Phenotypic and functional characterization of human blood BDCA3+ subsets

There has been a recent focus on human blood DCs expressing BDCA3 (CD141). However, these cells are not fully characterized. Our study aimed to understand whether they constitute a homogeneous population or if they are related to two other DC subsets: plasmacytoid DCs (BDCA2+) and myeloid DCs (BDCA1+). We constructed multi-color panels for surface phenotyping of blood BDCA3+ DCs by flow cytometric analysis. We observed that lineage- PBMC heterogeneously distribute into BDCA3hi and BDCA3int subsets, with the latter subset expressing higher levels of CCR5 and CXCR4. Surface phenotyping of lineage- PBMC for the BDCA2 (found on pDC) and BDCA3 surface antigens yielded a subset of BDCA2+/BDCA3int cells, representing 15-30% of BDCA2+/lin- pDC. To assess functional significance of this population, we stimulated PBMC with TLR9 agonists CpGA or HSV-1 and stained for intracellular IFN-α and TNF-α in cells expressing BDCA2 and/or BDCA3. The BDCA2+/BDCA3int subpopulation produced IFN-α and TNF-α and upregulated the transcription factor IRF-7 more strongly than the BDCA2+/BDCA3- subpopulation, while the BDCA2-/BDCA3+ subpopulation constitutively expressed the lowest level of IRF-7 and barely produced IFN-α. However, similarly lower levels of TNF-α were found in the BDCA2-/BDCA3+ and BDCA2+/BDCA3- subpopulations when compared to the BDCA2+/BDCA3int subpopulation. These results suggest that the BDCA2+/BDCA3int subset has major functions associated with classical pDCs.