Figure 2 from IFNγ Production by Functionally Reprogrammed Tregs Promotes Antitumor Efficacy of OX40/CD137 Bispecific Agonist Therapy

https://doi.org/10.1158/2767-9764.26538478

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posted on 2025-03-28, 22:23 authored by Charlotte J. Imianowski, Paula Kuo, Sarah K. Whiteside, Teresa von Linde, Alexander J. Wesolowski, Alberto G. Conti, Alexander C. Evans, Tarrion Baird, Benjamin I. Morris, Nicole E. Fletcher, Jie Yang, Edmund Poon, Matthew A. Lakins, Masahiro Yamamoto, Neil Brewis, Michelle Morrow, Rahul Roychoudhuri

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 Foxp3EGFP-Cre-ERT2RosaflSTOPfl-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.

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DOI - Is supplement to IFNγ Production by Functionally Reprogrammed Tregs Promotes Antitumor Efficacy of OX40/CD137 Bispecific Agonist Therapy

ARTICLE 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. 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.

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Keywords

Immunology Tumor immunology animal models Regulatory T cells Immune responses to cancer Immunotherapy Agonist antibodies

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