Lytic Granule Exocytosis Research Articles

A High-Throughput Phenotypic Screen of Cytotoxic T Lymphocyte Lytic Granule Exocytosis Reveals Candidate Immunosuppressants

We screened the National Institutes of Health’s Molecular Libraries Small Molecule Repository for inhibitors of cytotoxic T lymphocyte (CTL) lytic granule exocytosis by measuring binding of an antibody in the extracellular solution to a lysosomal membrane protein (LAMP-1) that is transferred to the plasma membrane by exocytosis. We used TALL-104 human leukemic CTLs stimulated with soluble chemicals. Using high-throughput cluster cytometry to screen 364,202 compounds in a 1536-well plate format, we identified 2404 initial hits: 161 were confirmed on retesting, and dose–response measurements were performed. Seventy-five of those compounds were obtained, and 48 were confirmed active. Experiments were conducted to determine the molecular mechanism of action (MMOA) of the active compounds. Fifteen blocked increases in intracellular calcium >50%. Seven blocked phosphorylation of extracellular signal-regulated kinase (ERK) by upstream mitogen-activated protein kinase kinases >50%. One completely blocked the activity of the calcium-dependent phosphatase calcineurin. None blocked ERK catalytic activity. Eight blocked more than one pathway. For 8 compounds, we were unable to determine an MMOA. The activity of 1 of these compounds was confirmed from powder resupply. We conclude that a screen based on antibody binding to CTLs is a good means of identifying novel candidate immunosuppressants with either known or unknown MMOAs.

LYST controls the biogenesis of the endosomal compartment required for secretory lysosome function.

Chediak-Higashi syndrome (CHS) is caused by mutations in the gene encoding LYST protein, the function of which remains poorly understood. Prominent features of CHS include defective secretory lysosome exocytosis and the presence of enlarged, lysosome-like organelles in several cell types. In order to get further insight into the role of LYST in the biogenesis and exocytosis of cytotoxic granules, we analyzed cytotoxic T lymphocytes (CTLs) from patients with CHS. Using confocal microscopy and correlative light electron microscopy, we showed that the enlarged organelle in CTLs is a hybrid compartment that contains proteins components from recycling-late endosomes and lysosomes. Enlargement of cytotoxic granules results from the progressive clustering and then fusion of normal-sized endolysosomal organelles. At the immunological synapse (IS) in CHS CTLs, cytotoxic granules have limited motility and appear docked while nevertheless unable to degranulate. By increasing the expression of effectors of lytic granule exocytosis, such as Munc13-4, Rab27a and Slp3, in CHS CTLs, we were able to restore the dynamics and the secretory ability of cytotoxic granules at the IS. Our results indicate that LYST is involved in the trafficking of the effectors involved in exocytosis required for the terminal maturation of perforin-containing vesicles into secretory cytotoxic granules.

Midline 1 controls polarization and migration of murine cytotoxic T cells.

Midline 1 (MID1) is a microtubule-associated ubiquitin ligase that regulates protein phosphatase 2 A levels. Loss-of-function mutations in MID1 lead to the human X-linked Opitz G/BBB (OS) syndrome characterized by defective midline development during embryogenesis. We have recently shown that MID1 is strongly up-regulated in murine cytotoxic T lymphocytes (CTLs), and that it has a significant impact on exocytosis of lytic granules and the killing capacity of CTLs. The aims of the present study were to determine the localization of MID1 in migrating CTLs, and to investigate whether MID1 affects CTL polarization and migration. We found that MID1 mainly localizes to the uropod of migrating CTLs and that it has a substantial impact on CTL polarization and migration in vitro. Furthermore, analysis of contact hypersensitivity responses supported that MID1 controls effector functions of CTLs in hapten-challenged skin in vivo. These results provide significant new knowledge on the role of MID1 in CTL biology.

Midline 1 directs lytic granule exocytosis and cytotoxicity of mouse killer T cells.

Midline 1 (MID1) is a microtubule-associated ubiquitin ligase that regulates protein phosphatase 2A activity. Loss-of-function mutations in MID1 lead to the X-linked Opitz G/BBB syndrome characterized by defective midline development during embryogenesis. Here, we show that MID1 is strongly upregulated in murine cytotoxic lymphocytes (CTLs), and that it controls TCR signaling, centrosome trafficking, and exocytosis of lytic granules. In accordance, we find that the killing capacity of MID1(-/-) CTLs is impaired. Transfection of MID1 into MID1(-/-) CTLs completely rescued lytic granule exocytosis, and vice versa, knockdown of MID1 inhibited exocytosis of lytic granules in WT CTLs, cementing a central role for MID1 in the regulation of granule exocytosis. Thus, MID1 orchestrates multiple events in CTL responses, adding a novel level of regulation to CTL activation and cytotoxicity.

