Perforin Pathway Research Articles

Perforin, Fas/Fas ligand, and TNF-alpha pathways as specific and bystander killing mechanisms of hepatitis C virus-specific human CTL.

In chronic hepatitis C, Fas expression is up-regulated in the hepatocytes, especially near liver-infiltrating lymphocytes, and Fas ligand is expressed on the lymphocytes. The presence of hepatitis C virus (HCV)-specific CTLs has been demonstrated both in peripheral blood and among liver-infiltrating lymphocytes of patients with chronic hepatitis C. We studied the killing mechanisms of HCV-specific human CTLs using target cells that were sensitive or resistant to agonistic anti-Fas Abs and TNF-alpha. We show that HCV-specific CTL clones kill non-Ag-bearing bystander cells as well as Ag-bearing cells, although the bystander killing is less efficient than the specific target cell killing, and the efficacy of the bystander killing of anti-Fas- and soluble TNF-alpha-sensitive cells is greater than that of resistant cells. We also show that the killing of Ag-presenting, sensitive cells is mediated by Fas ligand and TNF-alpha as well as perforin, although the latter plays a major role in the killing at a low E:T ratio, and that the killing of sensitive bystander cells is primarily mediated by Fas ligand and TNF-alpha on CTLs expressed upon specific Ag stimulation, which may be relevant to the bystander lysis by HCV-specific CTLs of uninfected hepatocytes, in which Fas expression is up-regulated. Activated CTLs also kill bystander cells by the perforin-based mechanism, although it requires a high E:T ratio. The effective bystander killing requires a close intercellular contact between CTLs and target cells, although TNF-alpha released from the CTLs mediates lysis of the bystander cells without a close cell-cell contact.

A broad cytotoxic T lymphocyte response to influenza type B virus presented by multiple HLA molecules.

The HLA restriction and epitope specificity of cytotoxic T lymphocytes (CTL) involved in recovery from influenza type B infection have not been extensively characterized. Here lymphocytes obtained from a healthy individual contained virus-specific CTL restricted by class I HLA molecules, HLA-A1, A2, B7 and B8, and the class II HLA molecules, HLA-DR1 and DR3. Four conserved viral epitopes were predicted from allele-specific motifs for peptides interacting with HLA-B8 and HLA-DR1. Bulk CTL recognized three 9mer HLA-B8-restricted peptides from nucleoprotein, residues 30-38, 263-271 and 413-421, and a 13mer HLA-DR1-restricted peptide from hemagglutinin, residues 308-320. The epitopes presented by HLA-A1, HLA-B7 and HLA-DR3 remain undefined. Peptide-specific CTL lines recognized influenza type B virus-infected cells indicating the peptides are representative of naturally processed epitopes. A hemagglutinin peptide-specific CD4 CTL clone expressed approximately 200 molecules of perforin mRNA/cell, suggestive of a functional perforin pathway for target cell lysis. The results indicate a broad CTL response composed of both CD8 CTL and CD4 CTL recognizing viral epitopes presented by multiple HLA molecules.

Islet rejection in perforin-deficient mice: the role of perforin and Fas.

Perforin and Fas are the two main pathways by which cytotoxic T lymphocytes (CTLs) mediate target cell lysis in vitro. The perforin pathway is predominantly used by CD8+ cells, which comprise the majority of CTLs. The Fas pathway has been demonstrated to be the principal cytolytic mechanism in CD4+ CTLs. CTLs have been shown to play an important role in allograft rejection. In this study, we examined the relevance of perforin and Fas to allograft rejection by transplanting pancreatic islets from fully allogeneic C3H/HeJ (C3H) or Fas-deficient C3H/lpr donors into perforin-deficient (P0) mice or controls with intact perforin genes (P2). P0 or P2 mice that were rendered diabetic with streptozotocin at 300 mg/kg i.p. received approximately 350 islets obtained from C3H or C3H/lpr donors by in situ collagenase digestion and Ficoll density centrifugation of the pancreas. Four groups of animals were studied: C3H to P2 (group 1), C3H to P0 (group 2), lpr to P0 (group 3), and syngeneic P2 to P2 (group 4). Graft survival monitored by blood sugar levels was compared among the groups. At the time of rejection (blood sugar >300 mg/100 ml), grafts were harvested and analyzed by histopathology, immunocytochemistry, and reverse transcriptase-polymerase chain reaction. Primary splenic T cells of the recipients, harvested at the time of rejection, were tested for cytotoxicity against 51Cr-labeled donor cells. The mean graft survival for groups 1, 2, and 3 was 10.2+/-1.4, 12.2+/-6.0, and 13.2+/-0.8 days, respectively. Syngeneic grafts survived indefinitely. Rejecting grafts from all groups (1, 2, and 3) showed an intense infiltration by both CD4+ and CD8+ cells and complete islet destruction. Reverse transcriptase-polymerase chain reaction revealed granzyme B in rejecting grafts from all three groups. Perforin and Fas pathways alone or in combination are not required for islet rejection, suggesting that these pathways may not play a crucial role in allograft rejection.

