CD40-mediated regulation of human B-cell responses

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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).

THE CD4O/CD4OL Stimulation

with anti-CD40

Initial studies utilized monoclonal antibodies (mAb) to CD40 to delineate its function. Anti-CD40 alone did not induce proliferation of resting B cells, but potentiated proliferation of B cells stimulated by anti-k or anti-CD40 (Clark and Ledbetter, 1986; Paulie et al., 1989; Ledbetter et al., 1987 ; Gordon et al., 1988). Without costimulation, however, anti-CD40 induced resting tonsilar B cells to enlarge and become more buoyant (Gordon et al., 1988). In addition, anti-CD40 induced homotypic adhesion of dense tonsilar B cells by aCDl1 a/CD1 8 (LFAl) dependent and an LFAl-independent mechanism (Gordon et al., 1900; Barrett et al., 1991). These results suggest that engagement of CD40 itself can directly stimulate some aspects of B-cell activation. Engagement of CD40 with mAb, however, did not increase the level of c-myc expression, increase intracellular calcium or induce B cells to leave Go (Clark and Ledbetter, 1986; Clark et al., 1989). In addition, anti-CD40 did not costimulate with B-cell growth factor, IL1 or IL2, suggesting that anti-CD40 alone could not activate B cells to enter the cell cycle or acquire responsiveness to some cytokines (Ledbetter et al., 1987). However, the combination of anti-CD40 and IL4 could promote proliferation of resting B cells (Rousset et al., 1991). Moreover, resting B cells could be induced to secrete small amounts of IgM in response to anti-CD40 and IL4 (Gordon et al., 1989). In addition, engagement of CD40 by antiCD40 bound to fibroblasts expressing Fc receptors and IL4 caused sustained proliferation of human tonsil B cells (Banchereau et al., 1991). The cells that were induced to proliferate continuously in this model system remained IgD+ (Galibert et al., 1994). The combination of antibody to CD40 and IL4 also induces the secretion of IgE by purified IgE-negative B cells and by naive neonatal B cells (Splawski et al., 1993; Jabarra et al., 1990; Zhang et al., 1991; Gascan et al., 1991). However, activation of the B cells with polyclonal activators such as formalinized Staphylococcus aureus Cowan I (SA) enhances the Ig secreted in response to anti-CD40 (Splawski et al., 1993). In addition, antibody to CD40 has been shown to promote the proliferation and prevent apoptosis by germinal centre B cells (Liu et al., 1989; Butch and Nahm, 1992; Knox et al., 1993). Taken together, these results indicate that engagement of CD40 provides specific activation signals to B cells that, in the presence of costimulation with IL4, can result in proliferation and Ig isotype switch to IgE in the absence of T cells. The CD40 ligand expressed by activated T cells, therefore, has become a major candidate molecule to account for the cell contact-dependent activation signals delivered to B cells during T cell/B cell collaboration.

