Functional analysis of HLA-DP polymorphism: a crucial role for DPß residues 9, 11, 35, 55, 56, 69 and 84–87 in T cell allorecognition and peptide binding

Gema Díaz1, Massimo Amicosante2, Dolores Jaraquemada3, Richard H. Butler4, M. Victoria Guillén1, Miguel Sánchez1,5, César Nombela1 and Javier Arroyo1

1 Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain 2 Department of Biology and Division of Respiratory Diseases of the University of ‘Tor Vergata’ at the IRCCS ‘L. Spallanzani’, 00149 Rome, Italy 3 Unitat d'Immunologia, Departamento de Biologia Cellular, Fisiologia e Immunologia. Institut de Biología Fundamental (UAB), 08193 Barcelona, Spain 4 Institute of Cell Biology, National Research Council, 00016 Monterotondo (Rome), Italy 5 Present address: Departamento de Microbiología y Genética, Instituto de Microbiología Bioquímica, USAL, 37007 Salamanca, Spain

Correspondence to: J. Arroyo; E-mail: jarroyo{at}farm.ucm.es
Transmitting editor: T. Sasazuki


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The information available on the specific function of HLA-DP and the structure–function relationships is very limited. Here, single amino acid substitutions of HLA-DPB1*02012 have been used to analyze the role of polymorphic residues of the DPß1 domain on DP-mediated T cell allorecognition and peptide binding. Using a panel of specific anti-HLA-DP mAb, we identified the HLA-DP residues involved in the recognition by these mAb, with a crucial role for DPß56 for most of the mAb assayed. Individual substitutions at residues 9, 11, 35, 55, 56 and 69 completely abrogated T cell recognition mediated by two different HLA-DPw2-allospecific T cell clones (8.3 and 8.9). Interestingly single changes at positions 9, 11, 35 and 55 of HLA-DPß also altered the binding of peptides AAII(12–27) and IIP(53–65), natural ligands of the HLA-DPB1*02012 molecule. Individual changes at residues located in pocket 1 (84, 85, 86 and 87 from HLA-DPß) led to a partial reduction in cytotoxic T lymphocyte-mediated lysis and also partially affected peptide binding. However, the simultaneous substitution of these positions completely abolished both T cell allorecognition and peptide binding, suggesting a major role for polymorphisms at pocket 1 in HLA-DP function. Molecular modeling, used to predict changes induced by amino acid substitutions, supported the functional data. Taken together, these results strongly suggest that polymorphic residues 84, 85, 86 and 87 at pocket 1, residues 9, 35 and 55 at pocket 9, and residues 11 and 69 at pockets 6 and 4 respectively play a key role in HLA-DP function, probably by modifying the way the peptide is bound within the groove of HLA-DP2 and determining changes in the conformation of the MHC–peptide complex recognized by the TCR.

Keywords: allorecognition, HLA-DP antigen, molecular modeling, peptide binding, polymorphism, T cell


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Molecules encoded within the class II region of the MHC are polymorphic, non-covalently associated {alpha}ß heterodimers whose function is to bind peptide fragments derived from foreign protein antigens and display them on the cell surface for interaction with the antigen-specific receptors of CD4+ T lymphocytes (1). Characterization of MHC class II peptide binding and T cell recognition, at the molecular level, is essential to understand a central event of the immune response that could have important applications in clinical practice.

Human MHC class II molecules are classified in three isotypes: HLA-DR, -DQ and -DP. Specifically, HLA-DR and -DQ are the best structurally and functionally characterized. The three-dimensional structures of HLA-DR and -DQ bound to different peptides have been determined by X-ray crystallography (24), and reveal that HLA class II molecules bind peptides in a polyproline type II-like conformation through two different types of interactions: (i) side chains of polar conserved class II residues form hydrogen bonds with the peptide backbone and (ii) peptide side chains are accommodated in the pockets of the peptide binding groove as anchor residues. Five main pockets (pockets 1, 4, 6, 7 and 9), whose size, hydrophobicity and charge vary in an allele-specific manner, accommodate the anchor residues of the peptide in the majority of class II molecules so-far characterized. Polymorphic residues of class II molecules line these pockets and are responsible for the peptide-binding specificity of each allele of the different MHC isotypes (2,5). Identification of MHC class II-bound naturally processed peptides (6) and peptide-binding assays has permitted the definition of peptide motifs, especially for HLA-DR (7). In addition, polymorphic residues of class II molecules are critical for T cell recognition, by affecting direct MHC–TCR or MHC–peptide interactions (8,9). Polymorphism also plays a crucial role in T cell recognition of allogenic MHC molecules, mainly by affecting the repertoire of peptides available for recognition by alloreactive T cells (10,11). The recent determination of the HLA-DR–hemagglutinin–TCR{alpha}ß structure has been crucial for better understanding TCR recognition of the MHC–peptide complex, enabling the identification of polymorphic residues in the MHC class II molecule that are contacted by the TCR and peptide contacts with the TCR (12,13).

The importance of polymorphic residues in peptide binding and T cell recognition, mainly in the HLA-DR molecule but also in the HLA-DQ molecule, has been the subject of intense investigation (9,1419). In contrast, considerably less is known about HLA-DP molecules. Studies investigating pool sequences of peptides eluted from HLA-DP molecules (6,20,21) resulted in the description of putative anchor residues, but this information is very limited, mainly due to the difficulty in obtaining sufficient amounts of purified HLA-DP protein (21). Only limited information is available on HLA-DP peptide binding (21) and antigen-specific T cell recognition (22). Little is known about the relevance of the polymorphism in the function of HLA-DP (23,24). Previous work from our group could establish the importance of polymorphic residue 69 of HLA-DPB1*02012 in the function of this molecule (25,26), especially for HLA-DP allorecognition. The change of Glu -> Lys at this position abrogates the recognition by several HLA-DPw2-alloreactive CD4+ cytotoxic T lymphocytes (CTL) (26).

