Fig. 4.

CD1e facilitates antigen loading and unloading on CD1d. (A) CD1e promotes α-GalCer presentation after a short time pulse. APCs were pulsed for different lengths of time with α-GalCer (10 ng/mL) and fixed before addition of human iNKT cells. (B) CD1e shortens the half-life of CD1d–α-GalCer complexes. APCs were pulsed for 2 h with α-GalCer (2 ng/mL) and chased for different lengths of time before the addition of human iNKT cells. (A and B) THP-1 cells transfected with the CD1D gene (○) or with the CD1D and CD1E genes (●) were used. Mean release of IL-4 is shown (±SD) (n = 3). (C) Plate-bound CD1d was pulsed with α-GalCer (1 μg/mL) in the presence of different soluble LTPs, BSA, or CD1e or with PBS before addition of human iNKT cells. hGM-CSF release in the absence of α-GalCer was below detection limits of the assay. (D). Plate-bound CD1d was loaded with α-GalCer (1 μg/mL), washed, then incubated overnight with different LTPs or PBS before addition of human iNKT cells. (C and D) Bars show mean hGM-CSF release (+SD) (n = 3). *P < 0.05, **P < 0.01. Data shown are representative of at least three experiments. (E and F) CD1e mediates the transfer of anionic lipids to CD1d (E) and lipid unloading from CD1d–GD3 complexes (F). (E) IEF gel of the products after incubation of CD1d (10 μM) for 1 h at pH 5.0 with phosphatidylcholine/phosphatidylethanolamine/sphingomyelin/cholesterol liposomes incorporating the indicated anionic lipid (15% molar ratio, 200 μM final concentration) in the presence or absence of CD1e (10 μM). The first two lanes show a control experiment using liposomes devoid of anionic lipids. (F) IEF gel of the products when purified CD1d–GD3 complexes were incubated with or without CD1e and the indicated lipids (15% molar ratio in liposomes). The lipids used were sulfatide (SLF), GD3, bis-(monoacylglycero)phosphate (BMP), and phosphatidylserine (PS).