Multiparametric Analysis of Host Response to Murine Cytomegalovirus in MHC Class I–Disparate Mice Reveals Primacy of D^k-Licensed Ly49G₂⁺ NK Cells in Viral Control

Natural killer cells confer essential innate immunity and host defense against viral infection (1). Human NK cell deficiency results in severe susceptibility to viral infections, especially herpesviruses (2, 3). Likewise, NK cell deficiency in the mouse due to selective immunodepletion, mAb blocking, or genetic mutation of NK cells results in susceptibility and much less efficient viral control. NK cell detection and killing of infected cells require highly efficient and precise recognition of viral targets. Regulation of NK cell cytokines and cytotoxicity involves numerous cell surface stimulatory and inhibitory receptors that are needed to scrutinize cellular targets for evidence of viral infection or transformation (4). Although the NKRs are highly polymorphic, genetic approaches to investigate the mechanistic bases of NK-mediated resistance to infection and viral control have yielded significant insight.

Many NK cell receptors reportedly enhance recognition of viral targets and occasionally elicit viral control (5–14). Prominent examples are Ly49H and Ly49P, two stimulatory receptors that are known to bind the viral protein m157 and MHC I D^k on murine CMV (MCMV)-infected targets, respectively (9, 15–18). In contrast, inhibitory receptors bind class I MHC molecules as ligands and render NK cells self-tolerant by preventing lytic attack against autologous cells. Cellular changes that result in missing-self MHC class I display, as sometimes occurs as a result of viral infection or cell transformation, also evoke NK detection and target cell lysis (19). Indeed, viruses have evolved intriguing and rather selective strategies to evade NK cell detection via missing-self class I MHC cues (6, 20–23).

Inhibitory receptors also serve to educate NK cells through “licensing” or “arming” (24, 25). Although licensed NK cells can be readily stimulated ex vivo via activating receptor cross-linkage, unlicensed NK cells display hyporesponsiveness. Less severe disease features due to chronic viral infection observed in patients with combined genes for inhibitory killer Ig-like receptor and HLA class I molecules hint that human licensed NK cells might also contribute to viral control (26, 27). In support of this concept are several human studies demonstrating expansion and/or activation of select inhibitory receptor–expressing NK cells following exposure to certain viral infections (28–30). The results suggest that self-licensed NK cells may be poised to efficiently respond to missing self-MHC cues following viral exposure, although direct evidence of a role for human inhibitory NKRs in viral control is lacking.

Genetic analysis of MCMV resistance in MA/My and C57L hybrid mice showed that NK cells with the inhibitory Ly49G₂ (G₂) receptor provide critical protection in offspring with MHC I D^k, a major MCMV resistance factor and G₂-licensing ligand (31–34). Because D^k resistance is heightened in C57L mice compared with MA/My mice (35), C57L alleles outside of the MHC may enhance NK-mediated viral clearance. Altogether, the results suggested that self-licensed NK cells, possibly regulated by background genetic effects, contribute to highly efficient detection and elimination of viral targets. However, several reports showed that G₂⁺ NK cells in B6 mice rapidly expanded in response to proliferation cues (36) or exposure to one of several different viruses (37–39). Moreover, licensed Ly49C/I⁺ NK cells were since shown to diminish viral control in MCMV-infected B6 mice (40). Thus, the precise role of self-licensed NK cells and inhibitory receptors in viral control and clearance is still uncertain.

To address this question, we combined classical genetics with flow cytometry and examined distinct NK cell subset features before and after MCMV exposure to visualize the NK cell response to infection in a large cohort of MHC I D^k-disparate mice. The results demonstrate that G₂⁺ NK cells underwent highly selective expansion in D^k mice, which directly corresponded with significant viral protection. We infer that licensed NK cells are essential to efficiently detect and then eliminate MCMV-infected targets, a response that is undeniably shaped by class I MHC polymorphism.