Although the function of the HLA-DP molecule in the immune response has not been fully elucidated, some diseases like juvenile chronic arthritis (27), juvenile rheumatoid arthritis (28), berylliosis (29) or hard metal lung disease (30) have been associated with specific HLA-DP alleles. In particular, the polymorphic position HLA-DPß69 has been specifically associated with chronic immune disorders like berylliosis (29) by playing a direct role in driving the immune response against beryllium (31,32). Moreover, HLA-DP has also received attention as a possible target for allospecific T cell recognition in the context of graft versus host disease (33). Detailed studies on the structure–function relationship of HLA-DP molecules, defining peptide-binding properties and important residues for T cell recognition, would, therefore, help to establish the function of HLA-DP in the immune response and associated diseases, particularly in the absence of a crystallographically determined HLA-DP structure.

In this report, the involvement of HLA-DP polymorphism in the function of this molecule was investigated. Differences among HLA-DP alleles define 13 main polymorphic residues within the ß1 domain: residues 8, 9, 11, 35, 36, 55, 56, 69, 76, 84, 85, 86 and 87. B-lymphoblastoid cell line (LCL) transfectants expressing the wild-type and site-directed mutants at each of these 13 polymorphic positions were used to evaluate their role in T cell recognition and peptide binding, as well as to define the implication of these residues in the epitopes recognized by HLA-DP-specific antibodies. Molecular modeling of the wild-type and mutant HLA-DPB1*02012 alleles on the basis of the crystallized HLA-DR4 molecule, together with the functional results, allowed us to define the participation of the polymorphic HLA-DPß residues in the function of this molecule.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell lines and culture
Lymphoblastoid cell line 45.EM1 was derived from a mutant cell line haploid for HLA, 45.1 [remaining haplotype A2,B5,DR1,DQw1,DPw2 (DPA1*0301, DPB1*02012) (34)] by ICR191 treatment and subsequently selected by resistance to lysis by HLA-DPw2-allospecific CTL clones. This cell line is HLA-DPw2. It does express normal levels of DPA mRNA, but is not able to transcribe DPB1. Production of 45.EM1 transfectants expressing the wild-type DPB1*02012 and the site-directed mutant DPB1*02012-A36 and DPB1*02012-K69 alleles has been previously described (26). The Epstein–Barr virus-transformed B cell line WT47, consanguineous and homozygous for HLA-DP (DPA1*0101, DPB1*1601), was obtained from the European Collection of Animal Cell Culture (Porton Down, UK). LCL were cultured in RPMI 1640 medium (Life Technologies, Paisley, UK) supplemented with 10% heat-inactivated FCS (Life Technologies), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies). Cells were incubated at 37°C in a humidified incubator with 5% CO2.

Antibodies and peptides
The mAb used in this work and consensus specificities were: B7/21: DP monomorphic (35); ILR1: DPw2, 3, 4.2, DR5 (36); NFLD.M58: DPB1*0201, 0301, 0402, 0601, 0901, 1001, 1401, 1601, 1701, DRB1*1101 (37); NFLD.M60: DPB1*0201, 0301, 0402, 0601, 0901, 1001, 1401, 1701, DRB1*1101 (37); NFLD.M64: DPB1*02012, 0301, 0402, 0901, 1001, 1401, 1601, 1701 (W. Marshall, pers. commun.); NFLD.M66: all DPB1* except 0202, 0401, 0401 and 1501; weak on DPB1*02012 (37); NFLD.M68: DP monomorphic (38,39); NFLD.M69: DP and DR monomorphic (39); NFLD.M70: DPA1*0201 (37); NFLD.M73: DPB1*0101, 0201, 0301, 0402, 0601, 0901, 1001, 1401, 1601, 1701, DRB1*1101 (37); NFLD.M75: DPB1*0301, 0402, 0601, 0901, 1401, DRB1*1101 (W. Marshall, pers. commun.); NFLD.M77: DPB1*0301, 0601, 0901, 1001, 1401, 1701 (39).

Peptides were synthesized using standard Fmoc chemistry. The IIP(53–65) peptide (VPDHVVWSLFNTL) was synthesized at the Servicio de Proteómica at UAM (Madrid, Spain) and the AAII(12–27) peptide (LQSLVSQFYQTVQDYA) was purchased from Genemed Synthesis (San Francisco, CA). Both synthetic peptides were biotinylated at the N-terminus using standard procedures and purified by reverse-phase HPLC to >=90% purity. The sequence and purity were confirmed by mass spectrometry.

