STAT1 and STAT4 responsiveness to type 1 IFN in CD8 T cells during infection. WT mice were uninfected (D0) or infected with LCMV for 8 days (D8). CD8 T cells were purified for Western blot analysis or identified in mixed populations by flow cytometric analyses. Cells were examined after control or IFNα treatments for 90 minutes ex vivo (A) or in vivo (B). The results of ex vivo treatments are shown in panel A. Western blot analyses of pSTAT1, pSTAT4, IFNAR, or β-actin proteins extracted from purified CD8 T cells are presented. Flow cytometric analyses of pSTAT1 or pSTAT4 in CD8 T-cell subsets of mixed populations are to the right. Solid dark lines show staining of IFNα-treated cells. Gray areas show staining of untreated cells, and dotted lines show staining with isotype controls for cytokine-treated samples. (B) The results using purified CD8 T cells isolated from mice treated with either control or IFNα for Western blot analysis of pSTAT1, pSTAT4, IFNAR, and β-actin proteins are shown. Results are representative of 2 or more independent experiments, with percentages in flow panels showing means ± SEMs of 3 independent samples analyzed within 1 experiment. To analyze STAT1 and STAT4 levels in CD8 T cells responding to LCMV infection, cells were prepared from uninfected mice or mice infected with LCMV for the indicated times. Total spleen yields were measured, and the percentages and numbers of CD8 T cells was determined with flow cytometry (C). (D) Cytoplasmic staining of total STAT1 was evaluated in total cells and in CD8 T-cell subsets. (E) Cytoplasmic staining of total STAT4 was determined in total cells and in CD8 T-cell subsets. Numbers given are positive averages, with isotype control staining subtracted, and SEMs. Arrows identify peak STAT intensities in CD8 T-cell subsets (D-E). Results are representative of 2 or more independent experiments, with percentages in flow panels that show means ± SEMs of 3 independent samples analyzed within 1 experiment.