MA/My and C57L breeder mice were purchased from The Jackson Laboratory. C57L-derived strains, known as R2, R2-NKC^het, R2-NKC^mamy, R7, R7-NKC^het, R7-NKC^mamy, R12, and Tg-D^k, and MA/My-derived strains, known as M.H2^b and M.Tg1, were reported previously (32, 35). We bred and studied 38 (R7 × M.H2^b)F₂, 62 (MA/My × C57L)F₂, 72 [(MA/My × C57L)F₁ × M.H2^b]N₂ and 61 [(MA/My × C57L)F₁ × C57L]N₂ hybrid offspring. Mice were managed with the Jackson Laboratory's Colony Management System (Version 4.1.2). All mice used in this study were maintained in a dedicated animal care facility under specific pathogen–free conditions and treated in accordance with the regulations and guidelines of the Animal Care and Use Committee of the University of Virginia.

Spleen and liver genomic DNA samples were prepared using a Gentraprep kit. To ensure sample identity, all mice were “footprinted” by genotyping MHC, NK gene complex (NKC), and chromosome 19 loci using gene-specific PCR and high-resolution melting (HRM) analysis, as described (41), prior to genome-wide single nucleotide polymorphism (SNP) typing by DartMouse (Lebanon, NH) using the Illumina medium-density SNP panel. Footprint analysis was 100% concordant with the genome-wide SNP analysis for all mice (data not shown).

mAbs and isotype controls used for flow cytometry were purchased from BD Biosciences, BioLegend, and R&D Systems and included anti-CD16/32 (2.4G2), G₂ (4D11), Ly49I/U (I/U; 14B11), CD3 (145-2C11), CD19 (6D5), and NKp46 (29A1.4). These were conjugated to FITC, PE, PerCP, PerCP-Cy5.5, allophycocyanin, or biotin, followed by streptavidin conjugated to allophycocyanin-Cy7 (BioLegend). Dead cells were excluded using a Live/Dead Fixable Violet Dead Cell stain kit with compensation based on the ArC Amine Reactive Compensation Bead Kit (both from Invitrogen). Compensation for tandem dyes on Abs that stain rare cell subpopulations used the AbC anti-Rat/Hamster Bead Kit (Invitrogen) with uninfected blood leukocytes and single-stain controls on spleen leukocytes postinfection. Blood (4 d preinfection) and spleen (3.5 d postinfection) leukocytes were stained as described previously (34). Live splenocytes were calculated after manual counting of cells excluding trypan blue. Flow cytometric analysis of stained cells was performed on a FACSCanto II (BD Biosciences). Data acquired using FACSDiva software (BD Biosciences) were analyzed with FlowJo software (Versions 8.0 and 9.2; TreeStar, Ashland, OR). NK cell subpopulation frequencies and NKR geometric means (median fluorescence intensity [MFI] values) were calculated in FlowJo and exported into Excel tables.

Primary flow cytometry data files, grouped by experimental cohort, are publicly available through FlowRepository (http://flowrepository.org/), as identified by the following public experiment tags: FR-FCM-ZZ4W, FR-FCM-ZZ4J, FR-FCM-ZZ4K, FR-FCM-ZZ4L, FR-FCM-ZZ4N, FR-FCM-ZZ4X, FR-FCM-ZZ4V, FR-FCM-ZZ5Z, FR-FCM-ZZ5Y, FR-FCM-ZZ52, FR-FCM-ZZ53, FR-FCM-ZZ54, FR-FCM-ZZ5C, FR-FCM-ZZ5B, FR-FCM-ZZ5D, FR-FCM-ZZ5E, FR-FCM-ZZ5F, FR-FCM-ZZ5H, FR-FCM-ZZ5W, FR-FCM-ZZ5J, FR-FCM-ZZ5K, FR-FCM-ZZ5L, FR-FCM-ZZ5M, FR-FCM-ZZ5N, FR-FCM-ZZ5X, FR-FCM-ZZ5P, FR-FCM-ZZ5S, and FR-FCM-ZZ5R. The primary data include pre- and postinfectio analyses for F₂ cohorts 1, 2, 4, 5, and 6 and backcross cohorts 1–8. F₂ cohort 3 was excluded from the FACS analysis as the result of experimental error associated with loss of postinfection splenocyte samples. Public identification tags FR-FCM-ZZ5B and FR-FCM-ZZ5W include analysis of only one experimental animal. Individual sample details (e.g., source, treatment, mAb staining panel) are also available on selection of “Flow Sample/Specimen Details.”

