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PubMed1995

Analysis of perforin expression in human peripheral blood lymphocytes, CD56+ natural killer cell subsets and its induction by interleukin-2.

In this study new evidence is obtained by the use of an anti human perforin monoclonal antibody (mAb), anti P1, concerning the number of perforin positive cells in human peripheral blood lymphocytes (PBL). It is shown that about 23% of PBL is perforin positive and that this percent increases by the treatment in RPMI 1640 medium alone to 33% and with 1000 U r hIL-2 to 46%. Assessment of the cytotoxicity potential of NK cells from PBL, freshly isolated and treated, against tumor cell line K562 by the standard NK cell 4-hr 51-chromium release assay, indicates a significant enhancement in their cytotoxicity. By FACStar sorting and analysis of the CD56+ NK cell population new evidence is obtained which shows that about 25-30% of this population represents the CD56bright+ subset, while 70-75% represents the CD56dim+ subset. As the two subsets were shown to differ functionally they were stained with anti P1 for the evaluation of perforin content and it was found that both of them are positive for perforin from 97-99%, suggesting that the functional difference is not due to perforin content. In this sense, as NK cells are constitutively positive for perforin, the increase in the cytotoxicity of NK cells induced by IL-2 is most likely due to the synthesis and expression of various adhesion molecules on NK cells which increase their cytotoxic potential, as well as, that the detected increase in the number of perforin positive cells by this lymphokine does not belong to NK, but to the T lymphocyte population. The data obtained in this study indicate the possibility of perforin detection in human lymphocytes by an anti human perforin mAb and the change in the number of perforin positive cells after stimulation with interleukin-2.

Antibodies, Monoclonal↗
PubMed2000

Interferon-gamma-induced membrane PAF-receptor expression confers tumor cell susceptibility to NK perforin-dependent lysis.

Perforin is known to display a membranolytic activity on tumor cells. Nevertheless, perforin release during natural killer (NK)-cell activation is not sufficient to induce membrane target-cell damage. On the basis of the ability of perforin to interact with phospholipids containing a choline phosphate headgroup, we identify the platelet-activating factor (PAF) and its membrane receptor as crucial components in tumor cell killing activity of human resting NK cells. We demonstrate for the first time that upon activation, naive NK cells release the choline phosphate-containing lysolipid PAF, which binds to perforin and acts as an agonist on perforin-induced membrane damage. PAF is known to incorporate cell membranes using a specific receptor. Here we show that interferon-gamma (IFN-gamma) secreted from activated NK cells ends in PAF-receptor expression on perforin-sensitive K562 cells but not on perforin-resistant Daudi cells. In order to prove the capacity of PAF to interact simultaneously with its membrane PAF receptor and with perforin, we successfully co-purified the 3 components in the presence of bridging PAF molecules. The functional activity of this complex was further examined. The aim was to determine whether membrane PAF-receptor expression on tumor cells, driven to express this receptor, could render them sensitive to the perforin lytic pathway. The results confirmed that transfection of the PAF-receptor complementary DNA into major histocompatibility complex class I and Fas-receptor negative tumor cells restored susceptibility to naive NK cells and perforin attack. Failure of IFN-gamma to induce membrane PAF receptor constitutes the first described mechanism for tumor cells to resist the perforin lytic pathway.

Adult↗
PubMed1998

[Perforin protein and gene expression in the different phases of endometrium].

OBJECTIVE: To study the expression of perforin protein and gene in the different phases of endometrium and the role of perforin in the reproductive system. METHODS: Immunohistochemical and in situ hybridization techniques were employed to demonstrate the population, phenotype of the perforin positive cell in endometrum of different phases of menstrual cycle, first-trimester decidua and postmenopausal endometrium. RESULTS: Perforin positive cells were found to be located in the endometrial stroma and expressed natural killer-like cell surface marker (CD56 antigen, also named NK-like cell or CD56+ lymphocyte). The quantitative analysis of perforin protein cells demonstrated an increase in number from proliferative endometrium to secretory endometrium through first-trimester decidua (P < 0.05). These cells were absent in the postmenopausal endometrium. Perforin mRNA positive cells formed islands in proliferative endometrium stroma, but the perforin protein expression cells were scattered. There was difference between the perforin gene expression and protein expression in proliferative endometrium. In first-trimester decidua, perforin mRNA positive cells incresed. Some perforin mRNA positive cells located around spiral arterioles were observed. CONCLUSIONS: Expression of perforin in the different phases of endometrium is specific and plays an important role in the reproductive function. They probably exert some influence on embryo implantation and may be involved in endometrial stromal breakdown during menstruation.

