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CORRESPONDENCE Scott R. Burrows: scottb{at}qimr.edu.au OR Jamie Rossjohn: Jamie.rossjohn{at}med.monash.edu.au
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F. Tynan and D. Elhassen contributed equally to this work.
The CD8+ T cell response to an infecting pathogen is generally focused toward a limited subset of antigenic peptides presented on the surface of infected cells. Furthermore, a hierarchy of immunodominance that is maintained in unrelated individuals is often observed between those peptides that are the targets of CTL recognition (1). There appear to be three major factors that control the immunogenicity of a foreign peptide: the specificity of the antigen processing machinery, the peptide binding preferences of MHC class I molecules, and limitations in the diversity of the TCR repertoire (1). How these parameters focus the CTL response toward a limited number of determinants within an antigen is not completely understood.
The dominant factor controlling the magnitude of the CTL response to a foreign peptide is the quantity of peptide presented on the surface of the APC. MHC class I molecules show strict binding specificity because of the high level of polymorphism concentrated in the antigen-binding cleft (2). The pockets (A–F) of the peptide-binding groove vary in their depth, electrostatic potential, and hydrophobicity, thereby determining the individual specificity of the peptide–MHC interaction (3). For most MHC alleles, two of these pockets display a marked preference for one or two amino acids, termed anchor residues, at certain positions within the peptide. For example, the common class I molecule HLA-B*3501 prefers peptide ligands, with proline as a dominant anchor residue at position 2 (P2) and tyrosine (or less commonly phenylalanine, methionine, leucine, or isoleucine) at P
(the COOH terminus) (4, 5). Peptide amino acids at other secondary anchor positions can also influence allele-specific binding (6).
Although immunodominance of antigenic CTL epitopes usually correlates with the abundance of peptide presented on the surface of the APC (7), there have been several reports where this is clearly not the case (8, 9). In these instances, the major factors controlling immunodominance have been proposed to be limitations and bias in the TCR repertoire imposed by thymic or peripheral selection (1, 10–12). There has also been a suggestion that some immunodominant determinants may be intrinsically more immunogenic because of an innate propensity to interact with TCRs, perhaps through the orientation or nature of side chains available for interaction (1); however, no evidence for this theory has been presented to date.
We have defined a CTL epitope (77APQPAPENAY86, referred to as APQP) from the BZLF1 or Z EBV replication activator protein of EBV that binds well to both HLA-B*3501 and HLA-B*3508, two closely related molecules that differ by a single amino acid at position 156 (156Leucine vs. 156Arginine, respectively). This epitope was found to be immunogenic in individuals expressing HLA-B*3508 but, unexpectedly, no response could be detected in HLA-B*3501+ EBV-exposed individuals, indicating that factors other than the level of peptide presentation are influencing immunogenicity. Structural analysis revealed major differences in the peptide conformation when bound to each HLA-B35 allotype but, surprisingly, there were no important differences in the MHC class I heavy chain conformation. These data indicate that T cell responsiveness to a foreign peptide can be influenced by its MHC-bound conformation.
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To determine if other EBV-sero+ HLA-B35+ donors respond to this BZLF1 epitope, CTL lines were raised against the APQP peptide using PBMCs from a panel of such donors to test for reactivity with the peptide. The donors expressed either HLA-B*3501 or HLA-B*3508, two allotypes of the HLA-B35 serotype that diverge by a single amino acid difference at position 156. Surprisingly, this micropolymorphism appeared to have a major impact on responsiveness to the APQP epitope, because no evidence of a response to the peptide could be seen with these cytotoxicity assays in any of eight HLA-B*3501+ donors. In contrast, three donors expressing the much less common HLA-B*3508 responded well to the peptide, as did a donor positive for both HLA-B*3501 and HLA-B*3508 (Fig. 1 C).
