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The Journal of Immunology, 2004, 173: 6312-6318.
Copyright © 2004 by The American Association of Immunologists

Cmv1-Independent Antiviral Role of NK Cells Revealed in Murine Cytomegalovirus-Infected New Zealand White Mice1

Marisela Rodriguez*, Pearl Sabastian{dagger}, Patricia Clark{dagger} and Michael G. Brown2,*,{dagger}

Departments of * Microbiology and {dagger} Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ly49H+ NK cells play a critical role in innate antiviral immune responses to murine CMV (MCMV). Ly49Hb6 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Studies of genetic variation in host susceptibility to lethal murine CMV (MCMV)3 infection now extend over several decades (1) with both MHC and non-MHC genetic contributions to the outcome of virus infection (2). Cmv1, an autosomal dominant locus, represented the first major non-MHC genetic determinant of MCMV resistance studied and later mapped (3, 4, 5). MCMV infection is limited in resistant C57BL/6 (B6) mice by NK cells that express Ly49H (Cmv1r) activation receptors, but virus resistance in this strain was eliminated by anti-Ly49H mAbs given before MCMV infection (3, 4, 5, 6). However, infected BALB/c or BXD8 (contains a Ly49h deletion mutation) strains do not effectively limit MCMV replication because their NK cells lack Ly49H receptors (Cmv1s). After MCMV infection of B6 mice, Ly49H+ cells increase IFN-{gamma} production and rapidly proliferate (7, 8). The role of Ly49Hb6 in MCMV resistance was recently explained by its recognition of the MCMV-encoded, MHC class I-like m157 ligand expressed on infected target cells (9, 10). Further, Ly49hb6 bacterial artificial chromosome transgenes transferred virus resistance in non-Ly49H-expressing strains, including FVB and BALB/c (11), and DAP12 coreceptor signaling-deficient mutant B6 mice displayed incomplete MCMV resistance even though Ly49H expression was apparent (12). Therefore, Ly49H expression was required for acute MCMV resistance in B6 mice.

However, formal Cmv1 genetic mapping experiments led to the identification of Mrc, a distal NK gene complex (NKC) locus separated from Ly49h that also potentially controlled virus levels in acute MCMV infection (13, 14). Although Mrcb6 did not directly lower MCMV replication in a novel NKC recombinant congenic strain (14), other investigators have also obtained data implicating NKC loci other than Ly49H in acute MCMV immunity (15). Moreover, while Cmv1r was defined in strains that severely limited MCMV replication through natural killing, Cmv1s-type strains showed wide variation in virus levels after acute infection (16), thereby suggesting other loci might modify virus titers in MCMV-infected mice.

A role for the NKG2D NK cell activation receptor in MCMV resistance was also recently investigated (17, 18). Mouse NKG2D binds MHC class I-like ligands including retinoic acid early inducible transcript (Rae1) family members and the minor histocompatibility Ag H-60. Their expression is mostly restricted to early development, but transformed, infected, or heat-shocked adult tissues also display these ligands (19, 20). NKG2D ligands can, in fact, regulate NKG2D expression levels and in some genetic backgrounds, this may have important functional consequences for natural killing (21). Interestingly, although MCMV infection increased Rae1 gene expression in macrophages, Rae1 protein expression was actually reduced in infected 3T3 cells by the MCMV-m152 gene product, gp40 (18). Deletion of m152 from MCMV curtailed its capacity for the replication in BALB/c mice without altering its growth characteristics in B6 mice, suggesting that gp40 might serve to help evade NK cell immunity during acute MCMV infection of some inbred mouse strains (17, 18). Together these findings implicated the potential existence of acute MCMV immunity modifiers in certain inbred strains.