INTERACTION

227

The CD40 ligand The murine ligand for CD40 was cloned and found to be a 260 a.a. type II transmembrane glycoprotein related to TNFa (Arrnitage et al., 1992). The ligand is expressed on activated but not resting T cells. Both Thl and Th2 clones express the CD40 ligand (CD40L) (Armitage et al., 1992). CVl/EBNA cells transformed with the murine CD40L induced proliferation of both murine and human B cells in the absence of costimulation, which contrasted with the inability of anti-CD40 alone to promote B-cell proliferation (Armitage et al., 1992). These results are, however, consistent with the ability of cellassociated anti-CD40 to support proliferation of human B cells, suggesting that the degree of crosslinking of CD40 may determine its mitogenic potential (Banchereau et al., 1991). In addition, the CVl/EBNA cells transfected with the murine CD4OL were able to support the secretion of IgE by both human and murine B cells in combination with IL4 (Armitage et al., 1992). In the absence of IL4, however, no IgE production was noted, emphasizing the differences in the signals required for B-cell proliferation, on the one hand, and Ig isotype switching and secretion on the other. The corresponding human ligand for CD40 is a 33-kDa protein expressed by activated T cells (Lane et al., 1992; Spriggs et al., 1992; Gauchat et al., 1993a; Graf et al., 1992). Cells transfected with the human CD40L also supported the proliferation of human B cells in the absence of additional stimuli, and in combination with IL4 promoted the secretion of IgE (Spriggs et al., 1992). Both the murine and human ligands are expressed early after T-cell activation. The human CD4OL is expressed mainly by CD4+ T cells, although there may be a subpopulation of CDS+ T cells that also express the ligand. No particular correlation with CD45 phenotype was found in initial studies of human CD40L expression, suggesting that CD40L is expressed by both naive and memory T cells after activation (Lane et al., 1992). By contrast, an increase in the expression of CD40L by murine memory T cells has been noted (Roy et al., 1993). Expression of CD4OL by murine T cells stimulated by immobilized anti-CD3 was found to be more stable than that induced by phorbol esters and ionomytin (Castle et al., 1993). In addition, the expression of the murine CD40L was inhibited by IFNy, whereas cyclosporin completely inhibited expression by both activated human and murine T cells, suggesting that the expression of CD40L may be regulated by cytokines (Gauchat et al., 1993a; Roy et al., 1993; Fuleihan et al., 1994). Of note, TGFP inhibited the expression of CD40L by murine Th2 cells, suggesting that there may be differential regulation of this molecule by some T-cell subpopulations (Roy et al.,

228

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1993). Some of the differences between the murine and human studies may be related to the mode of stimulation of the T cells, since most of the human studies employed PMA and ionomycin that may provide a more optimal signal. CD40L has also been shown to be expressed on mast cells, and these cells were able to induce IgE secretion by B cells (Gauchat et al., 1993b). X-linked hyper-IgM syndrome (HIgiWXL). The importance of the CD40/CD40L interaction for humoral immunity is demonstrated by HIgM-XL, which is characterized by the production of IgM but the nearly complete absence of IgG, IgA and IgE Ig isotypes. The defect in HIgM-XL was mapped to the q24-27 region of the X chromosome (Padayachee et al., 1992). Recently, the human gene for the CD40L was also mapped to the long arm of the X-chromosome at Xq26.3-27.1, implicating the CD40L as a candidate gene for HIgM-XL (Graf et al., 1992). Recently, the defect in the HIgM-XL has been shown to be in the expression of the CD40L by activated T cells (Aruffo et al., 1993; DiSanto et al., 1993; Korthauer et al., 1993 ; Fuleihan et al., 1993 ; Allen et al., 1993). Multiple mutations of the gene have been described resulting in complete absence of the ligand or expression of a defective ligand that does not bind CD40. Initially, patients with HIgM-XL were felt to have a defect in the B cells because their T cells could support Ig production by normal B cells stimulated with pokeweed mitogen (PWM) (Levitt et al., 1993). In addition, HIgM-XL B cells could not secrete IgG or IgA in response to SA, Epstein-Barr virus (EBV) or PWM and normal T cells. PWM is known to stimulate Ig production largely from IgD- postswitch memory B cells (Jelinek et al., 1986). IgM production originates largely from IgD+ B cells that have not undergone switch recombination. As shown in table I, PWM-stimulated normal adult T cells only induced IgM production by HIgM-XL B cells. These

Table I. Pattern of Ig production

by HIgM-XL

results can now be interpreted with the knowledge that the defect in HIgM-XL is the expression of an abnormal CD40L by the T cells. The inability of the HIgM-XL B cells to secrete IgA or IgG in vitro upon PWM stimulation would appear to be the result of the lack of previous in vivo CD40 engagement, and resultant switch to downstream Ig isotypes and generation of memory B cells. Neonatal B cells and IgD + adult naive B cells behave similarly to HIgM-XL B cells in that they are not stimulated to secrete IgG or IgA in response to PWM and normal T cells (Jelinek et al., 1986; Splawski and Lipsky, 1991). The production of IgM by PWM stimulated HIgM-XL B cells differs from the inability of neonatal B cells to secrete this Ig isotype in response to PWM stimulation and implies the presence of IgM memory cells, that presumably were generated in vivo in response to environmental stimuli without the influence of CD40 engagement. The possibility that HIgM-XL B cells also have defect in switch recombination cannot be assessed in these studies. The ability of the HIgM-XL T cells to support the production of some Ig by normal B cells and PWM (table I) suggests that the CD40/CD40L interaction is not absolutely necessary for Ig secretion by some of the post-switch memory B cells that are responsive to PWM, but clearly this interaction plays an important role, as responses of normal B cells supported by HIgM-XL T cells and PWM are markedly diminished compared to that supported by normal T cells. Some of this difference might be related to the young age of the HIgM-XL donor (3 years). Despite this, the data with PWM stimulation are consistent with the conclusion that engagement of CD40 by CD40L is likely to play a role in amplifying responses of normal memory B cells stimulated with PWM. HIgM-XL B celIs have been reported to proliferate in response to recombinant CD40L or anti-CD40 and IL4 (Aruffo et al., 1993; Allen et al., 1993). In

