activating signals to monocytes and likely monocyte precursors. Since expression of CD137 is strictly activation-dependent,

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Herbert Schwarz

monocytes would encounter CD137 in tissues where activated immune cells or endothelial cells are present (Schwarz et al., 1995; Broll et al., 2001). Therefore, reverse signaling by CD137 ligand into monocytes is expected to amplify ongoing immune responses.

1.2. CD137 Ligand Activities on Dendritic Cells CD137 ligand is expressed at low levels on murine and human dendritic cells which were derived in vitro from monocytes or hematopoietic progenitor cells or were isolated from tonsil or spleen. Its expression on dendritic cells is enhanced by proinflammatory stimuli, including IL-1, CD40 ligand, LPS, and double stranded RNA (Table 3.2). Crosslinking of the CD137 ligand enhances the expression of CD11c, CD80, CD86 and MHC class II and induces cellular adherence and the release of IL-6 and IL-12 (Futagawa et al., 2002; Kim et al., 2002; Langstein et al., 1999). Different investigations come to different conclusions on whether the CD137 signal alone is sufficient to induce dendritic cell activation or whether it only works in combination with additional activating signals such as CD40 ligand. Interestingly, the CD137 ligand signal also upregulates CD137 ligand expression, pointing to a positive feed back loop (Kim et al., 2002). These activities on dendritic cells are very similar to CD137 activities on monocytes. The physiological function of the CD137 ligand signal in dendritic cells is an induction or enhancement of their antigen-presenting capacity, and consequently an initiation or enhancement of immune responses.

1.3. CD137 Ligand Activities on B Cells B cells, another group of APC, can also express CD137 ligand. Human and murine transformed B cells express CD137 ligand protein constitutively while activation may be required for primary B cells (Table 3.2). Unlike in the case of monocytes and potentially dendritic cells, signals through CD137 ligand do not initiate activation of B cells. Rather, they enhance proliferation and immunoglobulin synthesis of preactivated B cells (Pauly et al., 2002; Pollok et al., 1994). In contrast to these in vitro data, constitutive expression of CD137 ligand on APC in transgenic mice causes a gradual elimination of peripheral B cells (Zhu et al., 2001). The CD137 ligand-transgenic mice have normal B cell numbers and functions up to the age of three months, and develop B cell deficiencies only later in life. This could indicate that initially the signals through CD137 ligand are activating for B cells, while prolonged CD137 ligand signals may be deleterious for B cell numbers and functions, possibly due to overstimulation. An alternative explanation would be that activated T cells or other CD137-expressing cells receive too much CD137 stimulation by being exposed to the CD137 ligand-expressing transgenic B cells, and in turn lead to an elimination of the B cells. Indeed, it has been shown that crosslinking of CD137 on monocytes causes them to induce apoptosis in B cells (Kienzle and von Kempis, 2000).

Significance of Reverse Signal Transduction for the Biology First antigen CD4+ encounter T-cell

B-cell

35

Costimulation of primary antibody response

affinity maturation Second antigen encounter FDC

CD40 ligand

B-cell

CD40

CD137

Costimulation of secondary antibody response CD137 ligand

Figure 3.2. Depiction of the suggested roles of the CD40 and CD137 receptor/ligand systems in B cell maturation and the costimulation of humoral immune responses. At the first antigen encounter the CD40 receptor/ligand system costimulates B cell proliferation and immunoglobulin class switch. At the second antigen encounter during affinity maturation the CD137 receptor/ligand system costimulate B cell proliferation and immunoglobulin synthesis. FDC: follicular dendritic cell.

These functional data on CD137 ligand-mediated B cell activation are supported by histological findings. CD137 is expressed by follicular dendritic cells in germinal centers (Lindstedt et al., 2003; Pauly et al., 2002). B cells accumulate after first antigen encounter in these anatomical structures and undergo the process of affinity maturation. Follicular dendritic cells present the antigens in form of iccosomes to the B cells and play an essential role in the clonal selection of B cells with high affinity B cell receptors. Its expression on follicular dendritic cells allows CD137 to provide costimulatory and survival signals to those B cells, which have rearranged their immunoglobulin genes resulting in a high affinity binding to the antigen (Pauly et al., 2002). The activities of CD137 and its ligand in B cell activation and development are reminiscent of those of the CD40 receptor/ligand pair, also members of the TNF receptor and ligand families. The CD40 receptor/ligand system mediates T cell help to B cells, which have encountered their specific antigen for the first time. It can be hypothesized that after somatic hypermutation of the complementary determining region, the second antigen encounter takes place on the surface of follicular dendritic cells, and here costimulation is mediated—at least in part—by the CD137 receptor/ligand system (Figure 3.2).

1.4. CD137 Ligand Activities on Bone Marrow Cells Expression of CD137 ligand on murine bone marrow cells has been documented at the mRNA level, and CD137 ligand signaling takes place not only in differentiated hematopoietic cells but also in bone marrow cells. Proliferation of murine bone marrow cells treated with M-CSF is enhanced when the cells are exposed to immobilized CD137 protein. CD137 ligand signals also inhibit receptor activator of nuclear factor-κB (RANK) ligand-induced differentiation of bone marrow cells towards osteoclasts (Saito et al., 2004). As mentioned above, a missing differentiation signal may also be the cause for the amplification of

36

Herbert Schwarz

myeloid progenitors in CD137 ligand-deficient mice (Kwon et al., 2002). Taken together, these data suggest that the CD137 ligand signal directs differentiation of bone marrow cells away from osteoclast and into the myeloid lineage. And the CD137 ligand signal remains important even after differentiation of the bone marrow cells to monocytes and macrophages, as CD137 ligand provides activating and growth-enhancing signals to these cells.

1.5. CD137 Ligand Activities on T Cells Expression of CD137 ligand protein could either not be detected on primary T cells, or only at low levels. But CD137 ligand is present on human and murine T cell lines (Table 3.2). It may be that CD137 ligand is expressed on primary T cells at such low levels that detection by commonly used detection methods such as flow cytometry is difficult. T cell lines may acquire stronger CD137 ligand expression during the transformation process. The activities of CD137 ligand on T cells stand in contrast (1) to its activities on APC, and (2) to the activities of CD137 on T cells. The signal through CD137 ligand downregulates T cell activity. Coculture of anti-CD3-activated human PBMC with CD137-expressing transfected CHO or COS-7 cells completely inhibits proliferation and induces cell death by apoptosis. Anti-CD137 ligand antibodies and recombinant CD137 protein have the same effects. Recombinant CD137 protein works only when it is immobilized on the tissue culture plates, or crosslinked via secondary antibodies while it is inactive when added as a soluble protein, demonstrating also for T cells that these effects are mediated by crosslinking CD137 ligand (Ju et al., 2003; Schwarz et al., 1996). The inhibitory effect of CD137 ligand on T cells is also evident in vivo. Splenocytes from CD137-deficient mice respond with an increased proliferation to stimulation with mitogens or anti-CD3. Addition of a CD137 ligand signal by cocultivating CD137-deficient splenocytes with CD137-expressing cells reduces their proliferation to the level of wild-type cells (Kwon et al., 2002). At present it is not clear whether CD137 exerts this inhibitory effects directly by crosslinking CD137 ligand on T cells, or indirectly via other CD137 ligand-expressing cells. In contrast to signals through CD137 which are generally costimulatory for T cells, signals through CD137 ligand are inhibitory. CD95 and CD95 ligand, two other members of the TNF receptor and TNF families, respectively, exert similar opposite effects on T cells. Although the signal through CD95 induces apoptosis in CD8-positive T cells, the signal through CD95 ligand is costimulatory (Suzuki and Fink, 1998). Not much is known about the underlying mechanism of the T cell inhibitory activities of CD137 ligand. Although CD137 ligand signaling induces expression of CD95 on CD4- and CD8-positive T cells and on B cells, induction of apoptosis does not seem to involve CD95 (Michel et al., 1999). For one, antagonistic CD95 antibodies do not block CD137 ligand-induced apoptosis, and secondly, CD137 ligand induces apoptosis in resting as well as activated lymphocytes, while CD95induced apoptosis is restricted to activated lymphocytes. Also, apoptosis signals through CD137 ligand are less potent and have a slower kinetic than apoptosis

Significance of Reverse Signal Transduction for the Biology

37

(A)

DC

T cell Costimulation

CD137-expressing cell

Apoptosis (B)

T cell

CD137-expressing cell

Apoptosis

CD137 ligand CD137 Other costimulatory receptor//ligand pair

Figure 3.3. Role of the CD137 receptor/ligand system in (A) the initiation and maintenance, and (B) the downregulation of T cell responses. (A) At the beginning of an immune response T cells receive activating signals from dendritic cells (DC) through CD137 and other costimulatory molecules. The inhibitory signals through CD137 ligand are blocked by activating signals. (B) Once the cause of the immune response has been eliminated, the dendritic cell will no longer provide activating signals and inhibit CD137 ligand-mediated apoptosis in T cells. Among the CD137-expressing cells, which can crosslink CD137 ligand on T cells and initiate apoptosis are also activated T cells. Therefore, in this phase of an immune response the number and density of activated T cells correlates with the inhibitory signals through CD137 ligand.

signals through CD95, taking days rather than hours until apoptosis is significantly noticeable (Schwarz et al., 1996; Michel et al., 1999). What could be the physiological function of the inhibitory activities of CD137 ligand on T cells? No studies have been reported addressing this question. It may be that CD137 ligand provides a negative feedback signal to T cells. CD137 expression on T cells is strictly activation-dependent. With a mounting immune response and an accompanying increasing density of CD137-expressing T cells, the inhibitory signals through CD137 ligand would also increase. But as long as the antigen is present, activated APC will express CD137 ligand and other costimulatory molecules, which may block the inhibitory CD137 ligand signals (Figure 3.3A). However, when the antigen has been cleared, APC will no longer provide costimulation and the inhibitory activities of CD137 ligand may become predominant, thereby contributing to the downsizing of a no longer needed antigen-specific T cell response (Figure 3.3B).

1.6. CD137 Ligand Activities on Non-hematopoietic Cells CD137 ligand expression could be detected on human carcinoma cell lines of colon, lung, breast, ovarian, and prostate origin (Table 3.2). Crosslinking of CD137 ligand induces release of IL-8 from tumor cells (Salih et al., 2000). It is

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(A)

Tumor cell

T cell

APC

T cell Costimulation

Inhibition

(B)

Inhibition

CD137 ligand

CD137

Figure 3.4. Activities of the CD137 receptor/ligand system in tumor-immune interactions. Ectopic expression of CD137 ligand on tumor cells inhibits an anti-tumor immune response by inducing inhibitory signal through CD137 in T cells (A). The tumor cell would here imitate an APC in a situation in which it downregulates T cell activity via the CD137 receptor/ligand system (B). The interaction of an APC with a T cell can also enhance an anti-tumor T cell response in a physiological context which favors transmission of costimulatory signals to T cells (B).

at present not known whether CD137 ligand is also present on the corresponding healthy tissues or whether the tumor cells acquire CD137 ligand expression during the transformation process. It used to be puzzling why tumor cells would express CD137 ligand, which has been shown to induce an anti-tumor immune response upon transgenic expression on tumor cells (Guinn et al., 1999; Melero et al., 1998). Further, IL-8 which is a proinflammatory chemokine and is induced by reverse signaling through CD137 ligand in the tumor cells is also expected to enhance an anti-tumor immune response. However, recently it has become evident that CD137 not only costimulates T cells, but under certain circumstances can inhibit T cell activity and immune responses (Al-Shamkhani, 2004; Foell et al., 2003; 2004). It is at present not clear under which circumstances the CD137 signal is activating or inhibitory for T cells. But it is conceivable that the local environment in the tumor favors the inhibitory over the costimulatory activity of CD137 on T cells. The anti-tumor activity of infiltrating T cells would then be inhibited by tumor-expressed CD137 ligand. Thus, expression of CD137 ligand—possibly as a neoantigen—may contribute to escape of tumor cells from immunosurveillance (Figure 3.4A). In that case the CD137 ligand-expressing tumor cell would imitate an APC in a situation where it delivers inhibitory CD137 signals to T cells (Figure 3.4B). It needs to be emphasized that the same interaction can enhance T cell activity and anti-tumor immunity in a different physiological context. Signaling through CD137 ligand into the tumor cell would not be required for this explanation of ectopic CD137 ligand expression on tumors.

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1.7. CD137 Ligand Signal Transduction Pathway Signal transduction pathways which mediate reverse signaling through CD137 ligand have been studied using M-CSF or RANK ligand-induced osteoclast differentiation of murine bone marrow cells. The CD137 ligand signal suppressed phosphorylation of Akt, whereas it had no effect on the phosphorylation of inhibitor of receptor activator of nuclear factor-κB (I-κB) and extracellular signal-regulated kinase (ERK) 1/2, p38 and jun kinase. The CD137 ligand signal also suppressed nuclear factor of activated T cells (NFAT)-2 induction but did not affect the expression of TNF receptor-associated factor (TRAF) 6 and c-Fos. Casein kinase is also implicated in CD137 ligand signaling since its inhibition blocked CD137 ligandmediated osteoclast differentiation and proliferation of bone marrow cells (Saito et al., 2004). A recognition site for casein kinase is present in the cytoplasmic domain of CD137 ligand (Watts et al., 1999).

1.8. Regulation of CD137 Ligand Signaling Expression of both, CD137 and CD137 ligand is regulated activationdependently. CD137 ligand is expressed constitutively on many cells, and most if not all immune cells. Cell activation however may increase CD137 ligand expression. In contrast, expression of CD137 seems to be more tightly regulated. Except for dendritic cells (Wilcox et al., 2002) there is no constitutive expression of CD137 on primary cells known. Cell activation is more critical for CD137 expression. Therefore, the interaction of CD137 and CD137 ligand, and the initiation of signaling through both molecules is mainly regulated through CD137 expression.

1.9. Influence of Soluble CD137 and Soluble CD137 Ligand on CD137 Ligand Signaling Soluble forms of CD137 (sCD137) and CD137 ligand exist. Soluble CD137 is generated by differential splicing and released by activated T cells (Michel et al., 1998). Levels of sCD137 are enhanced in sera of autoimmune, leukemia and lymphoma patients (Furtner et al., 2005; Jung et al., 2004; Michel et al., 1998; Sharief, 2002). Release of sCD137 does not correlate with proliferation but rather with activation induced cell death (Michel and Schwarz, 2000), implying that sCD137 forms a negative feedback loop to reduce further activation through membrane-bound CD137 and to prevent activation induced cell death. Indeed in soluble forms of CD137 proteins antagonize the costimulatory activities of the membrane-bound CD137 and reduce immune activity (DeBenedette et al., 1995; Hurtado et al., 1995). It has been demonstrated in the case of monocytes (Langstein and Schwarz, 1999; Langstein et al., 1998; 1999) and T cells (Schwarz et al., 1996) that soluble CD137 protein is not able to crosslink CD137 ligand and to initiate signaling. Soluble forms of CD137 seem therefore to antagonize CD137 as well as CD137 ligand signaling.

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Soluble CD137 ligand is generated by proteolytic cleavage and levels of sCD137 ligand are enhanced in sera of patients suffering from hematological malignancies and autoimmune disease (Salih et al., 2001; Jung et al., 2004; Salih et al., 2004). In contrast to sCD137 which seems to be purely antagonistic, sCD137 ligand is active and can provide costimulation to T cells (Salih et al., 2001). This implies that sCD137 ligand only inhibits reverse signaling through CD137 ligand while initiating signaling through CD137.

2. Bidirectional Signaling in Other Receptor/Ligand Systems Bidirectional signal transduction is not a rare phenomenon. It has been documented for several other members of the TNF and TNFR families, Eph receptors and ephrins, integrins and cell adhesion molecules, and B7 and its receptors. Among the members of the TNF family reverse signaling has been demonstrated for membrane-integrated TNF, CD27 ligand, CD30 ligand, CD40 ligand, CD95 ligand, CD137 ligand, OX40 ligand, LIGHT, TRAIL, and TRANCE. These receptor/ligand systems can therefore signal bidirectionally and so exert their manifold and diverse activities on activation, proliferation, differentiation, and survival or death of immune cells and non-immune cells (Eissner et al., 2004). The Eph receptors are a large family of receptor protein tyrosine kinases and are highly conserved during evolution. They participate in the regulation of embryo patterning, neural development, axon guidance, angiogenesis, and vascular network assembly. Their ligands are the ephrins and some ephrins are transmembrane proteins. Together with their Eph receptors these ephrins can signal bidirectionally and activate respective downstream signaling cascades simultaneously, leading to the above mentioned biological activities (Cowan and Henckemeyer, 2002; Wilkinson, 2000). Integrins transduce signals into the cells they are expressed on upon binding to a ligand. Among the integrin ligands are cell adhesion molecules, such as ICAM1, which themselves are expressed as cell surface molecules and can transmit signals into the cells they are expressed on (Chirathaworn et al., 1995; Holland and Owens, 1997). In addition, another kind of two-way signaling has been described for integrins, referred to as inside-out and outside-in signaling (Qin et al., 2004). Ligands for integrins can also be components of the extracellular matrix and binding transmits a signal into the integrin-expressing cell (outside-in signaling). Cell activation can change the conformation of the extracellular domain of the integrin and thereby the affinity to its ligand (inside-out signaling). This type of two-way signaling of integrins is not identical to the bidirectional signaling discussed here since it often affects only one cell, the one which expresses the integrin. The CD137 receptor/ligand system shares some features with the B7/CD28 costimulatory system. Both, B7 and CD137 ligand are expressed constitutively on dendritic cells but expression is enhanced upon activation. Both engage a receptor on T cells which provides costimulation. Interestingly, it has recently been demonstrated that the two receptor/ligand systems also share bidirectional signaling, since

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B7 also is able to transduce a signal into dendritic cells. The functional activities of B7 signaling are however more complex since two ligands (B7.1 and B7.2) and two receptors (CD28 and CTLA-4) exist. The reverse signal through B7 can either tolerize dendritic cells or activate then, depending on whether B7 proteins are engaged by CTLA-4 or by CD28, respectively (Greenwald et al., 2005; Orabona et al., 2004).

3. Concluding Remarks Bidirectional signaling provides considerable complexity to the biology of the CD137 receptor/ligand system. Reverse signaling feeds back to the CD137 ligand-expressing cells and allows a more coordinated immune response and its fine-tuning. One of the important tasks for future research will be to identify which activities are mediated by CD137 and which by CD137 ligand signaling. Studies using knock-out mice or antagonists, such as antibodies are not expected to bring clarity since they inhibit signaling in both directions. However, CD137 and CD137 ligand mutants lacking the cytoplasmic domains would disrupt signaling in only one direction. Due to trimerization of CD137 and its ligand, truncated forms of these molecules should also exert a dominant negative effect and inactivate trimers of intact CD137 and CD137 ligand, respectively. Alternatively, once the signal transduction pathways of CD137 and CD137 ligand have been elucidated it may be possible to use inhibitors which block only one of the two pathways. The reverse signaling pathways initiated by CD137 ligand has only been partly elucidated, but it seems reverse signaling does not employ a new set of signal transducing molecules, rather, CD137 ligand signaling relies on molecules which are well known from the study of other signaling pathways. CD137 has been reported to costimulate as well as to inhibit T cell activity. It is at present not yet clear how these opposite functions of CD137 are regulated and in which situations they are utilized. It would however be interesting to determine whether the inhibitory effects of CD137 ligand and CD137 on T cells are coordinated and employed simultaneously. If that were the case this would open up the possibility of fratricide through CD137 and CD137 ligand, as has already been documented for the CD95 receptor/ligand system (Callard et al., 2003). Sometimes the concept of reverse signaling is still being met with skepticism, and it is being questioned whether the observed effects cannot be explained otherwise. The quantity of data contributed by a number of different laboratories makes incidental findings highly unlikely. Also, identical results are obtained when recombinant CD137 protein, anti-CD137 ligand antibodies or CD137-transfected cells are used to activate CD137 ligand, ruling out the possibility that observed effects may be due to contaminations in protein batches. Also, the phenotypes of CD137 ligand-transgenic and CD137-deficient mice confirm the data obtained in vitro. In addition, a signaling cascade initiating from the cytoplasmic domain of CD137 ligand is being identified. These independent lines of evidence form a solid basis for the acceptance of reverse signaling through CD137 ligand.

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It becomes increasingly evident that the CD137 receptor/ligand system mediates essential cross talk between many leukocyte subpopulations, and also between immune and non-immune cells. The basis for this extensive cross talk and many diverse activities is the ability of the CD137 receptor/ligand system to transduce signals in both directions. The identification of which activities are mediated by CD137 or by CD137 ligand signaling will enhance our understanding of the regulation of immune responses, and should also be helpful in the development of therapeutics targeting the CD137 receptor/ligand system.

References Alderson, M.R., Smith, C.A., Tough, T.W., Davis-Smith, T., Armitage, R.J., Falk, B., Roux, E., Baker, E., Sutherland, G.R., Din, W.S., and Goodwin, R.G. (1994). Molecular and biological characterization of human 4-1BB and its ligand. Eur. J. Immunol., 24, 2219–2227. Al-Shamkhani, A. (2004). CD137: Costimulator turns suppressor? Immunology, 113, 23–25. Arens, R., Nolte, M.A., Tesselaar, K., Heemskerk, B., Reedquist, K.A., van Lier, R.A., and van Oers, M.H. (2004). Signaling through CD70 regulates B cell activation and IgG production. J. Immunol., 173, 3901–3908. Broll, K., Richter, G., Pauly, S., Hofstaedter, F., and Schwarz, H. (2001). CD137 Expression in Tumor Vessel Walls: High Correlation with Malignant Tumors. Am. J. Clin. Path., 115, 543–549. Callard, R.E., Stark. J., and Yates, A.J. (2003). Fratricide: A mechanism for T memory-cell homeostasis. Trends Immunol., 24, 370–375. Chirathaworn, C., Tibbetts, S.A., Chan, M.A., and Benedict, S.H. (1995). Cross-linking of ICAM-1 on T cells induces transient tyrosine phosphorylation and inactivation of cdc2 kinase. J. Immunol., 155, 5479–5482. Cowan, C.A. and Henkemeyer, M. (2002). Ephrins in reverse, park and drive. Trends Cell. Biol., 12, 339–346. Croft, M. (2003). Co-stimulatory members of the TNFR family: Keys to effective T-cell immunity? Nat. Rev. Immunol., 3, 609–620. DeBenedette, M.A., Chu, N.R., Pollok, K.E., Hurtado, J., Wade, W.F., Kwon, B.S., and Watts, T.H. (1995). Role of 4-1BB ligand in costimulation of T lymphocyte growth and its upregulation on M12 B lymphomas by cAMP. J. Exp. Med., 181, 985–992. DeBenedette, M.A., Shahinian, A., Mak, T.W., and Watts, T.H. (1997). Costimulation of CD28− T lymphocytes by 4-1BB ligand. J. Immunol., 158, 551–559. Eissner, G., Kolch, W., and Scheurich, P. (2004). Ligands working as receptors: Reverse signaling by members of the TNF superfamily enhance the plasticity of the immune system. Cytokine Growth Factor Rev., 15, 353–366. Foell, J., Strahotin, S., O’Neil, S.P., McCausland, M.M., Suwyn, C., Haber, M., Chander, P.N., Bapat, A.S., Yan, X.J., Chiorazzi, N., Hoffmann, M.K., and Mittler, R.S. (2003). CD137 costimulatory T cell receptor engagement reverses acute disease in lupus-prone NZB × NZW F1 mice. J. Clin. Invest., 111, 1505–1518. Foell, J.L., Diez-Mendiondo, B.I., Diez, O.H., Holzer, U., Ruck, P., Bapat, A.S., Hoffmann, M.K., Mittler, R.S., and Dannecker, G.E. (2004). Engagement of the CD137 (4-1BB) costimulatory molecule inhibits and reverses the autoimmune process in collagen-induced arthritis and establishes lasting disease resistance. Immunology, 113, 89–98. Futagawa, T., Akiba, H., Kodama, T., Takeda, K., Hosoda, Y., Yagita, H., and Okumura, K. (2002). Expression and function of 4-1BB and 4-1BB ligand on murine dendritic cells. Int. Immunol., 14, 275–286. Furtner, M., Straub, R.H., Kruger, S., and Schwarz, H. (2005). Levels of soluble CD137 are enhanced in sera of leukemia and lymphoma patients and are strongly associated with chronic lymphocytic leukemia. Leukemia 19, 883–885.

Significance of Reverse Signal Transduction for the Biology

43

Gravestein, L.A. and Borst, J. (1998). Tumor necrosis factor receptor family members in the immune system. Semin. Immunol., 10, 423–434. Green, D.R., Droin, N., and Pinkoski, M. (2003). Activation-induced cell death in T cells. Immunol. Rev., 193, 70–81. Greenwald R.J., Freeman G.J., and Sharpe A.H. (2005). The B7 Family Revisited. Annu. Rev. Immunol., 23, 515–548. Guinn, B.A., DeBenedette, M.A., Watts, T.H., and Berinstein, N.L. (1999). 4-1BBL cooperates with B7-1 and B7-2 in converting a B cell lymphoma cell line into a long-lasting antitumor vaccine. J. Immunol., 162, 5003–5010. Holland, J. and Owens, T. (1997). Signaling through intercellular adhesion molecule 1 (ICAM-1) in a B cell lymphoma line. The activation of Lyn tyrosine kinase and the mitogen-activated protein kinase pathway. J. Biol Chem., 272, 9108–9112. Hurtado, J.C., Kim, S.H., Pollok, K.E., Lee, Z.H., and Kwon, B.S. (1995). Potential role of 4-1BB in T cell activation. Comparison with the costimulatory molecule CD28. J. Immunol., 155, 3360– 3367. Ju, S.W., Ju, S.G., Wang, F.M., Gu, Z.J., Qiu, Y.H., Yu, G.H., Ma, H.B., and Zhang, X.G. (2003). A functional anti-human 4-1BB ligand monoclonal antibody that enhances proliferation of monocytes by reverse signaling of 4-1BBL. Hybrid. Hybridomics, 22, 333–338. Jung, H.W., Choi, S.W., Choi, J.I., and Kwon, B.S. (2004). Serum concentrations of soluble 4-1BB and 4-1BB ligand correlated with the disease severity in rheumatoid arthritis. Exp. Mol. Med., 36, 13–22. Kienzle, G. and von Kempis, J. (2000). CD137 (ILA/4-1BB), expressed by primary human monocytes, induces monocyte activation and apoptosis of B lymphocytes. Int. Immunol., 12, 73–82. Kim, Y.J., Li, G., and Broxmeyer, H.E. (2002). 4-1BB ligand stimulation enhances myeloid dendritic cell maturation from human umbilical cord blood CD34+ progenitor cells. J. Hematother. Stem Cell Res., 11, 895–903. Kwon, B.S., and Weissman, S.M. (1989). cDNA sequences of two inducible T-cell genes. Proc. Natl. Acad. Sci. USA, 86, 1963–1967. Kwon, B., Moon, C.H., Kang, S., Seo, S.K., and Kwon, B. (2000). 4-1BB: Still in the midst of darkness. Mol. Cells, 10, 119–126. Kwon, B.S., Hurtado, J.C., Lee, Z.H., Kwack, K.B., Seo, S.K., Choi, B.K., Koller, B.H., Wolisi, G., Broxmeyer, H.E., and Vinay, D.S. (2002). Immune responses in 4-1BB (CD137)-deficient mice. J. Immunol., 168, 5483–5490. Laderach, D., Wesa, A., and Galy, A. (2003). 4-1BB-ligand is regulated on human dendritic cells and induces the production of IL-12. Cell. Immunol., 226, 37–44. Langstein, J., Michel, J., Fritsche, J., Kreutz, M., Andreesen, R., and Schwarz, H. (1998). CD137, (ILA/4-1BB), a member of the TNF receptor family regulates monocyte activation via reverse signaling. J. Immunol., 160, 2488–2494. Langstein, J. and Schwarz, H. (1999). Identification of CD137 as a potent monocyte survival factor. J. Leuk. Biol., 65, 829–833. Langstein, J., Michel, J., and Schwarz, H. (1999). CD137 Induces Proliferation and Endomitosis in Monocytes. Blood, 94, 3161–3168. Langstein, J., Becke, F.M., Soellner, L., Krause, G., Brockhoff, G., Kreutz, M., Andreesen, R., and Schwarz, H. (2000). Comparative analysis of CD137 and LPS effects on monocyte activation, survival, and proliferation. Biochem. Biophys. Res. Com., 273, 117–122. Lee, P.K., Chang, C.J., and Lin, C.M. (2003). Lipopolysaccharide preferentially induces 4-1BB ligand expression on human monocyte-derived dendritic cells. Immunol. Lett., 90, 215– 221. Lindstedt, M., Johansson-Lindbom, B., and Borrebaeck, C.A. (2003). Expression of CD137 (4-1BB) on human follicular dendritic cells. Scand. J. Immunol., 57, 305–310. Lotz, M., Setareh, M., von Kempis, J., and Schwarz, H. (1996). The nerve growth factor/tumor necrosis factor receptor family. J. Leuko. Biol., 60, 1–7. Melero, I., Shuford, W.W., Newby, S.A., Aruffo, A., Ledbetter, J.A., Hellstrom, K.E., Mittler, R.S., and Chen, L. (1997). Monoclonal antibodies against 4-1BB T cell activation molecule eradicate established tumors. Nat. Med., 3, 682–685.

44

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Melero, I., Bach, N., Hellstrom, K.E., Aruffo, A., Mittler, R.S., and Chen, L. (1998). Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: Synergy with the CD28 co-stimulatory pathway. Eur. J. Immunol., 28, 1116–1121. Michel, J., Langstein, J., Hofst¨adter, F., and Schwarz, H. (1998). A soluble form of CD137 (ILA/4-1BB) is released by activated lymphocytes and is detectable in sera of patients with rheumatoid arthritis. Eur. J. Immunol., 28, 290–295. Michel, J., Langstein, J., Krammer, P., and Schwarz, H. (1999). CD137-induced apoptosis is independent of CD95. Immunology, 98, 42–46. Michel, J. and Schwarz, H. (2000). Soluble CD137 is selectively expressed by T lymphocytes and correlates with activation induced cell death of lymphocytes. Cytokine, 12, 742–746. Orabona, C., Grohmann, U., Belladonna, M.L., Fallarino, F., Vacca, C., Bianchi, R., Bozza, S., Volpi, C., Salomon, B.L., Fioretti, M.C., Romani, L., and Puccetti, P. (2004). CD28 induces immunostimulatory signals in dendritic cells via CD80 and CD86. Nat Immunol., 5, 1134–1142. Palma, C., Binaschi, M., Bigioni, M., Maggi, C.A., and Goso, C. (2004). CD137 and CD137 ligand constitutively coexpressed on human T and B leukemia cells signal proliferation and survival. Int. J. Cancer, 108, 390–398. Pauly, S., Broll, K., Giegerich, G., and Schwarz, H. (2002). CD137 regulates B lymphocyte proliferation and differentiation in germinal centers. J. Leuk. Biol., 72, 35–42. Pollok, K.E., Kim, Y.J., Hurtado, J., Zhou, Z., Kim, K.K., and Kwon, B.S. (1994). 4-1BB T-cell antigen binds to mature B cells and macrophages, and costimulates anti-mu-primed splenic B cells. Eur. J. Immunol. 24, 367–374. Qin, J., Vinogradova, O., and Plow, E.F. (2004). Integrin bidirectional signaling: A molecular view. PLoS Biol., 2, e169. Saito, K., Ohara, N., Hotokezaka, H., Fukumoto, S., Yuasa, K., Naito, M., Fujiwara, T., and Nakayama, K. (2004). Infection-induced up-regulation of the costimulatory molecule 4-1BB in osteoblastic cells and its inhibitory effect on M-CSF/RANKL-induced in vitro osteoclastogenesis. J. Biol. Chem., 279, 13555–13563. Salih, H.R., Kosowski, S.G., Haluska, V.F., Starling, G.C., Loo, D.T., Lee, F., Aruffo, A.A., Trail, P.A., and Kiener, P. (2000). Constitutive expression of functional 4-1BB (CD137) ligand on carcinoma cells. J. Immunol., 165, 2903–2910. Salih, H.R., Schmetzer, H.M., Burke, C., Starling, G.C., Dunn, R., Pelka-Fleischer, R., Nuessler, V., and Kiener, P.A. (2001). Soluble CD137 (4-1BB) ligand is released following leukocyte activation and is found in sera of patients with hematological malignancies. J. Immunol., 167, 4059–4066. Salih, H.R., Nuessler, V., Denzlinger, C., Starling, G.C., Kiener, P.A., and Schmetzer, H.M. (2004). Serum levels of CD137 ligand and CD178 are prognostic factors for progression of myelodysplastic syndrome. Leuk. Lymphoma. 45, 301–308. Schwarz, H., Tuckwell, J., and Lotz, M. (1993). A receptor induced by lymphocyte activation ILA: A new member of the human nerve growth factor/tumor necrosis factor receptor family. Gene, 134, 295–298. Schwarz, H., Valbracht, J., Tuckwell, J., Kempis, J., and Lotz, M. (1995). ILA, the human 4-1BB homologue is inducible in lymphoid and other cell lines. Blood, 85, 1043–1052. Schwarz, H., Blanco, F., Valbracht, J., Kempis. J., and Lotz, M. (1996). ILA, a member of the human NGF/TNF receptor family regulates T lymphocyte proliferation and survival. Blood, 87, 2839– 2845. Sharief, M.K. (2002). Heightened intrathecal release of soluble CD137 in patients with multiple sclerosis. Eur J Neurol., 9, 49–54. Sica, G. and Chen, L. (2000). Modulation of the immune response through 4-1BB. Adv. Exp. Med. Biol., 465, 355–362. Summers, K.L., Hock, B.D., McKenzie, J.L., and Hart, D.N. (2001). Phenotypic characterization of five dendritic cell subsets in human tonsils. Am. J. Pathol., 159, 285–295. Suzuki, I. and Fink, P.J. (1998). Maximal proliferation of cytotoxic T lymphocytes requires reverse signaling through Fas ligand. J. Exp. Med., 187, 123–128. Watts, A.D., Hunt, N.H., Wanigasekara, Y., Bloomfield, G., Wallach, D., Roufogalis, B.D., and Chaudhri, G. (1999). A casein kinase I motif present in the cytoplasmic domain of members of the tumour necrosis factor ligand family is implicated in ‘reverse signalling.’ EMBO J., 18, 2119–2126.

Significance of Reverse Signal Transduction for the Biology

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Wilcox, R.A., Chapoval, A.I., Gorski, K.S., Otsuji, M., Shin, T., Flies, D.B., Tamada, K., Mittler, R.S., Tsuchiya, H., Pardoll, D.M., and Chen, L. (2002). Cutting edge: Expression of functional CD137 receptor by dendritic cells. J. Immunol., 168, 4262–4267. Wilkinson, D.G. (2000). Eph receptors and ephrins: Regulators of guidance and assembly. Int. Rev. Cytol., 196, 177–244. Zhou, Z., Kim, S., Hurtado, J., Lee, Z.H., Kim, K.K., Pollok, K.E., and Kwon, B.S. (1995). Characterization of human homologue of 4-1BB and its ligand. Immunol. Lett., 45, 67–73. Zhu, G., Flies, D.B., Tamada, K., Sun Y., Rodriguez, M., Fa, Y-X., and Chen, L. (2001). Progressive depletion of peripheral B lymphocytes in 4-1BB (CD137) ligand/Ealpha-transgenic mice. J. Immunol., 167, 2671–2676.

4 CD137 Signal in the Regulation of Innate Immunity Lieping Chen

1. Introduction In contrast to our ever increasing knowledge regarding the role of CD137 signaling in the induction and development of adaptive immunity, our understanding with regard to the effect of CD137 in innate immunity is still in it’s earliest stages. Experimental evidence supports that CD137 engagement is capable of triggering activity of natural killer (NK) cells, macrophages, dendritic cells (DC), and granulocytes. The majority of results, however, are obtained in cell culture systems which raise the questions regarding their roles in ongoing immune responses in vivo and in human diseases. The hypothesis that is presently being pushed forward is that the CD137 pathway plays a critical role in the regulation of innate immunity and may bridge innate and adaptive immunity. This chapter will assemble available data with an attempt to delineate the functions of CD137 in individual components of innate immunity.

