We show in this study that human T cells purified from peripheral blood, T cell clones, and Jurkat T cells release microvesicles in the culture medium. These microvesicles have a diameter of 50–100 nm, are delimited by a lipidic bilayer membrane, and bear TCR β, CD3ε, and ζ. This microvesicle production is regulated because it is highly increased upon TCR activation, whereas another mitogenic signal, such as PMA and ionomycin, does not induce any release. T cell-derived microvesicles also contain the tetraspan protein CD63, suggesting that they originate from endocytic compartments. They contain adhesion molecules such as CD2 and LFA-1, MHC class I and class II, and the chemokine receptor CXCR4. These transmembrane proteins are selectively sorted in microvesicles because CD28 and CD45, which are highly expressed at the plasma membrane, are not found. The presence of phosphorylated ζ in these microvesicles suggests that the CD3/TCR found in the microvesicles come from the pool of complexes that have been activated. Proteins of the transduction machinery, tyrosine kinases of the Src family, and c-Cbl are also observed in the T cell-derived microvesicles. Our data demonstrate that T lymphocytes produce, upon TCR triggering, vesicles whose morphology and phenotype are reminiscent of vesicles of endocytic origin produced by many cell types and called exosomes. Although the exact content of T cell-derived exosomes remains to be determined, we suggest that the presence of TCR/CD3 at their surface makes them powerful vehicles to specifically deliver signals to cells bearing the right combination of peptide/MHC complexes.

Activated human T lymphocytes exposed to apoptotic stimuli targeting mitochondria (i.e. staurosporine), enter an early, caspase-independent phase of commitment to apoptosis characterized by cell shrinkage and peripheral chromatin condensation. We show that during this phase, AIF is selectively released from the intermembrane space of mitochondria, and that Bax undergo conformational change, relocation to mitochondria, and insertion into the outer mitochondrial membrane, in a Bid-independent manner. We analyzed the subcellular distribution of cathepsins (Cat) B, D, and L, in a search for caspase-independent factors responsible for Bax activation and AIF release. All were translocated from lysosomes to the cytosol, in correlation with limited destabilization of the lysosomes and release of lysosomal molecules in a size selective manner. However, only inhibition of Cat D activity by pepstatin A inhibited the early apoptotic events and delayed cell death, even in the presence of bafilomycin A 1 , an inhibitor of vacuolar type H ؉ -ATPase, which inhibits acidification in lysosomes. Small interfering RNA-mediated gene silencing was used to inactivate Cat D, Bax, and AIF gene expression. This allowed us to define a novel sequence of events in which Cat D triggers Bax activation, Bax induces the selective release of mitochondrial AIF, and the latter is responsible for the early apoptotic phenotype.

Naive T cells continuously recirculate between secondary lymphoid tissue via the blood and lymphatic systems, a process that maximizes the chances of an encounter between a T cell and its cognate antigen. This recirculation depends on signals from chemokine receptors, integrins, and the sphingosine-1-phosphate receptor. The authors of previous studies in other cell types have shown that Rac GTPases transduce signals leading to cell migration and adhesion; however, their roles in T cells are unknown. By using both 3-dimensional intravital and in vitro approaches, we show that Rac1-and Rac2-deficient T cells have multiple defects in this recirculation process. Racdeficient T cells home very inefficiently to lymph nodes and the white pulp of the spleen, show reduced interstitial migration within lymph node parenchyma, and are defective in egress from lymph nodes. IntroductionThe continuous recirculation of naive T lymphocytes between secondary lymphoid organs (SLOs), such as lymph nodes (LNs) and spleen, through the blood and lymphatic systems is vital for an efficient adaptive immune response. 1 T cells in the blood enter LNs through a specialized vasculature termed high endothelial venules (HEVs). Fast-moving T cells in the bloodstream initially interact with HEVs via L-selectin (CD62L) on the surface of the lymphocyte binding to PNAd on endothelial cells, causing the T cells to slow down and roll on the endothelium. 1 Subsequently, binding of either the CCL19 or CCL21 chemokines presented on endothelial cells to their receptor CCR7 on T cells results in activation of the integrins lymphocyte function-associated antigen-1 (LFA-1; ␣L␤2) and VLA-4 (␣4␤1). These in turn bind to their ligands, intercellular adhesion molecule (ICAM)-1 and ICAM-2 (for LFA-1) and vascular cell adhesion molecule-1 (for VLA-4), on endothelial cells, leading to arrest and firm attachment of the lymphocytes. T cells then migrate laterally on the luminal surface of the endothelial cells before transmigrating through the endothelium, onto its basal side. From here the T cells migrate into the cortical zone of the LN, again under the influence of CCR7-binding chemokines. Intravital microscopy has shown that once within the interstitium of the LN, T cells continue to migrate at high speeds (10-15m/min) in response to CCR7-binding chemokines. [2][3][4][5][6][7] In contrast to the requirement for integrins in firm adhesion to HEV, these molecules appear to be dispensable for interstitial migration. 8 If T cells fail to encounter an antigen-presenting cell with cognate antigen and are not activated, they eventually migrate out of the LN by moving into the medulla, crossing a lymphatic endothelial barrier and entering the efferent lymphatic vessels. From here the T cells are able to migrate through the lymphatic system back into the blood vasculature. This egress from LNs is under the control of sphingosine-1-phosphate (S1P), which binds to the S1P receptor 1 (S1P 1 ) on T cells. 9 The Rac GTPases (Rac1, Rac2, and Rac3) are members of the Rho-fami...