TCF1 and LEF1 promote B-1a cell homeostasis and regulatory function

Abstract

B-1 cells are innate-like immune cells abundant in serosal cavities with antibodies enriched in bacterial recognition, yet their existence in humans has been controversial^1-3. The CD5⁺ B-1a subset expresses anti-inflammatory molecules including IL-10, PDL1 and CTLA4 and can be immunoregulatory^4-6. Unlike conventional B cells that are continuously replenished, B-1a cells are produced early in life and maintained through self-renewal⁷. Here we show that the transcription factors TCF1 and LEF1 are critical regulators of B-1a cells. LEF1 expression is highest in fetal and bone marrow B-1 progenitors, whereas the levels of TCF1 are higher in splenic and peritoneal B-1 cells than in B-1 progenitors. TCF1-LEF1 double deficient mice have reduced B-1a cells and defective B-1a cell maintenance. These transcription factors promote MYC-dependent metabolic pathways and induce a stem-like population upon activation, partly via IL-10 production. In the absence of TCF1 and LEF1, B-1 cells proliferate excessively and acquire an exhausted phenotype with reduced IL-10 and PDL1 expression. Furthermore, adoptive transfer of B-1 cells lacking TCF1 and LEF1 fails to suppress brain inflammation. These transcription factors are also expressed in human chronic lymphocytic leukaemia B cells and in a B-1-like population that is abundant in pleural fluid and circulation of some patients with pleural infection. Our findings define a TCF1-LEF1-driven transcriptional program that integrates stemness and regulatory function in B-1a cells.

Fig. 1. TCF1 and LEF1 are highly expressed in B-1 cells and B-1a cells are reduced in TCF1–LEF1-deficient mice.

a, Gene expression in the different clusters projected on uniform manifold approximation and projection (UMAP) of scRNA-seq from total peritoneal B cells. b,c, Flow cytometry plots showing TCF1 or LEF1 expression (b) or both (c) in peritoneal cavity B cells. d, Flow cytometry plots showing gating strategy of B220^low, B220^intermediate and B220^high cells from peritoneal cavity B cells, CD5 and PtC expression and histogram of TCF1 and LEF1. FMO, fluorescence minus one. e–g, Flow cytometry plots showing CD38⁺ and CD38⁻ B cells, CD43, CD5, TCF1 and LEF1 expression from the indicated compartments (e,f) and quantification of the percentage of CD43⁺CD5⁺ cells within the CD19⁺ cells in healthy donor (HD; n = 7), CLL (n = 11), pleural infection (n = 3) and sleep disordered breathing/otitis media with effusion ((SDB/OME); n = 6; g). CTV, CellTrace Violet; FSC, forward scatter; PBMC, peripheral blood mononuclear cell; PF, pleural fluid. h, Contour plots (top) and quantification (bottom) showing PtC reactivity in the indicated subsets from freshly processed PBMC of HD (n = 3). i, Fold change of expression of TCF1 and LEF1 between CD43⁺CD5⁺ cells and CD43⁻CD5⁻ cells in patients with CLL. j–m, Contour plots and quantification of the percentage of B-1, B-2, B-1a and B-1b cells from the peritoneal cavity (j,k); B-1 and follicular B cells from the spleen (l,m) from TCF1^WTLEF1^WT (n = 4 or 6), TCF1^Δ (n = 4), LEF1^Δ (n = 6) or TCF1^ΔLEF1^Δ (n = 4 or 5). n, Frequency of the indicated peritoneal and splenic B cell populations in sublethally irradiated Rag1^−/− recipient mice 6 weeks after reconstitution with fetal embryonic day 14.5 liver cells from mice of TCF1^WTLEF1^WT (n = 4) or TCF1^ΔLEF1^Δ (n = 7). Each symbol represents an individual mouse and bars represent median values. Data are representative of n = 5 (e), n = 3 (k–m) and n = 4 (n) experiments. Data are from n = 5 (g) experiments and n = 3 donors (h). Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey multiple-comparison test (g,k,m) and two-tailed Mann–Whitney U-test (n) or two-way ANOVA (h). The exact P values are shown. Source data

