STAT1 and STAT4 levels in proliferating CD8 T cells responding to LCMV infection. Cells were prepared from uninfected mice or mice infected with LCMV for the indicated times. The percentages of CD8 T cells were determined with flow cytometry to stain the subsets (A). To identify the proliferating subsets, BrdU was administered in vivo 2 hours before harvest, and CD8 T-cell subsets were examined for expression of STAT1 or STAT4 along with BrdU (B). Numbers given are positive averages ± SEMs, with isotype control staining subtracted, of 3 independent sample analyzed within 1 experiment. Results are representative of 2 or more independent experiments. Arrows mark areas of peak BrdU incorporation. The consequences of LCMV infection for STAT expression in CD8 T cells subsets from WT mice were evaluated by Western blot analysis. Studies were performed with WT mice either uninfected (D0) or infected with LCMV for 8 days (D8). (C) STAT1 and STAT4 expression levels were determined on D0 or D8 by Western blot analysis with the use of total, CD8, and non-CD8 cell preparations. Cell lysates were made and analyzed as indicated in “Western blot analysis.” Samples were tested for STAT1, STAT4, and IFNAR levels. The specificity was confirmed with samples from IFNAR⁻, STAT1⁻, and STAT4⁻ mice as controls. Results are representative of 2 or more independent experiments with 3 animals individually tested. (D) The LCMV-specific CD8 T cells were separated from total and nonspecific CD8 T cells on D8 of infection according to staining with pooled class 1 tetramers presenting the 3 immunodominant LCMV peptides for the B6 mice, NP396-404, GP267-286, and GP33-41 (Tet⁺ or Tet⁻). The total and sorted CD8 T-cell populations were analyzed for STAT1, STAT4, and IFNAR expression as described earlier. Results are representative of 2 or more independent experiments.