CD4+ T-cell-mediated cytotoxicity against staphylococcal enterotoxin B-pulsed synovial cells.

Apoptosis of synovial cells in rheumatoid arthritis (RA) synovium determined in vivo is suggested to counteract the overgrowth of synovium. Immunohistological examination has revealed the infiltration of activated CD4+ T cells, which express Fas ligand (FasL), in RA synovium. The presence of a putative antigen (Ag) of autoimmune disorders in a target organ may induce the activation of specific T cells in the inflammatory region such as RA synovium. We examined the possible role of CD4+ T cells activated by synovial cells in a staphylococcal enterotoxin B (SEB)-dependent manner, inducing synovial cell apoptosis. Synovial cells were cultured with or without interferon-gamma (IFN-gamma) and further incubated with CD4+ T cells in the presence of SEB. After the cocultivation, both the cytotoxicity and FasL expression of CD4+ T cells were investigated. Constitutive Fas expression was detected on both unstimulated and IFN-gamma-stimulated synovial cells. CD4+ T cells did not kill SEB-pulsed unstimulated synovial cells efficiently. In contrast, when CD4+ T cells were incubated with IFN-gamma-stimulated synovial cells with SEB whose human leucocyte antigen (HLA)-DR and -DQ expression was markedly induced, significant cytotoxicity by these cells against synovial cells was detected. The addition of anti-HLA-DR and -DQ monoclonal antibodies (mAbs) or human Fas chimeric protein (hFas-Fc) reduced this cytotoxicity. FasL expression of CD4+ T cells cocultured with IFN-gamma-stimulated synovial cells with SEB was significantly induced. Furthermore, the addition of mAbs against CD54, CD58 and CD106 inhibited both the cytotoxicity and FasL expression of CD4+ T cells induced by IFN-gamma-stimulated synovial cells in the presence of SEB, indicating the importance of costimulatory molecules on synovial cells in activating CD4+ T cells. Our results suggest that CD4+ T cells are activated by synovial cells by an SEB-dependent manner and express FasL, inducing Fas-mediated apoptosis of the latter cells. These phenomena may regulate the overgrowth of synovial cells in RA synovium.     

Molecular and cellular mechanisms regulating T and B cell apoptosis through Fas/FasL interaction

Fas (CD95) and Fas ligand (FasL) are a receptor/ligand pair critically involved in lymphocyte homeostasis and peripheral tolerance such that genetic defect in either Fas or FasL results in an autoimmune lymphoproliferative syndrome. Fas is a type I transmembrane protein and a member of the tumor necrosis factor receptor (TNFR) family whereas FasL is a type II transmembrane protein and a member of TNF family. Binding of Fas by FasL induces apoptosis of the Fas-expressing cells. In the past few years, Fas/FasL interaction has been connected to a series of important phenomena previously viewed as independent immune processes. The activation-induced T cell death (AICD) and the FasL-mediated cytotoxicity by activated T cells are two critical mechanisms that can account for most of these phenomena. It is in the context of the two mechanisms that we discuss in this review the molecular and cellular events that occur during T/T and T/B interactions that account for the down-regulation of the immune response. We have also discussed recent advances in the areas of FasL gene regulation, lymphokine regulation of AICD, and regulation of B cell susceptibility to FasL. Investigation in these areas should help elucidate the role of Fas/FasL in the complex network of regulatory mechanisms that control immune response and autoimmunity.     

Molecular and Cellular Mechanisms RegulatingTand B Cell Apoptosis through Fas/FasL Interaction

Fas (CD95) and Fas ligand (FasL) are a receptor/ligand pair critically involved in lymphocyte homeostasis and peripheral tolerance such that genetic defect in either Fas or FasL results in an autoimmune lymphoproliferative syndrome. Fas is a type I transmembrane protein and a member of the tumor necrosis factor receptor (TNFR) family whereas FasL is a type II transmembrane protein and a member of TNF family. Binding of Fas by FasL induces apoptosis of the Fas-expressing cells. In the past few years. Fas/FasL interaction has been connected to a series of important phenomena previously viewed as independent immune processes. The activation-induced T cell death (AICD) and the FasL-mediated cytotoxicity by activated T cells are two critical mechanisms that can account for most of these phenomena. It is in the context of the two mechanisms that we discuss in this review the molecular and cellular events that occur during T/T and T/B interactions that account for the down-regulation of the immune response. We have also discussed recent advances in the areas of FasL gene regulation, lymphokine regulation of AICD, and regulation of B cell susceptibility to FasL. Investigation in these areas should help elucidate the role of Fas/FasL in the complex network of regulatory mechanisms that control immune response and autoimmunity.     

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.     

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.     