Adult↗
PubMed2001

Reduction of viral myocarditis in mice lacking perforin.

Cellular immunity plays an important role and contributes to morbidity and mortality in the development of cardiomyopathy secondary to acute myocarditis. Lymphocytotoxicity has been proposed as due to infiltrated cytotoxic T lymphocytes (CTLs). Perforin is thought to be a major factor responsible for the cytolytic properties of the CTLs. To investigate whether mice lacking perforin have enhanced severity of viral myocarditis and acceleration of myocardial apoptosis, perforin knockout (KO) mice were infected with encephalomyocarditis virus. The average cardiac viral titer in perforin KO mice was not significantly different from that in the wild type mice. The heart weight/body weight ratio in perforin KO mice on days 4 and 12 were the same in the wild type mice (n=4 of each). Hearts in perforin KO mice were smaller than that in perforin + mice and grades of myocardial necrosis and lymphocyte infiltration were significantly reduced than in perforin + mice on day 12 after viral inoculation. In the perforin KO mice, the number of apoptotic cells was significantly higher than in wild type on day 4. Perforin blockade induced the early induction of myocardial apoptosis and suppressed the late onset of myocardial injury by cellular autoimmunity. Blockade of perforin gene expression at an early stage may limit viral myocarditis for therapeutic intervention.

Animals↗
PubMed1995

Morphologic and functional characterization of perforin-deficient lymphokine-activated killer cells.

Mice deficient in perforin, a key mediator of lymphocyte-mediated cytolysis, have recently been generated using the gene knockout technique. CTL and NK cells derived from these mice have been shown to be defective in the granule-dependent cytolytic pathway. To investigate whether the granule-formation process has been altered in these perforin-deficient cytotoxic cells, rendering them defective in using the other granule mediators, we have examined in the present study the morphologic and functional characteristics of perforin-deficient LAK cells. Perforin-deficient LAK cells, similar to wild-type LAK cells, were shown to contain a large number of granules in their cytoplasm. By electron microscopy, the morphology of the granules present in these two cell populations appeared indistinguishable. The complete depletion of perforin in LAK cells derived from perforin gene-knockout mice was further confirmed by immunoelectron microscopy using anti-perforin antiserum. The expression of other cytolytic mediators, present either within the granules (granzymes A and B) or elsewhere (Fas ligand), appeared to be unperturbed, as investigated using the reverse transcription-PCR technique. Like the CTL and NK cells isolated from perforin-deficient mice, perforin-deficient LAK cells could lyse only target cells that express high levels of Fas molecule. Furthermore, these perforin-deficient LAK cells, similar to wild-type LAK cells and a CTL clone, were resistant to perforin-mediated cytolysis.

Animals↗
PubMed1996

Expression and characterization of functionally active recombinant perforin produced in insect cells.

A key cytolytic mediator used by killer lymphocytes, perforin (also known as pore-forming protein or cytolysin), has been shown to be capable of undergoing polymerization to form pores in cell membranes and cause osmotic lysis of target cells. Although perforin has been purified from killer lymphocytes and the coding gene has been cloned and sequenced, information concerning the domain structure of the perforin molecule has remained scarce. To overcome the difficulty in obtaining sufficient amounts of perforin and to further assess the functional relevance of the N-terminal portion of the perforin molecule in its lytic activity, we have attempted in the present study to produce recombinant perforins. Three forms of recombinant mouse perforin, a full-length form and two N-terminal truncated forms, have been expressed in insect (Spodoptera frugiperda (Sf9)) cells using recombinant baculovirus. Biochemical and functional characterization showed the purified full-length recombinant perforin to be capable of lysing target cells, inducing Ca2+ influx into target cells, and forming structural pores in target membranes. Significant lytic activities were also detected for the two truncated recombinant perforins lacking, respectively, the N-terminal 21 amino acid residues and 121 amino acid residues. Time course study showed that the latter acted less efficiently than the former. These results suggest the N-terminal portion of the perforin molecule to be an important, but not the only, domain responsible for the lytic function of the perforin molecule.

