Regulation of IFN-γ expression in reprogrammed regulatory T Cells through IL-23 receptor and Ifng distal cis-regulatory elements

Abstract

Regulatory T (Treg) cells are critical for maintaining immune homeostasis, but they also contribute to immune suppression and hinder antitumor immune responses and immunotherapy in the tumor microenvironments of human cancer patients and mouse models of cancer. However, based on our lab’s previous work, the inflammatory cytokines IL-18 and IL-12 can synergistically reprogram a fraction of tumor-infiltrating Treg cells to adopt a Th1-like phenotype and secrete IFN-g, and this process is greatly amplified by disruption of TCR signaling via the Carma1/Bcl10/Malt1 (CBM) signalosome. The molecular mechanisms governing this transition remain poorly understood. Here, we investigate the role of the IL-23/IL-23R axis and the epigenetic regulation of the Ifng locus during Treg reprogramming. We demonstrate that IL-23R is preferentially expressed on Treg that do not secrete IFN-γ, and its overexpression reduces IFN-γ production in IL-12/IL-18 driven reprogrammed Tregs dependent on its signaling function. By deleting cis-regulatory elements (CREs) of the Ifng gene, we furthermore identify several distal elements that contribute to its transcriptional regulation in reprogrammed Tregs. Most notably, deletion of CNS -34 and CNS +40 significantly reduce IFN-γ production. CUT&RUN-qPCR analysis reveals distinct recruitment patterns of RelA to these CNS regions, suggesting differential transcriptional control of Ifng in Th1 and reprogrammed Treg cells. Our findings provide novel insights into the mechanisms controlling IFN-γ expression in Tregs and highlight potential targets for modulating Treg in tumor immunotherapy.