Supplementary Figure 7: An anti-HER2 EVIR enhances EV uptake by DCs and cross-dressing with MHCI/antigen complexes.

(a) Flow cytometry analysis of CD8⁺ OT-I proliferation, assessed by CellTrace dilution, after co-culture with DC-CtrlR or anti-HER2 DC-EVIR cells, and EV-OVA (not containing HER2). The top panels show representative flow cytometry dot plots. The bottom panel shows the percentage of CD8⁺ OT-I cells that completed 1 to 3 (P1-3) or more than 3 (P>3) cell cycles. Data show mean percentage values ± SEM (n=3 independent cell cultures per condition).

(b, c) Flow cytometry dot plots showing B2M (b) and H-2Kb (c) expression in the indicated cell types. In (b), “B2m KO DC” refers to DCs isolated from the bone marrow of B2m KO mice (analysis performed once to verify a genetically-determined phenotype). In (c), “FVB DC” indicates DCs that were differentiated from the bone marrow of FVB/n mice (analysis performed once to verify a genetically-determined phenotype), whereas “Total CD45⁺ cells (NSG)” indicates all hematopoietic (CD45⁺) cells retrieved from a matrigel-embedded tumor containing H2kb KO MC38 cells and FVB/n DCs (analysis performed on 4 independent cell suspensions, of which one is shown). MHCI-competent bone marrow-derived DCs of C57Bl/6 mice (“DC”) and MC38 cancer cells (“MC38”) are shown for comparison (analysis performed once to verify a genetically-determined phenotype).

(d) Expression of H-2Kb in DCs transduced as indicated and exposed to the indicated EV preparations. Cells were analyzed 24 h after exposure to EVs. Data show mean values ± SEM (n=3 independent cell cultures per condition); statistical analysis by two-way ANOVA with Sidak's multiple comparison test.

Numerical values for the experiments with quantitative data are presented in Supplementary Table 2.