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PubMed2001

Activation of natural killer cells with interleukin 2 (IL-2) and IL-12 increases perforin binding and subsequent lysis of tumour cells.

Natural killer (NK) cells can lyse a variety of different tumour cells by exocytosis of perforin, subsequent binding of perforin to the target cell membrane and formation of lytic pores. Some tumour cells, however, are resistant to cellular cytotoxicity. Using the NK-resistant tumour cell lines ML-2, MONOMAC-1, RPMI and L540Cy, we demonstrated that activation of NK cells with interleukin 2 (IL-2) and IL-12 resulted in significant lysis of these tumour targets. To investigate the underlying mechanisms, we isolated the cytotoxic granules from non-activated and IL-2-/IL-12-activated NK cells and compared the killing of K562 leukaemia cells (sensitive to NK cell-mediated lysis) and ML-2 leukaemia cells (resistant to NK cell-mediated lysis). In contrast to K562 cells, which were easily killed by NK-cell granules, ML-2 cells were resistant to granules from non-activated NK cells. However, granules from NK cells activated with IL-2 and IL-12 were able to induce significant tumour cell lysis. Cell death of both K562 and ML-2 cells by granules from activated NK cells was completely blocked by anti-perforin antibodies, indicating that perforin mainly accounts for the lysis induced by NK granules. Comparing granules from non-activated and IL-2-/IL-12-activated NK cells, the increased cell death of ML-2 cells was caused by an improved binding of perforin to the target cell membrane. Functional assays, however, indicated that the differences in perforin binding were not as a result of an augmented production of perforin by activated NK cells. We conclude that activation of NK cells results in an increased binding of perforin and subsequent lysis of tumour cells.

Antibodies, Monoclonal↗

PubMed2002

Evidence for a perforin-mediated mechanism controlling cardiac inflammation in Trypanosoma cruzi infection.

CD8+ T lymphocytes are considered an important cell population involved in the control of parasitaemia and mortality after Trypanosoma cruzi infection. However, despite recent developments in this field, the mechanism whereby this control is exerted is still not completely understood. Here we have used perforin knockout (-/-) mice infected with Y strain T. cruzi in order to evaluate specifically the participation of the perforin-based cytotoxic pathway in the destruction of cardiomyocytes, cellular inflammatory infiltration, and control of parasitaemia and mortality. We observed that although parasitaemia was equivalent in perforin (+/+) and (-/-) groups, survival rate and spontaneous physical performance were significantly lower in the perforin deficient mice. The cardiac inflammatory cell infiltration, mostly composed of CD8+ cells, was more evident in perforin (-/-) mice. Ultrastructural and immunofluorescence analysis, as well as plasma creatine kinase activity, revealed cardiomyocyte damage and necrosis, more evident in perforin (-/-) mice. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assays performed in heart samples revealed similar and modest levels of apoptosis in both perforin (+/+) and (-/-) mice. These results indicate that perforin does not play a pivotal role in the control of parasitaemia and direct lysis of cardiomyocytes, but seems to be an important molecule involved in the control of cardiac inflammation and pathology induced by a highly virulent strain of T. cruzi.

Animals↗

PubMed1998

Interleukin-12 and perforin mRNA expression is augmented in blood mononuclear cells in multiple sclerosis.

Cytokines are suggested to orchestrate an abnormal immune response in multiple sclerosis (MS). The regulatory cytokine interleukin (IL)-12 induces T-helper (Th) cell switch to the Th1 type and the production by cytotoxic T cells of perforin, a cell lysis-inducing factor. It has been suggested that Th1-like cytokines may promote the development of MS, and the production of perforin to induce oligodendrocyte damage. In-situ hybridization with radiolabelled synthetic oligonucleotide probes was used to detect and enumerate mononuclear cells (MNC) expressing IL-12 and perforin mRNA in blood and cerebrospinal fluid (CSF) from patients with MS and controls. Plasma and CSF levels of IL-12 (p70) were evaluated by ELISA. Higher numbers of IL-12 and perforin mRNA-expressing MNC were registered in blood in MS and also in controls with aseptic meningoencephalitis (AM) compared to healthy subjects. There were a few patients with other non-inflammatory neurological diseases who also had high levels of IL-12 or perforin mRNA expressing blood MNC. A parallel elevation was observed for IL-12 (p70) concentrations in plasma. In the MS patients' CSF, there was a further augmentation of IL-12 mRNA expressing MNC. To evaluate autoantigen-induced IL-12 and perforin mRNA expression, blood MNC were cultivated +/- myelin basic protein (MBP), a candidate autoantigen in MS. Higher numbers of MBP-reactive IL-12 and perforin mRNA expressing blood MNC were detected in MS than controls. The augmentation of both IL-12 and perforin in MS might reflect ongoing inflammatory processes in MS and could represent targets for future treatments.

