CD40 ligand and autoantigen are involved in the pathogenesis of low-grade B-cell lymphomas of mucosa-associated lymphoid tissue

Low-grade MALT-type lymphomas are malignancies of mucosal marginal-zone B cells and preceded by reactive inflammatory lymphoid tissue. Experimental observations suggest that antigen and CD40 Ligand act during cognate T/B cell interaction and are crucial for germinal center B-cell maturation generating marginal-zone B cells. To investigate the mechanisms underlying the development of extranodal MALT-type lymphomas, the immunoglobulin receptor was sequenced and analyzed for antigen specificity using heterohybridoma technology. Furthermore, CD40 ligand expression was evaluated by immunohistochemistry and by semiquantitative RT-PCR, and ligand binding to the CD40 of tumor B cells was studied using the CD40 system. Hypermutations were found in low-grade lymphomas throughout CDR1-CDR3 suggestive of positive selection through their antigen receptor. Different VH families were used and more than 69% of tumor immunoglobulins bound different mucosal antigens. CD40L expression was found in the tumor marginal zone in substantial amounts. The in vitro proliferation response of all low-grade MALT-type lymphomas was dependent on anti-CD40-mediated signals and cytokines. Our data provide evidence that autoantigen as well as the CD40L expressed by activated nonneoplastic T cells may drive the evolution of low-grade MALT-type lymphomas either directly or by paracrine mechanisms and that antigen may contribute to lymphoma pathogenesis.     

CD154/CD40 and CD70/CD27 interactions have different and sequential functions in T cell-dependent B cell responses: enhancement of plasma cell differentiation by CD27 signaling

CD40, a TNF receptor family member, plays a central role in T cell-mediated B cell activation. We have recently demonstrated that CD27, another TNF receptor family member, was also involved in B cell regulation and enhanced Ig production. In this report we compare CD27 and CD40 signals in B cell function. We selectively mimicked the effect of T cell help by addition to peripheral blood B cells activated with Staphylococcus aureus Cowan I strain and IL-2 of irradiated 300-19 cells transfected with either the CD70 (CD27 ligand) gene or the CD154 (CD40 ligand) gene, the vector alone, or both CD70 and CD154 genes. CD27 ligation induced only a slight increase in B cell proliferation compared with the dramatic enhancement induced by CD40 ligation; double ligation proved to be less efficient than CD40 ligation alone. In contrast, IgG production was increased only by CD27 ligation alone. Moreover, the CD27 signal was more efficient when it was given on day 2 of the culture rather than on day 0. Phenotypic analysis of the activated cells showed that CD27 ligation increased the percentage of cells showing a plasma cell profile (CD19-, CD38+), whereas upon CD40 ligation most of the cells still had a germinal center-like phenotype (CD19+, CD38+). Our results suggest that the CD27 and CD40 signals are not synergistic but, rather, are complementary and involve distinct steps of T cell-dependent B cell activation. CD27 may be more important in the induction of plasma cell differentiation at a time when the expansion phase has already occurred.     

The expression of CD26 and CD40 ligand is mutually exclusive in human T-cell non-Hodgkin's lymphomas/leukemias

CD26 and CD40 ligand (CD40L) are surface molecules on human activated T lymphocytes that play a critical role in the regulation of lymphopoiesis. Both molecules are expressed on a restricted fraction of human T-cell non-Hodgkin's lymphomas (NHL)/leukemias; however, little is known about their functional and/or clinical significance in these disorders. In this study, the pattern of expression of CD40L was compared with that of the CD26 molecule. A series of 67 human T-cell NHL/leukemias and a panel of leukemia/lymphoma T-cell lines were evaluated by immunohistochemistry, flow cytometry, and RNA studies. The overall frequency of CD26+ and CD40L+ samples was rather similar (25/67 [37%] v 18/67 [27%]). However, the majority of CD26-expressing cases clustered in the lymphoblastic lymphomas (LBL)/T-acute lymphoblastic leukemias (ALL; 12/23) and CD30+ anaplastic large-cell (ALC) lymphomas (5/8), whereas CD40L+ lymphomas included a large fraction of mycosis fungoides (11/21 [52%]). CD26 and CD40L coexpression was found only in 2 myocosis fungoides cases and 1 small lymphocytic lymphoma. Thus, the expression of the two antigens was mutually exclusive in almost all T-cell lymphomas/leukemias. Accordingly, lymphoma cell lines expressed either one of the molecules or the relative amounts of CD26 and CD40L were inversely proportional. In contrast, reactive T lymphocytes from patients with non-neoplastic T-cell expansions and in vitro activated CD3+ or CD4+ normal T cells were found to coexpress CD40L and CD26. Results of a multivariate analysis showed that the expression of CD26 in T-cell LBL/ALL patients was associated to a worse outcome in terms of survival, as compared with patients with CD26- tumors (P < or = .0001). Based on our results, it can be concluded that, (1) as opposed to activated or reactive normal T cells, the expression of CD26 and of CD40L is mutually exclusive in human T-cell lymphomas/leukemias; (2) expression of CD26 is restricted to aggressive pathologic entities, such as T-cell LBL/ALL and T-cell CD30+ ALC lymphomas, whereas CD40L is expressed on slow progressing diseases such as mycosis fungoides; and (3) within the T-cell LBL/ALL group of tumors, CD26 may identify a subset of poor prognosis patients.     

