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

• Published in: Frontiers in immunology 2021-10, Vol.12, p.705422-705422
• Main Authors: Draghi, Arianna, Chamberlain, Christopher Aled, Khan, Shawez, Papp, Krisztian, Lauss, Martin, Soraggi, Samuele, Radic, Haja Dominike, Presti, Mario, Harbst, Katja, Gokuldass, Aishwarya, Kverneland, Anders, Nielsen, Morten, Westergaard, Marie Christine Wulff, Andersen, Mads Hald, Csabai, Istvan, Jönsson, Göran, Szallasi, Zoltan, Svane, Inge Marie, Donia, Marco
• Format: Article
• Language: English
• Subjects: Antigens, CD - analysis; Antigens, Neoplasm - immunology; Apyrase - analysis; Cancer and Oncology; Cancer och onkologi; CD137 (4-1BB); CD4-Positive T-Lymphocytes - chemistry; CD4-Positive T-Lymphocytes - immunology; CD8-Positive T-Lymphocytes - chemistry; CD8-Positive T-Lymphocytes - immunology; Clinical Medicine; Datasets as Topic; Flow Cytometry; Humans; immune-monitoring; immune-responses to cancer; Immunology; Integrin alpha Chains - analysis; Interferon-gamma - analysis; Interferon-gamma - biosynthesis; Interferon-gamma - genetics; Klinisk medicin; Lymphocyte Activation - genetics; Lymphocytes, Tumor-Infiltrating - chemistry; Lymphocytes, Tumor-Infiltrating - immunology; Medical and Health Sciences; Medicin och hälsovetenskap; Neoplasm Proteins - analysis; Neoplasm Proteins - biosynthesis; Neoplasm Proteins - genetics; Single-Cell Analysis; single-cell technologies; Transcriptome; Tumor Microenvironment - immunology; Tumor Necrosis Factor Receptor Superfamily, Member 9 - analysis; Tumor Necrosis Factor-alpha - analysis; Tumor Necrosis Factor-alpha - biosynthesis; Tumor Necrosis Factor-alpha - genetics; tumor-infiltrating lymphocytes (TILs); tumor-specific activation; tumor-specific reactivity
• ISSN: 1664-3224; 1664-3224
• DOI: 10.3389/fimmu.2021.705422

Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current assays identifying tumor-specific functional activation measure the upregulation of surface molecules, 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 , using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and 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 compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4 and CD8 tumor-specific reactive TILs. , the combined detection of , , and 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 , which can be rapidly adopted in most cancer immunology laboratories.