Xenospecific cytotoxic T lymphocytes: potent lysis in vitro and in vivo.

As little is known about the molecular mechanisms responsible for lymphocyte-mediated rejection of xenografts, we have studied the relative contribution of perforin and Fas pathways in cytotoxic lymphocytes generated in mice transplanted with human cell lines. Responder lymphocytes generated in immunocompetent mice displayed significant lysis of human target cells, which suggests that mice can generate a strong lymphocytotoxic response to human cells. Effector cells generated in immunocompetent and gld (Fas ligand mutant) mice predominantly use a perforin-mediated cytotoxic mechanism. By contrast, a Fas-mediated pathway could be stimulated in perforin-deficient or beta2-microglobulin-deficient mice, providing the human target cells were sensitive to Fas-mediated lysis. In vitro depletion of effector CD3+ CD8+ T cells, but not CD4+ T or NK1.1+ cells, completely inhibited lysis of human target cells. This suggests that CD3+ CD8+ T cells were responsible for perforin-mediated xenospecific cytotoxicity. In vivo depletion of NK1.1+ cells and CD4+ T cells before the final immunization abrogated the capacity of lymph node cells to generate xenospecific CD8+ cytotoxic T lymphocytes. By contrast, in vitro depletion of CD4+ T cells was most effective in abrogating the xenospecific Fas-mediated cytotoxicity of perforin-deficient effector cells. Xenospecific cytotoxic T cells were also capable of mediating tumor rejection when adoptively transferred into scid/scid mice bearing established human COLO 205 xenografts. Overall, these data suggested that xenospecific cytotoxic T lymphocytes can lyse target cells via either perforin- or Fas-mediated pathways and that these cells can provide protective and specific immunity against tumor xenografts in the absence of an intact humoral immune system.

Functions of CD8 T-cell subsets secreting different cytokine patterns

CD8+T cells can differentiate into two effector phenotypes, Tc1 and Tc2, secreting different cytokine patterns. Both subsets are cytotoxic via the perforin and Fas pathways, and both kill resting and activated B cells, ruling out the possibility of cognate help, although Tc2 cells may provide bystander help. Both subsets induce inflammation with similar cellular infiltrates. Tc1 cytokine synthesis is limited by two mechanisms — IL-4 induces a permanent deficiency in cytokine secretion, and rapid killing of target cells limits CD8+T-cell activation and cytokine production. These multiple CD8 T-cell activities provide a versatile set of immune functions.

The role of the perforin and Fas pathways of cytotoxicity in skin graft rejection.

Cell-mediated cytotoxicity is believed to play an important role in the mechanisms of rejection following organ and tissue transplantation. We have investigated the role of the perforin and Fas pathways of cell-mediated cytotoxicity on the survival of xenogeneic, allogeneic and hemiallogeneic skin grafts. Wild type C57BL/6 (B6), perforin deficient C57BL/6 (B6 PKO) and Fas ligand deficient (B6 gld/gld) mice were grafted with xenogeneic Lewis rat skin grafts, or allogeneic B10.BR, class I disparate B6 bml, class II disparate B6 bm12, and Fas deficient allogeneic C3H lpr/lpr grafts. No significant difference was observed in the timing of rejection with B6 PKO mice as recipients when compared with normal B6 controls. All grafts were also rejected by B6 gld/gld recipients, but with a significantly delayed pattern of rejection, showing, therefore, that Fas, although not critical, may participate to the mechanisms of rejection. Neither perforin nor Fas, however, seem to be essential for rejection of a skin graft, and it is likely that alternative mechanisms assume an important role in the effector phase of rejection once these cell-cytotoxic pathways are abrogated.

Recipient CD8+ cells are responsible for the rapid elimination of allogeneic donor lymphoid cells.