Peptide conformation, not peptide binding efficiency, controls the immunogenicity of a viral CTL epitope
To determine if these dramatic differences in responsiveness to this 10-mer epitope in individuals expressing HLA-B*3501 or HLA-B*3508 are related to how well the peptide associates with each HLA-B35 subtype, MHC–peptide binding assays were conducted. These assays used the T2 cell line transfected with either HLA-B*3501 or HLA-B*3508. These antigen-presenting mutant cells express stable class I HLA molecules on their surface upon addition of exogenous HLA binding peptide (15). As positive controls for these experiments, peptides known to be highly immunogenic EBV epitopes in HLA-B*3501+ or HLA-B*3508+ individuals were used for comparison. These included the BZLF1 11-mer EPLPQGQLTAY (13, 14), which is immunogenic in HLA-B*3501+ individuals, and the unusually long 13-mer peptide LPEPLPQGQLTAY was included for HLA-B*3508 (13, 16). An earlier study has shown that this 13-mer epitope binds to HLA-B*3508 more efficiently than the completely overlapping EPLPQGQLTAY 11-mer peptide. Consistent with this observation, EBV-exposed individuals expressing HLA-B*3508 show a strong CTL response to the 13-mer epitope in preference to the overlapping 11-mer peptide (13, 16).
As shown in Fig. 2 A, the APQP peptide stabilized the expression of both HLA-B*3501 and HLA-B*3508 on the surface of T2 cells, indicating efficient binding to each class I molecule. In comparison to the immunogenic 11- or 13-mer BZLF1 epitopes, the APQP 10-mer bound with similar efficiency to both HLA-B*3501 and HLA-B*3508.
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To determine if the T cells that respond to the APQP peptide in HLA-B*3508+ donors have the capacity to recognize the peptide bound to HLA-B*3501, peptide-specific CTL clones were raised from an HLA-B*3508+ donor and screened for recognition of the epitope presented on target cells expressing either HLA-B*3501 or HLA-B*3508 (Fig. 3 A). These peptide dose-response cytotoxicity assays show that five out of five CTL clones from donor SB recognized the peptide several orders of magnitude more efficiently in the context of self–HLA-B*3508 compared with HLA-B*3501, suggesting that the peptide binds with distinct conformations on these closely related HLA molecules. To examine this same issue with a broader range of APQP-specific T cells, multiple CTL microcultures were generated from donors SB (HLA-B*3508+) and MB (HLA-B*3501+ and HLA-B*3508+) by stimulating PBMCs at a limiting dilution with the APQP peptide. On day 13, the microcultures were screened for cross-recognition of the peptide presented by either HLA-B*3501+ or HLA-B*3508+ PHA blast target cells (peptide concentration = 0.02 µg/ml). The data shown in Fig. 3 B are from CTL microcultures raised from responder PBMC densities from which <50% of the wells produced CTL specific for the EBV epitope; thus, most were likely to have been generated from a single peptide-specific CTL. Consistent with data presented in Fig. 3 A, the vast majority of these CTLs recognized the APQP peptide much more efficiently when bound to HLA-B*3508+ target cells compared with HLA-B*3501+ target cells. These data indicate that the HLA-B*3501–APQP and HLA-B*3508–APQP structures are quite distinct targets for T cell recognition.
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release in an ELISPOT assay where the effector T cells were challenged with LCLs expressing HLA-B*3501 and infected with either WT EBV (BZLF1+ LCLs) or a recombinant EBV in which the BZLF1 gene was deleted (BZLF1– LCLs). As shown in Fig. 3 C, the 2D6 CTLs responded strongly to the BZLF1+ LCLs but not the BZLF1– LCLs, demonstrating that the APQP peptide is processed from endogenously expressed antigen and presented by HLA-B*3501 at sufficient levels for T cell activation. This data supports the notion that factors other than peptide presentation levels control the immunogenicity of this viral CTL epitope.
To examine levels of the APQP peptide processed and presented from endogenously expressed viral antigen in HLA-B*3501+ versus HLA-B*3508+ cells, we acid eluted class I–bound peptides from EBV-infected cells expressing each of these HLA alleles and subjected extracts to HPLC fractionation. As mentioned in the previous paragraph, only a small proportion of cells in LCLs express the BZLF1 antigen, which posed a potential problem for these experiments. Because levels vary slightly between different cell lines, we used flow cytometry and an mAb for BZLF1 to test several HLA-B*3501+ and HLA-B*3508+ LCLs from our stored panel, and those expressing the highest levels were selected for further analysis (HLA-B*3501+ LCL, 7.3% BZLF1+; B*3508+ LCL, 3.8% BZLF1+). Ionizing radiation has been shown to increase the amount of viral reactivation in LCLs (18); thus, to ensure that levels of BZLF1 expression were sufficient for the peptide elution experiments, the LCLs were -irradiated (200 rads) and incubated at 37°C overnight before peptide recovery. This procedure nearly doubled the percentage of cells in our cell lines that were positive for BZLF1 expression (HLA-B*3501+ LCL, 12.3% BZLF1+; B*3508+ LCL, 6.5% BZLF1+). Additional control experiments using ELISPOT assays similar to those shown in Fig. 3 C showed that processing and presentation of the APQP peptide were not inhibited by the irradiation step (unpublished data).
