DUSP4 inhibition ameliorates TCR signaling in senescent CD4⁺ T cells. (A and B) After 2 rounds of stimulation with anti-CD3/anti-CD28 Abs, activated CD4⁺ T cells from 3 independent healthy individuals were nucleoporated with 1.5 μg SCR or DUSP4 siRNAs. Twelve hours after transfection, T cells were stimulated on plates coated with anti-CD3/anti-CD28 Abs for 2 days. DUSP4 and DUSP6 transcript or protein levels were then evaluated by quantitative PCR (left and middle) or immunoblot (right). The ratios of DUSP4 over α-tubulin proteins are indicated below the gels. pERK content was determined by PhosphoFlow in siRNA-transfected T cells restimulated by CD3 cross-linking (B). Representative plots of the MFI of pERK in transfected CD4⁺ T cells are shown (left). Results show the mean pERK content in transfected CD4⁺ T cells ± SD (right). (C-E) CD4⁺ T cells from 3 independent elderly or ICL subjects were nucleoporated with 1.5 μg SCR or DUSP4 siRNAs and then stimulated for 2 days, as described earlier. siRNA-transfected cells were recovered and tested for DUSP4 and DUSP6 expression (C) and for their pERK content after CD3 cross-linking (D). On day 3 after stimulation, the frequencies of CD27-expressing cells CD4⁺ T cells and levels of CD40L expression were determined by flow cytometry and shown as the percentage increase after DUSP4 silencing (E). Results represent the mean ± SD or are representative of 3 independent experiments. *P < 0.05 and **P < 0.005 compared with SCR siRNA-transfected CD4⁺ T cells (as determined using the Mann-Whitney U test).