Fig. 2. TCF1 and LEF1 are required for B-1a self-renewal.

a,b, Contour plots (a) and quantification (b) of positive cells and TCF1 or LEF1 expression in peritoneal B-1a cells from 4 (n = 5), 8 (n = 4) and 16 (n = 3) weeks of age mice. c, Contour plots showing B-1a cells from TCF1^WTLEF1^WT and TCF1^ΔLEF1^Δ mice at 4 (top) and 14 (bottom) weeks of age, gated on peritoneal CD19⁺B220⁻ cells (left) and quantification (right). d, Experimental diagram (top left), dot plots (right) and quantification (bottom left) of BrdU-labelled donor-derived B-1a or B-2 cells: TCF1^WTLEF1^WT (n = 3) and TCF1^ΔLEF1^Δ (n = 4). e,f, Circular plots show cell numbers of the different clonotypes (e) with corresponding heatmap (number of clones) and quantification of peritoneal B cell clones with a particular V_H–V_L pairing from TCF1^WTLEF1^WT and TCF1^ΔLEF1^Δ mice. Clones composed of Ighv11-2/Igkv14-126, Ighv12-3/Igkv4-91 and Ighv9-3/Iglv2 were labelled as c1, c2 and c3, respectively (f, top) and their frequency quantified (f, bottom). g, Contour plots (left) and quantification (right) of PtC liposome-binding CD5⁺ PC B-1 cell numbers from TCF1^WTLEF1^WT (n = 6) and TCF1^ΔLEF1^Δ (n = 4) mice. h, Quantification of the percentage of peritoneal and splenic B-1 cells from ETS1^WT (Ets1^+/+.Cre^Cd19; n = 5) and ETS1^Δ (Ets1^flox/flox.Cre^Cd19; n = 4) mice. Each symbol represents an individual mouse, and the bars indicate median values. Results are representative of n = 2 (a–d,g,h) experiments. Statistical analysis was performed using two-tailed Welch’s t-test (d), Mann–Whitney U-test (g,h), one-way ANOVA with Tukey multiple-comparison test (b) or two-way ANOVA (c). The exact P values are shown. Source data

Fig. 3. TCF1 and LEF1 in B-1a cell mitosis, development and gene expression.

a, Gene set enrichment analysis of bulk RNA-seq from TCF1^WTLEF1^WT and TCF1^ΔLEF1^Δ B-1a cells shows enrichment for the indicated signalling pathways. The black dotted lines indicate neutral enrichment. b, Correlation analysis between expression of TCF1 or LEF1 and expression of MYC (top) or BCL2 (bottom) in peritoneal B-1a, B-1b and B-2 cells (n = 6). gMFI, geometric mean fluorescence intensity. c, UMAP of scRNA-seq profiles from the four indicated sorted cell subsets (FL B1P and BM B1P), B-1 cells from the spleen (SP B1) and peritoneal cavity (PC B1) barcoded and pooled for sequencing. d,e, Developmental trajectory of the combined (d) or individual (e) FL B1P, BM B1P, SP B1 and PC B1 populations constructed by Monocle2. f, Comb plots displaying the incidence and amplitude of the indicated genes in each subset shown in panel d. g, Dot plot presentation of hallmark gene sets upon gene set enrichment analysis for differentially expressed genes in B-1a cells of the indicated genotypes. h, Dot plot presentation of expression of the indicated genes in human prenatal B-1 cells according to single, double or no TCF1 and LEF1 expression. Panel h was adapted with permission from ref. , AAAS. Data are representative of n = 2 experiments. Statistical analysis was performed using two-tailed Pearson correlation analysis (b). The significant hallmark gene sets in MSigDB (adjusted P < 0.05) for each contrast based on the hypergeometric testing using cluster profiler package (g). Source data

Fig. 4. TCF1 and LEF1 promote IL-10 production by B-1a cells and their control of CNS inflammation.

