<abstract xmlns="http://www.w3.org/1999/xhtml"><p>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---lt---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.</p></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---lt---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.

Variability of <italic>ex-vivo</italic> stimulated T-cells secretory profile in healthy subjects

George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures

Revista Romana de Medicina de Laborator

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Variability of  stimulated T-cells secretory profile in healthy subjects

Peripheral blood lymphocytes (PBL) are able to synthesize various cytokines that play key roles in the immune response and intercellular signaling. Since...

Variability of ex-vivo stimulated T-cells secretory profile in healthy subjects

Publié en ligne: 07 févr. 2020

Pages: 75 - 89

Reçu: 02 oct. 2019

Accepté: 25 nov. 2019

DOI: https://doi.org/10.2478/rrlm-2020-0004

Mots clés
peripheral blood lymphocytes, ex-vivo T-cells stimulation, cytokines, flow cytometry, reference intervals

© 2020 Ion Bogdan Manescu et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Variability of ex-vivo stimulated T-cells secretory profile in healthy subjects

Publié en ligne: 07 févr. 2020

Pages: 75 - 89

Reçu: 02 oct. 2019

Accepté: 25 nov. 2019

DOI: https://doi.org/10.2478/rrlm-2020-0004

Mots clésperipheral blood lymphocytes, ex-vivo T-cells stimulation, cytokines, flow cytometry, reference intervals

© 2020 Ion Bogdan Manescu et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Mots clés
peripheral blood lymphocytes, ex-vivo T-cells stimulation, cytokines, flow cytometry, reference intervals