Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin stabilization directs splenic DC progenitors towards CD8α+ DC development. (A) Intracellular β-catenin expression in naïve Ex3fl/fl and Ex3DC−/− splenic CD11c+ cells. (B) Intracellular β-catenin levels in splenic CD4+ T cells isolated from Ex3fl/fl and Ex3DC−/− mice. The data show results from an individual mouse that is representative of of at least 3 experiments with 3–5 mice per group. (C) Western blot analysis of β-catenin in bone marrow-derived DC from Ex3fl/fl and Ex3DC−/− mice following cytoplasmic (C) and nuclear (N) fractionation. Antibodies against PARP and Rab5 were used for nuclear and cytoplasmic loading controls, respectively. The data are from 1 independent trial. (D and E) Comparison pre-cDC populations from the (D) bone marrow and (E) spleen of Ex3fl/fl and Ex3DC−/− mice by flow cytometry. Numbers in representative plots represent percentages of relevant populations within the indicated gate. Bar graphs show mean percentages plus standard error (S.E.) of relevant populations. The data represent the combination of 2 independent experiments (n=10 mice per group). (F) Levels of splenic pre-CD8α+ DC, defined as CD11c+CD8α−B220−CD24+, in Ex3fl/fl and Ex3DC−/− mice by flow cytometry. The data are representative of 3 independent experiments, each involving 4–5 mice per group. **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Expressing, Isolation, Western Blot, Derivative Assay, Fractionation, Comparison, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin stabilization expands splenic CD8α+ and plasmacytoid DC populations. (A–G) Mature DC subset analysis of naïve Ex3fl/fl and Ex3DC−/− splenocytes by flow cytometry. (A and B) Percentage and total number of CD11c+ cells in Ex3fl/fl and Ex3DC−/− spleens. (C–E) Percentage and total number of (C and D) CD8α+ DC and (C and E) CD11b+ DC in naïve Ex3fl/fl and Ex3DC−/− spleens. The data are representative of at least 3 independent experiments (n=3–5 mice per group). (F and G) Percentage and total number of B220+PDCA-1+ plasmacytoid DC in naïve Ex3fl/fl and Ex3DC−/− spleens. (H and I) Mature DC subset analysis of naïve Ex3fl/fl and Ex3DC−/− lung tissue by flow cytometry. (H) Plots from representative mice and (I) percentage of CD103+CD11b− lung DC for multiple mice are shown. (J–L) Mature DC subset analysis of naïve Ex3fl/fl and Ex3DC−/− intestinal lamina propria by flow cytometry. (J) Plots from representative mice and percentages of (K) CD103+CD11b− and (L) CD103+CD11b+ intestinal DC for multiple mice are shown. Dots in relevant graphs represent results from individual mice. Bar graphs display means and standard errors of individual mice. The data are representative of at least 2 independent experiments (n=3–5 mice per group). *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin signaling controls Irf8 expression. (A) Semi-quantitative PCR analysis of Nfil3, Batf3, Id2, and Irf8 mRNA in CD11c+ splenocytes magnetically purified from naïve Ex3fl/fl and Ex3DC−/− mice. mRNA levels were normalized to GAPDH. The data are representative of 2 independent experiments (n=2–3 mice per group) (B) Representative flow cytometric plots of IRF8 expression by Ex3fl/fl and Ex3DC−/− CD8α− and CD8α+ splenic DC. (C) MFI of IRF8 within Ex3fl/fl and Ex3DC−/− CD8α− and CD8α+ DC and (D) the percent of CD8α+ DC expressing IRF8 are shown. Dots represent results from individual mice. The data are the combined results of 2 experiments, and the experiment was independently performed at least 3 times (n=4–5 mice per group). (E) Representative FACS plot of IRF4 expression and IRF4 MFI in Ex3fl/fl and Ex3DC−/− CD11c+ splenocytes. The data are representative of 3 independent experiments (n=4 mice per group). (F) Chromatin immunoprecipitation of naïve Ex3DC−/− Flt3L DC cultures with control IgG or β-catenin antibody followed by quantitative PCR to determine Irf8 promoter occupancy. DNA levels were normalized to 1% input chromatin. The data are representative of 2 independent experiments. (G) Quantitative PCR analysis of Axin2 and Irf8 gene expression in BMDC following 5 hr culture with DMSO or ICG-001. Fold change is relative to DMSO control. The data are from one independent trial. (H and I) Intracellular expression of IRF8 and β-catenin following ICG-001 treatment of BMDC (H) or MutuDC1940 cells (I). The data are representative of 2 (MutuDC1940 cells) and 4 (BMDC) independent experiments with 3 replicates per treatment per experiment. *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Purification, Chromatin Immunoprecipitation, Control, Gene Expression

