Human dendritic cells (DCs) are derived from distinct circulating precursors that are precommitted to become CD1c+ or CD141+ DCs

Abstract

In humans, conventional dendritic cells (cDCs) exist as two unique populations characterized by expression of CD1c and CD141. cDCs arise from increasingly restricted but well-defined bone marrow progenitors that include the common DC progenitor that differentiates into the pre-cDC, which is the direct precursor of cDCs. In this study, we show that pre-cDCs in humans are heterogeneous, consisting of two distinct populations of precursors that are precommitted to become either CD1c⁺ or CD141⁺ cDCs. The two groups of lineage-primed precursors can be distinguished based on differential expression of CD172a. Both subpopulations of pre-cDCs arise in the adult bone marrow and can be found in cord blood and adult peripheral blood. Gene expression analysis revealed that CD172a⁺ and CD172a^- pre-cDCs represent developmentally discrete populations that differentially express lineage-restricted transcription factors. A clinical trial of Flt3L injection revealed that this cytokine increases the number of both CD172a^- and CD172a⁺ pre-cDCs in human peripheral blood.

Figure 1.

Heterogeneity in human pre-cDCs revealed by single-cell mRNA sequencing. (a) Gating strategy for isolation of CD1c⁺ cDCs and CD141⁺ cDCs in blood. CD1c⁺ cDCs (blue) and CD141⁺ cDCs (red) are identified in live CD3⁻ CD20⁻ CD335⁻ CD66b⁻ cells, i.e., T, B, NK, and neutrophil cell depleted based on the expression of CD1c and CD141, respectively, and the absence of CD14 expression (n = 5). Gate frequencies from the parent population are shown. (b) Unsupervised clustering reveals transcriptomic signatures of single differentiated blood cDCs, i.e., CD1c⁺ cDCs (77 single cells; blue) and CD141⁺ cDCs (87 single cells; red; three samples). Shown are the top 30 genes exhibiting the strongest differential expression for each subset (P < 10⁻⁵; likelihood ratio test for single-cell differential expression; see the Single-cell mRNA sequencing section of Materials and methods and Table S1). (c) Gating strategy for isolation of human pre-cDCs in cord blood. Live CD3⁻ CD20⁻ CD19⁻ CD335⁻ CD66b⁻ CD14⁻ CD1c⁻ CD141⁻ CD303⁻ cells, i.e., T cell, B cell, NK cell, neutrophil, monocyte, and DC depleted, were stained for CD34, CD117, CD123, CD135, CD116, CD115, and CD45RA markers. CD34⁻ CD117⁺ CD123^−/+ CD135⁺ CD116⁺ CD115⁻ CD45RA⁺ pre-cDCs are shown. Gate frequencies from the parent population are shown. SSC, side scatter. (d) Single-cell clustering of individual cord blood pre-cDCs (three samples). This analysis shows that cord blood pre-cDCs cluster in CD1c⁺ lineage–primed (266 single cells; blue), CD141⁺ lineage–primed (29 single cells; red), and non-DCs (157 single cells; gray). Shown are the top 30 genes exhibiting the strongest differential expression for each lineage-primed subset (P < 10⁻⁵; likelihood ratio test for single-cell differential expression; see the Single-cell mRNA sequencing section of Materials and methods and Table S2). (e) Expression of selected genes in CD1c⁺ lineage–primed cells (blue), CD141⁺ lineage–primed cells (red), and non-DCs (gray) presented as violin plots (y axis, gene expression; x-axis, abundance of cells expressing the gene).

Figure 2.

