CD40 Ligand

CD40 Ligand Edward A. Clark* Primate Center, University of Washington, Box 357330, Seattle, WA 98195-0001, USA * corresponding author tel: 206-543-8706, fax: 206-685-0305, e-mail: [email protected] DOI: 10.1006/rwcy.2000.05006.

SUMMARY CD40L is a member of the tumor necrosis factor (TNF) family expressed on activated T cells after T cell antigen receptor ligation. It binds to the CD40 receptor on B cells and dendritic cells and thereby provides a critical `helper T cell' signal necessary for germinal center formation, isotype class switching, and production of, for example, IgG and IgE antibodies. CD40L may also transmit a signal back to T cells, to promote short-term T cell proliferation. CD40L and CD40 are not simply restricted to cells regulating immune responses. The CD40LÿCD40 signaling pathway may also play an important role in the regulation of epithelial cell, fibroblast, and smooth muscle cell proliferation.

the signaling pathways and biologic functions of this receptor.

Discovery CD40L/CD154 is a 33±35 kDa type 2 membrane glycoprotein expressed primarily on activated CD4+ T cells but also on CD8+ T cells. It is a member of the TNF family of ligands. The cDNA encoding CD40L was initially described by Armitage et al. (1992), while the CD40L protein was identified by two groups using monoclonal antibody to mouse CD40L/ gp39 (Noelle et al., 1992) and human CD40L/T-BAM (5C8, Lederman et al., 1992) respectively. The CD40L+ T cells are found principally in the outer zone of germinal centers (GCs) and within T zones that are rich in dendritic cells (Casamajor-Palleja et al., 1995). Mature T cells and not thymocytes express CD40L (Fuleihan et al., 1995).

BACKGROUND The MedLine database from 1990 to May 1999 listed over 800 references for CD40L, and in 1998 there were over 180 studies published in which CD40L was mentioned. This chapter will, therefore, simply summarize key information about CD40L (CD154) and the most exciting recent studies on it. There are several excellent reviews discussing CD40L and its historical background (Callard et al., 1993; Banchereau et al., 1994; Foy et al., 1996; Grewal and Flavell, 1998). This chapter emphasizes the expression of CD40L and the biologic consequences of CD40L deficiency or interfering with CD40L binding to CD40. In contrast, the chapter on CD40 principally deals with the expression of CD40 and

Alternative names CD40L (CD154) is also called gp39, T-BAM or TRAP.

Main activities and pathophysiological roles Patients with X-linked hyper-IgM syndrome (HIM) have mutations in a gene mapping to Xq24 and encoding for CD40L (Callard et al., 1993; DiSanto et al., 1993; Korthauer et al., 1993). Patients

458 Edward A. Clark with HIM do not isotype class-switch and produce IgG antibodies, and have few or no GCs in their lymphoid tissues. Similarly, CD40L-deficient mice fail to mount secondary antibody responses to T celldependent antigens and undergo isotype class-switching (Xu et al., 1994; Renshaw et al., 1994). CD40L-deficient individuals have not only deficient antibody responses, but also defective antigen-specific T cell responses (Grewel et al., 1995). The T cell defect is apparently not caused simply by a lack of signaling to antigen-presenting cells (APCs), but is also due to lack of signaling directly to the T cell via CD40L (Armitage et al., 1993; Blair et al., 2000, see below). These findings, together with those of a large series of in vitro studies, have shown that CD40LÿCD40 interactions are usually but not invariably essential for isotype class-switching and germinal center development in responses to T celldependent antigens.

GENE AND GENE REGULATION

Accession numbers GenBank: Mouse CD40L: CAA46448 Human CD40L: NP_00065, P29965 The CD40L gene has also been cloned and sequenced in cattle (Mertens et al., 1995; GenBank accession numbers P51749 and Q28203) and in dogs (GenBank accession number AAD04375).

PROTEIN

Sequence See Figure 1.

Figure 1 Amino acid sequences for mouse CD40L (Armitage et al., 1992) and human CD40L (Hollenbaugh et al., 1992). Gene Mouse CD40 (Armitage et al., 1992, Genebank Assession number CAA46448): 1 MIETYSQPSP RSVATGLPAS MKIFMYLLTV FLITQMIGSV LFAVYLHRRL DKVEEEVNLH 61 EDFVFIKKLK RCNKGEGSLS LLNCEEMRRQ FEDLVKDITL NKEEKKENSF EMQRGDEDPQ 121 IAAHVVSEAN SNAASVLQWA KKGYYTMKSN LVMLENGKQL TVKREGLYYV YTQVTFCSNR 181 EPSSQRPFIV GLWLKPSIGS ERILLKAANT HSSSQLCEQQ SVHLGGVFEL QAGASVFVNV 241 TEASQVIHRV GFSSFGLLKL

