MHC-class II tetramers in allergy research

The prevalence of Type I allergies, i.e. IgE-mediated hypersensitivity reactions to innocuous substances is constantly rising. At present, the only causative treatment of this type of allergy is specific immunotherapy, i.e. repeated injections of high doses of allergen extracts. However the administration of entire allergens into sensitized individuals bears the risk of inducing severe IgE-mediated side effects. Therefore, safer and more effective approaches have been proposed. In this respect, the use of allergen-derived, short, linear peptides representing T cell epitopes has been considered as promising alternative to conventional immunotherapy. Such peptides are not able to cross-link IgE, but still capable of inducing tolerance in disease-eliciting CD4+ Th2- lymphocytes. The large diversity of T cell epitopes and the polymorphism of peptide-presenting HLA-molecules limited the prospect of success with this strategy for relevant allergens until now. We have recently identified mugwort pollen allergy as an ideal candidate for peptide immunotherapy. Art v 1 is recognized by >95% of mugwort sensitised individuals. It contains one single immunodominant T cell epitope presented by HLA-DRB1*01. In turn, the HLA-DRB1*01 haplotype is expressed by the majoritiy of mugwort-allergic patients. These preconditions make mugwort pollen allergy not only a useful model to develop peptide immunotherapy, but also to study allergen-specific immune responses ex vivo in freshly isolated cells from peripheral blood by using MHC-classIIetramers. These reagents are only recently available and allow the identification, characterization and enrichment of peptide-specific T cells. The major objectives of this project are: i) to introduce MHC-classII/peptide tetramers technology into allergy research in order to investigate allergen-specific T cell responses and immune mechanisms underlying tolerance. Fluorescence-labelled HLA-DRB1*01/peptide tetramers will be used to compare Art v 1-specific T cell responses of allergic and non-allergic individuals and to study alterations of the T cell response in patients undergoing conventional immunotherapy; ii) to develop a peptide-based vaccine for immunotherapy of mugwort pollen allergy. For this purpose, candidate peptides containing the immunodominant T cell epitope will be evaluated for immunological features, e.g. lack of IgE-binding and mediator release activity, but presence of T cell activating capacity. The most promising peptide candidates will be analyzed in vitro for their potency to induce tolerance in Art v 1-specific Th2 cells. In summary, the use of MHC-classIIetramers for ex vivo analysis of antigen-specific CD4+ T cells in allergic and non-allergic individuals will provide new insights in the pathomechanisms of Type I allergy. Monitoring the T cell response during conventional desensitization will elucidate immune mechanisms operative in peripheral tolerance such as deletion, anergy, active suppression or immune deviation.. In addition, a candidate vaccine for peptide immunotherapy in mugwort pollen allergy will be developed. These studies will also have implications for the development of tolerogenic vaccines in other branches of medicine.

Type I allergies are hypersensitivity reactions of the immune system to harmless substances (allergens) which are caused by allergen-specific IgE-mediated processes. Allergic diseases such as allergic rhinoconjunctivitis, atopic asthma and atopic dermatitis affect more than 20% of the population in Austria and other industrialized countries. Allergen-specific T lymphocytes play a central role in IgE-mediated allergies. In allergic individuals the T cell response to allergens is dominated by so-called T helper (h) 2 cells. Th2 cells produce high amounts of mediators that induce the production of allergen-specific IgE antibodies. Allergen-specific immunotherapy (SIT) is the only curative treatment for Type I allergy that down-regulates the disease-eliciting allergen-specific Th2 response. SIT consists in the repeated administration of increasing doses of allergens in order to achieve clinical tolerance to natural allergen exposure. At present, vaccines used for SIT consist of protein extracts of allergenic sources, e.g. pollen extract. Recombinant allergens (i.e. proteins produced in bacteria) and hypoallergenic variants (i.e. allergens with no/low IgE-binding and retained T cell activating capacity) are new therapeutic approaches towards safer and more effective vaccines for SIT. In this project we studied allergen-specific IgE- and T cell responses to important allergens in mugwort and ragweed-pollen, which are the major elicitors of pollen allergy in late summer. We found that the T cell response to the major allergen in mugwort pollen is strikingly uniform to only one single peptide region of the allergen and preferentially observed in individuals ex-pressing HLA-DR1 (a molecule that presents the peptide to the T cell antigen receptor, TCR). This finding allowed us to use mugwort pollen allergy as a model to investigate activation of T cells in detail (interactions of HLA, peptide and TCR), both on a molecular basis in vitro and in silico by computer simulation. We also established and optimized a method which applies a new tool, so-called HLA-class II tetramers, to identify allergen-specific T cells, and are now using it to study the immunological mechanisms underlying SIT. It is known that individuals sensitized to ragweed or mugwort pollen show clinical cross-reactivity to either allergen source. We found that the major ragweed pollen allergen has a higher allergenic potential than its related homolog in mugwort pollen at both, IgE antibody and T cell level. Nevertheless, the latter has sensitization capacity of its own and in regions with predominant mugwort pollen exposure the mugwort allergen could also be the primary sensitizing allergen. These immunological findings emphasize the strong allergenicity of the studied weed allergens. For the major ragweed- and mugwort allergens we developed variants and peptides which may be applied in SIT of weed pollen allergy in the future and may prove safer and more effective than the conventional allergen extracts being used at the moment.