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At least 19 recordsLinked to original sources

PubMed1992

Perforin expression in human peripheral blood mononuclear cells. Definition of an IL-2-independent pathway of perforin induction in CD8+ T cells.

Perforin gene expression upon in vitro stimulation was studied at the mRNA level in normal human PBMC and in subpopulations. Freshly isolated PBMC express low levels of perforin mRNA. Increased perforin expression is rapidly induced by the calcium ionophore A23187 and by rIL-2. Phorbolesters (PMA), by comparison, are poor inducers of perforin RNA. Perforin induction by Ca-ionophore, unlike granzyme 2 and IL-2 induction, did not synergize with phorbolesters in PBMC or in purified T cells. Instead, perforin mRNA induction by A23187 in purified T cells requires the presence of adherent cells. Ca-ionophore plus adherent cell-induced perforin occurred in CD8+ T cells and was abolished by depletion of CD8+ T cells but not by depletion of CD4+ T cells. Adherent cells alone did not express perforin under any condition. Perforin mRNA induction by both A23187 and by rIL-2 is independent of de novo protein synthesis. The half-life of perforin mRNA induced by either stimulus is approximately 100 min. Cyclosporin A completely abrogates perforin induction by A23187 but only slightly inhibits the effect of rIL-2 on perforin mRNA expression. These data show that A23187 activates perforin gene expression in CD8+ cells by an IL-2-independent pathway and that the molecular mechanism of perforin expression may be different from the one induced by IL-2. Granzyme 2 (human leukocyte protease-HLP, homologous to murine granzyme B) mRNA expression was studied in comparison to perforin. Granzyme 2 in contrast to perforin responds to the synergistic action of phorbolester and Ca-ionophore in PBMC. In addition, the kinetics of the induction of granzyme and perforin mRNA, by various signals are different. Our data suggest that situations in vivo may exist that allow perforin expression in CD8+ cells in the absence of cytokines by a combination of Ca signals and accessory receptor ligation. The same signals may not be sufficient for granzyme 2 expression in any T cell subpopulation.

Base Sequence↗

PubMed2003

Essential roles of perforin in antigen-specific cytotoxicity mediated by human CD4+ T lymphocytes: analysis using the combination of hereditary perforin-deficient effector cells and Fas-deficient target cells.

Although the cytotoxic mechanisms of murine CTLs have been investigated extensively using various mutant and knockout mice, those of human CTLs, especially CD4+ CTLs, are still obscure. To clarify the roles of perforin in Ag-specific cytotoxicity mediated by human CD4+ CTLs, alloantigen-specific and HSV-specific human CD4+ T lymphocyte bulk lines and clones were established from a patient with hereditary perforin deficiency and her healthy father, and their cytotoxic activities were investigated. Alloantigen-specific CD4+ T lymphocytes expressing perforin exerted cytotoxicity against Fas-negative as well as Fas-positive allogeneic B lymphoblastoid cell lines established from members of a family with hereditary Fas deficiency. Perforin-deficient, but not perforin-expressing, CD4+ T lymphocytes failed to show strong cytotoxicity against HSV-infected autologous B lymphoblastoid cells. Perforin-deficient CD4+ T lymphocytes could exert relatively low level cytotoxicity against allogeneic IFN-gamma-treated keratinocytes. Although cytotoxicity mediated by perforin-expressing CD4+ CTLs was almost completely inhibited by concanamycin A, a potent inhibitor of the perforin-mediated cytotoxic pathway, cytotoxicity against IFN-gamma-treated keratinocytes mediated by perforin-deficient CD4+ T lymphocytes was inhibited only partially by concanamycin A, but was inhibited significantly by antagonistic anti-Fas Ab and anti-Fas ligand Ab. The combination of perforin-deficient effector T lymphocytes and Fas-negative target cells used in the present study provides a novel experimental system for studying the detailed mechanisms of human CTL-mediated cytotoxicity. The present data demonstrate that perforin-negative CD4+ CTLs can exert cytotoxicity against Fas-sensitive target cells; however, perforin plays essential roles in Ag-specific cytotoxicity mediated by human CD4+ as well as CD8+ CTLs.

