Natural variations at position 93 of the invariant Vα24‐Jα18 α chain of human iNKT‐cell TCRs strongly impact on CD1d binding

Abstract

Human invariant natural killer T (NKT) cell TCRs bind to CD1d via an ''invariant'' Va24-Ja18 chain (iNKTa) paired to semi-invariant Vb11 chains (iNKTb). 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 (4-30%). 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 b-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.Key words: CD1d . iNKT cells . Molecular modeling . Surface plasmon resonance . TCR IntroductionInvariant natural killer T (iNKT) lymphocytes are a versatile and potent subset of T cells which play important roles in both host defense and tolerance. In contrast to conventional T cells, they recognize the non-polymorphic lipid-antigen-presenting molecule CD1d via highly conserved TCRs. In fact, all iNKT-cell TCRs use an ''invariant'' TCRa chain (iNKTa), Va24-Ja18 in humans, Va14-Ja18 in mice, paired to ''semi-invariant'' TCRb chains (iNKTb), which in humans solely utilize Vb11 rearranged with diverse TCR Ã These authors contributed equally to this work. 248Jb segments. For all iNKT-cell TCRs, binding to CD1d is primarily mediated by the Va-Ja rearranged ''invariant'' CDR3a loop as well as the two germline-encoded CDR1a and CDR2b loops [1,2]. In addition, we have recently shown that the only variable region of human iNKT-cell TCRs, i.e. the CDR3b loop, strongly modulates the affinity to CD1d [3].Human iNKT-cell clones with single-amino acid variations in the ''invariant'' Va24-Ja18 iNKTa chain have been previously identified [4,5], and a recent study found that 4-30% of human iNKT-cell clones carried such variations [4]. However, the effect of these variations on iNKT-cell TCR binding to CD1d has not been examined. All of the described variations to date are located immediately upstream of the CDR3a loop region and most of these involve residue 93 (p93). The most commonly used amino acid at this position is serine (Ser93), which is encoded for by the germline sequence of human Va24S1 (AE00521.1), but other identified natural variations at this position inclu...

Effective functional maturation of invariant natural killer T cells is constrained by negative selection and T-cell antigen receptor affinity

“…Moreover, these TCR-α variations strongly influenced the affinity of human iNKT TCRs for various antigenCD1d complexes, irrespective of the TCR-β chain with which they were paired (39). However, in contrast with our results where variations were most common at position 94, the variations in human iNKT TCR-α chains occurred most often at position 93 of the CDR3α.…”

“…A serine residue is found at position 93 in the majority of human iNKT TCR-αs, whereas in the mouse iNKT TCRs this position is a glycine residue. Although this position 93 does not directly contact CD1d, dynamic modeling studies suggested that it can nonetheless affect the conformation of the CDR3α loop (39). The reason for this difference between species is currently unclear.…”

The hypervariable region 4 (HV4) and position 93 of the α chain modulate CD1d‐glycolipid binding of iNKT TCRs

“…This is in line with the pivotal role of the V-J transition for iNKT TCR function as has been shown before for position 93 in human [10,11]. In mouse, we could confirm this influence for the naturally occuring G93A substitution, where previously only variations at position 94 had been addressed [16].…”

“…Despite demonstration of the pivotal role of invariant CDRαs, the V-J transition, which is the only source of natural variation within the invariant α chain, has been shown to significantly influence the binding to several antigens in humans. Most human iNKT TCRα chains harbor a serine at position 93 that can be replaced either by threonine, asparagine, or isoleucine concomitant with loss of avidity [11], explaining the importance of a canonical iNKT TCRα chains sequence after rearrangement. Several natural variations of position 93 have also been described for the mouse iNKT TCRα chain, where the canonical glycine has been replaced by alanine, isoleucine, or valine [5,6,[12][13][14][15].…”

“…3A). To assess the influence of position 93 on CD1d binding of rat iNKT TCRs, we analyzed different amino acids at this position as this finding seems to be specific for the AV14/AJ18 rearrangement and previous reports have shown natural variations of the V-J transition to indirectly influence CD1d binding by the human iNKT TCR [11]. Additionally to the most frequently found alanine and glycine at position 93, we also analyzed valine, which had been found in a rat AV14S8 (type 1) α chain, cloned from F344 IHL cDNA [15] and serine, which is present in the canonical human iNKT TCRα chain sequence [5][6][7].…”