All mice were weighed and bled 4 d prior to i.p. MCMV infection (10⁵ PFU Smith strain salivary gland virus stock) and then weighed again 3.5 d postinfection, just before euthanization. Approximately three quarters of each individual spleen was used for flow cytometric analysis. Small spleen and liver tissue fragments were processed for genomic DNA genotyping and quantitative real-time PCR analysis of MCMV genome level, as described previously (42).

To ensure flow cytometric data integrity, all FlowJo cell frequencies and NKR display values exported into Excel tables were verified against raw data. To further qualify the data set, one quarter of all mice, chosen at random, were retyped for spleen MCMV and genetic footprint using quantitative real-time PCR and HRM, respectively. Another quarter of randomly selected individual mice were retyped for liver MCMV. In addition, H-2 class I D exon 5 genotypes were verified using intron 8–specific PCR, followed by HRM analysis (41). “Atypical” mice also were screened with H-2 D intron 1– and intron 3–specific markers (41). All H-2 D gene makers were fully concordant in all mice.

Statistical analyses included paired and unpaired Student t tests, Pearson correlations, and multiple linear-regression tests performed using the R (versions 2.15.0 and 2.15.2) statistical computing environment, with select plots drawn using ggplot2 (43) and Corrplot (44) packages. The p values were corrected for multiple comparisons using both the Benjamini–Hochberg false discovery rate (45) and the Bonferroni correction.

Strain-specific variance in viral control and splenocyte recovery following MCMV infection (35) prompted our genetic analysis of the NK cell response to MCMV. We first analyzed peripheral blood and spleen NK cells (NKp46⁺, CD3⁻, CD19⁻) from naive mice with different MHC and NKC genotypes (Fig. 1A, 1B, Table I). G₂⁺ and I/U⁺ NK cell subsets were analyzed with mAbs 4D11 and 14B11, as described (17, 32, 34, 46). Without a monospecific-staining reagent, Ly49P⁺ NK cells were not examined. As expected of D^k-licensed G₂⁺ NK cells (33, 34), each of our C57L-derived strains with D^k had less frequent G₂⁺ NK cells, with significantly reduced G₂ receptor display intensity (MFI), compared with C57L (Fig. 1C, Table I), which supports that the subset was licensed. The effect of D^k was specific to G₂⁺ NK cells because the frequency of I/U⁺ NK cells was unaffected in C57L- and M.H2^b-derived Tg-D^k mice (Table I, Supplemental Table I). Nonetheless, a reduction in I/U⁺ NK cells in R2 and R7 compared with C57L revealed further MHC control of NK cell subsets (Table I, Supplemental Table I). The data confirmed that H-2^k regulates the homeostatic composition of NK subsets and receptor display features in mice with relevant Ly49 receptors, as shown previously for NKR expression in other strains (47, 48).

Likewise, NKR polymorphism is known to affect NK cell features and their role in MCMV resistance (49). Analysis of R2-NKC congenic strains revealed that both the frequency of G₂⁺ NK cells and I/U MFI were impacted by NKC polymorphism (Table I). Interestingly, a lower percentage of G₂⁺ NK cells in MA/My mice compared with R7-NKC^m mice suggested that a genetic factor(s) outside the MHC and NKC regions also shapes Ly49⁺ NK subsets and receptor display (Supplemental Table I). A difference in the frequency of I/U⁺ NK cells in M.H2^b, M.Tg1, and C57L mice further supports non-MHC, non-NKC genetic control of NK subset composition. Although D^k-mediated MCMV resistance was shown to be more effective in C57L-derived mice than in MA/My mice (35), the above results hinted that NK-mediated viral control may depend on quantitative, as well as qualitative, NKR expression features.

Previous studies implicated both Ly49P⁺ and G₂⁺ NK cells with MCMV resistance (16, 32). However, disentangling the contribution of either subset has been difficult without a monospecific reagent to detect Ly49P. A genetic approach to assess the role of the different NKRs is equally challenging because of the complexity of disrupting a singular Ly49 gene (50), as well as the rare frequency of cross-over events (51) within the Ly49 gene cluster. Moreover, NKC haplotypes associated with MCMV resistance in D^k-expressing MA/My and C57L-derived mice encode both Ly49P and a D^k-binding G₂ allele, whereas NKC types in less-resistant strains (e.g., BALB.K) lack both of the relevant Ly49 receptor genes/alleles.