B-Lymphocytes↗

PubMed1992

Structure and function of the murine perforin promoter and upstream region. Reciprocal gene activation or silencing in perforin positive and negative cells.

Gene expression of the cytolytic protein perforin is restricted to and tightly regulated in cytolytic lymphocytes. To begin to understand the molecular basis of perforin gene transcription, we cloned and analyzed 5.1 kb of the genuine murine perforin promoter and upstream region. The murine perforin promoter is located approximately 2.1 kb upstream of the translation start codon in the genomic DNA due to an intron in the 5' untranslated sequence. Although the sequenced murine promoter and upstream region was found to be quite homologous to that of the human gene, most of the interspecies conserved sequences lacked obvious consensus to known regulatory elements. Functional analysis of this region, however, indicated that it contains regulatory elements that may determine the cell-type-specific expression of this killer protein. After transient transfection into several cell lines, the perforin promoter and upstream region was used to drive the expression of the chloramphenicol acetyltransferase (CAT) reporter gene. High levels of CAT activities, exceeding 110 times the expression of a promoterless reporter gene construct, were expressed in CTL. In contrast, in perforin-negative cell types the perforin promoter and upstream region mediated barely detectable transcription of the CAT gene. Analysis of the immediate proximal perforin promoter, -120 to +2, revealed that it was ubiquitously active and that it expressed in all cells tested 20- to 50-fold higher CAT activity than the promoterless reporter gene construct. The cell-type restricted transcriptional activity of the perforin promoter and upstream region, however, was controlled by at least four negative and positive cis-acting upstream regions that spread over the entire 5 kb of the cloned DNA and acted reciprocally in different cells. Thus, in perforin-negative cells, the transcriptional activity of the immediate proximal perforin promoter was dominantly suppressed by several upstream negative regulatory elements, whereas in perforin-positive cells, the promoter activity was enhanced more than fivefold by several upstream regulatory elements.

Animals↗

PubMed1994

Resistance of CTL to perforin-mediated lysis. Evidence for a lymphocyte membrane protein interacting with perforin.

Cytotoxic T lymphocytes are highly resistant to killing by their own cytolytic protein perforin. We have investigated the molecular basis of this self-protection mechanism. CTL withstood high dosages of perforin, when complete lysis was obtained with various tumor target cell lines. A peptide-specific CTL clone readily lysed tumor targets presenting the peptide, but was unable to kill the peptide-presenting CTL in spite of equal degranulation. With streptolysin O, a pore-forming lytic protein of bacterial origin, no difference in susceptibility between the various targets was detected. Perforin was radioactively labeled by using the Bolton and Hunter method without any loss of its lytic activity. Experiments performed at 4 degrees C revealed that, on all of the cells studied, binding of the labeled perforin to the membrane is reversible and strictly Ca(2+)-dependent. After a short exposure to 37 degrees C, perforin was no longer dissociated from cell membranes by EDTA. Although no correlation between susceptibility and the extent of perforin binding was detected, differences in the conformation of membrane-bound perforin were observed. Perforin adsorbed to resistant cells was cleaved by trypsin into a 55-kDa (C-terminal) and a 10- to 15-kDa (N-terminal) fragment, whereas this cleavage was not found on tumor cell-bound perforin. A portion of perforin was included in the Triton X-114 detergent phase at 4 degrees C on resistant CTLs only. Our results are compatible with the notion that a protective molecule, specifically expressed on CTLs, interacts with perforin, thereby rendering it lytically inactive.

Animals↗

PubMed1997

Perforin-enhancing protein, a low molecular weight protein of cytotoxic lymphocyte granules, enhances perforin lysis.

