Genetic deletion of RBPJ does not rescue Ik^L/L pDC differentiation.

<p><b>(A)</b> Representative analysis of Flt3L-supplemented cultures of BM cells from compound mutant mice with Ik^L/L and/or RBPJ KO alleles, after addition of DMSO or GSI at day 0. Cultures were analyzed 8d later. <b>(B)</b> Numbers of total cells and pDCs obtained from cultures described in (A) (mean±SD from 3–5 mice per genotype; p values were obtained by paired Student’s t-test). <b>(C)</b> Analysis and <b>(D)</b> relative numbers of MDPs and CDPs from the BM of Ikaros-RBPJ compound mutant mice (representative of 2 independent experiments with 2–5 mice per genotype and per experiment; p values were obtained by Student’s t-test). *p≤0.05; **p≤0.01; ***p≤0.001.</p

Inhibition of γ-secretase rescues Ik^L/L pDC differentiation in vitro.

<p><b>(A)</b> Percentage of CD11c⁺CD317⁺CD11b^- pDCs after 8 days of Flt3L-supplemented WT and Ik^L/L BM cultures, treated with GSI or vehicle (DMSO) at the indicated days of culture. Representative of >5 independent experiments. <b>(B)</b> Percentage of pDCs from Flt3L-supplemented WT and Ik^L/L BM cultures, treated with GSI or DMSO at day 0 and analyzed at day 8. <b>(C, D)</b> Numbers of pDCs (C) and total cell numbers (D) obtained from cultures described in (B). *p≤0.05; **p≤0.01; ***p≤0.001 (Student’s t-test). <b>(E)</b> CCR9, Ly49Q and B220 expression on pDCs cultured as in (B). Representative of 3 independent experiments. <b>(F)</b> RT-qPCR analysis of <i>Ifna</i> expression induced from pDCs after in vitro culture. WT and Ik^L/L pDCs were sorted at d8 of culture, after GSI treatment at day 0, and stimulated for 16h with CpG ODN 1585. <i>Ifna</i> mRNA levels were measured by RT-qPCR and normalized to <i>Ubb</i> mRNA. nd: not done.</p

The TGFβ1 pathway is activated in Ik^L/L CDPs.

<p>CD45.2⁺ WT or Ik^L/L MDPs, and CDPs, were co-cultured with supporting CD45.1⁺ WT BM cells for 24h with Flt3L and GSI (or DMSO). CD45.2⁺ cells were then re-purified and their transcriptomes analyzed. 2 mice per condition. <b>(A)</b> Heat map representing K-means clustering of 963 genes differentially expressed between WT or Ik^L/L CDPs [fold change (FC)>1.5]. Clusters I and II are deregulated specifically in Ik^L/L CDPs. Clusters III and IV are deregulated in all Ik^L/L DC progenitors. Eng indicates the Endoglin gene. Red and green indicate high and low expression, respectively. <b>(B)</b> Hierarchical clustering of the genes from clusters II and IV in (A), using Immgen transcriptome data for DC progenitors and mature subsets (GSE15907). Clusters of genes similarly expressed between Ik^L/L CDPs and DC progenitors (Imm) or mature DCs (DC) are indicated. <b>(C)</b> K-means clustering of 70 genes differentially expressed between WT and Ik^L/L CDPs, and deregulated by GSI (FC>1.5). <b>(D)</b> Top 5 putative upstream regulators related to the 70 genes from (C), as identified by the Ingenuity Pathways Analysis software. <b>(E)</b> GSEA enrichment plots of genes specifically down-regulated [clusters I and III in (A)] and <b>(F)</b> up-regulated (clusters II and IV) in Ik^L/L CDPs. The ranked gene list corresponds to TGFβ1-regulated genes in CDPs, as identified by Felker et al (2010). NES: normalized enrichment score; FDR: false discovery rate. <b>(G)</b> Genome browser tracks showing Ikaros binding to loci associated with TGFβ activation in pre-B cells (BH1-Ik1-ER-Bcl2 cell line) and immature DN3 thymocytes (GEO GSE114629 and GSE61148 accession numbers).</p

Notch pathway activation in Ik^L/L pDCs.

<p><b>(A)</b><i>Hes1</i> and <i>Ptcra</i> mRNA expression in BM pDCs from 4 WT and 5 (Ik^L/L) mice, as analyzed by RT-qPCR and normalized to <i>Hprt</i> mRNA levels (mean±SD of triplicate data). (<b>B)</b> GFP reporter expression (black line) in total BM cells and BM pDCs (CD11c⁺CD317⁺) from Hes1-GFP⁺ WT and Ik^L/L mice, by flow cytometry. Grey histograms correspond to control cells from mice lacking the Hes1-GFP reporter. <b>(C)</b> Percentage of GFP⁺ BM pDCs (CD11c⁺CD317⁺) from Hes1-GFP⁺ WT and Ik^L/L mice, as analyzed in (B). *p≤0.05 (Student’s t-test). <b>(D)</b> CCR9 and SiglecH vs. GFP expression in BM pDCs from Hes1-GFP⁺ WT and Ik^L/L mice. Representative of 3 independent experiments.</p

Ikaros regulates DC progenitor development.

<p><b>(A)</b> Representative analysis of MDPs, CDPs, <b>(B)</b> pre-cDCs, and <b>(C)</b> pDCs from Ik^L/L (L/L) and WT BM, by flow cytometry. <b>(D)</b> Representative analysis of splenic cDCs (CD11c⁺MHCII⁺). <b>(E)</b> Relative numbers of BM MDPs, CDPs and pre-cDCs (as gated in A and B), <b>(F)</b> BM pDCs (as gated in C), and <b>(G)</b> splenic cDCs (as gated in D). Mean±SD of 4–12 animals per group. *p≤0.05; **p≤0.01; ***p≤0.001 (Student’s t-test).</p

TGFβ1 activation inhibits pDC development from Ik^L/L CDPs.

<p><b>(A)</b> CD105 expression on WT and Ik^L/L DC progenitors. <b>(B)</b> SiglecH vs. CD105 expression on BM pDCs (CD317⁺) and non-pDCs (CD317^-). Representative of 4 independent experiments. <b>(C)</b> Effect of the TGFβR1 inhibitor SB431542 on pDC differentiation in Flt3L-supplemented WT and Ik^L/L BM cultures. Cells were treated at d0 with SB431542 and/or GSI and analyzed at d8. Percentages of cells in the corresponding gates are indicated. Representative of 4 independent experiments. <b>(D)</b> Number of pDCs obtained from experiments described in (C). Data of the SB431542 treatments are shown at a larger scale in the lower panel. Representative of 4 independent experiments; 2 mice per genotype per experiment; p values were obtained with a Student’s t-test. *p≤0.05. <b>(E)</b> Schematic representation of Ikaros function during DC development in the BM. Ikaros and Notch signaling are required for the onset of DC differentiation and the appearance of MDPs and CDPs. Later, in CDPs, Ikaros promotes pDC development by antagonizing TGFβ1 signaling and by repressing the cDC gene expression program. HSPC: Hematopoietic stem/progenitor cell.</p