Structure-based design and evaluation of MHC class II binding peptides.

Structural information regarding binding of peptides to the major histocompatibility complex (MHC) class II molecule is of great use for the design of compounds that intervene in the interaction between the MHC-peptide-T-cell receptor (TCR) complex. These compounds can be applied in the treatment of T-cell-mediated auto-immune disease for specific modulation of the disease process. In case no crystal structure of the MHC molecule is available, homology models of the MHC molecule can be of importance. Here we describe the construction of a homology model of the MHC class II molecule and binding of the peptide, that are involved in experimental auto-immune encephalomyelitis, a rat model for human multiple sclerosis. The validity of the model was investigated using experimental data of peptides binding to this MHC molecule.

Induction of T cell anergy by liposomes with incorporated major histocompatibility complex (MHC) II/peptide complexes.

PURPOSE: The aim of this study was to use small unilamellar liposomes with incorporated MHC II/peptide complexes as a carrier system for multivalent antigen presentation to CD4 + T cells. METHODS: Purified peptide pre-loaded MHC II molecules were incorporated into small unilamellar liposomes and tested for their ability to activate A2b T cells. The outcome of T cell activation by such liposomes in the absence of accessory cells was tested via flow cytometry and a T cell anergy assay. RESULTS: Provided the presence of external co-stimulation, MHC II/ peptide liposomes were able to induce proliferation of the A2b T cell clone. More importantly incubation of these T cells with MHC II/ peptide liposomes in the absence of co-stimulation did not induce proliferation, however, a MHC/peptide ligand-density dependent down-regulation of the TCR was observed. Interestingly, when T cells after incubation with the MHC II/peptide liposomes were restimulated with their specific antigen in the presence of professional APC, these cells were anergic. CONCLUSIONS: We propose MHC II/peptide liposomes as a novel means to induce T cell anergy. The possibility to prepare 'tailor-made' liposomal formulations may provide liposomes with an important advantage for applications in immunotherapy.

Selection of T-cell epitopes from foot-and-mouth disease virus reflects the binding affinity to different cattle MHC class II molecules.

The major histocompatibility complex (MHC)-restricted selection of T-cell epitopes of foot-and-mouth disease virus (FMDV) by individual cattle MHC class II DR (BoLA-DR) molecules was studied in a direct MHC-peptide binding assay. By in vitro priming of T lymphocytes derived from animals homozygous for both MHC class I and II, five T-cell epitopes were analyzed in the context of three MHC class II haplotypes. We found that the presentation of these T-cell epitopes was mediated by DR molecules, since blocking this pathway of antigen presentation using monoclonal antibody TH14B completely abolished the proliferative responses against the peptides. To study the DR-restricted presentation of these T-cell epitopes, a direct MHC-peptide binding assay on isolated cattle DR molecules was developed. Purified cattle MHC class II DR molecules of the BoLA-DRB3*0201, BoLA-DRB3*1101, and BoLA-DRB3*1201 alleles were isolated from peripheral blood mononuclear cells. For each allele, one of the identified T-cell epitopes was biotinylated, and used as a marker peptide for the development of a competitive MHC-peptide binding assay. Subsequently, the T-cell epitopes of FMDV with functionally defined MHC class II specificity were analyzed in this binding assay. The affinity of the epitopes to bind to certain DR molecules was significantly correlated to the capacity to induce T-cell proliferation. This demonstrated at the molecular level that the selection of individual T-cell epitopes found at the functional level was indeed the result of MHC restriction.

Liposomes with incorporated MHC class II/peptide complexes as antigen presenting vesicles for specific T cell activation.

PURPOSE: The purpose of this study was to design a well-characterized liposomal carrier system for multivalent antigen presentation in order to activate T cells. METHODS: MHC class II molecules were loaded with peptide and subsequently reconstituted into liposomes. A FACS assay was developed to monitor peptide loading and MHC class II incorporation in the liposomes. For in vitro testing of the resulting MHC class II/peptide liposomes, a T cell hybridoma assay was employed. RESULTS: The FACS assay provided a qualitative means to visualize the amount of incorporated MHC class II and peptide molecules that were oriented in the appropriate way for antigen presentation to the T cells. Interestingly, when MHC class II molecules were loaded with the appropriate peptide prior to liposome incorporation, such liposomes were fully capable of inducing IL-2 production of a T cell hybridoma. CONCLUSIONS: This is the first article showing that MHC class II/peptide liposomes can serve as 'artificial antigen presenting cells' for activation of a CD4+ T cell hybridoma. As compared to soluble MHC class II/peptide complexes, the multivalency of liposomal complexes may be an important advantage when studying possible applications in immunotherapy.

The specificity of anti-HLA class II monoclonal antibodies in cattle.

