Figure 1.

CD1d-restricted NKT cell development is decreased in α-GalCer-treated thymic lobes. (a) Thymic lobes were harvested from gestational day 15 B6 mice and incubated with solvent (0.1% DMSO), α-GalCer (10⁻⁶ M), or β-GalCer (10⁻⁶ M). After 18 d of culture, thymocytes were harvested and stained with FITC-anti-TCRβ and PE-CD1d/α-GalCer tetramers. The plots are representative of three experiments involving a total of 4 thymic lobes for each condition, with the percentage of tetramer⁺ cells indicated. No significant differences in the total number of thymocytes were observed between α-GalCer–treated lobes and controls. In addition, α-GalCer treatment did not affect the levels of CD1d surface expression. (b) Thymic lobes were incubated with α-GalCer at indicated concentrations, and the numbers of tetramer⁺ cells were analyzed by flow cytometry. Representative data from one of two experiments are shown. (c) Thymocytes from each treated group (n = 4) were pooled and stimulated with plate-bound anti-CD3 (2C11, 10 μg/ml) for 48 h. The amount of IL-4 secretion was determined by sandwich ELISA. (d) α-GalCer (10⁻⁶ M) was added to FTOC at different time points after the beginning of the culture. After 20 d, thymic lobes were harvested and analyzed by flow cytometry for the presence of tetramer⁺ cells. Representative data from one of two experiments are shown.