HIF-1α and ID2, but not HIF-2α, limit pDC development in vivo. (A-D) BM, blood, and spleen of age-matched WT, HIF-1α^fl/fl LysM-Cre (HIF-1-KO), or HIF-2α^fl/fl LysM-Cre (HIF-2-KO) mice was analyzed by flow cytometry as indicated in supplemental Methods and supplemental Figure 6. (A) Quantification of the indicated cell populations per microliter of blood is shown. Individual data points correspond to 1 animal each (6-8 animals were analyzed in groups of 2 or 3 in 3 independent experiments. Red represents mean values. (B) Quantification of the indicated cell populations per milligram of spleen is shown. Individual data points correspond to 1 animal each (6 animals were analyzed in groups of 2 in 3 independent experiments. Red represents mean values. (C) Quantification of the indicated cell populations in the entire BM harvested from both hind limbs. Individual data points correspond to 1 animal (6 animals were analyzed in groups of 2 in 3 independent experiments. Red bars represent mean values. (D) The sum of total pDCs from BM, spleen, and peripheral blood from 6 animals of each genotype. (E) Mammary tumors were extracted from 100-day-old HIF-1α^fl/fl LysM-Cre (HIF-1-KO) or WT mice expressing the PyMT oncogene. Single-cell suspensions were analyzed by polychromatic flow cytometry as indicated in supplemental Figure 6. Quantitative analysis of relative pDC amounts within the total immune cell population. Individual data points corresponding to 1 animal each (9 animals were analyzed in at least 6 independent experiments. Red bars represent mean values. (F) pDCs were isolated from spleens and BM of WT and HIF-1α^fl/fl LysM-Cre mice. Spleen pDCs were isolated from single-cell suspensions using untouched magnetic sorting and BM pDCs by untouched magnetic sorting followed by further enrichment using FACS sorting. Relative mRNA expression of exon 2 of the HIF-1α gene transcript and of ID2 quantified by quantitative PCR. pDCs of 6 animals of each genotype were analyzed in groups of 2 in 3 independent experiments. (G) MDPs and CDPs were isolated from whole BM of WT or HIF-1-KO mice (see supplemental Methods and supplemental Figure 4), pooled and analyzed for relative ID2 mRNA expression. MDPs/CDPs of 6 animals of each genotype were analyzed in groups of 2 in 3 independent experiments. (H) BM, blood, and spleen of age-matched ID2^−/− (ID2-KO) mice and the respective WT control animals were analyzed by flow cytometry as indicated in supplemental Methods and supplemental Figure 6. Quantification of the indicated cell populations per microliter of blood, per milligram of spleen and in the entire hind limb BM. Individual data points correspond to 1 animal (6-8 animals were analyzed in groups of 2 or 3 in 3 independent experiments. Red bars represent mean values. (I) The sum of total pDCs from BM, spleen, and peripheral blood from 6 WT or ID2-KO. Data were analyzed using GraphPad Prism Version 5.0 for Windows. P values were calculated using Student t test (C-E, G-I) or 1-way ANOVA (A,B,F) with Bonferroni correction. Significant differences between experimental groups: *P < .05, **P < .01, ***P < .001.