Natural killer (NK) cells express inhibitory and activation receptors that recognize MHC class I-like molecules on target cells. These receptors may be involved in the critical role of NK cells in controlling initial phases of certain viral infections. Indeed, the Ly49H NK cell activation receptor confers in vivo genetic resistance to murine cytomegalovirus (MCMV) infections, but its ligand was previously unknown. Herein, we use heterologous reporter cells to demonstrate that Ly49H recognizes MCMV-infected cells and a ligand encoded by MCMV itself. Exploiting a bioinformatics approach to the MCMV genome, we find at least 11 ORFs for molecules with previously unrecognized features of predicted MHC-like folds and limited MHC sequence homology. We identify one of these, m157, as the ligand for Ly49H. m157 triggers Ly49H-mediated cytotoxicity, and cytokine and chemokine production by freshly isolated NK cells. We hypothesize that the other ORFs with predicted MHC-like folds may be involved in immune evasion or interactions with other NK cell receptors.

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Natural killer (NK) cells are lymphocytes that can be distinguished from T and B cells through their involvement in innate immunity and their lack of rearranged antigen receptors. Although NK cells and their receptors were initially characterized in terms of tumor killing in vitro, we have determined that the NK cell activation receptor, Ly-49H, is critically involved in resistance to murine cytomegalovirus in vivo. Ly-49H requires an immunoreceptor tyrosine-based activation motif (ITAM)-containing transmembrane molecule for expression and signal transduction. Thus, NK cells use receptors functionally resembling ITAM-coupled T and B cell antigen receptors to provide vital innate host defense.

Recent studies have suggested that the retention of selectable marker cassettes (like PGK-Neo, in which a hybrid gene consisting of the phosphoglycerate kinase I promoter drives the neomycin phosphotransferase gene) in targeted loci can cause unexpected phenotypes in ''knockout'' mice due to disruption of expression of neighboring genes within a locus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the ␤-like globin gene cluster. The insertion of PGK-Neo into the granzyme B gene, the most 5 gene in the granzyme B gene cluster, severely reduced the normal expression of multiple genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK-Neo cassette into the ␤-globin locus control region (LCR) abrogates the expression of multiple globin genes downstream from the cassette. In contrast, a targeted mutation of the promyelocyte-specific cathepsin G gene (which lies just 3 to the granzyme genes in the same cluster) had minimal effects on upstream granzyme gene expression. Although the mechanism of these long distance effects are unknown, the expression of PGK-Neo can be ''captured'' by the regulatory domain into which it is inserted. These results suggest that the PGK-Neo cassette can interact productively with locus control regions and thereby disrupt normal interactions between local and long-distance regulatory regions within a tissue-specific domain.

Adaptive immune responses are critical for the control and clearance of influenza A virus (IAV) infection. However, in recent years, it has become increasingly apparent that innate immune cells, including natural killer cells, alveolar macrophages (aMphi), and dendritic cells (DC) are essential following IAV infection in the direct control of viral replication or in the induction and regulation of virus-specific adaptive immune responses. This review will discuss the role of these innate immune cells following IAV infection, with a particular focus on DC and their ability to induce and regulate the adaptive IAV-specific immune response.

