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. 2022 May 11;605(7911):728–735. doi: 10.1038/s41586-022-04718-w

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© The Author(s) 2022

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. 7 — (a) Proliferation of HNSCC-derived CD4+ T responder cells from tumor (left) or from blood (right) in an in vitro suppression assay with IL-1R1- Tregs (light red) and IL-1R1+ Tregs (dark red) from tumor. Representative histograms show dilution of Cell Trace Violet (CTV) after 4 days. n = 4. (b) Concentration of Granzyme B, IL-2 and IFN-g in the culture supernatants of the 4-day suppression assays shown in main Figure 4a. (c) Proliferation of HNSCC-derived CD8+ T responder cells in an in vitro suppression assay with titrated amounts of IL-1R1- Tregs (left plot) and IL-1R1+ Tregs (right plot). Histograms show CTV dilution after 4 days of culture with Treg-to-Teff ratios of 1:4, 1:2 and 1:1 (top to bottom). (d) Plots depict representative post-sort purity of HNSCC-Treg populations used for the stimulation experiments in main Figure 4b. (e) Expression of IL-1R1 after two days of in vitro culture either unstimulated or in the presence of Gibco anti-CD3/CD28 beads for Tregs sorted from peripheral blood (blue), IL-1R1- (light red) and IL-1R1+ Tregs (dark red) from HNSCC. n = 3. (f) Volcano plots showing differential gene expression of SMART-Seq bulk RNAseq data (250 sorted cells) of blood Tregs, HNSCC IL-1R1- and IL-1R1+ after 2 days of culture with anti-CD3/CD28/CD2 beads with (light and dark red) or without IL-1 (grey). (g) Transcripts per million for FOXP3, Helios (IKZF2) and CTLA4 from the bulk RNAseq data for the indicated time points and culture conditions. n = 4 for the d2 time point, n = 6 for the d1 time point. (h) CD25 median fluorescence intensity (MFI) on the indicated Treg populations after 2 days of culture with anti-CD3/CD28/CD2 beads +/− IL-1. n = 3 for blood and n = 4 for the HNSCC samples. (i) Volcano plots showing differential gene expression of SMART-Seq bulk RNAseq data (250 sorted cells) of blood Tregs and HNSCC IL-1R1+ Tregs after 2 days either unstimulated or with anti-CD3/CD28/CD2 beads. Number of DE genes upregulated is highlighted in bold.