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ARTICLE |
CORRESPONDENCE Vincenzo Cerundolo: vincenzo.cerundolo{at}imm.ox.ac.uk
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Ji-Li Chen and Guillaume Stewart-Jones contributed equally to this work.
A major challenge for the design of cancer vaccines is that natural tumor antigens elicit relatively weak T cell responses. One approach currently being investigated is the optimization of the MHC class I anchor residues in tumor epitopes to enhance binding of the peptide to the MHC class I molecule. Vaccination of cancer patients with this type of peptide analogue has resulted in larger antigen-specific CTL expansions than vaccination with wild-type peptides (1), indicating that the use of peptide analogs should be considered for future clinical trials. However, questions remain as to how alterations of anchor residues, while enhancing the peptideMHC (pMHC) class I interaction, may affect TCR recognition.
The three-dimensional structures underlying TCR recognition have important implications for the design of molecular vaccines (2, 3). Structural studies have established the molecular characteristics determining peptide binding to MHC class I molecules (4). By comparison, relatively few structures of TCRpMHC complexes have been solved (5), a particular problem having being production of stable, soluble TCRs suitable for crystallization. One, potentially generic, solution to this problem is to engineer a novel interchain disulfide bond into the interface between the TCR constant domains (6), an approach exploited in the work reported here. Previous studies have compared the structures of pMHC complexes with those of altered peptide ligands, for example (79). These comparisons have demonstrated that substitutions of anchor residues can cause slight structural alterations that may indirectly impact on TCR recognition. Whereas the structural effects of changing surface exposed peptide residues have been studied directly in TCRpMHC complexes (10, 11), there have been no TCRpMHC structures determined that address the effect on TCR recognition of only anchor residue modifications. However, an example of the impact of changes at buried MHC class I residues on TCR recognition has been provided by Luz and colleagues (12).
Several studies have sought to relate the structural characteristics of the TCRpMHC interface with TCR binding affinities (13, 14). TCR binding is characterized by low affinities (0.157 µM) and a degree of cross-reactive recognition (15, 16). Although peptide analogs bearing substitutions at anchor residues have been used in several clinical trials (1), neither the structural effect of these changes, nor how such features correlate with enhanced immunogenicity in vivo are well understood. It is important to analyze the molecular basis for the improved immunological potency of peptide analogs, because such information may provide guidelines for the rational design of T cell vaccines. Ideally, the properties of superagonist peptide analogs would blend (a) high affinity and stability of binding to the MHC class I molecule, (b) TCR binding properties that elicit potent and effective T cell expansions, and (c) high levels of TCR cross-reactivity to the endogenous target epitope on vaccination in vivo.
NYESO-1 is one of the most promising tumor-specific antigens, which was identified by the application of serological analysis of recombinant cDNA libraries from human tumors (17, 18). NYESO-1 is expressed by a broad range of tumor types (17). Vaccines designed to boost CTL responses against NYESO-1 epitopes may therefore be useful in the treatment of many tumors. The A2-restricted NYESO-1 T cell response has been identified using NYESO-1specific CTL lines derived from melanoma patients and shown to be specific for the epitope NYESO-1157165 (18). To investigate the structural and kinetic features of TCR recognition of A2 loaded with the wild-type NYESO-1157165 peptide (SLLMWITQC; hereafter referred to as "ESO 9C peptide") we produced a soluble version of a NYESO-1157165specific TCR. Our initial analysis revealed that TCR interactions with this native epitope are dominated by two highly exposed residues, methionine and tryptophan, whose side chains protrude from the central part of the peptide (at positions 4 and 5). The presence of this prominent hydrophobic feature suggests that for this system TCR binding in general will focus on the central region of the peptide and be relatively tolerant of changes in the pMHC surface near the peptide carboxyl terminus. To test this hypothesis and its implications for the immunogenic characteristics of the system, we performed a series of studies to provide structural, solution binding, in vitro functional and in vivo immunogenic data. Because we had previously demonstrated that substitution of the cysteine to valine at position 165 (19) enhances the ability of the epitope to be recognized in vitro by NYESO-1157165specific CTL, we focused on this peptide analogue (SLLMWITQV; hereafter referred to as "ESO 9V peptide"). Our results provide insights into the mechanisms controlling the enhanced immunogenicity of superagonist peptides by demonstrating a correlation between the stability of the peptideMHCTCR complex, its ability to stimulate a faster formation of conjugates, resulting in polarization of lytic granules, killing of target cells, and in vivo proliferation.
