Fig. 2

Blocking ATM-associated DNA damage response prevents human Treg-induced T-cell senescence. a, b Pretreatment of naive CD4⁺ (in a) and CD8⁺ (in b) T cells with ATM inhibitor KU55933 significantly decreased senescent T-cell populations in responder T cells induced by nTreg cells. Anti-CD3 activated T cells were pretreated with or without KU55933 (10 μM) for 1 day, and then co-cultured with human nTreg cells for 3 days. SA-β-Gal expression in naive CD4⁺ and CD8⁺ T cells was determined with SA-β-Gal staining. The SA-β-Gal positive T cells were identified with dark blue granules as indicated by the arrows. Scale bar: 20 μm. Data shown in the right panels are mean ± SD from three independent experiments, and paired t test was performed. **p < 0.01, compared with the control CD4⁺CD25⁻ and KU55933 treatment groups. ^#p < 0.01, compared with the control CD4⁺CD25⁻ T cell treatment group. c, d Pretreatment of responder T cells with ATM inhibitor KU55933 markedly restored CD27 and CD28 expression in Treg-treated naive CD4⁺ (in c) and CD8⁺ (in d) T cells. CD27 and CD28 expression in treated naive CD4⁺ T cells as described in a and b were analyzed by the flow cytometry. e, f DNA damage response molecules and cell cycle regulatory molecules are co-expressed in human Treg-induced senescent CD4⁺ T cells as described in Fig. 1. Immunofluoresence double staining with antibodies against p-ATM, p-H2AX, p-53BP1, or p-CHK2 with anti-P16, P53, or P21antibodies in the same slide from responder T cells treated with Treg or control CD4⁺CD25⁻ effector T cells. Scale bar: 20 μm