Presence of LMP1 (Fig. 1B). Of note, the low or undetectable LMP1 levels in DG75-EBV cells and DG75-EBVex, respectively, are in agreement with a previous study (24). A big body of proof indicates that exosomes play a significant function in intercellular communication and, thereby, influence the outcome of an immune response (1, 35). To contribute to intercellular communication, exosomes must interact with and deliver their content towards the recipient cell. In a prior study, we observed that DC- and breast milkNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptJ Immunol. Author manuscript; readily available in PMC 2014 September 24.Gutzeit et al.Pagederived exosomes had a different binding pattern within PBMC cultures compared with exosomes from a gp350-expressing LCL (LCL1) (25). Our data demonstrate that the various DG75 exosomes bound with equivalent efficiency to B cells and monocytes inside PBMC cultures (Fig. 3B). Moreover, the detection of LMP1 shuttled through LCL1ex in B cell lysates indicated exosome binding and recommended their uptake (Fig.Diversity Library manufacturer 3C).Zearalanone MedChemExpress Confocal microscopy analysis demonstrated internalization of DG75 exosomes by B cells (Fig. 3D). Recently, fusion in the exosomal membrane with all the plasma membrane was demonstrated as a mechanism by which functional miRNA shuttled by DC-derived exosomes is delivered to the acceptor DC (36). Pegtel et al. (29) demonstrated the functional delivery of mature EBV-encoded microRNAs by way of exosomes released from EBV-infected B cells to monocytederived DCs.PMID:24318587 Even so, it nevertheless must be elucidated which uptake mechanism, direct fusion or endocytosis, enables B cell erived exosomes to provide their content into the cytoplasm in the recipient B cell. Apoptosis plays a crucial function in B cell improvement and homeostasis, plus the T cellderived cytokine IL-21 was shown in vitro to induce apoptosis of resting and activated primary murine B cells (37). Constant with that, unstimulated and IL-21 + anti-CD40stimulated main human B cells also showed signs of apoptosis and necrosis already at 24 h, plus the addition of DG75 exosomes didn’t guard resting B cells from apoptosis (Fig. 4A). Ectopic LMP1 expression within a B cell line and EBV infection of IgD+ B cells in vitro provide B cell survival signals via upregulation of autocrine BAFF and a proliferationinducing ligand (APRIL) expression (27, 38). Nonetheless, the concentration of LMP1 in B cells right after delivery by way of DG75-LMP1ex is substantially reduce compared with ectopically expressed LMP1 applied inside the above-described study (27). Future studies will investigate whether or not exosomes carrying high amounts of LMP1 induce autocrine BAFF and APRIL expression that supply survival and proliferation signals to B cells. This could be of distinct interest in offering a hyperlink between EBV exosomes and SLE, in which enhanced BAFF expression rescues self-reactive B cells from peripheral deletion (39). The addition of DG75 exosomes to PBMC cultures induced proliferation in B cells, whereas no proliferation was noticed for T cells (Fig. 4C). It has to be stressed that the absence of T cell proliferation may possibly be as a result of the extremely low binding efficiency of DG75 exosomes to T cells (3 ; information not shown). A dose-dependent proliferation was observed when isolated B cells had been exposed to DG75 exosomes, with a trend toward elevated proliferation for DG75LMP1ex (Fig. 5B). We would prefer to point out that these data had been generated in two laboratories with constant result.
