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Originally published In Press as doi:10.1074/jbc.M300628200 on May 28, 2003

J. Biol. Chem., Vol. 278, Issue 33, 30961-30970, August 15, 2003
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Production, Characterization, and Immunogenicity of a Soluble Rat Single Chain T Cell Receptor Specific for an Encephalitogenic Peptide*

Rachel H. McMahan {ddagger} §, Lisa Watson ¶, Roberto Meza-Romero ¶, Gregory G. Burrows ¶ ||, Dennis N. Bourdette {ddagger} ¶ and Abigail C. Buenafe {ddagger} ¶ **

From the Department of Neurology and ||Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97201 and {ddagger}Neuroimmunology Research, Veterans Affairs Medical Center, Portland, Oregon 97201

Received for publication, January 21, 2003 , and in revised form, May 20, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The encephalitogenic rat T cell clone C14 recognizes the myelin basic protein 69–89 peptide in the context of the RT1B major histocompatibility complex (MHC) class II molecule. Modeling of the C14 TCR molecule indicated that previously identified CDR3 motifs are likely to be central to interaction with MHC class II-presented peptide. Here we report the cloning and expression of C14-derived single chain TCR (scTCR) molecules in an Escherichia coli expression system. The recombinant molecule consists of the V{alpha}2 domain connected to the V{beta}8.2 domain via a 15-residue linker. Soluble C14 scTCR was purified using conventional chromatography techniques and refolded by a rapid dilution procedure. C14 scTCR was able to bind soluble rat MHC class II molecules bearing covalently coupled Gp-BP-(69–89) peptide, as analyzed using surface plasmon resonance. Immune recognition of the C14 scTCR protein as an antigen revealed that limited regions of the TCR may be more likely to induce responsiveness.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Antigen-specific activation of T lymphocytes occurs through the binding of the T cell receptor (TCR).1 In order to initiate an immune response, antigenic peptides must be presented in the context of a major histocompatibility complex (MHC) on the surface of antigen-presenting cells. In addition, co-receptors on the antigen-presenting cells must bind accessory molecules of the T cell in order for activation to occur. The TCR is a heterodimer consisting of {alpha} and {beta} subunits containing variable and constant domains that are structurally similar to immunoglobulin molecules. Because TCR interaction with the MHC-peptide complex defines the basis of specific recognition, an understanding of the structural elements of this recognition is of importance. Structural data regarding both human and mouse TCRs, both with (1–9) and without (10–12) the MHC-peptide complex, has been limited to a small but growing data base of information obtained through crystallographic (1–11) and NMR spectroscopic (12) studies. TCR recognition of specific MHC-peptide complexes occurs through the binding of complementarity-determining regions (CDRs) within the variable (V) regions of the {alpha} and {beta} chains. Analysis of the three-dimensional structure suggests that, in general, the CDR1 and CDR2 regions contact the {alpha}-helices of the MHC molecule, whereas CDR3 regions contact the peptide (3–9).

The immunoreactive properties of the TCR molecule as an antigen have been of long standing interest but remain poorly understood. The most compelling evidence that the TCR itself can elicit immune responsiveness has been demonstrated in numerous studies, most notably in models of T cell-mediated autoimmunity. The protective effects of vaccination with attenuated, pathogenic T cells were first demonstrated in the model of experimental autoimmune encephalomyelitis (EAE) and then extended to models of arthritis, thyroiditis, and diabetes (13, 14). Protection coincided with T cells expressing TCRs specific for the disease-inducing antigen. Subsequent studies showed that TCR protein (15–17) as well as TCR peptides (18, 19) could also induce protection from the induction of EAE and collagen-induced arthritis. The mechanism of action is unclear but may involve the induction of a regulatory T cell population. The observation of biased TCR gene expression in pathogenic T cell populations of certain mouse and rat models of EAE also led to the demonstration that antibodies to the TCR could successfully treat EAE (20, 21).

EAE is an autoimmune demyelinating disease with similar neuropathology to the human disease multiple sclerosis. EAE can be induced by a number of myelin component proteins and their derived peptides including myelin basic protein (MBP), proteolipid protein, and myelin oligodendrocyte glycoprotein (22, 23). In the Lewis rat model of EAE, active immunization with guinea pig basic protein (Gp-BP) resulted in the expansion and migration of MBP-reactive CD4+, MHC class II-restricted T cells into the central nervous system. These T cells were shown to be specific for an epitope corresponding to MBP residues 69–89 and demonstrated biased expression of V{beta}8.2 and V{alpha}2 TCR genes with the presence of specific motifs (24–26). Importantly, these V{beta}8.2 and V{alpha}2 TCR genes were also present in T cell clones capable of transferring EAE to naive rats (27, 28).