Adult↗

PubMed1998

Efficient nuclear targeting of granzyme B and the nuclear consequences of apoptosis induced by granzyme B and perforin are caspase-dependent, but cell death is caspase-independent.

The secretory lysosomes of cytolytic lymphocytes house the principal apoptotic molecules for eliminating virus-infected cells: a membranolytic agent, perforin, and the serine protease, granzyme B. Perforin allows granzyme B access to cytosolic and nuclear substrates that, when cleaved, result in the characteristic apoptotic phenotype. Key among these substrates is a family of cytoplasmic caspases that mediate cell suicide. We have examined the caspase dependence of several nuclear and cytoplasmic parameters of apoptosis induced by purified perforin and granzyme B. Cell membrane leakage in response to perforin and granzyme B was independent of caspase activation; however, nuclear events such as DNA fragmentation and nuclear condensation and disintegration were abolished by the broad-acting caspase inhibitor, z-VAD-fmk. Despite being spared from nuclear damage, z-VAD-fmk-treated cells exposed to both cytotoxins uniformly died when they were re-cultured, while cells exposed to perforin or granzyme alone survived and proliferated as readily as untreated cells. Pretreatment of cells with z-VAD-fmk also resulted in reduced granzyme B nuclear uptake following addition of perforin; however, its uptake into the cytoplasm in the absence of perforin was unaffected. We conclude that cell death in response to perforin and granzyme B does not require caspase activation and still proceeds efficiently through non-nuclear pathways when nuclear substrate cleavage is inhibited.

Amino Acid Chloromethyl Ketones↗

PubMed1989

In vivo expression of perforin by CD8+ lymphocytes during an acute viral infection.

CTL and NK cells cultured in vitro have been shown to contain a cytolytic pore-forming protein (PFP/perforin/cytolysin). To date, it has not been determined whether perforin is expressed by CTL that have been primed in vivo. Here, we have infected mice with two strains of lymphocytic choriomeningitis virus (LCMV), one of which mainly produces choriomeningitis and, the other, hepatitis. Brain and liver cryostat sections obtained from LCMV-infected mice were stained for various lymphocyte markers, including perforin. We were able to detect a large accumulation of perforin antigen in CD8+/Thy-1+/asialo GM1+/CD4- lymphocytes, which in fact represent the main infiltrating cell type found in brain and liver sections obtained during the late acute stage of LCMV infection. Perforin was also detected in a smaller population of CD8-/asialo GM1+/NK 1.1+/F480- cells, presumably corresponding to NK cells. Perforin-positive cells were found to have the morphology of blasts or large granular lymphocytes (LGL). These observations, together with in vitro studies performed in the past, indicate that perforin may be associated exclusively with LGL-like CTL blasts and NK cells. Our results demonstrate for the first time the presence of perforin in CTL that have been primed in vivo and suggest that perforin-positive CTL may be directly involved in producing the immunopathology associated with the LCMV infection.

Acute Disease↗

PubMed1996

Development of insulitis without diabetes in transgenic mice lacking perforin-dependent cytotoxicity.

