Perforin-independent Mechanism Research Articles

Reassessing granzyme B: unveiling perforin-independent versatility in immune responses and therapeutic potentials

The pivotal role of Granzyme B (GzmB) in immune responses, initially tied to cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, has extended across diverse cell types and disease models. A number of studies have challenged conventional notions, revealing GzmB activity beyond apoptosis, impacting autoimmune diseases, inflammatory disorders, cancer, and neurotoxicity. Notably, the diverse functions of GzmB unfold through Perforin-dependent and Perforin-independent mechanisms, offering clinical implications and therapeutic insights. This review underscores the multifaceted roles of GzmB, spanning immunological and pathological contexts, which call for further investigations to pave the way for innovative targeted therapies.

Granule-Derived Granzyme B Mediates the Vulnerability of Human Neurons to T Cell-Induced Neurotoxicity

Multiple sclerosis (MS) is considered an autoimmune disease of the CNS and is characterized by inflammatory cells infiltrating the CNS and inducing demyelination, axonal loss, and neuronal death. Recent evidence strongly suggests that axonal and neuronal degeneration underlie the progression of permanent disability in MS. In this study, we report that human neurons are selectively susceptible to the serine-protease granzyme B (GrB) isolated from cytotoxic T cell granules. In vitro, purified human GrB induced neuronal death to the same extent as the whole activated T cell population. On the contrary, activated T cells isolated from GrB knockout mice failed to induce neuronal injury. We found that following internalization through various parts of neurons, GrB accumulated in the neuronal soma. Within the cell body, GrB diffused out of endosomes possibly through a perforin-independent mechanism and induced subsequent activation of caspases and cleavage of α-tubulin. Inhibition of caspase-3, a well-known substrate for GrB, significantly reduced GrB-mediated neurotoxicity. We demonstrated that treatment of neurons with mannose-6-phosphate prevented GrB entry and inhibited GrB-mediated neuronal death, suggesting mannose-6-phosphate receptor-dependent endocytosis. Together, our data unveil a novel mechanism by which GrB induces selective neuronal injury and suggest potential new targets for the treatment of inflammatory-mediated neurodegeneration in diseases such as MS.

Serpina3n attenuates granzyme B-mediated decorin cleavage and rupture in a murine model of aortic aneurysm

Granzyme B (GZMB) is a proapoptotic serine protease that is released by cytotoxic lymphocytes. However, GZMB can also be produced by other cell types and is capable of cleaving extracellular matrix (ECM) proteins. GZMB contributes to abdominal aortic aneurysm (AAA) through an extracellular, perforin-independent mechanism involving ECM cleavage. The murine serine protease inhibitor, Serpina3n (SA3N), is an extracellular inhibitor of GZMB. In the present study, administration of SA3N was assessed using a mouse Angiotensin II-induced AAA model. Mice were injected with SA3N (0–120 μg/kg) before pump implantation. A significant dose-dependent reduction in the frequency of aortic rupture and death was observed in mice that received SA3N treatment compared with controls. Reduced degradation of the proteoglycan decorin was observed while collagen density was increased in the aortas of mice receiving SA3N treatment compared with controls. In vitro studies confirmed that decorin, which regulates collagen spacing and fibrillogenesis, is cleaved by GZMB and that its cleavage can be prevented by SA3N. In conclusion, SA3N inhibits GZMB-mediated decorin degradation leading to enhanced collagen remodelling and reinforcement of the adventitia, thereby reducing the overall rate of rupture and death in a mouse model of AAA.

