Project description:DNAX accessory molecule-1 (DNAM-1, also known as CD226) is an activating receptor expressed on subsets of natural killer (NK) and T cells, interacts with its ligands CD155 or CD112, and has co-varied expression with inhibitory receptors. Since inhibitory receptors control NK-cell activation and are necessary for MHC-I-dependent education, we investigated whether DNAM-1 expression is also involved in NK-cell education. Here we show an MHC-I-dependent correlation between DNAM-1 expression and NK-cell education, and an association between DNAM-1 and NKG2A that occurs even in MHC class I deficient mice. DNAM-1 is expressed early during NK-cell development, precedes the expression of MHC-I-specific inhibitory receptors, and is modulated in an education-dependent fashion. Cd226-/- mice have missing self-responses and NK cells with a normal receptor repertoire. We propose a model in which NK-cell education prevents or delays downregulation of DNAM-1. This molecule endows educated NK cells with enhanced effector functions but is dispensable for education.
Project description:Ly49-mediated recognition of MHC-I molecules on host cells is considered vital for natural killer (NK)-cell regulation and education; however, gene-deficient animal models are lacking because of the difficulty in deleting this large multigene family. Here, we describe NK gene complex knockdown (NKC(KD)) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKC(KD) NK cells exhibit defective killing of MHC-I-deficient, but otherwise normal, target cells, resulting in defective rejection by NKC(KD) mice of transplants from various types of MHC-I-deficient mice. Self-MHC-I immunosurveillance by NK cells in NKC(KD) mice can be rescued by self-MHC-I-specific Ly49 transgenes. Although NKC(KD) mice display defective recognition of MHC-I-deficient tumor cells, resulting in decreased in vivo tumor cell clearance, NKG2D- or antibody-dependent cell-mediated cytotoxicity-induced tumor cell cytotoxicity and cytokine production induced by activation receptors was efficient in Ly49-deficient NK cells, suggesting MHC-I education of NK cells is a single facet regulating their total potential. These results provide direct genetic evidence that Ly49 expression is necessary for NK-cell education to self-MHC-I molecules and that the absence of these receptors leads to loss of MHC-I-dependent "missing-self" immunosurveillance by NK cells.
Project description: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.
Project description:The programmed death-1 receptor is expressed on a wide range of immune effector cells, including T cells, natural killer T cells, dendritic cells, macrophages, and natural killer cells. In malignancies and chronic viral infections, increased expression of programmed death-1 by T cells is generally associated with a poor prognosis. However, its role in early host microbial defense at the intestinal mucosa is not well understood. We report that programmed death-1 expression is increased on conventional natural killer cells but not on CD4(+), CD8(+) or natural killer T cells, or CD11b(+) or CD11c(+) macrophages or dendritic cells after infection with the mouse pathogen Citrobacter rodentium. Mice genetically deficient in programmed death-1 or treated with anti-programmed death-1 antibody were more susceptible to acute enteric and systemic infection with Citrobacter rodentium. Wild-type but not programmed death-1-deficient mice infected with Citrobacter rodentium showed significantly increased expression of the conventional mucosal NK cell effector molecules granzyme B and perforin. In contrast, natural killer cells from programmed death-1-deficient mice had impaired expression of those mediators. Consistent with programmed death-1 being important for intracellular expression of natural killer cell effector molecules, mice depleted of natural killer cells and perforin-deficient mice manifested increased susceptibility to acute enteric infection with Citrobacter rodentium. Our findings suggest that increased programmed death-1 signaling pathway expression by conventional natural killer cells promotes host protection at the intestinal mucosa during acute infection with a bacterial gut pathogen by enhancing the expression and production of important effectors of natural killer cell function.
Project description:The PH-BEACH-WD40 (PBW) protein family members play a role in coordinating receptor signaling and intracellular vesicle trafficking. LPS-Responsive-Beige-like Anchor (LRBA) is a PBW protein whose immune function remains elusive. Here we show that LRBA-null mice are viable, but exhibit compromised rejection of allogeneic, xenogeneic and missing self bone-marrow grafts. Further, we demonstrate that LRBA-null Natural Killer (NK) cells exhibit impaired signaling by the key NK activating receptors, NKp46 and NKG2D. However, induction of IFN-? by cytokines remains intact, indicating LRBA selectively facilitates signals by receptors for ligands expressed on the surface of NK targets. Surprisingly, LRBA limits immunoregulatory cell numbers in tissues where GvHD is primed or initiated, and consistent with this LRBA-null mice also demonstrate resistance to lethal GvHD. These findings demonstrate that LRBA is redundant for host longevity while being essential for both host and donor-mediated immune responses and thus represents a unique and novel molecular target in transplant immunology.
