Perforin Expression Research Articles

Peculiar Phenotypic and Cytotoxic Features of Pulmonary Mucosal CD8 T Cells in People Living with HIV Receiving Long-Term Antiretroviral Therapy.

People living with HIV have high burdens of chronic lung disease, lung cancers, and pulmonary infections despite antiretroviral therapy (ART). The rates of tobacco smoking by people living with HIV vastly exceed that of the general population. Furthermore, we showed that HIV can persist within the lung mucosa despite long-term ART. As CD8 T cell cytotoxicity is pivotal for controlling viral infections and eliminating defective cells, we explored the phenotypic and functional features of pulmonary versus peripheral blood CD8 T cells in ART-treated HIV+ and uninfected controls. Bronchoalveolar lavage fluid and matched blood were obtained from asymptomatic ART-treated HIV+ smokers (n = 11) and nonsmokers (n = 15) and uninfected smokers (n = 7) and nonsmokers (n = 10). CD8 T cell subsets and phenotypes were assessed by flow cytometry. Perforin/granzyme B content, degranulation (CD107a expression), and cytotoxicity against autologous Gag peptide-pulsed CD4 T cells (Annexin V+) following in vitro stimulation were assessed. In all groups, pulmonary CD8 T cells were enriched in effector memory subsets compared with blood and displayed higher levels of activation (HLA-DR+) and exhaustion (PD1+) markers. Significant reductions in proportions of senescent pulmonary CD28-CD57+ CD8 T cells were observed only in HIV+ smokers. Pulmonary CD8 T cells showed lower perforin expression ex vivo compared with blood CD8 T cells, with reduced granzyme B expression only in HIV+ nonsmokers. Bronchoalveolar lavage CD8 T cells showed significantly less in vitro degranulation and CD4 killing capacity than blood CD8 T cells. Therefore, pulmonary mucosal CD8 T cells are more differentiated, activated, and exhausted, with reduced killing capacity in vitro than blood CD8 T cells, potentially contributing to a suboptimal anti-HIV immune response within the lungs.

MiRNA Regulation of NK Cells Antiviral Response in Children With Severe and/or Recurrent Herpes Simplex Virus Infections.

Severe and/or recurrent infection with Herpes simplex virus (HSV) is observed in a large group of patients treated in clinical immunology facilities. Atypical and prolonged HSV infection is the most common clinical manifestation of disturbed NK cell development and functions, yet the molecular basis of these disorders is still largely unknown. Since recent findings indicated the importance of miRNA in regulating NK cell development, maturation and functions, the aim of our study was to investigate miRNA expression pattern in NK cells in patients with severe and/or recurrent infections with HSV and analyze the role of these miRNAs in NK cell antiviral response. As a result, miRNA expression pattern analysis of human best known 754 miRNAs revealed that patients with severe and/or recurrent HSV infection had substantially upregulated expression of four miRNAs: miR-27b, miR-199b, miR-369-3p and miR-491-3p, when compared to healthy controls. Selective inhibition of miR-27b, miR-199b, miR-369-3p and miR-491-3p expression in NK-92 cells resulted in profound upregulation of 4 genes (APOBEC3G, MAP2K3, MAVS and TLR7) and downregulation of 36 genes taking part in antiviral response or associated with signaling pathways of Toll-like receptors (TLR), NOD-like receptors, the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and type I IFN-related response. Additionally, flow cytometry analysis revealed that miR-369-3p and miR-491-3p inhibitors downregulated NK cell intracellular perforin expression, while the expression of granzyme B and IFNγ remained unchanged. Taken together, our study suggests a novel mechanism which may promote recurrence and severity of HSV infection, based on miRNAs-dependent posttranscriptional regulation of genes taking part in antiviral response of human NK cells.

