Regulation of Fas(Apo-1/CD95)- and perforinmediated lytic pathways of primary cytotoxic T lymphocytes by the protooncogene bcl-2

Cytotoxic T cells (CTL) induce cell death of their target cells either by the surface interaction between Fas ligand and Fas or by the release of perforin and granzymes. Both lytic pathways induce apoptosis yet it is not known whether identical or distinct apoptotic pathways are activated. The protooncogene bcl-2 is known to protect various hematopoietic cells from apoptosis induced by diverse agents. Here we show that overexpression of the Bcl-2 protein in the murine mastocytoma line P815 or in concanavalin A-activated splenocytes suppresses apoptotic cell death induced by allospecific primary cytotoxic T lymphocytes (CTL) in which only the Fas lytic pathway was functional. Bcl-2 also reduced target cell killing induced by CTL whose lytic activity was dependent on the perforin/granzyme pathway only. These data provide evidence that, in the target cells studied here, both perforin/granzyme and Fas apoptotic pathways are modulated by Bcl-2 and suggest that these two pathways converge at a step prior to Bcl-2 inhibition.     

Switch from perforin-expressing to perforin-deficient CD8+ T cells accounts for two distinct types of effector cytotoxic T lymphocytes in vivo

Although CD8⁺ cytotoxic T lymphocytes (CTL) exhibit both Fas ligand (FasL) -based and perforin-based lytic activities, the accepted hallmark of a fully active CTL remains its perforin killing machinery. Yet the origin, rationale for possessing both a slow-acting (FasL) and a fast-acting (perforin) killing mechanism has remained enigmatic. Here we have investigated perforin expression in CTL directly involved in acute tumour (i.e. leukaemias EL4 and L1210) allograft rejection occurring within the peritoneal cavity. We show that at the height of the immune response, the majority of conjugate-forming CD8⁺ CTL express high levels of perforin messenger RNA and protein, and kill essentially via perforin. Later however, coinciding with complete rejection, fully cytocidal CTL emerge which exhibit a stark decrease in perforin and now kill preferentially via constitutively expressed FasL. Although late in emergence, and persistent, these powerful CTL are neither effector-memory nor memory CTL. This finding has implications for the monitoring of anti-transplant responses in clinical settings, based on assessing perforin expression in graft infiltrating CD8⁺ T cells. The results show that as the immune response progresses in vivo, targeted cellular suicide mainly prunes high perforin-expressing CD8⁺ cells, resulting in the gradual switch in effector CTL, from mostly perforin-based to largely Fas/FasL-based killers. Hence, two kinds of CD8⁺ CTL have two killing strategies.     

Independent roles of perforin, granzymes, and Fas in the control of Friend retrovirus infection

Cytotoxic T-cells (CTL) play a central role in the recovery of mammalian hosts from retroviral infections. However, the molecular pathways that mediate the antiretroviral activity of CTL are still elusive. Here we explore the protective role of the two main cytolytic pathways of CTL, that is, granule exocytosis and Fas/Fas ligand (FasL), in acute and persistent Friend retrovirus (FV) infection of mice. For this purpose, we have used mutant mouse strains with targeted gene defects in one or more components of the two cytolytic pathways including perforin, granzyme A, granzyme B, Fas, and FasL. The important function of CTL in resistance of C57BL/6 (B6) mice to FV is emphasized by the finding that depletion of CD8+ T-cells prior to virus infection resulted in severe splenomegaly and high viral loads in blood and spleen tissue. Analysis of primary FV infection in knockout mice revealed that acute infection was readily controlled in the absence of functional Fas. Most notably in the presence of Fas/FasL each of the three effector molecules of the exocytosis pathway (i.e., perforin, granzyme A, and granzyme B) was capable on its own to mediate suppression of virus replication and protection from leukemia. However, triple knockout mice lacking perforin and the two granzymes were fully susceptible to FV-induced leukemia. In contrast to acute infection the Fas/FasL pathway was mandatory for effective control of FV replication during persistent infection. These findings suggest novel pathways of CTL-mediated viral defense and contribute towards a better understanding of the molecular mechanisms of CTL activity in retroviral infections.     

