STAT4 promotes Bhlhe40 induction to drive protective IFN-g from natural killer cells during viral infection [STAT4 CUT&RUN]

NK cells represent a cellular component of the mammalian innate immune system, and mount rapid responses against viral infection, including the secretion of the potent anti-viral effector cytokine IFN-g. Following mouse cytomegalovirus (MCMV) infection, Bhlhe40 was the most highly induced transcription factor in NK cells among the basic helix-loop-helix family. Bhlhe40 upregulation in NK cells depended upon IL-12 and IL-18 signals, with the promoter of Bhlhe40 enriched for STAT4 and the permissive histone H3K4me3, and STAT4-deficient NK cells showing an impairment of Bhlhe40 induction and diminished H3K4me3. Transcriptomic and protein analysis of Bhlhe40-deficient NK cells revealed a defect in IFN-g production during MCMV infection, resulting in diminished protective immunity following viral challenge. Finally, we provide evidence that Bhlhe40 directly promotes IFN-g by binding throughout the Ifng loci in activated NK cells. Thus, our study reveals how STAT4-mediated control of Bhlhe40 drives protective IFN-g secretion by NK cells during viral infection. Overall design: To investigate the binding sites of STAT4 in NK cells during homeostasis and 3 hour IL-12 + IL-18 cytokine stimulation, we performed STAT4 CUT&RUN using WT and STAT4-/-(STAT4KO) mice. All cells for each conditions were sorted and cultured in complete IMDM + IL-15 media for 5 days prior. These cells were then washed and incubated in complete IMDM media without IL-15 for 1 hour prior to addition of appropriate stimulus, unstimulated or 3 hour IL-12 + IL-18. After stimulation, these CD49b+ NK cells were FACS sorted and CUT&RUN was performed, where replicates are biologically paired across stimulation conditions.