Transcriptional regulation of Munc13-4 expression in cytotoxic lymphocytes is disrupted by an intronic mutation associated with a primary immunodeficiency.

Autosomal recessive mutations in UNC13D, the gene that encodes Munc13-4, are associated with familial hemophagocytic lymphohistiocytosis type 3 (FHL3). Munc13-4 expression is obligatory for exocytosis of lytic granules, facilitating cytotoxicity by T cells and natural killer (NK) cells. The mechanisms regulating Munc13-4 expression are unknown. Here, we report that Munc13-4 is highly expressed in differentiated human NK cells and effector CD8(+) T lymphocytes. A UNC13D c.118-308C>T mutation, causative of FHL3, disrupted binding of the ETS family member ELF1 to a conserved intronic sequence. This mutation impairs UNC13D intron 1 recruitment of STAT4 and the chromatin remodeling complex component BRG1, diminishing active histone modifications at the locus. The intronic sequence acted as an overall enhancer of Munc13-4 expression in cytotoxic lymphocytes in addition to representing an alternative promoter encoding a novel Munc13-4 isoform. Mechanistically, T cell receptor engagement facilitated STAT4-dependent Munc13-4 expression in naive CD8(+) T lymphocytes. Collectively, our data demonstrates how chromatin remodeling within an evolutionarily conserved regulatory element in intron 1 of UNC13D regulates the induction of Munc13-4 expression in cytotoxic lymphocytes and suggests that an alternative Munc13-4 isoform is required for lymphocyte cytotoxicity. Thus, mutations associated with primary immunodeficiencies may cause disease by disrupting transcription factor binding.

The Perforin Pore Facilitates the Delivery of Cationic Cargos

Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (granzymes). The pore-forming protein perforin is essential for delivery of granzymes into the cytoplasm of target cells; however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex/perforin (MACPF) domain and oligomerizes to form an aqueous pore in the plasma membrane; therefore the simplest (and best supported) model suggests that granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favors the delivery of cationic molecules, and efficiently delivers human granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerized MACPF proteins, which may include an anionic lumen.

T Lymphocytes Expressing a CD16 Signaling Receptor Exert Antibody-Dependent Cancer Cell Killing

To expand applications for T-cell-based immunotherapy in cancer, we designed a receptor that binds the Fc portion of human immunoglobulins and delivers activation signals. The construct included the high-affinity CD16 (FCGR3A) V158 variant, CD8α hinge, and transmembrane domains, along with signaling domains from CD3ζ and 4-1BB (TNFRSF9), forming a chimeric receptor termed CD16V-BB-ζ. After retrovirus-mediated expression in human T cells, CD16V-BB-ζ bound humanized antibodies with higher affinity than a control receptor containing the more common F158 variant. Engagement of CD16V-BB-ζ provoked T-cell activation, exocytosis of lytic granules, and sustained proliferation, with a mean cell recovery after 4-week coculture with Daudi lymphoma cells and rituximab of nearly 70-fold relative to input cells. In contrast, unbound antibody alone produced no effect. CD16V-BB-ζ T cells specifically killed lymphoma cells and primary chronic lymphocytic leukemia cells in combination with rituximab at a low effector:target ratio, even when assayed on mesenchymal cells. Trastuzumab triggered CD16V-BB-ζ-mediated killing of HER2 (ERBB2)(+) breast and gastric cancer cells; similar results were obtained with an anti-GD2 antibody in neuroblastoma and osteosarcoma cells. Furthermore, coadministration of CD16V-BB-ζ T cells with immunotherapeutic antibodies exerted considerable antitumor activity in vivo. Signaling mediated by 4-1BB-CD3ζ induced higher T-cell activation, proliferation, and cytotoxicity than CD3ζ or FcεRIγ, and the receptor was expressed effectively after mRNA electroporation without viral vectors, facilitating clinical translation. Our results offer preclinical proof of concept for CD16V-BB-ζ as a universal, next-generation chimeric receptor with the potential to augment the efficacy of antibody therapies for cancer.