Granzyme (Gzm) B-deficient mice obtained by gene targeting were used to assess the role of Gzm B in the mechanisms used by natural killer (NK) and lymphokineactivated killer (LAK) and fas-based pathways are the two most important mechanisms used by CTL in mediating cytotoxicity (3-6). In the perforin/Gzm pathway (also known as the granule exocytosis pathway), cytolytic effector cells deliver lethal hits by directionally releasing their granule contents after specific recognition and conjugate formation with a target cell (7, 8). The major granule components involved in this process are the membrane pore-forming protein called perforin and serine proteases termed Gzms (9-13). The pores formed by perforin in the target cell membrane may allow for the entry of Gzms into target cells. Although purified perforin can cause cytolysis under certain conditions, the membrane damage caused by perforin alone does not induce apoptosis, a hallmark of CTL and NK cell attack (14,15). Several lines of evidence now favor the Gzms, in particular Gzm A and Gzm B, as the proteins responsible for triggering the target cell "internal disintegration" pathway leading to DNA breakdown and apoptosis (16)(17)(18)(19)(20)(21)(22)(23). Specifically, studies with the noncytotoxic rat basophilic leukemia (RBL) cells have shown that doubletransfected RBL lines need to express both perforin and Gzm A or B to mediate target cell lysis and DNA damage (20, 21); similarly, purified rat Gzm A has been shown to synergize with purified rat Gzm B to induce DNA fragmentation and apoptosis in permeabilized target cells (17,18). Finally, the deliveryThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.of aprotinin (a serine protease inhibitor) to target cells protects them from CTL-induced apoptosis (20).Using perforin-deficient mice, several groups have now shown that perforin is crucial for the granule exocytosis mechanism used by both activated CTL and NK cells (3-6, 24, 25). To better understand the contribution(s) or Gzms in this perforin/Gzm pathway, we recently produced Gzm Bdeficient mice by gene targeting and demonstrated that Gzm B-/-CTL derived from primary mixed lymphocyte cultures have a severe defect in their ability to rapidly induce DNA fragmentation and target cell apoptosis (22). In the present study, we have investigated the role of Gzm B in NK and lymphokine-activated killer (LAK) cell effector functions by using Na2[5tCr]CrO4 (5tCr) release and 5-[125I]iododeoxyuridine (125IdUrd)-labeled DNA release assays. The release of 5tCr, which labels the cytoplasmic proteins of target cells, reflects the loss of target cell membrane integrity (probably due to pore formation by perforin). The release of 125IIdUrd, which labels the nuclear DNA of target cells, reflects DNA fragmentation and directly correlates with apoptotic death as defined by chromatin condensation and nuclear memb...

The American College of Medical Genetics and Genomics (ACMG) recommends that clinical sequencing laboratories return secondary findings in 56 genes associated with medically actionable conditions. Our goal was to apply a systematic, stringent approach consistent with clinical standards to estimate the prevalence of pathogenic variants associated with such conditions using a diverse sequencing reference sample. Candidate variants in the 56 ACMG genes were selected from Phase 1 of the 1000 Genomes dataset, which contains sequencing information on 1,092 unrelated individuals from across the world. These variants were filtered using the Human Gene Mutation Database (HGMD) Professional version and defined parameters, appraised through literature review, and examined by a clinical laboratory specialist and expert physician. Over 70,000 genetic variants were extracted from the 56 genes, and filtering identified 237 variants annotated as disease causing by HGMD Professional. Literature review and expert evaluation determined that 7 of these variants were pathogenic or likely pathogenic. Furthermore, 5 additional truncating variants not listed as disease causing in HGMD Professional were identified as likely pathogenic. These 12 secondary findings are associated with diseases that could inform medical follow-up, including cancer predisposition syndromes, cardiac conditions, and familial hypercholesterolemia. The majority of the identified medically actionable findings were in individuals from the European (5/379) and Americas (4/181) ancestry groups, with fewer findings in Asian (2/286) and African (1/246) ancestry groups. Our results suggest that medically relevant secondary findings can be identified in approximately 1% (12/1092) of individuals in a diverse reference sample. As clinical sequencing laboratories continue to implement the ACMG recommendations, our results highlight that at least a small number of potentially important secondary findings can be selected for return. Our results also confirm that understudied populations will not reap proportionate benefits of genomic medicine, highlighting the need for continued research efforts on genetic diseases in these populations.

Murine natural killer cells (NK) express lectin-like activation and inhibitory receptors, including the CD94/NKG2 family of receptors that bind Qa-1, and the Ly-49 family that recognizes major histocompatibility complex class I molecules. Here, we demonstrate that cross-linking of NK cells with a new specific anti–Ly-49H mAb induced NK cell cytotoxicity and cytokine production. Ly-49H is expressed on a subset of NK cells and can be coexpressed with Ly-49 inhibitory receptors. However, unlike Ly-49 inhibitory receptors, Ly-49H is not detectable on naive splenic CD3+ T cells, indicating that Ly-49H may be an NK cell–specific activation receptor. In further contrast to the stochastically expressed Ly-49 inhibitory receptors, Ly-49H is preferentially expressed with the Ly-49D activation receptor, and expression of both Ly-49H and Ly-49D is augmented on NK cells that lack receptors for Qa-1 tetramers. On developing splenic NK1.1+ cells, Ly-49D and Ly-49H are expressed later than the inhibitory receptors. These results directly demonstrate that Ly-49H activates primary NK cells, and suggest that expression of Ly-49 activation receptors by NK cells may be specifically regulated on NK cell subsets. The simultaneous expression of multiple activation receptors by individual NK cells contrasts with that of T cell antigen receptors and is relevant to the role of NK cells in innate immunity.