Inhibition of phosphatidylinositol 3-kinase- and ERK MAPK-regulated protein synthesis reveals the pro-apoptotic properties of CD40 ligation in carcinoma cells

CD40, a member of the tumor necrosis factor receptor superfamily, is frequently expressed in carcinomas where its stimulation results in induction of apoptosis when de novo protein synthesis is inhibited. The requirement of protein synthesis inhibition for efficient killing suggests that CD40 transduces potent survival signals capable of suppressing its pro-apoptotic effects. We have found that inhibition of CD40 signaling on the phosphatidylinositol 3-kinase (PI3K) and ERK MAPK but not on the p38 MAPK axis disrupts this balance and sensitizes carcinoma cells to CD40-mediated cell death. The CD40-mediated PI3K and ERK activities were found to converge on the regulation of protein synthesis in carcinoma cells via a pathway involving the activation of p90 ribosomal S6 kinase (p90Rsk) and p70S6 kinases, upstream of the translation elongation factor eEF2. In addition, CD40 ligation was found to mediate a PI3K- and mammalian target of rapamycin (mTOR)-dependent phosphorylation of 4E-BP1 and its subsequent dissociation from the mRNA cap-binding protein eIF4E as well as an ERK-dependent phosphorylation of eIF4E, thus promoting translation initiation. Concomitantly, the antiapoptotic protein cFLIP was found to be induced in CD40 ligand-stimulated carcinoma cells in a PI3K-, ERK-, and mammalian target of rapamycin (mTOR)-dependent manner and down-regulation of cFLIPS expression sensitized to CD40-mediated carcinoma cell death. These data underline the significance of the PI3K and ERK pathways in controlling the balance between CD40-mediated survival and death signals through the regulation of the protein synthesis machinery. Pharmacological agents that target this machinery or its upstream kinases could, therefore, be exploited for CD40-based tumor therapy.     

A novel mechanism of CD40-induced apoptosis of carcinoma cells involving TRAF3 and JNK/AP-1 activation

Membrane-presented CD40 agonists can induce apoptosis in carcinoma, but not normal homologous epithelial cells, whereas soluble agonists are growth inhibitory but not proapoptotic unless protein synthesis is blocked. Here we demonstrate that membrane-presented CD40 ligand (CD154) (mCD40L), but not soluble agonists, triggers cell death in malignant human urothelial cells via a direct mechanism involving rapid upregulation of TNFR-associated factor (TRAF)3 protein, without concomitant upregulation of TRAF3 mRNA, followed by activation of the c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) pathway and induction of the caspase-9/caspase-3-associated intrinsic apoptotic machinery. TRAF3 knockdown abrogated JNK/AP-1 activation and prevented CD40-mediated apoptosis, whereas restoration of CD40 expression in CD40-negative carcinoma cells restored apoptotic susceptibility via the TRAF3/AP-1-dependent mechanism. In normal human urothelial cells, mCD40L did not trigger apoptosis, but induced rapid downregulation of TRAF2 and 3, thereby paralleling the situation in B-lymphocytes. Thus, TRAF3 stabilization, JNK activation and caspase-9 induction define a novel pathway of CD40-mediated apoptosis in carcinoma cells.     

Primary and malignant cholangiocytes undergo CD40 mediated Fas dependent apoptosis, but are insensitive to direct activation with exogenous Fas ligand

Introduction

Cholangiocarcinoma is a rare malignancy of the biliary tract, the incidence of which is rising, but the pathogenesis of which remains uncertain. No common genetic defects have been described but it is accepted that chronic inflammation is an important contributing factor. We have shown that primary human cholangiocyte and hepatocyte survival is tightly regulated via co-operative interactions between two tumour necrosis family (TNF) receptor family members; CD40 and Fas (CD95). Functional deficiency of CD154, the ligand for CD40, leads to a failure of clearance of biliary tract infections and a predisposition to cholangiocarcinoma implying a direct link between TNF receptor-mediated apoptosis and the development of cholangiocarcinoma.

Aims

To determine whether malignant cholangiocytes display defects in CD40 mediated apoptosis. By comparing CD40 and Fas-mediated apoptosis and intracellular signalling in primary human cholangiocytes and three cholangiocyte cell lines.

