Until today little is known on the expression pattern of NCR ligands. Their cellular distribution has been detected only on a limited number of tumor cells. A comprehensive analysis showed NKp30L expression on normal and neoplastic cells by staining with NKp30-Ig fusion constructs. More specifically, NKp30-Ig staining was observed in intra-cellular vesicular compartments very similar to early endosomal marker which corresponds to the formation of exosomes. This supports our observation that the cellular ligand BAT3 is localized in endosomal vesicles and secreted in exosomal form. However, our data cannot rule out the existence of different isoforms of BAT3 that are released in exosomal or in soluble form. For instance, the release of soluble inhibitory forms could be mediated through shedding activity, as described for the ligands engaging the NKG2D receptor. The inhibitory role of soluble recombinant BAT3 observed in our experiments might be due to a passive blocking or an PI-103 371935-74-9 active repression of NKp30-mediated signalling. To assess directly the expression of BAT3 on the surface of exosomes, we used flow cytometric analysis. Purified exosomes from iDCs and 293T cells were incubated with latex beads of 4 mm in diameter and stained with antibodies directed against BAT3 and several exosomal proteins. The expression of BAT3 and CD-9 was clearly detectable on the surface of both exosomal fractions. iDC-derived exosomes expressed also CD86, HLA class-I and HLA-DR as previously described. Positive staining for Hsp70 and Lamp-2 on 293Tderived exosomes revealed the purity of exosomes. Ligands for the NKG2D receptor were not detectable using NKG2D-Ig staining, suggesting that NKG2D engagement is independent of exosomes derived from 293T cells. We then used BAT3-overexpressing 293T cells to directly prove the binding of NKp30 to BAT3 on the exosomal surface. As determined by flow-cytometry, exosomes derived from BAT3- transfected 293T cells express high levels of BAT3 on their surface. Purified exosomes were stained with Ig fusion proteins. The binding of NKp30-Ig to BAT3 on exosomes was specific and not observed for human-Ig and CD30-Ig. These experiments suggest that exosomal BAT3 acts as a membrane associated molecule which is presented to NKp30. Interestingly, Screening Libraries recognition of so far unknown ligands for NKp46 was detectable using NKp46-Ig. A coordinated expression of ligands for different NCRs on exosomes may explain the cross-stimulation of NCRs in response to selective activation. Staining of iDC-derived exosomes with human Ig constructs was not feasible since Fc-receptors are present on exosomes derived from human dendritic cells. Next we analyzed whether extracellular BAT3 was detectable in a physical association with Hsp70.