2. NK Cells NK cell is an important component of the innate immune response against virally infected and malignant cells. Originated from hematopoietic stem cells, the development of NK cells requires less interactions with bone marrow stromal cells, which are not completely understood. (Hamerman et al., 2005). In peripheral tissue, two major functions of NK cells have been described. First, NK cells are effector cells which directly kill target cells through a largely perforin-dependent mechanism. Second, NK cells express arrays of cell surface molecules and secrete cytokines, which regulate immune responses in the site of infection or inflammation (Zingoni et al., 2005). As a consequence of target cell cytolysis by NK cells, more bacterial, viral, or tumor antigens may be provided to professional antigenpresenting cells for initiation of adaptive immunity. Finally, through cell–cell interaction or cytokine, NK cells could enhance the functions of antigen-presenting Lieping Chen • Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Jefferson 1-121, Baltimore, MD 21205 47 CD137 Pathway: Immunology and Diseases. Edited by Lieping Chen, Springer, New York, 2006

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cells and lymphoid cells. Therefore, an important role of NK cells appears to bridge innate and adaptive immunity. While resting NK cells express negligible CD137, activation of NK cells by IL-2 or IL-15 leads to surface expression of high level CD137 (Melero et al., 1998). The number of NK and NKT cells decreases significantly in CD137-deficient mice (Kwon et al., 2002), suggesting that CD137 may affect the development of NK cells. Stimulation by CD137L-transfected cells or CD137 agonistic monoclonal antibodies (mAb) induced vigorous proliferation of NK cells and IFN-γ secretion in vitro (Wilcox et al., 2002). However, cytolicic activity of NK cells against NK-sensitive target cells, was not changed in the same stimulation condition. There are several important implications from these findings. First, cytolytic and immune regulatory functions of NK cells could be segregated and controlled by distinct mechanisms. Second, manipulation of CD137 pathway provides a unique opportunity to selectively regulate immune regulatory functions of NK cells. Thus, these findings may provide explanations for several previous observations which could not be satisfactorily interpreted. Administration of either CD137 agonist mAb or recombinant CD137L has been shown to induce complete or partial regression of established tumors in various mouse models (Hellstrom and Hellstrom, 2003; also see summary in Chapter 8). In tumor models in which the role of NK cells were analyzed, antitumor immunity was either completely, or at least partially dependent on NK cells. This is proven by showing decreased tumor immunity CTL activity by depletion of NK cells using either a polyclonal or mAb (Melero et al., 1998, Wilcox et al., 2002b, Ye et al., 2002). However, for a NK-resistant P815 mouse tumor, anti-CD137 mAb does not inhibit tumor growth in immunodeficient BALB/c nu/nu mice, in which NK activity is elevated. However, in immunocompetent syngeneic mice, treatment by anti-CD137 mAb induced the regression, but the depletion of NK cells completely eliminated the induction of CTL and tumor immunity (Melero et al., 1998). In this system, the effect of NK cells could be entirely attributed to their immune regulatory function because P815 is resistant to NK cell lysis. In a liver metastasis model of MCA206 mouse colon cancer, adenovirus-mediated gene transfer of both CD137L and IL-12 gene into tumor cells led to the regression of established tumors, accompanied with increased NK activity (Martinet et al., 2000). Depletion of NK or CD8+ T cells in this system also largely abolished the antitumor immunity. Similarly, NK cells are also partially required for CD8+ CTL induction and tumor immunity in a C3 tumor model (Wilcox et al., 2002b). These results thus support a regulatory function of NK cells after activation by CD137. Although the immune regulatory function of NK cells has been a subject of research for a long period of time, the mechanisms underlying these observations are not yet elucidated. CD137 is a potent inducer of IFN-γ from T cells and NK cells and blocking IFN-γ eliminated various effects of anti-CD137 mAb, including induction of T cell activation and tumor immunity (Wilcox et al., 2002a). Therefore, the effect of NK cell activation by CD137 signal is at least partially dependent upon secretion of cytokines. Several recent studies support a critical role of NK cells in activation of dendritic cells that are critical antigen presenting cells (DegliEsposti and Smyth, 2005). In MCA206 tumor model, antitumor effect and NK

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cell activation by CD137L and IL-12 are accompanied by an increased influx of DC infiltrated into tumors and enhanced T cell stimulatory function (Pan et al., 2004). Therefore, enhancing regulatory function of NK cells in vivo may represent a useful approach to enhance adaptive immunity. The study of the role of CD137 signal in development, activation, and functional modulation of NK cells is essential for understanding mechanisms of CD137 in regulating immune responses. Current data has established a role of CD137 signal in the regulation of NK activity and supports that CD137 signaling may represent a critical link between innate and adaptive immune responses.

3. Macrophages/Monocyte Macrophages are phagocytic cells that accumulate in inflammatory sites. In addition to phagocytosis and subsequent antigen processing and presentation, macrophages also express cell surface molecules and secrete a wide variety of different cytokines, including IL-1, IL-6, IL-8, IL-12, and TNF-α, upon exposure to pathogens and cancer cells. Therefore, macrophages could have local and systemic effects and serve not only to amplify the innate immune response, but also to trigger adaptive immunity. Although resting human monocytes do not express CD137 mRNA, their expression could be upregulated rapidly by IL-1 or PMA (Schwarz et al., 1995). Another study has shown that cell surface CD137 could be detected by only a brief culture of monocytes in media (Kienzle and von Kempis, 2000). Of importance is the stimulation of monocytes by anti-CD137 mAb which upregulated mRNA expression of TNF-α and IL-8, whereas it also downregulated IL-10 expression (Kienzle and von Kempis, 2000). In a CD137+ monocytic line THP-1, cross-linking by anti-CD137 mAb upregulated CD54 and CD11b and increased adhesion of THP-1 cells to extracellular matrix proteins, in addition to increased secretion of IL-8 and TNF-α. Increased adhesion could be suppressed by an inhibitor of mitogen-activated protein kinase kinase (MEK), but not by a p38 kinase inhibitor (Choi et al., 2005). These results suggest that CD137 on monocytes is functional. Interestingly, CD137-stimulated monocytes were found to promote B cell apoptosis in a cell-contact dependent fashion (Kienzle and von Kempis, 2000). Therefore, CD137 signal may inhibit humoral immune response through monocyte activation. This observation is consistent with the finding that B cells were progressively deleted in CD137L transgenic mice, in which the expression of CD137L is under the control of MHC class II I-Eα promoter (Zhu et al., 2001). It has also been shown that administration of agonistic mAb to CD137 could inhibit T cell-dependent antibody response to sRBC (Mittler et al., 1999) and autoantibodies in several systemic autoimmunity models (Foell et al., 2003; Mittler et al., 2004). In addition to CD137, monocytes and macrophages also express high levels of CD137L and its expression is tightly controlled by cytokines. In several in vitro studies using human monocyte culture systems, CD137 protein is shown to be a potent monocyte activation factor. The plate-bound CD137 protein induced the

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expression of IL-6, IL-8, and TNF-α, and inhibits expression of IL-10, therefore supporting that the effect of CD137 is mediated through CD137L as a counterreceptor for monocytes/macrophages (Ju et al., 2003). CD137 engagement also induced expression of ICAM-1 and reduced expression of Fcγ receptor III. The effect does not seem to be non-specific since levels of HLA-DR remain constant. Importantly, the engagement of CD137L on macrophages promotes vigorous proliferation and survival. This effect is found in part due to, the elevated macrophage colony-stimulating factor (Langstein and Schwarz, 1999), an essential monocyte survival factor. Monocytes/macrophages express both CD137 and CD137L and are both capable of transducing activation signals. Therefore, this may represent a unique amplification system for boosting macrophage functions during inflammation and pathogen infection. However, there is not yet direct evidence to validate this mode in the activation of monocytes. Experiments thus far are all performed in a cell culture system, and how this mode contributes to the overall process of immune responses or inflammation is not yet clear, especially in vivo. Another area to be explored is the interactions through CD137-CD137L among macrophages with other cells which are key players of innate immunity. These cells include NK cells, DCs, and granulocytes, which are capable of expressing CD137 and/or CD137L. These issues could be solved by reconstitution experiments employing CD137 or CD137L deficient cellular components in the near future.

4. Dendritic Cells Dendritic cells (DCs) are among the most potent antigen-presenting cells for the priming of T cell responses, thus contributing to adaptive immunity. In addition, DCs also participate in innate immunity. First, DCs express abundant molecules which are responsible for the activation of NK cells (Degli-Esposti and Smyth, 2005). Second, DCs can release an array of cytokines, upon activation, which are important in the regulation of immune responses, including innate immunity (Steinman et al., 2005). Third, activated DCs are shown to directly kill tumor cells, largely due to their capacity to release TNF-α or to suppress the growth of tumor cells in vitro by a poorly understood “cytostasis” mechanism (Chapoval et al., 2000). Freshly isolated mouse splenic DCs and bone marrow-derived DCs express CD137 on the cell surface (Futagawa et al., 2002; Wilcox et al., 2002). In addition, the soluble form of CD137 could also be detected in DC culture (Wilcox et al., 2002). This may represent an alternative splicing form of CD137 and a potential decoy receptor. Co-incubation of CD137L transfected cells with DCs induces secretion of IL-6 and IL-12 (Futagawa et al., 2002; Wilcox et al., 2002). Furthermore, splenic DCs isolated from mice that were given a CD137 mAb were better able to stimulate proliferation of antigen-specific T cells when compared to DCs isolated from mice that had received a control led antibody (Wilcox et al., 2002). Collectively, this data has implicated CD137 as an important receptor capable of stimulating functional maturation of DCs.

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In addition to constitutive expression of CD137, DCs could also express CD137L, which has been shown to contribute to T cell costimulatory function (see Chapter 3). Therefore, interaction between CD137 and CD137L may represent one of molecular mechanisms of DC-DC interaction and may be a new mode of DC activation. IL-12 production by anti-CD40-stimulated DCs could be partially inhibited by an anti-CD137L mAb (Futagawa et al., 2002), suggesting a role of CD137 pathway in the regulation of DC-DC interaction. In addition to the role of CD137 in the activation of DCs in cell culture system, administration of CD137 mAb in RAG-1-deficient mice was shown to enhance the ability of CD137-expressing splenic DCS isolated from these mice to stimulate T cell proliferation (Wilcox et al., 2002). These results suggest that activation of DCs through CD137 modulate DC function in vivo. This experiment also shows that CD137 engagement can directly activate DCs independently on T and B cells. The roles of endogenous CD137 signals through DCs in the modulation of DC function have also been implicated in several studies. Administration of CD137Ig as a blocking agent impaired the accumulation of recipient DCs within mouse spleen in an intestinal allograft transplantation model (Wang et al., 2003). In a collagen-induced arthritis mouse model, administration of agonist CD137 mAb inhibited the development of rheumatoid arthritis in a susceptible DBA/1 model, accompanied with accumulation of indoleamine 2,3-dioxygenase in CD11b monocytes and CD11c+ DC (Seo et al., 2004). One caveat is, that although these findings could be interpreted as direct engagement of CD137 on DC, it is also possible that this is a consequence of T cell activation by CD137 antibody. Therefore, definitive experiments to address this issue have yet to be completed. In summary, although current experimental results support stimulatory function of CD137 signal for functional maturation of DCs, our knowledge in this area is very limited. For example, the DCs have multiple subsets that possess diverse immunological functions. The DC subset used in current studies is myeloid DCs and the expression pattern and functionality in other subsets such as the plasmatoid DC has not yet been explored. In addition, DCs express a large number of molecules with diverse functions; it is possible that the functions of CD137 signal may be replaceable by other molecules. These issues are yet to be addressed.

5. Granulocytes Granulocytes, including neutrophils, eosinophils, and basophils, are important effector cells in the innate immune response against bacterial, fungal, and parasitic pathogens. Because granulocytes are terminally differentiated cells, the accumulations of granulocytes at sites of inflammation are determined by cell migration and delay/suppressed apoptosis. These processes are believed to be predominantly regulated by cytokines. G-CSF and GM-CSF are important surviving factors for neutrophils and IL-5 is associated with increased survival of eosinophils (Simon, 2001, 2003). A small fraction of neutrophils is found to constitutively express CD137 (Simon, 2001). Eosinophils isolated from a healthy donor do not express CD137

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(Simon, 2001). There is no report as of now regarding the expression of CD137 on basophils. CD137 expression on eosinophils could be observed in patients suffering from IgE-mediated allergic responses, but not in normal subjects or those patients suffering from non-IgE-mediated eosinophilic disorders (Heinisch et al., 2001). In both neutrophils and eosinophils, CD137 stimulation promoted apoptosis in these cells, even in the presence of GM-CSF and/or IL-5 survival factors (Heinisch et al., 2000). In this regard, CD137 stimulation may play an important role in regulating granulocyte survival during the initiation and resolution of an inflammatory response. Combined with a report showing CD137 transcript that was frequently found in mast cells, a key type of cells storing and releasing inflammatory mediators for allergy, CD137 may also participate in the control of asthma induced by extrinsic allergens.

6. Summary Our knowledge of CD137 in the regulation of innate immunity is rudimentary. CD137 and its ligand are expressed on a wide variety of cell types in both transmembrane and soluble forms. Signaling through CD137 pathway is capable of activating DC, NK and T cells. Therefore, CD137 pathway may provide a critical link between innate and adaptive immunity. Understanding the mechanisms underlying the regulation of CD137 pathway in innate immunity may lead to the development of new and improved forms of immunotherapy for a broad spectrum of disease states, including the prevention and therapy of autoimmunity, graft rejection, and stimulation of tumor-specific immune response.

References Chapoval, A.I., Tamada, K., and Chen, L. (2000). In vitro growth inhibition of a broad spectrum of tumor cell lines by activated human dendritic cells. Blood, 95, 2346–2351. Choi, J.W., Lee, H.W., Roh, G.S., Kim, H.H., and Kwack, K. (2005). CD137 induces adhesion and cytokine production in human monocytic THP-1 cells. Exp. Mol. Med., 37, 78–85. Degli-Esposti, M.A., and Smyth, M.J. (2005). Close encounters of different kinds: dendritic cells and NK cells take centre stage. Nat. Rev. Immunol., 5, 112–124. Foell, J., McCausland, M., Burch, J., Corriazzi, N., Yan, X.J., Suwyn, C., O’Neil, S.P., Hoffmann, M.K., and Mittler, R.S. (2003). CD137-mediated T cell co-stimulation terminates existing autoimmune disease in SLE-prone NZB/NZW F1 mice. Ann. N.Y. Acad. Sci., 987, 230–235. Futagawa, T., Akiba, H., Kodama, T., Takeda, K., Hosoda, Y., Yagita, H., and Okumura, K. (2002). Expression and function of 4-1BB and 4-1BB ligand on murine dendritic cells. Int. Immunol., 14, 275–286. Hamerman, J.A., Ogasawara, K., and Lanier, L.L. (2005). NK cells in innate immunity. Curr. Opin. Immunol., 17, 29–35. Heinisch, I.V., Bizer, C., Volgger, W., and Simon, H.U. (2001). Functional CD137 receptors are expressed by eosinophils from patients with IgE-mediated allergic responses but not by eosinophils from patients with non-IgE-mediated eosinophilic disorders. J. Allergy Clin. Immunol., 108, 21–28. Heinisch, I.V., Daigle, I., Knopfli, B., and Simon, H.U. (2000). CD137 activation abrogates granulocytemacrophage colony-stimulating factor-mediated anti-apoptosis in neutrophils. Eur. J. Immunol., 30, 3441–3446.

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Hellstrom, K.E., and Hellstrom, I. (2003). Therapeutic vaccination with tumor cells that engage CD137. J. Mol. Med., 81, 71–86. Ju, S.W., Ju, S.G., Wang, F.M., Gu, Z.J., Qiu, Y.H., Yu, G.H., Ma, H.B., and Zhang, X.G. (2003). A functional anti-human 4-1BB ligand monoclonal antibody that enhances proliferation of monocytes by reverse signaling of 4-1BBL. Hybrid Hybridomics, 22, 333–338. Kienzle, G., and von Kempis, J. (2000). CD137 (ILA/4-1BB), expressed by primary human monocytes, induces monocyte activation and apoptosis of B lymphocytes. Int. Immunol., 12, 73–82. Kwon, B.S., Hurtado, J.C., Lee, Z.H., Kwack, K.B., Seo, S.K., Choi, B.K., Koller, B.H., Wolisi, G., Broxmeyer, H.E., and Vinay, D.S. (2002). Immune responses in 4-1BB (CD137)-deficient mice. J. Immunol., 168, 5483–5490. Langstein, J., and Schwarz, H. (1999). Identification of CD137 as a potent monocyte survival factor. J. Leukoc. Biol., 65, 829–833. Martinet, O., Ermekova, V., Qiao, J.Q., Sauter, B., Mandeli, J., Chen, L., and Chen, S.H. (2000). Immunomodulatory gene therapy with interleukin 12 and 4-1BB ligand: Long-term remission of liver metastases in a mouse model. J. Natl. Cancer Inst., 92, 931–936. Melero, I., Johnston, J.V., Shufford, W.W., Mittler, R.S., and Chen, L. (1998). NK1.1 cells express 4-1BB (CDw137) costimulatory molecule and are required for tumor immunity elicited by anti-4-1BB monoclonal antibodies. Cell. Immunol., 190, 167–172. Mittler, R.S., Bailey, T.S., Klussman, K., Trailsmith, M.D., and Hoffmann, M.K. (1999). Anti-4-1BB monoclonal antibodies abrogate T cell-dependent humoral immune responses in vivo through the induction of helper T cell anergy. J. Exp. Med., 190, 1535–1540. Mittler, R.S., Foell, J., McCausland, M., Strahotin, S., Niu, L., Bapat, A., and Hewes, L.B. (2004). Anti-CD137 antibodies in the treatment of autoimmune disease and cancer. Immunol. Res., 29, 197–208. Pan, P.Y., Gu, P., Li, Q., Xu, D., Weber, K., and Chen, S.H. (2004). Regulation of dendritic cell function by NK cells: Mechanisms underlying the synergism in the combination therapy of IL-12 and 4-1BB activation. J. Immunol., 172, 4779–4789. Schwarz, H., Valbracht, J., Tuckwell, J., von Kempis, J., and Lotz, M. (1995). ILA, the human 4-1BB homologue, is inducible in lymphoid and other cell lineages. Blood, 85, 1043–1052. Seo, S.K., Choi, J.H., Kim, Y.H., Kang, W.J., Park, H.Y., Suh, J.H., Choi, B.K., Vinay, D.S., and Kwon, B.S. (2004). 4-1BB-mediated immunotherapy of rheumatoid arthritis. Nat. Med., 10, 1088– 1094. Simon, H.U. (2003). Neutrophil apoptosis pathways and their modifications in inflammation. Immunol. Rev., 193, 101–110. Simon, H.U. (2001). Evidence for a pro-apoptotic function of CD137 in granulocytes. Swiss Med. Wkly., 131, 455–458. Steinman, R.M., Bonifaz, L., Fujii, S., Liu, K., Bonnyay, D., Yamazaki, S., Pack, M., Hawiger, D., Iyoda, T., Inaba, K., and Nussenzweig, M.C. (2005). The innate functions of dendritic cells in peripheral lymphoid tissues. Adv. Exp. Med. Biol., 560, 83–97. Wang, J., Guo, Z., Dong, Y., Kim, O., Hart, J., Adams, A., Larsen, C.P., Mittler, R.S., and Newell, K.A. (2003). Role of 4-1BB in allograft rejection mediated by CD8+ T cells. Am. J. Transplant., 3, 543–551. Wilcox, R.A., Chapoval, A.I., Gorski, K.S., Otsuji, M., Shin, T., Flies, D.B., Tamada, K., Mittler, R.S., Tsuchiya, H., Pardoll, D.M., and Chen, L. (2002). Cutting edge: Expression of functional CD137 receptor by dendritic cells. J. Immunol., 168, 4262–4267. Wilcox, R.A., Flies, D.B., Wang, H., Tamada, K., Johnson, A.J., Pease, L.R., Rodriguez, M., Guo, Y., and Chen, L. (2002a). Impaired infiltration of tumor-specific cytolytic T cells in the absence of interferon-gamma despite their normal maturation in lymphoid organs during CD137 monoclonal antibody therapy. Cancer Res., 62, 4413–4418. Wilcox, R.A., Flies, D.B., Zhu, G., Johnson, A.J., Tamada, K., Chapoval, A.I., Strome, S.E., Pease, L.R., and Chen, L. (2002b). Provision of antigen and CD137 signaling breaks immunological ignorance, promoting regression of poorly immunogenic tumors. J. Clin. Invest., 109, 651–659. Wilcox, R.A., Tamada, K., Strome, S.E., and Chen, L. (2002c). Signaling through NK cell-associated CD137 promotes both helper function for CD8+ cytolytic T cells and responsiveness to IL-2 but not cytolytic activity. J. Immunol., 169, 4230–4236.

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Ye, Z., Hellstrom, I., Hayden-Ledbetter, M., Dahlin, A., Ledbetter, J.A., and Hellstrom, K.E. (2002). Gene therapy for cancer using single-chain Fv fragments specific for 4-1BB. Nat. Med., 8, 343– 348. Zhu, G., Flies, D.B., Tamada, K., Sun, Y., Rodriguez, M., Fu, Y.X., and Chen, L. (2001). Progressive depletion of peripheral B lymphocytes in 4-1BB (CD137) ligand/I-Ealpha)-transgenic mice. J. Immunol., 167, 2671–2676. Zingoni, A., Sornasse, T., Cocks, B.G., Tanaka, Y., Santoni, A., and Lanier, L.L. (2005). NK cell regulation of T cell-mediated responses. Mol. Immunol., 42, 451–454.

5 Regulation of T Cell-Dependent Humoral Immunity Through CD137 (4-1BB) Mediated Signals Robert S. Mittler, Liguo Niu, Becker Hewes, and Juergen Foell

CD137 (4-1BB), a member of the tumor necrosis factor receptor (TNFR) superfamily is an activation-inducible type I transmembrane protein receptor expressed on activated T cells, Natural Killer cells (NK), macrophages (Mφ), and dendritic cells (DC). Its ligand, 4-1BB-L, is a member of the tumor necrosis factor (TNF) superfamily a type II transmembrane protein that is expressed on antigen presenting cells (APC). Ligand-induced trimerization of CD137 activates multiple signaling pathways in T cells that result in transcriptional activation, enhanced T cell activation and survival. Agonistic anti-CD137 mAbs can replace the need for 4-1BB ligand-mediated receptor crosslinking either in vitro or in vivo. 4-1BB-L specific mAbs can drive cytokine production in APCs; anti-4-1BB ligand mAbs that can block receptor-ligand binding also prevent CD137-mediated T cell costimulation. However, unlike agonistic mAbs to other T cell costimulatory receptors, signaling by anti-CD137 specific mAbs can also suppress the T cellmediated immune responses. CD137 receptor-proximal and downstream signals in T cells have been largely elucidated. However, this is not the case for anti-CD137induced immune suppression. Given that NK cells and dendritic cells express this receptor, it is not clear whether T cells are the cell targets during anti-CD137 mAb induced immune suppression. In this review we discuss CD137-mediated immune suppression and its role in regulating T-dependent B cell responses and to some extent APC function in normal, autoimmune, and tumor bearing mice.

Robert S. Mittler • Department of Surgery, Emory Vaccine Center, Emory University School of Medicine, 954 Gatewood Road, Atlanta, GA 30329. Liguo Niu • Emory Vaccine Center, Emory University School of Medicine, 954 Gatewood Road, Atlanta, GA 30329. Becker Hewes • Department of Pediatric Hematology and Oncology, Emory University School of Medicine, 954 Gatewood Road, Atlanta, GA 30329. Juergen Foell • Division of Pediatrics, Hematology, Oncology, and Immunology, Martin Luther University, Halle-Wittenberg, 06097 Halle, Germany. 55 CD137 Pathway: Immunology and Diseases. Edited by Lieping Chen, Springer, New York, 2006

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1. Introduction The concept that activation of lymphocytes requires two signals was first proposed by Bretcher and Cohn in an attempt to explain how mature B cells become activated and differentiate into antibody secreting cells on the one hand, or enter into a state of anergy on the other (Bretscher and Cohn, 1970). In a broader sense, the model addresses the capacity of the immune system to discriminate between self and nonself, and mechanisms that control lymphocyte activation versus tolerance. In 1975 Lafferty and Cunningham extended the two signal hypothesis to include T cell activation (Lafferty and Cunningham, 1975). This model now forms the basis for our current perception of how T cells become activated, immunocompetent, and develop memory. It also explains how peripheral tolerance to self-antigens can be maintained, and why lymphocytes having received activation signals through their antigen receptors can become anergic, or enter a pathway leading to apoptotic cell death. The decision between immune activation and immune silencing is often based on whether the T cell receives an additional signal, known as a costimulatory signal, following antigen recognition. This “second signal” is typically provided by an APC that expresses the ligand for a costimulatory receptor expressed on the T cell. Some of costimulatory receptors and their ligands are constitutively expressed on the cell surface while others are activation inducible. Our review focuses on the function of the CD137 T cell costimulatory pathway as it broadly relates to the regulation of T cell-dependent B cell mediated immunity. B cells, however, do not express CD137, and therefore, CD137-mediated regulation of B cell function must be indirect. In addition to T cells, a number of cell lineages, including DC, macrophages, and NK cells express CD137. It remains to be seen how these cells regulate T cell or B cell-mediated immune responses following CD137 crosslinking. To study T-dependent humoral immunity following CD137 signaling we vaccinated or virus infected normal mice and treated them with agonistic (for T cells) anti-CD137 mAbs. In addition, we treated autoimmuneprone and tumor-bearing mice with these antibodies.

2. T and B Cell Activation and Costimulation It is generally accepted that T cells require two signals in order to undergo activation and clonal expansion. The first of these activating signals, “signal-1” is initiated when the T cell receptor (TCR) is crosslinked following engagement with MHC bound peptides expressed on antigen presenting cells (APC). The second signal, “signal-2,” is provided when one of several receptor proteins expressed on a T cell is crosslinked by its ligand expressed on the APC. Collectively, these T cell surface receptors are referred to as T cell costimulatory receptors, of which CD28 and the CD28-CTLA-4-CD80/CD86 signaling pathway is the prototype. CD28 and CTLA-4 are members of the immunoglobulin (Ig)-superfamily (Brunet et al., 1987; Linsley et al., 1991; Salomon and Bluestone, 2001), and both share the same ligands CD80 (B7.1) or CD86 (B7.2). CD28 is constitutively expressed

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ACTIVATION//CLONAL EXPANSION

(Signal 1 only)

(Signal 1 + Signal 2)

+

CD4 T CELL

CD4 Signal 1

TCR Signal 2

CD28 CD80

MHC class II

APC

CD86 CD40

Figure 5.1. T cells require two signals for activation. T cell activation is initiated when the TCR binds a complex composed of pathogen-derived peptides and MHC molecules displayed on the surface of an APC (Signal-1). In the absence of additional signals the T cell becomes anergic (Gimmi et al., 1993) or dies by apoptosis (Wolf et al., 1994). Cognate recognition of the APC by the T cell allows CD28 to bind its ligand on the APC thus initiating Signal-2. The combination of both signals drives T cell clonal expansion, differentiation and memory.

on T cells where it functions as a T cell costimulatory molecule. CTLA-4 is an activation inducible receptor that functions as a negative regulator of T cell activation (Waterhouse et al., 1995). The ligands for these receptors are differentially expressed on APC. For instance, CD80 is not expressed on resting APC, while CD86 can, but only at low levels. CD80 and CD86 upregulation is secondary to TCR binding to peptide MHC complexes, and their appearance is dependent upon rapidly induced expression of CD40 ligand (CD154) on the T cell. CD154 is an activation inducible type II transmembrane protein that binds CD40 a costimulatory receptor constitutively expressed at varying levels on APCs and B cells. The CD154-CD40 signaling pathway induces APC activation that drives the subsequent expression, or upregulation of CD80 and CD86 on the APC, and this in turn facilitates CD28 receptor crosslinking (Figure 5.1). Since the recognition of CD28 as a T cell costimulatory receptor other costimulatory receptors belonging to the Ig, CD2, and TNFR superfamilies have been identified. Within the TNFR superfamily CD27, CD30, CD134 (OX40), CD137 (4-1BB), herpes virus entry mediator (HVEM), and glucocorticoid-induced tumor necrosis factor receptor family related gene (GITR) have all been shown to serve as costimulatory receptors for T cells (Watts, 2005). CD28 engagement with its CD80/CD86 ligands, lowers the activation threshold needed for antigen receptor-induced activation (Viola and Lanzavecchia, 1996), increases the half-life of IL-2 mRNA in the T cell (Chen et al., 1998; Linsley et al., 1991), upregulates transcription of anti-apoptotic genes (Boise et al., 1995a), and protects the T cell from activation induced cell death (AICD) (Boise et al., 1995a, 1995b; Gribben et al., 1995; Mueller et al., 1996; Noel et al., 1996; Radvanyi et al., 1996; Van Parijs et al., 1996; Walunas et al., 1996). Once activated through

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CD4 T Cell

Signal 2

Signal 1 Ag-induced BCR Aggregation

CD154 CD40

BCR (sIg)

Y

Antigen

Y

Y B Cell

Y

Ig Isotype switch Anti-apoptotic genes

Figure 5.2. CD4 T cell-mediated Ig class switching. CD4 T cells provide help to B cells that drives them into the cell cycle, protects them from apoptosis, and allows the B cell to class switch the antibody it produces from IgM to IgG, IgA, or IgE (Snapper and Mond, 1993). This process of help occurs in germinal centers and requires cognate recognition of CD40 with its ligand CD154 and usage of T cell derived cytokines such as IL-4, IL-5, TGF-β, and IFN-γ by the B cell.

antigen-dependent and costimulatory signals, CD4 T and CD8 T cells undergo clonal expansion and differentiate into effector helper and cytolytic T cells, respectively. Activated CD4 T effector cells produce either pro-inflammatory cytokines (Th1), or anti-inflammatory cytokines (Th2). CD4 T cells also provide help to antigen-activated B cells through cytokine and CD154-CD40 dependent pathways (Durie et al., 1994). CD154-mediated CD40 crosslinking on B cells induces transcription of anti-apoptotic genes and drives immunoglobulin class switching. CD4 T cells, in addition to crosslinking CD40, produce cytokines needed to promote B cell differentiation. Cytokines also help determine which class of antibody the B cell will produce, i.e., they help drive class switch recombination events that permit B cells to switch from IgM production to producing IgG, IgA, or IgE class antibodies (Foy et al., 1993; Kawabe et al., 1994; Kelsoe, 1996; Renshaw et al., 1994; van Essen et al., 1995; Xu et al., 1994). Thus, like T cells, B cells require two distinct signals for their full activation and survival. The first is generated through antigen-induced BCR crosslinking and the second, through CD154-induced crosslinking of CD40 (Figure 5.2). In humans, mutations in the CD154 protein leads to a heritable disease called hyper-IgM syndrome. Individuals afflicted with this disease can have elevated levels of IgM in their serum but have low to non-detectable levels of IgG, IgA, or IgE (Allen et al., 1993; Aruffo et al., 1993; DiSanto et al., 1993; Korthauer et al., 1993). Following infection, B cells respond by producing IgM class antibodies. The majority of B cells producing these antibodies respond to bacterial wall polysaccharides and lipopolysaccharides whose structure is highly repetitive. Antigens of this type elicit antibody responses independent of T cell help because their repetitive antigenic determinants extensively crosslink the BCR; antigens of this type are

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said to be T-independent (TI) antigens (Snapper and Mond, 1996). As the infection proceeds and the immune system expands its response, the breadth of the B cell response broadens and the majority of antigens that B cells encounter lack highly repetitive subunit structures. These antigens do not crosslink BCR sufficiently to drive B cell expansion and differentiation. In order for B cells to respond to these antigens they require T cell help, thus antigens of this type are classified as Tdependent antigens (TD). The distinction between T-independent and T-dependent antigens is important in the context of this review because interference with T cell costimulation blocks the induction of T-dependent humoral immunity but not T-independent humoral immune responses.

3. CD137 Expression and T Cell Costimulation CD137 is an activation inducible member of the TNFR superfamily and is found on activated thymocytes, T cells (Kwon et al., 1994; Pollok et al., 1993; Schwarz et al., 1995), and NK cells (Melero et al., 1998b). It has recently been found on granulocytes (Heinisch et al., 2000), eosinophils (Heinisch et al., 2001), macrophages (Kienzle and von Kempis, 2000; Langstein et al., 1998), DC (Futagawa et al., 2002; Pauly et al., 2002; Summers et al., 2001; Wilcox et al., 2002), and within the intra-tumor vasculature of patients with metastatic disease (Broll et al., 2001). The cognate ligand for CD137, 4-1BB ligand, a member of the TNF superfamily (Goodwin et al., 1993; Zhou et al., 1995), is constitutively expressed at low levels on B cells, increases with B cell activation, and can be activation induced on macrophages and DC following CD40 or LPS mediated APC activation. The kinetics of expression of CD137 varies depending upon the experimental system under study. For instance, CD137 expression is evident within 24 hours of anti-CD3/anti-CD28 mediated in vitro activation of mouse T cells, or in acute graft versus host disease (GVHD). We find that CD8 T cells achieve maximal expression of CD137 within 24–36 hours following anti-CD3 induced in vitro activation, whereas CD4 T cells tend to lag behind. However, within 48 hours both subsets reach equivalent levels of expression (Mittler et al., 2004). In LCMV infected mice we find no evidence of CD137 expression on either CD4 or CD8 T cells until 72 hours post-infection (unpublished observations) and the appearance of CD137 is first observed on splenic and lymph node CD4 T cells in these mice. In contrast, expression of CD137 on CD8 T cells occurs much more rapidly in superantigen (sAg) injected mice and can be seen within 6–12 hours after injection of sAg (Takahashi et al., 1999). The difference in the rate of expression most likely reflects, or is at least dependent upon the kinetics of induction of prerequisite activation events such as the expression of CD154 on T cells and upregulation of CD80 and CD86 on antigen presenting cells. Crosslinking CD137 with anti-CD137 mAbs, soluble 4-1BB ligand fusion proteins, or 4-1BB ligand transfected cell lines induces T cell costimulation in antigen or mitogen activated T cells (DeBenedette et al., 1995; Shuford et al., 1997). Whereas costimulation of T cells by anti-CD28 preferentially activates CD4 T cells, we have found that anti-CD137 mAbs preferentially induces CD8+ T cell

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activation (Shuford et al., 1997) and drives their production of IFN-γ (Shuford et al., 1997) and TNF-α (our unpublished observations). However, others have not found this to be the case (Cannons et al., 2001) and the discrepancy between these two reports has not been resolved. In mice, injection of anti-CD137 mAbs resulted in accelerated rejection of skin or cardiac allografts and exacerbated acute GVHD (Shuford et al., 1997). The underlying cellular and molecular events by which CD137-mediated T cell costimulation regulates the function of the immune system are rapidly coming into focus (Watts, 2005). Furthermore, the potential for targeting the CD137 signaling pathway in treating diseases such as autoimmunity and cancer seems promising (Foell et al., 2003, 2004; Melero et al., 1997; Seo et al., 2004; Sun et al., 2002). In the following sections of this review we describe the effects of anti-CD137 treatment of mice and how these antibodies regulate T-dependent humoral immunity. We will also discuss how B cells may suppress the development of anti-tumor immunity and how this condition can be reversed with anti-CD137 mAbs. The studies reported in this review were based on our early studies of antiCD137 mediated suppression of humoral immunity in mice (Mittler et al., 1999) and the anti-tumor properties of anti-CD137 mAbs (Melero et al., 1997, 1998a).

4. Anti-CD137-Mediated Suppression of Humoral Immunity Blocking the CD28 or CD40 costimulatory pathways using soluble ligands to CD80/CD86 or antibodies to CD154 suppresses the development of T cell dependent but not T-independent humoral immunity. Therefore, it was not surprising that we found that anti-CD137 mAbs also suppressed T-dependent but not T cell-independent B cell responses. What was surprising was that suppression was not mediated by blocking receptor-ligand binding but appeared to require CD137-mediated signaling. Classic T-dependent antigens include allogeneic cells and soluble proteins foreign to the host. In our studies we used sheep red blood cells (SRBC), human IgG (huIgG), or Keyhole Limpet hemocyanin (KLH) to immunize mice. We also employed a commonly used T-independent antigen, Ficoll-TNP. Mice injected with anti-CD137 mAbs were unable to generate CD4 T cell-dependent humoral immune responses to each of these three T-dependent antigens, and we found that as little as 50μg of anti-CD137 mAbs was sufficient to induce suppression. Pharmacokinetic analysis of circulating rat anti-CD137 mAb in mice revealed a serum half-life of 7.5 days, nevertheless, the mice remained non-responsive to further challenge with antigen for a period of 3–6 months (Mittler et al., 1999; Figure 5.3). Characterization of the kinetics of anti-CD137-mediated suppression of T-dependent humoral immunity showed that the induction of suppression is restricted to an early phase of B cell activation. For example, we found that when mice were injected with anti-CD137 during antigen priming or within several days thereafter they failed to generate antibody responses to the immunizing antigen. However, when anti-CD137 injection was delayed until 10 days after vaccination,

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Figure 5.3. (A) Anti-CD137-mediated suppression of anti-SRBC humoral immunity. BALB/c mice were injected intravenously (I.V.) with 50 μl of a 1% suspension of washed sheep erythrocytes (SRBC) in PBS containing 200 μg of anti-CD137 (filled triangles) or an isotype matched control mAb (filled squares). Seven weeks later the mice were challenged with a second I.V. injection of SRBC. The mice were bled on a weekly basis and anti-SRBC serum titers were measured by ELISA. (B) CD137-mediated suppression of anti-huIgG humoral immunity. BALB/c mice were injected with 20 μg of human IgG in 50 μl of PBS containing 200 μg of anti-CD137 mAbs I.V. Six weeks later the mice were challenged with a second injection of huIgG. The mice were bled on a weekly basis and serum anti-huIgG titers were measured by ELISA.

no suppression was observed and the mice responded normally to subsequent antigen challenge almost two months later (Figure 5.4). In later studies employing LCMV-infected mice, we found that anti-CD137 mAbs had to be injected within 48 hr of infection in order to exert maximal immune suppression. When anti-CD137 mAbs were injected into virus-infected mice after 72 hr, injection of anti-CD137 enhanced rather than suppressed anti-viral immunity. Besides defining the time limitation of anti-CD137 induced suppression, the LCMV infection studies resolved a longstanding paradox concerning the in vivo biological effects of anti-CD137 mAbs. We, and others, have found that injection of anti-CD137 mAbs into tumor-bearing and allografted mice induced potent CD8 T cell immunity. Yet, when these antibodies were injected into immunized mice they suppressed development of T-dependent humoral immunity. The reasons for this dichotomy were not known and led us to speculate that CD137-mediated signaling imparted different effects on CD4 and CD8 T cells. However, this notion was abandoned following the LCMV infection experiments in which we found that suppression was not restricted to CD4 T cells but that CD8 T cell mediated

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Weeks Figure 5.4. Anti-CD137-mediated suppression of Ag-priming. Two groups (n = 5) of BALB/c mice were immunized with SRBC on day 0 as previously described. One group received a 200 μg I.V. injection of anti-CD137 the same day (filled squares) and the second group on day 10 post-SRBC priming (filled triangles). Seven weeks later the mice were challenged with a second injection of SRBC and serum anti-SRBC titers measured as before.

immunity was equally suppressed. It seems from these studies that suppression is dependent upon antigen priming and not on MHC restriction. Mice bearing tumors as well as allografted mice, display tumor associated or alloantigens in MHC class I molecules, and T cells are most likely primed to these antigens long before anti-CD137 is administered. In contrast, this situation does not occur in immunized or virus infected mice. A series of adoptive cell transfer experiments were then carried out in SRBC immunized mice to determine whether anti-CD137 treated T cells were deleting antigen-specific B cells. This proved not to be the case. SRBC-specific B cell function in anti-CD137 treated mice was unimpaired; instead T cell function was. This was shown in experiments in which mice were immunized to SRBC and injected with anti-CD137 mAbs. The mice were later challenged with SRBC and found to be non-responsive. Two weeks later splenic T cells or B cells from these mice were purified and mixed with B cells from na¨ıve mice or T cells from na¨ıve mice, respectively, and injected into C.B-17 SCID recipients. In addition, a group of SCID mice were reconstituted with B cells and T cells from na¨ıve mice. The SCID recipients were then injected with SRBC 4 weeks later and challenged 9 weeks after adoptive cell transfer. Ten weeks after cell transfer the SCID mice were immunized with KLH to measure their capacity to generate T-dependent humoral immunity to a second antigen (Figure 5.5). We then determined whether CD8

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Figure 5.5. Anti-CD137 mAbs suppress T cell help. T cells and na¨ıve B cells (inverted triangles) or B cells and na¨ıve T cells (filled triangles) from SRBC immunized, anti-CD137 treated mice, or T and B cells from non-vaccinated na¨ıve BALB/c mice (filled squares) were adoptively transferred into na¨ıve recipient C.B-17 SCID mice. 2 × 107 BALB/c B cells, or 1 × 107 T cells, or a combination of both were adoptively transferred into SCID mice two weeks following SRBC challenge. All recipients were bled on a weekly basis, and at weeks 4 and 9 post-adoptive transfer the mice were challenged with SRBC. Nine weeks post-adoptive transfer the recipients were immunized with KLH to show they could be immunized to a second antigen. Serum anti-SRBC and KLH IgG titers were measured by ELISA.

