The peptide binding motif of the disease associated HLA-DQ (α 1* 0501, β 1* 0201) molecule

To identify the binding motifs of peptides which bind to the celiac disease and insulin-dependent-diabetes-mellitus (IDDM)-associated DQ2 molecule, peptides were eluted from affinity-purified DQ2 molecules. The eluted peptides were separated by reverse-phase HPLC. Prominent peptide peaks and the remaining pool of peptides were sequenced by Edman degradation. Truncated variants of eight different peptides with a length of 9–19 amino acids were identified; among them class II-associated invariant chain peptides (CLIP) and peptides that stem from HLA class I α, HLA-DQα1*0501, Ig and CD20 molecules. Data from the pool sequencing and the biochemical binding analyses of synthetic variants of an eluted high-affinity ligand (HLA class I α 46–60), indicate that the side chains of amino acid residues at relative position P1 (bulky hydrophobic), P4 (negatively charged or aliphatic), P6 (Pro or negatively charged), P7 (negatively charged) and P9 (bulky hydrophobic) are important for binding of peptides to DQ2. Computer modeling of the DQ2 with variants of the high-affinity ligand in the groove suggests that peptides bind to DQ2 through the primary anchors P1, P7 and P9 and making additional advantageous interactions using the P4 and P6 positions.     

Naturally processed peptides from HLA-DQ7 (α1*0501-β1*0301): influence of both α and β chain polymorphism in the HLA-DQ peptide binding specificity

Self peptides bound to HLA-DQ7 (α1*0501-β1*0301), one of the HLA molecules associated with protection against insulin-dependent diabetes mellitus, were characterized after their acid elution from immunoaffinity-purified HLA-DQ7 (α1*0501-β1*0301) molecules. The majority of these self peptides derived from membrane-associated proteins including HLA class I, class II, class II-associated invariant chain peptide and the transferrin-receptor (TfR). By in vitro binding assays, the specificity of these endogenous peptides for HLA-DQ7 (α1*0501-β1*0301) molecules was confirmed. Among these peptides, the binding specificity of the TfR 215 – 230 self peptide was further examined on a variety of HLA-DQ and DR dimers. Several findings emerged from this analysis: (1) this peptide displayed HLA-DQ allelic specificity, binding only to HLA-DQ7 (α1*0501-β1*0301); (2) when either the DQα or DQβ chain was exchanged, little or no binding was observed, indicating that specificity of HLA-DQ peptide binding was determined by polymorphic residues of both the α and β chains. (3) Unexpectedly, the TfR 215 – 230 self peptide, eluted from DQ, was promiscuous with regard to HLA-DR binding. This distinct DR and DQ binding pattern could reflect the structure of these two molecules as recently evidenced by crystallography.     

The peptide-binding motif of HLA-DR8 shares important structural features with other type 1 diabetes-associated alleles

The objective of this study was to characterize the peptide-binding motif of the major histocompatibility complex (MHC) class II HLA-DR8 molecule included in the type 1 diabetes-associated haplotype DRB1(*)0801-DQA1(*)0401/DQB1(*)0402 (DR8-DQ4), and compare it with that of other diabetes-associated MHC class II alleles; DR8-bound peptides were eluted from an HLA-DR homozygous lymphoblastoid cell line. The repertoire was characterized by peptide sequencing using a LTQ ion trap mass spectrometer coupled to a multidimensional liquid chromatography system. After validation of the spectra identification, the definition of the HLA-DR8 peptide-binding motif was achieved from the analysis of 486 natural ligands, based on serial alignments of all possible HLA-DR-binding cores. The DR8 motif showed a strong similarity with the peptide-binding motifs of other MHC class II diabetes-associated alleles, HLA-DQ8 and H-2 I-A(g7). Similar to HLA-DQ8 and H-2 I-A(g7), HLA-DR8 preferentially binds peptides with an acidic residue at position P9 of the binding core, indicating that DR8 is the susceptibility component of the DR8-DQ4 haplotype. Indeed, some DR8 peptides were identical to peptides previously identified as DQ8- or I-A(g7) ligands, and several diabetes-specific peptides associated with DQ8 or I-A(g7) could theoretically bind to HLA-DR8. These data further strengthen the association of HLA-DR8 with type I diabetes.     

