Academic literature on the topic 'Munc-13'

Abstract Cytotoxic T lymphocytes (CTLs) function to kill bacterial and viral infected target cells by releasing cytotoxic components such as perforin and granzymes that are contained within lytic granules, into the infected cell. Release occurs via the fusion of lytic granules at the contact zone between infected cell and CTL, the immunological synapse (IS). Defect in the fusion of lytic granules results in the dysfunction of CTLs, the underlying cause of the fatal disease Familial Hemophagocytic Lymphohistiocytosis (FHL). In order for fusion to occur, lytic granules must first arrive, dock and prime at the IS. Mutations within Munc 13-4, a priming factor, for lytic granules, results in FHL subtype-3. We have identified the presence of additional priming factors in CTLs. Targeted gene knockouts of these priming factors resulted in defects in lytic granule fusion at the immunological synapse. These findings demonstrate the presence of more regulatory mechanisms for the most critical event in CTLs, lytic granule fusion. Using high-resolution microscopy and evanescent wave imaging we aim to dissect the precise regulation of lytic granule fusion, which in turn would give us more insight into the regulation and function of CTLs.

Hemophagocytic lymphohistiocytosis is a multisystem inflammation, generated by the uncontrolled and excessive activation of cytotoxic T lymphocytes and natural killer cells. Severe immunodeficiency and generalized macrophage activation can often be detected in the background of this life threatening disorder. It is classified as a primary immunodeficiency. Functional abnormalities of the perforin protein or defects in granule secretory mechanisms are caused by gene mutations in most cases. Diagnostic criteria of hemophagocytic lymphohistiocytosis are the following: fever, splenomegaly, cytopenias affecting at least two of the 3 lineages in peripheral blood, hypertriglyceridemia and hyperferritinemia, elevated serum level of soluble interleukin-2 receptor (sCD25), hypofibrinogenemia, hemophagocytosis in bone marrow and decreased cytotoxic T cell and natural killer cell activity. In this case report the authors summarize the utility of functional flow cytometry in the diagnosis of hemophagocytic lymphohistiocytosis. Using flow cytometry, elevated intracellular perforin content, decreased killing activity of cytotoxic T cells and natural killer cells, and impaired cell surface expression of CD107a (LAMP1 protein) from in vitro stimulated blood lymphocytes were detected. Abnormal secretion of perforin was also demonstrated. Genetic testing revealed mutation of the MUNC 13-4 gene, which confirmed the base of the abnormal flow cytometric findings. This case report demonstrates the value of functional flow cytometry in the rapid diagnosis of genetically determined hemophagocytic lymphohistiocytosis, a condition in which early diagnosis is critical for optimal management. The authors emphasize the significance of functional flow cytometry in the differential diagnosis of immunodeficiencies. Orv. Hetil., 2014, 155(10), 389–395.

Abstract Familial hemophagocytic lymphohistiocytosis (FHL) is the genetic form of hemophagocytic lymphohistiocytosis with an autosomal recessive form of inheritance and early onset. FHL is characterized by prolonged fever, hepatosplenomegaly and pancytopenia. Type 3 of FHL (FHL3) which accounts for 30-35 % of all FHL patients, results from mutations in UNC13D gene encoding Munc13-4 protein. Munc13-4 controls fusion of lytic granules with the plasma membrane in cytotoxic lymphocytes and its defect leads to impaired cytotoxic activity in T and NK lymphocytes. The only curative method for FHL3 is allogenic hematopoietic stem cell (HSC) transplantation. For those patients without compatible bone