Antigen-Induced T Cell Death Is Regulated by CD4 Expression

Activation induced cell death (AICD) is a major physiologic pathway that regulates T cell homeostasis. In CD4 T cells, AICD is mediated mainly through Fas/FasL interactions. Although TCR occupancy triggers AICD, the contribution of its tightly associated CD4 coreceptor to the process that leads to AICD is not known. Here we show that CD4 molecule plays an essential regulatory role of TCR dependent AICD. Loss of CD4 rendered activated 5kc T cell hybridoma resistant to AICD. The resistance of CD4 negative 5kc T cells to AICD was due to selective inhibition of FasL expression and it could be reversed by addition of recombinant FasL. Furthermore, a direct functional link between CD4 and FasL was demonstrated by induction of FasL upon CD4 cross-linking in a TCR independent fashion. The importance of CD4 interaction with MHC/peptide complex in mediating AICD was also evident in normal T cells that could survive chronic stimulation with anti-CD3 but died after short period of proliferation after stimulation with MHC/peptide. Thus it appears that AICD is controlled by the CD4 molecule via regulation of FasL expression. These findings have important implications for our understanding of mechanisms of peripheral tolerance as well as pathogenesis of autoimmune diseases.     

IFN-gamma regulates Fas ligand expression in human CD4+ T lymphocytes and controls their anti-mycobacterial cytotoxic functions

Fas and Fas Ligand (FasL) expression, activation-induced cell death (AICD) and mycobacterial antigen-specific cytotoxicity of peripheral T cells from patients with complete inherited IFN-gamma receptor 1 binding chain deficiency (IFN-gammaR1-/-) were investigated. Fas was equally expressed in both normal and deficient T lymphoblasts and they underwent apoptosis when stimulated with agonist anti-Fas mAb. By contrast, T lymphoblasts and CD4+ T cell clones (TCC) from deficient patients displayed a reduced surface FasL expression and resistance to AICD. CD8+ TCC from healthy and deficient patients displayed similar high level of FasL and susceptibility to AICD. In Jurkat CD4+ T cells competent to transduce IFN-gamma signaling, IFN-gamma induced surface FasL export and their Fas-dependent apoptosis. Effector T cells generated from a patient with a dominant negative mutation of IFN-gammaR1 (IFN-gammaR1DN) following stimulation with mycobacterial antigens were unable to kill MHC class II-matched, mycobacterial antigen-pulsed macrophages. Normal Fas expression in T cells and FasL in CD8+ cells may account for the absence of autoimmune disorders in these patients. Conversely, defective FasL expression on IFN-gammaR1DN CD4+ T cells impairs their cytotoxic functions and highlights a novel role for IFN-gamma signaling in the control of mycobacterial infection in humans.     

Taurine attenuates CD3/interleukin-2-induced T cell apoptosis in an in vitro model of activation-induced cell death (AICD)

Interleukin (IL)-2 immunotherapy is used for the treatment of metastatic melanoma and renal cell carcinoma and mediates its effects through the clonal expansion of lymphocytes. Although IL-2 remains the most effective form of therapy for these cancers, response rates are poor and dose escalation is hampered by side effects, which include vascular leak and lymphopenia. The mechanism underlying T cell loss is currently unidentified but could be the induction of activation-induced cell death (AICD) mediated by FasL. Our previous studies have shown that the amino acid taurine can attenuate apoptosis induced by a number of factors in different cell types. Here, we induced T cell AICD via CD3 and IL-2 stimulation and investigated the effect of taurine on lymphocyte apoptosis. Anti-CD3-activated Jurkat T cells treated with IL-2 significantly increased FasL expression, which was associated with increased apoptosis. Treatment with taurine prior to stimulation down-regulated FasL protein expression and partially inhibited apoptosis. Inhibition of FasL-signalling resulted in an identical reduction in apoptosis. As the kinetics of AICD are completely different in circulating T cells, we repeated these experiments in such cells to confirm our finding. Stimulation of CD4(+) circulating T cells induced apoptosis in sensitized, but not freshly isolated T cells, which was abrogated partially by taurine. In Jurkat cells it was determined that taurine-mediated down-regulation of FasL protein expression was associated with decreased FasL mRNA expression and reduced NFkappaB activation. These results reveal one possible mechanism underlying the lymphopenia observed with IL-2 immunotherapy, involving increased FasL expression leading to apoptosis. Taurine may be of use in reversing the lymphopenia associated with IL-2, thereby augmenting its immunotherapeutic potential.     

