Fig. 2. Reduced ability of Batf3-/- DC to cross-prime CTLs against tumor antigens both in steady state and after treatment with anti-CD137 and anti-PD-1 mAbs.
(A-C) CD11c+ DCs from WT and Batf3-/- mice bearing MC38-OVA tumors were magnetically sorted from tumor-draining LNs and cocultured (see Methods) with purified naïve CD8+ OT-I TCR transgenic T cells over a range of DC:T cell ratios. (A) Left: representative flow cytometry dot plots of intracellular IFN-γ staining in OT-I T cells cultured at a 1:4 DC:T cell ratio. Right: percentages of IFN-γ-positive OT-I T cells at all ratios tested. (B) IFN-γ concentrations in the culture supernatants. (C) Percentages of proliferating OT-I cells by dilution of Cell Violet dye (D-F) WT and Batf3-/- mice grafted with MC38-OVA cells were treated with anti-CD137 (days 5 and 7) and tumor-draining LN analyzed on day 9 (see Methods). (D) Frequency of H-2Kb-OVA-tetramer+ cells among CD8+ T cells. (E) Intracellular IFN-γ production induced by restimulation with OVA257-264 peptide in CD8+ T cells from tumor-draining LN. (F) PD-1 surface staining on tumor-draining LN CD8+ T cells. (G) Frequency of PD-1+ lymphocytes among CD8+ TILs in mice treated as in D. (H) WT and Batf3-/- mice grafted with MC38 cells were treated with anti-CD137 and anti-PD-1 mAbs on days 12 and 14 and tumor-infiltrating lymphocytes were analyzed on day 16 to detect CD8 T lymphocytes specific for gp70 antigen (A-C) Two-way and (D-H) one-way ANOVA with Bonferroni post-hoc test. * p < 0.05; ** p< 0.01; *** p < 0.001