View larger version (45K): [in this window] [in a new window]   Figure 1. Structural and functional integrity of recombinant soluble NKT TCRs. (A) Purified bacterial NKT15 TCR was analyzed by SDS-PAGE under reducing (+DTT, dithiothreitol) and nonreducing (–DTT) conditions demonstrating ß heterodimers. (B) Native gel electrophoresis of folded ß heterodimeric NKT15 and control LC13 TCRs. (C) Recombinant soluble NKT12, NKT15, NKT18, and control LC13 TCRs were tested for their ability to block binding of mouse CD1d/-GalCer tetramers to murine thymocytes. Phycoerythrin-conjugated mCD1d/-GalCer tetramers were preincubated with the indicated soluble TCRs over a range of TCR concentrations before staining mouse thymocytes. Cells were analyzed by two-color flow cytometry showing mCD1d/-GalCer tetramer staining on the vertical axis and FITC-CD3 (mAb 145-2C11) staining on the horizontal axis. Cells staining positively with mCD1d/-GalCer tetramer and FITC-CD3 are indicated with a circle.

View larger version (33K): [in this window] [in a new window]   Figure 2. Overview of NKT cell TCR structure showing the conformation of the CDR loops at the Ag-binding interface. (A) Overview of the structure of NKT12 with the -chain and ß-chain shown in gray and pink, respectively. (B) The CDR3 loops of NKT12 are color coded according to their genetic origin. (C) Superimposition of the CDR loops of the NKT12 and NKT15 TCRs depicting the difference in the conformation of CDR3ß (NKT12, CDR loops colors are indicated; NKT15, orange).

The sequences encoded by the invariant J and hypervariable Dß-N-Jß region create the CDR3 loops (Fig. 2, A and B). Accordingly, the interface differs slightly between the NKT12 and NKT15 TCRs as a result of the conformation of the different CDR3ß loops (Fig. 2 C). Notably, the CDR1 and CDR2 loops adopt a conformation that is different from previously determined canonical CDR conformations (40), whereas CDR1ß and CDR2ß adopt canonical conformations ß1-1 and ß2-1, respectively (Table S2, available at http://www.jem.org/cgi/content/full/jem.20051777/DC1). The conformation of the CDR3 loop is well ordered and stabilized by interloop interactions as shown for other highly selected TCRs (34).

The ligand binding surface contains a preformed cavity suited to Ag binding
The ligand-binding surface of NKT12 contains a central water-filled cavity that is markedly electropositive and 120 ų in volume (Fig. 3, A and B). The cavity is lined by residues from CDR1 and CDR3 of the invariant NKT -chain, as well as CDR1ß and CDR3ß of the semi-invariant ß-chain that collectively make several direct and water-mediated interactions with each other (Fig. 3 B). The residues from the invariant V24-N-J18 include the following: Ser30 and Asn31 from CDR1; Arg33 from the V-framework close to CDR1; Ser93 from the CDR3 V-J junctional region; and Gly96, Ser97, and Leu104, all from the CDR3 J segment. Remarkably, Arg33 that forms the electropositive base of the cavity is unique to V24 among all the human V genes and is also conserved in mouse V14 and in only one other V family (TRAV7). Similarly, the J residues Gly96, Ser97, and Leu104 are only found in J18. The Vß framework residue Tyr33ß, adjacent to the CDR1ß, also lines the cavity and is conserved in mouse and human NKT Vß regions. The NKT15 TCR is virtually identical in structure to NKT12, apart from the amino acid sequence and conformation of its CDR3ß regions. These differences include two extra residues encoded by the Dß-N region, thereby introducing a charged, surface-exposed "RDR" motif that forms the tip of CDR3ß in the NKT15 TCR. Thus, the hypervariable CDR3ß residues that line the cavity of NKT12 include Thr95ß, Ser96ß, Ser100ß, and Tyr101ß; whereas, in NKT15, the corresponding residues lining the cavity are Ser95ß, Gly96ß, Leu97ß, and Gly101ß (Fig. 3, C and D; Table I). Although the positively charged cavity at the Ag-binding interface is conserved between NKT12 and NKT15, in the trigonal crystal form, the entrance to the cavity is occluded by the bulky Tyr103ß (Jß) from the CDR3ß loop of NKT15 (Fig. 3 C). Notably, however, this region of the CDRß loop is relatively mobile, such that Tyr103ß was unresolved in the orthorhombic crystal form. This suggests that Tyr103ß could be easily displaced, allowing access of potential ligands to this conserved region (Fig. 3 D). The cavity at the ligand interface could potentially accommodate small polar moieties such as the galactose ring of -GalCer that projects out of the antigen-binding cleft of CD1d (14, 15) (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20051777/DC1). This would be consistent with a direct interaction between NKT TCRs and the glycosyl head group of -GalCer bound to CD1d, as predicted from many studies and analogous to what has been proposed for CD1b/glycolipid recognition (41). The volume of the cavity would be predicted to vary according to the structure and flexibility of the CDR3ß sequences; this might facilitate interaction with other glycolipid head groups.