Human CD40 (Hollenbaugh et al., 1992, Genebank Accession number NP_00065 or P29965): 1 MIETYNQTSP RSAATGLPIS MKIFMYLLTV FLITQMIGSA LFAVYLHRRL DKIEDERNLH 61 EDFVFMKTIQ RCNTGERSLS LLNCEEIKSQ FEGFVKDIML NKEETKKENS FEMQKGDQNP 121 QIAAHVISEA SSKTTSVLQW AEKGYYTMSN NLVTLENGKQ LTVKRQGLYY IYAQVTFCSN 181 REASSQAPFI ASLCLKSPGR FERILLRAAN THSSAKPCGQ QSIHLGGVFE LQPGASVFVN 241 VTDPSQVSHG TGFTSFGLLK L

CD40 Ligand 459

Discussion of crystal structure A crystal structure of the extracellular region of CD40L has been described (Karpusas et al., 1995), and a three-dimensional structure has been produced showing how CD40L interacts with the CD40 receptor (Singh et al., 1998).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Although the expression of CD40L on activated T cells is emphasized in the literature, CD40L is in fact expressed on a number of cell types, including  T cells, macrophages, Kupffer cells in the liver, dendritic cells, eosinophils, endothelial cells, and smooth muscle cells (Armitage et al., 1992; Gauchat et al., 1995; Horner et al., 1995; Pinchuk et al., 1996; Mach et al., 1997; Gaweco et al., 1999). Some human B cell lines (Grammer et al., 1995), chronic lymphocytic leukemias (Schattner et al., 1998), and B cells from autoimmune (Blossom et al., 1997) or normal mice (Wykes et al., 1998) express CD40L. Ligating CD40 itself can upregulate the expression of its ligand on dendritic cells (Pinchuk et al., 1996) and induce the release of soluble CD40L from mouse B cells expressing cytosolic CD40L (Wykes et al., 1998). Activated NK cells also express CD40L, which may in fact function to facilitate the NK-mediated killing of CD40+ target cells (Carbone et al., 1997). Thus, the `T cell centric' models in the literature almost certainly oversimplify how CD154ÿCD40 interactions are regulated in vivo.

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Again, most of the studies on CD40L expression have been carried out using T cells. Ligating the TCR is the major means by which CD40L is induced on T cells (e.g. de Boer et al., 1993; Roy et al., 1995; Jaiswal et al., 1996), but costimulatory signals such as those via CD28 or cytokines such as IL-2 can sustain CD40L expression on T cells (de Boer et al., 1993; Klaus et al., 1994; Johnson-L'eger et al., 1998a). CD40L expression is reduced on T cells upon contact with CD40+ B cells. This may be induced by

decreasing CD40L mRNA expression, soluble CD40 binding CD40L or the rapid internalization of CD40L (van Kooten et al., 1994; Yellin et al., 1994). A subset of CD4+ memory T cells, like some B cells (Wykes et al., 1998), have preformed cytosolic CD40L (Casamajor-Pallaja et al., 1995). After TCR ligation, this preformed CD40L is rapidly expressed on the cell surface. Based on a combination of findings suggesting that, for example, (a) CD40 ligation upregulates the CD28 ligands CD80 and CD86, and (b) CD28 stimulation promotes the expression of CD40L, we proposed a `reciprocal dialogue' model for the activation of T cells and APCs (Clark and Ledbetter, 1994). This model emphasizes the interrelationship between these two receptor/ligand systems and shows that the signaling pathways are reciprocal, i.e. transmitted not only via CD40 and CD28 but also via their so-called ligands, CD40L and CD80/CD86 (Figure 2). Figure 2 The `reciprocal dialogue' model for the reciprocal activation of antigen-presenting cells (APCs) and T cells (Clark and Ledbetter, 1994). The engagement of MHC plus peptide on the APC by the TCR complex on a T cell activates APCs to express CD80/CD86, and the ligation of TCRs on T cells induces the expression of CD40L. The engagement of CD28 on T cells or CD40 on APCs induces a similar activation pathway including the activation of NFB and pro-growth signals in T and B cells. The reciprocal engagement of CD40L on T cells or CD80 and CD86 on APCs also transmits key signals to cells to promote sustained T cell/APC activation. An additional receptor for CD80/CD86, CTLA4, is also induced via T cell activation and functions in a feedback inhibitory pathway. Another receptor ligand pair, ICOS and ICOSL, which are related to CD28 and CD80 respectively, are also involved in T cell/ APC activation and IL-10 regulation. • proliferation • cytokines for isotype switch

