MHC class I molecules co-stimulate NK1.1 signaling and enhance Ca2+ flux in murine NK cells.

Simultaneous triggering of NK1.1 and MHC class I on NK cells gives a higher Ca2+ flux response compared to triggering the NK1.1 receptor alone. The data suggest a novel costimulatory role for MHC class I molecules on NK cell responses.

Short-term IL-15 priming leaves a long-lasting signalling imprint in mouse NK cells independently of a metabolic switch.

IL-15 priming of NK cells is a broadly accepted concept, but the dynamics and underlying molecular mechanisms remain poorly understood. We show that as little as 5 min of IL-15 treatment in vitro, followed by removal of excess cytokines, results in a long-lasting, but reversible, augmentation of NK cell responsiveness upon activating receptor cross-linking. In contrast to long-term stimulation, improved NK cell function after short-term IL-15 priming was not associated with enhanced metabolism but was based on the increased steady-state phosphorylation level of signalling molecules downstream of activating receptors. Inhibition of JAK3 eliminated this priming effect, suggesting a cross talk between the IL-15 receptor and ITAM-dependent activating receptors. Increased signalling molecule phosphorylation levels, calcium flux, and IFN-γ secretion lasted for up to 3 h after IL-15 stimulation before returning to baseline. We conclude that IL-15 rapidly and reversibly primes NK cell function by modulating activating receptor signalling. Our findings suggest a mechanism by which NK cell reactivity can potentially be maintained in vivo based on only brief encounters with IL-15 trans-presenting cells.

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

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 H2Dd lowered the capacity of Ly49A to deliver inhibitory signals after antibody crosslinking, suggesting that the cis interaction between H2Dd 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.

Independent control of natural killer cell responsiveness and homeostasis at steady-state by CD11c+ dendritic cells.

During infection and inflammation, dendritic cells (DC) provide priming signals for natural killer (NK) cells via mechanisms distinct from their antigen processing and presentation functions. The influence of DC on resting NK cells, i.e. at steady-state, is less well studied. We here demonstrate that as early as 1 day after DC depletion, NK cells in naïve mice downregulated the NKG2D receptor and showed decreased constitutive phosphorylation of AKT and mTOR. Subsequently, apoptotic NK cells appeared in the spleen concomitant with reduced NK cell numbers. At 4 days after the onset of DC depletion, increased NK cell proliferation was seen in the spleen resulting in an accumulation of Ly49 receptor-negative NK cells. In parallel, NK cell responsiveness to ITAM-mediated triggering and cytokine stimulation dropped across maturation stages, suggestive of a functional deficiency independent from the homeostatic effect. A role for IL-15 in maintaining NK cell function was supported by a gene signature analysis of NK cell from DC-depleted mice as well as by in vivo DC transfer experiments. We propose that DC, by means of IL-15 transpresentation, are required to maintain not only homeostasis, but also function, at steady-state. These processes appear to be regulated independently from each other.

Dynamic Regulation of NK Cell Responsiveness.

Natural killer (NK) cells deliver cytotoxic granules and immunomodulatory cytokines in response to transformed and virally infected cells. NK cell functions are under the control of a large number of germline-encoded receptors that recognize various ligands on target cells, but NK cells also respond to cytokines in the surrounding environment. The interaction between NK cell receptors and their ligands delivers either inhibitory or activating signals. The cytokine milieu further shapes NK cell responses, either directly or by influencing the way inhibitory or activating signals are perceived by NK cells. In this review, we discuss how NK cell function is controlled by inhibitory receptors and MHC-I molecules, how activating receptors contribute to NK cell education, and finally, how cytokines secreted by the surrounding cells affect NK cell responsiveness. Inputs at these three levels involve different cell types and are seamlessly integrated to form a functional NK cell population.