Type I allergy either results from a pathological inappropriate Th2 immune response or from the defective induction of immune tolerance to otherwise harmless antigen : the allergen. Recent advances have underlined the major role played by allergen-specific CD4+ T cells in the control of allergic response. However, immunological mechanisms involved remain to be better documented and could provide important information regarding peripheral tolerance induction observed in healthy individuals. Examination of such T cells responses seem to be critical to improve current allergy vaccines with the assumption that allergen-specific immunotherapy should recapitulate such naturally protective immune response. In this doctoral thesis, we assess at single cell level allergen-specific CD4+ T cell responses both in allergic and healthy individual by using MHC class II peptide tetramer made with high-affinity immunodominant allergen T cell epitope. To this aim, we determine experimental conditions needed to ensure efficient and specific detection of rare allergen-specific T cell using this technology. Focusing on either the major allergen from birch pollen (Bet v 1) or major allergens from mite (Der p 1 and Der p 2), we demonstrate by using this approach, that peripheral tolerance to allergen observed in healthy individuals is associated with the presence and expansion of allergen-specific CD4+ T lymphocytes. We also describe methods to functionally characterize tetramer-positive cells and demonstrated that allergenspecific CD4+ T cells from healthy individuals mostly secrete IFN-у and IL-10 in response to the allergen and express high level of CTLA-4, a marker associated with regulatory T cells. In contrast, allergen-specific CD4+ T cells from allergic patients are bona fide Th2 cells confirming the role of such cells in the induction and amplification of allergic inflammatory responses. Developments of such technology provide valuable insight regarding the role of allergen-specific CD4+ T cells in both allergic and healthy individuals but also during desensitisation. This technology might be also extremely useful as patient-monitoring tools to assess the efficacy of various immunotherapeutic strategies in humans, possibly helping to identify surrogate biological markers of clinical efficacy.