View larger version (52K): [in this window] [in a new window]   Figure 3. The Ag-binding interface of NTK12 and NKT15 TCRs contains a preformed cavity created by invariant residues of the -chain, a species-conserved residue from the ß-chain and CDR3ß. (A) NKT12 TCR -chain residues that are conserved between human and mouse are red; -chain residues that differ between these species are gray. The TCR ß-chain residue Tyr33ß is conserved across species, whereas the CDR3ß residues are divergent between species. ß-chain side chains are green. (B) Close-up of residues forming the putative ligand-binding cavity of the NKT12 binding surface. Residues are labeled according to the single amino acid code. (C) The putative ligand-binding cavity of NKT15 is obstructed by the bulky side chain of residue Tyr 103ß from CDR3ß in the trigonal form of the crystal structure. (D) Tyr103ß could not be visualized in the NKT15 TCR crystal comprising the orthorhombic space group. The high mobility of Tyr103ß could easily allow displacement of this side chain, exposing the putative Ag-binding cavity. Accordingly, the Tyr103ß side chain has been omitted, revealing the cavity.

We next studied the binding of several hybrid TCRs with switched or mutated CDR3ß regions (Fig. 4 and Table S4, available at http://www.jem.org/cgi/content/full/jem.20051777/DC1). The native integrity of these chimeric receptors was intact based on their binding with a conformation-dependent mAb 12H8 (34) and their behavior by gel filtration and SDS-PAGE (unpublished data). First, we examined the relative roles of the CDR1ß and CDR2ß, versus the hypervariable CDR3ß residues (encoded by Dß-N-Jß gene segments) of NKT15, by swapping the entire NKT15 TCR ß-chain for that of the classical MHC I–restricted TCR LC13 (36, 37) (Fig. 4 B). This TCR contains a different Vß chain (Vß6.2 or TRBV7-8*03) to NKT15, but uses the same Jß segment to create the CDR3ß loop (TRBD1/D2; TRBJ2-7*01). The hybrid NKT15/LC13ß TCR lost virtually all CD1d/-GalCer binding as measured by both SPR and by failure to inhibit NKT cell staining by CD1d/-GalCer tetramer (Fig. 4 B). Although this finding suggests that either CDR1ß and/or CDR2ß may be crucial for CD1d/-GalCer binding, it could also be explained by an incompatibility in the LC13 CDR3ß region. Therefore, a chimeric TCR was generated where just the CDR3ß loop of NKT15 was replaced with the corresponding loop of LC13 (also encoded by Jß TRBJ2-7) (Fig. 4 B). This swap shortens the CDR3ß loop of NKT15 by three residues and alters the sequence of buried amino acids involved in stabilizing the TCR -chain as well as changing the solvent-exposed residues at the tip of the loop (Table S4). The hybrid TCR had an 5–10-fold lower affinity by SPR measurements than NKT15 bound to both human and mouse CD1d/-GalCer. In addition, the hybrid TCR required 60-fold higher concentrations than NKT15 to achieve comparable inhibition of staining of mouse NKT cells with mCD1d/-GalCer tetramers (Fig. 4 B). However, the structures of the LC13 (36, 37) and NKT15 TCRs suggested that the orientation of the LC13 CDR3ß was likely to sterically impact on the conformation of the NKT15 CDR3 loop, thereby indirectly affecting the putative binding cavity and disrupting ligand recognition by the hybrid TCR (Fig. 4 C). Notwithstanding these findings, the CDR3ß loops of other NKT TCRs show substantial variation (26, 27, 42), suggesting that sequence variation at the solvent-exposed tips of the CDR3ß loops might be well tolerated. To this end, we made a triple alanine mutant in the ⁹⁸RDR¹⁰⁰ sequence (encoded by Dß-N), which is highly solvent accessible and, as such, their mutation is not predicted to affect the conformation of the CDR3 loop (Fig. 4 A). SPR-binding studies of this "AAA" mutant NKT15 TCR revealed its affinity for both human and mouse CD1d/-GalCer was comparable to the wild-type NKT15 (Fig. 4 B), indicating these three exposed residues are not critical to the affinity of the interaction. In addition, this hybrid TCR inhibited staining of mouse NKT cells by mCD1d/-GalCer tetramers at the same concentration as NKT12 and NKT15 TCRs (Fig. 4 B).

