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NK cells possess inhibitory receptors that are responsible for self-MHC class I recognition; beyond their inhibitory function, accumulating evidence indicates that such receptors confer NK cell functional competence through an unclear process termed "licensing." Ly49C is the main self-specific inhibitory Ly49 receptor in H-2(b) C57BL/6 (B6) mice. We used B6 Ly49C-transgenic and B6 β2 microglobulin (β2m)-knockout Ly49C-transgenic mice to investigate the impact of licensing through this inhibitory receptor in precursor and mature NK cells. We found that self-specific inhibitory receptors affected NK cell precursor survival and proliferation at particular developmental stages in an MHC class I-dependent manner. The presence of Ly49C impacted the NK cell repertoire in a β2m-dependent manner, with reduced Ly49A(+), Ly49G2(+), and Ly49D(+) subsets, an increased DNAM-1(+) subset, and higher NKG2D expression. Licensed NK cells displayed a skewed distribution of the maturation stages, which was characterized by differential CD27 and CD11b expression, toward the mature phenotypes. We found that Ly49C-mediated licensing induced a split effect on NK cell functions, with increased cytokine-production capabilities following engagement of various activating receptors while cytotoxicity remained unchanged. Analysis of licensed NK cell functions in vivo, in a system of mouse CMV infection, indicated that licensing did not play a major role in the NK cell antiviral response during acute infection, but it strongly impaired the generation and/or persistence of memory NK cells. This study unravels multifaceted effects of licensing on NK cell populations and their functions., (Copyright © 2016 by The American Association of Immunologists, Inc.)

The most frequent cause of sporadic viral encephalitis in western countries is Herpes simplex virus (HSV). Despite treatment, mortality rates reach 20-30% while survivors often suffer from significant morbidity. In mice, resistance to lethal Herpes simplex encephalitis (HSE) is multifactorial and influenced by mouse and virus strain as well as route of infection. The ability to restrict viral spread in the brain is one factor contributing to resistance. After infection of the oral mucosa with HSV type 1 (HSV-1), virus spreads throughout the brains of susceptible strains but is restricted in resistant C57BL/6 mice. To further investigate restriction of viral spread in the brain, mendelian analysis was combined with studies of congenic, intra-natural killer complex (intra-NKC) recombinant and antibody-depleted mice. Results from mendelian analysis support the restriction of viral spread as a dominant trait and consistent with a single gene effect. In congenic mice, the locus maps to the NKC on chromosome 6 and is provisionally termed Herpes Resistance Locus 2 (Hrl2). In intra-NKC recombinants, the locus is further mapped to the segment Cd69 through D6Wum34; a different location from previously identified loci (Hrl and Rhs1) also associated with HSV-1 infection. Studies with antibody-depleted mice indicate the effect of this locus is mediated by NK1.1(+) expressing cells. This model increases our knowledge of lethal HSE, which may lead to new treatment options.

Natural Killer (NK) cells are crucial in early resistance to murine cytomegalovirus (MCMV) infection. In B6 mice, the activating Ly49H receptor recognizes the viral m157 glycoprotein on infected cells. We previously identified a mutant strain (MCMVG1F) whose variant m157 also binds the inhibitory Ly49C receptor. Here we show that simultaneous binding of m157 to the two receptors hampers Ly49H-dependent NK cell activation as Ly49C-mediated inhibition destabilizes NK cell conjugation with their targets and prevents the cytoskeleton reorganization that precedes killing. In B6 mice, as most Ly49H+ NK cells do not co-express Ly49C, the overall NK cell response remains able to control MCMVm157G1F infection. However, in B6 Ly49C transgenic mice where all NK cells express the inhibitory receptor, MCMV infection results in altered NK cell activation associated with increased viral replication. Ly49C-mediated inhibition also regulates Ly49H-independent NK cell activation. Most interestingly, MHC class I regulates Ly49C function through cis-interactions that mask the receptor and restricts m157 binding. B6 Ly49C Tg, β2m ko mice, whose Ly49C receptors are unmasked due to MHC class I deficient expression, are highly susceptible to MCMVm157G1F and are unable to control a low-dose infection. Our study provides novel insights into the mechanisms that regulate NK cell activation during viral infection.

Activating and inhibitory receptors on natural killer (NK) cells have a crucial role in innate immunity, although the basis of the engagement of activating NK cell receptors is unclear. The activating receptor Ly49H confers resistance to infection with murine cytomegalovirus by binding to the 'immunoevasin' m157. We found that m157 bound to the helical stalk of Ly49H, whereby two m157 monomers engaged the Ly49H dimer. The helical stalks of Ly49H lay centrally across the m157 platform, whereas its lectin domain was not required for recognition. Instead, m157 targeted an 'aromatic peg motif' present in stalks of both activating and inhibitory receptors of the Ly49 family, and substitution of this motif abrogated binding. Furthermore, ligation of m157 to Ly49H or Ly49C resulted in intracellular signaling. Accordingly, m157 has evolved to 'tackle the legs' of a family of NK cell receptors.

