Secretion Of Cytotoxic Granules Research Articles

Abstract 3968: Class I HLA-independent lysis of cancer cells by tumor-infiltrating CD8 T cells

Abstract T cell-based therapies have transformed cancer treatment, although intrinsic or acquired resistance develops in nearly half of the patients. Tumor-infiltrating CD8+ T lymphocytes (TILs) are key determinants of anti-tumor immunity by secretion of cytotoxic granules, such as granzymes and perforin, and effector cytokines, such as TNFa and IFNg. A deep understanding of how CD8+ TILs eliminate cancer cells will help identify resistance mechanisms and develop new therapeutic strategies. Here, we demonstrate that TILs eliminated matched melanoma cells in a time and dose-dependent manner but in a class I HLA-independent fashion. Specifically, blocking class I HLA-TCR interaction either by the deletion of B2M (resulting in loss of class I HLA surface expression) or TCR genes did not alter the activity of these TILs. We further confirmed that TIL-mediated killing is contact-dependent. After excluding the potential roles of CD1d and MR1-restrictive T cells, gamma delta T cells, and NKG2D in TIL-mediated killing, a whole-genome CRISPR/Cas9 screen was performed in cancer cells. We identified that genes involved in JAK-STAT and cell-extrinsic apoptosis pathways are necessary for TIL-mediated killing. Together, these findings demonstrate that expanded CD8 TILs from melanoma patients are capable of eliminating melanoma cells via a novel, class I MHC-independent mechanism by coactivation of both JAK-STAT and cell-extrinsic apoptosis pathways. Citation Format: Hongyan Xie, Aiping Jiang, Jonathan Perera, Aonkon Dey, Neal Smith, Moshe Sade-Feldman, Robert T. Manguso, Nir Hacohen, Russell W. Jenkins. Class I HLA-independent lysis of cancer cells by tumor-infiltrating CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3968.

3-Dimensional Model to Study Apoptosis Induction of Activated Natural Killer Cells Conditioned Medium Using Patient-Derived Colorectal Cancer Organoids.

Natural killer (NK) cells are innate lymphocytes that can kill tumor cells via different pathways, including the secretion of cytotoxic granules in immunological synapses and the binding of apoptosis-inducing ligands with cognate death receptors on tumor cells. These ligands are also soluble in NK cells conditioned medium (NK-CM). However, novel preclinical in vitro models are required for solid tumors such as colorectal cancer (CRC) to investigate apoptosis induction of activated NK-CM in a tissue-like structure. In the present study, we established a patient-derived CRC organoid culture system as a new tool for CRC research in the last decade. Tumor organoids were stained with hematoxylin and eosin (H&E) and compared with the original tumor taken from the patient. Goblet cell differentiation and mucus secretion were evaluated using periodic acid–Schiff and alcian blue histochemical staining. Moreover, tumor organoids were stained for CDX2 and Ki67 markers with immunohistochemistry (IHC) to investigate gastrointestinal origin and proliferation. Histopathological evaluations indicated tumor organoids represent patient tumor characteristics. Primary NK cells were isolated and characterized using CD56 marker expression and the lack of the CD3 marker. Flow cytometry results showed the purity of isolated CD3−and CD56 + NK cells about 93%. After further ex vivo expansion, IL-2-activated NK-CM was collected. Secretions of IFN-γ and TNF-α were measured to characterize activated NK-CM. Cytokines levels were significantly elevated in comparison to the control group. Soluble forms of apoptosis-inducing ligands, including TNF-related apoptosis-inducing ligand (TRAIL) and FasL, were detected by western blot assay. Colon cancer organoids were treated by IL-2-activated NK-CM. Apoptosis was assessed by Annexin V-FITC/PI staining and quantified by flow cytometry. In conclusion, despite the activated NK-CM containing apoptosis-inducing ligands, these ligands’ soluble forms failed to induce apoptosis in patient-derived colon cancer organoids. Nevertheless, we report a reliable in vitro assessment platform in a personalized setting.