B and T cells cultured with normal B or T cells and stimulated with PWM. Ig isotype secreted (rig/ml)

B cells

T cells

IgM

IgGl

IgG2

IgG3

IgG4

IisA

I@

NL NL HIgM HIgM

NL HIgM NL HIgM

2,989 576 1,415 < 24

280 50 23 < 12

628 29 < 12 < 12

62 < 12 < 12 < 12

74 < 12 < 12 < 12

5,322 511 < 12 < 12

<8 <8 <8 <8

B cells (2.5 x 104/well) from either normal adult peripheral blood (NL) or from a patient with HIgM-XL (HlgM) were incubated with mitomycin C-treated T cells (1 .Ox 10s/well) from either normal adult peripheral blood or from the patient with HIgM-XL and stimulated with PWM. Supernatants were harvested for quantitation of secreted Ig by ELISA on day 15.

THE CD4O/CD4OL

INTERACTION

originates from memory B cells, these results are consistent with a central role for CD40/CD40L interaction in the stimulation of memory B cells. The data therefore indicate that CD40L plays a central role in the T-cell-dependent activation of both naive and memory B cells. The requirement for CD40Lmediated interaction appears to be more absolute for the activation of naive compared to post-switch memory B cells.

addition, HIgM-XL B cells have been shown to be induced to secrete IgE in response to IL4 and either recombinant CD40L or antibody to CD40, implying that the mechanisms for switch recombination are intact and functional. Consistent with this, secretion of IgG and IgA has been observed after stimulation with the combination of anti-CD40, SA and IL10 (Korthauer et al., 1993). This has not been a consistent finding, however, suggesting that other combinations of cytokines and signals might be required for switch recombination to IgG and IgA or that HIgM-XL B cells might have an additional defect in switch recombination to these particular downstream isotypes (Callard et al., 1993). In the current studies, anti-CD40 and IL4 induced the secretion of all Ig isotypes by HIgM-XL B cells. IgG, IgG4 and IgE were the predominant downstream isotypes secreted, but switching to all isotypes was observed (table II). Of note, the responses of HIgM-XL B cells were greater than those of normal B cells. Normal antiCD3-activated T cells also supported responses of HIgM-XL B cells (table III). Production of all downstream isotypes was observed. These results indicate that the switch recombination mechanism for all downstream isotypes is intact in HIgM-XL B cells and, moreover, that CD40 coupling to switch recombination is intact in HIgM-XL B cells. These results indicate that HIgM-XL B cells can be induced to switch to all Ig isotypes in the presence of normal T cells or by engagement of CD40 with mAb. By contrast, HIgM-XL T cells induced no Ig from HIgM-XL B cells, indicating an essential role for CD40/CD40L interaction in the activation of naive B cells. These results are consistent with the ability of anti-CD40 to inhibit T-cell-dependent Ig production by neonatal B cells completely (Splawski et al., 1993). Of note, anti-CD3-stimulated HIgM-XL T cells could support the secretion of some Ig by normal adult peripheral blood B cells, but did so much less effectively than normal T cells (table III). However, IgM production and secretion of all downstream isotypes were markedly diminished. Since a large part of the Ig produced in this model system