The HPLC fractions from extracts from the two cell lines that were predicted to include the APQP peptide (based on a parallel HPLC run with synthetic APQP peptide) were tested at varying dilutions for recognition by an APQP-specific CTL clone (Fig. 4, A and B). To allow an estimate of the concentration of APQP in each fraction, synthetic APQP was tested in parallel for CTL recognition at varying concentrations (Fig. 4 C). Half-maximum lysis was observed with 48 pg/ml of synthetic peptide (Fig. 4 C), and similar levels of lysis were recorded when HLA-B*3501 fraction 32 was used at a dilution of 1:32 (Fig. 4 A), suggesting that the concentration of APQP in this fraction was 
1,540 pg/ml. Similar extrapolation with data from the other fractions allowed an estimate of the APQP concentration in each (Fig. 4 D). The total amount of APQP peptide eluted off each cell line (from 8 x 108 cells) was estimated at 90 pg for HLA-B*3501 and 40 pg for B*3508. Because there were approximately twice as many BZLF1-expressing cells in the original HLA-B*3501+ population, these data suggest that similar levels of the APQP peptide are presented by virus-infected cells expressing these highly homologous HLA alleles, indicating that other factors must control the immunogenicity of this EBV T cell epitope. Further analysis by liquid chromatography–mass spectrometry of the peptide repertoire in fraction 32 from the B*3501+ LCLs confirmed that the APQP 10-mer peptide was present (not depicted).
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/P-C distance in the HLA-B*3508–APQP complex compares closely with that observed in the previously determined HLA-B35 complexes (19, 20). The binding pockets for P2-Pro and P-Tyr, primary anchor residues for HLA-B35, are conserved between this structure and that previously described (20, 21). Positions P4-Pro, P5-Ala, P6-Pro, P8-Asn, and P9-Ala of the peptide are solvent exposed and thus represent possible TCR contact points. Interestingly, only 21.4% of the 10-mer is solvent exposed when bound to HLA-B*3508, compared with 25% for the previously described nonameric HLA-B35 complex. This may be attributed to the apparent secondary anchor residue in the HLA-B*3508–APQP complex, where the P7-Glu side chain points down toward the antigen-binding cleft, forming an H bond with Tyr 74 and salt bridging to Arg 97 and the polymorphic Arg 156. This tethering of the central-bulged region is distinct from several previously determined bulged peptides where the central bulge participates minimally in contacts with the antigen-binding cleft (16, 21).
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root mean square deviation = 0.25 Å2). However, the polymorphism at position 156 greatly affects the conformation of the peptide. Somewhat surprisingly, in the HLA-B*3501–APQP complex, the P7-Glu/Arg 97 salt bridge is maintained despite the loss of the Arg 156 salt bridge. However, it is the loss of the heavy chain interaction with the P3-Gln that considerably affects the peptide conformation. This polymorphism-induced change in peptide interactions culminates in a dramatic shift of residues P4-Pro, P5-Ala, and P6-Pro of the peptide (Fig. 5, C and D). The side chain of P3-Gln within the HLA-B*3501–APQP complex swings upwards, forming H bonds to P5-AlaN and P5-AlaO. To accommodate the new conformation of the P3-Gln, the P4-Pro is pushed away toward the 1 helix of HLA-B*3501. The movement of the P4-Pro also includes a dramatic flip in the main chain carbonyl group P4-Pro peptide main chain, which then results in a marked shift in the main chain conformation of P5-Ala, such that the P5-Ala Cß groups are 6.5 Å apart in the HLA-B*3501 and HLA-B*3508 structures. This change in peptide conformation also moves the P5-Ala and P6-Pro closer toward the 2 helix of HLA-B*3501 (Fig. 5 D) and results in a switch from cis–P6-Pro to trans–P6-Pro. These conformational changes are important enough to drastically reduce recognition of the HLA-B*3501–APQP complex by the majority of T cells raised against HLA-B*3508–APQP.
