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.

Mechanisms controlling granule-mediated cytolytic activity of cytotoxic T lymphocytes

“…The multifocal nature of the nascent CS suggests that granules may be released at many different locations at the synaptic interface, which are most likely associated with the foci containing aggregated cognate pMHC and adhesion molecules. This is consistent with the most recent observation that a fraction of cytolytic granules are paired with TCR-containing endosomes and that the pairing is mediated by SNARE protein Vti1b (99). Pairs of endosomes are enriched at the CTL contact surface suggesting that the pairing may facilitate granule transport to the synaptic interface.…”

A Comparative Antibody Analysis of Pannexin1 Expression in Four Rat Brain Regions Reveals Varying Subcellular Localizations

“…Lysates from matched Panx1 wild type and KO tissues were generated from animals from Genentech (Qu et al, 2011) and KOMP (Qiu et al, 2011; Santiago et al, 2011). Methods for the preparation of tissues lysates and Western blots for the KOMP KO are described in (Santiago et al, 2011).…”

An initial and rapid step of lytic granule secretion precedes microtubule organizing center polarization at the cytotoxic T lymphocyte/target cell synapse

“…This analysis showed that lytic granules and MTOC dynamics were heterogeneous and clearly not synchronized in the various cells analyzed. The dynamics of lytic granules and MTOC was recently studied using TIRFM in human CTL (18) and mouse NK cells (19) stimulated by immobilized anti-CD3 or anti-NKp30 and anti-CD18, respectively. In human CTLs, it was found that the MTOC did not enter in the TIRF plane (18); conversely, in mouse NK cells, it was found that MTOC appeared in the TIRF plane and lytic granules mostly associated with MTOC (19).…”

“…The dynamics of lytic granules and MTOC was recently studied using TIRFM in human CTL (18) and mouse NK cells (19) stimulated by immobilized anti-CD3 or anti-NKp30 and anti-CD18, respectively. In human CTLs, it was found that the MTOC did not enter in the TIRF plane (18); conversely, in mouse NK cells, it was found that MTOC appeared in the TIRF plane and lytic granules mostly associated with MTOC (19). In line with the latter observation, superresolution microscopy analysis of the NK secretory domain showed that MTOC polarizes toward the granule penetrable synaptic areas of the actin meshwork (20).…”