Table II. Anti-CD4O+IL4

229

CD40-mediated

switch recombination

The lack of IgG-, IgA- and IgE-expressing B cells in patients with the HIgM-XL suggests that the CD4O/CD4OL interaction is crucial for in vivo switch to all downstream Ig heavy chain genes. Multiple studies have documented a role for CD40 and IL4 in Ig isotype switch to IgE (Gascan et al., 1991; Gauchat et al., 1990; Shapira et al., 1992). The ability of CD40 engagement to promote switch to other Ig isotypes has been more controversial. When the response of purified B cells to anti-CD40 and IL4 has been investigated, various results were observed, including secretion of IgE only, or secretion of small amounts of IgM, IgG and IgG4 in addition to IgE (Rousset et al., 1991; Zhang et al., 1991; Gascan et al., 1991). Our own studies demonstrated the secretion of IgM, IgG, including all IgG subclasses, but predominantly IgGl and IgG4, and IgE by neonatal B cells. IgA secretion was not observed (Splawski et al., 1993). Cell-associated anti-CD40 has been reported to promote the secretion of IgA in response to IL2 and IL4. However, the secretion of IgA did not necessarily reflect switch recombination, as a population of tonsil B cells which may contain post-switch memory cells was examined (Rousset et al., 1991; Banchereau et al., 1991). Induction of the secretion of IgA from IgDf B cells, which implies Ig isotype switch, has been reported with cell-associated antiCD40 in combination with TGFP and IL10 (Defiance et al., 1992). TGFP inhibited the production of IgA by IgD- B cells, implying that TGFP may be a

supports the production

of all Ig isotypes by HIgM-XL

B cells.

Ig isotype secreted (rig/ml) B-cell source

W

Normal B HIgM-XL B

145 491

IgGl 34 621

IgG2

IgG3

< 12 34

< 12 58

IgG4

&A

I@

19 192

14 124

1,374 1,809

B cells (2.5 x 104/well) were incubated with monoclonal antibody to CD40 (626.1, IgGl, 5 pg/ml) or a control antibody of the same isotype with 100 U/ml IL4. Supernatants were harvested on day 15 for quantitation of the Ig secreted by ELISA. The data indicate the Ig secreted in response to anti-CD40 and IL4 minus the control response, which was minimal.

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230 Table III. Anti-CD3-activated

HIgM-XL

IN IMMUNOLOGY

T cells are deficient in their ability to support Ig secretion by normal adult B cells. Ig isotype secreted (rig/ml) IgM

IgGl

IgG2

IgG3

IgG4

IgA

IiS

0 IL-2 IL-4

< 24 55 100

22 c 12 14

< 12 < 12 < 12

< 12 < 12 < 12

< 12 20 < 12

< 12 < 12 18

<8 < 12 < 12

NL

0 IL-2 IL-4

653 4,074 861

101 752 128

101 93 26

< 12 82 < 12

< 12 40 < 12

656 2,209 367

<8 14 <8

NL

NL

0 IL-2 IL-4

19,035 15,536 11,348

2,498 2,219 1,539

570 444 320

718 540 299

372 198 104

6,318 4,382 4,493

<8 <8 136

NL

HIgM

0 IL-2 IL-4

28,966 43,910 21,506

1,277 1,164 921

43 46 54

814 442 174

74 55 91

199 251 207

<8 <8 156

T cells

B cells

HIgM

HIgM

HIgM

Addition

B cells (2.5 x 104/well) from normal adult peripheral blood (NL) or a patient with HIgM-XL (HIgM) were incubated with T cells (1.0~ IOs/well) from either normal adult peripheral blood or T cells from the same patient with HIgM-XL and stimulated with immobilized anti-CD3 in the presence or absence of IL2 (50 U/ml) or IL4 (100 U/ml). Supernatants were harvested for quantitation of secreted Ig by ELISA on day 15.

