Phenotypic and Functional Analysis of CD4 T Cells

For phenotypic analysis of CD4 T cells, PBMCs were stained with CD4-FITC, CD8-PE, CD57-APC (BioLegend, San Diego, CA), CD28-PE (Invitrogen, Carlsbad, CA), CD45RA-PerCP710, PD1-FITC (eBioscience, San Diego, CA), or isotype control antibodies. To quantify cell apoptosis, PBMCs were stained with CD4-A647 and CD45RA-FITC for naïve or memory cell populations, and then stained with Annexin V-PE and 7-AAD using BD Pharmingen™ PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA). CD4⁺ T cells were also stained for caspase-3 expression following a protocol from CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit (Invitrogen). Levels of reactive oxygen species (ROS) in CD4 T cells were measured using the DCFDA-based Cellular ROS Detection Kit (Abcam, Cambridge, MA) or CellROX Green ROS Detection kit (ThermoFisher Scientific, Waltham, MA) according to manufacturer's protocol. For intracellular staining, the cells were fixed and permeabilized with Foxp3 Transcription Factor Staining Buffer Set (eBioscience), and stained with pATM (Ser1981)-PE antibody (BioLegend), pCHK2 (Thr68)-PE antibody, γH₂AX-PE (eBioscience), IL-2-PE, and IFN-γ-PE (Invitrogen). Flow cytometry was carried out as described previously (19 (link), 20 (link)).