IFNγ Production by Functionally Reprogrammed Tregs Promotes Antitumor Efficacy of OX40/CD137 Bispecific Agonist Therapy

Abstract

Regulatory T cells (Treg) are highly enriched within many tumors and suppress immune responses to cancer. There is intense interest in reprogramming Tregs to contribute to antitumor immunity. OX40 and CD137 are expressed highly on Tregs, activated and memory T cells, and NK cells. In this study, using a novel bispecific antibody targeting mouse OX40 and CD137 (FS120m), we show that OX40/CD137 bispecific agonism induces potent antitumor immunity partially dependent upon IFNγ production by functionally reprogrammed Tregs. Treatment of tumor-bearing animals with OX40/CD137 bispecific agonists reprograms Tregs into both fragile Foxp3+ IFNγ+ Tregs with decreased suppressive function and lineage-instable Foxp3- IFNγ+ ex-Tregs. Treg fragility is partially driven by IFNγ signaling, whereas Treg instability is associated with reduced IL2 responsiveness upon treatment with OX40/CD137 bispecific agonists. Importantly, conditional deletion of Ifng in Foxp3+ Tregs and their progeny partially reverses the antitumor efficacy of OX40/CD137 bispecific agonist therapy, revealing that reprogramming of Tregs into IFNγ-producing cells contributes to the anti-tumor efficacy of OX40/CD137 bispecific agonists. These findings provide insights into mechanisms by which bispecific agonist therapies targeting costimulatory receptors highly expressed by Tregs potentiate antitumor immunity in mouse models.

Significance: The bispecific antibody FS120, an immunotherapy currently being tested in the clinic, partially functions by inducing anti-tumor activity of Tregs, which results in tumor rejection.

Figure 1

OX40 and CD137 are highly coexpressed on tumor-associated Tregs. A, Representative plots showing expression of OX40 and CD137 on resting and activated CD4⁺ Tregs, CD4⁺ Tconv cells, and CD8⁺ T cells from the spleens and tumors of mice inoculated with MC38 cells. B and C, Quantification of resting CD44⁻ CD62L⁺ (left) and activated CD44⁺ CD62L⁻ (right) cells within the indicated T-cell populations in the spleen (B) and tumor (C). Data in B and C were analyzed by one-way ANOVA with Tukey correction for multiple comparisons. Bars and error are mean and SEM. ****, P ≤ 0.0001.

Figure 2

OX40/CD137 bispecific agonist (FS120m) drives functional fragility and lineage instability of Tregs. A, Schema (top) showing tamoxifen and FS120m treatment schedule. Tumor measurements (bottom) at indicated timepoints after MC38 tumor implantation. (Solid line, mean values; dotted lines, individual mouse tumor curves.) B, Representative plots showing percentages of CD4⁺ GFP⁺ RFP⁺ Tregs and CD4⁺ RFP⁺ GFP⁻ exTregs of total CD4⁺ T cells in the spleens and MC38 tumors of Foxp3^{EGFP-Cre-ERT2}Rosa^flSTOPfl-RFP reporter mice after treatment. C, Quantification of the percentage of RFP⁺ single-positive exTregs (GFP⁻) out of total RFP⁺ cells in spleens and tumors. D, Representative plots showing the production of IFNγ and TNF by indicated cell subsets from the spleens and tumors of reporter mice. E, Quantification of IFNγ and TNF production by indicated cell types from the spleens and tumors of MC38 tumor–bearing mice at day 21 after tumor implantation. F, Schema showing setup of Treg suppression assay (left), representative flow plots (center), and replicate measurements (right) showing percentage of dividing responders in indicated conditions. Data were analyzed by two-way ANOVA with Šídák correction for multiple comparisons (A), unpaired Student t test (C), with Bonferroni–Dunn correction for multiple comparisons (E), and ordinary one-way ANOVA with Tukey correction for multiple comparisons (F). Bars and error are mean and SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Ctrl, control.

