Fig. 5. Reduced FOXO1 reexpression in old individuals favors differentiation into granzyme B–expressing effector cells and promotes secretion of B cell cytotoxic exosomes.

(A and B) Naïve CD4⁺ T cells from young healthy individuals were activated with anti-CD3/anti-CD28 beads for 3 days followed by 2-day culture with vehicle or 50 nM FOXO1 inhibitor (AS1842856). Representative histograms (left) of intracellular granzyme B after restimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (A). The shaded histogram represents isotype control. Data (right) from six young healthy individuals (two-tailed paired t test). Expression of GZMB, PRF1, PRDM1, and RUNX3 determined by RT-PCR (B). Results are presented relative to vehicle-treated cells (two-tailed paired t test). (C) Naïve CD4⁺ T cells were activated with anti-CD3/anti-CD28 beads for 5 days. Representative histograms (left) of intracellular granzyme B after restimulation with PMA and ionomycin and cumulative data (right) in cells from seven 20- to 35-year-old and seven 65- to 85-year-old healthy individuals (two-tailed unpaired t test). (D and E) Unstimulated naïve CD4⁺ T cells from six 65- to 85-year-old healthy individuals were transfected with a TFEB expression vector or a control vector and then stimulated with anti-CD3/CD28 beads for 5 days. Representative histograms (left) of and cumulative data (right) of intracellular granzyme B after restimulation with PMA and ionomycin (two-tailed paired t test) (D). The shaded histogram represents isotype control. Gene expression was measured by RT-PCR (E). Results are expressed relative to control vector–transfected cells (n = 6; two-tailed paired t test). **P < 0.01, ***P < 0.001, ****P < 0.0001. (F) Naïve CD4⁺ T cells from young individuals were cultured for the past 24 hours of the 5-day culture in exosome-depleted medium with vehicle or 50 nM FOXO1 inhibitor (AS1842856). Exosomes were isolated and assessed for granzyme B by immunoblotting. Western blots representative of three experiments. (G and H) Freshly isolated peripheral blood B cells were treated with exosomes isolated as in (F) for 24 hours. Caspase-3 activity in B cells was determined by immunoblotting using an antibody recognizing both uncleaved and cleaved caspase-3. Western blots representative of two experiments (G). Relative frequencies of Annexin V⁺ 7-AAD⁺ B cells (H); n = 3, two-tailed paired t test. (I and J) Exosomes from young and old naïve CD4⁺ T cells were isolated as in (F). Exosomal granzyme B was detected by immunoblotting. Representative Western blots of cells from three young and three old individuals (I) and frequencies of apoptotic B cells induced by exosomes isolated from four young and four old individuals (J); two-tailed unpaired t test, **P < 0.01.