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. 2021 Oct 6;12(2):522–541. doi: 10.1158/2159-8290.CD-20-1513

Figure 7.

Figure 7. BPDCN-associated mutations in hematopoietic progenitors affect dendritic cell differentiation, RNA splicing, and activation signatures in vivo. A, Schematic of the in vivo experiment. sgRNA-transduced Cas9 knock-in bone marrow c-kit+ progenitors were injected into lethally irradiated wild-type recipient mice. Each guide was marked with either GFP or tagRFP. The percentage of double-positive cells in recipient bone marrow 8 weeks after transplantation is shown. B, Flow cytometry analysis of dendritic progenitor and mature pDCs in sgRNA-positive marrow cells, with groups compared by t test. C, GSEA of RNA-seq in control (NTG) and Zrsr2/Tet2-targeted (DKO) pDCs showing changes in signatures related to DC activation and IFNα receptor-dependent gene expression in the setting of viral infection. Heat map shows expression of the leading-edge genes in GSEA from low (blue) to high (red). MCMV, murine cytomegalovirus. D, Missplicing events in Zrsr2-, Tet2-, and Zrsr2/Tet2-targeted (DKO) pDCs. Events in each condition were calculated by pairwise comparisons between control knockout (n = 3) and Zrsr2-targeted (n = 2), Tet2-targeted (n = 3), and Zrsr2/Tet2-targeted (DKO; n = 3) biologically independent replicates. The number (and percentage) of events in each condition is shown in a single bar. The colors indicate different event types, intron retention (red), cryptic splice site (green), and exon skip (blue). E, Representative RNA-seq reads in Zrsr2-, Tet2-, and Zrsr2/Tet2-targeted (DKO) pDCs visualized on the same scale for Cecr5 (intron retention associated with Zrsr2 loss) and Adam19 (intron retention in Zrsr2- and Tet2-targeted pDCs with additive retention in Zrsr2/Tet2-targeted DKO pDCs). F, CD86 upregulation (ΔMFI) in vivo after systemic treatment with R848 compared with vehicle in control (NTG) or Zrsr2/Tet2-targeted DKO bone marrow pDCs (n = 3 independent animals/group, compared by t test). G, Left two panels, percentage of GFP+/tagRFP+ cells representing the indicated genotypes from in vitro cultures of Cas9 transgenic bone marrow expressing the indicated sgRNAs after vehicle or R848 treatment. Right, CD80 upregulation (ΔMFI) on pDCs of the indicated genotypes from R848 compared with vehicle-treated cultures. N = 3 independent cultures per genotype, groups compared by t test.

BPDCN-associated mutations in hematopoietic progenitors affect dendritic cell differentiation, RNA splicing, and activation signatures in vivo. A, Schematic of the in vivo experiment. sgRNA-transduced Cas9 knock-in bone marrow c-kit+ progenitors were injected into lethally irradiated wild-type recipient mice. Each guide was marked with either GFP or tagRFP. The percentage of double-positive cells in recipient bone marrow 8 weeks after transplantation is shown. B, Flow cytometry analysis of dendritic progenitor and mature pDCs in sgRNA-positive marrow cells, with groups compared by t test. C, GSEA of RNA-seq in control (NTG) and Zrsr2/Tet2-targeted (DKO) pDCs showing changes in signatures related to DC activation and IFNα receptor-dependent gene expression in the setting of viral infection. Heat map shows expression of the leading-edge genes in GSEA from low (blue) to high (red). MCMV, murine cytomegalovirus. D, Missplicing events in Zrsr2-, Tet2-, and Zrsr2/Tet2-targeted (DKO) pDCs. Events in each condition were calculated by pairwise comparisons between control knockout (n = 3) and Zrsr2-targeted (n = 2), Tet2-targeted (n = 3), and Zrsr2/Tet2-targeted (DKO; n = 3) biologically independent replicates. The number (and percentage) of events in each condition is shown in a single bar. The colors indicate different event types, intron retention (red), cryptic splice site (green), and exon skip (blue). E, Representative RNA-seq reads in Zrsr2-, Tet2-, and Zrsr2/Tet2-targeted (DKO) pDCs visualized on the same scale for Cecr5 (intron retention associated with Zrsr2 loss) and Adam19 (intron retention in Zrsr2- and Tet2-targeted pDCs with additive retention in Zrsr2/Tet2-targeted DKO pDCs). F, CD86 upregulation (ΔMFI) in vivo after systemic treatment with R848 compared with vehicle in control (NTG) or Zrsr2/Tet2-targeted DKO bone marrow pDCs (n = 3 independent animals/group, compared by t test). G, Left two panels, percentage of GFP+/tagRFP+ cells representing the indicated genotypes from in vitro cultures of Cas9 transgenic bone marrow expressing the indicated sgRNAs after vehicle or R848 treatment. Right, CD80 upregulation (ΔMFI) on pDCs of the indicated genotypes from R848 compared with vehicle-treated cultures. N = 3 independent cultures per genotype, groups compared by t test.