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The Journal of Immunology, 2002, 169: 5098-5108.
Copyright © 2002 by The American Association of Immunologists

The HLA Molecules DQA1*0501/B1*0201 and DQA1*0301/B1*0302 Share an Extensive Overlap in Peptide Binding Specificity1

John Sidney, Marie-France del Guercio, Scott Southwood and Alessandro Sette2

Epimmune, Inc., San Diego, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Assays to measure the binding capacity of peptides for HLA-DQA1*0501/B*0201 (DQ2.3) and DQA1*0301/B*0302 (DQ3.2) were developed using solubilized MHC molecules purified from EBV-transformed cell lines. These quantitative assays, based on the principle of the inhibition of binding of a high-affinity radiolabeled ligand, were validated by examining the binding capacity of known DQ-restricted epitopes or ligands. The availability of these assays allowed an investigation of patterns of cross-reactivity between different DQ molecules and with various common DR molecules. DQ2.3 and DQ3.2 were found to have significantly overlapping peptide binding repertoires. Specifically, of 13 peptides that bound either DQ2.3 or DQ3.2, nine (69.2%) bound both. The molecular basis of this high degree of cross-reactivity was further investigated with panels of single substitution analogs of the thyroid peroxidase 632–645Y epitope. It was found that DQ2.3 and DQ3.2 bind the same ligands by using similar anchor residues but different registers. These data suggest that in analogy to what was previously described for HLA-DR molecules, HLA-DQ supertypes characterized by largely overlapping binding repertoires can be defined. In light of the known linkage of both HLA-DQ2.3 and -DQ3.2 with insulin-dependent diabetes mellitus and celiac disease, these results might have important implications for understanding HLA class II autoimmune disease associations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Class II HLA-DQ molecules are {alpha}/{beta} (A/B) transmembrane heterodimers encoded within the MHC. Both the DQA and DQB chains are highly polymorphic. Approximately 20 different DQA chains and 40 different DQB chains have been sequenced to date (1). Because DQ specificities that are identical on the basis of serology may be composed of different A and B chains when associated with different DR haplotypes, a heterozygous individual can potentially express four distinct HLA-DQ molecules.

Based on studies in streptococcal and leprosy disease systems, it has been suggested that DQ molecules may play a role in immnosupression, as opposed to the helper function of DR molecules (2, 3, 4, 5, 6). However, in many other systems, Ag-specific DQ-restricted T cells can be found (6). In either case, it is apparent that DQ alleles are of immunological relevance.

The ability to measure the capacity of peptides to bind various common HLA-DQ molecules is a potentially valuable component in the characterization of protein-based products intended for human use. For example, in some cases it may be important to identify DQ-restricted epitopes or analogs to allow engineering of products, such as vaccines, with increased immunogenicity. Alternatively, it may be necessary to identify peptides, or peptide analogs, with reduced DQ binding capacity as a means of reducing the potential immunogenicity of candidate products.

Two common HLA DQ molecules, DQA1*0501/DQB1*0201 (DQ2.3)3 and DQA1*0301/DQB1*0302 (DQ3.2), are of particular interest because their expression has been associated with the human autoimmune disorders type 1 diabetes and celiac disease (7, 8, 9, 10, 11), respectively. The molecular basis for association between autoimmune disease and HLA molecules has been the subject of considerable debate. The peptide-binding capacity of a given class II MHC molecule has been hypothesized to play a causal role by allowing the binding of autoimmune epitopes and/or influencing the repertoire of peptides available during positive and negative selection. At the structural level, the association of a specific class II molecule with resistance to diabetes has been mapped to the presence of an aspartic acid residue (D) in position 57 of the class II {alpha} chain. Molecules associated with the presence of a noncharged serine, valine, or alanine residue at the same position confer susceptibility (12, 13). For example, DQ3.2, associated with susceptibility to type I diabetes, has serine in position 57. The same position is occupied by D in the closely related molecule DQA1*0301/B*0301 (DQ3.1), for which no association with diabetes predisposition has been reported. Interestingly, D is absent in position 57 of IAg7, the only class II molecule expressed by nonobese diabetic mice associated with a high incidence of spontaneous development of diabetes (14). Recently, the crystal structures of DQ3.2 and IAg7 have been reported (15, 16, 17, 18, 19, 20).

