Fig 2: Failure in FOXO1 reexpression promotes MVB expansion and exosome secretion in old naïve CD4+ T cells.(A to E) Naïve CD4+ T cells were activated, silenced for FOXO1 expression, or treated with FOXO inhibitor as described in Fig. 1C. (A) LAMP1 protein expression was determined by Western blotting. Representative Western blots (left) and results relative to those of cells from control-silenced or vehicle-treated samples (right) (n = 5, mean, two-tailed paired t test). (B) Western blot results for CD63, expressed relative to vehicle-treated cells (n = 5, two-tailed paired t test). (C) Flow cytometry of LysoTracker staining. MFI from five experiments (two-tailed paired t test). (D) Cells were stained with anti-LAMP1 (green) and anti-CD63 (red). Confocal images representative of three independent experiments show accumulation of LAMP1 and CD63 in FOXO1-inhibited naïve CD4+ T cells. DAPI, 4',6-diamidino-2-phenylindole. (E) Flow cytometry of cell surface CD63 expression. Representative histograms (left) and results from five experiments (right; two-tailed paired t test). The shaded histogram represents isotype control. (F) Naïve CD4+ T cells were cultured for the past 24 hours of the 5-day culture in exosome-depleted medium. Exosomes were isolated and assessed for exosomal markers (CD63, CD9, and ALIX) by immunoblotting. Representative Western blots and results from four experiments (expressed relative to vehicle-treated cells, two-tailed paired t test). (G) TFEB expression was restored in old naïve CD4+ T cells by transfection with a TFEB construct (fig. S5). CD63 cell surface expression in control- and TFEB-transfected activated CD4+ T cells was compared (n = 6, two-tailed paired t test). (H to L) Naïve CD4+ T cells from 20- to 35- and 65- to 85-year-old healthy individuals were stimulated with anti-CD3/anti-CD28 beads for 5 days. (H) LAMP1 and FOXO1 protein expression were determined by Western blotting. Representative Western blots and cumulative data from 11 young and 11 old healthy individuals (two-tailed unpaired t test, left); Pearson correlation plot of FOXO1 and LAMP1 (right). (I) CD63 Western blots from experiments of 16 young and 16 old individuals (two-tailed unpaired t test). (J) Acidic organelles were quantified as in (D). Cumulative data from 13 young and 13 old healthy individuals (two-tailed unpaired t test). (K) Flow cytometry of cell surface CD63. Representative histograms (left) and cumulative data from 15 young and 15 old healthy individuals (right; two-tailed unpaired t test). The shaded histogram represents isotype control. (L) Western blots for CD63 in exosomes isolated as described in (F). Representative Western blots (left) and results from nine young and nine old individuals (right; two-tailed unpaired t test). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: The effect of RSV on SIRT1 activation; FOXO1-SIRT1 interaction; and FOXO1, acetyl-FOXO1, and SIRT1 expression in SAP. Western blot analysis was employed on the expression of SIRT1, FOXO1, and acetyl-FOXO1. (a) Representative results for Western blot analysis of SIRT1, FOXO1, and acetyl-FOXO1 in the pancreases. (b) Semiquantitative analysis of 10 animals studied in each group. The relative amounts of SIRT1, FOXO1, and acetyl-FOXO1 in each group of rats were normalized by ß-actin (a). Bars represent the means ± SE (n = 10). ***P < 0.05 (SAP vs. sham); #P < 0.05 (RSV vs. SAP); ^P < 0.05 (EX vs. RSV). (c) Immunoprecipitation was employed in the interaction of SIRT1 and FOXO1. (d) The activity of SIRT1.