The variable endogenous retroviral insertion in the human complement C4 gene: a transmission study in type I diabetes mellitus

A variable endogenous retroviral element has been identified in intron 9 of the complement C4 gene [HERV-K(C4)], which maps to the class III region of the major histocompatibility complex (MHC) on chromosome 6p21.3. Genetic susceptibility to type I diabetes is mainly conferred by the MHC locus and the complement C4 region has been implied to contribute to human leukocyte antigen DQ (HLA-DQ) mediated disease risk. As the HERV-K(C4) insertion has been suggested to modulate expression of homologous genes, we investigated its transmission in 220 families with an offspring affected by type I diabetes as a potential disease susceptibility marker. There was no preferential transmission of the HERV-K(C4) insertion to affected offspring (P(TDT) = 0.79). Although 77.7% of HLA-DQ8 carried the HERV-K(C4) insertion, only 52.9% of -DQ2 haplotypes did (P(chi(2)) < 0.01). However, its insertion or deletion did not modulate the risk conferred by HLA-DQ8 (DQA1*0301-DQB1*0302) (P(chi(2)) = 0.27) or -DQ2 (DQA1*0501-DQB1*0201) (P(chi(2)) = 0.46). Thus, the HERV-K(C4) insertion is not associated with type I diabetes in Germans.     

The HLA-DRB1*0405 haplotype is most strongly associated with IDDM in Algerians.

Insulin-dependent diabetes mellitus (IDDM) in Caucasians is strongly associated with HLA-DR3-DQ2 and DR4-DQ8. In order to investigate the HLA class II associations with IDDM in Algerians, we have used polymerase chain reaction (PCR) and sequence specific oligonucleotide analysis (SSO) to identify DQA1, DQB1, and DRB1 alleles, haplotypes and genotypes in 50 unrelated IDDM patients and 46 controls from a homogeneous population in Western Algeria. Both DRB1*0301-DQA1*0501-DQB1*0201 (DR3-DQ2) and DRB1*04-DQA1*0301-DQB1*0302 (DR4-DQ8) haplotypes were found at increased frequencies among the patients compared to controls (45% vs. 13%, RR = 5.5, Pc < 10(-5) and 37% vs. 4%, RR = 12.9, Pc < 10(-4), respectively). Among the latter, in contrast to other Caucasian populations, only DRB1*0405-DQA1*0301-DQB1*0302 was significantly increased in the Algerian patients (25% vs. 1% in controls, RR = 30.3, Pc < 10(-3). Accordingly, the highest risk of disease was observed in DRB1*0301-DQA1*0501-DQB1*0201/DRB1*0405-DQA1+ ++*0301-DQB1*0302 heterozygotes (34% in patients vs. 0% in controls; RR = 49; Pc < 10(-3). This observation and its comparison with DR-DQ haplotypes in other ethnic groups suggest that the DRB1*0405 allele which encodes an Asp57-negative beta chain may contribute to IDDM susceptibility in a similar way as Asp57-negative DQ beta chains.     

A Peptide-Binding Assay for the Disease-Associated HLA-DQ8 Molecule

The study of peptide binding to HLA class II molecules has mostly concentrated on DR molecules. Since many autoimmune diseases show a primary association to particular DQ molecules rather than DR molecules, it is also important to study the peptide-binding properties of DQ molecules. Here we report a biochemical peptide-binding assay for the type I diabetes-associated DQ8, i.e. DQ (α1*0301, β1*0302), molecule. Affinity-purified DQ8 molecules were tested in peptide-binding assays using a radiolabelled influenza haemagglutinin (Ha) peptide encompassing positions 255–271(Y) as an indicator peptide. The Ha 255–271(Y) peptide bound to DQ8 in a pH-dependent fashion showing optimal binding around pH 5. The association kinetics were relatively slow and the resulting complexes were heat labile. The specificity of peptide binding to DQ8 was investigated in competitive inhibition experiments with a panel of 43 peptides of different lengths and sequences. The DQ8 molecules showed a different pattern of peptide binding compared to a previously studied DQ2 molecule. Peptides derived from thyroid peroxidase, HLA-DQ(α1*0301), HLA-DQ(α1*0302), retinol receptor and p21ras were among the high-affinity binders, whereas peptides derived from myelin basic protein were among the low-affinity binders. The sequence of the high-affinity peptides conformed with a previously published peptide-binding motif of DQ8.     

