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Contact-mediated signals and cytokines involved in B-cell activation and isotype switching in pre-B and mature B cells
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IN IA4MUNOLOGY
Contact-mediated signals and cytokines involved in B-cell activation and isotype switching in pre-B and mature B cells G. Aversa, B.C. Cocks, J. Punnonen, J.M. Carballido and J.E. de Vries Human
Immunology
Department, DNAX Research Institute, Palo Alto, CA 94304-1104 (USA)
Introduction
It has long been recognized that B-cell responses to antigen resulting in Ig synthesis require help from T cells (Okumura and Tada, 1971). More recently, it has become clear that this helper activity of T cells resides in the CD4+ subset. Cytokines produced by these cells promote B-cell growth and differentiation. In addition, they have been shown to direct Ig isotype switching both in mouse and human B cells (Coffman et al., 1988; de Vries et al., 1991). For example, IL4 and IL13 were found to induce human B cells to switch to IgG4 and IgE synthesis (P&e et al., 1988; Punnonen, 1993a), whereas TGF-P mediates IgA switching (Van Vlasselaer et al., 1992 ; Defiance et al., 1992). In addition to cytokines, a contact-mediated signal provided by activated CD4+ T cells was required for productive T-B cell interactions, resulting in Ig isotype switching and Ig production. This signal was delivered by molecules expressed on the surface of activated CD4+ T cells, as membranes of these cells were sufficient to stimulate B cells to proliferate and secrete Ig in the presence of cytokines (Brian et al., 1988; Hodgkin et al., 1990; Gascan et al., 1992). Resting T cells were ineffective. This T-cell derived contact-mediated signal could be substituted by mAb against CD40, a molecule constitutively expressed on B cells (Zhang et al., 1991; Gascan et al., 1991). The cloning and characterization of the mouse and subsequently, the human CD40 ligand (CD4OL) (Armitage et al., 1992; Spriggs et al., 1992; Hollenbaugh et al., 1992; Cocks et al., 1993) established this early T-cell activation antigen as a major component required for productive T-B cell interactions, resulting in Ig isotype switching and Ig synthesis. The CD4OL appears to be the major component of the T-cell signal provided by activated CD4+ T cells to B cells, and transfectants expressing CD40L, or anti-CD40 mAb, can replace activated CD4+ T helper cells in providing B-cell help. Strong
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synergistic effects on Ig production, however, were obtained when activated CD4+ T-cell clones were added together with optimal concentrations of antiCD40 mAb and IL4 to cultures of purified human B cells (Gascan et al., 1991), indicating that activated CD4+ T cells may provide, in addition to CD40L, additional costimulatory signals for B cells. Cross-linking of CD40 in the presence insufficient for pre-B-cell differentiation
of IL4 is
The differences between T-cell-derived costimulatory signals and CD40 engagement alone by antiCD40 mAb are more apparent in activation and isotype switching of pre-B cells. In contrast to mature B cells (Gascan et al., 1991), highly purified sp-, cp+, CDlO+, CD19+ pre-B cells obtained from foetal bone marrow stimulated with anti-CD40 mAb, either in soluble form or cross-linked on L cells transfected with CD32/FcyRII were unable to proliferate, switch isotype or produce Ig in the presence of IL4 (Punnonen et al., 1993b). Interestingly, activated Tcell clones or their plasma membranes were effective in inducing isotype switching and Ig production by pre-B cells in the presence of IL4 (Punnonen et al., 1993b), indicating that in addition to CD40 crosslinking, other contact-mediated signals were provided by the T-cell clones or their membranes. It may be argued that the failure of anti-CD40 mAb to stimulate pre-B cells may be due to the low expression of CD40 on this precursor population compared to mature B cells. Indeed, stimulation of pre-B cells with activated T-cell clones and IL4, enhanced CD40 expression on these cells (Punnonen et al., 1993b) and this enhancement may be important in rendering the B cells more responsive to the CD4OL expressed on activated T helper cells. Whether the initial stimulus that induces B cells to enhance their CD40 expression may be the CD40L itself, or other cell surface molecules, remains to be determined. It
THE CD4O/CD4OL is clear, however, that simply cross-linking CD40 on pre-B cells is insufficient for the induction of differentiation of these cells into antibody-secreting cells in the presence of IL4. From these observations, it appears that B-cell progenitors can be induced via CD40 to differentiate into Ig-secreting cells, only after they have progressed beyond the pre-B-cell stage. The above in vitro observations of pre-B cells are compatible with recent reports of hyper-IgM syndrome patients. In these individuals, the intrinsic defect in isotype switching is a consequence of defective CD40L expression (Aruffo et al., 1993; Allen et al., 1993 ; DiSanto et al., 1993 ; Korthauer et al., 1993 ; Fuleihan et al., 1993). The number of B cells in these patients, however, is in the normal range, indicating that functional CD40L is not a necessary component for B-cell development. Furthermore, these patients have normal or elevated serum IgM levels, indicating that B cells still can be induced to proliferate and differentiate to IgM-secreting cells despite the absence of functional CD40L. This suggests that additional molecules are involved in the proliferation and differentiation of immature and mature B cells and that the CD40L does not play a major role in B-cell ontogeny.
