Deficient major histocompatibility complex-linked innate murine cytomegalovirus immunity in MA/My.L-H2b mice and viral downregulation of H-2k class I proteins

NK cells are key effectors of innate immunity and host survival during cytomegalovirus (CMV) infection. Innate murine CMV (MCMV) resistance in MA/My mice requires Ly49H/m157-independent H-2k-linked NK cell control. Here we show that replacement of MA/My H-2k with C57L H-2b susceptibility genes led to a remarkable loss of innate virus immunity, though NK gamma interferon was induced in H-2b and H-2k strains shortly after infection. Thus, H-2b genes expressed in C57L or MA/My.L-H2b are sufficient in alerting NK cells to intrusion but fail to support NK restraint of viral infection. In addition, novel H-2 recombinant strains were produced and utilized in a further refinement of a critical genetic interval controlling innate H-2k-linked MCMV resistance. Importantly, this analysis excluded the gene interval from Kk class I through class II. The responsible gene(s) therefore resides in an interval spanning Dk class Ia and more-distal major histocompatibility complex (MHC) nonclassical class Ib genes. Recently, the NK activation receptor Ly49P and MHC class I Dk proteins were genetically implicated in MCMV resistance, in part because Ly49P-expressing reporter T cells could specifically bind Dk molecules on MCMV-infected mouse embryonic fibroblasts (MEFs). However, as we found that H-2k innate resistance differs in the C57L or MA/My backgrounds and because MCMV very efficiently downregulates H-2k class I proteins in L929 cells and primary MEFs shortly after infection, a Ly49P/Dk model should not fully explain H-2k-linked MCMV resistance.     

Requisite H2k role in NK cell-mediated resistance in acute murine cytomegalovirus-infected MA/My mice

Human CMV infections are a major health risk in patients with dysfunctional or compromised immunity, especially in patients with NK cell deficiencies, as these are frequently associated with high morbidity and mortality. In experimental murine CMV (MCMV) infections, Ly49H activation receptors on C57BL/6 (B6) NK cells engage m157 viral ligands on MCMV-infected cells and initiate dominant virus control. In this study, we report that MCMV resistance in MA/My relies on Ly49H-independent NK cell-mediated control of MCMV infection as NK cells in these mice do not bind anti-Ly49H mAb or soluble m157 viral ligands. We genetically compared MA/My resistance with MCMV susceptibility in genealogically and NK gene complex-Ly49 haplotype-related C57L mice. We found that MCMV resistance strongly associated with polymorphic H2k-linked genes, including MHC and non-MHC locations by analysis of backcross and intercross progeny. The H2b haplotype most frequently, but not absolutely, correlated with MCMV susceptibility, thus confirming a role for non-MHC genes in MCMV control. We also demonstrate a definite role for NK cells in H2k-type MCMV resistance because their removal from C57L.M-H2k mice before MCMV infection diminished immunity. NK gene complex-linked polymorphisms, however, did not significantly influence MCMV control. Taken together, effective NK cell-mediated MCMV control in this genetic system required polymorphic H2k genes without need of Ly49H-m157 interactions.     

Cmv4, a new locus linked to the NK cell gene complex, controls innate resistance to cytomegalovirus in wild-derived mice

CMV can cause life-threatening disease in immunodeficient hosts. Experimental infection in mice has revealed that the genetically determined natural resistance to murine CMV (MCMV) may be mediated either by direct recognition between the NK receptor Ly49H and the pathogen-encoded glycoprotein m157 or by epistatic interaction between Ly49P and the host MHC H-2D(k). Using stocks of wild-derived inbred mice as a source of genetic diversity, we found that PWK/Pas (PWK) mice were naturally resistant to MCMV. Depletion of NK cells subverted the resistance. Analysis of backcrosses to susceptible BALB/c mice revealed that the phenotype was controlled by a major dominant locus effect linked to the NK gene complex. Haplotype analysis of 41 polymorphic markers in the Ly49h region suggested that PWK mice may share a common ancestral origin with C57BL/6 mice; in the latter, MCMV resistance is dependent on Ly49H-m157 interactions. Nevertheless, PWK mice retained viral resistance against m157-defective mutant MCMV. These results demonstrate the presence of yet another NK cell-dependent viral resistance mechanism, named Cmv4, which most likely encodes for a new NK activating receptor. Identification of Cmv4 will expand our understanding of the specificity of the innate recognition of infection by NK cells.     

