EPIGENETIC CHARACTERIZATION OF TUMOR INFILTRATING CD4+ T REGULATORY CELLS

In recent years, the role of CD4+ regulatory T cells (Treg cells) in inhibiting the anti cancer activity of effector T cells has become increasingly evident and they are therefore currently considered promising targets for cancer immunotherapy. Despite Treg cell depletion has been reported to increase anti-tumor specific immune responses and to reduce tumor burden, some relevant issues still remain to be addressed, for a safer, more effective clinical application of these therapies. Previous findings in our lab identified unique transcriptional profiles of human Treg cells infiltrating colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) supporting the existence of an underlying regulatory hubs that specifically shape tumor Treg identity and represent potential target for their functional modulation specificially in the tumor microenvironment. Treg cell transcriptome represents just a layer of the machinery that drive the acquisition of their tumor cell state. Ultimately, a comprehensive understanding of the regulatory networks that govern tumor Treg gene expression programs can provide insights on how a more selective inhibition can be achieved by interfering with the hubs that translate the cues coming from the tumor microenvironment. To define the epigenetic blueprints specific for tumor infiltrating Treg cells we have integrated distinct histone marks (H3K4me3, H3K4me1, H3K36me3, H3K27ac, H3K27me3) with ATAC-seq derived chromatin accessibility data we generated in Treg cells isolated from peripheral blood, normal and tumor tissues. To infer genome chromatin states we have employed ChromHMM a machine learning based approach that predicts the molecular structure of promoters and enhancers based on data from chromatin accessibility assays and a set of histone modification. We focused in particular on active enhancer regions, characterized by the co-presence of H3K27Ac and H3K4me1. Transcription factor footprinting analysis on the identified regulatory regions showed TF groups with distinct binding activity profile across Treg populations that are clearly lost or gained specifically in tumor Treg cells. Based on our findings we are implementing CRISPR based epigenetic modulation of selected enhancers that represent a novel strategy to reverse Treg mediated immunosuppression in the tumor microenvironment. The new knowledge coming from this project cues will lead to a better understanding of tiTreg plasticity and to the identification of novel potential targets that can guide the rational development of tiTreg reprograming and innovative therapeutics for cancer with increased efficacy and reduced adverse effects.