Is CD40L alone activation ?
sufficient
for
mature
B-cell
Engagement of CD40 on mature B cells by antiCD40 mAb in the presence of IL4 or IL13 induces B-cell proliferation and Ig synthesis (Gascan et al., 1991; Zhang et al., 1991; Punnonen et al., 1993). Cross-linking CD40 on human B cells with human CD40L, expressed on CV-l/EBNA or COS cells, induces B-cell proliferation in the absence of cytokines and Ig synthesis in the presence of cytokines (Spriggs et al., 1992; Cocks et al., 1993). Soluble CD40L, cross-linked by a second step reagent or in a multimerit form, was reported to be biologically active in directly stimulating B-cell proliferation (Lane et al., 1993 ; Armitage et al., 1993a,b), indicating that crosslinking of CD40 is required and is sufficient for Bcell triggering. It appears, therefore, that the CD40L is able, by itself, to stimulate proliferation of mature B cells. It has been shown recently that CD40L expression on activated murine T-cell clones can be stabilized by plastic-bound stimuli, or by CD40 engagement using CD40 Ig, whereas soluble stimuli induce a rapid turnover of this molecule (Castle et al., 1993). In addition, the levels of newly synthesized CD40L on membranes from activated T-cell clones did not correlate with their ability to induce B-cell proliferation (Castle et al., 1993). The authors sug-
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gested that de nova synthesized CD40L is not efficient in activating B cells and that modification or assembly of the CD40L, or additional accessory signals, play a role in augmenting T-dependent B-cell responses. We recently reported that the transmembrane form of TNFa (mTNFa), which shares sequence and structural homologies with the CD40L (Peitsch and Jongeneel, 1993), is involved in T-B cell interactions, leading to IgG4 and IgE synthesis (Aversa et al., 1993). mAb to TNFa blocked ILCdependent IgG4 and IgE synthesis by highly purified or sIgD+ B cells, stimulated by activated T-cell clones or their plasma membranes (Aversa et al., 1993). Membrane TNFa was also reported to be an important molecule for help given by unstimulated HIV+ T cells to B cells, resulting in B-cell proliferation and Ig synthesis, and this help was suggested-to be CD40Lindependent (Macchia et al., 1993). The inability of anti-TNFR mAb to directly stimulate B cells in the presence of cytokines suggests that these mAb do not act like the anti-CD40 mAb, and that the costimulatory signal provided by the mTNFa is qualitatively different from that of the CD40L. Thus, CD40L alone is sufficient for adult B-cell activation. However, its optimal functional expression on T cells does not seems to be directly dependent on its level of expression, but rather on its post-synthetic modification/assembly or association with other surface molecules.
Role of CD40 and CD40L
expression
on T cells
The role that CD4OL plays in B-cell activation has been extensively studied. The implications for its function on T cells, however, is much less understood. A recent study shows that human CD4+ T cells can be costimulated via the CD40L by mouse cells transfected with CD40, in combination with anti-CD3 mAb (Cayabyab et al., 1994). This study and a previous report showing functional expression of CD40 on thymic epithelial cells (Galy et al., 1992) suggests a role for the CD40L in T-cell differentiation and activation. Thus, CD40L-CD40 interactions should not be considered a one-way signal from activated T to B cells, but rather a two-way interaction between T cells and APC or between other cell types expressing this receptor-ligand combination. We have previously shown that anti-CD40 mAb synergize with T-cell clones in inducing Ig production by B cells (Gascan et al., 1991). In subsequent studies, we substituted membranes from T-cell clones for the intact T cells and we consistently found a dose-dependent cross-inhibition of anti-CD40 mAb
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and T-cell membranes, rather than synergy (G. Aversa et al., unpublished data). This puzzling finding could be partly explained by a recent report showing expression of CD40 on resting as well as activated T cells (Armitage et al., 1993a), which is consistent with the ability of CD40L to costimulate T cells (Armitage et al., 1993b). The CD40 molecules present on the membrane preparation could sequester the anti-CD40 mAb used for stimulating the B cells, thus inactivating it. The large amounts of T-cell membrane equivalents, compared to the small number of intact T-cell clones necessary for stimulating B cells (Gascan et al., 1992) may account for the neutralization of anti-CD40 mAb by membranes, but not by the intact T-cell clones. This explanation, however, does not explain why the membrane signal itself would be affected by the anti-CD40 mAb. The physiological function of CD40 on T cells is not clear, although its presence on these cells suggests a role in T-T cell interactions, and a possible function as an additional accessory component in T-cell activation.