Genomic Modifiers of Natural Killer Cells,Immune Responsiveness and Lymphoid Tissue Remodeling Together Increase Host Resistance to Viral Infection

The MHC class I D^k molecule supplies vital host resistance during murine cytomegalovirus (MCMV) infection. Natural killer (NK) cells expressing the Ly49G2 inhibitory receptor, which specifically binds D^k, are required to control viral spread. The extent of D^k-dependent host resistance, however, differs significantly amongst related strains of mice, C57L and MA/My. As a result, we predicted that relatively small-effect modifier genetic loci might together shape immune cell features, NK cell reactivity, and the host immune response to MCMV. A robust D^k-dependent genetic effect, however, has so far hindered attempts to identify additional host resistance factors. Thus, we applied genomic mapping strategies and multicolor flow cytometric analysis of immune cells in naive and virus-infected hosts to identify genetic modifiers of the host immune response to MCMV. We discovered and validated many quantitative trait loci (QTL); these were mapped to at least 19 positions on 16 chromosomes. Intriguingly, one newly discovered non-MHC locus (Cmv5) controlled splenic NK cell accrual, secondary lymphoid organ structure, and lymphoid follicle development during MCMV infection. We infer that Cmv5 aids host resistance to MCMV infection by expanding NK cells needed to preserve and protect essential tissue structural elements, to enhance lymphoid remodeling and to increase viral clearance in spleen.     

Gain of virulence caused by loss of a gene in murine cytomegalovirus

Mouse strains are either resistant or susceptible to murine cytomegalovirus (MCMV). Resistance is determined by the Cmv1(r) (Ly49h) gene, which encodes the Ly49H NK cell activation receptor. The protein encoded by the m157 gene of MCMV has been defined as a ligand for Ly49H. To find out whether the m157 protein is the only Ly49H ligand encoded by MCMV, we constructed the m157 deletion mutant and a revertant virus. Viruses were tested for susceptibility to NK cell control in Ly49H+ and Ly49H- mouse strains. Deletion of the m157 gene abolished the viral activation of Ly49H+ NK cells, resulting in higher virus virulence in vivo. Thus, in the absence of m157, Ly49H+ mice react like susceptible strains. 129/SvJ mice lack the Ly49H activation NK cell receptor but express the inhibitory Ly49I NK cell receptor that binds to the m157 protein. The Deltam157 inhibitory phenotype was weak because MCMV encodes a number of proteins that mediate NK inhibition, whose contribution could be shown by another mutant.     

NK gene complex and chromosome 19 loci enhance MHC resistance to murine cytomegalovirus infection

An H-2^k MHC locus is critical for murine cytomegalovirus (MCMV) resistance in MA/My mice and virus control is abolished if H-2^k is replaced with H-2^b MHC genes from MCMV-susceptible C57L mice. Yet, H-2^k resistance varies with genetic background; thus, modifiers of virus resistance must exist. To identify non-MHC resistance loci, spleen and liver MCMV levels and genome-wide genotypes were assessed in (C57L × MA/My) and (MA/My × C57L) F₂ offspring (representing 550 meioses). Significantly, a non-Mendelian frequency of MHC genotypes was observed for offspring of the latter cross. Quantitative trait loci (QTL) and their interaction potential in MCMV resistance were assessed in R/qtl; QTL on chromosomes 17, 6, and 19 affected MCMV levels in infected animals. A chromosome 6 QTL was linked with the NK gene complex and acted in an additive fashion with an H-2^k MHC QTL to mitigate spleen MCMV levels. We provide biological confirmation that this chromosome 6 QTL provided MCMV control independent of H-2^k via NK cells. Importantly, both chromosome 6 and 19 QTLs contribute to virus control independent of H-2^k. Altogether, MHC and non-MHC MCMV-resistance QTL contribute in early resistance to MCMV infection in this genetic system.     

NK gene complex haplotype variability and host resistance alleles to murine cytomegalovirus in wild mouse populations

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.     