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Results |
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Previous studies that have compared TCR structures in unbound and complexed states (2529) report remarkably large (up to 15 Å) conformational changes in the CDR3 and ß loops. Although the 1G4 TCR uses a substantial cavity to accommodate the MW peg between its CDR1
, CDR3
, and CDR3ß loops (Fig. 1 C), this cavity is preformed (Fig. 1 E). In contrast with the mechanisms of induced TCR fit reported for other pMHCTCR complexes, a well optimized binding surface is achieved by relatively small structural changes. Comparison of the unliganded and liganded 1G4 TCR structures reveals only relatively minor main chain shifts in all the CDRß loops (for example, <1.6 Å for V96ß at the tip of the CDR3ß; Fig. 1, C and E). The largest main chain shifts (up to 4.2 Å) in the V
domain also occur at the tip of the CDR3 loop resulting from a change in the main chain conformation of residues 9599. Only subtle adjustments in preexisting (unliganded) side chain conformations appear to be required to optimize the interaction of Y100
with the peptide at M4 and ring-stacking interactions between Y31
and the W5 side chain. However, these TCR residues show a considerable reduction in flexibility on engagement with the MW peg (average crystallographic temperature factors for the side chain atoms of Y31
are 36 Å2 unliganded and 23 Å2 liganded, and for Y100
the values are 50 Å2 unliganded and 22 Å2 liganded).
Comparison of the 1G4 TCRESO 9CA2 and 1G4 TCRESO 9VA2 complexes
Because the 1G4 TCR recognition appears to be so focused on the central region of the peptide, we wished to assess whether substitutions of the carboxyl-terminal residue of the NYESO-1 peptide could alter the peptide structure and affinity of interaction with the 1G4 TCR. Because we have previously shown that substitution of cysteine to valine at position 165 of the NYESO-1157165 epitope enhanced its ability to sensitize target cells for killing by the 1G4 CTL clone (19), we decided to focus further structural studies on the 9V NYESO-1 peptide analogue (ESO 9V).
The crystal structure of the 1G4 TCRA2ESO 9V complex was determined at 1.7 Å resolution from crystals of the same space group and essentially identical unit cell dimensions to those used for the 1G4A2ESO 9C structure. To a first approximation the two complex structures are very similar. Structural comparison reveals that substitution of the cysteine to valine causes little difference in the general orientation of the side chain within the F pocket of the A2 peptide binding groove. Changes in the positions of the A2 side chains constituting the F pocket are also minimal. However, the relatively high resolution of both the 1G4A2ESO 9C and 1G4A2ESO 9V crystal structures permits a detailed analysis (Fig. 1 F). It is evident that the ESO 9V side chain is buried deeper into the F-pocket and occupies a slightly larger volume than the cysteine side chain. Both the shape complementarity and the buried surface within the F-pocket is greater for ESO 9V compared with ESO 9C (side chain surface areas: ESO 9V = 191 Å2, ESO 9C = 162 Å2 and shape complementarities: ESO 9V = 0.81, ESO 9C= 0.72). These changes are sufficient to enhance significantly the van der Waal's contacts between the altered peptide ligand and A2, in line with this MHC class I molecule's reported preference for valine as COOH-terminal anchor residue (30).
