FIG 1:. Impaired terminal NK cell maturation in STAT5b-deficient patients. A, Western blot analysis of STAT5b on total extracts from peripheral blood mononuclear cells (PBMCs) from healthy donors and STAT5b-deficient patients. Polyclonal STAT5a and STAT5b antibodies were used. B, NK cells were identified as CD56brightCD3− and CD56dimCD3− NK cells gated on lymphocytes. C, Frequency of total NK cells in peripheral blood. D, CD56dim NK cells from healthy donors and STAT5b-deficient patients were studied by flow cytometry. The Median fluorescence intensity (MFI) data from age- and sex-matched healthy donor are included (light blue circles) in addition to data from unmatched controls acquired at the time of the experiment (dark blue circles). Horizontal bars represent the median, and the vertical bars indicate the standard deviation. Statistical significance was analyzed using the unpaired Student’s t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. HD, healthy donors (dark blue circle); previously reported healthy female age and gender matched controls (light blue circle); Pt, STAT5b-deficient patients, Pt 1 (red box); Pt 2 (green box).

FIG 2:. Stat5b−/− mice have impaired terminal NK cell maturation. A, Bone marrow was stained for lineage (LIN; CD3, CD19, Ly6G, Ter119), CD122, NK1.1, and NKp46. Flow cytometry was used to analyze the percentage of NK precursor cells (NKp), immature NK cells (iNK), and mature NK cells (mNK) gated on lymphocytes. Box plots depict mean ± Min/Max (n≥10 per genotype). One-way ANOVA and Bonferroni’s Multiple Comparison Test were used for statistical analysis of each developing stage. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. B, Splenocytes of mice were stained for CD3, NK1.1, CD49b, and NKp46, and then analyzed by flow cytometry for the percentage of CD3−NK1.1+, CD3−CD49b+ and CD3−NKp46+ NK cells gated on lymphocytes, respectively. Box plots depict mean ± Min/Max (n≥10 per genotype). C, Splenic CD3−NK1.1+ NK cells were analyzed for CD27 and CD11b expression by flow cytometry. Box plots depict mean ± Min/Max (n≥10 per genotype). One-way ANOVA and Bonferroni’s Multiple Comparison Test were applied for statistical analysis of each maturation stage.

FIG 3:. CD56bright NK cell subset with impaired cytokine production and higher VEGF-A expression. A, Median fluorescence intensity (MFI) for each cytokine and receptor in CD56bright NK cell after stimulation with PMA and ionomycin. Median fluorescence intensity (MFI) data from age- and sex-matched healthy donor are included (light blue circles) in addition to data from unmatched controls acquired at the time of the experiment (dark blue circles).Horizontal bars represent the median, and the vertical bars indicate the standard deviation. Statistical significance was analyzed using the unpaired Student’s t-stest. *p<0.05, **p<0.01, ****p<0.0001. HD, healthy donors (dark blue circle); previously reported healthy female age and gender matched controls (light blue circle); Pt, STAT5b-deficient patients, Pt 1 (red box); Pt 2 (green box). B, Expression of VEGF-A gene validated by qPCR of total NK cells from patient 1 and a healthy donor. Stat5b+/− and WT mice were challenged with RMA lymphoma cell line subcutaneously for 10 days, **p<0.01. C, Representative pictures of tumors and D, VEGF-A expression in tumor lysates, **p<0.01.