Several active components within MSCs secretome inhibit the IL-2-mediated effector functions of NK cells

Abstract Mesenchymal stem cells (MSCs) are well-known for their immunomodulatory functions in innate and adaptive immunity. Their functional diversity was verified in various disease model through paracrine mechanisms. MSCs-derived secretome consists of a variety of bioactive components such as nucleic acids, cytokines, chemokines, and small molecules. However, the effect of MSCs on natural killer (NK) cells activity is still controversial. In this study, we tried to identify active components within MSCs secretome possessing ability to affect NK cells activity and to investigate their action mechanisms. We found that MSCs secretome had no effect on survival and proliferation of NK-92 cells but specifically diminished cell activity, as evidenced by lower secretion of cytotoxic granules and effector cytokines and also reducing killing ability. This effect was reproduced in human peripheral NK cells equally. Moreover, seven candidates for modulating inflammation within MSCs secretome were identified via LC-MS/MS proteomic analysis. Interestingly, they are totally different factors from those known as MSC’s immunomodulatory factor. After treatment of neutralizing antibodies to each candidate, we observed that only three of the groups with neutralizing antibodies recovered cytokine secretion and cytotoxic ability of NK-92 cells. These findings suggest that MSCs secretome can inhibit the effector functions of NK cells via some particular molecules, which are increasing expression of inhibitory receptor CD96 on NK cells, consequently activating inhibitory signal. This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (No. 2021R1F1A1049630).

Immunomodulatory effects of flavonoids: An experimental study on natural-killer-cell-mediated cytotoxicity against lung cancer and cytotoxic granule secretion profile

Background: A new approach involving immune-cell-mediated cancer therapy has been adopted extensively for the sake of lung cancer treatments by utilizing natural killer (NK) cells. NK cell activity can be enhanced with certain agents, and among them are flavonoids. Thus, this study was conducted to investigate the immunomodulatory roles of apigenin, luteolin and quercetin on NK cell activity against lung cancer cells and on the secretions of perforin and granulysin profile. Methods: The NK-92 cells were grown in complete α-Minimum Essential Medium (MEM). NCI-H460 lung cancer cells were cultured in Roswell Park Memorial Institute 1640 media. NK cell activity against lung cancer cells were done using MTT(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The secretions of perforin and granulysin profiles were then analysed using enzyme-linked immunosorbent assay. Results: Apigenin, luteolin and quercetin significantly increased the NK-cell-mediated cytotoxic activity against lung cancer cells at concentrations 12.5 µg/ml and 25µg/ml ( P < 0.001). The secretion levels of perforin and granulysin from NK cells were also significantly enhanced with apigenin and luteolin treatment but not with quercetin. Conclusions: All three flavonoid compounds possessed some significant immunomodulatory actions on NK cell cytotoxic activity and granule secretion profiles towards lung cancer therapy.

Glucuronoxylomannan in the Cryptococcus species capsule as a target for Chimeric Antigen Receptor T-cell therapy

