Human and murine SH2B3 protein functionality and gross phenotypes of Sh2b3Δ, Sh2b3E372K, and Sh2b3R530Qmice and transitional B cell phenotypes in Sh2b3E372Kand Sh2b3R530Qmice, and chimeras of Sh2b3E372Kmice. (A) Relative GAS activity of unstimulated HEK293 cells overexpressing WT or SH2B3 variants identified in SLE and HC cohorts. Sample numbers for each condition are listed as follows: empty (n = 21), WT (n = 21), R43C (n = 9), C133Y (n = 12), E208Q (n = 21), E400K (n = 15), A536T (n = 18), Q540X (n = 18), and R566Q (n = 21). Means are shown as bars, and all conditions were compared to cells transfected with WT SH2B3. lmer with emmeans using the experiment as a blocking factor was used for the statistical analyses. *: P < 0.05, ***: P < 0.001, ****: P < 0.0001. (B) Relative GAS activity of HEK293T cells overexpressing WT or SH2B3 variants listed in gnomAD database at MAF > 0.001 in the presence of IFN-γ (50 ng/ml) stimulation. Results are pooled from four independent experiments and shown as the percentage of GAS activity compared to the EV control. Each dot is a biological replicate and the mean of three technical replicates. Sample numbers for each condition are listed as follows: empty (n = 30), WT (n = 11), W262R (n = 9), T165S (n = 9), F182L (n = 12), S186I (n = 12), P242S (n = 12), R80C (n = 12), A36E (n = 9), E78K (n = 9). lmer with emmeans using the experiment as the blocking factor was used for the statistical analyses. ****: P < 0.0001. (C) Representative direct binding curves for the WT and E372K SH2B3 SH2 domains to a JAK2 pY813 peptide. Results are representative of two independent experiments. (D) Melting temperatures for WT (unfilled) and E372K (filled) SH2B3 SH2 domains in their apo form and after the addition of the phosphomimetic phenyl phosphate (PP), JAK2 pY813, JAK3 pY785, and EPOR pY454 peptides. Results are pooled from at least three independent experiments. All WT: n = 3; E372K: apo n = 3, JAK2 n = 7, JAK3 n = 6, EPOR n = 4, PP n = 6. Means are shown as bars. Two-way ANOVA with Šídák’s adjustment was used for statistical analyses. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001. (E) Representative flow cytometric plot showing the gating strategy of B cells (CD19+CD3−) and T cells (CD19−CD3+). (F and G) Dot plots showing frequencies of B (F) and T cells (G) as percentages of total splenic lymphocytes in Sh2b3E372K (left panel) and Sh2b3R530Q (right panel) mice. (H and I) Total numbers of splenic transitional B cells (H) and T1 B cells (I) in Sh2b3E372K (left) and Sh2b3R530Q (right) mice. (J) CD45.2+/CD45.1+ T1 B cell ratios among splenic B cells in 50:50 BM chimeras of CD45.1-Sh2b3+/+ and CD45.2-Sh2b3+/+/Sh2b3E372K/E372K mice. (K) Frequencies of T2 B cells as percentages of splenic B cells in Sh2b3Δ (left), Sh2b3E372K (middle), and Sh2b3R530Q (right) mice. (L) Total numbers of splenic T2 B cells in Sh2b3E372K (left) and Sh2b3R530Q (right) mice. (M) CD45.2+/CD45.1+ T2 B cell ratios among splenic B cells in 50:50 BM chimeras of CD45.1-Sh2b3+/+ and CD45.2-Sh2b3+/+/Sh2b3E372K/E372K mice. (N) Total numbers of splenic T3 B cells in Sh2b3E372K (left), and Sh2b3R530Q (right) mice. (O) Frequencies of of splenic T3 B cells in Sh2b3Δ (left), Sh2b3E372K (middle), and Sh2b3R530Q (right) mice. (P) Frequencies of (P) CD45.2+/CD45.1+ T3 B cell ratios among splenic B cells in 50:50 BM chimeras of CD45.1-Sh2b3+/+ and CD45.2-Sh2b3+/+/Sh2b3E372K/E372K mice. Results in F–P are representative of two to three independent experiments. Each dot represents one mouse. Sample numbers for each group in F–I, K, L, N, and O are listed as follows: Sh2b3Δ panel: +/+ (n = 5), Δ/+ (n = 7), Δ/Δ (n = 6); Sh2b3E372K panel: +/+ (n = 5 in F and G, n = 4 in others), E372K/+ (n = 5 in F and G, n = 3 in others), E372K/E372K (n = 4); Sh2b3R530Q panels: +/+ (n = 6 in F and G, n = 5 in others), R530Q/+ (n = 6 in F–H, n = 7 in others), and R530Q/R530Q (n = 5–9). n = 10 in J and P. Means are indicated as bars. Student’s t tests were used for the statistical analysis in J, M, and P. One-way ANOVA was used for the statistical analyses in F–I, K, L, N, and O. Significance levels in lmer ANOVAs are indicated with asterisks, while those in multiple Student’s t tests are indicated with hashes. *: P < 0.05, **: P < 0.01, ***/###: P < 0.001, ****/####: P < 0.0001.
Figure S1.

