Flow Cytometry Analysis of PBMC Subsets

For flow cytometry, whole-blood specimens were processed within 4 hours of collection to isolate PBMCs, using Lymphoprep (Axis-Shields-Diagnostics) as previously described [27 (link)]. Cells were analyzed using a CyAn ADP 9 color flow cytometer (Beckman Coulter). The T-cell panel included CD3 BV510, CD4 V450, CD38 PE Cy7, HLA-DR AF700, PD-1 APC, and CD57 FITC (all from BD Biosciences) and CD8 PE (Biolegend). The monocyte panel included HLA-DR AF700, CD14 PE Cy7, and CD16 PE (all from BD Biosciences). Anti-mouse Igk isotype control and negative control particles (BD Biosciences) were used for compensation. A standardized gating strategy was followed for T cells (Supplementary Figure 1A) and monocytes (Supplementary Figure 1B). Monocyte subsets were identified as previously described [28 (link)].