It is widely accepted that T cells play an important role in the destruction of beta cells leading to autoimmune type I diabetes, but the involved effector mechanisms have remained unclear. We addressed this issue by testing the role of perforin-dependent cytotoxicity in a disease model involving transgenic mice expressing glycoprotein of lymphocytic choriomeningitis virus (LCMV-GP) in the beta cells of the endocrine pancreas. In such mice, LCMV infection leads to a potent LCMV-GP-specific T cell response resulting in rapid development of diabetes. We report here that in perforin-deficient LCMV-GP transgenic mice, LCMV infection failed to induce diabetes despite the activation of LCMV-GP-specific T cells. Deletion of nu beta 6+ T cells in Mls-1a perforin-deficient mice and the activation of LCMV-GP-specific T cells in perforin-deficient LCMV-GP transgenic mice, however, indicated that thymic tolerance induction by negative selection was not affected by the disruption of the perforin gene and that there is no fundamental difference between the T cell repertoires of normal control and perforin-deficient mice. In addition, adoptive transfer of LCMV-GP-specific TCR transgenic perforin-deficient T cells activated by LCMV-GP recombinant vaccinia virus led to marked insulitis with infiltration of CD4+ and CD8+ T cells without the development of diabetes. These findings indicate that perforin-dependent cytotoxicity is not required for the initiation of insulitis but is crucial for the destruction of beta cells in the later phase of the disease process. Other mechanisms or soluble factors present in the inflammatory islet infiltrate apparently lack the ability to efficiently induce diabetogenic beta cell damage.

Animals↗

PubMed1997

Granzyme B (GraB) autonomously crosses the cell membrane and perforin initiates apoptosis and GraB nuclear localization.

Granzyme B (GraB) induces apoptosis in the presence of perforin. Perforin polymerizes in the cell membrane to form a nonspecific ion pore, but it is not known where GraB acts to initiate the events that ultimately lead to apoptosis. It has been hypothesized that GraB enters the target cell through a perforin channel and then initiates apoptosis by cleaving and activating members of the ICE/Ced-3 family of cell death proteases. To determine if GraB can enter the cell, we treated YAC-1 or HeLa cells with FITC-labeled GraB and measured intracellular fluorescence with a high sensitivity CCD camera and image analyzer. GraB was internalized and found diffusely dispersed in the cell cytoplasm within 10 min. Uptake was inhibited at low temperature (4 degrees C) and by pretreatment with metabolic inhibitors, NaF and DNP, or cytochalasin B, a drug that both blocks microfilament formation, and FITC-GraB remained on the cell membrane localized in patches. With the simultaneous addition of perforin and FITC-GraB, no significant increase in cytoplasmic fluorescence was observed over that found in cells treated only with FITC-GraB. However, FITC-GraB was now detected in the nucleus of apoptotic cells labeling apoptotic bodies and localized areas within and along the nuclear membrane. The ability of GraB to enter cells in the absence of perforin was reexamined using anti-GraB antibody immunogold staining of ultrathin cryosections of cells incubated with GraB. Within 15 min, gold particles were detected both on the plasma membrane and in the cytoplasm of cells with some gold staining adjacent to the nuclear envelope but not in the nucleus. Cells internalizing GraB in the absence of perforin appeared morphologically normal by Hoechst staining and electron microscopy. GraB directly microinjected into the cytoplasm of B16 melanoma cells induced transient plasma membrane blebbing and nuclear coarsening but the cells did not become frankly apoptotic unless perforin was added. We conclude that GraB can enter cells autonomously but that perforin initiates the apoptotic process and the entry of GraB into the nucleus.

Animals↗

PubMed2004

The functional basis for hemophagocytic lymphohistiocytosis in a patient with co-inherited missense mutations in the perforin (PFN1) gene.

About 30% of cases of the autosomal recessive immunodeficiency disorder hemophagocytic lymphohistiocytosis are believed to be caused by inactivating mutations of the perforin gene. We expressed perforin in rat basophil leukemia cells to define the basis of perforin dysfunction associated with two mutations, R225W and G429E, inherited by a compound heterozygote patient. Whereas RBL cells expressing wild-type perforin (67 kD) efficiently killed Jurkat target cells to which they were conjugated, the substitution to tryptophan at position 225 resulted in expression of a truncated ( approximately 45 kD) form of the protein, complete loss of cytotoxicity, and failure to traffic to rat basophil leukemia secretory granules. By contrast, G429E perforin was correctly processed, stored, and released, but the rat basophil leukemia cells possessed reduced cytotoxicity. The defective function of G429E perforin mapped downstream of exocytosis and was due to its reduced ability to bind lipid membranes in a calcium-dependent manner. This study elucidates the cellular basis for perforin dysfunctions in hemophagocytic lymphohistiocytosis and provides the means for studying structure-function relationships for lymphocyte perforin.

