Data_Sheet_1_Inhibitory Receptor Crosslinking Quantitatively Dampens Calcium Flux Induced by Activating Receptor Triggering in NK Cells.PDF

Natural killer (NK) cell function is regulated by a balance between activating and inhibitory receptors, but the details of this receptor interplay are not extensively understood. We developed a flow cytometry-based assay system in which Ca²⁺ flux downstream of antibody-mediated activating receptor triggering was studied in the presence or absence of inhibitory receptor co-crosslinking. We show that the inhibitory influence on activating receptor-induced Ca²⁺ flux is quantitatively regulated, both on murine and human NK cells. Furthermore, both activating and inhibitory receptors operate in an additive way, suggesting that a fine-tuned balance between activating and inhibitory receptors regulate proximal NK cell signaling. We also demonstrate that murine NK cell expression of H2D^d lowered the capacity of Ly49A to deliver inhibitory signals after antibody crosslinking, suggesting that the cis interaction between H2D^d and Ly49A reduce the signaling capacity of Ly49A in this setting. Finally, we show that priming of NK cells by IL-15 rapidly augments Ca²⁺ flux after activating receptor signaling without attenuating the potential of inhibitory receptors to reduce Ca²⁺ flux. Our data shed new light on NK cell inhibition and raises new questions for further studies.