Propidium iodine

Blood samples were centrifuged at 1,150 g for 5 minutes at room temperature to separate the plasma from the cell pellets. Plasma was removed and frozen at −20 °C for future analysis. The cell pellets were re-suspended in 1.1 ml of 1X RBC lysis buffer (Biolegend) and incubated on ice for 10 minutes to remove the red blood cells. After lysis, samples were pelleted at 1,150 g in a microcentrifuge for 5 minutes at room temperature, and then stained with 65 μl of an antibody cocktail containing 5 μl anti-human CD3-FITC, 5 μl anti-human CD4-PE, 5 μl anti-human CD8a-APC antibodies (Biolegend, Catalog #300406, 300508, 301014 respectively) and 50 μl of phosphate buffered saline supplemented with 2% fetal bovine serum (PBS+). Samples were washed once with 1 ml PBS+ and pelleted again at 1,150 g for 5 minutes. Pelleted cells were re-suspended in 200 μl of PBS+ supplemented with 2 μg ml⁻¹ propidium iodine (Invitrogen) and analyzed on MACSQuant flow cytometer (Miltenyi Biotec). Samples were first gated by CD3 expression before determining the ratio of CD4 to CD8 cells within this subset. Samples containing fewer than 20 CD3+ events were excluded from the analysis.