In lieu of the challenges, we attempted to clarify the role of distinct NK subsets by analyzing a variety of immune response traits in a cohort of genetically diverse mice segregating D^k. Although Ly49p is expressed in G₂⁺ and G₂⁻ NK cells (33), we reasoned that both subsets may contribute MCMV protection and that key NK cell features must coincide with low viral burden in animals with the essential genetic make-up. We established a basis for the analysis by assessing NK cells in our D^k-disparate strains after MCMV infection. Because these were essentially equivalent in peripheral blood and spleen pre- and postinfection (Fig. 1B, Table I), we concluded that the subset composition of naive spleen NK cells could be estimated and used to gauge MCMV responsiveness.

We next examined 200+ MA/My × C57L hybrid offspring segregating D^k. MHC and NKC congenic blocks were included in the genetic crosses to help resolve non-MHC, non-NKC genetic effects. Before infection, the mice were weighed and bled. Blood leukocytes were stained with fluorochrome-conjugated mAbs for CD3, CD19, and NKRs and then analyzed by flow cytometry. Lymphocyte and NK cell subset light-scattering features, absolute number, frequency, and receptor display intensity were determined and recorded as individual preinfection traits. Subsequently, the mice were infected with MCMV. Spleen and liver tissues were collected 3.5 d later for analysis of postinfection traits, including genomic genotypes (data not shown) and viral load. Spleen leukocytes from infected animals were analyzed as for preinfection traits. The change in trait values was calculated from the preinfection (blood) and postinfection (spleen) trait values. In total, 110 distinct traits were assessed (Supplemental Fig. 1).

All trait-to-trait comparisons were performed in the R computing environment using an automated correlation analysis, followed by a strict Bonferroni correction for multiple tests (Supplemental Table II). From a heat map depicting the results of all correlations, we observed ≥15 hierarchical clusters that revealed trait-association relationships (Supplemental Fig. 1). For example, postinfection forward scatter (cluster 2) and side scatter (cluster 3) traits were clustered separately but also generally corresponded positively with one another. In contrast, clusters 2 and 3 were generally negatively associated with traits grouped together in cluster 1. To reduce the complexity of the analysis, 40 representative traits for the 15 clusters were selected and reanalyzed separately (Fig. 2).

Detailed trait associations, including those that best explain viral control, were then ascertained. Remarkably, the postinfection percentage of both total G₂⁺ (ρ = −0.8, p < 10⁻¹⁶) and G₂⁺ I/U⁻ (G₂ single-positive [SP]; ρ=-0.75, p < 10⁻¹⁶) NK cells was highly negatively correlated with log spleen MCMV (Fig. 2, Supplemental Table II). In fact, of all traits analyzed, only the change in percentage of body weight had a higher inverse correlation with MCMV in spleen (Supplemental Fig. 1, Supplemental Table II). Although the postinfection percentage of total I/U⁺ NK cells also correlated negatively (ρ = −0.43, p_Bonferroni [p_B] < 5 × 10⁻⁸), I/U⁺ G₂⁻ (I/U SP) NK cells showed no significant association (Fig. 2). The results suggest that the I/U⁺ NK correlation with spleen MCMV likely was due to overlapping G₂ expression. In contrast, postinfection G₂⁻ (ρ = +0.8, p < 10⁻¹⁶) and G₂⁻ I/U⁻ (double-negative [DN]; ρ = +0.75, p < 10⁻¹⁶) NK cells corresponded directly with spleen viral load better than any other single trait (Fig. 2, Supplemental Table II). Thus, we infer that G₂⁺ NK cells were linked inextricably with viral control, more so than any other lymphocyte feature, and that high viral load exacerbated malaise in mice with an abundance of G₂⁻ NK cells.