Two signaling pathways can lead to Fas ligand expression in CD8+ cytotoxic T lymphocyte clones

As shown previously, a given cytotoxic T lymphocyte (CTL) clone (KB5.C20) could be induced to express the Fas ligand (FasL) by either T cell receptor (TCR) engagement or phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation. In contrast, another CTL clone (BM3.3) has now been found to exert Fas-based cytotoxicity only after TCR engagement, but not after PMA/ionomycin stimulation. This suggested the existence of a PMA-insensitive, antigeninduced pathway leading to FasL expression. The inability of PMA to promote Fas-based cytotoxicity in BM3.3 cells was correlated with a defect in expression of the classical protein kinase C (PKC) isoforms α and βI. In KB5.C20 cells depleted of PMA-sensitive PKC isoforms and thus no longer responsive to PMA, Fas-based cytotoxicity could still be induced via the TCR/CD3 pathway. On the other hand, a requirement for phosphatidylinositol-3 kinase (PI3K) selectively in this TCR/CD3-induced pathway was demonstrated by specific inhibition with wortmannin. These results suggest that FasL expression when induced via the TCR/CD3 involves PI3K, and when induced by PMA/ionomycin requires the expression of PMA-sensitive PKC isoforms absent in clone BM3.3. Additional data suggest that in neither case was NF-χB activation implicated in FasL expression.     

Activation-induced apoptosis of mature T cells is dependent upon the level of surface TCR but not on the presence of the CD3 zeta ITAM

Activation-induced cell death (AICD) occurs primarily in recently activated T cells after a second TCR triggering. Since a threshold in the activation status may be critical for AICD, it is likely that the CD3 ITAM, docking sites for tyrosine kinases, regulate AICD. A 'threshold model' for AICD was tested by using two targeted mutant mouse strains lacking either the zeta chain (CD3zeta-/-) or the ITAM of the zeta chain (CD3zeta-/-:Tgzetadelta67-150). Although the T cells from the CD3zeta-/- mice express extremely low levels of surface TCR, a subpopulation (approximately 18%) of activated T cells could be induced to express TCR/FcepsilonRI gamma by using a powerful polyclonal activation protocol. These activated TCR/FcRI gamma T cells were capable of undergoing AICD, but its induction required 10 times as much anti-CD3epsilon mAb as that required for AICD of wild-type T cells. Thus, the intensity of AICD correlated with the level of CD3 expression and was less efficient with activated, CD3zeta(-/-)-derived T cells. By contrast, AICD of T cells from the CD3zeta-/-:Tgzetadelta67-150 mice could be induced with low doses of anti-CD3epsilon mAb and the extent of AICD was comparable to T cells from wild-type mice. The AICD induced in T cells from CD3-/-, CD3zeta-/-:Tgzetadelta67-150 and normal controls was specifically inhibited by Fas-Ig fusion proteins. Our data support the 'threshold model' of AICD by demonstrating that AICD is controlled by the strength of T cell activation.      

Protection of T cells from activation-induced cell death by Fas+ B cells

Naive CD4+ T cells proliferate strongly in response to stimulation by superantigens such as staphylococcal enterotoxin B (SEB). However, when these same cells revert to a resting phenotype and are subjected to restimulation with either SEB or anti-CD3, the majority of these SEB-responsive cells undergo Fas ligand (FasL)-mediated activation-induced cell death (AICD). We investigated the impact of Fas expression on T cell AICD by utilizing B cell stimulators that lacked functional FasL and either expressed or did not express the Fas receptor. Our results indicate that B cells play an important role in modulating the level of T cell AICD via the Fas/FasL pathway. Activated B cells expressing high levels of Fas receptor can redirect the FasL expressed by T cells primed to undergo AICD away from the T cells and prevent the induction of AICD in these cells. Furthermore, B cells stimulated through both the CD40 receptor and membrane IgM appear to mediate a stronger protective effect on T cells by virtue of their resistance to FasL-mediated cytolysis. These observations suggest a mechanism by which normal B cell and T cell responses to foreign antigen are maintained, while responses to self antigen are not.     

Murine AIDS requires CD154/CD40L expression by the CD4 T cells that mediate retrovirus-induced disease: Is CD4 T cell receptor ligation needed?

