Fig 2: Protocol for the induction of IL-10/IFN-γ–co-producing CD4+Foxp3− Tr1 cells.(A) D665 (250 μg per mouse) and G3c (250 μg per mouse) were intraperitoneally injected on days −3 and 0 sequentially. (B) Representative flow cytometry (FCM) analysis and frequency of Treg cells in the splenocytes of D665- and immunoglobulin G (IgG)–treated mice on day 0 (n = 6 for each group). ****P < 0.0001. (C and D) The median fluorescence intensity (MFI) of the GITR on CD4+Foxp3+ Treg cells and CD4+Foxp3− Teff cells in the splenocytes of D665- and IgG-treated mice on day 0 (n = 5 for each group). **P < 0.01 and ****P < 0.0001. (E) Representative FCM analysis of CD4+Foxp3+ Treg cells and CD4+Foxp3−IL-10+ Tr1 cells in the splenocytes of each treatment group day 3 (D3) and D7 (n = 4 for each group). (F) Frequency of CD4+Foxp3−IL-10+ Tr1 cells in the splenocytes of each treatment group on day 3 and day 7 (n = 4 for each group). *P < 0.05, **P < 0.01, and ****P < 0.0001. (G) Frequency of CD4+Foxp3+ Treg cells in the splenocytes of each treatment group on day 3 and day 7 (n = 4 for each group). *P < 0.05, ***P < 0.001, and ****P < 0.0001. Values are shown as the mean ± SEM.

Fig 3: Hypoxia and HIF-1α, but Not HIF-2α, Support CD8+ T Cell Acquisition of Effector Phenotype(A) CD8+ T cells were isolated from spleens of HIF-1αfl/fldlckCRE (maroon) or HIF-2αfl/fldlckCRE (green) and littermate control (black) mice, and activated with αCD3/CD28 for 48 hr, then expanded for 5 days in IL-2 and subjected to 21% or 1% O2 for 24 hr. Expression of CD44 and CD62L by flow cytometry is shown.(B) Intracellular expression of IFNγ, TNFα, and granzyme B in HIF-1αfl/fldlckCRE (maroon) and HIF-1αfl/fl control littermates (black) by flow cytometry after restimulation (n = 3, error bars represent SD).(C) Intracellular expression of IFNγ, TNFα, and granzyme B in HIF-2αfl/fldlckCRE (green) and HIF-2αfl/fl control littermates (gray) by flow cytometry after restimulation (n = 3, error bars represent SD).(D) Surface expression of costimulatory molecules/checkpoint receptors CD137, OX40, GITR, PD-1, TIM-3, and LAG3 on CD8+ T cells isolated from HIF-1αfl/fldlckCRE (maroon) HIF-1αfl/fl control littermates (black), activated and expanded as in (A) (n = 3, error bars represent SD).(E) Same as in (D) on CD8+ T cells isolated from HIF-2αfl/fldlckCRE (green) or HIF-2αfl/fl control littermate mice (gray) (n = 3, error bars represent SD).(F) Relative mRNA levels of Vegfa (left, n = 3) and amount of VEGF-A in media (right, n = 4, error bars represent SD) on CD8+ T cells for the indicated time points after αCD3/CD28 activation.(G) Amount of VEGF-A in media from CTLs expanded as in (A) and cultured under 21% O2 or 1% O2 for 24 hr (n = 4, error bars represent SD).(H) Relative expression of Vegfa in total LLC tumor lysate and immune populations isolated from subcutaneous Lewis lung carcinoma (LLC) tumors 11 days after implantation (n = 10, error bars represent SD).Grouped data were assessed by two-way ANOVA for multiple comparisons with Bonferroni correction. ∗∗∗∗p < 0.00005, ∗∗∗p < 0.0005, ∗∗p < 0.005, ∗p < 0.05. MFI, mean fluorescence intensity. See also Figure S2.

Fig 4: IL23R is expressed on tumor-infiltrating (TIL) Treg. (A) IL23R staining of spleen and tumor-infiltrating Treg (CD4+ CD25+ FoxP3+ lymphocytes) following tumor challenge with 2 × 105 MC38 colon carcinoma cells, as described in Materials and Methods. (B) CD44 and (C) Helios staining on IL23R+ vs. IL23R− TIL Treg identified in A. (D) FoxP3 expression and (E) IFNγ expression of incubated Treg after 5 d of culture in the presence of the indicated antibodies (GITR, glucocorticoid-induced TNFR-related; GARP, glycoprotein A repititions predominant; IL7r, Interleukin 7 receptor; TIGIT, T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif domains). Statistical tests in A, B, and C are paired Wilcoxon tests. Data are pooled from three or four independent experiments (n = 15/group [A–C] or 6/group [D and E]). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, non significant.

Fig 5: IL23R antibody treatment combined with GITR activation to promote tumor immunity. (A) Tumor growth curves in C57BL/6 mice treated intraperitoneally with anti-IL23R (100 μg/treatment), anti-GITR (100 μg/treatment), isotype controls (100 μg/treatment), or a combination of both IL23R and GITR antibodies (50 μg each/treatment), as in Fig. 4B. (B) Tumor inhibition of each antibody monotherapy and combination therapy, shown as a percentage of isotype-treated mice, where 0% inhibition indicates tumors that grew equal to isotype treatment and 100% inhibition indicates no tumor growth. (C) Tumor growth curves as in (A) using a higher dose (5 × 105) of MC38. (D) Survival analysis of high-dose–challenged mice as in (C). (E–H) Flow cytometry analysis of tumor-infiltrating Treg high-dose challenged mice including those with (E) Treg IFNγ expression, (F) Treg IL12Rβ2 expression, (G) CD8 T-cell IFNγ expression, and (H) CD8 T-cell Fas expression. Data are pooled from three independent experiments (n = 14 to 15/group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, non significant. Iso, isotype.