marrow donor, gene therapy could represent a therapeutic option. Our study is based on a comparative analysis to investigate the efficacy and safety of stem cell and T-cell gene therapy for FHL3. To this end we have generated SIN-lentiviral constructs expressing Munc13-4 and used them to produce either VSVG or T-cell specific pseudotyped lentiviral vectors. We also could obtain functionally mature T-cells in vitro, derived from umbilical cord blood HSCs which respond to TCR stimulation and show cytotoxic effect. Using these approaches, first we demonstrated that our SIN-lentiviral constructs are able to transduce efficiently human T-cells and HSCs. We used then these vectors to complement the FHL3 patient’s cells. Transduction of FHL3 CD8 effector cells restored their cytotoxic function that was comparable to that of control cells. In addition we noted that the overexpression of Munc 13-4 in normal human HSCs didn’t alter in vitro differentiation of these cells towards T-cells. The effect of this overexpression on B-cells and myeloid differentiation is currently under investigation. These preliminary results will be followed by ex vivo gene transfer experiments in Munc13-4 deficient “Jinx” mice to further investigate the functional restoration as well as toxic effects. This strategy, if approved, could offer a safe therapeutic method not only for FHL3 patients but also for other genetic or acquired dysfunctions of T-lymphocytes. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2021 Protein kinase D (PKD) is a subfamily of serine/threonine specific family of kinases, comprised of PKD1, PKD2 and PKD3 (PKCm, PKD2 and PKCn in humans). They are part of alternate DAG receptors along with RasGRPs, Munc 13s, chimerins and DAG kinases. Members of the novel class of PKC isoforms such as PKCd, PKCh, and PKCe associate with PKD in smooth muscle cells and COS-7 cells. The mechanism of activation of PKD and the specific PKC isoforms required for its activation are not known to date. This study is aimed at investigating the pathways involved in the activation of PKD in platelets. We show that human as well as murine platelets express PKD. PKD could be activated with PAR4 agonist AYPGKF, PAR1 agonist SFLLRN and GPVI agonist convulxin. AYPGKF and SFLLRN induced PKD phosphorylation as early as 30 sec and convulxin induced PKD phosphorylation at 1 minute. PKD phosphorylation induced by AYPGKF and convulxin were sustained for 5 minutes but phosphorylation induced by SFLLRN was attenuated after 2 minutes of stimulation. AYPGKF-induced PKD phosphorylation was reduced with a calcium chelator, dimethyl BAPTA, indicating that calcium-mediated signals might play a role in activation of PKD. PKD phosphorylation in response to AYPGKF was abolished with a Gq inhibitor, YM-254890, but was not affected by Gi-coupled P2Y12 receptor antagonist ARC-69931MX, indicating that PKD phosphorylation is Gq-, but not Gi- or G 12/13-dependent. PKD phosphorylation was abolished with pan-PKC inhibitors, GF109203X and Ro31-8220, indicating that PKCs are required for PKD activation in platelets. PKD phosphorylation was significantly inhibited with a PKC delta inhibitor, rottlerin, but was not affected by the classical PKC inhibitor, Go6976, suggesting that novel PKC isoforms are important for PKD activation. In addition, 2MeSADP that fails to activate PKCd did not induce phosphorylation of PKD in platelets. Furthermore, phosphorylation of PKD induced by AYPGKF was significantly reduced in PKCd-deficient platelets compared to that of wild type platelets. Hence, we conclude that PKD is a common signaling target downstream of various agonist receptors in platelets, and Gq-mediated signals and novel PKC isoforms, in particular PKCd is required for activation of PKD. Disclosures: No relevant conflicts of interest to declare.