View larger version (48K): [in this window] [in a new window]   Figure 4. NKT cell receptor CDR3ß regions impact on the recognition of CD1d/-GalCer. (A) Conformation of the CDR3 loops of the NKT15 TCR revealing the highly exposed "RDR" motif at the tip of the CDR3ß loop. (B) Inhibition of NKT cell staining by recombinant wild-type and chimeric NKT TCRs. TCRs comprised LC13ß; NKT15ß; NKT15-chain/LC13 ß-chain; NKT15-chain/NKT15 ß-chain engrafted with the LC13 CDR3ß-loop; and NKT15-chain/NKT15ß-chain with "RDR" to "AAA" substitution within the CDR3ß loop. Graded concentrations of soluble recombinant TCRs were preincubated with phycoerythrin-labeled tetramers of mCD1d-GalCer. These were used to costain mouse thymocyte cells with anti-CD3 mAb and analyzed by flow cytometry. Shown are flow cytometry plots with tetramer-staining after preincubation with 500 µg/ml TCRs. *, the inhibition observed with maximal concentrations of the NKT+LC13(CDR3ß) (500 µg/ml) was only partial, and equivalent to the inhibition observed with 8 µg/ml (not depicted) of NKT15. The indicated KD values are calculated from SPR studies of TCR binding to immobilized human and mouse CD1d-GalCer (Table S3 and Fig. S2 for NKT12 and NKT15). LC13 is a control TCR from an HLA-B8–restricted, virus-specific CTL; ND, KD not measurable. (C) Superposition of the CDR3ß loop of LC13 in the context of the NKT CDR3 based on the known structure of LC13 (references 36, 37). The LC13 CDR3ß loop is predicted to impact on the conformation of CDR3 and to disrupt access to the putative Ag-binding cavity.

Cross-species reactivity of NKT cells with CD1d/-GalCer
Unlike the highly restricted recognition of MHC I molecules by conventional ß TCRs, NKT cell TCRs (including NKT12, 15, and 18) show a remarkable cross-species reactivity, implying evolutionary conservation of an important immune specificity and function (33). The invariant TCR -chain from human NKT cells has 54% sequence identity with its mouse counterpart, whereas the human Vß11 and mouse Vß8.2 share 65% sequence identity. This cross-species identity is particularly marked in the CDR3 and CDR1 loops in which 10/13 and 2/6 residues, respectively, are identical between human and mouse sequences (Fig. 5). Moreover, the two identical residues in the CDR1 are surface exposed. The CDR3 and CDR1 residues that are conserved across species form part of an extensive, contiguous surface at the ligand-binding interface of the NKT TCRs (Fig. 5 A). Based on the mode of recognition of MHC–peptide complexes (35), this region of the TCR would be expected to make interactions with the CD1d–glycolipid complex. In addition, the CDR3 and CDR1 loops make numerous interloop interactions involving residues that are largely conserved between the human and mouse semi-invariant NKT TCRs. These interactions are important in stabilizing the conformation of the CDR loops.

View larger version (29K): [in this window] [in a new window]   Figure 5. Conserved residues from human and mouse NKT TCR CDR1, CDR3, and CDR2ß loops form a contiguous conserved surface that is adjacent to the putative Ag-binding cavity. The different conserved CDR regions are colored to correspond to the sequence alignment of mouse and human CDR loops. All other conserved and semi-conserved residues are brown and orange, respectively, in the alignment and are not shown in the structure. The TcR -chain is white and the TCR ß-chain is green.

