Effective control of MCMV proliferation in BALB mice depends on the H-2 haplotype. (A) BALB.By (H-2^b), BALB/c (H-2^d), and BALB.K (H-2^k) mice were infected with either 2.5 × 10³ PFU/mouse (BALB.By) or 5 × 10³ PFU/mouse (BALB/c and BALB.K) of MCMV. Viral titers were assessed in the spleen at days 2, 4, 6, and 10 p.i. (B) Day-4 viral titers in the spleen and liver of BALB.By and BALB.K mice infected with 2.5 × 10³ PFU/animal are shown. The data are presented as median viral titer ± interquartile range (IQR). The number of animals used at each time point is between 4 and 12 and represents four pooled experiments.

H-2 and m04/gp34-specific proliferation of Ly49L⁺ NK cells in BALB mice during MCMV infection. (A) 2B4 reporter cells expressing Ly49 receptors from the BALB repertoire were stained with a panel of Ly49-specific antibodies as well as with anti-M2 (DAP12) antibody. MFI is shown. (B) Freshly extracted splenocytes from BALB congenic mice were stained for NK cells and different Ly49 receptors. YE1/48 and 12A8 antibody staining is shown. YE1/48 stains a double population of NK cells with either high or intermediate intensity. The intermediate population is not stained by the anti-Ly49A–specific clone (12A8) and represents the fraction of NK cells expressing the activating Ly49L receptor. BALB.By, BALB/c, or BALB.K mice were infected with WT or Δm04 MCMV. Their spleens were collected at different time points p.i. (C and D) The number of NK cells (C) and the percentages of Ly49A-, Ly49C-, Ly49G-, or Ly49L-positive NK cell fractions (D) were determined. Asterisks denote p-values <0.05. (E) Representative dot plots of Ly49A and Ly49L expression on NK cells from BALB.By, BALB/c, or BALB.K mice infected with WT MCMV at day 4 and 6 p.i. Data are from three experiments (mean ± SD) with three to four mice per group. n/a, not available.

Adoptive transfer of naive Ly49L⁺ NK cells into BALB.K neonates improves their survival after MCMV infection. (A) Adoptive transfer scheme. (B) Sorting gate position and purity. (C) Survival of BALB.K neonate mice receiving Ly49L⁺ NK cells (1 × 10⁴, 4 × 10⁴, or 7 × 10⁴), Ly49L⁻ NK cells (7 × 10⁴), total MA/My NK cells (1 × 10⁵), or PBS as a control, followed by MCMV infection at a dose of 1,000 PFU/pup. Data represent four pooled experiments. (D) Viral titers at day 3 p.i. in neonate pups transferred with 1 × 10⁵ of either Ly49L⁺ or Ly49L⁻ NK cells or PBS only (median viral titer ± IQR). Three pups per condition were tested. Data represent one of two experiments performed.

Several activating Ly49 receptors recognize an MCMV-infected cell based on the presence of the m04/gp34 viral peptide within a specific H-2 context. (A) Percentages (mean ± SD) of GFP expression by reporter cells carrying individual Ly49 receptors upon co-culture with MEF cells of the H-2^f (top), H-2^k (middle), or H-2^d (bottom) haplotype under the following conditions: uninfected (∅), IFN-γ pretreated (200 U), or infected at an MOI of 0.5 with WT, Δm04, or Δm04Δm06Δm152 MCMV. (B) GFP expression by reporter cells carrying individual Ly49 receptors upon co-culture with H-2^k MEF cells that are either uninfected (thin line) or WT MCMV infected (gray filled line) in the absence (top) or presence of H-2D^k or H-2K^k blocking antibody (middle) or isotype control (bottom). (C) Sequence alignment of the predicted H-2-binding region of Ly49 NKDs based on the Ly49A:H-2D^d crystal structure. Conserved residues between the Ly49 receptors are shown as dashes. Secondary structure elements are denoted by loops (white rectangles) and β-strands (gray ovals). Downward arrows represent putative MHC class I contact sites, whereas boxed residues delimit the NXT N-glycosylation motif. Residues in red represent amino acids unique in Ly49 receptors recognizing the infection in an m04/gp34–H-2–dependent manner. Sequences were retrieved from GenBank with the following accession nos.: Ly49D^B6, AF349733.1; Ly49D^NOD, AF218078.1; Ly49D1^PWK, AY860975.1; Ly49L2^BALB, AF204266; Ly49M^NOD, AF283252.1; Ly49P^MA/My, AY971807.1; Ly49P1^NOD, AF218080.1; Ly49R^MA/My, AF288377.1; and Ly49W^NOD, AF283250.1. Results from one of at least three experiments are shown.

Effective early and late control of MCMV proliferation in BALB.K mice depends on NK cells, whereas CD8⁺ T cells become involved later. (A and B) BALB.K mice were depleted of NK cells (top), CD8 T cells (bottom), or both (C) before WT MCMV infection. Viral titers were assessed at different time points p.i. in the spleen (A and B, top) and liver (A and B, bottom). The data represent three pooled experiments (median viral titer ± IQR) with three to four mice per experiment.

Preferential IFN-γ secretion of the Ly49L⁺ NK cell subpopulation at day 6 p.i. in BALB.K mice. BALB.By, BALB/c, and BALB.K mice were infected with 2,500 PFU/mouse of MCMV i.p. At 36 h and 6 d p.i., the spleens were collected, negatively enriched for NK cells, and activated with plate-bound α-NKp46 and α-NKG2D antibody followed by CD3, DX5, YE1/48, 12A8, and IFN-γ staining. Percentages of total IFN-γ⁺ NK cells gated on CD3⁻DX5⁺ are shown at 36 h (A) and 6 d (B) p.i. (C) Representative contour plots of 12A8⁻YE1/48^INTIFN-γ⁺ NK cells. Data represent one of two experiments performed (mean ± SD) with three to four mice per group.