Abstract FHL is a rare fatal disease of early infancy characterized by hyperinflammatory sindrome, fever, hepatosplenomegaly, cytopenia, hypertriglyceridemia, hypofibrinogenemia, central nervous system alteration. Defective cellular cytotoxicity results in pathogen persistence, hypercytokinemia, disseminated infiltration of lymphocytes and histiocytes and hemophagocytosis. Differential diagnosis between FHL due to PRF1 (FHL2) or MUNC13–4 (FHL3) mutations, and additional unknown genetic subtypes is not easy unless mutation analysis is performed. Furthermore, some infection-associated cases of HLH (“secondary”) may mimic FHL. We investigated natural killer (NK) cells function and phenotype in patients with FHL2 (n=5) or FHL3 (n=8). NK cells were cultured in IL-2 prior to their use in the various assays. B-EBV cell lines and dendritic cells are suitable targets in 51Cr-release assays to reveal the FHL3 NK cell defect. Tumor targets were partially lysed by FHL3 NK cells expressing even trace amounts of Munc13–4 protein. FHL2 NK cells were completely unable to lyse target cells. Cytokine production induced by mAb-crosslinking of triggering receptors was similar in patients and controls; under co-culture with 721.221 B-EBV cells, FHL NK cells were high producers, whereas control cells were almost ineffective. This could reflect persistence versus elimination of the source of stimulation in patients vs healthy controls, mimicking the pathophysiology of FHL. Finally, we tested cell surface CD107a expression in a degranulation assay, co-culturing 2x105 polyclonal NK cell or 24-hr IL-2 activated PBMCs with 2x105 target cells (K562, FO-1, 721.221 or P815 and anti-NKp30). CD107a expression was tested in CD56+ cell of either CD3− PBMC or polyclonal activated NK cell populations. CD107a was expressed in 11.8±7.5% cells in 6 FHL3 patients vs 48.5±11.5% in 9 controls (p<0.001). MFI was also lower in FHL3 patients vs. controls (9.3±3.1 vs. 42.6±21.9; p<0.001). Differently from FHL3, mean values of CD107a expression in 3 FHL2 patients were even higher than controls both in terms of percentage (63.3%) and MFI (86.6). Altogether, these flow-cytometry data show that the pattern of CD107a expression represents a novel tool to identify the defect of degranulation characteristic of Munc 13–4 deficiency. Importantly, in FHL2 NK cells, whose granules lack perforin, degranulation pattern is normal. Although use of purified activated NK cells may increase discrimination power of the test, even PBMC (24-hr IL-2 activated) could be used to reveal a defect of granule exocytosis. In addition to providing information useful for a better understanding of the pathophysiology of FHL and functional correlation with different gene mutations, our data may impact on FHL diagnosis. Combined use of surface expression of CD107a, together with intracytoplasmic staining with anti-perforin mAb, allows to promptly dissect FHL3 and FHL2 defects by the simple analysis of PBMC, so directing genetic analysis.

Abstract Abstract SCI-9 Hemophagocytic lymphohistiocytosis (HLH) is a group of immunodeficiencies characterized by clinical signs and symptoms of extreme inflammation. HLH is now more widely recognized and no longer viewed as a disorder of young children only, as more adults are being diagnosed and treated. HLH is defined by a unique pattern of clinical findings. In addition to fevers, cytopenias, hepatitis, and splenomegaly, markedly increased levels of inflammatory markers in the blood (ferritin and sCD163 reflecting activation of antigen presenting cells; sIL2Ra and neopterin reflecting activation of T cells) constitute the collection of diagnostic criteria. Activation of inflammatory cells within the central nervous system (CNS) is found in approximately 50 percent of children at diagnosis and requires targeted therapy. In many cases immune defects affecting cytotoxicity of T cells and natural killer cells underlie the susceptibility to HLH. Autosomal recessive disorders include perforin deficiency (the major cytotoxin of the immune system), or defects in proteins involved in degranulation and exocytosis of perforin and granzyme B (MUNC 13–4, MUNC18-2, STX11, Rab27a). The latter proteins are involved in degranulation generally within the hematopoietic system, thus impacting the function of neutrophils and platelets as well. A rare defect of granulogenesis, Chediak Higashi syndrome, is also associated with a high incidence of HLH. Two forms of X-linked lymphoproliferative syndrome (XLP1 – SAP deficiency, and XLP2 – XIAP deficiency), as well as the rare autosomal recessive disorder ITK (IL-2 inducible T cell kinase) deficiency, are characterized by a high incidence of Epstein-Barr virus-driven HLH and lymphoproliferation. A common pathogenic mechanism underlying these consequences has not yet been elucidated. Effective initial treatment for HLH consists of cytotoxic and anti-inflammatory agents. The most widely used over the past 20 years has been a combination of CNS-penetrating steroid (Decadron) and etoposide. Another approach has been to use anti-thymocyte globulin (ATG) as induction therapy. Both treatments have resulted in approximately 60 percent responses during the first month of therapy. Supportive care with broad-spectrum antimicrobials is a critical adjunct. More recently, a new induction protocol—hybrid immunotherapy for HLH, combining the features of early ATG followed by etoposide, with steroids—has been opened in the United States (http://clinicaltrials.gov/ct2/show/NCT01104025) and Europe. However, HLH persists or reactivates in nearly half of patients as immune suppression is reduced. While a common approach to reactivation is to reintensify previous therapy, no clear guidelines have been developed for this complication. The use of Campath, a humanized monoclonal anti-CD52 antibody, as salvage therapy prior to hematopoietic cell transplantation (HCT) is being tested, as both activated T cells and activated monocyte/macrophages (histiocytes) are targeted through CD52. Historically, the three-year survival after HCT in patients treated with HLH-94 was 60 percent. More recently, use of Campath-based reduced intensity conditioning protocols have led to improved results after HCT. Campath has the advantage of reducing graft-versus-host disease if properly timed prior to HCT. In a recent contemporaneous series of HCT from unrelated adult donors, three-year posttransplant survival improved from 43 percent to 92 percent with no early transplant-related mortality. Disclosures: No relevant conflicts of interest to declare.