In the TCR ß-chain, only the CDR2ß shows significant sequence identity between mouse and human TCRs that share 4/6 residues at the tip of this loop, and 4 amino-terminal residues (⁴⁵LIHY⁴⁸) and 5 carboxy-terminal residues (⁵⁵TEKGD⁵⁹) either side of the tip of the loop. Notably, the surface exposed CDR2ß loop, and the highly conserved "hot spot" formed by the CDR3 and CDR1 loops merge to create an even more extensive species-conserved region at the ligand interface (Fig. 5). Accordingly, this region of the TCR is likely to play a crucial role in CD1d–glycolipid interactions by mediating restricted recognition and cross-species reactivity. In contrast with the CDR2ß loop, the CDR1ß loop is only moderately conserved across human and mouse sequences. However, as outlined earlier, there are five further cross-species conserved residues that line the cavity at the ligand interface of the NKT TCR: Tyr33ß, Arg33, Gly96, Ser97, and Leu104.

Collectively, the structures of NKT12 and NKT15 suggest that the species conservation of CDR3, CDR1, and CDR2ß loops creates a contiguous conserved hot spot. It is highly likely that this hot spot and the collection of conserved residues forming the cavity at the ligand-interface are crucial in mediating the reciprocal cross-species reactivity between human and mouse NKT TCRs.

Proposed docking of NKT TCR onto CD1d/-GalCer
The NKT TCR structure, together with the recently published CD1d/-GalCer structures and previous mutagenesis experiments, has allowed us to propose a model for the interactions between the NKT TCR and CD1d/-GalCer. For several reasons, the model was based on the LC13–HLA–B8–FLR complex. First, we examined all the TCR–MHC-I-p complexes solved to date and the LC13 complex provided the best approximation for docking of the central TCR pocket over the -GalCer head group. Second, like -GalCer, the peptide determinant recognized by LC13 is a minimally protruding Ag and is similarly located to the glycosyl head group of -GalCer. Moreover, LC13 also has a central pocket that accommodates the main side chain of the ligand as proposed for NKT TCRs. Despite the known caveats of any structural estimation, the model provides plausible insight into NKT TCR recognition of hCD1d/-GalCer and is consistent with several published observations. Thus, in both the mouse and human CD1d molecules, the conserved residues Arg79, Asp80, Glu83, and Asp153 (Asp 151 in hCD1d) are critical for presentation of -GalCer by CD1d (44). Of these amino acids, Arg79 and Asp153, but not Glu83, make interactions with -GalCer (14, 15). However, mutagenesis studies suggest that all three can influence recognition by the NKT TCRs (17, 44, 45), perhaps directly or by affecting the orientation of the -GalCer glycosyl head group. Taking these issues into consideration, the model is consistent with diagonal docking of the NKT TCR over the ligand-binding domain of CD1d (Fig. 6). Because -GalCer protrudes only minimally from the CD1d cleft, the NKT TCR forms considerable contacts with CD1d. Thus, the CDR1 and CDR2 loops are primarily positioned over the 2 helix, interacting with hCD1d residues spanning Trp153–Trp160. The aromatic residue Phe51 (His51 in mouse), located within the CDR2 loop, could potentially stack neatly between the two bulky aromatic rings of Trp153 and Trp160 of hCD1d. The CDR1ß and CDR2ß loops docks over the 1 helix of CD1d, interacting with a localized stretch of CD1d residues (75–83). Although CDR2ß is located on the periphery, our model suggests that the prominent Tyr48ß packs against Ser75 and the aliphatic moiety of Arg79 in CD1d. Moreover, our model suggests a potential salt bridge between Arg79 and Asp30ß of the CDR1ß loop. This is consistent with a role for Arg79 independent of its interactions with -GalCer, as an important residue for recognition of CD1d/-GalCer by many NKT TCRs. Notably, however, substitution of this residue can be tolerated by certain NKT cells indicating a variable contribution of Arg79 contingent upon the nature of the glycolipid Ag (44). The conserved TCR residues Asp94, Arg95, and Arg103 are solvent exposed and proximal to the binding interface cavity, where their charged nature provides the potential for complementary electrostatic interactions with one or more of these CD1d residues.