T

CD40L CD40

CD28 CD80/86

B or DC

• proliferation • isotype switch

460 Edward A. Clark As described below, this model has been substantiated in a number of studies both in vivo and in vitro. However, although CD40L expression is further increased principally by ligating CD28 (Klaus et al., 1994), it is also regulated via CD28independent signals from APCs (Ding et al., 1995). Furthermore, several studies have shown that CD40L/ CD40 signaling is not essential for T cell-dependent B cell responses. For example, Life et al. (1994) found that T cells from HIM patients can stimulate IgE production via a CD40L-independent mechanism; similarly, Lane et al. (1995) showed that CD40Lnegative T cells can still induce human B cells to proliferate and become plasma cells. Wu et al. (1995) also found that CD40L T cells can induce the expression of CD80 and CD86 on APCs. More recently, Yu et al. (1999) reported that CD40deficient mice can be induced to class-switch their B cells and produce IgG and IgE. Although the CD40L/CD40 and CD28/CD80ÿCD86 systems play a key role in T and APC activation and B cell development, they are under some conditions not essential. The induction of CD40L on CD3-stimulated T cells can be blocked by inhibitors of phosphatidylinositol 3-kinase (PI-3 kinase; Aagaard-Tillery and Jelinek, 1996), indicating that this kinase plays a key role in the regulation of CD40L. It is also blocked by cyclosporin A (Klaus et al., 1994), suggesting that two distinct pathways via calcineurin and PI-3 kinase work together to promote CD40L expression. IL-12 upregulates CD40L on T cells (Peng et al., 1998), while glucocorticoids reduce its expression (Bischof and Melms, 1998). Whereas IFN can inhibit the expression of CD40L on both TH1 and TH2 T cells, TGF effectively blocks CD40L expression only on TH2 cells (Roy et al., 1993). Studies using TCR transgenic mice suggest that the induction of CD40L does not require costimulatory molecules on APCs, or at least is less dependent on costimulation than is IL-2 production in T cells (Jaiswal et al., 1996). However, Croft et al. (1997) found, in a screen of a number of different T cell-associated molecules, that CD40L and IL-2 expression in particular were dependent on costimulation. Furthermore, Johnson-L'eger et al. (1998b) reported that CD3-stimulated T cells, although initially expressing CD40L, do not produce sufficient IL-2 to sustain CD40L expression; these authors propose that CD4+ helper T cells may need to encounter APCs expressing CD80/86 so that sufficient costimulation occurs, leading to the stable expression of CD40L and maximal secretion of IL-2.

RECEPTOR UTILIZATION CD40L has just one receptor, CD40, a member of the TNF receptor family expressed on B cells, dendritic cells, activated macrophages, follicular dendritic cells, epithelial cells, and smooth muscle cells.

IN VITRO ACTIVITIES

In vitro findings Signaling via CD40L on Activated T Cells There is now good evidence that CD40L can both receive and transmit signals to CD40L+ cells as predicted by the `reciprocal dialogue' model (Clark and Ledbetter, 1994). Armitage et al. (1993) initially reported that CD40L can stimulate T cells to make more IL-2, TNF and IFN . However, whether this occurred via a direct signal or an indirect effect was not clear. Then Cayabyab et al. (1994) found that CD40+ transfectants can augment anti-CD3-induced T cell proliferation via an IL-2-dependent mechanism. The crosslinking of CD40L and CD28 may also promote thymocyte proliferation (Ruggerio et al., 1996). Just as ligating CD40 on B cells activates JNK and p38 MAP kinase so too does ligating CD40L on T cells activate these kinases (Brenner et al., 1997). Peng et al. (1996) found that CD40L/CD40 interactions can upregulate the expression of IL-2 and IFN , as well as of TH2 cytokines such as IL-4, IL-5, and IL-10, by a direct effect on T cells. While it remains possible that some of the effects observed required signaling via CD28 on the T cells, van Essen et al. (1995) found that rudimentary GCs can develop in CD40ÿ/ÿ mice that are injected with a CD40immunoglobulin fusion protein. This suggests that CD40L is essential for transducing a signal to T cells in vivo that is important for the process of GC formation. Likewise, Grewal et al. (1995) found that CD40L was required to induce antigen-specific helper T cells to proliferate; this was probably the result of a direct effect on the T cells as well as via the indirect regulation of `costimulation' on the APCs (Grewal and Flavell, 1998). Blotta et al. (1996) found that crosslinking CD40L on T cells upregulates IL-4 synthesis, and Poudrier et al. (1998) found that CD40L costimulation promotes IL-4 production by T cells in vivo. Most recently, Blair et al. (2000) showed that ligating CD40L promoted CD3-induced T cell proliferation. However, unlike CD28 costimulation, CD40L costimulation does not upregulate Bcl-X and