A Cmv2 QTL on chromosome X affects MCMV resistance in New Zealand male mice

NK cell-mediated resistance to viruses is subject to genetic control in humans and mice. Here we used classical and quantitative genetic strategies to examine NK-mediated murine cytomegalovirus (MCMV) control in genealogically related New Zealand white (NZW) and black (NZB) mice. NZW mice display NK cell-dependent MCMV resistance while NZB NK cells fail to limit viral replication after infection. Unlike Ly49H⁺ NK resistance in C57BL/6 mice, NZW NK-mediated MCMV control was Ly49H-independent. Instead, MCMV resistance in NZW (Cmv2) involves multiple genetic factors. To establish the genetic basis of Cmv2 resistance, we further characterized a major chromosome X-linked resistance locus (DXMit216) responsible for innate MCMV control in NZW × NZB crosses. We found that the DXMit216 locus affects early MCMV control in New Zealand F₂ crosses and demonstrate that the NZB-derived DXMit216 allele enhances viral resistance in F₂ males. The evolutionary conservation of the DXMit216 region in mice and humans suggests that a Cmv2-related mechanism may affect human antiviral responses. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pathogenesis of acute murine cytomegalovirus infection in resistant and susceptible strains of mice.

We have characterized the progress of acute murine cytomegalovirus (MCMV) infection in the spleen, liver, and salivary gland of susceptible (BALB/c) and resistant (C3H) strains of mice after intraperitoneal inoculation. Viral replication was analyzed by virus titration, infectious-center assays, and in situ cytohybridization with cloned subgenomic fragments of the MCMV genome. The most striking differences between strains were observed in the spleen. At 24 h postinfection (p.i.), both strains had a similar number of infected spleen cells. At 48 h p.i., BALB/c mice showed marked dissemination of the splenic infection which continued until 96 h p.i. In contrast, the number of infected C3H spleen cells did not increase from the 24-h level but declined later on. This early block in dissemination of MCMV infection in C3H mouse spleens was not a result of the H-2k haplotype, as BALB.K (H-2k) mice, which show an intermediate level of resistance to MCMV infection, exhibited dissemination of the infection between 24 and 48 h p.i., albeit at a reduced level. However, between 72 and 96 h p.i., we observed a decline in the number of infected spleen cells in BALB.K mice similar to that observed in C3H mice. We also demonstrated by Southern blot analysis of DNA from the infected spleen cells that the termini of the MCMV genome fuse after in vivo infection.     

Genetically determined resistance to lethal murine cytomegalovirus infection is mediated by interferon-dependent and -independent restriction of virus replication.

Susceptibility of 4-week-old mice of different strains to lethal murine cytomegalovirus (MCMV) infection was studied. Strains homozygous for H-2k and C57BL strains were resistant to greater than or equal to 10(5.5) PFU. B10.BR mice congenic for C57BL background genes and H-2k were about 10-fold more resistant than either C3H/HeN or C57BL strains. BALB/c mice (H-2d) were susceptible (50% lethal dose, 10(5.05) PFU). This susceptibility was dominant over resistance associated with H-2k but not that associated with C57BL background genes. The dominant susceptibility trait segregated in backcross mice as if carried by a single gene. Virus replication in spleen cells in vivo correlated with susceptibility to lethal infection. A similar trend was found in tests of salivary glands. Replication of MCMV in vitro in cultures of adherent spleen cells and primary mouse embryo cells correlated with replication in vivo. Neutralization of interferon (IFN) in cultures of adherent spleen cells reversed H-2k-linked restriction of viral replication but had minor effects on cells of other strains. Natural killer cell responses to infection were often higher in more resistant strains, but B10.BR mice developed minimal natural killer cell responses. Specific antibody and cytotoxic T cell responses in B10.BR mice were similar or lower than in other strains. Thus, resistance to lethal MCMV infection was not immunologically mediated, was dependent on and reflected by the capacity of cells from a given mouse strain to support replication in vivo and in vitro, and was IFN dependent and recessive if linked to H-2k but IFN independent when associated with C57BL background genes.     

Molecular genetic characterization of the distal NKC recombination hotspot and putative murine CMV resistance control locus

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.The nucleotide sequence data reported in this paper were assigned GenBank accession numbers AF462604, AY145449 and AY145450.     

