Protein Kinase Cδ Regulates Antigen Receptor-Induced Lytic Granule Polarization in Mouse CD8+ CTL

Abstract

T cell receptor (TCR)‐induced activation of protein kinase C (PKC) has long been known to be critical for regulation of granule exocytosis mediated cytotoxicity in CD8+ T cells. However, the mechanism by which PKC regulates this effector function is not clear. In addition, it is not known which PKC family members are involved in the regulation of this process. By combining the use of pharmacological inhibitors and mice with targeted gene deletions, we showed that Protein Kinase C □elta is required for granule exocytosis mediated lytic function in mouse CD8+T cells. We studied lysosomal/lytic granule movements in CD8+ T cells responding to target cell recognition by confocal microscopy and live imaging and demonstrated that PKC □elta regulates TCR induced lytic granule polarization. In summary, our studies identified Protein Kinase C □elta, a member of the family of diacylglycerol dependent, calcium independent PKC isoforms, as a novel regulator of TCR mediated lytic granule release in mouse CD8+CTL. This work was supported by the NIH grants RO1AI48837 and RO1AI41573 to SV and by NIH, NCI F32CA101449‐02 and Research Advisory Council grant to SR.

Rottlerin Inhibits Chlamydial Intracellular Growth and Blocks Chlamydial Acquisition of Sphingolipids from Host Cells

“…Although the inclusion recruitment of PKC␦ may prevent PKC␦ from exerting its proapoptotic activity in the mitochondria (20,27), the localization of PKC␦ near the inclusions may also serve as a platform for Chlamydia to utilize the PKC␦ activity to facilitate chlamydial acquisition of host lipids. This hypothesis is supported by the finding that PKC␦ plays an essential role in various exocytosis processes, including antigen receptor-induced lytic degranulation (19), insulin secretion (28), and sphingomyelin transportation from the Golgi apparatus to the plasma membrane (29). In addition, PKC␦ can also activate the Raf-MEK-ERK-cPLA2 signaling pathway (17), which is required for chlamydial acquisition of glycerophospholipids from host cells (25).…”

Protein Kinase C δ Localizes to Secretory Lysosomes in CD8+ CTL and Directly Mediates TCR Signals Leading to Granule Exocytosis-Mediated Cytotoxicity

“…ϩ CTL, we have recently shown that PKC ␦ is required for lytic granule exocytosis but is dispensable for activation, cytokine production, and expression of cytolytic molecules in response to TCR stimulation (16). We also showed that PKC ␦ does not regulate MTOC reorientation but is required for TCR-induced lytic granule polarization.…”

“…The cells in complete RPMI 1640 (10 7 cells/ml) were incubated for 30 min on ice in the presence or absence of 10 g/ml of purified anti-CD3 Ab followed by addition of 25 g/ml of goat anti-hamster IgG at 37°C for indicated periods of time. Cells were immediately lysed with ice-cold Lysis buffer (20 mM Tris-HCl (pH 7.5), 137 mM NaCl, 10% glycerol, 1% nonidet P-40, 2 mM EDTA, 1 mM PMSF, 2 mM Na 3 VO 4 , 10 mM NaF, and protease inhibitor mixture (Sigma-Aldrich)) for 30 min on ice and subjected to SDS-PAGE followed by immunobloting, as previously described (16).…”

“…To test whether PKC ␦-GFP is physiologically relevant for the studies concerning the role of PKC ␦ in regulation of lytic granule exocytosis, we ectopically expressed the fusion protein in PKC ␦-deficient CD8 ϩ CTL and assessed its ability to reverse the cytolytic defect characteristic of PKC ␦-deficient CTL. The PKC ␦-GFP expression vector was introduced into polyclonally activated primary mouse CD8 ϩ T cells by nucleofection, followed by re-directed chromium release assay using Fas-resistant L1210 cells as targets to specifically assess granule exocytosis-mediated cytotoxicity (16). We have previously used this transfection system to achieve high frequency of ectopic gene expression in polyclonal population of highly potent CD8 ϩ CTL (16).…”

Cell Death Mechanisms Induced by Cytotoxic Lymphocytes

“…In exocytosis or degranulation, there is microtubules mobilization that leads the preformed granules or lysosomes of the cytotoxic cell towards the point of contact with the target cell, releasing stored lytic molecules (51,52). Degranulation can be detected by exposure of the lysosomal-membrane-associated glycoproteins, CD107a, CD107b, and CD63, on the lymphocyte surface (53).…”