Clinical Implication of PD-L1 Expression in Gallbladder Cancer

Introduction: Programmed death ligand-1 (PD-L1) expression has been established as a prognostic factor for various solid tumors, but is not well understood in gallbladder cancer (GBC). This study was designed to evaluate PD-L1 expression in GBC and investigate its association with clinicopathological factors, tumor-infiltrating immune cells and survival outcomes. Methods: The expression of PD-L1 was detected by immunohistochemistry from 94 GBC patients who underwent R0/R1 resection at our institution between 2003 and 2016. Tumor-infiltrating CD8+ T cells or CD163+ positive macrophages were assessed at invasive front of tumor, tumor stroma and intraepithelial, respectively. To evaluate the cytotoxic activity of CD8+ T cell, the expression of Perforin and Granzyme B was also assessed using immunohistochemistry. Results: The expression of PD-L1, Perforin and Granzyme B was identified in 38 patients (40.4%), 29 patients (30.9%) and 20 patients (21.3%), respectively. High PD-L1 expression was associated with male gender, high preoperative CA19-9 level, lymphatic invasion, venous invasion, lymph node metastasis and liver metastasis. High PD-L1 expression was related to poorer OS (p=0.0046) and DFS (p=0.0011). In addition, CD163+ macrophage infiltration of tumor was positively correlated with PD-L1 expression. Moreover, high CD8+ TIL group at invasive front of tumor had better OS (p=0.011) compared to low CD8+ TIL group. However, high PD-L1 was also related to poor prognosis in this subgroup. Conclusions: PD-L1 expression is a useful biomarker for advanced GBC. The subgroup of high PD-L1 with high CD8+ TIL at invasive front of tumor would be potential therapeutic target for PD-1/PD-L1 checkpoint inhibitors.

Diagnostic and prognostic value of perforin-1 mRNA expression in acute myeloid leukemia

ObjectiveTo study diagnostic value of perforin-1 gene mRNA expression and its relation to patients' outcomes in acute myeloid leukemia (AML).BackgroundAML is a molecularly heterogeneous hematological malignancy with variable response to treatment and characterized by bone marrow and tissue infiltration with abnormally differentiated cells of hematopoietic origin. Perforin-1 is a glycoprotein responsible for pore formation in cell membranes of target cells. It has a role in immune regulation. Without perforin-1, cytotoxic T cells and natural killer cells show reduced or no cytolytic effect on target cells.Patients and methodsExpression levels of perforin-1 mRNA were assessed by reverse transcriptase PCR and correlated with patients' features of response and survival.ResultsPatients with AML had significantly lower perforin expression compared with the control group (P < 0.001). Only seven patients, representing 11.7% of the studied patients had low perforin expression which was significantly related to poor disease cytogenetics and poor response (P = 0.018 and 0.043, respectively). However, logistic regression analysis revealed that older age and poor cytogenetics were considered as independent risk predictors for poor response in studied AML cases. At the end of follow-up period, 55% of patients were alive. The relation between perforin expression and overall survival revealed that there was no significant relation between perforin expression and overall survival in the studied patients (P = 0.09).ConclusionLower perforin-1 mRNA expression was significantly associated with some poor prognostic features in patients with AML, such as unfavorable cytogenetics and poor response. It is also associated with shorter survival; however, this relation was not statistically significant.

In-vitro Evaluation of Effects of Mesenchymal Stem Cells on TLR3, TLR7/8 and TLR9-activated Natural Killer Cells

Objectives: In this study, it was aimed to investigate the immunomodulatory effects of Mesenchymal stem cells (MSCs) on Natural Killer (NK) cells activated by Toll-like receptor (TLR) agonists. Methods: MDA-MB-231, MCF-7 and NK-92 cells were induced with TLR3, TLR7/8 and TLR9 agonists and co-cultured with MSCs. Alterations in IFN-?, TNF-?, Granzyme-b and Perforin expressions were determined by qPCR method, CD69 and CD107a expressions were determined by flow cytometry, and cytotoxicity was determined by MTT-assay. Results: All TLR agonists significantly increased the expressions of the IFN-?, TNF-?, Granzyme-b, Perforin, CD69 and CD107a in-vitro. We determined that the cytokine, cytotoxic molecules, and activation markers of NK-92 cells interacting with breast tumor cells significantly increased by TLR3 and TLR9 agonists. However, suppression rather than activation occurred on the NK-92 cells due to the simultaneous induction of the immunosuppressive effects of MSCs by these agonists. On the other hand, the TLR7/8 agonists provided a low NK-92 induction, however, the inhibitory effects of MSCs were not triggered. Therefore, it provided a more significant activation than TLR3 and TLR9 agonists. Conclusion: Our findings suggested that TLR7/8 agonists may be a better choice to induce antitumor effects of NK cells in a tumor tissue rich in MSCs. Keywords: Toll like receptor agonists, mesenchymal stem cells, natural killer cells, immunomodulation, anti-tumor immunity

Gastric cancer mesenchymal stem cells inhibit natural killer cell function by up-regulating FBP1.

