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Originally published In Press as doi:10.1074/jbc.M409427200 on November 4, 2004

J. Biol. Chem., Vol. 280, Issue 3, 1882-1892, January 21, 2005
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Design of Soluble Recombinant T Cell Receptors for Antigen Targeting and T Cell Inhibition*{boxs}

Bruno Laugel{ddagger}, Jonathan M. Boulter§, Nikolai Lissin¶, Annelise Vuidepot¶, Yi Li¶, Emma Gostick{ddagger}, Laura E. Crotty||, Daniel C. Douek||, Joris Hemelaar**, David A. Price||{ddagger}{ddagger}, Bent K. Jakobsen¶, and Andrew K. Sewell{ddagger}§§

From the {ddagger}The T-cell Modulation Group, The Peter Medawar Building for Pathogen Research, University of Oxford, South Parks Rd., Oxford OX1 3SY, United Kingdom, §Department of Medical Biochemistry and Immunology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom, Avidex Ltd., 57 Milton Park, Abingdon, Oxon OX14 4RX, United Kingdom, ||Human Immunology Section, Vaccine Research Center, NIAID, National Institutes of Health, Bethesda, Maryland 20892, and **Magdalen College, University of Oxford, Oxford OX1 4AU, United Kingdom

Received for publication, August 17, 2004 , and in revised form, November 1, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The use of recombinant T cell receptors (TCRs) to target therapeutic interventions has been hindered by the naturally low affinity of TCR interactions with peptide major histocompatibility complex ligands. Here, we use multimeric forms of soluble heterodimeric {alpha}{beta} TCRs for specific detection of target cells pulsed with cognate peptide, discrimination of quantitative changes in antigen display at the cell surface, identification of virus-infected cells, inhibition of antigen-specific cytotoxic T lymphocyte activation, and identification of cross-reactive peptides. Notably, the A6 TCR specific for the immunodominant HLA A2-restricted human T cell leukemia virus type 1 Tax11–19 epitope bound to HLA A2-HuD87–95 (KD 120 µM by surface plasmon resonance), an epitope implicated as a causal antigen in the paraneoplastic neurological degenerative disorder anti-Hu syndrome. A mutant A6 TCR that exhibited dramatically increased affinity for cognate antigen (KD 2.5 nM) without enhanced cross-reactivity was generated; this TCR demonstrated potent biological activity even as a monomeric molecule. These data provide insights into TCR repertoire selection and delineate a framework for the selective modification of TCRs in vitro that could enable specific therapeutic intervention in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Peptide major histocompatibility complex (pMHC)1 antigens displayed on the surface of target cells are recognized by T cells via their specific T cell receptor (TCR) (1). The TCR coreceptors CD8 or CD4 bind to invariant domains of pMHC class I (pMHCI) or pMHC class II (pMHCII) and are known to facilitate the process of antigen recognition by T cells (2). Recent advances have enabled the generation of high quality soluble TCR, pMHCI, pMHCII, CD8, and CD4 proteins; these in turn have allowed the biophysical characterization of the interactions between these molecules. Accordingly, the TCR and CD4/8 co-receptors have been shown to have very low affinities (KD ~ 10–3–10–6 M) for cognate pMHC. Despite these low affinity interactions, however, the process of antigen engagement can initiate T cell recognition of antigen-presenting cells (APCs) bearing fewer than 10 copies of a specific pMHC complex (3, 4). The mechanisms by which these weak recognition events result in such exquisite sensitivity are not fully understood.

The production of soluble recombinant {alpha}{beta} T cell receptors has proved challenging. The main technical pitfall is heterodimeric instability in the absence of anchoring transmembrane domains and {alpha}/{beta} pairing through an interchain disulfide bond. One of the commonest protein engineering strategies used in TCR studies to date has been the construction of single-chain TCRs. This technique, which takes advantage of the structural similarities between antibodies and TCRs, is based upon the single-chain Fv technology used to generate antibody fragments (5). In short, for the TCR it involves the cloning and expression of a unique chimerical open reading frame where the variable (V) {alpha} and V{beta} domains are paired with a protein linker (6, 7). These reagents have been successfully used in structural and biophysical studies (7, 8), but widespread application of the method has proven more difficult. Alternative approaches have included shuffling the variable and constant (C) domains of the TCR to the C-region of an immunoglobulin {kappa} light chain to generate a soluble heterodimeric protein (9); the resultant TCR was shown to react with several anti-TCR antibodies but was not shown to bind specific antigen.

In this study, we generate soluble versions of the human HLA A2-restricted JM22 and A6 TCRs specific for dominant viral epitopes derived from the influenza matrix and human T cell leukemia virus type 1 (HTLV-1) Tax proteins, respectively. Unlike most of the recombinant TCRs described to date, these proteins comprise an {alpha} and {beta} chain that are expressed separately and paired by a non-native disulfide bond (10). We overcome limitations imposed by the intrinsically low binding affinity and correspondingly short half-life of the monomeric TCR/pMHCI interaction by building multimeric forms of these proteins. The exquisite binding sensitivity and specificity exhibited by these multimeric TCRs allowed us to monitor quantitative modifications of antigen display on APCs and, in the case of A6, to investigate the binding parameters of the TCR with several syngeneic cross-reactive ligands. These latter data, which represent the first biophysical demonstration of the self-reactivity of a human TCR, have important implications for our understanding of T cell repertoire selection by thymic editing. Furthermore, we characterize these soluble multimeric TCRs functionally and show that such reagents can be used to inhibit antigen-specific CD8+ T cell activation. Finally, we employ a novel method that allows the generation of TCRs with cognate ligand affinities in the antibody range; these latter reagents hold the potential to revolutionize immunotherapeutics by enabling targeted drug delivery and antigen-selective immunosuppression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
B Cell Lines—LBL 721.174 cells, T1 cells, and Epstein-Barr virus-immortalized HLA A2+ B cells (PK) were maintained at 37 °C in RPMI medium supplemented with 10% fetal calf serum, L-glutamine (2 mM), penicillin (100 units/ml), and streptomycin (100 µg/ml) (R10 medium).

