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The V14 NKT Cell TCR Exhibits High-Affinity Binding to a Glycolipid/CD1d Complex¹

Stéphane Sidobre^2,*, Olga V. Naidenko^2,3,*, Bee-Cheng Sim^4,, Nicholas R. J. Gascoigne, K. Christopher Garcia and Mitchell Kronenberg^5,*

^* Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121; Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037; and Departments of Microbiology and Immunology and Structural Biology, Stanford University School of Medicine, Stanford, CA 94305

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Most CD1d-dependent NKT cells in mice have a canonical V14J18 TCR rearrangement. However, relatively little is known concerning the molecular basis for their reactivity to glycolipid Ags presented by CD1d. Using glycolipid Ags, soluble forms of a V14 NKT cell-derived TCR, and mutant and wild-type CD1d molecules, we probed the TCR/CD1d interaction by surface plasmon resonance, tetramer equilibrium staining, and tetramer staining decay experiments. By these methods, several CD1d -helical amino acids could be defined that do not greatly alter lipid binding, but that affect the interaction with the TCR. Binding of the V14⁺ TCR to CD1d requires the agonist -galactosylceramide (-GalCer), as opposed to the nonantigenic -galactosylceramide, although both Ags bind to CD1d, indicating that the carbohydrate moiety of the CD1d-bound Ag plays a major role in the TCR interaction. The TCR has a relatively high-affinity binding to the -GalCer/CD1d complex, with a particularly slow off rate. These unique properties are consistent with the coreceptor-independent action of the V14 TCR and may be related to the intense response to -GalCer by NKT cells in vivo.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Conventional T lymphocytes are reactive to peptide Ags presented by MHC class I or class II molecules. In addition to these cells, a number of nonconventional T lymphocyte subsets have been described. One of the best-studied cell types in this category is the diverse group of T lymphocytes that respond to glycolipid Ags presented by CD1 molecules (1, 2). NKT cells constitute the largest population of CD1-reactive T cells identified in vivo so far (3). In mice, NKT cells represent up to 30% of the total lymphocytes in the liver, 20% of the TCR⁺ T cells in the bone marrow, and 3% of the TCR⁺ T cells in the spleen (4, 5). The majority of NKT cells in the mouse express a canonical or invariant V14J18 rearrangement, which is typically paired with V8.2, V7, or V2, although there is apparently little or no selection for the complementarity-determining region (CDR)⁶3 of the associated -chain (6, 7, 8). V14⁺ NKT cells are reactive to CD1d, a nonclassical class I Ag-presenting molecule (3). The natural ligand or ligands presented by mouse CD1d to these T cells are not known. However, a model Ag, -galactosylceramide (-GalCer), stimulates nearly all of these cells and has proven useful in the analysis of their potential functions (5, 9, 10, 11). Indeed, the injection of -GalCer triggers a rapid, transient, and massive response of mouse NKT cells, which includes the secretion of IFN- and IL-4 (9, 11, 12). NKT cell activation propagates rapidly to other cell types, among which are NK cells, dendritic cells, and subsets of B and conventional T cells. This NKT cell-mediated activation is accompanied by the induction of both costimulatory molecules and cytokines by the responding cell types (5, 13, 14, 15). There is no evidence for clonal expansion or a memory response by NKT cells (11, 16, 17). In fact, they are not detectable soon after -GalCer stimulation, and they may undergo activation-induced cell death. Therefore, the quick and vigorous response of NKT cells to -GalCer is more similar to an innate rather than an adaptive immune response (4, 5, 11, 18). This response may allow NKT cells to regulate adaptive immunity in several contexts, including the protection against autoimmune diseases (19, 20, 21), parasites (22, 23, 24), and bacteria (25, 26), and in antitumor responses (27, 28).

In multiple studies of the interaction of the TCR with either MHC class I or class II molecules, a correlation has been observed between the outcome of the T cell response in developing or mature cells and the t_1/2 of the interaction of the TCR with the peptide-MHC complex (29, 30, 31). However, so far relatively little is known about the molecular basis of NKT cell TCR reactivity.

In this work we present the first biophysical investigation of the characteristics of the trimolecular interaction between a CD1 molecule, a glycolipid Ag, and an TCR. This has been done using soluble mouse CD1d molecules, the model Ag -GalCer, and a soluble TCR obtained from an NKT cell hybridoma. The results indicate that NKT cell TCR has properties distinct from the TCRs expressed by conventional T cells. These properties may be responsible for the unusual nature of the response to -GalCer and perhaps also for some of the singular features of NKT cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Baculovirus expression of CD1d mutants

CD1d mutants (F10A, R79E, R79E-D80A, and L150I-D153Y) were subcloned into the 5' SalI and 3' BamHI sites in a previously described baculovirus transfer vector for expression of wild-type (wt) CD1d tetramers (11). This cloning strategy placed the mutant CD1d H chains under the control of the polyhedrin promoter in a two-promoter vector and in front of a BirA/His tag cassette. Virus production and protein purification were performed as previously described (11). Briefly, High Five (BTI-TN-5B1-4) cells (Invitrogen, San Diego, CA) were infected with baculovirus at a multiplicity of infection of 5–10. After 4–5 days, supernatants were harvested and dialyzed against 0.15 M sodium phosphate buffer (pH 7.4) and CD1d proteins were purified by affinity chromatography on Ni-NTA columns (Qiagen, Valencia, CA), followed by anion exchange chromatography on monoQ column (Amersham Pharmacia Biotech, Piscataway, NJ). To assess the reactivity of the soluble proteins with conformation-specific CD1d mAbs, an anti-penta-His-Tag mAb (Qiagen) was immobilized (1 µg/well) on a 96-well plate by incubation for 1 h at 37°C. The plates were then washed five times with PBS and blocked by incubation for 1 h at 37°C with 10% FCS in PBS. wt or mutated CD1d molecules (at different concentrations, see Fig. 1) were then captured by incubation for 1 h at 37°C. After washing, 1 µg/well 11 anti-CD1d mAb (32) or 5C6 anti-CD1d mAb (gift of Dr. C.-R. Wang, University of Chicago, Chicago, IL) (33) were added to protein-captured wells and to control wells without CD1d. After incubation for 1 h at 37°C, bound mAbs were detected using a HRP-conjugated secondary Ab (Jackson ImmunoResearch Laboratories, West Grove, PA).