Animals↗

PubMed2006

Effect of antibody to CD134 on perforin-mediated cytolysis in human peripheral blood mononuclear cells.

CD134 (OX40) is a new member of the tumor necrosis factor receptor (TNFR) superfamily. The influence of CD134 on perforin-mediated cytolysis remains obscure. In this study, human peripheral blood mononuclear cells (PBMC) were induced with phytohemagglutinin (50 microg/mL), by using anti-CD134 blocking monoclonal antibody (MAb; 1, 5, and 10 microg/mL) for 6, 12, 24, and 48 h. Perforin-mediated cytolysis against human erythrocytes of PBMC was detected. The expression of perforin mRNA and protein was also measured by reverse transcription-polymerase chain reaction and flow cytometry. Our data showed that the perforin-mediated cytolysis of PBMC was downregulated by various concentrations of anti-CD134 blocking MAb for different times and reached a minimum at 24 h at any concentration. Anti-CD134 blocking MAb induced downregulation of perforin-mediated cytolysis of PBMC in a dose-dependent manner in the range of 1-5 microg/mL. Perforin- mediated cytolysis of PBMC reached a plateau when the concentration of anti-CD134 blocking MAb exceeded 5 microg/mL. Anti-CD134 blocking MAb also induced the inhibition of the expression of perforin protein and mRNA in PBMC. In conclusion, anti-CD134 blocking MAb inhibits perforin-mediated cytolysis of PBMC by, at least in some part, decreasing the production of preforin, which may be useful in treating several autoimmune diseases.

Antibodies, Blocking↗

PubMed2006

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.

Animals↗

PubMed2004

Perforin expression in peripheral blood lymphocytes and skin-infiltrating cells in patients with lichen planus.

BACKGROUND: Current evidence suggests that lichen planus is a T-cell-mediated autoimmune disease in which cytotoxic mechanisms have been poorly investigated. OBJECTIVES: We investigated the expression of perforin in subpopulations of peripheral blood lymphocytes (PBL) in exacerbation and remission phases of the disease as well as in skin lesions. METHODS: We performed a simultaneous detection of perforin (intracellular molecule) and cell surface antigens on PBL by flow cytometry, and skin lesions were investigated by immunohistochemistry. RESULTS: The most interesting finding was a significant increase of perforin expression in cytotoxic T lymphocytes (CD3+ perforin+ cells) in the exacerbation phase of disease (P < 0.05), which was mostly located in the CD8+ subpopulation (CD8+ perforin+) (P < 0.01). Using immunohistochemistry we confirmed the infiltration of T lymphocytes in skin lesions, especially of CD4+ and CD8+ phenotypes, compared with uninvolved (P < 0.05) and healthy skin (P < 0.01). The expression of perforin was also significantly higher in lesional skin compared with nonlesional and healthy skin (P < 0.05). CONCLUSIONS: Our results clearly show the upregulation of perforin expression in peripheral blood as well as in lesions of patients with lichen planus and therefore suggest an important role for perforin in this autoimmune disease.

Adult↗

PubMed1999

Progesterone directly and indirectly affects perforin expression in cytolytic cells.

