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.

During virus infection, NK cells are needed in the body to provide early immune defense. Their activity is regulated by MHC class I-binding cell surface inhibitory and stimulatory receptors that include mouse Ly49, human killer Igrelated receptor (KIR), 3 and NKG2/CD94 receptors (1-3). NK cell-mediated virus control is subject to genetic factors that can influence viral replication and host mortality (4). For instance, the Ly49H activation receptor displayed on the surface of NK cells in C57BL/6 mice binds murine CMV (MCMV) m157 ligands at the surface of infected cells to impart MCMV resistance (5, 6). In New Zealand White, MA/My, and PWK strains, MCMV resistance also requires NK-mediated virus control, but Ly49H-independent defense mechanisms are key (7-11). It remains unclear which genetic factors are at work and how such factors mediate virus resistance through NK cells.Attempts have been made to identify genetic factors that underlie MCMV resistance/susceptibility traits in the offspring of genetic crosses between MCMV-resistant (Cmv r ) MA/My mice and strains that appear to be more MCMV susceptible (Cmv s ). To date, major MCMV control loci have been mapped to the MHC and NK gene complex (NKC) on chromosomes 17 and 6, respectively (9, 10). We have further shown that MHC polymorphism is respon- Materials and Methods Animalsmice were bred and maintained in a specificpathogen-free vivarium at the University of Virginia (Charlottesville, VA), which is fully certified by the American Association for Accreditation of Laboratory Animal Care. All recombinant congenic strains generated and used in this study were backcrossed to the progenitor strain C57L for at least 10 generations. All animal studies were approved by and conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of the University of Virginia. Recombinant congenic strain generation, genetic markers, and genotyping (C57L.M-H2k ϫ C57L)F 1 or (MA/My.L-H2 b ϫ MA/My)F 1 were brother ϫ sister mated and screened for recombination crossovers within the D17Mit16-D17Uva09 interval using several simple sequence-length polymorphism (SSLP) markers to distinguish MA/My and C57L alleles (Table I). SSLP-amplified PCR products were resolved in POP7-filled capillaries and analyzed on a Genetic Analyzer 3130xl using Data Collection (version 3.0) and GeneMapper software (version 4.0; Applied Biosystems) as described previously (10). Some unlabeled SSLP amplicons were fractionated in 4% agarose gel electrophoresis and visualized on an UV transilluminator after ethidium bromide staining.For generation of novel SSLP markers, chromosome 17 sequence data available from the National Center for Biotechnology Information were manually inspected for microsatellite repeat sequences. Sequence-specific

MHC class I (MHC I) IntroductionNatural killer (NK) cells are essential mediators of virus immunity 1 ; their deficiency in humans or depletion from mice leads to uncontrolled viral replication and poor clinical outcome. 2-4 MHC class I (MHC I) molecules, which are ligands for polymorphic human KIR and mouse Ly49 receptors, play a critical role in NK-cell activation and selftolerance. The absence of self-MHC I renders cells susceptible to NK-cell cytotoxicity. [5][6][7] Moreover, the interaction between MHC I and NK-cell inhibitory receptors is critical in NK-cell acquisition of selftolerance and functional competence. [8][9][10][11] Indeed, NK inhibitory receptor recognition of virus-infected cells displaying reduced or altered self-MHC I is a paradigm for the field. Despite this, only activation receptors have so far been shown to confer specific recognition and control of virus infection by NK cells; the significance of NK inhibitory receptors in virus recognition therefore is debated. 12 In MA/My mice and other H-2 k strains, the MHC provides vital protection against lethal murine CMV (MCMV) infection. [13][14][15] Classic genetics studies to examine MA/My virus resistance mapped genes to the MHC I D region on chromosome 17 and the NK gene complex (NKC) on chromosome 6. [15][16][17][18] Recently, we identified MHC I D k as a critical genetic factor, 19 and further demonstrated a requisite role of licensed Ly49G2 ϩ NK cells needed in MCMV resistance. 18,19 Consistent with a requirement for NK stimulatory signals, Ly49P has been shown to bind MHC I D k on infected cells displaying MCMV glycoprotein gp34. 20 Thus, virus sensing via Ly49G2 and Ly49P receptors likely combine to drive potent antiviral NK responses after MCMV infection in H-2 k mice. The intriguing finding of Babic et al recently demonstrating how MCMV gp34 associated with MHC I on infected cells may enhance interaction with NK inhibitory receptors and immune evasion strategies, further highlights that NK missing-self MHC I surveillance mechanisms contribute to virus control in vivo. 21 As major effectors in front line antiviral defenses, NK cells further impact adaptive immunity through interactions with dendritic cells (DCs). NK-DC "crosstalk" can enhance NK cytotoxicity and proliferation, sustain splenic DC subsets after virus infection, 22,23 and stimulate DC maturation and Ag-presenting functions. [22][23][24][25][26][27][28][29][30] NK cells may thus provide needed signals during NK-DC crosstalk that promote tumor-or virus-specific CD8 T-cell immunity. 23,31 In accord with this suggestion, high-affinity Ly49H recognition of MCMV m157 displayed on infected cells caused NK cells to affect the regulation of type I IFNs, DC subset retention and induction of virus-specific CD8 T-cell effectors. 22,23,26 However, rapid containment of MCMV and Ag availability via Ly49H-mediated NK responses may also inadvertently contribute to viral persistence by reducing initial CD4 and CD8 T-cell responses. 32 Whether NK inhibitory receptor recognition of virus i...

MHC I Dk and Ly49G2 inhibitory receptor-expressing NK cells are essential to murine (M)CMV5 resistance in MA/My mice. Without Dk, Ly49G2+ NK cells in C57L mice fail to protect against MCMV infection. As a cognate ligand of Ly49G2, Dk licenses Ly49G2+ NK cells for effector activity. These data suggested that Dk-licensed Ly49G2+ NK cells might recognize and control MCMV infection. However, a role for licensed NK cells in viral immunity is uncertain. We combined classical genetics with flow cytometry to visualize the host response to MCMV. Immune cells collected from individuals of a diverse cohort of MA/MyxC57L offspring segregating Dk were examined before and after infection, including Ly49+ NK subsets, receptor expression features and other phenotypic traits. To identify critical NK cell features, automated analysis of 110 traits was performed in R using Pearson’s correlation followed with a Bonferroni correction for multiple tests. Hierarchical clustering of trait-associations and principal component analyses were used to discern shared immune response and genetic relationships. The results demonstrate that Ly49G2 expression on naïve blood NK cells was predictive of MCMV resistance. However, rapid Ly49G2+ NK cell expansion following viral exposure selectively occurred in Dk offspring; this response was more highly correlated to MCMV control than all other immune cell features. We infer that Dk-licensed Ly49G2+ NK cells efficiently detected missing-self MHC cues on viral targets, which elicited cellular expansion and target cell killing. MHC polymorphism therefore regulates licensing and detection of viral targets by distinct subsets of NK cells required in innate viral control.