The encephalitogenic BP-(69–89)-specific T cell clone C14 (29) expresses TCR V{alpha}2 and V{beta}8.2 chain sequences identical to those found clonally expanded in the central nervous system of rats with EAE (24–26). We undertook the cloning and expression of a soluble single chain form of the C14 TCR (scTCR) so that we could investigate specific interactions at the combining site that culminate in pathogenesis. We also wished to use the C14 scTCR to investigate immunogenic properties of the scTCR itself. Unlike antibody molecules, the production of large amounts of soluble, nonaggregated TCR molecules has been difficult to achieve. The fact that the TCR exists mainly as a cell surface molecule in association with a number of other surface molecules probably contributes to the problem of poor solubility. A number of cloning strategies and in vitro refolding techniques have been reported to greatly improve the yield of soluble TCR, both as scTCR molecules (30–33) and as {alpha}{beta}-dual chain heterodimers (34, 35). Using an Escherichia coli expression system and a rapid dilution refolding method, we were able to produce 5–8 mg/l quantities of soluble C14 scTCR molecules. We report here the production, characterization, and antigenic properties of a two-domain scTCR derived from a pathogenic Lewis rat T cell clone.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Homology-based Modeling of the C14 TCR—Structure-based homology modeling was based on the refined crystallographic coordinates of the murine D10 TCR engaged with a peptide-MHC class II complex (7) obtained from the Brookhaven Protein Data Bank (Brookhaven National Laboratories, Upton, NY). Sequence alignment of the {alpha} and {beta} chains from the C14 and D10 scTCRs provided a starting point for our studies. The program Sybyl (Tripos Associates, St. Louis, MO) was used to generate graphic images using an O2 work station (Silicon Graphics, Mountain View, CA). Amino acid residues in the scTCR of D10 (Protein Data Bank accession number 1D9) were substituted with the rat C14 BV8S2 and AV2 TCR side chains, and the polypeptide backbone was modeled as a rigid body during structural refinement using local energy minimization. After 100 iterations of energy minimization, the total energy calculated for the C14 scTCR was 6568.5 kcal/mol. This compares favorably with a total energy of 1937 kcal/mol obtained after a parallel minimization of the D10 scTCR from a starting energy of 111,393 kcal/mol for the D10 crystal structure.

Assembly of scTCR Genes—Reverse transcriptase-PCR analysis for TCR gene usage (26, 36) and sequencing analysis demonstrated that only a single V{alpha} gene and a single V{beta} gene were expressed in the A1 hybridoma, derived from the C14 encephalitogenic Lewis rat T cell clone specific for the BP-(69–89) peptide (29). To construct the scTCR gene, primers containing appropriate restriction sites were designed to flank sequences encoding the V and J regions as follows: V{alpha} primers, RV{alpha}2NcoI (5'-CGACCCATGGCTCAGCAGGTCAAACAAAGTCC) and RJ{alpha}SmaI (5'-CGGACCCGGGCTTCACCACCAGTTGCG); V{beta} primers, RV{beta}8Bam (5'-ATCAGGATCCGAAGCTGCAGTCACACAAAGC) and RJ{beta}SacI (5'-GATCGAGCTCAACCGTGAGCTTGGTGCCG). V{alpha}- and V{beta}-amplified genes were individually cloned into pCR2.1 using the TA cloning kit (Invitrogen), and the sequences were verified. The purified plasmids were digested with the appropriate restriction enzymes, and the inserts were gel-purified on glass beads (Bio 101, Inc., Vista, CA) and then ligated into NcoI- and XhoI-digested pET21d (Novagen, Madison, WI) together with a linker region. The double-stranded linker regions, DAK (GSADDAKKDAAKKGS) (33) or G4S (GGGGSGGGGSGGGGS) (7), encoded 15-amino acid residue spacers and were flanked by corresponding restriction sites (SmaI and BamHI) that connected the V{alpha} and V{beta} segments. Constructs were transformed into AD494-competent cells (Novagen). Colonies were screened by SDS-PAGE for the production of an isopropyl-{beta}-D-thiogalactoside-inducible protein band of ~28 kDa.

scTCR Expression and Purification—A 4-ml overnight culture of the C14 scTCR was grown in Luria broth containing carbenicillin (50 µg/ml) and kanamycin (15 µg/ml). The overnight culture was diluted into 1 liter of the same medium and grown to midlogarithmic phase (A600 = 0.6) at 37 °C, and protein production was induced by the addition of 1 mM isopropyl-{beta}-D-thiogalactoside for 3–4 h. The cells were harvested by centrifugation at 5000 x g, resuspended in binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), sonicated on ice, and repelleted at 10,000 x g for 15 min. After sonicating and washing two more times, the pellet was resuspended in binding buffer that contained 6 M urea and stirred for 72 h at 4 °C. Insoluble material was removed by centrifugation at 39,000 x g for 45 min. The supernatants from 4 liters of bacterial culture were filtered (0.22 µm) and loaded onto a nickel-agarose column (HiTrap chelating column; Amersham Biosciences) at a flow rate of 1 ml/min. The column was washed with 10 volumes of binding buffer followed by 10 volumes of wash buffer (60 mM imidazole, 0.5 M NaCl, 6 M urea, 20 mM Tris-HCl, pH 7.9). The protein was eluted with 6 volumes of binding buffer containing 500 mM imidizole and then dialyzed against PBS, pH 7.4, containing 6 M urea and 0.5 mM EDTA. The protein was incubated with 10 mM dithiothreitol overnight at 4 °C with stirring and further purified by gel filtration on a HiLoad 16/60 Superdex 75pg column (Amersham Biosciences) equilibrated with PBS, pH 7.4, containing 1 mM dithiothreitol, 6 M urea, and 0.5 mM EDTA. The column was loaded with 1–2 ml of dialyzed, dithiothreitol-treated protein and run at a flow rate of 0.5 ml/min.