Animals↗
PubMed2004

A pore-forming protein, perforin, from a non-mammalian organism, Japanese flounder, Paralichthys olivaceus.

A perforin cDNA of Japanese flounder, Paralichthys olivaceus, was cloned from a cDNA library of kidney stimulated with ConA/PMA. The full-length cDNA is 2,157 bp, which encodes 587 amino acids. The Japanese flounder perforin gene consists of five exons and four introns, with a length of approximately 3 kb. The amino acid sequence of the Japanese flounder perforin is 36% identical to that of rat perforin and 37% identical to amino acid sequences of mouse and human perforin. The Japanese flounder perforin also showed low homology to human and mouse complement components (C6, C7, C8 and C9), ranging from 19% to 24%. However, the membrane attack complex/perforin domain is conserved. A phylogenetic analysis placed the Japanese flounder perforin in the same cluster with other known mammalian perforins. RT-PCR analysis revealed that the perforin gene was expressed in the peripheral blood leukocytes, head kidney, trunk kidney, spleen, heart, gill and intestine of healthy fish. Recombinant perforin produced in insect cells using the baculovirus expression system showed calcium-dependent hemolytic activity.

Amino Acid Sequence↗
PubMed1996

Fractionation of perforin and granzymes by immobilized metal affinity chromatography (IMAC).

Cytotoxic lymphocytes and natural killer cells kill their targets by releasing pore-forming granules or by Fas ligand-Fas initiated death. The granules contain the pore-forming protein perforin, proteoglycan and multiple serine proteases termed granzymes. In this paper we describe two options for isolating perforin and granzymes. Both options separate the proteins by their ability to bind to immobilized metal affinity chromatography (IMAC) columns. The first option, with Cu2+ as the metal (Cu2+-IMAC), separates both perforin and granzymes while the second, with Co2+ as the metal (Co2+-IMAC), separates only perforin. After Cu2+-IMAC perforin is > 20-fold enriched with excellent recovery of lytic activity. Only two proteins are substantial contaminants. After Cu2+-IMAC, the perforin is dilute and requires concentration before additional steps of purification. The second option, with Co2+ as the metal Co2+-IMAC), yields perforin that is concentrated in a sharp peak. The concentrated perforin is immediately suitable for further purification. The first option, with Cu2+, isolates the granzymes while the second option, Co2+-IMAC, does not. After isolation, the perforin lytic and granzyme activities are stable for weeks at 4 degrees C, an advantage to previous isolation methods for these proteins. The excellent recoveries of perforin and granzymes also indicate that these proteins are less than 4% and 15% of the total lymphocyte granule protein, respectively.

Animals↗
PubMed1998

Identification of an astrocyte cell population from human brain that expresses perforin, a cytotoxic protein implicated in immune defense.