View larger version (34K): [in this window] [in a new window]   Figure 6. Proposed docking of NKT TCR onto CD1d/-GalCer. (A) In the model, the NKT12 and ß chain are shown in gray and pink, respectively, and the CDR loops are red (CDR1), green (CDR2), blue (CDR3), magenta (CDR1ß), yellow (CDR2ß), and cyan (CDR3ß). -galactosylceramide is shown in orange within the antigen-binding domain of hCD1d (purple). (B) View of the antigen-binding cleft of human CD1d–presenting -GalCer (orange). The CDR footprint of the proposed docking model of NKT12 is shown for reference and the CDR loops are colored as in A. Surface-exposed residues on the 1 and 2 helices of hCD1d are shown. Residues in red had an impact on NKT activation by mCD1d in the published literature. Residues in white either had no impact or were not investigated. Residues in yellow are those residues likely to be important in the docking of NKT12 onto CD1d/-galactosylceramide according to our proposed model.

Upon ligation, the surface-exposed galactose of -GalCer is "walled" by the CDR3 and CDR3ß loops such that the galactose is positioned within the electropositive preformed cavity. The galactose head group of -GalCer is likely to interact with residues lining the pocket, including Asn31, Arg33, Gly96, Ser97, and Lys31ß. The role of Arg33 may involve specificity interactions analogous to those observed in the LC13 TCR binding pocket, where His33 at the base of the pocket makes a water-mediated bond to a critical peptide side-chain from the ligand (37).

Larger Ag headgroups, such as the tri-hexosyl moiety of the glycosphingolipid iGb3 (7, 8), are likely to be partially accommodated via movement in the CDR3 loops. This is consistent with the observed plasticity of ßTCRs in engaging with pMHC molecules. We predict that the flexible CDR3ß loop will allow modulation of NKT cell recognition of different glycolipids. Therefore, the extent of the preformed cavity volume is likely to change, depending on the position of the CDR3ß loop. This is consistent with a high degree of structural variation being tolerated in this region without loss of affinity to ligands such as -GalCer.

	DISCUSSION

Top Abstract RESULTS DISCUSSION MATERIALS AND METHODS References

 
NKT cells straddle the roles of both innate and adaptive cellular immune responses (46). They respond rapidly upon antigen recognition by their semi-invariant TCR, producing a variety of cytokines that can regulate tumor immunity, autoimmunity, and allergy and can orchestrate protective responses to infectious agents (2, 47–49). Despite expressing a semi-invariant TCR, NKT cells are reported to be selected or activated by different glycolipid molecules such as the marine sponge-derived -GalCer (6), a phosphoethanolamine (50), PIM4 (11), -glycuronosyl-ceramides (8–10, 51, 52), sulfated variants thereof (10), a tumor-derived ganglioside GD3 (31), self-antigens including some forms of ß-galactosylceramide (53, 54), and isoglobotrihexosylceramide (iGb3) (7). This capacity to recognize a variety of foreign and self-derived glycolipid Ags reflects the increasingly diverse functional roles ascribed to NKT cells (47–49) and highlights the need for a structural explanation as to how the semi-invariant NKT TCR can achieve such a diversity of recognition. Abundant evidence suggests that the glycosyl head group of the glycolipid sugar moiety is a critical determinant recognized by the NKT TCR (14–16, 29, 44, 45, 55, 56). Thus, the NKT TCR binds efficiently to CD1d complexed with -GalCer but not with ß-GalCer containing similar sphingosine and acyl chains (56), consistent with the lipid chains being buried in the Ag-binding cavity, whereas the glycosyl head group protrudes out of the cleft (14, 15). It is notable that the small target created by the polar head of -GalCer induces a highly selected "immunodominant" ß TCR repertoire. This bias is reminiscent of the highly restricted CTL repertoires toward minimally exposed (36, 57, 58) or highly unusual viral determinants (59–61).