switch factor but subsequently may not promote the secretion of IgA, but rather may inhibit it. Recombinant CD4OL expressed by CVl /EBNA cells in combination with IL2 or IL10 has been reported to elevate the levels of IgM, IgGl and IgA produced from tonsil B cells. Significantly, IL4 did not enhance the amount of these Ig isotypes produced. However, the secretion of IgG4 and IgE was dependent on costimulation with IL4. TGFP strongly inhibited the ability of IL4 to promote IgG4 and IgE secretion (Armitage et al., 1993). In these studies, however, it is unclear whether the Ig produced in response to the recombinant CD40L resulted from Ig heavy chain isotype switch because of the post-switch memory cells in the tonsilar B cells examined. Ig isotype switch involves a DNA rearrangement that transfers the VDJ segment, which confers antigen specificity, from its association with the Cu gene to another downstream constant gene with resultant depletion of the intervening DNA (Honjo and Kataoka, 1978 ; Kataoka et al., 1980). This recombination occurs between regions of repetitive DNA sequences called switch regions which are located 5’ of each heavy chain constant region gene except delta. Although the mechanisms directing these events are

not well-understood, it is widely accepted that before recombination occurs, transcripts are initiated in the DNA sequences 5’ of the switch region of the heavy chain constant region gene to be expressed (Stavnezer et al., 1985; Sideras et al., 1989; Blackwell et al., 1986; Lutzker et al., 1988; Bet-ton et al., 1989). Since these transcripts are not translated, they are referred to as sterile. Evidence is accumulating that certain cytokines induce sterile transcription of specific downstream H chain isotypes, and are therefore thought to be involved in switch recombination to specific heavy genes (Lutzker et al., 1988; Berton et al., 1989). Our own studies indicated that anti-CD40 alone was sufficient to induce sterile germline transcription of all downstream Ig isotypes with the possible exception of IgG4 and IgE. By contrast, IL4 alone was not sufficient to induce sterile transcripts of any downstream isotype genes including IgG. The combination of anti-CD40 and IL4, however, routinely induced IgG4 and IgE sterile transcripts (Jumper et al., 1994). This contrasts with a number of other studies which demonstrated that IL4 alone was sufficient to induce IgE sterile transcripts, which were increased by anti-CD40 (Gascan et al., 1991; Callard et al., 1993; Gauchat et al., 1990). Our studies utilized highly purified B cells from adult peripheral

THE CD4O/CD4OL

blood rather than splenic B cells which could explain the differences in these results. We additionally noted that even small numbers of T cells supported the induction of sterile transcripts in the presence of IL4, presumably by providing CD4OL-mediated signalling. Our results indicate that in the absence of stimulation via CD40, IL4 is an insufficient signal for the induction of sterile IgE transcripts. However, IL4 clearly costimulates with CD40L sterile transcription of IgE and IgG4. However, anti-CD40 alone was sufficient to induce sterile IgG transcripts from naive neonatal B cells, indicating that CD40 engagement alone is sufficient for the induction of the first step in switch recombination. Cytokine

specificity of CD40 stimulation

Our previous studies and those of others suggest that the combination of anti-CD40 and IL2 did not result in the secretion of Ig (Splawski et al., 1993). Of note, studies with recombinant CD40L contrast with those utilizing antibody to CD40, in that IL2 promotes proliferation and Ig secretion (Armitage et al., 1993). This may be particularly important for T-cell-dependent Ig production by neonatal T cells and Thl cells, which secrete little if any IL4. It should be noted that activated ThO, Thl and Th2 cells all can express CD4OL, suggesting that they can promote T-cell-dependent Ig secretion (Noelle et al., 1992). It is unclear whether this difference between ligand and antibody is caused by enhanced cross-linking of the CD40 molecule, resulting in more effective activation or the involvement of accessory molecules on the CVl/EBNA cells which, in combination with the CD40L, promote the IL2 response. In addition, recombinant CD40L-mediated stimulation induced IL 1O-dependent proliferation and Ig secretion (Armitage et al., 1993). It is not known whether the Ig secretion supported by the combination of IL2 or IL10 and CD4OL represents Ig isotype switch or not. However, anti-CD40 did not alter the Ig production or the Ig isotypes secreted in response to SA and IL2, suggesting that it did not alter the IL2 responsiveness or induce switch by B cells stimulated with SA and IL2 (Splawski et al., 1993). The role of CD40 in T-cell-dependent

