Fig 2: Binding affinity and ligand blocking measurements of Pembrolizumab from mRNA or CHO sources to PD-1 by SPR (Biacore) and cell-based assays. (A-B) Association and dissociation of Pembrolizumab to hPD-1 by SPR. (C) Affinity of Pembrolizumab to CHO-hPD-1 cells. (D) Affinity of Pembrolizumab to activated CD4+ T cells. CHO-hPD1 cells-based ELISA with fixed concentrations of human PD-L1 (E) or PD-L2 (F). All the data were showed as Mean ± S.E.M, n=3.

Fig 3: PD-L1 protein transferred onto the surface of negative cells via EVs is functionally active: A) Schematic for functional analysis of EV-mediated PD-L1 transfer: Ai) PD-1 receptor binding assay and Ai) T cells suppression assay. B) Histogram showing the PD1-Fc-PE binding on the PD-L1+EV treated 293 T or H1299 cells with respective controls. C) Calculated MFI for PD1-Fc-PE receptor binding on PD-L1+EVs treated or respective control cells. D-G) T cells suppression assay and T reg phenotype induction: Following the post CD3/CD28 activation, the CFSE labeled T cells from three independent donors were co-cultured in duplicate with either 293T or PD-L1+EVs pre-treated 293T cells and analyzed by multiparameter flow cytometry after 4 days of incubation. D) Flow histogram for CFSE dye intensity in CD3+ cells and E) total MFI for CD3+CFSE cells cultured with 293T or PD-L1+EVs pre-treated 293T cells. F) Histogram for FoxP3 expression of CD3+CD4+CD25+ cells and H) MFI for the FoxP3 expression in T cells cultured with 293T or PD-L1+EVs pre-treated 293 T cells. Data are shown as the mean ± SD from one of two independent experiments (*p < 0.05, **p < 0.01)