T cell-intrinsic STING signaling promotes regulatory T cell induction and immunosuppression by upregulating FOXP3 transcription in cervical cancer

Abstract

Background: Stimulator of interferon genes (STING) is an innate immune sensor of cytoplasmic double-stranded DNA originating from microorganisms and host cells. The activation of cytosolic DNA-STING pathway in tumor microenvironments is usually linked to more robust adaptive immune responses to tumors, however the intracellular function of STING in regulatory T cells is largely unknown. In the present study, we aimed to explore the contribution of intracellular STING activation to regulatory T cell induction (iTreg) in cervical cancer (CC) microenvironments.

Methods: Blood samples and tumor specimens were obtained from patients with CC. The intratumoral STING, CCL22, CD8 and forkhead box P3 (FOXP3) expression levels were measured by immunohistochemistry. T cell-specific STING conditional knockout mice (CD4-Cre/STING^flox/flox, TKO) were generated, and syngeneic TC-1 tumor model were investigated. The differentiation and molecular regulatory pathway of human and murine iTreg under different treatments were investigated by ex vivo assays, immunoblotting and quantitative PCR. Tumor-associated exosomes (T-EXO) were isolated from CC cell lines and exosomal contents were identified by ELISA and Western blot analysis. The impact of T-EXO on T cell differentiation was tested in in vitro cell culture.

Results: Increased STING, CCL22 level, FOXP3⁺ cells but decreased CD8⁺ cells in tumor tissues predicted poor survival. Tumor-bearing CD4-Cre-STING^flox/flox (TKO) mice displayed slower tumor growth tendencies as well as fewer FOXP3⁺ cells but higher CD8⁺ cell proportion in tumor tissues than wild-type (WT) mice. Activating of STING signaling cooperated with T cell receptor, interleukin-2 receptor and transforming growth factor-beta (TGF-β) signals to promote CD4⁺CD25^highFOXP3⁺ iTreg differentiation from both human and murine CD4⁺-naïve T cells from WT and IFNAR^-/- mice but not TKO or IRF3^-/- mice in vitro. Ectopic STING, TBK1 or IRF3 expression promoted iTreg differentiation from human CD4⁺-naïve T cells. T cell-intrinsic STING activation induced FOXP3 transcription through TBK1-IRF3-mediated SMAD3 and STAT5 phosphorylation independent of interferon-β. In CC, tumor-derived exosomes activated STING signaling in tumor-infiltrated T cells by exosomal TGF-β, cyclic GMP-AMP synthase and 2'-3'-cGAMP, leading to iTreg expansion.

Conclusions: These findings highlight a novel mechanism for iTreg expansion mediated by tumor-derived exosome-activated T cell-intrinsic STING signal, and provide a rationale for developing immunotherapeutic strategies targeting STING signal in CC.

Keywords: CD4-positive T-lymphocytes; T-lymphocytes; biomarkers, tumor; immunotherapy.