Effective T cell responses entail the coproduction of IFN-γ, TNF-α, and IL-2. Cytokine production is determined by transcriptional and posttranscriptional events. However, increased transcript levels do not always translate into protein production, and therefore simultaneous transcripts and protein measurement are essential for the appropriate analysis of T cell responses. In this study, we optimized flow cytometry–based fluorescence in situ hybridization (Flow-FISH) for IFN-γ to multicolor flow cytometry that allows for single-cell measurement of mRNA and protein levels. This high-throughput analysis detected Ag-specific human T cells of low frequency. We also employed Flow-FISH for single-tube analysis of IFN-γ transcript and protein profile to simultaneously study the responsiveness of different T cell subsets, that is, naive, effector, and memory T cells. Importantly, the simultaneous transcript and protein analysis of IFN-γ and of TNF-α and IL-2 revealed that T cell responses consist of two types: one subtype loses mRNA expression during activation, whereas the other maintains high transcript levels throughout stimulation. High cytokine transcript levels correlated with increased protein production. Intriguingly, this mRNAhi-expressing T cell population also produced higher levels of other cytokines, indicating that Flow-FISH helps identify the best cytokine producers during T cell activation. We conclude that Flow-FISH is a rapid, sensitive, and cost-effective method to determine the quality of T cell responses induced by, for instance, T cell vaccines.
This work was supported by the Landsteiner Foundation of Blood Transfusion Research and by the Dutch Science Foundation (LSBR- Fellowship 1373 and VIDI Grant 917.14.214 to M.C.W.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- fluorescence in situ hybridization
- flow cytometry–based FISH
- geometric mean fluorescence intensity
- intracellular cytokine staining
- quantitative PCR.
- Received September 1, 2016.
- Accepted November 7, 2016.
- Copyright © 2017 by The American Association of Immunologists, Inc.