The changes in the position of the COOH-terminal anchor residue, resulting from the C9V substitution, propagate through the peptide main chain resulting in the P6P8 region being positioned slightly deeper (0.3 Å) within the A2 peptide binding groove. Although the P6P8 region of the peptide has fewer contacts with the 1G4 TCR than the MW peg at P4 and P5, the structural difference in the peptide is clearly transmitted to the TCR (Fig. 1 F). The angle of engagement made by the 1G4 TCR differs between the peptide complexes by some 0.9°. This is essentially a change in the TCR's tilt, relative to an axis drawn perpendicular to the pMHC binding surface, in response to the lower position of the main chain COOH-terminal half of the ESO 9V peptide. Numerous small changes are discernible at the complex interface. These changes are focused on residue Q155 of the A2 2 helix. For the A2ESO 9V complex the Q155 side chain is well ordered and makes hydrogen bonds to the O
atoms of S52 and S53 side chains. In contrast, for the A2ESO 9C complex the Q155 side chain is relatively disordered and appears to be sampling an additional conformation, forming a hydrogen bond to T95
on the CDR3
loop of the 1G4 TCR. The change in TCR tilt of the A2ESO 9V complex precludes the latter option and the CDR3
loop adopts a less well ordered structure. The average B-factor for residues in the tip of CDR3
loop (all atoms in Ser96
and Gly97
) are 33.93 and 42.46 for the A2ESO 9C and A2ESO 9V complexes, respectively. Analysis of the shape complementarity between the 1G4 TCR and the pMHCs suggests a slightly better overall fit is achieved with A2 /ESO 9V (Sc = 0.73) compared with A2ESO 9C (Sc = 0.71). The significance of this difference in Sc is discussed in Materials and methods. The difference includes tighter interactions between TCR and variant peptide (ESO 9V peptide Sc = 0.85; ESO 9C peptide Sc = 0.83).
Enhanced binding affinity of ESO 9V peptide for A2 molecules and slower 1G4 TCR dissociation rate from the ESO 9VA2 complex
Our comparison of the ESO 9C- and ESO 9V-based complex structures revealed the substitution of cysteine at position 9 by valine provides an improved fit to the A2 F pocket, a result that might be expected on the basis of established anchor preferences. However, this substitution, at a position buried within the binding groove, was also revealed to trigger subtle, but significant, changes at the TCRpMHC interface. To correlate these structural features of the TCRpMHC complexes with kinetics of binding, we assessed the affinity of binding of the ESO 9V and ESO 9C peptides to A2 molecules, and the affinity of 1G4 TCR binding to A2 molecules loaded with either the ESO 9V peptide or the wild-type ESO 9C peptide. Binding of ESO 9V peptide to A2 molecules was compared with that of ESO 9C by measuring peptide binding to metabolically labeled A2 molecules in T2 cells (31). Measurement of the optical density of metabolically labeled A2 SDS gel bands showed that ESO 9V peptide can stabilize a higher proportion of A2 molecules than the ESO 9C peptide (Fig. 2 A).
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Surface plasmon resonance affinity measurements were performed using the soluble form of 1G4 TCR in order to compare the affinity of binding of a NYESO-1157165specific TCR to A2 molecules loaded with either the wild-type ESO 9C peptide or the ESO 9V analogue. The experiments demonstrated a higher affinity of binding for the 1G4 TCR to the A2ESO 9V complex than to the A2ESO 9C complex (Fig. 2 C). Analysis of the binding curves of conformation-specific anti-A2peptide and anti-ß2M antibodies confirmed that equivalent amounts of correctly refolded A2peptide complexes were immobilized to the Biacore chips (Fig. S1, A and B). Good agreement was observed between the affinities determined kinetically (the ratio of Koff to Kon) and those determined by equilibrium measurements. The equilibrium binding constants (Kd) at 25°C for the soluble 1G4 TCR binding to A2 loaded with the ESO 9V or ESO 9C peptide were determined to be 5 and 13.3 µM, respectively (Fig. 2 C). Kinetic measurements demonstrated that the higher affinity of binding was predominantly due to a slower off rate for the 1G4 TCR when bound to the A2ESO 9V complex compared with the A2ESO 9C complex. The slower off-rate is in line with the structural data, which indicate that more pMHCTCR contacts form at the ESO 9V interface than at the ESO 9C interface.