Perforin is a 68 kD protein found in the granules of cytotoxic lymphocytes and is used by lymphocytes to form lethal pores in the membranes of the cells they kill. We and others have found that when perforin is purified, its lytic activity is markedly reduced. ELISAs indicated that our final recovery of perforin protein was excellent. We decided to determine if depletion of other granule proteins contributed to the loss of lytic activity. We isolated perforin to the point where lytic activity was diminished and added back granule proteins that had no lytic activity or detectable (antigenic) perforin. Perforin was isolated by Cu2+-immobilized metal affinity chromatography (IMAC) followed by phenyl-Superose hydrophobic interaction chromatography (HIC). Its lytic activity was enhanced by a low molecular weight (<15 kD) protein, perforin enhancing protein (PEPr). We have isolated PEPr by two methods, HIC and MonoQ. Nonlytic PEPr restored perforin to close to its original lytic activity. A protein similar if not identical to PEPr was also detectable as an 125I-labeled protein associated with lytic perforin. We propose that PEPr acts in conjunction with perforin to form lethal pores and suggest that PEPr may be the rat equivalent of the human cytotoxic lymphocyte protein, granulysin.

Animals↗

PubMed1990

Resistance of cytolytic lymphocytes to perforin-mediated killing. Inhibition of perforin binding activity by surface membrane proteins.

The mechanism whereby cytolytic lymphocytes protect themselves from killing mediated by their own cytotoxic protein, perforin, was studied. By using a competition assay, we demonstrated that the resistance of cells to perforin-mediated cytolysis is inversely correlated with their ability to absorb perforin, with tumor cells and noncytotoxic lymphocytes that are susceptible to perforin-mediated lysis being able to absorb perforin from the supernatant much better than CTL. The evidence implies that there is molecule on cytolytic lymphocytes that interferes with perforin-binding activity, resulting in the inability of perforin to lyse these cells. The molecule is most likely a surface protein or complex of proteins because its activity decreases after CTL treatment with the proteolytic enzymes trypsin and papain, and the activity can be recovered by incubation of the treated CTL cells at 37 degrees C for 6 h. The recovery can be blocked by emetine, cycloheximide, and actinomycin D, inhibitors of protein and RNA/DNA synthesis. The protein contains carbohydrate groups that play an important role in the function of the protein, as indicated by the fact that inhibition of glycosylation by tunicamycin and cleavage of sialic acid from the protein with neuraminidase result in a significant increase of perforin binding to CTL. Cross-linkage of CTL membrane proteins with glutaraldehyde and formaldehyde and blockage of the functional domains of the protein with an antiserum against CTL also inhibit the activity of this protein. Temperature-dependence studies that allow for a dissociation of the binding and pore-forming stages of perforin-mediated hemolysis suggest that the protective protein interferes at the perforin-binding stage.

Animals↗

PubMed1997

Channel formation and [Ca2+]i accumulation induced by perforin N-terminus peptides: comparison with purified perforin and whole lytic granules.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells express the pore forming protein perforin, which contributes to lymphocytotoxicity. The hallmark of perforin action is opening high-conductance transmembrane channels that enable massive influx of Ca2+ ions (deleterious to many cell types), as well as granzymes, which may trigger the apoptotic pathway. To explore the functional domains in the perforin molecule, we investigated in PN71 lymphocytes, the ability of perforin N-terminus synthetic peptides (compared to purified perforin and perforin-containing lytic granules), to cause intracellular Ca2+ ([Ca2+]i) accumulation and open transmembrane channels. To this end, we used the whole cell recording technique and Indo 1 fluorescence to measure membrane currents and [Ca2+]i, respectively. We have demonstrated that the N-terminus peptide Hu-34 (amino acids 1-34) closely resembled perforin action, reflected by [Ca2+]i accumulation and channel activity, while shorter peptides (e.g., Hu-16) generated mostly short-lived channels but no [Ca2+]i elevation. Hence, the first 34 amino acids of the perforin N-terminus sequence are sufficient for the perforin action.

Amino Acid Sequence↗

PubMed1991

Killing of cells by perforin. Resistance to killing is not due to diminished binding of perforin to the cell membrane.

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.

Animals↗

PubMed2005

An inframe perforin gene deletion in familial hemophagocytic lymphohistiocytosis is associated with perforin expression.

