Cytotoxic T lymphocytes kill virus-infected and tumorigenic target cells through the release of perforin and granzymes via fusion of lytic granules at the contact site, the immunological synapse. It has been postulated that this fusion process is mediated by non-neuronal members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex protein family. Here, using a synaptobrevin2-monomeric red fluorescence protein knock-in mouse we demonstrate that, surprisingly, the major neuronal v-SNARE synaptobrevin2 is expressed in cytotoxic T lymphocytes and exclusively localized on granzyme B-containing lytic granules. Cleavage of synaptobrevin2 by tetanus toxin or ablation of the synaptobrevin2 gene leads to a complete block of lytic granule exocytosis while leaving upstream events unaffected, identifying synaptobrevin2 as the v-SNARE responsible for the fusion of lytic granules at the immunological synapse.

VAMP8 is associated with the recycling endosome compartment rather than with cytotoxic granules and is required for a fusion step between recycling endosomes and the plasma membrane that brings syntaxin-11 to the immune synapse for cytotoxic granule exocytosis.

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.

SNARE proteins are essential fusion mediators for many intracellular trafficking events. Here, we investigate the role of Syntaxin7 (Stx7) in the release of lytic granules from cytotoxic T lymphocytes (CTLs). We show that Stx7 is expressed in CTLs and is preferentially localized to the region of lytic granule release, the immunological synapse (IS). Interference of Stx7 function by expression of a dominant-negative Stx7 construct or by small interfering RNA leads to a dramatic reduction of CTLmediated killing of target cells. Real-time visualization of individual lytic granules at the IS by evanescent wave microscopy reveals that lytic granules in Stx7-deprived CTLs not only fail to fuse with the plasma membrane but even fail to accumulate at the IS. Surprisingly, the accumulation defect is not caused by an overall reduction in lytic granule number, but by a defect in the trafficking of T cell receptors (TCRs) through endosomes. Subsequent high-resolution nanoscopy shows that Stx7 colocalizes with Rab7 on late endosomes. We conclude from these data that the accumulation of recycling TCRs at the IS is a SNARE-dependent process and that Stx7-mediated processing of recycling TCRs through endosomes is a prerequisite for the cytolytic function of CTLs.

CTLs kill target cells via fusion of lytic granules (LGs) at the immunological synapse (IS).Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) function as executors of exocytosis. The importance of SNAREs in CTL function is evident in the form of familial hemophagocytic lymphohistiocytosis type 4 that is caused by mutations in Syntaxin11 (Stx11), a Qa-SNARE protein. Here, we investigate the molecular mechanism of Stx11 function in primary human effector CTLs with high temporal and spatial resolution. Downregulation of endogenous Stx11 resulted in a complete inhibition of LG fusion that was paralleled by a reduction in LG dwell time at the IS. Dual color evanescent wave imaging suggested a sequential process, in which first Stx11 is transported to the IS through a subpopulation of recycling endosomes. The resulting Stx11 clusters at the IS then serve as a platform to mediate fusion of arriving LGs. We conclude that Stx11 functions as a t-SNARE for the final fusion of LG at the IS, explaining the severe phenotype of familial hemophagocytic lymphohistiocytosis type 4 on a molecular level.Keywords: Cytotoxic T lymphocytes r FHL r Lytic granule fusion r Syntaxin11 clusters r TIRFM Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionCTLs kill their target cells through the polarized fusion of lytic granules (LGs) at the immunological synapse (IS). The IS is initiated when the TCRs recognize specific MHC peptide complexes on the target cell. The recognition results in the clustering of molecules that leads to the formation of supramolecular activation clusters (SMACs) [1]. TCRs and associated signaling proteins accumulate to form a central SMAC while adhesion proteins Correspondence: Prof. Jens Rettig e-mail: jrettig@uks.eu needed for stable target cell contact form the peripheral SMAC. After IS formation, LGs polarize, dock, prime, and finally fuse to release their contents into the cleft between CTL and target cell [2].Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins are known executors of almost all intracellular fusion events [3]. The SNARE proteins that execute synaptic vesicle fusion in neurons and neuroendocrine cells have been well characterized [4,5]. Vesicle fusion is initiated by the formation of a four helical bundle that consists of one R-SNARE (v-SNARE) that resides on one membrane (mostly vesicle membranes) and three Q-SNAREs (Qa, Qb, Qc; t-SNARE) that reside on another membrane (mostly the plasma membrane) of the cell [6]. paired Student t-test). (B) Cell lysates from naïve CTLs (N), CTLs stimulated for 3 days by anti-CD3/anti-CD28 antibody-coated beads (S) andCTLs overexpressing TFP-Stx11 (OE) were assessed for Stx11 expression by Western blot using a polyclonal anti-Stx11 antibody. (C) Lysates from CTLs transfected with either control or three different Stx11 siRNAs (#1, #2, and #6, respectively) were blotted for Stx11 (top) and GAPDH (bottom) as loading control. (D) Qua...