Refolding of scTCR—scTCR protein was refolded using a rapid dilution protocol similar to that of Pecorari et al. (34). Briefly, elutes containing the column-purified protein peak were combined and added to refolding buffer (1 M L-arginine, 2 mM EDTA, 0.2 mM reduced glutathione, 0.2 mM oxidized glutathione, 100 mM Tris, pH 8.0) at a concentration of <=60 µg/ml and stirred at 4 °C for 48 h. scTCR was dialyzed three times against 10 liters of PBS, pH 7.4, at 4 °C and concentrated in Amicon 8400 and 8050 chambers using YM10 ultrafiltration membranes (Millipore Corp., Billerica, MA). scTCR protein was further concentrated to ~1 mg/ml using Centriprep 10 concentrators (Millipore) and centrifuged at 15,000 x g for 15 min followed by 0.22-µm filtration. All refolded protein was stored at –80 °C.

Electrophoresis and Western Blotting—Purified scTCR protein was resolved using SDS-PAGE in 15% acrylamide under both reducing and nonreducing conditions. The gel was either stained with Coomassie Blue or transferred onto polyvinylidene difluoride membrane for Western analysis. For Western blots, nonspecific protein was blocked by incubating for 1 h with 3% BSA in Tris-buffered saline. The membrane was incubated with a monoclonal antibody specific for the rat V{beta}8.2 T cell receptor (PharMingen, San Diego, CA) followed by an alkaline phosphatase-conjugated secondary antibody. Detection was performed using Western Blue substrate (Promega, Madison, WI). Alternatively, the membrane was incubated with monoclonal antibody supernatants (1:500 dilution) followed by a horseradish peroxidase-labeled secondary antibody and detection was performed using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) substrate (BioFX Labs, Owings Mills, MD).

Analytical Size Exclusion Chromatography—Analytical size exclusion chromatography was performed using a Superdex 75 (10/30) column attached to an AKTA FPLC system (Amersham Biosciences). The column was calibrated using a low molecular weight gel filtration kit (Amersham Biosciences) followed by 2 volumes of PBS, pH 7.4. scTCR protein in PBS, pH 7.4, was injected at a flow rate of 1 ml/min and eluted using the same buffer.

Surface Plasmon Resonance—OX-6 mAb specific for the rat MHC class II RT1.B molecule was coupled to a CM5 biosensor chip by standard amine coupling in 10 mM NaOAc, pH 5.0, with a resulting final resonance of ~10,000 resonance units. Rat RTL201, consisting of the {alpha}1 and {beta}1 domains of RT1.B with MBP-(69–89) peptide covalently attached (37, 38), was immobilized on the chip by the OX-6 mAb at an Rmax of 100–400 resonance units for binding experiments. Kinetic measurements were performed on a Biacore 3000 in HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20) at a flow rate of 50 µl/min to minimize the effects of mass transfer limitation. scTCR proteins were injected in a series of concentrations with regeneration (10 mM glycine-HCl, pH 2.0) of the OX-6-coated chip and immobilization of fresh RTL201 before each injection of scTCR. Binding of scTCR to a control flow cell with OX-6 alone was subtracted for each concentration. Fitting of kinetic data to a 1:1 binding model with baseline drift was performed using Biaevaluation 3.0 software.

Mice—BALB/c and B10.PL female mice were obtained from Jackson Laboratories (Bar Harbor, ME) at 8–10 weeks of age. Mice were housed in the Animal Resource Facility at the Portland Veterans Affairs Medical Center in accordance with institutional guidelines.

Antibody Production—For production of serum and hybridoma antibodies, BALB/c mice were injected intraperitoneally with 100 µg of scTCR protein emulsified with Complete Freund's adjuvant (Sigma) containing 100 µg of Mycobacterium tuberculosis (Difco). Two weeks later, mice were boosted intraperitoneally with 50 µg of scTCR in incomplete Freund's adjuvant. For the production of monoclonal Abs, mice were rested for 2 months after the boost and then injected intravenously with 10 µg of scTCR in saline. Three days after intravenous boost, spleens were obtained and fused with SP2/0 myeloma cells as described (39). After 10–14 days in HAT selection medium, supernatants were screened for scTCR-specific antibody by enzyme-linked immunosorbent assay using 96-well polystyrene plates (Costar, Cambridge, MA) coated with 10 µg/ml scTCR. Plates were blocked with PBS plus 1% BSA, incubated with supernatants, and then incubated with alkaline phosphatase-labeled rabbit anti-mouse IgM + IgA + IgG (Zymed Laboratories Inc., South San Francisco, CA). Positive wells were detected by the addition of p-nitrophenyl phosphate substrate, and absorbance was read at 450 nm.