T Cell Lines—The E6 cytotoxic T lymphocyte (CTL) line was derived from peripheral blood mononuclear cells obtained from an HTLV-1-infected donor. Cells specific for the HLA A2-restricted HTLV-1 Tax11–19 epitope were initially expanded by exposure to peptide-pulsed autologous peripheral blood mononuclear cells in R10 and then further stimulated with peptide-pulsed PK B cells and mixed irradiated allogeneic feeder peripheral blood mononuclear cells from three unrelated donors in Iscove's modified Dulbecco's minimum essential medium (Sigma) supplemented with fetal calf serum and antibiotics as above together with phytohemagluttinin (4 µg/ml) and T-STIM (10%; BD Biosciences). The specificity of the line was confirmed by pMHCI tetramer staining. The 003 CTL clone specific for the HLA A2-restricted HIV-1 epitope SLYNTVATL (p17 Gag; residues 77–85) was isolated and maintained as previously described (11).

Manufacture of Soluble Heterodimeric TCRs—The generation of soluble TCR heterodimers was based on the procedure described by Boulter et al. (10). Each TCR chain was individually cloned in the bacterial expression vector pGMT7 and expressed in Escherichia coli BL21-DE3(pLysS). Residues threonine 48 and serine 57, respectively, of the {alpha}- and {beta}-chain TCR constant region domains were both mutated to cysteine. A biotinylation target motif (12) was also fused to the C terminus of the TCR {beta} chain to allow tetramerization with extravidin. Expression, refolding, purification, and biotinylation of soluble TCR heterodimers have been described previously (13). Development and production of the high affinity A6c134 TCR are described elsewhere.2

Multimerization of T Cell Receptors—Tetramerization of TCR heterodimers was performed by the addition of extravidin or R-phycoerythrin-labeled extravidin (Sigma) in aliquots to saturate its binding sites to a total TCR:extravidin molar ratio of 4:1.

Flow Cytometry—106 B cells per staining were pelleted and pulsed with the appropriate concentration of HLA A2-restricted peptides diluted in RPMI for 90 min at 37 °C. Cells were then washed once in 5 ml of fluorescence-activated cell sorter buffer (phosphate-buffered saline with 2 mM EDTA and 2% fetal calf serum) and resuspended in 100 µlof TCR tetramer solution at 100 µg/ml (with respect to TCR). Incubation was carried out at 37 °C for 30 min except when specified. Cells were washed twice in 5 ml of fluorescence-activated cell sorter buffer before analysis. All samples were collected on a FACSCalibur flow cytometer, and data were analyzed with CellQuest (BD BioSciences) software; a minimum of 5000 live cells was analyzed per sample.

Peptides—All HLA A2-restricted peptides (>95% purity; Invitrogen) were dissolved in Me2SO and diluted in RPMI medium to the desired concentrations. Binding affinity of peptides to HLA A2 was estimated using the algorithm developed by Rammensee et al. (14).

CTL Inhibition Assay—HLA A2+ B cells (PK) were pulsed with the indicated peptide concentrations as described above or infected with vaccinia virus. 5 x 103 target cells were then incubated with TCR tetramers or monomers or bovine serum albumin at a final concentration of 10 µg/ml for 4 h at 37 °C in 50 µl of R5 medium. CTLs maintained overnight in R5 were then added to the targets at a 5:1 (Tax-specific CTL) or 3:1 (HIV-1 Gag-specific CTL) effector:target ratio in a 100-µl final volume, and incubation was carried out for a further4hat 37 °C. Sample supernatant was then harvested, and macrophage inflammatory protein (MIP) 1{beta} concentration was quantified in duplicate assays by enzyme-linked immunosorbent assay according to the manufacturer's instructions (R&D Technologies, Abingdon, UK). Absorbance was measured at 450 nm with a Bio-Rad 550 microplate reader; standard curves were constructed with each assay.

Vaccinia Virus—The construction of the recombinant vaccinia virus expressing the pX region of HTLV-1 used in this study was reported in Parker et al. (15). PK B cells were infected with viral particles at a multiplicity of infection 5 in Iscove's modified Dulbecco's minimum essential medium with 0.1% bovine serum albumin for 90 min at 37 °C. Cells were then washed once, resuspended in Iscove's modified Dulbecco's minimum essential medium supplemented with 10% fetal calf serum and antibiotics as above, and then incubated for 12 h to allow for protein expression.