Concerted action of perforin and granzymes is critical for the elimination of Trypanosoma cruzi from mouse tissues,but prevention of early host death is in addition dependent on the FasL/Fas pathway

CTL and NK cells are critical for resistance to acute Trypanosoma cruzi infection, but are also implicated in the pathology induced by this intracellular protozoan parasite. Here we explore to what extent the two main cytolytic pathways of CTL and NK cells, i.e. the granule exocytosis and the Fas ligand (FasL)/Fas pathways, are responsible for the elimination of parasites from mouse tissues and control of organ pathology. For this purpose we have employed mouse strains with targeted gene defects in one or more components - including perforin, granzyme A and granzyme B, and Fas - of either of the two cytolytic pathways, and we used the highly pathogenic T. cruzi strain Tulahuen. We show that parasites are effectively cleared from infected tissues independently of the FasL/Fas pathway by the concerted action of perforin and the two granzymes. However, prevention of pathology and early host death is critically dependent in addition on an operational FasL/Fas interaction. Thus, in contrast to C57BL/6 (B6) wild-type mice, mouse strains with deficiencies in either the FasL/Fas or the perforin/granzyme pathway similarly suffer from early death, independently of their differential capacity to control parasite growth; this finding indicates that the two cytolytic pathways control distinct but vital processes during infection with T. cruzi.     

Perforin and Fas/Fas ligand-mediated cytotoxicity in acute and chronic woodchuck viral hepatitis.

The Fas ligand (FasL)/Fas and the perforin-granzyme cytotoxic pathways presumably play a central role in the development of hepatocellular injury in viral hepatitis. To recognize the potential contribution of FasL and perforin-based cell killing in hepadnaviral infection, we adopted a cytotoxic assay using murine Fas+ P815 and human Fas- K562 cells as targets. Freshly isolated peripheral blood mononuclear cells (PBMC) from woodchucks with newly acquired woodchuck hepatitis virus (WHV) infection (n = 6), with chronic WHV hepatitis (n = 9), and from healthy animals (n = 11) were used as effector cells. We have found that woodchuck lymphoid cells kill cell targets via both the FasL/Fas and the perforin death pathways. The contribution of Fas-dependent cytolysis was ascertained in blocking experiments with anti-Fas antibody and by incubation of PBMC with cyclohexamide to prevent de novo synthesis of FasL. The involvement of the perforin pathway was confirmed by treatment of K562 cells with colchicine to inhibit the microtubule-dependent perforin release. Comparative analysis showed that peripheral lymphoid cells from acute WHV hepatitis, but not those from chronic WHV infection, are more cytotoxic and that this increase seems to be entirely due to activation of perforin-mediated killing. The data indicate that acute infection in woodchucks is associated with the augmented capacity of lymphoid cells to elicit perforin-dependent killing, but in chronic infection, independent of the severity of liver disease and duration of chronicity, these cells have the same or lower cytotoxic potential as PBMC from healthy controls. These findings suggest a role for non-specific cellular immunity, presumably natural killer (NK) cells, in the control of early WHV infection and in the progression of chronic hepatitis.     

CD95 (Fas) ligand expression of Epstein-Barr virus (EBV)-infected lymphocytes: a possible mechanism of immune evasion in chronic active EBV infection

The Epstein-Barr virus (EBV) induces infectious mononucleosis (IM) and can be associated with chronic active EBV infection (CAEBV). Cytotoxic T lymphocytes (CTL) play an important role in excluding EBV-infected cells. Two cytotoxic mechanisms of CTL have been demonstrated: one perforin/granzyme-based and the other Fas (CD95)/Fas ligand (FasL)-based. To clarify these two pathways in CAEBV, we analyzed six patients with CAEBV and four patients with IM using immunohistochemical staining of the lymph nodes. In both CAEBV and IM, CD8⁺ T-cells increased in number, but CD56⁺ natural killer cells were rare. In four of six cases with CAEBV, approximately half the lymphocytes were positive for T cell-restricted intracellular antigens (TIA-1), which were recognized by the cytolytic granules of CTL. In IM, the number of TIA-1 positive cells was smaller than that in CAEBV. Fas-positive lymphocytes were frequently encountered in both CAEBV and IM. However, FasL-positive lymphocytes increased in three of six patients with CAEBV, but not in patients with IM. Except for one case with CAEBV, the number of perforin- and/or granzyme-positive cells was small in number in both CAEBV and IM cases. In double-staining FasL and EBV in situ hybridization, FasL-positive EBV-infected lymphocytes were detected in CAEBV but not in IM. In CAEBV, the Fas/FasL pathway and not perforin pathways appears to play an important role in the pathogenesis. The data suggest that EBV-infected lymphocytes may evade immune attack through the expression of FasL.     