IL-12 up-regulates CD40 ligand (CD154) expression on human T cells

IL-12 is a heterodimeric cytokine produced by APC that promotes the development of CD4+ Th1 cells and their IFN-gamma production after TCR/CD3 triggering. We here investigated the capacity of IL-12 to modify the expression on T cells of CD40 ligand (CD40L or CD154), a molecule transiently expressed on activated T cells and known to be of utmost importance for cognate interaction with B cells and for activation of dendritic cells and macrophages. Our data demonstrate that IL-12 up-regulates CD40L expression on anti-CD3-activated human peripheral blood T cells. For optimal induction of CD40L, IL-12 synergizes with IL-2 as well as with other costimulatory interactions, such as B7/CD28. The effect of IL-12 was observed at both the protein and the mRNA level. T cells costimulated by IL-12 provided more efficient help for IL-4-dependent B cell proliferation and for IgG production than when activated in the absence of IL-12. This helper activity was blocked by an mAb against CD40L, indicating that the effect of IL-12 on B cells is mediated indirectly through CD40L. The data thus suggest that the effects of IL-12 on cellular and humoral immune responses are partly mediated through CD40L induction.     

Blocking OX-40/OX-40 ligand interaction in vitro and in vivo leads to decreased T cell function and amelioration of experimental allergic encephalomyelitis

The OX-40R is a member of the TNF receptor family and is expressed primarily on activated CD4+ T cells. When the OX-40R is engaged by the OX-40 ligand (OX-40L), a potent costimulatory signal occurs. We have identified a population of CD11b+ cells, isolated from the central nervous system (CNS) of mice with actively induced experimental allergic encephalomyelitis (EAE), that expresses OX-40L. Moreover, the expression of OX-40L was found to be associated with paralytic episodes of EAE and was reduced or absent at disease recovery. These CD11b+ cells also coexpressed B7 and MHC class II. Therefore, to address the relative contributions of OX-40R/OX-40L and CD28/B7 to the costimulation of myelin-specific T cells, blocking studies were performed using soluble OX-40R and/or soluble CTLA-4. CD11b+ cells isolated from the CNS of mice with actively induced EAE were able to present Ag to proteolipid protein 139-151-specific T cell lines in vitro. The addition of soluble OX-40R:Ig to CD11b+ brain microglia/macrophages inhibited T cell proliferation by 50-70%. The addition of CTLA-4:Ig inhibited T cell proliferation by 20-30%, and the combination inhibited T cell proliferation by 95%. In vivo administration of soluble OX-40R at the onset of actively induced or adoptively transferred EAE reduced ongoing signs of disease, and the mice recovered more quickly from acute disease. The data imply that OX-40L, expressed by CNS-derived APC, acts to provide an important costimulatory signal to EAE effector T cells found within the inflammatory lesions. Furthermore, the data suggest that agents designed to inhibit the OX-40L/OX-40R complex may be useful for treating autoimmune disease.     