Immune response of HLA-DQ transgenic mice to house dust mite allergen p2: identification of HLA-DQ restricted minimal epitopes and critical residues

HLA-DQ8 (HLA-DQA1*0301; HLA-DQB1*0302) and HLA-DQ6 (HLA-DQA1*0103; HLA-DQB1*0602) genes were introduced into mouse class II (H-2A(o)(beta)) knockout mice. Transgenic HLA-DQ8 and HLA-DQ6 mice were individually immunized and challenged using synthetic peptides representing HDM (Dermatophagoides pteronyssinus) allergen p2. HLA-DQ8 mice responded to p2 peptides 1-20, 41-60, 51-70, 61-80, 91-110, and 101-120. HLA-DQ6 mice responded to peptides 1-20, 11-30, 21-40, 41-60, and 51-70. Using single amino acid truncated 30-mer peptides, residues necessary for HLA-DQ8 recognition were identified spanning regions 3-12, 50-70, and 91-120. A synthetic peptide comprising residues 3-12 was synthesized and a series of single alanine substitutions was introduced into the minimal peptide. Introduction of alanine residues at positions 3, 11, and 12 resulted in a significant loss of immune recognition. It was concluded that residues 4, 5, 7, 11, and 12 are critical for immune recognition by HLA-DQ8 mice.     

An allo-specific peptide derived from HLA-A2 is presented by HLA-DQ7

The peptide motifs of two HLA class II molecules, DR11 and DQ7, were determined from natural peptides. The EBV transformed B cells JVM (HLA-A2-DRB1^*1102-DQA1^*0501-DQB1^*0301) were cultured to a final yield of 2 10¹⁰ cells. DR11 and DQ7 molecules were immunopurified and peptides were extracted after acid elution and separated by reverse-phase HPLC. Five peptides from DR11 and five from DQ7 were sequenced using Edman degradation and other peptides were analysed by pool sequencing. Peptides were from 11 to 15 amino acids in length and P often occurred at the second residue for both DR5 and DQ7. The peptide motif for DR11 was I at position i and K or R at position i + 4. For DQ7 the most significant signal was A in the middle of the peptide as also described by Falk et al. The source of one peptide eluted from DQ7 was a polymorphic part of the HLA-A2 heavy chain (from 56th to 69th amino-acid). It was also exactly the same peptide than the synthetic peptide used by Krensky et al in the 10th international workshop to modulate lysis by HLA-A2-specific cytotoxic T lymphocytes. The biochemical characterisation of this peptide from HLA-DQ7 strongly supports functional tests showing an indirect presentation of alloantigens by MHC molecules.     

Structure of celiac disease-associated HLA-DQ8 and non-associated HLA-DQ9 alleles in complex with two disease-specific epitopes

The association of celiac disease (CD) with HLA-DQ2 and HLA-DQ8 is indicative of preferential mucosal T cell recognition of gluten fragments bound to either DQ allele. We have recently identified two gluten-derived, HLA-DQ8-restricted T cell stimulatory peptides, one each from gliadin and glutenin, recognized by specific T cell clones derived from the small intestine of CD patients. We have now performed molecular modeling and examined the fine specificity of these peptides in complex with HLA-DQ8. There is only one binding register for both peptides, with glutamine residues at the p1 and p9 anchor positions. Both T cell clones recognize substituted peptides at p1 and p9, but poorly so at p2-p8, especially the gliadin-specific clone. Contrasting patterns of recognition of p9Gln --> Glu peptide variants (both predicted as better DQ8 binders by modeling) were observed: enhancement of recognition for the gliadin peptide, yet complete absence thereof for the glutenin peptide. The double-substituted gliadin peptide variant p1/9Gln --> Glu, which can also arise by pepsin/acid/transglutaminase treatment, shows a considerable increase in sensitivity of recognition, consistent with better binding of this peptide to DQ8, as predicted by energy minimization. Surprisingly, the two native peptides are also recognized by their respective T cell clones in the context of the related molecule HLA-DQ9 (beta57Asp(+)). The p1/9Gln --> Glu gliadin peptide variant is likewise recognized, albeit with a 10-fold lower sensitivity, the first reported p9Glu binding in a beta57Asp(+) MHC II allele. Our results have important implications for the pathogenesis of autoimmune disease and the possible manipulation of aberrant responses thereof.     

