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.

CD137 Pathway: Immunology and Diseases. Edited by Lieping Chen, Springer, New York, 2006

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, andCD30/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 etal., 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 4-1BB 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 integrin-mediated cell adhesion, and increasing T-cell cytolytic potential (Kwon et al., 2000; Melero etal., 1997; Shuford etal., 1997; Tan etal., 2000; Vinay and Kwon, 1998). In an experiment involving 4-1BBL-/- mice infected with influenza virus, the knockout mice had lower numbers ofDb/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 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-y, which in turn acts on DCs or macrophages in a paracrine manner. IFN-y-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 monomelic 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 lig-and. 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 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-kB and AP-1 (Saoulli et al., 1998). Studies to date have shown that 4-1BB produces signals through TNF receptor-associated factor-NF-KB inducing kinase-NF-KB (TRAF-NIK-NF-kB) (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 4-1BB-mediated NF-kB activation that provides CD8+ lymphocytes with prolonged survival via up-regulation of Bcl-XL and Bfl-1 expression. The critical importance of NF-kB activation by 4-1BB for Bcl-XL and Bfl-1 induction in our study is consistent with recent studies showing NF- KB-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-kB activation is essential for Bcl-XL induction and anti-apoptotic effects in primary human CD4+ T lymphocytes (Khoshnan et al., 2000). Similarly, NF-KB-mediated up-regulation of Bcl-XL and Bfl-1 is important for CD40 survival signaling in B lymphocytes (Lee etal., 1999). Although it has been shown that the PI-3 kinase/Akt pathway plays a key role in NF-kB 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 andBfl-1 in primary CD8+ Tlympho-cytes. LY294002, a PI-3 kinase blocker, abolished 4-1BB-mediated T cell proliferation to the same extent as did PDTC, an NF- kB 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-kB signals have separate

Cd137 Signal

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-kB, intracellular [Ca2+], and phosphorylation of ERK1/2. 4-1BB-mediated activation of NF-kB 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-y 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-y. 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.

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-kB, intracellular [Ca2+], and phosphorylation of ERK1/2. 4-1BB-mediated activation of NF-kB 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-y 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-y. 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-kB 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-kB 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 -pathway-mediated down-regulation of p27kip1 (Appleman et al., 2002). Mitogenic ligands 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, p27kip1 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 ofERK1/2, 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 Gi/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 etal., 1997; Moon and Nelson, 2001; Schluns etal., 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 thanPD98059, rapamycin, wortmannin, oranti-IL-2mAb. 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-sensitivePI3K/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 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 p21kip1, 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 anti-apoptotic genes bcl-XL 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 knock-out/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 p815-m4-1BBL cells. TCR pathway proteins such as Lck, pTyr, PKC-0 and SLP-76 were also redistributed to the lipid rafts, and other TCR pathway proteins such as PKC-0 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-y1 to the lipid rafts and its subsequent activation. Methyl-p-cyclodextrin, which disrupts raft formation (Marwali et al., 2003), blocked translocation of PLC-y1 to the detergent-insoluble fraction, redistribution of lipid rafts and of Lck, and degradation of IKB-a, 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 blocks 4-1BB-induced IL-2 and IFN-y 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-kB, 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 4-1BB 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 phophory-lation 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- kB 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 co-stimulatory molecule in both adaptive and innate immunity, its signal transduction 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-2004-003-100349) and the International Cooperation Research Program from KOSEF and the Korean Ministry of Science and Technology.

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