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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. 9 — pMHCII-NP-induced formation of TR1-like and terminally differentiated TR1 cells from PD-1^hiCXCR5^hi precursors. A Cartoon showing the experimental approach used to track the development of TR1 cells from PD-1^hiCXCR5^hi precursors. Briefly, we transferred FACS-sorted PD-1^hiCXCR5^hi CD4⁺ T cells from the spleens of female NOD mice treated with 5 doses of BDC2.5 mi/I-A^g7-NPs (1.5 × 10⁵) into female NOD.Scid hosts and treated the hosts with 10 additional doses of pMHCII-NPs. B UMAP-based feature plots for representative TFH-associated gene transcripts (and Foxp3) corresponding to the PD-1^hiCXCR5^hi CD4⁺ T cells used for transfer. C FACS profile of BDC2.5 mi/I-A^g7 tetramer⁺ CD4⁺ cells arising in PD-1^hiCXCR5^hi cell-transfused NOD.Scid hosts upon BDC2.5 mi/I-A^g7-NP treatment (10 doses over 5 weeks). The data correspond to cells pooled from 2 hosts from 1 experiment. D UMAP plots showing the Tet⁺ TFH, TR1-like and TR1 subsets within the BDC2.5 mi/I-A^g7 tetramer⁺ CD4⁺ cell pool from NOD.Scid hosts (C), after sorting and scRNAseq analysis. E Feature plots for representative TFH and TR1-associated gene transcripts in the UMAP dimensionality reduction analysis from (D). F FACS profiles for BDC2.5 mi/I-A^g7 tetramer⁺ CD4⁺ cells arising in PD-1^hiCXCR5^hi cell-transfused NOD.Scid hosts upon treatment with BDC2.5 mi/I-A^g7-NP (10 doses over 5 weeks) and the anti-CD25 mAb or rat IgG (10 doses of 500 μg i.p). The data correspond to cells pooled from 2 hosts for each treatment group from 1 experiment. The value shown corresponds to the percentage of tetramer⁺ cells within the CD4⁺ gate. G scRNAseq profiles for the tetramer⁺ cells from (F). Left, UMAP plots; right, relative percentages of TFH, TR1-like and TR1 cells within the tetramer⁺ cell pool. H FACS profiles for BDC2.5 mi/I-A^g7 tetramer⁺ CD4⁺ cells arising in NOD.Scid hosts transfused with PD-1^hiCXCR5^hi cells from NOD.Cd4-Cre or NOD.Cd4-Cre.Prdm1^loxP/loxP mice upon treatment with BDC2.5 mi/I-A^g7-NPs (10 doses over 5 weeks). The data correspond to cells pooled from 2 hosts for each treatment group from 1 experiment. The value shown corresponds to the percentage of tetramer⁺ cells within the CD4⁺ gate. I scRNAseq profiles for the tetramer⁺ cells from (H). Left, UMAP plots; right, relative percentages of TFH, TR1-like and TR1 cells within the tetramer⁺ cell pool