Figure 2.

Gating strategy for the identification of the different subsets of circulating monocytes and dendritic cells (DCs) in a representative peripheral blood (PB) sample from a healthy donor. Coloured events in Panels a and b correspond to all the antigen-presenting cell (APC) subsets under study (that is, total PB monocytes and DCs), firstly gated from leukocytes (grey dots) according to their typical expression pattern of sideward scatter (SSC^int) together with CD45 (Panel a) and HLADR (Panel b). Next, monocytes were identified within total APCs, based on their expression of CD33^+/++ (not shown), together with their expression profile for CD14 and CD16 (coloured dots included in the gate in Panel c). Their differential expression of CD14 and CD16 further allowed the identification of three major subpopulations of monocytes (Panel c): CD14⁺⁺/CD16⁻ classical monocytes (cMo), CD14^+/lo/CD16^lo intermediate monocytes (iMo) and CD14^lo/−/CD16⁺non-classical monocytes (ncMo). Within this latter monocytic subset, both SLAN⁻ and SLAN⁺ ncMo were identified, as shown in Panel d: the region including SLAN⁺ ncMo (blue dots included in the gate) was set based on internal negative controls for SLAN (grey dots in Panel d). Myeloid DCs (mDCs, Panel e) were identified based on their high expression of CD33 and HLADR in the absence of CD14 and CD16, to exclude monocytes, while plasmacytoid DCs (pDCs, Panel f) were identified by the strong CD123 expression and positivity for HLADR, also in the absence of CD14 and CD16. Colour codes: yellow dots correspond to cMo, iMo are painted in green, whereas blue dots correspond to ncMo; dark purple and orange dots represent mDC and pDC, respectively.