Background aimsThe genus Cryptococcus comprises two major fungal species that cause clinical infections in humans: Cryptococcus gattii and Cryptococcus neoformans. To establish invasive human disease, inhaled cryptococci must penetrate the lung tissue and reproduce. Each year, about 1 million cases of Cryptococcus infection are reported worldwide, and the infection's mortality rate ranges from 20% to 70%. Many HIV+/AIDS patients are affected by Cryptococcus infections, with 220,000 cases of cryptococcal meningitis reported worldwide in this population every year (C. neoformans infection statistics, via the Centers for Disease Control and Prevention, https://www.cdc.gov/fungal/diseases/cryptococcosis-neoformans/statistics.html). To escape from host immune cell attack, Cryptococcus covers itself in a sugar-based capsule composed primarily of glucuronoxylomannan (GXM). To evade phagocytosis, yeast cells increase to a >45-µm perimeter and become titan, or giant, cells. Cryptococci virulence is directly proportional to the percentage of titan/giant cells present during Cryptococcus infection. To combat cryptococcosis, the authors propose the redirection of CD8+ T cells to target the GXM in the capsule via expression of a GXM-specific chimeric antigen receptor (GXMR-CAR). ResultsGXMR-CAR has an anti-GXM single-chain variable fragment followed by an IgG4 stalk in the extracellular domain, a CD28 transmembrane domain and CD28 and CD3-ς signaling domains. After lentiviral transduction of human T cells with the GXMR-CAR construct, flow cytometry demonstrated that 82.4% of the cells expressed GXMR-CAR on their surface. To determine whether the GXMR-CAR+ T cells exhibited GXM-specific recognition, these cells were incubated with GXM for 24 h and examined with the use of brightfield microscopy. Large clusters of proliferating GXMR-CAR+ T cells were observed in GXM-treated cells, whereas no clusters were observed in control cells. Moreover, the interaction of GXM with GXMR-CAR+ T cells was detected via flow cytometry by using a GXM-specific antibody, and the recognition of GXM by GXMR-CAR T cells triggered the secretion of granzyme and interferon gamma (IFN-γ). The ability of GXMR-CAR T cells to bind to the yeast form of C. neoformans was detected by fluorescent microscopy, but no binding was detected in mock-transduced control T cells (NoDNA T cells). Moreover, lung tissue sections were stained with Gomori Methenamine Silver and evaluated by NanoZoomer (Hamamatsu), revealing a significantly lower number of titan cells, with perimeters ranging from 50 to 130 µm and giant cells >130 µm in the CAR T-cell treated group when compared with other groups. Therefore, the authors validated the study's hypothesis by the redirection of GXMR-CAR+ T cells to target GXM, which induces the secretion of cytotoxic granules and IFN-γ that will aid in the control of cryptococcosis ConclusionsThus, these findings reveal that GXMR-CAR+ T cells can target C. neoformans. Future studies will be focused on determining the therapeutic efficacy of GXMR-CAR+ T cells in an animal model of cryptococcosis.

Histidine-rich glycoprotein augments natural killer cell function by modulating PD-1 expression via CLEC-1B.

Augmentation of natural killer (NK) cell cytotoxicity is one of the greatest challenges for cancer immunotherapy. Although histidine‐rich glycoprotein (HRG), a 75‐kDa glycoprotein with various immunomodulatory activities, reportedly elicits antitumor immunity, its effect on NK cell cytotoxicity is unclear. We assessed NK cell cytotoxicity against K562 cells. We also measured concentrations of cytokines and granzyme B in the cell supernatant. The proportion of CD56bright NK cells and NK cell surface PD‐1 expression was assessed with flow cytometry. The neutralizing effects of anti‐C‐type lectin‐like receptor (CLEC) 1B against HRG were also measured. NK cell morphological changes were analyzed via confocal microscopy. HRG significantly increased NK cell cytotoxicity against K562 cell lines. HRG also increased the release of granzyme B and the proportion of CD56bright NK cells. Further, HRG was able to decrease NK cell surface PD‐1 expression. The effects of HRG on NK cells were reversed with anti‐CLEC‐1B antibodies. Additionally, we confirmed NK cell nuclear morphology and F‐actin distribution, which are involved in the regulation of cytotoxic granule secretion. Because both PD‐1 and CLEC‐1B are associated with prognosis during malignancy, HRG incorporates these molecules to exert the antitumor immunity role. These facts indicate the potential of HRG to be a new target for cancer immunotherapy.

Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression patterns

The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56hi versus CD56dim defined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation stages. Such comprehensive cytotoxic profiling may identify distinct patterns of cytotoxic potential relevant for specific infections, autoimmunity or tumor settings.

A Novel Carcinoembryonic Antigen T-Cell Bispecific Antibody (CEA TCB) for the Treatment of Solid Tumors.