Ig production

The data are consistent with the conclusion that the interaction of CD40 on B cells and CD40L on activated T cells accounts for much, if not alI, T-celldependent B-cell responses. The demonstration that anti-CD40 or recombinant CD40L promotes B-cell activation and proliferation in the absence of T cells supports this assumption (reviewed in Noelle et al., 1992). In addition, inhibition of the CD40/CD40L interaction has been shown to inhibit T-cell-

INTERACTION

231

dependent B-cell proliferation (Lane et al., 1992; Grabstein et al., 1993). The importance of the CD40/CD40L interaction in the development of a primary antigen-specific response has been delineated in a murine system. Antigen in combination with murine CD40L expressed on fixed CVl/EBNA cells and IL2 resulted in the induction of an antigenspecific IgM response by unimmunized murine splenic B cells. In contrast, IL4 and IL5 were able to induce polyclonal Ig secretion, but were unable to induce a primary antigen-specific response (Grabstein et al., 1993). These results suggest that CD40 engagement was important in the development of a primary immune response that was dependent on IL2 rather than IL4. The production of IgM by patients with the HIgM-XL suggests that primary responses are not dependent on the CD40/CD40L interaction. However, almost all of the reports of Ig secretion induced by anti-CD40 or CD40L demonstrate some IgM secretion, suggesting that the CD40/CD40L interaction may play a role in T-cell-dependent IgM secretion and the induction of a primary response (table II) (Splawski et al., 1993). Since naive B cells can secrete IgM in response to SA or anti-u and IL2 in the absence of T celIs, it is likely that the IgM secretion seen in the patients with HIgM-XL is not T-celldependent (Splawski and Lipsky, 1991). The analysis of the function of HIgM-XL T and B cells presented above strongly suggests that CD40L plays an essential role in the T-cell-dependent activation of naive B cells as well as an important role in responses of memory B cells. Consistent with this, both primary and secondary responses were found to be depressed in HIgM-XL patients immunized with a T-cell dependent antigen (Nonoyama et al., 1993). Moreover, there is experimental data to suggest a role for the CD40KD4OL interaction in the induction of a secondary immune response. Human IgD- B cells from previously immunized donors stimulated with antigen, anti-CD40 and IL10 secreted antigen-specific Ig for a T-cell-dependent antigen which was comparable to the response supported by antigen-specific T cells (Nonoyama et al., 1993). In addition, in vivo administration of anti-gp39 (CD40L) mAb to mice dramatically reduced both the primary and secondary humoral responses to a T-cell-dependent antigen, without altering the response to a T+$independent antigen (Boy et al., 1993). These results support the conclusion that the CD40KD40L interaction is important in T-cell-dependent responses of both memory and naive B cells rather than only the promotion of Ig isotype switch. The mechanism whereby CD40 exerts its effects on B cells is still not known. Engagement of CD40 by antibody leads to enhanced tyrosine phosphorylation of several distinct phosphoproteins in pro-B, pre-pre-B and activated mature B, but not resting mature B cells, and also a rapid increase in inositol trisphosphate (Uckun et al., 1991). In addition, CD40

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ligation markedly increased the activity of several distinct serine/threonine-specific protein kinases. A recent study suggests that CD40 engagement by CD40L promotes the induction of the truns-acting transcription factor, NF-KB. The induction of NF-KB was not dependent on PKC signalling, in contrast to Igreceptor-mediated induction (Lalmanach-Girard et al., 1993). The binding site of NF-KB is found in the promoter of several genes of immunological interest including, IRK, IL2R and IL6 (Baeurle, 1991). Of note, triggering B cells with anti-CD40 leads to the secretion of IL6, and IL6 induces increased phosphorylation of the CD40 molecule (Clark and Shu, 1990). Whether this plays a role in B-cell responses induced by ligation of CD40 remains to be determined. Conclusion In summary, CD40 is expressed early in B-cell development and appears to play an active role in T-cell-dependent responses at many stages of B-cell differentiation. The reagents that have been developed to study this interaction in the absence of other signals can now be utilized to further our knowledge of T-cell-dependent and -independent responses at different stages of B-cell differentiation and to explore the molecular cascade initiated by CD40 engagement. In addition, these reagents should be helpful in correcting the genetic defect and in exploring the potential of CD40 engagement to boost the humoral response to immunization.

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