Surface Plasmon Resonance—A Biacore 3000TM machine and CM-5 sensor chips were used. Approximately 5000 response units of streptavidin were covalently linked to the chip surface in all four flow cells using the amino-coupling kit according to manufacturer's instructions. Biotinylated pMHCI proteins and biotinylated control protein (OX68) were bound to the sensor surfaces by flowing dilute solutions (50 µg/ml) of protein over the relevant streptavidin-coated flow cell. 500 response units (for kinetics measurements) or 1000 response units (equilibrium affinity measurements) of protein ligand were bound to each flow cell. If biotinylated TCR monomers were to be used, surfaces were blocked with 1 mM biotin in HEPES-buffered saline. Soluble A6, JM22, and 1G4 TCRs were then flowed over the relevant flow cells at a rate of 5 µl/min (equilibrium) or 50 µl/min (kinetics) at the concentrations indicated. The 1G4 HLA A2-restricted, NY-ESO-specific TCR was used as a negative-binding control. All measurements were performed at 25 °C using HEPES-buffered saline. Responses were recorded in real time and analyzed using BIAevaluation software (BIAcore, Uppsala, Sweden). Equilibrium dissociation constants (KD values) were determined assuming a 1:1 interaction (A + B {leftrightarrow} AB) by plotting specific equilibrium binding responses against protein concentrations followed by non-linear least squares fitting of the Langmuir binding equation, AB = B x ABmax/(KD + B), and were confirmed by linear Scatchard plot analysis using Origin 6.0 software (Microcal; Northampton, MA). Kinetic binding parameters (kon and koff) were determined using BIAevaluation software. koff values for TCR tetramers were estimated using dissociation phase data at least 10 min after reagent binding to prevent interference from the small fraction of weakly bound TCR tetramers on the BIAcoreTM chip surface.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Comparative Kinetic Parameters of A6 and JM22 TCRs in Monomeric and Multimeric Form—The TCRs derived from the CTL clones A6 and JM22 were selected for this study. Each of these TCRs is specific for a peptide presented in the context of HLA A*0201 (HLA A2 from here on). The A6 TCR recognizes an antigenic peptide that comprises residues 11–19 of the lymphotrophic retrovirus HTLV-1 transcription factor Tax (16). The co-crystal structure of the A6 TCR and its cognate ligand has been determined (17). This TCR/pMHCI interaction has the highest affinity for a human TCR and a syngeneic ligand measured to date (18). The JM22 TCR is specific for residues 58–66 of influenza A matrix protein (19). The affinity and kinetic parameters of this interaction have been characterized (20), and the crystal structure of the TCR complexed to its cognate ligand has recently been determined (21). Whereas dissociation rates are similar for both TCRs and fall within the spectrum of values observed for all TCR/pMHC interactions studied to date (22), the on-rate measured for the A6 TCR is unusually fast and accounts for most of the difference in affinity between these two TCR/pMHCI interactions. Equilibrium binding analyses of biotinylated A6 and JM22 TCRs in monomeric and multimeric forms are shown in Fig. 1. The multimers took longer than the monomers to reach equilibrium binding. This difference is believed to reflect the steric rearrangements required for binding of the tetrameric molecules. Importantly, binding specificity of the multimers was not affected, and no response was monitored with irrelevant pMHCI complexes. The most striking feature of this data is the substantial reduction in dissociation rate exhibited by the multimerized TCRs compared with monomers. TCR tetramers exhibited complex dissociation kinetics, with a fraction of TCR tetramers (14% for A6 and 43% for JM22) exhibiting a more rapid off-rate (koff); this likely represents those reagents binding via only two pMHCI molecules or those binding in a sterically compromised manner. The vast majority of the TCR tetramers, presumably those bound to three pMHCI molecules, have a considerably slower (true) koff. The greatly reduced koff of tetramerized TCR results in reagents that bind with a half-life of 154 and 43 min for the A6 and JM22 TCR tetramers, respectively (Fig. 1). Thus, the A6 and JM22 TCR tetramers exhibit a 1339- and 444-fold slower dissociation rate than the respective monomeric interactions. The dissociation of A6 TCR tetramers did not exhibit concentration dependence (Supplemental Fig. 2). The greatly increased half-life of tetramerized TCRs provides a basis for the use of such reagents in cellular binding assays where the half-life of interaction is critical for stable adhesion to the cell surface.



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  		FIG. 1.
Binding of A6 and JM22 TCR monomers and tetramers to HLA A2-Tax and HLA A2-influenza complexes. Streptavidin was linked to a BIAcoreTM CM-5 chip by amine coupling, biotin-tagged pMHCI was loaded onto each flow cell, and data were collected at 25 °C with a flow rate of 5 µl/min. 5 µl of each biotinylated TCR monomer at 1 mg/ml was flowed over all flow cells as was 25 µl of each TCR tetramer at 50 µg/ml. Negligible response was observed to non-cognate pMHCI for both TCR monomers and tetramers. To facilitate visual comparison of monomer and tetramer binding events, the much larger monomer response values were normalized to the peak values for the tetramers. Kinetic binding parameters for the tetramers were estimated using BIAEvaluationTM software (see table). Monomer KD values were taken from experiments with unbiotinylated A6 TCR (Supplemental Fig. 1) or JM22 TCR (20). There are two apparent off-rates for the TCR tetramers, (i) a minority fast off-rate thought to correspond to those tetramers binding less than three antigens and (ii) a slow (true) off-rate for those tetramers likely binding three pMHCI molecules. The latter off-rate is shown in the table. Some irreversible binding of biotinylated TCR monomers is observed because of the incomplete blocking of the streptavidin-coated chip surface with soluble biotin. RU, response units.

 
Soluble A6 and JM22 T Cell Receptors Bind Antigen-pulsed B Cells Specifically—Experimental parameters with multimerized TCRs were tested systematically to define the optimal staining conditions described under "Experimental Procedures." Fig. 2 shows a typical staining of peptide-pulsed HLA A2+ B cells with the A6 and JM22 TCR multimers under optimal conditions. Background staining of unpulsed cells was negligible as the mean fluorescence intensity was similar to the value obtained for unstained cells (data not shown). Antigen-specific staining was observed with peptide concentrations of 10–5 M and more in all cases. The A6 TCR multimer stained cognate peptide-loaded APC more efficiently than the JM22 TCR multimer (compare Figs. 2, A and B). This difference in staining at high concentrations of exogenous peptide probably reflects ligand affinity and off-rate differences (Fig. 1).



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  		FIG. 2.
TCR multimer staining of APCs. HLA A2+ B-cells were pulsed with exogenous peptides at the indicated concentrations and stained with A6 or JM22 TCR multimers as described under "Experimental Procedures." Flu, influenza.

 
TCR Multimers Do Not Exhibit Cross-reactivity with Unrelated Self- and Non-self Peptides—Multimerized A6 and JM22 TCRs do not stain HLA A2+ B cells without the addition of exogenous antigen, implying that they do not cross-react with the repertoire of self-peptides displayed by HLA A2 molecules on the cell surface at physiological densities. In addition, experiments in which B cells pulsed with either the HTLV-1 Tax11–19 peptide or the influenza matrix58–66 peptide were stained with the irrelevant TCR tetramer failed to show detectable cross-staining with non-cognate ligand (Fig. 3). These results indicate that the recombinant proteins bind to the surface of APC in a peptide-dependent manner and that nonspecific binding does not occur. Thus, in vitro protein engineering and refolding do not seem to affect the binding specificity of these TCRs. However, the possibility remained that these reagents could bind high amounts of other unrelated, non-self peptides displayed by HLA A2 molecules on the cell surface. To rule out this possibility, we stained HLA A2+ B cells pulsed with high concentrations (200 µM) of several HLA A2-restricted peptides derived from different pathogens, some of them exhibiting a degree of sequence similarity with HTLV-1 Tax11–19 and influenza matrix58–66 at non-anchor residues. No nonspecific binding of TCR multimers to cells pulsed with any of these peptides was detected (Fig. 3). Even though this does not entirely rule out the possibility of cross-reactivity, these results confirm that the A6 and JM22 TCRs retain cognate antigen specificity in soluble multimeric form.