The development and function of natural killer (NK) cells is regulated by the interaction of inhibitory receptors of the Ly49 family with distinct peptide-laden major histocompatibility complex (MHC) class I molecules, although whether the Ly49 family is able bind to other MHC class I-like molecules is unclear. Here we found that the prototypic inhibitory receptor Ly49A bound the highly conserved nonclassical MHC class I molecule H2-M3 with an affinity similar to its affinity for H-2D(d). The specific recognition of H2-M3 by Ly49A regulated the 'licensing' of NK cells and mediated 'missing-self' recognition of H2-M3-deficient bone marrow. Host peptide-H2-M3 was required for optimal NK cell activity against experimental metastases and carcinogenesis. Thus, nonclassical MHC class I molecules can act as cognate ligands for Ly49 molecules. Our results provide insight into the various mechanisms that lead to NK cell tolerance.

The Ly49H activating receptor on C57BL/6 (B6) NK cells plays a key role in early resistance to murine cytomegalovirus (MCMV) infection through specific recognition of the MCMV-encoded MHC class I-like molecule m157 expressed on infected cells. The m157 molecule is also recognized by the Ly49I inhibitory receptor from the 129/J mouse strain. The m157 gene is highly sequence variable among MCMV isolates, with many m157 variants unable to bind Ly49H(B6). In this study, we have sought to define if m157 variability leads to a wider spectrum of interactions with other Ly49 molecules and if this modifies host susceptibility to MCMV. We have identified novel m157-Ly49 receptor interactions, involving Ly49C inhibitory receptors from B6, BALB/c, and NZB mice, as well as the Ly49H(NZB) activation receptor. Using an MCMV recombinant virus in which m157(K181) was replaced with m157(G1F), which interacts with both Ly49H(B6) and Ly49C(B6), we show that the m157(G1F)-Ly49C interactions cause no apparent attenuating effect on viral clearance in B6 mice. Hence, when m157 can bind both inhibitory and activation NK cell receptors, the outcome is still activation. Thus, these data indicate that whereas m157 variants predominately interact with inhibitory Ly49 receptors, these interactions do not profoundly interfere with early NK cell responses.

The design of effective antiviral immunotherapies depends on a detailed understanding of the cellular and molecular processes involved in generating and maintaining immune responses. Control of cytomegalovirus (CMV) infection requires the concerted activities of both innate and adaptive immune effectors. In the mouse, immunity to acute murine CMV (MCMV) infection depends on natural killer (NK) cells and/or CD8(+) T cells. The relative importance of NK and CD8(+) T cells varies in different mouse strains. In C57BL/6 mice, early viral infection is controlled by Ly49H(+) NK cells, whereas in BALB/c mice, CD8(+) T cells exert the principal antiviral activities. Although the role of NK and CD8(+) T cells is defined, the molecular mechanisms they utilize to limit acute infection are poorly understood. Here, we define the specific roles of perforin (pfp) and interferon-gamma (IFN-gamma) in the context of NK- or T-cell-mediated immunity to MCMV during acute infection. We show that pfp is essential for both NK- and T-cell-mediated antiviral immunity during the early stages of infection. The relative importance of IFN-gamma is more pronounced in Ly49H(-) mice. Using BALB/c background mice congenic for Ly49H and lacking pfp, we show that Ly49H-regulated NK-cell control of MCMV infection is dependent on pfp-mediated cytolysis.

Natural killer (NK) cells play important roles in innate defense against infectious agents particularly viruses and also tumors. They mediate their effects through direct cytolysis, release of cytokines and regulation of subsequent adaptive immune responses. NK cells are equipped with sophisticated arrays of inhibitory and activation receptors that regulate their function. In this review we illustrate some of the major evolutionary relationships between NK cell receptors among different animal species and what some of the major mechanisms are that give rise to this diversity in receptor families, including the potential roles of pathogens such as viruses in driving receptor evolution.

