The receptor tyrosine kinase Flt3 is required for dendritic cell development in peripheral lymphoid tissues

Abstract

Dendritic cell (DC) development begins in the bone marrow but is not completed until after immature progenitors reach their sites of residence in lymphoid organs. The hematopoietic growth factors regulating these processes are poorly understood. Here we examined the effects of signaling by the receptor tyrosine kinase Flt3 on macrophage DC progenitors in the bone marrow and on peripheral DCs. We found that the macrophage DC progenitor compartment was responsive to superphysiological amounts of Flt3 ligand but was not dependent on Flt3 for its homeostatic maintenance in vivo. In contrast, Flt3 was essential to the regulation of homeostatic DC development in the spleen, where it was needed to maintain normal numbers of DCs by controlling their division in the periphery.

Figure 1

Identification of cDC progenitors. (a) Flow cytometry of Lineage-negative (Lin⁻ = CD3⁻CD19⁻B220⁻NK1.1⁻CD11c⁻CD11b⁻Ter119⁻Gr-1⁻) BM (top) and spleen (bottom) cells analyzed for the expression of Kit and Sca-1 (left column). Sca-1⁻ (middle column) but not Sca-1⁺ (right column) cells in the BM express CSF1R. This analysis was performed twice on spleen cells and over 50 times on BM cells. (b) Colony assays in semisolid medium. Total BM (squares), Lin⁻CSF1R⁺ (triangles), or Lin⁻CSF1R⁻ (circles) cells were cultured in the presence of erythropoietin and IL-3 (burst forming unit-erythroid cell, BFU-E), or erythropoietin alone (colony forming unit-erythroid cell, CFU-E). Colony growth in GM-CSF, CSF1, and Flt3L was analyzed after 7 days. Colony assays were performed 4 times in duplicates. (c) Flow cytometry of spleen cells 14 days after adoptive transfer of purified Lin⁻CSF1R⁺Kit⁻ (middle), or Lin⁻CSF1R⁺Kit⁺ (bottom) cells. Spleen cells were gated on live (DAPI⁻) CD3⁻CD19⁻NK1.1⁻Ter119⁻, donor cells (CD45.2⁺), and analyzed for expression of Gr-1 (polymorphonuclear neutrophils, PMN), F4/80 (red-pulp macrophages, RP-Mp), PDCA1 (pDC), CD11b, CD11c, and CD8 (monocytes and cDC). CD8⁺ cDC are depicted in dark green dots. Data are representative for 10 transfers. (d) Flow cytometry showing Lin⁻ BM cells analyzed for the expression of CX₃CR1, Kit and CSF1R (top), or CSF1R, Kit and Flt3 (bottom) showing phenotype correlation between Lin⁻Kit⁺CX₃CR1⁺ (MDP* 5), Lin⁻CSF1R⁺ cells (MDP^Δ, this paper), and CDP. The original MDPs were defined as Lin⁻Kit⁺CX₃CR1⁺ cells (MDP* top left). Analysis was repeated twice. (e) Flow cytometry of Lin⁻CX₃CR1⁺Kit⁺ (MDP5) (top), Lin⁻CX₃CR1⁺Kit⁻ (middle), and Lin⁻CSFR1⁺Kit⁻ (bottom) cells analyzed 14 days after transfer. Dot plots were gated on donor–derived cells (CD45.2⁺). Transfers were performed twice.

Figure 2

Effects of Flt3 ligand (Flt3L) stimulation. (a) Flow cytometry of spleen cells (top) gated on CD3⁻CD19⁻NK1.1⁻Ter119⁻ and BM cells (bottom) gated on Lin⁻ (CD3⁻CD19⁻B220⁻NK1.1⁻CD11b⁻CD11c⁻Gr1⁻Ter119⁻) Sca-1⁻ cells from mice injected with Flt3L or PBS daily for 8 days. Colors represent distinct myeloid cell types as described above dot plots including percentages of indicated populations (top). Analysis was performed 4 times. (b) Absolute number of spleen cells, cDC, pDC, monocytes, PMN and red pulp macrophages (top), and MDPs in BM (bottom) in response to sustained Flt3L stimulation over a time course of 8 days. Data are representative for 2 independent experiments. (c) Developmental potential of Lin⁻CSF1R⁺ cells with and without stimulation of Flt3L. Mice were stimulated with Flt3L over a time course of 6 days before purification of Lin⁻CSF1R⁺ cells. cDC yield was calculated based on 10⁵ injected cells. Data are representative for 4 independent experiments. (d) Injection of Flt3L results in mobilization of myeloid progenitors from the BM into the spleen. Mice were stimulated with Flt3L over a time course of 6 days. Spleen cells from Flt3L treated (CD45.2⁺) and congenic untreated mice (CD45.1⁺) were mixed, depleted for cDC, and injected into irradiated recipients (CD45.1⁺CD45.2⁺). Contribution of either donor to CD3⁻CD19⁻B220⁻NK1.1⁻Ter119⁻CD11b⁻CD11c⁻Gr1⁻ immature spleen cells is depicted for ‘Input’. Contribution of spleen cells from Flt3L treated (open bars) and untreated (closed bars) to spleen cDC, PMN, and monocytes was analyzed after 6 days. Data are representative for 2 independent experiments.

