Defining the immunoglobulin (Ig) switch to IgE in glycan-specific B cell responses using the model disease of anaphylaxis to galactose-alpha-1,3-galactose

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Up to now, carbohydrate-specific immune responses are insufficiently understood. In the scope of this proposal, we investigate the immunological mechanisms of carbohydrate-specific B cell responses using the model antigen galactose-alpha-1,3-galactose ("alpha-gal"). Alpha-is ubiquitously expressed and all mammals except of old-world monkeys and humans express this carbohydrate. Consequently, alpha-gal is recognized as non-self by the latter and it is known that after tick bites, alpha-gal specific IgE antibodies can be induced potentially eliciting life-threatening allergic reactions upon contact with alpha-gal expressing drugs or red meat. Interestingly, a substantial amount of alpha-gal specific IgM and IgG is present in all humans independent of this type of alpha-gal sensitization and is believed to contribute to the protection e.g. against parasite infections and to belong to the pool of ‘natural’ antibodies derived from B-1 cells. For more detailed investigations, we established a mouse model in which alpha-gal specific IgE antibodies and anaphylaxis after alpha-gal exposure develop upon percutaneous sensitization to alpha-gal. Using this model, however, we could show an expansion of alpha-gal specific B-2 cells as well as germinal center B cells with dominant IgG1 but absent IgE expression, indicating for the first time that carbohydrate-specific IgE responses arise through sequential class switching involving an IgG1 intermediate stage. Following up on these observations, we will perform in-depth analysis of alpha-gal specific B cells and, among others, decipher the role of specific B cell populations using transfer into B cell deficient mice. B cell receptor repertoire analyses and ex vivo class switching approaches of different isotypes in the mouse model as well as in alpha-gal allergic patients and controls will be used to elucidate the generation of alpha-gal IgE via an IgG1 intermediate stage. In the second part of the proposal, we will for the first time investigate the role of type 2 immunity in carbohydrate antigen, in our case alpha-gal, induced IgE class switching. To this end, we will use IL-4 reporter mice to decipher the type 2 immune cascade using single-cell RNA sequencing. The role of follicular and type 2 helper T cells in alpha-gal specific IgE responses and anaphylaxis will be investigated using adoptive transfer of T cell subsets into T cell deficient mice. In summary, this project will help us to decipher the so far insufficiently understood immunological mechanisms underlying high affinity carbohydrate-specific humoral immune responses