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

Arianna Draghi, Christopher Aled Chamberlain, Shawez Khan, Krisztian Papp, Martin Lauss, Samuele Soraggi, Haja Dominike Radic, Mario Presti, Katja Harbst, Aishwarya Gokuldass, Anders Kverneland, Morten Nielsen, Marie Christine Wulff Westergaard, Mads Hald Andersen, Istvan Csabai, Göran Jönsson, Zoltan Szallasi, Inge Marie Svane, Marco Donia*

*Corresponding author for this work
5 Citations (Scopus)


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.

Original languageEnglish
Article number705422
JournalFrontiers in Immunology
Pages (from-to)705422
Publication statusPublished - 11 Oct 2021


  • Antigens, CD/analysis
  • Antigens, Neoplasm/immunology
  • Apyrase/analysis
  • CD4-Positive T-Lymphocytes/chemistry
  • CD8-Positive T-Lymphocytes/chemistry
  • Datasets as Topic
  • Flow Cytometry
  • Humans
  • Integrin alpha Chains/analysis
  • Interferon-gamma/analysis
  • Lymphocyte Activation/genetics
  • Lymphocytes, Tumor-Infiltrating/chemistry
  • Neoplasm Proteins/analysis
  • Single-Cell Analysis
  • Transcriptome
  • Tumor Microenvironment/immunology
  • Tumor Necrosis Factor Receptor Superfamily, Member 9/analysis
  • Tumor Necrosis Factor-alpha/analysis


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