CD40 Ligand 461 long-term proliferation, suggesting that CD40L may function as a short-term or effector cell costimulator.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Role in experiments of nature and disease states Clinical Effects of Blocking the CD40L/CD40 Pathway In Vivo Grewal et al. (1996) found that CD40Lÿ/ÿ myelin basic protein (MBP) transgenic mice cannot be induced to make IFN or develop experimental allergic encephalomyelitis with MBP unless they are given CD80+ APCs. The lack of CD40L apparently leads to an insufficient induction of functional APCs so that autoimmune disease cannot be induced. The acceptance of allografts in anti-CD40L-treated mice requires the presence of IFN (Markees et al., 1998), but the mechanism behind this requirement is unknown. Antigen-induced airway inflammatory responses are dramatically reduced in CD40Lÿ/ÿ mice, perhaps because IL-4 and TNF levels are reduced (Lei et al., 1998). Given that CD40L/CD40 interactions are essential for many T cell-dependent processes, including the formation of certain autoantibodies, blockade of the CD40L/CD40 pathway with monoclonal antibody or soluble fusion proteins is an attractive immunotherapeutic strategy. Indeed, CD40L (gp39) monoclonal antibodies have been shown in a number of murine models to block the development of autoimmune diseases, especially if given prior to onset of the disease, including collagen-induced arthritis (Durie et al., 1993), spontaneous lupus nephritis (Mohan et al., 1995), autoimmune oophoritis (Griggs et al., 1996), and insulitis/diabetes (Balasa et al., 1997). The effect of anti-CD154 on disease progression is more variable. Whereas the progression of diabetes in nonobese mice is not blocked by anti-CD154 (Balasa et al., 1997), anti-CD40L can block the progression of relapsing experimental autoimmune encephalomyelitis in mice when given either at the peak of the acute disease or during remission (Howard et al., 1999). Furthermore, Larsen et al. (1996a) blocked murine cardiac allograft rejection with anti-CD40L monoclonal antibody in both unimmunized and sensitized mice. Thus, CD40L blockage can be efficacious even

in already sensitized individuals. Balashov et al. (1997) reported that CD40L is expressed at higher levels on activated T cells from patients with multiple sclerosis compared with controls. They suggest that CD40L-induced IL-12 production by these cells may play a key role in the pathogenesis and disease progression of multiple sclerosis. CD40L-positive T cells accumulate in atheroma, and ligating CD40 on atheroma-associated cells induces proinflammatory responses. Thus, Mach et al. (1998) tested whether CD154 monoclonal antibody could influence atherogenesis in vivo; they found that CD40L monoclonal antibody treatment reduced the size of aortic atherosclerotic lesions in mice developing atherosclerosis. Consistent with this, the same group reported later that the CD40Lmediated ligation of CD40 on endothelial cells induces the de novo expression of matrix metalloproteinases and promotes angiogenic functions (Mach et al., 1999). Noelle's group, although pioneers in the area of CD154 monoclonal antibody therapy, have also emphasized that the effect of CD154 monoclonal antibody in vivo may vary depending on a number of factors, including the animal's underlying condition. Autoimmune CD95-deficient mice, for example, treated with anti-CD40L have accelerated renal disease and lymphadenopathy (Russell et al., 1998). Ligating CD40L on lpr/lpr thymocytes apparently inhibits apoptosis. A combination of CD40/CD40L and CD28/ CD80ÿCD86 blockade, as predicted by the model in Figure 2, has been found to be specially effective in blocking deleterious immune responses. Larsen et al. (1996b) found that combined blockade promotes the long-term survival of fully allogeneic skin grafts and vascularized cardiac allografts. This has been substantiated by others (Sun et al., 1997), and again IFN is required for CD40/CD40L and CD28/ CD80±CD86 blockade in order to prevent allograft rejection (Konieczny et al., 1998). This combined blockade can also prevent the development of murine lupus (Daikh et al., 1997). However, the additive or synergistic effects of CD40/CD40L and CD28/CD80±CD86 blockade are not universal. For instance, Bumgardner et al. (1998) suggest that allogeneic responses to hepatocytes require CD40L/CD40 interactions but are not affected by an CTLA4-Ig blockade of CD28 signaling. Furthermore, the CD154 and CD28 blockades may work by affecting independent pathways rather than by simply affecting each other (Judge et al., 1999). Clearly, much remains to be learned about how the selective disruption of CD40L/CD40

462 Edward A. Clark interactions, either alone or in combination with blockade or other receptor systems (e.g. ICOS/ ICOSL, 41BB/41BBL, CD30/CD30L, RANK/ RANKL, etc.), affect T cell anergy or the quality of in vivo immune responses.

Knockout mouse phenotypes CD40L-deficient mice fail to mount secondary antibody responses to T cell-dependent antigens and undergo isotype class-switching (Xu et al., 1994; Renshaw et al., 1994).

