FOXO3 Regulates CD8 T Cell Memory by T Cell-Intrinsic Mechanisms
Figure 2
Absence of FOXO3 does not affect the phenotype or function of effector CD8 T cells.
(A) At 8 days after LCMV infection, splenocytes from +/+ and FOXO3−/− mice were isolated and stained for expression of CD44, CD62L, CD27 and CD122 on NP396- (top) and GP33- (bottom) specific CD8 T cells. Representative histograms in panel A are gated on CD8 and MHC-I tetramer positive populations with the numbers indicating MFI for the indicated protein in either +/+ or FOXO3−/− mice. (B) Total splenocytes were stained with MHC-I tetramer, anti-CD8, anti-CD127 and anti-KLRG-1, and the total number of SLECs (KLRG-1high/CD127low) and MPECs (CD127high/KLRG-1low) were quantified by flow cytometry. Data are from 4 to 6 independent experiments with 3–6 mice/group/experiment; error bars represent the SEM and * indicates statistical significance at p<.05. (C) On day 8 PI, Splenocytes from +/+ or FOXO3−/− mice were stimulated with LCMV epitope peptides for 5 hours directly ex-vivo. Following stimulation, cells were stained for cell surface CD8 and intracellular IFNγ, IL-2 and TNFα. Panel C shows cytokine production by effector CD8 T cells. Representative dot plots (left) are gated on total lymphocytes with the top number indicating observed MFI for IFNγ staining in peptide-stimulated CD8 T cells and the bottom number indicating percentage of total splenocytes that are CD8 and IFNγ positive. Dot plots (right) represent the percentage of IL-2 and/or TNFα producing cells among IFNγ+ve CD8 T cells. (D) Intracellular staining for Granzyme B. The FACS histograms are gated on LCMV-specific CD8 T cells from +/+ (BLUE) and FOXO3−/− (RED) mice. The green histogram represents staining with isotype control antibodies. The data are MFIs for Granzyme B expression +/− SD.