To examine the effect of class I MHC polymorphism on NK cell features, a principal component analysis (PCA) was performed (Fig. 3). As shown, all offspring, marked by MHC genotype, were plotted based on differences in the first two principal components, distilling all 110 features down to two dimensions to enable visualization (Fig. 3). The primary principal component (horizontal axis), which explains the majority of the variation among all 110 features, very clearly separates D^k and D^k/b mice from D^b offspring, which were generally marked by higher values in the first principal component. Interestingly, three “atypical” D^b mice clustered separately from other D^b offspring, primarily due to a major difference in the first principal component. Altogether, the PCA results clearly demonstrated the essential impact of MHC genotype on traits that distinguished the offspring.

The above results suggested that G₂⁺ NK expansion postinfection was defined by MHC genotype. To examine this, we focused on those NK features most significantly (Bonferroni-corrected) associated with viral control. Before infection, naive NK cell features varied broadly in the genetically diverse cohort (Fig. 4A, data not shown). As expected and consistent with a D^k-licensing effect, G₂⁺ NK cells were less frequent (ρ = −0.44, pB < 1.5 × 10⁻⁸) with lower G₂ receptor display (ρ = −0.38, p_B < 5 × 10⁻⁶) in naive D^k offspring, whereas DN NK cells were slightly more frequent (Supplemental Fig. 1). In contrast, MHC type had no impact on the percentage of I/U⁺ NK cells or I/U MFI in the hybrid offspring (Supplemental Fig. 1).

Remarkably, only G₂ MFI on peripheral blood NK cells in naive mice significantly predicted (ρ = +0.33, p_B = 0.002) viral load in spleen postinfection (Figs. 2, 4A). This result suggests that G₂⁺ NK cells were pre-equipped to handle MCMV in mice with a G₂-licensing ligand D^k. Interestingly, NKp46 MFI on naive NK cells was higher in H-2^k offspring (Supplemental Fig. 1). However, preinfection NKp46 by itself was not a predictive index of viral control (Fig. 4A). Altogether, the results from naive mice suggested that D^k-licensed G₂⁺ NK cells marked by relatively lower G₂ MFI were essential to provide protection following MCMV infection.

We further addressed the question by comparing the composition and features of NK cells in the D^k-disparate offspring postinfection. The percentage of postinfection G₂⁺ NK cells was much higher in D^k offspring, whereas DN NK cells were more abundant in infected offspring without D^k (Fig. 4B). Selective expansion of the G₂⁺ NK subset in response to MCMV likely explains the dichotomy, because the average G₂⁺ NK frequency in infected spleen was ∼45% higher than in naive blood, whereas the average I/U⁺ NK frequency revealed little, if any, response to MCMV (Fig. 4B, 4C, data not shown). A severe reduction in G₂⁺ NK frequency was especially striking in offspring lacking D^k, although the percentage of I/U⁺ NK cells also declined in the same animals (Fig. 4C). We infer from the results that, in addition to shaping NK subsets at rest in naive mice, MHC class I polymorphism had a definite effect on NK subset composition and effector function during the response to MCMV. The most parsimonious interpretation of the data is that G₂⁺ NK cells provided critical viral control in the spleen of mice with a self-D^k ligand but failed to elicit the same protection in mice without D^k, in which DN NK cells were most abundant.

MCMV infection highlights the plasticity of NK subsets and the profound role for MHC polymorphism in shaping innate immunity and viral control. We next used multiple-regression analysis to investigate whether any traits other than MHC type significantly affected viral load in spleen. Each continuous variable trait plus MHC type was tested (full model) and compared with test results using MHC type alone (reduced model); this accounted for ∼63% of the variation in spleen viral load (Supplemental Table III). Full models based on postinfection percentage of total G₂⁺ (77%) and G₂ SP (70%) NK cells, but not G₂ MFI, explained significantly more viral load variation than did the reduced MHC-type–alone model (Fig. 5). MHC type plus the postinfection percentage of I/U⁺ NK cells also explained 71% of the variation in viral load (statistically significantly better prediction than MHC alone), likely due to overlap with G₂⁺ NK cells because any difference in the reduced and full model with I/U SP NK cells was insignificant (Fig. 5). Full models with the postinfection percentage of G₂⁻ or DN NK cells explained slightly >77% of viral load variation (Fig. 5, Supplemental Table III). However, both subsets correlated significantly with high viral load (Fig. 2, Supplemental Table II). Hence, as with the results in Fig. 4, multiple-regression analysis confirms that the postinfection balance of G₂⁺ and DN NK cells correlated significantly with MCMV protection. Multiregression analysis further implicates that another genetic factor(s) beyond the MHC regulates the frequency/composition of NK cells in the response to MCMV. It is unlikely that the effect was due to NKC polymorphism, because the Ly49g genotype was not significantly correlated (Supplemental Fig. 1). The results demonstrate that the best index of MCMV control involved a balance of more frequent G₂⁺ NK cells, which coincided with less frequent DN NK cells; together with MHC type, these traits explained the majority of viral load variation.

