Natural variations at position 93 of the invariant Va24-Ja18 a chain of human iNKT-cell TCRs strongly impact on CD1d binding

Human invariant natural killer T (NKT) cell TCRs bind to CD1d via an invariant Va24-Ja18 chain (iNKTa) paired to semi-invariant V beta 11 chains (iNKT beta). Single-amino acid variations at position 93 (p93) of iNKTa, immediately upstream of the invariant CDR3a region, have been reported in a substantial proportion of human iNKT-cell clones (430%). Although p93, a serine in most human iNKT-cell TCRs, makes no contact with CD1d, it could affect CD1d binding by altering the conformation of the crucial CDR3a loop. By generating recombinant refolded iNKT-cell TCRs, we show that natural single-nucleotide variations in iNKTa, translating to serine, threonine, asparagine or isoleucine at p93, exert a powerful effect on CD1d binding, with up to 28-fold differences in affinity between these variants. This effect was observed with CD1d loaded with either the artificial a-galactosylceramide antigens KRN7000 or OCH, or the endogenous glycolipid beta-galactosylceramide, and its importance for autoreactive recognition of endogenous lipids was demonstrated by the binding of variant iNKT-cell TCR tetramers to cell surface expressed CD1d. The serine-containing variant showed the strongest CD1d binding, offering an explanation for its predominance in vivo. Complementary molecular dynamics modeling studies were consistent with an impact of p93 on the conformation of the CDR3a loop.