These observations would be consistent with the cavity at the ligand interface of the NKT12 and NKT15 TCRs acting as a preformed Ag pocket. The structures of CD1d/GalCer have led to speculation that the CDR3 of the NKT TCR would be placed over the galactose ring (14). This mode of interaction fits the idea that the cavity might envelope the polar glycosyl head group of -GalCer and some related antigens. Supporting this interpretation, the walls of the cavity are formed from unique amino acids present in the V-domain (CDR1 and Arg33) and CDR3 regions of the invariant TCR -chain. Moreover, these regions are highly conserved between mouse and human NKT TCRs that are functionally cross-reactive (62). In addition, CDR1ß and CDR3ß residues also contribute to the cavity wall, providing a mechanism by which the CDR3ß could modulate Ag specificity. Many studies have reported heterogeneous CDR3ß usage by NKT cells, suggesting that these loops do not play a key role in NKT cell recognition (25–28). However, our data indicate that not all CDR3ß sequences support recognition of CD1d–glycolipid complexes and this is probably the result of steric hindrance by some CDR3ß loops that obstruct the cavity interface and/or interfere with the conformation of the highly conserved CDR3 loops.

The binding kinetics and affinities of the human NKT12, NKT15, and NKT18 TCRs for hCD1d/-GalCer are typical of conventional ßTCR–MHCp interactions with K_D values of 8–20 µM. This contrasts with the high affinity binding of recombinant mouse NKT TCRs, where the K_D values are reported to be 0.1–0.3 µM (28, 45). These differences presumably reflect species variability despite the remarkable reciprocal cross-reactivity of human and mouse NKT cells. This cross-reactivity correlates with a conserved hot spot at the NKT TCR interface that comprises a contiguous surface suitable for interacting with CD1d–glycolipid complexes. Additional conserved residues forming the cavity at the ligand interface are also likely to contribute to the reciprocal cross-species reactivity. Despite the cross-species interactions, the glycosyl head group of -GalCer is shifted by up to 3 Å between the mouse and human structures, which is likely to impact on the recognition of the galactose moiety itself (63). Human CD1d has a tryptophan residue at position 153 that pushes the galactose head group away, whereas, in mouse, the equivalent amino acid (position 155) is a much smaller glycine residue (14, 15). The previously reported lack of temperature dependence in the binding affinity of mouse NKT TCRs with CD1d-GalCer suggests a rigid "lock and key" mode of binding (28, 56). However, the cross-reactivity of human and mouse NKT TCR for CD1d–-GalCer implies that NKT TCR recognition has considerable structural plasticity more typical of ßTCR–MHC-I interactions and potentially important in the recognition of different glycolipid antigens.

How can NKT cells, with a semi-invariant TCR, recognize a diversity of glycolipid antigens with structurally distinct carbohydrate head groups? Although this can only be definitively answered with direct structural studies, the recently defined -glucuronosylceramides (8–10, 51, 52) are structurally very similar to -GalCer and -glucosylceramide and thus conceivably would fit into the same TCR cavity. It is also possible, or even likely, that only a minor subset of fresh human NKT cells recognize some of these more recently defined antigens. For example, <10% of NKT cells were labeled by PIM4-loaded CD1d tetramers (11) and the labeling was 10–100-fold weaker than for -GalCer-loaded CD1d tetramers. Similarly, although in mice most NKT cells depend on iGb3 for thymic selection (7), it is not proven that iGb3 is a key selecting ligand for human NKT cells (64). Moreover, given that mouse NKT cells do not stain with CD1d/iGb3 tetramers (7), their affinity for NKT TCRs is likely to be very low, suggesting that the tri-hexosyl head group of this Ag might be poorly recognized in comparison to the galactosyl head group of -GalCer. Furthermore, the extent to which fresh human NKT cells recognize other glycolipid ligands such as GD3 (31) and phosphoethanolamine (50) is unknown. Notwithstanding these concerns, it is not inconceivable that different sugar groups could be accommodated within the pocket by a degree of plasticity in the CDR3 loops and in particular the CDR3ß loop, which was shown to exhibit a degree of flexibility in our study.

