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

We report the capacity of CD40 ligand (CD40L)-negative T cell clones to activate human B cells. CD40L-negative T cells induce a level of B cell proliferation 10–20% of that seen with normal T cells. The signal provided by the negative clones is synergistic with that derived from a CD40L transfectant, and restores B cell proliferation to normal levels, showing that CD40L-negative T cell clones are not inherently inhibitory for B cells. Although their capacity to induce proliferation was much reduced, CD40L-negative T cell clones were still strong inducers of B cell differentiation to plasma cells. This differentiation to plasma cells was inhibited by a CD40L transfectant. The data are discussed with regard to the normal in vivo mechanism for maintaining B cell memory and memory antibody responses to T-dependent antigens.     

IL-10 interrupts memory B cell expansion in the germinal center by inducing differentiation into plasma cells

Germinal center (GC) B cells undergo proliferation, somatic hypermutation and isotype switching in the course of differentiation into plasma cells to produce high-affinity antibodies. To understand the molecular mechanism regulating the expansion of memory B cells and the termination of expansion by differentiation into plasma cells, we investigated the effect of interleukin-2 (IL-2), IL-4, IL-10 and CD40 ligand (CD40L) on the differentiation of GC B cells in the defined culture system containing a follicular dendritic cell (FDC)-like cell line. IL-2, IL-4 and CD40L are required for the optimum proliferation and differentiation of GC B cells. When IL-10 was added to this culture condition, CD20⁺ CD38⁺ GC B cells sequentially differentiated into CD20⁺ CD38⁻ memory B cells and then CD20⁻ CD38⁺ plasma cells. In the absence of IL-10, the resulting CD20⁺ CD38⁻ memory B cells continued to proliferate and retained its phenotype. The proliferation of memory B cells was interrupted by addition of IL-10 which induced the differentiation into plasma cells. The expression of CD80 and CD86 was up-regulated in the memory B cells, compared to naive B cells and plasma cells. The identity of memory B cells generated in vitro from GC B cells was further substantiated since memory B cells generated in vivo displayed the identical pattern of proliferation and differentiation under the same culture condition. These results highlight the potent role of GCT helper cells in the expansion and differentiation of memory B cells by regulating different cytokine production.     

Regulation of murine B cell growth and differentiation by CD30 ligand

A ligand for CD30 has been recently cloned, and has been shown to have sequence homology with the tumor necrosis factor family of cytokines. CD30 ligand (CD30L) was found to be induced on helper T cell clones, and its receptor was expressed on freshly isolated and activated murine B cells. Recombinant murine CD30L was found to share many functional properties with CD40 ligand (CD40L) in the regulation of murine B cell growth and differentiation in vitro. CD30L stimulated B cell proliferation, antigen-specific antibody production, and polyclonal immunoglobulin secretion in a cytokine-dependent manner. In particular, the stimulation of B cell proliferation by CD30L required interleukin (IL)-4 and IL-5, induction of anti-sheep red blood cell antibody-secreting B cells by CD30L required IL-2 and IL-5, and optimal induction of polyclonal immunoglobulin secretion required IL-4 and IL-5. Under these conditions, the polyclonal secretion of IgGl, IgA, IgG3 and IgE was induced. The induction of immunoglobulin secretion by CD30L was independent of CD40L, as B cells from CD40L deficient-mice responded normally to CD30L treatment. We conclude that CD30L is a potent mediator of B cell growth and differentiation in vitro and may play a role in cognate T cell-B cell interactions.     

Induction of LFA-1 Independent Human B Cell Proliferation and Differentiation by Binding of CD40 with its Ligand

Engagement of CD40 on resting B cells in the presence of IL-4 triggers B cell proliferation, differentiation and homotypic adhesion. This study was designed to investigate the role of LFA-1/ICAM-1 interactions in homotypic adhesion and proliferation of CD40-activated human B lymphocytes. Freshly isolated B cells were cultured in vitro in the presence of IL-4 and of L cells expressing CD40L, the CD40 ligand. The addition to the culture medium of LFA-1 and ICAM-1 antibodies inhibited homotypic B lymphocyte adhesion. However, these antibodies failed to affect B lymphocyte proliferation and antibody production. These results indicate that aggregation and proliferation are independent events although both induced by CD40 activation.     

