Fig. 5. Enhanced sMAC formation with age.

(a) Immunoblots (left) and data (right) of endogenous sestrin proteins in lysates from young (20-35 years) versus old (70-85 years) primary human CD4⁺ T cells. Age-dependent expression of sestrins was normalized to endogenous GAPDH and presented relative to that of young donors, set as 1. (b) Flow-cytometry assessing sestrin2⁺ cells among CD4⁺ T_erl and CD4⁺ T_sen subsets from 8 young and 8 old individuals. (c) Age-dependent sMAC formation by Image Stream in cells as in (b); representative of several images from 4 different individuals per each age-group. (d) Sestrin2⁺-p-MAPKs⁺ co-localization scores (BDS) of cells as in (c) (n = 4). (e) VZV-specific proliferation in CD4⁺ T_erl and CD4⁺ T_sen cells from 3 young and 3 old donors. Pooled data (n = 3) presented relative to those of CD4⁺ T_erl from young donors, set as 1. (f) Restored VZV-specific sensitivity (proliferation) in CD4⁺ T_sen from old humans where one or two Sestrins were silenced by shRNA (n = 3), presented relative to that of cells transduced with shCtrl and stimulated with the lowest VZV antigen dilution (1:100), set as 1. (g) Restored IL2 synthesis in sestrin-silenced CD4⁺ T_sen reactivated with autologous APCs loaded with a VZV antigen dilution of 1:100 and presented as in (f) (n = 3). Data are pooled from three experiments with three separate donors (a, e, f, g) or four experiments with eight (b) or four (c, d) donors. *p<0.05 **p<0.01 and ***p< 0.001, ANOVA for repeated measures with Bonferroni post-test correction. Error bars indicate s.e.m. throughout.