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. 2024 Feb 14;25(3):512–524. doi: 10.1038/s41590-024-01755-7

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© The Author(s) 2024

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. 4 — a–f, Foxp3^Cre-YFP/+Il23r^fl/fl female mice were inoculated i.d. with B16 cells and T_reg cells from tumors, and tdLNs were analyzed by flow cytometry on day 9 or 14 after injection. Data are shown from one representative experiment out of two independent experiments with n = 5–6. a, UMAP and FlowSOM clustering displaying T_reg cell subsets in tumors. b, Frequency plots of Il23r-KO and WT T_reg cells out of total T_reg cells in tumors. c, Spiral plot displaying the effect size of differential marker expression between Il23r-KO and WT T_reg cells in tumors. d, Representative contour plots depicting T_reg cells on day 14 after tumor inoculation. e, UMAP and FlowSOM clustering displaying T_reg cells in tdLNs on day 14 after tumor inoculation (left). A heat map depicting relative marker expression is shown on the right. f, Frequency plots of subsets of Il23r-KO and WT T_reg cells in tdLNs on day 14 after tumor injection. Statistical significance was determined by two-way ANOVA with a Sidak’s post hoc test. g,h, Scatter plot (g) and violin plots (h) displaying frequencies of IL-23R⁺ T_reg and eT_reg cells among total T_reg cells. Data are pooled from one to two experiments with n = 3–5. i, Il23r^fl/fl and Foxp3^Cre-YFPIl23r^fl/fl mice were inoculated i.d. with MC38 tumor cells. T_reg cells were analyzed by flow cytometry on day 14 after inoculation. Violin plots display the median expression (median fluorescence intensity (MFI)) of cytokines. The data shown are from one experiment with n = 6–12. j, Extracellular acidification rate (ECAR) measurement of T_reg cells from spleens and LNs of Foxp3^Cre-YFPIl23r^fl/fl or Foxp3^Cre-YFPmice under the specified conditions after stimulation with anti-CD3/anti-CD28, IL-2 and IL-23 for 72 h. Data are representative of the results of two independent experiments with n = 4. k, Quantification of glycolysis in Il23r-KO and WT T_reg cells. l–n, Ex vivo suppression of CellTrace Violet-labeled CD4⁺ conventional T (T_con) cell proliferation by Il23r-KO and WT T_reg cells (l and m) or WT T_reg cells ± anti-IL-23R (n). Data shown are from two independent experiments with n = 5. Statistical significance was assessed by two-way ANOVA with a Sidak’s post hoc test. Data in f, i, k, l and n are displayed as mean ± s.e.m. Statistical significance in c and i–k was determined using two-tailed t-tests; 2-DG, 2-deoxyglucose; NS, not significant.

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