IntroductionThe dysfunction of natural killer (NK) cells has been widely reported in malignancies, including in solid tumours. Gastric cancer mesenchymal stem cells (GCMSCs) are one of the vital elements of stromal cells in the tumour environment (TME) which possess immunosuppressive activity. This study aimed to determine whether GCMSCs are involved in the inhibition of NK cell immune function and explore its underlying mechanism.Material and methodsCD107a and perforin expression of GCMSCs conditioned medium (GCMSCs-CM)-primed NK cells were detected by flow cytometry. To determine NK cell cytotoxicity, the CytoTox96 Non-Radioactive Cytotoxicity Assay kit was used. Glucose uptake and lactate production assay were performed to evaluate the metabolism state of NK cells treated with GCMSCs-CM. The expression of FBP1 in NK cells was analysed by immunoblotting.ResultsGCMSCs inhibited the degranulation capacity, perforin production and cytotoxicity of NK cells. GCMSCs-CM restrained NK cell glucose uptake and lactate production, thus weakening their glycolytic metabolism. FBP1 expression of NK cells was upregulated in the presence of GCMSCs-CM. Using FBP1 inhibitor could reverse the dysfunctional state of NK cells.ConclusionsThis study indicated that GCMSCs could exert immunosuppressive effects on NK cells by up-regulating FBP1 expression, opening up new avenues for NK cell-based GC immunotherapy.

The exosomes derived from CAR-T cell efficiently target mesothelin and reduce triple-negative breast cancer growth

Genetically engineered T cells expressing a chimeric antigen receptor (CAR) have rapidly developed into a powerful and innovative therapeutic modality for cancer patients. However, the problem of dose-dependent systemic toxicity cannot be ignored. In this study, exosomes derived from mesothelin (MSLN)-targeted CAR-T cells were isolated, and we found that they maintain most characteristics of the parental T cells, including surface expression of the CARs and CD3. Furthermore, CAR-carrying exosomes significantly inhibited the growth of both endogenous and exogenous MSLN-positive triple-negative breast cancer (TNBC) cells. The expression of the effector molecules perforin and granzyme B may be a mechanism of tumor killing. More importantly, a highly effective tumor inhibition rate without obvious side effects was observed with the administration of CAR-T cell exosomes in vivo. Thus, the use of CAR-T cell exosomes has great therapeutic potential against MSLN-expressing TNBC.

Cellular immunotherapy with multiple infusions of in vitro-expanded haploidentical natural killer cells after autologous transplantation for patients with plasma cell myeloma

Background aimsTo investigate the feasibility and safety of haploidentical natural killer (NK) cell infusions as consolidation immunotherapy after autologous stem cell transplant (ASCT) in patients with plasma cell myeloma. MethodsTen patients (median age, 59 years) received induction treatment followed by high-dose melphalan (200 mg/m2) at day –1, ASCT at day 0 and increasing NK cell doses (1.5 × 106, 1.5 × 107 and multiple doses of 1.0 × 108 cells/kg body weight) from day +1 to day +30 after ASCT. NK cells were harvested and purified from peripheral blood of haploidentical donors and expanded for 19 days with interleukin (IL)-2 and IL-15 under Good Manufacturing Practice conditions. ResultsNK cell numbers increased 56.0-fold (37.4- to 75.5-fold). Patients received a median of 3.8 × 108 (0.9–5.7 × 108) NK cells/kg body weight in six (three to eight) infusions. Multiparametric mass cytometry analysis demonstrated an altered surface receptor repertoire of expanded NK cells with increased degranulation and cytokine production activities but diminished expression of perforin. Donor NK cells were detectable in the peripheral blood, peaking 1 h after each dose (up to 90% donor NK cells). The treatment was safe and well tolerated, without evidence of graft-versus-host disease. Comparison with a control patient population receiving ASCT without NK cell infusions showed no significant difference in relapse, progression-free survival and overall survival. ConclusionsThis study demonstrates reliable manufacturing of high numbers of activated NK cells for multiple-dose infusions and safe administration of these cellular products. The trial was registered at ClinicalTrials.gov (identifier no. NCT01040026).