References Aagaard-Tillery, K. M., and Jelinek, D. F. (1996). A role for phosphatidylinositol 3-kinase in generating T cell help for B cell growth and differentiation. J. Immunol. 157, 2769±2778. Armitage, R. J., Fanslow, W. C., Strockbine, L., Sato, T. A., Clifford, K. N., Macduff, B. M., Anderson, D. M., Gimpel, S. D., Davis-Smith, T., Maliszewski, C. R., Clark, E. A., Smith, C. A., Grabstein, K. H., Cosman, D., and Spriggs, M. K. (1992). Molecular and biological characterization of a murine ligand for CD40. Nature 357, 80±82. Armitage R. J., Tough, T. W., Macduff, B. M., Fanslow, W. C., Spriggs, M. K., Ramsdell, F., and Alderson, M. R. (1993). CD40 ligand is a T cell growth factor. Eur. J. Immunol. 23, 2326±2331. Balasa, B., Krahl, T., Patstone, G., Lee, J., Tisch, R., McDevitt, H. O., and Sarvetnick, N. (1997). CD40 ligandCD40 interactions are necessary for the initiation of insulitis and diabetes in nonobese diabetic mice. J. Immunol. 159, 4620±4627. Balashov, K. E., Smith, D. R., Khoury, S. J., Hafler, D. A., and Weiner, H. L. (1997). Increased interleukin 12 production in progressive multiple sclerosis: induction by activated CD4+ T cells via CD40 ligand. Proc. Natl Acad. Sci. USA 94, 599±603. Banchereau, J., Bazan, F., Blanchard, D., Briere, F., Galizzi, J. P., van Kooten, C., Liu, Y. J., Rousset, F., and Saeland, S. (1994). The CD40 antigen and its ligand. Annu. Rev. Immunol. 12, 881± 922. Bischof, F., and Melms, A. (1998). Glucocorticoids inhibit CD40 ligand expression of peripheral CD4+ lymphocytes. Cell. Immunol. 187, 38±44. Blair, P. J., Riley, J. L., Harlan, D. M., Abe, R., Tadaki, D. K., Hoffmann, S. C., White, L., Francomano, T., Perfetto, S. J., Kirk, A. D., and June, C. H. (2000). CD40 Ligand (CD154) Triggers a short-term CD4+ T cell activation response that results in secretion of immunomodulatory cytokines and apoptosis. J. Exp. Med. 191, 651±660. Blossom, S., Chu, E. B., Weigle, W. O., and Gilbert, K. M. (1997). CD40 ligand expressed on B cells in the BXSB mouse model of systemic lupus erythematosus. J. Immunol. 159, 4580±4586. Blotta, M. H., Marshall, J. D., DeKruyff, R. H., and Umetsu, D. T. (1996). Cross-linking of the CD40 ligand on human CD4+ T lymphocytes generates a costimulatory signal that up-regulates IL-4 synthesis. J. Immunol. 156, 3133±3140. Boer, M. de, Kasran, A., Kwekkeboom, J., Walter, H., Vandenberghe, P., and Ceuppens, J. L. (1993). Ligation of B7 with CD28/CTLA-4 on T cells results in CD40 ligand

expression, interleukin-4 secretion and efficient help for antibody production by B cells. Eur. J. Immunol. 23, 3120±3125. Brenner, B., Koppenhoefer, U., Grassme, H., Kun, J., Lang, F., and Gulbins, E. (1997). Evidence for a novel function of the CD40 ligand as a signalling molecule in T-lymphocytes. FEBS Lett. 417, 301±306. Bumgardner, G. L., Li, J., Heininger, M., and Orosz, C. G. (1998). Costimulation pathways in host immune responses to allogeneic hepatocytes. Transplantation 66, 1841±1845. Callard, R. E., Armitage, R. J., Fanslow, W. C., and Spriggs, M. K. (1993). CD40 ligand and its role in X-linked hyper-IgM syndrome. Immunol. Today 14, 559±564. Carbone, E., Ruggiero, G., Terrazzano, G., Palomba, C., Manzo, C., Fontana, S., Spits, H., Karre, K., and Zappacosta, S. (1997). A new mechanism of NK cell cytotoxicity activation: the CD40±CD40 ligand interaction. J. Exp. Med. 185, 2053±2060. Casamayor-Palleja, M., Khan, M., and MacLennan, I. C. (1995). A subset of CD4+ memory T cells contains preformed CD40 ligand that is rapidly but transiently expressed on their surface after activation through the T cell receptor complex. J. Exp. Med. 181, 1293±1301. Cayabyab, M., Phillips, J. H., and Lanier, L. L. (1994). CD40 preferentially costimulates activation of CD4+ T lymphocytes. J. Immunol. 152, 1523±1531. Clark, E. A., and Ledbetter, J. A. (1994). How B and T cells talk to each other. Nature 367, 425±428. Croft, M., Joseph, S. B., and Miner, K. T. (1997). Partial activation of naive CD4 T cells and tolerance induction in response to peptide presented by resting B cells. J. Immunol. 159, 3257± 3265. Daikh, D. I., Finck, B. K., Linsley, P. S., Hollenbaugh, D., and Wofsy, D. (1997). Long-term inhibition of murine lupus by brief simultaneous blockade of the B7/CD28 and CD40/gp39 costimulation pathways. J. Immunol. 159, 3104±3108. Ding, L., Green, J. M., Thompson, C. B., and Shevach, E. M. (1995). B7/CD28-dependent and -independent induction of CD40 ligand expression. J. Immunol. 155, 5124±5132. 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., Fava, R. A., Foy, T. M., Aruffo, A., Ledbetter, J. A., and Noelle, R. J. (1993). Prevention of collagen-induced arthritis with an antibody to gp39, the ligand for CD40. Science 261, 1328±1330. Foy, T. M., Aruffo, A., Bajorath, J., Buhlmann, J. E., and Noelle, R. J. (1996). Immune regulation by CD40 and its ligand GP39. Annu. Rev. Immunol. 14, 591±617. Fuleihan, R., Ahern, D., and Geha, R. S. (1995). Expression of the CD40 ligand in T lymphocytes and induction of IgE isotype switching. Int. Arch. Allergy Immunol. 107, 43±44. Gauchat, J. F., Henchoz, S., Fattah, D., Mazzei, G., Aubry, J. P., Jomotte, T., Dash, L., Page, K., Solari, R., Aldebert, D., et al. (1995). D40 ligand is functionally expressed on human eosinophils. Eur. J. Immunol. 25, 863±865. Gaweco, A. S., Wiesner, R. H., Yong, S., Krom, R., Porayko, M., Chejfec, G., McClatchey, K. D., and Van Thiel, D. H. (1999). Kupffer cell expression of CD40L (CD154) in human chronic liver allograft rejection. Transplant. Proc. 31, 560± 561. Grammer, A. C., Bergman, M. C., Miura, Y., Fujita, K., Davis, L. S., and Lipsky, P. E. (1995). The CD40 ligand expressed by human B cells costimulates B cell responses. J. Immunol. 154, 4996±5010. Grewal, I. S., and Flavell, R. A. (1998). CD40 and CD154 in cellmediated immunity. Annu. Rev. Immunol. 16, 111±35.

