Autoreactive CD1d-restricted clones are influenced by CD1e. Type 1 (A) and type 2 (B) NKT cell clones were incubated with single-transfected (CD1D only) and double-transfected (CD1E and CD1D) THP-1 cells. The difference in activation is shown as fold increase (ratio of cytokine release in response to double- and single-transfected cells). In total, 5 × 10⁴ T cells were stimulated with 10⁵ THP-1 cells. Released hGM-CSF is expressed as mean ng/mL ± SD (n = 3). Data shown are representative of at least three experiments.

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).

DCs from Tg mice induce a better response after stimulation with S. paucimobilis. (A) DCs from Tg mice (●) or non-Tg littermates (○) were incubated for 2 h with heat-killed S. paucimobilis at different bacteria:APC ratios and then used to stimulate iNKT hybridoma cells. mIL-2 (ng/mL ± SD) was determined by ELISA. (B) DCs from Tg (●) or non-Tg littermates (○) were incubated for different lengths of time with heat-killed S. paucimobilis (bacteria:APC 200:1), washed, and then fixed before addition of iNKT hybridoma cells. After 24 h, mIL-2 (ng/mL ± SD) was determined. (C and D) DCs from Tg or non-Tg littermates were infected at a multiplicity of infection (MOI) of 1:1 for 2 h with S. paucimobilis or were not infected, then washed and incubated with gentamicin for 1 h, and finally cocultured with freshly isolated WT mouse splenocytes. CD69 mean fluorescence intensity (MFI) (C) and percentage (D) of CD69⁺ CD1d–α-GalCer dimer⁺ iNKT cells as measured by flow cytometry. *P < 0.05. (E) Tg (●) or WT (○) DCs were infected for different lengths of time with S. paucimobilis at a low MOI (1:1), incubated for 1 h with gentamicin, and then used to stimulate freshly isolated WT iNKT cells. Up-regulation of CD69 on CD1d–α-GalCer dimer⁺ iNKT cells was measured by flow cytometry. ■ indicates CD69 expression on iNKT cells incubated with noninfected DCs. Data shown are representative of at least three experiments.

CD1e participates in the presentation of CD1b- and CD1c-restricted lipid antigens. (A–C) Self-antigens. (A) CD1b-restricted clone GG123b response to THP-1 CD1b and CD1e (●) and THP-1 CD1b only (○). (B and C) CD1c-restricted clones K34B27.f (B) and DN4.99 (C) response to THP-1 CD1c and CD1e (●) and THP-1 CD1c only (○). (D–F) CD1b-dependent exogenous antigens. GG33a clone response to GM1 (D), Z4B27 clone response to SGL12 (E), and DS1C9b clone response to sulfatide (F) presented by THP-1 CD1b and CD1e (●) and THP-1 CD1b only (○). Human GM-CSF (hGM-CSF) release is expressed as mean ng/mL ± SD (n = 3). *P < 0.05, **P < 0.01. Data shown are representative of at least three experiments.

CD1e influences the type of cytokines secreted in response to the presentation of exogenously added antigen. THP-1 cells transfected with the CD1D gene (○) or with the CD1D and CD1E genes (●) were incubated for 2 h with different concentrations of α-GalCer (A–C), α-LacCer (D–F), or GSL′ (G–I) before addition of the human iNKT clone VM-D5. Released cytokines are expressed as mean ng/mL ± SD (n = 3). Data shown are representative of at least three experiments.

CD1e expressed in Tg DCs influences presentation of self-lipids. (A) DCs from Tg mice (●) or non-Tg littermates (○) were incubated for different lengths of time with α-GalCer (10 ng/mL) and then fixed before addition of iNKT mouse hybridoma cells. (B) The half-life of CD1d–α-GalCer complexes is reduced in Tg DCs. DCs from Tg mice (●) or non-Tg littermates (○) were pulsed for 2 h with α-GalCer (2 ng/mL) and chased for different lengths of time before addition of iNKT mouse hybridoma cells. (A and B) Released mIL-2 (ng/mL ± SD) was determined in triplicate. (C) Detection of CD1d–α-GalCer complexes by L363 antibody staining on the surface of Tg (●) or WT (○) DCs incubated for 24 h with α-GalCer. (D) Detection of CD1d–α-GalCer complexes by L363 antibody on Tg (●) or WT (○) DCs pulsed for 24 h with different doses of α-GalCer and chased for 24 h before staining. Data shown are representative of three independent experiments.