Sited-directed mutagenesis and transfection
The mutagenesis strategy was used to produce DPB1*02012 cDNAs in which specific codons encoding polymorphic residues were replaced by the codons that encode the amino acid found at the corresponding position in other HLA-DPB1 alleles. Eleven mutagenic oligonucleotides were designed to generate substitutions at positions 8, 9, 11, 35, 55, 56, 76, 84, 85, 86 and 87. Substitutions at 36 and 69 have been described elsewhere (25,26). Mutagenesis was performed as previously described (25), using the Muta-Gene M13 in vitro mutagenesis kit (Bio-Rad, Richmond, CA) following the manufacturer’s instructions. All mutations were confirmed by DNA sequencing using an automated DNA sequencer ABI Prism 377 (Applied Biosystems, Foster City, CA). Full-length DPB1 cDNAs (wild-type and site-directed mutants) were subcloned into the plasmid pREP4 (Invitrogen, San Diego, CA), as described (26). B-LCL 45.EM1 was transfected with each of these constructs by electroporation with a BTX-600 electro cell manipulator (BTX, San Diego, CA) as described (26). Transfectants expressing the highest levels of HLA-DP were isolated by flow cytometry. The expression of similar HLA-DP levels in all transfectants was confirmed by flow cytometry using B7/21 antibody.

T cell clones
Human T cell clones 8.3 and 8.9 had been previously generated in mixed lymphocyte culture from the same batch of DPw2-specific primed lymphocyte-typing reagents used originally to defined HLA-DP (40), in which responder and stimulator cells were matched for HLA-A, -B, -C, -DR and -DQ, but mismatched for -DP. Both clones had been previously shown to be HLA-DPw2 allospecific in population analyses (41). However, whereas clone 8.9 lysed all DPw2+ LCL tested, clone 8.3 showed reduced or no lysis to particular LCL expressing DPw2, but not HLA-DR (41). T cell clones were maintained in culture using IMDM (Life Technologies) containing 10% human serum, 50 U/ml penicillin (Life Technologies) and 0.05 mg/ml streptomycin (Life Technologies). The clones were stimulated approximately once a week with HLA-DPw2+ irradiated peripheral blood mononuclear leukocytes plus 1 µg/ml of phytohemagglutinin (Difco, Detroit, MI).

Cytotoxicity assays
A standard 51Cr-release assay was used to measure CTL responses (26,40). Radiolabeled target cells were incubated at 37°C for 4 h with CTL at different effector/target ratios in round-bottomed 96-well plates (Costar, Cambridge, MA). All assays were performed in triplicate. The percentage of specific cytotoxicity was calculated by the formula: (experimental 51Cr release – spontaneous 51Cr release)/(maximum 51Cr release – spontaneous 51Cr release) x 100. Spontaneous release was determined in the absence of T cells, while maximal release was determined incubating target cells in the presence of 5% Triton X-100.

Peptide binding assays
B-LCL (5 x 105) were incubated in RPMI/1% FCS (Life Technologies), at 37°C for 4 h, with the biotinylated peptide (20 µM) at pH 5–6. Cells were washed twice at 4°C with PBS containing 1% FCS. Then, 0.5 µg/100 µl of streptavidin–phycoerythrin (Caltag, Burlingame, CA) was added and incubated for 30 min at 4°C. The cells were then washed twice using PBS/1% FCS and finally resuspended in 300 µl of PBS/1% FCS. Stained cells were analyzed by flow cytometry on a FACScan analyzer (Becton Dickinson, San Jose, CA), excluding dead cells from the analysis by propidium iodide staining. Control cells were incubated in the absence of peptide under the same conditions and the mean fluorescence from these samples was taken as background. The parental cell line 45.EM1 which expresses the same HLA-DR and -DQ alleles, but not -DP, was treated in the same way and used as a negative control. The fluorescence intensity obtained with these two controls was subtracted from the total fluorescence obtained in each experiment. The fluorescence data were then normalized with respect to the level of HLA-DP expression in each transfectant by dividing the difference between the mean fluorescence in the presence and absence of the biotinylated peptide by the number of HLA-DP molecules expressed in each transfectant. HLA-DP expression was quantified by flow cytometry using mAb B7/21 and the amount of HLA-DP molecules per cell determined by using standard size beads (Quantum MESF kit), as described in the manufacturer’s protocol (Bangs Laboratories, Fishers, IN).

Immunofluorescence labeling and flow cytometric analysis
For immunofluorescence analysis, cells were stained as described (26) and analyzed on a FACScan (Becton Dickinson, San Jose, CA). Briefly, the cells were incubated with the specific mAb, washed 3 times with PBS, and incubated with fluorescein-conjugated goat anti-mouse Ig (G,M,A) (Sigma, St Louis, MO) and analyzed by flow cytometry. The staining of each transfectant with the different mAb was normalized as a percentage of the binding to the monomorphic anti-HLA-DP B7/21 using the following formula: (MCF for mAb – MCF for negative control)/(MCF for B7/21 – MCF for negative control), where MCF is mean channel fluorescence. Transfectants were incubated with fluorescein-conjugated goat anti-mouse Ig (G,M,A) (Sigma) as negative control. The binding levels are expressed in six categories determined by the normalized percent MCF values: – – (no binding), <5% of normalized percent MCF; – (negative), <20%; + (low binding), 20–39%; ++ (intermediate binding), 40–59%; +++ (high binding), 60–79%; ++++ (maximal binding), >=80%.

Molecular modeling of HLA-DP2 and mutated HLA-DP molecules
Sequence and domain structure information of the HLA-DP2 molecule were obtained from the Swiss-Prot database. Sequences corresponding to the extracellular parts of the {alpha} and ß chain respectively were individually submitted to the Swiss-Model Protein Modeling Server (http://www.expasy.ch/swissmod/) via the ‘first approach mode’. Both resulting models were subsequently re-submitted via the ‘combine mode’ allowing the Swiss-Model Protein Modeling Server to choose the best crystal structure template available for modeling the HLA-DP2 molecule. The resulting three-dimensional model of the extracellular domain of HLA-DP2 was modeled on the crystal structure of the HLA-DR4 molecule (42). Further processing was carried out using InsightII/Discover (Accelrys, San Diego, CA). The coordinates of the CLIP peptide in the HLA-DR3 crystal structure (43) were superimposed onto the HLA-DP2 model (HLA-DP2-CLIP). Optimization of the model was performed using the conjugate gradients method and the consistent-valence force field (c.v.f.f.). The effects of solvent were simulated using a distance-dependent dielectric constant 4.0 x r.

