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. 2024 May 18;15:4232. doi: 10.1038/s41467-024-48611-8

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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 — Immune cells from different lymphoid tissues (as indicated) in Tlr9^fl/fl/Cd19Cre^+/- B6 (KO) and Tlr9^fl/fl/Cd19Cre^-/- B6 (Ctr) mice (all males, 18–22-week-old) were prepared and analyzed using flow cytometry after staining with different markers and Zombie Dye. All the samples were first gated on Zombie Dye negative cells followed by further gating on different immune cell subsets. A–F Immune-phenotype of B cells (gated on B220⁺) in KO and Ctr mice. A B cells were first gated on B220⁺ cells. The proportion of Pro-Pre B cells (CD24⁺,CD43^-, IgD^-, IgM^-), immature-Pre B cells (CD24⁺,CD43^-, IgD^-, IgM^mid), immature B cells (CD24⁺,CD43^-, IgD^-, IgM⁺), early mature B cells (CD24⁺,CD43^-, IgD⁺, IgM⁺) and late mature B cells (CD24⁺,CD43^-, IgD⁺, IgM^-), in the bone marrow (KO: n = 4 mice, Ctr: n = 5 mice). B The proportion of CD5⁺CD1d⁺ B cells in SPL, MLN, PP of the KO and the Ctr mice (KO: n = 5 mice, Ctr: n = 3 mice). C The proportion of B cells expressing IL-10 (KO: n = 4 mice s, Ctr: n = 3 mice). D The proportion of CD21⁺CD23^- marginal zone (MZ) B cells (KO: n = 5 mice, Ctr: n = 3 mice). E The proportion of CD21^midCD23⁺ follicular (FO) B cells (KO: n = 5 mice, Ctr: n = 3 mice). F The proportion of IgD^lowGL7⁺CD95⁺ germinal center (GC) B cells (KO: n = 5 mice, Ctr: n = 3 mice). G–M Immune-phenotype of T cells (gated on TCRβ⁺) in KO mice and Ctr mice by flow cytometric analysis. G The proportion of total TCRαβ⁺ T cells (KO: n = 4 mice, Ctr: n = 3 mice). H Frequency of CD4⁺ T cells (KO: n = 4 mice, Ctr: n = 3 mice). I The proportion of CD8⁺ T cells (KO: n = 4 mice, Ctr: n = 3 mice). J The proportion of CD44⁺CD62L^- memory CD4⁺ T cells (KO: n = 4 mice, Ctr: n = 3 mice). K The proportion of CD44⁺CD62L^- memory CD8⁺ T cells (KO: n = 4 mice, Ctr: n = 3 mice). L The proportionof CXCR5⁺ PD-1⁺ follicular T helper (T_FH) CD4⁺ T cells (KO: n = 5 mice, Ctr: n = 5 mice). M The proportion of CXCR5^-PD-1⁺ peripheral helper (T_PH) CD4⁺ T cells (KO: n = 4 mice, Ctr: n = 3 mice). All the experiments were repeated three times with similar results. Data were analyzed using two-tailed Student’s t-test. The data are presented as mean ± SD. The data presented in the figures are from one of the experiments. The total cell numbers of the immune cells in each subset is presented in Supplementary Fig. 3.