Fig 2: Lung-resident Mgl2+/IDO1+ R2D2 population generates T-regs in the lung.a Flow cytometry plots (left) and quantification (right) of CD4+Foxp3+ T-reg cells in in WT, Batf3-/-, and IRF4fl/flCD11ccre mice treated with one dose of OVA (1 µg) intranasally (i.n.). Lungs were harvested on day 14; n = 4–6 mice/group. Data are representative of two independent experiments. b, c Flow cytometry plots (left) and quantification (right) of T-regs in TNFR2fl/flCD11ccre b and RelBfl/flCD11ccre mice c treated with one dose of OVA (1 µg) i.n. Lungs were harvested on day 14; n = 3–5 mice/group. Data are representative of two independent experiments. d IRF4fl/flCD11ccre mice were adoptively transferred (i.n.) with lung TNFR2+ and TNFR2- cDC2 from WT mice lung and treated with one dose of OVA (1 µg) i.n. Flow cytometry analysis (left) and quantification (right) of T-regs at day 14; n = 3–6 mice/group. Data are representative of two independent experiments. e, f Flow cytometry analysis of TNFR2+ cDC2 at steady state. Data are representative of three independent experiments; n = 3 mice/group. g IRF4fl/flCD11ccre mice were adoptively transferred (i.n.) with lung Mgl2+TNFR2+ and Mgl2-TNFR2+ cDC2 and treated with one dose of OVA (1 µg) i.n. Flow cytometry analysis (left) and quantification (right) of T-regs at day 14; n = 3–4 mice/group. Data are representative of two independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by one-way ANOVA Tukey’s multiple comparison test a, d, g or unpaired student t-test b, c. *P < 0.05, **P < 0.001, and ***P < 0.0001.

Fig 3: IDO vaccine reduces frequency of Tregs and enhances therapeutic ratio in the TME. C57BL/6J mice were treated as in Fig. 1A and Fig. 2A, except 3 days after second vaccination, mice were sacrificed, and tumors were harvested for immune response study. The frequency of B16F10 tumor-infiltrating CD4+ (A), FoxP3+CD4+ (B), CD8+/Tregs (C), Dextra-gp100+ CD8+/Tregs (D), and Penta-IDO+CD8+/Tregs (E) was determined by flow cytometry. The frequency of TC-1 tumor-infiltrating CD4+ (F), FoxP3+CD4+ (G), CD8+/Tregs (H), Dextra-E7+ CD8+/Tregs (I), and Penta-IDO+CD8+/Tregs (J) were also measured by flow cytometry. Data are shown as an average of two independent experiments (n = 8–13 per group). Error bars indicate the SEM. Statistical analysis was performed by unpaired, one-tailed Student t test. NS, nonsignificant; *, P = 0.05; **, P = 0.01; ***, P = 0.001; ****, P = 0.0001.

Fig 4: IDO vaccine modulates phenotypic characteristics of the myeloid population in the TME. Gating strategy in Supplementary Fig. S4. Phenotypic characteristics of myeloid populations were analyzed by flow cytometry in the B16F10 and TC-1 tumor models (see Fig. 1A and Fig. 2A, respectively, except 3 days after second vaccination, mice were sacrificed, and tumors were harvested for immune response evaluation. The frequency of B16F10 tumor-infiltrating IDO+B220+ DCs and IDO+B220- DCs (A), total B220+ DCs (B), B220- DCs (C) were determined. The frequency of TC-1 tumor-infiltrating IDO+B220+ DCs and IDO+B220- DCs (D), B220+ DCs (E), B220- DCs (F) were also assessed. Data are shown from one representative experiment (n = 4–7 per group) of two independent experiments. Error bars indicate the SEM. Statistical analysis was performed by unpaired, one-tailed Student t test. NS, nonsignificant; *, P = 0.05; **, P = 0.01; ***, P = 0.001.

Fig 5: IDO vaccine reduces IDO-expressing suppressive myeloid cell populations in the TME. B16F10-bearing or TC-1–bearing mice were treated as in Fig. 1A and Fig. 2A, respectively, except 3 days after second vaccination, mice were sacrificed, and tumors were harvested for immune response evaluation. The frequency of B16F10 tumor-infiltrating IDO+ MDSCs (A), IDO+ macrophages (B), and IDO+DCs (C) were examined by flow cytometry. Data are shown from one representative experiment (n = 4–6 per group) of two independent experiments. The frequency of TC-1 tumor-infiltrating IDO+ MDSCs (D), IDO+ macrophages (E), IDO+DC (F) were also examined by flow cytometry. Data are shown as an average of two independent experiments (n = 10–15 per group). Error bars indicate the SEM. Statistical analysis was performed by unpaired, one-tailed Student t test. NS, nonsignificant; *, P = 0.05; **, P = 0.01; ***, P = 0.001; ****, P = 0.0001.