The Vα14 NKT cell TCR exhibits high-affinity binding to a glycolipid/CD1d complex

Most CD1d-dependent NKT cells in mice have a canonical Valpha14Jalpha18 TCR rearrangement. However, relatively little is known concerning the molecular basis for their reactivity to glycolipid Ags presented by CD1d. Using glycollpid Ags, soluble forms of a Valpha14 NKT cell-derived TCR, and mutant and wild-type CD1d molecules, we probed the TCR/CD1d interaction by surface plasmon resonance, tetramer equilibrium staining, and tetramer staining decay experiments. By these methods, several CD1d a-helical amino acids could be defined that do not greatly alter lipid binding, but that affect the interaction with the TCR. Binding of the Valpha14(+) TCR to CD1d requires the agonist alpha-galactosyleeramide (alpha-GalCer), as opposed to the nonantigenic beta-galactosylceramide, although both Ags bind to CD1d, indicating that the carbohydrate moiety of the CD1d-bound Ag plays a major role in the TCR interaction. The TCR has a relatively high-affinity binding to the alpha-GalCer/CD1d complex, with a particularly slow off rate. These unique properties are consistent with the coreceptor-independent action of the Va14 TCR and may be related to the intense response to a-GalCer by NKT cells in vivo.