TheCmv1Host Resistance Locus Is Closely Linked to theLy49Multigene Family within the Natural Killer Cell Gene Complex on Mouse Chromosome 6

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 theLy49andLy55(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 localizeCmv1more 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 × C57BL/6J)F₁× BALB/c and 550 (A/J × C57BL/6J)F₁× A/J progeny. A total of 25 polymorphic genes or microsatellite markers were analyzed over a region of 10 map units fromD6Mit134toD6Mit59.TheCmv1phenotypes 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,Cmv1maps distal to, but very closely linked to, theLy49multigene family (<0.2 map units), suggesting that MCMV resistance may be conferred by MHC class I-specific NK cell receptors.     

Ly49h-deficient C57BL/6 mice: a new mouse cytomegalovirus-susceptible model remains resistant to unrelated pathogens controlled by the NK gene complex

Cmv1 was the first mouse cytomegalovirus (MCMV) resistance locus identified in C57BL/6 mice. It encodes Ly49H, a NK cell-activating receptor that specifically recognizes the m157 viral protein at the surface of MCMV-infected cells. To dissect the effect of the Ly49h gene in host-pathogen interactions, we generated C57BL/6 mice lacking the Ly49h region. We found that 36 h after MCMV infection, the lack of Ly49h resulted in high viral replication in the spleen and dramatically enhanced proinflammatory cytokine production in the serum and spleen. At later points in time, we observed that MCMV induced a drastic loss in CD8(+) T cells in B6.Ly49h(-/-) mice, probably reflecting severe histological changes in the spleen. Overall, our results indicate that Ly49H(+) NK cells contain a systemic production of cytokines that may contribute to the MCMV-induced pathology and play a central role in maintaining normal spleen cell microarchitecture. Finally, we tested the ability of B6.Ly49h(-/-) mice to control replication of Leishmania major and ectromelia virus. Resistance to these pathogens has been previously mapped within the NK gene complex. We found that the lack of Ly49H(+) NK cells is not associated with an altered resistance to L. major. In contrast, absence of Ly49H(+) NK cells seems to afford additional protection against ectromelia infection in C57BL/6 mice, suggesting that Ly49H may recognize ectromelia-infected cells with detrimental effects. Taken together, these results confirm the pivotal role of the Ly49H receptor during MCMV infection and open the way for further investigations in host-pathogen interactions.     

Expression of m157, a murine cytomegalovirus-encoded putative major histocompatibility class I (MHC-I)-like protein, is independent of viral regulation of host MHC-I

A murine cytomegalovirus (MCMV)-encoded protein, m157, has a putative major histocompatibility complex class I (MHC-I) structure and is recognized by the Ly49H NK cell activation receptor. Using a monoclonal antibody against m157, in this study we directly demonstrated that m157 is a cell surface-expressed glycophosphatidylinositol-anchored protein with early viral gene kinetics. Beta-2 microglobulin and TAP1 (transporter associated with antigen processing 1) were not required for its expression. MCMV-encoded proteins that down-regulate MHC-I did not affect the expression of m157. Thus, m157 is expressed on infected cells in a manner independent of viral regulation of host MHC-I.     

<Emphasis Type="Italic">Ly49h</Emphasis> is necessary for genetic resistance to murine cytomegalovirus

Natural killer (NK) cells play critical roles in antiviral immunity. While the importance of effector mechanisms such as interferons has been demonstrated through knockout mice, specific mechanisms of how viruses are recognized and controlled by NK cells are less well defined. Previous genetic studies have mapped the resistance genes for murine cytomegalovirus (MCMV), herpes simplex virus-1 (HSV-1), and ectromelia virus to the NK gene complex on murine chromosome 6, a region containing the polymorphic Ly49 and Nkrp1 families. Genetic resistance to MCMV in C57BL/6 has been attributed to Ly49H, an activation receptor, through susceptibility of the recombinant inbred strain BXD-8 that lacks Ly49h (also known as Klra8) but derived about half of its genome from its DBA/2 progenitor. However, it remained possible that epigenetic effects could account for the MCMV phenotype in BXD-8 mice. Herein, we report the generation of a novel congenic murine strain, B6.BXD8-Klra8 ( Cmv1-del )/Wum, on the C57BL/6 genetic background to evaluate the effect of deletion of a single NK activation receptor, Ly49H. Deletion of Ly49H rendered mice much more susceptible to MCMV infection. This increase in susceptibility did not appear to be a result of a difference in NK cell expansion or interferon-gamma (IFN-gamma) production between the C57BL/6 and the B6.BXD8 strains. On the other hand, the deletion of Ly49h did not otherwise affect NK cell maturation or Ly49D expression and had no effect on susceptibility to HSV-1 or ectromelia virus. In conclusion, Ly49h is necessary for genetic resistance to MCMV, but not HSV-1 or ectromelia virus.     