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  		FIG. 3.
TCR multimers exhibit exquisite staining specificity. HLA A2+ B cells were pulsed with a variety of A2-restricted peptides at 200 µM or incubated with medium only (unpulsed cells) and stained with the indicated TCR tetramers as described under "Experimental Procedures." Bars show the S.D. from the mean fluorescence intensity values from two independent stainings. Flu, influenza.

 
TCR Multimers Can Sense Quantitative Modifications of Antigen Display at the Cell Surface and Stain APCs Processing Endogenous Antigen—Monoclonal antibodies have been used previously to examine the presentation of specific pMHC (23–33). Generation of such reagents is laborious and involves extensive screening of monoclonal antibodies, although some recent technical advances may facilitate this process (34, 35). The use of soluble TCRs may prove to be advantageous in this respect. O'Herrin et al. (36) used soluble TCR dimers to monitor quantitatively and qualitatively the up-regulation of pMHC molecules by IFN{gamma}. We repeated this result in our systems; HLA A2+ B cells cultured in medium supplemented with 50 units/ml IFN{gamma} for 48 h, pulsed with peptide, and then stained with soluble TCR multimers exhibited higher levels of specific staining than cells grown in IFN{gamma}-free medium (data not shown). Because the staining of TCR multimers seemed to reflect the up-regulation of MHC molecules induced by IFN{gamma} at the cell surface, we examined whether we could monitor similar effects induced by other biomolecules. {beta}2-Microglobulin ({beta}2m) is known to enhance the activation of CTLs when added exogenously in bioassays (37, 38). Although the precise molecular events responsible for this effect are not completely understood, it is clearly associated with an increase in the number of antigenic pMHCI complexes on the cell surface. The effect of {beta}2m on TCR multimer staining was monitored using cells pulsed with varying concentrations of peptide in the presence or absence of high concentrations of exogenous {beta}2m (Fig. 4). In the absence of {beta}2m, the minimum peptide concentration for which specific staining occurred above background (detection threshold) was 10 µM (Figs. 2, 4, A and B). At 10 µM peptide concentration, the mean fluorescence intensity values were substantially increased in the presence of exogenous {beta}2m (from 10.43 to 36.28 for the A6 TCR multimer and from 10.76 to 21.00 for the JM22 TCR multimer in the experiment shown) (Fig. 4, A and B). Furthermore, the addition of exogenous {beta}2m lowered the detection threshold for both the A6 and JM22 TCR multimers, to allow specific staining of cells pulsed with 1 µM cognate peptide (Fig. 4, A and B). These data are consistent with previous observations from studies using conformation-specific antibodies and highlight the dramatic effect of exogenous {beta}2m on the density of cell surface pMHCI complexes. The effect of {beta}2m on TCR binding was dose-dependent, with a dynamic range from 1 to 100 µg/ml (data not shown). The stainings shown in this study were carried out with higher {beta}2m concentrations to ensure optimal effect. In Fig. 4C, similar concentrations of exogenous {beta}2m were used to facilitate staining of HLA A2+ cells infected with recombinant vaccinia virus grown in medium supplemented with IFN{gamma}. Cells were stained 12 h post-infection by vaccinia expressing the HTLV-1 Tax protein or an irrelevant vaccinia. The A6 TCR multimer stained only HLA A2+ B cells infected with vaccinia expressing the Tax protein (Fig. 4C). That the detection of naturally processed ligand required the presence of exogenous {beta}2m indicates that {beta}2m-mediated enhancement of pMHCI antigen density on the cell surface does not operate through peptide exchange catalysis but likely reflects an effect on conformational stabilization.



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  		FIG. 4.
Characterization of TCR binding by flow cytometry. LBL721.174 cells were incubated for 90 min with the HLA A2-HTLV-1 Tax (A) or HLA A2-influenza (Flu) matrix. (B) peptides at the indicated concentrations or with medium only in the presence of either 200 µg/ml bovine serum albumin (BSA, empty bars) or 200 µg/ml {beta}2m (filled bars) and stained with the relevant TCR tetramer conjugated with extravidin-PE as described under "Experimental Procedures." Flu, influenza. C, PK B cells (HLA A2+) infected with recombinant vaccinia virus (see "Experimental Procedures") were stained 12 h after infection in the continuous presence of 200 µg/ml {beta}2m.

 
Increasing the Order of Multimerization Does Not Enhance the Sensitivity of Antigen Detection—The ability of TCR multimers to detect APCs bearing cognate peptide on their cell surface is a function of the reduced dissociation rate compared with the monomeric molecule. We, therefore, reasoned that increasing the order of multimerization might lead to further incremental increases in detection threshold. However, microbeads coated with fluorochrome-conjugated TCRs were not able to distinguish lower levels of cell surface antigen than TCR tetramers; the addition of a cross-linker induced further increases in background staining (Supplemental Fig. 3 and data not shown).

