Supplementary Figure 6: Identification of subset-specific DC progenitors in vitro and in vivo.

Percentage of proliferating cells among total Siglec-H⁺ BM pre-DC subsets and total Siglec-H⁻ BM pre-DCs as determined by expression of Azami green expression in Fucci mice measured in vivo (data representative of 3 independent experiments with 7 mice in total, mean ± SEM, p: 0.0003, unpaired, two-tailed t-test, *p < 0.001, (a)). Flow cytometric analysis of B220 and MHC II expression on the progeny of CD45.2⁺ pre-DC subsets (CD45.2⁺Siglec-H⁺Ly6C⁻, CD45.2⁺Siglec-H⁺Ly6C⁺, CD45.2⁺Siglec-H⁻Ly6C⁺ or CD45.2⁺Siglech-H⁻Ly6C⁻ pre-DCs) added individually into day 2 in vitro CD45.1⁺ FLT3L-stimulated BM cultures. Cells depicted were harvested on day 3 or 6 and are gated CD45.2⁺CD11c⁺ expression (data representative of 3 independent experiments with 1 analyte per population (b)). Pie charts depict flow cytometric analysis of single Siglec-H⁺Ly6C⁻ pre-DC or Siglec-H⁺Ly6C⁺ pre-DC progeny cultured for 6 days on OP9 feeder cells in day 9 FLT3L-stimulated BM culture conditioned media. Progeny of single Siglec-H⁺Ly6C⁻ pre-DC or Siglec-H⁺Ly6C⁺ pre-DC was analyzed for the presence of pDC, cDC1 and cDC2 (n depicts number of clones analyzed, Siglec-H⁺Ly6C⁻ pre-DC n=24, Siglec-H⁺Ly6C⁺ pre-DC n=35 (c)). Flow cytometric analysis of phenotypes of splenic progeny from CD45.2⁺Siglec-H⁺Ly6C⁻, CD45.2⁺Siglec-H⁺Ly6C⁺, CD45.2⁺Siglec-H⁻Ly6C⁺ or CD45.2⁺Siglech-H⁻Ly6C⁻ pre-DCs injected into femurs of recipient mice 5 days earlier (data representative of 3 independent experiments with 1 analyte per pre-DC population, (d)). Schematic depiction of the DC development cascade in the bone marrow; CDPs as well as Siglec-H⁺Ly6C⁻ pre-DCs show potential to give rise to cDCs as well as pDCs, while acquisition of Ly6C on Siglec-H⁺Ly6C⁻ pre-DCs leads to the loss of pDC potential. Siglec-H⁺Ly6C⁺ pre-DCs are further differentiating in Siglec-H⁻Ly6C⁺ as well as Siglec-H⁻Ly6C⁻ pre-DC with selective potential for cDC2 or cDC1 respectively (e).