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. These are plasmacytoid DC (pDC), myeloid/conventional DC1 (cDC1) and myeloid/conventional DC2 (cDC2).

This application note describes a multicolour 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. This enabled the distinction of not only the classical and pDC but also unique ones like the Axl⁺ Siglec6⁺ DC (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.

The study demonstrates the versatility of the BD FACSymphony™ A3 Cell Analyzer in performing a flow experiment with a 20 fluorochrome multiparametric panel to analyse major human DC subsets. As shown in the application note, the panel is suitable for both conventional and unsupervised analysis of human DCs.

Identification of AS DC and pDC population in human peripheral blood

A. DCs were identified following a gating strategy. 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 (Villani et al.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.

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Reference

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

This application note is not peer-reviewed.

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