a, UMAP showing clonal BCR usage in clusters of peritoneal B cells and expression of Tcf7, Cd5 and Ctla4. b, Gene module scores associated with gene signatures. c, PDL1 histograms and gMFI in the indicated cell subsets (n = 8). d,e, Flow cytometry plots and quantification of IL-10⁺ cells in TCF1^high or TCF1^low PC B1 cells (n = 6; d) and IL-10⁺ cells within PC B1 cells from TCF1^WTLEF1^WT (n = 11) and TCF1^ΔLEF1^Δ mice (n = 5; e). f, Expression of CD5, CD21, CD1d and B220 by CD19^lowIL-10⁺ (blue), CD19^highIL-10⁺ (red) and IL-10⁻ (grey) cells with or without stimulation. g, TCF1 and LEF1 histograms (left) and quantification (right) in the cell subset shown in panel f (n = 3). h, Pseudocolour plots and quantification of IL-10⁺ splenic B cells from TCF1^WTLEF1^WT (n = 6) and TCF1^ΔLEF1^Δ (n = 4) mice. i,j, Correlation analysis between TCF1 or LEF1 expression and the percentage of IL-10⁺ cells (n = 6; i) or expression of CD5 and the percentage of IL-10⁺ in peritoneal B-1a cells from TCF1^WTLEF1^WT (n = 5) and TCF1^ΔLEF1^Δ (n = 5; j). k, Mean clinical scores of mice treated with either PBS (n = 5) or adoptively transferred with peritoneal B-1 cells from TCF1^WTLEF1^WT (n = 5) or TCF1^ΔLEF1^Δ (n = 5). l–n, Contour plots of GRM2A-positive cells within the indicated cell populations from the peritoneal cavity (l,m) and the spleen (n). o, Identification of GRM2A-positive B-1 cells in the EAE mouse brain; representative cells are from the area labelled ‘i’. Representative cells from the areas labelled ‘ii–iv’ are shown in Extended Data Fig. 7h. Each symbol represents an individual mouse, and the bars represent the median values (c–e,g,h). Data are presented as mean ± s.e.m. (k). Data are representative of n = 2 experiments (c–e,g–k) and n = 3 mice (l). Statistical analysis was performed using two-tailed Pearson correlation analysis (i,j), two-tailed Mann–Whitney t-test (h), one-way ANOVA with Tukey multiple-comparison test (c–e,g) and two-way ANOVA (k). The exact P values are shown. Source data

Fig. 5. TCF1 and LEF1 deficiency in B-1 cells prevents emergence of stem-like B-1 cells and promotes exhaustion.

Purified peritoneal cavity TCF1^WTLEF1^WT or TCF1^ΔLEF1^Δ B-1 cells were cultured with LPS, IL-10R-Fc antibody or IgG1 isotype for 3 days. a,b, Density plots (a) and quantification (b) of the mean ratio of plasma cells (Pl.Cs; CD138⁺):non-plasma cells (CD138⁻; b). c, Percentage of non-plasma cells undergone more than two divisions. d, Density plots (left) and mean ratio of plasma cells:non-plasma cells (right) after 3 days of LPS stimulation with or without IL-10. e, Representative gating strategy of MYC^high and MYC^low cells at division 3 (D3). f,g, Density plots of MYC expression in proliferating cells (f) and quantification of the percentage of MYC^high and MYC^low in each division measured by CTV dilution (g). h, Representative gating strategy of MYC^high and MYC^low cells at undivided (D0) and D3 (top) and the ratio of MYC^low:MYC^high (bottom). i, Histograms of CD86, FCRL5, CD11b and CD19 expression among MYC^high and MYC^low cells. j, Histograms (left) and quantification (right) of CD86, FCRL5 and CD19 expression on TCF1^WTLEF1^WT or TCF1^ΔLEF1^Δ B-1 cells stimulated with LPS for 3 days that are either D0 or have undergone D3 or D5. k, Histogram of PDL1 expression in D0 and D1 of TCF1^WTLEF1^WT or TCF1^ΔLEF1^Δ B-1 cells stimulated with or without LPS for 3 days. l, Chromatin immunoprecipitation followed by sequencing (ChIP–seq) analysis of TCF1 binding in CD4⁺CD8⁺ thymocytes and ATAC-seq analysis at the Pdl1 locus on B-1a cells from Vh12/Vk4 transgenic mice. Panel l was adapted from refs. ^,, Springer Nature Ltd. Data are presented as mean ± s.d. Data are representative of n = 2 experiments with a total of 9 mice per genotype, with peritoneal cavity cells from 3 mice being pooled together. Statistical analysis was performed using one-way ANOVA with Tukey multiple-comparison test (b–d,h) or two-way ANOVA (j). The exact P values are shown. Source data