At the Eleventh International HLA Histocompatibility Workshop, numerous anti-HLA class II monoclonal antibodies (mAb) were tested. For several of the polymorphic mAb, one epitope for binding has been mapped within the antigen-binding site of the class II molecules. Screening of the available bovine DRB3 and DQB exon 2 sequences revealed that some of the key amino acid (AA) motifs of these epitopes were present in cattle as well, and the question was raised whether this sharing of key AA motifs might cause interspecies cross-reactivity. Eight polymorphic anti-HLA class II mAb (seven anti-HLA DRB1 and one anti-HLA DQB) were selected for analysis of their reactivity towards bovine lymphocytes. In addition, the monomorphic anti-HLA class II mAb, 7.5.10.1, was selected for analysis, as this mAb was described to detect class II polymorphism in cattle. Flow cytometry and lymphocyte microcytotoxicity testing revealed that five of the polymorphic anti-HLA mAb were reactive with bovine lymphocytes. Furthermore, the anti-bovine reactivity of 7.5.10.1 was confirmed. These findings were supported by biochemical analysis. The anti-bovine reaction of the anti-HLA mAb did not correspond with the expected reaction, which was based on the presence of the AA, postulated to be responsible for recognition. Therefore, we suggest that the patterns of reactivity of the anti-HLA mAb are not always determined by one epitope.

Definition of an extended MHC class II-peptide binding motif for the autoimmune disease-associated Lewis rat RT1.BL molecule.

The Lewis rat, an inbred rat strain susceptible to several well-characterized experimental autoimmune diseases, provides a good model to study peptide-mediated immunotherapy. Peptide immunotherapy focussing on the modulation of T cell responses by interfering with TCR-peptide-MHC complex formation requires the elucidation of the molecular basis of TCR-peptide-MHC interactions for an efficient design of modulatory peptides. In the Lewis rat most autoimmune-associated CD4+ T cell responses are MHC class II RT1.BL restricted. In this study, the characteristics of RT1.BL-peptide interactions were explored. A series of substitution analogs of two Lewis rat T cell epitopes was examined in a direct peptide-MHC binding assay on isolated RT1.BL molecules. Furthermore, other autoimmune-related as well as non-disease-related T cell epitopes were tested in the binding assay. This has led to the definition of an extended RT1.BL-peptide binding motif. The RT1.BL-peptide binding motif established in this study is the first described rat MHC-peptide binding motif based on direct MHC-peptide binding experiments. To predict good or intermediate RT1.BL binding peptides, T cell epitope search profiles were deduced from this motif. The motif and search profiles will greatly facilitate the prediction of modulatory peptides based on autoimmune-associated T cell epitopes and the identification of target structures in experimental autoimmune diseases in Lewis rats.

THE SPECIFICITY OF ANTI-HLA CLASS II MONOCLONAL ANTIBODIES IN CATTLE.

At the Eleventh International HLA Histocompatibility Workshop, numerous anti-HLA class II monoclonal antibodies (mAb) were tested. For several of the polymorphic mAb, one epitope for binding has been mapped within the antigen-binding site of the class II molecules. Screening of the available bovine DRB3 and DQB exon 2 sequences revealed that some of the key amino acid (AA) motifs of these epitopes were present in cattle as well, and the question was raised whether this sharing of key AA motifs might cause interspecies cross-reactivity. Eight polymorphic anti-HLA class II mAb (seven anti-HLA DRB1 and one anti-HLA DQB) were selected for analysis of their reactivity towards bovine lymphocytes. In addition, the monomorphic anti-HLA class II mAb, 7.5.10.1, was selected for analysis, as this mAb was described to detect class II polymorphism in cattle. Flow cytometry and lymphocyte microcytotoxicity testing revealed that five of the polymorphic anti-HLA mAb were reactive with bovine lymphocytes. Furthermore, the anti-bovine reactivity of 7.5.10.1 was confirmed. These findings were supported by biochemical analysis. The anti-bovine reaction of the anti-HLA mAb did not correspond with the expected reaction, which was based on the presence of the AA, postulated to be responsible for recognition. Therefore, we suggest that the patterns of reactivity of the anti-HLA mAb are not always determined by one epitope.

A high recombination frequency within the chicken major histocompatibility (B) complex.

Chickens of a commercial pure White Leghorn line were typed for B-F and B-G by serological, biochemical and molecular biological methods. Amongst 287 typed animals of one particular line, three animals with recombinant haplotypes were identified. Compared to earlier reports this revealed a statistically significant (P < 0.05), tenfold higher recombination frequency in this chicken line.

Biochemical identification of B-F and B-G allelic variants of the chicken major histocompatibility complex.

Biochemical methods were used to analyse B-F and B-G antigens of the chicken major histocompatibility complex (MHC). In a panel of 12 inbred or partially inbred chicken lines the MHC haplotypes, originally defined by serological and histogenetical methods, were compared. Using monoclonal 18-6G2, allele-specific B-G patterns were obtained by immunoblotting. Comparison of B-G12 and B-G2 revealed a shared banding pattern, but additional products were detected for B-G12. The B-F products of B2 and B12 had identical IEF patterns. The identical B-F products and partially shared B-G products might explain the serological cross-reaction between these haplotypes. In addition, the IEF pattern of B-F21 appeared similar to B-F2 and B-F12, but the partial proteolysis map showed a clear difference. Although two B-F bands could be detected per haplotype, no evidence for the expression of more than one B-F locus was found. The biochemical methods enabled a precise definition of expressed MHC products and can be a useful tool for the identification of B-alleles in other chicken lines or outbred chickens for their MHC antigens.