ESO 9V induces polarization of CTL granules more efficiently than the wild type
To assess the functional consequences of the differences in pMHC and TCRpMHC binding affinities, we analyzed the rate of polarization of cytotoxic T cell granules on stimulation of a NYESO-1157165specific CTL clone with target cells pulsed with either the ESO 9C or the ESO 9V peptides. These experiments were performed with the same 1G4 CTL clone for which the structural and kinetic studies had been derived.
When T2 cells are pulsed with 1 µM ESO 9C peptide (Video S1 and Fig. 3, AE), lytic granule polarization is slow. Fig. 3, AE, shows two CTLs from contact to polarization. In one case (white arrow) granule polarization from the periphery (Fig. 3 B) to the synapse (Fig. 3 E) occurs within 17 min after the initial contact. In the other example (red arrow), the CTL first crawls under one target (Fig. 3, A and B) before contacting a new target (Fig. 3 C) without polarization of the lytic granules toward either target in >22 min. In contrast, granule polarization is rapid when T2 targets are pulsed with 1 µM ESO 9V peptide (Video 2 and Fig. 3, FL). For one CTL, from the first encounter (Fig. 3 F, green arrowhead) to granule polarization (Fig. 3 L) the elapsed time is 4 min. Other CTLs in the field being filmed (Fig. 3 L, black arrowheads) had already polarized their granules toward targets by the time images were captured.
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Enhanced in vivo immunogenicity of ESO 9V peptide analogs
We assessed whether the ESO 9V peptide is capable of expanding in A2 transgenic mice greater numbers of NYESO-1157165specific CTL that are cross reactive to the native epitope. The results show that recombinant vaccines encoding the ESO 9V peptide are capable of inducing stronger NYESO-1157165 responses than vaccines encoding the wild-type ESO 9C peptide, both in terms of percentages of responding mice and frequency of the T cell response induced (Fig. 6 A). Furthermore, ESO 9Vprimed mice were capable of recognizing the wild-type ESO 9C peptide, as defined by ex vivo tetramer staining with A2Kb tetramers loaded with the ESO 9C peptide (Fig. 6 B). The proportion of NYESO-1157165specific CTL, stained by A2Kb tetramers loaded with either the ESO 9C or the ESO 9V peptide was very similar. Consistent with these findings, in vitro killing assays showed that NYESO-1157165specific CTL, generated from ESO 9Vimmunized mice, were capable of lysing target cells loaded with wild-type ESO 9C peptide, as well as target cells loaded with the ESO 9V analogue (Fig. 6 C). IFN- ELISPOT assay was used to assess further cross reactivity and the function of these CTL. CTL generated from animals immunized with the ESO 9V sequence were capable of producing IFN-
in response to ESO 9C peptide. As expected, responses were enhanced in the presence of the reducing agent TCEP, which prevents cysteinylation and dimerization of ESO 9C peptide (unpublished data (19, 34). Finally, a larger proportion of ESO 9Vprimed T cells was capable of secreting IFN-
, when stimulated by target cells pulsed with ESO 9C peptide, than ESO 9C primed T cells (Fig. 6 D). Enhanced IFN-
response by T cells from ESO 9Vprimed mice was not due to the higher numbers of ESO 9Vprimed T cells, because the numbers of splenocytes from ESO 9V and ESO 9C primed mice added to the ELISPOT plates were normalized for the percentage of ESO 9C tetramer positive T cells.
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DISCUSSION |
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To elicit effective antitumor responses, it is important to develop strategies capable of reversing T cell tolerance to tumor antigens and jump starting the immune response to expand rapidly greater numbers of tumor-specific CTL. Although previous papers have correlated higher affinity of peptide binding to MHC class I molecules and TCR with increased immunogenicity (3640), none of the previous studies have compared functional data to structural and kinetic analyses.