In terms of peptide-binding repertoires, peptide-binding assays and the sequencing of pools of eluted ligands have allowed definition of tentative motifs for DQ1, DQ2.3, DQ3.1, and DQ3.2 (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33). These experiments have also yielded the sequences of several endogenously processed ligands for various DQ molecules. Previous work in the HLA-DR system demonstrated that several different DR molecules are associated with largely overlapping peptide-binding repertoires and therefore can be grouped in at least two main DR supertypes (34). Until now, however, no quantitative data has been available regarding the degree, if any, of overlap in peptide-binding repertoires (cross-reactivity) among different DQ molecules and between DQ and DR molecules.

In the present report, the DQ2.3, DQ3.1, and DQ3.2 binding capacity of panels of DR- and DQ-restricted epitopes was determined. A surprisingly large degree of overlap between the peptide-binding repertoires of the DQ3.2 and DQ2.3 molecules was revealed. The basis for this high degree of overlap in peptide-binding repertoires was probed further by testing in parallel the DQ2.3 and DQ3.2 binding capacity of a large panel of single-substitution analogs of the thyroid peroxidase 632–645 epitope.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and Abs

The EBV-transformed homozygous cell lines shown in Table IGo were used as sources of purified MHC molecules (23, 29, 35, 36, 37). The HLA-DR and DQ types of each cell line were derived from the literature, as indicated in the World Health Organization panel of EBV-transformed homozygous cell lines (database provided by Dr. D. Mann, University of Maryland, College Park, MD) or as indicated online at the ImMunoGeneTics project Database (www.ebi.ac.uk/imgt/hla/). In most cases, HLA-DR specificity was also verified using high-resolution PCR typing performed by the Indiana University Histocompatibility Laboratory (Bloomington, IN). Cells were maintained in vitro by culture in RPMI 1640 medium (Flow Laboratories, McLean, VA) supplemented with 2 mM L-glutamine (Life Technologies, Grand Island, NY), 100 U (100 µg/ml) penicillin-streptomycin solution (Life Technologies), and 10% heat-inactivated FCS (Hazleton Biologics, Lenexa, KS). Large-scale cultures were maintained in roller bottles.


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  		Table I. Cell lines used as sources of purified HLA-DQ and -DP molecules

 
The Abs used for purification of MHC class II molecules are listed in Table IIGo (38, 39, 40, 41, 42). The HLA-DR-{alpha}-chain-specific mAb LB3.1 was provided by Dr. J. Gorga (Suntory Pharmaceutical Research Laboratories, Cambridge, MA). The mAb SPVL-3, specific for HLA-DQ, was provided by Dr. G. Nepom (Virginia Mason Research Center, Seattle, WA). Other Abs were obtained from the American Type Culture Collection (Manassas, VA). Purified Abs were produced from specific hybridoma cultured in roller bottles or injected in mice for ascites production. Culture supernatant or ascites were precipitated with ammonium sulfate and were purified using protein A-Sepharose CL4B. Purified Abs were covalently coupled to protein A-Sepharose CL4B at a concentration of 3 mg/ml.


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  		Table II. Monoclonal Abs

 
MHC purification

Class II molecules were purified from cell lysates as previously described (43). Briefly, cells were lysed at a concentration of 108 cells/ml in 50 mM Tris-HCl, pH 8.5, containing 1% (v/v) Nonidet P-40, 150 mM NaC1, 5 mM EDTA, and 2 mM PMSF. Alternatively, 1% Renex was used instead of Nonidet P-40. Lysates were passed through 0.45-µm filters and cleared of nuclei and debris by centrifugation at 10,000 x g for 20 min. MHC molecules were purified by mAb-based affinity chromatography.