LP-BM5, a retroviral isolate, induces a disease featuring an acquired immunodeficiency syndrome termed murine AIDS (MAIDS). Many of the features of the LP-BM5-initiated disease are shared with HIV/AIDS. Our lab has shown that the interaction of B and CD4 T cells that is central to MAIDS pathogenesis requires ligation of CD40 on B cells by CD154 on CD4 T cells. Despite this strict requirement for CD154 expression, whether CD4 T cell receptor (TCR) occupancy is essential for the induction of MAIDS is unknown. To block TCR engagement, Tg mouse strains with monoclonal TCR of irrelevant peptide/MHC specificities, all on MAIDS-susceptible genetic backgrounds, were tested: the study of a panel of TCR Tg CD4 T cells controlled for the possibility of serendipitous crossreactive recognition of virus-associated or induced-self peptide, or superantigen, MHC complexes by a given TCR. The results argue that TCR engagement is not necessary for the induction of MAIDS.     

CD4+ CD25+调节性T细胞AICD机制的研究

目的探讨CD4^＋CD25^＋调节性T细胞活化诱导的细胞死亡（AICD）发生的机制。方法CD4^＋CD25^＋T细胞以磁性细胞分离器（MACS）从BALB／c小鼠或DO11．10小鼠的静息T细胞分离纯化。体外细胞增殖抑制实验证实其免疫调节作用。CD4^＋CD25^＋T细胞的AICD以CD3／CD28单克隆抗体活化或以特异性OVA323-339肽、抗原提呈细胞活化等两种方法获得。CD4^＋CD25^＋T细胞凋亡相关基因的表达通过实时定量PCR检测。流式细胞仪检测细胞的凋亡率。进一步观察FasL中和抗体、TRAIL中和抗体及caspase抑制剂zVAD-fmk对CD4^＋CD25^＋T细胞凋亡的影响。结果MACS成功分离CD4^＋CD25^＋T细胞，纯度可达98％，该细胞可特异性表达Foxp3基因，能明显抑制效应性T细胞的体外增殖。CD3／CD28抗体以及OVA特异性抗原活化8d的CD4^＋CD25^＋调节性T细胞AICD达39％～45％。活化前后的CD4^＋CD25^＋调节性T细胞死亡受体家族表达发生明显变化；FasL、TRAIL中和抗体及zVAD-fmk可明显抑制CD4^＋CD25^＋调节性T细胞的凋亡。结论FasL／Fas及其他凋亡相关分子可能参与了CD4^＋CD25^＋调节性T细胞的凋亡。     

Loss of CD8 and TCR binding to Class I MHC ligands following T cell activation

The capacity of T cells to bind peptide/MHC ligands changes with T cell development and differentiation. Here we study changes in peptide/MHC multimer binding following T cell activation. Surprisingly, T cell activation caused a marked reduction in specific peptide/MHC Class I multimer binding, which was distinct from transient TCR down-regulation, and was especially dramatic for engagement with low-affinity peptide/MHC ligands. Direct CD8-Class I interactions were also profoundly and rapidly impaired following T cell stimulation, even though surface CD8alpha and CD8beta levels were unchanged after activation, suggesting that decreased CD8 co-receptor binding contributes to this effect. Finally, we show that enzymatic desialylation restores much of the multimer binding on activated T cells, suggesting that altered glycosylation may inhibit TCR/CD8 binding to peptide/MHC ligands. These radical changes in activated T cells' ability to perceive peptide/MHC ligands may contribute to selective outgrowth of clones with high affinity for the stimulatory ligand.     

Th1 and Th2 subsets equally undergo Fas-dependent and -independent activation-induced cell death

Stimulation of previously activated T cells results in apoptosis, termed activation-induced cell death (AICD). Recent analysis revealed that the Fas/Fas ligand (FasL) interaction is predominantly involved in AICD of T cells. Furthermore, based on the analysis of various T cell clones and lines, it has been reported that FasL is expressed mainly in Th1 but not in Th2 cells. However, the exact expression pattern of FasL and its function in normal activated T cells has not been determined. In the present study, by utilizing completely differentiated Th1 and Th2 cell populations obtained from ovalbumin-specific T cell receptor (TCR)-transgenic mice, the FasL expression on Th1 and Th2 was determined. Furthermore, involvement of Fas-FasL interaction in AICD of Th1 and Th2 cells was analyzed by two approaches: one was the inhibition of AICD by anti-FasL monoclonal antibodies, and the other AICD of Th1/Th2 subsets from TCR-transgenic mice backcrossed to lpr mice. We demonstrated that Th2 cells express FasL on the cell surface at a level similar to that expressed by Th1 cells, and that both subsets were equally susceptible to the Fas-mediated AICD. These observations suggest not only that the expression of FasL is not always correlated with Th subsets as defined by the cytokine-producing profile, but also that the responses of both Th1 and Th2 subsets are regulated by Fas-mediated AICD. Finally, analysis of the kinetics of AICD revealed a novel Fas/FasL-independent pathway in its initial stage. These findings revealed the precise function of Fas/FasL-mediated as well as Fas/FasL-independent AICD in the regulation of helper T cell responses.     