Figure 3

Myeloid compartments in Flt3^−/− mice. (a) Representative flow data showing reduced numbers of cDC, pDC in Flt3^−/− mice. Spleen cells from 4 week old mice were gated on CD3⁻CD19⁻NK1.1⁻Ter119⁻ cells (top), and the relative contribution of myeloid cells was determined in wild-type (left) and Flt3^−/− (right) mice. Spleen cells were counted (f) and absolute cell numbers calculated for pDC (CD3⁻CD19⁻NK1.1⁻Ter119⁻Gr-1⁻PDCA1⁺, b), cDC (CD3⁻CD19⁻NK1.1⁻Ter119⁻Gr-1⁻PDCA1⁻F4/80⁻CD11c⁺, c), Monocytes (CD3⁻CD19⁻NK1.1⁻Ter119⁻PDCA1⁻F4/80⁻CD11c⁻Gr-1^−/loCD11b⁺, d), and PMN (CD3⁻CD19⁻NK1.1⁻Ter119⁻Gr-1⁺, e). To determine frequencies of intrasplenic cDC progenitors medium density cells were counted (g), and numbers of pre-cDC calculated (CD3⁻CD19⁻NK1.1⁻Ter119⁻CD45RA^−/loCD11c⁺CD43⁺SIRP-α^lo, h). Absolute cell numbers of BM cells were counted (i) and MDP (Lin⁻CSF1R⁺) numbers calculated (j). Each point represents one mouse assayed at 2, 4 or 9 weeks of age as indicated.

Figure 4

DC development in Flt3^−/− bone marrow chimeras. (a) Absolute numbers of wild-type (closed squares) and Flt3^−/− (open squares) spleen PMN, RP–macrophages, monocytes, pDC, and cDC in bone marrow chimeras 4 months after injection of Lin⁻Kit⁺ cells into lethally irradiated recipients. Each square represents an individual mouse. (b) Mixed bone marrow chimeras. Relative contribution of Flt3^−/− (CD45.2⁺) cells to BM HSC (KSL, Kit⁺Sca–1⁺Lin⁻), MDP (Lin⁻CSF1R⁺), and spleen T cells (CD3⁺), B cells (CD19⁺), PMN, monocytes, RP–macrophages, pDC and cDC was determined 1.5 months after transfer. Data was pooled from 3 recipient mice and is representative for 2 independent experiments. (c) Parabiosis between wild-type controls and wild-type or Flt3^−/− mice for 5 weeks. Dot plots were gated on spleen T cells (CD3⁺) (orange, upper panels) and cDCs (CD3⁻CD19⁻Ter119⁻NK1.1⁻Gr1⁻PDCA1⁻F4/80⁻CD11c^hi) (blue, lower panels), and analyzed for contribution of either parabiotic partner. Origin of cells was determined using distinct CD45 isoforms (wild-type : wild-type CD45.1⁺ : CD45.2⁺; wild-type : Flt3^−/− CD45.1⁺ : CD45.2⁺).

Figure 5

Flt3 in peripheral expansion. (a) Adoptive transfer of mixed BM Lin⁻CSF1R⁺ (MDP, left) or white blood cells (WBC, right) from wild-type and Flt3^−/− mice into mildly irradiated wild-type (top) or Flt3L^−/− (bottom) recipient mice. Plots show percentage contribution of wild-type (open bars) and Flt3^−/− (closed bars) to the input and to reconstituted cDCs after 11 days. Data from 3 independent experiments are pooled. Contribution of Flt3^−/− cells after transfer into wild-type mice is significantly reduced (MDP in wild-type: P = 0.001, WBC in wild-type: P = 0.04, MDP in Flt3L^−/−: P = 0.2, WBC in Flt3L^−/− P = 0.2; analyzed by paired t-test). (b,c) Reduced BrdU incorporation into Flt3^−/− cDC compared to wild-type cells in the same microenvironment. (b) Wild-type versus Flt3^−/− mixed BM chimeras were generated and BrdU incorporation into cDC (CD3⁻CD19⁻Ter119⁻NK1.1⁻F4/80⁻PDCA1⁻CD11c⁺) measured. Dot plots show representative FACS analysis of BrdU incorporation into cDC after a pulse of 2 hours. WT : WT chimeras (top) were analyzed in parallel with Flt3^−/− : WT chimeras (bottom). Percentages indicate frequencies of BrdU incorporation into either CD45.2⁺ or CD45.2⁻ (WT : WT or WT : Flt3^−/−) cells in mixed BM chimeras. (c) Plots represent pooled data of BrdU incorporation into MDP (top) and cDC (bottom) after a pulse of 2 hours (left) or into pDC (top) and cDC (bottom) after a pulse of 4 days (right). BrdU incorporation into Flt3^−/− cDC was significantly reduced after a pulse of 2 hours (P = 0.018) or 4 days (P = 0.011). BrdU incorporation was not significantly different in Flt3^−/− MDP after a pulse of 2 hours (P = 0.26) or in Flt3^−/− pDC after a pulse of 4 days (P = 0.56). P values were determined by paired t-test comparing the increments in BrdU incorporation in either WT : WT or Flt3^−/− : WT cells in the same mouse. (d) Carboxyfluorescein diacetate succinimidyl diester (CFSE) dilution 5 days after transfer of mixed Lin⁻CSF1R⁺ wild-type (CD45.1⁺) and Flt3^−/− (CD45.2⁺) cells into irradiated recipient mice. Cells were gated on CD3⁻CD19⁻NK1.1⁻Ter119⁻Gr1⁻PDCA1⁻CD11c^hiMHCII⁺ cells (left dot plot), and contribution of both donor cell types determined after exclusion of recipient cDC (right dot plot). Histograms depict retention of CFSE in wild-type (green color, upper histogram) or Flt3^−/− (orange color, lower histogram) cells. Data is representative for three independent experiments. (e) CFSE dilution 6 days after transfer of wild-type spleen and LN cells (7 × 10⁶) into wild-type (left) or Flt3L^−/− (right) mice. Histograms were gated on donor CD11c^hi cells and depict level of CFSE labeling. Shown are 2 out of 6 different recipient mice for each group.