On the Origin of Immunoglobulin E's: Elucidating the Immunological Mechanisms Underlying Mucosal IgE Responses in type2 Disease

Work Package 1 Rationale: High levels of IgE can be detected locally in the nasal cavity and nasal polyps from patients with CRSwNP. Given the high levels of polyreactive IgE generated in NP, it is intriguing to speculate how NP IgE responses differ from allergic IgE B cell responses. For this, a high-resolution analysis of the mechanisms leading to IgE producing B cells in both settings is needed but has to date not been performed.

Milestone 1.1: To fully characterize the IgE B cell niche in CRSwNP and allergic rhinitis, the investigators will collect multiple samples from 25 CRSwNP, 25 CRSsNP and 25 allergic rhinitis patients with HDM allergy. In addition, tonsil biopsies as a proxy for systemic immune responses will be collected. From 5 HDM allergic patients with high IgE levels nasal tissue (inferior concha) and tonsil biopsies isolated and subject these tissues to thorough characterization of the IgE B cell niche.

Milestone 1.2: In this milestone, the investigators will set out to obtain a high detail map of IgE B cell responses and how they are governed in the type2 niche in CRSwNP and allergic rhinitis. To this end, a high-resolution single cell map of the nasal polyp , inferior concha and tonsil tissue will be generated via the 10x genomics 5'VDJ seq protocol. By creating a high-resolution map of the B cell subsets and their immune receptor profiles, the investigators aim to understand how chronic type2 IgE B cell responses are governed and driven in non-allergic and allergic type2 disease. For this analysis DALI (Digital Addressable lighting Interface) bioinformatic software package will be used. The investigators will here specifically aim to trace the origins of IgE+ plasmablast or plasma cell clones through germinal center responses, or directly from naïve B cells in extrafollicular responses. In addition, NicheNet analysis will allow for the detection of regulators of IgE B cell responses, such as other immune cells or structural cells. This software tool analyzes potential sender-receiver pairs for cytokines, chemokines, and cellular ligands, based on differential gene expression and curated receptor-ligand lists.

Work package 2: While high levels of polyreactive IgE can be detected in nasal secretions and tissues of patients with CRSwNP, it is unclear whether class switch recombination to IgE is driven by antigen or by damage associated factors.

Milestone 2.1: To date, the characteristics of IgE in NP remain enigmatic, yet these IgE's are key to understand their role in disease biology. To elucidate the characteristics of NP IgE, the investigators will generate 20 monoclonal IgE antibodies from each of 5 patients with CRSwNP and similarly 20 monoclonal IgE antibodies from each of 5 patients with allergic rhinitis will be generated. By this, the full-length heavy and light chain sequences of IgE+ clones will be retrieved, and have these produced as recombinant monoclonal antibodies. In addition, NP IgE antibodies possess polyreactivity will be assessed, rendering them able to bind to several antigens, a characteristic which has been attributed to so-called natural IgE.