Release of preformed Fas ligand in soluble form is the major factor for activation-induced death of Jurkat T cells.

Interaction of Fas/APO-1 (CD95) and its ligand (FasL) plays an important role in the activation-induced cell death (AICD) of T lymphocytes. In the present work, the contribution of soluble FasL to AICD of the human T-cell line Jurkat has been studied. Jurkat cells prestimulated with phytohaemagglutinin (PHA) induced the death of non-activated Jurkat cells, and also of L1210Fas, but not that of Fas-negative L1210 cells. Culture supernatants from prestimulated Jurkat cells were highly toxic to their non-activated counterparts. Time-course analysis revealed that PHA-stimulated Jurkat cells quickly release (less than 15 min) to the medium a toxic molecule following a biphasic pattern, with maximal cytotoxic activities at 1 hr and 7 hr after stimulation. The cytotoxic effect of those supernatants was prevented by the addition of a blocking anti-Fas monoclonal antibody, suggesting that PHA-stimulated Jurkat cells exert Fas-based cytotoxicity mainly through the release of soluble FasL. The constitutive intracellular expression of FasL in non-activated Jurkat cells and its release as a consequence of PHA activation were detected by immunostaining and immunoblotting using an anti-FasL antibody. These data indicate that, at least in Jurkat cells, AICD is mainly mediated by the rapid release of performed FasL in soluble form upon stimulation.     

Activation of a T cell hybridoma by an alloligand results in differential effects on IL-2 secretion and activation-induced cell death.

The molecular nature of the interaction of T cell receptors (TCR) with alloligands is not well understood. Although a role for groove-bound peptide(s) has been clearly demonstrated for major histocompatibility complex (MHC) class I alloreactivity, this has not been established for MHC class II-induced alloresponses. In the present study, we have analyzed the interaction of a nominal peptide-self MHC complex and of an alloligand with their cognate TCR (1934.4 TCR for autoantigen recognition and qCII85.33 TCR for allorecognition). Our results demonstrate that 1934.4 TCR recognition of the N-terminal epitope of myelin basic protein (Ac1-11, Ac=acetylated at position 1) complexed with the MHC class II molecule I-A(u) involves contacts with both chains of the MHC molecule.In contrast, qCII85.33 TCR recognition of an allopeptide:I-A(u) complex appears to predominantly involve the beta chain of the MHC molecule. Thus, the two TCR appear to have different footprints on the I-A(u) molecules. Unexpectedly, this differential involvement of the two chains of the I-A(u) molecule affects activation induced cell death, with allostimulation resulting in poor induction of FasL expression and relatively low levels of apoptosis. Significantly, stimulation of cognate T cells with alloantigen or autoantigen results in similar levels of IL-2 secretion. The reduced apoptosis of T cells in response to allostimulation may be one of the mechanisms that favors the expansion of a relatively large repertoire of alloreactive T cells.     

Soluble egg antigen-stimulated T helper lymphocyte apoptosis and evidence for cell death mediated by FasL(+) T and B cells during murine Schistosoma mansoni infection

Granuloma formation around schistosomal eggs is induced by soluble egg antigens (SEA) and mediated by the activity of CD4(+) Th lymphocytes and their cytokines. Regulation of the inflammatory Th cell response during infection is still insufficiently understood. The hypothesis of this study was that activation-induced cell death (AICD) of CD4(+) T cells is involved in the immune inflammatory response. This study investigated the dynamics of splenic and granuloma CD4(+) Th cell apoptosis and Fas ligand (FasL) expression during the acute and chronic stages of murine schistosomal infection. Enhanced apoptosis of freshly isolated CD4(+) Th lymphocytes commenced after egg deposition and persisted during the peak and modulated phases of granuloma formation. After oviposition, CD4(+), CD8(+), and CD19(+) splenocytes and granuloma cells expressed elevated levels of FasL but FasL expression declined during the downmodulated stage of infection. In culture, SEA induced splenic and granuloma CD4(+) T-cell apoptosis and stimulated expression of FasL on splenic but not granuloma CD4(+) T cells, CD8(+) T cells, and CD19(+) B cells. SEA-stimulated splenocytes and granuloma cells preferentially lysed a Fas-transfected target cell line. Depletion of B cells from SEA-stimulated splenic cultures decreased CD4(+) T cell apoptosis. Coculture of purified splenic B cells with CD4(+) T cells and adoptive transfer of purified B cells indicated that antigen-stimulated B cells can kill CD4(+) Th cells. However, CD4(+) T cells were the dominant mediators of apoptosis in the granuloma. This study indicates that AICD is involved in the apoptosis of CD4(+) T cells during schistosomal infection.     