View larger version (22K): [in this window] [in a new window]   FIGURE 1. wt and mutant CD1d molecules are recognized by conformation-sensitive CD1d mAbs. ELISA measurement of the ability of two conformation sensitive CD1d mAbs, 1B1 (A) and 5C6 (B), to bind to the indicated soluble CD1d molecules that were captured on a 96-well plate via their His tags. One representative experiment of two carried out in triplicate is shown.

A single-chain (sc)TCR construct was designed to include the V and V segments from the 2C12 (V14V8.2) hybridoma TCR separated by a 15-aa (Gly⁴Ser³) linker, as previously described (34). The invariant V14J18 segment was truncated after the last amino acid of J18 (Pro). Note that J18 was originally called Ja281 and in subsequent reclassifications was first called J15 (35) and then J18 (36). The V8.2J2.5 segment was truncated after the last amino acid of J8.2 (Leu). The primers used were V14 forward primer (5'-TCCAATTCCATATGAAGACCCAAGTGGAGCAGAGTCC-3' (NdeI site underlined)) and reverse primer, which introduced the GlySer linker (5'-CTCGGATCCACCGCCTCCGGAGCCACCTCCGCCTGAACCGCCTCCACCAGGTATGACAATCAGCTGAGTCCC-3' (BamHI site underlined)) and V8.2 forward primer (5'-GGTGGATCCGAGGCTGCAGTCACCCAAAGCCCAAG-3' (BamHI site underlined)) and reverse primer (5'-ATTGAATTCTTATAACACGAGGAGCCGAGTGCCTGG-3' (EcoRI site underlined, stop codon in bold)). V14 and V8.2 segments were cloned into pET28a(+) plasmid (Novagen, Madison, WI). V14 was cloned into NdeI/BamHI sites, and the V8.2 segment was cloned into BamHI/EcoRI sites, in frame with the N-terminal 6-His tag and a thrombin cleavage site. Two surface-exposed hydrophobic residues on the V8.2 domain (Ile⁷⁵ and Leu⁷⁸) were replaced by two serine residues to improve expression yield and solubility (34). Importantly, these mutations, located distal from the putative contact surface with the -GalCer/CD1d complex, have been shown in another system not to affect the binding of the scTCR to the antigenic complex (34).

scTCR expression and refolding

The pET28 scTCR plasmid was transformed into BL21(DE3) cells (Novagen) for high-level expression, and inclusion bodies were purified following standard protocol (37). scTCR refolding was performed following a published protocol (34). Briefly, inclusion bodies were dissolved in 7 M GnHCl with 10 mM 2-ME by addition of 1.1 g GnHCl powder to 1 ml of a slurry containing the inclusion bodies. Melted inclusion bodies were spun to remove undissolved material. A total of 50–100 mg of scTCR protein was added dropwise to 200 ml of refolding buffer (3 M urea in 50 mM Tris-HCl (pH 8), with 2 mM reduced glutathione and 0.2 mM oxidized glutathione) with stirring at 4°C. Stirring was continued for 2–4 h at 4°C and, subsequently, stirred refolding mixture was further diluted with 0.2 M NaCl, 0.05 M Tris-HCl (pH 8), added dropwise at a flow rate of 1 ml/min. After dilution, the final volume of 1.2 L was stirred overnight to allow for precipitation of aggregated protein, filtered through a 0.2-µm filter, and incubated with stirring with 1 ml Ni agarose beads (Qiagen) for 24 h. Ni agarose beads were harvested on a scintered glass funnel, and the refolded TCR was eluted with 0.5 ml of 0.5 M imidazole in 50 mM Tris-HCl (pH 8), 150 mM NaCl. The eluted material was immediately injected onto a Superdex 200 gel filtration column (Amersham Pharmacia Biotech) for size exclusion chromatography in 50 mM Tris (pH 8), 200 mM NaCl. The purity of the preparation was checked by SDS-PAGE followed by staining with Coomassie blue.

Tetrameric -GalCer/CD1d complexes

Tetramers of wt or mutant CD1d molecules were produced as described by Matsuda et al. (11). CD1d molecules were biotinylated with the BirA enzyme (Avidity, Denver, CO) following the manufacturer’s protocol. Biotinylated CD1d molecules were then incubated overnight at room temperature with a 3-fold molar excess of -GalCer (solubilized in 0.5% Tween 20, 0.9% sodium chloride, hereafter called vehicle) or with an equal amount of vehicle alone. -GalCer-loaded or unloaded monomers were then tetramerized using a 1:4 molar ratio of PE-conjugated streptavidin (BD PharMingen, San Diego, CA). This preparation was analyzed by gel filtration chromatography on Superdex 200 column (Amersham Pharmacia Biotech) equilibrated in PBS.