Role of CD40L on non-T cells CD4OL expression, detected by Northern blots and FACS analysis, was originally reported to be restricted to T cells (Spriggs et al., 1992). PCR analysis however, revealed that purified monocytes and NK cells also express the CD40L message (Cocks et al., 1993), and this was consistent with reports of NK cells providing costimulatory signals to B cells for Ig synthesis (Yuan et al., 1992). More recently, functional human CD40L expression was detected on mast cells and basophils, the latter expressing this molecule constitutively (Gauchat et al., 1993). Mast cells were able to induce IgE synthesis in B cells in the presence of IL4, whereas basophils did not require exogenous cytokines, probably because they produced IL4 (Gauchat et al., 1993). Recently, we found that activated, but not resting, U937 monocytoid cells, in the presence of IL4, induce proliferation and Ig secretion by highly purified B cells (Aversa et al., manuscript in preparation). CD40L expression on the surface of stimulated U937 cells could not be detected, suggesting the possibility that other activation-induced molecules on monocytes may provide B-cell help. It cannot be excluded, however, that a few CD40L molecules, below detection levels, may be present on the surface of activated U937 cells, and that these may be sufficient, by themselves or in synergy with other molecules, in inducing B-cell activation. The finding that CD40L is expressed on cells such as mast cells and basophils, which can also produce cytokines like IL4, suggests that B-cell help can oc-
cur in vivo at sites distinct from lymphoid organs, in tissues where B cells can come in contact with these non-T helper cells.
Concluding
remarks
The important role of CD40L in the activation and differentiation of mature B cells leading to Ig secretion is now well established. It is also clear that the CD4OL is not essential for B-cell development and is not sufficient for the activation and differentiation of pre-B cells, and that other surface molecules are involved in this process. The nature of these other molecules is still unknown, and their identification may provide a better insight into the process of B-cell development. The observation that the CD40L can function as a signalling molecule for T cells adds another piece to the puzzle of cellular interactions and lymphocyte function: the detection of CD40 on T cells and the CD40L on mast cells, basophils, NK cells and monocytes. In addition, the finding that cells other than T cells, which are located in non-lymphoid tissues, express CD40L, produce IL4 and can mediate B-cell activation and differentiation including IgE synthesis, has implications for the understanding the initiation and pathophysiology of allergic disorders, distinguishing the local events from systemic immune responses, and may help in the design of more effective therapies. The authors assistance.
wish to thank
Jo Ann Katheiser for secretarial
DNAX Research Institute of Molecular and Cellular Biology, Inc. is supported by Schering-Plough Corporation.
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K.N.,
Macduff,
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Anderson,
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Gimpel, S.D., Davis-Smith, T., Maliszewski, C.R., Clark, E.A., Smith, C.A., Grabstein, K.H., Cosman, D. & Spriggs, M.K. (1992), Molecular and biological characterization of a murine ligand for CD40. iVature (Lond.), 357, 80-82. Armitage, R.J., Macduff, B.M., Alderson, M.R. & Fanslow, W.C. (1993a), Induction of T-cell proliferation by antibodies from the CD40 panel. Tissue Antigens, 42, 424. Am-&age, R.J., Tough, T.W., Macduff, B.M., Fanslow, W .C., Spriggs, M.K., Ramsdell, F. & Alderson, M.R.
THE CD4O/CD4OL (1993b), CD40 ligand is a T-cell growth factor. Eur. J. Immunol., 23, 2326-233 1. Aruffo, A., Farrington, M., Hollenbaugh, D., Li, X., Milatovich, A., Nonoyama, S., Bajorath, J., Grosmaire, L.S., Stenkamp, R., Neubauer, M., Roberts, R.L., Noelle, R.J., Ledbetter, J.A., Francke, U. & Ocks, H.D. (1993), The CD40 ligand, gp39, is defective in activated T cells from patients with X-linked hyper-IgM syndrome. Cell, 12, 291-300. Aversa, G., Punnonen, J. & de Vries, J.E. (1993), The 26 kd transmembrane form of TNF-a on activated CD4+ T-cell clones provides a costimulatory signal for human B cell activation. J. Exp. Med., 177, 15751585. Brian, A.A. (1988), Stimulation of B-cell proliferation by membrane-associated molecules from activated T cells. Proc. Nati. Acad. Sci. USA, 85, 564-568. Castle, B.E., Kishimoto, K., Steams, C., Brown, M.L. & Kehry, M.R. (1993), Regulation of expression of the ligand for CD40 on T helper lymphocytes. J. Immunol., 151, 1777-1788. Cayabyab, M., Phillips, J.H. & Lanier, L.L. (1994), CD40 preferentially co-stimulates activation of CD4+ T lymphocytes. J. Immunol., 152, 1523-1531. Cocks, B., de Waal Malefyt, R., Galizzi, J.