For affinity purification, columns of inactivated Sepharose CL4B and protein A-Sepharose were used as pre-columns. Lysates were initially depleted of HLA-DR molecules by repeated passage over protein A-Sepharose beads conjugated with the LB3.1 (anti-HLA-DR A chain) mAb. DQ molecules were subsequently captured by passage over Protein A-Sepharose beads conjugated with either the anti-HLA-DR/DQ mAb HB180 or the anti-DQ mAb SPVL-3. In each case, after two to four passages of lysate, the specific columns were washed with 10-column volumes of 10 mM Tris-HCl, pH 8, with 1% (v/v) Nonidet P-40, two-column volumes of PBS, and two-column volumes of PBS containing 0.4% (w/v) n-octylglucoside. Class II molecules were eluted from the columns with 50 mM diethylamine in 150 mM NaCl containing 0.4% (w/v) n-octylglucoside, pH 11.5. A 1/100 volume of 2 M glycine, pH 2.5, was added to the eluate to reduce the pH to ~8. The eluate was then concentrated by centrifugation in Centriprep 30 concentrators at 2000 rpm (Amicon, Beverly, MA). Protein purity, concentration, and effectiveness of depletion steps were monitored by SDS-PAGE and bicinchoninic acid assay.

Peptide synthesis and radiolabeling

Peptides used were synthesized at Epimmune (San Diego, CA) as described elsewhere (44) or were purchased as crude material from Mimotopes (Minneapolis, MN). Peptides synthesized at Epimmune were typically purified to >95% homogeneity by reverse-phase HPLC. Purity of Epimmune-synthesized peptides was determined using analytical reverse-phase HPLC and amino acid analysis, sequencing, and/or mass spectrometry. Lyophilized peptides were resuspended at 4–20 mg/ml in 100% DMSO and then diluted to required concentrations in PBS 0.05% (v/v) Nonidet P-40 (Fluka Biochemika, Buchs, Switzerland). Peptides were 125I-radiolabeled using chloramine T methodology, as previously described (43).

Peptide-binding assays

Quantitative assays to measure the binding of peptides to soluble class II molecules are based on the inhibition of binding of a radiolabeled standard peptide. Binding assays were performed essentially as described previously (43). Briefly, 1–10 nM radiolabeled peptide was coincubated for 2 days at either room temperature or 37°C with 1 µM to 1 nM purified MHC in the presence of a cocktail of protease inhibitors. Assays were performed at various pH conditions, ranging from pH 4 to pH 7. The final pH of assay mixtures was adjusted using citrate buffer as described elsewhere (43).

After incubation, the percentage of MHC-bound radioactivity was determined by size exclusion gel filtration chromatography using a TSK 2000 column (Toso Haas, Montgomeryville, PA). Alternatively, the percentage of HLA-DR-bound radioactivity was determined by capturing MHC/peptide complexes on Optiplates (Packard Instrument, Meriden, CT) coated with the LB3.1 mAb and determining bound cpm using the TopCount microscintillation counter (Packard Instrument).

In preliminary assays, candidate labels were tested for direct binding to purified MHC molecules. The amount of MHC yielding 10–20% bound radioactivity was used in subsequent inhibition of binding assays in which the concentration of peptide yielding 50% inhibition of the binding of the radiolabeled peptide was calculated. Under the conditions used, in which [label] < [MHC] and IC50 >= [MHC], the measured IC50 values are reasonable approximations of the true Kd values. Each competitor peptide was tested in two to four independent experiments. As a positive control, the unlabeled version of the radiolabeled probe was also tested in each experiment. Peptides were initially tested at one or two high doses. The IC50 of peptides yielding positive inhibition were then determined in subsequent experiments, in which two to six further dilutions were tested. As in previous studies, peptides with an affinity for a specific class II molecule of 1000 nM or better are defined as binders for the respective molecules (34). The radiolabeled standard peptide used for each assay and its respective IC50 nanomolars are listed in Table IIIGo.


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  		Table III. HLA-DQ and DR binding assays

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment of an HLA DQ2.3 peptide binding assay

Previously reported DQ2 epitopes, natural DQ2 ligands, or DQ2 binding peptides were used to establish HLA-DQ2.3 assays. Various analogs of these candidate ligands were also designed to allow for radiolabeling. Optimal binding of a tyrosinated analog of the DQ2 natural ligand MHC Ia 50–63 (21, 22) (MHC Ia Y50–63; sequence YPFIEQEGPEFFDQE) to DQ2.3 molecules was obtained at pH 4.5 with incubation for 2 days at 37°C (Fig. 1GoA). In optimal conditions, as little as 15 nM MHC was required to obtain 15% binding of the input radioactivity (Fig. 1GoB).