Progression of spontaneous autoimmune diabetes is associated with a switch in the killing mechanism used by autoreactive CTL

Autoimmune (type 1) diabetes mellitus results from the destruction of insulin-producing pancreatic beta-cells by T lymphocytes. Beta-cell death that is induced by autoreactive CTL in diabetes involves both Fas/Fas ligand (FasL)- and perforin/granzyme-mediated pathways, although their relative contributions during the progression of the disease remain unknown. We demonstrate here that despite the preferential use of the Fas/FasL pathway for cytolysis of beta-cell targets, transgenic beta-cell-specific CTL were able to kill targets via the perforin pathway when triggered by a higher affinity stimulus. In addition, we show that the killing mechanism used by islet-associated CD8(+) T cells from non-obese diabetic mice changed as the mice aged and correspondingly, with the stage of diabetes. These results provide direct evidence for age-related changes in the cytotoxic pathways used by diabetogenic T cells during the progression of autoimmune diabetes.     

Perforin and Fas induced by IFNgamma and TNFalpha mediate beta cell death by OT-I CTL

Direct interaction between auto-reactive CTL and specific peptide-MHC class I complexes on pancreatic beta cells is critical in mediating beta cell destruction in type I diabetes. We used mice with genetic modifications in three major pathways used by CTL, perforin, Fas and pro-inflammatory cytokines to assess the relative contribution of these mechanisms to beta cell death. In vitro-activated ovalbumin (OVA)-specific CTL, from OT-I TCR-transgenic mice, specifically killed transgenic beta cells expressing OVA (from RIP-mOVA mice) in a 16-h cytotoxicity assay. Perforin-deficient CTL had a reduced ability to kill OVA-expressing islets in vitro (22.1 +/- 3.8%) compared with wild-type CTL (71.4 +/- 4.6%). Fas-deficient islets were only slightly protected from wild-type CTL but were completely protected from the residual killing observed with perforin-deficient CTL. Residual cytotoxicity in perforin-deficient CTL was also prevented by overexpression of SOCS-1, which blocks multiple cytokine signaling pathways. It was also prevented by pre-incubation with anti-tumor necrosis factor-alpha (anti-TNFalpha) antibody or by blocking IFNgamma responsiveness through expressing a dominant negative IFNgamma receptor. Perforin-deficient CTL produced IFNgamma and TNFalpha that was shown to directly induce islet Fas expression during the assays. This suggests that Fas-deficiency, SOCS-1 overexpression and blockade of IFNgamma and TNFalpha all protect beta cells from residual cytotoxicity of perforin-deficient CTL by blocking Fas upregulation. These findings indicate that wild-type CTL destroy antigen-expressing islets via a perforin-dependent mechanism. However, in the absence of perforin, the Fas/FasL pathway provides an alternative mechanism dependent on islet cell Fas upregulation by cytokines IFNgamma and TNFalpha.     

FD-891, a structural analogue of concanamycin A that does not affect vacuolar acidification or perforin activity, yet potently prevents cytotoxic T lymphocyte-mediated cytotoxicity through the blockage of conjugate formation

FD-891 belongs to a group of 18-membered macrolides, and is a structural analogue of a specific inhibitor of vacuolar type H+-ATPase, concanamycin A (CMA). In our previous work, we have shown that CMA specifically inhibits perforin-dependent cytotoxic T lymphocyte (CTL)-mediated cytotoxicity through the degradation and inactivation of perforin, although CMA does not affect Fas ligand (FasL)-dependent cytotoxicity. Here, we show that FD-891 potently prevents not only perforin-dependent but also FasL-dependent CTL-mediated killing pathways by blocking CTL-target conjugate formation. In contrast to CMA, FD-891 was unable to inhibit vacuolar acidification and only slightly decreased the perforin activity in lytic granules. FD-891 blocked granule exocytosis in response to anti-CD3, mainly owing to the lack of CTL binding to immobilized anti-CD3. The conjugate formation was markedly inhibited only when effector cells were pretreated with FD-891. Consistent with these observations, fluorescence-activated cell sorter (FACS) analysis for cell surface receptors revealed that FD-891 significantly reduced the expression of the T-cell receptor (TCR)/CD3 complex. These data suggest that the blockage of conjugate formation and subsequent target cell killing might be at least partly owing to FD-891-induced down-regulation of the TCR/CD3 complex.     