T Cell Lines—B10.Pl mice were immunized subcutaneously at four sites in the lower back with 100 µg of scTCR protein in CFA containing 100 µg of M. tuberculosis. Inguinal lymph nodes were obtained 10 days postimmunization, and a single cell suspension was prepared. Lymph node T cells (6 x 106 cells/ml) were stimulated with 25 µg/ml scTCR for 48 h in stimulation medium (RPMI containing 1 mM sodium pyruvate, 1 mM non-essential amino acids, 2 mM L-glutamine, 2-mercaptoethanol, 2% fetal calf serum) and then expanded in growth medium (RPMI containing 1 mM sodium pyruvate, 1 mM non-essential amino acids, 2 mM L-glutamine, 2-mercaptoethanol, 10% fetal calf serum, 20 units/ml recombinant interleukin-2) for 5 days. scTCR-specific T cell lines were maintained by weekly stimulation with 2–10 µg/ml scTCR followed by expansion in growth medium. For proliferation assay, T cells were plated at 2 x 104 cells/well in 96-well plates in the presence of 4 x 105 irradiated antigen-presenting cells (thymocytes plus splenocytes)/well plus antigen for 72 h. [3H]Thymidine was added for the last 18 h of culture, after which the wells were harvested, and the amount of incorporated label was determined using a Betaplate liquid scintillation counter (PerkinElmer Life Sciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning and Modeling of the Recombinant C14 scTCR—A schematic diagram of the V{alpha}2/V{beta}8.2 scTCR construct is shown in Fig. 1A. Two different linkers, DAK (33) and G4S (7), were used to connect the V{alpha} and V{beta} chains in case the linker region should have an effect on refolding and solubility of the scTCR protein. A sequence encoding the C-terminal 6x histidine (His6) tag was present on the pCR2.1 vector and allowed for the purification of the recombinant scTCR proteins by nickel chelation chromatography. The amino acid sequence of the C14 V{alpha}2 and V{beta}8.2 genes used to encode the scTCR construct is shown in Fig. 1B. We took this opportunity to compare these sequences to the V{alpha}2 and V{beta}8.2 genes of the murine D10 TCR, since the crystal structure coordinates of the D10 scTCR (7) were available. The C14 and D10 V{alpha}2 sequences were 75.5% homologous, and the V{beta}8.2 sequences were 80.4% homologous at the amino acid level (including residues up to the C-terminal Cys). Homology increased to 83.3 and 84.5%, respectively, when the CDR loops were excluded.



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  		FIG. 1.
Construction and modeling of the C14 scTCR molecule. A, schematic representation of the C14 scTCR construct in the pET21d expression vector. B, comparison of the C14 V{alpha} and V{beta} TCR sequences with those of the murine D10 TCR. C, model of the C14 scTCR based on the crystal structure coordinates of the D10 scTCR, showing the V{alpha} domain in red and the V{beta} domain in blue. Rendered (space-filled) residues (Asn95{alpha}, Asn96{alpha}, Asn97{alpha} (yellow); Asp96{beta} and Ser97{beta} (cyan); Ser98{beta} (green)) in the CDR3 loops depict amino acid motifs that were identified in previous studies (24–28) and proposed to contribute to antigen specificity. Cys residues participating in disulfide bond formation are seen as yellow bands in the {beta}-strand ribbons.

 

Coordinates of the D10 crystal structure were used to derive a molecular model (Fig. 1C) of the C14 TCR. The linkers and His6 tag are not shown due to the flexibility of their structure. The V{beta}8.2 CDR3 loop in the C14 TCR is one residue shorter than that in the D10 TCR, although the C14 and D10 V{alpha}2 CDR3 loops are the same length. Previous studies identified the presence of motifs in the V{beta}8.2 CDR3 (Asp96-Ser97) (24, 25, 27, 28) and V{alpha}2 CDR3 (Asn95-Asn96-Asn97) (26–28) regions of encephalitogenic T cell clones as well as sequences derived from the spinal cord of rats with EAE. In the C14 model (Fig. 1C), Asp96{beta}, Ser97{beta} (both cyan), and Ser98{beta} (green) were found at the apex of the CDR3{beta} loop. Ser98{beta} is contributed by the J{beta}2.7 segment and may partially explain the observed biased use of this J{beta} (24). Other contributing factors may involve the interaction of J{beta} with interfacing V{alpha} or J{alpha} residues. In the CDR3{alpha} loop, the central asparagine residue Asn96{alpha} (yellow) was positioned at the apex and appeared most accessible for interaction with peptide. The flanking asparagines (Asn95{alpha} and Asn97{alpha}, both yellow) may play important roles in the packing of CDR3 loop structures or in the interaction with CDR3{beta} contacts. Thus, the C14 TCR model supports a role for these CDR3 motifs in antigen specificity.