Involvement of LFA-1/ICAM-2 adhesive interactions and PKC in activation-induced cell death following SEB rechallenge.

Ligation of T-cell receptor (TCR) causes mature T cells to proliferate or, on re-exposure to antigen, can cause them to die by activation-induced cell death (AICD). In proliferative responses, costimulatory and adhesive interactions are required and activation of protein kinase C (PKC) has been shown to be essential. Whether or not interactions involving costimulatory signals and PKC have a role in facilitating AICD remains unclear. Here we have examined the role of CD28/B7 and leucocyte function associated antigen-1 (LFA-1)/intracellular adhesion molecule (ICAM) mediated interactions in AICD triggered by staphylococcal enterotoxin B (SEB) in murine lymph node T cells. We show that, after a primary proliferative response to SEB, LFA-1/ICAM-2 adhesive interactions can play a part in AICD following SEB rechallenge, while B7 and ICAM-1 mediated interactions are not essential for this process. In addition, using a highly selective PKC inhibitor, Ro31.8425, we show that PKC activation is essential for the regulation of AICD by SEB rechallenge.     

Role of mitogen-activated protein kinases in activation-induced apoptosis of T cells

A member of the mitogen-activated protein (MAP) kinase family, Jun N-terminal kinase (JNK), has been implicated in regulating apoptosis in various cell types. We have investigated the requirement for another type of MAP kinase, extracellular signal-regulated protein kinase (ERK) in activation-induced cell death (AICD) of T cells. AICD is the process by which recently activated T cells undergo apoptosis when restimulated through the T-cell antigen receptor. Here we show that both JNK and ERK are activated rapidly upon T-cell receptor (TCR) ligation prior to the onset of AICD. A chemical inhibitor of ERK activation, PD 098059, inhibits ERK activation and apoptosis, while JNK activation is not inhibited. This suggests that JNK activation is not sufficient for apoptosis. TCR cross-linking induces expression of the apoptosis-inducing factor, Fas ligand (FasL), and its expression correlates with ERK activation. In addition, apoptosis induced by direct ligation of the Fas receptor by anti-Fas antibody is not associated with ERK activation and is not inhibited by PD 098059. These data suggest that ERK activation is an early event during T-cell apoptosis induced by antigen-receptor ligation, and is not involved in apoptosis per se but in the expression of FasL. MAP kinase family members may be similarly involved in inducing apoptosis signals in other cell types.     

CD4+ T cell-mediated cytotoxicity toward thyrocytes: the importance of Fas/Fas ligand interaction inducing apoptosis of thyrocytes and the inhibitory effect of thyroid-stimulating hormone

The accumulation of activated CD4+ T cells and antigen (Ag)-dependent cellular interactions between thyrocytes and CD4+ T cells have been determined in thyroid gland from patients with Graves' disease. The Fas/Fas ligand (FasL) interaction between antigen-presenting cells and T cells regulates the apoptosis of the former cells triggered by the latter cells. The inhibition of Fas-mediated apoptosis in thyrocytes could be a underlying mechanism of hyperplasia of thyrocytes in patients with Graves' disease. We investigated the potential role of Fas/FasL interaction between thyrocytes and CD4+ T cells in the induction of Fas-mediated apoptosis of the former cells induced by the latter cells. The presence of only a few specific T cells responsive to a putative autoantigen has hampered the investigation of specific T cell activation toward antigen-presenting cells (APCs). Therefore, we used a superantigen, staphylococcal enterotoxin B (SEB), to examine specific T cell activation toward thyrocytes in vitro since it stimulates a large proportion of T cells with particular Vbeta elements. Spontaneous apoptosis of thyrocytes in culture was not found even in the presence of various kinds of cytokines. In contrast, a clear induction of Fas-mediated apoptosis by anti-Fas IgM was determined in interferon-gamma (IFN-gamma)-stimulated thyrocytes. In addition, a significant cytotoxicity of purified CD4+ T cells toward IFN-gamma-stimulated thyrocytes in the presence of SEB was induced, and the addition of anti-HLA-DR and -DQ monoclonal antibodies (mAbs) or blockade of the Fas/FasL interaction reduced this cytotoxicity. FasL expression of CD4+ T cells cocultured with IFN-gamma-stimulated thyrocytes in the presence of SEB was clearly induced. Furthermore, the addition of mAbs against CD54 and CD58 inhibited both cytotoxicity and FasL expression of CD4+ T cells. The cytotoxicity of CD4+ T cells toward IFN-gamma-stimulated, SEB-pulsed thyrocytes was markedly inhibited when we used thyrocytes cultured with IFN-gamma in the presence of thyroid-stimulating hormone (TSH) as target cells. Our results suggest that 1) CD4+ T cells were activated by thyrocytes expressing MHC class II molecules in an SEB-dependent manner and then expressed FasL. 2) These activated FasL+ CD4+ T cells killed thyrocytes by interacting with Fas on thyrocytes and FasL on activated CD4+ T cells. The presence of costimulating molecules such as CD54 and CD58 on thyrocytes was also necessary to generate activated FasL+ CD4+ T cells. 3) Since the actions of thyroid stimulating antibody (TSAb) toward thyrocytes are similar to those of TSH, one goitrogenic activity of TSAb may, in part, be due to the inhibitory effect on Fas-mediated apoptosis of thyrocytes triggered by activated CD4+ T cells.     