Flow Cytometry Enables a High-Throughput Homogeneous Fluorescent Antibody-Binding Assay for Cytotoxic T Cell Lytic Granule Exocytosis

We developed a homogeneous phenotypic fluorescence end-point assay for cytotoxic T lymphocyte lytic granule exocytosis. This flow cytometric assay measures binding of an antibody to a luminal epitope of a lysosomal membrane protein (LAMP-1) that is exposed by exocytosis to the extracellular solution. Washing to remove unbound antibody is not required. Confirming the assay’s ability to detect novel active compounds, we screened at a concentration of 50 µM a synthetic diversity library of 91 compounds in a 96-well plate format, identifying 17 compounds that blocked by 90% or more. The actions of six structurally related tetracyano-hexahydroisoindole compounds that inhibited by ~90% at a concentration of 10 µM were investigated further. Four reduced elevations in intracellular Ca2+; it is likely that depolarization of the cells’ membrane potential underlies the effect for at least two of the compounds. Another compound was found to be a potent inhibitor of the activation of the mitogen-activated protein (MAP) kinase ERK. Finally, we transferred the assay to a 384-well format and screened the Prestwick Compound Library using high-throughput flow cytometry. Our results indicate that our assay will likely be a useful means of screening libraries for novel compounds with important biological activities.

Comparison of primary human cytotoxic T-cell and natural killer cell responses reveal similar molecular requirements for lytic granule exocytosis but differences in cytokine production

AbstractCytotoxic lymphocytes, encompassing cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, kill pathogen-infected, neoplastic, or certain hematopoietic cells through the release of perforin-containing lytic granules. In the present study, we first performed probability-state modeling of differentiation and lytic granule markers on CD8+ T cells to enable the comparison of bona fide CTLs with NK cells. Analysis identified CD57bright expression as a reliable phenotype of granule marker–containing CTLs. We then compared CD3+CD8+CD57bright CTLs with NK cells. Healthy adult peripheral blood CD3+CD8+CD57bright CTLs expressed more granzyme B but less perforin than CD3−CD56dim NK cells. On stimulation, such CTLs degranulated more readily than other T-cell subsets, but had a propensity to degranulate that was similar to NK cells. Remarkably, the CTLs produced cytokines more rapidly and with greater frequency than NK cells. In patients with biallelic mutations in UNC13D, STX11, or STXBP2 associated with familial hemophagocytic lymphohistiocytosis, CTL and NK cell degranulation were similarly impaired. Therefore, cytotoxic lymphocyte subsets have similar requirements for Munc13-4, syntaxin-11, and Munc18-2 in lytic granule exocytosis. The present results provide a detailed comparison of human CD3+CD8+CD57bright CTLs and NK cells and suggest that analysis of CD57bright CTL function may prove useful in the diagnosis of primary immunodeficiencies including familial hemophagocytic lymphohistiocytosis.

Impairment of CD4+ cytotoxic T cells predicts poor survival and high recurrence rates in patients with hepatocellular carcinoma

The role of CD4(+) cytotoxic T cells (CTLs) in hepatocellular carcinoma (HCC) remains obscure. This study characterized CD4(+) CTLs in HCC patients and further elucidated the associations between CD4(+) CTLs and HCC disease progression. In all, 547 HCC patients, 44 chronic hepatitis B (CHB) patients, 86 liver cirrhosis (LC) patients, and 88 healthy individuals were enrolled in the study. CD4(+) CTLs were defined by flow cytometry, immunohistochemistry, and lytic granule exocytosis assays. A multivariate analysis of prognostic factors for overall survival was performed using the Cox proportional hazards model. Circulating and liver-infiltrating CD4(+) CTLs were found to be significantly increased in HCC patients during early stage disease, but decreased in progressive stages of HCC. This loss of CD4(+) CTLs was significantly correlated with high mortality rates and reduced survival time of HCC patients. In addition, the proliferation, degranulation, and production of granzyme A, granzyme B, and perforin of CD4(+) CTLs were inhibited by the increased forkhead/winged helix transcription factor (FoxP3(+) ) regulatory T cells in these HCC patients. Further analysis showed that both circulating and tumor-infiltrating CD4(+) CTLs were independent predictors of disease-free survival and overall survival after the resection of the HCC. The progressive deficit in CD4(+) CTLs induced by increased FoxP3(+) regulatory T cells was correlated with poor survival and high recurrence rates in HCC patients. These data suggest that CD4(+) CTLs may represent both a potential prognostic marker and a therapeutic target for the treatment of HCC.

