Distinguishing human peripheral blood CD16+ myeloid cells based on phenotypic characteristics

Myeloid lineage cells present in human peripheral blood include dendritic cells (DC) and monocytes. The DC are identified phenotypically as HLA‐DR+ cells that lack major cell surface lineage markers for T cells (CD3), B cells (CD19, CD20), NK cells (CD56), red blood cells (CD235a), hematopoietic stem cells (CD34), and Mo that express CD14. Both DC and Mo can be phenotypically divided into subsets. DC are divided into plasmacytoid DC, which are CD11c−, CD304+, CD85g+, and myeloid DC that are CD11c+. The CD11c+ DC are readily classified as CD1c+DC and CD141+ DC. Monocytes are broadly divided into the CD14+CD16− (classical) and CD14dimCD16+ subsets (nonclassical). A population of myeloid‐derived cells that have DC characteristics, that is, HLA‐DR+ and lacking lineage markers including CD14, but express CD16 are generally clustered with CD14dimCD16+ monocytes. We used high‐dimensional clustering analyses of fluorescence and mass cytometry data, to delineate CD14+ monocytes, CD14dimCD16+ monocytes (CD16+Mo), and CD14− CD16+DC (CD16+DC). We sought to identify the functional and kinetic relationship of CD16+DC to CD16+Mo. We demonstrate that differentiation of CD16+DC and CD16+Mo during activation with IFNγ in vitro and as a result of an allo‐hematopoietic cell transplant (HCT) in vivo resulted in distinct populations. Recovery of blood CD16+DC in both auto‐ and allo‐(HCT) patients after myeloablative conditioning showed similar reconstitution and activation kinetics to CD16+Mo. Finally, we show that expression of the cell surface markers CD300c, CCR5, and CLEC5a can distinguish the cell populations phenotypically paving the way for functional differentiation as new reagents become available. Human blood myeloid CD16+ dendritic cells are distinguished from CD14dimCD16+ monocytes by their surface phenotype, which reinforces the concept that heterogeneity of cell preparations can confound functional analysis.