Conserved and heterogeneous lipid antigen specificities of CD1d-restricted NKT cell receptors

CD1d-restrieted NKT cells use structurally conserved TCRs and recognize both self and foreign glycolipids, but the TCR features that determine these Ag specificities remain unclear. We investigated the TCR structures and lipid Ag recognition properties of five novel V alpha 24-negative and 13 canonical V alpha 24-positive/V beta 11-positive human NKT cell clones generated using alpha-galactosylceramide (alpha-GalCer)-loaded CD1d tetramers. The V alpha 24-negative clones expressed V beta 11 paired with V alpha 10, V alpha 2, or V alpha 3. Strikingly, their V alpha-chains had highly conserved rearrangements to J alpha 18, resulting in CDR3 alpha loop sequences that are nearly identical to those of canonical TCRs. V alpha 24-positive and V alpha 24-negative clones responded similarly to alpha-GalCer and a closely related bacterial analog, suggesting that conservation of the CDR3 alpha loop is sufficient for recognition of alpha-GalCer despite CDRIa and CDR2 alpha sequence variation. Unlike V alpha 24-positive clones, the Va24-negative clones responded poorly to a glucose-linked glycolipid (alpha-glucosylceramide), which correlated with their lack of a conserved CDR1 alpha amino acid motif, suggesting that fine specificity for alpha-linked glycosphingolipids is influenced by V alpha-encoded TCR regions. V alpha 24-negative clones showed no response to isoglobotrihexosylceramide, indicating that recognition of this mammalian lipid is not required for selection of J alpha 18-positive TCRs that can recognize alpha-GalCer. One alpha-GalCerreactive, Va24-positive clone differed from the others in responding specifically to mammalian phospholipids, demonstrating that semi-invariant NKT TCRs have a capacity for private Ag specificities that are likely conferred by individual TCR beta-chain rearrangements. These results highlight the variation in Ag recognition among CD1d-restricted TCRs and suggest that TCR alpha-chain elements contribute to alpha-linked glycosphingolipid specificity, whereas TCR beta-chains can confer heterogeneous additional reactivities.