Tetramer staining

All stainings and washes were performed in a buffer consisting of 2% FCS, 0.075% sodium bicarbonate, and 0.1% sodium azide in RPMI medium (Sigma-Aldrich, St. Louis, MO), according to Savage et al. (38). For equilibrium staining, hybridoma cells were stained for 3 h at room temperature, with -GalCer-loaded or unloaded wt or mutant CD1d tetramers, in concentrations ranging from 1.2 x 10^-10 to 5.8 x 10^-8 M and 10 µg/ml anti-TCR mAb (BD PharMingen) used to normalized the intensity of tetramer staining to the level of TCR present on the surface of the cells. The cells were then washed two times and fixed in 1% paraformaldehyde (Sigma-Aldrich) and 1 µg/ml propidium iodide (BD PharMingen) in PBS. The intensity of fluorescence on hybridoma cells was determined by flow cytometry analysis using a FACScan flow cytometer (BD Biosciences, Mountain View, CA). For tetramer staining decay, 2C12 hybridoma cells were stained for 45 min at room temperature with 10 µg/ml anti-TCR mAb and 0.5 nM -GalCer-loaded or unloaded wt CD1d tetramers. The cells were then either cooled at 4°C or warmed at 37°C. After 15 min of equilibration, the cells were washed and 100 µg/ml of the anti-CD1d mAb 1B1 were added to prevent rebinding of the tetramers. At various time points, an aliquot was washed two times and fixed in 1% paraformaldehyde and 1 µg/ml propidium iodide in PBS. The intensity of fluorescence was determined by flow cytometry analysis. For all analyses, TCR-negative and propidium iodide-positive cells were gated out. For tetramer staining decay on freshly isolated NKT cells, B10.A thymocytes were stained with a mixture of -GalCer-loaded CD1d tetramer (0.5 nM), anti-TCR mAb, anti-NK1.1, and anti-CD8 (10 µg/ml each) for 45 min at room temperature. The cells were washed and 100 µg/ml of the anti-CD1d mAb 1B1 were added to prevent rebinding of the tetramers. At various time points, an aliquot was washed two times and fixed in 1% paraformaldehyde in PBS. The intensity of fluorescence was determined by flow cytometry analysis gating on cells that were TCR^intNK1.1⁺CD8^-.

Hybridoma bioassays

The V14V8.2 NKT cell hybridomas N38-2C12 (2C12) and DN3A4-1.2 (1.2) and the V14V10 NKT cell hybridoma DN3A4-1.4 (1.4) have already been described (39, 40). Stimulation of NKT cell hybridomas in an APC-free assay was performed as described by Naidenko et al. (41), with some modifications. Briefly, wt or mutated CD1d molecules (5 µg/ml in PBS) were coated in a 96-well plate by incubation of 100 µl for 1 h at 37°C. The plates were then washed five times with PBS and blocked by incubation for 1 h at 37°C with 10% FCS in PBS. After washing, -GalCer (50 ng/well) was added and incubated for 1 h at 37°C. Control wells, in which protein had or had not been coated, were incubated with 1% DMSO in PBS. The plates were then washed using complete RPMI medium and hybridoma cells (3 x 10⁴ cells/well) were immediately added. IL-2 release was measured after 16 h of culture in a sandwich ELISA using rat anti-mouse IL-2 mAbs (BD PharMingen) and a rIL-2 standard (BD PharMingen).

Surface plasmon resonance

All real-time binding experiments were performed on a BIAcore X biosensor system (BIAcore, Uppsala, Sweden), using 10 mM HEPES (pH 7.4), 150 mM sodium chloride, 3 mM EDTA, and 0.005% polysorbate 20 (HBS-EP, BIAcore) as running buffer. Analysis of wt or mutant CD1d binding to immobilized biotin -GalCer was performed as already described (41). For analysis of TCR binding, -GalCer/CD1d complexes or -GalCer/CD1d complexes were preformed by incubation of biotinylated CD1d molecules with a 3-fold molar excess of the Ag overnight at room temperature. Glycolipid Ags were a gift of the Kirin Pharmaceutical Research Laboratory (Gunma, Japan). Ag-loaded or unloaded CD1d monomers were immobilized to a level of 1000 resonance units (RUs) on flow cell (FC)2 of a neutravidin (Molecular Probes, Eugene, OR)-modified CM5 sensor chip (BIAcore) at a flow rate of 5 µl/min. No ligand was immobilized on FC1, which therefore provided a control surface that allowed corrections for refractive index changes and nonspecific protein sticking. The V14V8.2 scTCR was injected at a flow rate of 25 µl/min at concentrations of 5, 2.5, 1.25, and 0.625 µM in HBS-EP. As a specificity control, an irrelevant V8.2⁺ scTCR, the 172.10 TCR (34), was injected at a flow rate of 25 µl/min at a concentration of 5 µM in HBS-EP. Specific binding sensograms were obtained by subtraction of the RU values for the control FC1 from the RU values for the FC with immobilized Ag/CD1d (FC2). Kinetic parameters and/or apparent equilibrium K_d were obtained by fitting the specific sensograms with the built-in models of the BIAeval 3.1 software (BIAcore).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ag presentation by soluble, mutant CD1d molecules

