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

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.

List of scientific papers

I. Development of classical Hodgkin's lymphoma in an adult with biallelic STXBP2 mutations. Machaczka M, Klimkowska M, Chiang SC, Meeths M, Müller ML, Gustafsson B, Henter JI, Bryceson YT. Haematologica. 2013 May;98(5):760-4.
https://doi.org/10.3324/haematol.2012.073098

II. An N-terminal missense mutation in STX11 causative of FHL4 abrogates syntaxin-11 binding to Munc18-2. Müller ML, Chiang SC, Meeths M, Tesi B, Entesarian M, Nilsson D, Wood SM, Nordenskjöld M, Henter JI, Naqvi A, Bryceson YT. Front Immunol. 2014 Jan 14;4:515.
https://doi.org/10.3389/fimmu.2013.00515

III. VAMP8-dependent fusion of recycling endosomes with the plasma membrane facilitates T lymphocyte cytotoxicity. Marshall MR, Pattu V, Halimani M, Maier-Peuschel M, Müller ML, Becherer U, Hong W, Hoth M, Tschernig T, Bryceson YT, Rettig J. J Cell Biol. 2015 Jul 6;210(1):135-51.
https://doi.org/10.1083/jcb.201411093