Polarity of the P1 anchor residue determines peptide binding specificity between HLA-A*3101 and HLA-A*3303

A previous pool sequence analysis showed that HLA-A*3101 and HLA-A*3303 binding peptides have the same anchor residues at P2 and the C-terminus, the only difference being that HLA-A*3303 binding peptides have two additional P2 anchor residues. Using a stabilization assay with RMA-S transfectants expressing HLA-A*3101 and human beta2-microglobulin, we tested the binding of 232 8- to 11-mer peptides carrying HLA-A*3303 anchor residues to HLA-A*3101. One hundred of these peptides (43.1%) bound to HLA-A*3101, confirming that these residues are also anchors for HLA-A*3101. Although aromatic hydrophobic P2 residues were previously shown to be stronger anchors than aliphatic hydrophobic P2 residues in HLA-A*3303 binding peptides, we detected no significant difference in HLA-A*3101 binding affinity between peptides carrying aromatic or aliphatic hydrophobic P2 residues. Statistical analysis previously showed a positive effect of negatively charged P1 residues and a negative effect of positively charged P1 residues for peptide binding to HLA-A*3303. In contrast such analysis demonstrated a positive effect of positively charged P1 residues and a negative effect of negatively charged P1 residues for peptide binding to HLA-A*3101. Analysis using mutated peptides confirmed these results. The present study therefore demonstrates that peptide binding specificity between HLA-A*3101 and HLA-A*3303 is determined by the polarity of the P1 anchor residue.     

Pathomechanisms in celiac disease

Celiac disease is a complex autoimmune disease which is characterized by a strong genetic association (HLA-DQ2 or -DQ8), gluten as nutritional etiological factor, and the enzyme tissue transglutaminase as endomysial autoantigen. Patients develop highly predictive IgA autoantibodies to tTG. Certain gluten peptides are presented by the disease-associated HLA-DQ2/DQ8 molecules leading to stimulation of gluten-specific T cells. This immune response which is driven in the lamina propria causes the mucosal transformation characteristic for celiac disease. Increased intestinal expression of tTG in patients with CD appears to play an important role in the pathogenesis of CD. Thus, modification of gluten peptides by tTG, especially deamidation of certain glutamine residues, can enhance their binding to HLA-DQ2 or -DQ8 and potentiate T cell stimulation. Furthermore, tTG-catalyzed cross-linking and consequent haptenization of gluten with extracellular matrix proteins allows for storage and extended availability of gluten in the mucosa. New therapeutic approaches aim at proteolytic destruction of immunodominant gliadin peptides that are resistant to intestinal enzymes by bacterial prolyl endopeptidases, the inhibition of tTG activity with highly specific enzyme inhibitors or at HLA-DQ2/DQ8 blocking peptide analogues.     

THE HLA-DRB1*0405 HAPLOTYPE IS MOST STRONGLY ASSOCIATED WITH IDDM IN ALGERIANS

Insulin-dependent diabetes mellitus (IDDM) in Caucasians is strongly associated with HLA-DR3-DQ2 and DR4-DQ8. In order to investigate the HLA class II associations with IDDM in Algerians, we have used polymerase chain reaction (PCR) and sequence specific oligonucleotide analysis (SSO) to identify DQA1, DQB1, and DRB1 alleles, haplotypes and genotypes in 50 unrelated IDDM patients and 46 controls from a homogeneous population in Western Algeria. Both DRB1*0301-DQA1*0501-DQB1*0201 (DR3-DQ2) and DRB1*04-DQA1*0301-DQB1*0302 (DR4-DQ8) haplotypes were found at increased frequencies among the patients compared to controls (45% vs. 13%, RR = 5.5, P_c < 10^-5 and 37% vs. 4%, RR = 12.9, P_c < 10^-4, respectively). Among the latter, in contrast to other Caucasian populations, only DRB1*0405-DQA1*0301-DQB1*0302 was significantly increased in the Algerian patients (25% vs. 1% in controls, RR = 30.3, P_c < 10^-3). Accordingly, the highest risk of disease was observed in DRB1*0301-DQA1*0501-DQB1*0201/DRB¹*0405-DQA1*0301-DQB1*0302 heterozygotes (34% in patients vs. 0% in controls; RR = 49; P_c < 10^-3). This observation and its comparison with DR-DQ haplotypes in other ethnic groups suggest that the DRB1*0405 allele which encodes an Asp57-negative β chain may contribute to IDDM susceptibility in a similar way as Asp57-negative DQβ chains.     

