Interaction between NKC alleles and H2^k locus is associated with resistance to MCMV

To validate the epistatic interaction between the NKC and H2 detected by linkage analysis [25], we used a marker-assisted strategy to construct congenic mouse lines in which a chromosome 6 segment (Cmv3) from MA/My MCMV-resistant mice was independently introgressed into BALB/c (H2^d) and BALB.K (H2^k) susceptible backgrounds. Congenic BALB.K mice have been described previously [30]. The correlations between the current physical maps and the genomic region introgressed in the respective single and double congenic strains BALB-Cmv3^MA/MyH2^d and BALB-Cmv3^MA/MyH2^k are shown in Figure 1 and Table 1.

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Figure 1. Generation, phenotype, and MCMV infection outcome of BALB mice congenic for the natural killer gene complex inherited from MA/My mice.

(A) Left, physical map of chromosome 6 markers used to determine the size of the MA/My fragment introgressed into the BALB background. The four versions of chromosome 6 indicate the genotypes of sub-congenic strains produced during the generation of BALB.Cmv3^MA/My congenic mice carrying a 10 Mb segment (between SNPs rs13479016 and rs13479061) spanning the NKC region from parental MA/My mice. Right, physical map of chromosome 17 markers used to characterize the 9.4 Mb segment (between D17Mit28 and D17Mit51) comprising the H2 region of BALB.K mice (H2^k). (B) NK cell receptor expression in MA/My parental mice and derived BALB-Cmv3^MA/MyH2^d and BALB-Cmv3^MA/MyH2^k congenic mice. The receptors (indicated on the bottom of the panel) were gated by FACS on NKp46⁺ splenic NK cells; the proportion of expression is indicated in each histogram. (C) Viral load in spleens (left) and livers (right) of mice of the indicated genotypes, as determined by plaque-forming assays 3 days p.i. (D) Spleen size (top) and weight and total cellularity (bottom) determined in MA/My and BALB-Cmv3^MA/MyH2^k mice at 7 days p.i. White bar, uninfected mice; black bar, MCMV-infected mice. Data were analyzed using two-way ANOVA analysis and the two-tailed Student's test. Data are presented as mean ± SEM and P values of significant differences between groups are indicated. Results shown in panel B are representative of three experiments using 2–3 mice per group; results shown in panels C and D are representative of five independent experiments using 3–8 mice per group.

https://doi.org/10.1371/journal.pgen.1001368.g001

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Table 1. Nomenclature, NKC/H2 genotype, and MCMV titer of mouse strains used in this paper.

https://doi.org/10.1371/journal.pgen.1001368.t001

To examine the effect of the genetic background on the expression of NKC-encoded receptors in the Cmv3^MA/My region, we used a panel of antibodies with known antigen specificities (Figure S1) [31]. Though several anti-Ly49 antibodies are cross-reactive [31], we observed variations among the mouse strains in terms of frequency of Ly49 subpopulations. Compared to BALB-Cmv3^MA/MyH2^d mice, BALB-Cmv3^MA/MyH2^k animals had a significantly increased frequency of NK cells stained with the monoclonal antibodies 12A8 (against Ly49R; P = 0.02) and 14B11 (against Ly49I/U; P = 0.007) (Figure 1B and Figure S2). Notably, 14B11-stained NK cells were also significantly increased in BALB-Cmv3^MA/MyH2^k mice compared to MA/My mice (P = 0.005). These results demonstrated the influence of H2 alleles [32], as well as the influence of an additional non-H2 mechanism, in the formation of the Ly49 repertoire. We also observed a highly significant increase in the frequency of NK cells labeled with 2F1 antibody (P = 0.004), which recognizes the maturation marker KLRG1, in BALB-Cmv3^MA/MyH2^d mice compared to their H2^k counterparts. Finally, we observed that NK cells from the three mouse strains that share the Cmv3^MA/My allele lacked expression of NKG2A/C/E and CD94-associated receptors at the protein but not mRNA level (Figure 1B and Figure S3B). By contrast, there was a normal expression of these receptors in the FVB/N mouse strain, which carries an NKC haplotype similar to that of MA/My mice (Figure S3A and S3B).

To evaluate the effect of the transferred MA/My chromosome 6 (Cmv3) segment on the response of MCMV-susceptible BALB/c and BALB.K mice, we infected congenic and parental control mice by intraperitoneal (i.p.) inoculation of MCMV sublethal doses (Figure 1C). By day 3 post-infection (p.i), uncontrolled MCMV replication was observed in the spleen of susceptible BALB/c mice (log₁₀ plaque-forming units [PFU] = 4.39±0.16), while MCMV-resistant MA/My mice had restricted viral replication, as shown by a >100-fold lower viral titer (log₁₀ PFU = 1.81±0.05) than that seen in BALB/c mice. Single congenic BALB.K and BALB-Cmv3^MA/MyH2^d mice had viral titers that were indistinguishable from those observed in BALB/c mice. More importantly, congenic mice with the BALB.K background, which carry only one copy of the MA/My NKC (BALB-NKC^BALBCmv3^MA/My.H2^k), were as susceptible to MCMV as BALB/c, BALB.K, and BALB-Cmv3^MA/MyH2^d mice. By contrast, double congenic BALB-Cmv3^MA/MyH2^k mice had restricted virus growth to the same extent as resistant MA/My mice. Viral titers in the liver correlated with those observed in the spleens. Furthermore, by day 7 p.i., the virus was cleared from the spleen (unpublished data), which had undergone a massive increase in weight and cell number in MA/My and BALB-Cmv3^MA/MyH2^k mice (Figure 1D). Collectively, these data demonstrate that the interaction between Cmv3^MA/My and H2^k confers MCMV resistance and is sufficient to explain the control of viral load observed in MA/My mice.

