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.