Substitutions at positions 8 (L -> V), 9 (F -> Y), 11 (G -> L), 35 (F -> Y), 36 (V -> A), 55 (D -> A), 56 (E -> A), 69 (E -> K), 76 (M -> V), 84 (G -> D), 85 (G -> E), 86 (P -> A) and 87 (M -> V) of the HLA-DP2 ß chain were introduced in all the optimized models of the HLA-DP2 molecule, and subsequently optimized for all protein residues within 10 Å of the peptide and the peptide itself. Each of these models was subjected to a molecular dynamics simulation at 600°C for 5000 iterations (plus 200 iterations for equilibration). Conformations selected as possible low-energy configurations were again optimized.


    Results
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 Abstract
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 Methods
 Results
 Discussion
 References
 
Preparation of site-directed mutant DPB1*02012 molecules and characterization of their binding to anti-HLA-DP mAb
To investigate the functional role of individual polymorphic positions of the DPß molecule, we generated 11 site-directed mutant DPB1*02012 alleles that together with the previously described DPß36 and 69 (26) cover most of the individual polymorphic positions at the DPß1 domain. For each position analyzed, the wild-type DPB1*02012 allele residue was replaced by the corresponding amino acid found at the same positions in other DPB1 naturally allelic variants. Using B-LCL 45.EM1 as a recipient for transfection, 14 B-LCL transfectants expressing the DPB1*02012 wild-type and site-directed mutant DPB1*02012 alleles were generated. B-LCL transfectants expressing comparable high levels of DP molecules were selected by flow cytometry and sorting with the mAb B7/21.

The binding of these transfectants to a panel of monomorphic and polymorphic anti-HLA-DP mAb was analyzed by flow cytometry. Monomorphic B7/21 was used as a reference antibody and the data for each transfectant expressed as a percentage of the binding to this mAb (Table 1). Recognition by the monomorphic antibodies, B7/21, NFLD.M68 and M69, was not affected by any of the mutations assayed (data not shown), ruling out the possibility of major structural alterations as a consequence of the changes introduced. The DPB1*02012-A56 allele did not bind to ILR1, in agreement with data from Yu et al. (36). Recognition by NFLD.M58, M60, M64, M73 and M75, as well as by M77, was also completely abolished in the A56 mutant, indicating that this residue is essential in the epitope recognized by these antibodies. These results are almost in agreement with the data from Marshall et al. (37), who proposed the epitope DE at 55–56 for these antibodies on the basis of the recognition of homozygous B cell lines and peripheral blood B cells, and indicate that the residue 56 and not 55 is the essential component of this epitope. In accordance with the previous proposed epitope for NFLD.M66 (residues 84–87), binding of this antibody was affected in mutants D84 and E85, although the variants A86 and V87 bound perfectly. In contrast to the previously suggested epitope for NFLD.M77 (37), our data showed that the binding to this antibody was not affected in the HLA-DPB1*02012-Y35 and A36 alleles. Moreover, the binding of this antibody to DPB1*02012-A56, and also to E85 and A86, was clearly diminished, indicating that residue 56 and probably residues 85 and 86 are part of the epitope recognized by NFLD.M77, but not L at 11 and FV at 35–36 as previously suggested (37). Interestingly, the binding of NFLD.M70 was clearly affected in mutant alleles DPB1*02012-L11, V8, Y35, E85 and A86.


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Table 1. Binding of the panel of polymorphic anti-HLA-DP mAbs to transfectants expressing wild type HLA-DPw2 (DPB1*02012) and site-directed mutant DPB1*02012 alleles
 
The role of HLA-DP2 polymorphism in T cell allorecognition
In order to analyze the relative contributions of each HLA-DPB1*02012 polymorphic residue to T cell recognition, transfectants expressing wild-type or mutant DPw2 molecules were used as targets in cytotoxicity assays with two HLA-DPw2-specific alloreactive T cell clones. As shown in Fig. 1 and as previously described (26,40), both allospecific T cell clones (8.3 and 8.9) had positive responses with the cell line 45.1 (HLA-DPw2 positive) and wild-type HLA-DPw2 transfectants (45.EM1/DPB1*02012), but were not able to lyse the B-LCL 127, which is haploid for HLA and expresses the DPB1*0401 allele.



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Fig. 1. Cytotoxicity reactivity of HLA-DPw2-specific alloreactive CTL clones 8.3 (circles) and 8.9 (crosses) for B cell lines expressing (i) wild-type DPB1*02012: LCL 45.1 and transfectant 45.EM1/DPB1*02012, (ii) wild-type DPB1*0401: LCL 127, (iii) wild-type DPB1*1601: LCL WT47 or (iv) site-directed DPB1*02012 mutants.