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin stabilization enhances IL-12 production by CD8α+ DC. (A) IL-12p40 production by naïve Ex3fl/fl and Ex3DC−/− splenocytes stimulated in vitro with LPS, STAg, or media control measured by ELISA. (B) IL-12p40 production by splenic CD11c+ DC magnetically purified from Ex3fl/fl and Ex3DC−/− mice stimulated in vitro with LPS, STAg, or media control measured by ELISA. (C) IL-12p40 production by CD8α+ and CD8α− DC DC purified from naïve Ex3fl/fl and Ex3DC−/− splenocytes following in vitro stimulation with media or STAg for 48 hr measured by ELISA. (D) IL-12p40 secretion by Ex3fl/fl splenocytes pre-treated with ICG-001 for 5 hr and then stimulated overnight with LPS or STAg measured by ELISA. (E) IL-12p40 production by splenocytes (106) from Ex3DC−/− mice cultured for 5 hr with 5 μM ICG-001 or DMSO and then stimulated with media, LPS (100 ng/ml), or STAg (25 μg/ml) overnight. (F) IL-12p40 production by MutuDC1940 cells (105) pre-treated with 20 μM ICG-001 or DMSO for 2 hr and then stimulated with media or STAg (25 μg/ml) overnight. The data are representative of at least 3 (A, F) and 2 (B–E) independent experiments, each involving 3–5 mice per group, except (C), which used pooled samples from 3 mice per experiment. *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: In Vitro, Control, Enzyme-linked Immunosorbent Assay, Purification, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: Constitutive DC β-catenin signaling increases the proinflammatory cytokine response to Toxoplasma. (A) Survival of Ex3fl/fl and Ex3DC−/− mice following i.p. infection with Toxoplasma Type II strain ME49 (25 cysts) (n=4–6 mice per group). The data are representative of at least 3 experiments. (B) Quantitative PCR amplification of parasite (B1 gene) and host DNA (ASL gene) isolated from Ex3fl/fl and Ex3DC−/− spleens 9 days post-infection. Parasite load is displayed as a ratio of parasite genomes to host genomes (n=3–4 mice per group). (C) IL-12p40 production by CD11c+ DC magnetically separated from Day-6 post-infection Ex3fl/fl and Ex3DC−/− splenocytes and cultured overnight without additional stimulation (n=3 mice per group). (D) IL-12p70 production by bulk splenocytes from Day-6 post-infection Ex3fl/fl and Ex3DC−/− mice (n=3 mice per group). The data are representative of 2 independent experiments. (E) IL-12p40 and IFN-γ production by splenocytes from Day-10 post-infection Ex3fl/fl and Ex3DC−/− mice cultured for 72 hr without additional stimulation (n=3–5 mice per group). The data are representative of 3 independent experiments. (F) IL-12p40, IFN-γ, and TNF-α levels in serum collected from Day-9 post-infection Ex3fl/fl and Ex3DC−/− mice (n=3–5 mice per group). The data are representative of 2 independent experiments. The means and S.E. of individual mice are shown. *, p<0.05; **, p<0.01.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Infection, Real-time Polymerase Chain Reaction, Amplification, Isolation, Cell Culture