Figure 3

OX40/CD137 dual agonism results in decreased CD25 expression and IL2 responsiveness of Tregs. A, Representative histograms and replicate measurements showing CD25 expression on CD4⁺ Tregs from the spleen (left) and tumor-draining lymph node (right). B, Schema showing setup of ex vivo IL2 assay (left), replicate measurements of Foxp3-GFP expression (center), and Treg count (right) after 4 days in culture with the indicated concentrations of IL2. C, Schema representing treatment schedule of tamoxifen, FS120m, and rIL2/anti-IL2 mAb complex. D, Representative plots (left) and replicate measurements (right) of the percentage of RFP⁺ SP exTregs (GFP⁻) out of total RFP⁺ cells in the tumors and spleens of reporter mice treated according to C. Data were analyzed by unpaired Student t test (A), unpaired Student t test with Bonferroni–Dunn correction for multiple comparisons (B), and one-way ANOVA with Tukey correction for multiple comparisons (D). Bars and error are mean and SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.0001; ****, P ≤ 0.0001. Ctrl, control; dLN, tumor-draining lymph node.

Figure 4

The antitumor efficacy of OX40/CD137 dual agonism is dependent upon IFNγ signaling. A, Schema representing treatment schedule of FS120m and anti-IFNγ. B, Tumor measurements at indicated timepoints after MC38 tumor implantation of the mice described in A. C, Representative plots from the tumor (left) and spleen (right) showing IFNγ⁺ cells in CD8⁺ T cells and indicated CD4⁺ T-cell populations from mice receiving the specified treatment. D, Replicate values of IFNγ⁺ cells in CD8⁺ T cells and the indicated CD4⁺ T-cell populations from the tumor, spleen, and tumor-draining lymph node of mice receiving the specified treatment. Data were analyzed by two-way ANOVA with Tukey correction for multiple comparisons (B) and ordinary one-way ANOVA with Tukey correction for multiple comparisons (D). Bars and error are mean and SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Ctrl, control; dLN, tumor-draining lymph node; MFI, mean fluorescence intensity.

Figure 5

Treg instability is induced by OX40/CD137 bispecific agonist (FS120m) and not anti-PD1 treatment. A, Representative plots showing the frequency of IFNγ⁺ cells of CD4⁺ Foxp3⁻ Tconv cells and Foxp3⁺ Tregs in the blood on day 18 after MC38 tumor implantation. B, Quantification and statistical analysis of data shown in A. Data were analyzed by ordinary one-way ANOVA (B) with Tukey correction for multiple comparisons. Bars and error are mean and SEM. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Figure 6

The antitumor efficacy of OX40/CD137 dual agonism is partially dependent upon IFNγ production by Tregs and/or their lineage-instable progeny. A, PCR genotyping of CD8⁺, CD4⁺ Foxp3-GFP⁻ Tconv, and CD4⁺ Foxp3-GFP⁺ Tregs sorted from the spleens of mice with the indicated genotypes after treatment with tamoxifen. Tregs have cell-specific excision of IFNγ only in mice possessing both the Ifng^fl/fl and Foxp3^{EGFP-Cre-ERT2} alleles. B, Schema representing treatment schedule of tamoxifen and FS120m. C, Tumor measurements at indicated timepoints after MC38 implantation of Ifng^fl/flFoxp3^{EGFP-Cre-ERT2} mice and controls given the indicated treatment. D, Replicate measurements of IFNγ⁺ cells in the indicated CD4⁺ T-cell populations from the tumor. E, Percentages (top) and counts per gram (bottom) of the indicated cell types in the tumors of mice of the indicated genotypes given the specified treatment. Data were analyzed by two-way ANOVA with Tukey correction for multiple comparisons (C) and ordinary one-way ANOVA with Tukey correction for multiple comparisons (D and E). Bars and error are mean and SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Ctrl, control.