Thus, we explored the role of soluble protein on the maturation of iDCs in NK-DC-co-culture experiments and analyzed the expression of the co-stimulatory molecule CD86. As expected co-cultivation of monocyte-derived iDCs with NK cells induced enhanced expression of CD86. In this setting the up-regulation of CD86 was reduced in the presence of soluble recombinant BAT3 indicating that BAT3 has a substantial impact on the NK cell-mediated iDC-maturation. The WZ4002 EGFR/HER2 inhibitor effect of recombinant BAT3 on NK cell cytotoxicity has not been addressed so far. Therefore we performed europium release assays using NK cells either pre-stimulated with soluble or with immobilized recombinant BAT3. We observed that NKdependent lysis of Raji cells was inhibited when NK cells were stimulated with purified soluble HisBAT3. Similar inhibition was achieved upon blocking the NKp30 receptor with a masking monoclonal antibody. The control protein did not alter NK cell cytotoxicity. A similar reduction in NKp30- mediated cytotoxicity was also reported for the viral ligand pp65. On the other hand, we observed that immobilized BAT3 had an opposite effect enhancing the cytotoxicity of NK cells against the target cells compared to the control protein HisBB4. This data confirm previous results indicating that recombinant BAT3 in a soluble form inhibits cytokine secretion of NK cells, whereas immobilized BAT3 activates TNF-a and IFN-c release. Taken together, it was demonstrated that the NKp30- mediated DC-killing and DC maturation is at least partly dependent on BAT3. To assess the underlying mechanisms of BAT3 on NK cell function as an inhibitor in soluble form and an activator when immobilized, we directly tested whether BAT3 is secreted in a complex structure such as exosomes. Exosomes are membrane microvesicles that direct the communication among cells in the immune system. Tumor cells PF-04217903 secrete exosomes similar to immune regulatory cells such as antigen-presenting cells, T cells, reticulocytes and platelets. Exosomes were purified by ultracentrifugation from supernatant of 293T cells and their presence was confirmed by electron microscopy. As expected, the exosomes appeared as clusters of vesicles each surrounded by a double layer membrane. A specific immunostaining for BAT3 was detectable indicating that the exosomes expressed BAT3. This was confirmed by Western blot analysis of exosomes secreted from tumor cells and from iDCs. Moreover, sucrose gradient analysis was performed proving that BAT3 was associated with membrane vesicles. The analysis of several control proteins revealed a differential expression for Hsp70, Lamp2 and CD9 between exosomes derived from tumor cells versus iDC-derived exosomes.