Characterization of steady state and inflammatory peripheral blood dendritic cell subsets by multicolor flow cytometry

Background Dendritic cells (DCs) are professional antigen-presenting cells crucial in initiating and shaping adaptive immune responses. The effects of DCs on adaptive immune responses depend on functional specialization of distinct DC subsets and on the state of DC activation, which is largely dictated by environmental signals. Whereas immunostimulatory DCs promote immune responses, DCs matured in immunosuppressive conditions counteract T-cell activation. Despite immune checkpoint inhibitors are increasingly used in cancer immunotherapy, the expression of immune checkpoints on DC subsets has been poorly characterized. Aim To optimize a multi-color flow cytometric method for the identification of 5 distinct DC subsets and their expression of costimulatory molecules and inhibitory immune checkpoints. Methods Whole blood samples were stained with standard procedures and acquired on a BD-FACSymphony. Data were compensated with FlowJo by using single-stained controls. DCs were identified as viable CD45br/lineage-/HLA-DR+cells. DC-lineage DCs were divided into CD11c+myeloid DCs (mDCs) and CD123+plasmacytoid DCs (pDCs). mDCs were further divided into CD141+and CD1c+mDCs. Inflammatory DCs were divided into monocyte-derived DCs (moDCs) and slanDCs, based on CD1a and M-DC8 expression, respectively. The expression of costimulatory molecules (CD40,CD80,CD86) and immune checkpoints (ILT2,PD-L1,TIM3) was assessed on each DC subset. Results The proportion of mDCs and pDCs was similar to that observed in previous studies with a 6-color validated method. Within mDCs, CD141+cells represented a minority of total mDCs. slanDCs were present at low levels and moDCs were almost undetectable in most samples, as expected. CD40, CD80 and CD86 expression was lower on pDCs than mDCs, thus confirming our previous studies. CD141+cells expressed higher CD40 levels but lower CD86 levels than CD1c+cells. Also immune checkpoint expression was heterogeneous among DC subsets. PD-L1 and ILT2 were negligible on CD141+mDCs, expressed at low levels on pDCs and CD1c+mDCs, and at higher levels on slanDCs. TIM3 was expressed at the highest levels on CD141+mDCs, and negligible on pDCs. Conclusions Our optimized 18-color method allows direct comparison of the activatory/regulatory phenotype of DC subsets, representing a valuable tool for the characterization of peripheral blood DCs. Applied to the study of DCs in the tumor microenvironment, this assay has the potential to provide unprecedented information for improving our comprehension of the role of DCs in cancer, and possibly identifying predictive biomarkers for cancer immunotherapy response.