Cytotoxic mechanisms of IL-4 polarized CTLs (101.28)

Abstract IL-4 down-regulates perforin and granzymes and depresses rapid (4 hr) cytotoxic T lymphocyte (CTL) killing. Curiously, IL-4 can increase anti-tumor immunity in animal models. We wanted to know how IL-4 polarized CTLs kill and found that they have potent delayed cytotoxicity. We cultured BALB/c mouse splenocytes with conA and 500 units/ml mouse IL-4 or IL-2 for 3 days and then re-cultured the cells for 3 days with cytokines without conA. The IL-4 CTLs were polarized as indicated by expression of the IL-4 inducible lipase PLRP2. By flow cytometry, IL-4 CTLs expressed Grz B at lower levels than IL-2 CTLs. We assayed cytotoxicity using anti-CD3-redirected death of P815 tumor cells. Death was measured at 48 hrs by loss of EGFP+ P815 cells using flow cytometry with cell count beads to account for dead cell disintegration. Cytotoxicity by the IL-4 CTLs was 80% at effector lymphocyte to P815 (E:T) ratios of 1:10. IL-4 CTLs had perforin-independent mechanisms, with similar activity for WT and Prf1-/- CTLs. We monitored T cells for death receptor ligands. Quantitative RT-PCR indicated that IL-4 cells maintained comparable levels of mRNA for LTβ, LIGHT, TRAIL and FasL, but had lower levels of TNFα and TNFβ, than IL-2 CTLs. We conclude that IL-4 polarized CTLs have potent cytotoxicity which could be mediated by death receptor ligands and/or by novel IL-4 inducible potential cytotoxins. Supported in part by NIH grants R01CA38942, T32CA09563, P20 RR-016464 & P20-RR015581.

Perforin-Independent Extracellular Granzyme B Activity Contributes to Abdominal Aortic Aneurysm

Granzyme B (GZMB) is a serine protease that is abundantly expressed in advanced human atherosclerotic lesions and may contribute to plaque instability. Perforin is a pore-forming protein that facilitates GZMB internalization and the induction of apoptosis. Recently a perforin-independent, extracellular role for GZMB has been proposed. In the current study, the role of GZMB in abdominal aortic aneurysm (AAA) was assessed. Apolipoprotein E (APOE)(-/-) x GZMB(-/-) and APOE(-/-) x perforin(-/-) double knockout (GDKO, PDKO) mice were generated to test whether GZMB exerted a causative role in aneurysm formation. To induce aneurysm, mice were given angiotensin II (1000 ng/kg/min) for 28 days. GZMB was found to be abundant in both murine and human AAA specimens. GZMB deficiency was associated with a decrease in AAA and increased survival compared with APOE-KO and PDKO mice. Although AAA rupture was observed frequently in APOE-KO (46.7%; n = 15) and PDKO (43.3%; n = 16) mice, rupture was rarely observed in GDKO (7.1%; n = 14) mice. APOE-KO mice exhibited reduced fibrillin-1 staining compared with GDKO mice, whereas in vitro protease assays demonstrated that fibrillin-1 is a substrate of GZMB. As perforin deficiency did not affect the outcome, our results suggest that GZMB contributes to AAA pathogenesis via a perforin-independent mechanism involving extracellular matrix degradation and subsequent loss of vessel wall integrity.

Pancreatic lipase-related protein 2 (PLRP2) induction by IL-4 in cytotoxic T lymphocytes (CTLs) and reevaluation of the negative effects of its gene ablation on cytotoxicity