Synaptobrevin2 is the v-SNARE required for cytotoxic T-lymphocyte lytic granule fusion

Cytotoxic T lymphocytes kill virus-infected and tumorigenic target cells through the release of perforin and granzymes via fusion of lytic granules at the contact site, the immunological synapse. It has been postulated that this fusion process is mediated by non-neuronal members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex protein family. Here, using a synaptobrevin2-monomeric red fluorescence protein knock-in mouse we demonstrate that, surprisingly, the major neuronal v-SNARE synaptobrevin2 is expressed in cytotoxic T lymphocytes and exclusively localized on granzyme B-containing lytic granules. Cleavage of synaptobrevin2 by tetanus toxin or ablation of the synaptobrevin2 gene leads to a complete block of lytic granule exocytosis while leaving upstream events unaffected, identifying synaptobrevin2 as the v-SNARE responsible for the fusion of lytic granules at the immunological synapse.

Development of classical Hodgkin's lymphoma in an adult with biallelic STXBP2 mutations

Experimental model systems have delineated an important role for cytotoxic lymphocytes in the immunosurveillance of cancer. In humans, perforin-deficiency has been associated with occurrence of hematologic malignancies. Here, we describe an Epstein-Barr virus-positive classical Hodgkin's lymphoma in a patient harboring biallelic mutations in STXBP2, a gene required for exocytosis of perforin-containing lytic granules and associated with familial hemophagocytic lymphohistocytosis. Cytotoxic T lymphocytes were found infiltrating the tumor, and a high frequency of Epstein-Barr virus-specific cytotoxic T lymphocytes were detected in peripheral blood. However, lytic granule exocytosis and cytotoxicity by cytotoxic T lymphocytes, as well as natural killer cells, were severely impaired in the patient. Thus, the data suggest a link between defective lymphocyte exocytosis and development of lymphoma in STXBP2-deficient patients. Therefore, with regards to treatment of familial hemophagocytic lymphohistocytosis patients with mutations in genes required for lymphocyte exocytosis, it is important to consider both the risks of hemophagocytic lymphohistocytosis and malignancy.

Ganglioside Inhibition of CD8+ T Cell Cytotoxicity: Interference with Lytic Granule Trafficking and Exocytosis

Granule exocytosis-mediated cytotoxicity by CD8(+) CTL plays a crucial role in adaptive immunity to tumors and to intracellular pathogens. This T cell effector function has been shown to be defective in various murine tumor models and in human cancer. However, factors and their mechanisms that cause inhibition of CD8(+) T cell lytic function in tumor-bearing hosts remain to be fully defined. We postulate that gangliosides, highly expressed on tumor cell membranes, actively shed into the tumor microenvironment, and having well-established immunosuppressive properties, may be such a factor. We exposed primary mouse CD8(+) CTL to gangliosides derived from three sources (tumors and normal brain). This significantly inhibited cytotoxicity-mediated by granule exocytosis, that is, cytotoxicity of alloantigen-specific and polyclonal CD8(+) CTL in vitro. These molecules did not interfere with the interaction of CD8(+) T cells with their cognate targets. Rather, they inhibited lytic granule release in response both to TCR engagement and to stimuli that induce granule release in a nonpolarized manner. At the subcellular level, confocal microscopic imaging identified inhibition of polarization of lytic granules to the immunological synapse upon target cell recognition. Thus, tumor-shed gangliosides suppress lytic activity of CD8(+) T cells by a novel mechanism, that is, inhibition of trafficking of lytic granules in response to TCR engagement, as well as by interfering with the process of granule exocytosis in CD8(+) T cells.