Role for TLR2 in NK cell-mediated control of murine cytomegalovirus in vivo

Natural killer (NK) cells are essential for the early control of murine cytomegalovirus (MCMV) infection. Here, we demonstrate that toll-like receptor 2 (TLR2) plays a role in the NK cell-mediated control of MCMV. TLR2 knockout (KO) mice had elevated levels of MCMV in the spleen and liver on day 4 postinfection compared to C57BL/6 mice. In vivo depletion of NK cells with anti-NK1.1 antibodies, however, eliminated the differences in viral titers between the two groups, suggesting that the effect of TLR2 on MCMV clearance on day 4 was NK cell mediated. The defect in early antiviral control was associated with a decreased NK cell population in the spleen and liver and reduced amounts of interleukin-18 and alpha/beta interferon secreted in the TLR2 KO mice. Our studies suggest that in addition to the reported involvement of TLR9 and TLR3, TLR2 is also involved in innate immune responses to MCMV infection.     

Suppression of murine cytomegalovirus (MCMV) replication with a DNA vaccine encoding MCMV M84 (a homolog of human cytomegalovirus pp65)

The cytotoxic T-lymphocyte (CTL) response against the murine cytomegalovirus (MCMV) immediate-early gene 1 (IE1) 89-kDa phosphoprotein pp89 plays a major role in protecting BALB/c mice against the lethal effects of the viral infection. CTL populations specific to MCMV early-phase and structural antigens are also generated during infection, but the identities of these antigens and their relative contributions to overall immunity against MCMV are not known. We previously demonstrated that DNA vaccination with a pp89-expressing plasmid effectively generated a CTL response and conferred protection against infection (J. C. Gonzalez Armas, C. S. Morello, L. D. Cranmer, and D. H. Spector, J. Virol. 70:7921-7928, 1996). In this report, we have sought (i) to identify other viral antigens that contribute to immunity against MCMV and (ii) to determine whether the protective response is haplotype specific. DNA immunization was used to test the protective efficacies of plasmids encoding MCMV homologs of human cytomegalovirus (HCMV) tegument (M32, M48, M56, M82, M83, M69, and M99), capsid (M85 and M86), and nonstructural antigens (IE1-pp89 and M84). BALB/c (H-2(d)) and C3H/HeN (H-2(k)) mice were immunized by intradermal injection of either single plasmids or cocktails of up to four expression plasmids and then challenged with sublethal doses of virulent MCMV administered intraperitoneally. In this way, we identified a new viral gene product, M84, that conferred protection against viral replication in the spleens of BALB/c mice. M84 is expressed early in the infection and encodes a nonstructural protein that shares significant amino acid homology with the HCMV UL83-pp65 tegument protein, a major target of protective CTLs in humans. Specificity of the immune response to the M84 protein was confirmed by showing that immunization with pp89 DNA, but not M84 DNA, protected mice against subsequent infection with an MCMV deletion mutant lacking the M84 gene. The other MCMV genes tested did not generate a protective response even when mice were immunized with vaccinia viruses expressing the viral proteins. However, the M84 plasmid was protective when injected in combination with nonprotective plasmids, and coimmunization of BALB/c mice with pp89 and M84 provided a synergistic level of protection in the spleen. Viral titers in the salivary glands were also reduced, but not to the same extent as observed in the spleen, and the decrease was seen only when the BALB/c mice were immunized with pp89 plus M84 or with pp89 alone. The experiments with the C3H/HeN mice showed that the immunity conferred by DNA vaccination was haplotype dependent. In this strain of mice, only pp89 elicited a protective response as measured by a reduction in spleen titer. These results suggest that DNA immunization with the appropriate combination of CMV genes may provide a strategy for improving vaccine efficacy.     