Host NK cells can reject MHC-incompatible (allogeneic) bone marrow cells (BMCs), suggesting their effective role for graft-vs leukemia effects in the clinical setting of bone marrow transplantation. NK cell-mediated rejection of allogeneic BMCs is dependent on donor and recipient MHC alleles and other factors that are not yet fully characterized. Whereas the molecular mechanisms of allogeneic MHC recognition by NK receptors have been well studied in vitro, guidelines to understand NK cell allogeneic reactivity under the control of multiple genetic components in vivo remain less well understood. In this study, we use congenic mice to show that BMC rejection is regulated by haplotypes of the NK gene complex (NKC) that encodes multiple NK cell receptors. Most importantly, host MHC differences modulated the NKC effect. Moreover, the NKC allelic differences also affected the outcome of hybrid resistance whereby F1 hybrid mice reject parental BMCs. Therefore, these data indicate that NK cell alloreactivity in vivo is dependent on the combination of the host NKC and MHC haplotypes. These data suggest that the NK cell self-tolerance process dynamically modulates the NK cell alloreactivity in vivo.

The dissection of genetic resistance to murine cytomegalovirus infection in inbred laboratory mouse strains led to the identification of a natural killer cell activation receptor that recognizes a virus-encoded protein. Herein, we summarize the genetic approach and findings that have provided novel insights into innate immune control of virus infections.

The NK gene complex (NKC) on mouse chromosome 6 encodes receptors that are expressed on NK cells, such as Ly49H, and is involved in regulating NK cell control of virus infections, such as murine cytomegalovirus (MCMV). In the present study, we investigated the level of allelic heterogeneity in NKC loci in populations of outbred wild mice. This work revealed extensive levels of heterogeneity within two wild mouse populations. Analysis of MCMV replication in a population of specific pathogen-free outbred wild mice revealed that low viral titres, which are normally associated with the Cmv1(r) allele of the Cmv1 host resistance locus, were not prevalent in the mice tested. Hence, NKC-mediated resistance associated with Cmv1(r)/Ly49H-like effects was rare in this population. Overall, these data indicate that the NKC region is highly polymorphic and thus it is very likely that it confers on mice sufficient variability to cope with infection by a range of pathogens.

The natural killer complex (NKC) is a genetic region of highly linked genes encoding several receptors involved in the control of NK cell function. The NKC is highly polymorphic, and allelic variability of various NKC loci has been demonstrated in inbred mice. Making use of BALB.B6-Cmv1r congenic mice, in which the NKC from disease-susceptible C57BL/6 mice has been introduced into the disease-resistant BALB/c background, we show here that during murine malaria infection, the NKC regulates a range of pathophysiological syndromes such as cerebral malaria, pulmonary edema, and severe anemia, which contribute to morbidity and mortality in human malaria. Parasitemia levels were not affected by the NKC genotype, indicating that control of malarial fatalities by the NKC cells does not operate through effects on parasite growth rate. Parasite-specific antibody responses and the proinflammatory gene transcription profile, as well as the TH1/TH2 balance, also appeared to be influenced by NKC genotype, providing evidence that this region, known to control innate immune responses via NK and/or NK T-cell activation, can also significantly regulate acquired immunity to infection. To date, NKC-encoded innate system receptors have been shown mainly to regulate viral infections. Our data provide evidence for critical NKC involvement in the broad immunological responses to a protozoan parasite.

Dendritic cells (DC), first characterized in 1973 by Steinman and Cohn, have been defined as the professional antigen presenting cells (APC), capable of activating naïve T cells much more efficiently than either B cells or macrophages. DC also capture and process antigen more efficiently than other APC, and offer MHC-antigen complexes to T cells at higher densities, and in the context of larger amounts of co-stimulatory molecules (i.e. CD40, CD80 and CD86) at the T cell-DC synapse. Although historically, the principal function of DC is the priming of naïve T cells, more recently they have also been shown to affect the functions of natural killer (NK) cells. Interactions between DC and NK cells may be critical in situations where immune surveillance requires efficient early activation of NK cells, as is the case during infections. This review aims to summarise the interactions that occur between DC and NK cells during viral infection.

Effective natural killer (NK) cell recognition of murine cytomegalovirus (MCMV)-infected cells depends on binding of the Ly49H NK cell activation receptor to the m157 viral glycoprotein. Here we addressed the immunological consequences of variation in m157 sequence and function. We found that most strains of MCMV possess forms of m157 that evade Ly49H-dependent NK cell activation. Importantly, repeated passage of MCMV through resistant Ly49H+ mice resulted in the rapid emergence of m157 mutants that elude Ly49H-dependent NK cell responses. These data provide the first molecular evidence that NK cells can exert sufficient immunological pressure on a DNA virus, such that it undergoes rapid and specific mutation in an NK cell ligand enabling it to evade efficient NK cell surveillance.