Three-color flow cytometric assay for the study of the mechanisms of cell-mediated cytotoxicity

Cytotoxic lymphocytes kill tumor or virus-infected target cells utilizing two mechanisms (1) release of lytic granules (containing perforin and granzymes) and (2) Fas ligand (FasL)/Fas or TNF initiated apoptosis. We have examined mechanisms of target cell lysis using a new Flow Cytometric Cytotoxicity Assay (FC Assay). Target cells were labeled with PKH 67 dye. Cell death was estimated by 7-amino-actinomycin (7-AAD) inclusion and annexin V-PE binding. A strong direct correlation has been found between the percentage of dead target cells in the FC Assay and the results of 51Cr release assay when human LAK and CTL were used as a model system. We have shown that both NK and CTL kill tumor cells mostly by granule-mediated mechanisms, as lysis was blocked by a perforin inhibitor Concanamycin A (Folimycin) but was significantly less sensitive to zVAD-FMK caspase inhibition. The FC assay allows accurate measurement of cell-mediated cytotoxicity as individual target cell death is detected directly.     

Serial killing by cytotoxic T lymphocytes: T cell receptor triggers degranulation,re-filling of the lytic granules and secretion of lytic proteins via a non-granule pathway

CD8⁺ cytotoxic T lymphocyte (CTL) clones begin to synthesize the lytic proteins granzyme A, granzyme B and perforin after stimulation with allogeneic target cells. The lytic proteins are stored in the secretory granules which are released after cross-linking of the T cell receptor (TcR) upon target cell recognition. During lytic granule biogenesis granzyme A protein synthesis can be detected between 2 and 10 days after allogeneic stimulation of the CTL. Although granzyme A is stored in the lytic granules over this period, the majority of granzyme A synthesized is secreted directly from the CTL. TcR triggering of degranulation also results in new synthesis of the lytic proteins, which can be inhibited by cycloheximide (CHX). Some of the newly synthesized lytic proteins can be stored in the cell and refill the granules. But up to one third of granzymes A and B can be secreted directly from the CTL via the constitutive secretory pathway as shown by granzyme A enzymatic activity and immunoblots of secreted granzyme B, where one third of the protein fails to acquire the granule targeting signal. Perforin is also secreted via the constitutive pathway, both from the natural killer cell line, YT, and from CTL clones after TcR cross-linking. Constitutive secretion of the lytic proteins can be blocked by both CHX and brefeldin A (BFA). While BFA does not affect the directional killing of recognized targets, it abrogates bystander killing, indicating that bystander killing arises from newly synthesized lytic proteins delivered via a non-granule route. These results demonstrate that the perforin/granzyme-mediated lytic pathway can be maintained while CTL kill multiple targets. We show that CTL not only re-fill their granules during killing, but also secrete lytic proteins via a non-granule-mediated pathway.     

Mechanism of lymphocyte-mediated cytolysis: functional cytolytic T cells lacking perforin and granzymes.

Involvement of the lytic protein perforin (c. 65,000 MW) and of granule proteases (granzymes) in cell lysis induced by cytolytic T lymphocytes (CTL) has been suggested, but is still controversial. For example, in vivo-primed peritoneal exudate CTL (PEL) have been found to express perforin and granzyme activity in amounts comparable to those found in non-lytic lymphocytes, although PEL are the most potent of all CTL. Exploiting several cloned CTL hybridomas developed in this laboratory and newly available molecular probes for detecting perforin, granzymes, protein and mRNA, we now directly demonstrate killer T lymphocytes which kill effectively and specifically, but are free from perforin, lytic granules and granzymes, all three of which have been postulated to be involved in lymphocyte-mediated killing. The CTL hybridomas are completely devoid of perforin and granzymes prior to, during, and after activation by antigen, mitogen or interleukin-2 (IL-2). The induction of lytic granules, perforin, and granzymes in the in vivo-primed PEL, but not in the cloned CTL hybridomas, upon cultivation in IL-2, further suggests the involvement of these constituents in antigen/lymphokine-induced CTL activation and differentiation rather than directly in their cytocidal activity. Together, these findings support a perforin- and granzyme-independent CTL lytic mechanism.     

