Abstract
The prevalence of allergic disease has increased at an alarming rate in the past few decades, affecting both children and adults. Despite numerous attempts to understand the mechanisms which drive sensitization to innocuous antigens, the factors that lead to the development of allergy remain unclear. Although a number of pharmacological agents have been developed to treat allergies, these treatments are largely aimed at reducing allergic symptoms. Additionally, the mechanisms which maintain allergic sensitization are not known, hampering the development of effective treatments for curing or even reducing allergic reactions. While it is clear that IgE+ B cells are vital for the pathogenesis of the allergic response, little is known about these cells. Here, we sought to elucidate the signals that drive the development of allergen specific IgE secreting B cells, as well as the mechanism by which these cells are maintained, in the context of galactose-alpha-1,3-galactose (alpha-gal), or red meat allergy.
Using mass cytometry (CyTOF) we have analyzed the expression of 23 cell surface markers in PBMCs from 19 alpha-gal-allergic patients and 20 non-allergic controls by CyTOF. Our data revealed previously unrecognized heterogeneity within traditional B cell subsets. Additionally, through the use of a novel bioinformatics pipeline, we identified a collection of B cell subsets, or clusters, which correlated with alpha-gal specific serum antibody titers. Functional analysis of these B cell clusters demonstrated that cells isolated from patients with red meat allergy preferentially secreted alpha-gal specific IgE following in vitro stimulation, implicating these novel B cell phenotypes in promoting IgE in alpha-gal allergy.
In addition to investigating circulating B cell subsets in patients with red meat allergy, we have developed a murine model of tick-induced IgE responses to investigate the signals that drive the development of the IgE response. Subcutaneous immunization with tick extract results in a robust, tick specific IgE response in C57BL/6 (B6) mice, however no significant induction in serum IgE resulted from intraperitoneal (I.P.) immunization with tick extract. The IgE response observed in mice immunized subcutaneously was accompanied by the concomitant induction of a germinal center (GC) response. This response is T cell dependent, as the inhibition of T cell help resulted in an attenuated IgE response. Utilizing MyD88 deficient mice, we have identified a requirement for MyD88 signaling in the production of IgE following subcutaneous immunization with tick extract, suggesting that toll-like receptor (TLR) signaling plays a role in the development of tick induced IgE responses. Additionally, through studies with MyD88 conditional knock out mice, we demonstrate that the requirement for MyD88 signaling is B cell intrinsic. Taken together, we have identified a role for MyD88 in the development of the IgE response to tick extract, and identified novel B cell subsets in red meat allergic patients that may play a role in the pathogenesis of food allergy, revealing new potential targets for effective treatment of prevention of allergic responses. The systems we have developed will provide tools for future investigations in the development and maintenance of IgE responses.