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  		FIGURE 1. Development of a DQ2.3 peptide-binding assay. A, Binding of MHC Ia Y50–63a and the Y7>F analog of the DR3 epitope MT 65-kDa 3–13 (sequence YKTIAFDEEARR) (45 46 47 ) to purified DQ2.3 molecules. Binding is shown as a function of final assay pH for a fixed concentration (0.2 mg/ml) of DQ molecules. Assays were performed at 37°C as described in Materials and Methods. DQ2.3 was purified from lysates of the EBV-transformed homozygous cell line MAT using the DR/DQ mAb HB180 as described in Materials and Methods. Similar data were obtained using DQ2.3 molecules purified from the EBV-transformed homozygous cell line STEINLIN using the DQ-specific mAb SPVL-3 (data not shown). B, Direct binding of MHC Ia Y50–63a to various concentrations of DQ2.3 molecules purified from MAT. Final assay pH was 4.5. C, Inhibition of the binding of MHC Ia Y50–63a to purified DQ2.3 molecules. Binding of MHC Ia Y50–63a was inhibited using excess amounts of unlabeled MHC Ia Y50–63a. In addition, the capacity of the DR3 high-affinity binder MT 65-kDa 3–13 and the DQ2 binder Mycobacterium bovis 65-kDa 243–255Y to inhibit the binding of MHC Ia Y50–63a was measured.

 
In the experiments above, DQ molecules were purified using the HLA DR/DQ-specific mAb HB180 and cell lysates of the MAT cell line previously depleted of DR molecules using the DR-specific mAb LB3.1. Similar binding data were observed using a different DQ2.3+ cell line (STEINLIN) or a different mAb (SPVL-3; DQ specific) (data not shown). Both MAT and STEINLIN cells are homozygous for HLA-DR3. Accordingly, functional specificity controls were performed using the Mycobacterium tuberculosis (MT) 65-kDa Y3–13 peptide (sequence YKTIAFDEEARR). This peptide is a good DR3 binder, also binding optimally at pH 4.5 (45, 46, 47). No binding of MT 65-kDa Y3–13 to DQ2.3 preparations was observed under any of the conditions examined (Fig. 1GoA). Conversely, no binding of MHC Ia Y50–63 to DR3 molecules purified from either MAT or STEINLIN was observed (data not shown). These data demonstrate that the binding detected in the case of MHC Ia Y50–63 could be attributed to DQ2.3 and was not due to the presence of contaminating amounts of DR3.

In inhibition of binding experiments, the binding of MHC Ia Y50–63 was readily inhibited by an excess of unlabeled peptide, with IC50 in the 10–30 nM range (Fig. 1GoC). The MT 65-kDa 243–255 peptide (sequence KPLLIAEDVEGEY), previously identified as a high-affinity DQ2 binder (22, 23, 48), also inhibited efficiently, with an IC50 in the 40–60 nM range. In contrast, the high-affinity DR3 binder MT 65-kDa Y3–13 inhibited only weakly and ~100-fold less well than did MHC Ia Y50–63, with an IC50 in the 1500–3000 nM range. In this context, it is important to point out that throughout this study, 1000 nM is used as the cutoff value to denote biologically significant affinity of binding. The use of this criterion is based on previous work that examined the distribution of binding affinities of known epitopes recognized by human T cells (34). Specifically, it was noted that 31 of 32 (97%) known epitopes bound their restricting element with an IC50 of 1000 nM or better.

As shown in Table IVGo, five of six (83%) peptides previously identified as DQ2 ligands (21, 22, 23, 48, 49) bound with affinities of 300 nM or better. In contrast, only 4 of 14 (29%) peptides identified as ligands for other DQ molecules (23, 24, 28, 29, 30, 45, 49, 50, 51, 52, 53) and 1 of 4 (25%) peptides previously identified as DR3 binders also bound DQ2.3 with and IC50 <= 1000 nM. Finally, four of four (100%) of the known DR3 epitopes (45) bound DR3 with an IC50 less than 250 nM, but only 1 of 20 (5%) DQ epitopes bound DR3 with an IC50 < 1000 nM.