Expression and Purification of the Recombinant C14 scTCR—Expression of recombinant C14 scTCR (~28 kDa) was induced in transformed bacterial cells with 1 mM isopropyl-{beta}-D-thiogalactoside for 3–4 h (Fig. 2A, lane 3). Recombinant scTCR proteins were isolated from the insoluble fraction of the bacterial lysate containing inclusion bodies. scTCR-containing pellets were solubilized in 6 M urea and purified via the His6 tag by nickel chelation chromatography (Fig. 2A, lane 4). Both the scTCRDAK and scTCRG4S proteins co-purified with a 14-kDa protein (Fig. 2A, lane 4). Western blot analysis demonstrated that both the 14-kDa protein and the full-length scTCR protein were recognized by a rat V{beta}8.2-specific mAb (not shown). Thus, given the size of the contaminating band and the fact that it contained the His6 tag and was bound by V{beta}8.2-specific mAb (Pharmingen), we concluded that the 14-kDa protein was generated by cleavage of the scTCR near the linker region. Following affinity purification, imidizole was removed by dialyzing into PBS (pH 7.4) containing 6 M urea and 0.5 mM EDTA. The protein was completely reduced by the addition of 10 mM dithiothreitol and further purified on a HiLoad 16/60 Sephadex size exclusion column (Amersham Biosciences) under reducing conditions in order to remove the cleavage product. The resulting chromatogram (Fig. 2B) and SDS-PAGE analysis (inset) demonstrated that this strategy for removal of the co-purifying cleavage product was successful. The cleavage product does not appear to be common to all scTCRs, since we have engineered and purified corresponding human scTCRs without the presence of a cleavage product.2



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  		FIG. 2.
A, SDS-PAGE analysis of C14 scTCRDAK at various stages of purification. Lanes 1 and 5, molecular mass markers; lane 2, preinduction bacterial lysate; lane 3, isopropyl-{beta}-D-thiogalactoside-induced bacterial lysate; lane 4, C14 scTCRDAK following purification by nickel chelation chromatography. The C14 scTCR is 28 kDa (arrow) and co-purifies with a 14-kDa cleavage product (*). B, size exclusion elution profile of C14 scTCRDAK run under reducing conditions. Inset, Coomassie Blue-stained SDS-PAGE gel of peak fractions. Lanes 1 and 2 correspond with elutes from the first peak containing C14 scTCR; lanes 3 and 4 correspond with the second peak containing only the 14-kDa cleavage product. C, analytical size exclusion chromatography of soluble C14 scTCRDAK following refolding and concentration to 1 mg/ml. The column was calibrated with the following molecular mass standards (arrows): BSA (66.7 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa), and myoglobin (17.3 kDa). All proteins were eluted with PBS, pH 7.4, at 1 ml/min. Inset, refolded C14 scTCRDAK run under reducing (R) and nonreducing (NR) conditions.

 

Size exclusion fractions containing purified scTCR protein were combined and refolded. Following dialysis into PBS (pH 7.4), the protein was concentrated down to ~1–1.5 mg/ml. 5–8 mg of soluble refolded protein per liter of bacterial culture was consistently obtained using this refolding method. The overall yields for both the scTCRDAK and scTCRG4S were similar, indicating that the linker region had no differential effect on the solubility of the scTCR.

Physical Properties of the Recombinant scTCR—Analytical size exclusion chromatography was performed on the scTCR proteins in order to determine the degree of aggregation following refolding. The major peak eluted at ~28 kDa for both scTCRDAK and scTCRG4S, indicating that the majority (75–85%, depending on batch) of refolded protein was present in monomeric form (Fig. 2C). Analysis of the chromatogram indicated that some dimeric and possibly trimeric forms were present. Preliminary experiments using dynamic light scattering indicated that larger aggregates of the refolded proteins were not present (not shown). Refolded protein analyzed on a nonreducing gel demonstrated a shift to lower apparent molecular weight, verifying the presence of folded protein with disulfide bond formation (Fig. 2C, inset). The presence of multiple bands (three bands shifted to a lower molecular weight) on the nonreducing gel, however, indicated that misfolded scTCR protein could be present. Further purification of the refolded C14 scTCR monomer fraction by preparative size exclusion chromatography may be required before further structural analysis can be completed. In order to ensure that only properly folded scTCR is present, affinity purification of C14 scTCR using V{beta}8.2- or V{alpha}2-specific antibody may be required as well. At present, one mAb (R78; Pharmingen) specific for rat V{beta}8.2 is commercially available. Contaminating lipopolysaccharide from the bacterial preparation appeared to be absent in the purified and refolded scTCR preparations, as indicated by the lack of ovalbumin-specific T cell proliferation to scTCR in in vitro control wells (not shown).

Binding of Recombinant Rat scTCR to Soluble MHC Class II Molecules—Despite the possible presence of misfolded protein, we conducted preliminary experiments to determine whether we could detect the binding of scTCR to a rat MHC class II-peptide complex. Surface plasmon resonance was used to analyze the binding of recombinant rat C14 scTCRDAK to a soluble rat MHC class II molecule consisting of the {beta}1 and {alpha}1 domains of the Lewis rat RT1.B molecule with covalently attached MBP-(69–89) peptide (recombinant TCR ligand or RTL201) (36, 37). Coupling of the C14 scTCR or RTL directly to the CM5 chip was tested in initial pilot experiments. Significant amounts of protein were coupled either by standard amine coupling or through a C-terminal Cys engineered into a mutagenized C14 scTCR (not shown). Unfortunately, scTCR and RTL molecules were prone to varying degrees of denaturation under all regeneration conditions tested (not shown), possibly due to the absence of stabilizing cell surface proximal domains. Thus, we employed a capture system where a mAb that could withstand regeneration conditions was used to immobilize the RTL and where regeneration would have no effect on subsequent RTL-scTCR interaction. Another study determined that kinetic data obtained from a capture experiment were preferable and gave fewer artifacts than data obtained from a comparable direct coupling experiment (40).