T cells mediated CD4 T cell suppression or deletion. CD8 T cell-deficient MHC class I deficient mice were immunized with SRBC and injected at the same time with anti-CD137 mAbs. Like wild type mice, in the absence of anti-CD137 SRBC immunized MHC class I deficient mice generated normal primary and secondary anti-SRBC antibody responses. In contrast, anti-SRBC humoral immunity was totally suppressed when the mice were injected with anti-CD137 mAbs during immunization (Mittler et al., 1999). Thus, CD8+ T cells are not required for the induction or maintenance of suppression. Rather, our results suggested that anti-CD137 treatment impaired CD4 T helper function. This could be caused by CD4 T cell death through antibody-dependent cell cytotoxicity (ADCC), AICD, induction of regulatory T cells, interference with T cell-APC costimulation or antigen priming of T cells. We found that rat IgG2A anti-CD137 mAbs do not deplete T cells and do not bind mouse Fc-receptors and cause ADCC. Furthermore, depleting rat anti-CD4 mAbs were found to suppress CD4 T cell-mediated help for a period of 4–5 weeks whereas anti-CD137 mAb treatment lasted 3–6 months. Since both anti-CD4 and anti-CD137 mAbs were of rat origin it is unlikely that they have very different clearance rates in mice. Therefore, we suspect that antiCD137 mAbs function by inducing long-term anergy, and that CD4 regulatory

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T cells enforce this state of anergy. This subject will be addressed further later on in this chapter. Although anti-CD137 treatment suppressed the induction of T-dependent humoral immune responses, it failed to suppress T-independent humoral immunity. When mice undergoing immunization to Ficoll-TNP were injected with antiCD137 mAbs we failed to suppress the development of Type II T-independent humoral immune responses (Mittler et al., 1999; Figure 5.6). This is not altogether surprising since CTLA-4 Ig and anti-CD154 mAbs, reagents that block the CD28 and CD40 signaling pathways, respectively, do not suppress T independent humoral immunity and B cells do not express CD137. Although some APC can express CD137, B cell activation is not dependent upon APC function.

5. Anti-CD137 Induced Suppression of Autoantibodies Self-reactive T cells escape from thymic deletion and become part of the normal T cell repertoire. In normal individuals these potentially autoreactive T cells are held in check by regulatory T cells and remain quiescent (Paust and Cantor, 2005). In individuals suffering from autoimmune disease these T cells escape the regulatory process, become activated and drive inflammatory processes, support autoantibody production and participate in tissue destruction. In diseases such

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as systemic lupus erythematosus and rheumatoid arthritis CD4 T cells drive the activation and differentiation of T-dependent autoantibody secreting B cells and their deletion or suppressed function blocks the development or progression of disease (Foell et al., 2003, 2004; Goronzy and Weyand, 1995; Mason et al., 2002; Seo et al., 2004; Sun et al., 2002; Wagner et al., 2004; Wofsy, 1988). Based on our studies showing that anti-CD137 mAb treatment suppressed T-dependent humoral immunity, several laboratories including ours have shown that anti-CD137 mAb treatment provides protection against the initiation, or progression of established antibody-dependent autoimmune diseases (Foell et al., 2003, 2004; Seo et al., 2004; Sun et al., 2002). Our initial studies in NZB/W F1 lupus-prone mice focused on the long-term effects of short periods of anti-CD137 treatment on the development of SLE. Subsequent studies examined the effects of this treatment in CIA susceptible DBA/1 mice. In both situations mice were treated at varying points during disease progression. We began by measuring the ability of anti-CD137 treatment to prevent the development of autoantibodies and disease progression in young pre-diseased NZB/W F1 mice. To do this, we injected 8 week-old female NZB/W F1 mice with 200 μg of anti-CD137 I.P. once every third week until the mice reached 24 weeks of age, altogether, the mice received seven injections (Figure 5.7A). DBA/1 mice were immunized twice on days 0 and 21 with CII and injected with anti-CD137 on days 0, 6, and 21. Disease progression was followed for 100 days and joint inflammation destruction were measured (Foell et al., 2004). The results of both studies were quite striking in that disease progression failed to occur or was markedly delayed and diminished. NZB/W F1 mice given just three 200 μg I.P. injections of anti-CD137 (one injection every third week), beginning at 26–32 weeks of age, ceased production of anti-dsDNA antibodies, and within 2–3 weeks of the first injection the level of serum anti-dsDNA IgG fell to near background levels (Figure 5.7B) and signs of disease progression were no longer evident. Impressively, the treated mice survived for over two years even though they eventually began to produce anti-dsDNA antibodies (Foell et al., 2003; Figure 5.7C). In another study we found that seventy percent of 41-week-old mice given a single I.P. injection of anti-CD137 mAbs responded to treatment in similar fashion and lived over two years (a normal life span for mice). Similarly, anti-CD137 mAbs suppressed established CIA in DBA/1 mice (Foell et al., 2004; Seo et al., 2004). Finding that anti-CD137 treatment reversed established T-dependent antidsDNA responses in lupus prone mice was unexpected because we had previously shown that it was not possible to suppress established T-dependent humoral immune responses in normal mice. To determine whether NZB/W F1 mice differed from normal mice in this regard we immunized NZB/W F1 mice with SRBC and then left them alone until they reached an age when they normally show signs of developing autoimmune disease. By the time the mice reached 26 weeks of age they had moderate to high levels of anti-dsDNA antibodies in their serum. We injected the mice once with 200 μg of anti-CD137 mAbs and challenged them with SRBC. The mice were bled periodically and serum anti-SRBC and anti-dsDNA antibody titers were measured by ELISA. As observed previously, serum levels of anti-dsDNA antibodies dropped precipitously following injection of anti-CD137

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Figure 5.7. (A) Anti-CD137 mAbs induce suppression of anti-dsDNA antibodies. Lupus prone NZB/W F1 mice were injected I.P. with 200 μg of non-depleting rat anti-CD137 mAbs (filled triangles) or rat IgG (filled squares) once every third week between 8–24 weeks of age and serum anti-dsDNA antibody levels measured at the indicated times by ELISA. By 46 weeks of age all of the mice that received rat IgG had died (). (B). Twenty-six week-old NZB/W F1 mice were injected with anti-CD137 mAbs (filled triangles) or rat IgG (filled squares) every third week until they reached 35 weeks of age and serum anti-dsDNA antibodies measured. By 46 weeks of age all of the mice that received rat IgG had died (). (C). Survival of anti-CD137 treated NZB/W F1 mice. Thirty-six week-old NZB/W F1 were injected twice 3 weeks apart with 200 μg of anti-CD137 mAbs and their survival followed.

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66 Robert S. Mittler, Liguo Niu, Becker Hewes, and Juergen Foell

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mAbs. Nevertheless, anti-SRBC responses remained elevated (Foell et al., 2003; Figure 5.8). The basis for this disparity is not yet clear. However, it has been shown that virus specific CD8 T cells in mice chronically infected with LCMV undergo clonal exhaustion due to constant stimulation. In this respect autoimmune mice bear a similarity to chronically infected mice in that their autoreactive T cells are constantly in a state of activation, and they too probably die through exhaustion. Therefore, disease progression can be viewed as a continuous cycle of dying exhausted autoreactive cells and an influx of newly minted na¨ıve autoreactive T and B cells lymphocytes. In such an environment anti-CD137 mAbs have no effect on existing activated autoreactive T cells, but they could suppress antigen priming of na¨ıve autoreactive T cells in the same way they suppress anti-SRBC responses. However, this is just a hypothesis and at present there is no evidence to support this notion. Furthermore, other factors must come into play in suppressing autoimmunity in NZB/W F1 mice because anti-CD137 mAbs do not suppress or reverse autoimmune diabetes, a CD4 T cell-dependent, B-cell independent disease. Perhaps the difference lies is the lifespan of autoantibody producing B cells and not autoreactive CD4 T cells. In a variation of the preceding scenario, autoreactive CD4 T cells are held in check following anti-CD137 treatment by DC induced CD4 regulatory T cells and no longer retain the capacity to provide help to B cells.

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Figure 5.9. (A) CD4 T cell depletion suppresses anti-dsDNA production. Thirty-three week-old NZB/W F1 mice were injected I.P. once with 500 μg with GK1.5, a depleting anti-CD4 mAb (gray triangles), rat IgG (inverted triangles), or left untreated, (filled squares). Anti-dsDNA serum antibodies were measured weekly. (B) CD8 T cells are not required for anti-CD137-mediated suppression of autoantibody production. Thirty-six week-old NZB/W F1 mice were injected I.P with 500 μg of YTS169.4, a depleting anti-CD8 mAb. One week later the mice were injected with 200 μg of antiCD137 (filled squares) or left untreated (filled triangles) and serum anti-dsDNA antibodies measured every 5 days by ELISA.

If antibody producing, pathogenic B cells do have a short lifespan, one would expect to see a loss of autoantibody production as they die off. Others have shown that anti-CD4 mAb mediated suppression or depletion of CD4 T cells causes a temporary loss of anti-dsDNA autoantibody production in NZB/W F1 mice and indicates the ever present need for CD4 T cell help in maintaining autoimmune disease (Wofsy, 1988, 1993; Wofsy et al., 1988; Wofsy and Seaman, 1985, 1987). This transient suppression is due to the removal of CD4 T cells followed by the clearance of anti-CD4 mAbs and the export of mature na¨ıve autoreactive CD4 T cells from the thymus (Figure 5.9A). Anti-CD137 nondepleting mAbs appear to affect the function of autoreactive CD4 T helper cells in maintaining autoantibody production, but whether this is a direct effect of antiCD137 binding to autoreactive CD4 T cells or an indirect effect on their priming is not yet known. We have found that injection of mice with anti-CD137 soon after LCMV infection suppressed anti-viral immunity in these mice. Dendritic cells in LCMV infected mice upregulate CD137 on their surface. DC taken from these mice display an immature phenotype, i.e., they had not upregulated CD40, CD80 or CD86 on their surface, phenotypic markers of DC maturation. Furthermore, in vitro crosslinking of CD137 on DC rapidly induced phosphorylation of Stat3, a negative

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regulator of DC and B cell maturation (Maris et al., submitted). These data suggest but do not conclusively prove that the immediate target of anti-CD137 mAbs in blocking SLE progression in lupus prone mice is the DC or other antigen presenting cells. CD8 T cells in lupus-prone mice become activated and express CD137 as disease progresses, however, it appears that CD8 T cells play a minor role in controlling autoimmune disease in this setting and depletion of these T cells slightly exacerbates disease (our unpublished observations). However, CD8 T cell depletion does not affect the ability of anti-CD137 mAbs to induce suppression of antidsDNA autoantibodies (Foell et al., 2003; Figure 5.9b). Furthermore, suppressed CD4 T cell helper function in anti-CD137 treated mice can be overcome by adoptively transferring CD4 T cells from untreated 26 week-old lupus involved mice into the anti-CD137 treated mice after they have cleared most of the anti-CD137 mAb from their circulation (Figure 5.10).

6. DC Function, CD137 Expression and Signaling Dendritic cells acquire, internalize, process, and present antigens in the form of protease cleaved peptides derived from extracellular or intracellular antigens (Steinman, 1991). Processed peptides are then displayed in the context of MHC molecules on the cell surface and prime T cells bearing the appropriate

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peptide-specific TCR. The acquisition and presentation of antigens by dendritic cells is restricted to different stages of their development, the former function being carried out by immature dendritic cells, while the latter is thought to be carried out by mature dendritic cells (Steinman et al., 1999). During maturation, DC increase their expression of MHC class II molecules as well as several ligands (CD40, CD80, and CD86) for costimulatory receptors expressed on T cells (Steinman et al., 1999). By doing so, DC acquire the ability to antigen prime and activate T cells. A key component in the establishment of immunity to pathogens is the ability of dendritic cells to recognize pathogenic microorganisms through a conserved family of Toll-like Receptors (TLR). TLR recognize specific ligands produced by bacteria such as lipopolysaccharides, flagellin, and CpG nucleotides (Medzhitov and Janeway, 1999); the triggering of these receptors represents a major physiological mechanism through which dendritic cells undergo maturation (Medzhitov and Janeway, 2000). It has been suggested that prior to infection or inflammation immature dendritic cells remain in a “steady state” and maintain peripheral tolerance self antigens and environmental proteins (Steinman and Nussenzweig, 2002). Support for this point of view stems from two quarters. Peripheral tolerance can be initiated following deletion of autoreactive T cells via a process that is akin to the establishment of central tolerance in the thymus. Many dendritic cells, particularly those in the T cell areas, express an adsorptive endocytosis receptor termed DEC-205 on their surface, the natural ligand for which is not known (Inaba et al., 1995). Hawiger and colleagues used an antigen (HEL) to target DC in the form of an HEL-DEC-205 fusion protein. The cytoplasmic domain of DEC-205 contains an EDE tri-acidic amino acid targeting sequence, thus allowing targeting of the receptor-HEL fusion protein to MHC class II-containing compartments. Compared with normal HEL uptake and antigen processing, fusion protein targeting of HEL was found to be 30–100 fold more efficient (Hawiger et al., 2001). When HEL specific TCR transgenic T cells were exposed to these immature dendritic cells the T cells proliferated during the first week post-stimulation. However, the majority of proliferating T cells were deleted, thus inducing a state of tolerance to HEL in the remaining antigen specific T cells even following vaccination with complete Freund’s adjuvant containing HEL peptides. However, tolerance could be overcome if, at the time of exposure of dendritic cells to antigen, the DC received a maturation signal (Hawiger et al., 2001). In agreement with these studies are several reports demonstrating that bone marrow dendritic cells can mediate peripheral deletion or anergy in situ (Steinman and Nussenzweig, 2002). Mouse spleen and bone marrow derived DC can express CD137 receptors on their surface (Futagawa et al., 2002; Wilcox et al., 2002). It has also been shown that human tonsil follicular DC express high levels of CD137 on their surface (Lindstedt et al., 2003). We find that all phenotypic subsets of mouse splenic DC have the capacity to express CD137 following viral infection, but that DC expressing the highest level of CD137+ bear the phenotype of a myeloidderived, immature dendritic cell (Maris et al., submitted). Following CD137 receptor crosslinking, either by 4-1BB ligand expressing tumor cells or an anti-mouse CD137 mAb, splenic dendritic cells produce and secrete IL-6 and IL-12 (Futagawa

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et al., 2002; Wilcox et al., 2002). IL-6 is an anti-inflammatory Th2 cytokine whose expression and function is prominent in myeloid cell development, humoral immune responses, and in certain autoimmune diseases. These include EAE (Ishihara and Hirano, 2002; Samoilova et al., 1998), RA (Hata et al., 2004; Hwang et al., 2004; Nishimoto and Kishimoto, 2004), juvenile diabetes (Scholin et al., 2004; Vozarova et al., 2001), and SLE (Ohteki et al., 1993; Suzuki et al., 1993; Tang et al., 1991). IL-12 is a pro-inflammatory cytokine that promotes the development of Th1 mediated immune responses, it induces T cells to produce IFN-γ and drives both innate and adaptive anti-tumor immune responses (Gao et al., 2003; Kodama et al., 1999; Martinet et al., 2002; Parihar et al., 2002; Smyth et al., 2000; Uekusa et al., 2002). How these two opposing cytokines are coordinately regulated following CD137 signaling, and what their physiological roles are in dendritic cell biology and T cell immunity remains to be determined. Anti-CD137 induced suppression of SLE can be reversed by adoptively transferring dendritic cells from untreated NZB/W F1 mice into anti-CD137 treated autoimmune mice (Foell et al., 2003; Figure 5.11). This observation supports the notion that CD4 helper T cells are not deleted or permanently impaired, but that they may have failed to receive the required sequence of signals from dendritic cells during antigen priming. If true, then dendritic cells rather than T cells are the primary targets of anti-CD137 mAbs, and signals delivered to the dendritic cell through CD137 block its ability to antigen prime CD4 T cells.

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7. Anti-tumor Immunity and B Cells The innate and adaptive arms of the immune system have the capacity to kill tumors. This cytolytic function is fulfilled by the innate immune system through NK cells and by the adaptive immune system through CD8 cytotoxic effector T cells (CTL). Initiation of anti-tumor immunity requires the participation of macrophages, dendritic cells and NK cells, all members of the innate immune system. The induction adaptive immunity and the generation of tumor specific CD8 T cell effector killers (CTL) generally is dependent upon CD4 T cell help (Melero et al., 1997, 1998b). Relative to effective cell-mediated anti-tumor responses evidence suggesting that B cells play an important role in curative antitumor immunity is sparse and there is some evidence that B cells may contribute to tumor progression. The notion that B cells might interfere with the induction of T cell mediated anti-tumor immunity was first suggested in experiments in which B cell development was blocked in neonatal mice that were subsequently inoculated with tumor cells. Compared with untreated littermates, tumor progression in these mice was markedly suppressed. More recently, Qin et al. demonstrated that it was possible to successfully vaccinate and protect BALB/c μMT B cell deficient mice against a variety of tumors (Qin et al., 1998). These studies strongly suggest that interplay between B cells and T cells suppress the development of anti-tumor immunity in μMT mice. For example, these transgenic B cell deficient mice have the capacity to develop anti-tumor immunity following vaccination with irradiated tumor cells whereas syngeneic wild type mice do not. Adoptive transfer of B cells into na¨ıve μMT mice renders them refractive to vaccination and the mice succumb to the tumor (Qin et al., 1998). However, the mechanisms through which B cells mediate suppression of anti-tumor immunity are poorly understood. It has been suggested that B cells may exert their suppressive effect on the induction of anti-tumor immunity by virtue of their capacity to capture low levels of tumor antigens through their high affinity antigen receptors and thus sequester antigen from dendritic cells that prime T cells (Qin et al., 1998). In humans, the prognosis in certain neoplastic diseases such as colorectal and ovarian cancer bears an inverse relationship between production of anti-oncogene antibody levels and disease progression (Houbiers et al., 1995; Morrin et al., 1994). If B cell-induced suppression of anti-tumor immunity occurs in humans as it has been observed in mice, novel treatment approaches based upon B cell depletion might find clinical applicability. Neuroblastoma, the most common non-cranial childhood cancer is one example that might benefit. Children with non-remitting neuroblastoma receive myeloablative whole body irradiation and chemotherapy followed by autologous bone marrow rescue. These procedures coupled with autologous T cell rescue might allow for successful patient vaccination against their tumors. For this reason we decided to study the phenomenon of B cell induced suppression of anti-tumor immune responses in mice. To pursue this study we followed the development of Lewis lung carcinoma (LLC) in C57BL/6 wild type, Ragdeficient, and μMT B cell deficient mice. Mice were vaccinated and/or inoculated

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Tumor Volume, mm3

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Days Post-Inoculation//Challenge Figure 5.12. LLC tumor progression and survival of C57BL/6 μMT mice. Two groups of 10 mice were inoculated subcutaneously with LLC cells. Each of 10 LLC inoculated na¨ıve mice are shown having varying sized tumors. The second group of mice had been vaccinated with irradiated LLC cells two weeks prior to challenge. None of these mice developed tumors as indicated by the arrow pointing to baseline. Tumor progression in each group was monitored several times weekly.

with LLC cells in order to identify the cellular and biochemical mediators that regulate B cell-mediated suppression of anti-tumor immunity, and to determine whether suppression can be reversed. Wild type C57BL/6 mice rapidly developed LLC tumors regardless of whether they had been vaccinated with irradiated LLC cells or not. C57BL/6 μMT B cell deficient mice also developed tumors but at a slower rate whereas vaccination with irradiated LLC cells uniformly protected the mice from subsequent challenge. However, when the mice were reconstituted with C57BL/6 splenic B cells prior to vaccination the mice were no longer protected when challenged several weeks later with LLC tumor cells. Furthermore, when the mice were first vaccinated and later reconstituted with B cells and subsequently challenged with LLC all of the mice were protected. Thus, in order for B cells to suppress the development of anti-tumor immunity they had to be present in the mice prior to, or during, LLC vaccination (Figure 5.12). Using this H-2b restricted LLC tumor model we confirmed earlier observations made by Qin et al. in BALB/c (H-2d ) B cell deficient mice. By showing that B cell-induced suppression of anti-tumor immunity occurs in an antigen-nonspecific manner, and is neither antibody-mediated or serum dependent (manuscript in preparation) we extended the findings of Qin and colleagues. Furthermore, suppression of anti-tumor immunity is not the sole province of B cells but can also be mediated by dendritic cells (Figdor et al., 2004; Munn et al., 2004) and regulatory T cells (Wei et al., 2004). We find that both dendritic cells, or CD4 T cells lacking CD25 expression, in addition to B cells from syngeneic wild type mice, can suppress the development of anti-tumor immunity to LLC in μMT B cell deficient, or Rag-deficient mice

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150 n=4

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50 3 mice

0 5

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Days Post-Challenge Figure 5.13. Anti-CD137 restores LLC immunity in B cell reconstituted mice containing μMT mice. Six C57BL/6 μMT mice were reconstituted with B cells from na¨ıve BL/6 mice. Three mice were injected with 200 μg of anti-CD137 mAbs I.P. on days 0, 3 and 5 (filled diamonds) and the remaining three received Rat IgG. The mice were vaccinated with irradiated LLC cells on day 0 and challenged with viable LLC cells on day 14, and tumor progression was followed periodically thereafter.

whereas CD4+ CD25+ T cells or CD8+ T cells cannot. In addition, we find that suppression of anti-tumor immunity to LLC in B cell reconstituted, LLC vaccinated μMT mice can be overridden by injecting them with anti-CD137 (4-1BB) mAbs (Figure 5.13). At present it is not clear just how B cells or DC suppress the development of anti-tumor immunity or how anti-CD137 mAb treatment overrides B cell-mediated suppression in μMT mice. One hypothesis that we favor is that tumor antigen-experienced APC fail to mature and cannot adequately prime tumor-specific T cells. Perhaps this is due to their failure to adequately up-regulate their cell surface expression of costimulatory ligands such as CD40, CD80/86, 4-1BB-L, or other costimulatory or adhesion ligands. Regardless, failure to prime T cells to tumor antigens renders them anergic and this may be due to a direct effect of DC on rendering the T cell anergic, or inducing it to develop regulatory properties.

8. Anti-CD137 mAbs Disrupt Hematopoiesis in Mice We have found that repeated anti-CD137 treated lupus prone NZB/W F1 mice develop multi-focal hepatitis. Cessation of anti-CD137 treatment led to the resolution of hepatitis. To determine whether development of hepatitis was or was not related to the susceptibility of NZB/W F1 mice to autoimmune disease, we repeatedly injected normal na¨ıve mice with therapeutic doses of anti-CD137. Like NZB/W F1 mice, we found that normal mice also developed hepatitis. Further

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Weekly Injections Figure 5.14. Loss of splenic B cells following repetitive injection of anti-CD137. BALB/c mice were injected weekly with 200 μg I.P. the indicated number of times. One week after the final injection the mice were euthanized and the frequency and absolute numbers (data not shown) of CD19+ B cells determined by flow cytometry. Control mice were injected with a non-crossreactive rat anti-human CD137 mAb.

analysis revealed other anomalies or pathologies in the treated animals. Included among these were splenomegaly, lymphadenopathy, hepatomegaly, anemia, extramedullary hematopoiesis, abnormal white blood cell counts, loss of CD19+ cells in the spleen and bone marrow, a marked increase in B cells in the lymph nodes, and marked elevation of Lin− Sca-1+ c-kit+ bone marrow cells (Niu et al., manuscript submitted). We further noted marked accumulation of CD8+ T cells in the lungs, liver, and bone marrow. Figure 5.14 illustrates the reduced frequency of CD19+ B cells in the spleens of na¨ıve C57BL/6 given two or five weekly intraperitoneal injections of anti-CD137. The reduced frequency of splenic B cells was mirrored by a loss in absolute numbers of B cells and not due to an increase in other cell elements. Accompanying the loss of CD19+ B cells from the spleen, anti-CD137 injected mice lost almost all antibody secreting cell function following five weekly injections as indicated by a precipitous drop in serum IgG levels (Figure 5.15). This was particularly evident in the bone marrow where greater than 98% of antibody secreting plasma cells were lost (Figure 5.16). The reason for this loss is not clear, but it is important to note that we observed a tenfold increase in the number of CD8 T cells in the bone marrow of these mice, and this suggests to us that their presence may be a contributing factor. The disruption of normal homeostasis of the hematopoietic system together with hepatomegaly and multi-focal hepatitis in anti-CD137 treated mice is T cell dependent. Repeated injections of anti-CD137 mAbs in nude or Rag-deficient mice have no observable ill effect. However, following T and B cell reconstitution,

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Serum IgG, μg//ml

10 3

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Figure 5.15. Loss of serum IgG following injection of anti-CD137. BALB/c mice were injected I.P. with 200 μg of anti-CD137 or rat IgG weekly for 5 weeks. At week 6 the mice were bled and serum IgG levels were determined by ELISA.

Plasma Cell Number//Femur

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10 4

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Anti-CD137

Figure 5.16. Loss of bone marrow plasma cells following multiple anti-CD137 injections. BALB/c mice were injected I.P. weekly for 5 weeks with 200 μg of anti-CD137 as previously described. One week later the mice were euthanized and femurs removed. Bone marrow cells were collected and antibody-secreting plasma cells were enumerated following 36 hr Elispot assay.

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Rag−/− mice develop the full range of disruptive and pathophysiological events observed in normal mice. Furthermore, na¨ıve TCR transgenic Rag−/− mice did not develop hepatomegaly or multi-focal hepatitis following multiple anti-CD137 injections. This observation suggests that a small pool of CD137+ antigen activated T cells in normal mice undergo oligoclonal expansion and directly, or indirectly give rise to the disruptive events noted. It remains to be determined whether the specificity of the activated T cell plays a role in pathogenesis or whether these events are activation and CD137 dependent but antigen independent responses. It is also important to note that transgenic mice that over-express 4-1BB ligand on antigen presenting cells develop many of the anomalies observed in anti-CD137 treated mice (Zhu et al., 2001). Therefore, it is very unlikely that these events are attributable to non-specific or inflammatory processes induced by rat anti-CD137 antibodies.

9. Concluding Remarks In this review we have highlighted the pluripotent effects of anti-CD137mediated costimulation with emphasis on T-dependent B cell function in normal and autoimmune prone mice. We have shown that anti-CD137 treatment of normal mice during antigen priming, but not thereafter, suppresses the induction of T-dependent humoral immunity as well as CD8 T cell immunity. We suggest that the mechanism through which this occurs may involve CD137-mediated signaling in dendritic cells and other CD137-expressing APC. In studying lupus prone mice, contrary to our observations in normal mice, we find that we can suppress established T-dependent humoral autoimmune responses. On the other hand, as in normal mice, we fail to suppress established T-dependent humoral immunity to conventional antigens in NZB/W F1 mice, and we speculate how this may occur. We have also included some of our unpublished studies on the regulatory function of B cells and their suppression of anti-tumor immunity. These data, while tangential to the main thrust of this review, highlight how B cell may mediate suppression of anti-tumor immunity and how anti-CD137 treatment can override suppression. Finally, we show that repeated treatment of normal mice with anti-CD137 mAbs has a dramatic T cell-dependent effect on lymphocyte trafficking, homeostasis, hematopoiesis and liver pathology. Many questions remain to be answered concerning the mechanisms that drive these events, and it is important to understand how dysregulation of immune cell homeostasis relates to the positive effects of anti-CD137 treatment of autoimmune disease and cancer.

References Allen, R.C., Armitage, R.J., Conley, M.E., Rosenblatt, H., Jenkins, N.A., Copeland, N.G., Bedell, M.A., Edelhoff, S., Disteche, C.M., Simoneaux, D.K., et al. (1993). CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome. Science, 259, 990–993.

78

Robert S. Mittler, Liguo Niu, Becker Hewes, and Juergen Foell

Aruffo, A., Farrington, M., Hollenbaugh, D., Li, X., Milatovich, A., Nonoyama, S., Bajorath, J., Grosmaire, L.S., Stenkamp, R., Neubauer, M., et al. (1993). The CD40 ligand, gp39, is defective in activated T cells from patients with X-linked hyper-IgM syndrome. Cell, 72, 291–300. Boise, L.H., Minn, A.J., Noel, P.J., June, C.H., Accavitti, M.A., Lindsten, T., and Thompson, C.B. (1995a). CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-XL. Immunity, 3, 87–98. Boise, L.H., Noel, P.J., and Thompson, C.B. (1995b). CD28 and apoptosis. Curr. Opin. Immunol., 7, 620–625. Bretscher, P., and Cohn, M. (1970). A theory of self-nonself discrimination. Science, 169, 1042–1049. Broll, K., Richter, G., Pauly, S., Hofstaedter, F., and Schwarz, H. (2001). CD137 expression in tumor vessel walls. High correlation with malignant tumors. Am. J. Clin. Pathol., 115, 543–549. Brunet, J.F., Denizot, F., Luciani, M.F., Roux-Dosseto, M., Suzan, M., Mattei, M.G., and Golstein, P. (1987). A new member of the immunoglobulin superfamily–CTLA-4. Nature, 328, 267–270. Cannons, J.L., Lau, P., Ghumman, B., DeBenedette, M.A., Yagita, H., Okumura, K., and Watts, T.H. (2001). 4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy. J. Immunol., 167, 1313–1324. Chen, C.Y., Del Gatto-Konczak, F., Wu, Z., and Karin, M. (1998). Stabilization of interleukin-2 mRNA by the c-Jun NH2-terminal kinase pathway. Science, 280, 1945–1949. DeBenedette, M.A., Chu, N.R., Pollok, K.E., Hurtado, J., Wade, W.F., Kwon, B.S., and Watts, T.H. (1995). Role of 4-1BB ligand in costimulation of T lymphocyte growth and its upregulation on M12 B lymphomas by cAMP. J. Exp. Med., 181, 985–992. DiSanto, J.P., Bonnefoy, J.Y., Gauchat, J.F., Fischer, A., and de Saint Basile, G. (1993). CD40 ligand mutations in x-linked immunodeficiency with hyper-IgM. Nature, 361, 541–543. Durie, F.H., Foy, T.M., Masters, S.R., Laman, J.D., and Noelle, R.J. (1994). The role of CD40 in the regulation of humoral and cell-mediated immunity. Immunol. Today, 15, 406–411. Figdor, C.G., de Vries, I.J., Lesterhuis, W.J., and Melief, C.J. (2004). Dendritic cell immunotherapy: mapping the way. Nat. Med., 10, 475–480. Foell, J., Strahotin, S., O’Neil, S.P., McCausland, M.M., Suwyn, C., Haber, M., Chander, P.N., Bapat, A.S., Yan, X.J., Chiorazzi, N., et al. (2003). CD137 costimulatory T cell receptor engagement reverses acute disease in lupus-prone NZB x NZW F1 mice. J. Clin. Invest., 111, 1505– 1518. Foell, J.L., Diez-Mendiondo, B.I., Diez, O.H., Holzer, U., Ruck, P., Bapat, A.S., Hoffmann, M.K., Mittler, R.S., and Dannecker, G.E. (2004). Engagement of the CD137 (4-1BB) costimulatory molecule inhibits and reverses the autoimmune process in collagen-induced arthritis and establishes lasting disease resistance. Immunology, 113, 89–98. Foy, T.M., Shepherd, D.M., Durie, F.H., Aruffo, A., Ledbetter, J.A., and Noelle, R.J. (1993). In vivo CD40-gp39 interactions are essential for thymus-dependent humoral immunity. II. Prolonged suppression of the humoral immune response by an antibody to the ligand for CD40, gp39. J. Exp. Med., 178, 1567–1575. Futagawa, T., Akiba, H., Kodama, T., Takeda, K., Hosoda, Y., Yagita, H., and Okumura, K. (2002). Expression and function of 4-1BB and 4-1BB ligand on murine dendritic cells. Int. Immunol., 14, 275–286. Gao, W., Kumar, S., Lotze, M.T., Hanning, C., Robbins, P.D., and Gambotto, A. (2003). Innate immunity mediated by the cytokine IL-1 homologue 4 (IL-1H4/IL-1F7) induces IL-12-dependent adaptive and profound antitumor immunity. J. Immunol., 170, 107–113. Gimmi, C.D., Freeman, G.J., Gribben, J.G., Gray, G., and Nadler, L.M. (1993). Human T-cell clonal anergy is induced by antigen presentation in the absence of B7 costimulation. Proc. Natl. Acad. Sci. USA, 90, 6586–6590. Goodwin, R.G., Din, W.S., Davis-Smith, T., Anderson, D.M., Gimpel, S.D., Sato, T.A., Maliszewski, C.R., Brannan, C.I., Copeland, N.G., Jenkins, N.A., et al. (1993). Molecular cloning of a ligand for the inducible T cell gene 4-1BB: A member of an emerging family of cytokines with homology to tumor necrosis factor. Eur. J. Immunol., 23, 2631–2641. Goronzy, J.J., and Weyand, C.M. (1995). T and B cell-dependent pathways in rheumatoid arthritis. Curr. Opin. Rheumatol., 7, 214–221.

Regulation of T Cell-Dependent Humoral Immunity Through CD137 (4-1BB)

79

Gribben, J.G., Freeman, G.J., Boussiotis, V.A., Rennert, P., Jellis, C.L., Greenfield, E., Barber, M., Restivo, V.A., Jr., Ke, X., Gray, G.S., et al. (1995). CTLA4 mediates antigen-specific apoptosis of human T cells. Proc. Natl. Acad. Sci. USA, 92, 811–815. Hata, H., Sakaguchi, N., Yoshitomi, H., Iwakura, Y., Sekikawa, K., Azuma, Y., Kanai, C., Moriizumi, E., Nomura, T., Nakamura, T., and Sakaguchi, S. (2004). Distinct contribution of IL-6, TNF-alpha, IL-1, and IL-10 to T cell-mediated spontaneous autoimmune arthritis in mice. J. Clin. Invest., 114, 582–588. Hawiger, D., Inaba, K., Dorsett, Y., Guo, M., Mahnke, K., Rivera, M., Ravetch, J.V., Steinman, R.M., and Nussenzweig, M.C. (2001). Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. J. Exp. Med., 194, 769–779. Heinisch, I.V., Bizer, C., Volgger, W., and Simon, H.U. (2001). Functional CD137 receptors are expressed by eosinophils from patients with IgE-mediated allergic responses but not by eosinophils from patients with non-IgE-mediated eosinophilic disorders. J. Allergy Clin. Immunol., 108, 21–28. Heinisch, I.V., Daigle, I., Knopfli, B., and Simon, H.U. (2000). CD137 activation abrogates granulocytemacrophage colony-stimulating factor-mediated anti-apoptosis in neutrophils. Eur. J. Immunol., 30, 3441–3446. Houbiers, J.G., van der Burg, S.H., van de Watering, L.M., Tollenaar, R.A., Brand, A., van de Velde, C.J., and Melief, C.J. (1995). Antibodies against p53 are associated with poor prognosis of colorectal cancer. Br. J . Cancer, 72, 637–641. Hwang, S.Y., Kim, J.Y., Kim, K.W., Park, M.K., Moon, Y., Kim, W.U., and Kim, H.Y. (2004). IL-17 induces production of IL-6 and IL-8 in rheumatoid arthritis synovial fibroblasts via NF-kappaBand PI3-kinase/Akt-dependent pathways. Arthritis Res. Ther., 6, R120–128. Inaba, K., Swiggard, W.J., Inaba, M., Meltzer, J., Mirza, A., Sasagawa, T., Nussenzweig, M.C., and Steinman, R.M. (1995). Tissue distribution of the DEC-205 protein that is detected by the monoclonal antibody NLDC-145. I. Expression on dendritic cells and other subsets of mouse leukocytes. Cell Immunol., 163, 148–156. Ishihara, K., and Hirano, T. (2002). IL-6 in autoimmune disease and chronic inflammatory proliferative disease. Cytokine Growth Factor Rev., 13, 357–368. Kawabe, T., Naka, T., Yoshida, K., Tanaka, T., Fujiwara, H., Suematsu, S., Yoshida, N., Kishimoto, T., and Kikutani, H. (1994). The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. Immunity, 1, 167–178. Kelsoe, G. (1996). Life and death in germinal centers (redux). Immunity, 4, 107–111. Kienzle, G., and von Kempis, J. (2000). CD137 (ILA/4-1BB), expressed by primary human monocytes, induces monocyte activation and apoptosis of B lymphocytes. Int. Immunol., 12, 73–82. Kodama, T., Takeda, K., Shimozato, O., Hayakawa, Y., Atsuta, M., Kobayashi, K., Ito, M., Yagita, H., and Okumura, K. (1999). Perforin-dependent NK cell cytotoxicity is sufficient for anti-metastatic effect of IL-12. Eur. J. Immunol., 29, 1390–1396. Korthauer, U., Graf, D., Mages, H.W., Briere, F., Padayachee, M., Malcolm, S., Ugazio, A.G., Notarangelo, L.D., Levinsky, R.J., and Kroczek, R.A. (1993). Defective expression of T-cell CD40 ligand causes X-linked immunodeficiency with hyper-IgM. Nature, 361, 539– 541. Kwon, B.S., Kozak, C.A., Kim, K.K., and Pickard, R.T. (1994). Genomic organization and chromosomal localization of the T-cell antigen 4-1BB. J. Immunol., 152, 2256–2262. Lafferty, K.J., and Cunningham, A.J. (1975). A new analysis of allogeneic interactions. Aust. J. Exp. Biol. Med. Sci., 53, 27–42. Langstein, J., Michel, J., Fritsche, J., Kreutz, M., Andreesen, R., and Schwarz, H. (1998). CD137 (ILA/4-1BB), a member of the TNF receptor family, induces monocyte activation via bidirectional signaling. J. Immunol., 160, 2488–2494. Lindstedt, M., Johansson-Lindbom, B., and Borrebaeck, C.A. (2003). Expression of CD137 (4-1BB) on human follicular dendritic cells. Scand. J. Immunol., 57, 305–310. Linsley, P.S., Brady, W., Grosmaire, L., Aruffo, A., Damle, N.K., and Ledbetter, J.A. (1991). Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. J. Exp. Med., 173, 721–730.