-P., de Vries, J.E. & Aversa, G. (1993), IL-13 induces proliferation and differentiation of human B cells activated by the CD40 ligand. ht. Immunol., 5, 657-663. Coffman, R.L., Seymour, B. W.P., Lebman, D.A., Hiraki, D.D., Christiansen, J.A., Shrader, B., Cherwinski, H.M., Savelkoul, H.F.J., Finkelman, F.D., Bond, M.W. & Mosmann, T.R. (1988), The role of helper T cell products in mouse B cell differentiation and isotype regulation. Immunol. Rev., 102. 5-28. De Vries, J.E., Gauchat, J.-F., Aversa, G.G., Punnonen, J., Gascan, H. & Yssel, H. (1991), Regulation of IgE synthesis by cytokines. Curr. Opin. Immunol., 3, 851-858. Defiance, T., Vanbervliet, B., Briere, F., Durand, I., Rousset, F. & Banchereau, J. (1992), Interleukin 10 and transforming growth factor p cooperate to induce antiCD40-activated naive human B cells to secrete immunoglobulin A. J. Exp. Med., 175, 671-682. DiSanto, J.P., Bonnefoy, J.Y., Fauchat, J.F., Fischer, A. & de Saint Basile, G. (1993), CD40 ligand mutations in X-linked immunodeficiency with hyper-IgM. Nature (Lond.), 361, 541-543. Fuleihan, R., Ramesh, N., Loh, R., Jabara, H., Rosen, R.S., Chatila, T., Fu, S.M., Stamenkovic, I. & Geha, R.S. (1993), Defective expression of the CD40 ligand in X chromosome-linked immunoglobulin deficiency with normal or elevated IgM. Proc. Nat!. Acad. Sci. USA, 90, 2170-2173. Galy, A.H.M. &Spits, H. (1992), CD40 is functionally expressed on human thymic epithelial cells. J. Immunol., 149, 715-782. Gascan, H., Aversa, G.G., Gauchat, J.-F., Van Vlasselaer, P., Roncaraolo, M.-G., Yssel, H., Kehry, M., Spits, H. & de Vries, J.E. (1992), Membranes of activated CD4+ T cells expressing T cell receptor (TcR) ap or TcR ~6 induce IgE synthesis by human B cells in the presence of interleukin-4. Eur. J. Immunol., 22, 1133-1141. Gascan, H., Gauchat, J.-F., Aversa, G.G., van Vlasselaer, P. & de Vries, J.E. (1991), Anti-CD40 monoclonal
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antibodies or CD4+ T cell clones and IL-4 induce IgG4 and IgE switching in purified human B cells via different signaling pathways. J. Immunol., 147, 8-13. Gauchat, J.-F., Henchoz, S., Mazzei, G., Aubry, J.-P., Brunner, T., Blasey, H., Life, P., Talabot, D., FloresRomo, L., Thompson, J., Kishi, K., Butterfield, J., Dahinden, C. & Bonnefoy, J.-Y. (1993), Induction of human IgE synthesis in B cells by mast cells and basophils. Nature (Lond.), 365, 340-343. Hodgkin, P.D., Yamashita, L.C., Coffman, R.L. & Kehry, M.R. (1990), Separation of events mediating B cell proliferation and Ig production by using T cell membranes and lymphokines. J. Immunol., 145, 2025-2034. Hollenbaugh, D., Grosmaire, L.S., Kullas, C.D., Chalupny, N.J., Braesch-Andersen, S., Noelle, R. J., Stamenkovic, I., Ledbetter, J.A. & 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 costimulatory activity. EMBO J., 11, 4313-4321. Korthauer, U., Graf, D., Mages, H.W., Briere, F., Padayachee, M., Malcolm, S., Ugazio, A.G., Motarangelo, L.D., Levinsky, R.J. & Kroczek, R.A. (1993), Defective expression of T-cell CD40 lifand causes X-linked immunodeficiency with hyper-IgM. Nature (Lond.), 361, 539-541. Lane, P., Brocker, T., Hubele, S., Padovan, E., Lanzavecchia, A. & McConnell, F. (1993), Soluble CD40 ligand can replace the normal T-cell-derived CD40 ligand signal to B cells in T-cell-dependent activation. J. Exp. Med., 177, 1209-1213. Macchia, D., Almerigogna, F., Parronchi, P., Ravina, A., Maggi, E. & Romagnani, S. (1993), Membrane tumor necrosis factor-a is involved in the polyclonal B-cell activation induced by HIV-infected human T cells. Nature (Lond.), 363, 464-466. Okumura, K. & Tada, T. (1971), Regulation of homocytotrophic antibody formation in the rat. - III. Effect of thymectomy and splenectomy. J. Immunol., 10, 1019-1026. Peitsch, M.C. & Jongeneel, C.V. (1993), A 3-D model for the CD40 ligand predicts that it is a compact trimer similar to the tumor necrosis factors. ht. Immunol., 5, 233-238. P&e, J., Rousset, F., Briere, F., Chretien, I., Bonnefoy, J.Y., Spits, H., Yokota, T., Arai, N., Arai, K., Banchereau, J. & de Vries, J.E. (1988), IgE production by normal human lymphocytes is induced by interleukin 4 and suppressed by interferons gamma and alpha and prostaglandin E2. Pioc. Nat/. Acad. Sci. USA, 85, 6880-6884. Punnonen, J., Aversa, G., Cocks, B.G., McKenzie, A.N.J., Menon, S., Zurawski, G., de Waal Malefyt, R. & de Vries, J.E. (1993a), Interleukin-13 induces interleukin-4-independent IgG4 and IgE synthesis and CD23 expression by human B cells. Proc. Natl. Acad. Sci. USA, 90, 3730-3734. Punnonen, J., Aversa, G. & de Vries, J.E. (1993b), Human pre-B cells differentiate into immunoglobulinsecreting plasma cells in the presence of interleukin-4 and activated CD4+ T cells or their membranes. Blood, 82, 2781-2789. Spriggs, M.K., Armitage, R.J., Strockbine, L., Clifford,
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K.N., Macduff, B.M., Sate, T.A., Maliszewski, C. R. & Fanslow, W.C. (1992), Recombinant human CD40 ligand stimulates B cell proliferation and immunoglobulin E secretion. J. Exp. Med., 176, 1543-1550. Van Vlasselaet, P., Punnonen, J. & de Vries, J.E. (1992), Transforming growth factor p directs IgA switching in human B cells. J. Zmmunol., 148, 2062.
