Rapid Identification of the Tumor-Specific Reactive TIL Repertoire via Combined Detection of CD137, TNF, and IFNγ, Following Recognition of Autologous Tumor-Antigens

Abstract

Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current <i>in vitro</i> assays identifying tumor-specific functional activation measure the upregulation of surface molecules, <i>de novo</i> production of antitumor cytokines, or mobilization of cytotoxic granules following recognition of tumor-antigens, yet there is no widely adopted standard method. Here we established an enhanced, yet simple, method for identifying simultaneously CD8⁺ and CD4⁺ tumor-specific reactive TILs <i>in vitro</i>, using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and <i>de novo</i> production of TNF and IFNγ after recognition of naturally-presented tumor antigens, we demonstrate that a larger fraction of tumor-specific and reactive CD8⁺ TILs can be detected <i>in vitro</i> compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4⁺ and CD8⁺ tumor-specific reactive TILs. <i>In situ</i>, the combined detection of <i>TNFRSF9</i>, <i>TNF</i>, and <i>IFNG</i> identified most of the tumor-specific reactive TIL repertoire. In conclusion, we describe a straightforward method for efficient identification of the tumor-specific reactive TIL repertoire <i>in vitro</i>, which can be rapidly adopted in most cancer immunology laboratories.