 
Six of the 13 substitutions in DPB1*02012 [9 (F -> Y), 11 (G -> L), 35 (F -> Y), 55 (D -> A), 56 (E -> A) and 69 (E -> K)] completely abrogated recognition by both 8.3 and 8.9 T cell clones (Fig. 1), suggesting a crucial role for these polymorphic residues in T cell recognition of HLA-DP. In contrast, the results revealed that the alloresponse of both anti-DPw2 clones was not affected by amino acid substitutions at positions 8, 36 and 76 (Fig. 1). Therefore, these residues, in spite of being located at the floor of the hypothetical HLA-DP binding groove (residues 8 and 36) or in the {alpha} helix (residue 76), are not relevant for the recognition by these T cell clones. With regard to the recognition of the mutants involving residues 84–87 by the T cells, there was an intermediate situation, since the change at 86 (P -> A) slightly affected T cell recognition mediated by 8.3 and 8.9 CTL, while changes at 85 (G -> E) and 87 (M -> V) led to a decrease of ~50% lysis with respect to the wild-type DPB1*02012. Although the T cell recognition is only partially affected by individual mutations in this region, when we tested the B-LCL WT47, expressing the DPB1*1601 allele which differs from DPB1*02012 only in these four positions at the DPß1 domain, none of the two CTL clones assayed were able to recognize this B-LCL (Fig. 1). These results indicate that residues 84, 85, 86 and 87 play a key role, and suggest an additive effect of these positions in T cell recognition.

In general, there was considerable homogeneity in the responses observed with both alloreactive anti-DPw2 clones with the whole panel of site-directed mutants. However, the substitution at 84 (G -> D) had no or little effect on the recognition mediated by the 8.9 clone, whereas the 8.3 response decreased drastically. We have previously described (26,41) a similar pattern observed with the B-LCL 139. Although this cell line is HLA-DPw2+, it is not recognized by clone 8.3. Probably, these differences in the recognition are related to differences in the MHC–peptide complex recognized by each clone.

Role of HLA-DP2 polymorphic positions in peptide binding
To obtain further insight into the polymorphism of the HLA-DP molecule, we examined the role of HLA-DPB1*02012 polymorphic positions in peptide binding. Two peptides, IIP(53–65) and AAII(12–27), eluted from HLA-DPB1*02012 by Chicz et al. (21), were chosen based on their high affinity for HLA-DPw2. Their ability to bind to different HLA-DP molecules was measured using the cell-surface-binding assay described above. We confirmed that both peptides were unable to bind to HLA-DR and -DQ, since the HLA-DP-deficient cell line 45.EM1 (DRw1, DQw1, DP negative), used as a control, did not significantly bind either of these peptides (data not shown). To facilitate comparisons between HLA-DP alleles, the binding of peptides IIP(53–65) and AAII(12–27) to the wild-type DPB1*02012 was considered as 100%, and the binding of other transfectants are with reference to this after taking into account variations in HLA-DP expression (see Methods).

Substitutions at positions DPß 11 and 35 completely abolished the binding of IIP(53–65) peptide, while mutations at residues 9 and 55 led to a clear increase in their peptide-binding ability (Fig. 2). This strongly suggests that residues at these positions affect the way that IIP(53–65) is bound in the HLA-DP groove. Less dramatic, but still significant, effects were found with positions 8, 69 and 84–87, where modifications partially inhibited IIP(53–65) binding. Substitutions at positions 36, 56 and 76 were observed to be less important, since these mutations had very little or not effect on IIP(53–65) binding.



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Fig. 2. Binding of peptides IIP(53–65) (solid bars) and AAII(12–27) (open bars) to 45.EM1 transfectants expressing wild-type or site-directed mutant HLA-DP molecules. The amount of peptide bound by each transfectant was obtained as described in Methods and compared to the amount of peptide bound by cells expressing the wild-type DPB1*02012 allele. Peptide-binding values for each transfectant are expressed as percentages referred to the binding of the same peptide to the 45.EM1 transfectant cell line expressing the wild-type DPB1*02012 allele.

 
In the case of the AAII(12–27) peptide, the majority of the HLA-DPB1*02012 substitutions had little significant effect on binding, their binding abilities ranking from 70 to 100% of the wild-type DPB1*02012 allele. Only changes in residues V76 and Y35 clearly increased the binding ability for this peptide. In particular, the DPB1*02012-Y35 allele doubled the binding of the wild-type, suggesting an important role for this residue in the binding of AAII. Similar to IIP peptide binding, mutations at pocket 1, especially A86, partially reduced the binding of AAII(12–27) to HLA-DPB1*02012. Interestingly, and in accordance with the results previously shown for T cell recognition, simultaneous substitution of residues in pocket 1 (D84, E85, A86 and V87) completely abrogated the binding of both peptides to HLA-DP2 as WT47 B-LCL, carrying the DPB1*1601 allele (which differs from DPB1*02012 only at these positions), is unable to bind these peptides (data not shown). This suggest, as previously shown for T cell recognition, an additive effect of individual polymorphic positions in pocket 1 for peptide binding.

Molecular modeling of wild-type and site-directed mutant HLA-DPB1*02012 molecules
In an attempt to interpret the functional data, we modeled the DPB1*02012 molecule carrying CLIP in the peptide-binding cleft based on the crystal structure of HLA-DR4 as described in Methods. CLIP was oriented in the HLA-DP2 molecule superimposing the coordinates of this peptide in the HLA-DR3/CLIP crystal.