The CD4-related molecule,LAG-3 (CD223), regulates the expansion of activated T cells

The lymphocyte activation gene-3 (LAG-3, CD223) is a CD4-related, activation-induced cell surface molecule that binds to MHC class II with high affinity. The function of murine LAG-3 on T cells is unclear. Here, we show that V beta 7/8(+)LAG-3(-/-) T cells expand poorly following staphylococcal enterotoxin B (SEB) stimulation in vitro. LAG-3(-/-) T cells proliferate at a normal rate, but exhibit increased cell death. Similar observations were made with LAG-3(-/-)CD4(+)OT-II TCR transgenic T cells following peptide stimulation. Despite reduced T cell expansion and increased cell death, LAG-3(-/-)OT-II(+) T cells secrete more IL-2 and IFN-gamma following stimulation. Antigen-driven expansion of LAG-3(-/-) T cells was restored by constitutive expression of LAG-3 via retroviral-mediated stem cell gene transfer. We further show that LAG-3 function is mediated via its cytoplasmic domain, for which a conserved 'KIEELE' motif is essential. Our data support a role for LAG-3 in regulating the expansion of activated T cells.     

TCR ligand avidity determines the mode of B-Raf/Raf-1/ERK activation leading to the activation of human CD4+ T cell clone

The interactions between peptide/MHC complexes and their cognate TCR are essential for various T cell responses. However, the relationship between the avidity of TCR ligand and the subsequent intracellular signaling through the TCR is still unclear. To investigate the effects of TCR ligand avidity on TCR-mediated signaling, we established L cells expressing HLA-DR4 molecules covalently linked with agonistic peptide (high-affinity ligand) or altered peptide ligand (APL; low-affinity ligand) at various densities as APC for a cognate human CD4(+) T cell clone. Using this system, we demonstrated that the T cell clone stimulated with APL/HLA-DR4 complexes presented at an excessive density provoked the up-regulation of CD69, IL-2 production and proliferation, but no detectable phosphorylation of ZAP-70/LAT/SLP-76. Furthermore, in contrast to the high-affinity stimulation, the low-affinity stimulation evoked delayed and sustained activation of the B-Raf/extracellular signal-regulated kinase (ERK) pathway without Raf-1 activation. The strength and duration of B-Raf/ERK activations closely correlated with the density of the TCR ligand. A knockdown approach confirmed that B-Raf activation was indispensable for the APL-induced T cell responses. These observations suggest that the differences in TCR-peptide/MHC interactions reflect the strength and duration of B-Raf/Raf-1/ERK activation in the human CD4(+) T cells.     

Activated T cells induce rapid CD83 expression on B cells by engagement of CD40

The conserved transmembrane glycoprotein CD83 was originally described as highly specific marker for mature dendritic cells in the peripheral circulation. Besides its regulatory role in thymic T cell maturation and peripheral T cell activation, recent studies suggest, that CD83 is also involved in the regulation of B cell maturation, homeostasis and function. Here we show, that antigen-specific T cell stimulation leads to CD83 induction predominantly on B cells. In vivo activation of T cells by injection of cognate antigenic peptide into T cell receptor transgenic mice induced strong expression of the early activation marker CD69 but only low levels of surface CD83 on T cells. In contrast CD83 was induced on 80% of B cells in the draining lymph node. This T cell mediated induction of CD83 expression on B cells was not mediated by soluble factors but was contact dependent because separation of B cells from an ongoing T cell stimulation in a transwell system abrogated CD83 expression. Since CD83 expression was induced on both MHC-matched and MHC-mismatched B cells present in cultures of activated T cells, cell contact via TCR/MHC binding was not essential. The application of an antibody to the CD40 ligand of T cells, however, strongly interfered with the induction of CD83 expression on bystander B cells. Taken together we provide evidence that activated T cells induce CD83 on B cells via CD40 engagement but independent of TCR/MHC binding and thus independent of antigen-specificity of B cells.     