Immunological Assessment of Pediatric Multisystem Inflammatory Syndrome Related to Coronavirus Disease 2019.

Background Recently, cases of multisystem inflammatory syndrome in children (MIS-C) associated with COVID-19 have been reported worldwide. Negative RT-PCR testing associated with positive serology in most cases suggests a post-infectious syndrome. Because the pathophysiology of this syndrome is still poorly understood, extensive virological and immunological investigations are needed.MethodsWe report a series of four pediatric patients admitted to Geneva University Hospitals with persistent fever and laboratory evidence of inflammation meeting published definition of MIS-C related to COVID-19, to whom an extensive virological and immunological workup was performed.ResultsRT-PCRs on multiple anatomical compartments were negative whereas anti-SARS-CoV-2 IgA and IgG were strongly positive by ELISA and immunofluorescence. Both pseudo- and full virus neutralization assays showed the presence of neutralizing antibodies in all children, confirming a recent infection with SARS-CoV-2. Analyses of cytokine profiles revealed an elevation in all cytokines, as reported in adults with severe COVID-19. Although differing in clinical presentation, some features of MIS-C show phenotypic overlap with haemophagocytic lymphohistiocytosis (HLH). In contrast to patients with primary HLH, our patients showed normal perforin expression and NK cell degranulation. The levels of soluble IL-2 receptor (sIL-2R) correlated with the severity of disease, reflecting recent T-cell activation.ConclusionOur findings suggest that MIS-C related to COVID-19 is caused by a post-infectious inflammatory syndrome associated with elevation in all cytokines, and markers of recent T-cell activation (sIL-2R) occurring despite a strong and specific humoral response to SARS-CoV2. Further functional and genetic analyses are essential to better understand the mechanisms of host-pathogen interactions.

Natural Killer (NK) Cell Expression of CD2 as a Predictor of Serial Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC).

NK cell ADCC supports monoclonal antibody anti-tumor therapies. We investigated serial ADCC and whether it could be predicted by NK phenotypes, including expression of CD16A, CD2 and perforin. CD16A, the NK receptor for antibodies, has AA158 valine or phenylalanine variants with different affinities for IgG. CD2, a costimulatory protein, associates with CD16A and can augment CD16A-signaling. Pore-forming perforin is essential for rapid NK-mediated killing. NK cells were monitored for their ADCC serial killing frequency (KF). KF is the average number of target cells killed per cell by a cytotoxic cell population. KF comparisons were made at 1:4 CD16pos NK effector:target ratios. ADCC was toward Daudi cells labeled with 51Cr and obinutuzumab anti-CD20 antibody. CD16A genotypes were determined by DNA sequencing. CD2, CD16A, and perforin expression was monitored by flow cytometry. Serial killing KFs varied two-fold among 24 donors and were independent of CD16A genotypes and perforin levels. However, high percentages of CD2pos of the CD16Apos NK cells and high levels of CD16A were associated with high KFs. ROC analysis indicated that the %CD2pos of CD16Apos NK cells can predict KFs. In conclusion, the extent of serial ADCC varies significantly among donors and appears predictable by the CD2posCD16Apos NK phenotype.

Inverse relationship between oligoclonal expanded CD69− TTE and CD69+ TTE cells in bone marrow of multiple myeloma patients