OX-6 mAb, specific for the rat MHC class II RT1.B molecule, was covalently coupled to a CM5 biosensor chip and used to capture RTL201 molecules. Soluble scTCR was then flowed over captured RTL201 or a control flow cell containing covalently captured OX-6 alone, and the binding interaction was measured. Fig. 3A demonstrates that scTCR showed significant binding to RTL201 (binding to control flow cell subtracted), whereas a control human MR3.2 scTCR protein3 showed no binding to RTL201 (Fig. 3B). The flow rate was maintained at 50 µl/min during the binding of scTCR to RTL in order to minimize the effects of mass transfer limitation. Minimal binding of the scTCR to OX-6 alone was detected (not shown). Affinity measurements were derived from several experiments where Rmax was estimated to range from 100 to 400 resonance units. Unlike the association rate kon (M–1 s–1), the dissociation rate koff (s–1) is independent of the analyte (scTCR) concentration. The measured dissociation rate for scTCRDAK binding to RTL201 ranged from 0.0062 to 0.0106 s–1, which is relatively slow for TCR-MHC binding (41). Although the affinity constant (KD = koff/kon) is likely to change once the scTCR is further purified, analysis of the affinity data as collected estimated an affinity constant of 2.7–2.9 µM. {chi}2 values ranged from 0.7 to 0.8.



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  		FIG. 3.
Surface plasmon resonance analysis of rat C14 scTCR (A) or human MR3.2scTCR (B) binding to a rat MHC class II molecule with covalent Gp-BP-(69–89) (RTL201). C14 scTCR demonstrated significant binding to RTL201, whereas MR3.2 showed no binding. Binding curves were globally fit to a 1:1 binding model with drifting base line taken into account. Residual plots are shown below each binding curve.

 

Immune Recognition of Recombinant scTCR—B cell and T cell recognition of recombinant rat C14 scTCR protein was investigated. Serum antibody responsiveness was analyzed after immunization of BALB/c mice with scTCR in CFA. Fig. 4A demonstrates a significant increase in serum antibody titer after boosting with scTCR protein on day 14. scTCR-specific serum antibody showed some cross-reactivity with the human scTCR MR3.2 protein (Fig. 4B), possibly due to the presence of a common linker region (DAK) and the histidine tag. As found with typical antibody responses to protein antigen, the level of IgM antibodies specific for scTCR peaked at day 10, whereas the levels of IgG antibodies peaked in the secondary response at day 35. B cell hybridomas were generated after a long term immunization regimen with rat scTCR protein, and four mAbs were tested for reactivity with a panel of proteins in an enzyme-linked immunosorbent assay. Table I shows the specific reactivity determined for each mAb. All mAbs reacted with the C14 scTCR protein used as the immunogen, as well as with a recombinant V{beta}8.2 fusion protein, V{beta}8.2-glutathione S-transferase (42). No reactivity was detectable to the control fusion protein glutathione S-transferase, ovalbumin, or BSA. In addition, the mAbs did not recognize two His6-containing recombinant proteins, rat V{alpha}2his6 and human V{beta}5.2his6, indicating that they did not recognize the His6 tag alone. Enzyme-linked immunosorbent assay blocking experiments performed on three of the four mAbs indicated that they did not bind to the same epitopes on the scTCR molecule (not shown). When tested for binding to C14 TCR-bearing hybridoma T cells, however, both serum and monoclonal antibodies demonstrated little or no binding as detected by flow cytometry, indicating that these antibodies may be recognizing epitopes not normally accessible on cell surface-associated full-length TCR. To determine whether C14 scTCR-specific antibodies could be recognizing new epitopes present only on multimeric forms of the scTCR molecule, Western blots were performed using two of the C14 scTCR-specific monoclonal antibodies (1E5:2 and 4D9:A2). Under nonreducing conditions, both monoclonals were found to bind monomeric as well as dimeric forms of the scTCR, as shown in Fig. 5 for the 4D9:A2 antibody. These monoclonals were highly specific and demonstrated no binding to MR3.2 human scTCR and human RTL control proteins. Since the monoclonals also bound the reduced form of the scTCR, it is likely that they recognize a contiguous epitope on the V{beta}8.2 chain.



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  		FIG. 4.
Immunization with scTCR induces specific serum and monoclonal antibodies. BALB/c mice were immunized with C14 scTCR in Complete Freund's adjuvant on day 0 and boosted with C14 scTCR in incomplete Freund's adjuvant on days 14 and 28. A, serum titers indicate a significant increase in antibody levels by day 21 after immunization. B, peak IgM levels were detected on day 10, whereas peak IgG levels were detected on day 35. Some cross-reactivity to a control human scTCR protein (MR3.2) was detected, indicating potential recognition of the common linker region and/or His6 tag.

