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. 2023 Jun 7;37(7):1572–1575. doi: 10.1038/s41375-023-01932-x

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© The Author(s) 2023

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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Fig. 2 — A Upper panel. UMAP plot with T-REX analysis of bone marrow and peripheral blood Tregs based on a statistical threshold of ≥ 95% change in cell phenotypes. Pink and red colour denote regions of phenotypic change identified by T-REX with a higher representation in bone marrow samples. Blue areas are enriched in peripheral blood. (Light colours: 85–95% change, dark colours: ≥95% change). Lower panel. DBSCAN clustering for the areas of ≥95% change. B Heatmap showing MEM enrichment of Treg markers in DBSCAN clusters. C Percentage of Tregs with high expression of CCR4, CCR6, CD161, CD39, HLA-DR in bone marrow and peripheral blood samples. Each patient is represented by a line. *p < 0.05 (two tailed paired Wilcoxon test). D PB-derived Tregs (CD4⁺CD25^highCD127^-/low), treated with both IL-6 and TGF-β, showed, after 10 days of culture, a significant higher expression of CD161 (One-way ANOVA, Tukey HSD test: *p < 0.05, **p < 0.01), if compared with cells untreated or treated with IL-6 or TGF-β alone. Untreated = cells only activated with T Cell TransAct™ and IL-2. Data are represented as mean ± SD of 3 independent experiments. E The graphs show the functional activity of CD161⁺ Tregs. Left panel. Suppression activity of CD161⁺ Tregs and expanded Tregs, referred as Tregs, on the proliferation of Trans Act stimulated-Tcons in a mixed lymphocyte reaction performed for 4 days. Data are presented as mean ± SD of 3 independent experiments (Two-way ANOVA, Sidak’s multiple comparison test; ****p < 0.0001; ***p = 0.0009). Right panel. Killing of the K562 cell line by Tcons, ratio Effector:Target 1:20. Where indicated, Tcons were co-coltured with CD161⁺ Tregs and Expanded Tregs, referred as Tregs, at different Tregs:Tcons ratio, 1:2 and 1:5 respectively (mean ± SD of 3 experiments; One-way ANOVA, Tukey’s multiple comparison test: Tcons alone vs + CD161⁺ Tregs ratio 1:2 **p = 0.001; vs + Tregs ratio 1:2 ****p < 0.0001; vs + CD161⁺ Tregs ratio 1:5 *p = 0.021; vs + Tregs ratio 1:5 ****p < 0.0001; + CD161⁺ vs Tregs ratio 1:2 *p = 0.011; + CD161⁺ vs Tregs ratio 1:5 *p = 0.015). F Flow cytometric analysis of the expression of RORγt, FOXP3, HLA-DR, CCR4, and CCR6 on CD161⁺ Tregs, defined as CD45⁺CD3⁺CD4⁺CD25^highCD127^-/lowCD161⁺ and Expanded Tregs, referred as Tregs and defined as CD45⁺CD3⁺CD4⁺CD25^highCD127^-/low (mean ± SD of 3 experiments; Two-way ANOVA, Sidak’s multiple comparison test, **p = 0.005; ****p < 0.0001).