Circulating precursors of human CD1c+ and CD141+ dendritic cells

Abstract

Two subsets of conventional dendritic cells (cDCs) with distinct cell surface markers and functions exist in mouse and human. The two subsets of cDCs are specialized antigen-presenting cells that initiate T cell immunity and tolerance. In the mouse, a migratory cDC precursor (pre-CDC) originates from defined progenitors in the bone marrow (BM). Small numbers of short-lived pre-CDCs travel through the blood and replace cDCs in the peripheral organs, maintaining homeostasis of the highly dynamic cDC pool. However, the identity and distribution of the immediate precursor to human cDCs has not been defined. Using a tissue culture system that supports the development of human DCs, we identify a migratory precursor (hpre-CDC) that exists in human cord blood, BM, blood, and peripheral lymphoid organs. hpre-CDCs differ from premonocytes that are restricted to the BM. In contrast to earlier progenitors with greater developmental potential, the hpre-CDC is restricted to producing CD1c(+) and CD141(+) Clec9a(+) cDCs. Studies in human volunteers demonstrate that hpre-CDCs are a dynamic population that increases in response to levels of circulating Flt3L.

Figure 1.

Expression of cytokine receptors on DCs and monocytes. Flow cytometry plots show expression of CD117, CD135, CD115, CD123, CD45RA, CD34, and CD116 on gated CD3⁻CD19⁻CD56⁻ cells (gray), differentiated monocytes (CD14⁺ orange), pDCs (CD303⁺ green), CD1c⁺ cDCs (blue), and CD141⁺ cDCs (red) from peripheral blood. Table summarizes results of flow cytometry.

Figure 2.

Screening of populations in the cord blood for committed cDC lineage potential. (a) Flow cytometry plots show gating of CD45⁺CD3⁻CD19⁻CD56⁻CD14⁻CD66b⁻CD1c⁻CD141⁻CD303⁻ cells in human cord blood can be divided into four populations based on CD117, CD34, and CD135: CD34⁺CD117⁺ (CD34⁺), CD34⁻CD117⁻ (CD117⁻), CD34⁻CD117⁺CD135⁻ (CD135⁻), and CD34⁻CD117⁺CD135⁺ (CD135⁺). Numbers indicate the frequency of respective gates. (b) Differentiation potential of 1,000 purified cells from each of 4 populations indicated in (a) in MS5+FSG cultures for 7 d. Flow cytometry plots show gated live human CD45⁺ cells. (c) Flow cytometry plots show CD135⁺ cells indicated as in (a) can be further separated into 4 populations based on CD116, CD115, and CD45RA: CD135⁺CD116⁻ (CD116⁻), CD135⁺CD116⁺CD45RA⁻(CD45RA⁻), CD135⁺CD116⁺CD45RA⁺CD115⁺ (CD115⁺), and CD135⁺CD116⁺CD45RA⁺ CD115⁻ (CD115⁻). (d and e) Differentiation potential of 100 purified cells from each of 4 populations indicated in (c) in MS5+FSG cultures for 7 d. (d) Flow cytometry plots of CD45⁺ cells gated as in (c), showing expression of CD141, CLEC9a, CD1c, and CD19. (e) Graphs show mean output of pDC, CD1c⁺ cDC, CD141⁺ cDC and monocytes from each population from three independent experiments. (f) Histograms show expression of CD11c, HLA-DR, CD123, CD135, CD117, CD45RA, CD116, and CD115 on indicated cell-type. (g) Morphology of purified cord blood hpre-CDCs by Giemsa staining of cytospin preparations (100×). Dotted lines indicate cropping out of white space between cells. Bar, 10 µm.

Figure 3.

hpre-CDCs in human lymphoid organs and blood. (a) Representative flow cytometry plots of gated Lin(CD3/19/56/14/66b)⁻DC(CD1c/141/303)⁻CD34⁻ cells show gating strategy and composition of hpre-CDCs (SSC^loCD117⁺CD116⁺CD135⁺ CD45RA⁺CD115⁻, red) and premonocytes (SSC^loCD117⁺CD116⁺CD135⁺ CD45RA⁺CD115⁺, green) in human cord blood (CB), BM, peripheral blood (PB), and tonsil. Numbers indicate frequency of cells of parent gate (CB, n = 5; BM, n = 4, PB, n = 5; Tonsil, n = 3; n, number of donors). (b) Differentiation potential of purified hpre-CDCs indicated in (a) in MS5+FSG cultures for 5 d. Flow cytometry plots of gated live human CD45⁺ cells show culture output of CD141⁺ cDC (red) and CD1c⁺ cDC (blue). Representative results of four (CB), three (BM), three (PB), and two (tonsil) independent experiments are shown. (c) Differentiation potential of purified premonocytes from cord blood (CB) and BM in MS5+FSG cultures for 5 d. FACs plots show phenotype of gated live human CD45⁺ culture-derived cells including monocytes (orange) and CD1c⁺ cDCs (blue). Representative of three independent experiments are shown.

