T Cell Senescence Assay with Treg Co-culture

Senescence-associated β-galactosidase (SA-β-Gal) activity in senescent T cells was detected as we previously described^{13 (link),14 (link)}. Naive CD4⁺ or CD8⁺ T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) (4.5 μM), and co-cultured with Treg or control T cells at a ratio of 4:1 in anti-CD3-coated 24-well plates for 3 or 5 days. Co-cultured naive T cells were then separated from co-cultures using fluorescence-activated cell sorting (FACS) gated on CFSE-positive populations, and then stained with SA-β-Gal staining reagent. For some experiments, the co-cultured naive T cells were determined for SA-β-Gal expression in the presence of the following inhibitors: ATM inhibitor KU55933 (10 μM, Tocris Bioscience); STAT1 inhibitor MTA (5 μM), STAT3 inhibitor S3I201 (10 μM), and JAK2 inhibitor AG490 (30 μM) (Sigma-Aldrich); MAPK inhibitors including U0126 (10 μM), SB203580 (10 μM) and SP600125 (10 μM) (Calbiochemistry). For blockade of glucose transport and glycolysis analyses, the Treg cells were pretreated with glucose transporter and glycolysis inhibitors phloretin (2 μM) or 2-deoxy-d-glucose (2-DG, 1 mM) (Cayman Chemical) for 24 h and then co-cultured with naive T cells in the presence of low concentrations of phloretin (0.5 μM) or 2-DG (300 μM) for 3 days.