Results

Primary cholangiocytes and cholangiocyte cell lines were relatively insensitive to direct Fas-mediated killing with exogenous FasL when compared with Jurkat cells, which readily underwent Fas-mediated apoptosis, but were extremely sensitive to CD154 stimulation. The sensitivity of cells to CD40 activation was similar in magnitude in both primary and malignant cells and was STAT-3 and AP-1 dependent in both.

Conclusions

1) Both primary and malignant cholangiocytes are relatively resistant to Fas–mediated killing but show exquisite sensitivity to CD154, suggesting that the CD40 pathway is intact and fully functional in both primary and malignant cholangiocytes 2) The relative insensitivity of cholangiocytes to Fas activation demonstrates the importance of CD40 augmentation of Fas dependent death in these cells. Agonistic therapies which target CD40 and associated intracellular signalling pathways may be effective in promoting apoptosis of malignant cholangiocytes.     

RKTS-33, an epoxycyclohexenone derivative that specifically inhibits Fas ligand-dependent apoptosis in CTL-mediated cytotoxicity

Cytotoxic T lymphocytes (CTLs) eliminate virus-infected cells and tumor cells by two distinct killing pathways, mediated by lytic granules containing perforin and by Fas ligand (FasL). ECH [(2R,3R,4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-6-(1E)-propenyl-cyclohex-5-en-1-one] has been shown to inhibit FasL-dependent apoptosis or the killing pathway in short-term culture. However, since ECH exhibited cell toxicity in long-term culture, we attempted the synthesis of less toxic epoxycyclohexenone derivatives. In the present study, we found that RKTS-33 [(2R,3R,4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-cyclohex-5-en-1-one] has cell toxicity lower than ECH in long-term culture, and further investigated the inhibitory effect of RKTS-33 on CTL-mediated killing pathways. RKTS-33 did not affect cell-surface expression of FasL upon CD3 stimulation, but profoundly inhibited the FasL-dependent killing pathway mediated by CD4+ and CD8+ CTLs, indicating that RKTS-33 specifically blocks target cell apoptosis but not CTL function. By contrast, RKTS-33 did not affect the perforin-dependent killing pathway in CD8+ CTLs. These results indicate that RKTS-33 is a specific inhibitor of the FasL-dependent killing pathway in CTL-mediated cytotoxicity.     

CD40 is overexpressed by HPV16/18-E6 positive cervical carcinoma and correlated with clinical parameters and vascular density

CD40 is expressed in many tumor cells, however, its role in tumor biology is yet to be demonstrated. In the present study, we investigated the role of CD40 in cervical carcinoma. In vivo, we evaluated CD40 expression in 56 cervical carcinoma tissues, 43 cervicitis and 38 normal cervix, and investigated the relationship between CD40 and HPV antigen, histopathological parameters, vascular density, and vascular endothelial growth factor (VEGF) expressions. The results clearly demonstrated that CD40 expression, including membranous and cytoplasmic staining, was significantly higher in cervical carcinoma than in the cervicitis and normal cervix. The expression of CD40 was significantly correlated with HPV and VEGF expressions and microvessel density (MVD). These observations provide evidence that CD40 may be involved in neovascularization of cervical carcinoma, they also suggest that CD40 and VEGF may be useful biomarkers for evaluating the risk of developing cervical carcinoma, and may also be used as a target for therapy.     

Does CD40 ligation induce B cell negative selection?

Binding of CD154 to its receptor, CD40, provides costimulation for mature B cell activation and differentiation in response to Ag receptor signals. In mice, early B cell precursors express CD40, but its function at this stage is unknown. We examined the effects of CD40 ligation during B cell ontogeny in transgenic mice constitutively expressing CD154 on B cells (kappaEP-CD154). Precursors beyond pro-B cells were absent in adult bone marrow but were increased in the fetal liver. Newborn kappaEP-CD154 mice had largely increased numbers of peripheral B cells, which were CD154+, and that 36 h after birth expressed high surface levels of CD23 and MHC class II, resembling activated mature B cells. Nevertheless, kappaEP-CD154 mice were hypogammaglobulinemic, indicating that the expanded population of apparently activated B cells was nonfunctional. Further analysis revealed that soon after birth, kappaEP-CD154 mice-derived B cells became CD5+/Fas+, after which progressively decreased in the periphery in a CD154-CD40-dependent manner. These results indicate that CD40 ligation during B cell ontogeny induces negative selection characterized by either hyporesponsiveness or an arrest in maturation depending on the time of analysis and the anatomic site studied.     

