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. 2023 Mar 27;20(5):489–511. doi: 10.1038/s41423-023-00989-z

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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 — pMHCII-NP-induced TR1-like cells are transcriptionally homogeneous but oligoclonal and coexist with a Tet⁺ TFH-like subpopulation that contains identical clonotypes. A t-SNE plot of Smartseq2-based scRNAseq data for sorted Tet⁺ and Tet⁻ cells from NOD mice treated with BDC2.5 mi or InsB_9-23/IA^g7-NPs (from n = 5 mice for Tet⁺ cells and 15 mice for Tet⁻cells; aliquots of the sorted cells were also used for other experiments). The data are from 4 experiments. B Seurat clustering analysis of the Tet⁺ pools from A showed the presence of two clusters for each pMHCII type. C Two-dimensional plot of the average log2FC values for the differentially expressed genes (adjusted P value < 0.05) between pMHCII-NP-induced Tet⁺ TR1-like, Tet⁺ TFH-like and Tet⁻ cell types pooled from samples from BDC2.5/IA^g7-NP-treated NOD mice (n = 5 mice for Tet⁺ cells and 15 mice for Tet⁻ cells) and samples from Fla_462-472/IA^b-NP-treated C57BL/6 mice (n = 6 mice for Tet⁺ cells and 5 mice for Tet⁻ cells). The data were obtained by SmartSeq2 scRNAseq in 2 experiments. The X-axis shows Tet⁺ TFH-like vs. Tconv cells, and the Y-axis shows Tet⁺ TR1-like vs. Tconv cells. The dot color represents the cell subset specificity of differential gene expression. Only the genes with the greatest differential expression are labeled. D, E Distribution of unique TCR sequences in cells in the Tet⁺ pools arising in response to treatment with two different pMHCII-NP types (BDC2.5mi- or InsB_9-23/IA^g7-NPs) in NOD mice. The histogram shows the distribution of the different TCRαβ clonotypes identified vs. the number of cells (clones) expressing each TCRαβ pair. The data are from 1 (D) and 3 (E) experiments. F tSNE plot from (B) showing the cluster locations for cells with TCRαβ pairs expressed by more than one cell (in black). The data correspond to BDC2.5mi- or InsB_9-23/IA^g7-NP-treated mice from 4 experiments. G Venn diagram from F showing the distribution of repeated TCRαβ pairs in clusters #1 (TFH-like) vs. #2 (TR1-like). Most (34/46) of the clonotypes found in the TFH-like cluster (#1) were also found in the TR1-like cluster (#2) (34/69)