AbstractCD8+CD57+ terminal effector T (TTE) cells are a component of marrow-infiltrating lymphocytes and may contribute to the altered immune responses in multiple myeloma (MM) patients. We analyzed TTE cells in the bone marrow (BM) and peripheral blood (PB) of age-matched controls and patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), and newly diagnosed (ND) MM using flow cytometry, mass cytometry, and FlowSOM clustering. TTE cells are heterogeneous in all subjects, with BM containing both CD69− and CD69+ subsets, while only CD69− cells are found in PB. Within the BM-TTE compartment, CD69− and CD69+ cells are found in comparable proportions in controls, while CD69− cells are dominant in MGUS and SMM and predominantly either CD69− or CD69+ cells in NDMM. A positive relationship between CD69+TTE and CD69−TTE cells is observed in the BM of controls, lost in MGUS, and converted to an inverse relationship in NDMM. CD69−TTE cells include multiple oligoclonal expansions of T-cell receptor/Vβ families shared between BM and PB of NDMM. Oligoclonal expanded CD69−TTE cells from the PB include myeloma-reactive cells capable of killing autologous CD38hi plasma cells in vitro, involving degranulation and high expression of perforin and granzyme. In contrast to CD69−TTE cells, oligoclonal expansions are not evident within CD69+TTE cells, which possess low perforin and granzyme expression and high inhibitory checkpoint expression and resemble T resident memory cells. Both CD69−TTE and CD69+TTE cells from the BM of NDMM produce large amounts of the inflammatory cytokines interferon-γ and tumor necrosis factor α. The balance between CD69− and CD69+ cells within the BM-TTE compartment may regulate immune responses in NDMM and contribute to the clinical heterogeneity of the disease.

Imperfect storm: is interleukin-33 the Achilles heel of COVID-19?

The unique cytokine signature of COVID-19 might provide clues to disease mechanisms and possible future therapies. Here, we propose a pathogenic model in which the alarmin cytokine, interleukin (IL)-33, is a key player in driving all stages of COVID-19 disease (ie, asymptomatic, mild–moderate, severe–critical, and chronic–fibrotic). In susceptible individuals, IL-33 release by damaged lower respiratory cells might induce dysregulated GATA-binding factor 3-expressing regulatory T cells, thereby breaking immune tolerance and eliciting severe acute respiratory syndrome coronavirus 2-induced autoinflammatory lung disease. Such disease might be initially sustained by IL-33-differentiated type-2 innate lymphoid cells and locally expanded γδ T cells. In severe COVID-19 cases, the IL-33–ST2 axis might act to expand the number of pathogenic granulocyte–macrophage colony-stimulating factor-expressing T cells, dampen antiviral interferon responses, elicit hyperinflammation, and favour thromboses. In patients who survive severe COVID-19, IL-33 might drive pulmonary fibrosis by inducing myofibroblasts and epithelial–mesenchymal transition. We discuss the therapeutic implications of these hypothetical pathways, including use of therapies that target IL-33 (eg, anti-ST2), T helper 17-like γδ T cells, immune cell homing, and cytokine balance.

NKG7 - regulating endosomal pathways?

Ng et al. have identified NKG7 as a regulator of inflammation in response to diverse immunological challenges. While NKG7 was required for the degranulation of cytotoxic cells, additional defects including reduced expansion and trafficking of CD8 T cells, and altered antigen presentation, were noted in NKG7-deficient mice. The precise mechanism by which NKG7 mediates its effects has not been resolved but may involve regulation of endosomal vesicle trafficking.

O15 Recognition of subclinical macrophage activation syndrome in an adolescent systemic juvenile idiopathic arthritis patient receiving tocilizumab: a case report

O15 Recognition of subclinical macrophage activation syndrome in an adolescent systemic juvenile idiopathic arthritis patient receiving tocilizumab: a case report

SLeX Expression Delineates Distinct Functional Subsets of Human Blood Central and Effector Memory T Cells.

Sialyl Lewis X (sLeX) regulates T cell trafficking from the vasculature into skin and sites of inflammation, thereby playing a critical role in immunity. In healthy persons, only a small proportion of human blood T cells express sLeX, and their function is not fully defined. Using a combination of biochemical and functional studies, we find that human blood sLeX+CD4+T cells comprise a subpopulation expressing high levels of Th2 and Th17 cytokines, chemokine receptors CCR4 and CCR6, and the transcription factors GATA-3 and RORγT. Additionally, sLeX+CD4+T cells exclusively contain the regulatory T cell population (CD127lowCD25high and FOXP3+) and characteristically display immune-suppressive molecules, including the coinhibitor receptors PD-1 and CTLA-4. Among CD8+T cells, sLeX expression distinguishes a subset displaying low expression of cytotoxic effector molecules, perforin and granzyme β, with reduced degranulation and CD57 expression and, consistently, marginal cytolytic capacity after TCR engagement. Furthermore, sLeX+CD8+T cells present a pattern of features consistent with Th cell-like phenotype, including release of pertinent Tc2 cytokines and elevated expression of CD40L. Together, these findings reveal that sLeX display is associated with unique functional specialization of both CD4+ and CD8+T cells and indicate that circulating T cells that are primed to migrate to lesional sites at onset of inflammation are not poised for cytotoxic function.