Collectively, our findings suggest a basis for the semi-invariant nature of NKT TCRs in which highly conserved residues in the - and ß-chains of the TCR contribute to binding of glycolipid head groups through a discrete cavity at the ligand interface. Other conserved features of the NKT TCR appear to play a role in CD1d binding, whereas plasticity of antigen recognition is preserved through variation in CDR3ß loops and the intrinsic adaptability of other CDR loops at the ligand interface. Furthermore, our proposed model of the complex suggests that the CDR3 loop plays a prominent role in determining Ag specificity as well as dictating the observed cross-species reactivity via interactions with CD1d. It will be intriguing to visualize this plasticity in complexes of NKT TCRs with a variety of CD1d/glycolipid antigens and to correlate the modes of interaction with functional diversity in NKT cells.

	MATERIALS AND METHODS

Top Abstract RESULTS DISCUSSION MATERIALS AND METHODS References

 
Protein expression, refolding, and purification.
cDNAs encoding NKT cell receptors and CD1d were derived as described in the Supplemental Materials and methods. Inclusion body protein of the NKT TCR and NKT TCRß chains were prepared essentially as described previously (36, 37, 65). 64 mg TCR and 32 mg TCRß inclusion body proteins were thawed, pulsed with 1 mM DTT, combined, and injected into 800 ml of stirring refolding buffer containing 100 mM Tris, pH 8.5, 5 M urea, 0.4 M arginine, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 2 mM EDTA, 0.2 mM PMSF, and 1 µg/ml Pepstatin A at 4°C. Equal amounts of the NKT TCR and NKT TCRß chain inclusion body proteins were added 16 h later. After 24 h, refolded protein was dialyzed against 10 mM Tris-HCl, 0.1 M urea, pH 8, and subsequently against 10 mM Tris-HCl. Dialyzed protein was captured on a column containing DEAE Sepharose Fast Flow (GE Healthcare), and eluted with 10 mM Tris-HCl, 200 mM NaCl. Eluted NKT TCR was concentrated and loaded unto a HiLoad Superdex 75 pg gel filtration column (GE Healthcare) in the presence of 10 mM Tris-HCl, 150 mM NaCl. Fractions containing the TCR were pooled and purified further on a Mono-Q column (GE Healthcare). Peak fractions were pooled and concentrated before one more purification by gel filtration as described earlier in this paragraph. Peak fractions were concentrated to 10–20 mg/ml and were used subsequently in crystal trials.

Crystallization.
Rod-shaped crystals of the NKT TCRs were grown at 12 mg/ml by the hanging drop vapor diffusion technique and at room temperature. NKT12 crystals grew in 18% PEG 3350, 0.1 M cacodylate, pH 6.3, 0.2 M lithium chloride, and NKT15 crystals grew in 9% PEG 3350, 0.1 M cacodylate, pH 6.4, 0.2 M ammonium acetate. The crystals belong to space group C222₁ and the unit cell dimensions were consistent with one molecule per asymmetric unit (Table II). NKT15 crystals were also grown in 0.2 M sodium sulfate. 20% PEG 3350, pH 6.6. These crystals belong to space group P3₂, with unit cell dimensions consistent with two molecules per asymmetric unit (Table II).

Structure determination and refinement.
The crystals were flash frozen before data collection using up to 15% glycerol as the cryoprotectant. The data were processed and scaled using the HKL package. Datasets of 2.2, 2.4, and 2.6 Å resolution were collected at the BioCars beamline using a Quantum 4 CCD detector (Table II). The crystal structures were solved using the molecular replacement method, as implemented in MOLREP (66), using the unliganded LC13 (37) structure as the search model, where the LC13 CDR loops were initially removed and all other nonidentical residues were mutated to alanine. Unbiased features in the initial electron density map confirmed the correctness of the molecular replacement solution. The progress of refinement was monitored by the R_free value with neither a sigma, nor a low resolution cut off being applied to the data. Initially, the structures were refined using rigid-body fitting of the individual domains followed by the simulated-annealing protocol implemented in CNS (version 1.0) (67). Later, translation, libration, and screw-rotation displacement (TLS) refinement was performed using REFMAC (68). Refinement was interspersed with rounds of model building using the program "O" (69). Tightly restrained individual B-factor refinement was used, and bulk solvent corrections were applied to the data set. Water molecules were included in the model if they were within hydrogen-bonding distance to chemically reasonable groups, appeared in F_o – F_c maps contoured at 3.5, and had a B-factor of <60 Ų. The structures have been deposited in the PDB (accession nos. 2EYR, 2EYS, 2EYT).

