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

Genes, Transcripts and Proteins of CD137 Receptor and Ligand

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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 (