IFN-γ upregulates survivin and Ifi202 expression to induce survival and proliferation of tumor-specific T cells

Background

A common procedure in human cytotoxic T lymphocyte (CTL) adoptive transfer immunotherapy is to expand tumor-specific CTLs ex vivo using CD3 mAb prior to transfer. One of the major obstacles of CTL adoptive immunotherapy is a lack of CTL persistence in the tumor-bearing host after transfer. The aim of this study is to elucidate the molecular mechanisms underlying the effects of stimulation conditions on proliferation and survival of tumor-specific CTLs.

Methodology/Principal Findings

Tumor-specific CTLs were stimulated with either CD3 mAb or cognate Ag and analyzed for their proliferation and survival ex vivo and persistence in tumor-bearing mice. Although both Ag and CD3 mAb effectively induced the cytotoxic effecter molecules of the CTLs, we observed that Ag stimulation is essential for sustained CTL proliferation and survival. Further analysis revealed that Ag stimulation leads to greater proliferation rates and less apoptosis than CD3 mAb stimulation. Re-stimulation of the CD3 mAb-stimulated CTLs with Ag resulted in restored CTL proliferative potential, suggesting that CD3 mAb-induced loss of proliferative potential is reversible. Using DNA microarray technology, we identified that survivin and ifi202, two genes with known functions in T cell apoptosis and proliferation, are differentially induced between Ag- and CD3 mAb-stimulated CTLs. Analysis of the IFN-γ signaling pathway activation revealed that Ag stimulation resulted in rapid phosphorylation of STAT1 (pSTAT1), whereas CD3 mAb stimulation failed to activate STAT1. Chromatin immunoprecipitation revealed that pSTAT1 is associated with the promoters of both survivin and ifi202 in T cells and electrophoresis mobility shift assay indicated that pSTAT1 directly binds to the gamma activation sequence element in the survivin and ifi202 promoters. Finally, silencing ifi202 expression significantly decreased T cell proliferation.

Conclusions/Significance

Our findings delineate a new role of the IFN-γ signaling pathway in regulating T cell proliferation and apoptosis through upregulating survivin and ifi202 expression.     

A live-cell assay to detect antigen-specific CD4+ T cells with diverse cytokine profiles

Recently activated, but not resting, CD4(+) T cells express CD154, providing costimulatory signals to B cells and antigen-presenting cells (APCs). Therefore, de novo CD154 expression after stimulation identifies antigen-specific CD4(+) T cells. Previous assays were limited by the transient nature of surface CD154 expression; we overcame this by including fluorescently conjugated CD154-specific antibody during stimulation. Our assay is fully compatible with intracellular cytokine staining, and can be used for stimulations as long as 24 h. Notably, it is nonlethal, providing a means to purify viable antigen-specific CD4(+) T cells for further analysis. Using this assay, we found that stimulated cells expressing tumor necrosis factor (TNF)-alpha, interleukin (IL)-2 or interferon (IFN)-gamma were predominantly CD154(+). Furthermore, some cells expressing none of these cytokines also expressed CD154, suggesting that CD154 marks cells with other effector functions. For vaccine- or pathogen-specific responses, we found substantial heterogeneity in expression of CD154 and cytokines, suggesting previously unrecognized diversity in abilities of responding cells to stimulate APCs through CD40.     

Caspase-3 is necessary and sufficient for cleavage of protein synthesis eukaryotic initiation factor 4G during apoptosis.

Induction of apoptosis BJAB cells is accompanied by the rapid cleavage of protein synthesis eukaryotic initiation factor 4G and the appearance of a fragment of approximately 76 kDa. Inhibition of apoptotic proteases (caspases) has previously been shown to prevent the cleavage of eukaryotic initiation factor 4G. In MCF-7 breast carcinoma cells, which are deficient in caspase-3, eukaryotic initiation factor 4G is not cleaved but in vivo expression of caspase-3 restores eukaryotic initiation factor 4G cleavage following induction of apoptosis. Recombinant caspase-3 can also cleave eukaryotic initiation factor 4G to yield the 76 kDa fragment both in cell extracts and when the eukaryotic initiation factor 4G is presented in a purified eukaryotic initiation factor 4F complex. These results indicate that caspase-3 activity is necessary and sufficient for eukaryotic initiation factor 4G degradation.     

