Interferon-γ Drives Treg Fragility to Promote Anti-tumor Immunity

Abstract

Regulatory T cells (T_regs) are a barrier to anti-tumor immunity. Neuropilin-1 (Nrp1) is required to maintain intratumoral T_reg stability and function but is dispensable for peripheral immune tolerance. T_reg-restricted Nrp1 deletion results in profound tumor resistance due to T_reg functional fragility. Thus, identifying the basis for Nrp1 dependency and the key drivers of T_reg fragility could help to improve immunotherapy for human cancer. We show that a high percentage of intratumoral NRP1⁺ T_regs correlates with poor prognosis in melanoma and head and neck squamous cell carcinoma. Using a mouse model of melanoma where Nrp1-deficient (Nrp1^-/-) and wild-type (Nrp1^+/+) T_regs can be assessed in a competitive environment, we find that a high proportion of intratumoral Nrp1^-/- T_regs produce interferon-γ (IFNγ), which drives the fragility of surrounding wild-type T_regs, boosts anti-tumor immunity, and facilitates tumor clearance. We also show that IFNγ-induced T_reg fragility is required for response to anti-PD1, suggesting that cancer therapies promoting T_reg fragility may be efficacious.

Keywords: PD-1; cancer immunotherapy; interferon-γ; interferon-γ receptor; neuropilin-1; regulatory T cells; tumor immunology; tumor microenvironment.

Figure 1. Decreased Nrp1 expression leads to tumor regression and enhanced survival

(A-C) Lymphocytes were harvested from PBL of healthy donors (n=8) or from PBL and TIL of Head and Neck Squamous Cell Carcinoma (HNSCC) and metastatic melanoma (3-5 experiments, n=16-23) and frozen or stained fresh. Frozen TIL and PBL were thawed and stained directly without stimulation. (D-G) Foxp3^{Cre-YFP/Cre-YFP}, Nrp1^L/LFoxp3^{Cre-YFP/Cre-YFP}, Nrp1^L/LFoxp3^{Cre-YFP/DTR-GFP}, Foxp3^{DTR-GFP/DTR-GFP}, and Foxp3^DTR-GFP/+ mice were injected with B16.F10 melanoma tumor cells ID on day 0. Tumor growth was measured with digital calipers every three days. Mice were removed from study when tumor growth reached a diameter of 2cm in any direction or when necrosis was observed, and survival plots were generated (4 experiments, n=9-18). (F-G) Foxp3^{DTR-GFP/DTR-GFP}, and Foxp3^DTR-GFP/+ mice were treated with 100μg Diptheria Toxin IP every three days starting on day 7. (H) T_regs were isolated on day 12 post B16 injection from ndLN and TIL of Foxp3^{Cre-YFP/Cre-YFP}, Nrp1^L/LFoxp3^{Cre-YFP/Cre-YFP}, and Nrp1^L/LFoxp3^{Cre-YFP/DTR-GFP} mice and cultured with effector T cells and APCs for 72 hours in a classical microsuppression assay. T_regs were pooled from 3 mice with 5-6 mice per group per experiment. Proliferation was measured and percent suppression was calculated as described in methods. Data represent 3-5 (A-C), 4 (D-G), or 3 (H) independent experiments. Error bars represent the mean ± SEM. Student unpaired t test (Fig. 1B, H), 2 way ANOVA (Fig. 1E, G), and Kaplan-Meier tests (Fig. 1E, G) were used (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 2. Nrp1 alters the T_reg transcriptome

(A-C) T_regs were purified based on CD4⁺, and GFP or YFP expression from Foxp3^{Cre-YFP/Cre-YFP}, Nrp1^L/LFoxp3^{Cre-YFP/Cre-YFP}, and Nrp1^L/LFoxp3^{Cre-YFP/DTR-GFP} mice on D12, cDNA and libraries were generated using the Clontech SmartER Ultra-Low and Illumina Nextera XT Library Prep kits. Samples were normalized to 2nM and sequenced on a NextSeq500. (A) Differentially expressed genes are determined by the genes that have q-value of 0.2 between any two of the four T_reg groups in the TIL. PCA was computed using the “prcomp()” R functions using the normalized voom data restricted to the same differentially expressed genes as shown in figure. (B-C) Significant genes were cross-referenced with those that were annotated to “plasma membrane” or “extracellular part” in the Cellular Component Gene Ontology. The Gene Ontology annotations were obtained from mSigDB. A number of genes associated with the Ifng/Il12/Il18 pathways were upregulated in the Nrp1^−/− samples. Data represent 5 independent experiments with 3-5 mice pooled per experiment.

Figure 3. Nrp1^−/− T_regs display increased IFNγ in the tumor microenvironment

(A-B) Foxp3^{Cre-YFP/Cre-YFP}, Nrp1^L/LFoxp3^{Cre-YFP/Cre-YFP}, and Nrp1^L/LFoxp3^{Cre-YFP/DTR-GFP} mice were injected with B16.F10 melanoma tumor cells ID on day 0 and sacrificed on day 12. Lymphocytes were isolated from ndLN and TIL of mice noted, stimulated and stained for IFNγ and IFNγR. (n=8-18). (C-E) C57BL/6 mice were injected with B16.F10 melanoma tumor cells ID on day 0. Mice were treated with either Sema4aIg or IgG1 every 3 days until sacrifice starting on day 5 (400ug, 200ug, 200ug, 200ug). (C) Tumors were measured on day 12 for prior to sacrifice (n=10-25). (D) Lymphocytes were isolated from ndLN and TIL, stimulated and stained for IFNγ (n=5-13). (E) Lymphocytes were isolated from ndLN and TIL, and stained for IFNγR (n=5). Data represent 3-4 independent experiments. Student unpaired t test was used. (*p<0.05, **p < 0.01, ***p <0.001, ****p < 0.0001).