CEA TCB is a novel IgG-based T-cell bispecific (TCB) antibody for the treatment of CEA-expressing solid tumors currently in phase I clinical trials (NCT02324257). Its format incorporates bivalent binding to CEA, a head-to-tail fusion of CEA- and CD3e-binding Fab domains and an engineered Fc region with completely abolished binding to FcγRs and C1q. The study provides novel mechanistic insights into the activity and mode of action of CEA TCB. CEA TCB activity was characterized on 110 cell lines in vitro and in xenograft tumor models in vivo using NOG mice engrafted with human peripheral blood mononuclear cells. Simultaneous binding of CEA TCB to tumor and T cells leads to formation of immunologic synapses, T-cell activation, secretion of cytotoxic granules, and tumor cell lysis. CEA TCB activity strongly correlates with CEA expression, with higher potency observed in highly CEA-expressing tumor cells and a threshold of approximately 10,000 CEA-binding sites/cell, which allows distinguishing between high- and low-CEA-expressing tumor and primary epithelial cells, respectively. Genetic factors do not affect CEA TCB activity confirming that CEA expression level is the strongest predictor of CEA TCB activity. In vivo, CEA TCB induces regression of CEA-expressing xenograft tumors with variable amounts of immune cell infiltrate, leads to increased frequency of activated T cells, and converts PD-L1 negative into PD-L1-positive tumors. CEA TCB is a novel generation TCB displaying potent antitumor activity; it is efficacious in poorly infiltrated tumors where it increases T-cell infiltration and generates a highly inflamed tumor microenvironment. Clin Cancer Res; 22(13); 3286-97. ©2016 AACR.

Polygenic mutations in the cytotoxicity pathway increase susceptibility to develop HLH immunopathology in mice

AbstractHemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory disease. Inherited forms of HLH are caused by biallelic mutations in several effectors of granule-dependent lymphocyte-mediated cytotoxicity. A small proportion of patients with a so-called “secondary” form of HLH, which develops in the aftermath of infection, autoimmunity, or cancer, carry a monoallelic mutation in one or more HLH-associated genes. Although this observation suggests that HLH may have a polygenic mode of inheritance, the latter is very difficult to prove in humans. In order to determine whether the accumulation of partial genetic defects in lymphocyte-mediated cytotoxicity can contribute to the development of HLH, we generated mice that were doubly or triply heterozygous for mutations in HLH-associated genes, those coding for perforin, Rab27a, and syntaxin-11. We found that the accumulation of monoallelic mutations did indeed increase the risk of developing HLH immunopathology after lymphocytic choriomeningitis virus infection. In mechanistic terms, the accumulation of heterozygous mutations in the two degranulation genes Rab27a and syntaxin-11, impaired the dynamics and secretion of cytotoxic granules at the immune synapse of T lymphocytes. In addition, the accumulation of heterozygous mutations within the three genes impaired natural killer lymphocyte cytotoxicity in vivo. The genetic defects can be ranked in terms of the severity of the resulting HLH manifestations. Our results form the basis of a polygenic model of the occurrence of secondary HLH.

Ubiquitin-dependent endocytosis of NKG2D-DAP10 receptor complexes activates signaling and functions in human NK cells.

Cytotoxic lymphocytes share the presence of the activating receptor NK receptor group 2, member D (NKG2D) and the signaling-competent adaptor DNAX-activating protein 10 (DAP10), which together play an important role in antitumor immune surveillance. Ligand stimulation induces the internalization of NKG2D-DAP10 complexes and their delivery to lysosomes for degradation. In experiments with human NK cells and cell lines, we found that the ligand-induced endocytosis of NKG2D-DAP10 depended on the ubiquitylation of DAP10, which was also required for degradation of the internalized complexes. Moreover, through combined biochemical and microscopic analyses, we showed that ubiquitin-dependent receptor endocytosis was required for the activation of extracellular signal-regulated kinase (ERK) and NK cell functions, such as the secretion of cytotoxic granules and the inflammatory cytokine interferon-γ. These results suggest that NKG2D-DAP10 endocytosis represents a means to decrease cell surface receptor abundance, as well as to control signaling outcome in cytotoxic lymphocytes.

Cytotoxic granule secretion by lymphocytes and its link to immune homeostasis.

