Six-colour flow cytometry for the immunophenotypic and functional characterization of peripheral blood dendritic cells

Dendritic cells (DCs) are widespread antigen-presenting cells with a prominent role in the activation, polarization and regulation of adaptive immunity. Accordingly, possible changes in their number and function may be relevant to the comprehension and monitoring of a wide range of pathologies. Peripheral blood is the most accessible source of DCs, and this renders flowcytometry analysis of peripheral blood DCs (PBDCs) a non invasive procedure that can be serially repeated in patient followup. Because they lack unique markers, PBDCs can be identified in whole blood in different ways, all needing multiple fluorescence channels to focalize analyses on myeloid (mDCs) or plasmacytoid (pDCs) PBDCs that represent functionally distinct subsets. Current methods have limitations in mutliparametric PBDC analysis, because based on 3- or 4-colour flow cytometry. We developed a 6-colour method that allowed the analysis of multiple parameters simultaneously on mDCs and pDCs. In the same tube, mDCs and pDCs were identified as lin-/HLA-DR+/CD11c+ and lin-/HLA-DR+/CD123+ cells, respectively, and analysed for concomitant expression of two costimulatory molecules or regulatory cytokines. mDCs and pDCs were enumerated in fresh whole blood samples from 20 adult volunteers; comparison with a 3-colour method previously used in our lab was used for validation. Analysis of unstimulated activation/maturation markers demonstrated, by direct comparison of subsets, that pDCs express significantly lower levels of CD80, CD86 and CD40, compatible with the tolerogenic role assigned to pDCs. To assess the subset-specificity of PBDC responses, whole blood samples and separated PBMCs were short-term stimulated with TLR-specific ligands (for TLR3, TLR4, TLR7, TLR9) or inflammatory cytokines. Responses of mDCs and pDCs were assessed as upregulation of activation/maturation markers and intracellular cytokines (IL-4, IL-6, IL-10, IL-12, TNFa, IFN-a). As expected, ligands for TLR3 and TLR4 preferentially stimulated mDCs, ligands for TLR7 and TLR9 pDCs, inflammatory cytokines both subsets. LPS (TLR4 ligand) and imiquimod (TLR7 ligand) adequately stimulated PBDCs in whole blood samples, while the other stimuli were only effective in PBMC suspensions, possibly due to the presence of blood inactivators. With this study we provide a new method for the analysis of PBDCs that may be particularly useful when a direct comparison between mDCs and pDCs is advisable.