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Blood, Vol. 111, Issue 4, 1942-1945, February 15, 2008
The BXH2 mutation in IRF8 differentially impairs dendritic cell subset development in the mouse
Blood Tailor et al.
111: 1942
Supplemental materials for: Tailor et al
Files in this Data Supplement:
- Figure S1. Analysis of CD4+ and CD8α+ DCs in spleens from IRF8WT and IRF8R294C mice (JPG, 29.2 KB)
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Splenic DC population is gated as CD11chigh subset and further analyzed for CD4+, CD8 + and CD4-CD8-DCs.
- Figure S2. Lack of CD8α+ DCs in MuLV producing BXH2 mice (JPG, 82.3 KB)
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(A) Splenic CD11c+ low density cells were tested for pDCs with B220 expression. (B) CD11chigh cells were analyzed for CD4 and CD8 expression. Genotyping protocol: IRF8R294C and IRF8WT mice were distinguished by PCR genotyping of the mouse tail DNA using Hot Star Taq master mix from Qiagen. Two separate PCR reactions were set up using the forward primer (5′-GCCTCTCATGGGCTGAGATTA-3′) and BXH2-RW primer (5′¬TGCCGCTGCAGAACACGCG-3′) or BXH2-RM (5′-TGCCGCTGCAGAACACGCA-3′) yielding PCR products from WT and R294C mutated IRF-8 allele respectively. Primers (PipWTS2:5′¬GCAATGGGAAACTCCGACAGT-3′ and PipWTAS2: 5′-CAGCGTCCTCCTCACGATTGT-3′) amplifying IRF-4 gene were included in each PCR reaction as internal positive control. Initial 15 min incubation at 95°C was followed by 28 cycles of 94°C-30 sec, 66°C-45 sec and 72°C for 1min along with final extension of 72°C for 10 min.
- Figure S3. Rescue of cytokine production (JPG, 32.6 KB)
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IRF8KO BM progenitor cells were transduced with MSCV-puro empty vector (control), MSCV-IRF8R294C and MSCV-IRF8WT, cultured in the presence of Flt-3L for 7 days and stimulated with TLR ligands. IRF8R294C could rescue the function of IFN production but not that of IL12p40 production correlating with the ability to support development of pDCs but not CD8+ DCs. Protocol for gene transfer experiment: Bone marrow mononuclear cells for IRF8−/− mice were incubated (day -3) at 2 × 106 cells/ml in 24 well plate in presence of stem cell factor (SCF, 50 ng/ml) and IL-3 (6 ng/ml) 24 hr. Cells were subjected to retrovirus (MSCV-control, MSCV-IRF8R294C, MSCV-IRF8WT) transduction (day -2 and -1, twice) by spinoculation as described before (Tamura T., et al., J Immunol 2005, Tsujimura H., et al., Blood 2003). On day 0 cells were washed and cultured in presence of 100 ng/ml of Flt3-L, and subjected to puromycin (2µg/ml) selection on day 2 till day 7. Cells were harvested and subjected to flow cytometry characterization and TLR stimulation.
- Figure S4. Binding of IRF8R294C to IFNa4 and IL12p40 promoters in vivo detected by ChIP analysis (JPG, 29.7 KB)
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Chromatin was prepared form BMDCs from IRF8WT and IRF8R294C mice stimulated with New castle Disease virus (NDV) and binding of IRF8WT and IRF8R294C to the two promoters was analyzed using anti-IRF8 antibody. Results show that after stimulation, IRF8R294C bound to the IFNa4 promoter after stimulation, although to a lower degree than IRF8WT. In contrast, IRF8R294C completely failed to bind to the IL12p40 promoter, although IRF8WT bound to the promoter. These data are in line with the functional phenotype of IRF8R294C DCs as well as EMSA results in Figure 2C. Procedures for ChIP analysis with NDV-stimulated DCs are described in Tailor, P et al, Immunity 27:228, 2007.
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