The use of a Fab antibody specific to the A2/NYESO-1157165 peptide complex (32) permits comparison of the efficiency of ESO 9C and ESO 9V binding to surface expressed A2 molecules (Fig. 2 B). The results of these experiments are consistent with 100-fold difference in binding affinity to A2 molecules between the ESO 9C and ESO 9V peptides. They also show that, within the range of peptide concentrations used for the analysis of granule polarization (Figs. 3 and 4) and cytokine secretion (Fig. 5), cells pulsed with ESO 9V peptide present to the 1G4 CTL clone approximately three- to fivefold more peptide than cells pulsed with the ESO 9C peptide. These results suggest that the enhanced effector functions of the 1G4 CTL clone stimulated by ESO 9V peptide can be accounted for by a combined effect of higher peptide affinity to A2 molecules and slower off rate of the 1G4 TCR from the ESO 9VA2 complex. These results are consistent with previously published data demonstrating enhanced antigen-specific antitumor immunity with altered peptide ligands (40), although for the system studied by Slansky and colleagues the effect is entirely due to a threefold slower pMHCTCR off rate. Although lytic granule polarization formed in response to maximal pMHC densities has been extensively characterized, fewer reports have examined variations in the efficiency of lytic granule polarization with peptide density (41). The results of our confocal microscopy experiments (Fig. 4) highlight variations in the efficiency of lytic granule polarization over a range of different peptide concentrations and different stability of TCRpMHC complexes.
Increased immunogenicity in A2 transgenic mice and enhanced expansion of NYESO-1specific CTL from melanoma patients' PBL demonstrate that the enhanced stimulation of the clone 1G4 can be extended to a polyclonal population. Although we cannot rule out the possibility that in vivo results can mainly be accounted for by the higher affinity of binding of the ESO 9V peptide to A2 molecules, it is of interest that NYESO-1157165specific T cells from ESO 9Vprimed mice have an enhanced ability to recognize target cells pulsed with the ESO 9C peptides, as compared with NYESO-1157165specific T cells from ESO 9C primed mice (Fig. 6 D). It is possible that this enhanced T cell activity could be due to the increased stability of the ESO 9VA2 complex resulting in differences at the level of the immunological synapse at the T cellDC interface and in the expansion of better quality T cells. Consistent with this possibility, differences in the molecular composition of the immunological synapse between CTL and target cells have recently been described at different peptide concentrations (41), resulting in different signaling pathways and activation thresholds in individual CTL.
It appears, therefore, that the "consensus optimization" of the NYESO-1157165 F-pocket anchor side chain to valine can achieve increased T cell stimulation by augmenting the peptide affinity to A2, while maintaining, and in at least one case enhancing TCR recognition by decreasing the dissociation rate constant for TCR binding. Although the enhanced stability of the A2ESO 9V variant was expected, our combined structural and functional analysis underscores the importance of also assessing the indirect effect of anchor residue changes on the TCR recognition surface.
The central role of the MW peg in our complex structures is consistent with results obtained for an alanine scan of the ESO 9C peptide using NYESO-1157165specific T cell lines (42). A recent paper by Webb and colleagues confirmed the presence of this feature in the isolated A2ESO 9C structure (43). We have extended the analysis of Webb et al. by determining a high resolution structure (1.5 Å) of unliganded A2 molecules loaded with the NYESO-1157165 peptide analogue bearing a leucine at position 165 (A2ESO 9L), which demonstrates that the overall structure of the ESO 9L epitope in A2 is similar to the structure of the A2ESO 9V and A2ESO 9C complexes (unpublished data). It is of interest, however, that for isolated structures the MW side chains are more disordered than in the TCR complexes, consistent with the possibility that binding of the 1G4 TCR stabilizes the MW peg. Our study has primarily focused on TCR binding in assessing factors to be considered when engineering peptide analogs. However, CD8 binding to class I molecules is important for stabilizing the interaction between low affinity TCR and class I molecules (44) and for controlling the dynamics of CTL activation and immunological synapse formation (45). Consistent with these findings, we have shown that higher affinity recognition of the ESO 9V peptide by the 1G4 TCR is less CD8 dependent than recognition of the ESO 9C peptide. This alteration in CD8 dependency does not require any change in TCR docking orientation and therefore does not conform to the model suggested by Buslepp and colleagues (46).
