STAT3 mutations K658N, K392R, and M394T in studied patients. (A) Schematic representation of STAT3 protein domains with the observed mutations marked as black lines. Germ-line and somatic mutation hotspots for HIES^5,6  and LGL leukemia^11-13  are indicated as green and blue bars, respectively, at top. (B) Crystallographic structure of STAT3 dimer (RCSB Protein Data Bank code 1BG1). K658N, K392R, and M394T mutations are indicated as red dots. (C-D) HEK293 cells containing STAT3-responsive luciferase were transfected with empty, WT, and mutant STAT3 overexpression plasmids with or without IL-6 stimulation. K392R and M394T significantly increased STAT3 transcriptional activity in basal and stimulated conditions. Error bars represent standard error of the mean (n = 6; C). The K658N mutant showed hypersensitivity to IL-6 stimulation in low concentrations. Error bars represent standard error of the mean (n = 3; D). Two-way analysis of variance, *P < .05, **P < .01, and ***P < .001. (E) No significant increase in pSTAT3^Y705 phosphorylation was observed when HEK293 cells were transfected with mutant STAT3-overexpression constructs. Equal amounts of parallel-derived whole cell lysates were loaded per condition. α-tubulin and STAT3 were used as loading and expression controls, respectively. +, presence of IL-6 stimulation; –, absence of IL-6 stimulation. (F) In peripheral blood, no significant increase in STAT3 phosphorylation was noted in studied patients. Color change indicates relative pSTAT3^Y705 expression. Forward panel, K392R; middle panel, K658N; back panel, healthy control (n = 3, value range presented in parentheses).