Project description:Natural killer (NK) cells are lymphocytes of the innate immune system able to recognize and kill tumors lacking self-MHC class I molecules. This "missing-self" recognition is mediated by the lack of engagement of MHC class I-specific inhibitory NK cell receptors that include the killer cell Ig-like receptors (KIR) in humans and Ly49 molecules in mice. A promising immunotherapeutic strategy against MHC class I(+) cancer cells is to block NK cell inhibitory receptors using monoclonal antibodies (mAb). However, interactions between MHC class I molecules and their inhibitory receptors are also required for the acquisition of NK cell functional competence, a process referred as to "education." In addition, inhibitory receptors are involved in self-tolerance on educated NK cells. Here, we developed a preclinical mouse model in which all NK cells are educated by a single transgenic inhibitory receptor, human KIR2DL3, through the engagement with its HLA-Cw3 ligand. This approach revealed that NK cells could be reprogrammed to control the development of mouse syngenic tumors in vivo. Moreover, in vivo anti-KIR mAb treatment induced the killing of HLA(+) target cells without breaking self-tolerance. Finally, the long-term infusion of anti-KIR mAb neither abolished NK cell education nor tumor cell recognition. Therefore, these results strongly support the use of inhibitory receptor blockade in cancer patients.
Project description:Class IA phosphotidylinositol-3-kinases (PI3Ks) are a family of p85/p110 heterodimeric lipid kinases that are important in regulating signaling events in B and T cells. However, their role in natural killer (NK) cells is not understood. Here, using mice that lack the regulatory p85alpha subunit and its alternatively spliced variants p55alpha/p50alpha (collectively termed as p85alpha(-/-)), we defined the role of PI3K in NK cell development and function. p85alpha(-/-) mice had impaired lineage commitment leading to reduced NK cellularity in the bone marrow and liver. p85alpha(-/-) NK cells showed a defective Ly49 subset specification and a decreased expression of CD43. Lack of p85alpha severely reduced the NK-mediated cytotoxicity against tumor cells representing 'induced-self' and 'missing-self'. More importantly, NKG2D and NK1.1 receptor-mediated cytokine and chemokine generation was significantly compromised in p85alpha(-/-) NK cells. These results reveal a previously unrecognized role of p85alpha in the development, terminal maturation, cytokine/chemokine generation and tumor clearance of NK cells.
Project description:Natural killer (NK) cells are innate lymphocytes that are thought to kill cells that down-regulate MHC class I (MHC-I) through "missing-self" recognition. NK cells from B2m-/- mice that lack surface MHC-I, however, are not autoreactive as predicted by the missing-self hypothesis. As a result, it is unclear if MHC-I down-regulation in vivo induces NK cell reactivity or tolerance to missing-self. Here, we generated a floxed B2m mouse to acutely down-regulate MHC-I in vivo in a host that normally expresses MHC-I. Global down-regulation of MHC-I induced NK cell hyporesponsiveness and tolerance to missing-self without overt missing-self reactivity. In contrast, down-regulation of MHC-I on a small fraction of hematopoietic cells triggered missing-self reactivity. Surprisingly, down-regulation of MHC-I only on CD4+ T cells predominately induced tolerance to missing-self without resetting NK cell responsiveness. In this setting, inflammation triggered substantial missing-self reactivity. These results show that MHC-I down-regulation can induce either NK cell tolerance or killing in vivo and that inflammation promotes missing-self reactivity.
Project description:Natural killer (NK) cells can mediate the rejection of bone marrow allografts and exist as subsets based on expression of inhibitory/activating receptors that can bind MHC. In vitro data have shown that NK subsets bearing Ly49 receptors for self-MHC class I have intrinsically higher effector function, supporting the hypothesis that NK cells undergo a host MHC-dependent functional education. These subsets also play a role in bone marrow cell (BMC) allograft rejection. Thus far, little in vivo evidence for this preferential licensing across mouse strains with different MHC haplotypes has been shown. We assessed the intrinsic response potential of the different Ly49(+) subsets in BMC rejection by using ?2-microglobulin deficient (?2m(-/-)) mice as donors. Using congenic and allogeneic mice as recipients and depleting the different Ly49 subsets, we found that NK subsets bearing Ly49s, which bind "self-MHC" were found to be the dominant subset responsible for ?2m(-/-) BMC rejection. This provides in vivo evidence for host MHC class I-dependent functional education. Interestingly, all H2(d) strain mice regardless of background were able to resist significantly greater amounts of ?2m(-/-), but not wild-type BMC than H2(b) mice, providing evidence that the rheostat hypothesis regarding Ly49 affinities for MHC and NK-cell function impacts BMC rejection capability.
Project description:In this study, we aim to establish the means to characterize cynomolgus macaque natural killer (NK) cells, and to investigate the CD27/CD11b NK maturation model in Macaca fascicularis NK cell subpopulations. The NK development studies performed mainly in mice models have demonstrated four functionally distinct subsets as defined by CD27 and CD11b, however this has not yet been established in nonhuman primates. The expression data of these macaque NK subsets will be associated with the antibody-dependent cellular cytotoxicity and missing-self responses produce by each subset. Overall design: Unstimulated NK cell subsets were profiled for gene expression after 10-colour flow cytometry cell sorting based on CD27 and CD11b surface expression.