In this investigation, host genetic factors that contribute to restriction of virus replication in acute MCMV infection were extensively studied in NZ (New Zealand) strains and their outcross and backcross offspring. Although NK cells were required for MCMV control early in infection, we did not observe a role for Cmv1-type NK cell resistance in New Zealand White (NZW) mice. NK cells in NZW apparently use a Cmv1-independent mechanism controlled through multiple genes to limit MCMV infection. The features of acute MCMV resistance in NZW, therefore, suggest that NK cells use an arsenal of distinct antiviral defenses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6J, BALB/cJ, NZB/B1NJ, and NZW/LacJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in the University of Virginia Medical School specific pathogen-free vivarium at the (Charlottesville, VA) which is fully accredited by the American Association for Accreditation of Laboratory Animal Care. NZW/BF1, NZW/BF2, (NZW/BF1 x NZW)N2, and BALB/NZWF1 mice were bred at University of Virginia. NZM2328 mice were provided by Dr. S. M. Fu (University of Virginia, Charlottesville, VA). Mouse inventory and breeding records were kept using MouSeek 1.0 (http://groups.yahoo.com/group/mouseek/) provided by Dr. C. F. Davis (Baylor College of Medicine, Houston, TX). All animal studies were approved by and conducted in accordance with Animal Care and Use Committee oversight.

Cell lines and tissue culture

Hybridomas (a gift from Dr. W. M. Yokoyama, Howard Hughes Medical Institute, Washington University, St. Louis, MO) 3D10 (IgG1 and anti-Ly49H) and 4E4 (IgG2a and anti-Ly49D), and NIH 3T12 (American Type Culture Collection, Manassas, VA) cells were grown in DMEM supplemented with 10% newborn calf serum, penicillin/streptomycin (100 U/ml per 100 µg/ml), and glutamine (2.0 mM). HEK293 cells (Tissue Culture Facility, University of Virginia) were grown in similarly supplemented DMEM-F12 plus sodium-pyruvate (1.0 mM) and sodium-bicarbonate (1.5 mg/ml). NK cells were prepared from nylon wool nonadherent mouse splenocytes and were grown in R10 supplemented with 800 U/ml IL-2 (Chiron, Emeryville, CA) for 6–10 days as described (22).

Abs and FACS analysis

mAbs were purified from spent cell-free hybridoma supernatants (Lymphocyte Culture Center, Department of Anatomy and Cell Biology, University of Virginia). Purified mAb 3D10 was FITC conjugated using a kit (Calbiochem, La Jolla, CA). Purchased mAbs included PK136 (IgG2a) PE (BD Pharmingen, La Jolla, CA), anti-human IgG FITC (Jackson Immunoresearch Laboratories, West Grove, PA), and mAb 9E10 (IgG1, anti-c-myc; Lymphocyte Culture Center, University of Virginia). Chimeric m157-IgG fusion protein was obtained from the spent cell-free supernatants of transiently transfected HEK293 cells essentially as described (9). Briefly, a modified CDM8 expression vector encoding chimeric m157-IgG fused to mouse CD150 leader sequence was kindly provided by Dr. L. Lanier (University of California, San Francisco, CA). Plasmid DNA (10 µg) purified from Escherichia coli (MC1061/P3) grown in Luria-Bertani broth plus kanamycin (25 µg/ml), ampicillin (50 µg/ml), and tetracycline (5 µg/ml) was transiently transfected into 107 HEK293 cells using 9 µl of cytofectene (Bio-Rad, Hercules, CA) per milliliter of culture medium. Chimeric m157-IgG containing supernatant was harvested from HEK293 cells on day 3 posttransfection. For flow cytometric analysis using a FACScan (BD Biosciences), IL-2-activated NK cells or mouse spleen leukocytes were bound with unlabeled mAb 2.4G2 (5 µg/ml) and subsequently stained with primary-labeled mAbs 3D10 FITC (5–10 µg/ml), PK136 PE (2 µg/ml), or with m157-Ig (50 µl of supernatant per 106 splenocytes) followed by anti-human IgG FITC (14 µg/ml) as described (22).

Virus assays

MCMV (Smith Strain) was serially passaged in weanling BALB/c mice and third passage salivary gland homogenate stocks were repeatedly titered on 3T12 cell monolayers by plaque assay as described (23, 24). Experimental mice (8–12 wk) were i.p. infected with MCMV (5–10 x 104 PFU) and subsequently euthanized on day 3 postinfection (typically 80 h). To study the role of NK cells during MCMV infection, mice were i.p. injected (200 µg mAb/200 µl PBS) 48 h before and/or immediately before MCMV infection.

