Defining human dendritic cell progenitors by multiparametric flow cytometry

Abstract

Human dendritic cells (DCs) develop from progressively restricted bone marrow (BM) progenitors: these progenitor cells include granulocyte, monocyte and DC progenitor (GMDP) cells; monocyte and DC progenitor (MDP) cells; and common DC progenitor (CDP) and DC precursor (pre-DC) cells. These four DC progenitors can be defined on the basis of the expression of surface markers such as CD34 and hematopoietin receptors. In this protocol, we describe five multiparametric flow cytometry panels that can be used as a tool (i) to simultaneously detect or phenotype the four DC progenitors, (ii) to isolate DC progenitors to enable in vitro differentiation or (iii) to assess the in vitro differentiation and proliferation of DC progenitors. The entire procedure from isolation of cells to flow cytometry can be completed in 3-7 h. This protocol provides optimized antibody panels, as well as gating strategies, for immunostaining of BM and cord blood specimens to study human DC hematopoiesis in health, disease and vaccine settings.

Figure 1

Schematic view of human dendritic cell (DC) hematopoiesis. DC hematopoiesis is initiated in the bone marrow (BM). A granulocyte, monocyte and DC progenitor (GMDP) develops into a monocyte and DC progenitor (MDP). MDPs give rise to monocytes and a common DC progenitor (CDP), which loses the potential to produce monocytes. CDPs give rise to plasmacytoid DCs (pDCs), as well as a circulating cDC precursor (pre-cDC). Pre-cDCs migrate from the BM through the blood to the periphery to produce the two major subsets of conventional DCs (cDCs)—i.e., BDCA-1⁺ cDCs and BDCA-3^hi cDCs.

Figure 2

Gating strategy for the identification of human DC progenitors. (a) Workflow for the identification of DC progenitors. (b,c) Human BM (b) and CB (c) MNCs were stained as indicated in Table 2 (panel #1). Progenitors are identified by first gating on live cells (FSC-A versus SSC-A), and then on singlets (SSC-A versus SSC-W) and live cells (Live/Dead blue versus SSC-A). Lin⁻ BDCA⁻ cells are obtained by successively gating out CD3⁺, CD19⁺ cells (CD3, CD19 versus SSC-A), CD14⁺, CD10⁺ cells (CD14 versus CD10), BDCA-1⁺, BDCA-3^hi cells (BDCA-1 versus BDCA-3) and CD66b⁺, CD335⁺, BDCA-2⁺ cells (CD66b, CD335, BDCA-2 versus CD123). Lin⁻BDCA⁻CD123^−/lo and Lin⁻BDCA⁻CD123^hi populations are further subgated on the basis of the expression of CD34 versus CD117, CD135 versus CD116 and CD45RA versus CD115. In these gated populations, GMDPs are CD123^−/loCD34⁺CD117⁺CD135⁺CD116⁻CD115⁻CD45RA⁺; MDPs are CD123^−/loCD34⁺CD117⁺CD135⁺CD116⁻CD115⁺CD45RA⁺; CDPs are CD123^hiCD34⁺CD117⁺CD135⁺CD116⁺CD115⁻CD45RA⁺; and pre-cDCs are CD123^−/lo D34⁻CD117⁺CD135⁺CD116⁺CD115⁻CD45RA⁺SSC-A^lo. FSC, forward scatter; SSC, side scatter. (d) Expression of CD45 by GMDPs, MDPs, CDPs and pre-cDCs from human BM. Histograms show the expression of stained (black line) and unstained (gray line) cells. (e) FMO control for CD115 staining used to discriminate between GMPs and MDPs in CB.

Figure 3

Characterization of human DC progenitors. (a) Workflow for the characterization of DC progenitors. (b) Expression of CD11c, HLA-DR, CD38 and CD33 by GMDPs, MDPs, CDPs and pre-cDCs from human BM. DC progenitors are defined as in Figure 2b. Histograms show expression (x axis) and number of cells (y axis) of stained (black line) and unstained (gray line) cells.

Figure 4

Gating strategy for the isolation of human DC progenitors. (a) Workflow for the isolation of DC progenitors. (b) Gate hierarchy for sorting. (c) Human BM MNCs were stained as indicated in Table 2 (panel #3). Progenitors are identified by first gating on live cells (FSC-A versus SSC-A). Lin⁻ BDCA⁻ cells are obtained by successively gating out CD3⁺, CD19⁺, CD14⁺ cells (CD3, CD19 and CD14 versus SSC-A), BDCA-1⁺, BDCA-3^hi cells (BDCA-1 versus BDCA-3) and CD66b⁺, CD335⁺, CD10⁺, BDCA-2⁺ cells (CD66b, CD335, CD10, BDCA-2 versus CD123). Lin⁻BDCA⁻CD123^−/lo and Lin⁻BDCA⁻CD123^hi populations are further subgated on the basis of the expression of CD34 versus CD117, CD135 versus CD116 and CD45RA versus CD115. In these gated populations, GMDPs are CD123^−/lo CD34⁺CD117⁺CD135⁺CD116⁻CD115⁻CD45RA⁺; MDPs are CD123^−/loCD34⁺CD117⁺CD135⁺ CD116⁻CD115⁺CD45RA⁺; CDPs are CD123^hiCD34⁺ CD117⁺CD135⁺CD116⁺CD115⁻CD45RA⁺; and pre-cDCs are CD123^−/loCD34⁻CD117⁺ CD135⁺CD116⁺CD115⁻CD45RA⁺SSC-A^lo. FSC, forward scatter; SSC, side scatter.

Figure 5

Gating strategy for culture output after hematopoietic differentiation on MS-5 stromal cells of human DC progenitors. (a) Workflow for isolation of DC progenitors. (b) CD34⁺ HSPCs from BM were isolated as in Figure 3c, and then cultured in MS5+FSG for 7 d. Culture output was assessed by flow cytometry using antibody panel #4, as indicated in Table 2. Cells developing in culture are identified by first gating on live cells (FSC-A versus SSC-A), and then on singlets (SSC-A versus SSC-W) and live cells (Live/Dead blue versus SSC-A). CD45⁺Lin⁻ cells are obtained by successively gating on CD45⁺ cells (CD45 versus SSC-A) and gating out CD3⁺ cells (CD3 versus SSC-A), CD19⁺ cells (CD19 versus SSC-A) and CD335⁺ cells (CD335 versus SSC-A). Flow cytometry plots show phenotype of output cells, including granulocytes (brown), BDCA-3^hi cDCs (red), BDCA-1⁺ cDCs (blue), monocytes (orange) and BDCA-2⁺ pDCs (green).

Figure 6

Proliferative capacity of human DC progenitors after hematopoietic differentiation on MS-5 stromal cells. (a) Workflow for culture proliferation of DC progenitors. (b) GMDPs, MDPs, CDPs and pre-cDCs were purified from CB, as shown in Figure 3c, labeled with CFSE, and cultured in MS5+FSG for 0.5, 1.5, 3 and 5 d; proliferation was assessed by flow cytometry using antibody panel #5, as indicated in Table 2. Flow cytometry plots show gated culture-derived cells, as defined in Figure 5b: CD45⁺ cells (gray), BDCA-1⁺ cDCs (blue) and BDCA-3^hi cDCs (red). (c) Graphs showing the output of BDCA-1⁺ cDCs (blue) and BDCA-3^hi cDCs (red) for 10³ GMDPs (5 d), MDPs (5 d), CDPs (3 d) and pre-cDCs (3 d) cultured in MS5+FSG, as illustrated in Figure 6b.