Impact of RUNX3 KO on H3K27ac abundance in human NK cells

The surface receptor CD8a is present on 20-80% of human (but not mouse) NK cells, yet its function on NK cells remains poorly understood. CD8a expression on donor NK cells was associated with a lack of therapeutic responses for leukemia patients in prior studies, thus we hypothesized that CD8a may impact critical NK cell functions. Here, we discovered that CD8a- NK cells had improved control of leukemia in xenograft models, compared to CD8a+ NK cells, likely due to an enhanced capacity for proliferation. Unexpectedly, CD8a expression was induced on approximately 30% of previously CD8a- NK cells following IL-15 stimulation. These ‘induced’ CD8a+ (‘iCD8a+’) NK cells had the greatest proliferation, responses to IL-15 signaling, and metabolic activity, compared to those that sustained existing CD8a expression (‘sustained CD8a+) or those that remained CD8a- (‘persistent CD8a-‘). These iCD8a+ cells originated from an IL-15Rb high NK cell population, with CD8a expression dependent on the transcription factor RUNX3. Moreover, CD8A CRISPR/Cas9 deletion resulted in enhanced responses through the activating receptor NKp30, possibly by modulating KIR inhibitory function. Thus, CD8a status identifies human NK cell capacity for IL-15-induced proliferation and metabolism in a time-dependent fashion and exhibits a suppressive effect on NK cell activating receptors CUT&TAG was performed on control and RUNX3 KO human NK cells, and abundance of histone modifications H3K27ac was quantified. Briefly, freshly isolated NK cells were electroporated with RUNX3 or TRAC sgRNA and Cas9 mRNA, cultured in vitro in 5 ng/mL IL-15 for 9 days, and processed using the Active Motif CUT&TAG-IT assay kit