Extended Data Fig. 1. TCF7 and LEF1 expression in mice and human B cells.

(a) UMAP plots showing the expression of Bhlhe41 after removing all BCR genes in peritoneal total B cells. (b) Expression of Tcf7 and Lef1 across the ImmGen Database. Follicular B cells (FO); memory B cells (Mem B); marginal zone B cells (MZ), germinal centre B cells (GC), transitional 1 (T1), transitional 2 (T2) and transitional 3 (T3) B cells. (c) Flow cytometry plots present TCF1 or LEF1 positive B cells in spleen. (d-e) Gating strategies of B-1a, B-1b and B-2 cells in peritoneal cavity and B-1, B-2, FO and MZ cells in spleen (d) and gMFI quantification of expression of TCF1 and LEF1 in B cell subsets from peritoneal cavity (n = 5) and spleen (n = 4) (e). (f-g) Contour plots (f) and gMFI quantification of TCF1 and LEF1 in B cells subsets from bone marrow (n = 5) (g). (h) Contour plots showing gating strategy and TCF1 and LEF1 expression in CD43⁺CD5⁺ B cells from PBMC and pleural fluid (PF) of patient. (i) Fold change of expression of TCF1 and LEF1 between CD43⁺CD5⁺ cells and CD43⁻CD5⁻ cells in each donor (n = 3). (j) Histogram and contour plots (top) and quantification (bottom) of expression of CD27, CD24, TCF1, LEF1, IgM and IgA in indicated cell subsets. Fold change of expression of TCF1 and LEF1 between CD43⁺CD5^int cells or CD43^lo/−CD5⁺ and CD43^-CD5^- cells in HD (n = 6). (k). Contour plot showing PtC reactive cells in total B cells from PBMC. Each symbol represents an individual mouse or donor, and bars represent median values. Data are representative of n = 2 experiments. Statistical analysis was performed using one way ANOVA with Tukey multiple-comparison test (e,g), two way ANOVA (j), Paired t test (i). The exact P values are shown. Source data

Extended Data Fig. 2. TCF1 and LEF1 are required for formation of normal peripheral B-1a numbers.

(a) Quantification of cell numbers of B-1 and B-1a from peritoneal cavity and B-1 cells from spleen in TCF1^WTLEF1^WT (n = 4 or 5), TCF1^Δ (n = 4), LEF1^Δ (n = 5), TCF1^ΔLEF1^Δ (n = 4 or 5) mice. (b) Quantification of percentage of pre-pro B, pro B, pre-B, immature B, mature B and B-1 progenitors (B-1P) from bone marrow in TCF1^WTLEF1^WT (n = 5) and TCF1^ΔLEF1^Δ (n = 5) mice. (c) gMFI of CD5 expression within B-1a cells in peritoneal cavity between TCF1^WTLEF1^WT (n = 7) and TCF1^ΔLEF1^Δ (n = 7) mice. (d) Frequency of bone marrow (BM) B cell populations from lethally irradiated Rag1^–/– recipient mice 6 weeks after transferring foetal liver cells (E14.5) from TCF1^WTLEF1^WT (n = 4) and TCF1^ΔLEF1^Δ (n = 7). (e) Frequency of the indicated peritoneal and splenic B cell populations in sub-lethally irradiated Rag1^–/– recipient mice 7 weeks after reconstitution with bone marrow cells from mice with TCF1^WTLEF1^WT (n = 5), TCF1^Δ (n = 6), LEF1^Δ (n = 7), TCF1^ΔLEF1^Δ (n = 6) mice. Each symbol represents an individual mouse and bars represent median values. Data are representative of n = 3 (a-c), n = 4 (d-e) experiments, Statistical analysis was performed using two-tailed Mann-Whitney U test (b-d), one way ANOVA with Tukey multiple-comparison test (a,e). The exact P values are shown. Source data