The brain is an immunoprivileged organ isolated from the peripheral immune system. However, it has been shown that resident cells, notably astrocytes and microglia, can express numerous innate immune molecules, providing the capacity to generate a local antipathogen system. Perforin is a cytolytic protein present in the granules of cytotoxic T lymphocytes and natural killer cells. Expression in cells other than those of the hemopoetic lineage has not been described. We report here that fetal astrocytes in culture (passages 2 to 15), astrocytoma, and adult astrocytes expressed perforin. Reverse transcriptase polymerase chain reaction followed by Southern blot was carried out using multiple specific primers and all cDNAs were cloned and sequenced. Human fetal astrocyte perforin cDNA sequence was approximately 100% identical to the reported perforin cDNA cloned from T cells. Western blot analysis using monoclonal and polyclonal antiperforin peptide antibodies revealed a protein of 65 kD in both human fetal astrocyte and rat natural killer cell lysates (n = 4). Immunostaining followed by FACS(R) and confocal and electron microscopy analysis revealed that perforin was expressed by 40-50% of glial fibrillary acidic protein positive cells present in the fetal brain culture (n = 11). Perforin was not localized to granules in astrocytes but was present throughout the cytoplasm, probably in association with the endoplasmic reticulum. Perforin was not detected in normal adult brain tissue but was present in and around areas of inflammation (white and grey matter) in multiple sclerosis and neurodegenerative brains. Perforin-positive cells were identified as reactive astrocytes. These findings demonstrate that perforin expression is not unique to lymphoid cells and suggest that perforin produced by a subpopulation of astrocytes plays a role in inflammation in the brain.

Adult↗
PubMed1995

Perforin expression in endometrium during the menstrual cycle.

Perforin is a marker of functionally active cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. We examined perforin expression in endometrium throughout the menstrual cycle. In addition, perforin expression was studied in endometrial leukocytes in women with anovulatory cycles with or without progesterone therapy and in postmenopausal women. In the endometrium, perforin-positive endometrial lymphocytes increased in numbers in the middle through late secretory phases. In women with anovulatory cycles, the proliferative endometrium, before estrogen withdrawal endometrial breakdown, contained less perforin-positive lymphocytes compared with the premenstrual endometrium of the normal menstrual cycle. The progestin-induced endometrial decidualization was accompanied by an extensive recruitment of perforin-positive cells. In contrast, the postmenopausal atrophic endometrium showed complete absence of perforin-positive cells. The lymphocytes isolated from secretory endometrium were CD3-, CD56+ (approximately 80%) or CD3+, CD8+ T-cell receptor (TCR)-gamma delta- (approximately 20%). Almost 90% +/- 5.5 of the CD3-, CD56+, and up to 25% +/- 7% of CD8+ T lymphocytes contained perforin. Perforin expression was further confirmed in endometrial lymphocytes by Northern blot analysis. In vitro, isolated endometrial lymphocytes exhibited NK-like cytotoxicity. After cyclic hormone withdrawal during normal menstrual cycle, these perforin-positive cytotoxic cells may be involved in endometrial stromal breakdown during menstruation.

Blotting, Northern↗
PubMed1997

CD4+ and CD8+ T cells each can utilize a perforin-dependent pathway to mediate lethal graft-versus-host disease in major histocompatibility complex-disparate recipients.

Perforin-deficient (-/-) mice were used as T-cell donors for infusion into irradiated major histocompatibility complex (MHC)-disparate recipients to investigate the requirement for perforin-mediated cytolysis during graft-versus-host disease (GVHD) generation. Administration of 5x10(6) C57BL/6 (H2b) perforin -/- splenocytes was significantly less effective in inducing GVHD lethality when given to MHC class I + II disparate B10.BR (H2k) recipients, as compared with wild-type (+/+) controls. Perforin expression by donor T cells was not required for GVHD induction because recipients given fivefold higher numbers of perforin -/- donor splenocytes uniformly succumbed to lethal GVHD. Because both CD4+ and CD8+ donor T cells are required for optimal GVHD lethality in this strain combination, to discern the relative contribution of perforin-mediated cytolysis by CD4+ and CD8+ T cells, additional studies were performed. For these latter studies, we used a sensitive assay involving the infusion of highly purified CD4+ or CD8+ T cells into sublethally irradiated MHC class II or I disparate recipients, respectively. As compared with recipients of perforin +/+ T cells, recipients of either CD4+ or CD8+ perforin -/- T-cell subsets had a significant reduction in GVHD-mediated lethality at T-cell doses that were uniformly lethal. T-cell dose titration studies established that GVHD lethality in recipients of perforin -/- CD4+ or CD8+ T cells was reduced by approximately threefold. These data are the first to indicate that approaches to limit perforin-mediated cytolysis should be similarly effective in situations in which CD4+ or CD8+ T cells dominate the GVHD response.

