CHARACTERIZATION OF THE EPIGENOMIC PROFILE SHAPING HUMAN FOXP3+ REGULATORY T CELL IDENTITY IN THE TUMOR MICROENVIRONMENT

One of the hallmarks of cancer is the evasion of the anti-tumor immune response. Cancer cells orchestrate several mechanisms to grow unchecked while promoting a suppressive tumor microenvironment (TME). In this context, CD4+ FOXP3+ regulatory T cells (Tregs) have emerged as a major immunosuppressive population sustaining cancer progression. In physiological conditions they mediate immune tolerance and maintain balance in immune responses, controlling their intensity and timing. Moreover, Tregs can acquire specific phenotypes to modulate distinct immunological contexts and regulate tissue homeostasis through mechanisms that extend beyond their canonical immune functions. Tumor-infiltrating Treg cells (TI-Tregs) are frequently found in solid tumors, and their ratio compared to CD8+ effector T cells often correlates with poor patient prognosis. This makes the selective depletion or reprogramming of TI-Tregs a promising strategy in cancer immunotherapy, aiming to enhance anti-tumor immunity without disrupting Treg-dependent homeostasis in healthy tissues. Previous studies have revealed specific transcriptional programmes of TI-Tregs compared to other immune subsets, including Tregs in the blood (PB-Tregs) and normal tissues adjacent to tumors (NAT- Tregs). These findings pave the way for discovering novel TI-Treg-specific targets. However, a deeper molecular profiling of this Treg cell state, including their regulatory landscape shaped by TME cues, is required to develop novel immunotherapy approaches. Here, we focus on epigenomic rewiring as a key process that orchestrates Treg adaptation and the acquisition of a tumor-promoting phenotype. The integration of external signals dynamically alters various layers of epigenomic regulation, mediating the establishment of tailored gene expression programs. In this regard, enhancers and DNA-binding transcription factors (TFs) are pivotal in forming specific regulatory networks. To map the TI-Treg epigenomic profile, CD4+ CD25+ CD127- Tregs were isolated from the blood (PB), tumors, and normal adjacent tissue (NAT) of colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) patients. Chromatin accessibility and different histone modifications were profiled using ATAC-seq and ChIPmentation-seq, respectively, to reconstruct the genome-wide active and repressed chromatin states and to precisely map open chromatin regions with enhancer features. Additionally, 5 single-cell RNA-seq data were leveraged to discover bidirectionally transcribed noncoding enhancer RNAs (eRNAs), another feature marking active enhancers and delineating the portion of the enhancer region most likely mediating enhancer function and TF binding. TF motif analysis and in silico TF footprinting were employed to assess TF binding dynamics and differential regulatory networks across Treg cell states. We map the comprehensive enhancerome of human Tregs through the integration of multiple epigenomic features assessed in independent omics datasets. This atlas includes 14,158 bona fide active enhancers in TI-Tregs and in normal tissue-Tregs. Notably, our combined multi-omics approach highlights that enhancers comprise a minor fraction of open chromatin regions in Tregs, underscoring the advantage of our approach in robustly annotating active enhancers compared to other accessible regions. We reveal distinct open chromatin landscapes across PB-, NAT-, and TI-Treg cell states, with altered accessibility in cis-regulatory elements including enhancers. Focusing on TI-Treg chromatin rewiring, we find that epigenomic changes at enhancer level reflect transcriptional differences with respect to NAT-Tregs. Moreover, the existing specificities between these two Treg populations, confirmed by our findings, are driven at a much greater extent by enhancers as opposed to promoters. Integration of epigenomic and transcriptomic data uncover different regulatory programs involving coordinated changes in enhancer activity and target gene expression across Treg cell states. We find diverse axes of Treg epigenomic regulation within the tumor. On one hand, the activation program, shared with NAT- Tregs, is further sustained in the highly suppressive TI-Tregs. On the other hand, a TI- Treg private regulatory program results from the adaptation to TME cues and involves potentially specific metabolic alterations. Interestingly, distinct Treg regulatory circuits are driven by both shared and specific TF families. We confirm BATF as a crucial regulator of the activation program, shared between TI- and NAT-Tregs. Additionally, we uncover potentially relevant TFs for TI- Treg specific adaptation, with factors from non-canonical NFkB pathway among the top drivers. Overall, comprehensive understanding and dissection of multilayer epigenomic regulation can inform on novel targets for specific modulation of Tregs in the TME, with potential clinical relevance for the design of novel immunotherapies.