Human anergic CD4+ T cells can act as suppressor cells by affecting autologous dendritic cell conditioning and survival.

T cell suppression exerted by regulatory T cells represents a well-established phenomenon, but the mechanisms involved are still a matter of debate. Recent data suggest that anergic T cells can suppress responder T cell activation by inhibiting Ag presentation by dendritic cells (DC). In this study, we focused our attention on the mechanisms that regulate the susceptibility of DC to suppressive signals and analyzed the fate of DC and responder T cells. To address this issue, we have cocultured human alloreactive or Ag-specific CD4+ T cell clones, rendered anergic by incubation with immobilized anti-CD3 Ab, with autologous DC and responder T cells. We show that anergic T cells affect either Ag-presenting functions or survival of DC, depending whether immature or mature DC are used as APC. Indeed, MHC and costimulatory molecule expression on immature DC activated by responder T cells is inhibited, while apoptotic programs are induced in mature DC and in turn in responder T cells. Ligation of CD95 by CD95L expressed on anergic T cells in the absence of CD40-CD40L (CD154) interaction are critical parameters in eliciting apoptosis in both DC and responder T cells. In conclusion, these findings indicate that the defective activation of CD40 on DC by CD95L+ CD154-defective anergic T cells could be the primary event in determining T cell suppression and support the role of CD40 signaling in regulating both conditioning and survival of DC.     

TCR-independent killing of B cell malignancies by anti-third-party CTLs: the critical role of MHC-CD8 engagement

We previously demonstrated that anti-third-party CTLs (stimulated under IL-2 deprivation against cells with an MHC class I [MHC-I] background different from that of the host and the donor) are depleted of graft-versus-host reactivity and can eradicate B cell chronic lymphocytic leukemia cells in vitro or in an HU/SCID mouse model. We demonstrated in the current study that human allogeneic or autologous anti-third-party CTLs can also efficiently eradicate primary non-Hodgkin B cell lymphoma by inducing slow apoptosis of the pathological cells. Using MHC-I mutant cell line as target cells, which are unrecognizable by the CTL TCR, we demonstrated directly that this killing is TCR independent. Strikingly, this unique TCR-independent killing is induced through lymphoma MHC-I engagement. We further showed that this killing mechanism begins with durable conjugate formation between the CTLs and the tumor cells, through rapid binding of tumor ICAM-1 to the CTL LFA-1 molecule. This conjugation is followed by a slower second step of MHC-I-dependent apoptosis, requiring the binding of the MHC-I α2/3 C region on tumor cells to the CTL CD8 molecule for killing to ensue. By comparing CTL-mediated killing of Daudi lymphoma cells (lacking surface MHC-I expression) to Daudi cells with reconstituted surface MHC-I, we demonstrated directly for the first time to our knowledge, in vitro and in vivo, a novel role for MHC-I in the induction of lymphoma cell apoptosis by CTLs. Additionally, by using different knockout and transgenic strains, we further showed that mouse anti-third-party CTLs also kill lymphoma cells using similar unique TCR-independence mechanism as human CTLs, while sparing normal naive B cells.     

The human B lymphocyte and carcinoma antigen, CDw40, is a phosphoprotein involved in growth signal transduction

The human B lymphocyte and carcinoma-associated Ag, CDw40, (p50, Bp50) is a receptor candidate for normal growth regulation. Interaction of mAb with this pan-B Ag, together with preactivating agents such as 12-O-tetradecanoylphorbol-13-acetate or anti-mu, deliver strong growth-promoting signals to the cells. We here demonstrate that signaling through this Ag is dependent on its aggregation on the cell surface. Thus, monovalent antibody fragments were relatively inefficient in this respect but effectively blocked stimulation by intact antibody. By using affinity purified CDw40 protein we have also demonstrated that it is antigenically distinct from other B cell-associated Ag, including the six differentiation clusters CD19 to CD24. The mAb S2C6 and G28.5, prepared by immunizing mice with human bladder carcinoma cells or tonsillar B-cells, respectively, were the only antibodies giving detectable binding. Either of these antibodies could also completely block the binding of the other, suggesting an identity or structural proximity of the epitopes recognized. The CDw40 Ag was shown to be a phosphoprotein lacking intrinsic protein kinase activity. The results provide further evidence for CDw40 being an important B cell growth factor receptor which may also have growth regulatory functions in the development of certain human carcinomas.     