Because we did not have antibodies that specifically recognize Ly49P receptors and to examine a possible role of CD94 heterodimers, we attempted to confirm the results obtained in the congenic mice in a new cross between two strains that independently carried the H2^k loci and the Ly49P gene at the NKC. We examined the segregation of MCMV viral load in the spleens of progeny mice from an F₂ cross between the MCMV-susceptible strains FVB/N and BALB.K. Although both parental strains sustained a relatively high viral titer (5.2 log₁₀ PFU), the 137 F₂ progeny showed a continuous distribution ranging from 2 to 6 log₁₀ PFU (Figure 2A). To evaluate the contribution of H2 and NKC genes to MCMV resistance in this cross, F₂ mice were genotyped and distributed according to their NKC (Ly49e) and H2 (IAA1) genotypes. Mice homozygous for H2^kk alleles from BALB.K and NKC^ff alleles from FVB/N had the lowest viral load (Figure 2B). The model that best fitted this phenotype/genotype distribution in the analysis of variance had a joint logarithm of odds (LOD) score of 9 (P<10⁻¹¹) and accounted for 29.6% of the phenotypic variation (Table 2). Thus, there was a highly significant association between NKC/H2 interaction and control of MCMV infection in this second cross, indicating that Cmv3 was also present in the FVB/N mouse strain and that its expression in the presence of H2^k was necessary and sufficient for viral control. Furthermore, these data suggest that the same gene encodes Cmv3 in both the MA/My and FVB/N NKC regions.

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Figure 2. MCMV resistance in FVB/N×BALB.K F₂ progeny dependent on specific combinations between the NKC and H2.

(A) Genetic analysis of MCMV infection control in FVB/N×BALB.K F₂ progeny. Mice (FVB/N and BALB.K, n = 11 per strain; FVB/N×BALB.K F₂, n = 137) were infected with 5,000 PFU of MCMV; the spleen viral titers were determined by plaque assay at day 3 p.i. (B) Box plots show the combined effect of the NKC and H2 loci derived from FVB/N (H2^qq and NKC^ff) or BALB.K (H2^kk and NKC^cc) parental mice on the viral loads in the spleens of the F₂ progeny. The median and interquartile ranges are shown. Solid dots denote outliers. The red box corresponds to the combination of NKC^ff and H2^kk loci (P<6×10⁻¹¹).

https://doi.org/10.1371/journal.pgen.1001368.g002

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Table 2. ANOVA of MCMV load in spleens from (FVB/N×BALB.K) F₂ mice.

https://doi.org/10.1371/journal.pgen.1001368.t002

Transgenic expression of H2-D^k has a modest effect on MCMV control in the presence of H2^q

Previously, we showed that activation of Ly49P-bearing reporter cells requires the H2-D^k host molecule on MCMV-infected cells [25]–[26]. Therefore, to better delineate the role of H2 in the host response against MCMV, we attempted in vivo rescue of the FVB/N susceptible phenotype by genetic transfer of an 11 kb H2-D^k genomic fragment cloned from AKR mice (Figure 3A). We monitored for the presence of a diagnostic 300 bp fragment corresponding to exon 3 of the H2-D^k gene to identify transgenic FVB-Tg(D^k)⁺ mice among the founder population (unpublished data). By surface staining of mouse embryo fibroblasts (MEF) from FVB-Tg(D^k)⁺ mice, we observed the normal low levels of H2-D^k expression under regular conditions (Figure 3B). However, IFN-β treatment up-regulated expression of H2-D^k on MEF cells from either FVB-Tg(D^k)⁺ mice or AKR mice (H2-D^k transgene donor mouse strain) to the same extent, indicating that the transgene promoter regulatory sequences were intact (Figure 3B). We also found that the level of expression of H2-D^k in splenocytes from the FVB/N transgenic mice was similar to the natural H2-D^k expression in splenocytes from MA/My or BALB.K strains (Figure 3C). Finally, we investigated the expression of transgenic H2-D^k and endogenous H2-D^q molecules in T and B cells isolated from the spleen and observed that the two MHC-I molecules were expressed in FVB-Tg(D^k)⁺ mice at levels similar to those found in H2^q- and H2^k-bearing inbred mice (Figure S4). To test whether the H2-D^k transgene is capable of stimulating the Ly49P receptor, we assessed the activation of Ly49P-bearing reporter cells and found that these cells were equally activated by MCMV-infected MEF cells from BALB.K or from FVB-Tg(D^k)⁺ (Figure S5A). Stimulation of Ly49P reporters was also observed upon challenge by MEFs infected with a mutant virus lacking m157 (the Ly49H ligand). However, Ly49P reporter cell stimulation was lost upon infection of MEF cells with a mutant virus lacking the m04 gene, indicating that the transgenic H2-D^k molecule also requires m04/gp34 to stimulate Ly49P, as reported (Figure S5B) [26].

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Figure 3. Functional characterization of H2-D^k transgenic mice.

