Protein Kinase C δ Regulates the Depletion of Actin at the Immunological Synapse Required for Polarized Exosome Secretion by T Cells

Abstract

Multivesicular bodies (MVB) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, ILVs are secreted as Fas ligand-bearing, pro-apoptotic exosomes following T cell receptor (TCR)-induced fusion of MVB with the plasma membrane at the immune synapse (IS). In this study we show that protein kinase C δ (PKCδ), a novel PKC isotype activated by diacylglycerol (DAG), regulates TCR-controlled MVB polarization toward the IS and exosome secretion. Concomitantly, we demonstrate that PKCδ-interfered T lymphocytes are defective in activation-induced cell death. Using a DAG sensor based on the C1 DAG-binding domain of PKCδ and a GFP-PKCδ chimera, we reveal that T lymphocyte activation enhances DAG levels at the MVB endomembranes which mediates the association of PKCδ to MVB. Spatiotemporal reorganization of F-actin at the IS is inhibited in PKCδ-interfered T lymphocytes. Therefore, we propose PKCδ as a DAG effector that regulates the actin reorganization necessary for MVB traffic and exosome secretion.

Actin reorganization at the centrosomal area and the immune synapse regulates polarized secretory traffic of multivesicular bodies in T lymphocytes

“…We have previously shown, by using a well-established IS model [31] [35] [32], that PKCδ-interfered Jurkat clones challenged with antigen on APC secreted a lower amount of exosomes upon IS formation [14]. We also defined that the deficient polarization of MVB containing the exosome precursors towards the IS underlies the decreased exosome secretion [14].…”

“…We have previously shown, by using a well-established IS model [31] [35] [32], that PKCδ-interfered Jurkat clones challenged with antigen on APC secreted a lower amount of exosomes upon IS formation [14]. We also defined that the deficient polarization of MVB containing the exosome precursors towards the IS underlies the decreased exosome secretion [14]. In the context of secretory traffic at the IS, it has been published that not always MTOC polarization is necessary or sufficient for the transport or certain secretory vesicles to the IS [7], such as lytic granules, and for the cytotoxic hit delivery [36][37][38].…”

“…In contrast, Th lymphocytes such as Jurkat cells generate stable, long-standing IS (from 10-30 min up to hours), since this appears to be necessary for both directional and continuous secretion of stimulating cytokines [4] [2]. Moreover, we have described in Th lymphocytes that cortical actin reorganization at the IS plays an important role during the polarized traffic of multivesicular bodies (MVB) containing intraluminal vesicles (ILV) [14], a different type of secretory vesicles involved in exosome secretion at the IS [15][16][17]. Exosome secretion in Th lymphocytes follows TCR stimulation, IS formation, MVB polarization and degranulation, and these exosomes can induce Fas ligand-dependent autocrine activation-induced cell death (AICD) [18] [16] [15], a key process in regulating T lymphocyte homeostasis [19].…”

“…Exosome secretion in Th lymphocytes follows TCR stimulation, IS formation, MVB polarization and degranulation, and these exosomes can induce Fas ligand-dependent autocrine activation-induced cell death (AICD) [18] [16] [15], a key process in regulating T lymphocyte homeostasis [19]. Regarding the intracellular signals controlling this specialized secretory pathway, we have shown that TCR-stimulated PKCδ regulates cortical actin reorganization at the IS ultimately controlling MTOC/MVB polarization leading to exosome secretion at the IS [14]. PKCδ-interfered T cell clones showed inhibition of cortical actin reorganization at the IS concomitantly to defective MTOC/MVB polarization [14].…”

“…Overall, this lead us to hypothesize that an altered actin reorganization at the IS may underlie the deficient MVB polarization occurring in PKCδ-interfered T cell clones [14]. However, it cannot be ruled out that PKCδ may govern other actin reorganization networks apart from actin reorganization at the IS, 6 that may also contribute to the diminished MTOC/MVB polarization efficiency observed in PKCδ-interfered clones [14].…”