CTLs specific for the APQP epitope interact with peptide residues that are oriented differently when bound to HLA-B*3501 and HLA-B*3508
To investigate why these peptide structural differences had such a major impact on CTL recognition, three APQP-specific CTL clones raised from the HLA-B*3508+ donor SB were tested for their ability to tolerate single amino acid substitutions throughout the length of the peptide (Fig. 7, A–C). To aid with interpretation of this data, peptide–MHC binding assays were also performed on selected peptides that were not well recognized by any of the CTL clones. These binding assays measured stabilization of HLA-B*3508 molecules on the surface of the T2.B*3508 cell line after exogenous peptide addition, and the concentration of peptide required for half-maximum stabilization of HLA-B*3508 was then calculated (Fig. 7 D). Considering that the primary anchor residues at P2 and P10 were maintained within all the analogues, a surprising number of amino acid substitutions at other positions within the peptide reduced binding to HLA-B*3508. Consistent with the structural analysis, the glutamate at P7 appears to be a particularly important secondary anchor residue for this peptide because most substitutions at this position resulted in reduced MHC binding.
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Discussion |
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Our data demonstrate that a single residue polymorphism between HLA-B*3501 and HLA-B*3508 controls responsiveness to the APQP epitope through a mechanism unrelated to peptide–MHC binding efficiency/stability (Fig. 2). Furthermore, HLA-B*3501+, EBV-infected lymphoblasts clearly present the APQP epitope from endogenously expressed antigen with similar efficiency to HLA-B*3508+ LCLs (Fig. 3 C and Fig. 4), ruling out differences in epitope processing within EBV-infected cells. It should be noted, however, that the response to this epitope could be initiated by cross-priming through dendritic cells that could process and present a different repertoire of EBV peptides compared with infected B cells. Nonetheless, direct stimulation by EBV-infected B cells is likely to have the major role in restimulating and maintaining these T cell populations during persistent infection. We also consider it highly unlikely that polymorphic antigen processing genes are influencing this response in our study subjects because our HLA-B*3501+ donors were all unrelated and the HLA-B*3508+ donors were from two unrelated families from different ethnic groups.
Another factor with the potential to influence the immunogenicity of a T cell epitope is immunodomination whereby the T cell response to an immunodominant determinant suppresses the response to another epitope (26, 27). It was therefore feasible that HLA-B*3501, but not HLA-B*3508, presents another unknown EBV epitope that is highly immunogenic, thereby suppressing the response to APQP in HLA-B*3501+ individuals. Arguing against this possibility are data presented in Fig. 1 C and Fig. 3 B showing that the HLA-B*3501/-B*3508–coexpressing donor MB responds to the APQP epitope.
The potential role of peptide conformation in controlling the differential responsiveness to this epitope became apparent after functional assays demonstrated that APQP-specific CTL clones from an HLA-B*3508+ donor were unable to recognize this peptide efficiently in the context of HLA-B*3501 (Fig. 3 A). Thus, although these two closely related HLA molecules present this identical peptide sequence very efficiently, they appear to be structurally distinct from the perspective of many T cells. This conclusion was strengthened by our analysis of multiple CTL microcultures, most of which recognized the peptide much more efficiently when presented on HLA-B*3508+ target cells compared with HLA-B*3501+ targets. It is notable that one of the donors used for this analysis coexpressed HLA-B*3501 and HLA-B*3508, yet there was no indication that T cells had been selected for the ability to cross-recognize the peptide on each of these HLA molecules (Fig. 3 B). Subsequent structural analysis confirmed major conformational differences between the peptide bound to HLA-B*3501 and HLA-B*3508 resulting from direct peptide interactions with the polymorphic MHC residue (Fig. 5 and Fig. 6). Furthermore, the conformational differences were particularly striking at highly exposed peptide residues that were shown to be important TCR contact residues for three out of three APQP-specific CTL clones (Fig. 7).
There are several possible mechanisms through which such peptide conformational differences could influence immunogenicity. It is possible that thymic and postthymic selection influence the relative number of naive T cells with the capacity to recognize each complex. For example, there may be an abundantly presented self-peptide that binds to both HLA-B*3501 and HLA-B*3508 that negatively selects CTLs with the potential to recognize the HLA-B*3501–bound conformation of the viral peptide or that positively selects for T cells that recognize the viral peptide presented by HLA-B*3508 but not HLA-B*3501. Alternatively, certain self-peptides may be presented preferentially by HLA-B*3508 that positively select T cells specific for HLA-B*3508–APQP (11, 12). In any case, the unique conformation of the APQP peptide when bound to HLA-B*3508 appears to be critical for recognition by most of the selected T cells and is, therefore, a major controlling influence over the immunogenicity of this EBV epitope.