Induction of LFA-1 Independent Human B Cell Proliferation and Differentiation by Binding of CD40 with its Ligand

Engagement of CD40 on resting B cells in the presence of IL-4 triggers B cell proliferation, differentiation and homotypic adhesion. This study was designed to investigate the role of LFA-1/ICAM-1 interactions in homotypic adhesion and proliferation of CD40-activated human B lymphocytes. Freshly isolated B cells were cultured in vitro in the presence of IL-4 and of L cells expressing CD40L, the CD40 ligand. The addition to the culture medium of LFA-1 and ICAM-1 antibodies inhibited homotypic B lymphocyte adhesion. However, these antibodies failed to affect B lymphocyte proliferation and antibody production. These results indicate that aggregation and proliferation are independent events although both induced by CD40 activation.     

Signaling via major histocompatibility complex class II molecules and antigen receptors enhances the B cell response to gp39/CD40 ligand

Activated T cells induce proliferation and differentiation of resting B cells in vitro through their CD40 molecules and lymphokine receptors. However, despite constitutive B cell expression of CD40 and lymphokine receptors, widespread nonspecific polyclonal B cell activation by activated T cells is seldom observed in vivo. The present study was designed to test the hypothesis that signals delivered via the B cell antigen (Ag) receptor (membrane immunoglobulin, mIg) and major histocompatibility complex (MHC) class II molecules enhance B cell responsiveness to CD40-mediated signals, providing specificity to the Ag-nonspecific, MHC-unrestricted CD40 signal. To test this hypothesis, both an Ag-specific mouse B cell clone CH12.LX, and freshly isolated resting splenic B cells were cultured with either soluble or membrane-bound forms of the T cell ligand for CD40 (CD40L), in the presence or absence of additional signals provided by Ag or anti-IgM, interleukin-4, and class II-specific monoclonal antibody (mAb). Differentiation of CH12.LX cells and proliferation of splenic B cells in response to both forms of CD40L was greatly enhanced by exposure to mIg-mediated signals, with greatest enhancement seen when cells were cultured with Ag prior to receiving other signals. Response to CD40L was further enhanced by concurrent culture with class II-specific, but not class I-specific mAb. Enhancement was greatest at limiting concentrations of CD40L. The ability of class II MHC-mediated signals to enhance Ag-specific B cell responsiveness to CD40-mediated signaling may selectively promote the activation of B cell clones capable of cognate interactions with helper T cells.     

Recombinant CD40 ligand stimulation of murine B cell growth and differentiation: cooperative effects of cytokines

The ligand for the B cell surface antigen CD40 was recently cloned from a murine thymoma cDNA library and shown to be expressed on activated T cells. In this study, we investigate the biological effects of murine recombinant CD40 ligand. The recombinant CD40 ligand expressed on the CV-1/EBNA monkey fibroblast cell line directly activated resting B cell to express elevated levels of cell surface class II major histocompatibility complex and CD23 molecules. CD40 ligand also stimulated B cell proliferation, reaching maximal levels on day 2 of culture and declining thereafter. This effect was positively regulated by other cytokines, most notably interleukin (IL)-4 and IL-5. By itself, CD40 ligand had no effect upon immunoglobulin secretion by B cells. However, when B cells were treated with CD40 ligand plus cytokines, immunoglobulin secretion was stimulated in a cytokine-dependent and isotype-specific manner. IL-4 was a potent co-stimulator of IgE and IgG1 in the presence of CD40 ligand, and IL-5 acted synergistically with IL-4 in these responses as well as in IgM and IgG3 production. Taken together, the results indicate that CD40 ligand is a potent regulatory molecule for B cell growth and differentiation, and its activities are potentiated in a cytokine-specific manner.     

Early pathogenesis during infectious bursal disease in susceptible chickens is associated with changes in B cell genomic methylation and loss of genome integrity

We propose a model by which an increase in the genomic modification, 5-hydroxymethylcytosine (5hmC), contributes to B cell death within the chicken bursa of Fabricus (BF) infected with infectious bursal disease virus (IBDV). Our findings indicate that, following an IBDV infection, Rhode Island Red (RIR) chickens have fewer surviving B cells and higher levels of 5hmC in the BF than the more resistant 15l line of birds. Elevated genomic 5hmC levels within the RIR BF are associated with markers of immune responses: infiltrating T cells and increased expression of CD40L, FasL and iNOS. Such changes correlate with genomic fragmentation and the presence of IBDV capsid protein, VP2. To explore the effects of CD40L, the immature B cell line, DT40, was exposed to recombinant chicken CD40L that resulted in changes in nuclear 5hmC distribution. Collectively, our observations suggest that T cell infiltration exacerbates early immunopathology within the BF during an IBDV infection contributing to B cell genomic instability and death to facilitate viral egress and immunosuppression.     

