Syntaxin 8 is required for efficient lytic granule trafficking in cytotoxic T lymphocytes

• Published in: Biochimica et biophysica acta 2016-07, Vol.1863 (7), p.1653-1664
• Main Authors: Bhat, Shruthi S., Friedmann, Kim S., Knörck, Arne, Hoxha, Cora, Leidinger, Petra, Backes, Christina, Meese, Eckart, Keller, Andreas, Rettig, Jens, Hoth, Markus, Qu, Bin, Schwarz, Eva C.
• Format: Article
• Language: English
• Subjects: Cell Degranulation; Cell Line; CTL; Cytoplasmic Granules - immunology; Cytoplasmic Granules - metabolism; Cytotoxicity, Immunologic; Endosomes - metabolism; Exocytosis; Human CD8 T cell; Humans; Killing; Lysosomal-Associated Membrane Protein 1 - metabolism; Lysosomes - metabolism; Lytic granule; Perforin - metabolism; Protein Transport; Qa-SNARE Proteins - genetics; Qa-SNARE Proteins - metabolism; RNA Interference; Secretory Vesicles - immunology; Secretory Vesicles - metabolism; SNARE; Syntaxin 8 (Stx8); T-Lymphocytes, Cytotoxic - immunology; T-Lymphocytes, Cytotoxic - metabolism; Time Factors; Transfection
• ISSN: 0167-4889; 0006-3002; 1879-2596
• DOI: 10.1016/j.bbamcr.2016.04.014

Cytotoxic T lymphocytes (CTL) eliminate pathogen-infected and cancerous cells mainly by polarized secretion of lytic granules (LG, containing cytotoxic molecules like perforin and granzymes) at the immunological synapse (IS). Members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family are involved in trafficking (generation, transport and fusion) of vesicles at the IS. Syntaxin 8 (Stx8) is expressed in LG and colocalizes with the T cell receptor (TCR) upon IS formation. Here, we report the significance of Stx8 for human CTL cytotoxicity. We found that Stx8 mostly localized in late, recycling endosomal and lysosomal compartments with little expression in early endosomal compartments. Down-regulation of Stx8 by siRNA resulted in reduced cytotoxicity. We found that following perforin release of the pre-existing pool upon target cell contact, Stx8 down-regulated CTL regenerate perforin pools less efficiently and thus release less perforin compared to control CTL. CD107a degranulation, real-time and end-point population cytotoxicity assays, and high resolution microscopy support our conclusion that Stx8 is required for proper and timely sorting and trafficking of cytotoxic molecules to functional LG through the endosomal pathway in human CTL. •Stx8 is among the most abundant Q-SNAREs in human cytotoxic T lymphocytes.•Stx8 is required for proper and timely sorting and trafficking of cytotoxic molecules.•Cytotoxicity in Stx8 down-regulated human cytotoxic T lymphocytes is impaired.