For quantitative analysis of MCMV, spleen and liver homogenates and/or tissue genomic DNA samples were obtained for all experimental mice. Virus levels were measured in plaque assays on 3T12 monolayers and/or quantitative real-time PCR (QPCR) using an iCycler (Bio-Rad) as described (24). For QPCR measurements, tissue DNA samples were purified from tissue fragments (~0.05 g) using a kit (Gentra Systems, Minneapolis, MN), spectrophotometrically quantified and resuspended in Tris-EDTA, pH 8.0 (50 µg/ml). DNA samples (50 ng) were amplified in 25 µl of 1x PCR buffer (0.875 U of Taq polymerase (Promega, Madison, WI), 2.5 mM MgCl2, 200 µM dNTPs (Amersham Biosciences, Piscataway, NJ), 0.13x SYBR Green (Molecular Probes, Eugene, OR), and 10 nM fluorescein (Bio-Rad)) using gene-specific forward and reverse oligonucleotides (10 pmol each) for MCMV immediate early gene (GenBank accession number M11788; 5'-tca gcc atc aac tct gct acc aac-3' and 5'-gtg cta gat tgt atc tgg tgc tcc tc-3) and murine {beta}-actin (GenBank accession number M12481; 5'-gct gta ttc ccc tcc atc gtg-3' and 5'-cac ggt tgg cct tag ggt tca-3') followed by an amplicon melt curve analysis as described (24). All sample measurements were performed in triplicate. Virus replication in the livers of infected mice served as an important control for MCMV infection because both resistant and susceptible strains typically display comparable liver virus levels through 4 days postinfection (dpi; Refs. 3 and 24). Infected mice with negligible MCMV in their spleens and livers that was beyond QPCR detection sensitivity were excluded from further study because traits could not be assigned. Results were reported as the log10 ((number of MCMV genome copies per number of {beta}-actin genomic copies) per 0.1 mg of tissue DNA).

Genotyping and genetic mapping of quantitative trait loci (QTL)

Fluorescent microsatellite markers (Table I) were used to determine genome-wide genotypes for genomic DNA samples that were also assessed for MCMV levels in QPCR. PCR amplification and genetic analysis on the Genetic Analyzer 3100 using GeneScan and Genotyper software (Applied Biosystems, Foster City, CA) were performed as described previously (14, 25). Marker regression was performed using MapManager QTX (K. F. Manly, University of Tennessee, Memphis, TN, www.mapmanager.org/mmQTX.html) (26). Permutation analysis and interval mapping were also performed using MapManager QTX. In the permutation test, 1000 permutations were used for all typed markers to determine likelihood ratio statistic (LRS) thresholds for significant linkage. The analyzed data set includes 119 genotyped F2 offspring.


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  		Table I. Microsatellite markers used for genotyping NZ hybrid offspring

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NK cell receptor expression and MCMV immunity in NZ mice

We surveyed NK cells for Ly49H expression in a panel of inbred strains. We reasoned that strains containing mAb 3D10-binding NK cells should display Cmv1r-type resistance after MCMV infection because mAb 3D10 can block MCMV resistance in B6 mice. NZW and NZB mice were further studied because of their genealogical relationship (27) and for the similarity of their proximal NKC haplotypes spanning the Nkrp1-Ly49 gene clusters without the distal NKC haplotype (includes Mrc) homology (25). Both strains contain NK1.1+ splenocytes (28) that also bound mAb 3D10 (see Fig. 1 and Fig. 4 for comparison with B6 and BALB/c), consistent with a previous report (29). However, paradoxically, NZB (Cmv1s) mice rapidly succumb to MCMV infection (16), suggesting that Ly49H-related NK cell receptors in these mice may not recognize MCMV infection or that NK receptor signaling was dysfunctional.



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  		FIGURE 1. Ly49H expression on NK1.1+ cells from NZ mice. IL-2-activated (6 days) NK cells (mAb 2.4G2 blocked) were labeled with anti-NK1.1 and anti-Ly49H mAbs and analyzed by flow cytometry. The data are representative of multiple independent experiments.

 


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  		FIGURE 4. Strain-specific NK recognition of MCMV-encoded m157. Fresh B6, NZW, NZB, and BALB/c splenocytes (mAb 2.4G2 blocked) were labeled with anti-NK1.1 PE and either anti-Ly49H FITC or m157-Ig plus anti-human IgG-FITC and analyzed by flow cytometry (105 total events per sample). Similarly gated lymphocytes of each strain are shown in FL1 vs FL2 dot plots. Gated NK cell percentages (of total lymphocytes) are also shown. Data are representative of three to five independently studied mice of each strain.