The ability of transfectants expressing the F10A, R79E, R79E-D80A, and L150I-D153Y mouse CD1d mutants to present Ags to NKT cells has already been described (42). Using insect tissue culture cells, we produced soluble versions of these mutant CD1d molecules, as reported for wt CD1d (11). Gel filtration chromatography on a Superdex 200 column (data not shown) indicated that the molecules were eluted in the elution volume expected for a CD1d H chain-₂-microglobulin heterodimer. We further investigated the conformation of the soluble mutant CD1d molecules using conformation-sensitive anti-CD1d mAbs. wt CD1d, F10A, R79E, and R79E-D80A are recognized by the 1B1 anti-CD1d mAb, although lower reactivity was obtained with the R79E-D80A double mutant (Fig. 1A). As already reported for A20 transfectants, the soluble L150I-D153Y double mutant is not recognized by 1B1 (42). However, the wt and CD1d mutants exhibited comparable reactivity with the 5C6 anti-CD1d mAb (Fig. 1B), with a slight reduction in the reactivity of the R79E-D80A double mutant, as observed using 1B1. These data are consistent with the ones obtained for the A20 transfectants expressing these CD1d mutants (42) and therefore suggest that the soluble CD1d proteins have a native conformation.

We then examined the ability of soluble CD1d mutants to present the antigenic glycolipid -GalCer to NKT cell-derived hybridomas in an APC-free assay (41). The F10A mutation alters an amino acid side chain buried deeply in the CD1d A' pocket (42, 43). A substantial level of IL-2 secretion in response to stimulation by -GalCer was obtained with plate-bound F10A CD1d for all three hybridomas tested. The response of the 2C12 hybridoma (V8.2V14) was not significantly affected (Fig. 2), whereas the responses from 1.2 and 1.4 hybridomas were partially reduced. The R79E mutation alters an amino acid near the end of the 1 helix pointing upwards toward the TCR (42, 43). When -GalCer was presented by R79E, the response was decreased for both the 1.2 (V8.2V14) and 1.4 (V10V14) hybridomas, but it remained the same for 2C12 (Fig. 2). The reason for this difference is not known, although 2C12 and 1.2 differ only for the CDR3 region of the -chain (42). This suggests that differences in TCR may be responsible for the differing abilities to recognize the mutant CD1d protein, although wt CD1d molecules exert no evident selection pressure on CDR3 sequences in vivo (6, 7, 8). Ag presentation by the two CD1d double mutants, R79E-D80A and L150I-D153Y, which alter amino acid side chains in the 2 helix that point across the F' pocket (42, 43), was also tested. When -GalCer was presented by the two double mutants, no IL-2 secretion above the background level was detected with any of the hybridomas (Fig. 2). Overall, these results are consistent with those previously reported using transfected A20 B cells expressing the mutant CD1d molecules as APCs (42).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Immobilized wt and mutant CD1d exhibit different abilities to present -GalCer. A total of 500 ng/well wt or mutant CD1d proteins were immobilized on a 96-well plate, incubated with -GalCer (50 ng/well), and cultured with NKT cell-derived hybridomas. IL-2 release was measured by ELISA. Data present the average of the normalized specific response; normalization was conducted to permit averaging of the values from five experiments. For this comparison, the response stimulated by -GalCer presented by wt CD1d was set to 100%. The specific response was obtained by subtracting the responses to both CD1d protein alone and -GalCer alone.

wt and mutant CD1d molecules bind to -GalSer

To determine whether the differences in the mutant CD1d molecules to present -GalCer are due to an impaired glycolipid binding, we used a previously described surface plasmon resonance assay to analyze the ability of the CD1d mutants to bind to a biosensor chip coated with an analog of -GalCer, biotin -GalCer (41). The ability of wt CD1d to bind this analog, biotinylated at the end of the acyl chain (41), and its antigenicity for NKT cells have already been established (41, 44).

At neutral pH, the wt and the four mutant CD1d molecules tested exhibited a dose-dependent binding to the immobilized glycolipid. Representative sensograms for two of the five CD1d molecules tested are shown in Fig. 3, and the data for all five molecules are summarized in Table I. The sensograms, obtained at five different concentrations of wt and mutant CD1d molecules, were then analyzed to obtain the kinetic parameters for the lipid-CD1d interaction (Table I). The association and dissociation portions of the sensograms were best fitted using the 1:1 Langmuir model. To asses the goodness of the fit, the residuals (difference between the theoretical value generated by the fitting algorithm and the experimental value) obtained for each time point are plotted against time (Fig. 3). The plot shows very low residual values and does not display any systematic deviation, which indicates that the experimental values are well fitted by the 1:1 Langmuir association model. This analysis leads to an on rate of 3.3 x 10⁴ M^-1 s^-1 and an off rate of 1.9 x 10^-2 s^-1, which therefore gives a calculated K_D of 0.5 µM for the binding of wt CD1d molecules to biotin -GalCer. The interactions of the four CD1d mutants with the immobilized -GalCer exhibit almost the same parameters (Table I). However, the slight differences we observed are unlikely to be responsible for the drastic changes observed in the ability of the mutant CD1d molecules to present -GalCer to NKT cell-derived hybridomas (Fig. 2).

View larger version (29K): [in this window] [in a new window]   FIGURE 3. wt and mutant mCD1 molecules bind to immobilized biotin -GalCer. Biotin -GalCer was immobilized on FC2 of a streptavidin sensor chip. wt CD1d (A) or the R79E-D80A double mutant (B) CD1d molecules (at concentrations of 1.25, 2.5, 5, 7.5, and 10 µM) were then successively injected on FC1 (control FC) and FC2 at 5 µl/min. The sensograms depict specific binding after subtraction of the RU values for the control FC (FC2 - FC1). Data from one representative experiment of three are shown.