Beyond the role of G₂⁺ NK cells in viral control, we unexpectedly found that postinfection NKp46 MFI on NK cells correlated negatively with spleen MCMV (Figs. 2, 4B). The results implied that NKp46 might help to limit MCMV spread. To test this, we first examined its relationship with different NK cell subsets postinfection. As expected, NKp46 MFI corresponded with more frequent G₂⁺ NK cells (ρ = +0.48, p_B < 7 × 10⁻¹¹). However, the postinfection percentage of I/U⁺ NK cells showed an even stronger relationship (ρ = +0.56, p < 10⁻¹⁶) (Figs. 2, 4B). In contrast, postinfection NKp46 MFI correlated negatively (ρ = −0.54, p < 10⁻¹⁶) with the percentage of DN NK cells (Figs. 2, 4B, Supplemental Table II). The results demonstrated that the expansion of DN NK cells best corresponded with lower NKp46 MFI.

Given its relationship to NK subset composition, we next examined postinfection NKp46 MFI on defined NK subsets. As expected, its expression on G₂ SP NK cells was inversely related to spleen viral load (Fig. 6A). However, its expression on I/U SP and DN NK cells corresponded even more favorably with viral control (Fig. 6A). The results indicated that distinct NK subsets must variably express NKp46 after MCMV exposure. In fact, DN NK cells in infected D^k mice had much higher expression than did either subset of Ly49⁺ NK cells (Fig. 6B). In contrast, spleen NK subsets from infected offspring without D^k displayed substantially lower NKp46 than did the D^k counterpart (Fig. 6B). Similar NKp46 disparity among G₂⁻ and G₂⁺ NK cells was observed in infected R7 mice (Fig. 6C). Altogether, the results suggest that a robust G₂⁺ NK response to MCMV in D^k mice was essential to regulate higher NKp46 MFI, and this was most pronounced on G₂⁻ NK cells.

The above results led us to question whether NKp46 expression, apart from the MHC effect, might also contribute to any of the variance in viral control. We assessed postinfection NKp46 MFI on NK cells by multiple-regression analysis comparing full (MHC plus postinfection NKp46 MFI) and reduced (MHC-alone) models. The full model significantly explained 75% of spleen viral load variance (Fig. 5, Supplemental Table III), a result equivalent in scale to a full model based on the post-G₂⁺ NK cell frequency. A full model based on NKp46 MFI on DN NK cells proved even better, whereas full models restricted to NKp46 MFI on G₂⁺ or G₂ SP NK cells accounted for less of the viral control variance (Fig. 5). Thus, the postinfection NKp46 MFI on NK cells explained significantly more of the variance in viral control than did MHC alone. MHC-independent genetic regulation of NKp46 MFI was most significantly evident on DN NK cells, whose frequency also explained significantly more viral load variance than did MHC alone. We infer that D^k-licensed G₂⁺ NK cells were able to efficiently detect infected targets, which led to their rapid activation and expansion. MHC-independent regulation further affects the balance of G₂⁺ and G₂⁻ NK cells, as well the differential NKp46 expression on distinct NK subsets after MCMV exposure (Fig. 7).

