4-1BB

4-1BB Dass S. Vinay1 and Byoung S. Kwon2,3,* 1

Department of General Surgery, University of Michigan Medical School, 1516 MSRB I, 1150 West Medical Center Drive, Ann Arbor, MI 48109, USA 2 The Immunomodulation Research Center, University of Ulsan, Ulsan, Korea 3 Department of Ophthalmology, LSUMC, 2020 Gravier Street Suite B, New Orleans, LA 70112, USA * corresponding author tel: 504-412-1200 ext 1379, fax: 504-412-1315, e-mail: [email protected] DOI: 10.1006/rwcy.2000.16007.

SUMMARY 4-1BB (CD137) is a member of the tumor necrosis factor (TNF) receptor superfamily expressed on activated CD4+, CD8+, as well as on NK cells (Vinay and Kwon, 1998). 4-1BB binds to 4-1BB ligand (41BBL) found on activated B cells, macrophages, and dendritic cells (Alderson et al., 1994; Goodwin et al., 1993; Pollok et al., 1994; DeBenedette et al., 1997). Data accumulated thus far show that signals generated through 4-1BB induce T cell activation and cytokine modulation (Vinay and Kwon, 1998). These observations are further substantiated by the findings that 4-1BBL-expressing cell lines or soluble 4-1BBL induce T cell proliferation and IL-2 secretion (Goodwin et al., 1993; DeBenedette et al., 1995, 1997; Hurtado et al., 1995; Saoulli et al., 1998). Also, the finding that addition of 4-1BB fusion proteins block T cell proliferation mediated by 4-1BBL (Goodwin et al., 1993; DeBenedette et al., 1995, 1997; Hurtado et al., 1995; Saoulli et al., 1998) further support a strong role for 4-1BB in T cell regulation. Evidence gathered also points out that antibodies to 4-1BB possess an increasing ability to activate and control CD8 responses (Shuford et al., 1997; Melero et al., 1997). This fact is further evidenced by recent findings using 4-1BBL-deficient mice (DeBenedette et al., 1999; Tan et al., 1999). Interestingly, literature also points out a role for 4-1BB in TH1 (Kim et al., 1998) and TH2 (Chu et al., 1997) and TC1 and TC2 development (Vinay and Kwon, 1999). Although, CD28 is considered as central to the T cell immune responses (Chambers and Allison, 1997), evidence available demonstrates that during T cell activation,

especially when stimulus through TCR is acute, 41BB can replace CD28 in the costimulation of naõÈ ve T cells (Watts and DeBenedette, 1999). The molecular basis underlying 4-1BB-mediated T cell activation is beginning to be appreciated. Although exhaustive studies are lacking, the available data show that this T cell-activating antigen transmits signals via interactions with the TNF receptorassociated (TRAF) family of molecules, such as TRAF1, TRAF2, and TRAF3, and activates NFB (Saoulli et al., 1998; Arch and Thompson, 1998; Jang et al., 1998).

BACKGROUND Optimal induction of T cell activation is incomplete without cognate interaction between T cells and antigen-presenting cell (APC)-derived cell surface molecules (Schwarz, 1990). A number of APC-derived cell surface determinants have been shown to possess the ability to potentiate/desensitize T cell effector functions. Although it is considered that B7/CD28 is central to this pathway, studies of CD28-deficient mice (Shahnian et al., 1993) have shown that this may not be the limiting factor in immune regulation. Recent studies indicate that several ligand/receptor pairs, beyond the B7/CD28 pathway, also have the ability to initiate and propel the ongoing immune reaction. Among these are three members of the expanding TNFR family: 4-1BB, Ox40, and GITR that may be involved in T cell function in the post CD28 phase.

1686 Dass S. Vinay and Byoung S. Kwon

Discovery The T cell-activating antigen (Ag) 4-1BB was initially discovered in screens for receptors on activated mouse lymphocytes (Kwon and Weissman, 1989).

Alternative names 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). Both ILA and 4-1BB recently received the human leukocyte differentiation antigen (HLDA) nomenclature CD137.

Figure 1 Organization of the 4-1BB gene. Exons are shown as boxes; UTRs are displayed as red boxes while the protein-coding regions are green boxes (Roman numerals). Lengths of exons and introns are shown in Arabic numerals (base pairs). (Full colour figure may be viewed online.) Exon I

Exon I

Exon II

Exon IV

Exon VI

Exon VII

Exon V

Exon VIII

Exon IX

Structure 4-1BB is a 30 kDa glycoprotein and exists both as a monomer and a 55 kDa dimer on the T cell surface. The entire gene spans approximately 13 kb of mouse chromosome 4.

Main activities and pathophysiological roles 4-1BB is shown to transmit costimulatory signals to T cells in synergy with anti-CD3. Functional studies reveal that antibodies to the 4-1BB molecule can increase graft-versus-host disease, accelerate the rejection of cardiac allografts and skin transplants, and eradicate established tumors. 4-1BB is also known to play an important role in HIV and Hodgkin's disease (see Vinay and Kwon, 1998 for a review).

