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.     

Apoptosis induced in T cells by human neuroblastoma cells: role of Fas ligand

The CD95/CD95L (Fas/Fas ligand) receptor/ligand system plays an important role in regulation of cell survival and induction of a programmed cell death. It is also involved in regulation of effector phase of T and NK cell cytotoxicity, establishment of immune privilege sites, and tumor escape from immune recognition. In this study, we assessed expression of CD95L in tumors obtained from patients with neuroblastoma (NB) and in established NB cell lines. We measured the presence of intratumoral T cell infiltrates and T cell survival in tumor tissue samples. High levels of apoptosis were observed in tumor-associated lymphocytes as well as in Jurkat T cells cocultured with NB cells in vitro. T cell death was reduced after treatment of NB cells (in vitro) with antibody to FAS ligand (FasL). Overall, our data suggest that NB-induced apoptosis of Fas-sensitive Jurkat T cells is mediated by functional FasL expressed on NB and Fas/FasL interaction may be responsible for the elimination of T cells in the NB microenvironment.     

Regulation of perforin-independent NK cell-mediated cytotoxicity

Natural killer (NK) cells have been thought to depend largely on perforin-mediated mechanisms for the induction of cell death in targets. However, this view has more recently been challenged. It is now clear that NK cells are capable of using death ligands like Fas ligand (FasL) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce cytotoxicity. Still, relatively little is known about the control of these "perforin-independent" cell death eliciting reactions, for example, the regulation of FasL expression on NK cells. In the present study, we confirm the ability of NK cells to mediate target cytotoxicity in the absence of perforin, in vivo and in vitro. We show that the induction of perforin-independent NK cell-mediated cell death is prevented by inhibiting signals mediated by MHC class I recognition. Furthermore, we demonstrate in vitro that cross-linking of the activation receptor NK1.1 on NK cells leads to the up-regulation of FasL on the cell surface. However, simultaneous engagement of an MHC class I binding inhibitory receptor prevents the externalization of FasL. These results provide a mechanistic explanation for theMHC class I-dependent regulation of perforin-independent cytotoxicity.     

Fas-induced apoptosis, and diseases caused by its abnormality

Homeostasis of multicellular organisms is controlled not only by proliferation and differentiation of cells but also by cell death. Physiological cell death mostly proceeds by apoptosis, a process which includes condensation and segmentation of the nuclei and cytoplasm, and often extensive fragmentation of chromosomal DNA into nucleosome units. Molecular and cellular characterization of Fas, a cell-surface protein recognized by cytotoxic monoclonal antibodies, has led to its definition as a receptor for a Fas ligand (FasL). FasL binds to Fas, which results in target cell apoptosis. A family of cysteine proteases seems to be sequentially activated during the Fas-mediated apoptosis. The phenotypes of the loss-of-function mutants of Fas and FasL indicate that this system is involved in T cell-mediated cytotoxicity, in down-regulation of immune responses, and probably in the turnover of senescent cells. Malfunction of the Fas system causes hyperplasia in peripheral lymphoid organs and the liver, accelerating autoimmune diseases and tumorigenesis, whereas exaggeration of the system leads to tissue disruption.     

B lymphocytes mediate Fas-dependent cytotoxicity in MRL/lpr mice

The Fas/Fas ligand (FasL) pathway is one of the two major effector mechanisms of T cell-mediated cytotoxicity. To prevent nonspecific killing by lymphoid cells, FasL expression on the cell surface of immune effector cells is strictly regulated. However, MRL/lpr autoimmune-prone mice massively overexpress FasL on their T lymphocytes, which render them able to kill Fas+ targets in vitro and in vivo. It is surprising that we show in the present work that B lymphocytes purified from MRL/lpr spleen cells express FasL to the same extent as T cells at the mRNA and protein level. These B cells are potent cytotoxic effectors against Fas+ but not Fas- targets. The B lymphocyte effectors were used ex vivo without any in vitro activation by B cell stimuli. Furthermore, we found that MRL/lpr B lymphocytes have the same cytotoxic potential as natural killer cells, which have been characterized as potent, Fas-mediated, cytotoxic effectors. The level of membrane-anchored FasL increases with the size of the B cell and cell-surface activation marker CD69 expression, indicating that the expression of FasL is up-regulated in parallel with the activation state of the B cell. The activated B cell population contained the major cytotoxic activity, and a minor part was associated with CD138/Syndecan-1+ plasma cells.     