Chronic lymphocytic leukemia B cells can express CD40 ligand and demonstrate T-cell type costimulatory capacity

Chronic lymphocytic leukemia (CLL) is characterized by a clonal expansion of CD5(+) B cells in the peripheral blood. Associated immune aberrations include abnormal Th-cell function and pathogenic autoantibodies. Under most circumstances, CLL B cells do not proliferate in culture and express a limited repertoire of surface antigens, including CD19, CD20, CD23, CD27, CD40, and CD70. In this report, we demonstrate that freshly isolated B cells from a subset of CLL cases constitutively express CD40 ligand (CD40L, CD154), a member of the tumor necrosis factor family which is normally expressed by activated CD4(+) T cells and mediates T-cell-dependent B-cell proliferation and antibody production. The degree of CD40L expression varied considerably among the CLL cases examined. CD40L was detected in purified CLL B cells by immunofluorescence flow cytometry, by RT-PCR, and by immunoprecipitation. To demonstrate that CD40L in the CLL B cells is functional, we used irradiated CLL cells to stimulate IgG production by target, nonmalignant B cells in coculture. The CLL B cells induced IgG production by normal B cells to a similar degree as did purified T cells in a process which was partially inhibited by monoclonal antibody to CD40L. This is one of the first reports of CD40L expression in a B-cell tumor. The data suggest that CD40L in the tumor cells may be a factor in the generation of pathologic antibodies by normal B cells in some patients with CLL.     

CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome

The ligand for CD40 (CD40L) is a membrane glycoprotein on activated T cells that induces B cell proliferation and immunoglobulin secretion. Abnormalities in the CD40L gene were associated with an X-linked immunodeficiency in humans [hyper-IgM (immunoglobulin M) syndrome]. This disease is characterized by elevated concentrations of serum IgM and decreased amounts of all other isotypes. CD40L complementary DNAs from three of four patients with this syndrome contained distinct point mutations. Recombinant expression of two of the mutant CD40L complementary DNAs resulted in proteins incapable of binding to CD40 and unable to induce proliferation or IgE secretion from normal B cells. Activated T cells from the four affected patients failed to express wild-type CD40L, although their B cells responded normally to wild-type CD40L. Thus, these CD40L defects lead to a T cell abnormality that results in the failure of patient B cells to undergo immunoglobulin class switching.     

A cytotoxic CD40/p55 tumor necrosis factor receptor hybrid detects CD40 ligand on herpesvirus saimiri-transformed T cells

The B cell activation molecule CD40 and the p55 tumor necrosis factor receptor (p55TNFR) belong to the same family of structurally conserved proteins. We constructed a chimeric receptor consisting of the CD40 extracellular and transmembrane domains and the p55TNFR intracellular domain. This receptor hybrid retained the biological activity and the ligand specificity of the respective wild-type receptor domains. Thus it exerted a marked cytotoxic effect in three different transfected cell lines after activation not only with anti-CD40 antibody but also with CD40 ligand (CD40L) in soluble and membrane-bound forms. Using hybrid-transfected baby hamster kidney cells we demonstrated that herpesvirus saimiri-transformed human CD4+ T lymphocytes constitutively express bioactive CD40 ligand on their surface. The hybrid receptor-based assay was highly specific for CD40 activating reagents and more sensitive than an assay measuring CD40-mediated B cell rescue from apoptosis. Hence CD40/p55TNFR transfectants may be useful for dissecting CD40L-mediated events in T-B cell interactions, and also to detect a defective CD40L molecule in putative hyper-IgM syndrome patients.     

Mucosally induced systemic T cell unresponsiveness to ovalbumin requires CD40 ligand-CD40 interactions

CD40 ligand (CD40L) gene-disrupted (CD40L-/-) mice were employed to examine the role of costimulatory signals via CD40L-CD40 interactions in mucosally induced tolerance. CD40L-/- and control (CD40L+/+) mice of the same C57BL/6 x 129/J background were immunized orally with 25 mg of OVA before systemic challenge with OVA in CFA. While CD40L+/+ mice showed reductions in Ag-specific T cell responses including delayed-type hypersensitivity (DTH) and proliferative responses, CD40L-/- mice underwent normal T cell responses. Further, cytokine analysis of splenic CD4+ T cells showed that both Th1-type (e.g., IFN-gamma and IL-2) and Th2-type (e.g., IL-4, IL-5, IL-6, and IL-10) responses were maintained in CD40L-/- mice orally immunized with OVA, whereas these cytokine responses in CD40L+/+ mice were significantly reduced. In addition, splenic CD4+ T cells from CD40L-/- mice orally immunized with OVA provided B cell help in Ag-specific Ab-forming cells when the cells were cultured with naive B cells in the presence of Ag and CD40L-transfected cell lines. In contrast, an identical culture condition containing splenic CD4+ T cells from orally tolerized CD40L+/+ mice did not exhibit helper activity. Taken together, these findings indicate that CD40L and CD40 interactions are essential for the induction of systemic T cell unresponsiveness to orally administered Ag.     