View this table: [in this window] [in a new window]   Table I. Kinetic analyses of the binding of -GalCer to wt and mutant CD1d molecules1

CD1d tetramers loaded with -GalCer are a powerful tool in the identification of V14⁺ CD1d-reactive T cells (10, 11, 45). Therefore, we produced tetrameric forms of the four soluble mutant CD1d molecules and used these to stain T cell hybridomas. Gel filtration chromatography on a Superdex G200 column demonstrated similar chromatograms for the wt and mutant mCD1d preparations, indicating adequate tetramer formation (data not shown). Consistent with a previous report by Matsuda et al. (11), we found that -GalCer-loaded wt CD1d tetramers bound to the three V14⁺ NKT cell-derived hybridomas tested (Fig. 4A and Table II), with little reactivity of the unloaded tetramers. Using a concentration range of tetramers from 1.2 x 10^-10 to 5.8 x 10^-8 M, we showed that this binding is saturable (Fig. 4B). A Scatchard transformation of the binding isotherms (Fig. 4B, inset) was conducted. The linear nature of the -GalCer/CD1d tetramer binding has also been observed in a previous study using peptide/MHC class II tetramers and conventional CD4⁺ T cells (38). Indeed, within a tetramer molecule, cooperativity triggers an increase of the global avidity of the tetramer compared with that of the monomer, which in fact permits staining with tetrameric form of -GalCer-loaded wt CD1d tetramers (10, 11). However, in this work we examined the binding of tetramer molecules taken as a whole entity. The linear Scatchard plot we obtained suggests that the binding of individual tetramers to T cells does not exhibit significant cooperativity between tetramers. The Scatchard transformation gives an apparent equilibrium K_D that is similar for the three V14⁺ hybridomas: 0.49, 1.6, and 1.14 nM for the binding to the 2C12, 1.2, and 1.4 hybridomas, respectively (Table II). Those values are much lower than the apparent K_D of 34 and 60 nM obtained for the binding of MCC/I-E^K tetramers to T cells from 5C.C7 or 2B4 from transgenic mice (38), suggesting a relatively stable interaction of -GalCer-loaded CD1d tetramers with the NKT cell TCR. The data also indicate that neither the V used nor the CDR3 region influences the interaction.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. Specific CD1d tetramer staining of the 2C12 hybridoma. A, 2C12 hybridoma cells were stained with different CD1d tetramers (3 nM) for 30 min at room temperature. Cells were then washed two times and analyzed by flow cytometry. One representative experiment of three is shown. B, Binding isotherm of wt CD1d tetramer to the 2C12 hybridoma. Tetramer fluorescence, normalized to the surface level of TCR staining, is plotted against tetramer concentration. 2C12 hybridoma cells were stained for 3 h at room temperature with an anti-TCR mAb and the indicated concentrations of wt CD1d tetramers. Cells were then washed two times and analyzed by flow cytometry. Inset, Scatchard transformation of the binding isotherm of wt CD1d tetramer to the 2C12 hybridoma. The ratio of the normalized fluorescence to the tetramer concentration is plotted against the tetramer concentration. One representative experiment of five is shown.

Production of a soluble NKT cell TCR

To further investigate the interaction of the NKT cell TCR with -GalCer/CD1d complexes, we produced a soluble form of the TCR from the 2C12 NKT cell-derived hybridoma. The protein was expressed in Escherichia coli as scVV polypeptide and refolded as previously described (34). The refolded scTCR was analyzed by gel filtration chromatography on a Superdex G200 column and the purity was assessed by SDS-PAGE (Fig. 5). The V14V8.2 scTCR was eluted at the expected elution volume for a monomer, under a symmetrical peak, which is a strong indication that the preparation is free of aggregated material (Fig. 5). Furthermore, and as expected, SDS-PAGE analysis showed no obvious contamination of the sample (Fig. 5).

View larger version (27K): [in this window] [in a new window]   FIGURE 5. Purification of the scTCR from the 2C12 hybridoma. Chromatogram showing the elution profile of the V14V8.2 scTCR from a Superdex G200 gel filtration column. Inset, SDS-PAGE analysis of the scTCR eluted from the Superdex G200 gel filtration column. The gel was stained with Coomassie blue. MW, m.w. markers.

Binding of the soluble NKT cell TCR to -GalCer/CD1d complexes

Surface plasmon resonance experiments were used to examine directly the binding of the NKT cell TCR to -GalCer/CD1d complexes. A specific, dose-dependent binding of the scTCR from 2C12 to immobilized -GalCer/wtCD1d complexes could be measured at either 25 or 37°C (Fig. 6 and Table III), suggesting that the strength of the interaction measured is relevant to in vivo conditions. By contrast, no binding was detected when either -GalCer/CD1d complexes (Fig. 6A) or unloaded wtCD1d molecules (data not shown) were immobilized. A control V8.2⁺ TCR, from a T cell hybridoma specific for a myelin basic protein peptide presented by the A^u class II molecule (34), did not bind to -GalCer/CD1d (data not shown). The same kind of dose-dependent binding of the scTCR from 2C12 to immobilized antigenic complexes could be measured with either -GalCer-loaded F10A or R79E CD1d molecules, while no binding was detected when -GalCer-loaded L150I-D153Y and R79E-D80A CD1d double mutants were immobilized (data not shown).