A surface analysis (top view) of the peptide-binding region of the HLA-DP2 molecule, after removing of the CLIP peptide is shown in Fig. 3(A). As expected from the homology with HLA-DR4, the binding cleft of HLA-DP2 presents four major contact sites. These four pockets interact with the peptide amino acid residues P1, P4, P6 and P9. The relative positions in the HLA-DP2 model of the mutations evaluated in this study are shown in Fig. 3(B). Due to the deletion of 2 amino acids at positions 23 and 24 of the HLA-DPß chain with respect to HLA-DRß, the relative homologous positions of the mutants on the HLA-DR molecule are positions 8, 9, 11, 37, 38, 57, 58, 71, 78, 86, 87, 88 and 89 respectively. Residues 84 and 85 of HLA-DPß should be located in pocket 1, residues 69 and 76 in the area of pocket 4, residues 8 and 11 in pocket 6, and residues 9, 35, 36, 55 and 56 in the area of pocket 9. Positions 86 and 87, although close to pocket 1, are mainly involved in the interaction area between the {alpha} and ß chains (Fig. 3B).



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Fig. 3. Model of the HLA-DPw2 molecule. The DPB1*02012 molecule carrying CLIP in the peptide-binding cleft was modeled as described in Methods based on the crystal structure of HLA-DR4. (A) Top view of the binding cleft showing the four pockets that should interact with the peptide amino acid residues P1, P4, P6 and P9 in the HLA-DPw2 molecule. (B) The mutations of the HLA-DP molecule characterized in this work with their localization in the modeled DP molecule (polymorphic mutated residues are marked).

 
The different individual amino acid substitutions characterized in this study were introduced in the HLA-DP2 model and the changes produced in the peptide-binding groove of the HLA-DP2 structure analyzed (Fig. 4).



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Fig. 4. Changes produced in the peptide-binding groove of the HLA-DPw2 (DPB1*02012) modeled molecule as a consequence of the different mutations introduced. The 13 individual amino acid polymorphic substitutions characterized in this study were introduced in the HLA-DP2 model and the changes produced in the peptide-binding groove of the HLA-DP2 structure analyzed. Details for the different parts of the molecule affected by each mutation and the corresponding area in the wild-type HLA-DPw2 (DPB1*02012) molecule are shown to compare mutations Y9, Y35 and A55 in pocket 9, A56 in the {alpha} helix of the ß chain, V8 and L11 affecting pockets 6 and 9, K69 and V76 affecting pockets 4 and 6, and individual (D84, E85, A86, V87) or simultaneous mutations (DPB1*1601) in pocket 1 affecting both pocket 1 and the contact area of peptide residues P–1 and P–2. Positive and negative charges at the cell surface of the HLA-DP are depicted in blue and red respectively.

 
Relative to pocket 9, the change of F -> Y at position 9 determines a reduction of the space with some more negative charge at the wall of this pocket (Fig. 4B versus A). This residue is only partially exposed at the surface. Residue ß35 is directly exposed at the surface of the molecule forming the base of pocket 9. Substitution of F -> Y at this position (Fig. 4C) produced minimal changes in the area of pocket 9. However, this position maps at the base of pocket 9 in close contact to residue D55 and therefore a change of F -> Y could permit the formation of a new hydrogen bond between Y35 and D55 closing the pocket from the bottom producing a shallow pocket 9. The presence of A instead of D at 55 in HLA-DP2 (Fig. 4D) determined minimal spatial changes in pocket 9. However, significant changes in charge distribution were observed with the loss of some negative charges and an increase of the positive surface area in the wall of the pocket. This residue is directly exposed at the surface of the molecule, forming a large part of pocket 9. In contrast, the mutation of V -> A at position 36 (data not shown) and the mutation E -> A at position 56 (data not shown) did not determine significant changes in the area of pocket 9 as these residues are not directly exposed, suggesting that they play a secondary role in peptide binding properties. However, some changes in conformation and in the charge distribution in the {alpha} helix of the ß chain were observed after substitution of E -> A at position 56 (Fig. 4F versus 4E).

Concerning pocket 6, the mutation L -> V at position 8 determined a reduction of the space in this pocket with a different charge distribution (Fig. 4H versus G), while no modification was observed in pocket 9. Residue DPß 11 is not directly exposed at the surface of the molecule. The mutation G -> L at this position (Fig. 4I) determined a reduction of the space together with the exposure of a negative charge in pocket 6 and a reduction in the accessibility to pocket 9.

Residues DPß69 and DPß76 are directly exposed at the surface of the molecule. Mutations at position 69 (E -> K) induced major changes to the shape and charge distribution of pocket 4, and also to the nearby pocket 6 (Fig. 4K versus J). In particular, HLA-DP2-K69 presented a deeper, slightly smaller pocket 4 with more neutral and positive charges, and a pocket 6 with modified charge distribution. Similar to the mutation at position 69, mutation M -> V at residue 76 (Fig. 4M versus L) determined a less negatively charged pocket 4 with respect to the wild-type molecule, and also a pocket 6 with a different shape and charge distribution.

