Single cell Paclitaxel clinical trial suspensions from spleen or cultured cells were labelled with biotinylated anti-CD4, anti-CD8, anti-B220, anti-CD11c, anti-CD11b and Gr1 for negative depletion. Cells were then magnetized with streptavidin-Microbeads (Miltenyi Biotech), and passed through the LS column to collect the flow through as DN T cells. For cultured cells, dead cell removal was performed before negative depletion using the dead cell removal kit from Miltenyi Biotech. Briefly, 4-day-cultured splenocytes from HBeAg × 7/16-5 dbl-Tg were harvested and labelled with propidium iodide, and subsequently mixed with anti-propidium iodide-Microbeads.
Propidium iodide-labelled dead cells were subsequently removed by magnetic selection. B cells were purified by positive selection with B220-microbeads. For DN progenitor depletion, cells were positively selected by biotinylated anti-CD4, anti-CD8, anti-B220, anti-CD11c, anti-CD11b and anti-Gr1 and were subsequently magnetized with streptavidin-Microbeads to exclude DN
T-cell progenitors. For antigen-specific stimulation, 4 × 105 splenocytes from 7/16-5 TCR Tg mice or HBeAg × 7/16-5 dbl-Tg mice were cultured in 4% fetal calf serum supplemented with Dulbecco’s modified Eagle’s medium in the presence of truncated HBcAg149, or the HBcAg-derived peptide p120–140 at concentrations of 0·2–2 μg/ml. Cells selleck chemical were placed in flat-bottom 96-well-plates for 1, 2, 3 or 4 days for further analysis. At day 2 and day 4, supernatants were collected for cytokine analysis. For the DN T-cell suppression assay, 4 × 105/well of naive 7/16-5 TCR-Tg splenocytes were used as target cells and 4-day cultured HBeAg-specific DN T cells were separated by negative enrichment as described above, and were added to the culture at given numbers. To analyse T-cell activation, antigen-specific T cells Idoxuridine (Vβ11+ CD4 T cells) were stained for CD25 as well as CD69 at given time points. For the surface staining, cells were incubated in 3% fetal bovine serum in PBS containing 10 μg/ml 2.4G2 to block FcR binding followed by the addition of fluorochrome-conjugated antibodies. All fluorochrome-conjugated antibodies were obtained from eBioscience as described above.
For the detection of CTLA-4, we performed intracellular staining and surface staining because of the nature of CTLA-4 expression. Foxp3 intracellular staining was performed using a commercially available kit from eBioscience. Flow cytometry was performed on an LSRII flow cytometer (Becton Dickinson, CA) available through the CCMI at the Salk Institute (La Jolla, San Diego, CA). Data were analysed using FlowJo software (Tree Star, Ashland, OR). Spleen cells from either unprimed or primed TCR-Tg, TCR × antigen dbl-Tg or wild-type mice were cultured (4 × 105/well) with various concentrations of a series of antigens. Culture supernatants were harvested at 48 hr for IL-2 determination and at 96 hr for interferon-γ (IFN-γ) determination.
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