Fas expression and apoptosis in human B cells

Mechanisms of B cell apoptosis are critical in reducing aberrant B cell proliferations such as those that arise in autoimmune disease and in B cell malignancies. The physiologic interaction of CD4+ helper T cells and B lymphocytes has been extensively studied over the past two decades. Although CD4+ T cells are considered primarily to offer positive costimulatory signals for B cell differentiation into active immunoglobulin-secreting cells, recent studies have shown that CD4+ T cells are crucial in downregulating the humoral immune response. In the course of cognate interaction between CD40 ligand (CD40L)-bearing CD4+ T cells and CD40-expressing germinal center B cells, CD40 ligation results in augmented Fas expression at the B cell surface. Like CD40L, Fas ligand is expressed on activated CD4+ Th1 cells and when bound to Fas receptor on the B cell surface, initiates an apoptotic signal in that cell. Thus, CD4+ T cells limit the growth of autologous germinal center B cells by first inducing Fas expression and then instigating a death signal via Fas ligand. In this work, we will consider these observations about CD4+ T-cell-induced, Fas-mediated B cell death in the context of other factors that affect apoptosis in B cells, normal and malignant.     

CD40 signalling in ileal Peyer's patch B cells: implications for T cell-dependent antigen selection

The ileal Peyer's patch (PP) plays a central role in B celldevelopment in young sheep and it is hypothesized that thisB cell development occurs independent of extrinsic antigen andT cells. Therefore, it was of interest to examine ileal PP folllcular(iPf) B cell responses to CD40 ligand, a molecule integral toT cell-dependent B cell development. A variable level of CD40expression was detected on a subpopulation of iPfB cells andJ558L cells, expressing a membrane form of mouse CD40 ligand(mCD40L), interacted specifically with the CD40 molecule oniPfB cells. In response to mCD40L the non-S phase iPfB cellswere rescued from apoptotic cell death and there was a markedproliferative response but viable cell number remained relativelyconstant. The mCD40L also induced decreased cytoplasmic cAMPlevels, blocked anti-Ig-induced iPfB cell death and inducedfunctional IL-2 receptor expression on a subpopulation of iPfBcells. Many of the mCD40L-induced responses of iPfB cells weresimilar to those reported for germinal centre and immature Bcells, and indicated that a cognate T cell-B cell interactioncould influence iPfB cell proliferation and differentiation.Finally, that mCD40L induced iPfB cell activation and differentiationwas evident as increased expression of CD5, the BAQ44A molecule,the CACT65A molecule and the expansion of surface IgG1⁺ B cells.These mCD40L-induced phenotypic changes were also observed onsubpopulations of freshly isolated iPfB cells and jejunal PPfollicular B cells. However, few iPfB cells had a phenotypesimilar to that observed in co-culture with mCD40L and thissuggested that T cell-dependent B cell development may playa minor role in ileal PP B cell development. The possible significanceof CD40 signalling is discussed in terms of the selection ofiPfB cells during development.     

Dysregulation of CD30+ T cells by leukemia impairs isotype switching in normal B cells

Chronic lymphocytic leukemia (CLL) is associated with impaired immunoglobulin (Ig) class-switching from IgM to IgG and IgA, a defect that leads to recurrent infections. When activated in the presence of leukemic CLL B cells, T cells rapidly up-regulate CD30 through an OX40 ligand and interleukin 4 (IL-4)-dependent mechanism. These leukemia-induced CD30+ T cells inhibit CD40 ligand (CD40L)-mediated S mu-->S gamma and S mu-->S alpha class-switch DNA recombination (CSR) by engaging CD30 ligand (CD30L), a molecule that interferes with the assembly of the CD40-tumor necrosis factor receptor-associated factor (TRAF) complex in nonmalignant IgD+ B cells. In addition, engagement of T cell CD30 by CD30L on neoplastic CLL B cells down-regulates the CD3-induced expression of CD40L. These findings indicate that, in CLL, abnormal CD30-CD30L interaction impairs IgG and IgA production by interfering with the CD40-mediated differentiation of nonmalignant B cells.     

Engagement of CD40 lowers the threshold for activation of resting B cells via antigen receptor

Cross-linking of surface Ig (sIg) on resting B cells can generate intracellular signals; however, for T-dependent antigens to promote growth and differentiation additional surface receptors must be engaged. Ligation of CD40 can stimulate B cell proliferation in the presence of interleukin-4. A recently identified counterstructure for CD40 is found on T helper cells and is believed to represent the cognate ligand for B cell activation. This study investigates the role of CD40 as an accessory molecule in sIg-dependent B cell activation. Simultaneous ligation of sIg and CD40 by monoclonal antibodies (mAb) in the presence of mouse L cells which express human Fey receptor type II (FcγRII-L cells) results in potent stimulation of small resting B cells. When CD40 is co-ligated, picomolar concentrations of mouse IgG1 anti-μ, and anti-δ mAb can stimulate B cell proliferation. This requires interaction of the anti-Ig mAb with the FcγRII-L cells: a mouse IgG2a anti-μ, mAb which is not recognized by FcγRII, was ≥ 1000-fold less effective. These findings suggest a mechanism for B cell activation whereby engagement of T cells via CD40 and its cognate ligand provides potent enhancement of signals delivered through sIg. Based on these observations, models for the activation of B cells by T-dependent antigens are presented.     