Pancreatic lipase-related protein 2 (PLRP2) is induced by IL-4 in vitro in cytotoxic T lymphocyte (CTL) clones and CTLs from immunized wild-type (WT) PLRP2(+/+) are more cytotoxic than PLRP2(-/-) CTLs, suggesting to previous investigators that the lipase PLRP2 might support CTL functions. Here, we further evaluate PLRP2 in CTLs. We found that PLRP2 was optimally induced in splenocytes by 3.5 x 10(-8) M IL-4 by day 6 after activation and was restricted to CD8(+) T cells. PLRP2 mRNA was detected inconsistently (and at low levels) after activation in the presence of IL-2. Cytotoxicity in 4 h (51)Cr assays of WT CTLs was approximately 3-fold the activity of PLRP2(-/-) CTLs cultured with IL-4 and, with IL-2, was unexpectedly approximately 2 fold the activity of PLRP2(-/-) CTLs. Thus, PLRP2 gene ablation affected short-term (perforin-dependent) cytotoxicity, even under the IL-2 conditions. Other variables failed to account for the reduced cytotoxicity. Granzyme B levels, activation markers, and CD8(+) T cell frequencies were similar for WT vs. PLRP2(-/-) CTLs (with either cytokine). Addition of rPLRP2 to IL-4 induced PLRP2(-/-) CTLs (or to cytotoxic granule extracts) failed to increase lysis, suggesting that the missing mediator is more than released PLRP2. Cytotoxicity of WT and PLRP2(-/-) CTLs was similar in 2-day tumor survival assays with IL-4, which can be mediated by perforin-independent mechanisms. We conclude that extracellular PLRP2 lipase is unable to directly augment the cytotoxicity that was lost by PLRP2 ablation and that after reevaluation, the question of what is PLRP2's role in CD8 T cells is still unanswered.

Mapping of Quantitative Trait Loci Determining NK Cell-Mediated Resistance to MHC Class I-Deficient Bone Marrow Grafts in Perforin-Deficient Mice

NK cells reject allogeneic and MHC class I-deficient bone marrow (BM) grafts in vivo. The mechanisms used by NK cells to mediate this rejection are not yet thoroughly characterized. Although perforin plays a major role, perforin-independent mechanisms are involved as well. C57BL/6 mice deficient in perforin (B6 perforin knockout (PKO)) reject class I-deficient TAP-1 KO BM cells as efficiently as normal B6 mice. In contrast, perforin-deficient 129S6/SvEvTac mice (129 PKO) cannot mediate this rejection while normal 129 mice efficiently reject. This suggests that in 129, but not in B6, mice, perforin is crucial for NK cell-mediated rejection of MHC class I-deficient BM grafts. To identify loci linked to BM rejection in perforin-deficient mice, we generated backcross 1 progeny by crossing (129 x B6)F(1) PKO mice to 129 PKO mice. In transplantation experiments, >350 backcross 1 progeny were analyzed and displayed a great variation in ability to reject TAP-1 KO BM grafts. PCR-based microsatellite mapping identified four quantitative trait loci (QTL) on chromosomes 2, 4, and 8, with the QTL on chromosome 8 showing the highest significance, as well as a fifth epistatic QTL on chromosome 3. This study describes the first important step toward identifying BM graft resistance gene(s).

Cutting Edge: Contact-Mediated Suppression by CD4+CD25+ Regulatory Cells Involves a Granzyme B-Dependent, Perforin-Independent Mechanism

CD4+CD25+ regulatory T cells (Treg) are potent immunosuppressive cells that are pivotal in the regulation of peripheral tolerance. In this report, we identify granzyme B (GZ-B) as one of the key components of Treg-mediated suppression. Induction of regulatory activity is correlated with the up-regulation of GZ-B expression. Proof of a functional involvement of GZ-B in contact-mediated suppression by Treg is shown by the reduced ability of Treg from GZ-B-/- mice to suppress as efficiently as Treg from WT mice. GZ-B-mediated suppression is perforin independent, because suppression by Treg from perforin-/- and WT is indistinguishable. Additionally, suppression mediated by Treg appears to be mediated, in part, by the induction of apoptosis in the CD4+CD25- effector cell. In summary, GZ-B is one of the key mechanisms through which CD4+CD25+ Treg induce cell contact-mediated suppression.