Journal: Scientific Reports

Article Title: Establishment of induced pluripotent stem cells from normal B cells and inducing AID expression in their differentiation into hematopoietic progenitor cells

doi: 10.1038/s41598-017-01627-1

Figure Lengend Snippet: Cell surface antigen analysis by two-color flow cytometry. ( a ) Phenotype analysis of lymphocytes of the lymph node by flow cytometric analysis with a combination of anti-CD3/-CD20 and anti-CD27/-CD20 antibodies. B cells are surrounded by a dotted line. ( b ) Phenotype analysis of B cells purified using CD19-microbeads by flow cytomeric analysis with a combination of anti-CD3/-CD20 antibodies. Purified B cells are surrounded by a dotted line.

Article Snippet: B cells were purified using magnetic-activated cell sorting CD19 Micro Beads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Purification

Journal: Scientific Reports

Article Title: Establishment of induced pluripotent stem cells from normal B cells and inducing AID expression in their differentiation into hematopoietic progenitor cells

doi: 10.1038/s41598-017-01627-1

Figure Lengend Snippet: Characterization of the BiPSCs. ( a ) Immunofluorescence staining of BiPSC13 and MIB2-6 for expression of the pluripotent markers Oct3/4, Nanog, SSEA4, TRA-1-60, and TRA-1-81. ( b ) Expression of endogenous Oct3/4, Sox2, Klf4, cMyc, Pax5, AID , and GAPDH in BiPSCs (BiPSC13, MIB2-6) and normal B cells (CD19) from the lymph node analyzed using RT-PCR. ( c ) RT-PCR analysis of the expression of retrovirus-derived Oct3/4, Sox2, Klf4 , and cMyc in BiPSCs (BiPSC13, MIB2-6). HUC-Fm, human umbilical cord fibroblast cells, (male; RIKEN, Tsukuba, Japan) infected with a retrovirus containing Oct3/4 , Sox2 , Klf4 , c-Myc , and GAPDH for 5 days were used as the positive control.

Article Snippet: B cells were purified using magnetic-activated cell sorting CD19 Micro Beads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Infection, Positive Control

Journal: Scientific Reports

Article Title: Establishment of induced pluripotent stem cells from normal B cells and inducing AID expression in their differentiation into hematopoietic progenitor cells

doi: 10.1038/s41598-017-01627-1

Figure Lengend Snippet: AID expression in BiPSCs induced with the doxycycline-controlled (Tet-off) system. ( a ) Induction of AID expression in the absence of doxycycline was confirmed in two clones (#1 and #2) derived from BiPSC13 by western blotting, and ( b ) qRT-PCR. In ( b ), the numbers on the Y axis are the relative ratio comparing the expression of AID mRNA of each sample standardized by the expression of GAPDH mRNA with that of CD19 + normal B cells purified from normal LN using CD19-microbeads. Raji, Burkitt lymphoma cell line. Data were analyzed in triplicates and normalized to glyceraldehyde 3-phosphate dehydrogenase. ( c ) Immunofluorescence analysis of the expression of Oct3/4 and Nanog in BiPSC13-AID (#1 and #2) after the induction of AID expression in the absence of doxycycline. ( d ) Induction of AID expression in the absence of doxycycline in two clones (#16 and #17) derived from MIB2-6 by western blotting, and ( e ) qRT-PCR as described in the legend to ( b ). ( f ) Immunofluorescence analysis of the expression of Oct3/4 and Nanog in MIB-AID (#16 and #17) after the induction of AID expression in the absence of doxycycline.

Article Snippet: B cells were purified using magnetic-activated cell sorting CD19 Micro Beads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Clone Assay, Derivative Assay, Western Blot, Quantitative RT-PCR, Purification, Immunofluorescence