Extended Data Fig. 3. TCF1 and LEF1 effect on B-1a development and maintenance.

(a) Diagram depicting key stages of B-1a development. (b-c) Gating strategy (b) and frequency (c) of CD19⁺B220^-Lin^-IgM^-CD93⁺ B-1 progenitors (B-1P) from E18.5 foetal liver cells (TCF1^WTLEF1^WT n = 3; TCF1^ΔLEF1^Δ n = 5) and bone marrow (BM) at postnatal days 1 (TCF1^WTLEF1^WT n = 7; TCF1^ΔLEF1^Δ n = 3), 3 (TCF1^WTLEF1^WT n = 2; TCF1^ΔLEF1^Δ n = 5) and 9 (TCF1^WTLEF1^WT n = 5; TCF1^ΔLEF1^Δ n = 4), and 8 weeks old (TCF1^WTLEF1^WT n = 8; TCF1^ΔLEF1^Δ n = 7). (d) Flow cytometry stains and quantification of TrB-1a identified as CD93⁺IgM⁺CD19⁺B220^loCD5⁺ from spleens of 9-day old mice of TCF1^WTLEF1^WT (n = 5) and TCF1^ΔLEF1^Δ (n = 4). (e) Flow cytometry stains and quantification of the CD5⁺ cells in B-1P from bone marrow at postnatal day 1 (TCF1^WTLEF1^WT n = 7; TCF1^ΔLEF1^Δ n = 3) and 3 (TCF1^WTLEF1^WT n = 2; TCF1^ΔLEF1^Δ n = 5) mice. (f) Gating strategy of CD5⁺ Ki67⁺ cells in CD19⁺ Lin^-CD93⁺IgM⁺ in TCF1^WTLEF1^WT spleen at neonatal day 9. (g) Ki67⁺ cells within TrB-1a cells and TrB cells as CD93⁺IgM⁺CD19⁺B220⁺CD5^– from spleens of 9-day old mice (n = 6). (h) Flow cytometry plots of Ki67 positive cells and histogram of TCF1 or LEF1 in splenic B-1a cells in 4 weeks old mice (Young, n = 5) and 8-16 weeks old mice (Adult, n = 7). (i) Treatment protocol of mixed sorted B-1a cells of (50% ly5b TCF1^WTLEF1^WT + 50% ly5ba TCF1^WTLEF1^WT) and (50% ly5b TCF1^ΔLEF1^Δ + 50% ly5a TCF1^WTLEF1^WT) in Rag1^–/– recipient mice at 2 months after intraperitoneal injection (left), Flow cytometry plots (middle) and quantification of percentage of B-1a cells in peritoneal cavity (TCF1^WTLEF1^WT n = 5; TCF1^ΔLEF1^Δ n = 5) and spleen (TCF1^WTLEF1^WT n = 4; TCF1^ΔLEF1^Δ n = 5) (right). Each symbol represents an individual mouse and bars represent median values. Data are representative of n = 2 experiments (c-e,g-i). Statistical analysis was performed using two-tailed Mann-Whitney t test (c-e,g-i). The exact P values are shown. Source data

Extended Data Fig. 4. TCF1 and LEF1 effect on BCR signalling of B-1 cells.

