Regulation of Granule Exocytosis-mediated Cytotoxicity in CD8+ T Lymphocytes.
Open AccessCD8+ cytotoxic lymphocytes (CTLs), a key player in adaptive immunity, provide a protective response by means of cell-mediated cytotoxicity - the direct elimination of infected or tumor cells by a regulated release of lytic granules. The degranulation process is a focal release of lytic granule contents towards target cell membrane within minutes of CTLs and target cell recognition. Upon TCR engagement, a well-defined sequence of subcellular events involving the reorganization and mobilization of intracellular structures is initiated within CD8+ T cells and eventually leads to the discharge of lytic granules. In contrast, the signaling cascade induced by TCR engagement that regulates CTL granule exocytosis remains to be fully characterized. PKC, a class of serine/threonine kinases, have long been implicated in lytic granule release. Yet, it was unclear which isoform(s) enabled degranulation in CTLs. Here, we identify PKCδ as critically involved in the regulatory process of granule exocytosis in primary mouse CD8+ T cells. Our studies exploiting pharmacological inhibitors and mice bearing targeted gene deletions reveal that PKCδ is required for lytic granule release in response to TCR stimulation, but is dispensable for other T cell effector functions - such as, T cell activation, cytokine production, and expression of cytolytic molecules. Importantly, a reversal in defective lytic function is achieved with reconstitution studies in PKCδ-deficient CTLs. Upon CTL and target cell interaction, initiated TCR signals induce a series of known intracellular events that involves the polarization and discharge of cytolytic granules at the immunological synapse. To establish the cellular mechanism of lytic function regulated by PKCδ in CD8+ T cells, we performed imaging studies with PKCδ-deficient CTLs and demonstrate that PKCδ is required for lytic granule polarization toward the T cell and target cell contact site. Studies to elucidate PKCδ intracellular localization in live CD8+ CTL reveal that it constitutively localizes to secretory lysosomes. On the other hand, the TCR-induced signaling molecules involved in CTL degranulation, leading up to or regulated by PKCδ, remains to be fully elucidated. Thus far, our biochemical and structure-function studies demonstrate that PKCδ facilitates the mobilization, but not localization, of lytic granules in a kinase-dependent manner, and that PKCδ is rapidly phosphorylated on the activation loop upon TCR ligation. Our data also supports a potential role of PKCδ as a signaling component of TCR-induced signaling pathway occurring on these cytolytic vesicles. Therefore, we chemically disrupted DAG generation in T cells, an activating and recruiting element of PKC, which resulted in a redistribution of PKCδ and subsequently a reduction in lytic activity. Furthermore, studies investigating likely sources of DAG on secretory lysosomes reveal PLD1 as a potential mediator of CTL cytotoxicity. However, in PLD1 knockdown studies, we observe an enhancement of cytolytic activity and degranulation, which suggest that PLD1 is not directly involved in PKCδ-mediated regulation of lytic function in CD8+ CTL. Instead, PLD1 may potentially mediate a negative feedback mechanism through its byproduct, phosphatidic acid (PA). Collectively, our studies reveal PKCδ as a novel and selective regulator of TCR-induced granule release-mediated cytotoxicity, which constitutively localizes to lytic granules in a DAG-dependent manner and directly transduces TCR signals to permit polarized granule secretion in a kinase-dependent manner.
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