CD40 Ligand 463 Grewal, I. S., Xu, J., and Flavell, R. A. (1995). Impairment of antigen-specific T-cell priming in mice lacking CD40 ligand. Nature 378, 617±620. Grewal, I. S., Foellmer, H. G., Grewal, K. D., Xu, J., Hardardottir, F., Baron, J. L., Janeway, C.A. Jr., and Flavell, R. A. (1996). Requirement for CD40 ligand in costimulation induction, T cell activation, and experimental allergic encephalomyelitis. Science 273, 1864±1867. Griggs, N. D., Agersborg, S. S., Noelle, R. J., Ledbetter, J. A., Linsley, P. S., and Tung, K. S. (1996). The relative contribution of the CD28 and gp39 costimulatory pathways in the clonal expansion and pathogenic acquisition of self-reactive T cells. J. Exp. Med. 183, 801±810. Hollenbaugh, D., Grosmaire, L. S., Kullas, C. D., Chalupny, N. J., Braesch-Andersen, S., Noelle, R. J., Stamenkovic, I., Ledbetter, J. A., and Aruffo, A. (1992). The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity. EMBO J. 11, 4313±4321. Horner, A. A., Jabara, H., Ramesh, N., and Geha, R. S. (1995). Gamma/delta T lymphocytes express CD40 ligand and induce isotype switching in B lymphocytes. J. Exp. Med. 181, 1239±1244. Howard, L. M., Miga, A. J., Vanderlugt, C. L., Dal Canto, M. C., Laman, J. D., Noelle, R. J., and Miller, S. D. (1999). Mechanisms of immunotherapeutic intervention by antiCD40L (CD154) antibody in an animal model of multiple sclerosis. J. Clin. Invest. 103, 281±290. Jaiswal, A. I., Dubey, C., Swain, S. L., and Croft, M. (1996). Regulation of CD40 ligand expression on naive CD4 T cells: a role for TCR but not co-stimulatory signals. Int. Immunol. 8, 275±285. Johnson-L'eger, C., Christenson, J., and Klaus, G. G. (1998a). CD28 co-stimulation stabilizes the expression of the CD40 ligand on T cells. Int. Immunol. 10, 1083±1091. Johnson-L'eger, C., Christenson, J. R., Holman, M., and Klaus, G. G. (1998b). Evidence for a critical role for IL-2 in CD40-mediated activation of naive B cells by primary CD4 T cells. J. Immunol. 161, 4618±4626. Judge, T. A., Wu, Z., Zheng, X. G., Sharpe, A. H., Sayegh, M. H., and Turka, L. A. (1999). The role of CD80, CD86, and CTLA4 in alloimmune responses and the induction of long-term allograft survival. J. Immunol. 162, 1947±1951. Karpusas, M., Hsu, Y. M., Wang, J. H., Thompson, J., Lederman, S., Chess, L., and Thomas, T. (1995). A crystal structure of an extracellular fragment of human CD40 ligand. Structure 3, 1031±1039. Klaus, S. J., Pinchuk, L. M., Ochs, H. D., Law, C. L., Fanslow, W. C., Armitage, R. J., and Clark, E. A. (1994). Costimulation through CD28 enhances T cell-dependent B cell activation via CD40±CD40L interaction. J. Immunol. 152, 5643±5652. Konieczny, B. T., Dai, Z., Elwood, E. T., Saleem, S., Linsley, P. S., Baddoura, F. K., Larsen, C. P., Pearson, T. C., and Lakkis, F. G. (1998). IFN-gamma is critical for long-term allograft survival induced by blocking the CD28 and CD40 ligand T cell costimulation pathways. J. Immunol. 160, 2059±2064. 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 Xlinked immunodeficiency with hyper-IgM. Nature 361, 539±541. Lane, P., Burdet, C., McConnell, F., Lanzavecchia, A., and Padovan, E. (1995). CD40 ligand-independent B cell activation revealed by CD40 ligand-deficient T cell clones: evidence for distinct activation requirements for antibody formation and B cell proliferation. Eur. J. Immunol. 25, 1788±1793.

