NK cell receptor NKG2D sets activation threshold for the NCR1 receptor early in NK cell development.

The activation of natural killer (NK) cells depends on a change in the balance of signals from inhibitory and activating receptors. The activation threshold values of NK cells are thought to be set by engagement of inhibitory receptors during development. Here, we found that the activating receptor NKG2D specifically set the activation threshold for the activating receptor NCR1 through a process that required the adaptor DAP12. As a result, NKGD2-deficient (Klrk1-/-) mice controlled tumors and cytomegalovirus infection better than wild-type controls through the NCR1-induced production of the cytokine IFN-γ. Expression of NKG2D before the immature NK cell stage increased expression of the adaptor CD3ζ. Reduced expression of CD3ζ in Klrk1-/- mice was associated with enhanced signal transduction through NCR1, and CD3ζ deficiency resulted in hyper-responsiveness to stimulation via NCR1. Thus, an activating receptor developmentally set the activity of another activating receptor on NK cells and determined NK cell reactivity to cellular threats.

NK cells link obesity-induced adipose stress to inflammation and insulin resistance.

An important cause of obesity-induced insulin resistance is chronic systemic inflammation originating in visceral adipose tissue (VAT). VAT inflammation is associated with the accumulation of proinflammatory macrophages in adipose tissue, but the immunological signals that trigger their accumulation remain unknown. We found that a phenotypically distinct population of tissue-resident natural killer (NK) cells represented a crucial link between obesity-induced adipose stress and VAT inflammation. Obesity drove the upregulation of ligands of the NK cell-activating receptor NCR1 on adipocytes; this stimulated NK cell proliferation and interferon-γ (IFN-γ) production, which in turn triggered the differentiation of proinflammatory macrophages and promoted insulin resistance. Deficiency of NK cells, NCR1 or IFN-γ prevented the accumulation of proinflammatory macrophages in VAT and greatly ameliorated insulin sensitivity. Thus NK cells are key regulators of macrophage polarization and insulin resistance in response to obesity-induced adipocyte stress.

NKG2D Promotes B1a Cell Development and Protection against Bacterial Infection.

NKG2D is a potent activating receptor that is expressed on cytotoxic immune cells such as CD8 T and NK cells, where it promotes cytotoxicity after binding stress ligands on infected or transformed cells. On NK cell precursors NKG2D modulates proliferation and maturation. Previously, we observed that NKG2D deficiency affects peripheral B cell numbers. In this study, we show that NKG2D regulates B1a cell development and function. We find that mice deficient for NKG2D have a strong reduction of B1a cell numbers. As a result, NKG2D-deficient mice produce significantly less Ag-specific IgM Abs upon immunization with T cell-independent Ags, and they are more susceptible to Gram-negative sepsis. Klrk1-/- B1a cells are also functionally impaired and they fail to provide protection against Francisella novicida upon adoptive transfer. Using mixed bone marrow chimeric mice, we show that the impact of NKG2D deficiency on B1a cell development is cell intrinsic. No changes in homeostatic turnover and homing of B cells were detectable, limiting the effects of NKG2D to modulation of the hematopoietic development of B1a cells. Using conditional ablation, we demonstrate that the effect of NKG2D on B1a cell development occurs at a developmental stage that precedes the common lymphoid progenitor. Our findings reveal an unexpected new role for NKG2D in the regulation of B1a cell development. The protective effects of this activating receptor therefore reach beyond that of cytotoxic cells, stimulating the immune system to fight bacterial infections by promoting development of innate-like B cells.

NKG2D stimulation of CD8+ T cells during priming promotes their capacity to produce cytokines in response to viral infection in mice.

Natural killer group 2 member D (NKG2D) is an activating receptor that is expressed on most cytotoxic cells of the immune system, including NK cells, γδ, and CD8+ T cells. It is still a matter of debate whether and how NKG2D mediates priming of CD8+ T cells in vivo, due to a lack of studies where NKG2D is eliminated exclusively in these cells. Here, we studied the impact of NKG2D on effector CD8+ T-cell formation. NKG2D deficiency that is restricted to murine CD8+ T cells did not impair antigen-specific T-cell expansion following mouse CMV and lymphocytic choriomeningitis virus infection, but reduced their capacity to produce cytokines. Upon infection, conventional dendritic cells induce NKG2D ligands, which drive cytokine production on CD8+ T cells via the Dap10 signaling pathway. T-cell development, homing, and proliferation were not affected by NKG2D deficiency and cytotoxicity was only impaired when strong T-cell receptor (TCR) stimuli were used. Transfer of antigen-specific CD8+ T cells demonstrated that NKG2D deficiency attenuated their capacity to reduce viral loads. The inability of NKG2D-deficient cells to produce cytokines could be overcome with injection of IL-15 superagonist during priming. In summary, our data show that NKG2D has a nonredundant role in priming of CD8+ T cells to produce antiviral cytokines.

NKG2D induces Mcl-1 expression and mediates survival of CD8 memory T cell precursors via phosphatidylinositol 3-kinase.

