Fig 1: EVs can be modified to harbor high levels of the immunomodulatory checkpoint proteins CD80, OX40L and PD-L1.(a) Hierarchical clustering of proteomes from WT and engineered cells (left) and EVs (right) using label-free MS-based proteomics, results from n=4 biological replicates. (b) Expression of murine CD80, OX40L and CD274 (PD-L1) in parental cells determined by RT-qPCR. Bar graph shows mean +/− s.e.m. of CT values from n=3–5 biological replicates. Expression of GΑΑPDH used as housekeeping gene. (c) Flow cytometry-based evaluation of overexpressing murine proteins at the surface of parental cells and EVs. Overlaid histograms for each protein show the profile for WT and engineered cells and EVs stained with isotype control and with the antibody of interest. The accompanying bar graphs show mean +/− s.e.m. of percentage of positive cells and of positive beads (for EV analyses). Individual data points from n=3–4 biological replicates are shown. (d) Representative NTA profile of WT and engineered EVs with murine proteins. Bar graph shows mean +/− s.e.m. of nanoparticle mode measurements from n=6 biological replicates. (e) Morphology of WT and engineered EVs with murine proteins determined by TEM. Scale bar = 100 nm. (f) On-beads flow cytometry-based evaluation of EV surface markers CD9, CD63 and CD81 in WT and engineered EVs. Heatmap shows percentage of positive beads. Results from n=3 biological replicates. (g) Flow cytometry-based evaluation of co-stimulatory (human OX40L, iCOSL, 4–1BBL, CD70), co-inhibitory (human Gal-9, HVEM, PD-L1, PD-L2) and dual-role (human CD80) immune checkpoint proteins at the surface of parental cells. Results from n=3–8 biological replicates. (h) Flow cytometry-based evaluation of co-stimulatory (human OX40L, iCOSL, 4–1BBL, CD70), co-inhibitory (human Gal-9, HVEM, PD-L1, PD-L2) and dual-role (human CD80) immune checkpoint proteins at the surface of EVs on beads. Results from n=3 biological replicates.
Fig 2: EVs can be modified to harbor high levels of the immunomodulatory checkpoint proteins CD80, OX40L and PD-L1.(a) Schematic representation of APC/cell-mediated and of EV-mediated engagement of the CD28/CTLA-4, OX40 and PD-1 pathways in T cells. (b) Relative expression of human CD80, OX40L and CD274 (PD-L1), in parental cells determined by RT-qPCR. Bar graph shows mean +/− s.e.m. of fold-change from n=3 biological replicates normalized to GΑΑPDH. Statistical significance was determined by two-tailed unpaired t-test, and p-values are shown. (c) Human CD80, OX40L and PD-L1 and loading control proteins in parental cells and EVs probed by western blot. (d) Flow cytometry-based evaluation of overexpressing proteins at the surface of parental cells and EVs. Overlaid histograms for each protein show the profile for WT and engineered cells and EVs stained with isotype control and with the antibody of interest. The accompanying bar graphs show mean +/− s.e.m. of percentage of positive cells and of positive beads (for EV analyses). Individual data points from n=3–4 biological replicates are shown. (e) Representative NTA profile of WT and engineered EVs. Bar graph shows mean +/− s.e.m. of nanoparticle mode measurements from n=6 biological replicates. (f) Morphology of WT and engineered EVs determined by TEM. Scale bar = 100 nm. (g) Schematic representation of the workflow utilized for the MS-based proteomics analysis of parental cells and EVs. (h) Bar graph shows mean +/− s.e.m. of number of proteins quantified in cells and EVs by MS, results from n=4 biological replicates. (i) Heatmap of CD80, OX40L and PD-L1 protein levels in parental cells and EVs determined by MS, results from n=4 biological replicates. Gray colored squares represent no detection. (j) Heatmap of EV markers and exclusion markers in parental cells and EVs determined by MS, results from n=4 biological replicates. Gray colored squares represent no detection. (k) PCA analysis of proteomes from parental cells and EVs, results from n=4 biological replicates. (l) Heatmaps show Pearson correlation of proteomes from parental cells (left) and EVs (right), results from n=4 biological replicates.
Supplier Page from Sino Biological, Inc. for Mouse CD80/B7-1 Gene ORF cDNA clone expression plasmid