Identification of a core gene-regulatory trajectory to terminal T cell dysfunction in human tumors and discovery of potential target genes involved in this process

Diseases such as cancer are often associated with suppression of the immune sys- tem. Thereby, cytotoxic CD8+ T cells, originally meant to kill the cancerous cells, tend to become dysfunctional, as they are struggling with long term exposure to antigens and the immunosuppressive tumor microenvironment. As a consequence, tumor-specific CD8+ T cells often fail to fully eliminate cancer cells. Much research has been conducted on T cell exhaustion in the context of chronic viral infection. However, it remains unknown, whether gene-regulation patterns causing terminal T cell exhaustion in cancer are specific to cancer type or tumor microenvironment or, whether they are governed by a universal exhaustion program. ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) is a great tool to identify cell states based on their open chromatin landscape. Therefore, we used this method to investigate chromatin states of CD8+ tumor-infiltrating lymphocytes, isolated from HCC (Hepatocellular Carcinoma), RCC (Renal Cell Carcinoma), and HNSCC (Head and Neck Squamous Cell Carcinoma) patients, on the single cell level. This approach enables unbiased identification of discrete TIL (tumor-infiltrating lymphocytes) subpopulations as well as key transcription factors and gene-regulatory elements that determine different cell states. Bioinformatic analysis verified terminal exhausted TIL populations in every cancer type except HNSCC and revealed candidate determinants of exhaustion common to RCC and HCC, as well as to BCC (Basal Cell Carcinoma, integrated data from a published study). Further, we sought to determine the role of these candidates using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) knock-out in expanded TILs from melanoma patients. We anticipate that our findings will improve understanding gene-regulatory mechanisms leading to terminal T cell dysfunction in different human cancer types and help to identify factors, which could improve immunotherapies such as CAR T-cell therapy.