Limited importance of CD40/CD40L interaction in the B7-dependent generation of anti-MOPC-315 cytotoxic T lymphocyte activity by tumor bearer splenic cells stimulated in vitro in the presence of tumor necrosis factor

We have previously illustrated the importance of B7-2 expression for the enhanced generation of cytotoxic T lymphocyte (CTL) activity by stimulation cultures of tumor bearer splenic cells to which tumor necrosis factor alpha (TNFalpha) has been added. Here we show that the B7-1 molecule is also important for CTL generation by such stimulation cultures, although to a much lesser extent than the B7-2 molecule. In addition, we show the importance of CD40/CD40L interaction for the expression of the B7-2 molecule, but not the B7-1 molecule, by tumor bearer splenic cells stimulated in vitro in the presence of TNF. The CD40/CD40L interaction is also shown to be important for the generation of CTL activity by tumor bearer splenic cells stimulated in vitro in the presence of exogenous TNF. However, the CD40/CD40L interaction is less important for the generation of enhanced CTL activity than for the expression of an elevated level of B7-2. Specifically, blockade of CD40/CD40L interaction, which reduced the level of B7-2 expressed by tumor bearer splenic cells stimulated in vitro in the presence of TNF to the level of B7-2 expressed by tumor bearer splenic cells stimulated in vitro in the absence of exogenous TNF, failed to reduce the level of CTL generated to the level generated by tumor bearer splenic cells stimulated in the absence of exogenous TNF. Finally, blockade of CD40/CD40L interaction was inferior to blockade of B7-2/CD28 interaction in inhibiting the generation of CTL activity by tumor bearer splenic cells stimulated in the presence of exogenous TNF. Thus, although CD40/CD40L interaction is important for the generation of enhanced CTL activity by stimulation cultures of tumor bearer splenic cells to which TNF has been added, TNF also mediates its potentiating effect for CTL generation by such stimulation cultures via other mechanisms that are independent of CD40/CD40L interaction but dependent on B7-2 expression.     

The T cell-B cell interaction via OX40-OX40L is necessary for the T cell-dependent humoral immune response

Recent in vitro studies have established that activated B cells express OX40 ligand (L), a member of the tumor necrosis factor/nerve growth factor family of cytokines, and become stimulated to proliferate and secrete immunoglobulin (Ig) after cross-linking of OX40L by its counterreceptor OX40, which is expressed on activated T cells. In the present study we investigated the in vivo role of this receptor-ligand pair for the interaction of T and B cells in the course of the T-dependent B cell response against 2,4,6 trinitro-phenyl-keyhole limpet hemocyanin. First, we showed that OX40 is maximally expressed by T cells in the periarteriolar lymphoid sheath (PALS) 3 d after primary immunization. These OX40+ cells are located in close proximity to antigen-specific, activated B cells. Second, we demonstrated that blocking of OX40-OX40L interaction with polyclonal anti-OX40 antibody or with antibodies against certain peptide sequences within its extracellular domain resulted in a profound decrease of the anti-hapten IgG response, whereas the antihapten IgM response was grossly unchanged. Third, we showed that this antibody treatment leads to an inhibition of the development of PALS-associated B cell foci, whereas the formation of germinal centers remained intact. Finally, our data suggest that, whereas B cell memory development was not impaired by anti-OX40 administration, OX40-OX40L interaction seems to be crucial in the secondary immune response. We conclude from these data that the OX40-OX40L interaction in vivo is necessary for the differentiation of activated B cells into highly Ig-producing cells, but is not involved in other pathways of antigen-driven B cell differentiation such as memory cell development in the germinal centers.     