80

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Martinet, O., Divino, C.M., Zang, Y., Gan, Y., Mandeli, J., Thung, S., Pan, P.Y., and Chen, S.H. (2002). T cell activation with systemic agonistic antibody versus local 4-1BB ligand gene delivery combined with interleukin-12 eradicate liver metastases of breast cancer. Gene. Ther., 9, 786–792. Mason, U., Aldrich, J., Breedveld, F., Davis, C.B., Elliott, M., Jackson, M., Jorgensen, C., Keystone, E., Levy, R., Tesser, J., et al. (2002). CD4 coating, but not CD4 depletion, is a predictor of efficacy with primatized monoclonal anti-CD4 treatment of active rheumatoid arthritis. J. Rheumatol., 29, 220–229. Medzhitov, R., and Janeway, C., Jr. (2000). The Toll receptor family and microbial recognition. Trends Microbiol., 8, 452–456. Medzhitov, R., and Janeway, C.A., Jr. (1999). Innate immune induction of the adaptive immune response. Cold Spring Harb. Symp. Quant. Biol., 64, 429–435. Melero, I., Bach, N., Hellstrom, K.E., Aruffo, A., Mittler, R.S., and Chen, L. (1998a). Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: Synergy with the CD28 co-stimulatory pathway. Eur. J. Immunol., 28, 1116–1121. Melero, I., Johnston, J.V., Shufford, W.W., Mittler, R.S., and Chen, L. (1998b). NK1.1 cells express 4-1BB (CDw137) costimulatory molecule and are required for tumor immunity elicited by anti4-1BB monoclonal antibodies. Cell. Immunol., 190, 167–172. Melero, I., Shuford, W.W., Newby, S.A., Aruffo, A., Ledbetter, J.A., Hellstrom, K.E., Mittler, R.S., and Chen, L. (1997). Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat. Med., 3, 682–685. Mittler, R.S., Bailey, T.S., Klussman, K., Trailsmith, M.D., and Hoffmann, M.K. (1999). Anti-4-1BB monoclonal antibodies abrogate T cell-dependent humoral immune responses in vivo through the induction of helper T cell anergy. J. Exp. Med., 190, 1535–1540. Mittler, R.S., Foell, J., McCausland, M., Strahotin, S., Niu, L., Bapat, A., and Hewes, L.B. (2004). Anti-CD137 antibodies in the treatment of autoimmune disease and cancer. Immunol. Res., 29, 197–208. Morrin, M., Kelly, M., Barrett, N., and Delaney, P. (1994). Mutations of Ki-ras and p53 genes in colorectal cancer and their prognostic significance. Gut, 35, 1627–1631. Mueller, D.L., Seiffert, S., Fang, W., and Behrens, T.W. (1996). Differential regulation of bcl-2 and bcl-x by CD3, CD28, and the IL-2 receptor in cloned CD4+ helper T cells. A model for the long-term survival of memory cells. J. Immunol., 156, 1764–1771. Munn, D.H., Sharma, M.D., Hou, D., Baban, B., Lee, J.R., Antonia, S.J., Messina, J.L., Chandler, P., Koni, P.A., and Mellor, A.L. (2004). Expression of indoleamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor-draining lymph nodes. J. Clin. Invest., 114, 280–290. Nishimoto, N., and Kishimoto, T. (2004). Inhibition of IL-6 for the treatment of inflammatory diseases. Curr. Opin. Pharmacol., 4, 386–391. Noel, P.J., Boise, L.H., Green, J.M., and Thompson, C.B. (1996). CD28 costimulation prevents cell death during primary T cell activation. J. Immunol., 157, 636–642. Ohteki, T., Okamoto, S., Nakamura, M., Nemoto, E., and Kumagai, K. (1993). Elevated production of interleukin 6 by hepatic MNC correlates with ICAM-1 expression on the hepatic sinusoidal endothelial cells in autoimmune MRL/lpr mice. Immunol. Lett., 36, 145–152. Parihar, R., Dierksheide, J., Hu, Y., and Carson, W.E. (2002). IL-12 enhances the natural killer cell cytokine response to Ab-coated tumor cells. J. Clin. Invest., 110, 983–992. Pauly, S., Broll, K., Wittmann, M., Giegerich, G., and Schwarz, H. (2002). CD137 is expressed by follicular dendritic cells and costimulates B lymphocyte activation in germinal centers. J. Leukoc. Biol., 72, 35–42. Paust, S., and Cantor, H. (2005). Regulatory T cells and autoimmune disease. Immunol. Rev., 204, 195–207. Pollok, K.E., Kim, Y.J., Zhou, Z., Hurtado, J., Kim, K.K., Pickard, R.T., and Kwon, B.S. (1993). Inducible T cell antigen 4-1BB. Analysis of expression and function. J. Immunol., 150, 771–781. Qin, Z., Richter, G., Schuler, T., Ibe, S., Cao, X., and Blankenstein, T. (1998). B cells inhibit induction of T cell-dependent tumor immunity. Nat. Med., 4, 627–630. Radvanyi, L.G., Shi, Y., Vaziri, H., Sharma, A., Dhala, R., Mills, G.B., and Miller, R.G. (1996). CD28 costimulation inhibits TCR-induced apoptosis during a primary T cell response. J. Immunol., 156, 1788–1798.

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Renshaw, B.R., Fanslow, W.C., 3rd, Armitage, R.J., Campbell, K.A., Liggitt, D., Wright, B., Davison, B.L., and Maliszewski, C.R. (1994). Humoral immune responses in CD40 ligand-deficient mice. J. Exp. Med., 180, 1889–1900. Salomon, B., and Bluestone, J.A. (2001). Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation. Annu. Rev. Immunol., 19, 225–252. Samoilova, E.B., Horton, J.L., Hilliard, B., Liu, T.S., and Chen, Y. (1998). IL-6-deficient mice are resistant to experimental autoimmune encephalomyelitis: Roles of IL-6 in the activation and differentiation of autoreactive T cells. J. Immunol., 161, 6480–6486. Scholin, A., Siegbahn, A., Lind, L., Berne, C., Sundkvist, G., Bjork, E., and Karlsson, F.A. (2004). CRP and IL-6 concentrations are associated with poor glycemic control despite preserved betacell function during the first year after diagnosis of type 1 diabetes. Diabetes. Metab. Res. Rev., 20, 205–210. Schwarz, H., Valbracht, J., Tuckwell, J., von Kempis, J., and Lotz, M. (1995). ILA, the human 4-1BB homologue, is inducible in lymphoid and other cell lineages. Blood, 85, 1043–1052. Seo, S.K., Choi, J.H., Kim, Y.H., Kang, W.J., Park, H.Y., Suh, J.H., Choi, B.K., Vinay, D.S., and Kwon, B.S. (2004). 4-1BB-mediated immunotherapy of rheumatoid arthritis. Nat. Med., 10, 1088– 1094. Shuford, W.W., Klussman, K., Tritchler, D.D., Loo, D.T., Chalupny, J., Siadak, A.W., Brown, T.J., Emswiler, J., Raecho, H., Larsen, C.P., et al. (1997). 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J. Exp. Med., 186, 47–55. Smyth, M.J., Taniguchi, M., and Street, S.E. (2000). The anti-tumor activity of IL-12: Mechanisms of innate immunity that are model and dose dependent. J. Immunol., 165, 2665–2670. Snapper, C.M., and Mond, J.J. (1993). Towards a comprehensive view of immunoglobulin class switching. Immunol. Today, 14, 15–17. Snapper, C.M., and Mond, J.J. (1996). A model for induction of T cell-independent humoral immunity in response to polysaccharide antigens. J. Immunol., 157, 2229–2233. Steinman, R.M. (1991). The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol., 9, 271–296. Steinman, R.M., Inaba, K., Turley, S., Pierre, P., and Mellman, I. (1999). Antigen capture, processing, and presentation by dendritic cells: Recent cell biological studies. Hum. Immunol., 60, 562–567. Steinman, R.M., and Nussenzweig, M.C. (2002). Avoiding horror autotoxicus: The importance of dendritic cells in peripheral T cell tolerance. Proc. Natl. Acad. Sci. USA, 99, 351–358. Summers, K.L., Hock, B.D., McKenzie, J.L., and Hart, D.N. (2001). Phenotypic characterization of five dendritic cell subsets in human tonsils. Am. J. Pathol., 159, 285–295. Sun, Y., Chen, H.M., Subudhi, S.K., Chen, J., Koka, R., Chen, L., and Fu, Y.X. (2002). Costimulatory molecule-targeted antibody therapy of a spontaneous autoimmune disease. Nat. Med., 8, 1405– 1413. Suzuki, H., Yasukawa, K., Saito, T., Narazaki, M., Hasegawa, A., Taga, T., and Kishimoto, T. (1993). Serum soluble interleukin-6 receptor in MRL/lpr mice is elevated with age and mediates the interleukin-6 signal. Eur. J. Immunol., 23, 1078–1082. Takahashi, C., Mittler, R.S., and Vella, A.T. (1999). Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal. J. Immunol., 162, 5037–5040. Tang, B., Matsuda, T., Akira, S., Nagata, N., Ikehara, S., Hirano, T., and Kishimoto, T. (1991). Ageassociated increase in interleukin 6 in MRL/lpr mice. Int. Immunol., 3, 273–278. Uekusa, Y., Gao, P., Yamaguchi, N., Tomura, M., Mukai, T., Nakajima, C., Iwasaki, M., Takeuchi, N., Tsujimura, T., Nakazawa, M., et al. (2002). A role for endogenous IL-12 in tumor immunity: IL-12 is required for the acquisition of tumor-migratory capacity by T cells and the development of T cell-accepting capacity in tumor masses. J. Leukoc. Biol., 72, 864–873. van Essen, D., Kikutani, H., and Gray, D. (1995). CD40 ligand-transduced co-stimulation of T cells in the development of helper function. Nature, 378, 620–623. Van Parijs, L., Ibraghimov, A., and Abbas, A.K. (1996). The roles of costimulation and Fas in T cell apoptosis and peripheral tolerance. Immunity, 4, 321–328. Viola, A., and Lanzavecchia, A. (1996). T cell activation determined by T cell receptor number and tunable thresholds. Science, 273, 104–106.

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Vozarova, B., Weyer, C., Hanson, K., Tataranni, P.A., Bogardus, C., and Pratley, R.E. (2001). Circulating interleukin-6 in relation to adiposity, insulin action, and insulin secretion. Obes. Res., 9, 414–417. Wagner, U., Pierer, M., Wahle, M., Moritz, F., Kaltenhauser, S., and Hantzschel, H. (2004). Ex vivo homeostatic proliferation of CD4+ T cells in rheumatoid arthritis is dysregulated and driven by membrane-anchored TNFalpha. J. Immunol., 173, 2825–2833. Walunas, T.L., Bakker, C.Y., and Bluestone, J.A. (1996). CTLA-4 ligation blocks CD28-dependent T cell activation. J. Exp. Med., 183, 2541–2550. Waterhouse, P., Penninger, J.M., Timms, E., Wakeham, A., Shahinian, A., Lee, K.P., Thompson, C.B., Griesser, H., and Mak, T.W. (1995). Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science, 270, 985–988. Watts, T.H. (2005). Tnf/Tnfr family members in costimulation of T cell responses. Annu. Rev. Immunol., 23, 23–68. Wei, W.Z., Morris, G.P., and Kong, Y.C. (2004). Anti-tumor immunity and autoimmunity: A balancing act of regulatory T cells. Cancer Immunol. Immunother., 53, 73–78. Wilcox, R.A., Chapoval, A.I., Gorski, K.S., Otsuji, M., Shin, T., Flies, D.B., Tamada, K., Mittler, R.S., Tsuchiya, H., Pardoll, D.M., and Chen, L. (2002). Cutting edge: Expression of functional CD137 receptor by dendritic cells. J. Immunol., 168, 4262–4267. Wofsy, D. (1988). The role of Lyt-2+ T cells in the regulation of autoimmunity in murine lupus. J. Autoimmun., 1, 207–217. Wofsy, D. (1993). Treatment of murine lupus with anti-CD4 monoclonal antibodies. Immunol. Ser., 59, 221–236. Wofsy, D., Chiang, N.Y., Greenspan, J.S., and Ermak, T.H. (1988). Treatment of murine lupus with monoclonal antibody to L3T4. I. Effects on the distribution and function of lymphocyte subsets and on the histopathology of autoimmune disease. J. Autoimmun., 1, 415–431. Wofsy, D., and Seaman, W.E. (1985). Successful treatment of autoimmunity in NZB/NZW F1 mice with monoclonal antibody to L3T4. J. Exp. Med., 161, 378–391. Wofsy, D., and Seaman, W.E. (1987). Reversal of advanced murine lupus in NZB/NZW F1 mice by treatment with monoclonal antibody to L3T4. J. Immunol., 138, 3247–3253. Wolf, H., Muller, Y., Salmen, S., Wilmanns, W., and Jung, G. (1994). Induction of anergy in resting human T lymphocytes by immobilized anti-CD3 antibodies. Eur. J. Immunol., 24, 1410–1417. Xu, J., Foy, T.M., Laman, J.D., Elliott, E.A., Dunn, J.J., Waldschmidt, T.J., Elsemore, J., Noelle, R.J., and Flavell, R.A. (1994). Mice deficient for the CD40 ligand. Immunity, 1, 423–431. Zhou, Z., Kim, S., Hurtado, J., Lee, Z.H., Kim, K.K., Pollok, K.E., and Kwon, B.S. (1995). Characterization of human homologue of 4-1BB and its ligand. Immunol. Lett., 45, 67–73. Zhu, G., Flies, D.B., Tamada, K., Sun, Y., Rodriguez, M., Fu, Y.X., and Chen, L. (2001). Progressive depletion of peripheral B lymphocytes in 4-1BB (CD137) ligand/I-Ealpha)-transgenic mice. J. Immunol., 167, 2671–2676.

6 CD137 in the Regulation of T Cell Response to Antigen Yuwen Zhu and Lieping Chen

CD4+ and CD8+ T cells express high level of CD137 on the cell surface upon activation. A well-documented function of CD137 is its costimulatory effect on both CD4+ and CD8+ T cell growth and differentiation in vitro. In vivo experiments, however, reveal a far more complex effect of CD137 signaling on T cell-mediated immunity. Administration of CD137 agonistic mAb delivers a potent stimulatory signal and leads to CD8 T cell-mediated viral clearance and tumor regression in various models. In sharp contrast, the same agonist mAb is also effective to prevent or even reverse ongoing autoimmune responses, largely mediated through inhibition of CD4+ T cell. In addition to T cells, CD137 is also found on other cells. Therefore, the effect of CD137 should be analyzed in the context of regulator of both innate and adoptive immunity in addition to simply be a T cell costimulator.

1. CD137 in Na¨ıve T Cell Costimulation 1.1. Effects of CD137 Engagement on CD8+ T Cells in vitro There is ample evidence that CD137 signal plays an important role in costimulation of T cell activation in the presence of T cell receptor (TCR) signal in vitro. Cross-linking of CD137 by anti-CD137 antibody resulted in an enhancement of purified splenic T cell proliferation compared to that stimulated by antiCD3 alone (Pollok et al., 1993). Similarly, CD137 engagement by immobilized CD137L fusion protein or CD137L transfectants augmented the T cell proliferation and cytokine production (Goodwin et al., 1993; DeBenedette et al., 1995). A soluble CD137 protein (CD137Fc) could significantly inhibit T cell proliferation and IL-2 production in both a culture of anti-CD3 stimulated splenocytes and primary mixed leukocyte reaction (MLR) (Hurtado et al., 1995). CD137L transfectant could costimulate CD28-deficient T cells, suggesting that CD137L/CD137 interaction represents a costimulatory pathway independent of CD28 (DeBenedette et al., 1997). Similar to CD28, CD137 engagement promotes T cell proliferation and IL-2 Yuwen Zhu and Lieping Chen • Department of Dermatology and Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. 83 CD137 Pathway: Immunology and Diseases. Edited by Lieping Chen, Springer, New York, 2006

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production by enhancing cell division. Furthermore, CD137 is believed to play a more important role in preventing activation induced cell death (AICD) (Lee et al., 2002; Takahashi et al., 1999). CD137 engagement was shown to increase expression of the antiapoptotic genes bcl-x(L), bfl-1 and c-FLIP via CD137-mediated phosphatidylinositol 3-kinase, the AKT/protein kinase B and/or NF-kappa B activation (Starck et al., 2005). Similar to mouse T cells, cross-linking of CD137 costimulates both CD4+ and CD8+ T cells in humans. Both monoclonal antibodies to human CD137 and cells transfected with human CD137 ligand induced a strong proliferative response in mitogen co-stimulated primary T cell response (Alderson et al., 1994). The presence of CD137L on the APC led to increased cell expansion, cytokine production, and the development of cytolytic effector function by human T cells (Bukczynski et al., 2003). CTLA-4 Ig partially blocked CD137L-dependent IL-2 production, suggesting CD28 signaling pathway may partially overlap with that of CD137. On the other hand, when cocultured with tumor cells expressing human CD137L in the presence of CD3 antibody, CD28-T cells freshly isolated from peripheral blood mononuclear cells (PBMC) clearly enhanced their proliferation, effector function as well as the expression of anti-apoptotic protein Bcl-XL , implying a CD28-independent costimulatory role of CD137 in human T cells (Wen et al., 2002). Reduced apoptosis is observed after co-stimulation by CD137 engagement, consistent with the increased levels of Bcl-x (L) in CD137 antibody-treated CD8+ T cells (Laderach et al., 2002). In addition, artificial antigen-presenting cells (APC) co-expressing ligands for CD28 and CD137 synergistically improved T cell proliferation and survival, compared with CD28 alone (Maus et al., 2002). All together, the data in humans suggest that CD137 signal contributes to the human T cell proliferation and effector T cell differentiation, and manipulation of CD137 signaling could be an effective tool to promote immune response in the aged or chronically infected individuals where CD28-T cells accumulate.

1.2. Effects of CD137 Engagement on CD8+ T Cells in vivo Ligation of CD137 on na¨ıve T cells by CD137L or CD137 antibody in the absence of TCR signal does not induce detectable responses (DeBenedette et al., 1997; Pollok et al., 1993). Because na¨ıve T cells do not express detectable CD137 (Cannons et al., 2001; Pollok et al., 1995), it is thus possible that TCR is required for upregulation of CD137 on T cell surface. Studies of CD137 expression in vitro indicate that CD137 is expressed 24 h after activation with peak expression at 48–72 h (Cannons et al., 2001; Pollok et al., 1995). After immunization, however, CD137 expression by CD8+ T cells could be detected from 12 to 24 hours postimmunization and quickly disappear 36 hours after antigen challenge (Dawicki et al., 2004). Therefore, CD137 expression in vivo has a much faster on-and-off rate than in vitro . The reason for this difference is still not clear. A fundamental understanding of the immunological functions of CD137 on T cell in vivo has been greatly improved by the use of knockout or transgenic mice. Consistent with in vitro findings, studies from various mouse models of tumors, virus infection, GVHD, and transplantation have clearly suggested that CD137

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delivers a potent costimulatory signal to CD8+ T cells. The role of CD137 on tumor and viral immunity will be discussed in detail in Chapter 8. Here our discussion will mainly focus on basic immunobiology of T cell responses to antigens. Two initial studies in 1997 demonstrated that engagement of CD137 in vivo by CD137 agonistic mAb enhances CD8 T cell proliferation and IFN-gamma production, therefore promoting acute graft versus host (GVH) disease, accelerating the cardiac and skin allograft rejection and tumor rejection (Melero et al., 1997; Shuford et al., 1997). In consistent with these findings, Cooper et al. showed that in vivo blockade of CD137 by CD137-Fc fusion protein inhibits CD8+ T cell expansion in the primary response to ovalbumin protein in adjuvant (Cooper et al., 2002). When adoptive transferred T cells are CFSE-labeled to trace cell division, administration of CD137-Fc in vivo significantly inhibits T cell division, demonstrating that CD137 functions to regulate CD8+ T cell clonal expansion by enhancing proliferation. In a tumor model, Ito et al. (2004) found that administration of CD137 mAb can enhance antitumor efficiency of dendritic cell-based vaccine. When CFSE-labeled OT-1 cells were adoptively transferred to assess the cell division by measuring CFSE profiles, the combination of anti-CD137 with DC vaccine significantly increased OT-1 T cell division in the draining lymph nodes. All these data suggest that CD137 engagement increases T cell proliferation at least partially through the enhanced cell division. In addition to the proposed direct role of CD137 signaling in the stimulation of T cell proliferation in vivo, several studies also support an effect of CD137 on T cell survival. Administration with superantigen is a popular model to study in vivo clonal expansion and subsequent clonal contraction, which is largely mediated by activation induced cell death (AICD) of peripheral T cells. Injection of anti-CD137 mAb together with SEA only slightly affects T cell expansion in the early phase. Instead, this mAb inhibits the deletion of superantigen-activated T cells, leading to a minimal contraction phase of activated T cells during the experiments. As a result, there are nearly 10-fold the number of Ag-specific CD8 T cells in the anti-CD137-treated mice in comparison to the control mice on day 21 (Takahashi et al., 1999). A similar effect of CD137 antibody on T cell survival was also seen in a peptide vaccination model (Diehl et al., 2002). When a CFSE-labeled transgenic T cell was transferred into normal B6 mice, systemic administration of anti-CD137 mAb together with peptide did not increase T cell division. However, massive accumulation of antigen-specific transgenic T cells was only observed in mice vaccinated with peptide in combination with CD137 mAb, but not in mice that received peptide alone. Analysis of CD137L or CD137 deficient mice also suggests a predominant role for CD137 in effector T cell survival. T cells from CD137L-deficient mice show normal proliferation and cell division after viral infection. The number of antigen-specific T cells is normal at the peak of the immune response against influenza but it was clearly decreased 21 days after infection (Bertram et al., 2002). Similarly, adoptively transferred OVA-specific T cells into CD137L deficient mice also show normal T cell expansion but defects in the maintenance of T cell survival (Dawicki and Watts, 2004). Thus, CD137 signaling may be involved in promoting the development of memory T cells from CD8+ effector T cells.

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1.3. Effects of CD137 on CD4+ T Cells: Positive or Negative? In contrast to stimulatory effects of CD137 on CD8+ T cells in vitro and in vivo, CD137 engagement could have both stimulatory and inhibitory effects on CD4+ T cells, especially in vivo, depending on disease models used or types of immune responses mounted in vivo. Like CD8+ T cells, CD4+ T cells upregulate CD137 upon activation, though there is evidence that CD137 expression level on CD4+ T cells is lower than that on CD8+ T cells (Taraban et al., 2002). In vitro studies show that CD137 delivers a stimulatory signal to purified polyclonal CD4+ T cells as well as antigen-specific CD4 transgenic T cells (Cannons et al., 2001; Chu et al., 1997; Gramaglia et al., 2000). Cross-linking CD137 on CD4+ T cells by CD137L or CD137 agonistic antibody increases cytokine production, proliferation and survival of T cells. As a consequence, CD4+ T cells have increased expression of the anti-apoptotic genes bcl-XL and bcl-2, as well as of cyclins D2 and E, and inhibited expression of the cyclin-dependent kinase (cdk) inhibitor p27kip1 . CD137-deficient DO11.10 TCR transgenic CD4+ T cells have less cell division and are more sensitive to activationinduced cell death (AICD) when cultured with antigen in vitro (Lee et al., 2003). Furthermore, several in vivo studies also support CD137 as a stimulatory coreceptor on CD4+ T cells. CD137 agonistic mAb increases CD4+ T cell-mediated GVHD as well as its effect on CD8+ T cells (Blazar et al., 2001). In an adoptive transfer model, CD4+ transgenic (OT-II) T cells in CD137L deficient mice have minor defects during the primary response but there is a clear decrease of CD4+ T cell recall response in CD137L deficient mice, which has a similar magnitude as CD8+ T cells both in primary and recall response (Dawicki and Watts, 2004). CD4+ T cells from mice constitutively expressing CD137 on T cell showed an increased proliferative capability versus normal T cells. The proliferation and antibody response against KLH (keyhole limpet hemocyanin), a CD4+ T celldependent antigen, were enhanced in the CD137 transgenic mice. And CD137 transgenic mice also exhibited less apoptotic and extensive CD4+ T cell expansion, leading to an elevated contact hypersensitivity response (Kim et al., 2003). Finally, Bansal-Pakala et al., found that in vivo delivery of the CD137 signal by the agonistic antibody can restore T cell response in aged mice, which have decreased immune response due to T cell deficiency. CD137 signals promote aged CD4 T cell response in vitro and rescue the defected T cell response in aged mice (Bansal-Pakala and Croft, 2002). In contrast to these observations, several recent studies show that CD137 agonist mAb might be an inhibitor for the CD4+ T cell-mediated immune response in vivo. An early study by Shuford and colleagues found that administration of agonistic CD137 mAb inhibits CD4+ T-dependent humoral immune responses (Shuford et al., 1997). The inhibitory effect of CD137 agonistic mAb on CD4+ T cell response has been confirmed in several autoimmune disease models by different laboratories (Foell et al., 2003; Seo et al., 2004; Sun et al., 2002a, 2002b). Experimental data to consolidate these findings is still lacking. As cells other than T cells, such as dendritic cells and natural killer cells, also express CD137, it is thus possible that CD137 signal on these cells may contribute to its suppressive effect.

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And there is no report so far showing that natural interaction between CD137 and CD137L could also inhibit the CD4+ T cell-mediated immune response in vivo yet. It is also unknown whether or not CD137L is the only ligand for CD137. Recently Lee and colleagues report that CD4+ T cells response in CD137 deficient mice increase and OT-II transgenic mice deficient of CD137 had enhanced cell division and expansion in a CD137-sufficient environment (Lee et al., 2005). Taken together, these findings suggest that the CD137 signal plays a negative role in CD4+ T cell responses in vivo.

2. CD137 and Regulatory T Cells (Treg) The concept of suppressor T cells exits for decades as a mechanism for the regulation of peripheral tolerance, though the molecular evidence for this phenomenon is largely unknown. During the last ten years, the identification of CD4+ CD25+ cells as a suppressor T cell subtype by Sakaguchi and colleagues have triggered explosive growth of knowledge in this field (Sakaguchi, 2004). In normal mice, approximately 10% of CD4+ T cells express CD25. These cells are anergic when stimulated via their TCR but can proliferate in presence of IL-2. CD4+ CD25+ cells can inhibit a variety of types of immune responses, both in culture and in vivo. The development of CD4+ CD25+ seems to be IL-2-dependent and requires endogenous B7-CD28 interaction, as the absence of IL-2, IL-2R, CD80/CD86, or CD28 results in a dramatic reduction of Treg number in peripheral lymphoid tissues (Bluestone and Abbas, 2003). Besides CD25, Foxp3, a Forkhead family transcriptional factor, is emerging as a promising signature for Treg, as recent studies have shown that the majority of Treg cells express Foxp3 and transfection of CD4+ CD25− T cells with Foxp3 confers regulatory activity (Fontenot et al., 2003; Hori et al., 2003; Khattri et al., 2003). Treg cells also constitutively express high-level of costimulatory molecules cytotoxic T lymphocyte-associated antigen (CTLA)-4, which has been shown to functionally inhibit immune response by initiating tryptophan catabolism in dendritic cells by binding to B7-1 and/or B7-2 (Fallarino et al., 2003). Recently gene array analyses have shown that several tumor necrosis factor (TNF) family members including CD137 were highly expressed in CD4+ CD25+ cells, which was confirmed by surface staining (Gavin et al., 2002; McHugh et al., 2002). The functions of CD137 on Treg was conducted by several independent groups both in vitro and in vivo. Although the results from different labs were sharply different, all data support a role of CD137 in the regulation of Treg cell functions. Choi et al. found that freshly isolated CD4+ CD25+ T cells constitutively express low-level CD137 and its expression upregulated upon activation (Choi et al., 2004). As CD4+ CD25+ Treg from CD137−/− mice suppressed responder T cells to the same extent as wild-type Treg do, CD137 did not appear to be a key player in the contact-dependent suppressor activity of Treg cells. However, signaling through CD137 on CD4+ CD25+ cells could neutralize the suppressive effect of Treg cells, although it did not affect Treg cell proliferation. When CD25− cells from CD137−/− mice were co-cultured with pre-activated CD25+ Treg cells, the

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presence of agonistic CD137 mAb efficiently antagonized the suppressive activity of Treg cells. However, CD137 stimulation could reverse the suppression of na¨ıve CD25+ Treg cells only in the presence of exogenous IL-2. Differential expression level of CD137 on na¨ıve versus activated Treg could be a possible explanation, as enough CD137 signaling is required to disrupt the suppressive effect of Treg cells. It should be noted that CD137 signals on Treg was transient, as Treg cells regained their suppressive ability upon removal of CD137 stimulation. In vivo GVHD experiments also supported that CD137 signals inhibit the suppressive activity of Treg cells: The co-transfering of CD25+ Treg cells together with purified CD25− CD4+ cells into MHC-mismatched, sublethally irradiated recipients significantly delayed death from GVHD. Co-injection of CD137 agonist mAbs neutralized Tregmediated suppression and accelerated death, even when the CD25− T cells were from CD137 deficient mice, supporting a direct role of CD137 signal on Treg. Interestingly, when CD25− T cells from wild type mice co-cultured with CD25+ Treg from CD137− deficient mice, CD137 signaling also abrogated Treg-mediated suppression, implying that CD137 stimulation renders CD25− T cells resistant to regulatory T cell-mediated suppression. Consistent to these findings, Morris et al., also found that CD137 signaling interferes with CD4+ CD25+ Treg-mediated tolerance in an experimental autoimmune thyroiditis (EAT) mouse model (Morris et al., 2003). In vivo depletion of CD25+ cells could abrogate established tolerance, indicating that CD4+ CD25+ Treg cells are essential for the tolerance induction. Administration of CD137 mAb inhibited the tolerance induction and interfered with the established tolerance to EAT. In vitro CD137 signals also inhibited the suppression of mouse thyroglobulinspecific T cell proliferation by CD4+ CD25+ Treg cells. In addition, CD137 stimulation did not increase Treg cells to proliferate. Thus, the authors suggested that signaling through CD137 on the autoreactive T cells directly overcomes suppression by CD4+ CD25+ Treg cells. In sharp contrast, Zheng et al. (2004) found that CD137 signal was a strong costimulator for CD4+ CD25+ cells both in vitro and vivo instead. Purified CD4+ CD25+ Treg cells proliferate actively in the presence of CD137L-Fc (both soluble and cross-linked by an Fc receptor). No detectable IL-2 was produced during Treg proliferation, suggesting that IL-2 did not involve in this process. Furthermore, the expanded Treg cells by CD137 retained their suppressive function. Although there is no straightforward answer for these seemly contradictory observations, it is not a total surprise in the context of previous observation that a CD137 signal can be either suppressive or stimulatory for CD4+ T cells. Further validation for these findings will be needed in the future.

3. CD137 and T Cell Anergy T cell anergy is a tolerant state that the lymphocyte is intrinsically unresponsive following prior exposure to an antigen, but remains alive for an extended period of time (Schwartz, 2003). T cell anergy can be prevented by costimulatory

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signals provided by accessory molecules like CD28 or the interleukin 2 receptor (IL-2R) in vitro. CTLA-4 also plays a role for CD4+ T cell anergy induction, as blockage of CTLA-4 pathway by blocking mAb or using CTLA-4−/− T cells can prevent the T cell anergy induction in vivo (Greenwald et al., 2001; Perez et al., 1997). Recent reports showed that T cell clonal anergy could be prevented by CD40 or OX40 mAb stimulation, highlighting the role of co-signaling molecules in the regulation of T cell anergy (Bansal-Pakala et al., 2001; Diehl et al., 1999). Importantly, the provision of OX40 signaling by agonist mAb could reverse established T cell anergy in vivo. Recent studies from our lab demonstrate an important role of CD137 in the regulation of CD8+ T cell anergy. The effect of CD137 agonist mAb in the prevention and reversal of CD8+ T cell tolerance was evaluated in three independent systems in vivo (Wilcox et al., 2004). In the OT-1 system, soluble chicken ovalbumin (OVA) peptide encoding an H-2Kb-restricted epitope was infused intravenously (i.v.) into the mice carrying OT-1 TCR transgenic T cells. A high dose OVA peptide injection led to express VLA-4 on nearly all OT-1 cells, indicating unexceptional antigen exposure of OT-1 cells in this system. Ten days after peptide injection, while the majority of activated OT-1 cells underwent contraction, a significant number of OT-1 cells persisted and was unresponsive to OVA rechallenge both in vitro and vivo, suggesting induction of OVA-specific T cell anergy. Administration of anti-CD137 mAb together with OVA peptide led to about ten-fold increase of OT-1 cells compared with the mice injected with peptide alone. Furthermore, in contrast to the rapid declining of effector OT-1 cells in control mice, the mice receiving anti-CD137 mAb did not have a clear contraction phase, leading to the persistence of a large number of OT-1 cells at least three weeks. Upon restimulation, the OT-1 cells from CD137 mAb treated group proliferated vigorously and produced large amount of IL-2. The level of proliferation and IL-2 secreting was significantly higher than na¨ıve OT-1 cells from PBS treated group, indicating that CD137 stimulation prevents anergy induction and therefore promotes memory T cell development. More importantly, CD137 mAb together with antigen also reverse established T cell anergy. When CD137 mAb together with OVA peptide was administered into mice containing pre-established anergic OT-1 cells 10 days earlier, a massive expansion of OT-1 cells was induced. These proliferating OT-1 cells expressed activation markers and regained antigen-specific cytotoxicity. Infusion of P1A peptide, which encodes a CD8+ T cell epitope for a dominant tumor antigen of P815 mastocytoma, could induce T cell tolerance and lead to growth of a regressive P815 clone (P815R) in syngeneic DBA/2 mice. The provision of anti-CD137 mAb together with P1A peptide prevented tumor growth, suggesting that CD137 stimulation could prevent the P1A peptide from induction of tolerance. Furthermore, even 13 days after peptide infusion, when tumor tolerance was already established, the injection of anti-CD137 mAb could still lead to tumor regression. While these data strongly support a potential role of CD137 signaling in reversal of T cell anergy, the possibility that CD137 stimulation increases anti-tumor response by broadening T cell recognition against subdominant epitopes cannot be excluded.

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In a bone marrow chimera model, 2C transgenic T cell, which recognizes H-2Ld alloantigen, was cotransferred with bone marrow into lethally irradiated BDF1 mice. The persistence of host antigens could lead to completely anergy of 2C. As a result, the persistent 2C T cells were unresponsive to stimulation by TCR cross-linking and could not lyse any transferred CFSE-labeled target cells. However, co-infusion of anti-CD137 mAb reversed the anergy state of 2C T cell, leading to the increased number of 2C T cell in the host, together with a marked reduction of transferred target cells. Additional evidence from other labs also supports the role of CD137 signal in the regulation of T cell anergy. In vivo triggering of CD137 by mAb could prevent the induction of peptide-specific tolerance, and result in the priming of a potent cytotoxic T lymphocytes (CTL) response instead. Furthermore, injection of anti-CD137 mAb could work as effectively as anti-CD40 mAb, replacing the need for CD4+ T cell help in the cross-priming of tumor-specific CTL immunity (Diehl et al., 2002). Similar to CD8+ T cells, peptide-induced CD4+ T cell tolerance could also be prevented by CD137 engagement during priming (Bansal-Pakala and Croft, 2002). In summary, these studies established a new function of CD137 signaling in preventing and/or breaking CD8+ T cell anergy. The effect is antigen-specific since at least breaking anergy requires co-administration of antigen. As tumor growth or chronic viral infection often induce T cell anergy, these findings provide a new approach for therapies.

