title: Docking of lytic granules at the immunological synapse in human CTL requires Vti1b-dependent pairing with CD3 endosomes.
abstract: Lytic granule (LG)-mediated apoptosis is the main mechanism by which CTL kill virus-infected and tumorigenic target cells. CTL form a tight junction with the target cells, which is called the immunological synapse (IS). To avoid unwanted killing of neighboring cells, exocytosis of lytic granules (LG) is tightly controlled and restricted to the IS. In this study, we show that in activated human primary CD8(+) T cells, docking of LG at the IS requires tethering LG with CD3-containing endosomes (CD3-endo). Combining total internal reflection fluorescence microscopy and fast deconvolution microscopy (both in living cells) with confocal microscopy (in fixed cells), we found that LG and CD3-endo tether and are cotransported to the IS. Paired but not single LG are accumulated at the IS. The dwell time of LG at the IS is substantially enhanced by tethering with CD3-endo, resulting in a preferential release of paired LG over single LG. The SNARE protein Vti1b is required for tethering of LG and CD3-endo. Downregulation of Vti1b reduces tethering of LG with CD3-endo. This leads to an impaired accumulation and docking of LG at the IS and a reduction of target cell killing. Therefore, Vti1b-dependent tethering of LG and CD3-endo determines accumulation, docking, and efficient lytic granule secretion at the IS.
authors.affiliation: Department of Biophysics, Saarland University, 66421 Homburg, Germany.
authors.forename: Bin
authors.initials: B
authors.lastname: Qu
authors.forename: Varsha
authors.initials: V
authors.lastname: Pattu
authors.forename: Christian
authors.initials: C
authors.lastname: Junker
authors.forename: Eva C
authors.initials: EC
authors.lastname: Schwarz
authors.forename: Shruthi S
authors.initials: SS
authors.lastname: Bhat
authors.forename: Carsten
authors.initials: C
authors.lastname: Kummerow
authors.forename: Misty
authors.initials: M
authors.lastname: Marshall
authors.forename: Ulf
authors.initials: U
authors.lastname: Matti
authors.forename: Frank
authors.initials: F
authors.lastname: Neumann
authors.forename: Michael
authors.initials: M
authors.lastname: Pfreundschuh
authors.forename: Ute
authors.initials: U
authors.lastname: Becherer
authors.forename: Heiko
authors.initials: H
authors.lastname: Rieger
authors.forename: Jens
authors.initials: J
authors.lastname: Rettig
authors.forename: Markus
authors.initials: M
authors.lastname: Hoth
mesh_terms: D017252:CD3 Complex; D002478:Cells, Cultured; D011992:Endosomes / Q000276:immunology*; D053804:Granzymes / Q000276:immunology*; D006801:Humans; D054992:Immunological Synapses / Q000276:immunology*; D008853:Microscopy; D011485:Protein Binding; D050766:Qb-SNARE Proteins / Q000276:immunology* / Q000378:metabolism; D022142:Secretory Vesicles / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D017252:CD3 Complex; D050766:Qb-SNARE Proteins; C491416:VTI1B protein, human; D053804:Granzymes