 

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  		TABLE I
Reactivity of anti-C14 scTCR mAbs

 


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  		FIG. 5.
Immunization with scTCR induces T cell lines specific for TCR determinants. A, increasing specificity in the proliferative responses of a C14-specific T cell line were observed after the first versus third stimulation round. B, epitope mapping of the V{beta}8.2 determinants recognized by a C14 scTCR-specific T cell line was carried out with the use of overlapping peptides. Determinants associated with both the V{beta}8.2 and the V{alpha}2 chain, but not ovalbumin control, were detected. V{beta}8.2 peptides are numbered according to the C14 V{beta} sequence in Fig. 1B.

 

T cell responses to recombinant rat scTCR protein were analyzed in B10.PL mice. Unlike the antibody response, T cell responsiveness to the scTCR would not be dependent on the presence of exposed, conformational epitopes. Ten days after subcutaneous immunization, draining LN cells were obtained and placed into culture with C14 scTCR protein for 2 days and then expanded in interleukin-2-containing growth medium for five additional days. T cells were cycled between stimulation and expansion multiple times. At each stimulation, T cells were tested for reactivity to antigen in a proliferation assay. Reactivity profiles in Fig. 6A demonstrated retained specificity for rat C14 scTCR and the V{beta}8.2H6 chain alone and a loss of nonspecific cross-reactivity to the human MR3.2 scTCR protein at the third stimulation compared with the first stimulation. Minimal reactivity to the MBP-(1–11) peptide was observed. In addition, mapping of reactivity to specific epitope regions of the V{beta}8.2 chain using overlapping peptide sequences demonstrated recognition of determinants close to the C terminus of the V{beta} chain (Fig. 6B). Recognition of V{alpha}2 determinants was also detected but this reactivity has not yet been mapped to specific epitopes within V{alpha}2.



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  		FIG. 6.
SDS-PAGE analysis (A) and corresponding Western blot analysis (B) of C14 scTCR protein and controls under reducing (R) and nonreducing (NR) conditions. The Western blot was performed using the C14 scTCR-specific monoclonal antibody 4D9:A2 and shows that this highly specific mAb recognizes both monomeric and dimeric forms of the scTCR. Lane 1, C14 scTCR (5 µg); lane 2, MR3.2 human scTCR (5 µg); lane 3, human RTL (5 µg).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study was initiated to lay the groundwork for investigating the biochemical and immunogenic properties of a T cell receptor known to be involved in T cell-mediated pathogenesis. MBP-specific T cell clones isolated from rats immunized with Gp-BP transferred disease to naive rats and were dominated by the expression of the V{beta}8.2 and V{alpha}2 TCR genes (27, 28). Curiously, the involvement of the V{beta}8.2 gene has been identified in several different models of rat as well as mouse autoimmunity (27). Subsequently, we demonstrated that T cells infiltrating the central nervous system in GpBP-(69–89)-immunized rats demonstrated biased expression of V{beta}8.2 and V{alpha}2 TCR genes displaying conserved CDR3 motifs (24–26). Our present model of the C14 scTCR indicates that the residues composing these motifs are present in a location central to the TCR combining site and are likely to interact with peptide antigen. The crystal structure of D10 scTCR in complex with peptide-MHC class II (7) was very similar to that of several TCRs binding peptide presented by MHC class I molecules as well as to the solution structure of free D10 scTCR as determined by NMR spectroscopy (12). However, whereas NMR analysis indicated that the CDR3{alpha} and CDR3{beta} loops were highly mobile in the unliganded D10, analysis of the D10 scTCR in the crystallized trimolecular complex indicated that the CDR3 loops packed together. Thus, it is likely that the CDR3 loops adopt a packed conformation when interacting with specific peptide presented by an MHC molecule. Since our model of the C14 TCR was based on the D10 scTCR crystal structure, the CDR3 loops are seen in close approximation to each other.

The approach we undertook to characterize the disease-associated TCR was to clone and express a solubilized, recombinant form of the TCR in order to generate sufficient quantities for purification to homogeneity. The refolding protocol adopted in our present paper allowed for the generation of purified, refolded rat scTCR that was largely monomeric but possibly not free of misfolded scTCR. The structural characteristics of a number of TCR molecules have been analyzed using various strategies that successfully produced soluble TCR molecules. Importantly, analyses of E. coli-derived scTCR molecules (7, 30–32) have demonstrated that specific binding interactions are conserved despite the absence of constant regions and glycosylation. However, the absence of constant regions and/or glycosylation on the scTCR may have contributed to the observed lack of scTCR-specific antibody binding to the cell surface-expressed form of the C14 TCR. Post-translational modifications are less likely to affect T cell recognition of scTCR epitopes, since the scTCR would first be processed and then presented as linear peptides in the context of MHC. In fact, we and others have used recombinant forms of TCRV{beta}8.2 protein to vaccinate mice and rats against the development of EAE (15, 17),4 which is characterized by pathogenic T cells expressing predominantly TCRV{beta}8.2.