Antitumor effect of CD40 ligand: elicitation of local and systemic antitumor responses by IL-12 and B7

The interaction between CD40 ligand (CD40L, CD154) and its receptor CD40 has been implicated in the establishment of cell-mediated immunity as well as humoral immune responses. To examine the role of CD40L in eliciting antitumor immunity, we introduced murine CD40L gene into P815 mastocytoma (CD40L-P815). CD40L-P815 cells underwent prompt rejection when inoculated s.c. into syngenic DBA/2 mice or athymic BALB/c nu/nu mice, which was mediated by NK cells and dependent on endogenous IL-12. The primary rejection of CD40L-P815 cells in DBA/2 mice elicited CD8+ T cell-mediated protective and systemic immunity against parental tumor cells, which was induced by CD4+ T cells and endogenous B7. These results indicated a potent antitumor effect of CD40L that is mediated by potentiation of host Ag-presenting cell functions, and introduction of CD40L will be useful as a new strategy of immuno-gene therapy against tumors.     

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OX40 (CD134) is a member of the tumor necrosis factor (TNF) receptor superfamily first identified as a rat T cell activation marker. In the present study, we identified the rat ligand for OX40 (OX40L) by molecular cloning. Rat OX40L cDNA was cloned from a HTLV-1-transformed rat T cell line by cross-hybridization with mouse OX40L cDNA. The predicted rat OX40L polypeptide is composed of 199 amino acids, showing 80.9 and 43.3% homology to mouse and human OX40L, respectively. Expression of rat OX40L mRNA was found in HTLV-1-transformed rat T cell lines. Expression of OX40L on the cell surface of these HTLV-1-transformed rat T cell lines was also demonstrated by flow cytometric analysis with a soluble fusion protein composed of the extracellular region of the Fc portion of human IgG (OX40-Ig). To explore the function of rat OX40L, we generated cDNA transfectants stably expressing rat OX40L. The rat OX40L transfectants exhibited a potent costimulatory activity for proliferation and IL-2 production of anti-CD3-stimulated rat T cells. These results indicated that rat OX40L can provide an efficient costimulation for rat T cells and that it may be involved in HTLV-1-associated pathologies in the rat system as has been suggested in the human system.     

CD40 induces resistance to TNF-mediated apoptosis in a fibroblast cell line

CD40, a member of the TNF receptor family, has been characterized as an important T-B cell interaction molecule. In B cells it co-stimulates isotype switching, proliferation, adhesion and is involved in cell death regulation. In addition to B cells, CD40 expression was found on transformed cells and carcinomas. However, little is known about its functions in these cell types. Recent studies show that CD40 mediates the production of pro-inflammatory cytokines in non-hematopoietic cells, inhibits proliferation or induces cell death. In some cell types the apoptotic program triggered by CD40 is only executed when protein synthesis is blocked, suggesting the existence of constitutively expressed resistance proteins. Here we demonstrate that CD40, similar to the 55-kDa TNF receptor (p55TNFR), has a dual role in the regulation of apoptosis in such cells. In the fibroblast cell line SV80 both CD40 and the p55TNFR trigger apoptosis when protein synthesis is blocked with cycloheximide (CHX). Simultaneous activation of both receptors results in markedly enhanced cell death. However, CD40 activation more than 4 h prior to a challenge with TNF/CHX paradoxically conferred resistance to TNF-induced cell death. Protection correlated with NF-kappaB induction and up-regulation of the anti-apoptotic zinc finger protein A20. Overexpression of A20 in turn rendered SV80 cells resistant to TNF cytotoxicity. In conclusion, our data provide evidence that CD40 may regulate cell death in non-hematopoietic cells in a dual fashion: the decision upon apoptosis or survival of a CD40-activated cell seems to depend on its ability to up-regulate resistance factors.     

Normal CD40-mediated activation of monocytes and dendritic cells from patients with hyper-IgM syndrome due to a CD40 pathway defect in B cells

Patients with X-linked hyper-IgM syndrome [CD40 ligand (CD40L) deficiency] are prone to infections by intracellular parasites. It has been suggested that this susceptibility is caused by defective macrophage activation through the CD40L-CD40 pathway. We studied the CD40-mediated activation of monocytes and dendritic cells from patients affected with a CD40L+ hyper-IgM syndrome characterized by a defect of B lymphocyte responses to CD40 agonists. We show that the CD40-induced production of IL-6, IL-8 and TNF-alpha by monocytes, and IL-12 by dendritic cells, and expression of the activation markers CD83, the costimulatory molecules CD86 and CD80, and HLA-DR antigens were all similar in patient and control cells. This observation is consistent with the clinical characteristics of the syndrome: a defect of immunoglobulin switch but no susceptibility to opportunistic infections, as observed in CD40L-deficient patients. These observations suggest that CD40-mediated activation pathways could be, at least in part, different in B and monocytic/dendritic cell lineages.     