4. CD137 and Memory T Cell (Tm) Response The generation and maintenance of immunological memory is the ultimate goal of vaccination. Upon exposure to antigen, antigen-specific T cells proliferate enormously and develop into effector T cells capable of immediate effector functions. Following a successful immune response, the effector T cell population undergoes significant contraction. Only small fraction (about 5–10%) of antigenspecific T-cell population survives and is maintained indefinitely. In comparison with extensive knowledge on the role of co-signal molecules in priming of immune responses, molecular mechanisms in the control and the development of memory T cells remain less clear. It has been shown that recall of memory T cells is less dependent or even independent of on CD28 costimulation (Byrne et al., 1988; Croft et al., 1994; London et al., 2000). However, recent emerging data indicate that several other co-signal interactions individually could may function in the secondary recall responses of Tm. For example, the secondary challenge exposure to antigen in CD27-deficient, 4-1BBCD137L-deficient, and CD30L-deficient mice led to significant inhibition at the peak of the recall response (Croft, 2003). In a model of collagen-induced arthritis, administration of an antagonist antibody to OX40L markedly suppresses arthritic symptoms when given at the time Tm are reactivated. Similarly, in experimental allergic lung inflammation, preventing OX40 signals at the challenge phase of asthmatic reactions markedly inhibits the recall response. Interestingly, OX40 does not regulate the initial division of memory T cells but

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controls the accumulation of high numbers of effector memory T cells (SalekArdakani et al., 2003). These data raise support to the possibility that signals from the co-signal molecules might be crucial for recall of Tm when re-exposure to antigens, although this has not yet been investigated. The importance of CD137 pathway in memory T cell response first comes from the studies of viral infection on CD137L deficient mice. Early data showed the response to lymphocytic choriomeningitis virus (LCMV) in CD137L−/− mice is relatively unimpaired, with no defect in the primary and secondary T cell response (DeBenedette et al., 1999). Another study also found that CD137L−/− mice generated effective primary anti-viral CD8+ T cell responses and were able to eliminate the infection. But quantization of these responses showed that CD137L−/− mice generated two- to three-fold fewer numbers of LCMV-specific cells compared with CD137L+/+ mice (Tan et al., 1999). In the absence of CD137L, CTL generation was reduced ten-fold and the number of antigen-specific CD8+ T cells generated was three- to ten-fold fewer than that in CD137L+/+ mice. Importantly, memory CD8+ T cell responses were also lower than those in CD137L+/+ mice. A high percentage of Ag-specific cells were generated after LCMV challenge of immunized CD137L+/+ mice, whereas immunized CD137L−/− mice generated lower percentages of Ag-specific cells and were impaired in their ability to eliminate the infection (Tan et al., 2000). In the case of influenza virus infection, there was no difference in the contraction of the CD8+ T cell response in the spleen between days 7 and 14. However, CD137L−/− mice showed decreased virus-specific CD8+ T cell number late in the primary response (days 21–38) as well as a defect in the secondary to influenza (Bertram et al., 2002). Interestingly, a single dose of anti-CD137 antibody delivered during priming fully corrected the defect in secondary CD8+ T cell response in CD28 deficient mice, suggesting that CD28 is not required for the recall of memory T cells. However, the same treatment failed to correct the defect in CD137L−/− mice; Instead, CD137L added during rechallenge resulted in greatly enhanced recall response (Bertram et al., 2004). Collectively, these studies suggest that CD137 signaling might have a significant impact late in the primary response or the secondary response. Studies from human T cells also suggest a potential role of CD137 in memory T cell development from effector CTLs. CD8+ CD28− cell in human usually represents functionally active CTL population. This subset of CD8 T cell expresses high levels of intracellular perforin and granzyme B. The massive increase in CD28− effector cells is often found during an acute antiviral immune response, such as HIV-1 infection and Epstein–Barr virus (EBV)-induced infectious mononucleosis (EBVIM) (Appay et al., 2002; Callan et al., 1998) . Similar findings have been reported in acute GVHD, in patients with common variable immunodeficiency (Garin et al., 1996; Werwitzke et al., 2000). The CD28− effector cell is sensitive to AICD and only a small population could survive as memory cell (Callan et al., 2000). It is still unclear, however, how the differentiation process is regulated. Interestingly, CD137 was preferentially induced in CD28− CTLs when CD8+ T cells from cord blood were stimulated with cytokines (Kim et al., 2002). Costimulation by anti-CD137 promoted survival and cytotoxic effect of those subset

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of T cells, leading to appearance of memory-like phenotypes, including restoration of CD28 expression and surface staining of CD45RO and CC chemokine receptor 6 (CCR6). Taken together, CD137 pathway involves in different stages of memory T cell development. CD137 signal might protect effector T cells from contraction, as CD137L deficient mice infected with influenza show no defect during T cell expansion but contraction phase. Human studies also showed that CD137 engagement prevents CD28− effector cell from apoptosis and promotes the differentiation to CD28+ post-effector memory T cell. However, recent studies suggest that early events during T cell priming dictate the subsequent contraction (Badovinac et al., 2002, 2004). It is still possible that CD137 signal during priming set the program to control effector T cell contraction. As the expression of CD137 or CD137L could be enhanced by various stimulations, it is interesting to investigate the potential role of CD137 pathway on pre-existing memory T cells response during a heterologous T-cell immunity.

5. Conclusions and Perspectives Here we assemble experimental data on the effect of CD137 on T cell responses. Besides as a potent costimulator for na¨ıve T cell, the CD137 pathway plays an important role on regulation of Treg cell function, memory T cell development, and prevention of T cell tolerance. A key issue to be addressed is that the same agonistic CD137 antibody promotes or inhibits CD4+ T cell potent antitumor response whereas inhibits humoral response. Eradication of this mechanism may help delineate complicated function of CD137 signal in the regulation of T cell development; priming, contraction, maintenance, and memory.

References Alderson, M.R., Smith, C.A., Tough, T.W., Davis-Smith, T., Armitage, R.J., Falk, B., Roux, E., Baker, E., Sutherland, G.R., and Din, W.S. (1994). Molecular and biological characterization of human 4-1BB and its ligand. Eur. J. Immunol., 24(9), 2219–2227. Appay, V., Dunbar, P.R., Callan, M., Klenerman, P., Gillespie, G.M., Papagno, L., Ogg, G.S., King, A., Lechner, F., Spina, C.A., et al. (2002). Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat. Med., 8, 379–385. Badovinac, V.P., Porter, B.B., and Harty, J.T. (2002). Programmed contraction of CD8(+) T cells after infection. Nat. Immunol., 3(7), 619–626. Badovinac, V.P., Porter, B.B., and Harty, J.T. (2004). CD8+ T cell contraction is controlled by early inflammation. Nat. Immunol., 5(8), 809–817. Bansal-Pakala, P., Jember, A.G., and Croft, M. (2001). Signaling through OX40 (CD134) breaks peripheral T-cell tolerance. Nat. Med., 7(8), 907–912. Bansal-Pakala, P., and Croft, M. (2002). Defective T cell priming associated with aging can be rescued by signaling through 4-1BB (CD137). J. Immunol., 169(9), 5005–5009. Bertram, E.M., Lau, P., and Watts, T.H. (2002). Temporal segregation of 4-1BB versus CD28-mediated costimulation: 4-1BB ligand influences T cell numbers late in the primary response and regulates the size of the T cell memory response following influenza infection. J. Immunol., 168(8), 3777– 3785.

CD137 in the Regulation of T Cell Response to Antigen

93

Bertram, E.M., Dawicki, W., Sedgmen, B., Bramson, J.L., Lynch, D.H., and Watts, T.H. (2004). A switch in costimulation from CD28 to 4-1BB during primary versus secondary CD8 T cell response to influenza in vivo. J. Immunol., 172(2), 981–988. Blazar, B.R., Kwon, B.S., Panoskaltsis-Mortari, A., Kwak, K.B., Peschon, J.J., and Taylor, P.A. (2001). Ligation of 4-1BB (CDw137) regulates graft-versus-host disease, graft-versus-leukemia, and graft rejection in allogeneic bone marrow transplant recipients. J. Immunol., 166(5), 3174– 3183. Bluestone, J.A., and Abbas, A.K. (2003). Natural versus adaptive regulatory T cells. Nat. Rev. Immunol., 3(3), 253–257. Bukczynski, J., Wen, T., and Watts, T.H. (2003). Costimulation of human CD28– T cells by 4-1BB ligand. Eur. J. Immunol., 33(2), 446–454. Byrne, J.A., Butler, J.L., and Cooper, M.D. (1988). Differential activation requirements for virgin and memory T cells. J. Immunol., 141, 3249–3257. Callan, M.F., Tan, L., Annels, N., Ogg, G.S., Wilson, J.D., O’Callaghan, C.A., Steven, N., McMichael, A.J., and Rickinson, A.B. (1998). Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus in vivo. J. Exp. Med., 187, 1395–1402. Callan, M.F., Fazou, C., Yang, H., Rostron, T., Poon, K., Hatton, C., and McMichael, A.J. (2000). CD8+ T-cell selection, function, and death in the primary immune response in vivo. J. Clin. Invest., 106(10), 1251–1261. Cannons, J.L., Lau, P., Ghumman, B., DeBenedette, M.A., Yagita, H., et al. (2001). 4-1BBL induces cell division, sustains survival and enhances effector function of CD4 and CD8 T cells with similar efficacy. J. Immunol., 167, 1313–1324. Choi, B.K., Bae, J.S., Choi, E.M., Kang, W.J., Sakaguchi, S., Vinay, D.S., and Kwon, B.S. (2004). 41BB-dependent inhibition of immunosuppression by activated CD4+CD25+ T cells. J. Leukoc. Biol., 75(5), 785–791. Chu, N.R., DeBenedette, M.A., Stiernholm, B.J., Barber, B.H., and Watts, T.H. (1997). Role of IL-12 and 4-1BB ligand in cytokine production by CD28+ and CD28– T cells. J. Immunol., 158(7), 3081–3089. Cooper, D., Bansal-Pakala, P., and Croft, M. (2002). 4-1BB (CD137) controls the clonal expansion and survival of CD8 T cells in vivo but does not contribute to the development of cytotoxicity. Eur. J. Immunol., 32(2), 521–529. Croft, M., Bradley, L.M., and Swain, S.L. (1994). Na¨ıve versus memory CD4 T cell response to antigen. Memory cells are less dependent on accessory cell costimulation and can respond to many antigen-presenting cell types including resting B cells. J. Immunol., 152, 2675–2685. Croft, M. (2003). Co-stimulatory members of the TNFR family: Keys to effective T-cell immunity? Nat. Rev. Immunol., 3(8), 609–620. Dawicki, W., and Watts, T.H. (2004). Expression and function of 4-1BB during CD4 versus CD8 T cell responses in vivo. Eur. J. Immunol., 34(3), 743–751. Dawicki, W., Bertram, E.M., Sharpe, A.H., and Watts, T.H. (2004). 4-1BB and OX40 act independently to facilitate robust CD8 and CD4 recall responses. J. Immunol., 173(10), 5944–5951. DeBenedette, M.A., Chu, N.R., Pollok, K.E., Hurtado, J., Wade W.F., Kwon, B.S., and Watts, T.H. (1995). Role of 4-1BB ligand in costimulation of T lymphocyte growth and its upregulation on M12 B lymphomas by cAMP. J. Exp. Med., 181(3), 985–992. DeBenedette, M.A., Shahinian, A., Mak, T.W., and Watts, T.H. (1997). Costimulation of CD28- T lymphocytes by 4-1BB ligand. J. Immunol., 158(2), 551–559. DeBenedette, M.A., Wen, T., Bachmann, M.F., Ohashi, P.S., Barber, B.H., Stocking, K.L., Peschon, J.J., and Watts, T.H. (1999). Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to influenza virus. J. Immunol., 163(9), 4833–4841. Diehl, L., den Boer, A.T., Schoenberger, S.P., van der Voort, E.I., Schumacher, T.N., Melief, C.J., Offringa, R., and Toes, R.E. (1999). CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy. Nat. Med., 5(7), 774– 779. Diehl, L., van Mierlo, G.J., den Boer, A.T., van der Voort, E., Fransen, M., van Bostelen, L., Krimpenfort, P., Melief, C.J., Mittler, R., Toes, R.E., and Offringa, R. (2002). In vivo triggering through 4-1BB

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enables Th-independent priming of CTL in the presence of an intact CD28 costimulatory pathway. J. Immunol., 168(8), 3755–3762. Fallarino, F., Grohmann, U., Hwang, K.W., Orabona, C., Vacca, C., Bianchi, R., Belladonna, M.L., Fioretti, M.C., Alegre, M.L., and Puccetti, P. (2003). Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol., 4(12), 1206–1212. Foell, J., Strahotin, S., O’Neil, S.P., McCausland, M.M., Suwyn, C., Haber, M., Chander, P.N., Bapat, A.S., Yan, X.J., Chiorazzi, N., Hoffmann, M.K., and Mittler, R.S. (2003). CD137 costimulatory T cell receptor engagement reverses acute disease in lupus-prone NZB × NZW F1 mice. J. Clin. Invest., 111(10), 1505–1518. Fontenot, J.D., Gavin, M.A., and Rudensky, A.Y. (2003). Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol., 4(4), 330–336. Garin, L., Rigal, D., Souillet, G., Bernaud, J., Merieux, Y., and Philippe, N. (1996). Strong increase in the percentage of the CD8bright+ CD28– T-cells and delayed engraftment associated with cyclosporine-induced autologous GVHD. Eur. J. Haematol., 56(3), 119–123. Gavin, M.A., Clarke, S.R., Negrou, E., Gallegos, A., and Rudensky, A. (2002). Homeostasis and anergy of CD4+CD25+ suppressor T cells in vivo. Nat. Immunol., 3(1), 33–41. Goodwin, R.G., Din, W.S., Davis-Smith, T., Anderson, D.M., Gimpel, S.D., Sato, T.A., Maliszewski, C.R., Brannan, C.I., Copeland, N.G., Jenkins, N.A., et al. (1993). Molecular cloning of a ligand for the inducible T cell gene 4-1BB: A member of an emerging family of cytokines with homology to tumor necrosis factor. Eur. J. Immunol., 23(10), 2631–2641. Gramaglia, I., Cooper, D., Miner, K.T., Kwon, B.S., and Croft, M. (2000). Co-stimulation of antigenspecific CD4 T cells by 4-1BB ligand. Eur. J. Immunol., 30, 392–402. Greenwald, R.J., Boussiotis, V.A., Lorsbach, R.B., Abbas, A.K., and Sharpe, A.H. (2001). CTLA-4 regulates induction of anergy in vivo. Immunity, 14(2), 145–155. Hori, S., Nomura, T., and Sakaguchi, S. (2003). Control of regulatory T cell development by the transcription factor Foxp3. Science, 299(5609), 1057–1061. Hurtado, J.C., Kim, S.H., Pollok, K.E., Lee, Z.H., and Kwon, B.S. (1995). Potential role of 4-1BB in T cell activation. Comparison with the costimulatory molecule CD28. J. Immunol., 155(7), 3360–3367. Ito, F., Li, Q., Shreiner, A.B., Okuyama, R., Jure-Kunkel, M.N., Teitz-Tennenbaum, S., and Chang, A.E. (2004). Anti-CD137 monoclonal antibody administration augments the antitumor efficacy of dendritic cell-based vaccines. Cancer Res., 64(22), 8411–8419. Khattri, R., Cox, T., Yasayko, S.A., and Ramsdell, F. (2003). An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol., 4(4), 337–342. Kim, Y.J., Brutkiewicz, R.R., and Broxmeyer, H.E. (2002). Role of 4-1BB (CD137) in the functional activation of cord blood CD28(−)CD8(+) T cells. Blood, 100(9), 3253–3260. Kim, J., Choi, S.P., La, S., Seo, J.S., Kim, K.K., Nam, S.H., and Kwon, B. (2003). Constitutive expression of 4-1BB on T cells enhances CD4+ T cell responses. Exp. Mol. Med., 35(6), 509–517. Laderach, D., Movassagh, M., Johnson, A., Mittler, R.S., and Galy, A. (2002). 4-1BB co-stimulation enhances human CD8(+) T cell priming by augmenting the proliferation and survival of effector CD8(+) T cells. Int. Immunol., 14(10), 1155–1167. Lee, H.W., Park, S.J., Choi, B.K., Kim, H.H., Nam, K.O., and Kwon, B.S. (2002). 4-1BB promotes the survival of CD8+ T lymphocytes by increasing expression of Bcl-xL and Bfl-1. J. Immunol., 169, 4882–4888. Lee, H.W., Nam, K.O., Seo, S.K., Kim, Y.H., Kang, H., and Kwon, B.S. (2003). 4-1BB cross-linking enhances the survival and cell cycle progression of CD4 T lymphocytes. Cell. Immunol., 223(2), 143–150. Lee, S.W., Vella, A.T., Kwon, B.S., and Croft, M. (2005). Enhanced CD4 T cell responsiveness in the absence of 4-1BB. J. Immunol., 174(11), 6803–6808. London, C.A., Lodge, M.P., and Abbas, A.K. (2000). Functional responses and costimulator dependence of memory CD4+ T cells. J. Immunol., 164, 265–272. Maus, M.V., Thomas, A.K., Leonard, D.G., Allman, D., Addya, K., Schlienger, K., Riley, J.L., and June, C.H. (2002). Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB. Nat. Biotechnol., 20(2), 143–148.

CD137 in the Regulation of T Cell Response to Antigen

95

McHugh, R.S., Whitters, M.J., Piccirillo, C.A., Young, D.A., Shevach, E.M., Collins, M., and Byrne. M.C. (2002). CD4+CD25+ immunoregulatory T cells: Gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity, 16, 311–323. Melero, I., Shuford, W.W., Newby, S.A., Aruffo, A., Ledbetter, J.A., Hellstrom, K.E., Mittler, R.S., and Chen, L. (1997). Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat. Med., 3(6), 682–685. Morris, G.P., Chen, L., and Kong, Y.C. (2003). CD137 signaling interferes with activation and function of CD4+CD25+ regulatory T cells in induced tolerance to experimental autoimmune thyroiditis. Cell. Immunol., 226(1), 20–29. Perez, V.L., Van Parijs, L., Biuckians, A., Zheng, X.X., Strom, T.B., and Abbas, A.K. (1997). Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity., 6(4), 411– 417. Pollok, K.E., Kim, Y.J., Zhou, Z., Hurtado, J., Kim, K.K., Pickard, R.T., and Kwon, B.S. (1993). Inducible T cell antigen 4-1BB. Analysis of expression and function. J. Immunol., 150(3), 771– 781. Pollok, K.E., Kim, S.H., and Kwon, B.S. (1995). Regulation of 4-1BB expression by cell-cell interactions and the cytokines, interleukin-2 and interleukin-4. Eur. J. Immunol., 25(2), 488– 494. Sakaguchi, S. (2004). Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol., 22, 531–562. Salek-Ardakani, S., Song, J., Halteman, B.S., Jember, A.G., Akiba, H., Yagita, H., and Croft, M. (2003). OX40 (CD134) controls memory T helper 2 cells that drive lung inflammation. J. Exp. Med., 198(2), 315–324. Schwartz, R.H. (2003). T cell anergy. Annu. Rev. Immunol., 21, 305–334. Seo, S.K., Choi, J.H., Kim, Y.H., Kang, W.J., Park, H.Y., Suh, J.H., Choi, B.K., Vinay, D.S., and Kwon, B.S. (2004). 4-1BB-mediated immunotherapy of rheumatoid arthritis. Nat. Med., 10(10), 1088–1094. Shuford, W.W., Klussman, K., Tritchler, D.D., Loo, D.T., Chalupny, J., Siadak, A.W., Brown, T.J., Emswiler, J., Raecho, H., Larsen, C.P., Pearson, T.C., Ledbetter, J.A., Aruffo, A., and Mittler, R.S. (1997). 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J. Exp. Med., 186(1), 47–55. Starck, L., Scholz, C., Dorken, B., and Daniel, P.T. (2005). Costimulation by CD137/4-1BB inhibits T cell apoptosis and induces Bcl-xL and c-FLIP(short) via phosphatidylinositol 3-kinase and AKT/protein kinase B. Eur. J. Immunol., 35(4), 1257–1266. Sun, Y., Lin, X., Chen, H.M., Wu, Q., Subudhi, S.K., Chen, L., and Fu, Y.X. (2002a). Administration of agonistic anti-4-1BB monoclonal antibody leads to the amelioration of experimental autoimmune encephalomyelitis. J. Immunol., 168(3), 1457–1465. Sun, Y., Chen, H.M., Subudhi, S.K., Chen, J., Koka, R., Chen, L., and Fu, Y.X. (2002b). Costimulatory molecule-targeted antibody therapy of a spontaneous autoimmune disease. Nat. Med., 8(12), 1405–1413. Takahashi, C., Mittler, R.S., and Vella, A.T. (1999). Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal. J. Immunol., 162(9), 5037–5040. Tan, J.T., Whitmire, J.K., Ahmed, R., Pearson, T.C., and Larsen, C.P. (1999). 4-1BB ligand, a member of the TNF family, is important for the generation of antiviral CD8 T cell responses. J. Immunol., 163(9), 4859–4868. Tan, J.T., Whitmire, J.K., Murali-Krishna, K., Ahmed, R., Altman, J.D., Mittler, R.S., Sette, A., Pearson, T.C., and Larsen, C.P. (2000). 4-1BB costimulation is required for protective anti-viral immunity after peptide vaccination. J. Immunol., 164(5), 2320–2325. Taraban, V.Y., Rowley, T.F., O’Brien, L., Chan, H.T., Haswell, L.E., Green, M.H., Tutt, A.L., Glennie, M.J., and Al-Shamkhani, A. (2002). Expression and costimulatory effects of the TNF receptor superfamily members CD134 (OX40) and CD137 (4-1BB), and their role in the generation of anti-tumor immune responses. Eur. J. Immunol., 32, 3617–3627. Wen, T., Bukczynski, J., and Watts, T.H. (2002). 4-1BB ligand-mediated costimulation of human T cells induces CD4 and CD8 T cell expansion, cytokine production, and the development of cytolytic effector function. J. Immunol., 168(10), 4897–4906.

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Werwitzke, S., Drescher, B., Schmidt, R.E., and Witte, T. (2000). CD8+ T cell populations in common variable immunodeficiency. Immunobiology., 202(2), 168–171. Wilcox, R.A., Tamada, K., Flies, D.B., Zhu, G., Chapoval, A.I., Blazar, B.R., Kast, W.M., and Chen, L. (2004). Ligation of CD137 receptor prevents and reverses established anergy of CD8+ cytolytic T lymphocytes in vivo. Blood., 103(1), 177–184. Zheng, G., Wang, B., and Chen, A. (2004). The 4-1BB costimulation augments the proliferation of CD4+CD25+ regulatory T cells. J. Immunol., 173(4), 2428–2434.

7 Autoimmune Diseases Yonglian Sun and Yang-Xin Fu

1. Introduction CD137 (4-1BB) is an inducible T cell co-stimulatory receptor and belongs to tumor necrosis factor receptor superfamily (TNFRSF9). In addition to its expression on activated T cells (Kwon and Weissman, 1989; Pollok et al., 1993), it is also expressed on other lymphocytes, including activated NK cells (Melero et al., 1998), NK T cells (Kwon et al., 2000) and CD4+ CD25+ regulatory T cells (Gavin et al., 2002; McHugh et al., 2002), as well as myeloid cells, such as monocytes, neutrophils, and dendritic cells (Futagawa et al., 2002; Heinisch et al., 2000; Kwon et al., 1997; Wilcox et al., 2002a). CD137L (4-1BBL), a member of TNF superfamily (TNFSF9), has been detected on professional antigen presenting cells (APCs) such as B cells, macrophages, and dendritic cells (Alderson et al., 1994; Goodwin et al., 1993; Pollok et al., 1994). This type of expression pattern indicates CD137/CD137L interactions may play a role in multiple steps in various innate and adaptive immune responses. It has been shown that CD137 engagement with either CD137L or agonistic monoclonal antibodies (mAbs) against CD137 together with TCR signaling results in increased T cell proliferation, cytokine production and prolonged CD8+ T cell survival (Hurtado et al., 1997; Pollok et al., 1993; Takahashi et al., 1999). In addition, experiments performed in both CD137 and CD137L-deficient mice suggest CD137 costimulation may play an important role in T cell-mediated immune responses (Blazar et al., 2001; DeBenedette et al., 1999; Tan et al., 1999, 2000). In accordance with its costimulatory function, agonist mAbs against CD137 have been shown to promote T cell cytolytic activity leading to increased allograft rejection (Shuford et al., 1997) and tumor eradication (Melero et al., 1997), broaden CD8+ T cell responses in viral immunity (Halstead et al., 2002). CD137/CD137L interaction deficit prevents the development of autoimmune disease in some animal models (Seo et al., 2003, 2004). These studies support the notion that CD137 signaling increases T cell function which may enhance immunity against tumors and infection, and CD137/CD137 pathway may participate in the pathogenesis of autoimmune disease.

Yonglian Sun and Yang-Xin Fu • The Department of Pathology and Committee in Immunology, The University of Chicago, Chicago, Illinois, USA. 97 CD137 Pathway: Immunology and Diseases. Edited by Lieping Chen, Springer, New York, 2006

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However, recent studies indicate a diametric outcome of CD137 signaling. Agonistic mAbs against CD137 that promoted tumor rejection in mice were also able to suppress T-dependent humoral immunity (Mittler et al., 1999) and ameliorate multiple organ-specific and systemic autoimmune diseases (Foell et al., 2003, 2004; Seo et al., 2004; Shao et al., 2005; Sun et al., 2002a, 2002b). In support of the possible regulatory role of CD137 signaling in T cell response, it has been shown that CD137-deficient T cells are hyperresponsive to mitogens compared with WT T cells (Kwon et al., 2002). These studies strongly suggest that CD137 signaling is capable of modulating immune responses differentially in vivo. Here, we review recent studies on endogenous and exogenous CD137 signal in the regulation of T cell-mediated autoimmune responses especially the application of agonistic mAbs against CD137 in different autoimmune disease models and the mechanisms involved.

2. Role of CD137/CD137L Interaction in the Pathogenesis of Autoimmune Diseases CD137/CD137L pathway is involved in the generation of a fully competent T cell response (Blazar et al., 2001; DeBenedette et al., 1999; Tan et al., 1999, 2000). Absence of CD137/CD137L interactions prevent the development of certain autoimmune diseases (Seo et al., 2003, 2004). Elevated serum levels of soluble forms of receptor and ligand were detected in patients with various autoimmune diseases (Jung et al., 2004; Michel et al., 1998; Sharief, 2002). These studies suggest CD137/CD137L interactions may play a role in the pathogenesis of autoimmune disease.

2.1. Lack of CD137/CD137L Interaction Prevents Autoimmune Diseases The studies performed by Seo et al. (2003) showed that interference in the interactions between CD137 and CD137L abolished the development of Herpetic stromal keratitis (HSK) in a murine model. HSK is an inflammatory disorder characterized by TH 1-mediated destruction of corneal tissues and is induced by HSV-1 infection (Deshpande et al., 2001; Russell et al., 1984). When the corneas of CD137−/− and CD137+/+ littermates on BALB/c background were infected with 1 × 105 PFU of HSV-1 RE strain, the incidence and severity of disease were significantly reduced in CD137−/− compared with CD137+/+ littermates. Leukocyte infiltration in corneas of the CD137−/− mice was absent as well. Similarly, when CD137/CD137L interactions were blocked with multiple doses of anti-CD137L mAb (TKS-1) (Futagawa et al., 2002), the disease development in CD137+/+ mice was also prevented. Herpes-infected CD137+/+ corneas manifested high levels of chemokines and cytokines which are characteristics of activated T cells and monocytes and implicated in HSK pathogenesis. In CD137+/+ mice, majority infiltrating CD3+ T cells in cornea expressed CD137 as well as other T cell activation markers such as CD44, CD25, and CD62L on their surfaces, indicating the possible requirement of CD137 expression for the induction of HSK.

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The authors proposed that the CD137 signaling may play a potential role on regulating CD62L (L-selectin) expression. L-Selectin is broadly expressed on most leukocytes and is required for na¨ıve lymphocyte homing to secondary lymphoid organs with high endothelial venules (HEV). In addition, it is involved in inflammatory leukocyte trafficking (Rosen, 2004). The lack of CD137/CD137L interactions may result in decreased expression of CD62L on certain T cells, which leads to reduced recruitment of effector T cells into peripheral tissue so that fewer T cells are available in cornea to stimulate inflammation and initiate HSK development. This is based on the facts that CD62L expression on draining lymph node (DLN) CD4+ T cell was significantly less in CD137−/− compared with CD137+/+ mice eight days post infection, a time point when T cells were beginning to infiltrate the corneas, but not at the earlier time points. And CD4+ T cells isolated from DLNs of CD137−/− mice eight days instead of three days PI, manifested diminished migratory potential. These studies indicate the CD137/CD137L interactions play a critical role in the pathogenesis of HSK through either facilitating the recruitment of pathogenic T cell from the draining lymph nodes to the corneal stroma or promoting the inflammatory responses in local tissue. CD137 signaling targeted immunotherapy may open a new window for treatment of human HSK. The same group (Seo et al., 2004) also tested the effect of CD137/CD137L pathway blockade on the development of collagen-induced arthritis (CIA), an experimental mouse model for the study of human rheumatoid arthritis (RA), which is a chronic and debilitating inflammatory autoimmune disease of joint (Feldmann et al., 1996). Type-II collagen (CII) is recognized as a prominent target of autoimmune destruction in RA and is the arthritogenic antigen in CIA (Luross and Williams, 2001; Myers et al., 1997). CIA is induced in genetically predisposed mice such as DBA/1 mice by immunization with bovine collagen II (bCII) emulsified in CFA. CD4+ T cells seem to play a central role in disease induction in arthritis (Ranges et al., 1985). Seo et al. (2004) found that administration of anti-CD137L (TKS-1) resulted in moderately ameliorated arthritis development than that seen with control IgG, and partially inhibited CII-specific CD4+ T cell recall response in CIA model. Such treatment reduced mRNA levels of inflammatory cytokine IL-6 but not IL-1β and TNF. Compared with agonistic anti-CD137 treatment which we are going to discuss later in this chapter, CD137/CD137L pathway blockade with anti-CD137L showed much milder suppressive effect on CIA. These studies indicate CD137/CD137L interaction may be involved in the activation of arthritogenic T cells.

2.2. Soluble CD137 and Autoimmune Diseases CD137 has been detected in both membrane-bound (mCD137) and soluble forms (sCD137). Expression level of soluble forms of CD137 does not always correlate with that of mCD137, since they are generated by alternative splicing (Setareh et al., 1995) instead of a proteolytic product of mCD137. Two splice variants of sCD137 has been identified (Michel et al., 1998). Expression of both membrane-bound and soluble isoforms is activation-dependent (Schwarz et al.,

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1996), while the production of sCD137 seems to be restricted to activated lymphocytes (Michel et al., 1998). The elevated levels of sCD137 have been observed in sera of patients with multiple autoimmune disorders such as rheumatoid arthritis (Jung et al., 2004; Michel et al., 1998), multiple sclerosis (Sharief, 2002), systemic lupus erythematosus (SLE) and Behcet’s disease (BD) (Jung et al., 2004). Michel et al. (1998) first observed dramatic increased levels of sCD137 in sera of patients with rheumatoid arthritis. Later studies (Jung et al., 2004) showed that both sCD137 and sCD137L serum levels were significantly higher in RA patients compared with healthy controls, and their concentration correlated with rheumatoid factor (RF) values and the disease severity. Moreover, the levels were markedly decreased at the quiescent stage following immunosuppressive therapy, compared with patients at disease onset (Jung et al., 2004). Sharief observed significantly higher levels of sCD137 in serum and the intrathecal compartment of patients with clinically active multiple sclerosis (MS), which is a demyelinating disease of the central nervous system (CNS), when compared with patients with clinically stable MS or healthy individuals (Sharief, 2002). It indicates that eminent release of sCD137 is related to clinically active MS. Substantially higher serum levels of sCD137 were also detected in patients with active SLE and BD (Jung et al., 2004). So far, elevated levels of sCD137 and sCD137L have been detected in multiple autoimmune disease and they may mirror the clinical symptoms of the disease. So the levels of sCD137 and sCD137L in sera may help the diagnosis and prognosis of certain autoimmune diseases. Although lots of evidence indicates CD137/CD137L interactions may be involved in the pathogenesis of autoimmune disease, their precise role is far from well dissected. Soluble CD137 and sCD137L may be implicated in the negative feedback control of the ongoing inflammation (Michel et al., 1998). Since activation of lymphocytes through mCD137 delivers a potent costimulatory signal, sCD137L may retard the function of mCD137 by competing the ligand and receptor, leading to restricted lymphocyte activation. In support of this, soluble recombinant CD137 protein has been shown to suppress the T cell responses (DeBenedette et al., 1995; Hurtado et al., 1995). On the other hand, CD137L is expressed on professional APCs, and crosslinking of the ligand by CD137 receptor induces activation of the ligand-expression cells (Alderson et al., 1994; Goodwin et al., 1993; Pollok et al., 1994). Interfering CD137/CD137L interaction by soluble receptor and ligand may also slake the activity of their stimulating cells. So for therapeutic purpose, the disruption of autoreactive lymphocyte activation by sCD137 may be helpful for treatment of deleterious inflammatory diseases.

3. Treatment of Autoimmune Disease with Agonistic Anti-CD137 T cells are implicated in the pathogenesis of many inflammatory diseases. To fully activate T cells, both TCR and costimulatory molecule delivered signals are required (Lanzavecchia and Sallusto, 2000). Costimulation blockade targeting

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various costimulatory molecules seems to be an attractive therapeutic approach for the treatment of T cell-dependent autoimmune diseases. However, these strategies appear to be prophylactic rather than therapeutic, since na¨ıve T cell activation are more dependent on costimulatory signaling while the function of preexisting autoreactive T cells are less costimulation-dependent. Another shortcoming of this approach is its requirement of repeated long-lasting treatments, which is costly. Promisingly, recent studies showed that costimulatory agonists of CD137 could prevent and have therapeutic effects on CD4+ T cell-mediated organ-specific autoimmune disease such as experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveitis (EAU) (Shao et al., 2005; Sun et al., 2002b), and both CD4+ T cell and autoantibody-involved systemic autoimmune diseases such as systemic lupus erythematosus (SLE), collagen type II-induced arthritis (CIA) and chronic graft-versus-host disease (cGVHD) (Foell et al., 2003, 2004; Kim et al., 2005; Seo et al., 2004; Sun et al., 2002a).

3.1. Experimental Autoimmune Encephalomyelitis (EAE) EAE is a primarily CD4+ T cell-mediated demyelinating disease of the CNS, and it is widely used as an animal model for human multiple sclerosis (MS). EAE can be induced in susceptible animal strains by immunization with various myelin proteins or immunodominant peptide epitopes derived from myelin basic protein (MBP), proteolipoprotein (PLP), or myelin oligodendrocyte glycoprotein (MOG) peptide emulsified in complete Freund’s adjuvant (CFA) together with pertussis toxin treatment (Gonatas et al., 1986; Wekerle, 1991). TH 1-type responses appear to be responsible for EAE pathogenesis, while TH 2 responses seem to be protective (Adorini and Sinigaglia, 1997; Liblau et al., 1995). The adjuvant and pertussis might be necessary to skew the systemic cytokine profile to TH 1 phenotype, affect the blood-brain barrier, and support prolonged inflammation. The therapeutic role of agonistic anti-CD137 on autoimmune disease was first evaluated in EAE model. Sun et al. (2002b) reported that agonistic anti-CD137 mAb (2A, rat IgG2a) (Wilcox et al., 2002b) treatment inhibited the development of EAE in various murine models. To induce EAE by active immunization, female C57BL/6 mice were immunized subcutaneously with 100 μg of MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) emulsified in an equal volume of CFA containing 1 mg/ml Mycobacterium tuberculosis H37 RA on days 0 and 7. When C57BL/6 mice were treated with a single dose of 150 μg of agonistic anti-CD137 monoclonal antibody (2A) i.p. on the day of the first s.c. immunization with MOG35–55 peptide emulsified in CFA, the development of EAE including disease incidence and severity was dramatically reduced with ablated spinal cord lymphocytic infiltration when compared with control rat IgG treated mice. These data suggest that CD137 engagement during the priming stage of an immune response strongly prevent the development of EAE. Further studies showed that, draining lymph node cells from anti-CD137treated mice failed to respond to antigen stimulation ex vivo and were not able to transfer disease to RAG-1 deficient recipient mice as that from control mice. In addition, 2A treatment inhibited delayed-type hypersensitivity (DTH) responses

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to MOG peptide. However, 2A treatment was not able to suppress disease development in an adoptive transfer model of EAE, which was established by transferring activated MOG-specific T cells by i.v. injection into sublethally irradiated C57BL/6 mice. Activated MOG-specific T cells were obtained by coculture for 4 days of MOG35–55 peptide with DLN cells from C57BL/6 mice immunized with MOG35–55 peptide emulsified in CFA. These results suggest that CD137 engagement during the priming stage of an immune response strongly inhibits the function of Ag-specific autoreactive T cells and prevent EAE development, and CD137 signaling plays a role before the effector phase of EAE pathogenesis. More impressively, agonistic anti-CD137 treatment showed therapeutic effect in a more clinically relevant EAE model. The relapsing-remitting clinical course is a characteristic feature of MS. Immunization of SJL mice with s.c. injection of PLP139–151 (HSLGKWLGHPDKF) peptide in CFA containing 500 μg of M. tuberculosis H37 RA results in the development of chronic relapsing-remitting EAE. When agonistic anti-CD137 treatment initiated on the days of immunization, it significantly reduced the severity of disease developed compared with control mice. When treatment was started after disease onset, EAE relapse was inhibited. As mentioned before, CD137 signaling plays a negative role during priming before the effector phase of EAE pathogenesis, why such treatment could still be efficient in suppressing EAE relapse after the onset of disease. One possibility is due to the “epitope spreading” during the chronic relapse-remitting EAE development. Epitope spreading is coined to describe the phenomenon that disease is induced in response to one epitope, however, T cell responses diversify to other epitopes within the same protein or to other proteins, due to autoimmune-mediated tissue damage and subsequent endogenous self-priming (Tuohy et al., 1998). It has been shown that, epitopes spread in a predictable fashion in PLP139–151 -induced EAE model in SJL mice: from PLP 139–151 to PLP 178–191 and then to MBP 84–104 (Vanderlugt et al., 2000; Yu et al., 1996). This spread appears related to relapse, since the incidence of relapse is reduced upon induction of tolerance to PLP 178–191 (Vanderlugt et al., 2000). It is possible that administration of agonistic anti-CD137 at the onset of first relapse inhibited the priming of neo-autoreactive T cells due to epitope spread leading to reduced later phase relapse. These studies support the notion that agonistic anti-CD137 mAb treatment tolerize autoreactive T cells and inhibited Th1-mediated autoimmune disease EAE.

3.2. Experimental Autoimmune Uveitis (EAU) In addition to EAE, experimental autoimmune uveitis (EAU), a widely used animal model of human uveitis, is another CD4+ T-cell mediated organ-specific autoimmune disease which can be inhibited by agonistic anti-CD137 treatment (Shao et al., 2005). Uveitis is a common cause of human visual disability and blindness. EAU can be induced in susceptible rodent strains by immunization with retinal proteins such as the receptor retinoid binding protein (IRBP) (Donoso et al., 1989; Gery et al., 1986), retinal S antigen (S-Ag) (Wacker et al., 1977) and melaninassociated Ag (MAA) (Bora et al., 1995; Broekhuyse et al., 1992) or by the adoptive transfer of uveitogenic T cells to syngeneic rodents. TH 1-like response promotes the

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development of EAU (Tarrant et al., 1998), whereas TH 2-like response manifests protective function (Rizzo et al., 1999; Wildner and Thurau, 1995), suggesting the TH 1 nature of this type of autoimmune disease. Similar as in EAE model, Shao and colleagues (Shao et al., 2005) observed that administration of agonistic anti-CD137 (2A) on the day of immunization prevented actively induced EUA in B10RIII mice by subcutaneous immmunization with 100 μg of IRBP161–180 peptide emulsified in an equal volume of CFA containing 500 μg/ml Mycobacterium tuberculosis H37 RA. T cells from 2Atreated mice showed deficit response to IRBP161–180 restimulation ex vivo and failed to transfer disease to na¨ıve syngeneic B10RIII mice. If anti-CD137 treatment was given six or twelve days after immunization, it did not protect against EAU. Such treatment did not affect adoptively transferred uveitis which was directly induced by activated IRBP peptide-specific T cells. These studies further support the idea that CD137 engagement inhibited CD4+ T cell-mediated autoimmune disease in the early stages of autoreactive T cell priming instead of the effector phases.