 
When we compared IL-2-activated NK cells, comparable levels of 3D10-staining were again observed for NZW, NZB, and B6 (Fig. 1). In fact, most NZB NK cells consistently displayed higher levels of Ly49H-related receptors than NZW NK1.1+ cells. Nonetheless, using either virus plaque assays or QPCR, we also observed only high MCMV in infected NZB spleens (Fig. 2), similar to virus replication in spleens of infected BALB/c mice. In contrast, we found infected NZW spleens contained low level MCMV, similar to infected B6 spleens (Fig. 2). It is important to note that virus levels in the livers of all of the infected mice of each strain served as important MCMV infection controls because both resistant and susceptible strains typically display comparable liver virus levels through 4 dpi (Fig. 2b and Refs. 3 and 24) and because distinct NK antiviral control mechanisms are operational in the spleen and liver (30, 31). Therefore, only NZW mice displayed B6-like host MCMV resistance early after virus infection.



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  		FIGURE 2. Assessment of anti-MCMV defenses in NZ strains and hybrid offspring. a, NZ and control inbred strains (as designated) were i.p. infected with MCMV (5 x 104 PFU). Plaque assay determined spleen virus titers (4 dpi) for individual mice and experimental group means are shown. b, NZ strains were i.p. infected with MCMV (1 x 105 PFU). Real-time QPCR determined spleen ({circ}) and liver (•) virus levels (3.5 dpi) for individual mice and experimental group means are shown. c, Control NZ strains and F1 hybrid offspring were i.p. infected with MCMV (5 x 104 PFU). QPCR determined spleen virus levels (4 dpi) for individual mice and experimental group means are shown. The results shown are representative of three independent experiments.

 
NK cell requirement for NZW MCMV immunity

To further study NZW MCMV resistance, NK cells were depleted from mice before infection using the opsonizing mAb PK136. This treatment removed NK cells (data not shown) and dramatically altered NZW virus resistance (Fig. 3a). Because anti-Ly49H mAb 3D10 treatment can also interfere with virus resistance in B6 mice without influencing NK cell numbers or subset distribution (4), we also assessed virus levels in similarly treated NZW spleens. However, anti-Ly49H-treated NZW mice did not differ from control mice even when they received two 3D10 mAb injections before MCMV infection (Fig. 3a). In contrast, 3D10 mAb singly injected B6 mice became completely susceptible to MCMV infection (Fig. 3b).



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  		FIGURE 3. Critical antiviral role of NZW NK cells in acute MCMV infection. a, NZW mice were i.p. injected with the indicated mAbs or PBS 48 h before and again immediately before infection with 1 x 105 PFU MCMV. QPCR determined spleen ({circ}) and liver (•) virus levels (3.5 dpi) are shown. The results shown are representative of three independent experiments although high virus was observed in 1 of 13 total anti-Ly49H-treated mice (not shown). b, B6 mice were i.p. injected with PBS or mAbs (as designated) 48 h before MCMV infection (1 x 105 PFU). QPCR determined spleen ({circ}) and liver (•) virus levels (4 dpi) are shown.

 
Although NZW and NZB NK cell effector functions, including activation signals transduced through the Ly49H coreceptor DAP-12, were previously reported intact (28), we tested whether NZW NK cells could bind to MCMV-encoded m157. Similar to IL-2-cultured NK cells (Fig. 1), freshly prepared splenocytes from NZW and NZB, but not BALB/c, were bound with anti-Ly49H mAb 3D10 (Fig. 4). However, we found that only control B6 and a minor subset of NZB NK1.1+ splenocytes specifically bound chimeric m157-Ig, whereas NZW splenocytes were not recognized by the same fusion protein (Fig. 4). Thus, NZW NK cells do not express a receptor for m157.

It is also important to note that while similar percentages of B6 NK cells were stained with either 3D10 or m157-Ig and displayed correspondingly similar mean fluorescence levels, we consistently observed 2- to 3-fold more NZB NK cells bound 3D10 with a mean fluorescence level significantly higher than those bound with m157-Ig (Fig. 4). Thus, m157 and 3D10 apparently also recognized distinct epitopes on NZB NK cells.

