Differential recognition of CD1d-alpha-galactosyl ceramide by the V beta 8.2 and V beta 7 semi-invariant NKT T cell receptors.

The semi-invariant natural killer T cell receptor (NKT TCR) recognizes CD1d-lipid antigens. Although the TCR alpha chain is typically invariant, the beta chain expression is more diverse, where three V beta chains are commonly expressed in mice. We report the structures of V alpha 14-V beta 8.2 and V alpha 14-V beta 7 NKT TCRs in complex with CD1d-alpha-galactosylceramide (alpha-GalCer) and the 2.5 A structure of the human NKT TCR-CD1d-alpha-GalCer complex. Both V beta 8.2 and V beta 7 NKT TCRs and the human NKT TCR ligated CD1d-alpha-GalCer in a similar manner, highlighting the evolutionarily conserved interaction. However, differences within the V beta domains of the V beta 8.2 and V beta 7 NKT TCR-CD1d complexes resulted in altered TCR beta-CD1d-mediated contacts and modulated recognition mediated by the invariant alpha chain. Mutagenesis studies revealed the differing contributions of V beta 8.2 and V beta 7 residues within the CDR2 beta loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR V beta usage in NKT cells.

Structure of the chicken CD3εδ/γ heterodimer and its assembly with the αßT cell receptor.

In mammals, the αßT cell receptor (TCR) signaling complex is composed of a TCRαß heterodimer that is noncovalently coupled to three dimeric signaling molecules, CD3εδ, CD3εγ, and CD3ζζ. The nature of the TCR signaling complex and subunit arrangement in different species remains unclear however. Here we present a structural and biochemical analysis of the more primitive ancestral form of the TCR signaling complex found in chickens. In contrast to mammals, chickens do not express separate CD3δ and CD3γ chains but instead encode a single hybrid chain, termed CD3δ/γ, that is capable of pairing with CD3ε. The NMR structure of the chicken CD3εδ/γ heterodimer revealed a unique dimer interface that results in a heterodimer with considerable deviation from the distinct side-by-side architecture found in human and murine CD3εδ and CD3εγ. The chicken CD3εδ/γ heterodimer also contains a unique molecular surface, with the vast majority of surface-exposed, nonconserved residues being clustered to a single face of the heterodimer. Using an in vitro biochemical assay, we demonstrate that CD3εδ/γ can assemble with both chicken TCRα and TCRß via conserved polar transmembrane sites. Moreover, analogous to the human TCR signaling complex, the presence of two copies of CD3εδ/γ is required for ζζ assembly. These data provide insight into the evolution of this critical receptor signaling apparatus.

CD1d-lipid-antigen recognition by the semi-invariant NKT T-cell receptor.

The CD1 family is a large cluster of non-polymorphic, major histocompatibility complex (MHC) class-I-like molecules that bind distinct lipid-based antigens that are recognized by T cells. The most studied group of T cells that interact with lipid antigens are natural killer T (NKT) cells, which characteristically express a semi-invariant T-cell receptor (NKT TCR) that specifically recognizes the CD1 family member, CD1d. NKT-cell-mediated recognition of the CD1d-antigen complex has been implicated in microbial immunity, tumour immunity, autoimmunity and allergy. Here we describe the structure of a human NKT TCR in complex with CD1d bound to the potent NKT-cell agonist alpha-galactosylceramide, the archetypal CD1d-restricted glycolipid. In contrast to T-cell receptor-peptide-antigen-MHC complexes, the NKT TCR docked parallel to, and at the extreme end of the CD1d-binding cleft, which enables a lock-and-key type interaction with the lipid antigen. The structure provides a basis for the interaction between the highly conserved NKT TCR alpha-chain and the CD1d-antigen complex that is typified in innate immunity, and also indicates how variability of the NKT TCR beta-chain can impact on recognition of other CD1d-antigen complexes. These findings provide direct insight into how a T-cell receptor recognizes a lipid-antigen-presenting molecule of the immune system.

A minimal binding footprint on CD1d-glycolipid is a basis for selection of the unique human NKT TCR.

Although it has been established how CD1 binds a variety of lipid antigens (Ag), data are only now emerging that show how alphabeta T cell receptors (TCRs) interact with CD1-Ag. Using the structure of the human semiinvariant NKT TCR-CD1d-alpha-galactosylceramide (alpha-GalCer) complex as a guide, we undertook an alanine scanning mutagenesis approach to define the energetic basis of this interaction between the NKT TCR and CD1d. Moreover, we explored how analogues of alpha-GalCer affected this interaction. The data revealed that an identical energetic footprint underpinned the human and mouse NKT TCR-CD1d-alpha-GalCer cross-reactivity. Some, but not all, of the contact residues within the Jalpha18-encoded invariant CDR3alpha loop and Vbeta11-encoded CDR2beta loop were critical for recognizing CD1d. The residues within the Valpha24-encoded CDR1alpha and CDR3alpha loops that contacted the glycolipid Ag played a smaller energetic role compared with the NKT TCR residues that contacted CD1d. Collectively, our data reveal that the region distant to the protruding Ag and directly above the F' pocket of CD1d was the principal factor in the interaction with the NKT TCR. Accordingly, although the structural footprint at the NKT TCR-CD1d-alpha-GalCer is small, the energetic footprint is smaller still, and reveals the minimal requirements for CD1d restriction.