Supplementary Figure 1: pDCs develop primarily from IL-7R⁺ lymphoid progenitors.

(a-c) CDPs, IL-7R⁺ LP and CSF1R⁻IL-7R⁻ NP progenitors as gated in Fig. 1a were cultured over 8 days in the presence of FLT3L (b) or FLT3L and OP9 stromal cells (a,c). Shown are two color histograms for the expression of SiglecH and CD19 at day 4 of culture (a). Relative cell output for pDCs (SiglecH⁺ CD317⁺CD19⁻), cDC1 (SiglecH⁻CD317⁻CD19⁻CD11c⁺MHCII⁺CD24⁺CD11b⁻), cDC2 (SiglecH⁻CD317⁻CD19⁻CD11c⁺MHCII⁺CD24⁻CD11b⁺) and B cells (CD19⁺) was determined at day 4, 6 and 8 as indicated (n=6 independent experiments, thin line represents the mean +/-s.d.). (d-l) Sort purified progenitors as defined in methods were isolated from CD45.1 and CD45.2 mice and used in a 1:1 ratio for in vitro or in vivo experiments as explained in Supplementary Fig. 1i. (d,e) Shown are representative two-color histograms pre-gated on SiglecH⁺CD11c⁺ pDCs (d) or pre-gated on SiglecH⁻CD11c⁺MHCII⁺ cDCs (e) of IL-7R⁺ LP (CD45.1) and CDPs (CD45.2) cultured over 8 days in the presence of FLT3L. (n=3 independent experiments). (f) Shown are representative two-color histograms for the expression of CD45.1 and CD45.2 pre-gated on CD19⁺ B cells of progenitor subsets, as indicated co-cultured for 4 days in the presence of FLT3L and OP9 stromal cells. (g,h) Percent output of CD45RA⁺ SiglecH⁺ pDCs (g) and CD19⁺ B cells (h) of progenitors cultured as in (f) (n=3 independent experiments, each dot represents a mouse, thin line represents the mean +/-s.d.). (j-k) Bone marrow and splenic SiglecH⁺CD317⁺pDC output was determined 4 days after in vivo i.v. co-transfer of CD45.1 and CD45.2 Lin⁻B220⁻Ly6C⁻CD117^hiCD135⁺ (c-kit^hi) progenitors. Shown are representative two-color histograms for the expression of CD45.1 and CD45.2 (j) or percent donor derived pDCs in BM and spleen (k) (n=3 independent experiments). (l) CD19⁺ BM B cells and splenic SiglecH⁻CD317⁻CD19⁻CD11c⁺MHCII⁺ cDC output was determined 4 days after in vivo i.v. co-transfer of CD45.1 CDPs and CD45.2 IL-7R⁺ LP (n=6 independent experiments). Shown are representative two-color histograms for the expression of CD45.1 and CD45.2. Statistical analysis was done using two-way ANOVA with Tukey post-test (g,h,k) (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).