Fig 1: 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.
Fig 2: OX40L EVs synergize with anti-CTLA-4 to promote anti-tumor immunity.(a) Schematic representation of experimental design employed to assess the therapeutic potential of OX40L EVs combined with anti-CTLA-4 in B16-F10 melanoma. (b) Kaplan–Meier curve of tumor-bearing mice treated with vehicle, WT or mOX40L EVs in the presence of isotype or anti-CTLA-4 antibody. Survival plot shows results from n=8–12 mice per group. Statistical analysis performed using log-rank Mantel–Cox test. (c) Kinetics of B16-F10 tumor growth treated with vehicle, WT or mOX40L EVs in the presence of isotype or anti-CTLA-4 antibody. Graph shows mean +/− s.e.m. of tumor volume from n=8–12 mice per group. Statistical significance was determined using two-way ANOVA with Šídák’s multiple comparison test. (d) Linear regression analysis to test for significant differences in slope of tumor growth curves between experimental groups. Statistical significance was determined using Simple linear regression and p-value is shown. Statistical significance defined as p < 0.05.
Fig 3: OX40L EVs promotes anti-tumor immunity in B16-F10 melanoma in a CD8+ T cell-dependent manner.(a) Schematic representation of experimental design employed to assess the contribution of CD4+ T cells in the anti-tumor phenotype elicited by OX40L EVs, using anti-CD4 antibody. (b) Kaplan–Meier curve of tumor-bearing mice treated with vehicle, WT or mOX40L EVs in the presence of isotype or anti-CD4 antibody. Survival plot shows results from n=6 mice per group. Statistical analysis performed using log-rank Mantel–Cox test. (c) Kinetics of B16-F10 tumor growth treated with vehicle, WT or mOX40L EVs in the presence of isotype or anti-CD4 antibody. Graph shows mean +/− s.e.m. of tumor volume from n=6 mice per group. Statistical significance was determined using two-way ANOVA with Šídák’s multiple comparison test. (d) Schematic representation of experimental design employed to assess the contribution of CD8+ T cells in the anti-tumor phenotype elicited by OX40L EVs, using anti-CD8 antibody. (e) Kaplan–Meier curve of tumor-bearing mice treated with vehicle, WT or mOX40L EVs in the presence of isotype or anti-CD8 antibody. Survival plot shows results from n=6 mice per group. Statistical analysis performed using log-rank Mantel–Cox test. (f) Kinetics of B16-F10 tumor growth upon treatment with vehicle, WT or mOX40L EVs in the presence of isotype or anti-CD8 antibody. Graph shows mean +/− s.e.m. of tumor volume from n=6 mice per group. Statistical significance was determined using two-way ANOVA with Šídák’s multiple comparison test. Statistical significance defined as p < 0.05.
Fig 4: 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.
Supplier Page from Sino Biological, Inc. for Human OX-40L / TNFSF4 / CD252 Gene ORF cDNA clone expression plasmid