The combined use of classical genetics and flow cytometric analysis of immune cells, collected first from naive animals and then infected animals, is an innovative and powerful approach to interrogate the immune response following virus exposure. However, a concern with multiple testing in large sets of immunological data is the possibility for random associations to occur by chance (52). With 110 trait values, multiple trait comparisons yielded >6000 correlation values. Thus, we applied the statistically conservative Bonferroni correction, as well as calculation of the false discovery rate (45), to exclude false-positive associations. Related trait associations (e.g., post %G₂⁺ and post %G₂⁻ NK cells or pre-I/U MFI and post-I/U MFI) were excluded. The report emphasizes presentation of significant data (i.e., p_B < 0.01) only after the more conservative Bonferroni correction so that some true associations may have been disregarded. Nonetheless, significant trait associations with viral load implicate either a direct or indirect effect on viral control. Significant variance in immune responsiveness and MCMV restraint facilitated our search to uncover key NK cell features and the responsible gene(s). Despite the focus on viral immunity, the approach is amenable to other settings in which genetic diversity has an influence on host immunity.

Most remarkable, the data in this study show that both the percentage and numbers of splenic G₂⁺ NK cells rapidly expanded in offspring with self-D^k, at the expense of G₂⁻ NK cells, within days after MCMV infection. The response profile was completely reversed in non-D^k mice in which innate control mechanisms were readily overwhelmed. Although no other single NK feature had a stronger or more significant correlation with viral control, we infer that licensed G₂⁺ NK cells were required to efficiently detect and elicit rapid killing of viral targets. However, this interpretation contrasts with the results of Orr et al. (40), who showed that licensed NK cells attenuated MCMV control in neonatal B6 mice. Although NK cells in B6 mice use the Ly49H activation receptor to detect MCMV m157 displayed on the surface of infected cells and render target cell killing (15), an H-2^b factor(s) is not known to significantly affect their capacity for MCMV recognition. In fact, Ly49C/I⁺ NK cells are not known to detect or respond to MCMV targets. Thus, the discrepancy might be explained by a difference in the primary mode of target recognition by NK cells in the different experimental settings. In the current work, licensed G₂⁺ NK cells were competent to detect missing-self MHC I cues on viral targets and responded more aggressively than did all other NK cells examined.

It was reported that G₂⁺ NK cells rapidly respond and undergo significant expansion in virus-infected B6 mice (36–39). However, depletion of G₂⁺ NK cells from B6 mice had no effect on viral control in spleen (38) and only a relatively modest effect in liver and salivary gland by day 7 after MCMV infection (40). Moreover, unlike MHC-independent G₂⁺ NK subset expansion following hematopoietic stem cell transplantation or in vivo cytokine stimulation in B6 mice (36), G₂⁺ NK cells in this study selectively expanded in offspring bearing self-D^k. This could have been due to a collapse of splenocytes and immune response capability in more MCMV-sensitive offspring as the result of a relatively high viral dose. However, similar differences in G₂⁺ NK responsiveness and expansion following lower-dose (10⁴ PFU/mouse) MCMV infection were observed in a separate cohort of MA/My × C57L offspring (data not shown). Together, the results establish that it was not an intrinsic proliferative potential of the NK subset itself, but an extrinsic D^k ligand that corresponded with subset expansion and viral control. When the frequency of licensed G₂⁺ NK cells increased, G₂⁻ NK cells were less frequent with higher NKp46 MFI, spleen MCMV was lower, and morbidity was reduced. Whether these effects are directly related to NK cell licensing, enhanced detection of viral targets, or both is an open question.

Curiously, a rare fraction of three “atypical” D^b mice with aberrantly low spleen MCMV and very little weight loss were clustered separately by PCA from the remainder of D^b mice. Naive G₂⁺ NK cells and G₂ MFI in the mice were very similar to other D^b offspring, consistent with the genetic typing (data not shown). Moreover, there was no evidence for allelic chimerism in MHC I D (41) (data not shown). However, unlike other D^b offspring, the aberrant mice retained G₂⁺ NK cells following MCMV exposure. Although a similar result was observed previously in related genetic crosses (53, 54) and weight loss and lymphocyte light scatter features were not unusual in the mice, we cannot verify whether inadequate infection, a “cage effect” for two of the three mice, or enhanced MCMV resistance explains the outcome. Interestingly, the Ly49G¹²⁹ allele, which is identical to the C57L Ly49G allele, was shown to bind MHC I D^b tetramers, but no evidence for NK licensing due to the interaction has been observed (A. Makrigiannis, personal communication). Therefore, it is tempting to speculate that G₂⁺ NK cells may, on rare occasion, be licensed by D^b or a nonclassical MHC class I (55) and be competent to detect MCMV targets via missing-self recognition. In fact, three additional D^b mice with aberrantly low spleen MCMV that were not distinguished by the PCA actually increased the G₂⁺ NK subset in response to MCMV (data not shown). Together, the data highlight the importance of D^k-licensed G₂⁺ NK cells in the response to MCMV and further hint that variations in the extent of NK licensing and capacity for detection of missing-self cues on viral targets might significantly influence NK-mediated viral control.