(A) Schematic representation of the H2-D^k gene within the 11.5 kb EcoRI genomic DNA fragment from AKR mice used to generate transgenic mice. (B) Expression of H2-D^k on MEFs from FVB/N nontransgenic (FVB-Tg(D^k)⁻, transgenic FVB-Tg(D^k)⁺, and AKR (H2^k) mice. MEFs from FVB-Tg(D^k)⁺ and wild-type littermates were prepared from PCR-typed embryos, untreated or incubated overnight with 100 U/ml IFN-β prior to analysis of H2-D^k expression by FACS. (C) H2-D^k staining on lymphocytes from FVB-Tg(D^k)⁻ (red peak), BALB.K (dashed peak), MA/My (black peak), and FVB-Tg(D^k)⁺ (dotted peak) mice. Bar graph shows quantification of the percentage of H2-D^k expression from 2–3 mice per group. (D) Splenocytes from B6.H2⁰ mice or NKC/H2 histocompatible mice were inoculated into either untreated or asialo-GM1–treated NK cell–depleted hosts. Ratio values indicate the relative survival in the test population (CFSE^high) compared to the histocompatible control population (CFSE^low) at 18 hours after injection. Three mice per group were analyzed. Statistical significance between untreated and NK-depleted mice is shown. (E) Viral load in spleens (left) and livers (right) of mice of the indicated genotypes was determined by plaque-forming assays at day 3 p.i. Data were analyzed using two-way ANOVA analysis and the two-tailed Student's test. Data are presented as mean ± SEM and P values of significant results between groups are indicated. Results shown are representative of 2–3 independent experiments.

https://doi.org/10.1371/journal.pgen.1001368.g003

To establish the contribution of H2-D^k to the MCMV response, we first investigated a possible modulation of the Ly49 receptor repertoire. No significant differences were found between transgenic and non-transgenic mice in terms of the frequency of the various NK cell populations tested (Figure S6). Since H2-D^k has the potential to influence licensing through its interactions with cognate Ly49 inhibitory receptors [16], we addressed the licensing status of wild-type and transgenic NK cells. To do this, we determined the ability of NK cells to mediate in vivo rejection of MHC class I deficient splenocytes isolated from B6.129P-H2-D1^tm1Bpe H2-K1^tm1Bpe (herein B6.H2⁰) mice using a quantitative CFSE-based method [33]. As previously described, B6.H2⁰ mice tolerated grafted syngeneic splenocytes (Figure 3D). By contrast, both FVB-Tg(D^k)⁺ and FVB-Tg(D^k)⁻ mice rejected B6.H2⁰ splenocytes with the same efficiency, suggesting that the presence of the H2-D^k transgene does not alter the licensing status of NK cells (Figure 3D). Finally, we monitored early viral replication following MCMV infection in FVB-Tg(D^k)⁺ mice, along with single and double BALB-Cmv3^MA/MyH2^d and BALB-Cmv3^MA/MyH2^k congenic mice and parental MA/My mice. We observed that FVB-Tg(D^k)⁺ mice had a statistically significant, albeit modest, reduction in MCMV replication in the spleen (but not in the liver) compared to nontrangenic littermates; this reduction represented only a small fraction (1∶7) of the reduction observed in BALB-Cmv3^MA/MyH2^k congenic mice (Figure 3E). Collectively, these data demonstrated that the H2-D^k transgene was fully expressed and able to recognize and activate the Ly49P receptor in vitro; however, it only provided partial control of MCMV infection.

Transgenic expression of H2-D^k has a major effect on MCMV control in the absence of H2^q

Classical MHC class I molecules are the prototype ligands for Ly49 receptors. Reporter cell assays and tetramer binding assays suggest that H2-D^k molecules elicit both activating signals, through Ly49P, and inhibitory signals, through Ly49I and Ly49V [25], [31]. By contrast, H2^q-encoded molecules can elicit strong inhibitory signals through Ly49I or Ly49C, but are inert to Ly49G2 [34], as well as to Cmv3-encoded activating receptors (Ly49P, Ly49R, and Ly49U) [25]. Therefore, we hypothesized that the poor MCMV infection control observed in FVB-Tg(D^k)⁺ mice resulted from competition between the inhibitory and activating signals emanating from H2^q- and H2-D^k-encoded ligands, respectively. To test this hypothesis, we crossed FVB-Tg(D^k)⁺ transgenic mice with B6.H2⁰ mice, which possess targeted deletions at the H2-D and H2-K genes. This cross produced F₃ progeny mice homozygous for the FVB/N NKC (Cmv3^FVB), but with a different assortment of MHC class I alleles. F₃ mice were either (1) deficient in endogenous MHC class I alleles in the presence or absence of the H2-D^k transgene (H2⁰/Tg(Dk)⁺ or H2⁰/Tg(Dk)⁻), (2) hemizygous for H2^q in the presence or absence of the H2-D^k transgene (H2^0/q/Tg(Dk)⁻ or H2^0/q/Tg(Dk)⁺), or (3) homozygous for H2^q in the presence of the H2-D^k transgene (H2^q/Tg(Dk)⁺) (Figure 4A and Table 1). Again, we monitored whether the frequencies of various NK cell populations were affected by the genetic makeup of F₃ mice and detected no major variations in the NK cell populations, the only exception being Ly49G⁺ NK cells, which were barely detectable in Cmv3^FVB/H2⁰/Tg(D^k)⁻ mice (Figure S7). Similarly, the level of H2-D^k and H2-K^k expression on splenocytes was equivalent in transgenic F₃ mice with different H2 genotypes (Figure S7, right panel). By contrast, we noted that levels of H2-D^q expression on lymphocytes from homozygous H2^q/Tg(D^k)⁺ transgenic mice were double those of hemizygous H2^0/q/Tg(D^k)⁺ transgenic mice (P = 0.001) (Figure 4B and bar graph). Despite this variation in MHC class I expression levels, licensing of NK cells from H2-D^k transgenic mice carrying no (0), one (0/q), or two (q/q) H2^q alleles was equivalent, as shown by their ability to reject CFSE-labeled splenocytes from B6.H2⁰ mice [Figure 4C right]. These results were confirmed using explanted, IL-2-activated NK cells in cytotoxicity assays against MCH class I–deficient RMA/S target cells [Figure 4C left], demonstrating that NK cells from transgenic mice with different H2 assortments sense equally the loss of MHC class I expression on target cells and therefore are equally educated [16].

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Figure 4. Functional characterization of F₃ mice carrying the NKC from FVB/N and different assortment of H2 molecules.

