Deficient NK cell response to WT and attenuated ECTV in aged mice. (A) Young (Y) and aged (A) mice were infected with 3,000 PFU WT ECTV or left uninfected (U) and euthanized 2 dpi. D-LNs were made into single cell suspensions, incubated with brefeldin A for 1–2 h, and stained for surface NK1.1 and CD3 and for intracellular IFN-γ and GzB. Column graph indicates mean ± SD for the proportion of NK cells identified as NK1.1⁺ and CD3⁻. Representative flow cytometry plots are also shown. (B) The samples in A were gated on NK cells and analyzed for expression of IFN-γ and GzB. Data correspond to the means ± SD for the proportion of NK cells expressing IFN-γ or GzB. Representative flow cytometry plots are also shown. Data are representative of at least five independent experiments. (C) Young and aged mice were infected with 3,000 PFU WT ECTV and euthanized 3 dpi. Virus titers in the indicated organs were determined by plaque assay. Gray bars, young mice; white bars, aged mice. Data correspond to the mean ± SD of six individual mice from two individual experiments. (D) As in A, but the mice were infected with ECTV Δ166. (E) As in B, except for the mice infected with ECTV Δ166 as in D. Data are representative of five independent experiments.