Indoleamine 2,3-dioxygenase is a critical resistance mechanism in antitumor T cell immunotherapy targeting CTLA-4

Abstract

The cytotoxic T lymphocyte antigen-4 (CTLA-4)-blocking antibody ipilimumab results in durable responses in metastatic melanoma, though therapeutic benefit has been limited to a fraction of patients. This calls for identification of resistance mechanisms and development of combinatorial strategies. Here, we examine the inhibitory role of indoleamine 2,3-dioxygenase (IDO) on the antitumor efficacy of CTLA-4 blockade. In IDO knockout mice treated with anti-CTLA-4 antibody, we demonstrate a striking delay in B16 melanoma tumor growth and increased overall survival when compared with wild-type mice. This was also observed with antibodies targeting PD-1-PD-L1 and GITR. To highlight the therapeutic relevance of these findings, we show that CTLA-4 blockade strongly synergizes with IDO inhibitors to mediate rejection of both IDO-expressing and nonexpressing poorly immunogenic tumors, emphasizing the importance of the inhibitory role of both tumor- and host-derived IDO. This effect was T cell dependent, leading to enhanced infiltration of tumor-specific effector T cells and a marked increase in the effector-to-regulatory T cell ratios in the tumors. Overall, these data demonstrate the immunosuppressive role of IDO in the context of immunotherapies targeting immune checkpoints and provide a strong incentive to clinically explore combination therapies using IDO inhibitors irrespective of IDO expression by the tumor cells.

Figure 1.

Delayed development of tumors and increased tumor-free survival with anti–CTLA-4 in IDO-deficient hosts. WT and IDO^−/− mice were challenged with B16F10 tumor cells i.d. and treated with anti–CTLA-4. Tumor growth and rejection were followed over time in the different groups. (A) Cumulative survival. Statistical significance was determined by Log-Rank test (*, P < 0.05). (B) Individual tumor growth. The numbers of mice/group rejecting tumors were: WT/untreated (0/10 mice), WT/anti–CTLA-4 (2/10), IDO^−/−/untreated (0/8), and IDO^−/−/anti–CTLA-4 (6/10). (C) Mean tumor size. Two-way ANOVA was used to evaluate statistical significance (p-value toward significance, P = 0.06). Data represent cumulative results from two independent experiments with four to five mice/group.

Figure 2.

IDO deficiency is associated with enhanced infiltration and accumulation of T cells in tumors after anti–CTLA-4 therapy. B16F10 tumors from untreated and anti–CTLA-4–treated WT and IDO^−/− mice were harvested 15 d after tumor challenge and analyzed by flow cytometry for their content of effector T cells and T reg cells. (A) Tumor weights. (B) Absolute numbers of CD45⁺, CD4⁺Foxp3⁻, and CD8⁺ cells per gram of tumor. (C) Percentage of CD4⁺Foxp3⁺ T cells of total CD45⁺ cells, and representative dot plots, for anti–CTLA-4-treated WT and IDO^−/− mice. (D) Ratio of CD4⁺Foxp3⁻ to CD4⁺Foxp3⁺ cells and CD8⁺ to CD4⁺Foxp3⁺ cells. (E) Percentage and representative dot plots of CD8⁺, CD4⁺Foxp3⁻ and CD4⁺Foxp3⁺ T cells expressing Ki67, PD-1 and ICOS, for untreated and treated IDO^−/− mice. Two-way ANOVA (A–B and D) and Student’s t test (C and E) were used to evaluate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, P < 0.1). Data shown are representative of two independent experiments with five independently analyzed mice/group.

Figure 3.

Antitumor effects of anti-PD-1/PD-L1 and anti-GITR are enhanced in IDO^−/− hosts. Survival curves for WT and IDO^−/− mice challenged with B16F10 tumor cells i.d. and treated with anti–PD-1/anti–PD-L1 (A) or anti-GITR (B). Data shown are cumulative results from two independent experiments with five mice/group and analyzed by Log-Rank Test (*, P < 0.05).

Figure 4.