(A) Breeding scheme for the generation of F₃ mice carrying the NKC loci from FVB/N parental mice and various combinations of H2 loci. The NKC, H2, and H2-D^k transgenic loci are represented by boxes, as indicated. The parental (P) FVB-Tg(D^k)⁺ and B6.H2⁰ strains were mated to generate the F₁ generation. Subsequently, F₂ mice carrying an homozygous FVB/N NKC locus and heterozygous for either the H2 or the H2-D^k transgene were kept and intercrossed to generate the F₃ mice with different H2 assortments (H2⁰: H2-K^b−/−D^b−/−). (B) H2-D^q staining of lymphocytes from Cmv3^FVB/H2⁰/Tg(D^k)⁺ (H2⁰ red peak), Cmv3^FVB/H2^0/q/Tg(D^k)⁺ (H2^0/q, dot peak), and Cmv3^FVB/H2^q/q/Tg(D^k)⁺ mice (H2^q/q, dashed peak). Histograms on the right represent the quantification of the level of H2-D^q expression analyzed in three mice per group. (C) Rejection of B6 MHC class I–deficient cells in vivo by the indicated hosts was assessed as in Figure 3, and statistically significant differences are shown. IL-2–derived NK cells from the indicated mouse strains were co-cultured with CFSE-labeled RMA/S cells. Specific lysis at the indicated effector/target ratios was assessed by staining with 7-AAD and analyzed by FACS. Values represent the mean of 2–3 mice per group. (D) Viral loads in spleens (left) and livers (right) of parental and F₃ mice of the indicated genotypes were determined by plaque assay at day 3 p.i. Results shown represent five pooled experiments. Data were analyzed using two-way ANOVA analysis and the two-tailed Student's test. Significant P values for differences between groups are indicated.

https://doi.org/10.1371/journal.pgen.1001368.g004

To determine the influence of the various MHC class I molecules on the NK cell immune response to MCMV infection, we examined F₃ mice and parental controls at early time points, particularly on day 3 p.i., when receptor-specific NK cell responses are established [18]. On day 3, the post-infection viral titers in the spleens and livers of Cmv3^FVB/H2^0/q/Tg(D^k)⁺ and Cmv3^FVB/H2⁰/Tg(D^k)⁻ mice were indistinguishable, demonstrating that the presence of H2^q dampens the effect of Cmv3^FVB/H2^k on the containment of virus replication (Figure 4D). By contrast, the presence of the transgene had a significant effect in the absence of endogenous class I molecules, as Cmv3^FVB/H2⁰/Tg(D^k)⁺ mice had close to 30-fold lower viral titers compared to Cmv3^FVB/H2^0/q/Tg(D^k)⁻ mice. In parallel, B6.H2⁰ control mice, which express Ly49H, also cleared the virus load despite lacking MHC-I molecules (Figure 4D). To investigate the role of NK cells in limiting viral spread, we found that, as in MA/My mice, the control of virus load was abrogated in BALB-Cmv3^MA/MyH2^k and Cmv3^FVB/H2⁰/Tg(D^k)⁺ mice if treated with anti-asialo GM1 antibody prior to MCMV infection, demonstrating that the resistance phenotype is NK cell-dependent (Figure S8A). Indeed, we observed uncontrolled virus growth not only when MA/My mice were pretreated with anti-asialo GM1 and anti-NK1.1 antibodies, but also after they were pretreated with YE1/48 (anti-Ly49PRTV), 12A8 (anti-Ly49R), or 4D11 (anti-Ly49GT) antibodies, indicating an overlap in Ly49 receptor expression on NK cells (Figure S9) [25]. At day 6 p.i., a time characterized by robust proliferation of receptor-specific NK cell populations responding to the virus [18], we found that mice expressing Cmv3 resistance (MA/My, BALB-Cmv3^MA/MyH2^k, and Cmv3^FVB/H2⁰/Tg(D^k)⁺) had cleared MCMV from the spleen (unpublished data). Furthermore, spleen cell numbers were increased 2–6-fold in these mice and BrdU uptake indicated a robust NK cell proliferation (Figure S8B). Together, our results indicate that expression of H2-D^k can rescue Cmv3-determined MCMV resistance in the absence of endogenous H2^q molecules and that Cmv3/H2-D^k-mediated resistance is associated with the expansion of NK cells in response to infection.

Host H2^q inhibitory signals quantitatively modulate MCMV resistance and restrict NK cell–specific proliferation upon MCMV challenge

To better define the role of H2^q alleles, we compared the kinetics of viral replication in Cmv3^FVB-Tg(D^k)⁺ transgenic mice carrying no, one, or two H2^q alleles. We observed that the number of H2 alleles correlated with a quantitative increase in viral load, as early as 36 hours p.i.. On days 3 and 5 p.i., differences in viral containment among mice of the three genotypes were significant (Figure 5A).

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Figure 5. H2^q expression interferes with NK cell antiviral responses.

(A) MCMV viral load in the spleens of F₃ transgenic mice at the indicated time-points was determined by plaque assay and P values of significant results between groups are indicated. (B) BrdU incorporation in NKp46-gated splenocytes stained with anti-BrdU and 4D5 antibodies. Splenoytes were isolated from mice of the indicated genotypes 5 days p.i. Graph bar represents the proportions of NK cells incorporating BrdU in total splenic leukocytes with standard deviations, using three mice per group. (C) Enriched NK cells from Cmv3^FVB/H2⁰/Tg(D^k)⁺ mice were labeled with CFSE then adoptively transferred into Cmv3^FVB/H2⁰/Tg(D^k)⁺ and Cmv3^FVB/H2^0/q/Tg(D^k)⁺ recipients 24 hours before infection with MCMV for 5 days. Analysis of CFSE dilution in NK cells from the spleens of infected (dashed peaks) or uninfected (solid peaks) mice. NK cell proliferation index (number of divisions of CFSE-labeled NK cells) in Cmv3^FVB/H2⁰/Tg(D^k)⁺ and Cmv3^FVB/H2^0/q/Tg(D^k)⁺ mice. Statistically significant differences between groups are indicated. Three mice per group were analyzed and results shown are representative of two experiments. Data were analyzed using two-way ANOVA analysis and the two-tailed Student's test. Significant P values for differences between groups are indicated.

