Fig 4.
Deletion of FOXO3 does not affect effector function during the early stages of chronic LCMV infection. (A) On day 8 p.i., splenocytes from WT and FOXO3L mice were stimulated with LCMV epitope peptides for 5 h directly ex vivo. Following stimulation, cells were stained for cell surface CD8 and intracellular cytokines (IFN-γ, IL-2, and TNF-α; P < .02 for GP33; P < .03 for GP276).(Left) Representative dot plots gated on total splenocytes show the percentage of splenocytes (top number) or of CD8 T cells (bottom number) positive for IFN-γ at 8 days p.i. (Right) Bar graphs show the total number of IFN-γ-producing LCMV-specific CD8 T cells (P < .02 for GP33; P < .04 for GP276) or the percentages of IFN-γ-producing cells that also produced TNF-α and IL-2 (triple cytokine producers). Data are from at least 4 independent experiments with 3 to 7 mice/group/experiment. (B) Splenocytes were stained with MHC-I tetramers and anti-CD8, followed by intracellular staining for granzyme B. Representative FACS histograms for granzyme B MFIs are gated from GP33 (top)- or GP276 (bottom)-specific CD8 T cells. The observed MFI for granzyme B staining on epitope-specific CD8 T cells is given in each case. (C) Splenocytes from WT and FOXO3L mice were stimulated with the LCMV-specific CD4 T-cell-specific peptide GP66. Following stimulation, cells were stained for cell surface CD4 (anti-CD4) and intracellular IFN-γ (anti-IFN-γ). The bar graph indicates the total numbers of IFN-γ-positive CD4 T cells. Data are from at least 3 independent experiments with 3 to 6 mice/group/experiment. (D) Titers of LCMV clone 13 from the sera, livers, and lungs of WT and FOXO3L mice were assessed by a plaque assay utilizing a Vero cell monolayer.