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

Abstract

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

Fig 1. Genomic profiling of MCMV immunity.

Multiple QTL chr maps for naïve and either LD (A) or HD (B) infected cohorts of mice. Shown are color-coded LOD profiles for the indicated pre- and postinfection traits for both cohorts.

Fig 2. Combined effect of NKC and MHC polymorphism on NK cell reactivity and MCMV resistance.

(A) Chr-6 interval mapping LOD plots for LD and HD mice. Shown are color-coded LOD profiles for I/U SP (i.e. G2^neg) NK cell traits. (B and C) The plots show allele effects for defined traits for LD and HD mice with genotypes (L = C57L, M = MA/My, and ML = heterozygous) indicated for peak SNP marker positions. (D) The plots show splenic G2⁺ NK cell features for naïve C57L and C57L.M-NKC^m congenic mice, in addition to spleen MCMV burden after LD-infection. Data are representative of 3 independent experiments.

Fig 3. MHC-independent regulation of NK cell accrual after MCMV exposure.

Color-coded chr-17 LOD plots for the indicated experimental traits in LD-infected (A) and HD-infected (B) cohorts of mice are shown. (C and D) Shown are allele effect plots for defined traits obtained for LD and HD mice with genotypes reported as in Fig 2. (E) A diagram of peak QTL positions (empty circles) with CIs (bracketed lines) for pre- and postinfection traits. A chr-17 physical map with defined marker loci and SNP positions is also shown. Two weight-related traits were not mapped (NM) in the LD cohort.

Fig 4. Verification of the Cmv5 locus on chr-17 in MA/My-related congenic strains.

(A and B) The plots show spleen weights (A) and percentages of NKp46⁺ NK cells (B) in uninfected, and LD- and HD-infected MA/My, M.H2^b and Tg1 (M.H2^b background) mice. The data are representative of 3 independent experiments. (C) The plot shows MCMV genome levels detected in spleen DNAs obtained from the indicated mice. The data are representative of 3 independent experiments. (D) The plots show spleen weights (left) and MCMV genome levels (right) for uninfected and HD-infected MA/My, M.H2^b, Tg1 and (MA/My x M.H2^b)F₁ cross mice. The data are representative of 2 independent experiments.

Fig 5. Cmv5-dependent protection of SLO structures and lymphoid remodeling in spleen during acute MCMV infection.

(A and B) H&E-stained tissue sections obtained from uninfected and HD-infected (d 3.5) spleens of MA/My-related mice (magnification, (A) X100; (B) X200 or X400 with X800 insets). Images of infected spleen are representative of 4 to 8 mice per group in two independent experiments. Diminished SLO structural integrity is evident by comparison of WP and MZ regions, in addition to severe necrosis apparent in RP regions of spleens from the different strains. Pseudo-nodules of leukocytes heterogeneous in size and found near the MZ in MA/My mice frequently contained activated and mitotic figures inside square at low magnification on left, and at higher magnifications on right. Cytomegalic inclusions (black arrow) and plasmacytoid-like cells (white arrow) frequently detected in TgD^k and M.H2^b spleens are indicated (insets in right panels). (C-E) Frozen spleen sections stained with fluorescence-conjugated mAbs against NKp46⁺ NK cells and MAdCAM⁺ marginal sinus lining cells (magnification X200 with X600 insets) (C), MAdCAM⁺ or CD169⁺ MZ macrophages (magnification X100) (D), and both SIGN-R1⁺ MZ macrophages and B220⁺ B cells or CD3⁺ T cells and B220⁺ cells (magnification X100) (E, see text for interpretations).