The NK gene complex (NKC) controls murine cytomegalovirus (MCMV) immunity through Cmv1-dependent natural killer (NK) cell responses. Ly49H expression correlates with Cmv1 phenotypes in different inbred strains, is required for MCMV resistance in C57BL/6 (B6) mice, and its interaction with the MCMV encoded m157 protein leads to NK cell-mediated destruction of virus-infected cells. However, genetic mapping studies have previously indicated that Cmv1 should reside in the D6Wum9-16 NKC interval, distal to Ly49h. Since these data suggested that multiple NKC-linked loci could regulate viral immunity, a putative MCMV resistance control ( Mrc) locus was pinpointed to within the D6Wum9-16 interval on a NKC-aligned bacterial artificial chromosome (BAC). Sequence analysis of BAC 151 revealed several novel G-protein coupled receptor genes, an HMG-1 remnant and many additional polymorphic microsatellites that were useful in determining the minimal genetic interval for the Mrc locus. Moreover, comparison of B6, BALB/c, A/J and recombinant Mrc alleles restricted the genetic interval to approximately 470 bp and showed that it was also a hotspot for recombination. MCMV challenge of novel NKC recombinant mice demonstrated that Mrc(B6) was not required for MCMV resistance nor could it directly complement the Ly49(BALB) haplotype to rescue MCMV susceptibility. Taken together, these data show that while Mrc apparently guides recombination, Ly49H expression is sufficient for MCMV resistance in B6 mice. A direct role for Mrc(B6) in virus resistance is excluded in the novel mice.

NKT cells are specialized cells coexpressing NK and T cell receptors. Upon activation they rapidly produce high levels of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) and are therefore postulated to influence T(H)1/T(H)2 immune responses. The precise role of the CD1/NKT cell pathway in immune response to infection remains unclear. We show here that CD1d-restricted NKT cells from distinct genetic backgrounds differentially influence T(H)1/T(H)2 polarization, proinflammatory cytokine levels, pathogenesis, and fatality in the P. berghei ANKA/rodent model of cerebral malaria. The functional properties of CD1d-restricted NKT cells vary according to expression of loci of the natural killer complex (NKC) located on mouse chromosome 6, which is shown here to be a significant genetic determinant of murine malarial fatalities.

Ly49H(+)NK1.1(+) natural killer (NK) cells are essential for the control of murine cytomegalovirus (MCMV) during the acute stage of infection. This cell subset expands at the later stages of infection in an MCMV-specific fashion. Here we demonstrate a critical interaction between Ly49H(+) NK cells and CD8alpha(+) dendritic cells (DCs) whereby the presence of Ly49H(+) NK cells results in maintenance of CD8alpha(+) DCs in the spleen during acute MCMV infection. Reciprocally, CD8alpha(+) DCs are essential for the expansion of Ly49H(+) NK cells by a mechanism involving interleukin 18 (IL-18) and IL-12. This study provides evidence for a functional interrelationship between DCs and NK cells during viral infection and defines some of the critical cytokines.

Natural killer (NK) cells are best known for their capacity to kill tumors but they are also critical in early innate responses to infection, especially herpesviruses. Recent studies indicate that NK cell receptors involved in tumor target specificity are also involved in responses to viral infections.

Natural killer (NK) cells play a crucial role in limiting the severity of diseases caused by a range of viruses. Recent data have shown that the effector functions of NK cells can be specifically stimulated when NK cell activation receptors engage cellular major histocompatibility complex (MHC) class I-like ligands induced after infection or by specific viral gene products. However, to counter this NK cell response viruses have evolved an array of strategies to subvert efficient NK cell activation. These data indicate that the balance of host NK cell responses and viral NK cell escape mechanisms can be strategically poised as each strives for survival.

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.

The susceptibility of certain inbred mouse strains to murine cytomegalovirus (MCMV) is related to their inability to generate a strong natural killer (NK) cell response. We addressed here whether the MCMV susceptibility of the BALB/c strain is due to viral functions that control NK cell activation in a strain-specific manner. MCMV expresses two proteins, gp48 and gp40, that are encoded by the genes m06 and m152, respectively; they down-regulate major histocompatibility complex (MHC) class I expression at the plasma membrane. Using MCMV deletion mutants and revertants, we found that gp40 but not gp48 controls NK cell activation. Absence of gp40 improved antiviral NK cell control in BALB/c, but not C57BL/6, mice. Down-regulation of H-60, the high-affinity ligand for the NKG2D receptor, was the mechanism by which gp40 modulates NK cell activation. Thus, a single herpesvirus protein has a dual function in inhibiting both the adaptive as well as the innate immune response.

