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 interrogate endogenous antitumor responses and to advance downstream therapeutic applications. Multiple 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. We evaluated these biomarkers in primary human ovarian tumor samples using single-cell mass cytometry to compare their relative phenotypic profiles, and examined their response to autologous tumor cells ex vivo. PD-1⁺ , CD103⁺ , and CD39⁺ TILs all contain a CD137⁺ cell subset, while CD137⁺ TILs highly co-express the aforementioned markers. CD137⁺ TILs exhibit the highest expression of cytotoxic effector molecules compared to PD-1⁺ , CD103⁺ , or CD39⁺ TILs. Removal of CD137⁺ cells from PD-1⁺ , CD103⁺ , or CD39⁺ TILs diminish their IFN-γ secretion in response to autologous tumor cell stimulation, while CD137⁺ TILs maintain high HLA-dependent IFN-γ secretion. CD137⁺ TILs exhibited an exhausted phenotype but with CD28 co-expression, suggesting possible 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 CD137-expressing TILs, implicating CD137 as a more selective biomarker for naturally occurring tumor-specific TILs.