PROBLEM: Decidual lymphocytes (DL) expressing the cytolytic molecule perforin represent approximately 55% of DL in the first trimester of human pregnancy. Progesterone dominates this phase of pregnancy and controls the production of uterine cytokines and growth factors. The aim of this study was to investigate the role of progesterone and progesterone-induced blocking factor (PIBF) on perforin expression in DL and peripheral blood lymphocytes (PBL). METHOD OF STUDY: Perforin expression was analyzed in PBL and DL incubated either in culture medium or with decidual adherent cells (DAC) and peripheral blood adherent cells (PBAC) and their supernatants with or without progesterone or PIBF. Perforin was detected by flow cytometry in PB and in decidual first trimester pregnancy lymphocytes. RESULTS: Progesterone in high concentrations directly affects perforin expression in DL but not in PBL. Progesterone in a concentration dependent manner indirectly blocks perforin expression in DL and PBL cultured with adherent cells or their supernatants. PIBF blocked upregulation of perforin expression of DL cultured with DAC, but none of those cultured with PBAC. Similarly, PIBF was inefficient when PBL or DL were cultured with PBAC. CONCLUSION: Progesterone present in a high concentration locally at the maternal-fetal interface modulates perforin expression in the first trimester pregnancy DL.

Cells, Cultured↗

PubMed2006

CD8+ T cells require perforin to clear West Nile virus from infected neurons.

Injury to neurons after West Nile virus (WNV) infection is believed to occur because of viral and host immune-mediated effects. Previously, we demonstrated that CD8+ T cells are required for the resolution of WNV infection in the central nervous system (CNS). CD8+ T cells can control infection by producing antiviral cytokines (e.g., gamma interferon or tumor necrosis factor alpha) or by triggering death of infected cells through perforin- or Fas ligand-dependent pathways. Here, we directly evaluated the role of perforin in controlling infection of a lineage I New York isolate of WNV in mice. A genetic deficiency of perforin molecules resulted in higher viral burden in the CNS and increased mortality after WNV infection. In the few perforin-deficient mice that survived initial challenge, viral persistence was observed in the CNS for several weeks. CD8+ T cells required perforin to control WNV infection as adoptive transfer of WNV-primed wild-type but not perforin-deficient CD8+ T cells greatly reduced infection in the brain and spinal cord and enhanced survival of CD8-deficient mice. Analogous results were obtained when wild-type or perforin-deficient CD8+ T cells were added to congenic primary cortical neuron cultures. Taken together, our data suggest that despite the risk of immunopathogenesis, CD8+ T cells use a perforin-dependent mechanism to clear WNV from infected neurons.

Animals↗

PubMed2004

The perforin mediated apoptotic pathway in lung injury and fibrosis.

AIMS: The perforin mediated pathway is the major pathway of cytotoxicity induced by activated T cells and natural killer cells, and may be involved in the development of pulmonary fibrosis. METHODS: Perforin and granzyme B expression were examined in idiopathic pulmonary fibrosis by means of immunohistochemistry, and perforin knockout mice were used to examine whether or not perforin mediated cytotoxicity participates in the pathophysiology of bleomycin induced pneumopathy. RESULTS: Perforin and granzyme B expression were upregulated in infiltrating lymphocytes in lung tissue from patients with idiopathic pulmonary fibrosis compared with normal lung parenchyma. Perforin and granzyme B expression were upregulated predominantly in infiltrating mononuclear cells after bleomycin instillation in wild-type mice. Although the development of bleomycin induced pneumopathy was not completely prevented, the pathological grade of inflammation and fibrosis, and the number of apoptotic cells in lung tissue, were significantly decreased in perforin knockout mice compared with wild-type mice. CONCLUSIONS: These results suggest that perforin mediated apoptosis may be associated with the pathophysiology of lung injury and fibrosis.

Animals↗

PubMed1996

Differential expression of perforin in muscle-infiltrating T cells in polymyositis and dermatomyositis.

Polymyositis (PM) and dermatomyositis (DM) are the prototypical inflammatory diseases of skeletal muscle. In PM, CD8+ T cells invade and destroy muscle fibers, whereas humoral effector mechanisms prevail in DM. We studied the expression of the cytotoxic mediator perforin in inflammatory cells in PM and DM muscle by semiquantitative PCR, immunohistochemistry and confocal laser microscopy. Similar levels of perforin mRNA were expressed in PM and DM, and abundant perforin-expressing CD3+CD8+ and CD3+ CD4+ T cells were observed in both diseases. However, there was a striking difference in the intracellular localization of perforin. In DM, perforin was distributed randomly in the cytoplasm of the inflammatory T cells. In contrast, 43% of the CD8+ T cells that contacted a muscle fiber in PM showed perforin located vectorially towards the target muscle fiber. The results suggest (a) that the random distribution of perforin in the cytoplasm of muscle-infiltrating T cells observed in DM reflects nonspecific activation, and (b) that the vectorial orientation observed only in PM reflects the specific recognition via the T cell receptor of an antigen on the muscle fiber surface, pointing to a perforin- and secretion-dependent mechanism of muscle fiber injury.

