MON-134 - Reprogramming Regulatory T Cells as Precision Immune Therapy for Endometriosis

Background: Endometriosis is characterized by ectopic growth of endometrial-like tissue, leading to chronic pelvic pain and infertility. Immune dysregulation promotes disease progression, with elevated regulatory T cells (Tregs) and reduced natural killer (NK) cells creating a permissive immune microenvironment. Thus, immune cell–based therapies may provide non-hormonal treatment options, but none are FDA-approved. Steroid Receptor Coactivator 3 (SRC 3) is essential for Treg-mediated immunosuppression. Deletion of SRC 3 reprograms Tregs into potent anti-tumor cells without inducing cytokine release syndrome. Given parallels between cancer and endometriosis pathophysiology, SRC 3–deficient Tregs may also suppress endometriosis.

Objective: To determine whether SRC 3 knockout (KO) Tregs inhibit endometriosis progression and characterize the immune microenvironment they generate.

Methods: Treg cell-specific SRC 3 KO mice by crossing floxed SRC-3 mice (SRC-3f/f) with Foxp3Cre/YFP mice, in which Cre recombinase is expressed under control of the Foxp3 promoter (SRC-3 KO:Treg, SRC 3f/f;Foxp3Cre/YFP) (n= 7) and SRC 3f/f controls (n= 7) underwent surgically induced endometriosis. On day 21, ectopic lesions were harvested for volume quantification and single-cell RNA sequencing (scRNA seq).

Results: SRC 3 KO:Treg mice developed significantly smaller ectopic lesions than controls, demonstrating that SRC 3 KO Tregs suppress endometriosis in vivo. scRNA seq revealed neutrophils as the predominant immune population in both genotypes, along with T, B, and NK cells. Neutrophil subclusters enriched for type I interferon (IFN), interferon-stimulated gene (ISG) programs, and oxidative pathways were markedly increased in endometriotic lesions in SRC 3 KO:Treg mice. Also, Treg subclustering showed strong upregulation of cytokine/chemokine-related genes, Sphingosine 1 Phosphate Receptor 1 (S1pr1) and CD40 Ligand (Cd40lg), in SRC 3 KO Tregs. These findings identify an SRC 3- S1pr1hi Cd40lghi Treg population that establishes an anti-endometriotic immune microenvironment.

Conclusion: S1pr1 promotes SRC-3 KO Treg trafficking into S1P-rich endometriotic lesions, while CD40lg activates dendritic cells to induce IFN and generate oxidative, pro-inflammatory neutrophils to suppress endometriosis progression. We hypothesize that SRC 3- S1pr1hi Cd40lghi Tregs migrate into lesions and reprogram local immune populations through S1pr1/S1P and CD40lg/CD40 signaling to suppress endometriosis progression. Ongoing studies aim to validate the mechanistic role of this axis in SRC 3 KO Treg–based therapy.

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