Matched sizes of activating and inhibitory receptor/ligand pairs are required for optimal signal integration by human Natural Killer cells

It has been suggested that receptor-ligand complexes segregate or co-localise within immune synapses according to their size, and this is important for receptor signaling. Here, we set out to test the importance of receptor-ligand complex dimensions for immune surveillance of target cells by human Natural Killer (NK) cells. NK cell activation is regulated by integrating signals from activating receptors, such as NKG2D, and inhibitory receptors, such as KIR2DL1. Elongating the NKG2D ligand MICA reduced its ability to trigger NK cell activation. Conversely, elongation of KIR2DL1 ligand HLA-C reduced its ability to inhibit NK cells. Whereas normal-sized HLA-C was most effective at inhibiting activation by normal-length MICA, only elongated HLA-C could inhibit activation by elongated MICA. Moreover, HLA-C and MICA that were matched in size co-localised, whereas HLA-C and MICA that were different in size were segregated. These results demonstrate that receptor-ligand dimensions are important in NK cell recognition, and suggest that optimal integration of activating and inhibitory receptor signals requires the receptor-ligand complexes to have similar dimensions

Impedance-based analysis of Natural Killer cell stimulation

The use of impedance-based label free cell analysis is increasingly popular and has many different applications. Here, we report that a real-time cell analyzer (RTCA) can be used to study the stimulation of Natural Killer (NK) cells. Engagement of NK cells via plate-bound antibodies directed against different activating surface receptors could be measured in real time using the label-free detection of impedance. The change in impedance was dependent on early signal transduction events in the NK cells as it was blocked by inhibitors of Src-family kinases and by inhibiting actin polymerization. While CD16 was the only receptor that could induce a strong change in impedance in primary NK cells, several activating receptors induced changes in impedance in expanded NK cells. Using PBMCs we could detect T cell receptor-mediated T cell activation and CD16-mediated NK cell activation in the same sample. Performing a dose-response analysis for the Src-family kinases inhibitor PP1 we show that T cells are more sensitive to inhibition compared to NK cells. Our data demonstrate that the RTCA can be used to detect physiological activation events in NK cells in a label-free and real-time fashion