https://doi.org/10.1371/journal.pgen.1001368.g005

To investigate the effect of H2^q molecules on NK cell specific responses against MCMV, we monitored BrdU incorporation on NK cells after MCMV infection in FVB/N WT and F₃ mice carrying no, one, or two copies of H2^q alleles. After 5 days p.i., NK cells were stained with the anti-Ly49ORV (4E5) monoclonal antibody, which stained around 50% of NK cells in these strains (Figure S6 and Figure S7), and with the anti-BrdU monoclonal antibody. In all mice, we observed that NK cells that incorporated BrdU were negative for the Ly49ORV antibody staining. Furthermore, the increase in BrdU incorporation was inversely proportional to the number of H2^q alleles (Figure 5B). This result suggests that there is a dose-dependent inhibition of NK cell proliferation by H2^q alleles in response to MCMV infection. Finally, to investigate whether host MHC-I molecules affect NK cell activity upon MCMV infection, we adoptively transferred CFSE-labeled NK cells enriched from Cmv3^FVB/H2⁰/Tg(D^k)⁺ donor mice into Cmv3^FVB/H2⁰/Tg(D^k)⁺ or Cmv3^FVB/H2^0/q/Tg(D^k)⁺ recipients (Figure 5C). After 5 days p.i., donor NK cells had undergone more rounds of division in the Cmv3^FVB/H2⁰/Tg(D^k)⁺ recipients than the NK cells that were transferred into Cmv3^FVB/H2^0/q/Tg(D^k)⁺ mice; this indicated that H2^q alleles limited NK cell proliferation induced by MCMV infection (Figure 6C bar graph). Thus, NK cells carrying H2-D^k as the sole MHC class I molecule were impaired in their ability to proliferate if the recipient mice carried H2^q alleles. Collectively, these results suggest that expression of host H2^q molecules dampens the capacity of NK cells to protect against MCMV.

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Figure 6. Model of H2-dependent, Cmv3-determined NK response against MCMV infection.

The strength of Ly49 inhibitory signals and the presence of H2-D^k-mediated activating signals modulate the NK cell response against virus infection. Our set of NKC congenic mice bore different assortments of Ly49 receptors, but carried an identical H2^k resistance haplotype. (A) NK cells from BALB.K mice had a high frequency and strong binding of inhibitory Ly49 receptors, which rendered BALB.K mice most susceptible to MCMV infection. (B) NK cells from congenic BALB.Cmv3^hetH2^k mice carried one copy of the activating Ly49p gene, which can activate the Ly49P/H2-D^k/m04 axis, allowing for intermediate viral loads in heterozygous mice. (C) NK cells from BALB.Cmv3^MA/MyH2^k mice had the lowest frequency (and/or weakest binding) of inhibitory Ly49 receptors for H2^k molecules and the highest frequency of activating Ly49P⁺ NK cells, resulting in strong control of MCMV infection. Our set of F₃ mice carried different MHC-I components, but an identical Cmv3-resistance haplotype, encoding seven inhibitory and three activating Ly49 receptors, including Ly49P. (D) Engagement of inhibitory receptors in FVB-Tg(D^k)⁻ mice resulted in inhibition of the NK cell response against MCMV. (E) In FVB-Tg(D^k)⁺ mice, activating signals mediated by the engagement of Ly49P by H2-D^k/m04, in the presence of inhibitory signals elicited by H2^q molecules, provided a marginal enhancement of the NK cell response, and intermediate virus control. (F) In the absence of inhibitory H2^q signals, H2-D^k-dependent activation of NK cells was more efficient, which resulted in strong control of MCMV infection in Cmv3^FVB/H2⁰/Tg(D^k)⁺ mice.

https://doi.org/10.1371/journal.pgen.1001368.g006

Combined effect of the NKC and MHC loci on NK cell antiviral responses

The role of the MHC has been studied using panels of congenic [27], sub-congenic, and transgenic mice or F₂ crosses with the same NKC haplotype [28]–[29], [35]. To the best of our knowledge, the present study is the first to provide formal proof of the impact of both NKC and MHC haplotypes on NK cell antiviral activities in vivo. Our study, through the use of single- and double-congenic mice, minimized differences in non-NKC or non-MHC genes. Thus, we established that the joint action of specific alleles at these two regions accounted for most of the overall phenotypic differences between the MCMV-susceptible BALB/c and MCMV-resistant MA/My mouse strains. BALB-Cmv3^MA/MyH2^k mice were indistinguishable from MA/My mice in terms of initial control of infection and late NK cell responses.

Although the MA/My NKC region in congenic mice encompasses more than just Ly49 genes, our data indicates that the influence of MHC alleles on MCMV-resistance stems from the capacity of MHC class I molecules to serve as ligands for Ly49 receptors. In support of this hypothesis, the F₂ cross between the MCMV-susceptible FVB/N (H2^q) and BALB.K (H2^k) mouse strains demonstrated that FVB/N mice carried a Cmv3 resistance allele that was conditional to H2^k and overridden by the H2^q susceptibility allele. Within the Cmv3 region, Ly49 receptors were responsive to MHC class I ligands. On the one hand, we noticed that none of the available anti-NKG2 or anti-CD94 antibodies recognized MA/My mouse NK cells, in contrast to NK cell recognition in FVB/N mice. Nevertheless, we observed that F₂ mice of the combined Cmv3^FVB/H2^k genotype restrained viral replication to an extent similar to that seen in MA/My mice. On the other hand, our haplotype studies [25], [36] and new public data (http://phenome.jax.org) indicate that FVB/N and MA/My share the same Ly49 gene repertoire, including Ly49P. Consequently, it seems that NK cell responsiveness during MCMV infection varies with different NKC-MHC combinations and is optimal only with a precise combination of Ly49 receptors inherited from MA/My (or FVB/N) mice and MHC class I H2^k molecules.