High DGK-α and Disabled MAPK Pathways Cause Dysfunction of Human Tumor-Infiltrating CD8+ T Cells That Is Reversible by Pharmacologic Intervention

CD8(+) tumor-infiltrating T cells (CD8-TILs) are found in many types of tumors including human renal cell carcinoma. However, tumor rejection rarely occurs, suggesting limited functional activity in the tumor microenvironment. In this study, we document that CD8-TILs are unresponsive to CD3 stimulation, showing neither lytic activity, nor lytic granule exocytosis, nor IFN-γ production. Mechanistically, no deficits in TCR proximal signaling molecules (lymphocyte-specific protein tyrosine kinase, phospholipase Cγ) were identified. In contrast, distal TCR signaling was suppressed, as T cells of TILs showed strongly reduced steady-state phosphorylation of the MAPK ERK and were unable to increase phosphorylation of ERK and JNK as well as AKT and AKT client proteins (IκB, GSK3) after stimulation. These deficits were tumor-specific as they were not observed in CD8(+) T cells infiltrating non-tumor kidney areas (CD8(+) non-tumor kidney-infiltrating lymphocytes; CD8-NILs). Diacylglycerol kinase-α (DGK-α) was more highly expressed in CD8-TILs compared with that in CD8-NILs, and its inhibition improved ERK phosphorylation and lytic granule exocytosis. Cultivation of TILs in low-dose IL-2 reduced DGK-α protein levels, increased steady-state phosphorylation of ERK, improved stimulation-induced phosphorylation of ERK and AKT, and allowed more CD8-TILs to degranulate and to produce IFN-γ. Additionally, the protein level of the AKT client molecule p27kip, an inhibitory cell cycle protein, was reduced, whereas cyclin E, which promotes G1-S phase transition, was increased. These results indicate that the tumor-inflicted deficits of TILs are reversible. DGK-α inhibition and provision of IL-2 signals could be strategies to recruit the natural CD8(+) T cells to the anti-tumor response and may help prevent inactivation of adoptively transferred T cells thereby improving therapeutic efficacy.

Function of Syntaxin11 in cytotoxic T lymphocytes (121.9)

Abstract Cytotoxic T Lymphocytes (CTLs) form immunological synapses (IS) with antigen presenting cells to kill them. Killing requires the fusion of lytic granules at the IS to release the containing cytotoxic molecules (perforin and granzymes). The fusion process is mediated by SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins. Since mutations within the coding region of syntaxin11 lead to Familial Hemophagocytic Lymphohistiocytosis (FHL4), an immune disorder caused by impaired granule exocytosis. We have investigated the contribution of syntaxin11 to lytic granule release. Using CD3 specific antibodies as a marker for a functional IS, we show that syntaxin11 accumulates at the IS in CTLs. Syntaxin11 was also co-localized with Munc18-2, a docking factor in agreement with the role of Munc18-2 as a syntaxin chaperone in other cell types. Using high resolution nanoscopy (structured illumination microscopy) we determined the subcellular localization of syntaxin11 in CTLs with a syntaxin11 specific antibody. Syntaxin11 was associated with recycling endosomes and mannose 6-phosphate receptor (M6PR) positive compartments. Functional assays using Syntaxin11 specific siRNA (knockdown studies) show a reduction in degranulation and killing efficiency in contrast to control siRNA transfected CTLs. These results support the conclusion that syntaxin11 is required for lytic granule exocytosis.

SNARE protein expression and localization in human cytotoxic T lymphocytes

The major function of cytotoxic T lymphocytes (CTLs) is to eliminate pathogen-infected and tumorigenic cells. This is mediated mainly through the exocytosis of lytic granules (LGs) containing cytotoxic components, such as perforin and granzymes at the immunological synapse (IS). The soluble NSF attachment receptor (SNARE) protein isoforms are well known to be required for vesicle exocytosis in neuronal synapses, but their potential function in CTLs is only partly understood. Here, we examined the expression of SNARE proteins before and after the activation of primary human CD8(+) T cells and determined their co-localization with LGs and CD3 after IS formation with target cells. We found that several key SNARE proteins in neuronal cells were not expressed in CTLs, such as syntaxin1B2 and SNAP-25. Vti1b, Stx8 and Stx16 had the highest degrees of co-localization with LGs while Stx3, Stx4, Stx6, Stx7, Stx8, Stx13, Vti1b, VAMP3 and VAMP4 co-localized with CD3. Our data provide the first complete expression profile and localization of SNAREs in primary human CD8(+) T cells, laying the groundwork for further understanding their potential role in T-cell function.

