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. 2020 Aug 18;53(2):353–370.e8. doi: 10.1016/j.immuni.2020.07.003

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© 2020 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Differential IRF8 Expression Defines the Two Trajectories of DC Development

(A–E) CyTOF analysis of FACS-purified CD45⁺lin(CD3,19,20,56,161)⁻ PB and BM progenitors, precursors, and mature DCs and monocytes using a panel of 33 surface antigens and two intracellular stains (IRF4 and IRF8). (A) tSNE visualization of lin⁻HLA-DR⁺ cells, down-sampled to select 75,000 cells (20,000 CD11b⁺CD14⁺ monocytes, 4,000 CD11b⁺CD16⁺ monocytes, and 50,000 non-monocyte cells). PB (red) and BM (gray) cells were distinguished by differential CD45⁺ conjugate staining and displayed across tSNE space. (B) Heatmap of DC or monocyte-subset-specific antigens displayed on tSNE plots as in (A) (blue-yellow-red scales represent channel values). “Mature cells” plot shows the location of DC and monocyte subsets and CD34⁺ progenitors, identified by back-gating from bivariate plots (Figures S5B–S5D). (C) The location in tSNE space of IRF8^hi (red) and IRF8^lo (orange) expressing cells identified by (1) standard gating on a bivariate plot of IRF8 versus CD304 and superimposition of these gated cells on tSNE space and (2) a heatmap of IRF8 expression across all cells. (D and E) Location in tSNE space of progenitors and precursors with pDC, cDC1, or DC2 (D) and DC3 or monocyte (E) potential as defined by previous experiments, identified by back-gating from bivariate plots (Figures S5B and S5C), and heatmaps of associated antigens.

(F) Diffusion map generated with 14,000 cells including GMPs, precursor and mature DCs, and monocytes. Populations were identified and color-coded according to Figures 3A (progenitors) and 4A (precursors, DCs, and monocytes), applied to CyTOF data as shown in Figures S5B and S5C. Heatmaps show the expression (log2) of IRF8 and key antigens superimposed across the diffusion map trajectories. See also Figure S5E. Diff C, diffusion component.

(G) Histograms summarizing IRF8 protein expression by flow cytometry (MFI) in progenitors, precursors, and mature cells of pDC, cDC1, DC2, and DC3 lineages from BM and PB. Bars show mean ± SEM. Circles show individual donors (BM progenitors, n = 4; BM and PB precursors and mature DCs, n = 3).

See also Figure S5.