Activation of the Ancestral Polarity Regulator Protein Kinase Cζ at the Immunological Synapse Drives Polarization of Th Cell Secretory Machinery toward APCs

Abstract

A key feature in T lymphocyte biology is that Th cells rapidly polarize their secretory machinery toward cognate APCs. The molecular mechanisms of these dynamic Th cell responses and their impact on APC biology remain to be elucidated. In this study, we demonstrate that protein kinase Cζ (PKCζ) is rapidly activated at the immunological synapse (IS) in human Th cells interacting with cognate dendritic cells (DCs) and that a functional PKCζ is required for the polarization of Th cell secretory machinery toward DCs. We also show that PKCζ-dependent Th cell polarization allows dedicated delivery of IFN-γ and CD40L at the IS and is required for the activation of cognate DCs to IL-12 production. PKCζ synaptic activation is a low-threshold phenomenon and, in Th cells interacting with multiple DCs, selectively occurs at the IS formed with the DCs offering the strongest stimulus leading to dedicated Th cell polarization. Our results identify the PKCζ signaling pathway as a key component of the Th cell polarization machinery and provide a molecular basis for T cell-dedicated activation of cognate DCs.

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

“…S2C), p-PKCζ staining was increased at the IS where it paralleled phospho-tyrosine (p-Tyr) staining. These results indicated that in CTLs, similarly to T H cells (14), the PKCζ pathway is active at the IS. We thus treated CTLs with a selective pseudosubstrate inhibitor of PKCζ (PKCζ-PS) (16) to investigate the impact of PKCζ signaling blockade on MTOC polarization.…”

“…We also provide further evidence that lytic granule secretion might be, under certain circumstances, uncoupled from MTOC dynamics by blocking MTOC polarization with a selective inhibitor of the ancestral polarity protein PKCζ (14,15). Our results show that in conditions in which MTOC polarization is inhibited, no major effect on cytotoxicity and on the activation of caspase-3 in target cells could be detected.…”

“…To do so, we targeted protein kinase Cζ (PKCζ) function in CTLs. Indeed, we and others recently showed that the activation of this ancestral polarizing enzyme at the IS is required for MTOC polarization in CD4 + helper T cells interacting with dendritic cells (14,15). To test whether this pathway is also implicated in MTOC polarization in CTLs, we monitored PKCζ activation at the IS by staining CTL/target cell conjugates with antibodies directed against the phosphorylated form of PKCζ (p-PKCζ) (14).…”

“…We therefore asked whether CTLs treated with PKCζ-PS might elicit cytotoxicity. For this analysis, we took advantage of the observation that in helper T-cell/antigen-presenting cell (APC) conjugates, treatment with PKCζ-PS resulted in an inhibition of the secretory machinery toward the APC that lasted at least 6 h, a time longer than a standard cytotoxicity assay (14). In TSST-1-stimulated Vβ2 + CTLs, inhibition of MTOC polarization did not affect cytotoxicity at the different effector:target (E:T) ratios (Fig.…”

L-Plastin Regulates Polarization and Migration in Chemokine-Stimulated Human T Lymphocytes

“…Our results strongly suggest that L-plastin regulates lymphocyte polarity and migration by enabling cells to establish an axis of asymmetry in response to chemokine signaling, in particular, the localization of the F-actin cytoskeleton to the leading edge of the cell. We also uncovered a novel mechanistic link between PKC z, an established regulator of lymphocyte polarity (40)(41)(42)(43), and L-plastin in human T lymphocytes, specifically via PKC z-mediated phosphorylation of L-plastin on its Ser 5 residue. L-plastin expression also influenced the Rac1 activation cycle, Akt phosphorylation, and migration on the integrin ligand ICAM-1, but in a manner that was independent of LFA-1 activation or adhesion.…”