B cell tolerance breach in Sh2b3 ^E372K mice. (A) Design of SW_HEL-mHEL^3× BM chimera experiment for assessing B cell tolerance in Sh2b3^+/+, Sh2b3^E372K/+, and Sh2b3^E372K/E372K mice. (B) Representative flow cytometric plots showing the gating of HEL-specific (self-reactive) and non-HEL-specific (non-self-reactive) BM mature B cells in WT or mHEL^3× mice receiving BM from SW_HEL-Sh2b3^+/+, SW_HEL-Sh2b3^E372K/+, or SW_HEL-Sh2b3^E372K/E372K donors. (C–J) Dot plot demonstrating the frequencies of HEL-specific (C) BM mature B cells (D) BM immature B cells, (E) BM pre-B cells, (F) splenic transitional B cells, (G) splenic mature B cells, and (H) splenic MZ B cells, (I) splenic FO B cells, and (J) splenic ABCs as percentages of total lymphocytes in WT or mHEL^3× recipient mice receiving BM from SW_HEL-Sh2b3^+/+, SW_HEL-Sh2b3^E372K/+, or SW_HEL-Sh2b3^E372K/E372K mice. +/+ WT (n = 4), +/+ mHEL^3× (n = 4), E372K/+ WT (n = 8), E372K/+ mHEL^3× (n = 6), E372K/E372K WT (n = 4), E372K/E372K mHEL^3× (n = 6). Means are shown as bars. Results are representative of two independent experiments. Two-way ANOVA was performed on log₁₀-transformed data to meet the variance homoscedasticity; *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001. (K–L) Ratios of BM Igκ⁺/Igλ⁺ (K) immature and (L) mature B cells in Sh2b3^E372K mice (+/+: n = 5, E372K/+: n = 3, E372K/E372K: n = 3). Means are shown as bars in C–L. One-way ANOVA was used for statistical analysis. Results are representative of two independent experiments. (M and N) Calcium influx into the cytoplasm of peripheral blood (M) transitional and (N) mature B cells of Sh2b3^E372K mice (+/+: n = 2, E372K/+: n = 3, E372K/E372K: n = 3) at steady state, following treatment with anti-IgM antibody and treatment with ionomycin measured by the ratios of bound/unbound indo-1 AM ester to Ca²⁺. Results are representative of two independent experiments.