Cytomegalovirus immunoevasin reveals the physiological role of "missing self" recognition in natural killer cell dependent virus control in vivo

Abstract

Cytomegaloviruses (CMVs) are renowned for interfering with the immune system of their hosts. To sidestep antigen presentation and destruction by CD8(+) T cells, these viruses reduce expression of major histocompatibility complex class I (MHC I) molecules. However, this process sensitizes the virus-infected cells to natural killer (NK) cell-mediated killing via the "missing self" axis. Mouse cytomegalovirus (MCMV) uses m152 and m06 encoded proteins to inhibit surface expression of MHC I molecules. In addition, it encodes another protein, m04, which forms complexes with MHC I and escorts them to the cell surface. This mechanism is believed to prevent NK cell activation and killing by restoring the "self" signature and allowing the engagement of inhibitory Ly49 receptors on NK cells. Here we show that MCMV lacking m04 was attenuated in an NK cell- and MHC I-dependent manner. NK cell-mediated control of the infection was dependent on the presence of NK cell subsets expressing different inhibitory Ly49 receptors. In addition to providing evidence for immunoevasion strategies used by CMVs to avoid NK cell control via the missing-self pathway, our study is the first to demonstrate that missing self-dependent NK cell activation is biologically relevant in the protection against viral infection in vivo.

Figure 1.

Δm04 is attenuated in vivo in an NK cell– and MHC I–dependent manner. (A–D) Indicated strains of mice were injected i.v. with 2 × 10⁵ PFU (except C57BL/6, which received a dose of 3 × 10⁵ PFU) of WT or Δm04 MCMV and 3 dpi viral titers in spleens were assessed. Where indicated, mice were depleted of NK cells by injection of 20 µl of anti-AGM1. P values, unpaired two-tailed Mann-Whitney test. A circle depicts the titer for each individual mouse; a small horizontal line indicates the mean. Data are representative of at least two independent experiments with four to five mice per group.

Figure 2.

m04 protects cells from NK cell–mediated killing. (A) Target B12 and SVEC4-10 cells were CFSE-labeled, infected with WT or Δm04 MCMV for 10 h, or were left uninfected, and co-cultured with splenocytes from naive BALB/c or C3H/J mice, respectively. Specific lysis of target cells was assessed by staining with 7-AAD and analyzed by flow cytometry. Data points on the graph represent the mean of a triplicate for each sample. Error bars represent SD. Data are representative of five (SVEC4-10) or three (B12) independent experiments. Statistical significance applies for the effector-to-target ratio of 16:1 and is indicated by asterisks. *, P < 0.05; **, P < 0.001; ***, P < 0.0001. (B) B12 and SVEC4-10 cells were infected with WT or Δm04 MCMV or left uninfected. Cells were harvested 10 hpi and stained for surface MHC I and NKG2D ligand expression. Data are representative of two independent experiments.

Figure 3.

m04 allows engagement of inhibitory Ly49 receptor. (A) Ly49A^CD3ζ or Ly49P^CD3ζ NFAT-GFP reporter cells were co-cultured with CBA/J, BALB/c, or C57BL/6 MEFs and either infected with indicated viruses or left uninfected. GFP expression was assessed (in the case of infected cells, overlay was made with uninfected cells presented by open histograms). Data are representative of two to four independent experiments. (B) Ly49A^HCTS reporter cells were co-cultured with the indicated MEF cells, left uninfected, or infected with WT or Δm04 MCMV. GFP expression was assessed. Data are representative of two to four independent experiments. (C) Ly49C NFAT-GFP reporter cells were co-cultured with BALB/c MEFs and BALB.F MEFs that were infected with indicated viruses or left uninfected and GFP expression was assessed. P values, unpaired two-tailed Student’s t test. Data are means of four independent experiments. Error bars represent SD.

Figure 4.

m04 antagonizes the function of m152 in the downmodulation of H-2D^k. (A) Ly49A NFAT-GFP reporter cells were co-cultured with CBA/J MEFs that were infected with indicated viruses or left uninfected, and GFP expression was assessed (shaded histograms, infected; open histograms, uninfected). Data are representative of three independent experiments. (B) CBA/J MEFs were infected with indicated viruses or uninfected. Cells were harvested at the indicated time points (hpi, hours post infection) and stained for H-2D^k expression (shaded histograms). Irrelevant isotype matched antibody was used as a control (open histogram). Shaded box represents the time window of cocultivation of target cells with reporter cells (experiment shown in A). Data are representative of three independent experiments.

Figure 5.

NK cells expressing inhibitory Ly49 receptors control Δm04 MCMV in vivo. (A) BALB/c mice were i.v. injected with 2 × 10⁵ PFU of WT or Δm04 MCMV and 3 dpi viral loads in spleen were assessed. Where indicated, mice were depleted of the indicated NK cell subsets by injecting 150 µg of the corresponding antibody. Mice in the control group received equivalent amount of PBS. P values, unpaired two-tailed Mann-Whitney test. A circle depicts the titer for each individual mouse; the horizontal line indicates the mean. Data are representative of two independent experiments with five mice per group. (B) BrdU incorporation by total NK cells in BALB/c and BALB.K mice that were left uninfected or infected with WT or Δm04 MCMV was assessed 2.5 d after infection. P values, unpaired two-tailed Student’s t test. Error bars represent SD. Data are means of three independent experiments with three mice per infection. (C) Proportions of CD3⁻NKp46⁺ cells expressing Ly49A, Ly49C, or Ly49G2 in BALB/c mice that were left uninfected or infected with WT or Δm04 MCMV were determined 2.5 dpi by flow cytometry. Data are means of two independent experiments. Error bars represent SD. (D) BrdU incorporation by CD3⁻NKp46⁺ cells expressing indicated Ly49 proteins in BALB/c and BALB.K mice infected with either WT or Δm04 MCMV was assessed 2.5 dpi. P values, unpaired two-tailed Student’s t test. Error bars represent SD. Data are representative of one (BALB.K) or two (BALB/c) experiments with three mice per infection.

Figure 6.

Control of Δm04 MCMV is partially mediated via NKG2D. The indicated strains of mice were i.v. injected with 2 × 10⁵ PFU of WT or Δm04 MCMV and 3 dpi viral loads in spleen were assessed. Mice were either untreated or NKG2D blockade was performed 6 h before infection by applying 300 µg of anti-NKG2D mAb. P values, unpaired two-tailed Mann-Whitney test. A circle depicts the titer for each individual mouse; the horizontal line indicates the mean. Data are representative of two independent experiments with five mice per group.