Fig 4: Age-associated failure in FOXO1 reexpression impairs lysosomal function in naïve CD4+ T cell responses.(A) Naïve CD4+ T cells were activated with anti-CD3/anti-CD28 beads. FOXO1 protein expression was determined by Western blotting. Representative Western blots of cells from one young (Y) and one old (O) adult and summary data from 13 young (20 to 35 years old) and 13 old (65 to 85 years old) healthy individuals. Intensities of FOXO1 protein expression were normalized to β-actin and are shown relative to the mean of unstimulated naïve CD4+ T cells from young individuals. The horizontal lines represent mean values; comparison by two-tailed unpaired t test. NS: not significant. (B) GSEA comparing fold transcript differences in young compared with old naïve CD4+ T cells on day 5 after stimulation (accession number: SRA: SRP158502) (19) with that of mouse Foxo1 knockout (KO) unstimulated T cells (46). NES, normalized enrichment score; FDR, false discovery rate. (C) Naïve CD4+ T cells from a young healthy adult were activated with anti-CD3/anti-CD28 beads, transfected with FOXO1 or control siRNA on day 2, and then cultured on plates coated with anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) antibodies. Alternatively, naïve CD4+ T cells were activated with anti-CD3/anti-CD28 beads; vehicle or 50 nM FOXO1 inhibitor (AS1842856) was added on day 3. TFEB mRNA was quantified by reverse transcription polymerase chain reaction (RT-PCR) on day 5 of culture. Results are normalized to control-silenced or vehicle-treated cells; mean of five experiments; two-tailed paired t test. (D) TFEB protein expression in cells treated as described in (C) was determined by Western blotting. Representative blots (left) and relative intensities, normalized to control samples (right), are shown; mean of five experiments, two-tailed paired t test. (E) Naïve CD4+ T cells were activated as described in (C) and transfected with indicated siRNA and plasmid. Lysosomal cathepsin gene expressions were quantified by RT-PCR. Results are normalized to control samples; mean ± SEM of four to six experiments; comparison by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. (F) TFEB and cathepsin transcripts from day 5–activated naïve CD4+ T cells from thirteen 20- to 35-year-old and thirteen 65- to 85-year-old healthy adults. Results are expressed relative to the mean of cells from young individuals. Horizontal lines represent mean values; comparison by two-tailed unpaired t test. (G) Transcriptome data from naïve CD4+ T cells from three young and three old healthy individuals stimulated with anti-CD3 and anti-CD28 beads for 5 days (accession number: SRA: SRP158502) (19). Violin plots and boxplots showing the fold gene expression differences of lysosomal genes, comparing the transcriptome of old to young T cells. Lysosomal gene sets were drawn from published experiments in human-derived cell lines (14, 18). Statistical analysis by Wilcoxon rank sum tests. (H and I) Naïve CD4+ T cells from a young healthy individual were stimulated and treated with FOXO1 inhibitor as in (C). Cells were then treated with DQ-BSA (5 μg/ml) for 5 hours. Fluorescence of cleaved DQ-BSA was analyzed by flow cytometry. (H) Representative histograms (left) and results of paired samples from seven young healthy individuals (right); two-tailed paired t test. (I) Representative histograms (left) and cumulative data from 12 young and 12 old healthy individuals; two-tailed unpaired t test. The gray histogram represents untreated naïve CD4+ T cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MFI: mean fluorescence intensity.

Fig 5: Disturbed signaling downstream of PI3K in CVID patients’ B cells. (A) Gating strategy for imaging and multicolor flow cytometry for nuclear FOXO1. Anti-?-stimulated naïve B cells were stained for ?, IgM, CD21 and CD38 to identify IgMpos, ?pos and ?neg CD21pos and CD21low B cells. Gating on ?pos B cells determined unstimulated populations, ?neg cells represented ?-stimulated populations. (B) Representative images of ?posCD21pos B cells of HD and ?posCD21low B cells of a CVID patient. (C) Representative histograms of the similarity score of FOXO1 and 7AAD in ?pos and ?neg CD21pos B cells of HD, CD21pos and CD21low B cells in CVID patients and statistics of the percentage of cells with nuclear FOXO1. (D) Expression of HIF1a in populations as indicated unstimulated and after stimulation with anti-IgM for 40 h. Data are representative for one of four independent experiments. (E) Numbers of living AnnexinVnegDAPIneg CD21low and CD21pos B cells after incubation of sorted cells for 48 h (n = 3–4). (F) Proportion of KI67pos, CD21low and CD21pos B cells after stimulation (n = 3). (G) Significantly altered hallmark pathways in CD21low or CD21pos B cells of CVID compared to CD21pos B cells of HD as determined by RNAseq of B-cell subsets ex vivo. * p < 0.05, ** p < 0.01.