Base Sequence↗

PubMed2003

Chronic active Epstein-Barr virus infection associated with mutations in perforin that impair its maturation.

Chronic active Epstein-Barr virus infection (CAEBV) is a rare disease in which previously healthy persons develop severe, life-threatening illness. Mutations in the perforin gene have been found in familial hemophagocytic lymphohistiocytosis, which shares some features with CAEBV. We studied a patient who died at age 18, 10 years after the onset of CAEBV. The patient had high titers of antibodies to EBV, EBV RNA in lymph nodes, T-cell lymphoproliferative disease, and hemophagocytic lymphohistiocytosis. DNA sequencing showed novel mutations in both alleles of the perforin gene that resulted in amino acid changes in the protein. The quantity of the native form of perforin from the patient's stimulated peripheral blood mononuclear cells (PBMCs) was extremely low and immunoblotting showed accumulation of an uncleaved precursor form of perforin. Stimulated PBMCs from the patient were defective for Fas-independent cytotoxicity. These data imply that mutations in this patient resulted in reduced perforin-mediated cytotoxicity by his lymphocytes. This is the first case in which perforin mutations have been shown to result in accumulation of the uncleaved, immature form of perforin. Mutations in the perforin gene are associated with some cases of CAEBV with hemophagocytic lymphohistiocytosis.

Adolescent↗

PubMed2005

A proportion of patients with lymphoma may harbor mutations of the perforin gene.

Perforin mutations have been demonstrated in a proportion of patients diagnosed with the familial form of hemophagocytic lymphohistiocytosis (HLH). In the present study, we evaluated whether some patients with lymphoma sharing clinical characteristics with HLH might harbor mutations of the perforin gene. We analyzed 29 patients and found that 4 patients, who developed either Hodgkin or non-Hodgkin lymphoma, had biallelic mutations of the perforin gene. One of these 4 patients, a 19-year-old female with T-cell lymphoma, had a brother carrying the same mutations who developed HLH. In 2 of the 4 patients with biallelic mutations of the perforin gene, we evaluated perforin expression by flow cytometry and natural killer (NK) activity and both were found to be absent. Moreover, we documented the presence of monoallelic mutations of the perforin gene in 4 more patients. One of these 4 latter patients also carried a mutation of the Fas gene. These data indicate that perforin deficiency, either alone or in combination with other mutations of genes involved in lymphocyte survival or functional activity, may be present in patients with lymphoma. These findings suggest that perforin also plays a key role in the mechanisms of immune surveillance that prevent tumor growth and/or development.

Adolescent↗

PubMed2005

A single amino acid change, A91V, leads to conformational changes that can impair processing to the active form of perforin.

Mutations in the perforin gene have been found in patients with hemophagocytic lymphohistiocytosis (HLH), a rare autosomal recessive disease. We describe a patient expressing perforin with amino acid changes A91V and W374X. The ability of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells to lyse target cells is greatly reduced. Here we demonstrate that perforin from this patient is not recognized using an antibody raised against native perforin (deltaG9), but is readily detected using an antibody raised against a peptide epitope (2d4), suggesting that the epitope recognized by deltaG9 is destroyed by the change at A91V. Immunoblotting reveals no protein corresponding to the truncated transcript encoded by W374X, revealing that only perforin with the A91V change is expressed in CTLs from the patient. Patient CTLs show bands corresponding to the immature and intermediate forms of perforin, but the mature active form of perforin is absent or barely detectable. The conformational changes and impaired cleavage of A91V perforin are likely to explain the reduced cytotoxicity in CTLs and NK cells from this patient and are likely to contribute to the pathogenesis of HLH.

Child, Preschool↗