STAT5B^N642H promotes cytokine independence of leukemic human NK cells. (A) IMC-1 and (B) KHYG-1 cell lines were transduced with nonmutant STAT5B (+STAT5B) or STAT5B^N642H (+STAT5B^N642H). As a control, cells were transduced with the empty vector, carrying only IRES-controlled eGFP (+GFP). After initial culture in presence of IL-2, transduced cells were completely deprived of IL-2. The percentage of transduced (GFP⁺) cells depicted as log2 fc relative to day 0 was monitored over time after cytokine withdrawal. (C) Schematic overview for transplantation of cytokine-independent STAT5B^N642H transduced, IL-2 dependent nonmutant STAT5B transduced, and nontransduced IMC-1 and KHYG-1 cells into immunodeficient NSG mice. (D) Survival analysis of NSG mice that received transplantation with IMC-1 (left) and KHYG-1 (right) (n ≥ 3 per cell line). (E) Relative quantification (percentages out of living cells) of nonmutant STAT5B or STAT5B^N642H transduced (GFP⁺) cells in blood, spleen, liver, and BM of NSG mice that received transplantation (n ≥ 2 per genotype; mean ± standard deviation). (F) Representative histograms for GFP signal within living cells in the liver (left) and BM (right) of NSG mice that received transplantation. (G) Representative images of hematoxylin and eosin stained liver and BM tissue from untransplanted (no NK) NSG mice and NSG mice that received transplantation with nonmutant STAT5B (KHYG1 + STAT5B) or STAT5B^N642H transduced KHYG-1 cells (KHYG1 + STAT5B^N642H). Levels of significance were calculated using the Mantel-Cox test (D) and the Mann-Whitney test (E). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.