Larsen, C. P., Alexander, D. Z., Hollenbaugh, D., Elwood, E. T., Ritchie, S. C., Aruffo, A., Hendrix, R., and Pearson, T. C. (1996). CD40-gp39 interactions play a critical role during allograft rejection. Suppression of allograft rejection by blockade of the CD40-gp39 pathway. Transplantation 61, 4±9. Larsen, C. P., Elwood, E. T., Alexander, D. Z., Ritchie, S. C., Hendrix, R., Tucker-Burden, C., Cho, H. R., Aruffo, A., Hollenbaugh, D., Linsley, P. S., Winn, K. J., and Pearson, T. C. (1996). Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381, 434±438. Lederman, S., Yellin, M. J., Krichevsky, A., Belko, J., Lee, J. J., and Chess, L. (1992). Identification of a novel surface protein on activated CD4+ T cells that induces contact-dependent B cell differentiation (help). J. Exp. Med. 175, 1091±1101. Lei, X. F., Ohkawara, Y., Stampfli, M. R., Mastruzzo, C., Marr, R. A., Snider, D., Xing, Z., and Jordana, M. (1998). Disruption of antigen-induced inflammatory responses in CD40 ligand knockout mice. J. Clin. Invest. 101, 1342±1353. Life, P., Gauchat, J. F., Schnuriger, V., Estoppey, S., Mazzei, G., Durandy, A., Fischer, A., and Bonnefoy, J. Y. (1994). T cell clones from an X-linked hyper-immunoglobulin (IgM) patient induce IgE synthesis in vitro despite expression of nonfunctional CD40 ligand. J. Exp. Med. 180, 1775±1784. Mach, F., Schonbeck, U., Sukhova, G. K., Bourcier, T., Bonnefoy, J. Y., Pober, J. S., and Libby, P. (1997). Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for CD40ÿCD40 ligand signaling in atherosclerosis. Proc. Natl Acad. Sci. USA 94, 1931±1936. Mach, F., Schonbeck, U., Sukhova, G. K., Atkinson, E., and Libby, P. (1998). Reduction of atherosclerosis in mice by inhibition of CD40 signalling. Nature 394, 200±203. Mach, F., Schonbeck, U., Fabunmi, R. P., Murphy, C., Atkinson, E., Bonnefoy, J. Y., Graber, P., and Libby, P. (1999). T lymphocytes induce endothelial cell matrix metalloproteinase expression by a CD40L-dependent mechanism: implications for tubule formation. Am. J. Pathol. 154, 229±238. Markees, T. G., Phillips, N. E., Gordon, E. J., Noelle, R. J., Shultz, L. D., Mordes, J. P., Greiner, D. L., and Rossini, A. A. (1998). Long-term survival of skin allografts induced by donor splenocytes and anti-CD154 antibody in thymectomized mice requires CD4(+) T cells, interferon-gamma, and CTLA4. J. Clin. Invest. 101, 2446±2455. Mertens, B., Muriuki, C., and Gaidulis, L. (1995). Cloning of two members of the TNF-superfamily in cattle: CD40 ligand and tumor necrosis factor alpha. Immunogenetics 42, 430±431. Mohan, C., Shi, Y., Laman, J. D., and Datta, S. K. (1995). Interaction between CD40 and its ligand gp39 in the development of murine lupus nephritis. J. Immunol. 154, 1470±1480. Noelle, R. J., Roy, M., Shepherd, D. M., Stamenkovic, I., Ledbetter, J. A., and Aruffo, A. (1992). A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells. Proc. Natl Acad. Sci. USA 89, 6550±6554. Peng, X., Kasran, A., Warmerdam, P. A., de Boer, M., and Ceuppens, J. L. (1996). Accessory signaling by CD40 for T cell activation: induction of Th1 and Th2 cytokines and synergy with interleukin-12 for interferon-gamma production. Eur. J. Immunol. 26, 1621±1627. Peng, X., Remacle, J. E., Kasran, A., Huylebroeck, D., and Ceuppens, J. L. (1998). IL-12 up-regulates CD40 ligand (CD154) expression on human T cells. J. Immunol. 160, 1166±1172. Pinchuk, L. M., Klaus, S. J., Magaletti, D. M., Pinchuk, G. V., Norsen, J. P., and Clark, E. A. (1996). Functional CD40 ligand expressed by human blood dendritic cells is up-regulated by CD40 ligation. J. Immunol. 157, 4363±4370.

