Journal: Gene therapy

Article Title: Dendritic cell functional improvement in a preclinical model of lentiviral-mediated gene therapy for Wiskott-Aldrich syndrome

doi: 10.1038/gt.2011.202

Figure Lengend Snippet: BMDCs of GT mice rescue T cell priming capacity. ( a-b ) Five ×10 5 BMDCs of BMT wt, BMT was −/− or GT mice were loaded with 0.05 nM of SIINFEKL OVA peptide and injected subcutaneously in C57BL/6 (CD45.2) wt recipient mice that had been transferred with 1.5×10 6 CFSE-labeled OT-I/CD45.1 cells 24 hours before. Draining LNs were collected 3 days after and T cell proliferation was analyzed by flow cytometry. ( a ) Representative histograms showing CFSE dilution profile of transferred OT-I cells, gated on CD8 + /CD45.1 + live cells. ( b ) Quantification of T cell expansion expressed as percentage of OT-I cells over the total CD8 + T cell population. Each symbol represents an individual mouse; horizontal lines indicate mean values. ( c-e ) Five ×10 5 BMDCs of BMT wt, BMT was −/− or GT mice were loaded with OVA (150 μg/ml) and injected subcutaneously in C57BL/6 (CD45.1) wt recipient mice that had been transferred with 1.5×10 6 CFSE-labeled OT-II/CD45.2 cells 24 hours before. Draining LNs were collected 3 days later and T cell proliferation and intracellular IFN-γ production was analyzed by flow cytometry. ( c ) Representative histograms showing CFSE dilution profile of transferred OT-II cells, gated on CD4 + /CD45.2 + live cells. ( d ) Data obtained from the analysis of CFSE dilution were expressed as the percentage of OT-II cells that remain undivided, that divided one to four times, or that divided five to eight times. ( e ) Quantification of T cell expansion expressed as percentage of OT-II cells over the total CD4 + T cell population. ( f ) Percentage of IFN-γ expressing OT-II cells, evaluated by flow cytometry. Each symbol represents an individual mouse; horizontal lines indicate mean values (n.s., not significant; *p<0.05, **p<0.005, ***p<0.001 student t test).

Article Snippet: C57BL/6 wt mice and OVA specific, MHC class II restricted, TCR transgenic OT-II mice were purchased from Charles River Laboratories Inc. (Calco, Italy).

Techniques: Injection, Labeling, Flow Cytometry, Expressing

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

Article Title: Opposing Roles for Complement Component C5a in Tumor Progression and the Tumor Microenvironment

doi: 10.4049/jimmunol.1200846

Figure Lengend Snippet: (A) Wildtype C57Bl/6 mice were injected with C5a-expressing lymphoma RMA-3CF4 cells or RMA CVA1 cells (n=10) and tumor growth was recorded. Data are shown as mean±s.e.m. ***p<0.001. (B) Spleen, TDLN, and tumor from tumor-bearing mice were prepared for single cell suspensions. Cells were then stained with Gr-1, CD11b, F4/80, or NK1.1. Representative dot plots and summarized data are shown. (C) Cells were stimulated with PMA/ionomycin and surface stained with CD8 and IFN-γ intracellularly. Representative dot plots (cells were gated on the CD8+ T cells), summarized IFN-γ-producing CD8+ T cells, and total CD8+ T cells are shown. (D) Cells were stimulated with PMA/ionomycin and surface stained with CD4 and IFN-γ intracellularly. Representative dot plots (cells were gated on the CD4+ T cells), summarized IFN-γ-producing CD4+ T cells, and total CD4+ T cells are shown.

Article Snippet: Naïve CD4 T cells from OT-II ovalbumin (OVA) TCR transgenic mice (Taconic) were purified by microbead separation (AutoMACS, Miltenyi Biotec).

Techniques: Injection, Expressing, Staining

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

Article Title: Opposing Roles for Complement Component C5a in Tumor Progression and the Tumor Microenvironment

doi: 10.4049/jimmunol.1200846

Figure Lengend Snippet: (A) C5a levels of RMA cells transfected with C5a or CV. Data indicate that RMA 3CF4 clone secreted higher level of C5a than RMA 1474 clone. (B) Wildtype C57Bl/6 mice were injected with low C5a-expressing lymphoma RMA-1474 cells (n=10) or RMA CVA1 cells (n=15) and tumor growth was recorded. Data are shown as mean±s.e.m. *p<0.05. (C) Cells from spleen and tumor from tumor-bearing mice were stained with Gr-1 and CD11b. Representative dot plots and summarized data are shown. (D) Cells were stimulated with PMA/ionomycin and surface stained with CD8 and IFN-γ intracellularly. Representative dot plots (cells were gated on the CD8+ T cells), summarized IFN-γ-producing CD8+ T cells, and total CD8+ T cells are shown. (D) Cells were stimulated with PMA/ionomycin and surface stained with CD4 and IFN-γ intracellularly. Representative dot plots (cells were gated on the CD4+ T cells), summarized IFN-γ-producing CD4+ T cells, and total CD4+ T cells are shown.

Article Snippet: Naïve CD4 T cells from OT-II ovalbumin (OVA) TCR transgenic mice (Taconic) were purified by microbead separation (AutoMACS, Miltenyi Biotec).

Techniques: Transfection, Injection, Expressing, Staining

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

Article Title: Opposing Roles for Complement Component C5a in Tumor Progression and the Tumor Microenvironment

doi: 10.4049/jimmunol.1200846

Figure Lengend Snippet: Peritoneal macrophages were stimulated with varying concentrations of C5a (0–500 ng/ml) for 24 h and then co-cultured with naïve CD4 OVA Tg T cells in the presence of OVA for 3 days. Cells were then stained intracellularly with IFN-γ (A) and IL-17A (B). For Treg induction assay, macrophages were co-cultured with naïve CD4 OVA Tg T cells in the presence of OVA with varying concentrations of C5a (0–500 ng/ml) for 4 days. Cells were stained intracellularly with Foxp3 (C). Representative dot plots and summarized data are shown. Cells were gated on the CD4+ T cells. Data are representative of at least three independent experiments.

Article Snippet: Naïve CD4 T cells from OT-II ovalbumin (OVA) TCR transgenic mice (Taconic) were purified by microbead separation (AutoMACS, Miltenyi Biotec).

Techniques: Cell Culture, Staining