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The Use of Flow Cytometry to Assess the State of Chromatin in T Cells
Published on: December 17, 2015
Expression and kinetics of cytokines determined by intracellular staining using flow cytometry
B Mascher1, P Schlenke, M Seyfarth
1Institute of Immunology and Transfusion Medicine, University of Lübeck School of Medicine, Germany.
Insights
Tumor necrosis factor alpha (TNFalpha) is the earliest cytokine produced by T-cells, peaking at 2 hours. Memory cells exhibit higher and faster cytokine production compared to naive cells.
Area of Science:
- Immunology
- Cell Biology
Background:
- Cytokine production is crucial for immune responses.
- Understanding cytokine kinetics in different lymphocyte subsets is vital.
Purpose of the Study:
- To characterize the kinetics of interleukin 2 (IL2), interferon gamma (IFNgamma), and tumor necrosis factor alpha (TNFalpha) production in human lymphocytes.
- To compare cytokine profiles between T-helper (Th) cells, cytotoxic T-cells (CTL), naive cells, and memory cells.
Main Methods:
- Intracellular cytokine staining via flow cytometry using the paraformaldehyde-saponin procedure.
- Stimulation of lymphocytes with phorbol-12-myristate-13-acetate (PMA) and ionomycin in the presence of monensin.
- Kinetic analysis of cytokine production over 24 hours, with an optimal time point at 5 hours.
Main Results:
- TNFalpha was the first cytokine produced, peaking at 2 hours, followed by IL2 and IFNgamma peaking at 8 hours.
- IL2 production exceeded IFNgamma in T-cells throughout the study period.
- Th cells primarily produced IL2 and TNFalpha, while CTL cells predominantly produced IFNgamma.
- Memory cells showed higher and faster cytokine production compared to naive cells, with naive cells mainly producing IL2.
Conclusions:
- TNFalpha exhibits rapid kinetics, while IL2 and IFNgamma production kinetics differ between T-cell subsets.
- Memory lymphocytes are more responsive cytokine producers than naive lymphocytes.
- The 5-hour stimulation time point is optimal for assessing cytokine production in healthy individuals.
Abstract:
Cytokine production by human lymphocytes was assessed by a flow cytometric procedure involving staining of intracellular cytokines by the paraformaldehyde-saponin procedure. The production of interleukin 2 (IL2), interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) was determined in T-helper (Th) and cytotoxic T-cells (CTL) as well as in naive and memory cells after stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin under the influence of monensin. Kinetic studies on IL2, IFNgamma and TNFalpha in lymphocyte subpopulations showed that TNFalpha was the first cytokine produced by T-cells. Production of this cytokine peaked at 2 h and declined rapidly thereafter. The peak of IL2 and IFNgamma production was at 8 h, and production of IL2 exceeded that of IFNgamma in T-cells at all times. IL2 production declined markedly after 8 h, while IFNgamma remained relatively stable for 24 h. IL2 and TNFalpha were mainly produced by Th cells while CTL primarily expressed IFNgamma. At all times a higher percentage of memory cells stained cytokine positive compared to naive cells and production of cytokines increased more rapidly in the memory cells. Naive cells produced primarily IL2, while memory cells expressed all the studied cytokines in substantial amounts. Kinetic studies between 1 and 24 h showed that 5 h was the optimal time point for evaluating the cytokines studied; hence normal values obtained from 50 healthy blood donors were evaluated after 5 h continuous PMA and ionomycin stimulation.