Genetics of NZ antiviral defenses in acute MCMV infection

Host immune responses after MCMV infection were next studied in NZ hybrid offspring to determine whether NZW resistance was dominant or recessive. Interestingly, infected NZW/BF1 spleens contained intermediate virus levels by comparison with infected control NZW and NZB spleens (Fig. 2c). An intermediate profile observed in F1 offspring was conceivably due to a gene dosage effect, codominant expression of distinct NZ alleles, or potential multigenic regulation of acute MCMV immunity. Therefore, virus levels were also determined for similarly infected NZW/BF2 and (NZW/B x NZW)N2 backcross offspring. Unlike the mostly clear cut 1:3 and 1:1 distributions of high and low virus titers in respective (BALB/c x B6)F2 and (BALB/c x B6)F1 x BALB/c backcross mice (3), a continuum of virus levels ranging from high to low were observed that readily distinguished MCMV immunity in individual NZ hybrid offspring (Fig. 5, a and b). Host resistance or susceptibility traits were assigned after careful examination of 256 offspring containing reliably measured MCMV. Low virus levels were observed in only 16% of F2 or 23.3% of NZW/BF1 x NZW backcross spleens (Fig. 5 and Table II). Because these values differ considerably from those predicted by Mendelian laws of gene segregation for a single recessive gene, NZW resistance was apparently regulated by multiple gene products. In further support of multigenic regulation of MCMV immunity in the hybrid offspring, high virus levels were observed in 39% of F2 or 26.7% (NZW/B x NZW)N2 backcross offspring,



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  		FIGURE 5. Genetics of host resistance and susceptibility in NZ hybrid offspring during acute MCMV infection. NZW/BF2 (a), (NZW/B x NZW)N2 (b), BALB/NZWF1 (c), and control NZW, NZB, and BALB/c mice were i.p. infected with MCMV (1 x 105 PFU). QPCR determined spleen and liver (not shown) virus levels (3.5 dpi) are shown. Results in 6c are representative of three independent experiments.

 

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  		Table II. NKC haplotype distribution in NZW/B hybrid offspring sorted by MCMV resistance/susceptibility trait

 
Although NZW resistance was unaffected by anti-Ly49H mAb 3D10 (Fig. 3), it also lacked genetic dominance and required multiple genes (Figs. 2 and 5). Thus, it was unlikely that Cmv1 controlled virus levels in infected NZW mice. To further address this issue, we also studied MCMV infection in NZM2328, an inbred strain containing a mixture of NZB- and NZW-type alleles that together contribute to the development of SLE, especially in females (32). We observed mostly high virus levels in infected NZM2328 spleens, while MCMV liver levels were similar to NZW and NZB (Fig. 2, a and b). Therefore, Cmv1 contributions were less plausible because NZM2328 mice retain a NKCnzw haplotype (P. Y. Clark and M. G. Brown, unpublished data). Moreover, we also enumerated virus in MCMV-infected BALB/NZWF1 tissues because dominant Cmv1r allele containing BALB/B6F1 mice display B6-like resistance after infection (3). Unexpectedly, both BALB/NZWF1 and control BALB/c-infected spleens contained high virus levels that greatly exceeded levels in infected NZW spleens (Fig. 5c). These data collectively demonstrated the unique features of NZW acute anti-MCMV resistance and, thereby, implicated a distinct antiviral pathway through NK cells in NZW mice.

Because both NKC and H-2 genetic determinants were known regulators in early MCMV infection in other genetic systems, NKC and H-2 haplotypes were determined for the NZW/BF2 and (NZW/B x NZW)N2 hybrid offspring (Table II). Although most resistant NZW/BF2 mice possessed an NKCnzw haplotype, five possessed only Ly49nzb-type alleles. Thus, Ly49nzw-type alleles were not required to limit virus replication. Intermediate to high virus levels observed in the spleens of numerous NKCnzw-type NZW/BF2 hybrids or four of eight NKCnzw-type (NZW/B x NZW)N2 backcross offspring likewise demonstrated that the NKCnzw haplotype was not necessarily associated with protection in MCMV infection. To determine H-2-types, we used H-2-flanking markers, D17Mit16 and D17Mit10, as well as several novel H-2 markers that distinguish H-2z- and H-2d-type alleles (P. Sabastian and M. G. Brown, unpublished data). However, H-2 types were not individually associated with MCMV levels in the F2 offspring spleens. To identify probable chromosome locations for MCMV control, we searched for putative QTL in marker regression analysis. Of the suggestive or significant chromosome associations observed, none coincided with NKC or H-2 locations (data not shown). Permutation analysis and interval mapping revealed putative QTL on chromosomes 17 (D17Mit152) and X (DXMit216) that were significant (Fig. 6). We conclude that NKC or H-2 haplotypes were not individually correlated with MCMV resistance or susceptibility traits in the hybrid offspring under study.



