A role for NKG2D in NK cell-mediated resistance to poxvirus disease

Abstract

Ectromelia virus (ECTV) is an orthopoxvirus (OPV) that causes mousepox, the murine equivalent of human smallpox. C57BL/6 (B6) mice are naturally resistant to mousepox due to the concerted action of innate and adaptive immune responses. Previous studies have shown that natural killer (NK) cells are a component of innate immunity that is essential for the B6 mice resistance to mousepox. However, the mechanism of NK cell-mediated resistance to OPV disease remains undefined. Here we show that B6 mice resistance to mousepox requires the direct cytolytic function of NK cells, as well as their ability to boost the T cell response. Furthermore, we show that the activating receptor NKG2D is required for optimal NK cell-mediated resistance to disease and lethality. Together, our results have important implication towards the understanding of natural resistance to pathogenic viral infections.

Figure 1. The Rapid Kinetics of the NK Cell Response Controls the Early Dissemination of ECTV to Central Organs by Using IFN-γ- and Perforin-Dependent Mechanisms

Intact or NK cell–depleted (treated with anti-NK1.1 mAb PK136) B6 mice were infected with 3,000 pfu ECTV. (A) 7 d PI, the absolute numbers of live lymphocytes in the spleen were determined by trypan blue exclusion. Data correspond to the average ± SD of pooled spleens of three mice from five independent experiments. (B) 7 d PI, virus titers in spleen and liver were determined by plaque assay. Data correspond to the average ± SD of six individual mice from two independent experiments. (C) B6 mice were infected with ECTV in the footpad. The NK cell response, as determined by the percentage of NK cells expressing intracellular IFN-γ, at the indicated times PI was determined in the indicated organs. Data correspond to pools of three mice and are representative of three similar experiments. (D) Representative flow cytometric analysis of D-LN from mice infected for 2 d with ECTV and from control uninfected mice. Upper panel: Dot plots indicating the proportion of NK cells (NK1.1⁺, CD3ɛ⁻). Lower panel: GzB and IFN-γ production by gated NK cells (NK1.1⁺/CD3ɛ⁻ gate of the upper panels). Data correspond to pools of three mice and are representative of at least five experiments. >98% of cells stained with control Ig were in the lower left quadrant of the dot plots (not shown). (E) B6 mice were infected with ECTV, and NK cell (NK1.1⁺, CD3ɛ⁻) proliferation was determined at different times PI in the indicated organs by using a 3-h BrdU incorporation assay. Data are representative of three independent experiments. (F) Flow cytometric analysis of D-LN from mice infected for 2 d with ECTV and from control uninfected mice. Upper panels: Plots are gated on NK cells (NK1.1⁺, CD3ɛ⁻). Lower panels: Gated on R1, R2, and R3 populations from the upper-right (infected) plot. Data are representative of three independent experiments. (G) Intact B6 mice and B6 mice depleted of NK cells (treated with anti-NK1.1) or T cells (treated with anti-CD4 and anti-CD8) were infected with ECTV and virus titers in spleen and liver were determined on day 3 PI. Data are the average ± SD of six individual mice from two experiments. (H) Wild-type and IFN-γ-deficient B6 mice were infected with ECTV in the footpad, and virus titers were determined 3 d PI. Data are the average ± SD of three individual mice and is representative of two individual experiments. (I) Wild-type and Pf-deficient B6 mice were infected with ECTV in the footpad and virus titers were determined 3 d PI. Data are the average ± SD of three individual mice and are representative of two individual experiments.

Figure 2. Reduced T Cell Responses in the Absence of NK Cells

(A) Intact or NK cell–depleted B6 mice were infected with 3,000 pfu ECTV. T cell proliferation on day 5 PI was determined in the D-LN by using a 3-h BrdU incorporation assay. Upper panel: gated on CD8⁺ T cells; lower panel: gated on CD4⁺ T cells. (B) Intact or NK cell–depleted B6 mice were infected with ECTV. Production of IFN-γ and GzB by CD8⁺ T cells in spleen on day 7 PI was determined. Plots are gated on CD8⁺ T cells. Data are representative of three independent experiments.

Figure 3. NKG2D Uses DAP10 or DAP12 Adapters to Help Resist Mousepox

(A) The indicated mice were infected with 3,000 pfu ECTV and the absolute number of lymphocytes in their spleens was determined on day 7 PI by trypan blue exclusion. An uninfected control is also shown. Data are the average ± SD of three pooled mice per group from at least three individual experiments. (B) Virus titers in spleen of the indicated mice on day 7 PI. Data are the average ± SD of three pooled mice per group from at least three individual experiments.

Figure 4. NK Cells Require NKG2D to Control Early Virus Dissemination and for Optimal Cytotoxicity but Not Activation

(A) B6 mice were infected with 3,000 pfu ECTV. On day 5 PI, mice were pulsed with BrdU for 3 h and their spleens analyzed by flow cytometry. Plots are gated on CD3ɛ⁻ NK1.1⁺ cells. Data correspond to pools of three mice from three individual experiments. (B) Increased viral titers of infected mice with NKG2D blockade in vivo. Intact B6 mice, B6 mice with NKG2D blockade, B6 mice depleted of NK cells, B6 mice depleted of T cells (treated with anti-CD4 + anti-CD8 mAbs), or depleted of T cells and with NKG2D blockade were infected with 3,000 pfu ECTV and the viral titers in spleen were determined on day 3 PI. Data are the average ± SD of three individual mice and are representative of two similar experiments. (C) The indicated mice were infected with 3,000 pfu ECTV, and the NK responses in the D-LN were determined at 2 d PI. Upper panel: Dot plot showing the proportion of NK cells (DX5⁺, CD3e⁻) in the D-LN of infected and control uninfected mice. Lower panel: GzB and IFN-γ production by NK cells. Cells correspond to the DX5⁺, CD3ɛ⁻ gate of the upper panels. Data correspond to pools of three mice and are representative of three similar experiments. (D) NK cells were purified from spleens (5 d PI) of ECTV-infected intact or NKG2D-blocked mice and stained as indicated. Filled histogram: isotype-matched control Ig; gray line: NKG2D-blocked mice; black line: intact mice. (E) NK cells were purified from spleens of intact or NKG2D-blocked mice as indicated and were used as effectors in a 4-h ⁵¹Cr release assay against the indicated targets. Data correspond to pools of three mice and are representative of three experiments. (F) As in (D), but the NK cells were purified from untreated mice and a neutralizing anti-NKG2D mAb was added to the in vitro cytotoxicity assays, as indicated.

Figure 5. ECTV Infection Induces Increased Expression of NKG2D Ligands In Vitro and In Vivo

(A) MEFs were infected with 0.5 pfu ECTV 189898-p7.5-EGFP for 18 h. Cells were analyzed for staining with the indicated reagents after gating for EGFP⁻ cells (uninfected) and EGFP⁺ cells (infected). Data correspond to one typical experiment from three similar experiments. Shaded area, infected cells stained with isotype-matched control Ig or secondary Ab alone; black line, infected cells stained with the indicated reagent; gray line, non-infected cells stained with the indicated reagent. (B) qRT-PCR was performed as described. Data were normalized to the amount of β-actin mRNA. Data are representative of two similar experiments.