P85/p110 phosphoinositide 3‐kinase (PI3K) is a heterodimer composed of a p85‐regulatory and a p110‐catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65‐PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85α‐protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.

Exosomes are extracellular vesicles (EV) of endosomal origin (multivesicular bodies, MVB) constitutively released by many different eukaryotic cells by fusion of MVB to the plasma membrane. However, inducible exosome secretion controlled by cell surface receptors is restricted to very few cell types and a limited number of cell surface receptors. Among these, exosome secretion is induced in T lymphocytes and B lymphocytes when stimulated at the immune synapse (IS) via T-cell receptors (TCR) and B-cell receptors (BCR), respectively. IS formation by T and B lymphocytes constitutes a crucial event involved in antigen-specific, cellular, and humoral immune responses. Upon IS formation by T and B lymphocytes with antigen-presenting cells (APC), the convergence of MVB towards the microtubule organization center (MTOC), and MTOC polarization to the IS, are involved in polarized exosome secretion at the synaptic cleft. This specialized mechanism provides the immune system with a finely-tuned strategy to increase the specificity and efficiency of crucial secretory effector functions of B and T lymphocytes. As inducible exosome secretion by antigen-receptors is a critical and unique feature of the immune system this review considers the study of the traffic events leading to polarized exosome secretion at the IS and some of their biological consequences.

We report here for the first time the detection of the ribosomal p70S6 kinase (p70S6K) in a hematopoietic cell, the neutrophil, and the stimulation of its enzymatic activity by granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF modified the V_max of the enzyme (from 7.2 to 20.5 pmol/min/mg) and induced a time- and dose-dependent phosphorylation on p70S6K residues Thr³⁸⁹ and Thr⁴²¹/Ser⁴²⁴. The immunosuppressant macrolide rapamycin caused either a decrease in intensity of phospho-Thr³⁸⁹ bands in Western blots, or as a downshift in the relative mobility of phospho-Thr⁴²¹/Ser⁴²⁴ bands (consistent with the loss of phosphate), but not both simultaneously. The immunosuppressant FK506 failed to inhibit p70S6K activation, but was able to rescue the rapamycin-induced downshift, pointing to a role for the mammalian target of rapamycin (mTOR) kinase. Rapamycin also caused an inhibition (IC₅₀ 0.2 nm) of the in vitro enzymatic activity of p70S6K. However, the inhibition of activity was not complete, but only a 40–50%, indicating that neutrophil p70S6K activity has a rapamycin-resistant component. This component was totally inhibited by pre-incubating the cells with the mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor PD-98059 prior to treatment with rapamycin. This indicated that a kinase from the MEK/MAPK pathway also plays a role in p70S6K activation. Thus, GM-CSF causes the dual activation of a rapamycin-resistant, MAPK-related kinase, that targets Thr⁴²¹/Ser⁴²⁴ S6K phosphorylation, and a rapamycin-sensitive, mTOR-related kinase, that targets Thr³⁸⁹, both of which are needed in cooperation to achieve full activation of neutrophil p70S6K.

Extracellular vesicles (EV) are a very diverse group of cell-derived vesicles released by almost all kind of living cells. EV are involved in intercellular exchange, both nearby and systemically, since they induce signals and transmit their cargo (proteins, lipids, miRNAs) to other cells, which subsequently trigger a wide variety of biological responses in the target cells. However, cell surface receptor-induced EV release is limited to cells from the immune system, including T lymphocytes. T cell receptor activation of T lymphocytes induces secretion of EV containing T cell receptors for antigen and several bioactive molecules, including proapoptotic proteins. These EV are specific for antigen-bearing cells, which make them ideal candidates for a cell-free, EV-dependent cancer therapy. In this review we examine the generation of EV by T lymphocytes and CAR-T cells and some potential therapeutic approaches of these EV.

T‐cell receptor stimulation induces the convergence of multivesicular bodies towards the microtubule‐organizing centre (MTOC) and the polarization of the MTOC to the immune synapse (IS). These events lead to exosome secretion at the IS. We describe here that upon IS formation centrosomal area F‐actin decreased concomitantly with MTOC polarization to the IS. PKCδ‐interfered T cell clones showed a sustained level of centrosomal area F‐actin associated with defective MTOC polarization. We analysed the contribution of two actin cytoskeleton‐regulatory proteins, FMNL1 and paxillin, to the regulation of cortical and centrosomal F‐actin networks. FMNL1 phosphorylation and F‐actin reorganization at the IS were inhibited in PKCδ‐interfered clones. F‐actin depletion at the central region of the IS, a requirement for MTOC polarization, was associated with FMNL1 phosphorylation at its C‐terminal, autoregulatory region. Interfering all FMNL1 isoforms prevented MTOC polarization; nonetheless, FMNL1 re‐expression restored MTOC polarization in a centrosomal area F‐actin reorganization‐independent manner. Moreover, PKCδ‐interfered clones exhibited decreased paxillin phosphorylation at the MTOC, which suggests an alternative actin cytoskeleton regulatory pathway. Our results infer that PKCδ regulates MTOC polarization and secretory traffic leading to exosome secretion in a coordinated manner by means of two distinct pathways, one involving FMNL1 regulation and controlling F‐actin reorganization at the IS, and the other, comprising paxillin phosphorylation potentially controlling centrosomal area F‐actin reorganization. Abbreviations: Ab, antibody; AICD, activation‐induced cell death; AIP, average intensity projection; APC, antigen‐presenting cell; BCR, B‐cell receptor for antigen; ^C , centre of mass; cent2, centrin 2; cIS, central region of the immune synapse; CMAC, CellTracker™ Blue (7‐amino‐4‐chloromethylcoumarin); cSMAC, central supramolecular activation cluster; CTL, cytotoxic T lymphocytes; DAG, diacylglycerol; DGKα, diacylglycerol kinase α; Dia1, Diaphanous‐1; dSMAC, distal supramolecular activation cluster; ECL, enhanced chemiluminescence; ESCRT, endosomal sorting complex required for traffic; F‐actin, filamentous actin; Fact‐low cIS, F‐actin‐low region at the centre of the immune synapse; FasL, Fas ligand; FMNL1, formin‐like 1; fps, frames per second; GFP, green fluorescent protein; HBSS, Hank's balanced salt solution; HRP, horseradish peroxidase; ILV, intraluminal vesicles; IS, immune synapse; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; MIP, maximal intensity projection; MVB, multivesicular bodies; MTOC, microtubule‐organizing centre; NS, not significant; PBL, peripheral blood lymphocytes; PKC, protein kinase C; PKCδ, protein kinase C δ isoform; PLC, phospholipase C; PMA, phorbol myristate acetate; Pol. Index, polarization index; pSMAC, peripheral supramolecular activation cluster; PSF, point spread function; ROI, region of interest; SD, standard deviation; shRNA, short hairpin RNA; SEE, Staphylococcus enterotoxin E; SMAC, supramolecular activation cluster; TCR, T‐cell receptor for antigen; T‐helper (Th); TRANS, transmittance; WB, Western blot.