CHARACTERIZATION AND SPATIAL LOCALIZATION OF REGULATORY SUBSETS IN HUMAN TUMORS

It is becoming increasingly clear that tumor immune microenvironment (TIME) plays a crucial role in cancer progression. In this contest T lymphocytes gained remarkable attention since they are often found to infiltrate tumors where they exert their functions to eradicate nascent tumor, but also in certain circumstances to promote its progression with the establishment of an immunosuppressive environment. CD8+ T cells were considered the predominant immune cellular component to infiltrate tumors, able to repress their proliferation through the release of cytotoxic molecules. Therefore several studies have been undertaken with the final goal to boost their activity against tumor cells. On the contrary, the role of CD4+ T cells has been largely underestimated in cancer immunity. They are known to provide helper signals to other immune subsets that lead to anti-tumoral responses, but also other subsets as regulatory T cells, have been described to promote the tumor growth by impairing effector cells activities. The immune cells that are found in tumor bed are continuously exposed to a plethora of signals, given by cancer cells, surrounding tissues or even by components of the immune system itself. All those different signals can affect cells identity, especially from the immune compartment. Cell plasticity is a peculiar feature of CD4+ T cells, which are known to shape and to adapt their phenotype in response to the different stimuli they are exposed to. This, together with their ability to interact with other cells from the immune system, can drastically affect tumor progression. Therefore the characterization of intratumoral CD4+ T cells could be fundamental in understanding the mechanisms leading to immunosurveillance failure. With this aim we performed single cell-RNAseq on CD4+ T cells isolated from Colorectal Cancer (CRC) and Non small cell lung cancer (NSCLC) and their respective adjacent healthy tissues. Our analysis revealed the enrichment of two subsets of CD4+ T cells in tumoral samples, T regulatory cells (Treg) and Type-1 regulatory T cells (Tr1). Since little is known about Tr1 cells’ role in cancer immunology, we focused on their characterization. We checked for 4 the production of IL-10, GMZK and for the expression of EOMES, the lineage transcription factor that has been recently identified in mouse and human peripheral Tr1 cells. Moreover, we sought to establish a correlation between Tr1 infiltration and patients’ outcome. We identified CHI3L2 as a specific Tr1 transcript and found that its expression correlates with a worse prognosis. All together, our data suggest that the CD4+ T regulatory cells are enriched in tumors and that Tr1 cells could largely contribute to the establishment of an immunosuppressive microenvironment that inhibits anti-tumor responses, making them suitable targets for novel immunotherapies.