Another possible explanation for our data, unrelated to TCR repertoire differences between HLA-B*3501+ and HLA-B*3508+ individuals, is that the conformation of this EBV epitope on HLA-B*3508 is intrinsically more immunogenic through structural features that enable it to interact favorably with a higher frequency of TCRs (1). It is now clear that certain antigenic peptides present challenging target structures for TCR recognition and are recognized by a very limited TCR repertoire (16, 28–30). It is therefore not unreasonable to expect that some peptide conformations will present docking surfaces complementary to a higher proportion of the TCR repertoire compared with others. Although it is impossible to decipher the exact mechanisms underlying the phenomenon, this study clearly demonstrates that peptide conformation can have a dramatic impact on the immunogenicity of an MHC–peptide complex and could therefore play a major role in controlling determinant selection and immunodominance in other T cell responses.
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Materials and Methods |
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CTL cultures.
CTL clones were generated by agar cloning as previously described (10) after initial stimulation of PBMCs for 1 h with 0.1 µM of the APQP peptide. Clones were maintained with biweekly restimulation with rIL-2 and the -irradiated (8,000 rads) autologous LCLs that had been prelabeled with the APQP peptide at 0.1 µM for 1 h and washed three times. Short-term CTL bulk cultures were also used as effectors in cytotoxicity assays. These were generated by culturing 2 x 106 PBMCs/2-ml well in growth medium with autologous PBMCs that had been precoated for 1 h with 0.1 µM of the APQP peptide (responder/stimulator = 2:1). Cultures were supplemented with 20 U/ml rIL-2 on day 3, split on day 7, and analyzed on day 10. CTL cultures were tested in duplicate or triplicate for cytotoxicity in the standard 5-h chromium release assay. In brief, CTLs were assayed against 51Cr-labeled LCL or PHA blast targets that were pretreated with synthetic peptide (Mimotopes Ltd.) or left untreated. The mean spontaneous lysis for target cells in culture medium was always <20%, and the variation from the mean specific lysis was <10%.
Short-term CTL microcultures were generated by limiting dilution as follows: PBMCs were distributed in roundbottom microtiter plates in growth medium at cell numbers ranging from 103 to 4 x 104 cells/well. Approximately 5 x 104 -irradiated (2,000 rads) autologous PBMCs that had been preincubated for 1 h with 1 µM of the APQP peptide were added to each well to give a total volume of 100 µl. Cultures were fed on days 4, 7, and 10 with 50 µl of medium supplemented with 20 U of rIL-2 and 30% supernatant from MLA-144 cultures. On day 13, each CTL microculture was split into multiple replicates and used as effectors in a standard 5-h 51Cr-release assay against target PHA blasts that had been treated with the APQP peptide or left untreated.
HLA class I peptide binding assays.
To assess peptide binding to the different HLA-B35 subtypes, T2.B*3501 and T2.B*3508 cells were incubated in AIM V serum-free medium (Invitrogen) with various concentrations (0.01, 0.1, 1, 10, and 100 µM) of peptides at 26°C for 14–16 h, followed by incubation at 37°C for 2–3 h and staining for HLA-B35 surface expression. To determine peptide dissociation rates, T2.B*3501 cells were pulsed with 100 µM of each peptide at 26°C for 14–16 h, followed by incubation at 37°C for 2–3 h. Cells were then either stained immediately for HLA-B35 surface expression (0 h) or were washed three times and incubated at 37°C for 1–6 h before staining. HLA-B35 surface expression was measured by a flow cytometer (FACSCalibur; Becton Dickinson) using an mAb to HLA-Bw6 (SFR8 Bw6). Data were expressed relative to the mean fluorescence intensity (MFI) measured using 100 µM of a reference peptide that was known to bind to HLA-B*3501 or HLA-B*3508, using the following formula: ([MFI with test peptide – MFI without peptide addition] x 100) / (MFI with reference peptide [at 100 µM] – MFI without peptide addition).
ELISPOT assays.
