RRML - Variability of ex-vivo stimulated T-cells secretory profile in healthy subjects
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Nr. 28(1)/2020 DOI:10.2478/rrlm-2020-0004
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Variability of ex-vivo stimulated T-cells secretory profile in healthy subjects

Ion Bogdan Manescu, Doina Ramona Manu, Georgiana Mihaela Serban, Minodora Dobreanu

Correspondence should be addressed to: Doina Ramona Manu

Abstract:

Peripheral blood lymphocytes (PBL) are able to synthesize various cytokines that play key roles in the immune response and intercellular signaling. Since alterations in cytokine production and/or activity occur in many pathological processes, the study of cytokine synthetic capacity of PBL is a valuable tool for assessing the immune profile. In this paper, we aimed to investigate the variability of interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α) and interferon gamma (IFN-γ) synthetic capacity of CD4+/CD8+ T-cells stimulated ex-vivo in healthy subjects, by means of a commercial intracellular cytokine staining (ICS) protocol. Peripheral blood mononuclear cells were isolated from 16 healthy subjects by Ficoll gradient centrifugation and activated ex-vivo with PMA/Ionomycin/Brefeldin-A for 4 hours. Activated PBL were surface-stained for CD3/CD4/CD8, fixed and permeabilized. ICS was performed using anti-human IL-2/TNF-α/IFN-γ and samples were analyzed on a BD-FACSAria-III flow cytometer. We recorded high post-isolation and post-activation mean viabilities: 82.1% and 82.4% respectively, p=0.84. Both CD4+/CD8+ subpopulations were found to partially produce each of the three cytokines, but in different proportions. On average, a significantly greater percentage of CD4+ cells was shown to produce IL-2 and TNF-α, compared with CD8+ cells (61.5%+/-5.8 vs. 25%+/-5.6 and 26.9%+/-11 vs. 7.5%+/-3.3 respectively, p<0.0001 for both). Contrarily, IFN-γ was produced by a higher proportion of CD8+ cells (8.4%+/-3.9 vs. 6.8%+/-3.2, p=0.01). These results show that the employed ICS protocol elicits a satisfactory and consistent cytokine response from PBL of healthy subjects. The collected data may be used to outline a preliminary reference range for future studies on both healthy/pathological subjects.

Keywords: peripheral blood lymphocytes, ex-vivo T-cells stimulation, cytokines, flow cytometry, reference intervals

Received: 2.10.2019
Accepted: 25.11.2019
Published: 19.12.2019

 
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How to cite
Manescu IB, Manu DR, Serban GM, Dobreanu M. Variability of ex-vivo stimulated T-cells secretory profile in healthy subjects. Rev Romana Med Lab. 2020;28(1):75-89. DOI:10.2478/rrlm-2020-0004