Allelic variability for mouse Chromosome 6 Nkc loci was assessed in 22 common laboratory strains of mice using selected natural killer gene complex (Nkc)-linked sequence tagged site markers. Most Nkc markers distinguished three or more alleles for a particular locus in the assessed mouse strains. Nkc locus alleles were highly conserved among genealogically related inbred strains, whereas far less similarity was observed among unrelated strains. Concurrent strain-to-strain comparisons for all Nkc-linked loci revealed common and uncommon Nkc haplotypes, including some that were likely recombinant. Nkc allele and haplotype assignments in inbred mouse strains and correlation with phenotypic traits should facilitate positional gene cloning strategies for unknown Nkc-linked trait modification loci.

CD1d-restricted Valpha14-Jalpha281 invariant alphabetaTCR(+) (NKT) cells are well defined in the C57BL/6 mouse strain, but they remain poorly characterized in non-NK1.1-expressing strains. Surrogate markers for NKT cells such as alphabetaTCR(+)CD4(-)CD8(-) and DX5(+)CD3(+) have been used in many studies, although their effectiveness in defining this lineage remains to be verified. Here, we compare NKT cells among C57BL/6, NK1.1-congenic BALB/c, and NK1.1-congenic nonobese diabetic mice. NKT cells were identified and compared using a range of approaches: NK1.1 expression, surrogate phenotypes used in previous studies, labeling with CD1d/alpha-galactosylceramide tetramers, and cytokine production. Our results demonstrate that NKT cells and their CD4/CD8-defined subsets are present in all three strains, and confirm that nonobese diabetic mice have a numerical and functional deficiency in these cells. We also highlight the hazards of using surrogate phenotypes, none of which accurately identify NKT cells, and one in particular (DX5(+)CD3(+)) actually excludes these cells. Finally, our results support the concept that NK1.1 expression may not be an ideal marker for CD1d-restricted NKT cells, many of which are NK1.1-negative, especially within the CD4(+) subset and particularly in NK1.1-congenic BALB/c mice.

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.

NK cells mediate early host defense against viral infection. In murine CMV (MCMV) infection NK cells play a critical role in controlling viral replication in target organs, such as spleen and liver. Until now it has not been possible to directly examine the role of NK cells in MCMV-induced inflammation in situ due to the inability to stain specifically for NK cells in infected tissues. In this study, we describe a method of in vivo fixation, resulting in the first identification of NK cells in situ using NK1.1 as the marker. Using this method, we characterize the NK1.1(+) cellular component of the inflammatory response to wild-type MCMV in the spleen, liver, and lung of genetically susceptible and resistant mice following i.p. infection. This study provides the first in situ description of the cellular response mediated specifically by NK cells following MCMV infection.

Overcoming the massive and rapid death of injected donor myoblasts is the primary hurdle for successful myoblast transfer therapy (MTT), designed as a treatment for the lethal childhood myopathy Duchenne muscular dystrophy. The injection of male myoblasts into female host mice and quantification of surviving male DNA using the Y-chromosome-specific (Y1) probe allows the speed and extent of death of donor myoblasts to be determined. Cultured normal C57BL/10Sn male donor myoblasts were injected into untreated normal C57BL/10Sn and dystrophic mdx female host mice and analyzed by slot blots using a 32P-labeled Y1 probe. The amount of male DNA from donor myoblasts showed a remarkable decrease within minutes and by 1 h represented only about 10-18% of the 2.5 x 10(5) cells originally injected (designated 100%). This declined further over 1 week to approximately 1-4%. The host environment (normal or dystrophic) as well as the extent of passaging in tissue culture (early "P3" or late "P15-20" passage) made no difference to this result. Modulation of the host response by CD4+/CD8+ -depleting antibodies administered prior to injection of the cultured myoblasts dramatically enhanced donor myoblast survival in dystrophic mdx hosts (15-fold relative to untreated hosts after 1 week). NK1.1 depletion also dramatically enhanced donor myoblast survival in dystrophic mdx hosts (21-fold after 1 week) compared to untreated hosts. These results provide a strategic approach to enhance donor myoblast survival in clinical trials of MTT.

The Cmv1 locus controls NK cell-mediated resistance to infection with murine CMV. Our recent genetic analysis of backcross mice demonstrated that the NK gene complex (NKC)-linked Cmv1 locus should reside between the Ly49 and Prp gene clusters on distal mouse chromosome 6. We have aligned yeast artificial chromosome (YAC) inserts in a contig spanning the interval between the Ly49 and Prp gene clusters. This YAC contig includes 13 overlapping YAC inserts that span more than 2 megabases (Mb) in C57BL/6 (B6) mice. Since we have identified genomic clones that span the Ly49-Prp gene region, we hypothesize that at least one should contain the Cmv1 locus. To narrow the Cmv1 critical region, we developed novel NKC genetic markers and used these to genotype informative backcross and intra-NKC recombinant congenic mouse DNA samples. These data suggest that Cmv1 resides on a single YAC insert within an interval that corresponds to a physical distance of approximately 390 kb. This high resolution, integrated physical and genetic NKC map will facilitate identification of Cmv1 and other NKC-linked loci that regulate NK cell-mediated immunity.