Biotinylation of CD1d and loading with -GalCer.
Biotinylated CD1d was mixed with -GalCer at a molar ratio of 1:3 (protein:lipid) at room temperature overnight. Loaded CD1d protein was then purified from free -GalCer and buffer exchanged into 10 mM Hepes-HCl, pH 7.4, 150 mM NaCl, and 0.005% surfactant P20 using a HiLoad Superdex 200 pg gel filtration column (GE Healthcare). Protein quality was monitored by its gel filtration profile and mobility on SDS-PAGE.

SPR binding studies.
All SPR experiments were conducted at 25°C on a Biacore 3000 instrument using HBS buffer (10 mM Hepes-HCl, pH 7.4, 150 mM NaCl, and 0.005% surfactant P20 supplied by the manufacturer) supplemented with 1% BSA. Approximately 2,000–3,000 RU of -GalCer–loaded human and mouse CD1d was immobilized onto streptavidin-coupled sensor chip (BiaCore). Corresponding levels of "unloaded" CD1d were also immobilized to act as controls. Equilibrium affinity was determined by injecting increasing concentrations of NKT TCR over all flow cells at 5 µl/min for 3 min. The final response was calculated by subtracting the response of the "unloaded" CD1d surface from the -GalCer CD1d surface. BIAevaluation version 3.1 (Biacore AB) was used to fit the equilibrium data.

CD1d/-GalCer tetramer inhibition assay and flow cytometry.
Anti-CD3–FITC (clone 145-2C11) was purchased from BD Biosciences. Fc-receptor block (anti-CD16/CD32, clone 2.4G2) was added to all staining cocktails. Mouse CD1d tetramer loaded with -GalCer was produced as described previously (70). CD1d/-GalCer tetramer was incubated with serially diluted (twofold dilutions from 500 µg/ml to 4 µg/ml) soluble human NKT cell TCR or an irrelevant TCR (LC13) control. Thymic cell suspensions were made by gently grinding the organ between the frosted ends of glass microscope slides in ice-cold PBS containing 2% FCS (FACS buffer). Cell suspensions were passed through 100 µm mesh before antibody staining. Equivalent inhibition of staining of thymocytes with CD1d/-GalCer tetramers was observed with NKT12, NKT15, NKT18, and NKT15‘AAA’ TCRs, and inhibition with these NKT TCRs was diminished equally upon dilution. Thymocytes were stained with anti-CD3 mAb, washed with FACS buffer, and stained with the CD1d/-GalCer tetramer–TCR mixtures. Cells were washed with FACS buffer to remove any unbound CD1d/-GalCer tetramer and analyzed by flow cytometry using a FACScalibur (Becton Dickinson) and analyzed using CELLQuest software (Becton Dickinson).

Modelling of the NKT TCR–CD1d–-GalCer complex.
The docking of NKT12 onto hCD1d was approximated in the following way. The MHC of each of the MHC–peptide–TCR complex structures available in the PDB was aligned onto the CD1d antigen-binding domain (residues 61–86 and 139–171). The TCRs from each of the complexes were examined for clashes with the helices of hCD1d and the -GalCer. The TCR with the best fit over CD1d/-GalCer was LC13 (PDB code: 1MI5) and so the C domain of LC13 was used to superpose NKT12 in the final CD1d–-GalCer–NKT12 model. NKT12 was adjusted manually using the program "O" to promote a better fit for our proposed model.

Online supplemental material.
Supplemental Materials and methods for cDNA cloning of NKT TCRs and CD1d are provided. Table S1 provides contacts at the V–Vß interface of NKT12 and the genetic origin of TCR residues. Table S2 describes the Phi psi torsion angles of CDR1 and CDR2 in NKT12. Table S3 describes the dissociation constants of natural and mutated NKT TCRs with CD1d/-GalCer. Table S4 gives the amino acid sequences of CDR3ß residues in natural and mutated NKT TCRs. Fig. S1 models the Ag-binding cavity of the NKT TCR to demonstrate how this can accommodate the galactose head group of -GalCer. Fig. S2 shows the SPR sensorgrams and affinity estimations of NKT12 binding to immobilized hCD1d-GalCer and mCD1d-GalCer. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20051777/DC1.