generated the bone marrow-derived dendritic cells and performed the antigen cross-presentation experiments. mechanism by which tumor cells systemically suppress the immune system, and provides a rationale for the application of exosomal PD-L1 as a predictor for anti-PD-1 therapy. EVs such as exosomes and microvesicles (a.k.a. shedding vesicles) carry bioactive molecules that influence the BMS-191095 extracellular environment and the immune system6C8. The exosomes from a panel of human primary and metastatic melanoma cell lines were purified by differential centrifugation9C11, and verified by transmission electron microscopy (EM) and nanoparticle tracking analysis (NTA) (Fig. 1a and 1b). Proteins associated with the exosomes were BMS-191095 then analyzed by reverse phase protein array (RPPA), a large-scale antibody-based quantitative proteomics technology12. The RPPA and western blot analysis revealed PD-L1 in exosomes, and its level was significantly higher in exosomes derived from metastatic melanoma cells compared to primary melanoma cells (Fig. 1c and d, Extended Data Fig. 1a). Iodixanol density gradient centrifugation further confirmed the association of PD-L1 with the exosomes (Extended Data Fig. 1b). PD-L1 was also detected in microvesicles, but at a lower level (Extended Data Fig. 1cCe). PD-L1 was also detected in EVs generated from mouse metastatic melanoma B16-F10 cells (Extended Data Fig. 1f). Open in a separate window Figure 1 Extrafacial expression of PD-L1 on melanoma cell-derived exosomes and its regulation by INF-a, A representative TEM image of purified WM9 cell exosomes. b, Characterization of purified exosomes by NanoSight nanoparticle tracking system. c, RPPA data showing the level of PD-L1 in the exosomes secreted by primary or metastatic melanoma cell lines (n = 3 for WM1552C, WM902B, A375, WM164, and n = 4 for WM35, WM793, UACC-903, WM9). See Extended Data Fig. 1a for statistical analysis. BMS-191095 d, Immunoblots for PD-L1 in the whole cell lysate (W) and purified exosomes (E) from different metastatic melanoma cell lines. The same amounts of proteins in whole cell lysates and exosome were loaded. e, A representative TEM image of WM9 cell-derived exosomes immunogold-labeled with anti-PD-L1 antibodies. Arrowheads indicate 5-nm gold particles. f, Diagram of ELISA of exosomal PD-L1 (left panel). PD-L1 on the surface of exosomes was determined. See Methods for details. g, Levels of PD-L1 on exosomes from melanoma cells, with or without IFN- treatment, as measured by ELISA. h, PD-l binding of exosomes. See Methods for details. i, Western blot analysis of PD-L1 in exosomes from IFN–treated cells (IFN) and control cells (C). The same amounts of BMS-191095 exosome proteins were loaded (left panel). Quantification of exosomal PD-L1 by western blotting (right panel). The experiments were repeated three (a, b) or two (d, e) times independently with similar results obtained. Data represent mean s.d. of three (f, h, i) or four (g) independent biological replicates. Statistical analyses were performed using two-sided unpaired which mediates the recognition and sorting of exosomal cargos15, led to a decrease in IP1 the level of PD-L1 in the exosomes and an increase of PD-L1 in the cell (Extended Data Fig. 1g, h). In addition, PD-L1 co-immunoprecipitated with Hrs from the cell lysates (Extended Data Fig. 1i). PD-L1 co-localized with Hrs and CD63, an exosome marker, in melanoma cells (Extended Data Fig. 1j, k). Knockdown of also blocked PD-L1 secretion via exosomes (Extended Data Fig. 1l). To test the secretion of exosomal PD-L1 by melanoma cells and knockdown tumors with indicated treatments (n = 7 mice per group). c, The proportions of Ki-67+PD-1+ CD8 TILs or splenic or lymph node CD8 T cells after indicated treatments (n = BMS-191095 6 for tumor samples of the EXO-IgG group, and n = 7 for all the other groups). See Extended Data Fig. 8d for representative contour plots. Data represent mean s.d. (aCc). Statistical analyses were performed using two-sided unpaired knockdown B16-F10 cells ( 0.05, Statistical analyses were performed using.