Precisely controlled cell-cell interactions with single-cell transcriptome readout identify a time-demand threshold response of human oncolytic NK cells

Cell communication via cell-cell interactions is a fundamental biological process shared by all multicellular organisms and broadly relevant to human health and disease. Activation and function of cells of the im-mune system particularly depends on direct cell-cell interactions, which are fast, transient and hard to study. To characterize NK-cell intrinsic gene expression programs that are activated upon target cell en-counter, we develop a cell interaction profiling system (CIPS) and investigate the genome-wide transcrip-tional responses of individual interacting cells with precise temporal control over the first minutes of inter-action. We utilize di-electrophoretic fields to initiate cell-cell contact of fluorescence microscopy-informed, selected single cells and separate them again with precise control of interaction time followed by single-cell transcriptomics as readout. Applying CIPS to the interaction of NK cells with cancer cells, we find that in-teractions shorter than 15 minutes lead to downregulation of a gene set including the tyrosine kinases TYK2 and LCK, required for NK-cell effector function and tumor surveillance. Dissecting the molecular effects associated with a rare genetic disease that compromises NK cell function, we show that this re-sponse is independent of target cell lysis. Exploiting our method’s ability to orchestrate sequential series of cell-cell interactions, we further demonstrate that the downregulation of the identified gene signature is overcome by repeatedly challenging NK cells with cancer cells. In conclusion, we introduce a method for precise dissection of cell-cell interactions with unprecedented temporal resolution; our observations sug-gest a model where NK cells downregulate genes relevant for engagement of lytic function if the interaction with a potential target cell is too short, but allows responsiveness to further interactions, representing a “time-demand threshold (TDT)”. 447 RNA-seq samples of cells analyzed with the cell interaction profiling system (CIPS) & FACS purified control samples