Animals↗
PubMed2003

Routine flow cytometric immuno-staining of T-cell perforin is preserved using diethylene glycol for erythrocyte-lysis but lost by the use of ammonium chloride.

The system of perforin-containing lytic granules of cytotoxic lymphocytes plays an important role in the immune defense machinery. Investigating the capacity and efficacy of this system in and ex vivo is helpful to understand immune responses and their modulation by therapeutic interventions. With regard to its pathophysiological function, we recently demonstrated a substantial increase of perforin-positive CD8+ T cells in the peripheral blood of patients with acute exacerbated psoriasis and severe generalized drug reactions, and, in marked contrast, a highly significant perforin-depletion and a perforin-hyperreleasability in atopic dermatitis (AD). To streamline the perforin staining procedure, isolation of peripheral blood mononuclear cells (PBMC) by Ficoll density centrifugation was to be replaced by lysis of erythrocytes. Ammonium chloride lysis, however, reduced the perforin content of CD8+ T cells substantially (up to 75-100%) as compared with Ficoll isolation of PMC. Incubation of cells in concanamycin A, a selective inhibitor of H+-ATPases, resulted in a similar loss of perforin staining pointing to the critical influence of lysosomal pH. Using diethylene glycol-mediated erythrocyte lysis, perforin was well preserved to be readily detectable by immuno flow cytometry. Representative examples of the application of this optimized perforin staining procedure as well as accumulated data are given for various dermatological disorders (psoriasis, atopic dermatitis, cutaneous drug reactions, graft-versus-host disease (GVHD) with strong involvement of the cytotoxic T-cell population. Our findings may help to explain recent conflicting reports about a widely varying range of the portion of perforin-positive cells in healthy individuals as a reflection of such artificial methodological influences.

Adenosine Triphosphatases↗
PubMed2006

Polymorphism in the proximal promoter region of the perforin gene and its impact on the course of HIV infection.

Cytotoxic T lymphocytes (CTLs) play an essential role in the control of viral replication during human immunodeficiency virus (HIV) infection. However, the efficacy of the CTL response varies between individuals. We tested the hypothesis that genetic polymorphisms in the lytic effector molecule perforin could influence the progression of HIV infection. The perforin gene was screened for single nucleotide polymorphisms (SNPs) by denaturing high-performance liquid chromatography (dHPLC). Correlations were sought between perforin genotype, perforin expression and lytic function of CD8+ T lymphocytes from HIV-positive patients. Association of perforin genotype with disease progression was investigated in 426 seroconverters enrolled in the French SEROCO cohort. AIDS-free survival curves were constructed using the Kaplan-Meier method and compared using the log-rank test. Three SNPs were found in the proximal promoter region of the perforin gene: 63G (allelic frequency 0.029), 112G (allelic frequency 0.071) and 1012T (allelic frequency 0.070). The presence of the 1012T genotype correlated with fewer perforin+ cells among circulating CD8+ CTL. However, CTL lines from HIV(-positive) individuals heterozygous for the perforin 1012T SNP displayed normal lysis of target cells, and within the SEROCO cohort, patients heterozygous for the 1012T SNP showed normal disease progression. However, 1012T/T homozygotes showed a tendency towards slower disease progression (P = 0.08). In conclusion, polymorphism in the perforin gene is limited, and although the 1012T genotype appears to influence perforin expression, it was not conclusively associated with disease progression in HIV infection.

Cytotoxicity, Immunologic↗
PubMed2003

Loss of the perforin cytotoxic pathway predisposes mice to experimental cytomegalovirus retinitis.

