NK cell-mediated murine cytomegalovirus (MCMV) resistance ( Cmv r ) is under H-2 k control in MA/My mice, but the underlying gene(s) is unclear. Prior genetic analysis mapped Cmv r to the MHC class I (MHC-I) D k gene interval. Because NK cell receptors are licensed by and responsive to MHC class I molecules, D k itself is a candidate gene. A 10-kb genomic D k fragment was subcloned and microinjected into MCMV-susceptible ( Cmv s ) (MA/My.L- H2 b × C57L)F 1 or (B6 × DBA/2)F 2 embryos. Transgenic founders, which are competent for D k expression and germline transgene transmission, were identified and further backcrossed to MA/My.L- H2 b or C57L mice. Remarkably, D k expression delivered NK-mediated resistance in either genetic background. Further, NK cells with cognate inhibitory Ly49G receptors for self-MHC-I D k were licensed and critical in protection against MCMV infection. In radiation bone marrow chimeras, NK resistance was significantly diminished when MHC-I D k expression was restricted to only hematopoietic or nonhematopoietic cells. Thus, MHC-I D k is the H-2 k -linked Cmv r locus; these findings suggest a role for NK cell interaction with D k -bearing hematopoietic and nonhematopoietic cells to shape NK-mediated virus immunity.

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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 (NKCKD) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKCKD NK cells exhibit defective killing of MHC-I–deficient, but otherwise normal, target cells, resulting in defective rejection by NKCKD mice of transplants from various types of MHC-I–deficient mice. Self–MHC-I immunosurveillance by NK cells in NKCKD mice can be rescued by self–MHC-I–specific Ly49 transgenes. Although NKCKD 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.

The Ly49 receptors are type II C-type lectin-like membrane glycoproteins encoded by a family of highly polymorphic and polygenic genes within the mouse natural killer (NK) gene complex. This gene family is designated Klra, and includes genes that encode both inhibitory and activating Ly49 receptors in mice. Ly49 receptors recognize class I major histocompatibility complex-I (MHC-I) and MHC-I-like proteins on normal as well as altered cells. Their functional homologs in humans are the killer cell immunoglobulin-like receptors, which recognize HLA class I molecules as ligands. Classically, Ly49 receptors are described as being expressed on both the developing and mature NK cells. The inhibitory Ly49 receptors are involved in NK cell education, a process in which NK cells acquire function and tolerance toward cells that express “self-MHC-I.” On the other hand, the activating Ly49 receptors recognize altered cells expressing activating ligands. New evidence shows a broader Ly49 expression pattern on both innate and adaptive immune cells. Ly49 receptors have been described on multiple NK cell subsets, such as uterine NK and memory NK cells, as well as NKT cells, dendritic cells, plasmacytoid dendritic cells, macrophages, neutrophils, and cells of the adaptive immune system, such as activated T cells and regulatory CD8+ T cells. In this review, we discuss the expression pattern and proposed functions of Ly49 receptors on various immune cells and their contribution to immunity.

The Nkrp1 (Klrb1)-Clr (Clec2) genes encode a receptor-ligand system utilized by NK cells as an MHC-independent immunosurveillance strategy for innate immune responses. The related Ly49 family of MHC-I receptors displays extreme allelic polymorphism and haplotype plasticity. In contrast, previous BAC-mapping and aCGH studies in the mouse suggest the neighboring and related Nkrp1-Clr cluster is evolutionarily stable. To definitively compare the relative evolutionary rate of Nkrp1-Clr vs. Ly49 gene clusters, the Nkrp1-Clr gene clusters from two Ly49 haplotype-disparate inbred mouse strains, BALB/c and 129S6, were sequenced. Both Nkrp1-Clr gene cluster sequences are highly similar to the C57BL/6 reference sequence, displaying the same gene numbers and order, complete pseudogenes, and gene fragments. The Nkrp1-Clr clusters contain a strikingly dissimilar proportion of repetitive elements compared to the Ly49 clusters, suggesting that certain elements may be partly responsible for the highly disparate Ly49 vs. Nkrp1 evolutionary rate. Focused allelic polymorphisms were found within the Nkrp1b/d (Klrb1b), Nkrp1c (Klrb1c), and Clr-c (Clec2f) genes, suggestive of possible immune selection. Cell-type specific transcription of Nkrp1-Clr genes in a large panel of tissues/organs was determined. Clr-b (Clec2d) and Clr-g (Clec2i) showed wide expression, while other Clr genes showed more tissue-specific expression patterns. In situ hybridization revealed specific expression of various members of the Clr family in leukocytes/hematopoietic cells of immune organs, various tissue-restricted epithelial cells (including intestinal, kidney tubular, lung, and corneal progenitor epithelial cells), as well as myocytes. In summary, the Nkrp1-Clr gene cluster appears to evolve more slowly relative to the related Ly49 cluster, and likely regulates innate immunosurveillance in a tissue-specific manner.

Natural killer (NK) cells express cell surface receptors that recognize class I major histocompatibility complex (MHC-I) molecules to distinguish between healthy and unhealthy cells. The multigenic and polymorphic nature of the MHC-I genes has influenced the convergent evolution of similarly polymorphic and diversified NK cell receptor families: the C-type lectin-like Ly49 receptors in mice, and the killer cell immunoglobulin-like receptors (KIRs) in humans. Although structurally distinct, both receptor families have similar functions in terms of MHC-I recognition and downstream signal transduction, and they regulate multiple aspects of NK cell biology during development and after maturation as fully differentiated and functionally competent cells. The Ly49 gene locus has undergone rapid, lineage-specific expansions and contractions resulting in multiple distinct haplotypes of variable gene number, allelic diversity, and MHC-I ligand specificity. This in turn has influenced the type and degree of Ly49 receptor expression on NK cells, and their contribution to immunity in different mouse strains. In this review, we have attempted to describe the evolutionary processes that have shaped strain-specific Ly49 receptor repertoires, and their impact on NK cell functions during health and disease.

Key Points NKR-P1B is involved in NK cell tolerance and MHC-I-independent missing-self recognition of Clr-b-deficient target cells. The NKR-P1B:Clr-b system plays a role in tumor surveillance and immune escape in the Eμ-myc transgenic mouse model of B-cell lymphoma.

The Nkrp1 (Klrb) family of NK cell receptors and their genetically linked Clr (Clec2) ligands are conserved between rodents and humans. Nonetheless, certain mouse and rat Nkrp1 genes exhibit significant allelic polymorphism between inbred strains. We previously demonstrated that the Nkrp1-Clr recognition system is genetically and functionally conserved between the B6 and BALB/c strains, with focused sequence divergence evident in certain genes (e.g., Nkrp1b,c). Here, we extend this finding by mapping the 129-strain Nkrp1-Clr gene cluster, which is structurally conserved yet displays significant sequence divergence relative to the B6 haplotype. In addition, we show that 129-strain NK cells possess comparable Nkrp1 and Clr transcript expression, and characterize several NKR-P1:Clr interactions that are functionally conserved between the B6 and 129 strains, including documented and novel receptor-ligand pairs. Thus, despite significant allelic polymorphism observed in the Nkrp1-Clr region, the overall genetic organization and functional repertoire appear to be conserved among mouse strains, in contrast to the striking variation observed in the corresponding Ly49 region. These data extend our knowledge of the complex genetically linked Nkrp1-Clr NK recognition system in mice.