CD4+CD25-T细胞凋亡机制的研究

目的：探讨CD4^＋CD25^-T细胞AICD发生的机制.方法：磁性细胞分离器（MACS）分离CD4^＋CD25^- T细胞.以CD3/CD28单克隆抗体活化BALB/c小鼠CD4^＋CD25^-T细胞或以OVA323-339肽、抗原递呈细胞活化DO11.10小鼠CD4^＋CD25^-T细胞两种方式建立AICD模型.基因芯片检测CD4^＋CD25^-T细胞和CD4＋CD25＋T细胞凋亡相关基因的表达.流式细胞仪检测细胞的凋亡率.并观察FasL中和抗体、TRAIL中和抗体及zVAD-fmk对CD4^＋CD25^-T细胞凋亡的影响.结果：MACS成功分离CD4^＋CD25^-T细胞,纯度可达98%.建立了CD3/CD28抗体以及OVA特异性抗原活化的CD4^＋CD25^-T细胞AICD模型,CD4^＋CD25^-T细胞凋亡率达35%～40%.基因芯片分析发现CD4^＋CD25^-T细胞相对高表达TRAIL、FAS,而CD4^＋CD25^-T细胞相对高表达DR5、FasL.FasL、TRAIL中和抗体及zVAD-fmk可明显抑制CD4＋CD25＋T细胞的凋亡.结论：FasL、TRAIL及其它凋亡相关分子可能参与了CD4^＋CD25^-T细胞的凋亡.     

Expression of inhibitory KIR is confined to CD8+ effector T cells and limits their proliferative capacity

A subset of effector/memory CD8(+) T cells expresses natural killer cell receptors (NKR). Expression of inhibitory NKR at that stage of T cell differentiation is poorly understood. Interestingly, recent studies in mice indicated that transgenic expression of an inhibitory NKR induced the accumulation of memory T cells by inhibiting activation-induced cell death (AICD). To further understand the role of inhibitory NKR on T cells, we characterized the subset of human peripheral T cells expressing the inhibitory NKR, CD158b, and studied the modulation of antigen-driven T cell expansion by an endogenous inhibitory NKR. We found that CD158b expression was confined to a population of CD8(+)TCRalphabeta(+) effector T cells as defined by a CD45RA(+)CCR7(-) phenotype and high constitutive expression of granzyme B1. Few cells expressed the activating form CD158j in the absence of CD158b. Functionally, engagement of CD158b by MHC ligands diminished early TCR signaling, as well as AICD. However, the reduced AICD did not rescue cells for proliferation, since T cell expansion in the presence of CD158b triggering was impaired. Expression of inhibitory NKR on effector CD8(+) T cells may explain in part the poor replicative capacity of T cells at that stage of differentiation.     

Regulation of cell surface expression of Fas (CD95) ligand and susceptibility to Fas (CD95)-mediated apoptosis in activation-induced T cell death involves calcineurin and protein kinase C, respectively

We show that an influenza hemagglutinin-specific CD4+ murine T cell hybridoma (IP-12-7) enters the apoptotic suicide program via the Fas ligand (FasL)/Fas-mediated pathway upon T cell receptor (TCR) stimulation. These cells express Fas and FasL mRNA, cell surface Fas and intracellular FasL, but do not enter apoptosis upon Fas ligation prior to TCR stimulation. TCR stimulation additionally results in protein synthesis-dependent cell surface expression of the preformed FasL. Addition of phorbol dibutyrate (PBu2) alone was sufficient to induce susceptibility to Fas ligation induced apoptosis, while addition of both PBu2 and calcium ionophore A23187 were required to induce FasL cell surface expression. Addition of cyclosporin A completely inhibited TCR-mediated death and FasL cell surface up-regulation, but had no effect on apoptosis induced directly by Fas ligation following TCR stimulation. Inhibitors of protein kinase C (PKC) (G? 6976 and GF 109203X) completely inhibited TCR-induced susceptibility to Fas ligation, but only partially inhibited TCR-induced cell surface expression of FasL. PKC isoenzymes alpha, beta, delta and zeta were expressed by this cell line and only the alpha and betaI isoforms translocated to the membrane fraction upon TCR stimulation. Our data suggest that in activation-induced T cell apoptosis PKC is involved in pathways that mediate the acquisition of Fas susceptibility, while calcineurin is required for cell surface expression of the preformed FasL.