Malignant counterpart of myeloid dendritic cell (DC) belonging to acute myelogenous leukemia (AML) exhibits a dichotomous immunoregulatory potential

Dendritic cells (DCs) play a central role in immune regulation. Some leukemic cells are argued to be malignant counterparts of DC because of their ability to differentiate into leukemic DC. We characterize DC-like leukemia homogenously expressing CD11c+CD86+ in acute myelogenous leukemia patients. They express the Wilms' tumor-1 antigen and common DC phenotypes (i.e., fascin+, CD83+, and DR+) directly. Purified leukemic cells produce interleukin-12 (IL-12) simultaneously with Fas ligand (FasL) and IL-6, which may suppress T cell-mediated immunity. These cells can elicit strong allogeneic T cell responses as well as induce tumor-specific CD8+ cytotoxic T cells, suggesting that they effectively present tumor-associated antigens. In contrast, they drive primary T cells toward apoptosis mediated in a tumor-specific way by a Fas-FasL interaction. Taken together, DC-like leukemia uniquely influences immune surveillance in contradictory ways, some of which may be involved in the mechanism of immune escape.     

Regulation of FasL expression in natural killer cells

Fas ligand (FasL)-mediated cytotoxicity is initiated in natural killer (NK) cells through ligation of their activating receptors. The CD16 receptor has been shown to induce FasL expression and cytotoxicity in NK cells. In this study, we made the novel observation that FasL expression was upregulated in NKL cells stimulated through 2B4 and LFA-1 activating receptors, implying a role for FasL-mediated cytotoxicity early in the immune response. Coligation with CD94/NKG2A human leukocyte antigen (HLA) class I inhibitory receptor did not block the induced FasL expression; therefore, these opposing pathways appear to function independently. We also showed, however, that FasL-mediated cytotoxicity was downregulated in CD94/NKG2A-expressing LAK cells in response to the HLA-E ligand, suggesting a mechanism by which aberrant cells expressing class I may evade FasL-mediated cytotoxicity. Thus we show for the first time that 2B4, LFA-1, and CD94/NKG2A receptors are involved in modulating FasL expression and, therefore, cytotoxicity mediated by NK 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.     

The multiple roles of Fas ligand in the pathogenesis of infectious diseases

Fas ligand (FasL) is a type II transmembrane protein that plays a critical role in immune homeostasis by binding to its receptor Fas (CD95) and inducing apoptosis. Fas/FasL dysregulation contributes to infectious disease pathogenesis. Microorganisms may inhibit Fas signal transduction to prolong intracellular survival and prevent killing by immune effector cells. FasL may be upregulated in directly infected cells to enhance killing of responding immune cells and facilitate immune evasion. The host response to infection may aim to induce apoptosis in directly infected cells, but immune cells that target directly infected cells can induce Fas-mediated apoptosis of uninfected bystander cells. FasL also contributes to the generation and regulation of the inflammatory response in infection. The multiple roles of FasL in infectious disease pathogenesis are discussed in the context of viral, bacterial and parasitic infections.     

Interferon-alpha (IFN-alpha) enhances cytotoxicity in healthy volunteers and chronic hepatitis C infection mainly by the perforin pathway.

Cell-mediated cytotoxicity is exerted via perforin and Fas ligand (FasL). We have recently shown that IFN-alpha up-regulates FasL expression in T cells isolated from healthy volunteers and augments activation-induced T cell death. Since the Fas/FasL system is implicated in the pathogenesis of hepatic failure and both molecules have been shown to be up-regulated in hepatitis C virus (HCV) infection, we studied whether cytotoxicity via the FasL system is enhanced by IFN-alpha and therefore could contribute to hepatic injury. We investigated FasL and perforin expression in peripheral blood mononuclear cells (PBMC) derived from HCV+ donors by Northern analysis and soluble FasL synthesis by ELISA. Natural killer (NK) cell and cytotoxic T lymphocyte (CTL) cytotoxicity was studied by 51Cr-release assays. IFN-alpha up-regulates FasL mRNA and protein synthesis in mitogen-activated PBMC of HCV+ individuals, as previously found in healthy subjects. Stimulation with IFN-alpha increases perforin mRNA levels in PBMC. In NK cytotoxicity assays, the enhancement of cytotoxicity by IFN-alpha is mainly due to the perforin pathway, while the FasL pathway plays only a minor role. In CTL cytotoxicity experiments neither the FasL nor the perforin pathway is further enhanced by IFN-alpha. Our data suggest that up-regulation of perforin by IFN-alpha results in elevated cytotoxicity, suggesting that IFN-alpha might support elimination of virally infected cells via this pathway. In contrast, the major effect of IFN-alpha on the Fas/FasL system might be the enhanced elimination of activated T cells as a means of finally limiting a T cell response.     

