Syntaxin 8 is required for efficient lytic granule trafficking in cytotoxic T lymphocytes.
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.
Biophysics, Center for Integrative Physiology and Molecular Medicine, School of Medicine, Saarland University, Building 48, 66421 Homburg, Germany. Electronic address: shruthi_s_bhat@yahoo.co.in.
Shruthi S
SS
Bhat
Biophysics, Center for Integrative Physiology and Molecular Medicine, School of Medicine, Saarland University, Building 48, 66421 Homburg, Germany. Electronic address: kim.friedmann@uks.eu.
Kim S
KS
Friedmann
Biophysics, Center for Integrative Physiology and Molecular Medicine, School of Medicine, Saarland University, Building 48, 66421 Homburg, Germany. Electronic address: arne.knoerck@uni-saarland.de.
Arne
A
Knörck
Biophysics, Center for Integrative Physiology and Molecular Medicine, School of Medicine, Saarland University, Building 48, 66421 Homburg, Germany. Electronic address: cora.hoxha@uks.eu.
Cora
C
Hoxha
Human Genetics, School of Medicine, Saarland University, Building 60, 66421 Homburg, Germany. Electronic address: p.leidinger@mx.uni-saarland.de.
Petra
P
Leidinger
Center for Bioinformatics, Saarland University, Building E2.1, 66123 Saarbrücken, Germany. Electronic address: c.backes@mx.uni-saarland.de.
Christina
C
Backes
Human Genetics, School of Medicine, Saarland University, Building 60, 66421 Homburg, Germany. Electronic address: hgemee@uniklinik-saarland.de.
Eckart
E
Meese
Center for Bioinformatics, Saarland University, Building E2.1, 66123 Saarbrücken, Germany. Electronic address: andreas.keller@ccb.uni-saarland.de.
Andreas
A
Keller
Physiology, Center for Integrative Physiology and Molecular Medicine, School of Medicine, Saarland University, Building 48, 66421 Homburg, Germany. Electronic address: jrettig@uks.eu.
Jens
J
Rettig
Biophysics, Center for Integrative Physiology and Molecular Medicine, School of Medicine, Saarland University, Building 48, 66421 Homburg, Germany. Electronic address: markus.hoth@uks.eu.
Markus
M
Hoth
Biophysics, Center for Integrative Physiology and Molecular Medicine, School of Medicine, Saarland University, Building 48, 66421 Homburg, Germany. Electronic address: bin.qu@uks.eu.
Bin
B
Qu
Biophysics, Center for Integrative Physiology and Molecular Medicine, School of Medicine, Saarland University, Building 48, 66421 Homburg, Germany. Electronic address: eva.schwarz@uks.eu.
Eva C
EC
Schwarz
D015550:Cell Degranulation; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000276:immunology / Q000378:metabolism*; D003602:Cytotoxicity, Immunologic; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D054353:Perforin / Q000378:metabolism; D021381:Protein Transport; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D022142:Secretory Vesicles / Q000276:immunology / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D052118:Lysosomal-Associated Membrane Protein 1; D050765:Qa-SNARE Proteins; D054353:Perforin
CTL; Exocytosis; Human CD8 T cell; Killing; Lytic granule; SNARE; Syntaxin 8 (Stx8)