Figure 1.
Rapid generation of murine invariant natural killer T (iNKT) cell lines in vitro. iNKT cells were purified from spleens of Vα14 transgenic (Tg) mice. 2 × 106 iNKT cells were co-cultured with 2 × 105 bone marrow dendritic cells (BMDCs) loaded with α-galactosylceramide (αGalCer) (100 ng/ml). Three to five days later, m (murine) IL-2 (10 U/ml) and mIL-7 (10 ng/ml) were added to the culture medium, and cells were stimulated with αGalCer-BMDCs every 2–3 weeks. (a) Fold increase in cell numbers of four independent cell lines (AC6, AC8, AC9 and AC10) at the indicated time-points. (b) Anti-CD3e-fluorescein isothiocyanate (FITC) and CD1d tetramer-allophycocyanin staining of an iNKT cell line at 8 weeks post in vitro culture. Results are representative of two separate experiments. (c) Vβ usage of iNKT cell lines. iNKT cell lines (AC3, AC4, AC6, AC7, AC8, AC9 and AC10) were stained with a panel of anti-Vβ monoclonal antibodies (mAbs). Frequencies of iNKT cells with indicated Vβ chains are expressed as percentages in the 100% stacked column chart. Each column represents the percentage of iNKT cells with the indicated Vβ chains contributing to the total iNKT cells.