NK Cells Contribute to the Control ofTrypanosoma cruziInfection by Killing Free Parasites by Perforin-Independent Mechanisms

The protozoan parasite Trypanosoma cruzi circulates in the blood as trypomastigotes and invades a variety of cells to multiply intracellularly as amastigotes. The acute phase leads to an immune response that restricts the proliferation of the parasite. However, parasites are able to persist in different tissues, which causes the pathology of Chagas' disease. Natural killer (NK) cells play an important role in innate resistance to a variety of pathogens. In the present study we analyzed whether NK cells participated in the control of experimental T. cruzi infection. NK cells were depleted from C57BL/6 mice by antiasialo antibodies. This treatment caused an increased parasitemia during the acute phase, but tissue parasite burdens were not significantly altered according to quantitative real-time PCR. Our results demonstrated that NK cells were activated during the initial phase of a T. cruzi infection and exhibited a contact-dependent antiparasitic activity against extracellular parasites that was independent from perforin. Thus, NK cells limit the propagation of the parasite by acting on circulating T. cruzi trypomastigotes.

In vivo rejection of tumor cells dependent on CD8 cells that kill independently of perforin and FasL.

Perforin/granzyme B- and Fas/FasL-mediated killing pathways are the main effector mechanisms of CTL and NK cells in antitumor immune responses. In this study, we investigated the relative role of these two lytic mechanisms in protection of the host from tumor progression, as well as spontaneous metastasis, using the D122 Lewis lung carcinoma and its gene-modified cells. Utilizing perforin knockout mice (B6-PKO) and Fas and FasL mutant (B6-MRL and B6-Smn) mice, we found that perforin expression in the host plays a crucial function in the prevention of metastasis. However, local tumor rejection of an H-2K(b) and B7-1 transfectant, 39.5-B7 cells, was not dependent either on perforin or Fas/FasL expression in vivo. In addition, CTL lysis of 39.5-B7 cells was independent of perforin and Fas/FasL interactions in 18-hour in vitro assays. We also confirmed that CD8 T-cells were responsible for rejecting 39.5-B7 local tumors, yet cytokines, TNF-alpha and gammaIFN were not involved in tumor rejection in vivo. Furthermore, blocking assays using caspase inhibitors (zVAD-fmk, zLETD-fmk and zLEHD-fmk) showed that, whereas caspase activation was partially required to induce 39.5-B7 lysis mediated by the perforin-dependent pathway, 39.5-B7 lysis by CTLs through the perforin-independent mechanism required caspase activation. Thus, these results suggested that perforin, Fas/FasL, gammaIFN and TNF-alpha independent lytic mechanisms, mediated by CD8 T cells, have a crucial role in rejection of 39.5-B7 cells in vivo. Caspase activation is a pre requisite for apoptosis of targets by CTLs.

Inhibition of the death receptor pathway by cFLIP confers partial engraftment of MHC class I-deficient stem cells and reduces tumor clearance in perforin-deficient mice.

NK cells mediate acute rejection of MHC class I-deficient bone marrow cell (BMC) grafts. However, the exact cytotoxic mechanisms of NK cells during acute BMC graft rejection are not well defined. Although the granule exocytosis pathway plays a major role in NK cell-mediated rejection, alternative perforin-independent mechanisms also exist. By analyzing the anti-apoptotic effects of cellular Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein (cFLIP) overexpression, we investigated the possible role of death receptor-induced apoptosis in NK cell-mediated cytotoxicity. In the absence of perforin, we found that cFLIP overexpression reduces lysis of tumor cells by NK cells in vitro and in vivo. In addition, perforin-deficient NK cells were impaired in their ability to acutely reject cFLIP-overexpressing TAP-1 knockout stem cells. These results emphasize the importance of NK cell death receptor-mediated killing during BMC grafts in the absence of perforin.