CD40-mediated
regulation J.B. Splawski
Yuan, D., Wilder, J., Dang, T., Bennett, M. & Kumar, V. (1992), Activation of B lymphocytes by NK cells. Znt. Zmmunol., 4, 1373-1380. Zhang, K., Clark, E.A. & Saxon, A. (1991), CD40 stimulation provides an IFN-y independent and IL-4 dependent differentiation signal directly to human B cells for IgE production. J. Zmmunol., 146, 1836-1842.
of human B-cell responses
(l) (*) and P.E.
Lipsky
(2)
(‘I The Departments of Pediatrics and ‘2) Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX 75235 (USA)
Interaction of the CD40 molecule expressed on B cells and the CD40 ligand on activated T cells plays
an important role in T-cell-dependent antibody production. The importance of CD40/CD40 ligand interaction in the induction of Ig isotype switch is demonstrated by the lack of IgG-, IgA- and IgEbearing B cells in patients with the hyper-IgM syndrome whose T cells lack a functional CD40 ligand. The studies described here examined the role of the CD40/CD40 ligand interaction in other T-celldependent functional responses of human B cells. Data are presented to suggest that CD40 engagement is not only important in the induction of Ig isotype switch, but also that it participates in the initiation of T-cell-dependent responses at all stages of B-cell maturation. The importance of CD40/CD40 ligand interactions in the development of T-celldependent humoral immunity suggests that manipulation of this interaction may provide new avenues for the regulation of humoral immune responses in vivo. CD40 is a 277-amino acid glycoprotein expressed by B cells, monocytes, follicular dendritic cells, thymic epithelial cells and certain carcinoma cells (Clark and Ledbetter, 1986; Paulie et al., 1989; Hart and McKenzie, 1988 ; Schriever et al., 1989 ; Alderson et al., 1993 ; Galy and Spits, 1992). The extracellular sequence of CD40 is homologous to a family
of molecules that includes the low avidity nerve growth factor receptor, the two TNFa receptors, fas, CD27 and CD30 (Braesch-Andersen et al., 1989; Stamenkovic et al., 1989; &hall et al., 1990; Smith et al., 1990; Mallett and Barclay, 1991). The CD40 molecule is a phosphoprotein lacking intrinsic protein kinase activity (Paulie et al., 1989). During Bcell ontogeny, CD40 expression occurs after CD10 and CDl9, but before CD20, CD21, CD22, CD24 and IgM (Uckun et al., 1990). CD40 appears to be functional as soon as surface IgM is expressed and may play a role in T-cell-dependent B-cell lymphopoiesis (Punnonen et al., 1992; Renard et al., 1994). CD40 is expressed at lower levels on peripheral blood B cells than on tonsil B cells (Ledbetter et al., 1987). Its expression is upregulated by anti-p or IL4, but not by IL2, although IL2 can increase the expression in combination with anti-p (Ledbetter et al., 1987; Valle et al., 1989; Gordon et al., 1988; Bjorck et al., 1991). Expression of CD40 is lost upon terminal differentiation of activated B cells to Ig-secreting cells (Ling et al., 1987). In summary, CD40 is expressed during most of the stages of B-cell maturation and differentiation, and its expression is regulated by cytokines and antigen receptor stimulation. Therefore, CD40 is a candidate to provide important regulatory signals to B cells at various stages of development .
(*) For correspondence: Judy B. Splawski, M.D., Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9063 (USA).