The granule-dependent cytotoxic activity of T and natural killer lymphocytes has progressively emerged as an important effector pathway not only for host defence but also for immune regulation. The analysis of an early-onset, severe, primary immune dysregulatory syndrome known as hemophagocytic lymphohistiocytosis (HLH) has been decisive in highlighting this latter role and identifying key effectors on the basis of gene mutation analyses and mediators in the maturation and secretion of cytotoxic granules. Studies of cytotoxicity-deficient murine counterparts have helped to define primary HLH as a syndrome in which uncontrolled T-cell activation in response to lymphocytic choriomeningitis virus infection results in excessive macrophage activation and inflammation-associated cytopenia. Recent recognition of late-onset HLH, which occurs in a variety of settings, in association with hypomorphic, monoallelic mutations in genes encoding components of the granule-dependent cytotoxic pathway or even in the absence of such mutations has broadened our view about the mechanisms that underlie the perturbation of immune homeostasis. These findings have led to the development of a model in which disease occurs when a threshold is reached through the accumulation of genetic and environmental risk factors. Nevertheless, validation of this model will require further investigations.

The Sorting Receptor Sortilin Exhibits a Dual Function in Exocytic Trafficking of Interferon-γ and Granzyme A in T Cells

Immunological control of infections or tumors depends on the release of effector cytokines and polarized secretion of cytotoxic granules from T cells and natural killer cells. Here we show that the sorting receptor Sortilin controlled both processes. In murine Sortilin-deficient cytotoxic T lymphocytes, regulated secretion of granzyme A and cytotoxic killing was enhanced and correlated with increased vesicle-associated membrane protein 7 availability. In contrast, loss of Sortilin reduced the release of interferon-γ upon infections and in autoimmune colitis. Exit of interferon-γ from the Golgi apparatus required the presence of Sortilin. Furthermore, we tracked the transport route of interferon-γ beyond this Sortilin-dependent Golgi to early endosome step. In wild-type T cells, trafficking of interferon-γ from the endosomal sorting platform to the plasma membrane proceeded independently of recycling endosomes, and interferon-γ remained excluded from late endosomes. Our results suggest that Sortilin modulates systemic immune responses through exocytic sorting of immunological effector molecules.

Characterization of the functional role of VAMP8 in granule exocytosis of T lymphocytes (69.12)

Abstract One salient event in the adaptive immune response is the activation of T lymphocytes by foreign antigens. They patrol our body for providing a defense against intracellular pathogens, viruses and malignant cells. Upon recognition, T lymphocytes exert their cytotoxic activity through secretion of cytotoxic granules at a specialized cell interface, the immune synapse (IS), or engage in stimulus-coupled release of immune mediators in pre-formed granules. To induce target cell death, T lymphocytes rely on exocytosis of granules, which is the result of a cascade of events consisting of docking, priming, and finally Ca2+-dependent fusion involving soluble NSF attachment receptor (SNARE) proteins. VAMP8 is a SNARE protein that is a key component of a variety of secretion in other immune cell types and whose function in T cell killing is unknown. VAMP8 re-localizes to the immunological synapse following CTL activation suggesting it plays an important role in granule fusion. VAMP8 has also been shown to localize abundantly at the membranes of early and late endosomes. Additionally, VAMP8 plays a role in the exocytosis of organelles formed in the secretory pathway, such as exocrine granules and secretory lysosomes. Hence, this research is aimed at elucidating the involvement of VAMP8 in T lymphocytes of secretory granule release allowing us to better understand the contribution of VAMP8 to the killing process of T lymphocytes.

Terminal transport of lytic granules to the immune synapse is mediated by the kinesin-1/Slp3/Rab27a complex

AbstractCytotoxic T lymphocytes kill target cells via the polarized secretion of cytotoxic granules at the immune synapse. The lytic granules are initially recruited around the polarized microtubule-organizing center. In a dynein-dependent transport process, the granules move along microtubules toward the microtubule-organizing center in the minus-end direction. Here, we found that a kinesin-1–dependent process is required for terminal transport and secretion of polarized lytic granule to the immune synapse. We show that synaptotagmin-like protein 3 (Slp3) is an effector of Rab27a in cytotoxic T lymphocytes and interacts with kinesin-1 through the tetratricopeptide repeat of the kinesin-1 light chain. Inhibition of the Rab27a/Slp3/kinesin-1 transport complex impairs lytic granule secretion. Our data provide further molecular insights into the key functional and regulatory mechanisms underlying the terminal transport of cytotoxic granules and the latter's secretion at the immune synapse.

