The early progenitors of mouse dendritic cells and plasmacytoid predendritic cells are within the bone marrow hemopoietic precursors expressing Flt3

Abstract

Flt3 ligand (Flt3L) is a growth factor for hemopoietic progenitors and can promote the expansion of both conventional dendritic cells (DCs) and plasmacytoid predendritic cells (p-preDCs). The cells responding to Flt3L treatment and the precursors for the DCs and p-preDCs had not been fully characterized. We examined different mouse bone marrow (BM) hemopoietic precursor populations for the surface expression of Flt3 and tested them for early DC and p-preDC precursor activity. Most DC precursor activity, other than that given by multipotent hemopoietic stem cells, was within the downstream precursors expressing Flt3. The majority of mouse BM common lymphoid precursors expressed high levels of Flt3 and these were the most efficient precursors of both DCs and p-preDCs. In contrast, only a small proportion of the common myeloid precursors (CMPs) expressed Flt3, but the precursor activity for both DCs and p-preDCs was within this minor Flt3+ CMP fraction. The granulocyte and macrophage precursors and pro-B cells did not express Flt3 and had no DC or p-preDC precursor activity. These findings demonstrate that the early precursors for all DC subtypes are within the BM Flt3+ precursor populations, regardless of their lymphoid or myeloid lineage orientation.

Figure 1.

Surface expression of Flt3 by mouse BM cells and by different hemopoietic precursor populations. Total BM cells were stained with PE-conjugated anti-Flt3 antibody together with other fluorescence-conjugated antibodies against different surface molecules expressed by BM cells including CD19, B220, CD3, Mac-1, Gr-1, MHC class II, and c-kit. The expressions of these molecules on the gated Flt3⁺ BM cells are shown in A. The solid lines represent the staining of different surface molecules and the dashed lines represent the isotype controls. The precursor populations from mouse BM or thymus were first enriched for lineage marker–negative cells and then distinguished by four color fluorescence staining as (B) CLP (c-kit⁺ CD127⁺), (C) CMP (CD34⁺ CD16/32^lo) and GMP (CD34⁺ CD16/32^hi), (D) intrathymic CD4^lo lymphoid precursors (c-kit⁺ Thy-1^lo), and (E) BM pro-B precursors (c-kit⁺ CD19⁺). The surface expression of Flt3 by each precursor population was then examined. The contour plots display the gating for each precursor population (cells within the boxes). The histograms show the Flt3 expression on each gated precursor population. The solid lines represent the Flt3 staining and the dashed lines represent the isotype controls. Data presented in this figure is representative of at least three similar experiments on each precursor population.

Figure 2.

Generation of DCs by BM Flt3⁺ and Flt3⁻ cell populations. BM cells from Ly5.2 mice were stained with PE-conjugated Flt3 antibody. The Flt3⁺ and Flt3⁻ cell fractions were purified by cytometric sorting. The purified cells were then i.v. transferred into lethally irradiated Ly5.1 recipient mice. The generation of DCs in the spleen of recipient mice was examined 2 wk after cell transfer. The donor-derived DCs were distinguished as Ly5.2⁺ CD11c⁺ cells from the DC-enriched spleen cell preparation. The data presented is typical of two such experiments.

Figure 3.

Generation of DCs and other hemopoietic lineage cells by Flt3⁺ and Flt3⁻ fractions of CMPs. The Flt3⁺ and Flt3⁻ fractions of CMPs were purified from the BM of Ly5.2 mice and i.v. transferred to lethally irradiated Ly5.1 recipient mice. The donor-derived cells were distinguished as Ly5.2⁺ cells. For DC production, recipient spleens were analyzed 2 wk after precursor transfer. Donor-derived DCs were identified as Ly5.2⁺ CD11c⁺ cells from DC-enriched spleen cell preparations (A). The subsets of donor-derived DCs were further segregated by their expression of CD4 and CD8 (A). For myeloid cell production, the recipient BM cells were analyzed 1 wk after precursor transfer for donor-derived Ly5.2⁺ Mac-1⁺ Gr-1⁺ cells (B). For B cell and T cell production, the recipient spleen and thymus were analyzed 2 wk after precursor transfer for donor-derived Ly5.2⁺ B220⁺ or Ly5.2⁺ CD4⁺ cells (B). The data presented is typical of three such experiments. Each experimental group contained two to three recipient mice.

Figure 4.

Comparison of the generation of DCs by different hemopoietic precursor populations. Different precursor populations were purified from C57BL/6 Ly5.2⁺ mice by FACS^® sorting and transferred i.v. into irradiated Ly5.1 recipient mice. 2 wk after precursor transfer, the recipient thymus and spleen were analyzed for donor-derived DCs. Each bar represents the average DC number produced by 10⁴ precursor cells from three experiments. Each experimental group contained two to three recipient mice.

Figure 5.

Generation of p-preDCs by different hemopoietic precursor populations. Different BM precursor populations were purified from C57BL/6 Ly5.2 mice and i.v. transferred to lethally irradiated Ly5.1 recipient mice. 2 wk after precursor transfer, recipient spleens were analyzed for donor-derived p-preDCs. The DC-enriched cell preparations were stained with anti–Ly5.2-FITC, anti–CD45RA-PE, and anti-CD11c-Alexa 594. The donor-derived cells were distinguished as Ly5.2⁺ cells (top). The donor-derived p-preDCs were identified as CD45RA⁺ CD11c^int cells within the gated Ly5.2⁺ cells (bottom, cells within the boxes). The data presented is representative of three similar experiments on each precursor population. Each experimental group contained two to three recipient mice.

Figure 6.

Comparison of the generation of p-preDCs by different hemopoietic precursor populations. Different precursor populations were purified from C57BL/6 Ly5.2⁺ mice by FACS^® sorting and transferred i.v. into irradiated Ly5.1 recipient mice. 2 wk after precursor transfer, the recipient thymus and spleen were analyzed for donor-derived p-preDCs. Each bar represents the average number of p-preDCs produced by 10⁴ precursor cells from three experiments. Each experimental group contained two to three recipient mice.

Figure 7.

The proposed developmental pathways for DCs and p-preDCs. Both conventional DCs and p-preDCs can develop from BM Flt3⁺ precursors regardless of whether this involves the “myeloid pathway” or the “lymphoid pathway.” The relationship of the Flt3⁺ lymphoid or myeloid precursors to the recently described common DC precursors currently remains unknown. HSC, hemopoietic stem cells; HPC, multipotent hemopoietic progenitor cells; CLP, common lymphoid precursors; CMP, common myeloid precursors; GMP, granulocyte and macrophage precursors; pro-T, intrathymic CD4^lo precursors; pro-B, B220⁺ CD19⁺ c-kit⁺ B cell precursors. The most efficient precursors of DCs and p-preDCs are the Flt3-expressing BM precursors, shown as cells within the boxes. The thickness of the lines is relative to the efficiency of the precursors in producing DCs or p-preDCs. The dashed lines represent proposed developmental pathways yet to be confirmed by direct evidence.