Abstract HLH is a rare immunoregulatory disorder characterized by widespread infiltration of histiocytes and T cells into organs, including the central nervous system (CNS), and is fatal in most cases without HCT. HLH can be inherited in an autosomal recessive pattern with unaffected sibling donors available for fewer than 20% of patients thus necessitating alternative donor HCT in the majority of cases. Data on 91 patients who underwent unrelated donor HCT and reported to the Center for International Blood and Marrow Transplant Research between 1989–2005 were analyzed. Fifty-one percent of patients were <1 yr at HCT and 29% had a Lansky performance score ≤80%. In a subset of patients (n=51) additional disease specific characteristics were available: 8 had a family history of HLH, of patients tested, 19 were confirmed to have either a perforin or MUNC-13 gene mutation; CNS disease was present at diagnosis in 21 patients and remained active in 4 at HCT; and 5 patients had active systemic disease at HCT. Most patients (80%) were conditioned with busulfan (BU), cyclophosphamide (CY), and etoposide (VP16) with or without anti-thymocyte globulin. Graft sources were bone marrow (86%), peripheral blood stem cells (4%) and cord blood (10%). Graft vs. host disease (GVHD) prophylaxis was cyclosporine or tacrolimus based in 89% of patients and T-cell depletion in 11%. Fifty-nine percent of grafts were matched at HLA A, B and DRB1, 34%, mismatched at 1-locus and 7%, mismatched at 2-loci. Neutrophil recovery was achieved by day 42 in 91% of patients. The probabilities of grades 2–4 acute at day-100 and chronic GVHD at 3-years were 41% and 23%, respectively. In multivariate analysis, the risk of overall mortality was 2-fold higher in patients who did not receive BU/CY/VP16 as their conditioning regimen (RR 1.99, p=0.03). In the sub-set of patients from whom disease-specific characteristics were available, overall mortality was higher in those with active systemic disease at HCT (RR 3.11, p=0.04). Early mortality was high, 35% at day-100 after HCT. Causes of early mortality included GVHD (n=5), infections (n=8), interstitial pneumonitis (n=8), organ failure (n=6), hemorrhage (n=3) and persistent disease (n=2). With a median follow-up of 49 months (range, 5–145) the 1- and 3-year probabilities of overall survival were 52% and 47%, respectively. For 46 patients with documented systemic remission at HCT, the 1- and 3-year probabilities of overall survival were 56% and 49%, respectively. These data represent the largest experience with unrelated donor HCT for HLH and demonstrate that a BU/CY/VP16 conditioning regimen provides cure in over 50% of patients. Outcome of HCT for patients with active systemic disease was poor with only 1 of 5 such patients surviving, demonstrating a need for novel therapies in patients who fail to respond to standard pre-transplant treatment. Unrelated donor HCT for HLH was associated with high early mortality and future studies should explore strategies to decrease early HCT-related mortality.