3.3. Systemic Lupus Erythematosus (SLE) In addition to CD4+ T cell-mediated organ-specific autoimmune disease, CD137 agonist is potent in suppressing T cell and autoantibody involved systemic autoimmune disease such as SLE. SLE is a CD4+ T cell-dependent, immune complex-mediated multisystem disorder and it most frequently affects young women. The most common clinical manifestations include skin rash, arthritis, renal disease, and CNS dysfunction (Hochberg et al., 1985). SLE is both CD4+ T cell and B cell dependent (Humbert and Galanaud, 1990; Sobel et al., 1994). Most commonly used animal models of SLE are MRL/lpr mice and NZB/NZW F1 mice. Agonistic anti-CD137 monoclonal antibodies have been shown to be both prophylactic and therapeutic in both animal models (Foell et al., 2003; Sun et al., 2002a). Mice carrying the lymphoproliferative (lpr) mutation in their fas gene fail to deplete autoreactive lymphocytes properly by activation-induced cell death (AICD), leading to progressive, spectacular lymphoadenopathy, multiple SLElike autoantibodies, and hypergammaglobulinemia (Cohen and Eisenberg, 1991; Theofilopoulos and Dixon, 1981). It is the same as for the other murine SLE models, that development of disease is highly dependent on background genes which are not well understood yet (Izui et al., 1984). MRL/lpr mice, which are the best-studied strain, develop a SLE-like autoimmune disease with severe glomerulonephritis and vasculitis and have a markedly shortened lifespan. C57BL6/lpr (B6/lpr) mice manifest a milder syndrome with lymphoadenopathy, autoantibodies, and hypergammaglobulinemia but without overt tissue pathology and with nearly normal longevity. Both CD4+ T cells and B cells are involved in disease pathogenesis. The lpr mutations result in accumulation of large numbers of non-malignant CD4 and CD8 double negative aberrant T lymphocytes (DNTC), which express both T cell markers such as CD3, TCR, Thy1 and B cell marker B220 (Morse et al., 1982; Wofsy et al., 1984) and are the major contributor of lymphadenopathy. T cells

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are required for the development of both lymphadenopathy and autoantibodies. CD4+ T cells provide help for autoantibody production while CD8+ T cells may contribute to the abnormal double negative T cells population (Jevnikar et al., 1994; Koh et al., 1995; Maldonado et al., 1995). B lymphocytes are involved in disease pathogenesis both as antigen-presenting cells and autoantibody producing cells (Chan et al., 1999). Sun et al. (2002a) reported that the short-term treatment of 9- to 10-weeks old MRL/lpr mice, which already manifested significant numbers of aberrant DNTCs and high serum levels of anti-DNA auto-antibody, with agonistic anti-CD137 monoclonal antibody (2A) dramatically prevented spontaneous autoimmune disease. Such treatment blocked lymphoadenopathy, autoantibody production, skin lesion, and kidney diseases, ultimately led to prolonged survival. By four to five months of age, all of the control MRL/lpr mice developed progressively severe lymphadenopathy. However, the 2A-treated mice showed drastically smaller spleens and peripheral lymph nodes (LNs) with dramatically diminished cellularity especially DNTC in peripheral lymphoid organ especially in LNs than the control mice. In addition, such treatment completely abrogated the development of cutaneous disease. Kidney diseases are the major cause of mortality in those afflicted with lupus. 2A treatment attenuated kidney pathology, IgG and complement C3 deposition in kidney as well as kidney function with significantly reduced proteinuria compared with control mice. Accordingly, such treatment significantly decreased the levels of autoantibodies against DNA. Most strikingly, 2A treatment significantly prolonged the survival of MRL/lpr mice. Most MRL/lpr mice died between 20 to 28 weeks, while 2A treated mice all remained healthy until at least 32 weeks when the experiments were terminated. Sun et al. (2002a) further explored the therapeutic effect of 2A on mice which already developed clinically testable autoimmune disease including high levels of auto-antibodies and significant levels of urine proteins. The results showed that administration of 200 μg 2A weekly for three doses attenuated the progression of lymphadenopathy, decreased the production of auto-antibody IgG anti-DNA levels and ameliorated renal function and pathology. Consistent with the above studies, Foell et al. (2003) found similar therapeutic effects using a distinct clone of agonistic mAb against CD137 (3H3, rat IgG2a) (Shuford et al., 1997) in a different animal model of human SLE, NZB × NZW F1 female mice. These mice develop systemic SLE-like disease affecting multiple organs with diffuse proliferative glomerulonephritis, high-titer antinuclear antibodies and premature death (Theofilopoulos and Dixon, 1985). They observed that administration of 200 μg of 3H3 at three weeks interval for 3 to 5 doses between 26 and 35 weeks of age reversed acute disease, blocked chronic disease, and extended the mice’s lifespan to normal level. Anti-dsDNA autoantibody production in treated mice, from age 8 to 40-week-old, and regardless of one or multiple doses, was rapidly suppressed without inducing systemic immunosuppression. These studies suggest agonistic anti-CD137 mAb could potentially be clinically valuable in treating well-established lupus-like diseases.

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3.4. Collagen-induced Arthritis (CIA) Rheumatoid arthritis is the most common chronic inflammatory disease targeting peripheral joints. It causes progressive cartilage destruction and bone erosion around joints (Palmer, 1995). CIA is an experimental mouse model used for the study of human RA and is induced in genetically predisposed mice by immunization with bovine collagen II (bCII) emulsified in CFA as mentioned before. CD4+ T cells play a central role in disease induction in arthritis (Ranges et al., 1985). Both antigen specific helper T cells and antibodies isolated from arthritic animals are capable of transferring disease within susceptible mouse strains (Seki et al., 1988; Taylor et al., 1995). Suggesting both B and T lymphocytes are involved in the pathogenesis of CIA. As previously mentioned, CD137/CD137L interaction blockade showed only mild effect in preventing the development of CIA, however, CD137 agonist was capable to both prevent and treat established CIA. Similar as SLE, CD137 engagement with agonistic monoclonal antibodies (3H3) not only suppressed the development of CIA, but also reversed disease progression in mice already afflicted with disease (Foell et al., 2004; Seo et al., 2004). Administration of agonistic anti-CD137 prevents arthritis development in a dose dependent manner when treatment started on the day of first immunization with bCII emulsified with CFA. Three to five doses of 200 μg 3H3 treatment completely prevented the development of arthritis with ablated clinical sign and joint pathology, anti-bCII autoantibody production was absent as well (Foell et al., 2004; Seo et al., 2004). Moreover, such treatment generated a long-term tolerance that protected the mice from subsequent rechallenge with the same antigen (Foell et al., 2004). Most strikingly, anti-CD137 treatment improves established CIA with quick and almost complete clearance of anti-bCII autoantibodies in sera. CD4+ T cells isolated from draining lymph node of bCII immunized and 3H3 treated mice lost recall response to bCII re-stimulation ex vivo as the control IgG treated mice did. These studies suggest that CD137 engagement induces potent immunosuppression in autoantibody production and autoreactive helper T cell function.

3.5. Chronic Graft-Versus-Host Disease (cGVHD) Chronic GVHD is a major clinical problem in human recipients of bone marrow, which leads to autoantibody production, fibrosis, and skin pathology (Vogelsang, 2001). Animals undergoing cGVHD develop a SLE-like systemic autoimmune syndrome including production of antinuclear autoantibodies and immune complex-mediated renal disease (van Rappard-Van der Veen et al., 1984). The etiology may be attributed to the breakdown of tolerance of autoreactive T and B cells resulting from alloresponse to minor recipient histocompatibility antigens. There is a preponderance of evidence that indicates both alloreactive donor CD4+ T cells and autoreactive host CD4+ T cells are essential for cGVHD (Chen et al., 1998; Morris et al., 1990). Chronic GVHD can be induced by transfer of parental lymphocytes into F1 hybrid mice.

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Kim et al. (2005) studied the role of CD137 agonist in cGVHD. cGVHD was induced by transfer of DBA/2 (H-2d ) parental lymphocytes into BDF1 mice (H2b/d ). They found that single dose of agonistic anti-CD137 mAb (3H3) (Shuford et al., 1997) injection was able to block the production of anti-DNA IgG and total IgE, abrogate immune complex deposition in kidney and prevent glomerulonephritis in the recipients, leading to increased survival. Additionally, such treatment also ameliorates advanced cGVHD.

4. Mechanisms Involved in CD137 Agonist-mediated Inhibition of Autoimmune Diseases Compelling data have been shown that agonistic anti-CD137 monoclonal antibodies displayed potent immunomodulatory function in various autoimmune disease models including CD4+ T cell-mediated and both CD4+ T cell and B cellinvolved, organ-specific and systemic autoimmune diseases. Multiple mechanisms are involved in CD137 engagement-induced inhibition of autimmunity, which includes: (1) increase of T lymphocyte apoptosis, (2) generation of CD8+ regulatory T cells, (3) CD4+ T cell anergy, (4) autoreactive B cell apoptosis. INF-γ plays an important role in various events.

4.1. Apoptosis of T Lymphocytes Agonistic anti-CD137 mAb treatment inhibited the function of pathogenic CD4+ T cells in various autoimmune disease models (Seo et al., 2004; Shao et al., 2005; Sun et al., 2002b). Such treatment could also reduce CD4+ T cell number in certain models (Kim et al., 2005; Sun et al., 2002a, 2002b). In lpr mice, CD137 engagement with anti-CD137 resulted in the diminishment of CD4+ T cell number (Sun et al., 2002a). Through tracking antigen-specific T cell response with DO11.10 TCR transgenic mice, which express transgenes encoding a TCR specific for chicken OVA peptide 329–339 bound to I-Ad class II MHC molecule, and can be detected with mAbs specific for CD4 and the clonotypic TCR (KJ1-26), Sun et al. (2002b) found the administration of agonistic anti-CD137 initially enhanced antigen-specific CD4+ T cell expansion, but subsequently promoted their depletion by inducing their apoptosis. In accordance with reduced CD4+ T cell number, in EAE and EAU models, anti-CD137 treatment initially promoted the function of autoreactive TH 1 cells followed by diminishment of their functions (Shao et al., 2005; Sun et al., 2002b). Four to five days after immunization, draining lymph node (DLN) T cells from antiCD137 treated mice showed increased proliferation and cytokine production such as IFN-γ, GM-CSF and IL-2 compared with control IgG treated mice. However, 10 days after immunization, DLN T cells from anti-CD137 treated mice showed reduced proliferation and cytokine production compared with control mice. This is different from CTLA blockade with CTLA-Fc, which inhibited T cell function at both time points (Shao et al., 2005). When cultured ex vivo, DLN T cells

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from anti-CD137 treated mice manifested significantly increased apoptosis rate compared with that of control mice (Shao et al., 2005). These studies suggest CD137 signaling plays diametric role in CD4+ T cell-mediated immune responses, it initially promoted CD4+ T cell activation and then increased their activation induced cell death (AICD) leading to diminished helper T cell function. This dual-phase outcome of CD137 signaling on CD4+ T cells was also observed in cGVHD model (Kim et al., 2005). Agonistic anti-CD137 treatment promoted the activation and proliferation of donor CD4+ T cells at the initial phase of cGVHD and followed by increased donor CD4+ T cell apoptosis, leading to the reduction of donor CD4+ T cell number in the spleen. In addition to CD4+ T cells, CD137 engagement induces drastic diminishment of DNTC population with increased apoptosis, which leads to the attenuation of lymphadenopathy in MRL/lpr mice (Sun et al., 2002a).

4.2. Regulatory T Cells and IFN-γ In a CIA model established in DBA/1 mice by immunization with bovine CII emulsified with CFA, Seo et al. (2004) observed a marked expansion of a unique CD8+ and IFN-γ-producing regulatory T cell population which express medium level of CD11c, a surface marker highly expressed on dendritic cells, in draining lymph node and spleen of anti-CD137 treated mice. The expansion of these CD11c+ CD8+ T cells required both CD137 crosslinking and antigen stimulation. When the CD11c+ CD8+ T cells isolated from CII immunized and anti-CD137 treated mice were adoptively transferred into na¨ıve DBA/1 mice, they suppressed CII-specific CD4+ T cell response and the development of CIA in the recipients, suggesting their dominant inhibitory function in regulating CD4+ T cell-mediated immune responses. CD11c+ CD8+ T cells produced abundant INF-γ but not regulatory cytokines such as TGF-β, IL-4 and IL-10. Blockade of INF-γ in anti-CD137 treated mice reversed the suppression of CII-specific CD4+ T cell function and amelioration of CIA induced by CD137 engagement. These data imply the key role played by INF-γ in the inhibition of CD4+ T cells mediated CIA (Seo et al., 2004). To link IFN-γ and the inhibition of CD4+ T cell function, Seo et al. (2004) further dissected the down stream biochemical events such as indoleamine 2,3-dioxygenase (IDO) and inducible nitric oxide synthetase (iNOS) production induced by IFN-γ. They found that anti-CD137 treatment increased the levels of IDO and iNOS mRNA in CD11b+ monocytes and CD11c+ DCs, and IFN-γ blockade prevents the expression of IDO and iNOS in macrophages and DCs induced by CD137 engagement. These studies suggest CD137 mediated production of IDO and iNOS by monocytes and DC is IFN-γ dependent. IDO has been shown to be an immune regulator which plays a critical role in maintaining maternal T cell tolerance to allogeneic fetuses during pregnancy (Munn et al., 1998), and in CTLA-4 mediated tolerance in transplantation and autoimmunity (Fallarino et al., 2003; Grohmann et al., 2002; Mellor et al., 2003). IDO is a tryptophan-catabolizing enzyme expressed by macrophages and other cell types; it has regulatory effects on T cells due to consuming of tryptophan

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which is an essential amino acids (Mellor et al., 2001; Munn et al., 1998, 1999). 1-methyl-tryptophan (1-MT) is a pharmacologic agent that competitively inhibits IDO enzyme activity (Cady and Sono, 1991). Administration of 1-MT in mice treated with anti-CD137 reversed the suppression of CIA, suggesting CD137mediated inhibition of CIA is IDO-mediated (Seo et al., 2004). In summary, administration of anti-CD137 in CIA model induces antigendependent clonal expansion of CD11c+ CD8+ T cells and promotes their IFN-γ production. IFN-γ further induces monocytes and DCs to produce IDO, which causes tryptophan catabolism and production of the metabolite kynurenine, which is thought to kill antigen-activated CD4+ T cells, leading to inhibition of autoimmune disease. Except for CIA model, in MRL/lpr mice, anti-CD137 treatment induced massive expansion of CD8+ T cells with drastic production of IFN-γ, and blockade of IFN-γ reversed the reduction of autoantibody production by anti-CD137 treatment (Sun et al., 2002a). However, whether CD137-mediated suppression of SLE in MRL/lpr mice is CD8+ T cell mediated need to be defined. In another SLE animal model, NZB × NZW F1 female mice, Foell et al. (2003) observed that elimination of CD8 T cells from these mice has no effect on anti-CD137-mediated blockade of disease, suggesting CD8+ T cell-independent mechanism involved. The inhibition of CD4+ T cell function by CD137 signaling activated CD8+ T cells has also been observed in other model. Myers et al. showed that Toll-like receptor (TLR) ligands and CD137 crosslinking massively promoted both CD4+ and CD8+ T cell clonal expansion in vivo, whereas CD137 costimulated-CD8+ T cells greatly inhibited CD4+ T cell recall responses through a type-β transforming growth factor-dependent mechanism (Myers et al., 2003). All these studies imply CD137 signaling activated CD8+ T cells could play a role in regulating CD4+ T cell function through different mechanism.

4.3. Helper T Cell Anergy It has been shown that agonistic anti-CD137 mAbs are potent suppressors of T cell-dependent humoral immunity (Mittler et al., 1999). When na¨ıve B cells were adoptively transferred into SCID recipient mice together with T cells, which were isolated from mice immunized with SRBC and treated with agonistic anti-CD137 mAb, the recipients generated deficit anti-SRBC humoral responses. However, when na¨ıve T cells were adoptively transferred into SCID recipients along with B cells, which were isolated from SRBC-immunized and anti-CD137-treated mice, the recipients developed normal humoral responses to SRBC (Mittler et al., 1999). These experiments indicate CD137 engagement in vivo with agonist antibodies preclude the generation of functional helper T cell populations. Agonistic anti-CD137 treatment of NZB × NZW F1 mice did not induce massive depletion of lymphocytes including CD4+ T cells as in MRL/lpr mice. And anti-CD137 mediated disease blockade is not CD8+ T cell dependent (Foell et al., 2003). These suggest other mechanisms than increased AICD of CD4+ T cells and induction of CD8+ regulatory T cells may be involved. Administration of anti-CD137 inhibited IL-2 and IL-4 production by CD4+ T cells, and adoptive

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transfer of antigen-primed CD4+ T cells isolated from untreated mice or bone marrow-derived dendritic cells (DCs) reversed CD137-mediated disease protection in NZB × NZW F1 mice. These data imply CD137-mediated signaling may anergize CD4+ T cells (Foell et al., 2003; Mittler et al., 1999).

4.4. B Cell Apoptosis In lpr mice, CD137 engagement with anti-CD137 resulted in massive diminishment of lymphocytes including DNTCs, CD4+ T cells and autoreactive B cells in a Fas and TNF-independent manner accompanied with increased apoptosis in those populations (Sun et al., 2002a). In MRL/lpr mice, agonistic anti-CD137 treatment dramatically decreased both the total B cell and anti-DNA-secreting B cell populations in an IFN-γdependent manner (Sun et al., 2002a). This was not likely due to a direct effect of the treatment, since CD137 was not detected on B cells. In B6/lpr mice, engagement of CD137 induced drastic IFN-γ production as well as expansion of CD11b+ /Gr-1+ macrophages/granulocytes population. Both B cell depletion and CD11b+ /Gr-1+ cell expansion are IFN-γ-dependent (Sun et al., 2002a). It has been shown that IFN-γ is able to activate macrophages, which in turn induces apoptosis of activated lymphocytes by direct or indirect mechanisms (Ding et al., 1988; Haendeler et al., 1999; Williams et al., 1998). In B6/lpr mice, IFN-γ activated macrophages indeed induced B-cell apoptosis in vitro (Sun et al., 2002a). In addition, anti-CD137 activated human monocytes induced B cell apoptosis as well (Kienzle and von Kempis, 2000). CD137 costimulation induced B cell apoptosis was also observed in cGVHD model (Kim et al., 2005). Anti-CD137 treatment increased host B cell apoptosis resulting in decreased host B cell number, and the deletion of both alloreactive CD4+ T cells and autoreactive B cells contributes to the inhibition of cGVHD by CD137 engagement. Similarly, consistent CD137/CD137L interaction led to progressive B cell diminishment in transgenic mice expressing CD137L on class II positive cells (Zhu et al., 2001). These transgenic mice showed selective depletion of B cells and increase of macrophages in the peripheral, low levels of circulating IgG, and defective humoral responses to antigen challenge. Above studies suggest that CD137 signaling augmented T cell IFN-γ production that promoted the activation of macrophages/granulocytes, which in turn induced the apoptosis of activated autoreactive B cells, thereby resulting in decreased autoantibody production. In the mean time, insufficient T cell help due to the decreased CD4+ T cell number or function may also contribute to the suppression of autoantibody production.

5. Summary Convincing studies have shown that CD137/CD137L interactions may play an important role in the pathogenesis of autoimmune disease. The serum levels of sCD137 and sCD137L in patients may help the diagnosis of certain autoimmune

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diseases, and CD137 targeted immunotherapy regimes especially CD137 agonists hold lots of promise in treating inflammatory diseases. From the therapeutic point of view, the agonistic anti-CD137 mAbs stands out for following reasons. First, they are both prophylactic and therapeutic. AntiCD137 treatment not just prevents the development of autoimmune disease, but also reverses the disease severity in afflicted mice, and it generates immunoregulatory mechanism protecting the mice from subsequent disease induction. Second, they allow for selective depletion or anergy induction of activated autoreactive lymphocytes without inducing global immunosuppression. Agonistic anti-CD137 treatment inhibits autoimmunity without overt interference with the normal immune response to exogenous antigens after the termination of treatment (Foell et al., 2003; Sun et al., 2002a). Third, Agonistic anti-CD137 treatment abrogates antibody production against itself due to its potential inhibition of T-dependent humoral immune responses, thereby making repeated administration possible (Foell et al., 2003; Mittler et al., 1999). The above feature of CD137 agonist allows shortterm therapy; therefore the treatment is cost-effective and beneficial to patients. So a therapeutic approach based on triggering CD137 is very attractive in clinical application of autoimmune and inflammatory disease treatment. However, CD137 crosslinking in vivo does not attenuate all autoimmune diseases. Experiments performed by Sytwu et al. (2003) showed that transgenic non-obese diabetic (NOD) mice, which overexpress membrane-bound agonistic single-chain anti-CD137 Fv (scFv) in pancreatic beta cells, developed earlier onset and more aggressive diabetes than their non-transgenic littermates with enhanced GAD-specific T-cell responses and higher mortality. NOD mice have been widely used as animal model for human type I autoimmune diabetes (T1D). Both CD4+ and CD8+ T cells are implicated in the pathogenesis of T1D. Since CD137 provides potent costimulatory signaling to CD8+ T cells and prolongs their survival, CD8+ T cell-involved autoimmune diseases may not be good targets of anti-CD137based immunotherapy. Combinatorial treatment with CD8+ T cell inhibitors may be necessary. Anti-CD137 based immunotherapy may be more effective in CD4+ T cell-dominant inflammatory diseases. When used properly, CD137-targeted immunotherapy strategy may provide an effective novel therapeutic avenue in the treatment of various autoimmune diseases.

References Adorini, L., and Sinigaglia, F. (1997). Pathogenesis and immunotherapy of autoimmune diseases. Immunol. Today, 18, 209–211. Alderson, M.R., Smith, C.A., Tough, T.W., Davis-Smith, T., Armitage, R.J., Falk, B., Roux, E., Baker, E., Sutherland, G. R., and Din, W. S. (1994). Molecular and biological characterization of human 4-1BB and its ligand. Eur. J. Immunol., 24, 2219–2227. Blazar, B.R., Kwon, B.S., Panoskaltsis-Mortari, A., Kwak, K.B., Peschon, J.J., and Taylor, P.A. (2001). Ligation of 4-1BB (CDw137) regulates graft-versus-host disease, graft- versus-leukemia, and graft rejection in allogeneic bone marrow transplant recipients. J. Immunol., 166, 3174– 3183.

Autoimmune Diseases

111

Bora, N.S., Kim, M.C., Kabeer, N.H., Simpson, S.C., Tandhasetti, M.T., Cirrito, T.P., Kaplan, A.D., and Kaplan, H.J. (1995). Experimental autoimmune anterior uveitis. Induction with melaninassociated antigen from the iris and ciliary body. Invest. Ophthalmol. Vis. Sci., 36, 1056–1066. Broekhuyse, R.M., Kuhlmann, E.D., and Winkens, H.J. (1992). Experimental autoimmune anterior uveitis (EAAU). II. Dose-dependent induction and adoptive transfer using a melanin-bound antigen of the retinal pigment epithelium. Exp. Eye. Res., 55, 401–411. Cady, S.G., and Sono, M. (1991). 1-Methyl-DL-tryptophan, beta-(3-benzofuranyl)-DL-alanine (the oxygen analog of tryptophan), and beta-[3-benzo(b)thienyl]-DL-alanine (the sulfur analog of tryptophan) are competitive inhibitors for indoleamine 2,3-dioxygenase. Arch. Biochem. Biophys., 291, 326–333. Chan, O.T., Madaio, M.P., and Shlomchik, M.J. (1999). The central and multiple roles of B cells in lupus pathogenesis. Immunol. Rev., 169, 107–121. Chen, F., Maldonado, M.A., Madaio, M., and Eisenberg, R.A. (1998). The role of host (endogenous) T cells in chronic graft-versus-host autoimmune disease. J. Immunol., 161, 5880–5885. Cohen, P.L., and Eisenberg, R.A. (1991). Lpr and gld: Single gene models of systemic autoimmunity and lymphoproliferative disease. Annu. Rev. Immunol., 9, 243–269. DeBenedette, M.A., Chu, N.R., Pollok, K.E., Hurtado, J., Wade, W.F., Kwon, B.S., and Watts, T.H. (1995). Role of 4-1BB ligand in costimulation of T lymphocyte growth and its upregulation on M12 B lymphomas by cAMP. J. Exp. Med., 181, 985–992. DeBenedette, M.A., Wen, T., Bachmann, M.F., Ohashi, P.S., Barber, B.H., Stocking, K.L., Peschon, J.J., and Watts, T.H. (1999). Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to influenza virus. J. Immunol., 163, 4833–4841. Deshpande, S.P., Lee, S., Zheng, M., Song, B., Knipe, D., Kapp, J.A., and Rouse, B.T. (2001). Herpes simplex virus-induced keratitis: Evaluation of the role of molecular mimicry in lesion pathogenesis. J. Virol., 75, 3077–3088. Ding, A.H., Nathan, C.F., and Stuehr, D.J. (1988). Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J. Immunol., 141, 2407–2412. Donoso, L.A., Merryman, C.F., Sery, T., Sanders, R., Vrabec, T., and Fong, S.L. (1989). Human interstitial retinoid binding protein. A potent uveitopathogenic agent for the induction of experimental autoimmune uveitis. J. Immunol., 143, 79–83. Fallarino, F., Grohmann, U., Hwang, K.W., Orabona, C., Vacca, C., Bianchi, R., Belladonna, M.L., Fioretti, M.C., Alegre, M.L., and Puccetti, P. (2003). Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol., 4, 1206–1212. Feldmann, M., Brennan, F.M., and Maini, R.N. (1996). Rheumatoid arthritis. Cell, 85, 307–310. Foell, J., Strahotin, S., O’Neil, S.P., McCausland, M. M., Suwyn, C., Haber, M., Chander, P.N., Bapat, A.S., Yan, X.J., Chiorazzi, N., et al. (2003). CD137 costimulatory T cell receptor engagement reverses acute disease in lupus-prone NZB × NZW F1 mice. J. Clin. Invest., 111, 1505–1518. Foell, J.L., Diez-Mendiondo, B.I., Diez, O.H., Holzer, U., Ruck, P., Bapat, A.S., Hoffmann, M.K., Mittler, R.S., and Dannecker, G.E. (2004). Engagement of the CD137 (4-1BB) costimulatory molecule inhibits and reverses the autoimmune process in collagen-induced arthritis and establishes lasting disease resistance. Immunology, 113, 89–98. Futagawa, T., Akiba, H., Kodama, T., Takeda, K., Hosoda, Y., Yagita, H., and Okumura, K. (2002). Expression and function of 4-1BB and 4-1BB ligand on murine dendritic cells. Int. Immunol., 14, 275–286. Gavin, M.A., Clarke, S.R., Negrou, E., Gallegos, A., Rudensky, A., and Howard Hughes Medical Institute, U. o. W. B. S. W. A. U. S. A. m. u. w. e. (2002). Homeostasis and anergy of CD4(+)CD25(+) suppressor T cells in vivo. Nature. Immunol., 3(1), 33–41. Gery, I., Wiggert, B., Redmond, T.M., Kuwabara, T., Crawford, M.A., Vistica, B.P., and Chader, G.J. (1986). Uveoretinitis and pinealitis induced by immunization with interphotoreceptor retinoidbinding protein. Invest. Ophthalmol. Vis. Sci., 27, 1296–1300. Gonatas, N.K., Greene, M.I., and Waksman, B.H. (1986). Genetic and molecular aspects of demyelination. Immunol. Today, 7, 121–126.

112

Yonglian Sun and Yang-Xin Fu

Goodwin, R.G., Din, W.S., Davis-Smith, T., Anderson, D.M., Gimpel, S.D., Sato, T.A., Maliszewski, C.R., Brannan, C.I., Copeland, N.G., Jenkins, N.A., et al. (1993). Molecular cloning of a ligand for the inducible T cell gene 4-1BB: A member of an emerging family of cytokines with homology to tumor necrosis factor. Eur. J. Immunol., 23, 2631–2641. Grohmann, U., Orabona, C., Fallarino, F., Vacca, C., Calcinaro, F., Falorni, A., Candeloro, P., Belladonna, M.L., Bianchi, R., Fioretti, M.C., and Puccetti, P. (2002). CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat. Immunol., 3, 1097–1101. Haendeler, J., Zeiher, A.M., and Dimmeler, S. (1999). Nitric oxide and apoptosis. Vitam. Horm., 57, 49–77. Halstead, E.S., Mueller, Y.M., Altman, J.D., and Katsikis, P.D. (2002). In vivo stimulation of CD137 broadens primary antiviral CD8+ T cell responses. Nat. Immunol., 3, 536–541. Heinisch, I.V., Daigle, I., Knopfli, B., and Simon, H.U. (2000). CD137 activation abrogates granulocytemacrophage colony-stimulating factor-mediated anti-apoptosis in neutrophils. Eur. J. Immunol., 30, 3441–3446. Hochberg, M.C., Boyd, R.E., Ahearn, J.M., Arnett, F.C., Bias, W.B., Provost, T.T., and Stevens, M.B. (1985). Systemic lupus erythematosus: A review of clinico-laboratory features and immunogenetic markers in 150 patients with emphasis on demographic subsets. Medicine (Baltimore), 64, 285– 295. Humbert, M., and Galanaud, P. (1990). [B-lymphocyte hyperreactivity and differentiation factors of T-lymphocytes in systemic lupus erythematosus]. Ann. Med. Interne., (Paris), 141, 213–216. Hurtado, J.C., Kim, S.H., Pollok, K.E., Lee, Z.H., and Kwon, B.S. (1995). Potential role of 4-1BB in T cell activation. Comparison with the costimulatory molecule CD28. J. Immunol., 155, 3360–3367. Hurtado, J.C., Kim, Y.J., and Kwon, B.S. (1997). Signals through 4-1BB are costimulatory to previously activated splenic T cells and inhibit activation-induced cell death. J. Immunol., 158, 2600–2609. Izui, S., Kelley, V.E., Masuda, K., Yoshida, H., Roths, J.B., and Murphy, E.D. (1984). Induction of various autoantibodies by mutant gene lpr in several strains of mice. J. Immunol., 133, 227–233. Jevnikar, A.M., Grusby, M.J., and Glimcher, L.H. (1994). Prevention of nephritis in major histocompatibility complex class II-deficient MRL-lpr mice. J. Exp. Med., 179, 1137–1143. Jung, H.W., Choi, S.W., Choi, J.I., and Kwon, B.S. (2004). Serum concentrations of soluble 4-1BB and 4-1BB ligand correlated with the disease severity in rheumatoid arthritis. Exp. Mol. Med., 36, 13–22. Kienzle, G., and von Kempis, J. (2000). CD137 (ILA/4-1BB), expressed by primary human monocytes, induces monocyte activation and apoptosis of B lymphocytes. Int. Immunol., 12, 73–82. Kim, J., Choi, W.S., La, S., Suh, J.H., Kim, B.S., Cho, H.R., Kwon, B.S., and Kwon, B. (2005). Stimulation with 4-1BB (CD137) inhibits chronic graft-versus-host disease by inducing activationinduced cell death of donor CD4+ T cells. Blood, 105, 2206–2213. Koh, D.R., Ho, A., Rahemtulla, A., Fung-Leung, W.P., Griesser, H., and Mak, T.W. (1995). Murine lupus in MRL/lpr mice lacking CD4 or CD8 T cells. Eur. J. Immunol., 25, 2558–2562. Kwon, B.S., and Weissman, S.M. (1989). cDNA sequences of two inducible T-cell genes. Proc. Natl. Acad. Sci. USA, 86, 1963–1967. Kwon, B.S., Tan, K.B., Ni, J., Oh, K.O., Lee, Z.H., Kim, K.K., Kim, Y.J., Wang, S., Gentz, R., Yu, G.L., et al. (1997). A newly identified member of the tumor necrosis factor receptor superfamily with a wide tissue distribution and involvement in lymphocyte activation. J. Biol. Chem., 272, 14272–14276. Kwon, B., Moon, C.H., Kang, S., Seo, S.K., and Kwon, B.S. (2000). 4-1BB: Still in the midst of darkness. Mol. Cells., 10, 119–126. Kwon, B.S., Hurtado, J.C., Lee, Z.H., Kwack, K.B., Seo, S.K., Choi, B.K., Koller, B.H., Wolisi, G., Broxmeyer, H.E., and Vinay, D.S. (2002). Immune responses in 4-1BB (CD137)-deficient mice. J. Immunol., 168, 5483–5490. Lanzavecchia, A., and Sallusto, F. (2000). Dynamics of T lymphocyte responses: intermediates, effectors, and memory cells. Science, 290, 92–97. Liblau, R.S., Singer, S.M., and McDevitt, H.O. (1995). Th1 and Th2 CD4+ T cells in the pathogenesis of organ-specific autoimmune diseases. Immunol. Today, 16, 34–38. Luross, J.A., and Williams, N.A. (2001). The genetic and immunopathological processes underlying collagen-induced arthritis. Immunology., 103, 407–416.

Autoimmune Diseases

113

Maldonado, M.A., Eisenberg, R.A., Roper, E., Cohen, P.L., and Kotzin, B.L. (1995). Greatly reduced lymphoproliferation in lpr mice lacking major histocompatibility complex class I. J. Exp. Med., 181, 641–648. McHugh, R.S., Whitters, M.J., Piccirillo, C.A., Young, D.A., Shevach, E.M., Collins, M., Byrne, M.C., Cellular Immunology Section, L. o. I. N. I. o. A., and Infectious Diseases, N. I. o. H. B. M. D., USA (2002). CD4(+)CD25(+) immunoregulatory T cells: Gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity, 16(2), 311–323. Melero, I., Shuford, W.W., Newby, S.A., Aruffo, A., Ledbetter, J.A., Hellstrom, K.E., Mittler, R.S., and Chen, L. (1997). Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat. Med., 3, 682–685. Melero, I., Johnston, J.V., Shufford, W.W., Mittler, R.S., and Chen, L. (1998). NK1.1 cells express 4-1BB (CDw137) costimulatory molecule and are required for tumor immunity elicited by anti-4-1BB monoclonal antibodies. Cell. Immunol., 190, 167–172. Mellor, A.L., Sivakumar, J., Chandler, P., Smith, K., Molina, H., Mao, D., and Munn, D.H. (2001). Prevention of T cell-driven complement activation and inflammation by tryptophan catabolism during pregnancy. Nat. Immunol., 2, 64–68. Mellor, A.L., Baban, B., Chandler, P., Marshall, B., Jhaver, K., Hansen, A., Koni, P.A., Iwashima, M., and Munn, D.H. (2003). Cutting edge: Induced indoleamine 2,3 dioxygenase expression in dendritic cell subsets suppresses T cell clonal expansion. J. Immunol., 171, 1652–1655. Michel, J., Langstein, J., Hofstadter, F., and Schwarz, H. (1998). A soluble form of CD137 (ILA/4-1BB), a member of the TNF receptor family, is released by activated lymphocytes and is detectable in sera of patients with rheumatoid arthritis. Eur. J. Immunol., 28, 290–295. Mittler, R.S., Bailey, T.S., Klussman, K., Trailsmith, M.D., and Hoffmann, M. K. (1999). Anti-4-1BB monoclonal antibodies abrogate T cell-dependent humoral immune responses in vivo through the induction of helper T cell anergy. J. Exp. Med., 190, 1535–1540. Morris, S.C., Cheek, R.L., Cohen, P.L., and Eisenberg, R.A. (1990). Autoantibodies in chronic graft versus host result from cognate T-B interactions. J. Exp. Med., 171, 503–517. Morse, H.C., 3rd, Davidson, W.F., Yetter, R.A., Murphy, E.D., Roths, J.B., and Coffman, R.L. (1982). Abnormalities induced by the mutant gene Ipr: Expansion of a unique lymphocyte subset. J. Immunol., 129, 2612–2615. Munn, D.H., Zhou, M., Attwood, J.T., Bondarev, I., Conway, S.J., Marshall, B., Brown, C., and Mellor, A.L. (1998). Prevention of allogeneic fetal rejection by tryptophan catabolism. Science, 281, 1191–1193. Munn, D.H., Shafizadeh, E., Attwood, J.T., Bondarev, I., Pashine, A., and Mellor, A.L. (1999). Inhibition of T cell proliferation by macrophage tryptophan catabolism. J. Exp. Med., 189, 1363–1372. Myers, L.K., Rosloniec, E.F., Cremer, M.A., and Kang, A.H. (1997). Collagen-induced arthritis, an animal model of autoimmunity. Life. Sci., 61, 1861–1878. Myers, L., Takahashi, C., Mittler, R.S., Rossi, R.J., and Vella, A.T. (2003). Effector CD8 T cells possess suppressor function after 4-1BB and Toll-like receptor triggering. Proc. Natl. Acad. Sci., USA, 100, 5348–5353. Palmer, D.G. (1995). The anatomy of the rheumatoid lesion. Br. Med. Bull., 51, 286–295. Pollok, K.E., Kim, Y.J., Zhou, Z., Hurtado, J., Kim, K.K., Pickard, R.T., and Kwon, B. S. (1993). Inducible T cell antigen 4-1BB. Analysis of expression and function. J. Immunol., 150, 771–781. Pollok, K.E., Kim, Y.J., Hurtado, J., Zhou, Z., Kim, K.K., and Kwon, B.S. (1994). 4-1BB T-cell antigen binds to mature B cells and macrophages, and costimulates anti-mu-primed splenic B cells. Eur. J. Immunol., 24, 367–374. Ranges, G.E., Sriram, S., and Cooper, S.M. (1985). Prevention of type II collagen-induced arthritis by in vivo treatment with anti-L3T4. J. Exp. Med., 162, 1105–1110. Rizzo, L.V., Morawetz, R.A., Miller-Rivero, N.E., Choi, R., Wiggert, B., Chan, C.C., Morse, H.C., 3rd, Nussenblatt, R.B., and Caspi, R.R. (1999). IL-4 and IL-10 are both required for the induction of oral tolerance. J. Immunol., 162, 2613–2622. Rosen, S.D. (2004). Ligands for L-selectin: Homing, inflammation, and beyond. Annu. Rev. Immunol., 22, 129–156. Russell, R.G., Nasisse, M.P., Larsen, H.S., and Rouse, B.T. (1984). Role of T-lymphocytes in the pathogenesis of herpetic stromal keratitis. Invest. Ophthalmol. Vis. Sci., 25, 938–944.

