In vivo analysis of dendritic cell development and homeostasis

Abstract

Dendritic cells (DCs) in lymphoid tissue arise from precursors that also produce monocytes and plasmacytoid DCs (pDCs). Where DC and monocyte lineage commitment occurs and the nature of the DC precursor that migrates from the bone marrow to peripheral lymphoid organs are unknown. We show that DC development progresses from the macrophage and DC precursor to common DC precursors that give rise to pDCs and classical spleen DCs (cDCs), but not monocytes, and finally to committed precursors of cDCs (pre-cDCs). Pre-cDCs enter lymph nodes through and migrate along high endothelial venules and later disperse and integrate into the DC network. Further cDC development involves cell division, which is controlled in part by regulatory T cells and fms-like tyrosine kinase receptor-3.

Fig. 1

Isolation of pre-cDCs. (A) Presence of MDPs and CDPs in the bone marrow (BM), blood or spleen (SP); not found (NF). (B) Identification of pre-cDCs in bone marrow, LNs, spleen and blood. Bar graph shows the percentage of pre-cDC in each organ. 1×10⁶ cells were acquired per sample. Lin: CD3/CD19/Ter119/NK1.1/B220. (C) Donor-derived spleen cells (CD45.2⁺) were analyzed for cDC (CD11c⁺ MHC class II⁺), pDC (CD11c^int B220⁺) and monocytes (CD11b⁺ CD11c^lo/−) 7 days after intravenous injection of 2×10⁶ bone marrow cells or 1×10⁵ pre-cDCs. (D) Analysis of donor-derived splenocytes after transfer with the indicated number of pre-cDCs from bone marrow, spleen and blood. (E) Chimerism in parabiotic mice. In (B) and (C) left side shows representative dot plots. Bar graphs summarize 2-4 independent experiments with 3-4 mice each. Error bars represent the mean ± SEM.

Fig. 2

Progenitor-product relationship among MDP, CDP and pre-cDC. (A) Phenotype of myeloid progenitor (MP, Lin⁻ Sca-1⁻ c-Kit^high CX₃CR1⁻), MDP (CX₃CR1⁺ c-Kit^hi) and CDP (CD115⁺CX₃CR1⁺c-Kit^lo) in Cx₃cr1^gfp/+ mice. (B) Donor-derived bone marrow cells analyzed by flow cytometry for presence of MDPs, CDPs and pre-cDCs two days after intra-bone marrow transfer of 5×10⁴ MPs, MDPs or CDPs. (C) Donor-derived splenocytes analyzed for the presence of cDCs, pDCs and monocytes 7 days after transfer of MDPs or CDPs. (D) EGFP expression in indicated cell populations in LysM-Cre x Rosa26-StopfloxEGFP reporter mice (fig. S8). EGFP index = % of EGFP⁺ cells / % of EGFP⁺ Gr1^high blood monocytes (20). In (C) and (D) left side shows representative dot plots. Bar graphs summarize 2-4 independent experiments with 3-4 mice. Error bars represent the mean ± SEM.

Fig. 3

Multi-photon imaging of pre-cDCs. (A) Transferred cells in inguinal LNs 5hrs (upper panel, green) and 6d (lower panel, cyan) after pre-cDC transfer. Panels at right show morphology of transferred pre-cDCs (arrowheads). Scale bars: 1 mm (left) and 30 μm (right). (B) Left: CD62L expression on pre-cDCs. Right: Graph shows number of LN cDCs or migratory DCs (mDCs) after treatment with anti-CD62L. Graph represents the mean ± SD, n=4 mice per group in 3 experiments. * p = 0.0286; ** p < 0.0001, Student’s T test. (C) Dynamic behavior, morphology, and position of pre-cDCs at indicated times. A cell track (yellow) is superimposed to visualize displacement. Scale bar = 50 μm. Far right shows superimposed 2D (XY) tracks with starting coordinates set to the origin. Number of cells analyzed (n) is indicated. (D) Graphs show velocity and confinement of pre-cDCs at indicated times. Differences between columns are significant by Kruskall-Wallis test (heterogeneity: speed, p =0.0006, meandering, p = 0.0055; * p<0.05, ** p<0.01, *** p<0.001, Dunn’s multiple comparison test). All multi-photon data represent at least two independent experiments.

Fig. 4

Tregs control DC expansion in the peripheral lymphoid organs. (A) Numbers of MDP, pre-cDCs and cDCs in the indicated organs after Treg depletion in Foxp3^DTR mice. (B) Pre-cDC and cDC division in spleen and LNs at indicated time after Treg depletion. (C and D) Effect of the Flt3 inhibitor, Sutent, on Treg-depletion induced cDC and B cell expansion. (E) Bar graph shows relative percentage of Flt3^−/− CD45.1 and Flt3^+/+ CD45.2 DCs and B cells after mixed bone marrow transfer into FoxP3^DTR CD45.1XCD45.2 mice after DT treatment. Panels are representative of two-four independent experiments. Bar graph represents the mean ± SEM, n=3-4.