AUTHOR=Gong Zheng , Li Qing , Shi Jiayuan , Ren Guangwen 

TITLE=An Artifact in Intracellular Cytokine Staining for Studying T Cell Responses and Its Alleviation

JOURNAL=Frontiers in Immunology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.759188

DOI=10.3389/fimmu.2022.759188

ISSN=1664-3224

ABSTRACT=<p>Intracellular cytokine staining (ICS) is a widely employed <italic>ex vivo</italic> method for quantitative determination of the activation status of immune cells, most often applied to T cells. ICS test samples are commonly prepared from animal or human tissues as unpurified cell mixtures, and cell-specific cytokine signals are subsequently discriminated by gating strategies using flow cytometry. Here, we show that when ICS samples contain Ly6G<sup>+</sup> neutrophils, neutrophils are <italic>ex vivo</italic> activated by an ICS reagent – phorbol myristate acetate (PMA) – which leads to hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) release and death of cytokine-expressing T cells. This artifact is likely to result in overinterpretation of the degree of T cell suppression, misleading immunological research related to cancer, infection, and inflammation. We accordingly devised easily implementable improvements to the ICS method and propose alternative methods for assessing or confirming cellular cytokine expression.</p>