View larger version (22K): [in this window] [in a new window]   FIGURE 6. Binding of the V14V8.2 scTCR to -GalCer/CD1d complexes. A, The scTCR was injected at 25°C at increasing concentrations (0.625, 1.25, 2.5, or 5 µM) at a flow rate of 25 µl/min over FC1 (control FC) and FC2 (where -GalCer-loaded (solid lines) or -GalCer (dotted line) CD1d molecules were immobilized). The sensograms plot the specific binding obtained from the subtracted RU values (FC2 - FC1). B, Steady state equilibrium fitting. Equilibrium response values obtained from fitting of the sensograms (A) with the 1:1 Langmuir association. One representative experiment of three is shown.

View this table: [in this window] [in a new window]   Table III. Biophysical parameters of the interaction of V14V8.2 scTCR with immobilized mCD1/-GalCer complexes1

The relatively high affinity displayed by the NKT cell TCR for -GalCer/CD1d complexes is driven by a markedly long t_1/2. Indeed, the on rate is in the average of the reported 10²–10⁴ M^-1 s^-1 range for conventional ⁺ TCRs (29). The surprising feature of this interaction is the slow off rate of 3.96 x 10^-3 s^-1, one order of magnitude slower than the published 10^-2–10^-1 s^-1 range (29, 46, 47). This leads to a t_1/2 (ln2/k_d) of the complex of 175 s (Table III), which is also much longer than the ones previously reported for conventional ⁺ TCR interaction with p-MHC complexes (29). The kinetic parameters for scTCR binding were similar when the two single mutants were compared with wt CD1d (Table III). It should be noted that the sensograms depicted in Fig. 6 were done with chips having the minimum coupling density of CD1d required to give a reproducible TCR binding signal. This was done to minimize TCR rebinding following dissociation from -GalCer/CD1d complexes, which could give an apparently increased t_1/2 of the interaction. At higher CD1d coupling densities, a greater absolute TCR binding signal is obtained and the calculated t_1/2 of binding is slightly longer (data not shown).

High TCR avidity indicated by measurement of decay of tetramer binding

The presence of a small amount of aggregated material in the soluble TCR preparation could give rise to an apparently longer t_1/2 for -GalCer/CD1d binding. However, the analysis by gel filtration chromatography indicated the absence of such aggregates (Fig. 5), and the equilibrium binding studies with -GalCer/CD1d tetramers suggested a particularly high avidity for 2C12 hybridoma cells (Table II). Nevertheless, we attempted to confirm the results suggesting a particularly long t_1/2 for the interaction of the NKT cell TCR with the -GalCer/CD1d complex by measuring the decay of tetramer staining (38). Because the scTCR we studied was cloned from the 2C12 hybridoma, we measured the decay of tetramer staining on 2C12 hybridoma cells. The cells were stained with -GalCer-loaded CD1d tetramers, and the rate of decay was then measured by flow cytometry after addition of a saturating amount of an anti-CD1d mAb to block rebinding of the tetramer to the hybridoma cells (Fig. 7). At 4°C, no significant decay was obtained over a period of 5 h, which is in sharp contrast with the reported t_1/2 for the decay of tetramer staining on conventional T cells, which varies from 5 to 240 min at this temperature (38, 48). At 37°C, we obtained a linear decay plot of the natural logarithm of the normalized fluorescence vs time, indicating that tetramer was occurring stochastically and that the resulting tetramer staining t_1/2 should be proportional to the t_1/2 of the -GalCer/CD1d complexes with the NKT cell TCR. The t_1/2 at 37°C for -GalCer-loaded CD1d tetramer binding to 2C12 hybridoma cells was 300 min (Fig. 7), longer than the reported tetramer decay values obtained at 4°C for conventional T cell (38, 48). Because TCRs on NKT cell-derived hybridomas might have physicochemical properties distinct from NKT cells in vivo, we analyzed the decay of tetramer staining on freshly isolated NKT cells. Thymocytes were isolated from B10.A mice and stained using a mixture of -GalCer-loaded CD1d tetramer, anti-TCR mAb, anti-NK1.1, and anti-CD8. The decay of tetramer staining on TCR^intNK1.1⁺CD8^- cells (NKT cells) was measured by flow cytometry after addition of an excess of blocking Ab to prevent the rebinding of the tetramer to the cells. As for the T cell hybridomas, at 4°C no decay of tetramer staining was observed with thymocytes (data not shown). At room temperature, the decay of tetramer staining was occurring stochastically, as attested by the linear decay plot of the natural logarithm of the normalized fluorescence (Fig. 7B). Therefore, the resulting tetramer staining t_1/2 should be proportional to the t_1/2 of the -GalCer/CD1d complexes with the NKT cell TCR. This t_1/2 for -GalCer-loaded CD1d tetramer binding to freshly isolated NKT cells was 348 min (Fig. 7B). This was clearly much longer than the ones reported for the decay of tetramer staining on conventional T cells at 4°C (38, 48). Thus, the data demonstrate that a stable interaction between the -GalCer/CD1d complex and the NKT cell TCR is likely to take place in vivo.

