Figure 1.

Flow cytometry gating strategy for monocytes. Analytic gating of flow cytometry data: (a) monocytes were identified from singlet cells; (b) monocytes were selected based on forward and side scatter; (c) cells expressing low levels of CD4 were selected; (d) cells co-expressing CD4^loCD11c were then identified; (e) gating on monocyte subsets was performed with classical monocytes as (CD14hi⁺⁺CD16⁻), intermediate monocytes as (CD14⁺⁺CD16⁺), and non-classical monocytes as (CD14⁺CD16⁺⁺); (f) classical monocyte activation shown by co-expression of programmed death ligand-1 (PD-L1) and CD25 is shown; (g) gating on the fluorescence minus one (FMO) controls is used to set gates for CD25 and PD-L1; (h) expression of interleukin (IL)-6 by classical monocytes following interferon (IFN)-γ stimulation for a representative patient; (i) expression of tumor necrosis factor (TNF)-α by classical monocytes following IFN-γ stimulation for a representative patient.