AIDS-related human cytomegalovirus (HCMV) retinitis continues to be a chronic ophthalmologic problem among human immunodeficiency virus type 1 (HIV-1)-infected patients who do not respond to highly active antiretroviral therapy. Although HCMV retinitis occurs during HIV-1-induced immunosuppression, the precise effector mechanism(s) that fails during the immunopathogenesis of AIDS to allow onset and progression of HCMV retinal disease remains unclear. We therefore performed a series of experiments to explore the relative roles of distinct pathways of lymphocyte-mediated cytotoxicity in either resistance or susceptibility to experimental murine cytomegalovirus (MCMV) retinitis in mice. Whereas mutant C57BL/6 mice deficient in the Fas/FasL cytotoxic pathway (gld mice) were identical to normal C57BL/6 mice and exhibited absolute resistance to retinal necrosis following subretinal MCMV inoculation, knockout C57BL/6 mice deficient in the perforin cytotoxic pathway (PKO mice) were susceptible to MCMV retinitis. Susceptibility of PKO mice to MCMV retinitis correlated with increased ocular MCMV titers when compared with ocular MCMV titers of gld and normal mice. Since mice with retrovirus-induced immunodeficiency syndrome (MAIDS) exhibited a frequency and severity of MCMV retinitis that were equivalent to those observed in PKO mice, we hypothesized that susceptibility to MCMV retinitis during MAIDS correlates with a decrease in the perforin cytotoxic pathway. To test this hypothesis, we developed a quantitative competitive reverse transcription-PCR assay to measure mouse perforin mRNA levels in the splenic T lymphocytes and MCMV-inoculated eyes of normal mice or mice with MAIDS. Perforin mRNA levels in splenic T lymphocytes were significantly decreased during MAIDS, by approximately 100-fold, from perforin mRNA levels in normal mice. Moreover, MCMV-inoculated eyes destined to develop retinitis during MAIDS also showed a significant decrease in perforin mRNA levels from the perforin mRNA levels of MCMV-inoculated eyes of normal mice destined to be resistant to retinitis. As expected, perforin mRNA could not be detected in unmanipulated and uninfected eyes of normal mice. These results provide the first evidence that the perforin cytotoxic pathway is more important than the Fas/FasL cytotoxic pathway in providing resistance to experimental MCMV retinitis and that loss of the perforin cytotoxic pathway predisposes to MCMV retinitis.

Animals↗
PubMed1998

Calreticulin, a component of the endoplasmic reticulum and of cytotoxic lymphocyte granules, regulates perforin-mediated lysis in the hemolytic model system.

Cytotoxic lymphocytes kill virally infected cells with specialized cytotoxic granules containing perforin, a protein that forms toxic pores in the target cell membrane. These specialized cytotoxic granules also contain calreticulin, an endoplasmic reticulum chaperone protein. The calcium-independent association of perforin and calreticulin prompted our evaluation of calreticulin's potential to function as a regulatory molecule that protects cytotoxic lymphocytes from their own perforin. We report here that 10(-7) M calreticulin blocked perforin-mediated lysis in the hemolytic model system using erythrocytes as targets. Previously, we found that millimolar levels of calcium in the hemolytic assays dissociate high-affinity perforin-calreticulin complexes, which makes it unlikely that perforin associates with calreticulin in solution when hemolysis is blocked. Calreticulin may affect perforin at the erythrocyte membrane. We observed calcium-dependent binding of calreticulin to erythrocyte membranes with a Kd of 2.7 x 10(-7) M and a saturation average of 10(5) molecules calreticulin per erythrocyte. At concentrations that blocked hemolysis, calreticulin occupied many of the calreticulin membrane-binding sites and was in molar excess of perforin. These observations open the possibilities that membrane-bound calreticulin prevents hydrophobic entry of perforin into membranes and (or) prevents perforin from assembling into polyperforin pores.

Calcium-Binding Proteins↗
PubMed2002

Perforin expression in cytotoxic lymphocytes from patients with hemophagocytic lymphohistiocytosis and their family members.

