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. 2023 Apr 20;4(5):665–681. doi: 10.1038/s43018-023-00547-6

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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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Extended Data Fig. 5 — a-e, Flow cytometry quantification of FOXP3⁺ Tregs from primary (P), RT, RT + Conc.IT and RT + Adj.IT treated tumor-bearing mice at indicated timepoints. a, Ki67⁺ Tregs (gated from CD45⁺CD11b⁻CD3⁺CD4⁺FOXP3⁺ T cells) in PDG-Ink4a tumors (Primary n = 5, RT n = 7, RT + Conc.IT n = 9, RT + Adj.IT n = 9 mice). b,c FOXP3⁺ Tregs (gated from CD45⁺CD11b⁻CD3⁺CD4⁺ T cells) in the superficial cervical lymph nodes (LN) (b) and blood (c) of PDG-Ink4a tumor bearing mice (b: Primary n = 4, d6 RT n = 9, d12 RT n = 10, d6 RT + Conc.IT n = 9, d12 RT + Conc.IT n = 7, d6 RT + Adj.IT n = 5, d12 RT + Adj.IT n = 6 mice. c: Primary n = 4, d6 RT n = 8, d12 RT n = 9, d6 RT + Conc.IT n = 10, d12 RT + Conc.IT n = 13, d6 RT + Adj.IT n = 10, d12 RT + Adj.IT n = 10 mice). d,e FOXP3⁺ Tregs (gated from CD45⁺CD11b⁻CD3⁺CD4⁺ T cells) in tumors (d) and LN (e) of PDG-p53 tumor-bearing mice (d: Primary n = 4, d6 RT n = 8, d12 RT n = 8, d6 RT + Conc.IT n = 7, d12 RT + Conc.IT n = 7, d6 RT + Adj.IT n = 9, d12 RT + Adj.IT n = 5 mice. e: d6 RT n = 5, d12 RT n = 4, d6 RT + Conc.IT n = 4, d12 RT + Conc.IT n = 2, d6 RT + Adj.IT n = 4, d12 RT + Adj.IT n = 3 mice). f, UMAP projection and unsupervised FlowSOM clustering of CD4⁺ T cell subpopulations in PDG-Ink4a glioblastoma 6d post treatment initiation identified 4 distinct subpopulations of CD4⁺ T cells (Pop 0-3). g, Heatmap depicting the MFI of activation markers for each subpopulation identified in (f). h, UMAP density projections plot of CD4 + T cell subpopulations from (f). i, UMAP projection and unsupervised FlowSOM clustering of CD4⁺ T cell subpopulations in PDG-Ink4a glioblastoma 12d post treatment initiation identified 4 distinct subpopulations of CD4⁺ T cells (Pop 0-3). j, Heatmap depicting the MFI of activation markers for each subpopulation identified in (i). k, UMAP density projections plot of CD4 + T cell subpopulations from (i) l-m, Stacked bar plot displaying the distribution of CD4⁺ T cells subpopulations in RT + Conc.IT and RT + Adj.IT treated tumors at d6 (l) and d12 (m) post treatment initiation. For f-h,l: d6 RT + Conc.IT n = 4, d6 RT + Adj.IT n = 4 mice. For i-k,m: d12 RT + Conc.IT n = 6, d12 RT + Adj.IT n = 7 mice. Statistics: one-way ANOVA with Benjamini, Krieger and Yekutieli correction for multiple testing (a-e) and two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli (m). Data are represented as mean ± S.E.M. (a-e) or - S.E.M. (l,m).

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