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  		FIGURE 6. Interval mapping for putative MCMV level control QTL on chromosomes 17 and X. Shown are LRS plots for chromsomes 17 (left) and X (right) containing significant putative QTL determined by Mapmanager QTX. Suggestive (8.0), significant (14.5), and highly significant (19.1) LRS thresholds determined in permutation analysis are shown for each interval map. Chromosome locus markers used in genotype analysis are shown at left.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A crucial role for NK cells in innate antiviral defenses has long been appreciated and perhaps best studied in MCMV-infected inbred mice (33). Although many NK cell receptors bind MHC class I molecules and inhibit natural killing, it is now known that NK cells also display germline-encoded receptors that can rapidly stimulate natural killing activities (34). Ly49H is one such activation receptor on NK cells in B6 mice that can provide vital anti-MCMV NK cell immunity through specific recognition of its virus-encoded ligand. Recent studies have also characterized important contributions of other nonpolymorphic innate determinants of anti-MCMV immunity including TLRs (35, 36), thereby suggesting that other NK receptors might also contribute to host defenses against virus infection.

We questioned whether robust NK cell control of MCMV infection was determined primarily through a Ly49H-dependent mechanism, or whether other major anti-MCMV NK cell mechanisms might also contribute. We chose the NZW and NZB genetic system because the strains exhibit marked differences following MCMV infection even though NZW and NZB NK cells displayed comparable levels of Ly49H-related (mAb 3D10+) receptors. As we hypothesized, NK cells were required for NZW control of MCMV, but MCMV resistance in this strain could reflect a complex mix of NK cell killing and cytokine production. More importantly, NK cell antiviral control in this genetic system differed from Ly49H-dependent, dominant control previously characterized for the B6 and BALB/c genetic system. Our conclusion is based on the following important observations: 1) mAb 3D10+ NK cells were consistently observed in the spleens of both NZ strains, including some NZB NK cells that displayed very high intensity staining; 2) anti-Ly49H mAb 3D10 injections given before virus infection did not eliminate NZW-type immunity; 3) NZW NK cells did not recognize the Cmv1 ligand m157; 4) NZW-type immunity did not exhibit genetic dominance in NZ hybrid offspring; and 5) MCMV levels in NZ hybrids were not influenced by NKC genes.

MCMV control through non-NKC-linked genes strongly suggested to us that Ly49H-related polymorphisms were not responsible. Still, a non-NKC gene(s) could influence NKC-encoded receptor gene expression and/or function so that its contribution in this complex trait is overshadowed by genetic variation outside the NKC. To this end, we have recently cloned Ly49H-related transcripts from NZB and NZW NK cells that encode identical activation-type lectin-like receptors (A. Dighe and M. G. Brown, unpublished data). In fact, the new sequences were more similar to Ly49Hb6 (~91% amino acid identity) than all other reported Ly49 sequences. Nevertheless, it was apparent that mAb 3D10-binding receptors on NZW NK did not contribute significantly to MCMV control from our in vivo mAb studies and because NZW NK failed to bind m157.

A continuum of virus levels observed for individually infected F1 and F2 progeny that ranged between levels observed for control parental types suggested multiple genetic loci (collectively termed Cmv2) should together impart MCMV control. Although we also did not observe a Mendelian 3:1 segregation of susceptibility and resistance profiles at the F2 generation, it was apparent that a singular dominant gene was not responsible. We were equally struck by the high virus levels observed for infected BALB/NZWF1 spleens because BALB/B6F1 offspring are entirely resistant to MCMV infection. Although BALB/c susceptibility has been attributed to the absence of Ly49H+ NK cells, our results indicate BALB/c susceptibility is also influenced by additional background genes because NZW resistance was recessive to a BALB/cgenome-encoded modifier that we provisionally termed CMV resistance inhibitor (Cri). Therefore, Cri dominantly attenuated Cmv2 resistance in NZW hybrid mice, but not Cmv1 resistance in B6 hybrid mice. Hence, multiple gene products apparently can modify antiviral defenses during acute MCMV infection.