In situ beta cell death promotes priming of diabetogenic CD8 T lymphocytes.

CTLs are important mediators of pancreatic beta cell destruction in the nonobese diabetic mouse model of type 1 diabetes. Cross-presentation of Ag is one means of priming CTLs. The death of Ag-bearing cells has been implicated in facilitating this mode of priming. The role of beta cell death in facilitating the onset of spontaneous autoimmune diabetes is unknown. Here, we used an adoptive transfer system to determine the time course of islet-derived Ag presentation to naive beta cell-specific CD8 T cells in nonobese diabetic mice and to test the hypothesis that beta cell death enhances the presentation of beta cell autoantigen. We have determined that beta cell death enhances autoantigen presentation. Priming of diabetogenic CD8 T cells in the pancreatic lymph nodes was negligible before 4 wk, progressively increased until 8 wk of age, and was not influenced by gender. Administration of multiple low doses of the beta cell toxin streptozotocin augmented in situ beta cell apoptosis and accelerated the onset and magnitude of autoantigen presentation to naive CD8 T cells. Increasing doses of streptozotocin resulted in both increased pancreatic beta cell death and significantly enhanced T cell priming. These results indicate that in situ beta cell death facilitates autoantigen-specific CD8 T cell priming and can contribute to both the initiation and the ongoing amplification of an autoimmune response.     

Dendritic cells are resistant to apoptosis through the Fas (CD95/APO-1) pathway.

Immunoregulation of lymphocytes and macrophages in the peripheral immune system is achieved in part by activation-induced cell death. Members of the TNF receptor family including Fas (CD95) are involved in the regulation of activation-induced cell death. To determine whether activation-induced cell death plays a role in regulation of dendritic cells (DCs), we examined interactions between Ag-presenting murine DCs and Ag-specific Th1 CD4+ T cells. Whereas mature bone marrow- or spleen-derived DCs expressed high levels of Fas, these DCs were relatively insensitive to Fas-mediated killing by the agonist mAb, Jo-2, as well as authentic Fas ligand expressed on the CD4+ T cell line, A.E7. The insensitivity to Fas-mediated apoptosis was not affected by priming with IFN-gamma and/or TNF-alpha or by blocking the DC survival signals TNF-related activation-induced cytokine and CD40L. However, apoptosis could be induced with C2-ceramide, suggesting that signals proximal to the generation of ceramide might mediate resistance to Fas. Analysis of protein expression of several anti-apoptotic mediators revealed that expression of the intracellular inhibitor of apoptosis Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein was significantly higher in Fas-resistant DCs than in Fas-sensitive macrophages, suggesting a possible role for Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein in DC resistance to Fas-mediated apoptosis. Our results demonstrate that murine DCs differ significantly from other APC populations in susceptibility to Fas-mediated apoptosis during cognate presentation of Ag. Because DCs are most notable for initiation of an immune response, resistance to apoptosis may contribute to this function.     

The role of CD40-CD154 interaction in cell immunoregulation

CD40, a member of the nerve growth factor/tumor necrosis factor receptor superfamily, and its ligand, CD154, play essential roles in cell immune responses. The results of many studies have indicated that CD40-CD154 interaction can upregulate costimulatory molecules, activate antigen-presenting cells (APCs), influence T-cell priming and T-cell-mediated effector functions as well as participate in the pathogenic processing of chronic inflammatory diseases, such as autoimmune diabetes, graft rejection, atherosclerosis, and cancer. Ligation of CD40 on cancer cells was also found to produce a direct growth-inhibitory effect through cell cycle blockage and/or apoptosis with no overt side effects on normal cells and treatment with CD154 can heighten tumor rejection immune response as well. However, systemic treatment with CD154 has some potential risks. Therefore, searching for efficient and safe strategies of CD154-based cancer therapy has been a hot topic in human cancer research. This review focuses on the latest discovered functions of CD40-CD154 interaction in cell immune responses and on the new findings of CD154-based human cancer therapy.     