Neural Route of CerebralListeria monocytogenesMurine Infection: Role of Immune Response Mechanisms in Controling Bacterial Neuroinvasion

The pathologic features of cerebral Listeria monocytogenes infection strongly suggest that besides hematogenous spread, bacteria might also spread via a neural route. We propose that after snout infection of recombination activating gene 1 (RAG-1)-deficient mice, L. monocytogenes spreads to the brain via a neural route. The neural route of invasion is suggested by (i) the immunostaining of L. monocytogenes in the trigeminal ganglia (TG) and brain stem but not in other areas of the brain; (ii) the kinetics of bacterial loads in snout, TG, and brain; and (iii) the increased resistance of mice infected with a plcB bacterial mutant (unable to spread from cell to cell). Gamma interferon (IFN-gamma) plays a protective role in neuroinvasion; inducible nitric oxide synthase (iNOS) accounts only partially for the protection, as shown by a comparison of the susceptibilities of IFN-gamma receptor (IFN-gamma R)-deficient, iNOS-deficient, and wild-type mice to snout infection with L. monocytogenes. The dramatically enhanced susceptibility of RAG-1-deficient, IFN-gamma R gene-deficient mice indicated the overall importance of innate immune cells in the release of protective levels of IFN-gamma. The source of IFN-gamma appeared to be NK cells, as shown by use of RAG-1-deficient, gamma-chain receptor gene-deficient mice; NK cells played a relevant protective role in neuroinvasion through a perforin-independent mechanism. In vitro evidence indicated that IFN-gamma can directly induce bacteriostatic mechanisms in neural tissue.

Altered pathological progression of diaphragm and quadriceps muscle in TNF-deficient, dystrophin-deficient mice.

We have previously demonstrated a role for T cells in Duchenne muscular dystrophy (DMD) using the mdx mouse and have shown that T cell killing of dystrophic muscle can occur through perforin-dependent and perforin-independent mechanisms. In this investigation, we explore the possibility that one perforin-independent mechanism utilized by the T cells is cytokine-based killing, specifically by tumor necrosis factor (TNF). We tested this hypothesis by generating mice that are TNF-deficient and dystrophin-deficient (TNF-/mdx). Body mass and muscle mass of the TNF-/mdx mice were significantly less than TNF+/mdx mice at 8 weeks of age. Creatine kinase levels and overall muscle strength were unchanged. Histopathology measurements showed different results in the diaphragm and quadriceps muscles. These data suggest that removal of TNF in vivo in dystrophic mice has differential effects on diaphragm and quadriceps suggesting that TNF is an unfavorable target for immunotherapy for DMD.

Eosinophilia of Dystrophin-Deficient Muscle Is Promoted by Perforin-Mediated Cytotoxicity by T Cell Effectors

Previous investigations have shown that cytotoxic T lymphocytes (CTLs) contribute to muscle pathology in the dystrophin-null mutant mouse (mdx) model of Duchenne muscular dystrophy through perforin-dependent and perforin-independent mechanisms. We have assessed whether the CTL-mediated pathology includes the promotion of eosinophilia in dystrophic muscle, and thereby provides a secondary mechanism through which CTLs contribute to muscular dystrophy. Quantitative immunohistochemistry confirmed that eosinophilia is a component of the mdx dystrophy. In addition, electron microscopic observations show that eosinophils traverse the basement membrane of mdx muscle fibers and display sites of close apposition of eosinophil and muscle membranes. The close membrane apposition is characterized by impingement of eosinophilic rods of major basic protein into the muscle cell membrane. Transfer of mdx splenocytes and mdx muscle extracts to irradiated C57 mice by intraperitoneal injection resulted in muscle eosinophilia in the recipient mice. Double-mutant mice lacking dystrophin and perforin showed less eosinophilia than was displayed by mdx mice that expressed perforin. Finally, administration of prednisolone, which has been shown previously to reduce the concentration of CTLs in dystrophic muscle, produced a significant reduction in eosinophilia. These findings indicate that eosinophilia is a component of the mdx pathology that is promoted by perforin-dependent cytotoxicity of effector T cells. However, some eosinophilia of mdx muscle is independent of perforin-mediated processes.

