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. 2024 Jan 5;15:324. doi: 10.1038/s41467-023-44382-w

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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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Cells were stimulated with phorbol 12-myristate 13-acetate/ionomycin/lipopolysaccharide (P/I/L) for 4-h and gated on singlets, lymphocytes, live/dead (L/D) stain^–CD45⁺CD4^–CD19⁺, followed by markers specific to each B-cell subset: B-1: B220^lo/–CD43⁺; and B-2: B220^hiCD43^–; B-2 cells were further subdivided as transitional-1 (T1): CD24^hiCD21^lo/–; transitional-2 (T2): CD24^hiCD21^int; transitional-2-marginal zone precursor (T2-MZP): CD24^hiCD21^hiCD23⁺; marginal zone (MZ): CD24^hiCD21^hiCD23^–; and follicular (FO): CD24^intCD21^intCD23⁺. The percent and total number of IL-10⁺ B cells in each B-cell subset in the spleen of WT and sIgM^–/– mice. (WT n = 11, sIgM^–/– n = 11; left, percent IL-10⁺ B cells: B1 ***p = 0.0002, T1 ****p < 0.0001, T2 ****p < 0.0001, T2-MZP ****p < 0.0001, MZ ****p < 0.0001, FO ****p < 0.0001; right, number of IL-10⁺ B cells: B1 ****p < 0.0001, T1 ****p < 0.0001, T2 ****p < 0.0001, T2-MZP ****p < 0.0001, MZ ****p < 0.0001, FO ****p < 0.0001). b Bone marrow (BM) cells were assessed after 4-h stimulation with P/I/L for B-cell fractions according to the Hardy scheme. Cells were gated on singlets, Live/Dead (L/D) stain^–CD3^–F4/80^–NK1.1^–Gr-1^–B220⁺. B220⁺ BM cells were further subdivided into Hardy Fractions (Fr.): Fr. A: B220⁺CD43⁺IgM^–BP1^–CD24^–; Fr. B: B220⁺CD43⁺IgM^–BP1^–CD24⁺; Fr. C-C’: B220⁺CD43⁺IgM^–BP1⁺CD24⁺; Fr. D: B220⁺CD43^–IgM^–IgD^–; Fr. E: B220⁺CD43^–IgM⁺IgD^–; and Fr. F: B220⁺CD43^–IgM^+/–IgD⁺. (IL-10^GFP n = 9, sIgM^–/–IL-10^GFP n = 10; percent IL-10⁺ B cells: Fr. A p = 0.3562, Fr. B p = 0.1823, Fr. C-C’ p = 0.3249, Fr. D p = 0.3555, Fr. E ****p < 0.0001, Fr. F ****p < 0.0001). c Breg phenotype identified by expression of TIM1⁺, CD1d^hiCD5⁺, or CD9⁺ among total splenic B cells without stimulation in sIgM^–/–IL-10^GFP mice. (TIM1⁺: IL-10^GFP n = 10, sIgM^–/–IL-10^GFP n = 10, ****p < 0.0001; CD1d^hiCD5⁺: IL-10^GFP n = 13, sIgM^–/–IL-10^GFP n = 13, *p < 0.0106; CD9⁺: IL-10^GFP n = 13, sIgM^–/–IL-10^GFP n = 13, ****p < 0.0001). d, e Splenocytes were stimulated for 4-h with P/I/L. d Frequency of IL-10⁺ B cells among each Breg phenotype. (TIM1⁺: IL-10^GFP n = 10, sIgM^–/–IL-10^GFP n = 10, ****p < 0.0001; CD1d^hiCD5⁺: IL-10^GFP n = 13, sIgM^–/–IL-10^GFP n = 13, ****p < 0.0001; CD9⁺: IL-10^GFP n = 9, sIgM^–/–IL-10^GFP n = 9, ****p < 0.0001). e Breg phenotype⁺ cells among IL-10⁺ B cells. (TIM1⁺: IL-10^GFP n = 10, sIgM^–/–IL-10^GFP n = 10, **p < 0.0089; CD1d^hiCD5⁺: IL-10^GFP n = 13, sIgM^–/–IL-10^GFP n = 13, ***p < 0.0003; CD9⁺: IL-10^GFP n = 9, sIgM^–/–IL-10^GFP n = 9, ***p < 0.0005). f Plasma cell (PC) identity was assessed by flow cytometry directly ex vivo on L/D stain^–CD45⁺CD3^–F4/80^–IgD^– lymphocytes using sIgM^–/– mice crossed with Blimp-1^GFP reporter mice (left) and among IL-10⁺ B cells after 4-h stimulation with P/I/L (right). (Blimp-1^GFPsIgM^{het or WT} n = 13, Blimp-1^GFPsIgM^–/– n = 8; Left, Percent Blimp-1⁺ of PCs, p = 0.8457; Right, percent Blimp-1⁺ of IL-10⁺ B cells: p = 0.2169. Data points indicate mean ± SD, and each symbol represents one mouse. P values were calculated using two-tailed Mann–Whitney U test (a–f); not significant (ns). Data are pooled from 2 (b; c–e: TIM1⁺/CD9⁺) or 3 (a; c–e: CD1d^hiCD5⁺; f) independent experiments. Source data are provided with this paper.