Extended Data Fig. 7: TCF1 and LEF1 effect on regulatory function of B-1 cells.
From: TCF1 and LEF1 promote B-1a cell homeostasis and regulatory function
(a) IL-10 secretion measured by cytometric bead Arrays (CBA) from TCF1WTLEF1WT (n = 7) and TCF1ΔLEF1Δ (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 TCF1WTLEF1WT (n = 5) or TCF1ΔLEF1Δ (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 (CD1dhi CD5+), immature transitional-2 (T2-MZP) (B220+ IgMhi CD21hi 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 TCF1WTLEF1WT 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 TCF1WTLEF1WT or TCF1ΔLEF1Δ 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 TCF1WTLEF1WT and TCF1ΔLEF1Δ 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.
