Immunophenotyping of Human Dendritic Cell Population

Dendritic cells (DCs) play an important role in innate and acquired immunity. They are the antigen-presenting cells that prime naive T cells and elicit memory T cell responses to foreign antigens. DC subsets can be classified into three types—plasmacytoid DC (pDC), myeloid/conventional DC1 (cDC1) and myeloid/conventional DC2 (cDC2).

Here, we feature a BD Biosciences application note that describes a multicolor flow cytometry panel featuring 20 fluorochrome-labeled antibodies, including BD Horizon™ Brilliant Reagents, used to evaluate the various DC subsets in peripheral blood mononuclear cells (PBMCs) taken from healthy human subjects.

The study was performed on a BD FACSymphony™ A3 Cell Analyzer. The five lasers of the instrument allowed the fluorochromes to be spread out, enabling distinction of not only the classical and pDCs but also unique DCs, like Axl⁺ Siglec6⁺ DCs (AS DC).

In the study, DC subsets were gated on lineage negative (LIN^-) HLA^-DR⁺CD14^- cells and further subtyped based on the known phenotypes and co-expressed markers. The cDC1 subset was identified with the CD141⁺CD16^- phenotype, co-expressing Clec9A and CD26. The cDC2 subset was derived from the CD16^- CD141^- population, co-expressing CD1c, CD11c, CD36 and CD172a/b. The AS DC subset was also gated from CD16⁻CD141⁻ cells but defined based on co-expression of Axl and Siglec6, the pDC population was gated from the CD16⁻CD141⁻ Axl⁻ Siglec6⁻ subset and identified by co-expression of CD123 and CD303.

In the study, DC subsets were gated on lineage negative (LIN^–) HLA-DR⁺CD14^– cells and further subtyped based on known phenotypes and co-expressed markers. The cDC1 subset was identified with the CD141⁺CD16^– phenotype, co-expressing Clec9A and CD26. The cDC2 subset was derived from the CD16^–CD141^– population, co-expressing CD1c, CD11c, CD36 and CD172a/b. The AS DC subset was also gated from CD16⁻CD141⁻ cells but defined based on co-expression of Axl and Siglec6. The pDC population was gated from the CD16⁻CD141⁻ Axl⁻ Siglec6⁻ subset and identified by co-expression of CD123 and CD303.

Identification of AS DC and pDC population in human peripheral blood
 

A. DCs were identified following a gating strategy. The AS DC subset was gated from CD16⁻CD141⁻ cells and defined based on co-expression of Axl and Siglec6. As shown, two major AS DC subsets, CD123high CD11c⁻ and CD123low CD11c⁺ were observed.¹ The pDC population was gated from the Axl⁻ Siglec6⁻ subset and identified by co-expression of CD123 and CD303. B. FMO controls for Axl and Siglec6 are shown, which follow a gating strategy as described in A.

Download the "Immunophenotyping of human dendritic cell population" application note.

Reference

1. Villani AC, Satija R, Reynolds G, et al. Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science. 2017;356(6335):eaah4573. doi:10.1126/science.aah4