Fig 2: The absence of IDO1 expression modifies the frequency of immunological cells in distinct mice tissues. Images of tumor masses and spleen from wild-type (WT) (A) and IDO-/-(B) mice subcutaneously injected with 1 × 105 TC-1 cells and euthanized 21 days later (n = 10 per group). Comparative analyses of (C) spleen and (D) tumor masses of WT and IDO-/- mice. Comparative analyses of (E) Treg, (F) DCs and (G) macrophages in spleens and tumor tissues of WT and IDO-/- mice. Comparative analyses of resident monocytes, inflammatory monocytes and MDSC in (H) spleens, (I) blood and (J) tumor tissues of WT and IDO-/- mice. Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001 by ANOVA or unpaired Student's t-test. (ns) Non-significant.

Fig 3: IDO expression impairs the antitumor effects of gDE7-based immunotherapy. (A) Wild-type (WT) and IDO-/-mice were subcutaneously injected with 1 × 105 TC-1 cells and immunized with two doses (7-day interval) of gDE7 (30 µg per animal) via subcutaneous (s.c.) route. Tumor growth was monitored until 60 day, when the animals were euthanized. Antitumor effects were measured by (B) tumor volume, (C) the percentage of tumor-free mice and (D) the percentage of mice survival. Vaccinated IDO-/- mice were significantly different than all other groups, highlighting that the gDE7-dependent antitumor effects are enhanced by inactivation of host IDO gene expression. Data from two identical experiments (n = 5) were pooled and analyzed by ANOVA or by Kaplan-Meyer test (exclusively for survival assay). *p < 0.05, **p < 0.01, and ***p < 0.001. (ns) Non-significant. When not signaled, * represents the statistical significance of one experimental group in relation to all others.

Fig 4: Immune microenvironment cells increase IDO1 expression during oncogenesis. Wild-type (WT) C57BL/6 mice (n = 7) were subcutaneously injected with 1 × 105 TC-1 cells and tumors were evaluated at 10 and 14 day after the tumor cell transplantation. Naïve mice were used as a reference of physiological parameters. The frequencies of DCs, macrophages, inflammatory monocytes and MDSC were evaluated in spleens, blood and tumor tissues. IDO1 expression was measured by intracellular staining and flow cytometry analyses. (A–D) Frequency of IDO1-expressing immune cells at 0 day (D0–naïve mice), 10 day (D10) and 14 day (D14) after TC-1 transplantation. Parameter used to characterize the different cell populations: (A) DCs (CD45+, CD11chigh, MCH-IIhigh), (B) macrophages (CD45+, MCH-II+, CD11b+, F4/80+), (C) inflammatory monocytes (CD45+, CD11bint, Ly6Chigh, Ly6G-), (D) MDSC (CD45+, CD11bhigh, Ly6Cint, Ly6G+). Data representative of two independently performed experiments. Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001 by ANOVA.