Figure 4.

Proliferative capacity of hpre-CDCs. (a) Expansion of purified hpre-CDCs or CD34⁺ HSPCs from peripheral blood (PB) or cord blood (CB) in MS5+FSG cultures for 7 d. Graph shows the mean fold change of live human CD45⁺ cells from 100 input cells from three independent experiments. *, P < 0.005, unpaired two-tailed Student’s t test. (b and c) CD34⁺ HSPCs and hpre-CDCs were purified from CB, labeled with CFSE, and cultured in MS5+FSG for 2 or 7 d; proliferation was assessed by flow cytometry. FACs plots show (b) gated CD45⁺ culture-derived cells, including CD34⁺ cells (purple), CD34⁻CD1c⁻CD141⁻ cells (orange), CD141⁺ cDCs (red), and CD1c⁺ cDCs (blue) and (c) their CFSE dilution. Dotted lines mark last division by hpre-CDCs. Plots are representative of three independent experiments.

Figure 5.

Clonal efficiency and potential of hpre-CDC. (a) Limiting dilution outgrowth assay show clonal efficiency of CD34⁺ HSPCs (n = 3) or hpre-CDCs from peripheral (PB, n = 4) and cord blood (CB, n = 3) in MS5+FSG cultures for 5 d (for hpre-CDCs) or 14 d (for CD34⁺ HSPCs; Materials and methods). **, P < 0.05, pairwise test by ELDA. (b) Representative flow cytometry plots of gated CD45⁺ cells derived from single hpre-CDC clones indicated in (a) show output of CD1c⁺ cDCs (blue) and CD141⁺ cDCs (red). (c) Graphs summarize the lineage output of single clones from CD34⁺ HSPCS, and hpre-CDCs from cord (CB) or peripheral blood (PB). G, granulocyte; M, monocyte; L, lymphocytes.

Figure 6.

hpre-CDCs descend from hCDPs. (a) Flow cytometry plots show comparison of hCDP (purple) and hpre-CDC (orange) in cord blood. (b) Flow cytometry plots of gated CD45⁺Lin(CD3/19/56/14)⁻CD34⁺ show phenotype and purity of magnetically enriched cord blood (CB) CD34⁺ cells (presorting, upper) and sorted cells (post-sorting, bottom). hCDPs were gated as CD34⁺CD38^hiCD45RA⁺CD10⁻CD123^hi. (c) Differentiation kinetics of hCDPs purified from CB in MS5+FSG cultures for 1, 2, or 4 d. FACs plots show culture output of live human CD45⁺ cells, including CD34⁺ cells (purple), pDC (green), CD1c⁺ cDC (blue), CD141⁺ cDCs (red), and hpre-CDCs (orange). Representative of four independent experiments are shown. Graphs summarize composition of indicated populations among total hCD45⁺ cells. Bars are mean values from four independent experiments, and error bars are SEM.

Figure 7.

Flt3L administration increases circulating DC subsets and hpre-CDCs in humans. PBMCs from Flt3L-treated volunteers (n = 3; 25 µg/kg for 10 consecutive days) were analyzed by flow cytometry over a 21-d period to assess the expansion of DC subsets (CD1c⁺ cDCs [blue], CD141⁺ cDCs [red], and pDCs [green]; hpre-CDCs (gray); monocytes (orange); and granulocytes (brown). (a) Representative flow cytometry dot plots show DC subsets and hpre-CDCs in blood (gating strategy in Fig. S4). (b) Graphs show the kinetics of cell number of cDC subsets, pDCs, and hpre-CDCs over 21 d in 3 Flt3L-treated volunteers. The absolute numbers per milliliter of blood were obtained by multiplying the number of cells (obtained by flow cytometry) by the total number of PBMCs per milliliter of blood. (c) Representative histograms show CD135 expression on CD141⁺ cDCs, CD1c⁺ cDCs, pDCs and hpre-CDCs over 21 d in one Flt3L-treated volunteer. (d) Differentiation potential of purified hpre-CDCs from blood of Flt3L-injected volunteers in MS5+FSG cultures after 7 d. Flow cytometry plots of gated CD45⁺ cells from culture show output of CD141⁺ cDCs (red), CD1c⁺ cDCs (blue), and lack of pDCs (green gate). (e) Representative flow cytometry dot plots show CD14⁺ monocytes and CD66b⁺ granulocytes in blood (gating strategy in Fig. S4). (f) Graphs show the kinetics of cell number of monocytes and granulocytes over 21 d in 3 Flt3L-treated volunteers. The absolute numbers per milliliter of blood were obtained by multiplying the number of cells (obtained by flow cytometry) by the total number of PBMCs per milliliter of blood. (g) Graph showing the mean fold change increase from the three patients (d1 vs. d14) of hpre-CDCs (gray), CD1c⁺ cDCs (blue), CD141⁺ cDCs (red), pDCs (green), monocytes (orange), and granulocytes (brown) per milliliter of blood. Error bars are SDs.