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. 2019 May 17;9:7526. doi: 10.1038/s41598-019-43899-9

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Flow cytometric analysis of cell activation markers and intra-cellular cytokine expression on CD4+ T-lymphocytes (TL): PBMCs were obtained prior to pulse therapy (day 0) and during pulse therapy (day 7) of patients with severe plaque psoriasis treated with anti-TNF agents. PBMC, cultured for 48 h in the presence of medium (unstimulated) or with anti-CD3 and anti-CD28 mAbs (stimulated). After recovered, PBMC were incubate with appropriate mAbs and isotype controls. Panels A to F show gates strategies for analysis. In panels C–E the red dots indicate the labeling of the control isotypes. In panel F, the blue dots indicate the CD25⁻ population. Panel G: CD69⁺ on CD4+ TL (Wilcoxon p < 0.05), panel H: IL-17⁺IFN-γ⁻ on CD4+ LT (Wilcoxon p > 0.05), panel I: IL-17⁻ IFN-γ⁺ on CD4+ LT (Wilcoxon p < 0.05), panel J: IL-17⁺IFN-γ⁺ on CD4+ LT (Wilcoxon p > 0.05) and panel K: CD25^highFoxP3⁺LAP⁺ on CD4+ LT (Wilcoxon p < 0.05). The results are expressed in percentage on the CD4+ TL. *Indicate statistical significance.