Interferon-dependent IL-10 production by Tregs limits tumor Th17 inflammation

Abstract

The capacity of IL-10 and Tregs in the inflammatory tumor microenvironment to impair anticancer Th1 immunity makes them attractive targets for cancer immunotherapy. IL-10 and Tregs also suppress Th17 activity, which is associated with poor prognosis in several cancers. However, previous studies have overlooked their potential contribution to the regulation of pathogenic cancer-associated inflammation. In this study, we investigated the origin and function of IL-10–producing cells in the tumor microenvironment using transplantable tumor models in mice. The majority of tumor-associated IL-10 was produced by an activated Treg population. IL-10 production by Tregs was required to restrain Th17-type inflammation. Accumulation of activated IL-10+ Tregs in the tumor required type I IFN signaling but not inflammatory signaling pathways that depend on TLR adapter protein MyD88 or IL-12 family cytokines. IL-10 production limited Th17 cell numbers in both spleen and tumor. However, type I IFN was required to limit Th17 cells specifically in the tumor microenvironment, reflecting selective control of tumor-associated Tregs by type I IFN. Thus, the interplay of type I IFN, Tregs, and IL-10 is required to negatively regulate Th17 inflammation in the tumor microenvironment. Therapeutic interference of this network could therefore have the undesirable consequence of promoting Th17 inflammation and cancer growth.

Figure 1. Tumor Tregs are a predominant source of Il10.

(A and C) Expression of IL-10eGFP in leukocyte populations (A) and CD4⁺ T cells (C) from MC38 tumor grown in VERT-X mice (black line) or B6 WT (gray) mice. Statistics show percentage of cells in each quadrant for 1 representative experiment of 3 or more. (B) Definition of tumor CD4⁺ Tregs by intracellular staining of FoxP3 (black line) or isotype (gray) staining. Percentage of gated cells is shown. (D) Real-time quantitative PCR for Il10 on sorted cell populations from MC38 tumor, normalized to Hprt. Experiment is representative of 3. (E) Il10 expression in MC38 tumor from T cell–conditional Il10-knockout mice (Il10^{T cellΔ/Δ}). RNA was extracted from unprocessed MC38 tumors from indicated strains of mice and real-time quantitative PCR performed for Il10. Relative expression of Il10 to Hprt is shown in a box-and-whiskers plot (ND, not detected). Data were combined from 2 independent experiments and statistical analysis performed by mixed-effects ANOVA (Tukey-Kramer). Mean ± SEM and n are also shown.

Figure 2. Expression of IL-10eGFP by CD4⁺CD25⁺ Tregs in tumor-associated tissue.

Expression of IL-10eGFP was analyzed in VERT-X mice with B16-F10 melanoma, Lewis lung carcinoma (LLC), or MC38 colon carcinoma in subcutaneous (A and B) or lung-associated (C) tissue by flow cytometry. Numbers indicate percentages of positive cells in the associated gate. FoxP3 (A) and IL-10eGFP (B) staining are indicated for subcutaneous tumor tissue, spleen, and tumor-draining (inguinal) lymph node. Results are representative of 2 experiments. (C) Lungs from mice with B16-F10 tumor nodules were dissected into tumor-associated and non–tumor-associated tissue, followed by processing, staining, and flow cytometry. Data show 1 tumor-bearing mouse of 2 analyzed (individually displaying 27% or 16% IL-10eGFP⁺ cells in the CD4⁺CD25⁺ Treg population of B16-F10 nodule-associated tissue).

Figure 3. Expression of Il17a and frequencies of IL-17–producing cells in MC38 tumor from T cell–conditional Il10-knockout mice.

(A) Tumor mass, leukocyte frequency, and FoxP3⁺ Treg frequencies are similar between WT and Il10^–/– mice. (B) Frequencies of CD4⁺ and CD8⁺ T cells, and frequencies of IL-17, TNF, or IFN-γ–producing cells following PMA and ionomycin stimulation of MC38 tumor cells from T cell–conditional Il10-knockout mice (Il10^{T cellΔ/Δ}), littermates (Il10^fl/fl), Il10^–/– mice, or WT mice. Cell frequencies as percentage of leukocytes and mean ± SEM are shown with statistics from 2-tailed Student’s t test with Welch’s correction. (C) Representative contour plot of tumor CD45⁺CD4⁺ T cells from FACS analysis used to identify IL-17A⁺ Th17 cells as quantitated in B. Percentages of gated cells are given. (D) Real-time quantitative PCR for Il17a on whole MC38 tumor tissue from indicated strains of mice. Box-and-whiskers plot is shown with mean ± SEM, n, and P values for mixed-effects ANOVA on combined data from 2 independent experiments (each showing significance).

Figure 4. Expression of IL-10 by Tregs is associated with a T cell activation signature.

