Perforin: structure and function
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When mouse target cells are subjected to cytolytic attack by mouse CTL cell lines that have been cultured for many months in high levels of IL-2, and have abundant perforin-rich secretory granules, they exhibit two prominent changes: 1) rapid and massive increase (greater than 10-fold) in intracellular Ca2+ concentration and 2) fragmentation of DNA into nucleosome-sized fragments. We show here that when the same target cells are subjected to cytolytic attack by perforin-deficient CTL, either human CTL or primary mouse CTL from peritoneal exudates, the same changes are observed, suggesting that perforin-rich and perforin-deficient CTL kill their target cells by similar (if not identical) mechanisms. It is possible that perforin-deficient CTL produce enough perforin to destroy target cells but not enough to be detected by currently available methods.
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IFN-gamma-secreting T1 and IL-5-secreting T2 subsets of CD8 effectors were generated in vitro using freshly isolated cells from wild-type and perforin knockout mice stimulated with allogeneic Ag-presenting cells. Both T1 and T2 effectors from wild-type mice exhibited perforin-mediated cytolysis. T1, but not T2, populations from perforin knockout mice displayed significant lysis by the Fas-mediated pathway. Th1 cells have recently been shown to be regulated by Fas and we speculate that Fas-mediated mechanisms are involved in the regulation of both Th1 and T1 populations of T cells.
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Abstract CD4 T cell effectors can promote survival against lethal influenza virus via perforin mediated cytolytic mechanisms, however; our understanding of how naïve CD4 cells differentiate into class II restricted killers remains obscure. To address this, TCR Tg CD4 cells were activated in vitro and examined for their ability to lyse target cells. We found that cytokine polarized CD4 T cell effectors displayed cytolytic activity with the hierarchy Th0>Th1>Th2>Th17; however, IL-6, TGF-β, IL-10, IL-12 or TNF-α were not required for inducing cytolytic activity in CD4 effectors. Antigen dose had a marked effect on cytotoxicity: low concentrations of peptide induced more potent cytolytic activity than relatively high concentrations. At low peptide concentration, exogenous IL-2 was necessary to drive granzyme B (GrB) expression and perforin mediated lysis. Using IL-2 deficient T cells, we found that IL-2 signaling was required in the first 24 h of culture, but increasing concentrations of IL-2 did not increase cytolytic activity. Studies are underway to determine whether Stat5 signaling is required for induction of cytolytic activity in CD4 T cells. Thus, low antigen dose and early activation signals via IL-2 direct the CD4 T cell response toward effectors with perforin mediated cytolytic potential.
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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-2s 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.
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Different cell types vary widely in their susceptibility to killing by the pore-forming cytolytic molecule perforin. In particular, the cells responsible for synthesis of perforin, i.e. cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, are very resistant to cytolysis by this molecule. It has previously been suggested that resistance is due, at least in part, to diminished binding of perforin to these cells. The purpose of the present study was to compare binding of perforin to sensitive and resistant cell types. To this end, perforin was biosynthetically labelled prior to purification. The purified labelled protein was then utilized to obtain a direct measure of the amount of perforin bound to cells during attack. Resistant cells (CTL, neutrophils) bound at least as much perforin as did sensitive cells (K562, HL60 etc.), indicating that resistance to perforin involves mechanisms operating after binding of the lytic molecule.
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Gene therapy approaches using first generation adenoviral vectors (RAds) can achieve long-term transgene expression in the brain, and therapeutic effects in animal models of brain tumors and neurodegeneration. However, long term expression and vectors' efficiency can be limited by immune responses, characterized by inflammation in the brain and the eventual elimination of transgene expression. Injection of first generation RAds into the striatum followed by peripheral immunization has allowed us to determine the cellular and molecular mechanisms by which the adaptive immune response regulates transgene expression in the CNS. Our experiments provided the following results: (1) systemic immunization against RAd reduced transgene expression by 30 days post-immunization; (2) the time course of entry of lymphocytes into the brain is as follows: first, CD4+, followed by CD45+ and F4/80+, and lastly, CD8+ T cells; (3) lymphocytes and macrophages contacted and phagocytosed transduced cells; (4) transgene expression persisted in the CNS of Rag1(|[minus]|/|[minus]|), CD4(|[minus]|/|[minus]|), IFN|[gamma]|(|[minus]|/|[minus]|) and perforin(|[minus]|/|[minus]|) animals; and (5) even in the absence of transgene expression, we could detect the presence of RAd genomes by quantitative TaqMan PCR. In summary, our data suggest the existence of a CD4-T cell response mediated by IFN|[gamma]| and potentially perforin that eliminates transgene expressing cells from the CNS. The continued presence of viral vector genomes, suggests the presence of non-cytolytic mechanisms as well. The balance of cytotoxic, and non-cytotoxic mechanisms is currently being investigated further.
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Our laboratory previously demonstrated that CD4 effectors generated in vitro could promote survival against a highly pathogenic influenza virus via an antibody independent mechanism that involved class II restricted, perforin‐mediated cytotoxicity. The present study set out to determine whether influenza specific CD4 cells could acquire cytolytic activity in vivo and the role of perforin in CD4 T cell responses to influenza. Surprisingly, influenza PR8 infection induced a population of cytolytic CD4 effectors that resided in the lung, but not the draining LN. These cells expressed granzyme B (GrB), were CD62L low and required perforin to lyse peptide pulsed targets. CD4 cells isolated from the lungs of infected mice were able to confer protection to a lethal dose of influenza PR8; however, cells isolated from the DLN were more effective at promoting survival. This was attributed to the ability of DLN CD4 cells to further differentiate to cytolytic GrB+ cells upon re‐infection. Lethally infected mice given flu specific CD4 cells deficient in perforin showed greater weight loss and slower time to recovery compared to mice given wildtype flu specific CD4 cells, suggesting that CD4 mediated cytolytic activity may play a role in protection to lethal infection. Studies are underway to determine whether perforin is required for B cell independent protection mediated by CD4 effectors in B cell deficient mice. Supported by PHS grants AI‐0672, AG‐21600 and the Trudeau Institute.
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