Antigen-specific Inhibition of CTL Activation by Soluble TCRs—The detection threshold of specific antigens on the cell surface using TCR multimers in combination with flow cytometry even under optimal conditions is limited to a relatively high peptide concentration (i.e. 10–6 M); this probably corresponds to a cell surface antigen density in excess of that produced by natural intracellular processing (34). To test whether binding could occur at lower antigen densities, a more sensitive experimental system was required. Previously, we have shown that phycoerythrin (PE)-conjugated pMHCI multimers can induce functional effects in CD8+ T cells at concentrations more than 2 orders of magnitude lower than can be detected by flow cytometry.3 We reasoned that TCR multimers could compete for antigens displayed on the cell surface of APCs with TCRs expressed by T cells specific for the same epitope and, therefore, inhibit activation of CTL. Fig. 5A shows the effect of preincubating target cells with multimerized TCRs on the activation of an HLA A2-restricted HTLV-1 Tax11–19-specific CTL line. The A6 TCRs inhibited MIP1{beta} release by over 2-fold for peptide concentrations ranging from 10–5 to 10–8 M as well as for peptide produced endogenously by recombinant vaccinia virus. CTL activation did not seem to be completely abrogated by preincubation with the TCRs, since for higher peptide concentrations (>10–7 M), MIP1{beta} release remained above background. Preincubation with the JM22 TCR tetramer failed to decrease MIP1{beta} production, indicating that the inhibition was antigen-specific. TCR binding, thus, occurs at low antigen density, more related to physiological levels, and with peptides produced in an endogenous manner, conditions in which flow cytometry fails to detect any binding of these reagents. This result highlights the limitations of the latter technique in terms of sensitivity when the amount of ligand on the cell surface is restricted. The limits of detection of PE on a FACS-Calibur are >150 molecules/cell (BD Biosciences). pMHCI tetramers are thought to cross-link at least three different TCRs on the T cell surface (39). Assuming the same kind of binding for TCR tetramers, >450 antigenic pMHCI molecules/cell will be required to observe positive staining by flow cytometry in our experiments. Interestingly, TCR monomers failed to mediate inhibition of CTL activation (Fig. 5C). This probably reflects a requirement for multimerization to achieve stable binding of soluble TCRs but could also indicate that inhibition of CTL activation is mediated through steric hindrance due to the large PE molecule in the complex rather than specific masking of the antigen. Such steric effects might be expected to prevent access of CTL to cognate and non-cognate pMHCI molecules on the APC surface. Fig. 5B shows the effect of A6 TCR multimers on the activation of a CTL clone specific for an HLA A2-restricted HIV-1 p17 Gag-derived peptide (SLYNTVATL; residues 77–85). In this experiment, HLA A2+ B cells pulsed with both the Tax11–19 peptide and the HIV-1 p17 Gag77–85 peptide were stained with A6 TCR tetramers and assayed for their ability to elicit activation of either HIV-1 Gag- or HTLV-1 Tax-specific CTLs. Incubation of the targets with A6 TCR multimers inhibited activation of the HTLV-1 CTL but did not inhibit activation of the HIV-1-specific CTLs by these same targets. We observed similar TCR-specific inhibition of CTL activation using tetramers made with unconjugated streptavidin, thus excluding steric hindrance due to the large PE moiety in the observed CTL inhibition (data not shown).



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  		FIG. 5.
TCR-mediated specific inhibition of CTL activation. A, HLA A2+ B cells (PK) were pulsed with the indicated concentrations of HLA A2-HTLV-1 Tax11–19 peptide or infected with vaccinia virus expressing the full-length Tax protein and used as targets in a CTL activation assay (see "Experimental Procedures"). Bars show the S.D. from the mean of two replicate assays. Data shown are representative of three experiments. BSA, bovine serum albumin. B, HLA A2+ B cells (PK) were pulsed with 1 µM HLA A2 HTLV-1 Tax peptide and either 0.1 µM (gray filling) or 10 µM (black filling) HLA A2-HIV Gag peptide or pulsed with 0.1 µM HTLV-1 Tax peptide (inset). Cells were then preincubated with 100 µg/ml A6 Tax TCR tetramer or bovine serum albumin and used in CTL activation assays with 003 human immunodeficiency virus Gag CTLs or with E6 Tax CTLs, as indicated. Bars show the S.D. from the mean of three replicate assays. Background MIP1{beta} release by E6 CTL was 60 pg/ml. C, HLA A2+ B cells (PK) were pulsed with 1 µM HLA A2-HTLV-1 Tax peptide and incubated with 10 µg/ml A6 wild type monomers/tetramers, A6c134 monomer or 1G4 TCR tetramer prior to CTL activation assay as described under "Experimental Procedures." Data are expressed as percentage of MIP1{beta} release compared with an assay performed in absence of TCR (100%). Bars show the S.D. from the mean of three replicate assays and are representative of two independent experiments. Background MIP1{beta} release without the addition of antigenic peptide was 91.8 pg/ml (or 14.3% of that with added antigen). The A6c134 monomer was able to inhibit CTL activation even at 0.1 µg/ml (data not shown).

 
The A6 Recombinant TCR Multimers Show Specificity Similar to the Native Cellular Protein—Given that functional effects can occur below the level of detection by flow cytometry, cross-reactivity becomes an even more significant concern when considering the application of these reagents for cellular targeting and therapeutic intervention in vivo. We, therefore, undertook experiments to characterize this potential confounding factor further. In an effort to understand the importance of individual amino acid side chains of a peptide in the TCR/pMHC interaction for T cell activation, Hausmann et al. (40) extensively tested the effect of single amino acid substitutions on the effector functions of CTL clones specific for the HLA A2-restricted HTLV-1 Tax11–19 antigen (40). From these substitutions they inferred different recognition motifs, which were used to search databases for protein sequences matching these motifs. Several peptides, all of them partial agonists eliciting significant levels of activation in A6 CTL, were identified. These "mimotopes" represented a good system to test whether the recombinant A6 TCR retained the specificity of the native molecule expressed on the T cell surface. Staining was carried out with seven of these peptides, chosen according to their activation potential. One was derived from the Tel1 protein of Saccharomyces cerevisiae (Tel1p549–557, MLWGYLQYV), and the other six were from human proteins (HuD87–95, LGYGFVNYI; BENE54–62, LLQGWVMYV; Phosphofructokinase572–580, TMGGYCGYL; protein-tyrosine phosphatase1073–1081, DLKGFLSYL; protein-tyrosine kinase32–40, SLHGYKKYL; HuR61–69, LGYG-FVNYV). Flow cytometry was used to determine that tetramerized A6 TCR bound to the surface of HLA A2+ APC pulsed with just two of these mimotopes (Fig. 6). These were HuD87–95, a nonameric peptidic fragment derived from a protein expressed in neurons, and Tel1p. Given the characteristically weak nature of TCR/pMHC interactions, the affinity of a particular TCR for cross-reactive, non-optimal ligands was expected to be very low. Accordingly, A6 TCR multimers only stained APC pulsed with very high concentrations of cross-reactive peptides. The detection threshold was 0.5 and 1 mM for Tel1p and HuD87–95, respectively; staining of HLA A2+ B-cells pulsed with 0.1 mM Tax11–19 peptide is shown for comparison (Fig. 6). Although weak, binding was peptide-specific and concentration-dependent. These results highlight the fact that the recombinant molecules retain the specificity of the native molecule and confirm the cellular data published by Hausmann et al. (40). The Tel1p and HuD87–95 peptides selected using the A6 TCR tetramers were taken forward for further analysis by surface plasmon resonance.



