Tivesicular bodies (MVBs), and microvesicles, (150-1000 nm diameter) budding straight from the plasma membrane [26], are membrane-bound vesicles naturally released from most cell sorts and recognized as potent vehicles of cell-to-cell communication. Nef-containing EVs happen to be reported to induce T-cell apoptosis [24], to make resting CD4+ T lymphocytes competent for HIV expression and replication, to reactivate cells latently infected with HIV-1 [270], too as to enhance the levels of cytokines and chemokines for instance IL-2, IL-8, IL-6, RANTES and IL-17A [31]. Though Nef has been regularly reported to enhance EV release [23,24,32] and to become itself secreted in EVs, it remains unclear which form of EV is primarily involved, considering that Nef has been detected in each exosomes [24,33,34] and microvesicles [35], in line with the cell form. Furthermore, both Nef and anti-Nef antibodies have been detected inside the serum of HIV-infected people [36,37], Neural Cell Adhesion Molecule L1 Proteins Species penicillin, one hundred /mL streptomycin (SigmaAldrich, Milan, Italy) and ten fetal bovine serum (FBS) (cat. 10270106, Gibco, Amarillo, TX, USA), previously inactivated at 56 C for 30 min. Circulating pDCs were isolated from PBMCs by constructive choice using an immunomagnetic-based kit (BDCA-four cell isolation kit, Miltenyi Biotec, Bologna, Italy), according to the manufacturer’s recommendations. The purified pDCs had been maintained in RPMI 1640 medium supplemented with two mM Lglutamine, one hundred Units/mL penicillin, 100 /mL streptomycin, 25 mM Hepes and 10 heatinactivated FBS. PBMCs depleted of monocytes (PBLs), PBLs depleted of pDCs (PBLs-.