Familial hemophagocytic lymphohistiocytosis is an autosomal recessive disease of early childhood manifested by hypercytokinemia and organ infiltration of macrophages and activated lymphocytes, and it is characterized by a fulminant clinical course. The molecular mechanism underlying this disease appears to be a deregulation of apoptosis of activated T cells and macrophages. Approximately 20-40% of patients with familial hemophagocytic lymphohistiocytosis reported worldwide had a perforin gene mutation. We report herein a novel perforin variant in the homozygous state in an Omani boy who was diagnosed 44 days after birth. Sequence analysis of the perforin gene coding region revealed a 12-base pair deletion (codon 284-287) resulting in the deletion of four amino acids in the membrane attack complex domain of the protein. This deletion maintains the reading frame of the perforin mRNA. Both parents were heterozygotes for this molecular defect. Flow-cytometric analysis revealed intracellular perforin expression at the lower end of the normal range in the cytotoxic T cells (CD3+/CD8+) and (CD3+/CD56+) and in around 50% of the natural killer cells (CD3-/CD56+). This is an additional example of a perforin variant which is associated with a significant level of cellular perforin expression and thus confirms that drastic reduction in its expression is not a constant feature in familial hemophagocytic lymphohistiocytosis type 2.

Amino Acid Sequence↗

PubMed2000

Trypanosoma cruzi: roles for perforin-dependent and perforin-independent immune mechanisms in acute resistance.

CD8+ T cells have been shown to be required for acute resistance to infection with the protozoan parasite, Trypanosoma cruzi, the causative agent of Chagas' disease. However, to date, the mechanism by which CD8+ T cells mediate protection in vivo has not been determined. While CD8+ T cells can exhibit cytolytic function, they also secrete cytokines such as IFN-gamma, which is known to mediate protection against T. cruzi infections. To determine whether cytolysis is an important effector function in vivo, we have compared outcomes of T. cruzi infection in normal and perforin-deficient mice. Our results indicate that while perforin-dependent cytolytic mechanisms clearly make a major contribution to acute resistance to T. cruzi infection, this contribution may be strain and challenge dose-dependent, since perforin-deficient mice challenged with lower doses of a less virulent strain survived and were subsequently resistant to challenge with virulent organisms. In vivo depletion studies demonstrated that survival of perforin-deficient mice challenged with low doses of T. cruzi requires both CD4+ and CD8+ T cells and is dependent on IFN-gamma secretion. These studies document the participation of both perforin-dependent cytotoxic and perforin-independent, IFN-gamma-dependent immune mechanisms in acute resistance to T. cruzi infection.

Animals↗

PubMed1997

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

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Animals↗

PubMed2006

[Effects of 5-azaC on methylation pattern of the perforin promoter of the perforin gene in normal human T cells].

OBJECTIVE: To investigate the effects of DNA hypomethylation on mRNA and protein expression of perforin promotor in T cells. METHODS: T cells were isolated from the peripheral venous blood of healthy donors by density gradient centrifugation. CD4(+) and CD8(+) subsets were isolated using Miltenyi beads and protocols provided by the manufacturer. Where indicated the T cells were stimulated with PHA for 24 h, then treated with 5-azaC for an additional 72 h. Genomic DNA, mRNA, and protein were isolated from untreated and 5-azaC-treated T cells. Purified DNA was treated with sodium bisulfite, the desired sequences were amplified in sequential fragments using nested PCR. The amplified fragments were cloned into bacteria DH5 alpha and 5 independent clones for each of the amplified fragments were sequenced. The expression of perforin was determined using real time RT-PCR and Western blot. RESULTS: The perforin mRNA and protein in the CD4(+) and CD8(+) subsets treated with 5-azaC were significantly higher than those in the untreated subsets (P<0.05). The results of bisulfite genomic sequencing showed that the methylation of perforin promotor was significantly reduced in the treated cells compared with the untreated cells (P<0.05). CONCLUSION: The mRNA and protein expression of perforin significantly increases in the CD4(+) and CD8(+) T cells treated with 5-azaC,which is associated with DNA hypomethylation of perforin promoter in T cells.