Human mesenchymal stem cells derived from induced pluripotent stem cells down-regulate NK-cell cytolytic machinery

AbstractA major issue in immunosuppressive biotherapy is the use of mesenchymal stem cells (MSCs) that harbor regulatory capacity. However, currently used bone marrow-derived MSCs (BM-MSCs) are short-lived and cannot assure long lasting immunoregulatory function both in vitro and in vivo. Consequently, we have generated MSCs from human induced pluripotent stem (IPS-MSCs) cells that share similar properties with embryonic stem cells (ES-MSCs). Herein, we compared the immunoregulatory properties of ES/IPS-MSCs with those of BM-MSCs and showed, for the first time, that IPS-derived MSCs display remarkable inhibition of NK-cell proliferation and cytolytic function in a similar way to ES-MSCs. Both MSCs disrupt NK-cell cytolytic machinery in the same fashion that BM-MSCs, by down-regulating the expression of different activation markers and ERK1/2 signaling, leading to an impairment to form immunologic synapses with target cells and, therefore, secretion of cytotoxic granules. In addition, they are more resistant than adult BM-MSCs to preactivated NK cells. IPS-MSCs could represent an attractive alternative source of immunoregulatory cells, and their capacity to impair NK-cell cytotoxicity constitutes a complex mechanism to prevent allograft rejection.

Cytokine Secretion Is Distinct from Secretion of Cytotoxic Granules in NK Cells

NK cells are renowned for their ability to kill virally infected or transformed host cells by release of cytotoxic granules containing granzymes and perforin. NK cells also have important regulatory capabilities chiefly mediated by secretion of cytokines, such as IFN-gamma and TNF. The secretory pathway for the release of cytokines in NK cells is unknown. In this study, we show localization and trafficking of IFN-gamma and TNF in human NK cells in compartments and vesicles that do not overlap with perforin or other late endosome granule markers. Cytokines in post-Golgi compartments colocalized with markers of the recycling endosome (RE). REs are functionally required for cytokine release because inactivation of REs or mutation of RE-associated proteins Rab11 and vesicle-associated membrane protein-3 blocked cytokine surface delivery and release. In contrast, REs are not needed for release of perforin from preformed granules but may be involved at earlier stages of granule maturation. These findings suggest a new role for REs in orchestrating secretion in NK cells. We show that the cytokines IFN-gamma and TNF are trafficked and secreted via a different pathway than perforin. Although perforin granules are released in a polarized fashion at lytic synapses, distinct carriers transport both IFN-gamma and TNF to points all over the cell surface, including within the synapse, for nonpolarized release.

Familial Hemophagocytic Lymphohistiocytosis Type 5 (FHL-5) Is Caused by Mutations in Munc18-2 and Impaired Binding to Syntaxin 11

Rapid intracellular transport and secretion of cytotoxic granules through the immunological synapse requires a balanced interaction of several proteins. Disturbance of this highly regulated process underlies familial hemophagocytic lymphohistiocytosis (FHL), a genetically heterogeneous autosomal-recessive disorder characterized by a severe hyperinflammatory phenotype. Here, we have assigned FHL-5 to a 1 Mb region on chromosome 19p by using high-resolution SNP genotyping in eight unrelated FHL patients from consanguineous families. Subsequently, we found nine different mutations, either truncating or missense, in STXBP2 in twelve patients from Turkey, Saudi Arabia, and Central Europe. STXBP2 encodes syntaxin binding protein 2 (Munc18-2), involved in the regulation of vesicle transport to the plasma membrane. We have identified syntaxin 11, a SNARE protein mutated in FHL-4, as an interaction partner of STXBP2. This interaction is eliminated by the missense mutations found in our FHL-5 patients, which leads to a decreased stability of both proteins, as shown in patient lymphocytes. Activity of natural killer and cytotoxic T cells was markedly reduced or absent, as determined by CD107 degranulation. Our findings thus identify a key role for STXBP2 in lytic granule exocytosis.