Activation-induced cell death in T cells

Summary: A properly functioning immune system is dependent on programmed cell death at virtually every stage of lymphocyte development and activity. This review addresses the phenomenon of activation‐induced cell death (AICD) in T lymphocytes, in which activation through the T‐cell receptor results in apoptosis. AICD can occur in a cell‐autonomous manner and is influenced by the nature of the initial T‐cell activation events. It plays essential roles in both central and peripheral deletion events involved in tolerance and homeostasis, although it is likely that different forms of AICD proceed via different mechanisms. For example, while AICD in peripheral T cells is often caused by the induction of expression of the death ligand, Fas ligand (CD95 ligand, FasL), it does not appear to be involved in AICD in thymocytes. This and other mechanisms of AICD are discussed. One emerging model that may complement other forms of AICD involves the inducible expression of FasL by nonlymphoid tissues in response to activated T lymphocytes. Induction of nonlymphoid FasL in this manner may serve as a sensing mechanism for immune cell infiltration, which contributes to peripheral deletion.     

Activation through CD40 ligation induces functional Fas ligand expression by Langerhans cells

Langerhans cells (LC) are professional antigen-presenting cells of dendritic cell (DC) lineage and are critical for the induction of primary immune responses in skin. Following antigenic stimulation, LC migrate to regional lymph nodes and induce antigen-specific activation of T cells. After primary expansion, the majority of T cells undergo Fas/Fas ligand (FasL)-mediated apoptotic cell death, thereby suppressing their excessive expansion. Although recent investigations have indicated an immunoregulatory function for DC, whether LC could be involved in Fas/FasL-mediated suppression of activated T cells is still unclear. In this study, we found that LC express FasL after activation triggered through CD40 molecules on their surface, but not by stimulation with LPS or IFN-gamma. The functional significance of FasL expression by LC was demonstrated using two different assays for apoptosis induced in Jurkat cells. The apoptosis in Jurkat cells was completely blocked by anti-FasL blocking antibody, suggesting a Fas/FasL-mediated mechanism. These results indicate a new feedback mechanism to down-regulate T cell activation by LC through the interaction of the TNF receptor/ligand superfamily, CD40/CD40L and Fas/FasL.     

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.     

Impaired apoptosis and immune senescence – cause or effect?

Summary: Aged animals and humans exhibit a decreased T‐cell activation response although they also exhibit increased susceptibility to responses to self‐antigens and a loss of self‐tolerance. The age‐related alteration in T‐cell reactivity, polyclonal expansion of T cells, and enhanced production of autoantibodies may reflect the numerous age‐associated alterations in the T‐cell arm of the immune system that have been revealed in numerous studies. These studies suggest that subpopulations of T cells are not deleted appropriately in older animals. They further suggest that an age‐related impairment of Fas/Fas ligand (FasL)‐mediated apoptosis – which plays a major role in activation‐induced cell death (AICD) of T cells – may contribute to compromised regulation of the immune system. The likely mechanisms that may lead to impaired induction of FasL in AICD senescent T cells include an age‐related shift from the apoptosis‐sensitive T‐helper 1 cell (Th1) response to the AICD‐resistant Th2 response, aberrant T‐cell receptor/CD3 downstream‐signaling pathways, and altered CD28/B7‐mediated T‐cell costimulatory signals. Pathologically, accumulation of AICD‐senescent T cells is associated with a defective cytotoxic T lymphocyte response and generation of autoreactive T cells. Based on the accumulating evidence, we propose that the emergence of the FasL^lo AICD‐senescent T cells is not only an effect of immune aging but also an important cause of T‐cell proliferative senescence in both humans and mice.     