Adult↗

PubMed1996

Negative cooperativity exhibited by the lytic amino-terminal domain of human perforin: implications for perforin-mediated cell lysis.

BACKGROUND: Cytolytic effector cells of the immune system recognize and lyse cells that carry non-self epitopes. One mechanism of cell lysis involves release of a 67-kDa pore-forming protein, perforin. The amino-terminal domain of perforin (>/= 19 residues) can account for most of the lysis activity, by a mechanism that is similar to that of holoperforin. Detailed mechanistic studies of this domain should yield useful insight into the factors underlying perforin activity in vivo. RESULTS: The mechanism of pore formation by the 22-residue amino-terminal domain of perforin was studied by kinetic and thermodynamic methods. Approximately 4 +/- 1 peptide monomers form an active pore by a mechanism that displays negative cooperativity. CONCLUSIONS: A negatively-regulated aggregation mechanism is likely to be common for pore-forming peptides. The positively-charged domain B of perforin (residues 7- 15) may mediate cooperativity through electrostatic interactions. Such a mechanism limits the number of protein molecules that are committed to any particular channel. This data supports smaller pores as the physiologically relevant aggregate, rather than the larger ring sizes identified by electron microscopy at higher, non-biological concentrations.

Humans↗

PubMed2003

The defect of the perforin granule system in cytotoxic T lymphocytes of atopic patients--are perforin reduction and hyperreleasability of clinical relevance?

Perforin-containing lytic granules are secretory lysosomes of cytotoxic lymphocytes. They act as a negative regulator of activated T cells, control immunoglobulin production, contribute to the regulation of the TH1/TH2 balance, and occupy a central role in anti-viral defense mechanisms. This review focuses on recent evidence for a fundamental defect in the lymphocytic perforin system of atopic patients, namely perforin reduction and the hyperreleasability of perforin granules. These findings are set in relation to the immune imbalance in atopy, which is characterized by a weakly restrained proliferation of allergen-specific T and B cells, a predominance of Ttype-2 cytokines, and an increased susceptibility to cutaneous infections. In the context of the wellknown defect of secretory lysosomes in mast cells and keratinocytes of atopic patients, the possibility of a cell type-independent major pathological factor in atopy is discussed: pan-cellular reduction and hyperreleasability of secretory lysosomes.

Animals↗

PubMed1999

A role for perforin in activation-induced T cell death in vivo: increased expansion of allogeneic perforin-deficient T cells in SCID mice.

Despite defective granule exocytosis, T cells from mice whose perforin gene was ablated by homologous recombination (pko mice) caused a similar degree of graft-vs-host disease as normal T cells after injection into sublethally irradiated C.B-17 SCID mice. Moreover host spleens contained significantly greater numbers of T cells from pko mice than from wild-type mice following their i.v. injection. This increase could not be explained by persistence of host APCs that were not cleared by defective donor cytotoxic effector cells. The absence of functional perforin-dependent suppressor cells or an altered cytokine profile of donor T cells could also not account for the behavior of pko cells. Spontaneous and Fas-mediated apoptosis of in vivo activated donor T cells were independent of donor origin. However, pko T blasts exhibited less growth inhibition and cell death after reactivation in vitro. The results are compatible with a model of a defective activation-induced cell death (AICD) pathway, controlled by perforin, accounting for the increased expansion of alloreactive pko T cells.

Animals↗

PubMed1999

IL-18 up-regulates perforin-mediated NK activity without increasing perforin messenger RNA expression by binding to constitutively expressed IL-18 receptor.