Blood transfusion can result in a variety of immunologic responses, including alloimmunization, transfusion-associated graft-versus-host disease, and immunosuppression that results in increased postoperative infection rate, and can also result in increased survival of allografts. One of the many factors influencing the resulting immunologic responses from a blood transfusion is the persistence of the donor leukocytes. A recent study has shown that almost all (99.9%) allogeneic leukocytes are removed within 2 days following transfusion but did not characterize the mechanism responsible for the rapid removal of allogeneic donor cells. Using a murine model these studies show that it is recipient CD8+ T cells that are responsible for the rapid elimination of allogeneic lymphocytes in naive recipients. Effective elimination was dependent on the donor/recipient combination and required that the donor cells express at least one MHC class I disparity to be recognized by the recipient CD8+ cells and that the donor cells also be able to induce additional responses that were needed by the CD8+ cells to manifest full activity. The perforin pathway was the predominant pathway used by the recipient CD8+ cells to mediate this elimination.

Perforin, Fas ligand, and tumor necrosis factor are the major cytotoxic molecules used by lymphokine-activated killer cells.

Lymphokine-activated killer (LAK) cells generated from perforin knockout mice possess significantly reduced cytotoxicity against a panel of tumor target cell lines, with some tumor cells being lysed exclusively by the perforin pathway. LAK cells are also capable of Fas ligand-mediated cytotoxicity. LAK cells generated from mice deficient in both perforin and Fas ligand (PKO/gld) were not cytolytic in short term cytotoxicity assays, demonstrating that perforin and Fas ligand are required for acute target cell lysis. However, PKO/gld LAK cells were cytotoxic in long term cytotoxicity assays against TNF-sensitive tumor lines, and this cytotoxicity was completely inhibited by neutralizing TNF Abs. This potent TNF cytotoxicity has not been fully appreciated previously because of the presence of dominant-acting perforin and Fas ligand in acute tumor cell lysis. TNF-based cytotoxicity by PKO/gld LAK was both soluble and membrane bound, and both forms of TNF were constitutively expressed. Thus, LAK cells are armed with at least three cytotoxic molecules: perforin, Fas ligand, and TNF.

Selective activation of Fas/Fas ligand-mediated cytotoxicity by a self peptide.

To study how MHC-associated self antigens may regulate the function of T cells in the periphery, we generated CD8+ T cell lines specific for a single residue variant of a self peptide. The self peptide (GAYEFTTL) was isolated from H-2-Kb class I MHC molecules immunopurified from tumor cells. CD8+ CTL lines from H-2b mice were generated against a variant peptide, pE4R, (arginine for glutamic acid at the TCR contact position 4). In short-term 51Cr-release assays, these CTL lysed H-2Kb targets that were pulsed with picomolar levels of pE4R but did not lyse target cells coated with the self peptide at micromolar levels. However, in overnight assays the CTL lysed Fas-positive target cells in the presence of nanomolar levels of the self peptide. This killing was shown to be entirely Fas/Fas ligand mediated by blocking with anti-Fas antibody and Fas-Fc chimeric molecules. While the self peptide was unable to induce serine esterase release from the CTL, it did induce secretion of IFN-gamma. By these criteria then, the unmodified self ligand served as a partial agonist for the CTL raised against a single-residue variant. CD8+ T cell lines raised by in vitro stimulation with the self peptide were likewise unable to kill self peptide-coated targets via the perforin pathway but did lyse targets via Fas. These and similar data from other groups show that self antigens (i.e., MHC/peptide complexes) may be recognized by mature peripheral T cells. The T cell population is tolerant of the self antigen in the sense that they do not respond to physiological levels of the MHC/peptide complex. However, when the level of self antigen is increased (by using synthetic peptide loading) CD8+ T cells may respond by proliferation, IFN-gamma secretion, Fas ligand upregulation, and Fas-mediated cytolysis but are still unable to respond by perforin-mediated cytolysis or granzyme release. The physiological significance of such partial activation in regulation of the immune system remains to be demonstrated.

Bcl-2 protects against Fas-based but not perforin-based T cell-mediated cytolysis.