Biochemical characterization of the human diabetes-associated HLA-DQ8 allelic product: Similarity to the major histocompatibility complex class II I-Ag7 protein of non-obese diabetic mice

The human HLA-DQ8 (A1*0301/B1*0302) allelic product manifests a strong association with insulin-dependent diabetes mellitus (IDDM). Previous biochemical studies of the major histocompatibility complex (MHC) class II I-A^g7 protein of IDDM-prone non-obese diabetic mice produced controversial results. To better define the biochemical properties of IDDM-associated MHC class II molecules, we analyzed DQ8 proteins, in comparison to other DQ allelic products, by partially denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We now report that DQ8 proteins have a normal peptide occupancy and lifespan in cells. Similar to I-A^g7, DQ8 proteins formed only a minor fraction of SDS-stable complexes with peptides. Although this phenotype was not unique to DQ8, some DQ allelic products such as IDDM-protective DQ6 proteins were SDS resistant. The DQ9 allelic product, differing from DQ8 only at position (P) β57, was SDS stable, suggesting that non-Asp residues at β57 might decrease the SDS stability of DQ proteins. We identified a single peptide which specifically induced an SDS-stable conformation in DQ8 as well as in I-A^g7 molecules. The residues at anchor P1 in this peptide were found to influence the SDS stability of both molecules. Together with our previous observation of similar binding motifs of I-A^g7 and DQ8, these results demonstrate an overall biochemical similarity of mouse and human diabetes-associated MHC class II molecules. This similarity might contribute to a common immunological mechanism of IDDM in both species.     

Binding of gluten-derived peptides to the HLA-DQ2 (α1*0501, β1*0201) molecule,assessed in a cellular assay

The nature of the immunopathogenic relationship underlying the very strong association of coeliac disease (CD) to the HLA-DQ (A1*0501, B1*0201) genotype is not known, but probably relates to binding of gluten-derived epitopes to the HLA-DQ (α1*0501, β1*0201) heterodimer (DQ2). These epitopes have not yet been defined. In this study we have tested the binding of various gluten-derived peptides to DQ2 in a cellular assay using Epstein–Barr virus (EBV)-transformed B lymphocytes and murine fibroblast transfectants. One of these peptides (peptide A), which has previously been shown to exacerbate the CD lesion in vitro and in vivo, was found to bind to DQ2, albeit only moderately, lending further credence to its possible role in the pathogenesis of CD. The nature of peptide A's binding to DQ2 was explored with truncated and conservative point substituted analogues and compared with the published DQ2 binding motif, the results of which explain the observed level of binding.     

Specificity and sequence comparison of naturally processed peptides from HLA-DQA10501-DQB10301 and HLA-DQA10301 DQB10301: Influence of the DQα chain?

Because HLA-DQα and β chains are polymorphic and the implication of certain DQαβ dimers in a variety of autoimmune diseases, sequence information of self peptides, bound to DQαβ dimers sharing β chain and linked to autoimmune disease, provides an approach toward understanding the mechanism of these diseases at the molecular level and the implication of DQα chain in peptide binding.Self peptides bound to immunoaffinity purified HLA-DQ7 molecules (DQA1^*0501-DQB^*0301) and (DQA^*0301-DQB1^*0301) expressed, respectively, in EBV cell lines Sweig and JHAF, were isolated and characterized. The bound peptides were released by acid elution and separated by HPLC. The specific predominant peaks of HLA-DQA1^*0501-DQB^*0301 were sequenced by Edman microsequencing. Twelve complete sequences were derived from 5 sources proteins including MHC associated molecules and other integral membrane proteins. The peptides varied in length from 9 to 25 amino acids and, contained nested sets with ragged N- and/or C-terminal ends. The alignment of peptides sequences according to the structure of HLA-DQA1^*0501-DQB^*0301 allowed the definition of a putative motif corresponding to aromatic residues (F, Y or W) at position i, and aliphatic residues (A, V or L) at position i+5 and/or i+6. The binding of these peptides to different DQ molecules on cell surface, by FACS analysis, is in progress. Differences in peptide motifs bound to (DQA1^*0501-DQB^*0301) and (DQA^*0301-DQB1^*0301) will be discussed in order to assess the influence of the DQα chain polymorphism on the motif and peptides bound to DQαβ dimers.     