IFN- ELISPOT assays were performed using cytokine capture and detection reagents according to the manufacturer's instructions (Mabtech). In brief, anti–IFN- antibodies were coated on the wells of a 96-well nitrocellulose plate, and duplicate wells were seeded with 1,000 CD8+ T cells and 50,000 target cells. Two LCLs raised from an HLA B*3501+ individual were used as target cells in these experiments: one carried a WT B95.8 virus genome (BZLF1+ LCL), and the other carried a B95.8 genome that had been rendered incapable of lytic cycle entry by disruption of the BZLF1 gene (BZLF1– LCL; a gift from A. Hislop and A. Rickinson, University of Birmingham, UK) (17). After incubation for 16 h, captured IFN- was detected with a biotinylated anti–IFN- antibody followed by development with streptavidin–horseradish peroxidase complex and chromogenic substrate, and spots were counted using an automated plate counter (AID).
Expression, purification, and crystallization of HLA-B35 alleles with APQPAPENAY.
Soluble HLA-B*3501 and HLA-B*3508 molecules (residues 1–276) and full-length ß2-microglobulin (residues 1–99) were expressed, refolded with the APQP peptide, purified, and concentrated to 10 mg/ml as previously described (33). Crystals were obtained by the hanging drop vapor diffusion technique. Block-shaped crystals grew within 4 d in a condition containing 0.2 M ammonium acetate and 16% wt/vol PEG 3350 (100 mM cacodylate, pH 7.6) at 4°C.
X-ray data collection and structure determination.
Crystals were soaked in reservoir solution containing increasing increments of glycerol as a cryoprotectant (5, 10, and 15%) and then flash frozen before data collection. Data were collected on an in-house radiation source and was processed and scaled using the HKL suite (34).
The HLA-B35 complex structures were refined from an HLA-B*3501 structure that was previously determined in our laboratory (unpublished data). The model was manually built using the program "O" (35) and improved through multiple rounds of refinement using the CNS suite (36). The progress of refinement was monitored by Rfactor and Rfree values. Rigid-body refinement and simulated annealing were used in the first instance, but in later rounds energy minimization and B-individual refinement were used to improve the quality of the model. See Table I for the final refinement and model statistics. The structures have been deposited in the Protein Data Bank under accession no. 2AXF (HLA-B*3508–APQP) and 2AXG (HLA-B*3501–APQP).
Purification of HLA complexes and peptide analysis.
Purification of HLA molecules was performed from 8 x 108 HLA-B*3501+ LCLs (donor MW: HLA A1, A3, B8, and B*3501) and HLA-B*3508+ LCLs (donor CA: HLA A30, A32, B42, and B*3508) that had been irradiated (200 rads) and incubated at 37°C overnight to enhance BZLF1 expression (18). These LCLs were selected for these experiments on the basis of relatively high levels of BZLF1 expression as measured using flow cytometry and a mouse mAb to BZLF1 (BZ.1). Peptides were recovered from cell lysates after treatment with 0.5% TFA and acid extraction from the cell pellet as described elsewhere (37). After ultrafiltration (3-kD cutoff) and preconcentration, peptides were collected in 50-µl fractions as described previously (38). Peptides were separated by RP-HPLC using a SMART system HPLC (GE Healthcare) with a vydac C18 column (1 mm [inside diameter] x 25 cm) and eluted using an optimized linear gradient of acetonitrile in aqueous 0.09% TFA. Peptide fractions were used in chromium release assays to sensitize HLA-B*3508+ LCL target cells for lysis by a CTL clone from the B*3508+ donor SB. In brief, 5 µl of each undiluted or diluted fraction was added to 15 µl of target cells for 90 min before CTL addition. To test for toxicity, each fraction was also added to chromium-labeled target cells without CTL addition, but no toxicity was detected, even at the lowest dilution (1:4; unpublished data). HLA-B*3501 fraction 32 was also characterized by liquid chromatography–mass spectrometry using an Ultra ion trap mass spectrometer coupled to an 1100 nanoLC (both Agilent Technologies) as described previously (38, 39) and found to contain the APQP 10-mer peptide after MS/MS fragmentation of the [M+2H]2+ species of target mass m/z = 529.2 D (unpublished data).
Online supplemental material.
Fig. S1 shows an alternate conformation of the polymorphic residue 156 in the HLA-B*3508–APQP complex. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20050864/DC1.
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Acknowledgments |
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This work was supported by grants from the Australian National Health and Medical Research Council (NHMRC), the Roche Organ Transplantation Research Fund, the Juvenile Diabetes Research Foundation, and the Australian Research Council. S.R. Burrows is a recipient of an NHMRC Career Development award, and J. Rossjohn is a Wellcome Trust Senior Research Fellow.
The authors have no conflicting financial interests.
Submitted: 2 May 2005
Accepted: 2 September 2005
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