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 CDP⁷. The original MDPs were defined as Lin⁻Kit⁺CX₃CR1⁺ cells⁵ (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 transfer³³. Dot plots were gated on donor–derived cells (CD45.2⁺). Transfers were performed twice.