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  		Table IV. HLA DQ2.3 binding capacity of a panel of HLA-DQ and -DR epitopes

 
Establishment of an HLA DQ3.2 peptide binding assay

As in the case of DQ2.3, candidate HLA-DQ3.2 ligands were radiolabeled and tested for their capacity to bind DQ3.2 molecules at both room temperature and 37°C and at pHs ranging from 4 to 7. HA 255–271Y (sequence FESTGNLIAPEYGFKISY), previously used by Straumfors et al. (24) as a ligand for DQ3.2 assays, bound at pH 4, but only poorly at higher pHs (Fig. 2GoA). The best binding was detected with a C-terminal E>Y analog of the DQ3.2 binder CD20 249–262 (26) (peptide CD20 249–262 E262>Y; sequence EEDIEIIPIQEEEY). Again, optimal binding was found at pH 4, after incubation for 2 days at 37°C. Good binding of CD20 249–262 E262>Y was observed with DQ3.2 molecules purified from the 145b (Fig. 2GoA), YAR, or PREISS cell lines (data not shown). Under optimal conditions, 15% binding of the input radioactivity was obtained with ~70 nM MHC (Fig. 2GoB).



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  		FIGURE 2. Development of a DQ3.2 peptide-binding assay. The CD20 249–262 and HA 255–271 peptides were previously described as DQ3.2 binders (24 26 ). Peptide 717.01 is a non-natural sequence that is used as the radiolabeled ligand for HLA-DR4w4, DR4w14, and DR4w15 binding assays (34 ). A, Binding of CD20 249–262 E262>Y and HA 255–271 to purified DQ3.2 molecules. Binding is shown as a function of final assay pH using a fixed concentration (0.2 mg/ml) of DQ molecules. Binding assays were performed at 37°C as described in Materials and Methods. DQ3.2 molecules were purified from the EBV-transformed homozygous cell line 145b using the DR/DQ mAb HB180 as described in Materials and Methods. Similar data were obtained using DQ3.2 molecules purified from the EBV-transformed homozygous cell lines YAR and PREISS (data not shown). B, Direct binding of CD20 249–262Y to various concentrations of DQ3.2 molecules purified from 145b. Final assay pH was 4.0. C, Inhibition of the binding of peptide CD20 249–262Y to DQ3.2 molecules purified from the 145B cell line. Binding of CD20 249–262Y was inhibited using excess amounts of unlabeled CD20 249–262Y. In addition, the capacity of the DQ3.2 high-affinity binder HA 255–271Y and the pan-DR4 binder 717.01 to inhibit CD20 249–262Y binding was measured.

 
Each of the cell lines used to isolate DQ3.2 molecules is homozygous for HLA-DR4. To demonstrate that the binding of CD20 249–262 E262>Y was not due to the presence of contaminating amounts of DR4, the high-affinity and degenerate DR4 (DR4w4, DR4w10, DR4w14, and DR4w15) binding peptide 717.01 (sequence YARFQSQTTLKQKT) (34, 54) was tested for its capacity to bind the same DQ3.2 preparations. No binding of 717.01 could be detected (see Fig. 2GoA), demonstrating that significant amounts of contaminating DR4 molecules were not present in the DQ3.2 preparations used. Conversely, CD20 249–262 E262>Y did not bind to purified DR4 molecules (DR4w4, DR4w14, and DR4w15) in direct binding assays (data not shown) and was found to be a poor inhibitor of DR4w4 and DR4w14 binding in competition assays (see Table VGo).


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  		Table V. HLA DQ3.2 binding capacity of a panel of HLA-DQ and -DR epitopes

 
Binding to DQ3.2 of CD20 249–262 E262>Y was readily inhibited using excess amounts of unlabeled peptide, with an IC50 in the 10–30 nM range. The natural DQ3.2 ligand thyroid peroxidase 632–645 (24, 48) (sequence IDVWLGGLAENFLPY) was also a good inhibitor, with an affinity in the 25–50 nM range. In contrast, the pan-DR4 binder 717.01 failed to inhibit CD20 249–262 E262>Y binding at doses up to 12,000 nM (Fig. 2GoC). When a larger panel of peptides was assayed, it was found that 9 of 13 (69.2%) of the peptides previously identified as DQ3.2 ligands bound DQ3.2 with affinities of 1000 nM or better (Table VGo) (24, 26, 28, 33, 37, 49, 50, 52, 53, 55). Six of 12 (50%) reported ligands for other DQ molecules (21, 23, 27, 29, 30, 48, 49) also bound DQ3.2. In contrast, a binding affinity of 1000 nM or better was observed in 15 of 39 (38.5%) instances in which a known DQ3.2 binder was tested for its capacity to bind to the three different DR4 molecules tested. Finally, another pan-DR4 binder peptide (717.10) (54, 56) failed to bind DQ3.2.