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  		FIG. 6.
A6 TCR tetramer staining of HLA A2+ B cells pulsed with cross-reactive peptides. PK B cells were pulsed with indicated concentrations of HuD87–95 (A) and Tel1p (B) peptides in the presence of 200 µg/ml {beta}2m for 90 min and stained as described under "Experimental Procedures." Staining of cells pulsed with 0.1 mM Tax11–19 peptide is shown for comparison.

 
Surface Plasmon Resonance Analysis of the Affinity between A6 TCR and Cross-reactive Ligands—A6 TCR tetramer staining of APCs pulsed with HuD87–95 and Tel1p was inefficient compared with staining of APCs pulsed with Tax11–19, likely reflecting differences in TCR/pMHCI binding affinities. However, other parameters not assessed in this study, such as the efficiency of peptide binding to HLA A2, could influence staining of APCs with TCR multimers. Estimations of relative binding affinities of both cross-reactive peptides and Tax11–19 peptide for HLA A2 were calculated. Predictive scores are shown in Table I and indicate that both peptides are likely to bind HLA A2, although more weakly in the case of HuD87–95. To measure the actual affinities of the interactions between the antigen and the A6 TCR, soluble HLA A2 molecules folded around the HuD87–95 and Tel1p peptides were synthesized and purified. Fig. 7, A and C, shows the binding response data of soluble A6 TCR at a range of concentrations (224 µM and 2-fold dilutions thereof) injected over, respectively, HLA A2-Tel1p and HLA A2-HuD87–95 complexes immobilized on the BIAcore flow cell. The equilibrium binding response of both interactions is plotted for each TCR concentration in a non-linear fit of the Langmuir isotherm (Figs. 7, B and D). Fits of three independent measurements gave a mean KD of 38.6 µM for HLA A2-Tel1p and 123.3 µM for HLA A2-HuD87–95. These values and the corresponding standard deviations are shown in Table I. A6 TCR/HLA A2-HuD87–95 binding did not reach saturation; this is likely to result in a lower accuracy of the KD estimation. The 1G4 TCR specific for the tumor epitope HLA A2-NY-ESO (10) failed to bind to any of these ligands (data not shown). Positive control data are shown for comparison (Figs. 7, E and F). Altogether, these results show that the cross-reactive ligands exhibit significant binding affinities with the A6 TCR.


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  		TABLE I
Summary of binding data of A6 TCR with various ligands measured by SPR

 



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  		FIG. 7.
Affinity of the A6 TCR for HLA A2-Tel1p and HLA A2-HuD87–95. Kinetic binding experiments for A6 TCR flowed over immobilized A2-Tel1p (A), A2-HuD87–95 (C), and A2-Tax11–19 (E). Equilibrium binding specific response is plotted against A6 TCR concentration and a non-linear fit of the Langmuir binding isotherm is shown for A2-Tel1p (B), A2-HuD87–95 (D), and A2-Tax11–19 (F). A6 TCR was flowed over the BIAcore chip at concentrations of 224 µM and 2-fold dilutions thereof down to 1.75 µM. Saturation binding with A2-Tax11–19 was reached at low concentrations. Only data with lower concentrations of TCR are shown in F.

 
A High Affinity Variant of the A6 TCR Inhibits CTL Activation as a Monomeric Molecule—Although multimeric TCRs could inhibit the activation of CTL with identical specificities, the detection of cross-reactivity with self-ligands is cause for concern in terms of translation of this technology into a therapeutic setting. Furthermore, the administration of multimeric TCRs may be problematic in vivo, and monomeric A6 TCR failed to inhibit the activation of HTLV-1-specific CTLs. The absence of a detectable biological effect with monomeric wild type TCR is presumably a consequence of the short half-life of the TCR/pMHCI interaction. We, therefore, developed a phage display system to select a mutant of the A6 TCR that binds to the cognate HLA A2-LLFGYPVYV antigen with extremely high affinity (KD 2.5 nM) without apparent loss of specificity; the affinity increment is due to a substantial decrease in off-rate (Supplemental Fig. 4).2 Previous studies show that the removal of unfavorable structural features can repair T cell recognition of antagonist peptides (18, 41). Similarly, structural analysis demonstrated that the enhanced affinity of our mutant TCR (A6c134) is due to contact optimization with bound peptide.2 The A6c134 TCR binds to HLA A2-LLF-GYPVYV antigen with a half-life of 3900s and can stain cells pulsed with as little as 10–8 M antigen.2 This enhanced affinity translated into a greatly increased biological potency compared with wild type recombinant TCR protein; indeed, the A6c134 TCR protein inhibited the activation of HTLV-1 Tax11–19-specific CTL even as a monomeric molecule (Fig. 5C). The inhibition of CTL activation by monomeric high affinity A6c134 TCR was comparable with that seen with multimeric wild type A6 TCR (Fig. 5C) for an identical peptide concentration. A6c134 TCR was also able to inhibit the release of lytic granules by an HTLV-1 Tax11–19-specific CTL line (Supplemental Fig. 5).

The A6 TCR Variant Exhibits Increased Affinity Selectively for the HLA A2-restricted Tax11–19 Parent Epitope—The A6c134 TCR exhibited minimal cross-reactivity with self peptides at physiological concentrations as demonstrated by the absence of background staining in flow cytometric assays; furthermore, there was no detectable staining of APCs pulsed with several unrelated peptides.2 To demonstrate formally that the increased affinity was specific for the wild type Tax11–19 epitope, we studied the interactions of A6c134 with the Tel1p and HuD87–95 epitopes to which cross-reactivity had been detected with the parent A6 TCR. Surface plasmon resonance showed that A6c134 and A6 TCRs bound to the Tel1p epitope with very similar affinity (KD = 46.5 and 38.6 µM, respectively; Fig. 7B and 8D). A6c134 bound the HuD87–95 epitope with an ~5-fold increased affinity compared with the A6 parent TCR (KD = 21 µM compared with 123 µM; Figs. 7D and 8E); however, this difference is minimal when compared with the 400-fold increased binding observed with HLA A2-Tax11–19 (KD = 2.5 nM compared with >0.9 µM2). Fig. 8A shows the comparative binding responses of A6c134 with HLA A2-Tax11–19, the cross-reactive ligand HLA A2-HuD, and a negative control. The association rate appears significantly slower in the case of HLA A2-Tax11–19. Yet, consistent with the findings of Li et al.,2 the most striking feature is the dramatically slower dissociation rate exhibited by the A6c134 monomers. This is illustrated in the first two injection cycles, during which dissociation of the TCR is barely detectable, and by the saturation of immobilized complexes as concentration of the analyte increases. Altogether, these results indicate that TCR affinity can be selectively increased for a specific peptide without concomitant major increases in the binding of related ligands.