Cutaneous T-cell lymphomas with pathogenic somatic mutations and absence of detectable clonal T-cell receptor gene rearrangement: two case reports

BackgroundCutaneous T-cell lymphomas (CTCL) are a heterogeneous group of extranodal non-Hodgkin lymphomas for which diagnosis can be challenging given the potential for overlap with inflammatory dermatoses. Current diagnostic criteria for CTCL incorporate clinical and histopathologic findings as well as results of T-cell receptor (TCR) gene sequencing. Molecular interrogation of TCR genes, TRG and TRB, has proven to be a critical tool for confirming diagnoses of CTCL and for disease tracking after initiation of therapy or after stem cell transplant. Methods for confirming a diagnosis of lymphoma in the absence of TCR gene clonality are lacking. We present two patients with CTCL with pathogenic somatic mutations in the absence of TRG and TRB clonality.Case presentationsCase 1: A 38-year-old male had a 19-year history of a diffuse skin rash with papulosquamous, granulomatous, and verrucous features and progressive ulcerated plaques and tumors demonstrating an atypical CD4+ T-cell infiltrate with expression of cytotoxic markers CD56, TIA-1, granzyme, and perforin on histopathology. No definitive evidence for T-cell clonality was detected by conventional PCR of 6 biopsies or by next-generation sequencing (NGS) of 14 biopsies. Somatic mutational profiling of a skin biopsy revealed pathogenic mutations in PIKC3D and TERT promoter hotspots, confirming the presence of a clonal process. Case 2: A 69-year-old male with a 13-year history of progressive, diffuse hypertrophic and eroded plaques showed an atypical CD4+ T-cell infiltrate with subset expression of TIA-1 and granzyme on histopathology. No TCR clonality was detected by TCR-NGS of 6 biopsies. Somatic mutational profiling of a skin biopsy detected a pathogenic mutation in TP53, confirming the presence of a clonal process.ConclusionsThese cases highlight how detection of pathogenic somatic mutations can confirm a diagnosis of lymphoma in a clinically and histopathologically suspicious cutaneous lymphoid proliferation without detectable TCR clonality.

Ethyl Pyruvate Promotes Proliferation of Regulatory T Cells by Increasing Glycolysis.

Ethyl pyruvate (EP), a stable form of pyruvate, has shown beneficial effects in animal models of shock, ischemia/reperfusion injury, and sepsis due to its potent anti-oxidant and anti-inflammatory properties. Our recent study demonstrated that EP application prevented the clinical manifestation of type 1 diabetes in mice by augmenting regulatory T cell (Treg) number and function. Our present study shows that EP increases Treg proliferation and suppressive function (perforin and IL-10 expression) during in vitro differentiation from conventional CD4+CD25− T cells. Enhanced expansion of Treg after EP treatment correlated with increased ATP levels and relied on increased glycolysis. Inhibition of oxidative phosphorylation did not attenuate EP stimulatory effects, suggesting that this metabolic pathway was not mandatory for EP-driven Treg proliferation. Moreover, EP lowered the expression of carnitine palmitoyltransferase I, an enzyme involved in fatty acid oxidation. Further, the stimulatory effect of EP on Treg proliferation was not mediated through inhibition of the mTOR signaling pathway. When given in vivo either intraperitoneally or orally to healthy C57BL/6 mice, EP increased the number of Treg within the peritoneal cavity or gut-associated lymphoid tissue, respectively. In conclusion, EP promotes in vitro Treg proliferation through increased glycolysis and enhances Treg proliferation when administered in vivo.

Expansion With IL-15 Increases Cytotoxicity of Vγ9Vδ2 T Cells and Is Associated With Higher Levels of Cytotoxic Molecules and T-bet.