View larger version (17K): [in this window] [in a new window]   FIGURE 7. Decay kinetics of tetramer staining. A, Decay plot of the natural logarithm of binding to the 2C12 hybridoma of -GalCer/CD1d tetramers. Normalized fluorescence is plotted vs time after addition of the CD1d blocking 1B1 Ab. Hybridoma cells were stained with -GalCer-loaded CD1d tetramer (0.5 nM) and anti-TCR mAb (10 µg/ml) at room temperature for 45 min then brought to either 4°C () or 37°C (•) and equilibrated for 15 min before addition of the 1B1 Ab (100 µg/ml). At consecutive time points, an aliquot was washed two times and analyzed by flow cytometry. B, Decay plot of the natural logarithm of binding of -GalCer/CD1d tetramers to freshly isolated NKT cells from the thymus. Normalized fluorescence is plotted vs time after addition of the CD1d blocking 1B1 Ab. B10.A thymocytes were stained with a mixture of -GalCer-loaded CD1d tetramer, anti-TCR mAb, anti-NK1.1, and anti-CD8 at room temperature for 45 min before addition of the 1B1 Ab (100 µg/ml). At consecutive time points, an aliquot was washed two times and analyzed by flow cytometry. One representative experiment of three is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Based on a docking model of -GalCer in the crystal structure of CD1d (49), it was suggested that charged amino acid side chains at several CD1d positions, including 79 (Arg), 80 (Asp), and 153 (Asp) mutated in this study, interact with hydrophilic portions of -GalCer, including the 2"-OH on the galactose (positions 79 and 80) the 3"-OH of galactose (position 79), and the amide group of the acyl chain (position 153). We and others have generated transfected cell lines expressing nonconservative mutations at positions 79 and 80 or 150 and 153 and were able to demonstrate that these changes have important effects on the ability to stimulate NKT cells (42, 49). To investigate these effects at a molecular level, we produced native soluble forms of the mutant CD1d molecules and, using a surface plasmon resonance assay, we demonstrated that they retain nearly equivalent ability to bind immobilized biotin -GalCer. However, when loaded with -GalCer, the R79E-D80A and L150I-D153Y CD1d mutants did not exhibit any detectable binding to the NKT cell TCR, with the R79E mutation having more subtle effects, depending upon the NKT cell TCR tested. Therefore, we conclude that the important effects on the ability of mutant CD1d molecules to stimulate NKT cell hybridomas are likely to be due an impaired TCR engagement of the -GalCer/CD1d complex rather than an impaired -GalCer loading in the binding pocket of the CD1d molecules. However, it remains possible that interaction between one or more of these CD1d amino acid side chains and -GalCer is required to orient properly the CD1d-bound glycolipid for TCR contact. This may be particularly true for the presentation of -GalCer by F10A. Our data suggest that this mutant retains the ability to bind to -GalCer in a nearly optimal way. However, it seems that this mutation of the floor of the binding pocket of the CD1d molecule might induce a nonoptimal orientation of -GalCer or, alternatively, an alteration in the CD1d helices. In either case, this apparently leads to a partially impaired TCR engagement of the -GalCer/CD1d complex by some NKT cell TCRs, although this was not observed with 2C12.

A combination of experimental methods was used to assess the interaction of the NKT cell TCR with -GalCer/CD1d complexes. Equilibrium tetramer staining and analysis of the decay of tetramer binding assess the avidity of the interaction of CD1d tetramers with the TCRs expressed on the surface of NKT cells, while surface plasmon resonance experiments measure the affinity of the interaction. The results of these assays were in general agreement, and they were also consistent with the results from Ag presentation assays using CD1d-coated plates. Most notable was the relative strength of the interaction of the TCR with the -GalCer/CD1d complex and the slow off rate observed. Using equilibrium tetramer staining, we showed that -GalCer-loaded wt CD1d tetramers bind to NKT cells with a high avidity, ranging from 0.5 to 1.6 nM. This high avidity is driven by a very long t_1/2, as shown by the absence of decay of the -GalCer-loaded CD1d tetramer staining on the surface of the 2C12 hybridoma at 4°C. The avidity is similar for all NKT cell hybridomas tested, although they have different -chains. A similar lack of decay of tetramer staining was observed at 4°C obtained by analyzing freshly isolated NKT cells from the thymus. The data suggest that differences either in the -chain expressed or in the CDR3 region are not likely to have a big effect on TCR avidity, at least when -GalCer is presented by wt CD1d. However, some differences, which may be -chain dependent, were evident when mutant CD1d molecules were used in tetramer binding experiments to the hybridomas.

Using surface plasmon resonance, we found that a K_D of 0.2 µM characterizes the interaction of -GalCer/CD1d complex with a V14V8.2 scTCR. This is near the lower limit of the published values for TCR interactions with p-MHC class I or class II complexes with only a few TCRs, falling in a similar range (29). However, the association rate of the interaction is near the average of what has been reported for conventional TCRs, unlike the fast on rate that characterizes the high-affinity interaction of 2C TCR with p2Ca/L^d complex (50). Instead, the low K_D we observed is due to the surprisingly slow dissociation rate of 3.96 x 10^-3 s^-1. Using surface plasmon resonance, such a slow k_d has never been reported for an TCR. However, dissociation rates of 5.5 x 10^-3 s^-1 and 3 x 10^-3 s^-1 were observed, using anti-TCR competition or labeled MHC methods, in studies of the interaction of 2C TCR with the p2Ca/L^d and QL9/L^d complexes, respectively (51, 52). However, in those studies whole T cells were used, and the presence of other surface molecules may be partially responsible for the values obtained. The structure of the natural Ag for the CD1d-dependent stimulation of NKT cells is not known. The majority of NKT cells expanded in vivo in the thymus and liver bind to -GalCer (10, 11); therefore, we can speculate that this natural Ag may be closely related at the structural level to -GalCer or may interact with the TCR in a similar way. It is noteworthy that the NKT cell TCR affinity for -GalCer-loaded CD1d molecules is higher that the affinity of any other published experimental Ags for CD4 and CD8 T cells (29, 30). Therefore, one could argue the opposite point of view, namely that -GalCer is a superagonist with properties that may not reflect those of the natural ligand.

