Interaction of Nf1 and Sh2b3 in mice.Mx1-Cre, Nf1^flox/flox, and Sh2b3^−/− mice were intercrossed and Cre expression was induced with polyinosinic-polycytidylic acid at birth. Mice were euthanized when moribund or at end of trial. (A) Combined homozygous mutations in Nf1 and Sh2b3 cause higher WBCs than either mutation does alone. Mean plus or minus standard error of the mean is shown for each time point, with a line for the trend predicted by a mixed-effects linear model.¹⁷  Significance by lmertest²⁹  yields P < 10⁻⁴ for Nf1^Δ/Δ Sh2b3^−/− vs Nf1^Δ/Δ and for Nf1^Δ/Δ Sh2b3^−/− vs Sh2b3^−/−. (B) A similar interaction was observed when Sh2b3 mutation was heterozygous, though with a lesser degree of leukocytosis. Data are presented as in panel A. P < 10⁻⁴ for Nf1^Δ/Δ Sh2b3^+/− vs Nf1^Δ/Δ and for Nf1^Δ/Δ Sh2b3^+/− vs Sh2b3^+/−. (C) Flow cytometry of peripheral blood taken at time of euthanization shows that leukocytosis involves both lymphoid (CD19⁺ or CD4/8⁺) and myeloid (Mac1⁺) lineages, with a trend toward myeloid expansion when Nf1 and Sh2b3 are both mutant. Means and standard errors are shown. In multivariate regression, Sh2b3 mutation increased B and myeloid cells (analysis of variance [ANOVA] P < 10⁻⁵), Nf1 mutation increased T (P < .05) and myeloid (P < 10⁻³) cells, and their interaction further increased B and myeloid cells (P < .05). (D) Splenomegaly is more severe in mice with mutations in both Nf1 and Sh2b3 than in mice with mutations in either gene alone. Individual mice are shown as dots, and the line for each genotype shows the linear regression trend for age. In multivariate linear regression, spleen size increased with time (P < .001 by ANOVA), with the number of mutant Sh2b3 alleles (P < 10⁻¹¹), and with Nf1 mutation (P < 10⁻¹³). Spleen size increased faster in Nf1^Δ/Δ mice (P < .01); other interaction terms were not significant. (E) Kaplan-Meier survival analysis shows that Sh2b3 mutation causes a dose-dependent acceleration of mortality in Nf1-mutant mice (log rank P < .01 for 0 vs 1 and for 1 vs 2 Sh2b3 alleles). (F) Extramedullary expansion of HSCs (Lin⁻c-kit⁺Sca1⁻CD150⁺CD48⁻) in the spleen is markedly increased when both Nf1 and Sh2b3 are mutated (P < .03 for Nf1^Δ/Δ Sh2b3^−/− vs Nf1^Δ/Δ and for Nf1^Δ/Δ Sh2b3^−/− vs Sh2b3^−/− by Student t test; assessed at time of euthanization).