HLA-DQ and DRB1 polymorphism and susceptibility to type 1 diabetes in Jamaica.

Genetic susceptibility to type 1 diabetes is determined by a combination of HLA-DQ and DRB1 alleles. In the present study, HLA associations with type 1 diabetes were investigated in the Jamaican population. DRB1 and DQ genotyping was performed on 45 type 1 diabetic patients and 132 control subjects born and resident in Jamaica. The small number of patients available for study reflected the low prevalence of type 1 diabetes in Jamaica. The results were compared with those from other African heritage populations and white Caucasians. The highest relative risk was associated with the DRB1*03-DQ2/DRB1*04-DQ8 genotype. Both DRB1*0401-DQ8 and DRB1*0408-DQ8 were positively associated with disease. DRB1*0408-DQ8 is uncommon amongst white Caucasians, where DRB1*0401-DQ8 is the major predisposing haplotype. The DRB1*1503-DQ6 haplotype was associated with protection from diabetes in the Jamaican population. This haplotype is rare amongst white Caucasians, where DRB1*1501-DQ6 is the protective haplotype. Data from African heritage populations suggest that DRB1*1503-DQ6 might be less protective than DRB1*1501-DQ6. DRB1*03-DQA1*0401-DQB1*0402 was associated with protection from diabetes in the Jamaican population, whereas in white Caucasians DRB1*08-DQA1*0401-DQB1*0402 is predisposing. These data demonstrate that comparison of genetic associations with type 1 diabetes in races with population-specific DRB1-DQ haplotypes provides new information as to the exact determinants of disease susceptibility. Further support is provided for roles of the DQ genes and the DRB1 gene (or a gene in linkage disequilibrium with it) in determining susceptibility to type 1 diabetes.     

T cell response pattern to glutamic acid decarboxylase 65 (GAD65) peptides of newly diagnosed type 1 diabetic patients sharing susceptible HLA haplotypes.

Autoantibodies and autoreactive T lymphocytes directed against several pancreatic beta cell proteins such as GAD65 have been identified in the circulation before and at the onset of clinical type 1 (insulin-dependent) diabetes. Using GAD65 synthetic peptides, we studied the proliferative response of peripheral blood mononuclear cells (PBMC) either from recently diagnosed type 1 diabetic patients, of whom the majority share the disease-associated HLA class II haplotype (DR4-DQB1*0201 or DR3-DQB1*0302), or from HLA-matched control subjects. We found that 67% (14/21) of the type 1 diabetic patients and 39% (9/23) of the control subjects exhibited a positive proliferative response. Compared with control subjects, however, PBMC from diabetic patients proliferated more frequently (P < 0.05) in the presence of peptide pools from the C-terminal region of GAD65 (amino acids 379-585). Diabetic patients with the same HLA-DQ or HLA-DR alleles showed partially identical T cell reactivity, but no clear correlation could be made between MHC class II specificity and T cell epitopes because of multiple combinations of class II alleles. In addition, by flow cytometry, we studied the direct binding of GAD65 peptides to MHC class II molecules of Epstein-Barr virus (EBV)-transformed B (EBV-B) cells obtained from a diabetic patient. We found that 11 GAD peptides were able to bind to the highly susceptible haplotype DRB1*0301/0401-DQA1*0301/0501-DQB1*0302/0201 on the surface of EBV-B cells in partial correlation with the results obtained in the proliferation assays.     

A peptide from the known HLA class I restricted melanoma-specific tumor antigen GP100 is presented by HLA-DR in the melanoma cell line FM3

A number of HLA-class I restricted melanoma tumor antigens such as MAGE-1, MAGE-3, MART-1/Melan-A or gp100 have been characterized but there is thus far no direct proof if peptides derived from these antigens are also presented in vivo by HLA-class II molecules. To examine this we cultured, up to 1 × 10¹⁰ cells of the constitutively class II and CD80 expressing melanoma cell line FM3 (DR B1^*0401, DR B1^*15x, DQ B1^*0301, DQ B1^*0602) that has been shown to be able to serve as antigen presenting cells, lysed the cells, isolated the HLA-DR and HLA-DQ by immunoaffinity chromatography and released the MHC-bound self peptides by a shift to pH 2. The peptide pool was separated by HPLC using a C18 reversed phase column. The peaks were collected and tested for purity by matrix assisted laser desorption spectrometry (MALDI). Homogeneous peaks were sequenced by Edman degradation, inhomogeneous peaks were further purified by rechromatography before submitting to the sequencer.