The regulation of FasL expression during activation-induced cell death (AICD)

Activation-induced cell death (AICD), a Fas ligand (FasL)-dependent pathway, is important for maintaining T-cell homeostasis. Interleukin-2 (IL-2), an enhancer of AICD, can also enhance FasL expression. However, we show that the level of FasL or FLIP protein did not correlate with the susceptibility to AICD. Some T cells expressed high levels of FasL yet failed to undergo AICD, while others expressed little FasL and were sensitive. AICD susceptibility did not correlate with the kinetics of FasL up-regulation or down-regulation. The down-regulation of FasL can be mediated by a metalloprotease. However, we describe an alternative mechanism for the loss of FasL by endocytosis. Endocytosis inhibitors such as cytochalasins, sodium azide, deoxyglucose, or low temperatures prevented the loss of FasL. KB8301, a metalloprotease inhibitor had no effect on the loss of FasL or AICD in the T cells. Enhancing FasL expression was not crucial for AICD and the down-regulation of FasL proceeded via endocytosis.     

Involvement of APO2 ligand/TRAIL in activation-induced death of Jurkat and human peripheral blood T cells

The interaction of Fas with Fas ligand (FasL) mediates activation-induced cell death (AICD) of T hybridomas and of mature T lymphocytes. The TNF/TNF receptor system also plays a significant role in AICD of mature T cells and in the maintenance of peripheral tolerance. We previously demonstrated that in human Jurkat leukemia cells, AICD is triggered mainly by the rapid release of preformed FasL upon TCR stimulation. In the present work, we show that the cytotoxic cytokine APO2 ligand (APO2L; also known as TRAIL) is constitutively expressed as an intracytoplasmic protein in Jurkat T cells and derived sublines. APO2L is also detected in fresh human peripheral blood mononuclear cells (PBMC) from a significant number of donors, and the amount of both FasL and APO2L substantially increases upon blast generation. A neutralizing anti-APO2L monoclonal antibody (mAb) partially suppresses the cytotoxicity induced by supernatants of phytohemagglutinin (PHA)-prestimulated Jurkat or human PBMC on non-activated Jurkat cells, indicating that APO2L is released by these cells and contributes to AICD. A combination of neutralizing anti-APO2L and anti-Fas mAb blocks around 60 % of the toxicity associated with supernatants from PHA-activated human PBMC. These results show that FasL and APO2L account for the majority of cytotoxic activity released during AICD, and suggest that additional uncharacterized factors may also contribute to this process.     

Receptors and ligands that mediate activation-induced death of T cells

Conclusion The critical roles played by Fas/FasL and TNF/TNFR in AICD, peripheral T cell deletion and clonal downsizing have become increasingly apparent over the last few years. Consequently, new approaches have become possible for manipulation of T cell-mediated immune responses in either a positive or negative manner. For example, in disease states where excessive AICD involving FasL and TNF is observed there lies the possibility of intervention using specific inhibitors of these apoptotic pathways such as soluble forms of Fas and TNFR or neutralizing antibodies to TNF/TNFR or Fas/FasL interactions. Alternatively, the recent demonstration that the apoptotic pathways induced by FasL and TNF involve ICE-like caspases has revealed an opportunity to regulate apoptosis using inhibitors of this pathway. Indeed, in vivo treatment with a tripeptide inhibitor of ICE-like proteases (Z-VAD.fmk) protects mice from Fas-mediated hepatitis and death [53]. Thus, a multi-faceted approach to the treatment of depletion of T cells arising from HIV infection may in the future include not only inhibitors of viral replication but also inhibitors of host cell apoptosis. In addition, treatment of tumors that express FasL may be aided by use of an inhibitor of Fas to allow generation of an anti-tumor T cell response or, alternatively, T cells reactive with the tumor may be expanded in vitro in the presence of Fas and TNF antagonists for use in adoptive immunotherapy. In the area of transplantation biology, transfection of the tissue to be transplanted with the gene for FasL may in some cases help prevent rejection of the graft by host T cells, although recent studies have indicated that so-called immune privilege does not result from simple expression of FasL. Moreover, the ability to induce selective elimination of antigen-reactive T cells by immunization in immune privileged sites (ACAID) may allow for induction of tolerance to antigens implicated in autoimmune disease. Finally, the recent demonstration that monocytes may be stimulated via CD4 cross-linking or treatment with M-CSF to induce AICD in T cells raises the possibility of loading these cells with antigen to induce tolerance by adoptive immunotherapy. Again, a strategy such as this might be used for the treatment of autoimmune disease or in the prevention of allograft rejection. On a cautionary note, however, it has become clear from recent studies that we are not in a position at present to manipulate T cell-mediated immune responses at will. Several studies in which the gene for FasL was transfected into cells to induce a state of immune privilege produced the opposite of the predicted effect. Further understanding of the apoptotic pathways that control the expansion and survival of T cells will undoubtedly allow the development of novel strategies for treatment of infectious disease, cancer, immunodeficiency and autoimmune disease.