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  		FIG. 8.
Affinity of the A6c134 TCR for HLA A2-Tel1p and HLA A2-HuD87–95. Kinetic binding experiments with A6c134 TCR flowed over immobilized ligands are shown. Overlay of the binding responses for A6c134 flowed over A2-Tax11–19 (solid line), A2-HuD87–95 (dotted line), and control A2-hTERT540 complexes (dashed line) (A). Kinetic binding experiments for A6c134 flowed over A2-Tel1p (B) and A2-HuD87–95 (C). Equilibrium binding specific response is plotted against A6c134 TCR concentrations, and a non-linear fit of the Langmuir binding isotherm is shown for A2-Tel1p (D) and A2-HuD87–95 (E). A6c134 TCR was flowed over the BIAcore chip at concentrations of 48 µM and 2-fold dilutions thereof down to 0.0937 µM.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we generated soluble human TCRs that, like membrane-bound TCRs, comprised two polypeptide chains; these were expressed separately from two distinct bacterial vectors and paired by a non-native disulfide bond. This strategy of protein engineering produces a protein similar to the native globular TCR that is extremely stable. The A6 and JM22 TCRs studied here appear structurally authentic, maintained their ligand specificity in this form, and were used to optimize the targeting of cells expressing cognate antigen. To overcome the intrinsically low affinity of TCR/pMHC interactions, we adopted two approaches. First, we increased overall avidity by multimerization to reduce the composite dissociation rate; this approach has been exploited successfully to allow identification of antigen-specific T cells with soluble pMHC molecules (39, 42). Second, we adopted mutational strategies to engineer a TCR from its A6 parent that had dramatically increased affinity for cognate ligand due to a reduction in off-rate. The TCR yielded by this latter approach was more sensitive than multimeric forms of the parent TCR in terms of its ability to detect cognate antigen on the target cell surface and exhibited potent biological activity with minimal cross-reactivity. The soluble heterodimeric TCRs manufactured in this study were utilized in a variety of functional assays including specific detection of target cells pulsed with cognate peptide, discrimination of quantitative changes in antigen display at the cell surface, identification of virus-infected cells, inhibition of antigen-specific CTL activation, and identification of cross-reactive peptides.

Part of this work has developed systems that utilize TCRs for the specific identification of cell surface pMHCI complexes. In this regard, several noteworthy features emerge from the data. First, PE-conjugated TCR multimers failed to emit a signal detectable by standard flow cytometric techniques when target cells were pulsed with low antigen concentrations (Fig. 2). However, functional assays clearly demonstrated specific and biologically relevant binding at antigen densities well below the fluorescence detection limit (Fig. 5). The requirement for high antigen density to visualize TCR multimer binding is, therefore, a methodological issue. In terms of detection sensitivity, the apparent discrepancy between TCR and pMHCI multimers, which are widely used to identify and characterize antigen-specific T cell populations directly ex vivo by flow cytometry, can be explained by respective ligand density differences on the target cell surface. A T cell is thought to bear ~30,000–100,000 TCRs (43), all of identical idiotypic specificity; at physiological antigen densities, however, only a small fraction of the 50,000 or so MHCI molecules on the surface of an APC (44) will present the epitope cognate for the TCR clonotype. Thus, it appears that flow cytometric detection of specific pMHCI at low antigen densities using soluble recombinant TCR-based reagents is limited by poor signal intensity. Second, despite these limitations, TCR multimers proved to be useful tools that enabled the discrimination of quantitative changes in antigen presentation on the target cell surface. Indeed, the effects of both {beta}2m and IFN{gamma} on the loading of exogenous peptide could be monitored by flow cytometry using these reagents. It is well documented that the addition of exogenous {beta}2m can enhance the apparent sensitivity of CTL to antigen (37). Other studies have shown that the addition of exogenous {beta}2m concomitant with peptide caused a vast increase in the number of conformationally correct pMHCI molecules on the cell surface as monitored with conformation-specific antibodies (38, 45). In the present study the addition of exogenous {beta}2m at a given concentration of peptide increased the number of TCR-cognate ligands by at least 2-fold and sufficiently increased the sensitivity of flow cytometric detection to allow the visualization of endogenously processed pMHCI antigen (Fig. 4). These observations could be extended to enable tracking of specific viral antigen expression in many settings. The synergistic effects of IFN{gamma}, which enhances processing and presentation of endogenously generated antigen (46), might further enhance the general applicability of either TCR multimers or high affinity variant TCRs for this purpose. Third, TCR multimers were used to screen for and identify syngeneic cross-reactive pMHCI ligands (Fig. 6); this approach eliminates the effects of adhesion/costimulatory molecular interactions that potentially confound cellular screening assays (47, 48) and has recently been validated in class II-restricted systems (49). Surface plasmon resonance studies confirmed binding, with measured affinities for both HLA A2-Tel1p and HLA A2-HuD87–95 lying at the lower end of the spectrum of previously defined TCR/pMHC interactions (22) (Fig. 7). To ensure an efficient adaptative immune response, it is advantageous that a single T cell clonotype can potentially engage in a functionally productive manner with several foreign peptides (50). On the molecular level, the corollary of this hypothesis is that a TCR clonotype will exhibit sufficient intrinsic conformational diversity to cross-react with several molecular mimics and less structurally related pMHCI complexes (51–53). Although alloreactive and/or xenoreactive ligands have been identified for other TCRs (51, 53–56), few cross-reactive syngeneic interactions between naturally occurring pMHCI and cognate TCR have been identified (57–60). Indeed, to the best of our knowledge, this study is the first to characterize the interaction between a pathogen-reactive human TCR and a cross-reactive self-peptide in biophysical terms.