Heterogeneous expression of CD172a in human cord blood pre-cDCs identifies populations with CD1c⁺ or CD141⁺ lineage potential. (a) Expression of CD172a receptor on cDCs and monocytes. The flow cytometry histogram shows expression of CD172a on live CD3⁻ CD20⁻ CD335⁻ CD66b⁻–gated monocytes (CD14⁺; orange), pDCs (CD303⁺; green), CD1c⁺ cDCs (blue), and CD141⁺ cDCs (red) from peripheral blood (n = 3). (b) Expression of CD172a receptor on pre-cDCs in human cord blood (CB), bone marrow, and peripheral blood (PB; cord blood, n = 6; bone marrow, n = 2; peripheral blood, n = 3). Flow cytometry plots show that Lin⁻ CD34⁻ CD117⁺ CD123^−/+ CD135⁺ CD115⁻ CD116⁺ pre-cDCs in human cord blood can be divided by CD172a into three populations: CD172a⁻ (red), CD172a^int (green), and CD172a⁺ (blue) pre-cDCs. Gate frequencies from parent population are shown. SSC, side scatter. (c) Heat map showing the top 41 differentially expressed genes selected by unsupervised hierarchical clustering (clustering analysis on all differentially expressed genes with p-value <0.05) between cord blood CD172a⁺ (n = 4), cord blood CD172a⁻ pre-cDCs (n = 4), peripheral blood CD141⁺ cDCs (n = 4), and peripheral blood CD1c⁺ cDCs (n = 4; Table S3). (d) Expression of specific surface markers and lineage-restricted transcription factors involved in DC development in CD1c⁺ cDCs, CD141⁺ cDCs, CD172a⁺ pre-cDCs, and CD172a⁻ pre-cDCs. (e) The graph shows mean output of CD141⁺ cDCs (red) and CD1c⁺ cDCs (blue) from the culture of CD172a⁻, CD172a^int, and CD172a⁺ pre-cDCs from five independent experiments. Error bars represent the standard deviation. (f) Gating strategy for culture output after hematopoietic differentiation on MS-5 stromal cells of human cord blood pre-cDCs. Total cord blood pre-cDCs were isolated and then cultured in MS5 + FSG for 7 d. Culture output was assessed by flow cytometry. Cells developing into culture are identified within the live Lin⁻ CD45⁺ cells. Output cells include CD66b⁺ granulocytes (Gran; brown), CD141⁺ cDCs (red), CD1c⁺ cDCs (blue), monocytes (Mono; orange), and CD303⁺ pDCs (green). Gate frequencies from the parent population are shown (n = 5). (g) Differentiation potential of 50–200 purified cells from cord blood CD172a⁻, CD172a^int, and CD172a⁺ pre-cDCs in MS5 + FSG cultures for 7 d. Flow cytometry plots show gated Lin⁻ CD45⁺ cells. CD141⁺ cDCs were identified as CD141⁺ CLEC9A⁺ and CD1c⁺ cDCs as CLEC9A⁻ CD14⁻ CD1c⁺ (n = 5). Gate frequencies from the parent population are shown. (h) Single-cell clonal assay of CD172a^int pre-cDCs. Representative flow cytometry of gated Lin⁻ CD45⁺ cells derived from single CD172a^int pre-cDC culture show clonal output of CD141⁺ cDCs (red) and CD1c⁺ cDCs (blue). For this representative experiment, 11 wells gave rise to CD141⁺ cDCs and 33 wells to CD1c⁺ cDCs (120 wells plated; 44 positive wells; n = 3). (i) Proliferative capacity of CD172a⁻, CD172a^int, and CD172a⁺ pre-cDCs. pre-cDC subsets were purified from cord blood, labeled with CFSE, and cultured in MS5 + FSG for 7 d. Proliferation was assessed by flow cytometry. Representative FACS plots show CD141⁺ cDCs (red) and CD1c⁺ cDCs (blue) CFSE dilution (n = 2).

Figure 3.

Human blood subsets of pre-cDCs. (a) Single-cell clustering for individual peripheral blood pre-cDCs. This analysis showed that peripheral blood pre-cDCs cluster in CD1c⁺ lineage–primed (282 single cells; blue), CD141⁺ lineage–primed (17 single cells; red), and non-DC (42 single cells; gray) populations (three samples). Shown are the top 30 genes exhibiting the strongest differential expression for each lineage-primed subset (P < 10⁻⁵; likelihood ratio test for single-cell differential expression; see the Single-cell mRNA sequencing section of Materials and methods and Table S4). (b and c) Expression of CD172a (b) and selected genes (c) in CD1c⁺ lineage–primed cells (blue), CD141⁺ lineage–primed cells (red), and non-DCs (gray) presented as violin plots (y axis, gene expression; x axis, abundance of cells expressing the gene).

Figure 4.

Flt3L mobilizes human CD172a⁻ and CD172a⁺ pre-cDCs into the blood. (a and b) PBMCs from Flt3L-injected volunteers (n = 3; 25 µg/kg for seven consecutive days) were analyzed by flow cytometry before (day 0) and after Flt3L treatment (day 12) to assess the expansion of CD1c⁺ and CD141⁺ cDC subsets as well as CD172a⁻, CD172a^int, and CD172a⁺ pre-cDC subsets in blood. Representative flow cytometry dot plots show CD1c⁺ cDCs (blue) and CD141⁺ cDCs (red; a) as well as CD172a⁻ (red), CD172a^int (green), and CD172a⁺ (blue; b) pre-cDC subsets (gating strategy in Fig. 1, a and c). Gate frequencies from parent population are shown. SSC, side scatter. (c) Representative histogram shows CD117 and HLA-DR expression on CD1c⁺ cDCs, CD141⁺ cDCs, CD172a⁻ pre-cDCs, and CD172a⁺ pre-cDCs in one of the Flt3L-treated volunteers.