The results from several studies suggest that the CD8 coreceptor stabilizes significantly the interaction of the TCR with the p-MHC complex (53, 54, 55). In one case, this was shown to result from a decrease in the off rate by an order of magnitude (54), although some studies did not find a CD8 enhancement of TCR affinity (56). However, V14⁺ NKT cells do not express CD8 (10, 11); they are either CD4⁺ or double negative. Because there is no evidence indicating that CD4 serves as a coreceptor for CD1d, or that the double negative mouse NKT cells are significantly less responsive to -GalCer, the data suggest that the NKT cell TCR operates independently of the known CD4 and CD8 TCR coreceptors. Indeed, the high affinity and the long t_1/2 of the NKT cell TCR may be required for its ability to function effectively when expressed by double negative T cells. The affinity of a TCR for the nonclassical class I molecules T10 and T22 has been measured, and interestingly this TCR also displays a high affinity and a slow off rate (57). This TCR is also expressed predominantly on double negative T cells. Therefore, considering these cases, there is a correlation between the ability of TCRs to work efficiently in a double negative environment and the fact that they display a high affinity and slow off rate. However, further work will be required to prove that alternative coreceptors are not used by NKT cells and to determine whether high TCR affinity is selected for in the absence of coreceptor function.

Despite the relatively high affinity of the interaction with -GalCer/CD1d, there is no affinity for unloaded CD1d molecules. Furthermore, we showed that -GalCer/CD1d complexes do not interact with the NKT cell TCR, although it was previously demonstrated that -GalCer binds to CD1d as well as -GalCer (41). These findings are in agreement with the results obtained from flow cytometric analyses using CD1d tetramers (10, 11), although these previous studies were not done in a way that would detect transient interactions or that would permit a quantitative analysis of the binding. The lack of binding of the TCR to unloaded CD1d molecules, or to CD1d molecules containing -GalCer, suggests an Ag- or ligand-driven selection of T cells expressing the canonical V14 rearrangement, consistent with a recent report that analyzed mice expressing a mutant CD1d molecule (58). The tight selection for a particular -chain rearrangement, and its clear dependence upon a CD1d-bound ligand, are consistent with the hypothesis that NKT cells are focused to recognize a structurally closely related set of natural ligands (4). Additionally, the inability of the TCR to bind to -GalCer/CD1d complexes confirms the hypothesis that TCRs reactive to glycolipid/CD1 complexes are focused primarily upon the exposed carbohydrate portion of the CD1-bound Ag (59).

Two mutations of the CD1d molecule, R79E and F10A, decrease the t_1/2 of the -GalCer/CD1d complex with the TCR by as much as four times. Despite this, these mutants retain the ability to present -GalCer to 2C12 hybridoma cells in an optimal or nearly optimal fashion. This may not be consistent with the implication of the serial triggering hypothesis, which proposes that there is a normal distribution of t_1/2 for TCR binding around a value that gives an optimal response (48). We consider this to be unlikely for NKT cells, because the t_1/2 is fairly long and the same degree of stimulation is observed when the t_1/2 decreases from 173 s to 48 s. However, it might be possible that the optimal t_1/2 has a much broader range for NKT cells than for conventional T cells.

In summary, in this work we have shown that the NKT cell TCR displays characteristic features when interacting with -GalCer presented by CD1d, including a high affinity and a long t_1/2. The in vivo response by NKT cells to -GalCer is unusually rapid and intense, and it is followed immediately by the apparent death of the responding T lymphocyte cells (11, 16, 17). This NKT cell response is observed even at the lowest doses of Ag that can stimulate these lymphocytes (J. Matsuda and M. Kronenberg, unpublished data). The correlation between high TCR affinity and the intense and unusual NKT cell response is noteworthy; therefore, we speculate that this atypical response could be due in part to the high affinity of the NKT cell TCR for -GalCer/CD1d complexes.

	Acknowledgments

 
We thank Dr. Frits Koning (Leiden University Medical Center, Leiden, The Netherlands) for critical reading of the manuscript and Lise Sidobre and Carlos Aguillera for technical support in protein production.

	Footnotes

 
¹ This work was supported by National Institutes of Health RO1 Grants AI45053, CA52511, and U54GM62116 (to M.K.), GM48002 and GM39476 (to N.R.J.G.), and AI48540 (to K.C.G.), Multiple Sclerosis Society Grant RG3148 (to K.C.G.), and a grant from the Human Frontiers of Science Program (to M.K.). S.S. was supported by a fellowship from La Ligue Nationale Contre le Cancer. This is manuscript no. 442 from the La Jolla Institute for Allergy and Immunology.

² S.S. and O.V.N. contributed equally to this work.

³ Current address: Department of Pathology and Immunology, Washington University, St. Louis, MO 63110.

⁴ Current address: Aurora Biosciences Corporation, 11010 Torreyana Road, San Diego, CA 92121.

⁵ Address correspondence and reprint requests to Dr. Mitchell Kronenberg, Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: mitch{at}liai.org

⁶ Abbreviations used in this paper: CDR, complementarity-determining region; -GalCer, -galactosylceramide; sc, single chain; wt, wild type; RU, resonance unit; FC, flow cell.

Received for publication February 6, 2002. Accepted for publication May 17, 2002.

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