Alternative identification of nonclassical monocytes for the study of the mechanism underling CD16 shedding. (A) Gating strategy used in a representative intracellular cytokine staining experiment on whole-blood stimulations. Cells are selected via sequential gating by the exclusion of doublets and dead cells followed by the positive gating on HLA-DR and CD11c (myeloid mononuclear cells, gate R1). A further gate on lineage-negative cells (R2) is set to select both mDCs and nonclassical monocytes. (B) R1 gated cells are plotted for control, LPS (0.5 μg/mL), and R848 (10 μM) (both reagents from Invivogen) stimulated tubes in a lineage vs CD16 plot to visualize the decrease in CD16 fluorescence intensity following activation in a visualization mode commonly used to identify monocyte subtypes: CMo, IMo, and NCMo. (C) Downregulation of CD16 in NCMo is concomitant with the production of TNFα. Red arrows emphasize the concomitant intracellular accumulation of TNFα and loss of surface CD16. (D-E) Distribution of CD38 in different myeloid mononuclear populations. mDC (red), NCMo (blue), and CMo (black) identified essentially as described in panels A through C are overlaid on total mononuclear cells (gray) in HLA-DR vs CD38 plots (D). (E) Segregation of DC subsets defined by the expression of CD16, slan, CD141, and CD1c into CD38-positive and -negative lineage^neg myeloid mononuclear cells. (F) In the top 2 panels, HLA-DR⁺CD11c⁺ cells were plotted in CD14 vs CD38 bivariate plots for both Ctrl- and R848- (10μM, 1 hour) stimulated tubes to show how mDC, NCMo, and CMo can be segregated one from another by the use of CD38. The bottom 2 plots illustrate the decrease in CD16 expression occurring following stimulation with R848; CD38, in contrast, remains unchanged. (G) Dose-dependent inhibitory effect of the panmetalloprotease inhibitor GM6001/Ilomastat (Sigma-Aldrich) on CD16 (□) and CD62L (▪) shedding induced by 1-hour incubation with R848 (both reagents were added at the same time at the starting of the culture; bars are means, and error bars represent SD of 3 experiments from 3 distinct donors). Cells are gated on CD11c⁺HLADR⁺; CMo are identified as being CD14⁺CD38⁺, while nonclassical monocytes are CD14^low/−CD38⁻. Cytometric off-line analysis was performed with FlowJo software (Tree Star), and generated numerical data graphed in Prism (GraphPad). Ctrl, control; CMo, classical monocyte; FSC, forward scatter; IMo, intermediate monocyte; LPS, lipopolysaccharide; mDC, myeloid dendritic cells; MFI, mean fluorescence intensity; NCMo, nonclassical monocyte; SSC, side scatter.