Development of real time PCR assays for the quantification of Fas and FasL mRNA levels in lymphocytes: studies on centenarians

Apoptosis plays a central role in the homeostasis of the immune system. During aging, there is an altered regulation of pivotal molecules that are responsible for the regulation of this type of cell death, such as those of the Fas/FasL system. Understanding the regulation of these genes can help to clarify, at least in part, the age-related changes that occur in immune cells. We have developed an original real time PCR assay for quantification of mRNA for Fas and FasL, and have studied a group of young donors, middle aged subjects and centenarians. We have found that the production of Fas mRNA is greatly increased in resting lymphocytes from centenarians; such an increase follows an age-related trend. On the contrary, the production of mRNA for the molecule, which is the natural ligand of Fas, i.e. FasL, is consistently reduced. Our preliminary results suggest that during aging a subtle balance in the production of molecules that cause apoptosis could exist, and that, in order to avoid an excessive death of immune cells, a still unknown mechanism could compensate the increase of Fas with the reduction of FasL.     

Effector cells derived from host CD8 memory T cells mediate rapid resistance against minor histocompatibility antigen-mismatched allogeneic marrow grafts without participation of perforin, Fas ligand, and the simultaneous inhibition of 3 tumor necrosis factor family effector pathways.

Reduced-intensity conditioning regimens for transplant recipients have heightened awareness of immunologic resistance to allogeneic bone marrow transplants (BMT). Although T cell-mediated cytotoxicity has been assumed to play a role in the resistance against donor allogeneic hematopoietic stem and progenitor cell grafts, several studies have reported relatively unimpaired resistance by recipients who lack perforin, Fas ligand (FasL), and other cytotoxic mediators. This study compared the early kinetics of T cell-mediated resistance in B6 (H2b) cytotoxically normal versus deficient recipients after transplantation with major histocompatibility complex-matched, minor histocompatibility antigen (MiHA)-mismatched allogeneic marrow grafts. Wild-type B6 or cytotoxic double-deficient perforin-/-/gld+/+ (B6-cdd) mice were sensitized against major histocompatibility complex-matched BALB.B or C3H.SW (H2b) MiHA and transplanted with a high dose (1 x 10(7)) of T cell-depleted bone marrow. CD8 T memory cells were shown to be present in recipients before BMT, and anti-CD8 monoclonal antibody infusion abolished resistance, thus demonstrating that CD8 T cells are the host effector population. Donor-committed and high proliferative potential progenitor numbers were markedly diminished by 48 hours after transplantation in both wild-type B6 and B6-cdd anti-donor MiHA-sensitized recipients. These observations indicate that the resistance pathway used in the cytotoxic deficient mice was both potent and rapidly induced--consistent with a CD8 memory T-cell response. To examine the role of Tumor necrosis factor-like weak inducer of apoptosis (TWEAK)- and TL1A-mediated cytotoxicity in this strong resistance, newly generated monoclonal antibodies specific for these ligands were administered to B6-cdd recipients sensitized to donor antigens. Recipients of syngeneic B6-gfp bone marrow exhibited significant donor colony-forming unit numbers after BMT. In contrast, low or absent colony-forming unit levels were detected in allogeneic recipients, including those that lacked perforin and FasL and that received anti-TWEAK, anti-tumor necrosis factor-related apoptosis-inducing ligand, and anti-TL1A monoclonal antibodies. These findings extend previous observations by demonstrating the existence of a rapidly effected resistance pathway mediated by memory CD8 effector T cells independent of the 2 major pathways of cytotoxicity. Together with previous findings, these results support the notion that effector cells derived from memory CD8 T-cell populations can mediate strong resistance against donor allogeneic MiHA-disparate hematopoietic engraftment by using a mechanism that is independent of the contribution of perforin, FasL, and the known death ligand receptor pathways.     