Fas ligand and perforin are the two key effector mechanisms in T cell-mediated cytotoxicity. These molecules mediate cytolysis of target cells by membrane damage and apoptosis. bcl-2 is known to protect cells against apoptosis induced by many stimuli including growth factor removal. However bcl-2's effect on Fas ligand and perforin-induced lysis has not been studied extensively. We investigated the effect of overexpression of bcl-2 alone, Fas alone or their combined overexpression on lysis of a commonly used target, P815, by perforin-sufficient, Fas ligand-sufficient and perforin-deficient or Fas ligand-deficient, allospecific cytotoxic T lymphocytes (CTL). Wild-type P815 are susceptible to lysis by perforin-sufficient CTL, regardless of the presence or absence (gld) of Fas ligand, but are poorly lysed by perforin-deficient CTL. Fas transfection of P815 makes target cells highly susceptible to lysis by both perforin-sufficient and -deficient CTL, indicating the presence of the Fas ligand-mediated cytotoxicity on both types of CTL. Co-transfection of P815-fas with bcl-2 abolishes their increased susceptibility to Fas-mediated lysis, even in the face of Fas overexpression on the cell membrane. The protective effect of bcl-2 against cell lysis is evident with perforin-deficient CTL as effector cells or when perforin activity is eliminated by the absence of extracellular calcium in perforin-sufficient CTL. bcl-2 overexpression by P815, however, does not protect against CTL lysis by the perforin pathway, regardless of Fas overexpression, as demonstrated by fas ligand mutated gld and wild-type perforin-sufficient CTL. Therefore bcl-2 can protect P815 target cells against Fas-mediated lysis when triggered by the Fas ligand on CTL, but not against perforin-mediated lysis.

Induction of lytic pathways in T cell clones derived from wild-type or protein tyrosine kinase Fyn mutant mice.

The OVA-reactive CD4+ Th1 clones and alloreactive CD8+ clones derived from wild-type or fyn-/- mice serve as model systems which have allowed us to investigate several aspects of the molecular events associated with T cell-mediated cytotoxicity, including 1) the differential utilization of two distinct cytolytic pathways by CD4+ Th1 clones and CD8+ CTL, 2) a comparison of the pathways of lysis induced by stimulation of the TCR or by alternative stimuli, 3) the requirement of Fyn for derivation of antigen-specific T-cell clones having properties of CD4+ Th1 and CD8+ CTL cells 4) the differential requirement of Fyn in the induction of responses by TCR and the alternative stimuli. Stimulation through the TCR, either by APC bearing relevant antigen or by immobilized anti-CD3 mAb, resulted in comparable levels of target cell lysis by clones from both wild-type and fyn-/- mice. These clones also utilize the Fas pathway to lyse target cells. Thus, Fyn does not appear to be required for expression of the Fas pathway when triggered through the TCR. In contrast, lysis of target cells by T-cell clones lacking Fyn was deficient when stimulated through Thy-1 or Ly-6C (using mAb) or with Con A or phorbol ester as compared to clones derived from wild-type mice. The basis for the defect in response to stimulation through the GPI-linked molecules appears to be a signaling defect which affects all of the functional responses we measured, while the defect in response to Con A stimulation appears to affect lysis but not lymphokine production. Thus, Fyn expression is selectively required for efficient activation of the Fas pathway of lysis through Thy-1, Ly-6C, and by Con A or phorbol ester in these T-cell clones. CD8+ clones derived from fyn-/- mutant mice, like clones derived from wild-type mice, display antigen-specific lysis, and appear to express perforin message and perforin protein. A Ca(++)-dependent (presumably perforin/exocytosis) component and Fas component of lysis was detected in CD8+ clones derived from fyn-/- mutant mice. Thus, Fyn is not required for expression of these components of antigen specific lysis by CD8+ alloreactive CTL clones. It appears that CD8+ clones that use multiple lytic mechanisms may selectively employ the perforin or Fas-based pathway depending on properties of the target cell or stimulus.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell-mediated cytotoxicity can be regulated by p53 tumor suppressor gene activity in vitro

The wild-type human p53 tumor suppressor gene was tested for its ability to modulate cytotoxic activity of in vitro activated peripheral blood lymphocytes. Peripheral blood mononuclear cells (PBMCs) were stimulated by phytohemagglutinin (PHA), interferon alpha 2b (IFN alpha 2b), interleukin 2 (IL-2) or their combinations to induce cytotoxicity. This stimulation significantly increased the percentage of cells expressing p53, which was at its maximum when induced by IL-2 combined with IFN alpha 2b. The role of p53 in the modulation of different aspects of cytotoxic activity of these cells was analyzed by studying the effects of p53 abrogation by antisense oligonucleotide (p53 AS) treatment in comparison with p53 sense or scrambled (missense) oligonucleotide (p53 S or p53 MS) treatment. We show that p53 plays a key role through induction of apoptosis in target cells (tumor necrosis factor pathway) rather than through osmolytic degeneration (perforin pathway) which is only slightly increased by p53 abrogation. Meanwhile, in vitro abrogation of p53 expression in PBL was found to be accompanied by an increase of CD8+ lymphocytes and an important increase of the CD56 'bright' NK cell sub-population.

Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways.

The recent generation of perforin knock-out mice has demonstrated a crucial role for the pore-forming perforin in cytolytic T-lymphocyte (CTL)-mediated cytolysis. Perforin-deficient mice failed to clear lymphocytic choriomeningitis virus in vivo, yet substantial killing activity still remained in perforin-free CTLs in vitro, indicating the presence of (a) further lytic pathway(s). Fas is an apoptosis-signalling receptor molecule on the surface of a number of different cells. Here we report that both perforin-deficient and Fas-ligand-deficient CTLs show impaired lytic activity on all target cells tested. The killing activity was completely abolished when both pathways were inactivated by using target cells from Fas-receptor-deficient lpr mice and perforin-free CTL effector cells. Fas-ligand-based killing activity was triggered upon T-cell receptor occupancy and was directed to the cognate target cell. Thus, two complementary, specific cytotoxic mechanisms are functional in CTLs, one based on the secretion of lytic proteins and one which depends on cell-surface ligand-receptor interaction.

Fas and Perforin Pathways as Major Mechanisms of T Cell-Mediated Cytotoxicity

Two molecular mechanisms of T cell-mediated cytotoxicity, one perforin-based, the other Fas-based, have been demonstrated. To determine the extent of their contribution to T cell-mediated cytotoxicity, a range of effector cells from normal control or perforin-deficient mice were tested against a panel of target cells with various levels of Fas expression. All cytotoxicity observed was due to either of these mechanisms, and no third mechanism was detected. Thus, the perforin- and Fas-based mechanisms may account for all T cell-mediated cytotoxicity in short-term in vitro assays.

CTL-mediated cytolysis: perforin and alternative pathways?

CTL-mediated cytolysis: perforin and alternative pathways?

Perforin-dependent and -independent pathways of cytotoxicity mediated by lymphocytes.

There is little doubt at the present time that both perforin-dependent and -independent pathways are important in mediating the cytotoxicity associated with lymphocytes. The cell distribution of perforin, initially thought to include both CTL and NK cells, now must be viewed with caution because all previous biochemical studies on CTL have been conducted with cell lines propagated in long-term cultures in the presence of T cell growth factors (IL-2 and perhaps some still undefined factors). Under these conditions, CTL are known to assume a broader, NK-like specificity in target cell killing and may thus differ significantly from primary CTL generated in the body. Accordingly, perforin does not seem to be present in primary CTL activated directly through mixed lymphocyte reactions. It remains to be shown how primary CTL lyse target cells in vivo. Initial studies conducted in several laboratories have already provided some clues. It now seems that even in cultured, perforin-containing CTL, the perforin pathway is not an obligatory mechanism required for target cell killing. Other pathways, possibly involving TNF/lymphotoxin-like molecules, may play a direct role in this type of cytotoxicity. Other still unidentified factors now also need to be sought, including membrane polypeptides that may develop cytotoxicity directly upon cell contact and binding. Although from the studies reviewed here it is clear now that perforin has a more limited role in cell killing than originally proposed, it is still intriguing that it should share structural and functional homologies with complement proteins, drawing paradoxical analogies between two systems (the cellular and the humoral immune systems) which have evolved to become specialized to carry out separate immunological tasks. The cloning of the genes for perforin and for all the C proteins that comprise the MAC should reveal important information on how these genes originated and then diverged during evolution. The cellular distribution of other granule products, such as serine esterases, also must be viewed with caution. A serine esterase activity was initially thought to be CTL-specific. This information stimulated an intensive research activity in many laboratories that resulted in both the purification of a serine esterase family and the cloning of several serine esterase transcripts. It is becoming clear from recent evidence that this group of enzymes is not truly CTL-specific and therefore would not be expected to develop any function rendered absolutely necessary for cytolysis.(ABSTRACT TRUNCATED AT 400 WORDS)