Angiogenesis,cell proliferation and apoptosis in progression of cervical neoplasia

OBJECTIVE: To investigate changes in angiogenesis, cell proliferation and apoptosis in the successive steps of cervical neoplasia and to analyze their interrelationship. STUDY DESIGN: A total of 182 cervical specimens, representing 12 normal epithelium, 33 cervical intraepithelial neoplasia (CIN) 1, 21 CIN 2, 30 CIN 3 and 86 squamous cell carcinomas, were evaluated. The microvessels were immunohistochemically labeled with CD34 antibodies. Computerized image analysis was used to evaluate microvessel density (MVD). The apoptotic cells were visualized by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique and proliferative cells by staining with Ki-67 antibodies. RESULTS: One-way analysis of variance showed that the MVD, Ki-67 labeling index and apoptotic index increased significantly with the progression of cervical neoplasia from normal epithelium, through CIN, to carcinoma (P <.001 for each index). All the indices, determined in all 182 cervical tissues, were significantly and positively associated with each other (P < .001 in all cases), with correlation coefficients ranging from .649 to .819. MVD in patients with recurrence or death was significantly higher than in disease-free patients (P < .05). CONCLUSION: The results suggest that tumor progression in the cervical epithelium is accompanied by angiogenesis and an increase in both cell proliferation and apoptosis. Angiogenesis may be a prognostic indicator in patients with squamous cell carcinoma of the cervix.     

Cell surface expression of CD154 inhibits alloantibody responses: A mechanism for the prevention of autoimmune responses against activated T cells?

Binding of CD40 by CD154 expressed on activated T cells is a pivotal event in T cell help to B cells, macrophages, and other antigen-presenting cells. Expression of CD154 by MHC mismatched cells, in contrast to expectations, strongly suppressed alloantibody responses against the cells. This was caused by a failure of priming of antibody responses by the CD154 expressing cells. We hypothesize that this lack of response against CD154 expressing cells may represent a mechanism that has evolved to prevent autoantibody responses being generated against the CD154 antigen itself, as B cells expressing antibody reactive with CD154 would probably escape deletion on binding antigen in the bone marrow due to rescue by the simultaneous ligation of CD40.     

Ligation or cross-linking of CD40 has different effects on AGS gastric cancer cells

A gastric cancer (GC) cell line, AGS, has high-level expression of CD40, a tumor necrosis factor receptor (TNFR) family member. CD40 is present on the surfaces of a large variety of cells, including B cells, endothelial cells, dendritic cells and some carcinoma cells, and delivers signals regulating diverse cellular responses, such as proliferation, differentiation, growth suppression, and cell death. In this research, we studied the effects of different forms of CD40 stimulation on AGS cells by flow cytometry, Western blotting and siRNA transfection. We found that different forms of CD40 stimulation, either recombinant soluble CD40L (sCD40L, ligation) or agonist anti-CD40 antibody (cross-linking), induced different effects in AGS gastric cancer cells, proliferation or apoptosis. We also showed that VEGF provided a significant contribution to sCD40L-induced proliferation, while agonist anti-CD40 antibody induced GADD45 upregulation and promoted apoptosis.     

B cell abnormalities in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic, multisystem autoimmune disease characterized by the differentiation of short- and long-lived immunoglobulin secreting plasma cells that secrete pathogenic autoantibodies. Ectopic germinal centers and plasma cells secreting autoantibodies have been observed in lupus nephritis kidneys. Candidate genetic susceptibility loci for SLE include genes that affect differentiation and survival of plasma cells, such as those that influence activation, proliferation, cytokine and chemokine secretion/responsiveness, and apoptosis of the T and B cells that are involved in humoral immunity generated in germinal centers, as well as genes that are involved in presentation and clearance of apoptotic material and autoantigens by antigen presenting cells and other phagocytes. Emerging data have demonstrated that B lymphocytes are active participants in humoral immune responses that lead to T-dependent and T-independent differentiation of immunoglobulin-secreting plasma cells by homotypic CD154-CD40 interactions as well as continued stimulation by B cell activating factor through B cell maturation antigen, B cell activating factor receptor and transmembrane activater.