The amount of signal that T cells receive by interacting with APCs is determined by several parameters including the concentration of pMHC complex (class Ipeptide binding affinity) and the duration of the interaction between T cells and APCs (TCR binding affinity). From this concept it follows that the success of vaccine strategies will be dependent on our ability to fine tune several parameters to induce strong T cell expansions. The use of superagonist analogs in clinical applications provides an opportunity to address some of the shortcomings of current vaccination strategies, as it has been shown that peptide analogs can induce stronger responses than wild-type peptides in vivo (3640). Peptide analogs are particularly relevant in the context of recombinant viral vector vaccination strategies, because immunodominance of viral vectorspecific responses often impairs their ability to elicit immune responses specific for recombinant epitopes. It is possible that the low frequency of recombinant epitopespecific CTL responses may be due to competition from immunodominant viral epitopespecific responses, underscoring the need for engineering high-affinity peptide analogs for recombinant virus vaccine approaches.
In conclusion, our results highlight the indirect effects of anchor residue substitutions on TCR binding, effects that are of substantial significance for the rational optimization of vaccination strategies. The sensitivity of TCR recognition can extend to detect differences in buried anchor side chains, resulting in altered TCR binding kinetics and different levels of activation signals. Our results suggest the importance of incorporating structural data into the process of identifying candidate superagonist peptide analogs. Identification of highly antigenic NYESO-1 peptide analogs is of importance for the development of vaccines capable of expanding NYESO-1specific CTL in cancer patients. Phase 1 clinical trials using NYESO-1 synthetic protein, peptides and recombinant viruses are already in progress aimed at eliciting tumor-specific T cell responses. The use of the NYESO-1 9V analogue should be considered in future clinical trials, because it could result in a more efficient induction of NYESO-1157165specific CTL in cancer patients.
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Materials and Methods |
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Expression and purification of the 1G4 NYESO-1 TCR. The 1G4 TCR was refolded and purified from E. coli derived inclusion bodies as described in reference 6.
Crystallization and x-ray diffraction data collection.
Initial crystallization screens for the 1G4 TCR (10 mg/ml), 1G4A2-ESO 9C or 1G4A2-ESO 9V complexes (1:1 TCRpMHC ratio at 10 mg/ml) used 100 nl + 100 nl drops dispensed by a Cartesian Technology Microsys MIC4000 (using Greiner 96-well protein crystallization plates), were stored in a automated vault and imaged with Veeco imaging systems. All crystallizations were done by the sitting drop vapor diffusion technique. Crystals of the isolated 1G4 TCR were grown at room temperature (21°C) from 2 µl + 2 µl protein + mother liquor (0.2 M sodium nitrate, 20% PEG 3350, pH 6.8) to dimensions of 180 µm x 130 µm x 70 µm. Crystals of the 1G4-A2-ESO 9C and 1G4-A2-ESO 9V complexes were grown at room temperature (21°C) from 2 µl + 2 µl protein + mother liquor (0.2 M potassium sodium tartarate tetrahydrate, 20% PEG 3350, pH 7.2) to dimensions of 130 µm x 80 µm x 70 µm.
Crystals were cryoprotected in reservoir solutions containing 10 and 20% glycerol; they were then flash-cooled, and maintained at 100 K, using a cryostream (Oxford Cryosystems). High-resolution data sets (1.4 Å for the 1G4 TCR, 1.9 Å for the 1G4A2-ESO 9C complex and 1.7 Å for the 1G4A2-ESO 9V complex) were collected at station ID14 EH2 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France) with an Area Detector Systems Corporation Quantum 4 CCD detector. The 1G4A2-ESO 9C and the 1G4A2ESO 9V complex crystals belonged to space group P21. Data were auto-indexed and integrated with the program DENZO (48), and scaled with the program SCALEPACK (48; Table I).
Crystal structure determination, refinement, and analysis.