The natural cytotoxicity (stimulatory) receptor (NCR) NKp46 is displayed on human and mouse NK cells; high-level expression corresponds with NK-mediated tumor killing (56, 57). NKp46 and other NCRs are known for binding viral hemagglutinins in cells infected with influenza or poxviruses (5, 13, 29). Recent work further claims NKp46 detection of an unknown ligand manipulated by human CMV infection (13, 29). Lower expression, as in NKp46^dull NK cells, corresponds with persistence in some chronic viral infections, including hepatitis C virus, HIV, and human CMV (28, 58–61) and in patients with pulmonary tuberculosis (62). Diminished NK functionality due to repressed NCR display in HIV-infected patients may explain reduced viral control (58, 59). However, low NKp46 display is not correlated with dysfunction in all NK subpopulations or in all HIV-infected patient groups (60), and it might even help to resolve HCV infection (61). However, this is an active area of investigation because NKp46^high cells with high cytotoxicity and cytokine potential were shown to control HCV infection (63). Thus, NKp46 is a critical marker to assess NK responsiveness in acute and chronic viral infections.

Interestingly, we found that high postinfection NKp46 MFI corresponded with reduced weight loss and lower viral load in the spleen after MCMV infection. To our knowledge, this is the first report linking NKp46 with viral control and morbidity in MCMV-infected mice. Diminished NKp46 display was an unmistakable effect of acute MCMV exposure, in accordance with human CMV infection (28). The result was most conspicuous in MCMV-sensitive D^b mice, but it also was evident even in more resistant D^k mice. High expression among MCMV-resistant D^k mice suggested that NKp46 MFI might simply represent a marker of activated NK cells. Yet, NK cells in all mice exhibited features consistent with cellular activation after MCMV exposure. More reasonably, NKp46 display is modulated in a way that corresponds to viral control. We envision two scenarios: NKp46 may help to detect MCMV targets or NKp46 may be an additional effector of anti-MCMV immunity, which requires a prior signal to unleash its full potential.

Although marked MCMV control was observed in offspring with postinfection NKp46^high G₂⁻ and DN NK cells, the relationship was still limited to offspring with self-D^k, which also had more abundant G₂⁺ NK cells. Moreover, post-NKp46 MFI plus MHC type accounted for ∼76% of the variation in viral load postinfection due to NKp46^high DN NK cells, which most significantly explained the association. These data illustrate the importance of G₂⁻ NK cells, in addition to G₂⁺ NK cells, in offspring with self-D^k and suggest that licensed NK detection of MCMV enhanced defined NK cell subsets needed to efficiently control MCMV.

In summary, we conclude that NK cells provided vital antiviral immunity in mice with the G₂-licensing ligand D^k. Their expansion following viral exposure clearly indicated significant immune cell responsiveness, reduced morbidity, increased MCMV control, and viral clearance. Moreover, detection of licensed NK features (i.e., G₂ MFI) prior to viral infection actually significantly predicted viral control, which implies that related human studies may likewise uncover predictive markers of NK-mediated viral control. The results foreshadow that predictive modeling is an important and promising approach to examine how protective human genotypes may correspond to NK cell–mediated responsiveness, immune enhancement, and viral clearance.

We thank Tim Bullock and members of the Brown laboratory for helpful discussion and critical reading of the manuscript. We also thank J. Han, T. Nguyen, and J. Cronk for technical support.

The authors have no financial conflicts of interest.