GENE

Accession numbers U022567

Sequence The 4-1BB gene consists of 10 exons and 9 introns in which there are two exons for 50 UTR and 8 for the coding region (Figure 1). Two types of UTR sequences were identified in the genomic sequence and found to be separated by an intron  2.5 kb in length. The cysteine-rich extracellular domain is divided into six exons. The signal sequence, transmembrane region,

and serine, threonine, proline (STP)-rich region immediately outside the transmembrane domain are contained in separate exons. Finally, the cytoplasmic domain that contains the p56lck-binding site is located in the last exon of the gene. The exon/intron boundaries are assigned by comparing the 4-1BB cDNA sequence with the genomic sequence. In the flanking sequence of the type I 50 UTR, no TATA box-related elements were found. Instead, they are very good matches of the consensus TPA-responsive element (AP-1) at positions ÿ18 to ÿ10, and of the NFB-binding sequence at positions ÿ49 to ÿ39. Upstream of these elements, this region contains a potential ets-binding site at positions ÿ169 to ÿ162, an activator protein 2 (AP-2)-binding site at positions ÿ460 to ÿ453, a typical CAAT element at positions ÿ498 to ÿ494, and an SP-1-binding site at positions ÿ522 to ÿ516. The 5' flanking region of the type II 50 UTR contains a TATA-related element at positions ÿ28 to ÿ23. Two potential ets-binding sites appear at positions ÿ15 to ÿ8 and ÿ139 to ÿ132, two potential AP-2-binding sites at positions ÿ89 to ÿ82 and ÿ331, and a very good match of an AP-1-binding site at positions ÿ311 to ÿ302.

PROTEIN

Accession numbers Murine 4-1BB: J04492 Human 4-1BB: U03397 Mouse 4-1BB ligand: L15435 Human 4-1BB ligand: U03398

4-1BB 1687

Description of protein The nucleotide sequence of murine 4-1BB revealed a single open reading frame which codes for a polypeptide of 256 amino acids with a calculated mass of 27,587 Da. The predicted protein contained an unusually large number (23) of cysteines. This region comprises four potential TNFR motifs, of which the first is partial and the third distinct 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 serine and threonines; potential sites for O-linked glycosylation. Amino acids 186±211 constitute the hydrophobic transmembrane domain followed by the stop-transfer sequence containing several basic residues. 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 human homolog of 4-1BB (h4-1BB) contains 255 amino acids with two potential N-linked glycosylation sites. The molecular weight of its protein backbone is calculated to be 27 kDa (Zhou et al., 1995) and is 60% identical to mouse 4-1BB. In the cytoplasmic domain, five regions of amino acid sequences are conserved between mouse and human, indicating that these residues might be important in 41BB function.

with a Kd of 1.86 nM. Western analysis showed that 4-1BBL has a molecular mass of approximately 18±25 kDa.

Cell types and tissues expressing the receptor The 4-1BB molecule is present on activated T cells and monocytes.

Regulation of receptor expression The 4-1BB is not detected (<3%) on resting T cells and T cell lines. However, when the T cells, in the presence of APCs, stimulated with a variety of agonists (plate-bound anti-CD3, Con A, PHA, IL-2, IL-4, anti-CD28, PMA, ionomycin alone or in combinations) upregulates and maintain its expression (Pollok et al., 1993). Upon activation, 4-1BB mRNA is detected within hours and cell surface expression can be detected between 10 and 16 hours. The expression peaks at 64 hours and is maintained until 120 hours after single stimulation (Hurtado et al., 1995). Interestingly, expression of 4-1BB is detectable on T cells derived from individuals with pathological conditions (Michel et al., 1998) indicating that its expression is indeed activation dependent.

Affinity for ligand(s)

Release of soluble receptors

Although the h4-1BB ligand was detected in both T and B cells of human peripheral blood, the ligand was preferentially expressed in primary B cells and B cell lines. Daudi, a B cell lymphoma, was one of the B cell lines that carried a higher number of ligands. Scatchard analysis showed that the Kd ˆ 1.4  109 M and the number of ligands in Daudi cells was 4.2  103. Primary B cells when stimulated with pokeweed mitogen showed enhanced ligand expression receptor binding. On the other hand, the ligand for murine 41BB (m4-1BBL) can be found at low levels on T cell lines (nonactivated and anti-CD3-activated), pre-B cell lines and a few immature macrophage cell lines. Also, the binding analysis with 4-1BB AP (a fusion protein consisting of the extracellular domain of 41BB fused to human placental alkaline phosphatase) exhibited no binding to glial tumor cell line, HeLa cells, or COS cells. On the other hand, the anti-IgMactivated primary B cells compared with anti-CD3activated primary T cells showed higher binding of 4-1BB AP. Scatchard analysis indicated that the A 20 B cell lymphoma expressed 3680 binding sites per cell

One of the interesting aspects of 4-1BB in immune regulation is its ability to occur in soluble form in tissue microenvironment. The 4-1BB shares this feature with certain other receptor forms like TNFR, NGFR, CD27, CD30, and CD95. Evidence documented till now suggests that the 4-1BB molecule is secreted in soluble form in sera and lymphocyte secretions in patients with rheumatoid arthritis (for details, see Michel et al., 1998). The significance of such a phenomenon remains to be explored.