114

Yonglian Sun and Yang-Xin Fu

Schwarz, H., Blanco, F. J., von Kempis, J., Valbracht, J., and Lotz, M. (1996). ILA, a member of the human nerve growth factor/tumor necrosis factor receptor family, regulates T-lymphocyte proliferation and survival. Blood, 87, 2839–2845. Seki, N., Sudo, Y., Yoshioka, T., Sugihara, S., Fujitsu, T., Sakuma, S., Ogawa, T., Hamaoka, T., Senoh, H., and Fujiwara, H. (1988). Type II collagen-induced murine arthritis. I. Induction and perpetuation of arthritis require synergy between humoral and cell-mediated immunity. J. Immunol., 140, 1477– 1484. Seo, S.K., Park, H.Y., Choi, J.H., Kim, W.Y., Kim, Y.H., Jung, H.W., Kwon, B., Lee, H.W., and Kwon, B.S. (2003). Blocking 4-1BB/4-1BB ligand interactions prevents herpetic stromal keratitis. J. Immunol., 171, 576–583. Seo, S.K., Choi, J.H., Kim, Y.H., Kang, W.J., Park, H.Y., Suh, J.H., Choi, B.K., Vinay, D.S., and Kwon, B.S. (2004). 4-1BB-mediated immunotherapy of rheumatoid arthritis. Nat. Med., 10, 1088–1094. Setareh, M., Schwarz, H., and Lotz, M. (1995). A mRNA variant encoding a soluble form of 4-1BB, a member of the murine NGF/TNF receptor family. Gene, 164, 311–315. Shao, H., Fu, Y., Liao, T., Peng, Y., Chen, L., Kaplan, H.J., and Sun, D. (2005). Anti-CD137 mAb treatment inhibits experimental autoimmune uveitis by limiting expansion and increasing apoptotic death of uveitogenic T cells. Invest. Ophthalmol. Vis. Sci., 46, 596–603. Sharief, M.K. (2002). Heightened intrathecal release of soluble CD137 in patients with multiple sclerosis. Eur. J. Neurol., 9, 49–54. Shuford, W.W., Klussman, K., Tritchler, D.D., Loo, D.T., Chalupny, J., Siadak, A.W., Brown, T.J., Emswiler, J., Raecho, H., Larsen, C.P., et al. (1997). 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J. Exp. Med., 186, 47–55. Sobel, E.S., Kakkanaiah, V.N., Kakkanaiah, M., Cheek, R.L., Cohen, P.L., and Eisenberg, R.A. (1994). T-B collaboration for autoantibody production in lpr mice is cognate and MHC-restricted. J. Immunol., 152, 6011–6016. Sun, Y., Chen, H.M., Subudhi, S.K., Chen, J., Koka, R., Chen, L., and Fu, Y.X. (2002a). Costimulatory molecule-targeted antibody therapy of a spontaneous autoimmune disease. Nat. Med., 8, 1405– 1413. Sun, Y., Lin, X., Chen, H.M., Wu, Q., Subudhi, S.K., Chen, L., and Fu, Y.X. (2002b). Administration of agonistic anti-4-1BB monoclonal antibody leads to the amelioration of experimental autoimmune encephalomyelitis. J. Immunol., 168, 1457–1465. Sytwu, H.K., Lin, W.D., Roffler, S.R., Hung, J.T., Sung, H.S., Wang, C.H., Cheng, T.L., Tsou, S.C., Hsi, S.C., and Shen, K.L. (2003). Anti-4-1BB-based immunotherapy for autoimmune diabetes: Lessons from a transgenic non-obese diabetic (NOD) model. J. Autoimmun., 21, 247– 254. Takahashi, C., Mittler, R.S., and Vella, A.T. (1999). Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal. J. Immunol., 162, 5037–5040. Tan, J.T., Whitmire, J.K., Ahmed, R., Pearson, T.C., and Larsen, C.P. (1999). 4-1BB ligand, a member of the TNF family, is important for the generation of antiviral CD8 T cell responses. J. Immunol., 163, 4859–4868. Tan, J.T., Whitmire, J.K., Murali-Krishna, K., Ahmed, R., Altman, J.D., Mittler, R.S., Sette, A., Pearson, T.C., and Larsen, C.P. (2000). 4-1BB costimulation is required for protective anti-viral immunity after peptide vaccination. J. Immunol., 164, 2320–2325. Tarrant, T.K., Silver, P.B., Chan, C.C., Wiggert, B., and Caspi, R.R. (1998). Endogenous IL-12 is required for induction and expression of experimental autoimmune uveitis. J. Immunol., 161, ¯ 122–127. Taylor, P.C., Plater-Zyberk, C., and Maini, R.N. (1995). The role of the B cells in the adoptive transfer of collagen-induced arthritis from DBA/1 (H-2q) to SCID (H-2d) mice. Eur. J. Immunol., 25, 763–769. Theofilopoulos, A.N., and Dixon, F.J. (1981). Etiopathogenesis of murine SLE. Immunol. Rev., 55, 179–216. Theofilopoulos, A.N., and Dixon, F.J. (1985). Murine models of systemic lupus erythematosus. Adv. Immunol., 37, 269–390.

Autoimmune Diseases

115

Tuohy, V.K., Yu, M., Yin, L., Kawczak, J.A., Johnson, J.M., Mathisen, P.M., Weinstock-Guttman, B., and Kinkel, R.P. (1998). The epitope spreading cascade during progression of experimental autoimmune encephalomyelitis and multiple sclerosis. Immunol. Rev., 164, 93–100. van Rappard-Van der Veen, F.M., Kiesel, U., Poels, L., Schuler, W., Melief, C.J., Landegent, J., and Gleichmann, E. (1984). Further evidence against random polyclonal antibody formation in mice with lupus-like graft-vs-host disease. J. Immunol., 132, 1814–1820. Vanderlugt, C.L., Neville, K.L., Nikcevich, K.M., Eagar, T.N., Bluestone, J.A., and Miller, S.D. (2000). Pathologic role and temporal appearance of newly emerging autoepitopes in relapsing experimental autoimmune encephalomyelitis. J. Immunol., 164, 670–678. Vogelsang, G.B. (2001). How I treat chronic graft-versus-host disease. Blood, 97, 1196–1201. Wacker, W.B., Donoso, L.A., Kalsow, C.M., Yankeelov, J.A., Jr., and Organisciak, D.T. (1977). Experimental allergic uveitis. Isolation, characterization, and localization of a soluble uveitopathogenic antigen from bovine retina. J. Immunol., 119, 1949–1958. Wekerle, H. (1991). Immunopathogenesis of multiple sclerosis. Acta. Neurol., (Napoli), 13, 197–204. Wilcox, R.A., Chapoval, A.I., Gorski, K.S., Otsuji, M., Shin, T., Flies, D.B., Tamada, K., Mittler, R.S., Tsuchiya, H., Pardoll, D.M., and Chen, L. (2002a). Cutting edge: Expression of functional CD137 receptor by dendritic cells. J. Immunol., 168, 4262–4267. Wilcox, R.A., Flies, D.B., Zhu, G., Johnson, A.J., Tamada, K., Chapoval, A.I., Strome, S.E., Pease, L.R., and Chen, L. (2002b). Provision of antigen and CD137 signaling breaks immunological ignorance, promoting regression of poorly immunogenic tumors. J. Clin. Invest., 109, 651–659. Wildner, G., and Thurau, S.R. (1995). Orally induced bystander suppression in experimental autoimmune uveoretinitis occurs only in the periphery and not in the eye. Eur. J. Immunol., 25, 1292–1297. Williams, M.S., Noguchi, S., Henkart, P.A., and Osawa, Y. (1998). Nitric oxide synthase plays a signaling role in TCR-triggered apoptotic death. J. Immunol., 161, 6526–6531. Wofsy, D., Hardy, R.R., and Seaman, W.E. (1984). The proliferating cells in autoimmune MRL/lpr mice lack L3T4, an antigen on “helper” T cells that is involved in the response to class II major histocompatibility antigens. J. Immunol., 132, 2686–2689. Yu, M., Johnson, J.M., and Tuohy, V.K. (1996). A predictable sequential determinant spreading cascade invariably accompanies progression of experimental autoimmune encephalomyelitis: A basis for peptide-specific therapy after onset of clinical disease. J. Exp. Med., 183, 1777–1788. Zhu, G., Flies, D.B., Tamada, K., Sun, Y., Rodriguez, M., Fu, Y.X., and Chen, L. (2001). Progressive depletion of peripheral B lymphocytes in 4-1BB (CD137) ligand/I-Ealpha)-transgenic mice. J. Immunol., 167, 2671–2676.

8 CD137/CD137 Ligand in Tumor and Viral Immunotherapy Ignacio Melero, Oihana Murillo, Inigo ˜ Tirapu, Eduardo Huarte, Ainhoa Arina, Laura Arribillaga, and Juan Jose´ Lasarte

1. CD137 (4-1BB) and CD137 Ligand (4-1BB-Ligand) Meet Tumor Immunology 1.1. The Arrival of Agonistic Anti-CD137 Monoclonal Antibodies Current tumor immunotherapy includes a series of approaches that intend to destroy malignant tissue by means of the cell destructive armamentarium with which the immune system is endowed. Priming and enhancing the cellular immune response against cancer cells or critical components of the tumor stroma is a difficult endeavor, but recent advances have achieved complete regressions of established transplantable rodent malignancies. Among these experimentally successful approaches we can list: (i) vaccinations with dendritic cells pulsed with tumor antigens and other vaccine formulations (Banchereau and Palucka, 2005); (ii) adoptive transfer of in vitro-preactivated lymphocytes into a tumor bearing host in whom lymphopenia has been induced (Dudley et al., 2002); (iii) systemic or local treatment with cytokines, including gene therapy approaches, that augment the cellular immune response (Murphy et al., 1996, 2005); (iv) transfection of costimulatory molecules for T cells into cancer cells; (v) molecular interference with physiological mechanisms that negatively regulate the immune response. The latter is a particularly fertile field of investigation that encompasses: (a) interference with T cell co-inhibitory molecules such as PD-1 (Dong et al., 2002; Hirano et al., 2005) and CTLA-4 (Egen et al., 2002), (b) the depletion or inactivation of regulatory T cells (Sakaguchi, 2005), and (c) the inhibition of enzymes whose activity locally interferes with T cell activation (Muller et al., 2005). Translation towards human therapy is slowly progressing although, in general, the results in mice tend to be more efficacious than the observations that are being made in the early clinical trials. Nonetheless, there is great hope that the Ignacio Melero, Oihana Murillo, Inigo ˜ Tirapu, Eduardo Huarte, Ainhoa Arina, Laura Arribillaga, and Juan Jose´ Lasarte • Centro de Investigaci´on M´edica Aplicada y Cl´ınica Universitaria. Universidad de Navarra. Pamplona, Spain. 117 CD137 Pathway: Immunology and Diseases. Edited by Lieping Chen, Springer, New York, 2006

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combination of several of these approaches in a stepwise fashion would optimize results and become standard clinical practise (Pardoll et al., 2004). In 1995, the field of costimulation in the immune response against tumors was a hot topic. Exciting experiments had shown that transfection of tumor cells with costimulatory ligands such as B7-1 (CD80) increased their immunogenicity in such a fashion that those tumor cells became rejected and were able to control the growth of untransfected tumor cells injected elsewhere in the mouse (Chen et al., 1992; Townsend and Allison, 1993). A key feature of these systems is that tumor cells had to present tumor rejection antigens in order to be enhanced by the artificial costimulation (Chen et al., 1994). Monoclonal antibodies directed toward lymphocyte membrane molecules can potentially act as artificial agonistic ligands for such receptors, providing signals that could mimic those received from the natural costimulatory ligands (Murillo et al., 2003). Following this simple reasoning, a number of experiments screened for an antitumor effect of monoclonal antibodies of this kind. This screening was performed by administering a series of this type of agents to mice bearing established mastocytomas in the peritoneal cavity. One of such antibodies, that was able to prevent tumor progression, was identified as directed against CD137 (4-1BB) (Melero et al., 1997a). Immediately after, two other monoclonal antibodies of the same specificity (Shuford et al., 1997) were tried in mice bearing the P815 mastocytoma either intraperitoneally or subcutaneously. The rapid progression of tumors in the control groups contrasted with the almost constant rejection in the treated groups. Some of the shrinking subcutaneous tumors were surgically removed to show a clear lymphocyte infiltrate entering the tumor nodules from the periphery. Tumor infiltrates chiefly contained T cells (both CD8+ and CD4+ ), as well as macrophages. Accordingly, a series of experiments clearly showed an increase of tumor specific cytotoxic T cell activity in the spleen of the treated mice (Melero et al., 1997a). It was surprising to observe that such cytotoxicity was detectable in the freshly isolated splenocytes even without restimulation with irradiated tumor cells in culture. Successful treatment and similar mechanistic observations were made in established tumors derived from the Ag104A sarcoma, a spontaneous malignancy considered of low intrinsic immunogenicity (Melero et al., 1997a). In the P815 mastocytoma model, it was noticed that selective depletion of either CD4 or CD8 T cells gave rise to a complete loss of the antitumor effect (Melero et al., 1997a). Subsequent studies have found that the requirement for CD4 T cell help is tumor model dependent (Miller et al., 2002). Selective depletion studies showed that NK cells were also absolutely necessary for the antitumor effect of agonistic anti-CD137 monoclonal antibodies (Melero et al., 1998b). CD137 is not detectable on the surface of resting NK cells but was found to be readily expressed on cytokine-activated Natural Killer cells (Melero et al., 1998b; Wilcox et al., 2002b).

1.2. A Comparison with Anti-CTLA-4 Monoclonal Antibodies During the time while these first observations were being made, the group of J.P. Allison described the antitumor effects of systemic administration of

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monoclonal antibodies that interfered with the function of CTLA-4 (CD152) (Leach et al., 1996). A side by side comparison with anti-CD137 mAb was made that indicated that the therapeutic activity against tumor mouse models was comparable, with certain superiority for the anti-CD137 in some tumors (Melero et al., 1997a). The obvious attempts at finding additive or synergistic effects have failed both at inducing stronger immune responses and at achieving more effective therapeutic activity. In other words, equal or even less tumor rejections were observed in the combined treatment group when compared to each antibody given separately (A. Arina et al. unpublished observations). The reasons underlying the lack of effectiveness of the combination are still unknown but might be related to the differential in vivo effects reported for anti-CD137 mAb on CD4 and CD8 T cells. With regard to potential toxicity, none of these antibodies appeared to cause any serious adverse effects in mice. However, the striking autoimmune phenotype of CTLA-4−/− mice (Tivol et al., 1995; Waterhouse et al., 1995) warned somehow about the serious autoimmnunity that was eventually reported in early clinical trials with anti-CTLA-4 mAb (Hodi et al., 2003; Phan et al., 2003; Sanderson et al., 2005). The soothing early safety observations with anti-CD137 mAb have received a further boost when it was repeatedly documented that anti-CD137 mAb not only did not induce autoimmunity, but on the contrary, prevented or successfully treated experimental autoimmune conditions in mice (Mittler, 2004; Seo et al., 2004; Sun et al., 2002a, 2002b). This became a puzzling paradox in the field of CD137 research because the observations of immune tumor rejections were difficult to reconcile with the prevention of autoimmune conditions by the very same therapeutic agents. Early observations had suggested that, although anti-CD137 mAb costimulated CD4+ T lymphocytes in culture, in vivo treatment with the antibodies resulted in a decrease of CD4 T cell helper functions, at least for antibody production against certain antigens (Shuford et al., 1997). Several hypotheses to explain this paradox have been raised including the ability of anti-CD137 agonistic antibodies to enhance the proliferation of regulatory T cells (Zheng et al., 2004), as well as the ability of anti-CD137 mAb to induce the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) expression (Seo et al., 2004), through a mechanism dependent on IFN-γ overproduction (Seo et al., 2004). Although the existence of these conflicting mechanisms reassures safety in clinical translational research, it provides a rationale to temporarily tamper with these anti-CD137dependent immunosuppresive mechanisms in order to further enhance antitumor immunity, even at the risk of inducing autoimmunity. Other specificities of mAb that enhanced antitumor responses were eventually described. Agonistic anti-CD40 mAbs are another set of promising agents of this kind (French et al., 1999). Side by side comparisons with anti-CD137 were difficult to perform and there were large differences depending on the tumor model, although in general anti-CD137 mAbs seemed to be more potent in the tested instances (A. Arina et al. unpublished observations). Unfortunately again, the antitumor effects of anti-CD137 and anti-CD40 mAbs were not mutually enhanced, as it had been anticipated based on the mechanism of action of anti-CD40 mAb that are postulated to act by licensing and activating dendritic cells for full CTL priming (French et al., 1999; Lanzavecchia, 1998).

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In contrast to our unpublished observations at the time of going to press two important studies have shown that anti-CD137 mAbs can synergize with antiCTLA-4 mAb (Kocak et al., 2006) and anti-CD40 (Uno et al., 2006). In the case of anti-CTLA-4 the synergy was observed against the MC38 colon carcinoma even with a human anti-CD40 mAb given to huCD40 knock-in mice. In the case of agonistic anti-CD40 mAbs the synergistic activity with anti-CD137 mAbs has been described against the 4T1 breast carcinoma. The therapeutic effect of the antiCD137 plus anti-CD40 combination is further enhanced by an anti-DR5 mAb that causes tumor cell apoptosis and presumably increase cross-presentation of tumor antigens.

1.3. Transfection of CD137 Ligand into Malignant Cells Gene therapy of cancer is an active field of research that was even more hope-raising at the late nineties. At the sight of the first results with the agonistic anti-CD137 monoclonal antibodies, it became a reasonable alternative to endow tumor cells with surface expression of the only characterized ligand for this surface costimulatory molecule (Goodwin et al., 1993). In order to test this idea a panel of retrovirus encoding the CD137 ligand cDNA were generated (Melero et al., 1998a). Transfected cells were able to bind a CD137 chimeric protein. Stable transfectants expressing high levels of CD137 ligand were selected in various tumors that included P815 mastocytoma and AG104A (Melero et al., 1998a). Transfected P815 cells were rejected after a transient growth in 92% of the cases raising systemic immunity against subsequent challenge. By contrast Ag104A CD137L+ transfectants progressed as lethal tumors regardless of expressing CD137 ligand (Melero et al., 1998a). This finding was reminiscent of the fact that this tumor cell line was able to graft as a lethal tumor even if transfected to express high levels of B7-1 (CD80) on its surface (Chen et. al., 1994). Interestingly, double transfectants co-expressing both CD137 ligand and CD80 were rejected in 60% of cases due to a stronger CTL response. This finding had important implications that suggested that the CD80-CD28 pathway and the CD137-CD137 ligand pathways of costimulation could be cross-talking (Melero et al., 1998a). Cross-talk was possible at several levels that importantly included the effect of CD28 ligation as enhancing the expression of CD137 on the plasma membrane of primed T lymphocytes (Melero et al., 1998a). Previous studies had nonetheless reported that CD137 and CD28 could operate independently (Chu et al., 1997). However, in vivo blockade of the CD28 pathway eliminated the stronger induction of CTLs displayed by the CD137L+ transfectants (Melero et al., 1998a). It is of note that in these models, rejection was dependent on CD8+ lymphocytes, but not on CD4+ T cells. From the beginning it became apparent that attempts of treatment with these CD137L+ transfectants for concomitant untransfected tumors were not successful, raising questions on the viability of this treatment option. It was therefore clear that the therapeutic effect of the agonistic anti-CD137 antibodies was more potent. Reasons are still unclear, although two explanations have been raised: higher

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affinity of the antibodies acting as artificial ligands; and the ability of the antibodies to chase antigen activated T cells everywhere in the organism without need for cell to cell contact to deliver costimulation (Murillo et al., 2003). The duration of the costimulatory interaction conceivably longer with mAbs could also be critical. One year later, these results were confirmed using CD137 ligand stable transfectants in the A20 lymphoma cell line (Guinn et al., 1999). It was found that CD137 ligand, CD80 and CD86 contributed to the generation of antitumor CTLs as deduced from the in vivo effect of blocking antibodies or chimeric proteins. These findings again indicated a clear role for the coexpression of the CD28 ligands (CD80 and CD86) along with CD137 ligand, a concept that was reinforced with subsequent studies (Guinn et al., 1999). Interestingly, some tumors that escaped rejection showed loss of CD137 ligand (Guinn et al., 1999), thus providing evolutionary evidence for selection against the expression of this costimulatory molecule. In the case of the A20 lymphoma, tumor cells expressed MHC class II, but the in vivo role of CD4 T cells was not explored. Nonetheless, indirect experiments suggested that the expression of CD137 ligand was not very efficient at costimulating the primary MLR reaction that is mainly mediated by alloreactive naive CD4+ T cells (Guinn et al., 1999). Surprisingly a latter report showed that human carcinoma cell lines and primary carcinomas expressed low but detectable levels of functional CD137 ligand (Salih et al., 2000). In subsequent studies by the same group it was found that CD137 ligand could become a soluble molecule with increased concentrations in patients with hematologic malignancies (Salih et al., 2001). Understanding the role of this soluble functional CD137 ligand, that is also found increased in chronic inflammation, might shed light to understand the reasons behind ectopic CD137 ligand expression in tumors. The still unclear reasons behind this observation cast further doubts on whether transfection of tumor cells with the natural ligand of CD137 holds any hope for clinical translation.

2. Developments and Improvements on CD137/CD137 Ligand Therapeutic Strategies 2.1. Immunization: CD137 Breaks Ignorance and Tolerance Agonistic anti-CD137 mAb need a certain degree of immunogenicity in the tumor in order to be able to exert their antitumor effects (Wilcox et al., 2002a). It became clear that transplantable tumors could be categorized into responsive and not responsive to anti-CD137 therapy, in a fashion that correlated with the intrinsic degree of immunogenicity of the tumor cells. It is still unknown if this basal immunization takes place upon presentation by endogenous dendritic cells (cross-presentation or surrogate presentation of the antigens) (Chen et al., 2004; van Mierlo et al., 2004) or directly by tumor cells reaching the draining lymph nodes (Ochsenbein et al., 1999). Whatever the case, it was conceivable that artificial

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vaccination with tumor antigens would be capable of starting the immune response generating antigen-specific activated T cells that would start to express CD137. A compelling study addressed this concept using peptide vaccination plus agonistic anti-CD137 mAb treatment (Wilcox et al., 2002a). It was demonstrated that several poorly immunogenic tumors, including C3 sarcoma, TC-1 lung carcinoma, and B16-F10 melanoma, once established as solid tumors or metastases, were refractory to treatment by anti-CD137 mAb due to immunological ignorance, rather than anergy or clonal deletion of tumor antigen-specific CTLs. Breaking CTL ignorance by immunization with tumor antigen-derived peptides, although insufficient to stimulate a curative CTL response, was necessary for anti-CD137 mAb to induce a CTL response leading to the regression of established tumors (Wilcox et al., 2002a). In this case, a well described epitope of the E7 oncogene of human papillomavirus 16 (HPV-16) was used as a surrogate tumor antigen (Wilcox et al., 2002a). There was evidence that under the conditions tested, this antigen remains ignored by the immune system without signs of immunization or tolerization (Melero et al., 1997b). This situation of immune ignorance is postulated to be a frequent type of relationship of potential tumor antigens and the immune system (Chen, 1998). However, in certain experimental examples the tumor antigens specifically cripple the immune system leaving it disabled to mount a proper immune response. This could be a result of deletion (physical elimination) of the lymphocytes with antigen receptors displaying relevant avidity for the antigen, but also a result of the induction of a paralysis in still viable specific lymphocytes, a status known as T-cell anergy. The deleting scenario is quite dire for immunotherapy, but not so much in the second case. It has been recently demonstrated that repeated treatment with anti-CD137 mAb can certainly revert a status of anergy induced against a tumor antigen. This remarkable effect was demonstrated for three surrogate tumor antigens towards which this status of anergy had been induced by peptide immunization (Wilcox et al., 2004). Active immunization using antigen-presenting dendritic cells as adjuvants has generated an impressive amount of preclinical and clinical results (Banchereau and Palucka, 2005). In these settings agonistic anti-CD137 mAb have been observed to display a truly synergistic effect (Ito et al., 2004; Tirapu et al., 2004) Important ongoing research will tell if anti-CD137 augments the efficacy of conventional treatments that destroy tumor cells releasing antigens, such as local radiotherapy or systemic chemotherapy. The concept of using an immunostimulating antibody and a procedure of immunization is not unique to anti-CD137 mAb. In the preclinical development of anti-CTLA-4 monoclonal antibodies, it became apparent that there was synergy of anti-CTLA-4 mAb with vaccination (Hurwitz et al., 1998), that even could lead to autoimmunity by breaking tolerance (i.e., vitiligo) (Phan et al., 2003). Clinical trials with a humanized anti-CTLA-4 mAb have only been conducted in combination with vaccination strategies such as GM-CSF transfected allogenic tumor cells (Hodi et al., 2003) and peptides (Phan et al., 2003). The overall concept of vaccination plus an immunostimulating monoclonal antibody could become very important for clinical translation (Murillo et al., 2003).

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2.2. CD137 as an Adjuvant for Adoptive T Cell Therapy and Bone Marrow Transplantation Adoptive transfer of antigen specific T lymphocytes is certainly a promising strategy for cancer treatment (Dudley and Rosenberg, 2003), as well as for chronic or latent viral infections (Moss and Rickinson, 2005). Several approaches are possible which share in common the obtainment of autologous lymphocytes that are stimulated with antigen in vitro and then artificially expanded for infusion. In these treatments interleukin-2 is commonly coadministered to keep injected lymphocytes alive and activated. There is strong experimental evidence that Interleukin15 would probably substitute with advantage these effects of IL-2 (Waldmann, 2003). Lymphodepletion of the host previously to lymphocyte infusion helps the antitumor effect correlating with less competition for T cell growth and survival factors, as well as with the removal of suppressor regulatory T cells (Treg cells) (Klebanoff et al., 2005). Activated T lymphocytes as those commonly infused in adoptive T cell therapies express CD137 and can receive costimulation by agonistic antibodies (Kim et al., 2001). Experimental proof of the concept for synergistic effects of adoptive T cell therapy and systemic treatment with agonistic anti-CD137 mAb is available (May et al., 2002), although there is controversy on whether the effect is exerted at promoting lymphocyte survival, activation and/or proliferation (May et al., 2002). Adoptive T cell therapy is potentiated by anti-CD137 mAb even in a transgenic model of spontaneous breast cancer in which the tumor expresses MUC-1 while the mouse is tolerant for this antigen (Mukherjee et al., 2004). CD137 can help adoptive T cell therapy also during in vitro expansions of T cells. An interesting paper using K562 as artificial antigen presenting cells shows much better in vitro induction of CTLs for adoptive therapy if the artificial antigen presenting cells are endowed with the ability to stimulate simultaneously the TCR, CD28, and CD137 (Maus et al., 2002). Allogeneic bone marrow transplantation is a procedure with success against many hematological malignancies. Its curative potential relies on high dose chemotherapy but importantly also on the function of the donor immune cells that attack the allogeneic malignant cells (Graft versus leukemia effect). However, this frequently pays the price of damage to certain host organs that are also attacked, giving rise to acute and chronic graft versus host disease. This is an area in which agonistic monoclonal antibodies against CD137 might find an important niche for application. As previously commented, anti-CD137 mAb can treat autoimmune conditions that share the property of a pathogenic role of autoreactive CD4 T cells. In this setting of bone marrow transplantation, it became apparent since the earliest studies with agonistic anti-CD137 mAb that they could find an application, because these antibodies enhanced CD8 dependent graft versus host reactions while inhibiting CD4 responses (Shuford et al., 1997). Indeed, subsequent reports point to the conclusion that anti-CD137 treatment can enhance acute graft versus host reactions including the graft versus leukemia effect (Blazar et al., 2001), while greatly ameliorating chronic graft versus host disease by inhibiting a pathogenic CD4+ mediated T-cell response (Kim et al., 2005).

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Infusion of donor T lymphocytes is common practice upon relapse of the malignancy after bone marrow transplantation and there are experimental indications that suggest that anti-CD137 mAb could also enhance this form of adoptive T cell therapy. With regard to NK cells, there is increasing evidence that these cells can mediate alloreactive recognition of target cells in certain mismatches of class I MHC alleles between donor and recipient through “missing self ” recognition (Ruggeri et al., 2005). This is known to be clinically efficacious in haploidentical bone marrow transplantation both to prevent graft versus host disease by killing the host dendritic cells that initiate the reaction, and by killing leukemia cells thus preventing the relapse (Ruggeri et al., 2002). These findings have raised considerable expectancy at exploiting the strategy for adoptive NK cell therapy with semiallogeneic NK cells (Miller et al., 2005). The fact that CD137 is expressed on cytokine-activated NK cells in such a way that its ligation further promotes NK activation, cytokine secretion and proliferation has not been explored in the alloreactive scenario (Melero et al., 1998b; Wilcox et al., 2002b). These investigations could become important for the future of these protocols of adoptive cell therapy with NK cells, that are already in clinical trials (Miller et al., 2005).

2.3. CD137 Acting in Synergy with Cytokines and Other Costimulatory Molecules Interleukin-12 as a recombinant protein has been one of the most promising anti-cancer agents arising from preclinical models. Unfortunately an underestimated maximal tolerated dose led to serious adverse effects in a phase II clinical trial that halted development. However systemic toxicity and local efficacy made IL-12 an excellent candidate for local gene therapy (Colombo and Trinchieri, 2002; Murphy et al., 2005). Tumor cell gene transfer of IL-12 has remarkable antitumor effects in mouse models that are dependent on increases of antitumor CTL, NK cells and on antiangiogenic effects triggered by this cytokine and its downstream mediators. A very exciting series of experiments demonstrated that IL-12 gene transfer by intratumor injection of a recombinant adenovirus synergizes with systemic agonistic monoclonal antibodies against CD137 (Chen et al., 2000). Although co-transduction of the gene encoding CD137 ligand with IL-12 genes had some effect, it was not as potent as the one achieved by systemic antiCD137 mAb (Martinet et al., 2000, 2002). The therapeutic effect in this case was in great part mediated by NK cells. These synergistic effects are also observed upon injection into the tumor tissue of dendritic cells that have been engineered to produce IL-12 by means of recombinant adenovirus (Tirapu et al., 2004). Direct intratumoral injection of both a recombinant adenovirus encoding IL-12 and dendritic cells engineered to produce IL-12 have been tested in pilot clinical trials demonstrating safety but modest clinical results (Mazzolini et al., 2005; Sangro et al., 2004). Hence, the road is paved for testing the combination of IL-12 gene therapy with anti-CD137 mAbs.

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The modest effect of CD137 ligand transfection either into tumor cells (Martinet et al., 2000, 2002) or into dendritic cells (Wiethe et al., 2003) has strikingly contrasted with the outstanding efficacy achieved by introducing into tumor cells a gene encoding for a transmembrane attached version of an agonist single chain (ScFv) antibody against CD137 (Ye et al., 2002). This artificial ligand promotes tumor rejection and generates systemic immunity in which both NK and CD4 T cells have a central role (Ye et al., 2002). CD137/CD137 ligand relatives in the TNF/TNF-Receptor family have also immunestimulating antitumor properties if stimulated with antibodies or if transfected into tumor cells. These include OX-40(CD134)/OX40 ligand, CD40/CD40 ligand, CD27/CD27 ligand (CD70). This issue has been recently reviewed (Croft, 2003; Watts, 2005) and exceeds by far the coverage of this chapter. It must be said that some of the most exciting experiments in the field of CD137 in immunotherapy involve the reported synergy of OX40 and CD137 at inducing cellular immune responses upon concomitant stimulation. Enforced dual costimulation through CD137 and OX40 induced profound specific CD8 T cell clonal expansions (Dawicki et al., 2004; Lee et al., 2004). The synergistic response of the specific CD8 T cells persisted for several weeks, and the expanded effector cells resided throughout lymphoid and nonlymphoid tissue. Dual costimulation through CD137 and OX40 did not augment the number of rounds of T cell division in comparison to single costimulators, but rather enhanced lymphocyte accumulation in a cell-intrinsic manner. It was shown that CD8 T cell clonal expansion and effector function did not require T cell help, but accumulation in non-lymphoid tissue was predominantly CD4 T cell-dependent (Lee et al., 2004). Dual costimulation mediated rejection of an otherwise resistant established murine sarcoma. Importantly, effector function directed towards established tumors was CD8 T cell dependent, while being entirely CD4 T cell independent. Available data suggest that OX40 seems to preferentially costimulate CD4 Th1, cells mirroring the effects of CD137 costimulation on CTLs (Lee et al., 2004). These observations with anti-OX40 mAb and OX40L are reminiscent of the synergistic effects reported upon cotransfection into tumor cells of two costimulatory molecules CD137 ligand and B7-1 (CD80) (Guinn et al., 1999; Melero et al., 1998a). In addition, some surface glycoprotein interactions are coinhibitory, as opposed to costimulatory for the T cell response. For instance the B7-H1 molecule that binds to PD-1 and some other non-identified surface T cell ligand can strongly inhibit the T cell response (Dong et al., 2002). In fact B7-H1 is expressed by many experimental and human tumors in vivo as an escape mechanism (Dong et al., 2002). Interestingly blocking B7-H1/PD-1 interactions with antibodies has antitumor effects (Dong et al., 2002) that are truly synergistic with the administration of anti-CD137 mAb to induce complete tumor eradication (Hirano et al., 2005). The mechanism involves a prevention of the resistance of tumor cells to the local killing by infiltrating lymphocytes (Hirano et al., 2005), but other mechanistic possibilities such as tolerance induction cannot be definitively excluded.

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Figure 8.1. Schematic representation of the different sites and modes of action of agonistic antiCD137 mAb, gene transfer of CD137 ligand, and combination strategies.

As mentioned before, there is no synergy between blocking the CTLA-4 inhibitory receptor and treatment with anti-CD137 mAb. Finding the reason could be informative for designing therapies based on CD137 and the concept of multiple costimulation. Combination immunotherapy, in other words, the sequential or simultaneous combination of several mechanisms can be one way to eventually reach curative treatments for humans (Pardoll et al., 2004). We have previously suggested that if immunotherapy were a car, the engine should be started (immunization), the gas pedal should be pressed (costimulation) and the brakes should be released (by disconnecting co-inhibitory receptors). This car can take us to success, but we will probably will have to face autoimmunity in our race (Tirapu et al., 2002). In Figure 8.1, a schematic representation of the explored combinations that exploit the CD137/CD137 ligand pathway has been represented.

3. CD137/CD137 Ligand in the Antiviral Immune Response and in Viral Vaccination Immunotherapy has potential for chronic and latent viral infections. Therapeutic vaccination and adoptive T-cell transfer are the strategies that are being explored for HIV and chronic viral hepatitis. Mice lacking CD137 or CD137 ligand show defects in CD8 T cell responses against viruses (DeBenedette et al.,

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1999; Kwon et al., 2002; Tan et al., 1999), with no defects in antibody or CD4 T cell responses to vesicular stomatitis virus (VSV), LCMV, or influenza virus (Kwon et al., 2002; Tan et al., 1999). Studies in CD137 ligand−/− mice suggest that the role of this molecule is mainly focused in the long term CTL response and in the induction and maintenance of memory in the CD8+ T cell compartment (Bukczynski et al., 2004). These features make the CD137/CD137 ligand pair a very interesting target for manipulation in antiviral immunotherapy. However, very few reports have explored this possibility. A pioneering study (Halstead et al., 2002) demonstrated increased CTL responses against experimental influenza. In vivo CD137 stimulation with an agonistic monoclonal antibody enhanced the primary CD8+ T cell response to influenza type A viral infection in mice. Stimulation of CD137 increased the absolute number of CD8+ T cells to influenza epitopes in the lungs of infected animals, preferentially expanding CD8+ T cells that recognized nondominant epitopes and greatly enhancing direct ex vivo cytotoxicity. The studies confirmed that the CD137 costimulatory pathway could operate independently from CD28 (Halstead et al., 2002). The effects enhancing memory to a broadened series of epitopes suggested potential to enhance the effect for vaccines relying on CTLs for prophylaxis or therapy. The induction of protective or therapeutic cellular immunity against hepatitis C virus (HCV) is a difficult goal. Immunization with a recombinant adenovirus encoding HCV-NS3 (RAdNS3) could partially protect mice from challenge with a vaccinia virus encoding HCV antigens. It was found that treatment with anti-CD137 mAb after the administration of a suboptimal dose of RAdNS3 enhanced cytotoxic and T helper cell responses against HCV NS3 (Arribillaga et al., 2005). Importantly, the ability of RAdNS3 to induce protective immunity against challenge with a recombinant vaccinia virus expressing HCV proteins was markedly augmented. Thus, combination of immunostimulatory anti-CD137 mAb with recombinant adenoviruses expressing HCV proteins might be useful in strategies of immunization against HCV. However in this case the epitope broadening seen with influenza viruses was not seen with subdominant peptides of the NS3 antigen, indicating that this might be not a general feature (Arribillaga et al., 2005). In another study, agonistic anti-CD137 mAb were shown to potentiate threefold the T-cell immunity raised by vaccination with poxviruses (Munks et al., 2004). These authors studied the murine CD8 T cell response to a DNA prime, poxvirus boost vaccine, similar to those used for human and simian immunodeficiency viruses vaccines. CD137 stimulation increased the number of functional memory CD8 T cells by two- to four-fold. Interestingly the enhancement was observed both at the peak of the response and in the memory phase. In this report the combination with anti-OX40 mAb was also tested. OX40 stimulation increased the number of antigen-specific CD4 T cells approximately three-fold. Stimulating both CD137 and OX40 enhanced the CD8 T-cell response more than CD137 alone (Munks et al., 2004). These studies agree to suggest a potential in vaccination against viral diseases in which protection is achieved by memory T cells and deserves attention for

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translation into clinical benefit. On the contrary, anti-CD137 mAb should not enhance the vaccines relying on the induction of neutralizing antibodies.

