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. 2018 Nov 15;8:16904. doi: 10.1038/s41598-018-35161-5

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© The Author(s) 2018

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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Fungi specific Th1 cytokine expression by T cells of different age groups. CD4⁺CD45RA⁺ T cells from neonates, infants, children, and adults were stimulated with C. albicans (orange) or A. fumigatus (blue) (as in Fig. 1) for 3 and 6 days respectively (A) Frequency of T cells expressing intracellular IL-2 (left panel), TNFα (middle panel) or IFNγ (right panel) was determined by flow cytometry. (B) Determination of IL-2 (left panel), TNFα (middle panel) or IFNγ (right panel) cytokine release of CD4⁺CD45RA⁺ T cells of neonates, infants and children or adults by LegendPlex which were either stimulated or not for 3 days. (C,D) CD4⁺CD45RA⁺ T cells were stimulated with C. albicans (C) or A. fumigatus (D) as in A, and the cells expressing single or multiple cytokines IL-2, TNFα, and IFNγ were determined by flow cytometry and analysed by Boolean gating and shown as fraction of all CD4⁺ T cells in a pie chart. The subsets that simultaneously express no (grey), one (blue), two (yellow) or three (red) different cytokines are grouped by colour. The data are representative of at least 5 donors. Cumulative results are shown and each dot in (A) and (B) represent a different donor. The error bars in figures denote ± SD. ^*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by one-way Anova with Tukey post hoc test (A) or Kruskal Wallis with Dunn’s post hoc test (B).