222
IN IA4MUNOLOGY
Contact-mediated signals and cytokines involved in B-cell activation and isotype switching in pre-B and mature B cells G. Aversa, B.C. Cocks, J. Punnonen, J.M. Carballido and J.E. de Vries Human
Immunology
Department, DNAX Research Institute, Palo Alto, CA 94304-1104 (USA)
Introduction
It has long been recognized that B-cell responses to antigen resulting in Ig synthesis require help from T cells (Okumura and Tada, 1971). More recently, it has become clear that this helper activity of T cells resides in the CD4+ subset. Cytokines produced by these cells promote B-cell growth and differentiation. In addition, they have been shown to direct Ig isotype switching both in mouse and human B cells (Coffman et al., 1988; de Vries et al., 1991). For example, IL4 and IL13 were found to induce human B cells to switch to IgG4 and IgE synthesis (P&e et al., 1988; Punnonen, 1993a), whereas TGF-P mediates IgA switching (Van Vlasselaer et al., 1992 ; Defiance et al., 1992). In addition to cytokines, a contact-mediated signal provided by activated CD4+ T cells was required for productive T-B cell interactions, resulting in Ig isotype switching and Ig production. This signal was delivered by molecules expressed on the surface of activated CD4+ T cells, as membranes of these cells were sufficient to stimulate B cells to proliferate and secrete Ig in the presence of cytokines (Brian et al., 1988; Hodgkin et al., 1990; Gascan et al., 1992). Resting T cells were ineffective. This T-cell derived contact-mediated signal could be substituted by mAb against CD40, a molecule constitutively expressed on B cells (Zhang et al., 1991; Gascan et al., 1991). The cloning and characterization of the mouse and subsequently, the human CD40 ligand (CD4OL) (Armitage et al., 1992; Spriggs et al., 1992; Hollenbaugh et al., 1992; Cocks et al., 1993) established this early T-cell activation antigen as a major component required for productive T-B cell interactions, resulting in Ig isotype switching and Ig synthesis. The CD4OL appears to be the major component of the T-cell signal provided by activated CD4+ T cells to B cells, and transfectants expressing CD40L, or anti-CD40 mAb, can replace activated CD4+ T helper cells in providing B-cell help. Strong
901 California
Avenue,
synergistic effects on Ig production, however, were obtained when activated CD4+ T-cell clones were added together with optimal concentrations of antiCD40 mAb and IL4 to cultures of purified human B cells (Gascan et al., 1991), indicating that activated CD4+ T cells may provide, in addition to CD40L, additional costimulatory signals for B cells. Cross-linking of CD40 in the presence insufficient for pre-B-cell differentiation
of IL4 is
The differences between T-cell-derived costimulatory signals and CD40 engagement alone by antiCD40 mAb are more apparent in activation and isotype switching of pre-B cells. In contrast to mature B cells (Gascan et al., 1991), highly purified sp-, cp+, CDlO+, CD19+ pre-B cells obtained from foetal bone marrow stimulated with anti-CD40 mAb, either in soluble form or cross-linked on L cells transfected with CD32/FcyRII were unable to proliferate, switch isotype or produce Ig in the presence of IL4 (Punnonen et al., 1993b). Interestingly, activated Tcell clones or their plasma membranes were effective in inducing isotype switching and Ig production by pre-B cells in the presence of IL4 (Punnonen et al., 1993b), indicating that in addition to CD40 crosslinking, other contact-mediated signals were provided by the T-cell clones or their membranes. It may be argued that the failure of anti-CD40 mAb to stimulate pre-B cells may be due to the low expression of CD40 on this precursor population compared to mature B cells. Indeed, stimulation of pre-B cells with activated T-cell clones and IL4, enhanced CD40 expression on these cells (Punnonen et al., 1993b) and this enhancement may be important in rendering the B cells more responsive to the CD4OL expressed on activated T helper cells. Whether the initial stimulus that induces B cells to enhance their CD40 expression may be the CD40L itself, or other cell surface molecules, remains to be determined. It
THE CD4O/CD4OL is clear, however, that simply cross-linking CD40 on pre-B cells is insufficient for the induction of differentiation of these cells into antibody-secreting cells in the presence of IL4. From these observations, it appears that B-cell progenitors can be induced via CD40 to differentiate into Ig-secreting cells, only after they have progressed beyond the pre-B-cell stage. The above in vitro observations of pre-B cells are compatible with recent reports of hyper-IgM syndrome patients. In these individuals, the intrinsic defect in isotype switching is a consequence of defective CD40L expression (Aruffo et al., 1993; Allen et al., 1993 ; DiSanto et al., 1993 ; Korthauer et al., 1993 ; Fuleihan et al., 1993). The number of B cells in these patients, however, is in the normal range, indicating that functional CD40L is not a necessary component for B-cell development. Furthermore, these patients have normal or elevated serum IgM levels, indicating that B cells still can be induced to proliferate and differentiate to IgM-secreting cells despite the absence of functional CD40L. This suggests that additional molecules are involved in the proliferation and differentiation of immature and mature B cells and that the CD40L does not play a major role in B-cell ontogeny.