HLA-DQ/DQ and HLA-DQ/DR cross-reactivity

Next, we sought to investigate in more detail the extent of overlap in the peptide-binding repertoires of different DQ and DR molecules. For these analyses, an arbitrary panel of DQ-binding peptides was assembled. This panel incorporates peptides shown in Tables IVGo and VGo and includes natural ligands of DQ2.3 and DQ3.2, but also for DQ1 and DQ3.1. Peptides previously identified as DR-restricted epitopes or high-affinity ligands were also included as controls.

This panel was then tested for its capacity to bind various DQ and DR molecules. The DQ molecules tested were DQ2.3 and DQ3.2, as described above, and DQ3.1, for which development of a peptide binding assay has been previously described (27). The DR molecules tested included those in linkage disequilibrium with the DQ alleles under investigation (DR3, DR4w4, DR4w14, DR7, and DR8w2). Binding to DR1, which is in linkage disequilibrium with DQ1 (DQB*05/06), was also tested because this molecule is a prototypical HLA-DR-supertype specificity. The data obtained are shown in Table VIGo. The frequency of cross-reactivity between different molecule pairs is summarized in Table VIIGo.


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  		Table VI. Cross-reactive binding between HLA-DQ and -DR alleles

 

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  		Table VII. Cross-reactivity between HLA-DQ and HLA-DR molecules

 
A high degree of cross-reactivity was noted between DQ2.3 and DQ3.2. Of the 21 peptides tested for both DQ2.3 and DQ3.2 binding, nine (42.9%) bound both molecules with an IC50 better than 1000 nM. It was also noted that eight other peptides bound neither DQ2.3 nor DQ3.2. Thus, concordant results were obtained in 17 of 21 cases (81.0%; p < 0.01). Of the 13 peptides that bound either molecule, nine (69.2%) bound both. This high degree of cross-reactivity is even higher than that detected among various DR-supertype molecules such as DR1, DR4w4, DR4w14, and DR7 (average 39.5%; data not shown).

A much less striking degree of cross-reactivity was noted between DQ3.1 and either DQ2.3 or DQ3.2, indicating that extensive repertoire overlap is not a generalized feature of DQ molecules. Specifically, it was found that 25% of the peptides that bound either DQ3.1 or DQ2.3 bound both molecules. Similarly, 31.6% of the peptides with the capacity to bind either DQ3.1 or DQ3.2 bound both. Interestingly, five of eight (62.5%) peptides that bound DQ3.1 and that were tested for DQ2.3 and DQ3.2 binding capacity bound both DQ2.3 and DQ3.2. Thus, most of the peptides that bind DQ3.1 also bind DQ2.3 or DQ3.2, whereas only a minority of the peptides binding DQ2.3 or DQ3.2 also bound DQ3.1. These data suggest that DQ molecules may, in general, recognize similar features in their peptide ligands, and that DQ2.3 and DQ3.2 molecules bind a broader repertoire of peptides that do DQ3.1 molecules.

The frequency of cross-reactivity among DQ2.3, DQ3.1, or DQ3.2 and various DR molecules was found to range between 7.1 and 40%, with an average of 23.9%. The lowest levels of DQ/DR cross-reactivity (7–13%) were observed between DR3 or DR8w2 and DQ2.3 or DQ3.2. These observations are of potential interest in that DR3 and DR8 are in linkage disequilibrium with DQ2.3 and DQ3.2 (57, 58), respectively, and may suggest that DQ/DR linkage disequilibrium, in some cases, could reflect selection for DQ/DR pairs with complementary specificity. Relatively low levels of cross-reactivity were also noted between DQ3.1 and DR4 molecules, which are in linkage disequilibrium, where only 15.8 and 16.7% of the peptides were cross-reactive between DQ3.1 and DR4w4 and DR4w14, respectively.