SIGNAL TRANSDUCTION

Associated or intrinsic kinases Though not studied in detail, the signal transduction pathways chosen by 4-1BB is beginning to be appreciated. Studies carried out thus far suggest that no apparent kinase activity is observed with 4-1BB, suggesting that 4-1BB-associated molecules may be involved in 4-1BB-mediated signal transduction.

1688 Dass S. Vinay and Byoung S. Kwon

Cytoplasmic signaling cascades Available data indicate that, like its relatives in the TNFR superfamily, signals by 4-1BB are relayed through TNF receptor-associated factors (TRAFs). Studies from our lab found that TRAF1, TRAF2, and TRAF3 all interact with the cytoplasmic domain of 4-1BB and mutation analysis showed the involvement of the runs of acidic residues in the cytoplasmic domain of 4-1BB (Jang et al., 1998). Jang et al. (1998) and Arch and Thompson (1998) demonstrated that 4-1BB crosslinking induces activation of NFB and is known to be inhibited by dominant negative TRAF2 and NFB-inducing kinase (NIK). Our laboratory also identified a putative binding site for the T cell-specific protein tyrosine kinase p56lck, in the cytoplasmic domain (Kim et al., 1993). The latter observation suggests that 4-1BB may be linked to the src tyrosine kinase family signaling pathway, but this has not been investigated further as yet. A recent study showed a role for apoptosis signal regulatory kinase (ASK-1) and c-Jun N-terminal kinase (JNK)/ stress-activating protein kinase (SAPK) in 4-1BBmediated T cell activation (Cannons et al., 1999).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY Following introduction of agonistic antibodies to 41BB to activate quiescent T cells it is generally 4±5 days before a response is noted (Kim et al., 1998), possibly because T cells require 2±3 days to bring out optimal cell surface expression of 4-1BB (Pollok et al., 1993). Once crosslinked either by anti-4-1BB or 4-1BB ligand (4-1BBL), the 4-1BB molecule transmits a distinct and potent costimulatory signal leading to the activation and differentiation of CD4+ and CD8+ cells in the context of anti-CD3 as first signal, resulting in the secretion of cytokines (IL-2, IL-4, and IFN ) (Chu et al., 1997). Stimulation through 41BB leading to T cell activation appears to work in both a CD28-dependent and -independent manner (DeBenedette et al., 1997). The capacity of 4-1BB to prevent activation-induced cell death (AICD) (Hurtado et al., 1997) and to rescue and sustain the ongoing immune reactions in the apparent absence of the CD28 molecule (Kim et al., 1998) is well documented in literature. It is interesting to note that 41BB-mediated signaling lies in its ability to potentiate

the cytolytic capacity of IL-2-activated intestinal intraepithelial cells (IELs) (Zhou et al., 1994). Our laboratory recently provided evidence that 41BB may be involved in the pathogenesis of AIDS. The level of 4-1BB expression and the percentage of 4-1BB-expressing T cells were higher in HIV-1positive individuals than in HIV-1-negative controls (Wang et al., 1998). In addition, crosslinking 4-1BB with agonistic monoclonal antibody enhanced HIV-1 replication. Administration of monoclonal antibodies to the 4-1BB molecule can completely eradicate established tumors (Melero et al., 1997). Shuford and colleagues (1997) demonstrated that in vivo administration of such monoclonal antibodies enhances graft-versus-host disease (GVHD), and increases the rapidity of cardiac allograft and skin transplant rejection. The role played by 4-1BB in Hodgkin's disease (HD) suggests that eosinophils bearing ligands for 4-1BB may act as an important element in the pathology of HD (Gruss et al., 1996).

Phenotypes of receptor knockouts and receptor overexpression mice Our laboratory has recently generated mice deficient in the 4-1BB molecule. Unpublished data from our laboratory point out that the absence of 4-1BB leads to dysregulated lymphocyte activation and abnormalities in the myeloid progenitor pool. These mice, under nonimmunized conditions, tend to secrete increased amounts of IgG2a, IgA; increased antigen-specific (T-dependent) IgG1, IgG2a, and offer no interference in the IgG class-switch.

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ACKNOWLEDGEMENTS SRC funds to IRC from the Korean Ministry of Science and Technology and NIH Grants (AI28125 and DE12156) are greatly appreciated.