464 Edward A. Clark Poudrier, J., van Essen, D., Morales-Alcelay, S., Leanderson, T., Bergthorsdottir, S., and Gray, D. (1998). CD40 ligand signals optimize T helper cell cytokine production: role in Th2 development and induction of germinal centers. Eur. J. Immunol. 28, 3371±3383. 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. Roy, M., Waldschmidt, T., Aruffo, A., Ledbetter, J. A., and Noelle, R. J. (1993). The regulation of the expression of gp39, the CD40 ligand, on normal and cloned CD4+ T cells. J. Immunol. 151, 2497±2510. Roy, M., Aruffo, A., Ledbetter, J., Linsley, P., Kehry, M., and Noelle, R. (1995). Studies on the interdependence of gp39 and B7 expression and function during antigen-specific immune responses. Eur. J. Immunol. 25, 596±603. Ruggiero, G., C'aceres, E. M., Voordouw, A., Noteboom, E., Graf, D., Kroczek, R. A., and Spits, H. (1996). CD40 expressed on thymic epithelial cells provides costimulation for proliferation but not for apoptosis of human thymocytes. J. Immunol. 156, 3737±3746. Russell, J. Q., Mooney, T., Cohen, P. L., MacPherson, B., Noelle, R. J., and Budd, R. C. (1998). Anti-CD40L accelerates renal disease and adenopathy in MRL-lpr mice in parallel with decreased thymocyte apoptosis. J. Immunol. 161, 729±739. Schattner, E. J., Mascarenhas, J., Reyfman, I., Koshy, M., Woo, C., Friedman, S. M., and Crow, M. K. (1998). Chronic lymphocytic leukemia B cells can express CD40 ligand and demonstrate T-cell type costimulatory capacity. Blood 91, 2689±2697. Singh, J., Garber, E., Van Vlijmen, H., Karpusas, M., Hsu, Y. M., Zheng, Z., Naismith, J. H., and Thomas, D. (1998). The role of polar interactions in the molecular recognition of CD40L with its receptor CD40. Protein Sci. 7, 1124±1135. Sun, H., Subbotin, V., Chen, C., Aitouche, A., Valdivia, L. A., Sayegh, M. H., Linsley, P. S., Fung, J. J., Starzl, T. E., and

Rao, A. S. (1997). Prevention of chronic rejection in mouse aortic allografts by combined treatment with CTLA4-Ig and anti-CD40 ligand monoclonal antibody. Transplantation 64, 1838±1843. van Essen, D., Kikutani, H., and Gray, D. (1995). CD40 ligandtransduced co-stimulation of T cells in the development of helper function. Nature 378, 620±623. van Kooten, C., Gaillard, C., Galizzi, J. P., Hermann, P., Fossiez, F., Banchereau, J., and Blanchard, D. (1994). B cells regulate expression of CD40 ligand on activated T cells by lowering the mRNA level and through the release of soluble CD40. Eur. J. Immunol. 24, 787±792. Wu, Y., Xu, J., Shinde, S., Grewal, I., Henderson, T., Flavell, R. A., and Liu, Y. (1995). Rapid induction of a novel costimulatory activity on B cells by CD40 ligand. Curr. Biol. 5, 1303±1311. Wykes, M., Poudrier, J., Lindstedt, R., and Gray, D. (1998). Regulation of cytoplasmic, surface and soluble forms of CD40 ligand in mouse B cells. Eur. J. Immunol. 28, 548±559. 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. Yellin, M. J., Sippel, K., Inghirami, G., Covey, L. R., Lee, J. J., Sinning, J., Clark, E. A., Chess, L., and Lederman, S. (1994). CD40 molecules induce down-modulation and endocytosis of T cell surface T cell-B cell activating molecule/CD40-L. Potential role in regulating helper effector function. J. Immunol. 152, 598±608. Yu, P., Morawetz, R. A., Chattopadhyay, S., Makino, M., Kishimoto, T., and Kikutani, H. (1999). CD40-deficient mice infected with the defective murine leukemia virus LP-BM5def do not develop murine AIDS but produce IgE and IgG1 in vivo. Eur. J. Immunol. 29, 615±625.