IFN-γ Is Required for Viral Clearance from Central Nervous System Oligodendroglia

Abstract Infection of the central nervous system (CNS) by the JHM strain of mouse hepatitis virus (JHMV) is a rodent model of the human demyelinating disease multiple sclerosis. The inability of effective host immune responses to eliminate virus from the CNS results in a chronic infection associated with ongoing recurrent demyelination. JHMV infects a variety of CNS cell types during the acute phase of infection including ependymal cells, astrocytes, microglia, oligodendroglia, and rarely in neurons. Replication within the majority of CNS cell types is controlled by perforin-dependent virus-specific CTL. However, inhibition of viral replication in oligodendroglia occurs via a perforin-independent mechanism(s). The potential role for IFN-γ as mediator controlling JHMV replication in oligodendroglia was examined in mice deficient in IFN-γ secretion (IFN-γ0/0 mice). IFN-γ0/0 mice exhibited increased clinical symptoms and mortality associated with persistent virus, demonstrating an inability to control replication. Neither antiviral Ab nor CTL responses were diminished in the absence of IFN-γ, although increased IgG1 was detected in IFN-γ0/0 mice. Increased virus Ag in the absence of IFN-γ localized almost exclusively to oligodendroglia and was associated with increased CD8+ T cells localized within white matter. These data suggest that although perforin-dependent CTL control virus replication within astrocytes and microglia, which constitute the majority of infected CNS cells, IFN-γ is critical for control of viral replication in oligodendroglia. Therefore, different mechanisms are used by the host defenses to control virus replication within the CNS, dependent upon the phenotype of the targets of virus replication.

Fas (CD95)-dependent cell-mediated immunity to Listeria monocytogenes.

Two distinct and complementary pathways, one mediated by perforin and the other dependent upon CD95 (Fas), effect cell-mediated cytotoxicity. We examined the relative roles of these pathways in host defenses against the intracellular bacterial pathogen Listeria monocytogenes by using murine listeriosis as a model system. Mice which lacked both perforin and Fas (P0L0) were generated, and their responses to primary and secondary listeriosis were compared to those of wild-type (WT), Fas-deficient (L0), and perforin knockout (P0) mice. Relative to WT mice during primary listeriosis, P0 mice exhibited a reduced capacity to clear the infection from their spleens but not their livers whereas L0 mice had elevated bacterial titers in their livers and a modestly increased titer in their spleens. In contrast, bacterial titers in P0L0 mice were increased approximately 50- to 560-fold in their spleens and 230- to 1, 000-fold in their livers; eventual clearance of listeriae from both organs was significantly delayed. Furthermore, the resistance of P0L0 mice to secondary listeriosis was significantly reduced in their spleens and livers compared to that of WT, P0, or L0 mice. In vitro experiments indicated that immune cytotoxic T lymphocytes (CTL) lysed L. monocytogenes-infected hepatocytes primarily via a Fas-dependent, perforin-independent mechanism. The absence of Fas severely abrogated the lysis of infected hepatocytes by immune CD8(+) CTL. Taken together, these results provide the first evidence for Fas-dependent CTL-mediated lysis of L. monocytogenes-infected hepatocytes and demonstrate complementary roles for Fas and perforin in host defenses against an intracellular bacterial pathogen.

Fas (CD95/Apo-1)-mediated damage to ventricular myocytes induced by cytotoxic T lymphocytes from perforin-deficient mice: a major role for inositol 1,4,5-trisphosphate.

