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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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Extended Data Fig. 1 — (a, b, h) Myeloid cell-scRNAseq data from murine B16 tumors (Mujal et al. 2022, GSE188548). (a) Heatmap depicting the expression of cell-type-defining genes. (b) Dotplot showing the expression of selected linage markers. (c) scRNAseq data of murine pan-tumor T cells (Andreatta et al. 2022, E-MTAB-9274). UMAP displaying T cell subsets (left) or Il23a⁺ T cells (right). (d) Representative FACS plot (left) of steady state murine skin CD45⁺cells (left) (pregated on live, CD45⁺cells). Representative FACS plots (right, pregated on live, CD45⁺, TCRγδ⁺) with gates of positive signal of respective IL23R-PE antibody clones of dendritic epidermal T cells (DETCs) (upper gate) and γδ^intermediate T cells (lower gate). Displayed data are from one experiment with n = 4. (e) Schematic illustration of the IL23R^tdTomatoallele. (f) Representative FACS plot (left) of murine skin T cells and histograms (right) depicting IL23R^tdTomato expression among T cell subsets from IL23R^tdTomato mice and total T cells (‘T cells’) from WT mice. Data shown from one representative experiment out of two independent experiments with n = 3. (g, k) T cells from i.d. inoculated B16 tumors in Foxp3^DTR-GFP IL23R^tdTomato mice were analyzed by flow cytometry on day 14. Data shown from one representative experiment out of two independent experiments with n = 5-6. (g) Heatmap displaying marker expression among tumor-infiltrating T cell subsets. (h) UMAP displaying Il23r⁺ myeloid cells. (i) Gating strategy used to FACS-sort γδ, CD8⁺, CD4⁺ T cells and Foxp3⁺ Treg cells from Foxp3^DTR-GFP mice. (j) Bar graph depicting relative Il23r mRNA expression level (qPCR) in FACS sorted T cells from steady state LNs or tdLNs, ndLNs and tumors from i.d. inoculated B16 tumor-bearing Foxp3^DTR-GFP mice. Pooled data from three independent experiments. Biologically independent samples: n = 2: ndLNs: γδ T cells, naïve CD4 Tcon; ndLNs: naïve CD8; tdLNs: CD8, CD4 Tcon; Tumor: CD8. n = 3: ndLNs: naïve Treg cell, naïve γδ T cells, γδ T cells; Tumor: γδ T cells, CD4 Tcon. n = 8: ndLNs Tumor Treg cell. n = 10: tdLNs: Treg cell; Tumor: Treg cell. Data are displayed as mean +/- SEM. (k) Heatmap displaying relative marker expression among IL23R⁺ and IL23R⁻Treg cellclusters. (l) Immunofluorescence-stainings of tdLNs from i.d. inoculated B16-tumor bearing IL23R^tdTomato mice. Foxp3 (green), IL23R^tdTomato (red), CD3 (white) DAPI (blue), merged (purple). Images (n = 4) are representatives from 2 independent experiments.

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