KIT D816V induces TNF expression. (A) TNF serum concentrations of patients with mastocytosis (n = 40) and of an age- and sex-matched control cohort were measured by ELISA. Results represent median and IQR. (B-C) TNF expression and secretion levels were measured in the human MC lines HMC-1.1 (KIT D816V^–), HMC-1.2 (KIT D816V⁺), ROSA^{KIT WT}, and ROSA^{KIT D816V}. ROSA cells were cultivated in medium containing 3 ng/mL SCF. Cells were seeded at a density of 0.5 × 10⁶/mL and incubated for 5 hours. TNF mRNA expression of the respective cells was analyzed by quantitative reverse transcription PCR (qRT-PCR) (B), TNF secretion into the supernatants was analyzed by ELISA after 24 hours of cultivation at a density of 1 × 10⁶/mL (C). (D-E) TNF expression levels in HMC-1.2 cells were determined after treatment with different concentrations of the KIT D816V-targeting drugs midostaurin (D) and avapritinib (E) for 5 hours. (F) Expression of TNF was measured in HMC-1.2 cells with doxycycline-inducible RNAi against KIT or a nontargeting control (NTC) after treatment with or without doxycycline (1 μg/mL) for 48 hours. (G-H) Mo7e cells, lentivirally transduced with an empty vector (CO), pWPI KIT WT, or pWPI KIT D816V, were seeded at a density of 1 × 10⁶ cells per mL and cultivated in presence or absence of granulocyte-macrophage colony-stimulating factor (GM-CSF; 2 ng/mL) for 24 hours. TNF mRNA expression levels in the cells were measured by qRT-PCR (G), and TNF secretion into the supernatants was analyzed by ELISA (H). Results represent the mean and standard error of the mean of 3 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.