Mutations in the perforin gene have been described in some patients with hemophagocytic lymphohistiocytosis (HLH), but the role of perforin defects in the pathogenesis of HLH remains unclear. Four-color flow cytometric analysis was used to establish normal patterns of perforin expression for control subjects of all ages, and patterns of perforin staining in cytotoxic lymphocytes (natural killer [NK] cells, CD8(+) T cells, CD56(+) T cells) from patients with HLH and their family members were studied. Eleven unrelated HLH patients and 19 family members were analyzed prospectively. Four of the 7 patients with primary HLH showed lack of intracellular perforin in all cytotoxic cell types. All 4 patients showed mutations in the perforin gene. Their parents, obligate carriers of perforin mutations, had abnormal perforin-staining patterns. Analysis of cytotoxic cells from the other 3 patients with primary HLH and remaining family members had normal percentages of perforin-positive cytotoxic cells. On the other hand, the 4 patients with Epstein-Barr virus-associated HLH typically had depressed numbers of NK cells but markedly increased proportions of CD8(+) T cells with perforin expression. Four-color flow cytometry provides diagnostic information that, in conjunction with evidence of reduced NK function, may speed the identification of life-threatening HLH in some families and direct further genetic studies of the syndrome.

CD8-Positive T-Lymphocytes↗
PubMed2004

Demethylation of promoter regulatory elements contributes to perforin overexpression in CD4+ lupus T cells.

Inhibiting DNA methylation in CD4+ T cells causes aberrant gene expression and autoreactive monocyte/macrophage killing in vitro, and the hypomethylated cells cause a lupus-like disease in animal models. Similar decreases in T cell DNA methylation occur in idiopathic lupus, potentially contributing to disease pathogenesis. The genes affected by DNA hypomethylation are largely unknown. Using DNA methylation inhibitors and oligonucleotide arrays we have identified perforin as a methylation-sensitive gene. Our group has also reported that DNA methylation inhibitors increase CD4+ T cell perforin by demethylating a conserved methylation-sensitive region that is hypomethylated in primary CD8+ cells, which express perforin, but is largely methylated in primary CD4+ cells, which do not. As lupus T cells also have hypomethylated DNA and promiscuously kill autologous monocytes/macrophages, we hypothesized that perforin may be similarly overexpressed in lupus T cells and contribute to the monocyte killing. We report that CD4+ T cells from patients with active, but not inactive, lupus overexpress perforin, and that overexpression is related to demethylation of the same sequences suppressing perforin transcription in primary CD4+ T cells and demethylated by DNA methylation inhibitors. Further, the perforin inhibitor concanamycin A blocks autologous monocyte killing by CD4+ lupus T cells, suggesting that the perforin is functional. We conclude that demethylation of specific regulatory elements contributes to perforin overexpression in CD4+ lupus T cells. Our results also suggest that aberrant perforin expression in CD4+ lupus T cells may contribute to monocyte killing.

Adult↗
PubMed1993

[Immunohistochemical study on the expression of perforin in tumor infiltrating lymphocytes of renal cell carcinoma].

Perforin is considered to be one of the important cytolytic factors found in the granules of killer cells. In this paper we attempted to demonstrate perforin in TIL (tumor infiltrating lymphocytes) of 55 human renal cell cancer cases and the perforin was proved by staining immunohistochemically with an anti-PFP monoclonal antibody. The result indicated that perforin was observed in 98% of the cases, while few positive cell was found in normal renal tissues. TILs were further characterized into their subsets including CD4+ T cells, CD8+ T cells, NK cells and gamma/delta T cells using antibodies specific to their surface markers. The antibodies used are anti-Lew3a, anti-Lew2a, anti-Lew19 and anti-delta chain antibody. The former two subsets were predominantly found to be infiltrating in the tumor tissue, while the latter two subsets were shown to be lesser than the former. For examination of the correlation between the presence of perforin and the characteristics of infiltrated lymphocyte subsets, double-staining of the cells was performed using anti-perforin and one of anti-surface marker antibodies, respectively. The perforin was found to be expressed on the four subsets at different extent. A stronger reactivity of the perforin was found in the CD4+ T cells and CD8+ T cells than that in the NK cells and the gamma/delta T cells. These findings suggest that perforin may play a role of effector functions in the immunological defense mechanism against cancer. To define the significance of the expression of the perforin not only on the CD8+ T cells but also on the CD4+ T cells is required for further study.

Adult↗