Systematic analysis of CD39, CD103, CD137 and PD-1 as biomarkers for naturally occurring tumor antigen-specific TILs

The detection of tumor-specific T cells in solid tumors is integral to the interrogation of endogenous antitumor responses and to the advancement of downstream therapeutic applications, such as checkpoint immunotherapy and adoptive cell transfer. A number of biomarkers are reported to identify endogenous tumor-specific tumor infiltrating lymphocytes (TILs), namely CD137, PD-1, CD103, and CD39, however a direct comparison of these molecules has yet to be performed. Here, we evaluate these biomarkers in primary human high-grade serous ovarian tumor samples using single-cell mass cytometry to characterize and compare their relative phenotypic profiles, as well as their response to autologous tumor cells ex vivo. CD137+, PD-1+, CD103+, and CD39+ TILs are all detectable in tumor samples with CD137+ TILs being the least abundant. PD-1+, CD103+, and CD39+ TILs all express a subset of CD137+ cells, while CD137+ TILs highly co-express the aforementioned markers. CD137+ TILs exhibit the highest expression of cytotoxic effector molecules, such as IFN{gamma} and Granzyme B, compared to PD-1+, CD103+ or CD39+ TILs. Removal of CD137+ TILs from PD-1+, CD103+, or CD39+ TILs results in lower secretion of IFN{gamma} in response to autologous tumor stimulation, while CD137+ TILs highly secrete IFN{gamma} in an HLA-dependent manner. CD137+ TILs exhibited an exhausted phenotype with CD28 co-expression, suggestive of antigen recognition and receptiveness to reinvigoration via immune checkpoint blockade. Together, our findings demonstrate that the antitumor abilities of PD-1+, CD103+, and CD39+ TILs are mainly derived from a subset of TILs expressing CD137, implicating CD137 is a more selective biomarker for naturally occurring tumor-specific TILs.