1. Rapid Identification of the Tumor-Specific Reactive TIL Repertoire via Combined Detection of CD137, TNF, and IFNγ, Following Recognition of Autologous Tumor-Antigens.
- Author
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Draghi A, Chamberlain CA, Khan S, Papp K, Lauss M, Soraggi S, Radic HD, Presti M, Harbst K, Gokuldass A, Kverneland A, Nielsen M, Westergaard MCW, Andersen MH, Csabai I, Jönsson G, Szallasi Z, Svane IM, and Donia M
- Subjects
- Antigens, CD analysis, Apyrase analysis, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Datasets as Topic, Flow Cytometry, Humans, Integrin alpha Chains analysis, Interferon-gamma biosynthesis, Interferon-gamma genetics, Lymphocyte Activation genetics, Lymphocytes, Tumor-Infiltrating immunology, Neoplasm Proteins biosynthesis, Neoplasm Proteins genetics, Single-Cell Analysis, Transcriptome, Tumor Microenvironment immunology, Tumor Necrosis Factor-alpha biosynthesis, Tumor Necrosis Factor-alpha genetics, Antigens, Neoplasm immunology, CD4-Positive T-Lymphocytes chemistry, CD8-Positive T-Lymphocytes chemistry, Interferon-gamma analysis, Lymphocytes, Tumor-Infiltrating chemistry, Neoplasm Proteins analysis, Tumor Necrosis Factor Receptor Superfamily, Member 9 analysis, Tumor Necrosis Factor-alpha analysis
- 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 in vitro assays identifying tumor-specific functional activation measure the upregulation of surface molecules, de novo 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 in vitro , using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and de novo 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 in vitro compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4+ and CD8+ tumor-specific reactive TILs. In situ , the combined detection of TNFRSF9 , TNF , and IFNG 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 in vitro , which can be rapidly adopted in most cancer immunology laboratories., Competing Interests: MD has received honoraria for lectures from Roche and Novartis (past two years). IMS has received honoraria for consultancies and lectures from Novartis, Roche, Merck, and Bristol-Myers Squibb; a restricted research grant from Novartis; and financial support for attending symposia from Bristol-Myers Squibb, Merck, Novartis, Pfizer, and Roche. HDR is currently an employee at Novo Nordisk. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Draghi, Chamberlain, Khan, Papp, Lauss, Soraggi, Radic, Presti, Harbst, Gokuldass, Kverneland, Nielsen, Westergaard, Andersen, Csabai, Jönsson, Szallasi, Svane and Donia.)- Published
- 2021
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