Fas and Fas ligand: lpr and gld mutations

Fas ligand (FasL) is a death factor that binds to its receptor, Fas, and induces apoptosis. Two mutations that accelerate autoimmune disease, lpr and gld, are known to correspond to mutations within genes encoding Fas and FasL, respectively. Here, Shigekazu Nagata and Takashi Suda summarize current knowledge of Fas and FasL, and discuss the physiological role of the Fas system in T-cell development, cytotoxicity and cytotoxic T lymphocyte (CTL)-mediated autoimmune disease.     

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.     

Expression of Fas and Fas ligand and apoptosis in tumor-associated lymphocytes and in tumor cells from malignant pleural effusions

The CD95 (APO-1/Fas)-Fas ligand (FasL) system is an important mediator of antitumor T cell cytotoxicity. The aim of the current study was to assess its significance in human cancer. Malignant effusions were selected as an environment allowing direct cell-to-cell contact in a fluid phase. Malignant pleural effusions collected from 23 patients with metastatic carcinoma of the bronchus, ovary, stomach or breast were examined by means of flow cytometry. The expression ofFas and FasL, probed with the appropriate antibodies, apoptosis of tumor cells and the characteristics of tumor-associated lymphocytes (TAL) were determined by TUNEL reaction in malignant and nonmalignant (control) effusions. All malignant cells had partially or completely lost the expression of CD95 and expressed an elevated level of FasL. In contrast, TAL obtained from malignant pleural effusions demonstrated a marked decrease in the expression of surface FasL and an increase in surface-bound Fas. The percentage of apoptotic malignant cells was significantly decreased, as compared to TAL and lymphocytes from nonmalignant pleural effusions. There were also differences in the expression of Fas and FasL among mononuclear cells from malignant and nonmalignant pleural effusions. The ability of TAL from malignant pleural effusions to induce apoptosis of K562 cells was diminished, as compared to peripheral blood lymphocytes. Taken together, these data suggest that tumor cells in the microenvironment of malignant pleural effusions can evade immune attack by downregulation of the CD95 receptor and by killing lymphocytes through the expression of FasL. These results confirm earlier reports which showed that lymphocytes from a tumor microenvironment appear to have a depressed cytotoxic action.     

Comparison of Fas(Apo-1/CD95)- and perforin-mediated cytotoxicity in primary T lymphocytes

Cytolytic T lymphocytes kill target cells by two independentcytolytic mechanisms. One pathway depends on the polarized secretionof granule-stored proteins including perform and granzymes,causing target cell death through membrane and DNA damage. Thesecond cytolytic effector system relies on the interaction ofthe Fas ligand (FasL) on the effector cell with its receptor(Fas) on the target cell, leading to apoptotic cell death. Usingmixed lymphocyte culture (MLC)-derived primary T lymphocytesof perforin-knockout and gld (with non-functional FasL) mice,the molecular basis of the two killing mechanisms was compared.The activity of both pathways was dependent on extracellularCa²⁺. Incubation of MLC-stimulated primary T cells with proteinsynthesis inhibitors prior to TCR triggering impaired FasL cellsurface expression and abolished cytolytic activity, althoughthe cells exhibited an intracellular pool of FasL. The perforin-dependentmechanism induced cell death more rapidly, although both pathwaysultimately showed similar killing efficiencies. Both pathwaysinduced comparable levels of DNA degradation, but Fas-inducedmembrane damage was less pronounced. We conclude that upon TCRtriggering FasL may be recruited in part from pre-existing intracellularstores. However, efficient induction of target cell death stilldepends on the continuous biosynthesis of FasL molecules.     

Agonist antibody and Fas ligand mediate different sensitivity to death in the signaling pathways of Fas and cytoplasmic mutants

We have produced three forms of human Fas: full-length Fas, Fas with a C-terminal deletion, and a chimera between extracellular Fas and the intracellular domain of the tumor necrosis factor receptor I p55 subunit. We transfected cell lines with these constructs to compare the relative capacity of antibody agonists and the physiological Fas ligand (FasL) to stimulate death. With two agonistic antibodies, the chimera is 100- to 1000-fold more sensitive to induction of death than the full-length Fas. The C-terminal deletion mutant also shows greatly enhanced death in comparison to the wild-type receptor. In contrast, when FasL is used to trigger the Fas pathway, wild-type Fas and the deletion mutant are similarly sensitive, whereas the chimera is 100-fold less susceptible to ligand-mediated killing than Fas. This demonstrates that antibody agonists and natural ligand can stimulate different signaling pathways and emphasizes the limitations of defining physiologically important signaling pathways solely by antibody agonists.     

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.