(A) Heat map showing genes with significant differences in expression between IL-10eGFP⁺ and IL-10eGFP^– Tregs from MC38 tumor in VERT-X mice. Genes are ranked according to fold change between IL-10eGFP⁺ and IL-10eGFP^– Tregs and expression level indicated by color. Expression of genes by CD4⁺FoxP3EGFP⁺ Tregs from MC38 tumor and tumor-free spleen in FoxP3EGFP mice is shown for comparison. (B) nCounter analysis (Nanostring) of gene expression on independent flow-sorted samples of Tregs from MC38 tumor in VERT-X and FoxP3-EGFP mice. Each point derives from a pool of tumors in an independent experiment. Graphs show mean ± SEM with P values from 2-tailed Student’s t test with Welch’s correction except for Ccr6 (Mann-Whitney).

Figure 5. Ifnar1 and Stat1 are required for expression of IL-10 and Stat1.

(A) Strategy for identification of CD4⁺ T cell subsets. Plots show intracellular staining of CD4, FoxP3, and either IL-10, IL-17A, or IFN-γ on single-cell suspensions of MC38 tumors following stimulation with PMA and ionomycin. Percentages of gated cells are given. (B) Frequencies of IL-10⁺FoxP3⁺CD4⁺ T cells, FoxP3⁺CD4⁺ T cells, and CD4⁺ T cells in MC38 tumor are given as percentage of CD4⁺ T cells or percentage of leukocytes from Ifnar1^–/–, Stat1^–/–, and WT mice. Mean ± SEM and P values compared with WT are shown for 5 independent experiments combined. (C) Real-time quantitative PCR for Il10 on whole MC38 tumor from Ifnar1^–/–, Stat1^–/–, and WT mice, normalized to Ptprc (CD45). Box-and-whiskers plots with mean ± SEM, n, and P values compared with WT using combined data from 4 (Ifnar1^–/–) or 1 (Stat1^–/–) independent experiments.

Figure 6. Type I IFN signaling is required for tumor Treg activation.

(A) nCounter gene expression analysis of flow-sorted tumor Tregs from Ifnar1^–/– or WT mice. Data points show tumor Tregs from pools of mice flow sorted in independent experiments. Data show mean ± SEM and P value. (B) Western analysis of STAT1 protein on whole spleen lysates of mice from indicated strains. Numbers above blot show STAT1 band intensities after normalization to β-actin. (C) Frequencies of Treg infiltrates in MC38 tumors from bone marrow chimeric mice and nonirradiated controls. Donor-to-host relationship (indicated with arrows) and cell frequencies are given with mean ± SEM. P values are given for indicated comparisons (by bar) of total Treg frequencies.

Figure 7. Ifnar1 and Il10 suppress Th17-associated gene expression by tumor CD4⁺ T cells.

CD4⁺CD25^– T cells were sorted from MC38 tumor of indicated strain, and gene expression was analyzed by nCounter analysis. Each data point derives from an independent experiment using pools of mice. Mean ± SEM and P-values are shown for Student’s t test with Welch’s correction compared with WT cells.

Figure 8. Th17-associated gene expression is limited by Il10, Ifnar1, and Stat1 in the TME.

(A) Real-time quantitative PCR for Ptprc (CD45) normalized to Hprt from whole-tumor tissue from indicated strains. Data from 4 to 6 independent experiments (4, Il10^–/–; 4, Ifnar1^–/–; 1, Stat1^–/–; 6, WT) were combined and analyzed by mixed-effects ANOVA on log-transformed data. (B) Day 13 tumor volume. Statistics show mean ± SEM and Student’s t test with Welch’s correction. (C) Real-time quantitative PCR on whole-tumor cDNA for indicated genes normalized to Ptprc. Data from 4 to 6 independent experiments (4, Il10^–/–; 4, Ifnar1^–/–; 1, Stat1^–/–; 6, WT) were combined and analyzed by mixed-effects ANOVA on log-transformed data. Geometric mean ± 95% CI and P values for mixed-effects ANOVA (using Dunnett’s method) are given for comparison with WT.

Figure 9. Frequencies of CD4⁺ T cells, Th1, Th17, and Tregs in spleens from tumor-free Il10^–/–, Ifnar1^–/–, and WT mice.

Single-cell suspensions from spleens of indicated strains of tumor-free mice were stimulated with PMA and ionomycin, and frequencies of IFN-γ⁺FoxP3^–CD4⁺ Th1 cells, IL17A⁺FoxP3^–CD4⁺ Th17 cells, and IL-10⁺FoxP3⁺ Tregs were enumerated. Data are combined from 2 independent experiments. Frequencies are given as percentage of CD45⁺ leukocytes. Statistics show mean and P values for 2-tailed unpaired Student’s t test with Welch’s correction.