Role of the MHC class I molecule H2-D^k

Our results confirmed that the H2 effect was due to the MHC class 1 molecule H2-D^k. Using an 11 kb genomic fragment containing a functional H2-D^k gene, we achieved a phenotypic rescue, although the rescue was incomplete if combined with H2^q alleles. The complete protective effect of H2-D^k was restored in F₃ mice lacking endogenous H2^q molecules. Although H2-D^k also affects the adaptive immune response, early containment of viral replication, massive NK cell proliferation, and reversal of the resistance phenotype by depletion of NK cells in FVB-H2⁰-Tg(D^k)⁺ clearly support a mechanism at the level of NK cells. Because of the presence of both inhibitory and activating Ly49 receptors, several nonexclusive scenarios could account for the precise mode of action of the combined MHC class I H2-D^k and Ly49 genotypes on the NK cell response against MCMV: (1)low threshold of NK cell activation through weak H2-D^k/Ly49 inhibitory signals, (2) effective NK cell activation through H2-D^k/Ly49 activating signals, and (3) interplay between H2-D^k/Ly49 activating and inhibitory signals.

Inhibitory signals

One possibility is that MHC class I/inhibitory Ly49 signals have a negative impact on the NK cell response to MCMV. In our study, mature NK cells in BALB.K mice (which are the most susceptible to MCMV infection) express three inhibitory receptors: Ly49A, Ly49C, and Ly49G2, which all bind to MHC-I H2^k molecules [31], [34], [37]. Thus, the majority of NK cells from BALB.K mice should be inhibited by a receptor for a self-ligand. Indeed, we have recently shown that deletion of the m04 gene renders BALB.K mice resistant to MCMV infection, as the protein it encodes abolishes NK cell activation via the “missing-self” recognition mechanism (Babic et al., 2010) [56]. The m04/gp34 protein escorts MHC class I molecules to the surface of infected cells, thus maintaining a level of surface MHC expression sufficient enough to trigger inhibitory NK cell receptors [38]. Thus, with only three (Ly49V, Ly49I, and Ly49G2) out of seven Ly49 inhibitory receptors able to recognize H2-D^k molecules, NK cells from BALB.Cmv3^MA/My H2^k mice should be less susceptible to inhibition by H2^k binding (Figure 6A–6C).

Activating signals

The existence of an H2-D^k–mediated activating axis to MCMV resistance is supported by the gain-of-function phenotype of FVB-H2-D^k transgenic mice, which presented itself despite their Ly49 repertoire that is virtually identical to that of their non-transgenic littermates (Figure S6A and S6B). Furthermore, the absence of NK cell triggering through inhibitory Ly49 receptors was not sufficient to allow efficient control of MCMV replication, as demonstrated by the F₃ Cmv3^FVB MHC class I–deficient mice. Most NK cells that develop in MHC class I–deficient hosts are unable to respond to MHC class I–deficient targets. However, a recent study demonstrated that, in the context of MCMV infection, NK cells eliminate virally infected cells in MHC class I–negative hosts, in addition to regaining the ability to eliminate MHC class I–deficient hematopoietic host cells [39]. This mechanism seems to be triggered by the inflammatory milieu induced by MCMV infection [39]. These observations suggest that the susceptibility of Cmv3^FVB MHC class I–deficient F₃ animals to MCMV infection is not due to a defect in education but to the absence of an activation axis, which is provided by H2-D^k.

Interplay of inhibitory and activating signals

Activating signals, mediated by the engagement of Ly49P by H2-D^k/m04, provided only a marginal enhancement of the NK cell response in the presence of H2^q. Interestingly, we observed a gene dosage effect in the inhibitory action of H2^q that correlated with the level of surface expression of this MHC class I molecule. However, H2^q copy number did not affect the ability of NK cells from H2-D^k transgenic mice (FVB or F3) to eliminate MHC class I deficient target cells; this indicates that H2^q gene dosage does not alter education/licensing of NK cells. By contrast, adoptive transfer experiments demonstrated that H2^q alleles expressed on host cells limit the ability of NK cells to respond to MCMV infection, indicating that the H2^q effect influences NK cell recognition of class I ligands on target cells. This suggests that H2^q inhibitory signals dominate over H2-D^k-dependent activating signals emanating from MCMV-infected cells. One possibility is that H2^q inhibitory signals are stronger and/or more frequent than H2^k-dependent activating signals. Indeed, it has been shown that both the density and the avidity of inhibitory Ly49-ligand pairs determine the strength of inhibition [40]. Alternatively, H2^q MHC class I molecules could compete with H2-D^k for binding with the m04 protein and thus blunt the m04/H2-D^k-Ly49P activating axis. We have noted that Ly49P reporter cells are equally stimulated by MCMV-infected MEFs of H2^k or H2^k/q genotype, which may indicate otherwise (Figure S5). However, these results might not reflect the effect of H2^q molecules on the H2-D^k/m04 complex under physiological conditions. While the molecular details of H2^q inhibition of NK cell function remain unclear, our results suggest a model in which two antagonistic mechanisms are at play in NKC-H2-determined resistance to virus infection (Figure 6). One involves enhanced NK cell responses through H2-D^k-mediated activating signals. The other involves dampened NK cell responses through inhibitory Ly49 receptors stimulated by class I H2^q (or H2^d) molecules, which override the effect of the H2-D^k construct.