Is CD40L alone activation ?
sufficient
for
mature
B-cell
Engagement of CD40 on mature B cells by antiCD40 mAb in the presence of IL4 or IL13 induces B-cell proliferation and Ig synthesis (Gascan et al., 1991; Zhang et al., 1991; Punnonen et al., 1993). Cross-linking CD40 on human B cells with human CD40L, expressed on CV-l/EBNA or COS cells, induces B-cell proliferation in the absence of cytokines and Ig synthesis in the presence of cytokines (Spriggs et al., 1992; Cocks et al., 1993). Soluble CD40L, cross-linked by a second step reagent or in a multimerit form, was reported to be biologically active in directly stimulating B-cell proliferation (Lane et al., 1993 ; Armitage et al., 1993a,b), indicating that crosslinking of CD40 is required and is sufficient for Bcell triggering. It appears, therefore, that the CD40L is able, by itself, to stimulate proliferation of mature B cells. It has been shown recently that CD40L expression on activated murine T-cell clones can be stabilized by plastic-bound stimuli, or by CD40 engagement using CD40 Ig, whereas soluble stimuli induce a rapid turnover of this molecule (Castle et al., 1993). In addition, the levels of newly synthesized CD40L on membranes from activated T-cell clones did not correlate with their ability to induce B-cell proliferation (Castle et al., 1993). The authors sug-
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gested that de nova synthesized CD40L is not efficient in activating B cells and that modification or assembly of the CD40L, or additional accessory signals, play a role in augmenting T-dependent B-cell responses. We recently reported that the transmembrane form of TNFa (mTNFa), which shares sequence and structural homologies with the CD40L (Peitsch and Jongeneel, 1993), is involved in T-B cell interactions, leading to IgG4 and IgE synthesis (Aversa et al., 1993). mAb to TNFa blocked ILCdependent IgG4 and IgE synthesis by highly purified or sIgD+ B cells, stimulated by activated T-cell clones or their plasma membranes (Aversa et al., 1993). Membrane TNFa was also reported to be an important molecule for help given by unstimulated HIV+ T cells to B cells, resulting in B-cell proliferation and Ig synthesis, and this help was suggested-to be CD40Lindependent (Macchia et al., 1993). The inability of anti-TNFR mAb to directly stimulate B cells in the presence of cytokines suggests that these mAb do not act like the anti-CD40 mAb, and that the costimulatory signal provided by the mTNFa is qualitatively different from that of the CD40L. Thus, CD40L alone is sufficient for adult B-cell activation. However, its optimal functional expression on T cells does not seems to be directly dependent on its level of expression, but rather on its post-synthetic modification/assembly or association with other surface molecules.
Role of CD40 and CD40L
expression
on T cells
The role that CD4OL plays in B-cell activation has been extensively studied. The implications for its function on T cells, however, is much less understood. A recent study shows that human CD4+ T cells can be costimulated via the CD40L by mouse cells transfected with CD40, in combination with anti-CD3 mAb (Cayabyab et al., 1994). This study and a previous report showing functional expression of CD40 on thymic epithelial cells (Galy et al., 1992) suggests a role for the CD40L in T-cell differentiation and activation. Thus, CD40L-CD40 interactions should not be considered a one-way signal from activated T to B cells, but rather a two-way interaction between T cells and APC or between other cell types expressing this receptor-ligand combination. We have previously shown that anti-CD40 mAb synergize with T-cell clones in inducing Ig production by B cells (Gascan et al., 1991). In subsequent studies, we substituted membranes from T-cell clones for the intact T cells and we consistently found a dose-dependent cross-inhibition of anti-CD40 mAb
224
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and T-cell membranes, rather than synergy (G. Aversa et al., unpublished data). This puzzling finding could be partly explained by a recent report showing expression of CD40 on resting as well as activated T cells (Armitage et al., 1993a), which is consistent with the ability of CD40L to costimulate T cells (Armitage et al., 1993b). The CD40 molecules present on the membrane preparation could sequester the anti-CD40 mAb used for stimulating the B cells, thus inactivating it. The large amounts of T-cell membrane equivalents, compared to the small number of intact T-cell clones necessary for stimulating B cells (Gascan et al., 1992) may account for the neutralization of anti-CD40 mAb by membranes, but not by the intact T-cell clones. This explanation, however, does not explain why the membrane signal itself would be affected by the anti-CD40 mAb. The physiological function of CD40 on T cells is not clear, although its presence on these cells suggests a role in T-T cell interactions, and a possible function as an additional accessory component in T-cell activation.