Memory formation of activated CD8 T cells is the result of a specific combination of signals that promote long-term survival and inhibit differentiation into effector cells. Much is known about initial cues that drive memory formation, but it is poorly understood which signals are essential during the intermediate stages before terminal differentiation. NKG2D is an activating coreceptor on Ag-experienced CD8 T cells that promotes effector cell functions. Its role in memory formation is currently unknown. In this study, we show that NKG2D controls formation of CD8 memory T cells by promoting survival of precursor cells. We demonstrate that NKG2D enhances IL-15-mediated PI3K signaling of activated CD8 T cells, in a specific phase of memory cell commitment, after activation but before terminal differentiation. This signal is essential for the induction of prosurvival protein Mcl-1 and precursor cell survival. In vivo, NKG2D deficiency results in reduced memory cell formation and impaired protection against reinfection. Our findings show a new role for PI3K and the NKG2D/IL-15 axis in an underappreciated stage of effector to memory cell transition that is essential for the generation of antiviral immunity. Moreover, we provide novel insights how these receptors control both effector and memory T cell differentiation.

Largely preserved functionality after the combined loss of NKG2D, NCR1 and CD16 demonstrates the remarkable plasticity of NK cell responsiveness.

Natural killer (NK) cells play an important role in the early defense against tumors and virally infected cells. Their function is thought to be controlled by the balance between activating and inhibitory receptors, which often compete for the same ligands. Several activating receptors expressed on virtually all NK cells lack an inhibitory partner, most notably CD16, NCR1 and NKG2D. We therefore hypothesized that a signal through at least one of these receptors is always required for full NK cell activation. We generated animals lacking all three receptors (TKO) and analyzed their NK cells. In vitro, TKO NK cells did not show reduced ability to kill tumor targets but displayed hyperresponsiveness to NK1.1 stimulation. In vivo, TKO animals had a minor reduction in their ability to control non-hematopoietic tumors and cytomegalovirus infection, which was the result of reduced NK cell activity. Together, our findings show that activating NK cell receptors without an inhibitory partner do not provide a 'master' signal but are integrated in the cumulative balance of activating and inhibitory signals. Their activity is controlled through regulation of the responsiveness and expression of other activating receptors. Our findings may be important for future development of NK cell-based cancer immunotherapy.

NKG2D: A Master Regulator of Immune Cell Responsiveness.

NKG2D is an activating receptor that is mostly expressed on cells of the cytotoxic arm of the immune system. Ligands of NKG2D are normally of low abundance, but can be induced in virtually any cell in response to stressors, such as infection and oncogenic transformation. Engagement of NKG2D stimulates the production of cytokines and cytotoxic molecules and traditionally this receptor is, therefore, viewed as a molecule that mediates direct responses against cellular threats. However, accumulating evidence indicates that this classical view is too narrow. During NK cell development, engagement of NKG2D has a long-term impact on the expression of NK cell receptors and their responsiveness to extracellular cues, suggesting a role in NK cell education. Upon chronic NKG2D engagement, both NK and T cells show reduced responsiveness of a number of activating receptors, demonstrating a role of NKG2D in induction of peripheral tolerance. The image that emerges is that NKG2D can mediate both inhibitory and activating signals, which depends on the intensity and duration of ligand engagement. In this review, we provide an overview of the impact of NKG2D stimulation during hematopoietic development and during acute and chronic stimulation in the periphery on responsiveness of other receptors than NKG2D. We propose that NKG2D interprets the context of the immunological environment through detection of cellular cues and in response sets the appropriate activation threshold for a large number of immune receptors. This perspective is of particular importance for future therapies that aim to exploit NKG2D signaling to fight tumors or infection.

NKG2D: A versatile player in the immune system.

NKG2D is known as a potent activating receptor of the immune system. It is expressed on a multitude of immune cells, including NK cells and different subsets of T cells. NKG2D recognizes various MHC I-like ligands that are induced on target cells exposed to stressors such as viral infection, DNA damage and oncological transformation. NKG2D drives or facilitates cytotoxic and cytokine responses towards cells expressing its ligands to eliminate the threat. Therefore, NKG2D is usually classified as a sensor that translates cellular stress into activation signals for immune cells. However, more recently it has become evident that NKG2D plays a role beyond direct killing of target cells. Lack of NKG2D affects development of NK cells in the bone marrow, resulting in hyperreactive NK cells. NKG2D deficiency on CD8 T cells affects the ability of effector cells to produce cytokines in response to T cell receptor engagement and reduces their capacity to establish immunological memory. Although NKG2D is not expressed on B cells subsets, lack of this receptor in hematopoietic precursors affects B cell development. Homing of mature B2 cells is altered in NKG2D-deficient mice and they have a strong reduction in peripheral B1a cell numbers, resulting in increased susceptibility to bacterial infections. The exact molecular mechanisms via which NKG2D mediates these versatile functions is still being explored, but appears to depend on the control of activation thresholds, either in hematopoietic precursors or mature immune cell subsets. In this review, we will elaborate on the underappreciated developmental and regulatory roles of NKG2D.