Regulation of calcium flux by activating and inhibitory receptor crosstalk in NK cells

Abstract: The birth of a new born child kicks off the immune system to be on call and to respond promptly to threats from the environment. Natural Killer cells, classified as a member of the innate lymphoid cell family, express various activating, inhibitory, adhesion and cytokine receptors as well as MHC class I molecules on their surfaces, by which they interact with surrounding cells and perform various immunological function. The balance between the stimulation of these receptors determines the functional output of NK cells. NK cells are critically important for tumor control and viral clearance, but several basic cellular mechanisms and pathways regulating their function, both in mice and humans, remain largely unknown. This PhD thesis tries to explore a few aspects of these by studying NK cell proximal signaling downstream of activating and inhibitory receptors, and also explores the phenomenon of MHC class I signaling in NK cells. The work focuses mainly on mouse NK cells, but with a few outlooks into experiments with human NK cells as well. The c-Abl non-receptor tyrosine kinase, a known pro-inflammatory protein in B and T cells, was proposed to function in inhibitory signaling in human NK cells. With the given parameters measured, we could find no role for c-Abl in murine NK cell education, using mice lacking c-Abl specifically in NK cells. The questions asked allowed us to design a novel real-time measurement of proximal inhibitory signaling using calcium flourometry as a read-out, which revealed no role of c-Abl in inhibitory signaling in murine NK cells. However, a possible role of c-Abl as a negative regulator of IFN-γ production was suggested. We further used this assay to show that upon in vivo dendritic cell depletion, NK cell calcium flux after triggering of the ITAM-dependent receptor NK1.1 was drastically reduced, suggesting that the DC crosstalk might be needed to maintain proximal signaling capacity in NK cells. Further exploration of this assay led us to generate data to determine that inhibitory signaling operates in a quantitative and additive way in murine and human NK cells. Furthermore, H2Dd ligand interaction in cis, in the NK cell membrane itself, compromised the ability of the Ly49A inhibitory receptor to transmit inhibitory signals inside the cell. We also provided evidence that IL-15, a vital cytokine for NK cell survival, ‘primes’ activating receptor signaling but does not directly affect the impact of inhibitory receptor signaling on these pathways. Finally, the MHC class I allele H2Kb was shown to function as a signalling mediator that both synergized with activating receptor signaling and triggered calcium release by itself. In this case, a potential cis interaction with Ly49C did not seem to affect H2Kb signal transmission. I believe that the studies included in my thesis help us to understand the regulation of NK cell activity better. They also emphasise the usefulness of modifying and developing in-house methods to explore NK cell signaling properties. In the future, as the mechanisms that fine-tune NK cell activation and inhibition continue to be identified, NK cells can hopefully be used better as a prominent therapeutic tool in the clinic.