Haghi, Leila

Abstract The molecular and functional diversity of regulatory T-cells (Tregs) in health and in disease remains unclear. We previously described in colorectal cancer (CRC) patients a subpopulation of RORγt+ Tregs with elevated expression of β-catenin and pro-inflammatory properties. Here we observed progressive expansion of RORγt+ Tregs in inflammatory bowel disease (IBD) patients during inflammation and early dysplasia. Activating Wnt/β-catenin signaling in human and murine Tregs was sufficient to recapitulate the disease-associated increase in frequencies of RORγt+ Tregs expressing IL-17, IFN-γ, and TNFa. We found that binding of the β-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt/β-catenin activation induced newly accessible chromatin sites in these genes and upregulated their expression. These findings indicate that TCF-1 and Foxp3 together limit the expression of pro-inflammatory genes in Tregs. Activation of ꞵ-catenin signaling interferes with this function and promotes the disease-associated RORγt+ Treg phenotype.

Abstract In the present study we provide a temporal and mechanistic explanation of how β-catenin signaling skews regulatory T cells (Treg) into a pathogenic, pro-inflammatory phenotype that co-express the Th17 lineage transcription factor RORγt along with Foxp3 during colon cancer (CC) development. We previously described this distinct subset of Tregs in the peripheral blood (PB) of established CC patients that suppresses effector T cell functions but simultaneously maintains inflammation by failing to regulate mast cell degranulation and production of IL-17. Moreover, we showed in mice that RORγt expression correlates with elevated β-catenin levels in T cells and that RORγt is a transcriptional target of β-catenin interaction partners TCF-1 (Tcf7) and HEB (Tcf12). To elucidate how Wnt/β-catenin signaling orchestrates the induction of pathogenic properties in Tregs specifically we analyzed inflammatory bowel disease (IBD) patients' tissue and PB. As IBD can progress to colon cancer, this disease presents a unique platform to study the temporal emergence of RORγt+/Foxp3+ Tregs during the course of the disease. To further mechanistically establish how activated β-catenin signaling facilitates re-programming of Tregs and acquisition of pro-inflammatory properties we over-expressed β-catenin specifically in Tregs and/or knocked out its interaction partners TCF-1 and HEB in a Foxp3-Cre mouse model. Indeed, in a specific sub-population of IBD patients we were able to detect RORγt+/Foxp3+ double positive Tregs in the patients' blood and tissue, expressing high levels of β-catenin and producing IL-17 upon re-stimulation. Mice expressing β-catenin Treg-specifically showed striking X-linked immune pathology. Males died 4 weeks after birth of a severe scurfy-like phenotype, indicating failure of Treg function. β-catenin overexpressing Tregs in mice displayed a reduced suppressive capacity, higher rate of apoptosis and produced effector cytokines IL-17 and IFN-γ. Furthermore, the chromatin accessibility of bona fide Treg genes was drastically reduced, whereas accessibility in T cell activation and IL-17 signature genes increased compared to wild-type Tregs in ATACseq analysis. An additional Treg-specific knock-out of HEB or TCF-1 reduced or delayed auto-immunity, respectively, with increased survival in both cases and partial rescue of systemic Treg numbers. We herewith provide evidence that induction of β-catenin signaling is indeed responsible for the pathogenic, pro-inflammatory conversion of Tregs through activation of RORγt expression. Further transcriptome analysis will reveal how the epigenetic changes in the chromatin translate to gene expression. We anticipate, that this mechanistic insight into β-catenin mediated pathogenic conversion of Tregs will reveal novel targets for cancer immuno-therapy. Citation Format: Jasmin Quandt, Leila Haghi, Akinola O. Emmanuel, Khashayarsha Khazaie, Fotini Gounari. β-catenin signaling mediates pathogenic pro-inflammatory conversion of Foxp3+ Treg cells early during colon cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-281.