DQ molecules bind the same peptide using different residues

The basis for the remarkable degree of cross-reactivity noted between DQ2.3 and DQ3.2 was examined next. For this analysis, a large panel of single-substitution analogs of the degenerate DQ binding peptide thyroid peroxidase 632–645Y was synthesized and tested for its binding to DQ2.3 and DQ3.2. The panel included at least five single-substitution analogs for each position, including conservative, semiconservative, and nonconservative residues. The binding data are summarized in Fig. 3Go.



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  		FIGURE 3. DQ2.3 and DQ3.2 binding capacity of single-substitution analogs of thyroid peroxidase 632–645Y. Binding data are expressed relative to the residue at each position with the highest binding affinity for the respective DQ molecule. A dash indicates relative binding <0.01. Relative binding >0.20 is indicated by bold font.

 
In the case of DQ2.3, at least one substitution at L8, E10, L13, and P14 was associated with a 10-fold reduction in binding capacity. Greater than fivefold reductions in binding capacity were noted with at least one substitution for the G7, A9, N11, and Y15 residues. A different pattern was noted in the case of DQ3.2, where at least one substitution for W4, G7, L8, A9, E10, and F12 resulted in a 10-fold or greater reduction in binding capacity and greater than fivefold reductions were noted at V3 and P14. Thus, the residues between G7 and E10 appear to have an important influence on both DQ2.3 and DQ3.2 binding capacity. However, the data also highlight how the pattern recognized by each molecule is unique. For example, in the case of DQ2.3, the C-terminal residues L13 and Y15 are important. Conversely, V3 and W4 at the N terminus are important in the case of DQ3.2. Thus, although DQ2.3 and DQ3.2 have overlapping peptide-binding repertoires, they bind their ligands using somewhat different motifs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Herein, we describe assays to measure the capacity of peptides to bind HLA-DQ2.3 and DQ3.2 molecules. These assays use MHC molecules purified from EBV-transformed homozygous cell lines and previously known DQ2.3 or DQ3.2 ligands as radiolabeled probes. The sensitivity of these assays is in the 20–30 nM range, which is similar to the sensitivity of other class II MHC-peptide binding assays developed using the same methodology (43). Competitive inhibition studies revealed a correlation between binding capacity and the known DQ restriction of various panels of peptides, validating the specificity and immunological relevance of each assay.

More extensive analysis of the peptide-binding specificity of DQ2.3 and DQ3.2 revealed that these molecules have largely overlapping peptide-binding repertoires. The degree of overlap is unprecedented, in that almost 70% of the peptides that had the capacity to bind (IC50 of 1000 nM or better) either DQ2.3 or DQ3.2 bound both molecules. By comparison, cross-reactivity between various molecules of the previously described HLA-DR-supertype (34) was found to average ~40%. This high degree of cross-reactivity between DQ2.3 and DQ3.2 had not been previously noted, and it is tempting to speculate that it might be somehow related to the fact that both molecules have been linked with autoimmune disorders in humans (7, 8, 9, 10, 11). This high degree of overlap was also surprising given the fact that distinct peptide-binding motifs for DQ2.3 and DQ3.2 have been reported (21, 22, 23, 24, 25, 28, 29, 30, 31, 52, 53). A dominant specificity for acidic residues in position 9 has been described for DQ3.2 (18, 24, 28, 29, 30, 52, 53). This observation has been supported by x-ray crystallographic studies (18, 19, 20). Conversely, the dominant specificity reported for DQ2.3 is for acidic residues in position 4 or 7 (21, 22, 23, 25, 28).

Our data are consistent with the x-ray crystallographic data, which indicates prominent P1 and P9 pockets with polar environments in the case of DQ3.2 (18). Examination of the sequence of thyroid peroxidase 632–645Y indicates that if E641 is located in the P9 pocket, then the preferred D635 substitution aligns with the P1 pocket. Interestingly, consideration of the preferences revealed by the single-substitution analyses suggests that large hydrophobic/aromatic residues might also serve as appropriate anchors. No structure is available for DQ2.3, but our data are consistent with the report of Quarsten et al. (22), which suggested that the P9 pocket in DQ2 does not require negative charges but rather accommodates large hydrophobic residues. It should also be emphasized that despite the large degree of repertoire overlap between DQ2.3 and DQ3.2, not all class II molecules with main anchor specificities for acidic residues share overlapping peptide-binding repertoires. Indeed, little overlap exists between the repertoires of DQ2.3 or DQ3.2 and the peptide-binding repertoire of DR3, which also hinges on the presence of negatively charged residues (46).

