Supplementary Figure 12: An anti-GD2 EVIR enhances EV uptake by DCs and presentation of a surrogate tumor antigen to T cells.

(a) Flow cytometry analysis of GD2 expression in MC38-mCh cells that had been transduced with the indicated GD2 synthase. The cells were analyzed once and employed in subsequent experiments.

(b) Flow cytometry analysis of mCh MFI in DC-CtrlR or anti-DG2 DC-EVIR untreated or treated with EV-mCh or EVs that had been isolated from GD2⁺ MC38-mCh cells (EV-GD2/mCh). Data show mean MFI values ± SEM (n=3 independent cell cultures per condition); statistical analysis by two-way ANOVA with Sidak's multiple comparison test.

(c) Flow cytometry analysis of GD2 expression in SM1-OVA cells that had been transduced with the indicated GD2 synthase. The cells were analyzed once and employed in subsequent experiments.

(d) Flow cytometry analysis of H-2Kb expression in SM1-GD2/OVA cells, untreated or treated with recombinant IFNγ as indicated. The experiment was performed once.

(e) Size distribution of EV-SM1-OVA, either with or without GD2, as determined by NTA. Data show mean values ± SEM (n=3 technical measurements of one EV preparation per type). Data are representative of 2 independent EV preparations.

(f) Flow cytometry analysis of CD8⁺ OT-I cell proliferation assessed by CellTrace dilution after co-culture with the indicated DCs and EVs. The left panels show representative flow cytometry dot plots (one cell culture of 3 performed per condition). The right panel shows the percentage of CD8⁺ OT-I cells that completed at least 1 cell cycle. Data show mean percentages ± 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.