The structures of the 1G4 TCR, 1G4A2-ESO 9C, and the 1G4A2-ESO 9V complexes were determined by molecular replacement with the program EPMR (49). For the two pMHCTCR complexes, initial solutions were obtained for the HLA-A2 heavy chain, and ß2M followed by a second round of molecular replacement with the A6 TCR used as a search probe while the A2 solution was kept fixed. The 1G4 molecular replacement was solved with the A6 TCR in a single round of molecular replacement. The program SWISS-MODEL (www.expasy.org/swissmod/SWISS-MODEL.html) was used to replace the A6 TCR sequence with that of 1G4 before refinement.
Using CNS (50), the models were refined as rigid body domains (1 and
2,
3, ß2M, peptide, V
, Vß, C
, and Cß for the complexes and the V
, Vß, C
, and Cß TCR domains for the 1G4 data). Subsequent minimization procedures included positional refinement, simulated annealing and restrained individual B factor refinement with bulk solvent correction and overall anisotropic B factor scaling. Manual refitting of the models was done with the interactive graphics program O (51), and water molecules were added at the last stages of refinement on the basis of peaks that were at least 3.0
in height in Fobs Fcalc electron density maps. The CDR loops of the isolated TCR showed significantly higher B factors than when complexed with pMHC, but the electron density was of sufficient quality to allow positioning of a single dominant main chain conformation for all of these loops.
We assessed the stereochemistry of the refined structures with the program PROCHECK (52). Surface complementarity calculations were done with the CCP4 program Sc (53; probe size 1.7 Å). To allow comparison of similar Sc values between structures it was necessary to estimate the standard uncertainty in atomic positions in the structures and how this translates to uncertainty in Sc value. For the 1G4A2-ESO 9C complex (the lowest resolution structure) the standard uncertainty in atomic positions derived by Refmac (54) using the Rfree was 0.148 Å. By running Sc 100 times on an ensemble of structures for this complex with the x, y, and z coordinates of each atom randomly perturbed using a Gaussian distribution of standard deviation 0.148 Å, we estimate the standard uncertainty in Sc to be 0.017. Thus, although the difference in Sc values for the two complexes is small, we believe it to be significant. Solvent accessibility calculations were done with Naccess (http://wolf.bms.umist.ac.uk/naccess/; probe size 1.4 Å). Graphical representations of the structure were prepared using programs Bobscript (55) and CSC Chemdraw (Cambridge Scientific Computing, Inc.).
Surface plasmon resonance
Surface plasmon resonance experiments were performed by using a Biacore3000 (Biacore). Biotinylated soluble HLA (ligand) was immobilized on Streptavidin-coated CM5 chip (Biacore) at the level of 1,000 RU (response units) per flow cell. Equilibrium binding was measured at the flow rate of 5 µl/min, starting from the lowest analyte concentration. The data points were plotted using Origin software. Kd values were calculated using the standard hyperbolic model fitting kinetics experiments. Kinetics of TCRpMHC interactions was measured at 30 µl/min. The curves were then fitted using a simultaneous kon/koff fitting model (Bioevaluation software; Biacore).
MHC class I assembly assay
This assay was performed as previously described (31).
Flow cytometry
3 x 104 T2 cells were pulsed with different concentrations of peptides for 2 h at 37°C. Cells were incubated with 40 µg/ml biotinylated Fab antibodies for 30 min at 4°C and visualized by Streptavidin-conjugated R-PE (Sigma-Aldrich) for 20 min at room temperature.
Confocal microscopy analysis.
T2 target cells were pulsed with 1 µM, 100 nM, 10 nM, 5 nM, and 1 nM of either ESO 9C or ESO 9V peptide for 1 h at 37°C and washed twice in PBS. 1G4 CTL and T2 cells were washed in PBS and each cell pellet was resuspended to a final concentration of 5 x 105 cells/ml in RPMI. 1G4 CTL and target were mixed 1:1, left for 5 min in suspension, and the plated on glass multiwell slides and incubated for 30 min at 37°C. Cells were fixed in 100% methanol precooled to 20°C, washed in PBS, and blocked in PBS, 2% BSA (Sigma-Aldrich). The slides were mounted in PBS containing 90% glycerol and 2.5% DABCO. Samples were examined using a Bio-Rad Radiance 2000 MP laser scanning microscope and the conjugation rate and granule polarization were quantified using an Axioplan 2 epifluorescent microscope (Zeiss).