Cancer immunotherapy has shown great advances during recent years, but it has yet to reach its full potential in all cancer types. Adoptive cell therapy (ACT) is now an approved treatment option for certain hematological cancers and has also shown success for some solid cancers. Still, benefit and eligibility do not extend to all patients. ACT with Vγ9Vδ2 T cells is a promising approach to overcome this hurdle. In this study, we aimed to explore the effect of different cytokine conditions on the expansion of Vγ9Vδ2 T cells in vitro. We could show that Vγ9Vδ2 T cell expansion is feasible with two different cytokine conditions: (a) 1,000 U/ml interleukin (IL)-2 and (b) 100 U/ml IL-2 + 100 U/ml IL-15. We did not observe differences in expansion rate or Vγ9Vδ2 T cell purity between the conditions; however, IL-2/IL-15-expanded Vγ9Vδ2 T cells displayed enhanced cytotoxicity against tumor cells, also in hypoxia. While this increase in killing capacity was not reflected in natural killer (NK) cell marker or activation marker expression, we demonstrated that IL-2/IL-15-expanded Vγ9Vδ2 T cells were characterized by an increased expression of perforin, granzyme B, and granulysin compared to IL-2-expanded cells. These cytotoxic molecules were not only increased in a resting state, but also released to a greater extent upon target recognition. In contrast, CD107a and cytokine expression did not differ between expansion conditions. However, IL-2/IL-15-expanded Vγ9Vδ2 T cells showed higher levels of transcription factor T-bet expression, which could indicate that T-bet and cytotoxic molecule levels confer the increased cytotoxicity. These results advocate the inclusion of IL-15 into ex vivo Vγ9Vδ2 T cell expansion protocols in future clinical studies.

Perforins Expression by Cutaneous Gamma Delta T Cells.

Gamma delta (GD) T cells are an unconventional T cell type present in both the epidermis and the dermis of human skin. They are critical to regulating skin inflammation, wound healing, and anti-microbial defense. Similar to CD8+ cytotoxic T cells expressing an alpha beta (AB) TCR, GD T cells have cytolytic capabilities. They play an important role in elimination of cutaneous tumors and virally infected cells and have also been implicated in pathogenicity of several autoimmune diseases. T cell cytotoxicity is associated with the expression of the pore forming protein Perforin. Perforin is an innate immune protein containing a membrane attack complex perforin-like (MACPF) domain and functions by forming pores in the membranes of target cells, which allow granzymes and reactive oxygen species to enter the cells and destroy them. Perforin-2, encoded by the gene MPEG1, is a newly discovered member of this protein family that is critical for clearance of intracellular bacteria. Cutaneous GD T cells express both Perforin and Perforin-2, but many questions remain regarding the role that these proteins play in GD T cell mediated cytotoxicity against tumors and bacterial pathogens. Here, we review what is known about Perforin expression by skin GD T cells and the mechanisms that contribute to Perforin activation.

Immunohistochemical study of perforin and apoptosis stimulation fragment ligand (FasL)in active vitiligo.

Several studies demonstrated a major pathological role of melanocyte-specific cytotoxic CD8+ T cells in the pathogenesis of vitiligo. It has been suggested that apoptosis, rather than necrosis, is the mechanism of melanocyte depletion in vitiligo. The aim of this study was to evaluate the expression and distribution of perforin and apoptosis stimulation fragment ligand (FasL) in the epidermis and dermis of the perilesional and non-lesional skin of vitiligo patients in comparison to controls, to assess their possible role in mediating apoptosis in vitiligo. Twenty patients with active non-segmental vitiligo and 20 healthy controls were enrolled in the study. Skin biopsies were taken from perilesional and non-lesional skin of patients with vitiligo, as well as covered skin of controls. Immunostaining for perforin and FasL was performed and the quantitative analysis for the expression of perforin and FasL was carried out in the epidermis and dermis of biopsied specimens. Epidermal perforin, dermal perforin, epidermal FasL, dermal FasL were significantly higher in perilesional as well as non-lesional skin than controls. There was a statistically significant positive correlation between epidermal and dermal perforin in perilesional skin. There was a statistically significant positive correlation between epidermal and dermal perforin, as well as epidermal and dermal FasL in non-lesional skin. In conclusion, the significant expression of perforin and FasL in the epidermis and dermis of both perilesional and non-lesional skin of active vitiligo patients suggests the role of cytotoxic granules and apoptotic cell death pathways in the pathogenesis of active vitiligo.