The T cell antigen receptor expressed by Valpha14i NKT cells has a unique mode of glycosphingolipid antigen recognition - PubMed (original) (raw)

The T cell antigen receptor expressed by Valpha14i NKT cells has a unique mode of glycosphingolipid antigen recognition

Stéphane Sidobre et al. Proc Natl Acad Sci U S A. 2004.

Abstract

Natural killer (NK) T cells with an invariant Valpha14 rearrangement (Valpha14i) are the largest population of lipid antigen-specific T lymphocytes identified in animals. They react to the glycolipid alpha-galactosyl ceramide (alpha-GalCer) presented by CD1d, and they may have important regulatory functions. It was previously shown that the Valpha14i T cell antigen receptor (TCR) has a high affinity for the alpha-GalCer/CD1d complex, driven by a long half-life (t(1/2)). Although this result could have reflected the unique attributes of alpha-GalCer, using several related glycolipid compounds, we show here that the threshold for full activation of Valpha14i NKT cells by these glycosphingolipids requires a relatively high-affinity TCR interaction with a long t(1/2). Furthermore, our data are consistent with the view that the mechanism of recognition of these compounds presented by CD1d to the Valpha14i NKT cell TCR is likely to fit a lock-and-key model. Overall, these findings emphasize the distinct properties of glycosphingolipid antigen recognition by Valpha14i NKT cells.

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Figures

Fig. 5.

Binding of the Vα14 Vβ8.2 scTCR to glycolipid/CD1d complexes. (A_–_D) The scTCR was injected at 25°C at increasing concentrations (1.25, 2.5, 5, 10, or 20 μM) at a flow rate of 10 μl/min over Fc1 (control flow cell) and Fc2, where α-GalCer-loaded (A), 4-deoxy-loaded (B), α-GlcCer-loaded (C), or α-ManCer-loaded (D) CD1d molecules were immobilized. The sensorgrams plot the specific binding obtained from the subtracted RU values (Fc2 - Fc1). (E) Steady-state equilibrium fitting. Normalized RU at equilibrium (Req) values obtained from fitting of the sensograms (A_–_D) with the 1:1 Langmuir association, expressed as percentage of the maximum for each compound, were plotted against the scTCR concentration. α-GalCer, ▴; α-GlcCer, •; 4-deoxy, ▪; and α-ManCer, ▾. One representative experiment of three is shown.

Fig. 1.

Structure of α-GalCer analogs. α-GlcCer is an α-

-glucopyranoside, in which the hydroxyl in position 4′ is in an equatorial position rather than the axial position for galactose. β-GalCer has a β glycosidic bound between the

-galactopyranoside unit and the ceramide. α-ManCer has an α-

-mannopyranoside unit, in which the hydroxyls in positions 2′ and 4′ of the carbohydrate are oriented differently when compared to

-galactopyranoside. The compound we named 4-deoxy, lacking the hydroxyl on the 4 carbon of the sphingosine, was referred to previously as AGL 514 (19). Similarly, 3,4-dideoxy was referred to as AGL 535 (19).

Fig. 2.

Stimulation of Vα14_i_ NKT cell-derived hybridomas with glycolipid antigens bound to plate-bound CD1d. One microgram per well of CD1d protein was immobilized on 96-well plates, incubated with the indicated amounts of α-GalCer (▴), 4-deoxy (▪), α-GlcCer (•), α-ManCer (X), 3,4-dideoxy (♦), and β-GalCer (○). 3C3 hybridoma cells were then cultured in the wells for 16 h. IL-2 release was measured by ELISA. Shown is a representative experiment of five, carried out in triplicate. The specific response was obtained by subtracting the responses to wells coated with CD1d protein alone and to the α-GalCer analogs alone. Similar results were obtained with the 1.2 and 1.4 hybridomas.

Fig. 3.

Cytokine secretion after stimulation of Vα14_i_ NKT cells with glycolipid antigens in vivo. Two (♦) and 14 h (▪) after i.p. injection with 2 μg of antigen, serum was harvested and analyzed for IL-4 (A) and IFN-γ (B) by ELISA. Each point represents the cytokine measured in an individual mouse. The dashed line indicates the limit of detection of the ELISA. There were three mice in each group, and data are representative of two separate experiments.

Fig. 4.

Specific CD1d tetramer staining. Binding isotherm of α-GalCer-loaded CD1d tetramer (▴), α-GlcCer-loaded CD1d tetramer (•), and 4-deoxy-loaded CD1d tetramer (▪) to the 2C12 hybridoma. Tetramer fluorescence, normalized to the surface level of TCRβ staining, is plotted against tetramer concentration. 2C12 hybridoma cells were stained for3hat room temperature with an anti-TCRβ mAb and the indicated concentrations of WT CD1d tetramers. Cells were then washed two times and analyzed by flow cytometry. One representative experiment of five is shown.

Fig. 6.

Correlation between t_1/2 of the interaction and the quantity of cytokine produced by Vα14_i NKT cells. The relative quantities of IL-4 (full symbols) and IFN-γ (open symbols) measured by ELISA 2 (♦) and 14 h (▪) after i.p. injection with 2 μg of antigen are plotted against the half-life of the interaction between the respective antigen/CD1d complexes and the NKT cell TCR.

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The T cell antigen receptor expressed by Valpha14i NKT cells has a unique mode of glycosphingolipid antigen recognition - PubMed (original) (raw)