IL-18 is a powerful inducer of IFN-gamma production, particularly in collaboration with IL-12. IL-18, like IL-12, also augments NK activity. Here we investigated the molecular mechanism underlying the up-regulation of killing activity of NK cells by IL-18. IL-18, like IL-12, dose dependently enhanced NK activity of splenocytes. This action was further enhanced by costimulation with IL-12. Treatment with anti-IL-2R Ab did not affect IL-18- and/or IL-12-augmented NK activity, and splenocytes from IFN-gamma-deficient mice showed enhanced NK activity following stimulation with IL-12 and/or IL-18. Splenocytes from the mice deficient in both IL-12 and IL-18 normally responded to IL-18 and/or IL-12 with facilitated NK activity, suggesting that functional NK cells develop in the absence of IL-12 and IL-18. IL-18R, as well as IL-12R mRNA, was constitutively expressed in splenocytes from SCID mice, which lack T cells and B cells but have intact NK cells, and in those from IL-12 and IL-18 double knockout mice. NK cells isolated from SCID splenocytes expressed IL-18R on their surface. IL-18, in contrast to IL-12, did not enhance mRNA expression of perforin, a key molecule for exocytosis-mediated cytotoxicity. However, pretreatment with concanamycin A completely inhibited this IL-18- and/or IL-12-augmented NK activity. Furthermore, IL-18, like IL-12, failed to enhance NK activity of splenocytes from perforin-deficient mice. These data suggested that NK cells develop and express IL-12R and IL-18R in the absence of IL-12 or IL-18, and that both IL-18 and IL-12 directly and independently augment perforin-mediated cytotoxic activity of NK cells.

Animals↗

PubMed2004

Perforin knockout mice, but not mice with MAIDS, show protection against experimental cytomegalovirus retinitis after adoptive transfer of immune cells with a functional perforin cytotoxic pathway.

Adoptive transfer studies were performed to test the hypothesis that the perforin cytotoxic pathway is more important than the Fas/FasL cytotoxic pathway in protection against experimental murine cytomegalovirus (MCMV) retinitis. Splenic immune cells from donor MCMV-immunized normal mice or gld mice deficient in Fas/FasL-mediated cytotoxicity significantly reduced the frequency and severity of MCMV retinitis following subretinal MCMV challenge when transferred into recipient PKO mice deficient in perforin-mediated cytotoxicity. In sharp contrast, splenic cells from donor MCMV-immunized PKO mice failed to provide protection against MCMV retinitis when transferred into recipient PKO mice. Protection was not achieved, however, in recipient mice with retrovirus-induced immunodeficiency (MAIDS), even when splenic cells originated from MCMV-immunized normal mice.

Adoptive Transfer↗

PubMed2002

Tumor-specific CTL kill murine renal cancer cells using both perforin and Fas ligand-mediated lysis in vitro, but cause tumor regression in vivo in the absence of perforin.

Kidney cancer is a devastating disease; however, biological therapies have achieved some limited success. The murine renal cancer Renca has been used as a model for developing new preclinical approaches to the treatment of renal cell carcinoma. Successful cytokine-based approaches require CD8(+) T cells, but the exact mechanisms by which T cells mediate therapeutic benefit have not been completely identified. After successful biological therapy of Renca in BALB/c mice, we generated CTLs in vitro using mixed lymphocyte tumor cultures. These CTL mediated tumor-specific H-2K(d)-restricted lysis and production of IFN-gamma, TNF-alpha, and Fas ligand (FasL) in response to Renca. CTL used both granule- and FasL-mediated mechanisms to lyse Renca, although granule-mediated killing was the predominant lytic mechanism in vitro. The cytokines IFN-gamma and TNF-alpha increased the sensitivity of Renca cells to CTL lysis by both granule- and FasL-mediated death pathways. Adoptive transfer of these anti-Renca CTL into tumor-bearing mice cured most mice of established experimental pulmonary metastases, and successfully treated mice were immune to tumor rechallenge. Interestingly, we were able to establish Renca-specific CTL from mice gene targeted for perforin (pfp(-/-)) mice. Although these pfp(-/-) CTL showed reduced cytotoxic activity against Renca, their IFN-gamma production in the presence of Renca targets was equivalent to that of wild-type CTL, and adoptive transfer of pfp(-/-) CTL was as efficient as wild-type CTL in causing regression of established Renca pulmonary metastases. Therefore, although granule-mediated killing is of paramount importance for CTL-mediated lysis in vitro, some major in vivo effector mechanisms clearly are independent of perforin.

Animals↗