Betv 1 specific immune response in healh and disease

In the Schroedinger project J 2107-MED, we are interested in the major birch pollen Betv1 (Betula verrucosa 1) specific immune responses of healthy individuals and Betv1 allergic patients. So far, the comparisons included serological examination, immunohistopathology of tonsillar sections and flow cytometry analysis. Healthy and allergic individuals show Betv1 specific antibodies titers in their sera, however, allergen specific plasma cells can only be detected in palatine tonsils of allergic patients, even out of the pollen season. Here we show for the first time Betv1 specific plasma cells in human tonsillar tissue sections. These plasma cells have the capability of secreting Betv1 specific antibodies which bind the fluorescence labeled recombinant allergen. In tonsillar sections of healthy individuals no specific cells could be seen, although the individuals had repeated contact with the Betv1 antigen. The milieu within the tonsils of allergic patients might function as a survivla niche for repeatedly activated plasma cells. IgE positive B cells could also be stained and are located int he perifollicular areas, outside the germinal centers and the mantle zones. Morphologically, these IgE positive cells are plasma cells with a characteristic large antibody containing cytoplasm and an eccentrical nucleus. In allergic patients the occurence of IgE plasma cells is higher compared to healthy controls. The positive staining in the mantle zone in the healthy control may be due to FceRII/ CD 23 bound IgE antibodies, and is comparable between both groups. Flow cytometry analysis was employed to isolate allergen specific B cells from freshly extomized tonsils, as well as from peripheral blood of healthy and allergic individuals. We established a method to label the recombinant Betv1 protein with a flurorochrome, which enabled us to detect and isolate Betv1-binding cells by flow cytometry. Allergen specific B cells were isolated as single cells for further molecular analysis. In the second year we plan to describe the Betv1 specific antibody repertoire and analyze the specific variable regions of the heavy (VH) and light (VL) antibody chains by molecular techniques. Therefore, we want to answer the question which VH/ VL gene segments are used for allergen specific immune responses in healthy people and allergic individuals. We plan to clone the Betv1 specific VH and VL chains into phagemids and express them as Fab fragments on the surface of filamentous phages. Using surface plasmon resonance technology we can measure the affinities of the expressed Fab fragments and answer the question, whether Betv1 allergic patients select for antibodies which differ in their affinity from antibodies produced by healthy individuals. We further want to answer the longstanding question, whether allergen specific responses are of a mono- or polyclonal origin and/or if a specific Betv1 memory pool is established over time.