Induction of FAS ligand expression in a human hepatoblastoma cell line by HCV core protein

Tumour cells and virus infected cells expressing Fas ligand (FasL) can evade immune surveillance by inducing apoptosis in T cells expressing Fas. In order to characterise a possible role of hepatitis C virus (HCV) core protein in similar mechanisms during HCV infection, we investigated Fas ligand expression and activity in a human hepatoblastoma cell line (HepG2) constitutively expressing this protein.Strong FasL induction was detected by immunoblotting and flow cytometry analysis in the core expressing cell lines Hep39. In contrast, vector transfected cells or cell lines expressing HCV E1-E2 proteins did not show FasL expression. Co-cultivation experiments of Hep39 cells with a Fas-sensitive T cell line indicated that FasL induced by the core protein had apoptotic activity toward target cells. Effect of the core protein on induction of FasL promoter was further examined by co-transfection of HepG2 cells with core-bearing plasmid and a vector in which luciferase gene expression is driven by human FasL promoter. Results of the luciferase assay indicated a positive regulation of FasL promoter by the core protein. In conclusion, HCV core protein plays a role in the induction of functional FasL in hepatoblastoma cell line and apoptosis in a target T cell line expressing Fas. Similar mechanisms may contribute, in vivo, to establishment of chronic infection and development of hepatocellular carcinoma (HCC).     

Comparison of Fas- versus perforin-mediated pathways of cytotoxicity in TCR- and Thy-1-activated murine T cells

T cell-mediated cytotoxicity can be triggered by cross-linking of TCR or Thy-1 surface proteins. While the TCR-triggered signaling initiates both perforin- and Fas ligand (FasL)-Fas-mediated mechanisms of cytotoxicity, it was not clear which mechanism was utilized by Thy-1-triggered signals and which pathway of cytotoxicity was triggered at low levels of antigen expression. It is shown that glycophosphatidylinositol-linked surface glycoprotein Thy-1 preferentially activates FasL-Fas- but not perforin-mediated cytotoxicity. This is explained by the lesser intensity of Thy-1-mediated signaling in T cells. The data suggest that Thy-1-triggered Fas-mediated cytotoxicity is completely dependent on cross-talk between Thy-1 and TCR signals since mutations in TCR-CD3 complex molecules or inhibition of tyrosine kinases or calcineurin abolished or strongly inhibited Thy-1-triggered FasL-Fas-mediated cytotoxicity. Lower concentrations of antigenic peptide or levels of cross-linking with anti-TCR-CD3 mAb are required to trigger Fas-mediated than perforin-mediated cytotoxicity by different cytotoxic T lymphocyte (CTL) lines and clones, and it is shown that cross-linking of Thy-1 is much less efficient in triggering accumulation of second messengers (intracellular Ca(2+)) than cross-linking of TCR on CTL. Taken together, these data reflect the possibility of differential activation of FasL and/or perforin pathways of cytotoxicity depending on the nature of activating stimuli and surface receptor.     

Nasal T/NK cell lymphomas commonly express perforin and Fas ligand: important mediators of tissue damage

 

Aims:

Two molecular mechanisms of T/natural killer (NK) cell-mediated cytotoxicity, one perforin based and the other Fas based, have been demonstrated, and both systems induce cytotoxicity in the target cells. The Fas-based mechanism involves the transducing molecule Fas and its ligand (FasL). In addition, perforin and/or FasL are also expressed in the cytotoxic T/NK cells. This study was thus designed to investigate the Fas and perforin pathways of the cytotoxic T/NK lymphoma cells in the nasal cavity.  

Methods and results:

Eight patients with nasal lymphoma were analysed using immunohistochemical staining methods. Two cases were CD3+ CD56+ (T/NK cell) type, and six were CD3− CD56+ (NK cell) type. All cases showed Epstein–Barr virus genomes by in-situ hybridization. In addition, all cases showed the expression of TIA-1 (GMP-17), which is a marker of cytotoxic T and NK cells. FasL was expressed in the majority of the lymphoma cells and some histiocytes, while Fas was found in lymphoma cells and many non-neoplastic cells. In addition, the expression of perforin was detected in almost all lymphoma cells. In the double stainings, lymphoma cells expressed both FasL and perforin. Based on these findings, both the perforin- and Fas-based pathway of the cytotoxic T/NK lymphoma cells are thus considered to play an important role in the clinical features.  

