Isolation of peripheral blood mononuclear cells (PBMCs)

PBMCs were isolated using the Ficoll-Paque Plus Method (GE Healthcare Bio-sciences AB) using a process that maintains the viability of B and T lymphocytes to account for relative lymphopenia among A-T versus control participants as previously described⁴⁷. Briefly, a 7-mL venous blood sample was transferred into a 50-mL conical tube and PBS was added to achieve a 20-mL volume. 15 mL of Ficoll-Paque Plus was transferred into a separate 50-mL conical tube using a syringe. The 20 mL of diluted blood was gently layered onto the Ficoll. The sample was spun at 400 ×g for 30 minutes at room temperature with no brake. The PBMC layer was then collected at the diluted plasma/Ficoll interface. PBS was added to the PBMCs to bring the volume up to 20 mL. The sample was then spun at 200 ×g for 10 minutes at room temperature. The supernatant was discarded and the cells were resuspended in 1 mL of PBS and counted using a TC20 automatic cell counter (Bio-Rad). 4 mL of PBS was added to the sample and spun at 200 ×g for 10 minutes to remove any contaminating Ficoll, platelets, and plasma proteins. For transcriptional and DNA methylation profiling, the sample was resuspended in 350 µL of RLT plus (Qiagen) containing 1% beta-mercaptoethanol, vortexed for 30 seconds, and then transferred to a −80 °C freezer.