Stepwise Maturation of Lytic Granules during Differentiation and Activation of Human CD8+ T Lymphocytes

During differentiation, cytotoxic T lymphocytes (CTL) acquire their killing potential through the biogenesis and maturation of lytic granules that are secreted upon target cell recognition. How lytic granule load in lytic molecules evolves during CTL differentiation and which subsets of lytic granules are secreted following activation remains to be investigated. We set up a flow cytometry approach to analyze single lytic granules isolated from primary human CTL according to their size and molecular content. During CTL in vitro differentiation, a relatively homogeneous population of lytic granules appeared through the progressive loading of Granzyme B, Perforin and Granzyme A within LAMP1+ lysosomes. PMA/ionomycin-induced lytic granule exocytosis was preceded by a rapid association of the docking molecule Rab27a to approximately half of the lytic granules. Activated CTL were found to limit exocytosis by sparing lytic granules including some associated to Rab27a. Our study provides a quantification of key steps of lytic granule biogenesis and highlights the potential of flow cytometry to study organelle composition and dynamics.

Tumor lactic acidosis suppresses CTL function by inhibition of p38 and JNK/c‐Jun activation

Lactic acidosis is common to most solid tumors and has been found to affect infiltrating immune cells. Here we document effector phase inhibition of cytotoxic T cells (CTLs) involving complete blockage of cytokine production and partial impairment of lytic granule exocytosis. Lactic acidosis impaired TCR-triggered phosphorylation of JNK, c-Jun and p38, while not affecting MEK1 and ERK. The select targeting of signaling proteins involved in IFNγ production (JNK/c-Jun, p38) without affecting those jointly used in cytokine regulation and granule exocytosis (MEK1/ERK) explains the observed split effect of lactic acidosis on the CTL responses. CTL inhibition by lactic acidosis showed fast dynamics with immediate onset and reversion. Functional recovery by neutralizing the extracellular pH despite continuous presence of lactate holds promise that CTL activity can be improved in the milieu of solid tumors with appropriate anti-acidosis treatment, thereby increasing the efficacy of adoptive T cell therapy.

The interplay betweenLeishmaniapromastigotes and human Natural Killer cellsin vitroleads to direct lysis ofLeishmaniaby NK cells and modulation of NK cell activity byLeishmaniapromastigotes

NK cells represent one of the first lines of defence in the immune reaction after invasion of Leishmania parasites. Depletion of mouse natural killer (NK) cells dramatically enhances susceptibility of normally resistant mice. In this study we evaluated the fate of NK cells and parasites after contact formation. The hydrophilic fluorescent dye CMFDA (chloro-methylfluorescin diacetate) that allows analysis of cytotoxicity in flow cytometry and microscopy was used. Furthermore, these findings were confirmed with scanning and transmission electron microscopy. Direct contact points were found between Leishmania promastigotes and naïve human NK cells. These contacts were associated with transfer of cytosol by membrane bridges and cytotoxicity of NK cells against Leishmania. However, in contrast to other target cells which allow repeated exocytosis of lytic granules, contact with Leishmania causes immediate destruction of NK cells in a non-apoptotic way. Our results give a reasonable explanation for ex vivo observations of reduced NK cell numbers and impaired NK response in patients with acute cutaneous leishmaniasis. Animal models have clearly shown that NK cells play a key role in the induction and direction of the immune response. Thus inhibition of NK cells at the onset of infection would be advantageous for the survival of the parasite.

A novel Dutch mutation in UNC13D reveals an essential role of the C2B domain in munc13‐4 function

UNC13D, encoding the protein munc13-4, is essential in intracellular trafficking and exocytosis of lytic granules. Mutations in this gene are associated with familial hemophagocytic lymphohistiocytosis type 3 (FHL3), a genetically heterogeneous, rare autosomal recessive immune disorder. How mutations affect function of munc13-4 is poorly understood. Since 2006 we genetically identified seven FHL patients with mutations in UNC13D. Here, we report for the first time a c.2695C>T (p.Arg899X) mutation in exon 28 of UNC13D in three young unrelated Dutch patients. The mutation causes a premature stop codon and encodes munc13-4(1-899), which lacks the C-terminal C2 domain. Genealogical research and haplotyping of the patient families demonstrated that a single ancestral founder introduced the mutation in the Netherlands. We then characterized the mutant protein phenotypically in cell biological and immunological assays. Munc13-4(1-899) was correctly targeted to CD63-positive secretory lysosomes, although its stability was reduced and dynamic turnover on the granule membrane became uncoupled from receptor signaling. In accord, and in contrast to wild-type munc13-4, ectopically expressed mutant failed to rescue degranulation in cells with silenced endogenous munc13-4. The functional and clinical data showed that this novel Dutch founder mutation leads to severe early onset of FHL3 due to misfolding and degradation of munc13-4(1-899).