Figure 4. Hypoxia sensitizes intratumoral T_regs to IFNγ-mediated fragility

(A-B) Foxp3^{Cre-YFP/Cre-YFP}, Nrp1^L/LFoxp3^{Cre-YFP/Cre-YFP}, and Nrp1^L/LFoxp3^{Cre-YFP/DTR-GFP} mice were injected with B16.F10 melanoma tumor cells ID on day 0 and sacrificed on day 12. Lymphocytes were isolated from TIL of mice noted, stimulated and stained for IFNγ (n=5). (C-D) Mice were treated with anti-CD8 or isotype (200ug) every 3 days starting on day 5. Tumor size was measured on day of sacrifice (D12), lymphocytes were isolated from TIL of mice noted, stimulated, and stained for IFNγ (n=7-10). (E) Lymphocytes were isolated from mice noted and used in a microsuppression assay. (F) T_regs were purified, processed, and analyzed as in Fig. 2 (n=5). Heatmap includes genes previously shown to be positive or negative regulators in the Hif1α pathway. Pathway analysis includes all genes in pathway. (G) Foxp3^{Cre-YFP/Cre-YFP} and Nrp1^L/LFoxp3^{Cre-YFP/Cre-YFP} mice were injected with B16.F10 melanoma tumor cells ID on day 0 and sacrificed on day 12. Lymphocytes were isolated from ndLN and TIL and stained for Hif1α (n=10). (H) T_regs were isolated from LN of Foxp3^{Cre-YFP/Cre-YFP} mice, stimulated for 3 days in hypoxia or normoxia, and stained (n=4-6). Data represent 2-5 independent experiments. Student unpaired t test was used. (*p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 5. IFNγ reduces T_reg suppression

(A, B) T_regs were isolated from ndLN and TIL of mice noted, stimulated with PMA and Ionomycin, cultured with IL-2 alone or with IL-2 and Nrp1^−/− T_regs for 72 hours and used in a microsuppression assay in absence of cytokine (n=6-7). (C, D) T_regs were isolated and stimulated as in (A), cultured in the bottom of a transwell plate with IL-2 alone or with IL-2 and Nrp1^−/− T_regs in the top well for 72 hours and used in a microsuppression assay in the absence of cytokine (n=6-7). (E) T_regs were isolated from ndLN and TIL of Foxp3^{Cre-YFP/Cre-YFP} mice, co-cultured with Nrp1^−/− T_regs and IL-2 in the presence or absence of anti-IFNγ, re-sorted and used in a microsuppression assay in the absence of cytokine (n=6). (F) T_regs were isolated from ndLN and TIL of Foxp3^{Cre-YFP/Cre-YFP} mice, treated with IL-2 and IFNγ for 72 hours, re-sorted and used in a microsuppression assay in the absence of cytokine (n=6). (G) T_regs were isolated from HNSCC PBL and TIL, cultured with IL-2 +/- IFNγ for 3 days, then used in a microsuppression assay in the absence of cytokine (n=2-14). Data represent 3-5 experiments. Error bars represent the mean ± SEM. Student unpaired t test (A-D) and 2 Way Anova (E-G) were used. (*p<0.05, **p <0.01, ****p <0.0001).

Figure 6. IFNγ uptake by T_regs is required for T_reg fragility and tumor clearance

(A-G) Foxp3^−/− mice were injected with 10⁶ T_regs on day 2 post-birth, and monitored for 28 days for the onset of any autoimmune symptoms [4 of 34 mice were removed from study], no more than 1 per experimental group. B16.F10 was injected ID on day 28 and tumor size was measured every 3 days. (H) Statistics of Foxp3^−/− mice tumor growth. Data represent 5-7 independent experiments with 5-7 mice per experimental group. Error bars represent the mean ± SEM. 2 way ANOVA was used. (ns, not significant, ***p < 0.001, ****p < 0.0001).

Figure 7. IFNγ-mediated T_reg fragility is required for antiPD1 response

(A-B) Foxp3^Cre-YFP and Ifngr1^L/LFoxp3^Cre-YFP mice were injected with MC38 SC on day 0 and treated with either anti-PD1 or isotype on days 6, 9, and 12 (200ug, 200ug, 200ug). (A) Tumor growth was measured with digital calipers every three days. Mice were removed from study when tumor growth reached a diameter of 2cm in any direction or when necrosis was observed, and survival plots were generated. (B) Lymphocytes were isolated from TIL on day 12 from Foxp3^Cre-YFP and Ifngr1^L/LFoxp3^Cre-YFP mice and were stimulated and stained for IFNγ. Data represent 2 independent experiments with 4-11 mice per experimental group. 2 way ANOVA (Fig. 7A), Kaplan-Meier test (Fig. 7A), and Student unpaired t test (Fig. 7B) were used (**p < 0.01, ***p<0.001, ****p < 0.0001).