Role of syntaxin-11 and munc18-2 in lymphocyte cytotoxic granule exocytosis

Sammanfattning: Mutations in genes required for the exocytosis of cytotoxic granules by NK cells and cytotoxic T lymphocytes are associated with early-onset familial hemophagocytic lymphohistiocytosis (FHL). In this project, we examined how certain missense mutations in genes encoding syntaxin-11 and Munc18-2 abolish exocytosis and cause disease. Furthermore, we dissected the role of another protein suspected to play a role in cytotoxic granule exocytosis, VAMP8. In the first study, we characterized an FHL5 patient (Munc18-2 p.Q432X and p.S545L) who developed Hodgkin lymphoma with underlying Epstein-Barr virus (EBV) infection. The tissue displayed high infiltrates of cytotoxic T lymphocytes responsive to EBV-derived peptides, yet the cells had dramatically reduced Munc18- 2 protein levels and were unable to undergo cytotoxic granule exocytosis. The patient is an important example of how Munc18-2 missense mutations impairing exocytosis can lead to late-onset HLH and, due to decreased immunosurveillance, might result in cancer. In the second study, we examined the pathophysiological mechanism underlying an N-terminal syntaxin-11 mutation (syntaxin-11 p.L58P) associated with FHL4 in three unrelated patients. These patients displayed defective cytotoxic granule exocytosis despite the functionally important SNARE domain of syntaxin-11 being intact. As the p.L58 is an evolutionary conserved amino acid, we hypothesized that the mutation interrupts interactions with Munc18-2. Further, we determined whether other conserved N-terminal syntaxin-11 residues also contribute to Munc18-2 binding. We found that the interaction is dependent on both an intact syntaxin-11 N-terminal peptide as well as Habc domain because mutations in either completely abolished binding to Munc18-2. It is thus plausible that syntaxin-11, analogous to the syntaxin- 1 / Munc18-1 interaction, employs distinct binding modes to different domains of Munc18-2. The interruption of the syntaxin-11 / Munc18-2 interaction could explain the reduced syntaxin-11 expression levels. Lastly, cytotoxic granule exocytosis is dependent on several proteins being part of the fusion complex between a cytotoxic granule and plasma membrane, yet the v-SNARE mediating granule fusion remains elusive. In the third study, we suspected VAMP8 to play a key role in this fusion process. However, VAMP8 did not localize to cytotoxic granules but instead to recycling endosomes. Upon T cell receptor stimulation, recycling endosomes were recruited to the immune synapse and fused with the plasma membrane. As VAMP8 knockdown blocked cytotoxic granule release, we hypothesized that VAMP8+ recycling endosomes might deliver an important component for the cytotoxic fusion machinery to the plasma membrane. Indeed, recycling endosomes transported syntaxin-11 to the plasma membrane, with VAMP8 knockdown hampering syntaxin-11 delivery. In summary, these data provide a deeper understanding of FHL and the molecular mechanisms of cytotoxic granule exocytosis. We describe a possible link between FHL and cancer which may have diagnostic, prognostic and treatment implications for other FHL patients. Further, we show how N-terminal syntaxin-11 mutations can cause disease, with data hinting towards a meticulous series of interaction modes necessary for syntaxin-11 maintenance and cytotoxic granule secretion. Finally, we find that VAMP8 delivers syntaxin-11 to the immunological synapse in human cytotoxic lymphocytes, demonstrating an unexpected role of recycling endosomes in cytotoxic granule exocytosis.