Live cell video microscopy.
2 x 104 1G4 CTL were incubated with 60 nM lysotracker green DND-26 (Molecular Probes) for 1 h at 37°C in RPMI, 5% human serum, and washed once in PBS and resuspended in 100 ml RPMI without phenol red, 5% human serum, 1 mM Hepes. T2 target cells were pulsed with 1 µM of either ESO 9C peptide or ESO 9V peptide for 1 h at 37°C and washed twice in PBS. 2 x 104 target cells were allowed to adhere on a glass coverslip mounted in a temperature-controlled chamber for 10 min at 37°C in RPMI without serum, without phenol red plus Hepes. Lysotracker green DND-26labeled 1G4 NYESO-1157165 CTL were added to the T2 cells in the chamber. Sequential confocal images were acquired every 20 s.
CD8-independent ELISA assays for soluble cytokines.
1G4 CTL and pulsed B cell targets were incubated at 37°C for 4 h at an effectortarget ratio of 1:1. After 4 h the supernatant was harvested and assayed for MIP-1ß and IFN- by ELISA (R&D Systems). Standard deviation from the mean of two duplicate assays is shown.
Vaccination of A2Kb mice and ex vivo tetramer staining.
A2Kb transgenic mice were vaccinated using a DNA-prime/recombinant vaccinia virusboost strategy (56). Mice were injected intramuscularly with 50 µg plasmid DNA encoding either ESO 9V or ESO 9C peptide followed by intravenous injection with 5 x 106 plaque forming units recombinant vaccinia virus containing the same antigens on day 10. 10 d after injection, fresh PBL were isolated from blood taken from the tail vein using red cell lysis buffer (Invitrogen). A2Kb tetramer for ex vivo staining were synthesized as previously described (57). Approval of care and use of animals was obtained from the Clinical Medicine Ethical Review Committee (University of Oxford, Oxford, UK) and by the UK Home Office.
Ex-vivo IFN- ELISPOT assay.
ELISPOT analysis for mouse IFN- secretion was performed with cells incubated overnight according to the protocol provided by the manufacturer (Mabtech). Mice splenocytes were generated using the method described above and rested in RPMI supplemented with 10% FCS at 4°C overnight.
In the experiment shown in Fig. 6 D, ESO 9C peptide was treated with 20 mM TCEP at room temperature for 1 h before being added to the wells at the final concentration of 200 µM.
Accession numbers
Atomic coordinates and structure factor amplitudes for the 1G4 TCRESO 9CA2 complex, 1G4 TCRESO 9VA2 complex, and the 1G4 TCR have been deposited in the Protein Data Bank under accession numbers 2BNR, 2BNQ, and 2BNU, respectively.
Online supplemental materials
For surface plasmon resonance, the antibody binding assay shown in Fig. S1, 10 serial dilutions in duplicates of BB7.2 and BBM.1 were made. For live cell video microscopy (Videos 1 and 2) a Nikon TE300 microscope attached to a Bio-Rad Radiance 200 MP laser scanning microscope was used with a 488-nm laser for epifluorescence and Nomarski differential interference contrast for transmitted light. The images were processed using MetaMorph version 4.5 software. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20042323/DC1.
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Acknowledgments |
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E.Y. Jones is a Cancer Research UK Principal Research Fellow. This work was funded by Cancer Research UK (C399-A2291), the US Cancer Research Institute, the UK Medical Research Council, and National Translational Cancer Research Network. G. Held was supported by the German Research Foundation. G. Griffiths is funded by the Wellcome Trust.
The authors have no conflicting financial interests.
Submitted: 12 November 2004
Accepted: 16 February 2005
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