In this way we were able to find amongst other peptides a HLA-DR-bound 16mer peptide derived from the melanocyte specific protein gp100 with the sequence WNRQLYPEWTEAQRLD. To prove the specific binding of this peptide to one of the two DR alleles we synthesized the peptide and submitted it to our in vitro peptide binding/competition assay using gel filtration, isolated HLA-DR alleles and a fluorescently labeled peptide. This is the first reported HLA-DR-restricted epitope of a known tumor-associated antigen in melanoma.     

The function of tissue transglutaminase in celiac disease

Celiac disease is a chronic small bowel disorder caused by an abnormal immune response to an array of epitopes of wheat gluten and related proteins of rye and barley in genetically susceptible individuals who express the HLA-DQ2/-DQ8 haplotype. Gluten peptides are efficiently presented by celiac disease-specific HLA-DQ2- and HLA-DQ8-positive antigen presenting cells to CD4(+) T-cells that, once activated, drive a T helper cell type 1 response leading to the development of the typical celiac lesion-villous atrophy, crypt hyperplasia and intraepithelial and lamina propria infiltration of inflammatory cells. Tissue transglutaminase (tTG) is a calcium dependent ubiquitous enzyme which catalyses posttranslational modification of proteins and is released from cells during inflammation. tTG is suggested to exert at least two crucial roles in celiac disease: as a deamidating enzyme, that can enhance the immunostimulatory effect of gluten, and as a target autoantigen in the immune response. Since glutamine-rich gliadin peptides are excellent substrates for tTG, and the resulting deamidated and thus negatively charged peptides have much higher affinity for the HLA-DQ2 and HLA-DQ8 molecules, the action of tTG is believed to be a key step in the pathogenesis of celiac disease. This review is focused on the function of tTG in celiac disease, although it also deals with novel advances in tTG-based therapies.     

Motif analysis of HLA class II molecules that determine the HPV associated risk of cervical carcinogenesis

In previous studies, the HLA class II haplotype HLA DRB1*0401-DQB1*0301 was shown to correlate with susceptibility to HPV infection, CIN and cervical cancer while DRB1*0101-DQB1*0501 indicated protection. The present study was designed to identify naturally processed peptide sequences bound to the susceptibility and protective HLA DR-DQ molecules, and use this for T-helper epitope prediction from HPV 16. The HLA class II molecules were obtained by immuno-affinity purification of Epstein-Barr virus B lymphoblastoid cell lines (BCL) homozygous for HLA DQA1*0301-DQB1*0301 and HLA DQA1*0101-DQB1*0501. Peptide pools eluted from the HLA molecules were sequenced by Edman degradation. On the basis of the peptide sequence data obtained, the E6, E7, L1 and L2 proteins of HPV 16 were examined to identify sequences which are likely to bind to HLA DQB1*0301 and DQB1*0501. In addition, motif prediction as well as the binding affinity of predicted peptide motifs for HLA DRB1*0401 and DRB1*0101, the DR alleles associated with susceptibility and protection respectively, was accomplished using published data and a prediction algorithm for the naturally processed peptide sequences bound to these molecules. The HLA DQB1*0501 peptide ligand sequence showed that proline gives an outstanding signal at position 2, Asn/Arg at P1, aliphatic/aromatic amino acids in the central portion, a hydrophobic cluster at P5 with a small contribution by small polar residues and another cluster of aromatic residues towards the C-terminus. The HLA DQB1*0301 sequence also showed that proline gives an outstanding signal at position 2, Thr/Arg at P1, aliphatic/aromatic amino acids in the central portion and an aliphatic cluster with a small contribution by small polar residues at P5. There were no differences in the number of HPV peptides that were predicted as being capable of binding to HLA DQB1*0301 and HLA DQB1*0501, but more HPV peptide motifs were predicted to bind with high affinity to HLA DRB1*0101 than DRB1*0401. The results suggest that HPV 16 peptide epitopes bind with higher affinity to the protective than to susceptible HLA DR-DQ molecules which may lead to a more effective immune response.