We provide the first demonstration, using experimental and spontaneous models of metastasis in C57BL/6 (B6) (RM-1 prostate carcinoma) and BALB/c (DA3 mammary carcinoma) mice, that tumor metastasis is primarily controlled by perforin-dependent cytotoxicity mediated by NK1.1+ cells. MHC class Ilow RM-1 and DA3 tumor cells were sensitive in vitro to Fas-mediated lysis or spleen NK cells in a perforin-dependent fashion. Perforin-deficient NK cells did not lyse these tumors, and perforin-deficient mice were 10-100-fold less proficient than wild-type mice in rejecting the metastasis of tumor cells to the lung. Fas ligand mutant gld mice displayed uncompromised protection against tumor metastasis. Depletion of NK subsets resulted in greater numbers of metastases than observed in perforin-deficient mice, suggesting that perforin-independent effector functions of NK cells may also contribute to protection from tumor metastasis.

Previously, we established that natural killer (NK) cells from C57BL/6 (B6), but not BALB/c, mice lysed Chinese hamster ovary (CHO) cells, and we mapped the locus that determines this differential CHO-killing capacity to the NK gene complex on chromosome 6. The localization of Chok in the NK gene complex suggested that it may encode either an activating or an inhibitory receptor. Here, results from a lectin-facilitated lysis assay predicted that Chok is an activating B6 NK receptor. Therefore, we immunized BALB/c mice with NK cells from BALB.B6-Cmv1(r) congenic mice and generated a mAb, designated 4E4, that blocked B6-mediated CHO lysis. mAb 4E4 also redirected lysis of Daudi targets, indicating its reactivity with an activating NK cell receptor. Furthermore, only the 4E4(+) B6 NK cell subset mediated CHO killing, and this lysis was abrogated by preincubation with mAb 4E4. Flow cytometric analysis indicated that mAb 4E4 specifically reacts with Ly-49D but not Ly-49A, B, C, E, G, H, or I transfectants. Finally, gene transfer of Ly-49DB6 into BALB/c NK cells conferred cytotoxic capacity against CHO cells, thus establishing that the Ly-49D receptor is sufficient to activate NK cells to lyse this target. Hence, Ly-49D is the Chok gene product and is a mouse NK cell receptor capable of directly triggering natural killing.

The molecular mechanisms underlying target recognition during natural killing are not well understood. One approach to dissect the complexities of natural killer (NK) cell recognition is through exploitation of genetic differences among inbred mouse strains. In this study, we determined that interleukin 2-activated BALB/c-derived NK cells could not lyse Chinese hamster ovary (CHO) cells as efficiently as C57BL/6-derived NK cells, despite equivalent capacity to kill other targets. This strain-determined difference was also exhibited by freshly isolated NK cells, and was determined to be independent of host major histocompatibility haplotype. Furthermore, CHO killing did not correlate with expression of NK1.1 or 2B4 activation molecules. Genetic mapping studies revealed linkage between the locus influencing CHO killing, termed Chok, and loci encoded within the NK gene complex (NKC), suggesting that Chok encodes an NK cell receptor specific for CHO cells. In vivo assays recapitulated the in vitro data, and both studies determined that Chok regulates an NK perforin-dependent cytotoxic process. These results may have implications for the role of NK cells in xenograft rejection. Our genetic analysis suggests Chok is a single locus that affects NK cell-mediated cytotoxicity similar to other NKC loci that also regulate the complex activity of NK cells.

Natural killer (NK) cells express C-type lectin-like receptors, encoded in the NK gene complex, that interact with major histocompatibility complex class I and either inhibit or activate functional activity. Human NK cells express heterodimers consisting of CD94 and NKG2 family molecules, whereas murine NK cells express homodimers belonging to the Ly-49 family. The corresponding orthologues for other species, however, have not been described. In this report, we used probes derived from the expressed sequence tag database to clone C57BL/6-derived cDNAs homologous to human NKG2-D and CD94. Among normal tissues, murine NKG2-D and CD94 transcripts are highly expressed only in activated NK cells, including both Ly-49A+ and Ly-49A- subpopulations. Additionally, mNKG2-D is expressed in murine NK cell clones KY-1 and KY-2, whereas mCD94 expression is observed only in KY-1 cells but not KY-2. Last, we have finely mapped the physical location of the Cd94 (centromeric) and Nkg2d (telomeric) genes between Cd69 and the Ly49 cluster in the NK complex. Thus, these data indicate the expanding complexity of the NK complex and the corresponding repertoire of C-type lectin-like receptors on murine NK cells.