Conclusion

It is puzzling that Ly49 receptors can sense MHC class I molecules on infected cells despite immune-evasion mechanisms elaborated by MCMV that downregulate surface expression of MHC class I molecules. Indeed, mouse strain–specific [41] and cell type–specific [42] differences have been reported in the ability of immunoevasins to inhibit lysis of infected cells by CTLs, indicating that the efficiency of MHC class I downregulation during MCMV infection [43] is context dependent. In vivo MCMV replication occurs in a multitude of cell types, and perhaps the ability of the virus to achieve immune avoidance selectively might contribute to the delicate equilibrium of coexistence it has established with the host.

The striking similarities between Ly49 and KIR interactions with their respective MHC-I ligands and how they both affect NK cell function prompted us and other researchers to use the mouse as a model to study NK cell antiviral responses. Our results lend support to clinical and epidemiological studies implicating KIR-HLA interactions of different strengths in determining a hierarchy of NK cell activation with varied effects on the host response against herpersviruses [44], HCV [45], and HIV [46]. Our work also highlights the ability of inhibitory signals to overcome NK cell activation. These regulatory mechanisms would be relevant in conditions where NK cell activation is undesirable during infection or immune disease. For example, activating KIR genotypes have been found to predispose to reactivation of quiescent, opportunistic infections associated with herpesvirus infections in HIV patients [47]), and to fatal outcome following Ebola virus infection [48]; furthermore, they may constitute a risk factor for susceptibility to autoimmunity and certain cancers [49], [50]. Ultimately, our data indicate that, as has been proposed for cancer and autoimmunity, manipulating the balance between inhibitory and activating NK receptor signals represents a possible avenue to harness the therapeutic potential of NK cells against virus infections.

Ethics statement

The animal protocols and experiments were approved by the Canadian Council on Animal Care (CCAC) and the McGill University Animal Resources Center.

Animals

MA/My, BALB.K, BALB/c, C57Bl/6 (B6), DBA/J, and AKR mice were purchased from The Jackson Laboratory. FVB/N mice were purchased from Charles Rivers Laboratories. The B6 mice deficient for H2-D^bK^b (B6.H2⁰) were kindly provided by Hidde L. Ploegh (Cambridge, Massachusetts).

Generation and genetic characterization of congenic mice

BALB-Cmv3^MA/MyH2^k and BALB-Cmv3^MA/MyH2^d were generated by backcrossing the (MA/MyXBALB.K) F1 or (MA/MyXBALB/c) F1 into BALB.K or BALB/c, respectively, for at least ten generations. At each backcross, inheritance of the NKC from parental MA/My mice was genotyped using either the Ly49e marker or the D6mit135 marker [51]. In the progeny, the introgressed portion from parental MA/My mice, which included the NKC, was analyzed using microsatellite markers or by detecting known SNPs. Once the genetic region was reduced from 34 Mb (between D6MIT36 and D6MIT59) to 10 Mb (between rs13479016 and rs13479061 SNPs), heterozygous mice were intercrossed to generate the homozygous congenic lines.

Generation of FVB-Tg(D^k) transgenic mice and derived F₃ strains

The H2-D^k genomic fragment cloned into the PBR22 plasmid was kindly provided by Bernd Arnold (Deutsches Krebsforschungszentrum [DKFZ], Heidelberg, Germany). The 11.5 kb fragment encompassing the D^k gene was subsequently purified and injected into fertilized FVB/N mouse eggs. Transgenesis was performed at the Quebec Transgenesis Research Network (QTRN). Transgenic founders were screened by PCR with the primers 5′-cacacgatccagcggctgt-3′ and 5′-ggcccggtctctctctgcag-3′, specific for H2-D^k exon 3. They were then bred to FVB/N WT mice. To generate F₃ mice, FVB-Tg(D^k) and B6.H2⁰ mice were bred to produce F₁ and F₂ progeny. To discriminate between the NKC and H2 regions inherited from the parental strains, the F₂ mice were genotyped at the NKC region with the D6Mit61 and D6Mit52 markers and at the H2 region with the D17Mit51 marker; they were also genotyped for the presence or absence of the H2-D^k transgene. Only the mice homozygous for the FVB/N NKC and heterozygous for either the H2 or the H2-D^k transgene were kept to generate the F₃ progeny, as listed in Table 1.

Antibodies and flow cytometry

To prepare splenic leukocytes, spleens were removed aseptically then gently mashed through a 70 µm nylon mesh (BD Bioscience). Red blood cells were lysed with ammonium chloride (Sigma). To isolate lymphocytes from mouse blood, mice were bled from the cheek; blood was collected in RPMI medium containing 15 mM EDTA. Lymphocytes were collected after gradient centrifugation using Histopaque-1077 (Sigma). Fc receptors were blocked with 2.4G2 antibody prior to staining with specific monoclonal antibodies. NK cells were incubated with NKp46 (conjugated to phycoerythrin [PE] or fluorescein isothiocyanate [FITC]) and specific monoclonal antibodies against Ly49A-Biot (YE148), Ly49A/D (12A8), Ly49CIH (14B11), Ly49D (4E5), Ly49G2 (4D11 or AT8), NKG2A/C/E (20d5) or NKG2A/B6 (16A11), CD94 (18D3), or KLRG1 (2F1). NK cells were also incubated with the following isotype control monoclonal antibodies: PE-conjugated golden syrian hamster IgG, FITC- or PE-conjugated mouse IgG2a K, or FITC-conjugated rat IgG2a K (e-Bioscience). H2-D^k and -D^q products were detected by anti-H2-D^k antibody (15-5-5) from BioLegend and anti-H2-D^q antibody (KH117) from e-Bioscience. To detect incorporated BrdU on NK cells, mice were scarified 5 or 6 days after MCMV infection; cells were first stained for surface antigens (anti-NKp46 and/or anti-Ly49 receptors) and then fixed, permeabilized, treated with DNase I, and stained with FITC- or allophycocyanin (APC)-conjugated anti-BrdU antibody (clone 3D4; BD Biosciences), according to the manufacturer's protocol. Flow cytometry analysis was performed with a FACSCalibur flow cytometer (BD Biosciences) and data were analyzed using CellQuest (BD Biosciences) or FlowJo (Tree Star). To assess NK cell proliferation in vivo, NK cells from the spleen were first enriched by negative selection (Miltenyi Biotec), then incubated with 5 µM CFSE for 15 minutes, washed, and resuspended in PBS. The purity of the NK cells (55%–70%) was evaluated by FACS using anti-NKP46 antibody; 2 million NK cells were then injected intravenously into recipient mice 24 hours before infection with MCMV. The proliferation index, indicating the number of divisions of CFSE-labelled NK cells, was determined using the FlowJo software.