CD40 signaling induces B cell responsiveness to multiple members of the gamma chain-common cytokine family.

CD40 signaling induces B cell proliferative and differentiation responses that can be modulated by many different cytokines. Cytokines in the IL-2 receptor gamma chain (gammac)-common family are known to play an integral role in B cell development. Therefore, we investigated the possibility that CD40 signaling induced B cell responsiveness to multiple gammac-common cytokines and that individual gammac-common cytokines induced distinct B cell responses. B cells were isolated from lymphoid follicles of sheep Peyer's patches (PP) and co-cultured with murine CD40 ligand (mCD40L). CD40 signaling induced PP B cell responsiveness to recombinant human IL-2, IL-4, IL-7 and IL-15. mCD40L-induced B cell growth was enhanced by combining IL-4 with a second gammac-common cytokine and sustained B cell growth required co-stimulation with IL-4 plus IL-2, IL-7 and IL-15. gammac-common cytokine responsiveness remained dependent upon CD40 signaling, and removal of mCD40L resulted in B cell differentiation and cell death. Similar proliferative responses to mCD40L and gammac-common cytokines were observed for both immature (ileal) and mature (jejunal) PP B cells. Finally, the capacity of CD40-activated B cells to respond to multiple gammac-common cytokines was analyzed with individual PP B cell clones. All B cell clones displayed similar proliferative responses to IL-2 but quantitatively different responses to IL-4, IL-7 and IL-15. The biological significance of B cell responsiveness to multiple gammac-common cytokines is discussed.     

CD40 ligand-positive CD8+ T cell clones allow B cell growth and differentiation

A fraction of activated CD8⁺ T cells expresses CD40 ligand (CD40L), a molecule that plays a key role in T cell-dependent B cell stimulation. CD8⁺ T cell clones were examined for CD40L expression and for their capacity to allow the growth and differentiation of B cells, upon activation with immobilized anti-CD3. According to CD40L expression, CD8⁺ clones could be grouped into three subsets. CD8⁺ T cell clones expressing high levels of CD40L (≥80% CD40L⁺ cells) were equivalent to CD4⁺ T cell clones with regard to induction of tonsil B cell proliferation and immunoglobulin (Ig) production, provided the combination of interleukin (IL)-2 and IL-10 was added to cultures. CD8⁺ T cell clones, with intermediate levels of CD40L expression (10 to 30% CD40L⁺ cells), also stimulated B cell proliferation and Ig secretion with IL-2 and IL-10. B cell responses induced by these CD8⁺ T cell clones were neutralized by blocking monoclonal antibodies specific for either CD40L or CD40. By contrast, CD40L^?? T cell clones (?5 % CD40L⁺ cells), only induced marginal B cell responses even with IL-2 and IL-10. All three clone types were able to activate B cells as shown by up-regulation of CD25, CD80 and CD86 expression. A neutralizing anti-CD40L antibody indicated that T cell-dependent B cell activation was only partly dependent on CD40-CD40L interaction. These CD40L^?? clones had no inhibitory effects on B cell proliferation induced by CD40L-expressing CD8⁺ T cell clones. Taken together, these results indicate that CD8⁺ T cells can induce B cell growth and differentiation in a CD40L-CD40-dependent fashion.     