We have constructed a physical map of a > 2-Mb region on mouse chromosome 6 that contains the natural killer gene complex (NKC). The map comprises a contig of 14 overlapping yeast artificial chromosomes onto which we positioned 25 NKC markers. NKC genetically linked genes encode > 17 proteins that directly control innate NK cell-mediated tumor lysis and disease resistance. Herein we show that Nkrp1 genes are clustered in a region flanked by A2m and Cd69 genes and that most Ly49 genes are clustered in a distal region -1 Mb distant. Importantly, syntenic intervals of mouse chromosome 6 and human chromosome 12p that include the NKC are conserved. NKC species conservation suggests that the human NKC may contain orthologues for the mouse viral disease resistance genes, Cmv1 and Rmp1. The high-resolution NKC map will facilitate investigation of NKC gene regulation and identification of phenotypically defined gene products that confer NK cell defense against viral pathogens.

Natural killer (NK) cells play important roles in controlling tumor cells and against a range of infectious organisms. Recent studies of mouse NK cell surface receptors, which may be involved in the specificity of NK cells, have shown that many of these molecules are encoded by the Ly49 and Ly55 (Nkrp1) multigene families that map to distal mouse chromosome 6. Also mapping to this NK cell gene complex (NKC) is the resistance locus, Cmv1, which is involved in genetically determined resistance to murine cytomegalovirus (MCMV). The aim of this study was to localize Cmv1 more precisely in relation to other NKC loci by generating a high-resolution genetic map of the region. We have analyzed 1250 backcross mice comprising panels of 700 (BALB/c x C57BL/6J)F1 x BALB/c and 550 (A/J x C57BL/6J)F1 x A/J progeny. A total of 25 polymorphic genes or microsatellite markers were analyzed over a region of 10 map units from D6Mit134 to D6Mit59. The Cmv1 phenotypes of mice recombinant in this interval were tested by infection with MCMV. The results obtained indicate that the functionally important NKC region is a tightly linked cluster of loci spanning at least 0.4 map units. Furthermore, Cmv1 maps distal to, but very closely linked to, the Ly49 multigene family (< 0.2 map units), suggesting that MCMV resistance may be conferred by MHC class I-specific NK cell receptors.

Herpesviruses, such as murine and human cytomegalovirus (MCMV and HCMV), can establish a persistent infection within the host and have diverse mechanisms as protection from host immune defences. Several herpesvirus genes that are homologous to host immune modulators have been identified, and are implicated in viral evasion of the host immune response. The discovery of a viral major histocompatibility complex (MHC) class I homologue, encoded by HCMV, led to speculation that it might function as an immune modulator and disrupt presentation of peptides by MHC class I to cytotoxic T cells. However, there is no evidence concerning the biological significance of this gene during viral infection. Recent analysis of the MCMV genome has also demonstrated the presence of a MHC class I homologue. Here we show that a recombinant MCMV, in which the gene encoding the class I homologue has been disrupted, has severely restricted replication during the acute stage of infection compared with wild-type MCMV. We demonstrate by in vivo depletion studies that natural killer (NK) cells are responsible for the attenuated phenotype of the mutant. Thus the viral MHC class I homologue contributes to immune evasion through interference with NK cell-mediated clearance.

The natural killer gene complex encodes proteins, some of which are structurally unrelated, that impact on NK-cell function. Detailed analyses have indicated that these molecules are involved in NK-cell recognition, activation, and inhibition. The importance of this genomic region is highlighted by studies indicating that NKC-associated genes significantly influence NK cell-mediated innate host defense against life-threatening pathogens and that the NKC is conserved among diverse species. Thus, further elucidation of the NKC and its gene products will provide a genetic basis for understanding innate immunity and NK-cell activity at the molecular level.

