Figure 5. CD103⁺ DC-derived CXCL9 is important for the efficacy of PD-1 blockade.

(A) Representative flow cytometry gating strategy for determination of CXCL9 and CXCL10 expression in tumor-infiltrating DC subpopulations (CD103⁺ DC and CD11b⁺ DC) within MC38 tumors harvested from the CXCL9-CXCL10 dual reporter (REX3) transgenic mice. (B-C) MC38 tumors were excised from REX3 transgenic mice on day 15 following treatment with isotype or anti-PD-1 antibodies according to the schedule presented in Figure 1A and were analyzed for CXCL9-RFP expression in CD103⁺ DC and CD11b⁺ DC (gated on CD45⁺ CD3⁻ CD19⁻ CD11c⁺ MHCII⁺ as demonstrated in Figure 5A). Bar graph represent the mean values of the indicated data points, and the error bars represent SEM; ***p<0.001, with statistical significance determined by one-way ANOVA with Tukey’s multiple comparison test. Representative data are shown from two independent experiments. (D-E) Bone marrow cells from CD45.1⁺ CD45.2⁺ Zbtb46^DTR mice were mixed in a 50:50 ratio with bone marrow cells from CD45.2⁺ WT, Cxcl9^−/− or Cxcl10^−/− mice and injected into lethally irradiated CD45.1⁺ WT recipient mice. Eight weeks after transplant, 1×10⁶ MC38 cells were injected subcutaneously into chimeric mice. Following tumor inoculation, administration of FTY720 (1mg/kg) and DT (20μg/kg) started one day before isotype or anti-PD-1 treatment (200μg; on day 8, 11 and 14), and continued until day 19 as indicated. Tumor growth and survival were monitored until the experimental endpoints (n=5–10 mice per group). Data are given as the mean ± SEM; ***p<0.001, with statistical significance determined by two-way ANOVA. Survival curves show the percentage of mice whose tumor sizes were <100mm² at each time point. Statistical differences in survival were assessed by a Mantel-Cox log-rank test (**p<0.01). Representative data are shown from two independent experiments. See also Figure S5 and S6.