Differential expression of CD40 ligand on T cell subsets. Implications for different roles of CD45RA+ and CD45RO+ cells in IgE production

Physical contact between human T lymphocytes and B lymphocytes is required for the induction of IgE production. In the present study, we examined the abilities of CD45RA+ and CD45RO+ human T cell subsets to provide help for IgE production by human peripheral blood B cells in the presence of IL-4. Purified peripheral CD45RA+ T cells are much better inducers of IgE synthesis than are CD45RO+ T cells. Activation of CD45RA+ T cells, but not CD45RO+ T cells, via the TCR/CD3 complex is sufficient to confer the ability to provide IgE help, suggesting that an inducible T cell surface molecule plays an important role in this system. The CD40 ligand, an inducible T cell surface molecule, is expressed at higher levels on CD45RA+ T cells as compared with CD45RO+ T cells following CD3-stimulation. Blocking of the CD40-CD40 ligand interaction in vitro by the addition of a soluble form of B cell CD40 Ag completely blocks IgE production induced by CD45RA+ T cells. Finally, the in vitro conversion of CD45RA+ T cells to the CD45RO+ phenotype is accompanied by a loss in the ability of these cells to express the CD40 ligand in response to anti-CD3 stimulation as well as a loss in their ability to provide IgE help. These results suggest that both CD45 subsets may play significant and distinct roles in the induction of IgE production under physiologic conditions: CD45RO+ T cells provide IL-4 and the CD45RA+ subset provides the second signal via the CD40 ligand.     

CD40 ligand expressed on B cells in the BXSB mouse model of systemic lupus erythematosus

Male BXSB mice, unlike female BXSB, develop a severe early onset lupus-like disease that has been linked to an intrinsic B cell defect. In investigating this B cell defect the present study showed that male, but not female, BXSB contained a higher percentage of large, activated splenic B cells that were more responsive to anti-CD40 mAb-induced proliferation. The hyperactivity of the large B cells from the male mice was also observed in the absence of anti-CD40 mAb or any other stimuli. In examining the mechanism of the B cell hyperactivity, it was found that 20% of unstimulated large B cells from male mice, unlike large B cells from female mice, expressed CD40 ligand (CD40L), a molecule normally expressed on activated CD4+ cells. The percentage of large B cells from the male BXSB that expressed CD40L was increased to 43% by stimulation with LPS. A functional role for CD40L expression on B cells was confirmed by showing that CD40-Ig blocked the spontaneous proliferation of the large B cells from male mice. In addition, the stimulatory capacity of the large B cells from the male mice was demonstrated by their ability to induce DNA synthesis in small B cells in a CD40L-dependent manner. These results demonstrated that large B cells from male BXSB expressed functionally active CD40L. It is likely that the B cell CD40L expression and increased susceptibility to CD40 signaling due to an intrinsic B cell hyperactivity promotes autoimmune disease in BXSB mice.     

CD4+ T cells inhibit growth of Epstein-Barr virus-transformed B cells through CD95-CD95 ligand-mediated apoptosis

Greater than 90% of the human population acquire Epstein-Barr virus (EBV) in infancy and retain a lifelong latent infection without any clinical consequences. Nevertheless EBV has been identified as the causal agent of infectious mononucleosis, and is associated with several tumours including endemic Burkitt's lymphoma and B cell lymphomas in immunosupressed patients. B cells infected with EBV are transformed in vitro and grow continuously as lymphoblastoid cell lines. The growth of EBV-transformed B cells in vivo is controlled by the immune system. Studies on immunity to EBV have mainly focused on MHC class I-restricted CD8+ cytotoxic T cells specific for viral latent antigens. Here it is reported that in vitro stimulation of peripheral blood lymphocytes by autologous EBV-infected B cells, which have been induced to express lytic cycle antigens, gives rise to a predominantly CD4+ T cell response. Furthermore, the growth of EBV-infected B cells can also be regulated by these activated CD4+ T cells through apoptosis mediated by CD95-CD95 ligand (CD95L). CD95-CD95L-mediated apoptosis is an important mechanism of normal B cell growth regulation. As EBV-transformed B cells remain susceptible to this mechanism, the control of EBV in vivo may be not only by virus-specific CD8+ cytotoxic T cell immunity but also by normal mechanisms of immune regulation of B cell growth.