Role of CD40L on non-T cells CD4OL expression, detected by Northern blots and FACS analysis, was originally reported to be restricted to T cells (Spriggs et al., 1992). PCR analysis however, revealed that purified monocytes and NK cells also express the CD40L message (Cocks et al., 1993), and this was consistent with reports of NK cells providing costimulatory signals to B cells for Ig synthesis (Yuan et al., 1992). More recently, functional human CD40L expression was detected on mast cells and basophils, the latter expressing this molecule constitutively (Gauchat et al., 1993). Mast cells were able to induce IgE synthesis in B cells in the presence of IL4, whereas basophils did not require exogenous cytokines, probably because they produced IL4 (Gauchat et al., 1993). Recently, we found that activated, but not resting, U937 monocytoid cells, in the presence of IL4, induce proliferation and Ig secretion by highly purified B cells (Aversa et al., manuscript in preparation). CD40L expression on the surface of stimulated U937 cells could not be detected, suggesting the possibility that other activation-induced molecules on monocytes may provide B-cell help. It cannot be excluded, however, that a few CD40L molecules, below detection levels, may be present on the surface of activated U937 cells, and that these may be sufficient, by themselves or in synergy with other molecules, in inducing B-cell activation. The finding that CD40L is expressed on cells such as mast cells and basophils, which can also produce cytokines like IL4, suggests that B-cell help can oc-
cur in vivo at sites distinct from lymphoid organs, in tissues where B cells can come in contact with these non-T helper cells.
Concluding
remarks
The important role of CD40L in the activation and differentiation of mature B cells leading to Ig secretion is now well established. It is also clear that the CD4OL is not essential for B-cell development and is not sufficient for the activation and differentiation of pre-B cells, and that other surface molecules are involved in this process. The nature of these other molecules is still unknown, and their identification may provide a better insight into the process of B-cell development. The observation that the CD40L can function as a signalling molecule for T cells adds another piece to the puzzle of cellular interactions and lymphocyte function: the detection of CD40 on T cells and the CD40L on mast cells, basophils, NK cells and monocytes. In addition, the finding that cells other than T cells, which are located in non-lymphoid tissues, express CD40L, produce IL4 and can mediate B-cell activation and differentiation including IgE synthesis, has implications for the understanding the initiation and pathophysiology of allergic disorders, distinguishing the local events from systemic immune responses, and may help in the design of more effective therapies. The authors assistance.
wish to thank
Jo Ann Katheiser for secretarial
DNAX Research Institute of Molecular and Cellular Biology, Inc. is supported by Schering-Plough Corporation.
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CD40-mediated
regulation J.B. Splawski
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of human B-cell responses
(l) (*) and P.E.
Lipsky
(2)
(‘I The Departments of Pediatrics and ‘2) Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX 75235 (USA)
Interaction of the CD40 molecule expressed on B cells and the CD40 ligand on activated T cells plays
an important role in T-cell-dependent antibody production. The importance of CD40/CD40 ligand interaction in the induction of Ig isotype switch is demonstrated by the lack of IgG-, IgA- and IgEbearing B cells in patients with the hyper-IgM syndrome whose T cells lack a functional CD40 ligand. The studies described here examined the role of the CD40/CD40 ligand interaction in other T-celldependent functional responses of human B cells. Data are presented to suggest that CD40 engagement is not only important in the induction of Ig isotype switch, but also that it participates in the initiation of T-cell-dependent responses at all stages of B-cell maturation. The importance of CD40/CD40 ligand interactions in the development of T-celldependent humoral immunity suggests that manipulation of this interaction may provide new avenues for the regulation of humoral immune responses in vivo. CD40 is a 277-amino acid glycoprotein expressed by B cells, monocytes, follicular dendritic cells, thymic epithelial cells and certain carcinoma cells (Clark and Ledbetter, 1986; Paulie et al., 1989; Hart and McKenzie, 1988 ; Schriever et al., 1989 ; Alderson et al., 1993 ; Galy and Spits, 1992). The extracellular sequence of CD40 is homologous to a family
of molecules that includes the low avidity nerve growth factor receptor, the two TNFa receptors, fas, CD27 and CD30 (Braesch-Andersen et al., 1989; Stamenkovic et al., 1989; &hall et al., 1990; Smith et al., 1990; Mallett and Barclay, 1991). The CD40 molecule is a phosphoprotein lacking intrinsic protein kinase activity (Paulie et al., 1989). During Bcell ontogeny, CD40 expression occurs after CD10 and CDl9, but before CD20, CD21, CD22, CD24 and IgM (Uckun et al., 1990). CD40 appears to be functional as soon as surface IgM is expressed and may play a role in T-cell-dependent B-cell lymphopoiesis (Punnonen et al., 1992; Renard et al., 1994). CD40 is expressed at lower levels on peripheral blood B cells than on tonsil B cells (Ledbetter et al., 1987). Its expression is upregulated by anti-p or IL4, but not by IL2, although IL2 can increase the expression in combination with anti-p (Ledbetter et al., 1987; Valle et al., 1989; Gordon et al., 1988; Bjorck et al., 1991). Expression of CD40 is lost upon terminal differentiation of activated B cells to Ig-secreting cells (Ling et al., 1987). In summary, CD40 is expressed during most of the stages of B-cell maturation and differentiation, and its expression is regulated by cytokines and antigen receptor stimulation. Therefore, CD40 is a candidate to provide important regulatory signals to B cells at various stages of development .
(*) For correspondence: Judy B. Splawski, M.D., Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9063 (USA).