Fig. 1.
Binding to CD1d and NKT cell recognition of mammalian lipids. (a) Binding of mammalian lipids and their lyso-forms to CD1d. Lipids were coated onto plastic wells, soluble CD1d-huFc was added, and binding was detected by a peroxidase-coupled anti-huFc antibody. (b) Dose-dependency of CD1d binding to PI. Different ratios of PI and TP were coated onto plastic wells, and various dilutions of soluble CD1d-huFc fusion protein were added, as described above. (c) Specificity of CD1d binding to PI and inhibition of CD1d–PI interaction by preloading of CD1d with free PI. Soluble CD1d–biotin was preincubated with different concentrations of free PI in 0.1 mg/ml Tween 20 and added subsequently to PI-coated plastic wells (filled bars, CD1d-biot). Biotinylated soluble MHC class I (H-2Kd) served as control (open bar). (d) Recognition of mammalian lipids by CD1d-restricted T cells. A20-CD1d or A20 were preloaded with the indicated lipids, splenic T cells from Vα14-Jα281 tg mice were added, and IFN-γ was determined after 48 h. Open bars, A20; filled bars, A20-CD1d. TP, tripalmitoylglycerol; LCL, lyso-CL; DLCL, dilyso-CL; LPI, lyso-PI. For all graphs, data are expressed as the mean OD ± SD of triplicates from one of three representative experiments.