Document Type

Article

Publication Date

1-1-2022

Publication Title

Front Immunol

Keywords

JMG, Animals, Artifacts, Biomarkers, Breast Neoplasms, Cell Line, Cytokines, Disease Models, Animal, Female, Flow Cytometry, Humans, Hydrogen Peroxide, Intracellular Space, Leukocyte Count, Lymphocyte Activation, Mice, Mice, Knockout, Models, Biological, Neutrophils, T-Lymphocytes

JAX Source

Front Immunol 2022 Jan 21; 13:759188

Volume

13

First Page

759188

Last Page

759188

ISSN

1664-3224

PMID

35126389

DOI

https://doi.org/10.3389/fimmu.2022.759188

Grant

CA188093, CA237307, CA251433, CA034196, Pyewacket Fund

Abstract

Intracellular cytokine staining (ICS) is a widely employed ex vivo 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+ neutrophils, neutrophils are ex vivo activated by an ICS reagent - phorbol myristate acetate (PMA) - which leads to hydrogen peroxide (H2O2) 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.

Comments

We appreciate Dr. Iiro Taneli Helenius for his critical editing of the manuscript and also thank the assistance from The Jackson Laboratory Scientific Service.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.

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