The last four polymorphic residues of HLA-DP2 molecule evaluated in this study [positions DPß84 (G -> D), 85 (G -> E), 86 (P -> A) and 87 (M -> V)] map in the area of pocket 1. However, only position 84, and minimally 85, seemed to be involved in the formation of the pocket 1 surface, determining a reduction of pocket 1 area and a minimal increase of the negative charge in the pocket itself (Fig. 4N–S). All the four residues seem to play a major role in forming the contact area between the HLA-DP {alpha} and ß chains, and in the part of the groove contacting peptide residues P–1 and P–2 (Fig. 4T–Y) that have been suggested to play a role in increasing the affinity of peptides for HLA class II molecules. Residues DPß84 and particularly DPß85 are exposed at the surface of the molecule mainly forming the interface of {alpha} chain interactions. Mutation G -> D at DPß84 caused an increase of negative charge in the contact area of P–1 while mutation G -> E at DPß85 increased the negative charge in the contact area of P–1 and P–2 (Fig. 4U and V versus T). A similar situation was observed in the models for mutants at positions 86 and 87 (Fig. 4W and X), which are not exposed at the surface of the molecule. Models with simultaneous substitutions at residues 84, 85, 86 and 87 from HLA-DP2 at pocket 1 revealed considerable changes in the conformation and a relevant increase of the negative charge in the contact area of P–1 and P–2 (Fig. 4Y).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The exact role played by HLA-DP in the immune response and disease remains to be defined (44). In fact, compared to other class II molecules, very limited information is available concerning peptide interactions and the role of HLA-DP polymorphic positions both in peptide binding and T cell recognition. We have investigated the functional role of HLA-DP polymorphism on mAb binding, T cell recognition and peptide binding. In addition, the functional data were supported by a detailed analysis of the possible molecular effects of each mutation using molecular models of HLA-DP2 and its mutants. The data reported here clearly indicates that individual HLA-DPß residues 9, 11, 35, 55 and 56 at pocket 9, 69 (26) at pocket 4, and 84, 85, 86 and 87 involved in pocket 1 play a key role in T cell allorecognition. Considering the mechanism by which HLA-DP mutations assayed here could affect T cell allorecognition, two possibilities could be envisioned: (i) an alteration of the interaction between the DP–peptide complex and the TCR or (ii) the involvement of these residues in MHC–peptide interactions. In the later case, mutations would prevent binding of the adequate peptide in the groove, change the nature of the peptide presented or affect the conformation of the peptide presented within the antigen-binding groove of the MHC without altering the overall affinity of the interaction. Interestingly, although the peptides assayed [IIP(53–65) and AAII(12–27)] are not known to be restricted by the alloreactive HLA-DPw2 T cells used in this study, single changes at positions 9, 11, 35 and 55, and simultaneous substitution of the polymorphic residues in pocket 1 (84–87), clearly affected peptide binding to HLA-DP2 molecules. In general, as previously described for HLA-DR (45), the effect of changing polymorphic residues of the molecule was not as well tolerated when judged by their effects on T cell recognition than on peptide binding.

Residues DPß 84, 85, 86 and 87 in pocket 1 play some role in HLA-DP function. Interestingly, a complete modification of the peptide-binding specificity and abrogation of T cell allorecognition was observed as a consequence of the simultaneous substitution of GGPM to DEAV at 84–87, indicating an additive effect of these individual polymorphic positions. The structural models for HLA-DP revealed that changes at positions DPß 84–87 produce alterations in the contact area between HLA-DP{alpha} and ß chains as well as in the part of the groove-contacting peptide residues P–1 and P–2. This should be sufficient to explain the importance of pocket 1 since the two TCR–pMHCII complex structures described both demonstrate that the peptide residue P–1 is involved in TCR binding (46). A functional role for pocket 1 has been previously shown for HLA-DR (8,47), although polymorphism in this molecule is limited to the residue 86 (G/V) and occasionally to 85 (V/A). Studies on DRß86 indicate that the effects of polymorphism in pocket 1 are not related to drastic changes in peptide affinity, but rather to the way peptides bind to the groove, modifying the MHC–peptide conformation (17). A similar situation could explain the effect of individual substitutions in the HLA-DP2 pocket 1. However, the additive effect of simultaneous changes at these positions has not been shown for DR (17).

Residues 9, 35, 36, 55 and 56 are predicted by the model to be located at pocket 9. Our data show that polymorphisms at residues 9, 35 and 55 significantly influence the HLA-DP function, probably due to the changes that these substitutions determine in the area and the charge distribution of pocket 9. The importance of residues 9, 37 and 57 in HLA-DR and -DQ molecules, corresponding to positions 9, 35 and 55 of -DP, both in peptide binding and T cell recognition, has been extensively reported (48,49). The model for the mutation F -> Y at 9 determines a reduction of the space in pocket 9 with a more negative charge in the wall of this pocket, suggesting a reduction in the capacity of binding large residues in pocket 9. This observation supports functional data for AAII(12–27) if previously predicted anchor residues with L15 fitting into pocket 1, and Q18, F20 and V23 in pocket 4, 6 and 9 respectively (21) are taken into account. In the case of IIP(53–65), it had been proposed (21) that F62 should reach into pocket 6, V57 in pocket 1, S60 in pocket 4 and L65 in pocket 9, although an alternative frame was also proposed with P54 in pocket 1 and V57, W59 and F62 in pockets 4, 6 and 9 respectively. The increased binding ability of the IIP(53–65) peptide for the DPß-Y9 mutant could relate to the presence of a more adaptable residue such as the flat F (with P in P1), in agreement with the alternative frame.

For the polymorphic residue at DPß55 the molecular models proposed significant changes in pocket 9 as consequence of the D -> A substitution with the lost of negative charge. This would mean a reduction in binding non-polar residues and an increase of polar or more adaptable residues, in agreement with the binding data for AAII(12–27) and IIP(53–65), if the alternative frame for IIP(53–65) is taken into account. In accordance with our functional data in HLA-DP, the replacement of Asp57ß in HLA-DQ (55 in HLA-DPß) by Ala increases the positive charge of pocket 9 and hence preference for negatively charged peptide residues, as a consequence of the loss of a hydrogen bond between Asp57ß and Arg76{alpha} (50). This salt-bridge hydrogen bond has been proposed to be conserved in most DR, DQ and DP molecules (4).