The cross-reactivity identified in this study has several important implications. First, it instructs on the limits of thymic selection. Of the cross-reactive human peptides capable of eliciting functional responses through the A6 TCR in the study by Hausmann et al. (40), only HuD87–95 bound the corresponding TCR tetramer in our hands by flow cytometry. Thus, this peptide likely ranks at the upper end of the A6 TCR affinity spectrum in terms of interactions with human peptides; indeed, this assumption was confirmed by surface plasmon resonance analysis for those peptides tested (data not shown). In a broader context, we have examined the binding of TCRs from several human immunodominant anti-viral CTL to their cognate ligand in addition to the A6 and JM22 TCRs studied here. All of these TCRs have a relatively high affinity for their cognate ligand (KD 0.9–10 µM). The TCR/pMHCI interaction of anti-tumor CTL appears to be weaker than this (KD 20–40 µM).4 Thus, the affinity of the interaction between HLA A2-HuD87–95 and the A6 TCR (KD 120 µM) is only 4 times weaker than that of TCRs capable of mediating functional T cell responses in the periphery. These data suggest that thymic selection operates within narrow limits and are consistent with studies in murine systems (61). Second, the cross-reactivity of A6 with HLA A2-HuD87–95 might be relevant in autoimmunity. Paraneoplastic neurological degenerations are disorders that develop in patients with coexistent malignancies. Paraneoplastic neurological degenerations are believed to be triggered by an anti-tumor immune response directed against neuronal antigens expressed inside tumor cells (62). Curiously, the HuD protein has been implicated as a source of such antigen in one form of paraneoplastic neurological degeneration called anti-Hu syndrome (62–64). It is believed that the expression of HuD by small cell lung cancer triggers an immune response against this protein, which is exclusively neuron-expressed in healthy individuals (63, 64). A recent study examined the recognition and processing of 14 computer-predicted HLA A*0201-restricted, HuD-derived epitopes (64). Only one of these peptides, comprising HuD residues 86–95, was generated through the MHCI antigen-processing pathway; HuD86–95 was found to be immunogenic in experimental systems (64). HuD86–95 contains the shorter 9mer HuD87–95 peptide (LGYGFVNYI) that cross-reacts with the A6 TCR in our study; HLA A2-HuD87–95 binds the A6 TCR tetramer to a slightly greater extent than the longer HuD86–95 peptide (data not shown). Cross-recognition of a neuron-derived epitope by a Tax11–19-specific TCR might be relevant to HTLV-1-associated pathology. 1–2% of HTLV-1 infected individuals, particularly those with a high viral load, develop HTLV-1-associated myelopathy/tropical spastic paraparesis. The HLA A2-restricted Tax11–19 epitope is highly immunodominant in HLA A2+ individuals infected with HTLV-1 and appears to elicit especially large CTL responses in the setting of HTLV-1-associated myelopathy/tropical spastic paraparesis (65, 66). Furthermore, HTLV-1-specific lymphocytes are enriched in the cerebrospinal fluid of individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (65). These findings suggest that such CTL might cause HTLV-1-associated myeloneuropathy (65–67); this could potentially result either from direct recognition of viral antigen in central nervous system tissue or from cross-recognition of central nervous system-derived self antigens such as HuD87–95. The latter hypothesis is supported by elegant studies showing that Tax11–19-stimulated T cell lines from two HLA A2+ individuals with HTLV-1-associated myelopathy killed targets pulsed with HuD86–95 peptide (40).

The ability of TCR multimers to target cells expressing specific antigens provides a vehicle for the delivery of therapeutic interventions selectively to sites of disease. In comparison to systemic delivery systems, targeted therapeutics might both enhance efficacy and minimize side effects. We show here, for example, that both wild type multimeric TCRs and engineered high affinity monomeric TCRs can be used to conceal specific antigens from cognate CTL (Fig. 5). In the future, it might be possible to use similar technology in vivo to down-modulate cellular autoimmune reactions. Alternatively, soluble TCRs could be used to deliver cytotoxic agents or to redirect pathogen-specific immune responses; this latter principle has recently been demonstrated using pMHCI-conjugated antibodies that allowed antiviral CTL to induce regression of human tumor xenografts in a murine model (68). However, the observation of TCR cross-reactivity is a potentially confounding factor in the context of targeted therapy. In this respect, the development of engineered TCRs with artificially enhanced binding affinities for cognate ligand represents a dramatic advance (69, 70).2 Importantly, this increased affinity is not accompanied by concomitant increases in cross-reactivity (Fig. 8).2 Thus, a strategy for the optimization of targeted therapeutics is defined that could be developed for multiple applications, most notably perhaps within the fields of tumor immunology and autoimmunity.


   FOOTNOTES
 
* This work was funded by the Wellcome Trust. 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

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. 1–5. Back

{ddagger}{ddagger} A Medical Research Council Clinician Scientist. Back

§§ A Wellcome Trust Senior Fellow. To whom correspondence should be addressed. Tel.: 44-1865-281539; Fax: 44-1865-281530; E-mail: andy.sewell{at}ndm.ox.ac.uk.

1 The abbreviations used are: pMHC, peptide major histocompatibility complex; TCR, T cell receptor; APC, antigen-presenting cell; MIP, macrophage inflammatory protein; HTLV-1, leukemia virus type 1; {beta}2m, {beta}2-microglobulin; CTL, cytotoxic T lymphocyte; PE, phycoerythrin. Back

2 Li, Y., Moysey, R., Molloy, P., Vuidepot, A.-L., Mahon, T., Baston, E., Dunn, S., Liddy, N., Rizkallah, P., Sami, M., Todorov, P., Jacob, J., Jakobsen, B. K., and Boulter, J. M. (2005) Nat. Biotechnol. in press. Back

3 A. K. Sewell, unpublished observations. Back

4 J. M. Boulter et al., unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Anton van der Merwe for helpful suggestions and critical reading of the manuscript.



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