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Project Description and Aims
Effective allergen-specific immunotherapy is known to induce several immunological changes that correlate well with clinical efficacy, including increased levels of allergen-specific IgG and decreased effector cell (basophil, mast cell) responsiveness. These changes precede any decrease in specific IgE and this correlation has led to the hypothesis that the induced IgG inhibits IgE-mediated effector cell activation. Two mechanisms of IgG-mediated inhibition, which are not mutually exclusive, have been proposed: blocking of IgE binding by direct competition for allergen epitopes and IgG-allergen complex engagement of the inhibitory FcγRIIb receptor on effector cells leading to down regulation of FcεRI signaling. Murine studies have suggested that IgG-mediated inhibition of anaphylaxis involves both mechanisms.
A third mechanism of effector cell hypo-responsiveness, termed ‘desensitization’, may also be clinically important, especially during the early phases of immunotherapy during which doses of allergen – initially below the threshold for eliciting reactions – are rapidly increased to an effective dose that is above that threshold. The induction of a transient refractory or ‘desensitized’ state by suboptimal doses of allergen has been well studied in vitro using both basophils and mast cells and this is thought to directly relate to the mechanism of in vivo desensitization.
Basophils are ideal for the study of immunotherapy mechanisms for several reasons. They are predominantly present in the peripheral blood, can be readily stimulated ex vivo, share expression of both receptor and signaling pathway molecules with mast cells, and can be measured by flow cytometry with minimal manipulation.
We have the opportunity to conduct a small, self-contained research project that complements an already funded large prospective clinical study of sublingual immunotherapy for food allergy. This project has the potential both to give new and unique insight into the mechanisms of sublingual immunotherapy and to establish innovative methods for the assessment of basophil activation by direct measurement of intracellular phosphorylation of signaling molecules.
Our central hypothesis is that immunotherapy down-regulates effector cell function by inhibiting FcεRI signaling. This will be tested in the following three aims.
Aim 1. We will test the hypothesis that IgG antibodies play an important function in suppressing basophil activation during sublingual immunotherapy in food allergic patients. We will test this by measuring ex vivo allergen activation of basophils from allergic subjects at serial time points with or without prior depletion of total or allergen-specific IgG. We expect that if allergen-specific IgG plays a role in basophil suppression, basophil activation will be enhanced following the depletion of specific IgG.
Aim 2. We will investigate the hypothesis that allergen-specific IgG inhibits basophil responsiveness in vivo via the FcγRIIb inhibitory receptor. We will test this by the direct measurement of the phosphorylation status of four key signaling molecules downstream of FcεRI and FcγRIIb – SYK, PI3K, BTK and PLCγ in patient basophils by intracellular staining and flow cytometry after activation with or without prior depletion of IgG. We will compare the activation status of these signaling molecules to degranulation. We expect that this novel method of measuring basophil activation will allow us to differentiate between an IgG-dependent global reduction of cell signaling activity, consistent with blocking of IgE binding, versus an IgG-dependent increase of inhibitory signaling, consistent with a role for FcγRIIb-mediated suppression of basophil activation.
Aim 3. We will test the hypothesis that basophil desensitization induces weak FcεRI signaling sufficient for proximal signaling events and homeostatic down regulation leading to a refractory state characterized by degradation of SYK. We will also test the hypothesis that SLIT-induced clinical desensitization leads to the same basophil phenotype ex vivo. We will use basophils that have been passively sensitized with NPspecific IgE to measure phosphorylation of SYK, SHP-2 and PI3K during induction of desensitization with sub-optimal stimulation. We will measure the levels of total SYK on a per cell basis over time following suboptimal stimulation. We will compare this to the phenotype of patient basophils ex vivo during clinical desensitization. We expect to determine whether clinical desensitization induces the same intracellular signaling pathways as in vitro desensitization.