Fig. 1: Foxo1 is gradually down-regulated in T cells with age.
a Cell suspensions from the spleen of a 3-month-old mouse (Young adult) and a 22-month-old mouse (Old) were first stained separately with anti-CD45 antibodies conjugated to different fluorochromes and then mixed before further staining. Foxo1 and Foxo3 fluorescence histograms of the indicated T-cell subsets are shown for representative mice (left panel). Relative Mean Fluorescence Intensities (MFIs) were calculated by dividing the MFI of a given T-cell subset of the old mouse by the MFI of the same T-cell subset of the barcoded young adult mouse (right panel). b A cohort of female mice was bled at different ages and blood cells were mixed with cells from 3-month-old female mice before revealing Foxo1 and Foxo3 expression. Of note, before mixing them, blood cells were barcoded with anti-CD45 antibodies conjugated to different fluorochromes. Relative MFIs are shown for the indicated T-cell subsets. c phospho-Foxo (pFoxo) and phospho-AKT (pAKT) fluorescence histograms of the indicated T-cell subsets are shown for representative mice (left panel). Relative MFIs were calculated by dividing the MFI of a given T-cell subset of the old mouse by the MFI of the same T-cell subset of the barcoded young adult mouse (right panel). d Relative expression of pFoxo by the indicated T-cell subsets from the spleen of old mice as a function of pAKT relative expression. e Volcano plot representation of the transcriptomic signature of CD4N cells from aged versus young adult mice. f The relative expression of Foxo1 and Foxo3 mRNAs was directly derived from the obtained signature and was plotted as Mean ± SEM (n = 3 Young adult mice; n = 4 old mice). g, h A list of genes up- and down-regulated in CD4N cells from Foxo1TKO mice compared with CD4N cells from Foxo1Ctrl mice was also determined (p value of < 0.05 and fold change > 1.5 or <−1.5, see Supplementary Data 1). The expression of these genes by CD4 TN cells as a function of mouse age was visualized using the Volcano (g) or GSEA (h) plot. Quantifications are represented as Means ± SEM. The significance of differences between two series of results was assessed using Student’s unpaired t test (a–c, f). For assessing correlations, Pearson correlation (two-sided test, coefficient, and 95% confidence intervals) was used (d). The p-values for the GSEA test statistics are calculated by permutation (h). Significant (p < 0.05) or almost significant (0.05 < p < 0.10) p-values are indicated. Source data are provided as a Source Data file.