The peptide-binding specificity of most HLA-DR and -DQ molecules for which motifs have been reported (34, 59, 60) is typically controlled by one or two dominant main anchors. Nonconservative or, in some cases, even semiconservative substitutions at these dominant anchors can preclude peptide binding. The peptide-binding specificities described herein for both DQ2.3 and DQ3.2 are somewhat unique in that although certain substitutions can be associated with significant reductions in binding capacity, in both cases binding capacity appears not to be conferred by clear main anchor residues. In this respect, it should be noted that a residue that provides little binding energy when the side chain is replaced by that of an alanine nevertheless may not allow other side chains, which could profoundly hinder the interaction. Future studies analyzing TCR contact residues may provide additional important information. However, taken together, our observations suggest that DQ2.3 and DQ3.2 binding capacity may be based on multiple interactions of lower specificity. Shared flexibility in the establishment of peptide side chain or backbone interactions for binding might also contribute to the cross-reactivity between these two molecules (27, 61)

A lower, but still appreciable, degree of cross-reactivity between DQ3.1 and DQ2.3 or DQ3.2 was also noted. Most of the peptides that bind DQ3.1 also bind either DQ2.3 or DQ3.2. Again, this cross-reactivity is apparently not due to shared motifs, but it reflects the fact that each molecule can accommodate the same peptide using a different register (data not shown). In any case, demonstration of a high degree of peptide-binding overlap between HLA DQ2.3 and DQ3.2 suggests that DQ-specific supertypes may be defined on the basis of functional binding capacity, as previously reported for HLA A and B class I (62, 63, 64) and HLA DR class II (34) molecules. This finding suggests that HLA DQ promiscuous epitopes can be identified, with implications for vaccine development and for the identification of potentially harmful epitopes linked to undesired immune reactions.

A broader analysis of the patterns of cross-reactivity between DQ2.3, DQ3.2, and DQ3.1 and various HLA-DR molecules was also performed. Lower than expected levels of cross-reactivity between selected DQ and DR specificities known to be in linkage disequilibrium were detected. These patterns would suggest that linkage disequilibrium may reflect, in some cases, selection for DQ/DR pairs with complementary specificity. This arrangement would have obvious functional benefits, allowing presentation of a more diverse set of epitopes, and thus potentially resulting in a broader and more diverse T cell repertoire.

Our analyses also revealed that, apart from some HLA-DQ/DR pairs in linkage disequilibrium, the peptide-binding repertoires of DQ molecules can also overlap with those of many DR molecules. This finding indicates that the binding specificity of DQ molecules is not, in general, mutually exclusive with the specificity previously defined for the HLA-DR supermotif. These results suggest that it should be possible to identify highly promiscuous helper T cell epitopes capable of mediating activation of T cells restricted by both HLA DR and DQ molecules. Studies are currently in progress in our laboratory to experimentally address this possibility, with obvious relevance for the design of epitope-based vaccines.


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  		Table VIA. Continued

 

   Acknowledgments
 
We thank Ram Madabushi and Jamie Ahner for their expert technical assistance and Dr. Manley Huang for his helpful comments.


   Footnotes
 
1 This work was supported in part by National Institutes of Health-National Institute of Allergy and Infectious Diseases Contract NO1-AI-95362. Back

2 Address correspondence and reprint requests to Dr. Alessandro Sette, Epimmune, Inc., 5820 Nancy Ridge Drive, Suite 100, San Diego, CA 92121. E-mail address: asette{at}epimmune.com Back

3 Abbreviations used in this paper: DQ2.3, DQA1*0501/DQB1*0201; DQ3.2, DQA1*0301/DQB1*0302; D, aspartic acid residue; DQ3.1, DQA1*0301/B*0301; MT, M. tuberculosis. Back

Received for publication July 1, 2002. Accepted for publication August 30, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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