Hrough association of endogenous LMP1 with endosomal tetraspanin CD63 and subsequent secretion through exosomes (18). Our results showed that principal EBV-infected B cells also released exosomes harboring LMP1, but expression levels have been a lot TLR7 Inhibitor Accession decrease compared with PARP1 Inhibitor Accession LCL-derived exosomes (Fig. 1A). Alternatively, the Burkitt’s lymphoma cell line stably transfected with LMP1 (DG75-LMP1) was a appropriate source to obtain human exosomes that harbored LMP1 at physiological concentrations and, hence, potentially mimic exosomes which are released in the course of major EBV infection (Fig. 1B). Major human B cells stimulated with IL-4 plus anti-CD40 secrete exosomes that reflect the activation state with the B cells (32). Consistent with these findings, DG75 exosomes reflected the phenotype of their corresponding B cell line (Fig. 2B). Ectopic LMP1 expression in EBV- Burkitt’s lymphoma cell lines was shown to increase MHC class I and II Ag expression (33, 34). In line with this, DG75-LMP1ex had considerably greater levels of HLA-ABC and HLA-DR than did DG75-COex (Fig. 2B). Generally, it has to be stressed that all 3 DG75 exosomes had a phenotypic profile that distinguished them, and these differences are most likely to influence biological effects. For instance, DG75-LMP1ex and DG75-EBVex had considerably larger levels of HLA-ABC molecules compared with DG75-COex, and it is actually tempting to speculate that they include EBV-specific peptides that might be presented on the surface of DCs or B cells following their uptake. Potentially, these exosomes could be an “additional source” of viral peptides, which improve the frequency of EBV-specific CTLs. In contrast, enhanced expression of HLA-DR molecules on DG75LMP1ex compared with DG75-COex and DG75-EBVex could be an more Ag supply used by DCs to license CD4+ Th cells that, in turn, can activate B cells, thereby inducing Ab responses. Also, LMP1 was detected only in DG75-LMP1ex; the diverse effects observed in this study between the various DG75 exosomes are clearly not only dependent around the 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 earlier study (24). A sizable body of evidence indicates that exosomes play a major role in intercellular communication and, thereby, influence the outcome of an immune response (1, 35). To contribute to intercellular communication, exosomes have to interact with and deliver their content material to the recipient cell. In a previous study, we observed that DC- and breast milk?NIH-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 various binding pattern inside PBMC cultures compared with exosomes from a gp350-expressing LCL (LCL1) (25). Our information demonstrate that the various DG75 exosomes bound with related efficiency to B cells and monocytes within PBMC cultures (Fig. 3B). Additionally, the detection of LMP1 shuttled through LCL1ex in B cell lysates indicated exosome binding and suggested their uptake (Fig. 3C). Confocal microscopy analysis demonstrated internalization of DG75 exosomes by B cells (Fig. 3D). Recently, fusion of the exosomal membrane using the plasma membrane was demonstrated as a mechanism by which functional miRNA shuttled by DC-derived exosomes is delivered towards the acceptor DC (36). Pegtel et al. (29) demonstrated the functional delivery of mature EBV-encoded microRN.