In our present paper, surface plasmon resonance analysis demonstrated that the rat C14 scTCR, but not a control human scTCR, was able to interact with rat RT1.B MHC class II molecules bearing covalently bound MBP-(69–89) peptide. The calculated affinity constant (KD) was well within the range observed for TCR interaction with MHC molecules (41). However, further purification of the C14 scTCR to homogeneity may result in a slightly modified KD value. Recent studies indicate that thermodynamic equilibrium (i.e. affinity) constants may demonstrate a poor correlation with T cell activation by specific peptide. Rather, T cell activation models showed a stronger correlation with kinetic stability or slower off rates for peptides interacting at the binding site (41, 43, 44). The slow dissociation rate (koff) observed for rat C14 scTCR bound to the MBP-(69–89)-MHC class II complex may represent a relatively stable interaction that leads to the activation of pathogenic T cells. In addition, distinct regions of the TCR combining site complement centrally located peptide residues and may play a major role in discriminating between energetically similar peptide ligands that mediate distinct biological effects (1). Thus, our future goals using purified recombinant scTCR molecules include determining the relationship between kinetic on and off rates for closely related peptide antigens and T cell activation leading to pathogenesis.

It is important to note that immunization with scTCR protein allows the identification of natural T cell epitopes rather than cryptic epitopes that may arise upon immunization with synthetic peptide fragments (45). Presentation of natural epitopes would be a critical factor in modulating T cell-mediated autoreactivity (46). In our mapping of TCR determinants recognized by a murine T cell line specific for the C14 scTCR, we identified significant reactivity to epitopes closer to the C terminus of the V{beta}8.2 chain. Although this was not a response to a syngeneic TCR protein, it was interesting to find that significant reactivity to other parts of the molecules, particularly the CDR1 and CDR2 regions, was not evident. The lack of reactivity could be a result of tolerance imposed by thymic mechanisms, since over 75% of the C14 TCR sequences are conserved between rat and mouse. It is also possible that a lack of natural MHC presentable peptides within certain regions in response to TCR protein has evolved as a mechanism to restrict reactivity to the TCR. Clearly, a broader survey of responses to multiple TCR molecules from various MHC backgrounds is of interest. For example, an analysis of the immune response to soluble D10 TCR molecules (47) demonstrated a clonotypic antibody response to D10 scTCR and a CD4+ T cell proliferative response in B10.PL (H-2u)- but not in AKR (H-2k)-immunized mice. However, immunization of both B10.PL and AKR mice with TCR molecules containing constant regions curiously induced much stronger B and T cell responses to constant region determinants.

Particular TCR regions, including determinants within framework 3, may be capable of eliciting a natural immuno-regulatory response. Kumar et al. (15) previously reported protection in the B10.PL model of EAE using recombinant TCR molecules, including scTCRs. Epitope mapping identified an immunodominant regulatory determinant associated with framework 3 of the V{beta}8.2 chain with core residues consisting of amino acids 85–92 (48). Our present data support the concept that this region contains naturally existing immunodominant determinant(s). Importantly, this stretch of core residues is identical between rat and mouse V{beta}8.2 and may bear a recently identified binding motif for I-Au (49). We are investigating whether T cells recognizing such determinants in our B10.PL model as well as in the Lewis rat model also possess regulatory activity. In our recent analysis of TCR determinants recognized by T cell lines specific for a human scTCR protein, the most highly reactive determinant was associated with the BV CDR3 region.2 Recognition of a BV CDR3 epitope coincident with the regulatory activity demonstrated by the human scTCR-specific T cells was supportive of a clonotypic regulatory mechanism, although downstream suppressive effects apparently were nonspecific. Recognition of the CDR3 region was also implicated in a study involving whole T cell vaccination of multiple sclerosis patients (50, 51). A mechanism proposed for TCR regulation of autoreactive T cells involves recognition of specific TCR determinants and release of regulatory cytokines by CD4+ T cells (16, 18, 52). Thus, immunization with scTCRs may prove to be very useful for the characterization of TCR-specific T cells with regulatory potential.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant NS35207 and NS041965, and National Multiple Sclerosis Society Grant RG3259A1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Integrated Dept. of Immunology, National Jewish Medical and Research Center and the University of Colorado Health Sciences Center, 1400 Jackson St., Denver, CO 80206. Back

** To whom correspondence should be addressed: Tykeson MS Research Laboratory UHS-46, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201. Tel.: 503-494-4424; Fax: 503-494-9537; E-mail: buenafea{at}ohsu.edu.

1 The abbreviations used are: TCR, T cell receptor; scTCR, single chain TCR; CDR, complementarity-determining region; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; RTL, recombinant TCR ligand; MHC, major histocompatibility complex; Gp-BP, guinea pig basic protein; PBS, phosphate-buffered saline; mAb, monoclonal antibody; BSA, bovine serum albumin. Back

2 A. C. Buenafe et al., submitted for publication. Back

3 A. C. Buenafe, manuscript in preparation. Back

4 A. C. Buenafe, unpublished observations. Back



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