Identification of a secretory immune regulatory ligand

Immunotherapies have transformed cancer treatment, but subsets of patients experience differential responses. Patients with squamous cell carcinomas (SCCs) in various tissues exhibit a high rate of relapse after initial responses to immunotherapies. Recent research highlighted the critical role of a group of stem cell-like tumor-initiating cells (TICs) in driving resistance to anti-tumor immunity and promoting relapse of both cutaneous (CSCCs) and head and neck (HNSCCs) SCCs despite intact antigen presentation. Although TICs drive SCC relapse, they compose less than 5% of the total tumor cell population, so their unique immune resistance mechanisms have been largely overlooked. We analyzed single-cell RNA-seq data profiling the gene signatures of various tumor populations in spontaneous GEMM CSCCs and identified that cytotoxic T lymphocyte-associated protein 2a ( Ctla2a ) was specifically activated in TICs. Ctla2a is a cysteine peptidase inhibitor with high homology to the I29 inhibitory domain of mouse and human cathepsins. Silencing Ctla2a in SCC cells reduced tumor growth, increased the frequency of granzyme B+ CD8+ T cells infiltrating the tumor, and sensitized SCC tumors to immunotherapy. We have found that the conditioned medium from SCC cells overexpressing Ctla2a is sufficient to blunt granzyme B production in CD8+ T cells and reduce CD8+ T cell antigen-specific cytotoxicity in vitro . Taken together, these results suggest that SCC TICs can secrete Ctla2a to modulate CD8+ T cell anti-tumor cytotoxicity. This interaction may serve as a target for next-generation immunotherapy that is able to blunt TIC-specific immune resistance.