4. Reflections on the Mechanism(s) of Action At this point of time many features converge in the potential of anti-CD137 mAb for human therapy. Enhancement of CTL responses while preventing autoimmunity seem to fulfill a new unexpected paradigm in immunotherapy. We must admit that we have only partial knowledge of the mechanisms operating behind this paradox. A better understanding of both kinds of apparently opposing effects would lead to improvements in future clinical strategies. Signaling through CD137 is an active area of study. Involvement of TRAF2 and 1, MAP kinases and NF-kB are well established (Watts, 2005). Recent evidence also shows that CD137 stimulation with agonistic antibodies amplifies many signaling events triggered through the T-cell receptors with data implicating molecular crosstalk at the immunological synapse (Nam et al., 2005). Stimulating the CD137 receptor with small molecules is an active and attractive area of research seeking to trigger similar signals. Signaling in these molecules of the TNF-Receptor family requires multimerization by ligand (Croft, 2003) a task in which the activity of bivalent agonistic antibodies could be improved by further crosslinking. The signaling molecular players through other costimulatory members of the TNFR family seem to be quite similar and there is hope to activate these signaling pathways with drugs (Croft, 2003). A key point to unravel in the future is why the antibodies have a differential effect on CD8 cells versus CD4 cells that is not observed with the natural ligand. Different tyrosine phosphorylation patterns have been reported in these two T lymphocyte subsets (Shuford et al., 1997), but these early investigations have not been pursued forward. Recent evidence has pinpointed to a pathway that could explain the beneficial effects in autoimmunity as observed in collagen induced arthritis (Seo et al., 2004). This study documents that a stronger CTL activation leads to an IFNγdependent induction of IDO, which in turn downregulates CD4 autoimmunity. This is possibly only part of the story because this mechanism should not account for the differential effects on CD4 and CD8, and other mechanisms will be probably unraveled (Mittler, 2004). Among other possibilities the effects of agonistic antibodies on the Foxp3+ natural regulatory T cells is going to be a productive area of research (Choi et al., 2004). Agonistic CD137 can costimulate proliferation of these lymphocytes (Zheng et al., 2004) and maybe on other cells with suppressor activity. For tumor and viral immunotherapy, it might be wise to tackle these check point mechanisms even at the risk of triggering autoimmunity or systemic inflammatory reactions.

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5. Using Preclinical Information for Clinical Development of Immunotherapy Taking anti-CD137 agonistic monoclonal antibodies to the clinic it is a difficult but not an impossible task. As represented in Figure 8.2, it first involves the production of a panel anti-human CD137 mAbs through classical production of mouse hybridomas or in transgenic mice that produce fully human antibodies. If the classical technology is chosen, genetic engineering will be required to humanize the sequence. At this time the antibodies should be selected for being able to costimulate the in vitro proliferation of human T cells ( ). However this property on its own will not ensure their therapeutic behaviour in vivo. To more accurately foresee this feature, two types of experiments would be feasible: (i) testing the effect of the antibody on CTL responses of SCID mice reconstituted with human lymphocytes, and (ii) generating Knock-in mice in which the human CD137 sequence would be regulated by the same factors that induce mouse CD137. In the latter case the effects of the selected humanized antibodies should match the ones already achieved against established transplanted tumors. Humanized monoclonal ROAD MAP TO CLINICAL TRANSLATION OF ANTI CD137 (4-1BB) MONOCLONAL ANTIBODIES CLASSICAL mAb PRODUCTION

PRODUCTION IN TRANSGENIC MICE REARRANGING HUMAN IMMUNOGLOBULINS

mAb HUMANIZATION BY GENETIC ENGINEERING

IN VITRO SELECTION: ASSESSING T CELL COSTIMULATION

IN VIVO SELECTION FOR CTL ENHANCEMENT : 1. HUMAN PBL RECONSTITUTED SCID MICE 2. KNOCK IN MICE FOR HUMAN CD137

GMP-BIOPROCESING

TROUBLE SHOOTING

QUALITY CONTROL CLINICAL-GRADE BATCHES RODENTS TOXICOLOGY REGULATORY MONKEYS -ACCUTE AFFAIRS -LONG TERM

DOSE FINDING PHASE I CLINICAL TRIALS (Advanced tumors)

RESULT EVALUATION: 1. PHARMACOKYNETICS 2. SAFETY 3. OPTIMAL DOSING 4. ASSESSMENT OF IMMUNITY

MELANOMA RENAL CELL CARCINOMA

INDUSTRIAL PRODUCTION SCALE-UP

MORE ADVANCED CLINICAL TRIALS (PHASE II) IMMUNIZATION PROCEDURES COMBINATIONS WITH RADIOTHERAPY/CHEMOTHERAPY OTHERS POTENCIAL INDICATIONS (CANCER, CHRONIC VIRAL INFECTION)

Figure 8.2. Stepwise development and decision making for development of anti-CD137 agonistic mAb for clinical application.

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antibodies are mandatory towards translation, but we should not forget that reported preclinical efficacy involves only rat immunoglobulins injected into mice. However to the best of our knowledge there is no reason to forecast any advantage for heterologous antibodies. Once a particular antibody is selected, bioprocessing experts would have to find a way to produce enough quantities for toxicology, quality control, and pilot clinical trials. Classical protein production in cell cultures is the easiest possibility, while other approaches to be considered could use more creative biotechnology in transgenic plants or mammals. A continuous improvement of these techniques is to be implemented to satisfy regulations and the potential demand. The clinicians designing and leading clinical development will have to perform the dose finding trials in patients with advance melanoma and renal cell carcinoma. Another possibility would be to include in these pilot trials patients with a broad spectrum of malignancies. Each alternative has potential advantages and drawbacks. Melanoma and renal cell carcinoma are probably the patients with the highest likelihood to detect some encouraging clinical efficacy at an early stage. Good early data would foster investment and will make the assessment of antitumor immunity an easier task. Combination with immunization strategies is a temptation and probably a wise alternative, since autoimmunity is not expected. Introducing model antigens for CTL and antibody responses would provide proof of the biological effects and give an idea of its intensity. However, artificial antigens might unbalance immunodominance (Yewdell and Del Val, 2004) and endanger the response to tumor antigens. Therefore the introduction of these model antigens in early trials is a debatable choice. We should bear in mind that autoimmunity with anti-CTLA-4 monoclonal antibodies was not anticipated before reaching the clinical arena already in combination with cancer vaccines. However in that case, the severe autoimmunity phenotype of CTLA-4−/− mice (Tivol et al., 1995; Waterhouse et al., 1995) should have advised a more prudent course for development. Nonetheless, careful assessment of autoimmunity is a must in any case in CD137 development. Dose finding is going to be very interesting. The antibodies would inhibit the response against themselves prolonging their half life even if not completely humanized. A humanized monoclonal antibody has already shown these properties in monkeys (Hong et al., 2000). A range from 0.1 mg/Kg to 40 mg/Kg of weight is to be tested with close follow up of the pharmacokinetics that is anticipated to be excellent. Readministration of doses should be decided from non-human primate pharmacokinetics but will probably consist in weekly or two-weekly repetitions. Studies in monkeys should have thoroughly pre-tested an overdose of these antibodies. Unfortunately, there are not reliable dosing studies in the mouse models, so we do not know the optimal preclinical regime of dosing for cancer treatments. If an IgG4 tail is chosen for the humanized antibody, no adverse effects are anticipated beyond the biological effects on the immune response. We should be confident that the prevention of autoimmunity as observed in mice would be conserved in human beings.

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Once pilot trials are finished and evaluated, the impetus should not fade away even in the absence of clinical efficacy or in the presence of a modest one. That would be the moment to try the best synergistic strategies defined in preclinical models, extending treatment to other diseases, that at some stage should include chronic viral infections resistant to conventional treatment. We are hopeful that this road-map (Figure 8.2) will be followed in the near future. Preclinical data doubtlessly support clinical development.

References Arribillaga, L., Sarobe, P., Arina, A., Gorraiz, M., Borras-Cuesta, F., Ruiz, J., Prieto, J., Chen, L., Melero, I., and Lasarte, J.J. (2005). Enhancement of CD4 and CD8 immunity by anti-CD137 (41BB) monoclonal antibodies during hepatitis C vaccination with recombinant adenovirus. Vaccine 23, 3493–3499. Banchereau, J., and Palucka, A.K. (2005). Dendritic cells as therapeutic vaccines against cancer. Nat. Rev. Immunol., 5, 296–306. Blazar, B.R., Kwon, B.S., Panoskaltsis-Mortari, A., Kwak, K.B., Peschon, J.J., and Taylor, P.A. (2001). Ligation of 4-1BB (CDw137) regulates graft-versus-host disease, graft-versus-leukemia, and graft rejection in allogeneic bone marrow transplant recipients. J. Immunol., 166, 3174–3183. Bukczynski, J., Wen, T., Ellefsen, K., Gauldie, J., and Watts, T.H. (2004). Costimulatory ligand 4-1BBL (CD137L) as an efficient adjuvant for human antiviral cytotoxic T cell responses. Proc. Natl. Acad. Sci. USA, 101, 1291–1296 (Epub 2004 Jan 1226). Colombo, M.P., and Trinchieri, G. (2002). Interleukin-12 in anti-tumor immunity and immunotherapy. Cytokine. Growth. Factor. Rev., 13, 155–168. Croft, M. (2003). Co-stimulatory members of the TNFR family: Keys to effective T-cell immunity? Nat. Rev. Immunol., 3, 609–620. Chen, L. (1998). Immunological ignorance of silent antigens as an explanation of tumor evasion. Immunol. Today, 19, 27–30. Chen, L., Ashe, S., Brady, W.A., Hellstrom, I., Hellstrom, K.E., Ledbetter, J.A., McGowan, P., and Linsley, P.S. (1992). Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4. Cell, 71, 1093–1102. Chen, L., McGowan, P., Ashe, S., Johnston, J., Li, Y., Hellstrom, I., and Hellstrom, K.E. (1994). Tumor immunogenicity determines the effect of B7 costimulation on T cell-mediated tumor immunity. J. Exp. Med., 179, 523–532. Chen, S.H., Pham-Nguyen, K.B., Martinet, O., Huang, Y., Yang, W., Thung, S.N., Chen, L., Mittler, R., and Woo, S.L. (2000). Rejection of disseminated metastases of colon carcinoma by synergism of IL-12 gene therapy and 4-1BB costimulation [In Process Citation]. Mol. Ther., 2, 39–46. Chen, W., Masterman, K.A., Basta, S., Haeryfar, S.M., Dimopoulos, N., Knowles, B., Bennink, J.R., and Yewdell, J.W. (2004). Cross-priming of CD8+ T cells by viral and tumor antigens is a robust phenomenon. Eur. J. Immunol., 34, 194–199. Choi, B.K., Bae, J.S., Choi, E.M., Kang, W.J., Sakaguchi, S., Vinay, D.S., and Kwon, B.S. (2004). 41BB-dependent inhibition of immunosuppression by activated CD4+CD25+ T cells. J. Leukoc. Biol., 75, 785–791 (Epub 2003 Dec 2023). Chu, N.R., DeBenedette, M.A., Stiernholm, B.J., Barber, B.H., and Watts, T.H. (1997). Role of IL12 and 4-1BB ligand in cytokine production by CD28+ and CD28− T cells. J. Immunol., 158, 3081–3089. Dawicki, W., Bertram, E.M., Sharpe, A.H., and Watts, T.H. (2004). 4-1BB and OX40 act independently to facilitate robust CD8 and CD4 recall responses. J. Immunol., 173, 5944–5951. DeBenedette, M.A., Wen, T., Bachmann, M.F., Ohashi, P.S., Barber, B.H., Stocking, K.L., Peschon, J.J., and Watts, T.H. (1999). Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking

132

Ignacio Melero et al.

both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to influenza virus. J. Immunol., 163, 4833–4841. Dong, H., Strome, S.E., Salomao, D.R., Tamura, H., Hirano, F., Flies, D.B., Roche, P.C., Lu, J., Zhu, G., Tamada, K., et al. (2002). Tumor-associated B7-H1 promotes T-cell apoptosis: A potential mechanism of immune evasion. Nat. Med., 8, 793–800. Dudley, M.E., Wunderlich, J.R., Robbins, P.F., Yang, J.C., Hwu, P., Schwartzentruber, D.J., Topalian, S.L., Sherry, R., Restifo, N.P., Hubicki, A.M., et al. (2002). Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science, 298, 850–854. Dudley, M.E., and Rosenberg, S.A. (2003). Adoptive-cell-transfer therapy for the treatment of patients with cancer. Nat. Rev. Cancer, 3, 666–675. Egen, J.G., Kuhns, M.S., and Allison, J.P. (2002). CTLA-4: New insights into its biological function and use in tumor immunotherapy. Nat. Immunol., 3, 611–618. French, R.R., Chan, H.T., Tutt, A.L., and Glennie, M.J. (1999). CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help. Nat. Med., 5, 548–553. Goodwin, R.G., Din, W.S., Davis-Smith, T., Anderson, D.M., Gimpel, S.D., Sato, T.A., Maliszewski, C.R., Brannan, C.I., Copeland, N.G., Jenkins, N.A., et al. (1993). Molecular cloning of a ligand for the inducible T cell gene 4-1BB: A member of an emerging family of cytokines with homology to tumor necrosis factor. Eur. J. Immunol., 23, 2631–2641. Guinn, B.A., DeBenedette, M.A., Watts, T.H., and Berinstein, N.L. (1999). 4-1BBL cooperates with B7-1 and B7-2 in converting a B cell lymphoma cell line into a long-lasting antitumor vaccine. J. Immunol., 162, 5003–5010. Halstead, E.S., Mueller, Y.M., Altman, J.D., and Katsikis, P.D. (2002). In vivo stimulation of CD137 broadens primary antiviral CD8+ T cell responses. Nat. Immunol., 3, 536–541. Hirano, F., Kaneko, K., Tamura, H., Dong, H., Wang, S., Ichikawa, M., Rietz, C., Flies, D.B., Lau, J.S., Zhu, G., et al. (2005). Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity. Cancer Res., 65, 1089–1096. Hodi, F.S., Mihm, M.C., Soiffer, R.J., Haluska, F.G., Butler, M., Seiden, M.V., Davis, T., Henry-Spires, R., MacRae, S., Willman, A., et al. (2003). Biologic activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients. Proc. Natl. Acad. Sci. USA, 100, 4712–4717. Hong, H.J., Lee, J.W., Park, S.S., Kang, Y.J., Chang, S.Y., Kim, K.M., Kim, J.O., Murthy, K.K., Payne, J.S., Yoon, S.K., et al. (2000). A humanized anti-4-1BB monoclonal antibody suppresses antigeninduced humoral immune response in nonhuman primates. J. Immunother., 23, 613–621. Hurwitz, A.A., Yu, T.F., Leach, D.R., and Allison, J.P. (1998). CTLA-4 blockade synergizes with tumor-derived granulocyte-macrophage colony-stimulating factor for treatment of an experimental mammary carcinoma. Proc. Natl. Acad. Sci. USA, 95, 10067–10071. Ito, F., Li, Q., Shreiner, A.B., Okuyama, R., Jure-Kunkel, M.N., Teitz-Tennenbaum, S., and Chang, A.E. (2004). Anti-CD137 monoclonal antibody administration augments the antitumor efficacy of dendritic cell-based vaccines. Cancer Res., 64, 8411–8419. Kim, J.A., Averbook, B.J., Chambers, K., Rothchild, K., Kjaergaard, J., Papay, R., and Shu, S. (2001). Divergent effects of 4-1BB antibodies on antitumor immunity and on tumor-reactive T-cell generation. Cancer Res., 61, 2031–2037. Kim, J., Choi, W.S., La, S., Suh, J.H., Kim, B.S., Cho, H.R., Kwon, B.S., and Kwon, B. (2005). Stimulation with 4-1BB (CD137) inhibits chronic graft-versus-host disease by inducing activationinduced cell death of donor CD4+ T cells. Blood, 105, 2206–2213 (Epub 2004 Nov 2202). Klebanoff, C.A., Khong, H.T., Antony, P.A., Palmer, D.C., and Restifo, N.P. (2005). Sinks, suppressors and antigen presenters: How lymphodepletion enhances T cell-mediated tumor immunotherapy. Trends Immunol., 26, 111–117. Kocak, E., Lute, K., Chang, X., May, K.F. Jr, Exten, K.R., Zhang, H., Abdessalam, S.F., Lehman, A.M., Jarjoura, D., Zheng, P., and Liu, Y. (2006). Combination therapy with anti-CTL antigen-4 and anti-4-1BB antibodies enhances cancer immunity and reduces autoimmunity. Cancer Res., 66, 7276–7284. Kwon, B.S., Hurtado, J.C., Lee, Z.H., Kwack, K.B., Seo, S.K., Choi, B.K., Koller, B.H., Wolisi, G., Broxmeyer, H.E., and Vinay, D.S. (2002). Immune responses in 4-1BB (CD137)-deficient mice. J. Immunol., 168, 5483–5490.

CD137/CD137 Ligand in Tumor and Viral Immunotherapy

133

Lanzavecchia, A. (1998). Immunology. Licence to kill [news; comment]. Nature, 393, 413–414. Leach, D.R., Krummel, M.F., and Allison, J.P. (1996). Enhancement of antitumor immunity by CTLA-4 blockade. Science, 271, 1734–1736. Lee, S.J., Myers, L., Muralimohan, G., Dai, J., Qiao, Y., Li, Z., Mittler, R.S., and Vella, A.T. (2004). 4-1BB and OX40 dual costimulation synergistically stimulate primary specific CD8 T cells for robust effector function. J. Immunol., 173, 3002–3012. Martinet, O., Ermekova, V., Qiao, J.Q., Sauter, B., Mandeli, J., Chen, L., and Chen, S.H. (2000). Immunomodulatory gene therapy with interleukin 12 and 4-1BB ligand: Long-term remission of liver metastases in a mouse model. J. Natl. Cancer Inst., 92, 931–936. Martinet, O., Divino, C.M., Zang, Y., Gan, Y., Mandeli, J., Thung, S., Pan, P.Y., and Chen, S.H. (2002). T cell activation with systemic agonistic antibody versus local 4-1BB ligand gene delivery combined with interleukin-12 eradicate liver metastases of breast cancer. Gene Ther., 9, 786–792. Maus, M.V., Thomas, A.K., Leonard, D.G., Allman, D., Addya, K., Schlienger, K., Riley, J.L., and June, C.H. (2002). Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB. Nat. Biotechnol., 20, 143–148. May, K.F., Jr., Chen, L., Zheng, P., and Liu, Y. (2002). Anti-4-1BB monoclonal antibody enhances rejection of large tumor burden by promoting survival but not clonal expansion of tumor-specific CD8+ T cells. Cancer Res., 62, 3459–3465. Mazzolini, G., Alfaro, C., Sangro, B., Feijoo, E., Ruiz, J., Benito, A., Tirapu, I., Arina, A., Sola, J., Herraiz, M., et al. (2005). Intratumoral injection of dendritic cells engineered to secrete interleukin12 by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas. J. Clin. Oncol., 23, 999–1010 (Epub 2004 Dec 1014). Melero, I., Shuford, W.W., Newby, S.A., Aruffo, A., Ledbetter, J.A., Hellstrom, K.E., Mittler, R.S., and Chen, L. (1997a). Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat. Med., 3, 682–685. Melero, I., Singhal, M.C., McGowan, P., Haugen, H.S., Blake, J., Hellstrom, K.E., Yang, G., Clegg, C.H., and Chen, L. (1997b). Immunological ignorance of an E7-encoded cytolytic T-lymphocyte epitope in transgenic mice expressing the E7 and E6 oncogenes of human papillomavirus type 16. J. Virol., 71, 3998–4004. Melero, I., Bach, N., Hellstrom, K.E., Aruffo, A., Mittler, R.S., and Chen, L. (1998a). Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: Synergy with the CD28 co-stimulatory pathway. Eur. J. Immunol., 28, 1116–1121. Melero, I., Johnston, J.V., Shufford, W.W., Mittler, R.S., and Chen, L. (1998b). NK1.1 cells express 4-1BB (CDw137) costimulatory molecule and are required for tumor immunity elicited by anti4-1BB monoclonal antibodies. Cell Immunol., 190, 167–172. Miller, R.E., Jones, J., Le, T., Whitmore, J., Boiani, N., Gliniak, B., and Lynch, D.H. (2002). 4-1BBspecific monoclonal antibody promotes the generation of tumor-specific immune responses by direct activation of CD8 T cells in a CD40-dependent manner. J. Immunol., 169, 1792–1800. Miller, J.S., Soignier, Y., Panoskaltsis-Mortari, A., McNearney, S.A., Yun, G.H., Fautsch, S.K., McKenna, D., Le, C., Defor, T.E., Burns, L.J., et al. (2005). Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood, 105, 3051– 3057 (Epub 2005 Jan 3054). Mittler, R.S. (2004). Suppressing the self in rheumatoid arthritis. Nat. Med., 10, 1047–1049. Moss, P., and Rickinson, A. (2005). Cellular immunotherapy for viral infection after HSC transplantation. Nat. Rev. Immunol., 5, 9–20. Mukherjee, P., Tinder, T.L., Basu, G.D., Pathangey, L.B., Chen, L., and Gendler, S.J. (2004). Therapeutic efficacy of MUC1-specific cytotoxic T lymphocytes and CD137 co-stimulation in a spontaneous breast cancer model. Breast Dis., 20, 53–63. Muller, A.J., DuHadaway, J.B., Donover, P.S., Sutanto-Ward, E., and Prendergast, G.C. (2005). Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory target of the cancer suppression gene Bin1, potentiates cancer chemotherapy. Nat. Med., 11, 312–319 (Epub 2005 Feb 2013). Munks, M.W., Mourich, D.V., Mittler, R.S., Weinberg, A.D., and Hill, A.B. (2004). 4-1BB and OX40 stimulation enhance CD8 and CD4 T-cell responses to a DNA prime, poxvirus boost vaccine. Immunology, 112, 559–566.

134

Ignacio Melero et al.

Murillo, O., Arina, A., Tirapu, I., Alfaro, C., Mazzolini, G., Palencia, B., Lopez-Diaz De Cerio, A., Prieto, J., Bendandi, M., Melero, I. (2003). Potentiation of therapeutic immune responses against malignancies with monoclonal antibodies. Clin Cancer Res., 9, 5454–5464. Murphy, A., Westwood, J.A., Teng, M.W., Moeller, M., Darcy, P.K., and Kershaw, M.H. (2005). Gene modification strategies to induce tumor immunity. Immunity, 22, 403–414. Murphy, W.J., Tian, Z.G., Asai, O., Funakoshi, S., Rotter, P., Henry, M., Strieter, R.M., Kunkel, S.L., Longo, D.L., and Taub, D.D. (1996). Chemokines and T lymphocyte activation: II. Facilitation of human T cell trafficking in severe combined immunodeficiency mice. J. Immunol., 156, 2104– 2111. Nam, K.O., Kang, H., Shin, S.M., Cho, K.H., Kwon, B., Kwon, B.S., Kim, S.J., and Lee, H.W. (2005). Cross-linking of 4-1BB activates TCR-signaling pathways in CD8+ T lymphocytes. J. Immunol., 174, 1898–1905. Ochsenbein, A.F., Klenerman, P., Karrer, U., Ludewig, B., Pericin, M., Hengartner, H., and Zinkernagel, R.M. (1999). Immune surveillance against a solid tumor fails because of immunological ignorance. Proc. Natl. Acad. Sci. USA, 96, 2233–2238. Pardoll, D., and Allison, J. (2004). Cancer immunotherapy: Breaking the barriers to harvest the crop. Nat. Med., 10, 887–892. Phan, G.Q., Yang, J.C., Sherry, R.M., Hwu, P., Topalian, S.L., Schwartzentruber, D.J., Restifo, N.P., Haworth, L.R., Seipp, C.A., Freezer, L.J., et al. (2003). Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma. Proc. Natl. Acad. Sci. USA, 100, 8372–8377. Ruggeri, L., Capanni, M., Urbani, E., Perruccio, K., Shlomchik, W.D., Tosti, A., Posati, S., Rogaia, D., Frassoni, F., Aversa, F., et al. (2002). Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science, 295, 2097–2100. Ruggeri, L., Capanni, M., Mancusi, A., Perruccio, K., Burchielli, E., Martelli, M.F., and Velardi, A. (2005). Natural killer cell alloreactivity in haploidentical hematopoietic stem cell transplantation. Int. J. Hematol., 81, 13–17. Sakaguchi, S. (2005). Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat. Immunol., 6, 345–352. Salih, H.R., Kosowski, S.G., Haluska, V.F., Starling, G.C., Loo, D.T., Lee, F., Aruffo, A.A., Trail, P.A., and Kiener, P.A. (2000). Constitutive expression of functional 4-1BB (CD137) ligand on carcinoma cells. J. Immunol., 165, 2903–2910. Salih, H.R., Schmetzer, H.M., Burke, C., Starling, G.C., Dunn, R., Pelka-Fleischer, R., Nuessler, V., and Kiener, P.A. (2001). Soluble CD137 (4-1BB) ligand is released following leukocyte activation and is found in sera of patients with hematological malignancies. J. Immunol., 167, 4059– 4066. Sanderson, K., Scotland, R., Lee, P., Liu, D., Groshen, S., Snively, J., Sian, S., Nichol, G., Davis, T., Keler, T., et al. (2005). Autoimmunity in a phase I trial of a fully human anti-cytotoxic Tlymphocyte antigen-4 monoclonal antibody with multiple melanoma peptides and Montanide ISA 51 for patients with resected stages III and IV melanoma. J. Clin. Oncol., 23, 741–750 (Epub 2004 Dec 2021). Sangro, B., Mazzolini, G., Ruiz, J., Herraiz, M., Quiroga, J., Herrero, I., Benito, A., Larrache, J., Pueyo, J., Subtil, J.C., et al. (2004). Phase I trial of intratumoral injection of an adenovirus encoding interleukin-12 for advanced digestive tumors. J. Clin. Oncol., 22, 1389–1397. Seo, S.K., Choi, J.H., Kim, Y.H., Kang, W.J., Park, H.Y., Suh, J.H., Choi, B.K., Vinay, D.S., and Kwon, B.S. (2004). 4-1BB-mediated immunotherapy of rheumatoid arthritis. Nat. Med., 10, 1088–1094 (Epub 2004 Sep 1026). Shuford, W.W., Klussman, K., Tritchler, D.D., Loo, D.T., Chalupny, J., Siadak, A.W., Brown, T.J., Emswiler, J., Raecho, H., Larsen, C.P., et al. (1997). 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J. Exp. Med., 186, 47–55. Sun, Y., Chen, H.M., Subudhi, S.K., Chen, J., Koka, R., Chen, L., and Fu, Y.X. (2002a). Costimulatory molecule-targeted antibody therapy of a spontaneous autoimmune disease. Nat. Med., 8, 1405– 1413.

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Sun, Y., Lin, X., Chen, H.M., Wu, Q., Subudhi, S.K., Chen, L., and Fu, Y. X. (2002b). Administration of agonistic anti-4-1BB monoclonal antibody leads to the amelioration of experimental autoimmune encephalomyelitis. J. Immunol., 168, 1457–1465. Tan, J.T., Whitmire, J.K., Ahmed, R., Pearson, T.C., and Larsen, C.P. (1999). 4-1BB ligand, a member of the TNF family, is important for the generation of antiviral CD8 T cell responses. J. Immunol., 163, 4859–4868. Tirapu, I., Arina, A., Mazzolini, G., Duarte, M., Alfaro, C., Feijoo, E., Qian, C., Chen, L., Prieto, J., and Melero, I. (2004). Improving efficacy of interleukin-12-transfected dendritic cells injected into murine colon cancer with anti-CD137 monoclonal antibodies and alloantigens. Int. J. Cancer., 110, 51–60. Tirapu, I., Mazzolini, G., Rodriguez-Calvillo, M., Arina, A., Palencia, B., Gabari, I., and Melero, I. (2002). Effective tumor immunotherapy: Start the engine, release the brakes, step on the gas pedal and get ready to face autoimmunity. Arch. Immunol. Ther. Exp. (Warsz), 50, 13–18. Tivol, E.A., Borriello, F., Schweitzer, A.N., Lynch, W.P., Bluestone, J.A., and Sharpe, A.H. (1995). Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity, 3, 541–547. Townsend, S.E., and Allison, J.P. (1993). Tumor rejection after direct costimulation of CD8+ T cells by B7-transfected melanoma cells. Science, 259, 368–370. Uno, T., Takeda, K., Kojima, Y., Yoshizawa, H., Akiba, H., Mittler, R.S., Gejyo, F., Okumura, K., Yagita, H., and Smyth, M.J. (2006). Eradication of established tumors in mice by a combination antibody-based therapy. Nat. Med., 12, 693–698. van Mierlo, G.J., Boonman, Z.F., Dumortier, H.M., den Boer, A.T., Fransen, M.F., Nouta, J., van der Voort, E.I., Offringa, R., Toes, R.E., and Melief, C.J. (2004). Activation of dendritic cells that cross-present tumor-derived antigen licenses CD8+ CTL to cause tumor eradication. J. Immunol., 173, 6753–6759. Waldmann, T.A. (2003). IL-15 in the life and death of lymphocytes: Immunotherapeutic implications. Trends Mol. Med., 9, 517–521. Waterhouse, P., Penninger, J.M., Timms, E., Wakeham, A., Shahinian, A., Lee, K.P., Thompson, C.B., Griesser, H., and Mak, T.W. (1995). Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science, 270, 985–988. Watts, T.H. (2005). Tnf/Tnfr family members in costimulation of T cell responses. Annu. Rev. Immunol., 23, 23–68. Wiethe, C., Dittmar, K., Doan, T., Lindenmaier, W., and Tindle, R. (2003). Provision of 4-1BB ligand enhances effector and memory CTL responses generated by immunization with dendritic cells expressing a human tumor-associated antigen. J. Immunol., 170, 2912–2922. Wilcox, R.A., Flies, D.B., Zhu, G., Johnson, A.J., Tamada, K., Chapoval, A.I., Strome, S.E., Pease, L.R., and Chen, L. (2002a). Provision of antigen and CD137 signaling breaks immunological ignorance, promoting regression of poorly immunogenic tumors. J. Clin. Invest., 109, 651–659. Wilcox, R.A., Tamada, K., Strome, S.E., and Chen, L. (2002b). Signaling through NK cell-associated CD137 promotes both helper function for CD8+ cytolytic T cells and responsiveness to IL-2 but not cytolytic activity. J. Immunol., 169, 4230–4236. Wilcox, R.A., Tamada, K., Flies, D.B., Zhu, G., Chapoval, A.I., Blazar, B.R., Kast, W.M., and Chen, L. (2004). Ligation of CD137 receptor prevents and reverses established anergy of CD8+ cytolytic T lymphocytes in vivo. Blood, 103, 177–184 (Epub 2003 Sep 2011). Ye, Z., Hellstrom, I., Hayden-Ledbetter, M., Dahlin, A., Ledbetter, J.A., and Hellstrom, K.E. (2002). Gene therapy for cancer using single-chain Fv fragments specific for 4-1BB. Nat. Med., 8, 343– 348. Yewdell, J.W., and Del Val, M. (2004). Immunodominance in TCD8+ responses to viruses: Cell biology, cellular immunology, and mathematical models. Immunity, 21, 149–153. Zheng, G., Wang, B., Chen, A., Choi, B.K., Bae, J.S., Choi, E.M., Kang, W.J., Sakaguchi, S., Vinay, D.S., and Kwon, B.S. (2004). The 4-1BB costimulation augments the proliferation of CD4+CD25+ regulatory T cells 4-1BB-dependent inhibition of immunosuppression by activated CD4+CD25+ T cells. J. Immunol., 173, 2428–2434.

Index A adaptive immunity CD137 (4-1BB) signaling through, 47–50, 98 T-cells mediated, 15, 16 antigen presenting cells, 15, 29. See also CD137 ligand autoimmune diseases, 96–110 autoimmune encephalomyelitis (EAE), 101, 102 autoimmune uveitis (EAU), 102, 103 CD137/CD137L role of, 98–106 chronic graft-versus-host disease (cGVHD), 105, 106 collagen-induced arthritis (CIA), 105 inhibitory mechanisms (CD137 agonist-mediated), 106–9 B cell, apoptosis, 109 helper T cell anergy, 108 T lymphocytes, apoptosis, 106, 107 soluble forms (CD137 and CD137L), 99, 100 systemic lupus erythematosus (SLE), 103, 104 treatment with anti-CD137, 100, 101 B B cell lymphocytes anti-tumor immunity, 72 apoptosis of, 109 CD40 and CD137 receptor/ligand systems, 35 bone marrow transplantation CD137/CD137 ligand system, 122, 123 C CD137 ligand, 29, 30–37 B cells, 34, 35 bone marrow cells, 35, 36 co-stimulatory ligands, 16 dendritic cells, 34 genomic organization (human and murine), 6–8 monocytes and macrophages, 30 non-hematopoietic cells, 37 protein expression, 7, 8 by tissue, 32

scatchard analysis, 7 signal transduction pathway, 39 by cell type, 31 influence of soluble CD137, 39 regulation of, 39 structure and expression, 6, 7 T cells, 36 western analysis, 7 CD137 receptor/CD137L system, 10, 29, 126, 127 action with cytokines, 124 antiviral responses, 126, 127 autoimmune diseases, 97 bidirectional signals, 10 bone marrow transplantation, 122 CD137/CD137L interactions effect on autoimmune diseases, 98, 99 HSK pathogenesis, 99 importance of, 9 health and disease, 9 immunization, 121, 122 improvements, 121 T cell responses, 37 T cell therapy, adoptive, 122, 123 adoptive cell transfer experiments, 62 therapeutic strategies, 121–26 tumor and viral immunotherapy, 117 CD137(4-1BB) receptors, 2, 3, 16. See also induced by lymphocyte activation (ILA) action with cytokines,124 anti-CD137 (4-1BB) anitgen-priming, 62 autoantibodies supression, 64–69 based immunotherapy, 110 hematopoiesis disruption, 74–77 mAb therapy, 11, 63, 110, 117, 118 of anti-SRBC humoral immunity, 61 treatment, 110 anti-tumor immunity B cells role, 72–74 autoimmune diseases, 17 CD8+ and CD4+ T cells, 16, 17 DC-DC interaction regulation, 51

137

138 CD137(4-1BB) receptors (cont.) effects on CD4+ T cells, 86 CD8+ T cell, 83, 85 memory T cell (Tm) response, 90 regulatory T cells (Treg), 87 expression, 59 genomic organization (human and murine), 4, 5 immune stimulator, 3, 16 RNA and protein expression, 8, 9 soluble forms, 39 T cell mediated responses, 3, 59, 97 tumor necrosis factor receptor (TNFR)/ligand family, 16 CD137(4-1BB) signal transduction pathways, 15–23, 29 bidirectional pathways, 40, 41 CD137 signaling, 47 CD137/CD137L interactions autoimmune diseases, 98, 99 HSK pathogenesis, 99 importance of, 9 CD8+ T lymphocytes, 18, 19, 21 costimulatory molecule, 22 developments and improvements, 121 health and disease, 9 immunization, 121, 122 innate immunity, 47 macrophages/monocyte, 49 SAPK/JNK pathways, 18 T cell responses, 37 therapeutic strategies, 121–26 TRAFs role, 18 tumor and viral immunotherapy, 117 chronic graft-versus-host disease, 105 CIA. See collagen-induced arthritis Class switching CD4 T cell Ig, 58 D dendritic cells, 34 E EAE. See autoimmune encephalomyelitis EAU. See autoimmune uveitis F follicular dendritic cells, 35 G genomic organization CD137 receptors, 5 CD137 ligands, 8

Index H herpetic stromal keratitis, 98 humoral immunity CD137(4-1BB) and CD137L T cells costimulation, 59 CD40 and CD137 receptor/ligand systems roles of, 35 expression and signaling, 69 mediated signals, 55 regulation of CD137 (4-1BB) signals, 55, 56 CD137 expression and signaling, 69 suppression of anti-CD137, 60–64 autoantibodies, 64 CD28 or CD40 costimulatory pathways, 60 DC Function, 69, 70 T and B cell activation, 56, 57 T cell-dependent immunity, 55 I ILA. induced by lymphocyte activation (CD137), 3, 16 immunotherapy preclinical information, 128 tumor immunotherapy agonistic anti-CD137 monoclonal antibodies, 117 anti-CD137 mAbs, 118 anti-CTLA-4 mAbs, 118 CD137/CD137 ligand, 117–21 viral immunotherapy CD137/CD137 L, 117, 126 indoleamine dioxygenase (IDO) process, 1 innate immunity CD137 pathway, 52 CD137 signal dendritic Cells, 50 granulocytes, 51 macrophages/monocyte, 49 NK Cells, 47–49 integrins, 40. See also bidirectional signal transduction M macrophages/monocyte, 30–34. See also macrophage colony stimulating factor (M-CSF) CD137-CD137L interactions effect, 50 innate immune response, 49 non-hematopoietic cells, 37 T cells, 36–37 memory T cell (Tm) CD137 pathway, 92 CD137 role, 90

Index monoclonal antibodies (anti-CD137), 117 anti-CD137 agonistic mAb clinical applications, 129, 130 anti-CTLA-4 mAbs, 118, 19 anti-dsDNA antibodies responses, 66, 68 anti-SRBC responses, 67 N NK Cells activity of, 47 dendritic cells activation, 48 immune regulatory function, 48 role of CD137 signal, 49 P PI-3 kinase/Akt pathway, 18 R regulatory T cells (Treg) and CD137, 87, 88 reverse signal transduction, 29, 30 biology of, 30–40 CD137 receptor/ligand system for, 29 S signal transduction pathway by cell type, 31 influence of soluble CD137, 39 regulation of, 39 CD137 ligand, 39 CD8+ T lymphocytes, progression, 18, 19, 21 costimulatory molecule, 22 innate immunity, 47 macrophages/monocyte, 49 NF-ˆeB activation and Bcl-XL expression, 18 role of CD137 signaling, 47 SAPK/JNK pathways, 18 TRAFs role, 18 systemic lupus erythematosus, 103, 104

139 T T cell receptor (TCR), 15, 56 T lymphocytes cell activation, 56 and IFN-α, 107 apoptosis of, 106 CD137, 88–90 costimulation, effects on (CD28), 57, 59 CD137-mediated, 29, 60 CD4+ T cells, 86 CD8+ T cells, 83–85 role of CD137, 83 humoral immunity regulation of, 55, 59 immunity, 15. See also CD137 signal transduction 4-1BB signal transduction pathways, 19 co-stimulatory signals, 15 tumor necrosis factor receptor (TNFR)/ligand family, 16 adoptive cell therapy CD137 adjuvant as, 122 TCR pathway proteins, 21 TNF receptor associated factor, 2, 17 TNFR superfamily, 97 CD 137(4-1BB), 17 TNFRSF9. See tumor necrosis factor receptor superfamily TRAF. See TNF receptor associated factor TRAF-NIK pathway, 1 tumor immunotherapy anti-CD137 mAbs,117, 118 anti-CTLA-4 mAbs, 118 CD137/CD137 ligand role of, 117–21 tumor necrosis factor (TNF) family, 1, 2, 29, 55 CD137 (4-1BB), 29, 55, 87, 97 CD137 and CD137L, 1, 2, 29 tumor necrosis factor receptor superfamily, 97 V viral immunotherapy CD137/CD137 L, 117, 126