Severely reduced T cell response to WT ECTV in aged mice. (A) Young (Y) and aged (A) B6 mice were infected with 3,000 PFU WT ECTV. 5 dpi they were inoculated i.p. with 2 mg BrdU and euthanized 3 h later. D-LNs were collected, made into single cell suspensions, and stained for surface CD3 and CD8 and for intracellular BrdU. Column graphs indicate the percentage of T_CD8+ cells in the D-LN that incorporated BrdU. Representative flow cytometry plots are shown together with those from uninfected controls (U) for comparison. Data are representative of three independent experiments. (B) Young and aged B6 mice were infected with 3,000 PFU WT ECTV and euthanized 7 dpi. Splenocytes were counted and restimulated with infected cells for 6 h with the addition of brefeldin A during the last hour. Next, the cells were stained for surface CD3 and CD8 and for intracellular IFN-γ and GzB, followed by flow cytometry. Column graphs indicate the calculated absolute number of T_CD8+ cells in the spleen that expressed GzB (left) or IFN-γ (right). Representative flow cytometry plots are shown together with a plot corresponding to an uninfected control mouse for comparison. (C) As in B but showing the calculated absolute numbers and representative plots of CD8⁺K^b- TSYKFESV⁺ cells in the spleen. Data correspond to the mean ± SD of pooled organs of two to three mice per group from five individual experiments.