Anti–CTLA-4 and 1MT synergize to mediate tumor rejection. Treatment schedule (A), Kaplan-Meier survival curves (B), mean tumor size (C) and individual tumor growths (D) for C57BL/6 mice challenged with B16F10 melanoma cells i.d. and treated with anti–CTLA-4 and/or 1MT. The numbers of mice/group rejecting tumors was: untreated (0/10 mice), anti–CTLA-4 (2/10), anti–CTLA-4/L-1MT (6/10), and anti–CTLA-4/D-1MT (5/10). Kaplan-Meier survival curves for C57BL/6 mice challenged with B16BL6 and treated with anti–CTLA-4, Gvax, and/or 1MT. 1MT was administered in the drinking water (E) or as time-release subcutaneous pellets (F). Data shown are pooled from two (C and D) or three (B, E, and F) independent experiments with five mice/group. Statistical significance was evaluated by Log-Rank Test(B, E, and F; *, P < 0.05; **, P < 0.01; ***, P < 0.001) and two-way ANOVA (C; *, P < 0.05; **, P < 0.01).

Figure 5.

Anti–CTLA-4/1MT treatment increases the ratio of effector T cells to T reg cells in tumor and elicits a tumor-specific T cell response. B16F10 tumors from untreated, anti–CTLA-4–treated, and anti–CTLA-4/1MT–treated C57BL/6 mice were harvested 15 d after tumor challenge and analyzed by flow cytometry for their content of effector T cells and T reg cells. (A) Tumor weights. (B) Percentage of CD8⁺, CD4⁺Foxp3⁻, and CD4⁺Foxp3⁺ T cells of total CD45⁺ cells. (C) Immune infiltrate analysis expressed as a percentage of total CD45⁺ cells. (D) Percentage of CD11b⁺Gr-1⁺ MDSCs of total CD45⁺ cells and representative dot plots. (E) Ratio of CD4⁺Foxp3⁻ to CD4⁺Foxp3⁺ cells and CD8⁺ to CD4⁺Foxp3⁺ cells. (F) Absolute numbers of CD8⁺ T cells per gram of tumor. (G) Frequency of CD8⁺GrB⁺ T cells of total CD45⁺ cells. TILs were restimulated for 4 h with PMA/Ionomycin (H) or overnight with DCs loaded with B16F10 tumor lysate or DCs with MC38 lysate as a nonmelanoma control tumor (I), and production of IFN-γ was determined by flow cytometry. Data were analyzed by two-way ANOVA (A, B, E, and F; *, P < 0.05; **, P< 0.01; NS, P = 0.06, P = 0.1) and Student’s t test (H; *, P < 0.05). Data represents one of three experiments with five independently analyzed mice/group.

Figure 6.

Anti–CTLA-4/1MT antitumor effect is T cell dependent. (A) Mean tumor growth and tumor-free survival curves for C57BL/6 mice challenged with B16F10 tumors i.d. and treated with anti–CTLA-4/1MT plus depleting antibodies for IFN-γ, CD8, CD4, or NK/NKT, or a corresponding dose of IgG isotype control (IgG). P-values are for two-way ANOVA and Log-Rank test (*, P < 0.05; ***, P < 0.001; ****, P < 0.0001), respectively. (B) Percentage of CD4⁺ and CD8⁺ T cells in peripheral blood after depletion assessed by flow cytometry. The horizontal bars indicate positive staining. Data represent cumulative results from two independent experiments with five mice/group.

Figure 7.

IDO impairs antitumor effects of anti-CTLA4 in the context of B16 tumors engineered to overexpress IDO. (A) Expression of GFP (black line) or IDO/GFP (gray) in B16F10 cells determined by flow cytometry. WT B16F10 cells were used for comparison (dotted line). (B) In vitro growth rate of GFP-B16F10 and IDO/GFP-B16F10 cells. (C) Mean growth of GFP-B16F10 and IDO/GFP-B16F10 tumors in naive and irradiated (450 cGy) mice. (D) Tumor-free survival curves for C57BL/6 mice challenged with GFP-B16F10 or IDO/GFP-B16F10 tumor cells i.d. and treated with anti–CTLA-4 or anti-CLTA-4/1MT. Data represent cumulative results from two independent experiments with five mice/group. Statistical significance was determined with Log-Rank test (*, P < 0.05; **, P < 0.01).

Figure 8.

Anti–CTLA-4 and 1MT synergize to mediate rejection of IDO-expressing breast tumor cells. Mean tumor growth (A) and cumulative survival (B) for BALB/c mice challenged with 4T1 mammary cancer cells i.d. and treated with anti-CLTA4 and/or 1MT. Data shown are pooled from two independent experiments with five mice/group and analyzed by two-way ANOVA (A) and Log-Rank test (B; *, P < 0.05; **, P < 0.01).