Figure 6. p38 activation by a specific AMPK agonist reproduces senescent features in non-senescent T cells.

(a) Telomerase activity by TRAP assay in CD27⁺ CD28⁺ CD4⁺ T cells activated by αCD3/CD28 and cultured as indicated for 3 days. AMPK was activated by A-769662 (150 μM); p38 was inhibited by BIRB 796 (500 nM). A DMSO vehicle solution was used as control. Experiments are from 3 different donors. (b) Replicative lifespan of long-term cultured CD27⁺ CD28⁺ CD4⁺ T cells assessed by cumulative PD from 3 separate donors. Cells were activated and cultured as described in (a). (c) Immunoblots showing the dose-dependent effect of the AMPK agonist A-769662 (0, 100 or 150 μM) on total Lck, Zap70 and SLP-76 expression, after 3 days, in CD27⁺ CD28⁺ CD4⁺ T cells activated by αCD3/CD28. A DMSO vehicle solution was used as control; detection of AMPKα ¹⁷²) phosphorylation served as pharmacological activation control. Immunoblots are representative of 4 independent experiments. (d) Pooled phospho-flow data from 3 independent experiments showing Lat and SLP-76 expression in CD27⁺ CD28⁺ CD4⁺ T cells cultured as described in (a-b) for 3 days. All *p< 0,05, **p<0.01, and ***p< 0.001 values were calculated using a one-way analysis of variance (ANOVA) for repeated-measures with a Bonferroni post-test correction. Error bars depict s.e.m.