The duration of TCR/pMHC interactions regulates CTL effector function and tumor‐killing capacity

Effector CTL contribute to tumoral immunity by killing tumor cells through secretion of cytotoxic granules and cytokines. Activation of CTL requires specific recognition of cognate peptide-MHC-I (pMHC) complexes on the tumor cell surface by the CTL TCR. It has been suggested that the half-life (t(1/2)) of the TCR/pMHC interaction modulates the activation of naïve CD8(+) T cells; however, it remains unknown whether CTL effector function can also be regulated by the TCR/pMHC t(1/2). Here, we have studied CTL activity in response to tumor cells loaded with pMHC that bind the TCR with different t(1/2). We observed that the TCR/pMHC t(1/2) can differentially regulate CTL effector function during the interaction with tumor cells and defines the nature of anti-tumoral CTL responses in vivo. Although prolonged TCR/pMHC t(1/2) promoted only partial expression of cytotoxic molecules, short t(1/2) induced partial polarization of lytic machinery toward target cells. In contrast, intermediate TCR/pMHC t(1/2) induced strong expression of cytotoxic molecules, efficient polarization of lytic machinery and subsequent release of toxic granules by CTL that killed tumor cells. Consistently, efficient in vivo CTL-mediated tumor clearance was only observed for tumors expressing intermediate t(1/2) pMHC ligands. These data suggest that there is an optimal TCR/pMHC t(1/2) for efficient CTL activity.

Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes

Granule-mediated cytotoxicity is the main effector mechanism of cytotoxic CD8+ T cells. We report that CD8+ T cells from acid sphingomyelinase (ASMase)-deficient (ASMase-KO) mice are defective in exocytosis of cytolytic effector molecules; this defect resulted in attenuated cytotoxic activity of ASMase-KO CD8+ T cells and delayed elimination of lymphocytic choriomeningitis virus from ASMase-KO mice. Cytolytic granules of ASMase-KO and wild-type CD8+ T cells were equally loaded with granzymes and perforin, and correctly directed to the immunological synapse. In wild-type CD8+ T cells, secretory granules underwent shrinkage by 82% after fusion with the plasma membrane. In ASMase-KO CD8+ T cells, the contraction of secretory granules was markedly impaired. Thus, ASMase is required for contraction of secretory granules and expulsion of cytotoxic effector molecules.

Characterization of SNARE proteins involved in granule exocytosis of cytotoxic T lymphocytes (35.25)

Abstract Cytotoxic T lymphocytes (CTLs) exert their cytotoxic activity through the polarized secretion of cytotoxic granules at the immunological synapse (IS). Because soluble NSF attachment receptor (SNARE) proteins mediate numerous intra- and intercellular fusion events and are essential effectors of exocytosis of synaptic granules in neurons, we examined the contribution of specific SNARE proteins to the formation of the IS. To determine the functional significance of specific SNAREs, our lab has examined the expression profile of more than 40 SNARE proteins by RT-PCR and quantitated various protein levels of these SNAREs in resting and activated CTLs. We have found that the expression profile of some SNARE proteins is up or down-regulated upon CTL maturation. In conjunction with these studies, we have also analyzed the sub-cellular localization and mobilization following IS formation of all SNAREs that are expressed in CD8+ T-cells for which specific antibodies are available. Some but not all SNARE proteins are translocated towards the immunological synapse and some co-localized with cytotoxic lytic granules at the immunological synapse. We found that syntaxin 4, syntaxin 6, VAMP 3, and VAMP 8, among other proteins, seem to polarize and colocalize with cytotoxic granule markers at the immune synapse suggesting they play important roles in cytotoxic granule secretion. Grant/ other support: DFG RE 1092/6-1