(a) Histogram (top) and gMFI quantification (bottom) of phosphorylation (p-) status of various BCR signaling components: p-BLNK (TCF1^WTLEF1^WT n = 6; TCF1^ΔLEF1^Δ n = 4); p-Btk (TCF1^WTLEF1^WT n = 6; TCF1^ΔLEF1^Δ n = 4); p-PLCγ2 (TCF1^WTLEF1^WT n = 6; TCF1^ΔLEF1^Δ n = 4); p-Syk (TCF1^WTLEF1^WT n = 5; TCF1^ΔLEF1^Δ n = 7); p-ERK1/2 (TCF1^WTLEF1^WT n = 5; TCF1^ΔLEF1^Δ n = 7) in B-1a cells. (b) Flow cytometry plots (top) and gMFI (bottom) of Igκ and Igλ in B-1a and B-2 cells from TCF1^WTLEF1^WT (n = 7) and TCF1^ΔLEF1^Δ (n = 5). (c) Histogram (left) and gMFI (right) of CD19 in B-1P from foetal liver (TCF1^WTLEF1^WT n = 3; TCF1^ΔLEF1^Δ n = 5) and B-1a cells (TCF1^WTLEF1^WT n = 4; TCF1^ΔLEF1^Δ n = 4) from peritoneal cavity. Each symbol represents an individual mouse and bars represent median values. Data are representative of n = 2 experiments (a-c). Statistical analysis was performed using two-tailed Mann-Whitney t test (a,c), one way ANOVA with Tukey multiple-comparison test (b). The exact P values are shown. Source data

Extended Data Fig. 5. TCF1 and LEF1 effect on specificity and clonotype of B-1 cells.

(a) Coverage-based diversity accumulation curve for Hill numbers of order (q = 0, 1 and 2), i.e., species richness, Shannon entropy and Simpson index, respectively in peritoneal B cell (top) or B-1 cells (bottom). (b) Circular plot of cell number of different clonotypes in TCF1^WTLEF1^WT and TCF1^ΔLEF1^Δ peritoneal B-1 cells. (c) Flow cytometry plots and quantification of frequency of PtC⁺B-1a cells in TCF1^WTLEF1^WT (n = 12) and TCF1^ΔLEF1^Δ (n = 8) mice. (d) Total serum IgM and IgG3 were determined by ELISA between TCF1^WTLEF1^WT (n = 5) and TCF1^ΔLEF1^Δ (n = 7) mice. (e) Flow cytometry plots and quantification of percentage of spleen B-1 cell derived plasma cells (B-1 PC) (Dump^–CD19^–CD43⁺IgM⁺IgD^–/loCD138⁺) (TCF1^WTLEF1^WT n = 5; TCF1^ΔLEF1^Δ n = 5) and B-1 plasmablasts (B-1 PB) (Dump^–CD19⁺CD43⁺IgM⁺IgD^–/lo CD138⁺) (TCF1^WTLEF1^WT n = 12; TCF1^ΔLEF1^Δ n = 10) in B-1 cells. (f) Flow cytometry plots and quantification of percentage of IgG3⁺ from peritoneal B-1a cells in TCF1^WTLEF1^WT (n = 5) and TCF1^ΔLEF1^Δ (n = 5) mice. Each symbol represents an individual mouse and bars represent median values. Data from n = 3 experiments in total (c, e), or representative of n = 3 experiments (d-f). The lines represent the diversity estimates. The shaded area around the curve represents the 95% confidence intervals (95% CI) (a). Statistical analysis was performed using two-tailed Mann-Whitney U test (c,e-f), one way ANOVA with Tukey multiple-comparison test (d). The exact P values are shown. Source data

Extended Data Fig. 6. Signalling pathways of TCF1 and LEF1 on mice and human B-1 cells.