Human and murine SH2B3 protein functionality and gross phenotypes of Sh2b3 Δ , Sh2b3 E372K , and Sh2b3 R530Q mice and transitional B cell phenotypes in Sh2b3 E372K and Sh2b3 R530Q mice, and chimeras of Sh2b3 E372K mice. (A) Relative GAS activity of unstimulated HEK293 cells overexpressing WT or SH2B3 variants identified in SLE and HC cohorts. Sample numbers for each condition are listed as follows: empty (n = 21), WT (n = 21), R43C (n = 9), C133Y (n = 12), E208Q (n = 21), E400K (n = 15), A536T (n = 18), Q540X (n = 18), and R566Q (n = 21). Means are shown as bars, and all conditions were compared to cells transfected with WT SH2B3. lmer with emmeans using the experiment as a blocking factor was used for the statistical analyses. *: P < 0.05, ***: P < 0.001, ****: P < 0.0001. (B) Relative GAS activity of HEK293T cells overexpressing WT or SH2B3 variants listed in gnomAD database at MAF > 0.001 in the presence of IFN-γ (50 ng/ml) stimulation. Results are pooled from four independent experiments and shown as the percentage of GAS activity compared to the EV control. Each dot is a biological replicate and the mean of three technical replicates. Sample numbers for each condition are listed as follows: empty (n = 30), WT (n = 11), W262R (n = 9), T165S (n = 9), F182L (n = 12), S186I (n = 12), P242S (n = 12), R80C (n = 12), A36E (n = 9), E78K (n = 9). lmer with emmeans using the experiment as the blocking factor was used for the statistical analyses. ****: P < 0.0001. (C) Representative direct binding curves for the WT and E372K SH2B3 SH2 domains to a JAK2 pY813 peptide. Results are representative of two independent experiments. (D) Melting temperatures for WT (unfilled) and E372K (filled) SH2B3 SH2 domains in their apo form and after the addition of the phosphomimetic phenyl phosphate (PP), JAK2 pY813, JAK3 pY785, and EPOR pY454 peptides. Results are pooled from at least three independent experiments. All WT: n = 3; E372K: apo n = 3, JAK2 n = 7, JAK3 n = 6, EPOR n = 4, PP n = 6. Means are shown as bars. Two-way ANOVA with Šídák’s adjustment was used for statistical analyses. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001. (E) Representative flow cytometric plot showing the gating strategy of B cells (CD19+CD3−) and T cells (CD19−CD3+). (F and G) Dot plots showing frequencies of B (F) and T cells (G) as percentages of total splenic lymphocytes in Sh2b3E372K (left panel) and Sh2b3R530Q (right panel) mice. (H and I) Total numbers of splenic transitional B cells (H) and T1 B cells (I) in Sh2b3E372K (left) and Sh2b3R530Q (right) mice. (J) CD45.2+/CD45.1+ T1 B cell ratios among splenic B cells in 50:50 BM chimeras of CD45.1-Sh2b3+/+ and CD45.2-Sh2b3+/+/Sh2b3E372K/E372K mice. (K) Frequencies of T2 B cells as percentages of splenic B cells in Sh2b3Δ (left), Sh2b3E372K (middle), and Sh2b3R530Q (right) mice. (L) Total numbers of splenic T2 B cells in Sh2b3E372K (left) and Sh2b3R530Q (right) mice. (M) CD45.2+/CD45.1+ T2 B cell ratios among splenic B cells in 50:50 BM chimeras of CD45.1-Sh2b3+/+ and CD45.2-Sh2b3+/+/Sh2b3E372K/E372K mice. (N) Total numbers of splenic T3 B cells in Sh2b3E372K (left), and Sh2b3R530Q (right) mice. (O) Frequencies of of splenic T3 B cells in Sh2b3Δ (left), Sh2b3E372K (middle), and Sh2b3R530Q (right) mice. (P) Frequencies of (P) CD45.2+/CD45.1+ T3 B cell ratios among splenic B cells in 50:50 BM chimeras of CD45.1-Sh2b3+/+ and CD45.2-Sh2b3+/+/Sh2b3E372K/E372K mice. Results in F–P are representative of two to three independent experiments. Each dot represents one mouse. Sample numbers for each group in F–I, K, L, N, and O are listed as follows: Sh2b3Δ panel: +/+ (n = 5), Δ/+ (n = 7), Δ/Δ (n = 6); Sh2b3E372K panel: +/+ (n = 5 in F and G, n = 4 in others), E372K/+ (n = 5 in F and G, n = 3 in others), E372K/E372K (n = 4); Sh2b3R530Q panels: +/+ (n = 6 in F and G, n = 5 in others), R530Q/+ (n = 6 in F–H, n = 7 in others), and R530Q/R530Q (n = 5–9). n = 10 in J and P. Means are indicated as bars. Student’s t tests were used for the statistical analysis in J, M, and P. One-way ANOVA was used for the statistical analyses in F–I, K, L, N, and O. Significance levels in lmer ANOVAs are indicated with asterisks, while those in multiple Student’s t tests are indicated with hashes. *: P < 0.05, **: P < 0.01, ***/###: P < 0.001, ****/####: P < 0.0001.

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