Mice lacking MHC class-I (MHC-I) display severe defects in natural killer (NK) cell functional maturation, a process designated as "education". Whether self-MHC-I specific Ly49 family receptors and NKG2A, which are closely linked within the NK gene complex (NKC) locus, are essential for NK cell education is still unclear. Here we show, using CRISPR/Cas9-mediated gene deletion, that mice lacking all members of the Ly49 family exhibit a moderate defect in NK cell activity, while mice lacking only two inhibitory Ly49 members, Ly49C and Ly49I, have comparable phenotypes. Furthermore, the deficiency of NKG2A, which recognizes non-classical MHC-Ib molecules, mildly impairs NK cell function. Notably, the combined deletion of NKG2A and the Ly49 family severely compromises the ability of NK cells to mediate "missing-self" and "induced-self" recognition. Therefore, our data provide genetic evidence supporting that NKG2A and the inhibitory members of Ly49 family receptors synergize to regulate NK cell education.

Natural killer (NK) cells are innate lymphocytes that play essential roles in mediating antitumor immunity. NK cells respond to various inflammatory stimuli including cytokines and stress-induced cellular ligands which activate germline-encoded activation receptors (NKRs), such as NKG2D. The signaling molecules activated downstream of NKRs are well defined; however, the mechanisms that regulate these pathways are not fully understood. IQ domain-containing GTPase-activating protein 1 (IQGAP1) is a ubiquitously expressed scaffold protein. It regulates diverse cellular signaling programs in various physiological contexts, including immune cell activation and function. Therefore, we sought to investigate the role of IQGAP1 in NK cells. Development and maturation of NK cells from mice lacking IQGAP1 (Iqgap1-/- ) were mostly intact; however, the absolute number of splenic NK cells was significantly reduced. Phenotypic and functional characterization revealed a significant reduction in the egression of NK cells from the bone marrow of Iqagp1-/- mice altering their peripheral homeostasis. Lack of IQGAP1 resulted in reduced NK cell motility and their ability to mediate antitumor immunity in vivo. Activation of Iqgap1-/- NK cells via NKRs, including NKG2D, resulted in significantly reduced levels of inflammatory cytokines compared with wild-type (WT). This reduction in Iqgap1-/- NK cells is neither due to an impaired membrane proximal signaling nor a defect in gene transcription. The levels of Ifng transcripts were comparable between WT and Iqgap1-/- , suggesting that IQGAP1-dependent regulation of cytokine production is regulated by a post-transcriptional mechanism. To this end, Iqgap1-/- NK cells failed to fully induce S6 phosphorylation and showed significantly reduced protein translation following NKG2D-mediated activation, revealing a previously undefined regulatory function of IQGAP1 via the mechanistic target of rapamycin complex 1. Together, these results implicate IQGAP1 as an essential scaffold for NK cell homeostasis and function and provide novel mechanistic insights to the post-transcriptional regulation of inflammatory cytokine production.

Natural killer (NK) cells contribute to the development of obesity-associated insulin resistance. We demonstrate that in mice obesity promotes expansion of a distinct, interleukin-6 receptor (IL6R)a-expressing NK subpopulation, which also expresses a number of other myeloid lineage genes such as the colony-stimulating factor 1 receptor (Csf1r). Selective ablation of this Csf1r-expressing NK cell population prevents obesity and insulin resistance. Moreover, conditional inactivation of IL6Ra or Stat3 in NK cells limits obesity-associated formation of these myeloid signature NK cells, protecting from obesity, insulin resistance, and obesity-associated inflammation. Also in humans IL6Ra+ NK cells increase in obesity and correlate with markers of systemic low-grade inflammation, and their gene expression profile overlaps with characteristic gene sets of NK cells in obese mice. Collectively, we demonstrate that obesity-associated inflammation and metabolic disturbances depend on interleukin-6/Stat3-dependent formation of a distinct NK population, which may provide a target for the treatment of obesity, metaflammation-associated pathologies, and diabetes.
Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Little is known on the control of lymphomas by NK cells. Here, we study the role of the NK group 2D (NKG2D) receptor for the immunosurveillance of lymphoma. By using transplantable tumors as well as a λ-myc-transgenic model of endogenously arising lymphoma and NKG2D-deficient mice, we show that NK cells eliminate tumor cells in vivo after receiving two signals. One step involved the activation of NK cells giving rise to IFN-γ expression, which was effected by MHCI(low) tumor cells or DCs. However, this was necessary but not sufficient to mediate cytotoxicity. Triggering cytotoxicity additionally required a second step, which could be mediated by engagement of the NKG2D receptor. Thus, NKG2D-deficient NK cells could become activated in vivo, but they were not able to reject transplanted lymphomas or to degranulate in animals bearing autochthonous lymphomas. Tumor growth in NKG2D-deficient λ-myc-transgenic mice was significantly accelerated compared to NKG2D-competent animals. Whereas the latter developed tumors that lost expression of NKG2D ligands (NKG2D-L) in late disease stages, this did not occur in NKG2D-deficient mice. This indicates that NK cells and the NKG2D receptor play a role for control of lymphomas and that selection for NKG2D-L loss mutants provides a mechanism of tumor escape.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.