Author:
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
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.
Funder
Kræftens Bekæmpelse
Lundbeckfonden
Danmarks Frie Forskningsfond
Sundhedsstyrelsen
Nemzeti Kutatási, Fejlesztési és Innovaciós Alap
Subject
Immunology,Immunology and Allergy
Cited by
15 articles.
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