The influence of host genotype on the relative importance of T cell subsets and natural killer (NK) cells in controlling murine cytomegalovirus (MCMV) replication has been investigated. Genetically susceptible BALB/c and A/J, moderately resistant C57BL/10, and resistant CBA/CaH mouse strains were treated with monoclonal antibodies (MAb) to the CD4 and CD8 markers and the extent of MCMV replication in major target tissues was determined. Both mouse strain-specific and tissue-specific effects were observed. CBA/CaH and C57BL/10 mice were found not to require CD4+ or CD8+ T cells for control of MCMV replication in the spleen or liver. In contrast, in A/J mice, as well as BALB/c mice, the CD8+ T cell population was primarily responsible for the clearance of virus from these tissues. However, in all strains of mice, CD4+ T cells were required for delayed type hypersensitivity and antibody responses, and for virus clearance in the salivary glands. The dependence of mice with the BALB genetic background on CD8+ T cells for limitation of acute MCMV infection was found to be negated in the BALB.B6-Cmv1(r) congenic strain, in which an effective NK cell response has been generated through the introduction of the resistant Cmv1(r) allele from C57BL/6 mice. Depletion of NK cells in the BALB.B6-Cmv1(r) strain using anti-NK1.1 MAb restored the role of CD8+ T cells in mediating viral clearance. These analyses demonstrate that some, but not all, strains of mice use CD8+ T cells to control MCMV replication and that even when CD8+ T cell-dependence exists, this can be circumvented by an appropriate NK cell response.

The Cmv1 resistance gene controls splenic replication of murine cytomegalovirus (MCMV) and confers natural killer (NK) cell-mediated resistance to otherwise lethal infection. The Cmv1 phenotypes of 13 inbred mouse strains have been assessed, and it was found that the Cmv1r resistance phenotype was restricted to the C57BL/6J and Ma/MyJ strains. We have further analyzed the linkage of Cmv1 to the NK gene complex (NKC) mapping to distal mouse chromosome 6 in 99 (BALB/c x C57BL/6J)F1 x BALB/c backcross mice using cloned gene probes and microsatellite markers from this region. No recombinants were observed between Cmv1 and the NKC-associated Ly49 and musNKR-P1 multigene families, nor the Kap locus, nor with 7 microsatellite markers, indicating that Cmv1 is closely linked (< 1 cM) to all of these markers. Analysis of the genotype of the MCMV-susceptible BXD8 RI strain around the NKC region revealed that it had C57BL/6J alleles at microsatellite markers immediately proximal and distal to Cmv1. This suggests that the Cmv1s phenotype of this strain is due to a germ-line mutation. Thus, the close linkage of Cmv1 to the Ly49 and musNKR-P1 multigene families suggests that it may represent an NK cell recognition structure encoded in the NKC region.

The resistance of mice to lethal infection by murine CMV (MCMV) is under complex host genetic control with contributions from both H-2 and non-H-2 genes. We have previously shown that an autosomal, non-MHC encoded gene, Cmv-1, controls MCMV replication in the spleen. We have investigated the mechanism by which the Cmv-1 resistance gene confers protection against MCMV infection. Using H-2 compatible irradiation bone marrow chimeras, the enhanced resistance to MCMV infection that is associated with the Cmv-1l allele in the C57BL background was shown to be mediated by an irradiation-sensitive bone marrow-derived cell population, or a factor produced by these cells. The lack of correlation between serum IFN titers and the strain distribution pattern of Cmv-1 in CXB recombinant inbred mouse strains suggests that IFN does not mediate resistance conferred by this gene. Similarly, the lack of effect of in vivo depletion of mature CD4+ and CD8+ T cells on virus replication in C57BL/6J mice indicates that T cells are unlikely to be involved. In contrast, in vivo depletion of NK cells by injection of the anti-NK1.1 mAb PK136 abrogated restricted splenic virus replication in C57BL/6J----BALB.B chimeric mice and in the Cmv-1l CXB strains. These data indicate that the effect of the Cmv-1 gene is mediated by NK cells. The significant augmentation in NK cell activity after MCMV infection of the susceptible Cmv-1h strains (BALB/cBy), CXBG/By, CXBH/By, CXBI/By, and CXBK/By) indicates the existence in these mice of NK cells that are functionally and phenotypically distinct from those in Cmv-1l strains. NK cells present in the Cmv-1h strains are unable to restrict efficiently splenic MCMV replication in vivo, possibly due to a lack of specificity for virus-infected target cells. Finally, flow cytometric analysis of NK1-1 expression in CXB and BXD RI mice together with MCMV replication studies in the BXD RI strains indicate that Cmv-1 is closely linked to NK1.1 and other loci that reside on a distal segment of murine chromosome 6 in a region that has recently been defined as the natural killer complex.

Site-directed in vitro mutagenesis followed by in vitro transcription and translation has been used to study structure/function relationships for murine interferon-alpha 1 (MuIFN-alpha 1). The mature form of the MuIFN-alpha 1 protein was expressed as well as analogue forms with amino acid substitutions at positions 33, 71, 72, 123 and 133. These positions were chosen on the basis of known human interferon-alpha structure/function relationships. Biological assays for antiviral activity on murine cells and natural killer cell activation have been performed for each of the proteins produced. The data obtained have been interpreted in the light of previous human and murine interferon-alpha structure/function work and the recently published three-dimensional structure of murine type I interferon.