Investigating the Epigenetic Landscape of the Ifng Locus During Proinflammatory Regulatory T Cell Reprogramming

Abstract

Regulatory T (Treg) cells are distinguished by the expression of the master transcription factor forkhead box P3 (Foxp3), which plays a pivotal role in facilitating their immunosuppressive function, thereby maintaining peripheral tolerance. However, mounting evidence underscores the dynamic adaptability of Treg cells known as plastic differentiation, usually characterized by the co-expression of lineage defining transcription factors of distinct pro-inflammatory effector T (Teff) cells subtypes (e.g. T-bet for Th1, GATA3 for Th2 and RORγt for Th17). However, while the plasticity enhances Treg cell’s suppressive function, complete differentiation with the loss of stable Foxp3 expression leads to overwhelming inflammatory syndrome. Previous studies show how environmental factors, including cytokines, alarmins and metabolites reprogram Treg to acquire Teff cell phenotypes. Within the complex milieu of the tumor microenvironment, there is a small subset of Foxp3+ Treg cells that secret IFNγ. Our previous research elucidated that by disrupting the CARMA1-BCL10-MALT1 (CBM) signalosome complex in Foxp3+ Treg cells amplify IFNγ secretion without enhancing T-bet expression, leading to curbing of tumor growth. We also demonstrated that IL-18 can promote IFNγ secretion in IL-12 stimulated reprogrammed Treg cells. Given that Foxp3 normally suppresses IFNγ expression through its interaction with AML1/Runx1, while T-bet facilitates IFNγ expression by direct binding to the Ifng locus in Th1 cells, we hypothesize that there is a specific epigenetic remodeling mechanism enables tumor infiltrating Treg to secret IFNγ in a T-bet independent manner. By characterizing the epigenetic changes of IFNγ producing Treg cells and comparing them with Th1 cells, coupled with paired deletion of cis-regulatory regions on IFNγ locus using CRISPR-Cas9 and the identification of the transcription factors that bind to these cis-regulatory regions, we delineated that a unique epigenetic regulatory program is required for Treg cells to facilitate IFNγ expression. By investigating the epigenetic mechanism of IFNγ expression and Treg reprogramming, we can potentially identify novel therapeutic strategies to rewire tumor-infiltrating Treg cells into IFNγ-secreting entities which hold the promise of bolstering their efficacy in controlling tumors and enhancing the anti-tumor immune response.