Figure 3.

OVM reactivates tumor-suppressed DC vaccines

(A–C) The expression of maturation markers (CD86, CD83, and MHC II) in mouse CD11c⁺ DCs after being cocultured at a 3:1 ratio with B16-F10 cells (A) or CT-26 cells (B) for 48 h or with Pan02 cells for 72 h (C); n = 3.

(D–F) The expression of CD86 and MHC II in human CD11c⁺ DCs after being cocultured at a 3:1 ratio with HCT-8 cells (D), HCT-116 cells (E), or SW-620 cells (F) for 24 h; n = 3.

(G and H) DCs were untreated or cocultured with B16-F10 cells for 48 h, separated with magnetic beads, and cocultured with spleen-derived T cells at a 1:5 ratio for 96 h; n = 3 in per group. (G) The MFI values of CD69 and CD44 on CD4⁺ (left) or CD8⁺ (right) T cells. (H) The concentrations of IFN-γ, perforin, and granzyme B in the coculture system.

(I) DCs were untreated or cocultured with B16-F10 cells and treated with 1 μg/mL OVA for 24 h. The MFI of H-2Kb OVA_257-264 on CD11c⁺ DCs was determined by flow cytometry; n = 4.

(J and K) DCs were untreated or cocultured with B16-F10 cells for 24 h and stimulated with 1 μg/mL OVA for another 24 h. DCs were separated with magnetic beads and cocultured with spleen-derived CD8⁺ T cells from OT-I mice at a 1:2 ratio for 48 h; n = 3 per group. (J) Schematic diagram. (K) Representative histograms (left) and MFI values (right) of CD69 and CD44 on OT-I CD8⁺ T cells are shown. The data in (A)–(G) are presented as the means ± SD, and p values were determined by unpaired Student’s t test (A)–(I) or one-way ANOVA (K).

(L–N) (L) C57BL/6J mice were implanted subcutaneously in the right flank with B16-F10-OVA cells on day 0 and administered one dose of vehicle, OVA-loaded DC vaccine (activated by OVA and LPS), or co-DC vaccine by intratumoral injection on day 6 (tumor volumes were approximately 50 mm³). OVA-loaded DCs were cocultured with B16-F10 cells for 48 h and sorted by magnetic beads to prepare the co-DC vaccine. (M) Tumor growth curves and (N) Kaplan-Meier survival curves are shown; n = 9. The p values were determined by one-way ANOVA at the final time point as indicated in the graphs or by the log rank test, as appropriate.

(O) The changes in CD86, CD83, and MHC II expression on mouse CD11c⁺ DCs cocultured with B16-F10 cells for 24 h and stimulated with control (Ctrl), LPS (1 μg/mL, as a positive control), or OVM (1 MOI) for another 24 h.

(P) The changes in CD86 and MHC II expression on human DCs cocultured with HCT-8 cells for 24 h and stimulated with control (Ctrl) or OVM (1 MOI) for another 24 h.

(Q) The concentrations of TNF-α, IL-12p70, and IFN-β in the coculture supernatant of graph (O).

(R and S) CD11c⁺ DCs were cocultured with B16-F10 cells for 24 h and stimulated with OVM (1 MOI) for another 24 h, separated with magnetic beads, and cocultured with spleen-derived T cells at a 1:5 ratio for 96 h. (R) The MFI of CD69 and CD44 on CD4⁺ (left) or CD8⁺ (right) T cells. (S) The concentrations of IFN-γ, perforin, and granzyme B in the co-DC or OVM-treated coculture system; n = 3. One-way ANOVA was used to determine the significance of differences between groups in the graphs of (O)–(S). All data are presented as the means ± SD. n.s., not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.