Viruses and infections

Stock MCMV from mouse salivary glands was prepared by passaging the virus (Smith strain ATCC VR-1399, lot 1698918) twice in BALB/c mice. The virus was prepared from a homogenate of salivary glands 21 days p.i.. Mice aged between 7 and 9 weeks were infected intraperitoneally with 2,000 PFUs of MCMV. The tissue culture-grown viruses [52] Δm157 MCMV, which lacks the m157 open reading frame (ORF), and Δm04 MCMV, which lacks the m04 ORF, have been previously described [23], [53] and were kindly donated by Ulrich H. Koszinowski (Max von Pettenkofer Institute, Munich, Germany) and Stepan Jonjic (Rijeka University, Rijeka, Croatia). Viral titers of the stock virus or mouse organs (spleen and liver) were evaluated in vitro by standard plaque assays on a confluent BALB/c MEF monolayer, as previously described [54].

MEFs: MCMV infection and reporter cell assay

The MEFs used in this work were generated as previously described [52], except for FVB-Tg(D^k)⁺ transgenic and nontransgenic MEF cells, which were generated from individuals embryos from the progeny of FVB-Tg(D^k)×FVB wild-type mouse crosses and then genotyped for the presence of the H2-D^k transgene. 2B4 reporter cells expressing Ly49H, Ly49P, Ly49C, or Ly49I were generated as previously described [22], [25], [55]. MEF cultures from AKR, FVB/N H2-D^k transgenic, FVB/N wild-type, and BALB.K mice were infected with MCMV (Smith strain) or Δm157 or Δm04 deletion viruses at a multiplicity of infection (MOI) of 1.0 for 24 hours; they were used to stimulate 2B4 reporter cells as previously described [25]. GFP was detected by flow cytometry and analyzed using the FlowJo software.

In vivo killing of CFSE-labeled MHC class I–deficient cells

Splenocytes from B6.H2⁰ mice were labeled with 0.4 mM CFSE (CFSE low) in RPMI medium containing 5% FCS; splenocytes from recipient mice were labeled with 4 mM CFSE (CFSE high) in RPMI containing 10% FCS. The splenocytes were then incubated at 37°C for 10 minutes before being washed three times in RPMI containing 10% FCS. Cells (5×10⁶) of each type were mixed, and the mixture (200 µl) was injected intravenously into recipient mice. After 18 hours, spleens were harvested and red blood cells were lysed. The relative percentage of cells in each CFSE population was measured by FACS as previously described [33].

In vitro killing of CFSE-labeled RMA/S cells

NK cells from the spleen were expanded for 5 days in RPMI medium supplemented with 1,000 U/ml human IL-2 (NCI Preclinical Repository). Cells were washed in RPMI and stained with NKp46 antibody to determine the purity of NK cells and to adjust the number of NK cells among strains. RMA/S cells were labeled with 0.4 mM CFSE in RPMI medium containing 5% FCS for 15 minutes at 37°C and washed three times. CFSE-RMA/S and NK cells were cocultured at effector/target ratios of 2∶1, 4∶1, 8∶1, 16∶1, and 32∶1 for 4 hours at 37°C. Specific lysis was determined by the measure of 7-aminoactinomycin D (7-AAD) incorporation (BD) in CFSE-RMA/S cells by flow cytometry, as previously described [56].

Statistical analysis

For the 137 (FVB/N×BALB/c) F₂ mice, the contribution of the NKC and H2 loci to the segregation of the phenotype was estimated with the linear model phenotype = m+NKC+H2+NKC:H2+e, where NKC and H2 represent factors that depend on the mode of inheritance proposed, m is the common mean value, NKC:H2 is an interaction term, and e is the independent, normally distributed random deviations. For the additive mode of inheritance, the NKC and H2 represent the number of FVB/N/BALB.K alleles at each locus. For the recessive mode of inheritance, the NKC and H2 are indicator variables of the homozygous FVB/N and BALB.K backgrounds, respectively. The four possible additive-recessive combinations of H2-NKC models, with and without an interaction term, were fitted. We assessed the magnitude of the contribution for each term in the model by its P value, obtained by 1 million bootstrapped samples, and partial η². Partial η² was computed as η² = SS_factor/(SS_factor+SS_error), where SS_factor is the type 3 associated sum of squares with the factor in the analysis of variance (ANOVA) table, and SS_error is the sum of squares corresponding to the residual variation. We carried out statistical and graphical analyses using R software. For other statistical analyses in this work, differences between groups were calculated with two-way ANOVA analysis, followed by Bonferroni after tests. For some of the analyses, unpaired, two-tailed Student's t-tests were conducted. Results with a P value of <0.05 were considered to be statistically significant.