Figure 5. Therapeutic immunity induced by Flt3L-poly I:C combined therapy requires CD103⁺ DCs and type I IFN.

(A-B) Tomato⁺ Braf-mutant tumor mice were treated as described in Figure S4B. Two days after the end of treatment, tumors were harvested and frozen. (A) Representative confocal images of CD3⁺ T cells (green) infiltrating tumors from the indicated treatment groups. (B) CD3⁺ T cell density was quantified from confocal images using Cell Profiler software. Shown is the mean ± SEM of 2 independent experiments (n= 3-4;12-16 tiled 20x images were analyzed per animal). (C-D) B16-YFP tumor-bearing mice were treated as described in Figure S4A. Two days after the end of treatment, tumors were harvested and frozen. (C) Shown are representative confocal images of CD3⁺ T cells (red) infiltrating tumors treated with PBS or FL-pIC. (D) CD3⁺ T cell density (left) and proportions of perivascular CD3⁺ T cells (<30um from CD31⁺ vessels; right) were quantified from confocal images using Cell Profiler software. Shown is the mean ± SEM of 4 independent experiments (n= 4-6; 12-16 tiled 20x images were analyzed per animal).

(E-H) B16 tumor-bearing mice were treated as in Figure S4A and tumor-infiltrating T cells were analyzed at day 15, unless specified otherwise. (E) Representative dot plots (left) and quantification (right) of the frequency of tumor-infiltrating CD8⁺ T cells expressing the cell cycle protein Ki67. Graph shows the mean ± SEM of 2 independent experiments (n= 4). (F) WT splenocytes were incubated in vitro with ovalbumin_257-264 peptide and labeled with high concentration of CFSE (CFSE^hi) or incubated with control medium and labeled with low CFSE concentration (CFSE^low). Same numbers of CFSE^hi and CFSE^low splenocytes were coinjected into B16 tumor-bearing mice treated with FL+pIC or PBS at day 16 and analyzed 24hrs later. Shown is the mean ± SEM of one experiment (n=4-5). (G) CD8⁺ T cells were analyzed for IFNγ and TNFα production after PMA/ionomycin stimulation. Shown is the mean ± SEM of 4 independent experiments (n=8). (H) Tumor-infiltrating CD8⁺ T cell/Treg cell ratio as the mean ± SEM of 4 independent experiments (n=8).

(I) Mice bearing B16-OVA tumors received daily FL injections from day 5 to 9 post- tumor challenge followed by one intratumoral injection of pIC at day 9. Two hours after the pIC injection, tumor-bearing mice were injected with 3×10⁶ naïve tumor antigen-specific CD8⁺ T cells (OT-I) in the presence or absence of the S1P receptor antagonist FTY-720 (from day 9, daily injection as described in the methods). Shown are representative dot plots (left) and quantification of the frequency and absolute numbers of tumor-infiltrating OT-I cells (right) four days after adoptive cell transfer. Graphs show the mean ± SEM of 2 independent experiments (n= 4-5).

(J-K) B16 tumors were injected into WT mice or Batf3^−/− mice and treated with FL+pIC. Here mice received only 5 injections because CD103⁺ DCs reappear in Batf3^−/− mice upon 9 FL injections. (J) Graph shows the mean tumor growth ± SEM of 2 independent experiments (n= 4-6). (K) B16 tumors were harvested on day 15. Bar graphs show the intratumoral T cell density as measured by flow cytometry (left) and CD8⁺ cell/Treg cell ratio (right) in Batf3^−/− versus WT treated mice. Results are shown as the mean ± SEM of one experiment (n=4-5).

(L) B16 tumor-bearing mice treated with FL-pIC with with anti-CD4, anti-CD8, anti-NK1.1 Ab or control IgG as described in the method section. Graph shows the mean tumor growth ± SEM of 2 independent experiments (n=3-9).

See also Figure S5.