Cytotoxic T lymphocytes (CTLs) that infiltrate the heart are important immune effectors implicated in heart transplant rejection, myocarditis, and other cardiomyopathies. To investigate the mechanism(s) underlying CTL damage to the myocardium through activation of the Fas receptor (Fas/CD95/Apo-1) by the Fas ligand, we explored the interaction between peritoneal exudate CTLs (PELs), derived from perforin gene-knockout (P-/-) mice, and murine ventricular myocytes. Fas expression on isolated ventricular myocytes was demonstrated immunohistochemically. Action potentials, [Ca2+]i transients, and contractions of myocytes conjugated to P-/- PELs or treated with the apoptosis-inducing anti-Fas monoclonal antibody Jo2 were recorded. Action potential characteristics of nonconjugated myocytes and myocytes conjugated with P-/- PELs were, respectively, as follows: Vm, -73.2+/-1.5 and -53.6+/-6.4 mV (mean+/-SEM); action potential amplitude, 117.9+/-3.9 and 74.3+/-21.2 mV; and action potential duration at 80% repolarization, 17+/-6 and 42+/-13 milliseconds (all P<.05). P-/- PELs also induced early and delayed afterdepolarizations as well as arrhythmogenic activity. Diastolic [Ca2+]i increased during the cytocidal interaction with P-/- PELs, from a fluorescence ratio of 0.82+/-0.05 (n=7) to 1.98+/-0.09 (n=13) (P<.05). All of the effects caused by P-/- PELs were reproduced by incubating the myocytes with Jo2. Heparin (50 microg/mL), an antagonist of inositol trisphosphate (IP3)-operated sarcoplasmic reticulum Ca2+ channels, or U-73122 (2 micromol/L), a phospholipase C inhibitor, but not the inactive agonist U-73343, prevented Fas-mediated myocyte dysfunction. Additionally, intracellular application (through the patch pipette) of the active IP3 analogue, inositol 1,4,5-trisphosphate, but not the inactive analogue, inositol 1,3,4-trisphosphate, caused electrophysiological changes resembling those resulting from P-/- PELs and Jo2, suggesting that CTL-induced Fas-based myocyte dysfunction is mediated by IP3. We conclude that a Fas-based perforin-independent mechanism of CTL action can account for the immunopathology seen in the allotransplanted heart, myocarditis, and dilated cardiomyopathy.

Fas- and perforin-independent mechanism of cytotoxic T lymphocyte.

Cytotoxic T lymphocytes (CTLs) play an important role in elimination of virus-infected cells (1). Recent studies revealed at least two distinct mechanisms that CTLs utilize to destroy their target cells. Both mechanisms induce target cell apoptosis specifically and directionally, but these processes are totally different. One is pore formation on target cell membrane by perforin secreted from CTLs (perforin-granzyme pathway), and the other is ligation of Fas, which is expressed on the surface of target cells and Fas ligand, on the surface of CTLs (Fas-FasL pathway) (2). Here we review our work and describe CTL clones that have novel lytic mechanisms derived from CD4-CD8- lymph node cells of gld mice.

Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes

To study the contribution of putative perforin-independent mechanism In the antigen-specific target destruction by cytotoxic T lymphocytes, CD8 + CTL lines were established from spleen cells of chimeric mice produced by injecting perforin (-/-) embryonic stem cells into blastocysts of RAG-2 (-/-) mice. When tested on normal concanavalin A blasts, these perforin-deficient cytotoxic T lymphocyte lines were found to be capable of inducing antigen-specific target cell lysis accompanied by DNA degradation. In contrast, with target cells carrying a mutation in Fas molecule, perforin-independent cytotoxicity was not detectable. These data not only confirmed the primary role of perforin but simultaneously revealed a major contribution of a perforin-independent Fas-mediated pathway in antigen-specific cytolysis.

Antigen-specific directional target cell lysis by perforin-negative T lymphocyte clones

Despite a number of reports indicating that perforin, a pore-forming protein, is the primary effector molecule mediating specific target cell lysis by cytotoxic T lymphocytes (CTL), several lines of evidence suggest the existence of perforin-independent mechanisms. We established class II-restricted, soluble protein-specific CD4+ T cell clones with killing function which do not express a detectable amount of perforin and perforin mRNA. Nevertheless, these clones induced cytolysis and DNA fragmentation of target cells in a specific and highly directional manner which was not inhibitable by antibody against TNF/lymphotoxin. These data not only indicate the existence of cytotoxic T cell subsets which do not utilize perforin, but also suggest that perforin is not mandatory for specific target lysis by T cells.