In the case of the F -> Y substitution at DPß35, the molecular modeling of HLA-DPw2 predicted the formation of a new hydrogen bond between Y35 and D55, closing the pocket 9 of the HLA-DP molecule and therefore reducing the depth of the pocket. In agreement with our peptide-binding data, long and relatively inflexible residues such as L in P9 of peptide IIP(53–65) [or even worse F in the alternative frame with P in P1 of IIP(53–65)] are less favored with DPß-Y35 than with DPß-F35. On the other hand, short bulky residues, such as V of AAII(12–27), fit better into a shallow pocket. A key role for the polymorphism of residue 37 in HLA-DQ and -DR molecules (35 in HLA-DPß) has also been described to influence the conformation of bound peptides that are recognized by the TCR (49). These effects have been explained by an occlusion of the base of the pocket 9 by the presence of a Tyr (48) or by the formation of a hydrogen bond between the Tyr at 37 and the side chain of P9 peptide residue (51), although the presence of an hydrogen bond when Asp is present at 57 can not be ruled out.

Residue DPß56 plays a crucial role in T cell allorecognition by clones 8.3 and 8.9, although binding of IIP(53–65) and AAII(12–27) peptides was not affected by mutations at this position. In contrast, the residue corresponding to DPß56 of the DR molecule (DRß58), presenting the same polymorphism (E/A), is not functionally relevant. Location in the HLA-DP2 model structure placed this residue at pocket 9, but at the end of the peptide-binding site with the side chain oriented to the outside. The effects of DPß56 substitution were not observed directly in pocket 9, but in the {alpha} helix of the ß chain. However, a direct TCR–MHC interaction that should explain inhibition of T cell recognition is not likely since the recent crystallization of several TCR–class II–peptide complexes (13,52,53) suggests that the main interactions between TCR and MHC–peptide complexes are placed in the central core of the MHC molecule.

The key relevant polymorphic position within pocket 6 was DPß11, while changes at DPß8 did not affect HLA-DP function. In accordance with the models which suggest reduction in binding for large residues such as W if the alternative frame for IIP(53–65) is considered, binding of IIP(53–65) to DPß-L11 was completely lost. The importance of residue 11 of HLA-DP agrees with the role of this residue in HLA-DR (10,45,54), where substitution of G by L affected peptide-binding properties.

In the case of pocket 4, position DPß69 is the crucial functional polymorphism within this pocket, while polymorphism at DPß76 had little effect on the functionality of HLA-DP. The change of M -> V at 76 in the HLA-DP model determined changes in the shape and charge distribution of pocket 4, and the nearby pocket 6, but no significant functional effects were caused by this mutation, probably because M is more flexible than V, compensating for the pocket size reduction. In contrast, the equivalent HLA-DR residue (residue 78) was involved both in peptide binding and T cell recognition (10,45,54). However, the DRß78 polymorphism is not M/V, but Y/V. As previously described, residue 69 plays a crucial role in T cell allorecognition (26,55). Binding of peptides IIP(53–65) and AAII(12–27) is slightly reduced as a consequence of the E -> K substitution. In agreement, mutations in DRß71 (corresponding to DPß69) had great effects on T cell recognition of DR7 and DR11 molecules, but little impact on quantitative peptide binding (8,18). In contrast, data obtained by Amicosante et al. (unpublished results) suggest that the charge of this residue should be important for the binding properties of pockets 4 and 6.

The data discussed above suggest that all HLA-DP substitutions analyzed in peptide binding may indirectly affect T cell recognition. Since none of the residues that we have mutated in the HLA-DP2 molecule are included in the 15 MHC II positions that are in direct contact with the TCR in the DR1–hemagglutinin–TCR complex (13), an alteration of the direct interaction between the DP molecule and the TCR to explain the absence of T cell allorecognition of several HLA-DP mutants is unlikely. Supporting this hypothesis, most of the residues of the HLA-DP2 molecule that we found to be involved in peptide binding plays a crucial role in T cell recognition. In some cases (DPß11 and 35) mutations could prevent binding of the adequate peptide in the groove or change the nature of the peptide presented, but the most likely situation should affect the conformation of the peptide presented within the antigen-binding groove of the MHC without substantial alteration of the overall affinity of the interaction. In agreement, two recent structural studies confirmed that subtle changes in allogenic MHC may alter the peptide conformation and location such that the same peptide is presented differently to the TCR (53).

In conclusion, the detailed analysis of the role of HLA-DP ß chain polymorphisms in the T cell recognition and peptide selectivity may help in understanding the role of this HLA class II isotype in the immune response as well as the mechanism of HLA-DP-associated susceptibility to immune disorders.


    Acknowledgements
 
This work was supported in part by the Comision Interministerial de Ciencia y Tecnología (grant SAF97-0134) and by the Comunidad Autónoma de Madrid (CAM)/Universidad Complutense (Proyectos de Grupos Estratégicos de la CAM-UCM). We would like to thank Dr Marshall for providing mAb, Dr Eric Long for providing cDNAs, and Rosa Pérez and María Isabel García for technical assistance. We are also in debt to Alberto Alvarez and Amalia Vázquez for their flow cytometry expertise. G. D. has a fellowship from the CAM.


    Abbreviations
 
CTL—cytotoxic T lymphocyte

LCL—lymphoblastoid cell line

MCF—mean channel fluorescence


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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