Conclusions:

Tissue damage is a common morphological feature in nasal T/NK cell lymphoma. The above findings therefore support the theory that tissue damage is due to both the cytotoxicity of T/NK lymphoma cells as well as to angiocentricity.     

Fas (CD95)/Fas ligand interactions regulate antigen-specific,major histocompatibility complex-restricted T/B cell proliferative responses

The effect of Fas ligand (FasL) cytotoxicity on T/B collaboration was examined in vitro using cloned T helper 1 cells and antigen-pulsed, activated B cells. We compared antigen-pulsed B cells that had been activated through different membrane receptors (IgM, CD14 and CD40) for their ability to induce T cell proliferation and to respond to T cell help. We also used a Fas-Ig fusion protein, an inhibitor of FasL-mediated cytotoxicity, to determine the effect of FasL cytotoxicity on the T and B cell proliferative responses. The data show that the extent of both T and B cell proliferative responses correlate with the relative resistance of activated B cell populations to FasL cytotoxicity. Moreover, both T and B cell proliferation could be enhanced by Fas-Ig. Our results demonstrate that FasL cytotoxicity is a negative regulatory mechanism for both T and B cell proliferative responses and that Fas-Ig can be an immunopotentiating agent for both T and B cell immunity.     

Combination of Human Fas （CD95/Apo-1） Ligand with Adriamycin Significantly Enhances the Efficacy of Antitumor Response

The prognosis of hepatocellular carcinoma （HCC） is poor, even with the combined treatment of curative resection and adjuvant chemoradiotherapy. To solve this problem, many biologic therapies have been investigated. Fas ligand （FasL, CD95L） is mainly expressed in activated T lymphocytes and natural killer （NK） cells, and plays a central role in both cell-mediated immunity and immune downregulation. Several studies have shown that FasL is expressed in HCC. In the present report, we prepared recombinant human pET-22b（＋）/FasL protein and investigated the effect of FasL on HCC cells in vitro and on tumor growth in a murine HCC tumor model. The well-known cytotoxic chemotherapeutic reagent adriamycin （ADM） served as a control. We found that FasL effectively suppressed the viability of H22 tumor cells and significantly induced the apoptosis of H22 cells. The apoptotic levels of cells treated with FasL-ADM were significantly higher than those treated with FasL or ADM alone, and the FasL-ADM combination resulted in a more than additive effect on tumor growth delay in this model. The results suggested that combined treatment of FasL and other chemotherapeutic agents may be a new approach to improve the efficacy of chemotherapy for HCC.     

IFNalpha2b stimulated release of IFNgamma differentially regulates T cell and NK cell mediated tumor cell cytotoxicity

Interferonalpha2b (IFNalpha2b) augments the suppressed immune functions and peripheral blood mononuclear cell (PBMC) cytotoxicity of head and neck squamous cell carcinoma (HNSCC) patients by differential regulation of IFNgamma, a pleotropic Th1 cytokine. In the present communication, we have examined the role of IFNgamma in IFNalpha2b initiated T and NK cell mediated cytotoxicity of tumor cells. IFNalpha2b activates both T and NK cells to release IFNgamma. IFNgamma plays a crucial role in enhancing tumor cell cytotoxicity by T cells, but not by NK cells, as evidenced by killing of a oral (KB) and breast (MCF7) cancer cells, without affecting the killing of NK sensitive erythroleukemic K562 cells by IFNalpha2b activated PBMC. IFNalpha2b driven tumor cell cytotoxicity is related to the rectification of the downregulated expression of cytotoxic molecules, perforin, granzyme B and FasL in CD8+ T and CD56+ NK cells. Expression of IFNalpha2b mediated perforin and granzyme B is dependent on IFNgamma in T cells, but not in NK cells. However, expression of FasL in both T and NK cells is not dependent on IFNgamma. In conclusion, IFNalpha2b enhances suppressed T cell cytotoxicity of HNSCC patients by stimulating perforin-granzyme B system, which is IFNgamma dependent. IFNalpha2b also induces the expression of perforin-granzyme B system in NK cells, but this NK mediated cytotoxicity is IFNgamma independent.