In further support, suggestive linkages for putative MCMV control QTL were gleaned from genetic analysis in the NZ hybrid progeny. Two moderate-strong QTL on chromosomes 17 and X were significant in permutation tests. We were somewhat surprised that the chromosome 17 QTL mapped outside the MHC because the H-2 regulation of MCMV infection has long been appreciated. We designed 13 additional H-2 markers that span the interval between D17Mit16-D17Mit10 to examine this issue in greater depth, but our results from an independent assessment of H-2 associations with MCMV control in all of the offspring tested and from our QTL study clearly distinguished it from the MHC. High virus levels in infected NZM2328 (H-2z) spleens that were substantially higher than in NZW support this conclusion. This and other related NZM strains should prove useful in further characterization of NZ alleles that determine viral immunity. Together, these findings highlight the importance of multiple independent antiviral mechanisms used by NK cells to rapidly and reliably control MCMV infection.

After careful examination of young and aged NZB mice, we found that only a small subset of NK1.1+ splenocytes also bound the m157-Ig fusion protein. NZB binding of m157-Ig also differed from Ly49Hb6 interaction with m157-Ig because a much smaller percentage of NZB NK cells bound m157 than bound mAb 3D10. NZW spleens containing comparable mAb 3D10+ NK cells, however, did not bind m157-Ig. These findings demonstrate that 3D10 and m157 must recognize distinct epitopes, but because mAb 3D10 can interfere with Ly49Hb6-m157 interaction, they should reside close together on Ly49H. Because Ly49H is the only B6 NK receptor known to interact with m157, perhaps a distinct m157-recognizing Ly49 receptor is expressed in NZB NK cells. Interestingly, Arase et al. (9) recently demonstrated that 129 NK cells also recognized m157 proteins through binding with NK inhibitory Ly49I receptors. Similar recognition by an inhibitory NZB NKR could potentially account at least in part for its MCMV susceptibility. Alternately, some unique subset of 3D10+ NZB NK cells could account for m157 binding. Our results also demonstrate that NZW NK cell recognition of MCMV infection must not require m157 expression. Further comparative analyses of the NZ Ly49 alleles now underway should be informative in definitive mapping of the m157 and 3D10 mAb epitopes and a better understanding of NK cell recognition of this important viral pathogen.

We conclude that a Cmv1-independent mechanism affords critical NK cell control of MCMV replication in NZW mice at early times after infection. This genetic system should provide important insight into NK cell function and further our understanding of innate mechanisms of antiviral immunity, especially those that are distinguished by genetic variation and polymorphism. High through-put genetic screening strategies to identify chromosomal locations harboring putative Cmv2-type and Cri modifiers of MCMV immunity represent important aims of our research program.


   Acknowledgments
 
We thank Dr. W. Yokoyama and Dr. L. Lanier for generously providing reagents. We thank Dr. S. M. Fu and Dr. S. Ju for helpful discussions and comments on the manuscript.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health, National Institute of Allergy and Infectious Diseases Grant AI50072 (to M.G.B.). M.R. was supported in part by National Institutes of Health, National Institute of Allergy and Infectious Diseases Training Grant 5T32AI007496. Back

2 Address correspondence and reprint requests to Dr. Michael G. Brown, Division of Rheumatology and Immunology, Department of Internal Medicine, Box 800412, University of Virginia Health Sciences Center, Charlottesville, VA 22908. E-mail address: mgb4n{at}virginia.edu Back

3 Abbreviations used in this paper: MCMV, murine CMV; Rae1, retinoic acid early inducible transcript; dpi, days postinfection; LRS, likelihood ratio statistic; NKC, NK gene complex; QPCR, quantitative real-time PCR; QTL, quantitative trait loci; Cri, CMV resistance inhibitor. Back

Received for publication January 13, 2004. Accepted for publication September 8, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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