(a) Expression-expression plot of transcriptomes of B-1a cells isolated from TCF1^WTLEF1^WT and TCF1^ΔLEF1^Δ mice. Superimposed B-1a signature genes from published RNA-seq data which over- or under-represented in peritoneal B-1a versus B-1b cells. (b) Expression heatmap of B-1a signature genes, with the colour representing normalized RPM for each gene across different samples. (c) Dot plot presentation of Hallmark gene sets according to GSEA for differentially expressed genes in TCF1^WTLEF1^WT and TCF1^ΔLEF1^Δ B-1a cells. (d) Cnet plot presentation of enriched Hallmark pathways (P < 0.05, FDR < 0.05) upon GSEA. Size = the number of differentially expressed gene which belongs to the enriched Hallmark pathway. Fold change difference between TCF1^WTLEF1^WT and TCF1^ΔLEF1^Δ B-1a cells is shown. (e) Frequency of TCF1 and LEF1 expressed cells in human B-1 clusters. (f) Dot plot representation of Tcf7 and Lef1 mRNA expression in different subsets of human prenatal tissues. Panels e,f were adapted from ref. , AAAS. (g) Flow cytometry plots and quantification of B-1-enriched cells from PBMCs from 4 SCID patients and 3 HD. (h) ChIP-seq analysis of TCF1 binding in CD4⁺CD8⁺ thymocytes and ATAC-seq analysis at Cd5, Myc and Ets1 on B-1a cells from Vh12/Vk4 transgenic mice. Panel h was adapted from refs. ^,, Springer Nature Ltd. The significant hallmark gene sets in MSigDB (adjustment p-value < 0.05) for each contrast based on the hypergeometric testing using cluster profiler package (c). Source data

Extended Data Fig. 7. TCF1 and LEF1 effect on regulatory function of B-1 cells.

(a) IL-10 secretion measured by cytometric bead Arrays (CBA) from TCF1^WTLEF1^WT (n = 7) and TCF1^ΔLEF1^Δ (n = 7) B-1 cells cultured in media with LPS for 3 days. (b) Flow cytometry plots and quantification of percentage of CD138⁺Lag3⁺cells in the spleen of TCF1^WTLEF1^WT (n = 5) or TCF1^ΔLEF1^Δ (n = 5) mice 3 days after injection with LPS. (c) gMFI quantification of TCF1 and LEF1 in B cells subsets from B-1a (CD19⁺ B220^– CD5⁺ CD23^–), B10 (CD1d^hi CD5⁺), immature transitional-2 (T2-MZP) (B220⁺ IgM^hi CD21^hi CD23⁺), marginal zone B cells (MZB) (CD19⁺ B220⁺ CD23^– CD21⁺), Follicular B cells (FO) (CD19⁺ B220⁺ CD23⁺ CD21⁻) from spleen of C57BL/6J mice (n = 6). (d) Flowchart of EAE immunization. (e) Expression of Gramd2a across the ImmGen Database. The figure was created using ImmGen (https://immgen.org). (f) Schematics of Gramd2a-T2A-iCre-P2A-mApple strain. (g) Frequency of GRM2A positive cells within B-1a, B-1b and B-2 populations from peritoneal cavity (n = 5) and B-1 and B-2 cells from spleen (n = 6). (h) Identification of GRM2A positive B-1 cells in EAE mouse brain. representative cells from each labelled areas from Fig. 4o (i) Flow cytometry plots of plasma cell (PC) (CD138⁺) and non-plasma cell (CD138⁻) cells from purified peritoneal cavity TCF1^WTLEF1^WT B-1 cells cultured with LPS and isotype control or anti-mouse IL-10R for 3 days. (j) Flow cytometry plots of CD138⁺ cells after magnetically purified splenic TCF1^WTLEF1^WT or TCF1^ΔLEF1^Δ B-2 cells were cultured with LPS and anti-mouse IL-10R or isotype control for 3 days. (k) Quantification of gMFI of MYC in each division of B-2 cells stimulated with LPS for 3 days. (l) Quantification of Zeb2 expression in TCF1^WTLEF1^WT and TCF1^ΔLEF1^Δ B-1a cells by RNA-seq. Each symbol represents an individual mouse, Data are presented as mean valued +/– S.D. Data are representative of n = 2 experiments (b-c,g), peritoneal cavity cells from three mice are pooled together (a,k). Images are representative of n = 3 mice (l). Statistical analysis was performed using two-tailed Mann-Whitney U test (a-b,l) and one way ANOVA (c,g). The exact P values are shown. Source data