Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

Supplementary Video 1: CMAC-labeled Raji cells (blue) were attached to fibronectin-coated MatTek chamber slides and SEE-pulsed (30 min). Double synapse formation by one GFP-CD63-expressing Jurkat cell was time-lapse imaged (right side). The video shows MVB traffic to the synapse contact areas in this cell and not in Jurkat cells that do not form synapses (left). One representative example is shown of 11 synapses recorded.Supplementary Video 7: Synapse formed by a DsRed2-PKD1-expressing Jurkat T lymphocyte (red) with a SEE-loaded (1 μg/ml) , CMAC-labeled Raji B lymphocyte (blue). Both fluorescence channels were captured simultaneously in the video. The red channel was deconvoluted using Huygens Essential Software. DsRed2-PKD1 accumulation was observed at the synaptic contact after 30 min.Supplementary Video 8. WT mouse T lymphoblasts were challenged with SEB-pulsed (1 μg/ml), CMAC-labeled EL-4 cells (blue) at a 1:1 ratio and transmittance and blue channels were acquired. EL-4 cells were large and blue; T lymphoblasts were smaller and irregular. White arrows indicate synaptic contacts, red arrows indicate cells that show apoptotic blebbing. T, apoptosis of effector T lymphoblasts (AICD); EL4, death of EL-4 target cells (CTL).Multivesicular bodies (MVBs) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these ILV contain Fas ligand (FasL) and are secreted as 'lethal exosomes' following activation-induced fusion of the MVB with the plasma membrane. Diacylglycerol (DAG) and diacylglycerol kinase α (DGKα) regulate MVB maturation and polarized traffic, as well as subsequent secretion of pro-apoptotic exosomes, but the molecular basis underlying these phenomena remains unclear. Here we identify protein kinase D (PKD) family members as DAG effectors involved in MVB genesis and secretion. We show that the inducible secretion of exosomes is enhanced when a constitutively active PKD1 mutant is expressed in T lymphocytes, whereas exosome secretion is impaired in PKD2-deficient mouse T lymphoblasts and in PKD1/3-null B cells. Analysis of PKD2-deficient T lymphoblasts showed the presence of large, immature MVB-like vesicles and demonstrated defects in cytotoxic activity and in activation-induced cell death. Using pharmacological and genetic tools, we show that DGKα regulates PKD1/2 subcellular localization and activation. Our studies demonstrate that PKD1/2 is a key regulator of MVB maturation and exosome secretion, and constitutes a mediator of the DGKα effect on MVB secretory traffic.Mark Ware (Nanosight Ltd, UK) for his support in NANOSIGHT studies, and Catherine Mark for excellent editorial assistance. RA received a doctoral fellowship from the Spanish Ministerio de Ciencia y Tecnología. This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (MINECO) Plan Nacional de Investigación Científica (BFU2011-22849 to MI). IM is funded by MINECO grant BFU2013-47640-P.Peer Reviewe