TRANSCRIPTOME ANALYSIS OF TUMOR INFILTRATING T REGULATORY CELLS UNVEILS SPECIFIC CODING AND NON CODING GENE SIGNATURE

Abstract

CD4+ Regulatory T cells (Treg cells) are a specialized subpopulation of T cells that act to suppress immune response, thereby maintaining homeostasis and self-tolerance. These cells play a critical role in preventing autoimmunity infections, and cancer. Treg cells can infiltrate tumor tissues where they suppress anti-tumor immune responses, contributing to the development of an immunosuppressive tumor microenvironment thus promoting immune evasion and cancer progression. Tumor infiltrating Treg cells can display function heterogeneity, depending on both the tumor type and the inflammatory milieu, therefore molecular characterization of Treg cells is crucial to understand how these cells can be modulated in the tumor to unleash effective anti-tumor T cell responses. To this aim, we investigated the transcriptional blueprints of Treg cells both in tumors and in the peripheral blood of healthy donors to define both coding and non-coding transcripts that best define the identity of these cells and might therefore represent novel prognostic markers or therapeutic targets. We performed a transcriptome analysis of both CD4+ Treg cells and effector cells (Th1 and Th17) infiltrating two of the most frequent types of human cancer defining the molecular signatures of tumor-infiltrating Treg cells in these two cancer types. We found tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine enriched in tumor infiltrating Treg cells compared to both the peripheral blood of patients and healthy donors. Given the high specificity of long non-coding RNA compared to coding sequences, we also performed bioinformatic analysis to assess the expression of known and novel non-coding transcripts. With this analysis we identified specific Treg cell non-coding transcript in proximity of CTLA4 locus. Since lncRNAs are by now considered as key regulatory elements in immune cell differentiation and maintenance of their identity, we characterized the identified lncRNAs in Treg cells in healthy donors, defining their epigenetic organization, expression level, localization and whether they contributed to the established Treg cell suppressive activity. All these findings unveiled another layer of the complexity of Treg cells biology and warrants for more detailed functional studies that can fully explain the pathways and the cellular networks that are affected by the identified coding and non-coding transcripts.