A) B6 (n=22) and B6.D2-D6 (n=23) mice were infected with ECTV and survival was monitored. B) Virus titers in spleen and liver 7 dpi (gray columns, B6 mice; white columns, B6.D2-D6 mice). Data correspond to the average ± SD of 3 individual mice.

A) Flow cytometry analysis of white blood cells in B6.D2-D6 mice. Upper panels: Gated on lymphocytes according to forward angle (FSC) and side angle (SSC) light scattering properties. Staining for NKp46 and CD3 is shown. Bottom panels: gated on NKp46⁺ CD3⁻ (NK) cells as indicated in the upper plots. Staining for CD94 and NKG2 is shown. B) Wild type B6, CD69-deficient, and CD94-deficient mice were infected with ECTV and survival was monitored. P value for wild type B6 or CD69-deficient vs. CD94-deficient mice. C) Virus titers in spleen and liver 7 dpi (gray columns, B6 mice; white columns, CD94-deficient mice). Data correspond to the average ± SD of 6 individual mice from two independent experiments.

A) Wild type B6 or [B6.D2-D6 × CD94-deficient] F1 mice were infected with ECTV and survival was monitored. B) CD94 transgenic (Tg⁺) and non-transgenic (Tg⁻) CD94-deficient X [CD94-deficient × B6-CD94Tg] F1 littermate mice and parental CD94-deficient mice were infected with ECTV and survival was monitored. C) Transgenic (Tg⁺) and non-transgenic (Tg⁻) B6.D2-D6 × [B6.D2-D6 × B6-CD94Tg] F1 littermate mice and parental B6.D2-D6 mice were infected with ECTV and survival was monitored. D) Flow cytometric analysis of white blood cells from the indicated mice. Plots are gated on NKp46⁺ CD3⁻ NK cells. Staining for CD94 and NKG2 is shown. Data are representative of three similar experiments. E) B6 mice were treated with 150 g 20D5HCmIgG1-Q mAb or an isotype-matched control mAb, infected with ECTV and survival monitored. Data are cumulative from three independent experiments.

A) B6 mice (gray columns) and B6.D2-D6 mice (white columns) were infected with ECTV and virus titers in spleen and liver were determined 3 dpi. Data correspond to the average ± SD of 5 individual mice and are representative of two similar experiments. B) Wild type B6 mice or CD94-deficient mice were depleted or not of NK cells with 15 l anti-Asialo GM antibody and infected with ECTV. Virus titers in spleen (gray bars) and liver (white bars) were determined 3 dpi. Data correspond to the average ± SD of 5 individual mice from two independent experiments. C) Wild type B6 mice treated with blocking 20D5HCmIgG1-Q mAb (gray columns) or an isotype-matched control mAb (white columns) were infected with ECTV and virus titers determined 3 dpi. Data correspond to the average ± SD of 6 individual mice from two independent experiments. D) Wild type and CD94-deficient B6 mice were infected with ECTV. NK cell proliferation was determined 5 dpi in the spleen by BrdU incorporation. Bar graph is the cumulative data for three similar experiments with three mice/group. Representative flow cytometry plots of gated DX5⁺ CD3⁻ NK cells are shown on the right. E) Wild type B6 and CD94-deficient mice were infected with ECTV or not and the draining popliteal lymph node was analyzed by flow cytometry 2 dpi. Shown are the cumulative data for 5 independent experiments as a column graph and representative plots indicating the proportion of NK cells (DX5⁺, CD3⁻) gated on the lymphocytes as determined by FSC and SSC. F) Column graphs show the cumulative data for GzB- and IFN-γ-positive cells in the DX5⁺ CD3⁻ NK cell gate (from D) for 5 independent experiments. Representative flow cytometry plots as used for the columns graphs are shown on the right. G) B6 Wild type or Qa-1b-deficient B6 mice depleted or not of NK cells with anti-NK1.1 mAb treatment were infected with ECTV and the virus titers in the liver and spleen were determined 3 dpi. Data correspond to the mean ± SD of 5 mice (bars as indicated).

A) Wild type B6 mice were infected with ECTV in the footpad or not and the expression of NKG2A and NKG2 in the draining popliteal lymph node was determined. Flow cytometry plots correspond to pools of LNs from three mice and are representative of two similar experiments. Data show cells gated on NK1.1⁺ CD3⁻ NK cells. B) As in A but showing histograms to highlight changes in mean fluorescence intensities (shaded histogram, uninfected mice, black line, infected mice). The numbers indicate the mean fluorescence intensity of the closest histogram. C) LN cells were magnetically sorted into DX5⁺ (NK cells, 40% pure) and DX5⁻ (non-NK, >99% pure). RNA was isolated, reverse transcribed, and the NKG2E message was quantified by qPCR using a specific primers/probe set. Data correspond to the mean ± SD of two wells and are representative of two similar experiments. D) RNA was obtained from the LNs of the indicated mice and analyzed by qPCR as in C. Data correspond to the mean ± SD of two wells and are representative of two similar experiments. E) L cells were infected for 4 hr with 1 PFU/cell of sucrose-purified ECTV virus. Surface expression of Qa-1 was analyzed by flow cytometry. Data are representative from three similar experiments (shaded gray: isotype-matched control Ig; thin line, anti-Qa-1 staining of uninfected L cells; Thick line, anti-Qa-1 staining of infected L cells.

A) L cells were infected with 1 PFU/cell purified ECTV virus for 2 hr, washed, and co-incubated with the indicated reporters for 18 hr at 37°C. The expression of GFP by the reporters was then analyzed by flow cytometry, gating on the reporter cells as distinguished by forward angle light scatter and side angle light scatter properties. Top histograms are representative data (shaded gray, reporter cells with uninfected L cells; dotted thick line: reporter cells with ECTV-infected L cells). The bottom panel displays the results for 2-5 individual experiments where the y-axis is the ratio of the geometric mean of the GFP fluorescence for the indicated reporter cells incubated with infected or uninfected L cells. B) L cells were treated as indicated or kept uninfected, incubated with CD94⁺NKG2E⁺NKG2D⁺ reporters for 16 hr, and the reporters' GFP fluorescence was determined by flow cytometry. Data represent the ratio of the geometric mean of the reporter cells incubated with L cells treated as indicated / GFP fluorescence of the reporter cells incubated with uninfected L cells. Data are representative of three similar experiments. C) 293T cells were treated as indicated or kept uninfected, incubated with CD94⁺NKG2E⁺NKG2D⁺ reporters for 16 hr, and the reporters' GFP fluorescence was determined by flow cytometry. Data represent the ratio of the geometric mean of the reporter cells incubated with 293T cells treated as indicated / GFP fluorescence of the reporter cells incubated with uninfected L cells. Data are representative of three similar experiments. D) Splenocytes from wild type B6 mice were incubated for 5 hr with the indicated bound mAbs in the presence of 1,000 U IL-2. Cells were analyzed by flow cytometry. Data show the proportion of NK cells (NK1.1⁺ CD3⁻) that expressed intracellular IFN-γ. Data correspond to the mean ± SD of three replicate samples and are representative of two similar experiments.