Peripheral blood mononuclear cell isolation and flow cytometry assay

Peripheral blood mononuclear cell (PBMC) isolation and flow cytometry assay was done as previously reported [30]. In brief, whole blood was drawn into EDTA tubes and cells were processed for PBMC isolation within one hour of collection and cryopreserved. Banked cryopreserved PBMCs were thawed and washed with warmed AIM-V liquid media (Invitrogen). Cells were then surface-stained with CD3, CD14, CD16, CD56, CD19, CD20, HLA-DR antibodies, and with Live/Dead fixable yellow dead cell stain (YARD). Data was acquired on a custom 4-laser BD LSRFortessa Cell Analyzer and all compensation and gating analyses were performed in FlowJo analytical software (gating strategy previously shown in figure 1 in reference [30]). Percentages of classical, intermediate, non-classical, and transitional monocyte subsets were determined based on CD14 and CD16 staining and absolute numbers of each subset were calculated from WBC and monocyte percent obtained from available clinical CBC performed on each participant.