Moflo legacy sorter

PBMCs were separated from whole blood and frozen. Freshly thawed cells were washed and incubated with CD3-FITC (Biolegend), CD4-PECy7 (Biolegend), CD8-APC (BD Biosciences), HLA-DR-PE (Biolegend), and CD38-BV421 (Biolegend) for 40 minutes at 4°C per the manufacturer’s recommendation. Immediately before sorting, plasma membrane compromised cells were labeled with propidium iodide (Sigma). Fluorescence-activated cell sorting (FACS) was performed on a MoFlo Legacy Sorter (Beckman-Coulter) at the Johns Hopkins School of Public Health Flow Cytometry Core Facility. Gates were set using fluorescence minus one plus isotype control. Because flow and sorting had to be performed over several sessions, the gates were set each session using a reference participant’s PBMCs (selected based on availability of cells). The population of interest was sorted directly into ≥4 volumes of Quick-RNA® MicroPrep lysis buffer (Zymo Research) per the manufacturer’s recommendation. Sorting was stopped when the number of sorted cells reached 125,000 cells, although many samples did not reach this number. Flow cytometry analysis on two randomly selected post-sort samples revealed >95% purity. Sorted samples were vortexed, incubated for 10 minutes at room temperature, vortexed again, and frozen at −80°C until isolation.