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. 2024 Feb 28;221(4):e20221080. doi: 10.1084/jem.20221080

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© 2024 Zhang et al.

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Figure S4. — Peripheral blood and splenic T cell phenotypes in Sh2b3^E372K and Sh2b3^R530Q mice. (A) Representative flow cytometric plot showing the gating of double-negative (DN; CD4⁻CD8⁻), CD4⁺ (CD4⁺CD8⁻), and CD8⁺ (CD4⁻CD8⁺) T cells. (B and C) Dot plots showing the ratios of (B) blood and (C) splenic CD4/CD8 T cells in Sh2b3^E372K mice and splenic CD4/CD8 T cells in Sh2b3^R530Q mice. (D) Frequencies of DN T cells in the peripheral blood of Sh2b3^E372K mice. (E) Representative flow cytometric plot showing the gating of CD4 naïve T (T_naïve: CD44^lo/−Foxp3⁻), T_EM (CD44^hiFoxp3⁻), and T_reg (Foxp3⁺) cells. (F and G) Frequencies (F) and total numbers (G) of CD4 T_EM cells in the spleens of Sh2b3^E372K and Sh2b3^R530Q mice. (H and I) Frequencies (H) and total numbers (I) of T_reg cells in the spleen of Sh2b3^E372K (left) and Sh2b3^R530Q (right) mice. (J) Percentage suppression of effector T cells (T_eff) by T_reg cells in culture at various T_eff/T_reg ratios by using T_reg cells sorted from the spleens of Sh2b3^+/+ (n = 3), Sh2b3^E372K/+ (n = 3), and Sh2b3^E372K/E372K (n = 4) mice. (K) Representative flow cytometric plot showing the gating of CD8 T_naïve (CD44^lo/−CCR7^lo) and T_EM (CD44^hiCCR7⁻) cells. (L and M) Frequencies of (L) peripheral blood and (M) splenic CD8 T_EM cells in Sh2b3^E372K and Sh2b3^R530Q mice. (N) Total numbers of splenic CD8 T_EM in Sh2b3^E372K, and Sh2b3^R530Q mice. (O–T) Ratios of CD45.2⁺/CD45.1⁻ (O) splenic and (P) peripheral blood CD4/CD8 T ratios, (Q) peripheral blood DN T cells, (R) splenic T_EM cells, (S) T_reg cells, and (T) peripheral blood CD8 T_EM cells in 50:50 BM chimeras of CD45.1-Sh2b3^+/+ and CD45.2-Sh2b3^+/+/Sh2b3^E372K/E372K mice (n = 8). Results in B–D, F–J, L–N, and S are representative of two independent experiments. Results in O–R and T are from a single experiment. Each dot represents one mouse, and sample numbers for each group in B–D, F–J, and L–N are listed as follows: Sh2b3^E372K panels: +/+ (spleen: n = 5, blood: n = 7), E372K/+ (spleen: n = 5, blood: n = 11), E372K/E372K (spleen: n = 4, blood: n = 9); Sh2b3^R530Q panels: +/+ (spleen: n = 6, blood: n = 12), R530Q/+ (spleen: n = 6, blood: n = 8), R530Q/R530Q (spleen: n = 5, blood: n = 9). One-way ANOVA was used for statistical analysis of immunophenotyping data (B–D, F–I, and L-N), while Student’s t test was used for analyzing data from BM chimera experiments (O–T). Significance levels of one-way ANOVAs are indicated with asterisks as follows: *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.