Cmv1-independent antiviral role of NK cells revealed in murine cytomegalovirus-infected New Zealand White mice

Ly49H(+) NK cells play a critical role in innate antiviral immune responses to murine CMV (MCMV). Ly49H(b6) recognition of MCMV-encoded m157 on infected cells activates natural killing required for host resistance. We show that mAb 3D10 (anti-Ly49H) recognizes comparable subsets of NK cells from New Zealand White (NZW), New Zealand Black (NZB), and C57BL/6 spleens. However, virus levels in the spleens of MCMV-infected NZW and NZB mice differed greatly. We found that MCMV replication in infected NZW spleens was limited through NK cells. Alternately, NZB mice were profoundly susceptible to MCMV infection. Although 3D10 mAb injections given before infection interfere with Cmv1-type resistance in C57BL/6 mice, similar mAb injections did not affect NZW resistance, likely because NZW NK cell receptors did not bind MCMV-encoded m157. Instead, anti-MCMV host defenses in hybrid NZ offspring were associated with multiple chromosome locations including several putative quantitative trait loci that did not overlap with H-2 or NK gene complex loci. This study revealed a novel pathway used by NK cells to defend against MCMV infection. Thus, the importance of Ly49H in MCMV infection may be shaped by other additional background genes.     

Ly49I NK cell receptor transgene inhibition of rejection of H2b mouse bone marrow transplants

The Ly49 family of genes encode NK cell receptors that bind class I MHC Ags and transmit negative signals if the cytoplasmic domains have immunoregulatory tyrosine-based inhibitory motifs (ITIMs). 5E6 mAbs recognize Ly49C and Ly49I receptors and depletion of 5E6+ NK cells prevents rejection of allogeneic or parental-strain H2d bone marrow cell (BMC) grafts. To determine the function of the Ly49I gene in the rejection of BMC grafts, we transfected fertilized eggs of FVB mice with a vector containing DNA for B6 strain Ly49I (Ly49IB6). Ly49IB6 is ITIM+ and is recognized by 5E6 as well as Ly49I-specific 8H7 mAbs. Normal FVB H2q mice reject H2b but not H2d BMC allografts, and the rejection of H2b BMC was inhibited partially by anti-NK1.1 and completely by anti-asialo GM1, but not by anti-CD8, Abs. In FVB mice, NK1.1 is expressed on only 60% NK cells. FVB. Ly49IB6 hosts failed to reject H2d or H2b BMC, but did reject class I-deficient TAP-1-/- BMC, indicating that NK cells were functional. Nondepleting doses of anti-Ly49I Abs reversed the acceptance of H2b BMC by FVB.Ly49IB6 mice. FVB.Ly49IB6+/- mice were crossed and back-crossed with 129 mice-H2b, 5E6-, poor responders to H2d BMC grafts. While transgene-negative H2b/q F1 or first-generation back-crossed mice rejected H2b marrow grafts (hybrid resistance), transgene-positive mice did not. Thus B6 strain Ly49I receptors transmit inhibitory signals from H2b MHC class I molecules. Moreover, Ly49IB6 has no positive influence on the rejection of H2d allografts.     

H-2 class I loci determine sensitivity to MCMV in macrophages and fibroblasts

Peritoneal (PM) and bone marrow-derived (BMM) macrophages and lung fibroblasts (LF) from inbred, intra-H-2 recombinant, H-2 mutant, and hybrid mice were infected with murine cytomegalovirus (MCMV) under centrifugal enhancement. At the concentration of virus employed, peritoneal macrophages from strains carrying Kd, Kb, D^d, KS and/or D^s, K4 and/or D4 alleles could be infected to a level of 80%–100%, as assessed by viral antigen expression or loss of Fc receptors. Cells lacking these haplotypes and carrying K^k, K^f, D^k, Df, or Db were resistant, yielding levels of infection below 20% . The background (non-H-2) and class II genotype and the S allele did not influence the proportions of cells infected. Furthermore, sensitivity was dominant in the F, progeny of H-2 b x H-2 k and H-2d x H-2 k crosses, and was not compromised by thebm1, bm3, bm10, or bm14 mutations in the al or2 regions of Kb orD ^b. The proportions of cells able to release infectious virus were low, but paralleled the frequencies of viral antigen expression. The class I genotype also determined susceptibility to MCMV infection in BMM and LF, although up to 35% of H-2 k BMM and 46% of H-2 k LF could be infected. The findings are consistent with an association between K and D antigens and a cellular receptor for MCMV on all three cell types.