Induction of B cell costimulatory function by recombinant murine CD40 ligand

T cell-dependent regulation of B cell growth and differentiation involves an interaction between CD40, a B cell surface molecule, and the CD40 ligand (CD40L) which is expressed on activated CD4⁺ T cells. In the current study, we show that recombinant membrane-bound murine CD40L induces B cells to express costimulatory function for the proliferation of CD4⁺ Tcells. CD40L- or lipopolysaccharide (LPS)-activated, but not control-cultured B cells were strong costimulators of anti-CD3 or alloantigen-dependent T cell responses. The molecular interactions responsible for the increased costimulatory functions were examined by analyzing the activated B cells for changes in the expression of two costimulatory molecules, B7 and heat-stable antigen (HSA), as well as by the use of antagonists of B7 and HSA (CTLA4.Fc and 20C9, respectively). The expression of both B7 and HSA was enhanced on B cells activated with LPS. As observed in previous studies, the costimulatory activity of the LPS-activated B cells was dependent on both B7 and HSA and was completely inhibited in the presence of a combination of CTLA4.Fc and 20C9. In contrast, activation of B cells with CD40L induced the expression of B7 but did not enhance the expression of HSA. In addition the costimulatory activity of the CD40L-activated B cells was partially, but not completely, inhibited by the combination of CTLA4.Fc and 20C9. These results demonstrate that CD40L regulates costimulatory function of B cells in part by inducing the expression of B7 and suggest that CD40L-activated B cells express an additional costimulatory activity that is not associated with LPS-activated B cells.     

Cell division number regulates IgG1 and IgE switching of B cells following stimulation by CD40 ligand and IL-4

CD40 ligand (CD40L) and IL-4 are sufficient to induce resting murine B cells to divide and switch isotypes from IgM and IgD to IgG1 and IgE. Tracking of cell division following (5-and 6) carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling revealed that B cells expressed IgG1 after three cell divisions, and IgE after five. The probability of isotype switching at each division was independent of both time after stimulation and of the dose of CD40L. IL-4 concentration regulated the number of divisions that preceded isotype switching. Loss of surface IgM and IgD was also related to cell division and appeared to be differentially regulated. B cell proliferation was typically asynchronous with the proportion of cells in consecutive divisions being markedly affected by the concentration of CD40L and IL-4. Simultaneous (5-bromo)-2 ′ -deoxyuridine labeling and CFSE staining revealed that B cells in each division cycle were dividing at the same rate. Therefore, division cycle asynchrony resulted from dose-dependent variation in the time taken to enter the first division cycle. These results suggest that T-dependent B cell expansion is linked to predictable functional changes that may, in part, explain why IgE is produced in response to prolonged antigenic stimulation.     

Enforced and prolonged CD40 ligand expression triggers autoantibody production in vivo.

CD40, a glycoprotein expressed on B lymphocytes plays an important role in B cell development, growth and differentiation. The ligand for the CD40 is a 39-kDa glycoprotein (CD154) expressed on the surface of activated T lymphocytes and is essential for thymus-dependent humoral immunity. The expression of CD154 is tightly regulated and its transient expression reduces the chances of potentially deleterious bystander activation of B cells. Stimulation through CD40 has been studied in vitro by using antibodies against CD40, by membranes of activated T cells or lately, by CD154 transfected cells. In this work we have evaluated the outcome of CD40-CD40 ligand interaction in vitro and in vivo by using CD154-transfected L929 cells. In vitro assays showed that CD154-L929 cells can induce on B cells: IL-4-dependent proliferation, up-regulation of CD23, CD54 and class II molecules and can also rescue WEHI-231 B cell lymphoma from anti-IgM-induced apoptosis. Interestingly, in vivo assays revealed that when CD154-L929 cells were inoculated into the spleen, mice developed a strong but transient production of anti-erythrocyte autoantibodies. Through B lymphocyte activation with CD154-transfected L929 cells both in vitro and in vivo, our data reveal that enforced and prolonged expression of CD40 ligand overcomes the tightly regulated mechanisms of B cell activation, triggering the production of autoantibodies. This system might be used to evaluate the early steps of an autoimmune response and the role of CD40-CD154 in the induction of primary responses in vivo.     

Suppression of apoptosis in normal and neoplastic human B lymphocytes by CD40 ligand is independent of Bcl-2 induction

The tendency of isolated germinal center (GC) B cells to undergo apoptosis was suppressed by recombinant cell-bound CD40 ligand (CD40L): after 2 days at 37°C, > 80 % of cells remained viable in the presence of CD40L as compared to < 1 % in control cultures. CD40L sustained a high rate of DNA synthesis in GC cells and was more effective than monoclonal antibody to CD40 in this regard. Group I Burkitt lymphoma (BL) cell lines induced to undergo apoptosis with anti-immunoglobulin or calcium ionophore were also protected by CD40L. In BL cells, this route of rescue was not accompanied by induction of Bcl-2 protein, the expression of which has been linked to hemopoietic cell survival. Bcl-2 was induced in GC cells responding to CD40L, but its appearance was a relatively late event not reaching significant levels over controls until day 2 of culture. Thus induction of Bcl-2 appears to be secondary to the survival signal imparted by CD40L. These findings are discussed in relation to a potential role for CD40L in supporting B cell tumors in vivo and the discovery that the molecular defect in the X-linked Hyper-IgM syndrome is targeted to the CD40L gene.