Regulatory T cells (Treg) are essential for the induction and maintenance of immunological tolerance to self and foreign antigens. The development of allergic asthma is mediated by T helper cell type-2 (Th2) inflammatory mechanisms and may also involve, based on murine and human studies of allergic asthma, compromised Treg immune regulation. Our overall objective was to more thoroughly elucidate the biology of Treg in allergic asthma, and to better understand their potential as a treatment for the disease. Initially, we characterized three different Treg phenotypes based on frequency and functional capacity. We showed both quantitative and functional heterogeneity in circulating Treg. Quantitative variability was also observed in circulating, but not airway, Treg when comparisons were made between healthy controls and asthmatic subjects. These findings emphasize the need for clear definitions of Treg phenotypes, and that interpretation of their frequency and function in health and disease needs to be phenotype-specific. Next, we assessed the Treg response in mild allergic asthmatic isolated early responders and dual responders after allergen inhalation challenge. We observed a reduced frequency of airway Treg after allergen challenge in DR, but not IER, associated with a smaller ratio of Treg to CD4+ cells. These data suggest that Treg to T effector cell (Teff) balance is important in the regulation of late asthmatic responses. Lastly, we evaluated the effects of two novel monoclonal anti-asthma therapies on circulating Treg after allergen inhalation challenge. We demonstrated that neither anti-OX40L nor anti-TSLP therapy altered circulating Treg frequency, while anti-TSLP, but not anti-OX40L, was effective in attenuating allergen-induced airway responses. These observations demonstrate the need for further investigation into the effects of anti-asthma therapies on Treg as well as the development of novel therapies aimed at manipulating Treg in order to better control immune responses. The findings of this thesis enhance our understanding of Treg in allergic asthma. Treg, utilized as stand-alone or adjunct therapy, may provide a novel therapy in the treatment of allergic asthma. / Thesis / Doctor of Philosophy (PhD)

Global prevalence of allergic diseases has been on the rise for the last 30 years. In Canada, this upward trend in allergic diseases has resulted in over 3 million Canadians being affected by allergic asthma. Allergic asthma is triggered by inhalation of environmental allergens resulting in bronchial constriction and inflammation, which leads to clinical symptoms such as wheezing, coughing and difficulty breathing. Asthmatic airway inflammation is initiated by the release of inflammatory mediators (-eg- histamine) released by granulocytic cells (-eg- mast cells and basophils). However, immunoglobulin E (IgE) antibody is also necessary for the initiation of the allergic cascade, and IgE is produced and released exclusively by memory B cells and plasma cells. Allergen crosslinking of IgE:FcεRI complexes on the surface of mast cells and basophils causes degranulation of pro-inflammatory mediators. Acute allergen exposure has also been shown to increase IgE levels in the airways of patients diagnosed with allergic asthma; however, more studies are needed to better understand local airway inflammation. Our group's work, in accordance with the literature, has shown an increase of IgE in the airways of subjects with mild allergic asthma following allergen inhalation challenge. Although regulatory B cells (Bregs) have been shown to modulate IgE-mediated inflammatory processes in allergic asthma pathogenesis, particularly in mouse models of allergic airway disease, the levels and function of these IgE+ B cells and Bregs remain to be elucidated in human models of asthma. The overall objective for this dissertation was to investigate the biology of B cells in allergic asthma pathogenesis, specifically investigating the frequency of IgE+ B cells and Bregs in allergic asthma, and the kinetics of these cells after allergen exposure. First, we characterized IgE+ B cells in the blood and sputum of allergic asthmatics and healthy controls with and without allergies (Chapter 2). We showed that IgE+ B cell levels were higher in sputum, but not blood, of allergic asthmatics compared to controls. We further demonstrated that these findings were consistent across airway IgE+ B cell subsets, which include IgE+ memory B cells and IgE+ plasma cells. Additionally, IgE+ B cells in sputum positively correlated with sputum eosinophils, total IgE and B cell activating factor (BAFF) measured in sputum fluid phase. These findings highlight the association of airway IgE+ B cells with allergic asthma, and suggest that local IgE+ B cell functions contribute to the pathogenesis of asthma. Second, we measured the trafficking of IgE+ B cells in periphery (blood, bone marrow and tonsil) and locally (sputum) in allergic asthmatics following whole lung allergen challenge (Chapter 3). IgE+ B cells only increased in the airways of allergic asthmatics following allergen inhalation challenge; there were no allergen-induced changes in IgE+ B cell levels in blood, bone marrow and tonsil. In addition, we showed allergen-induced increases in BAFF and total IgE, but not allergen-specific IgE in sputum fluid phase. Taken together, chapters 2 and 3 show that allergic asthmatics have elevated levels of IgE+ B cells in the airways, that can be further increased after allergen exposure. Therefore, local B cell production of IgE in the lungs may be an important source of IgE for initiation of acute inflammatory responses in allergic airways. Third, we evaluated the levels of Bregs in allergic asthmatics compared to controls, and examined the kinetics, function and distribution (bone marrow, blood and sputum) of Bregs following allergen inhalation challenge (Chapter 4). We showed that Bregs were 2-fold lower in the blood of allergic asthmatics compared to controls, highlighting a possible dysregulation of this regulatory cell type in allergic asthmatics, which may contribute to disease pathology. Furthermore, after whole lung allergen challenge Bregs decreased in the bone marrow with a co-incident increase in the blood and sputum of allergic asthmatics. This pattern reflects potential trafficking of these cells from bone marrow to the airways after exposure to allergic stimuli. Lastly, we stimulated CD19+ B cells purified from blood of allergic asthmatic with IL-4 in vitro. IL-4 is a type 2 cytokine known to isotype-switch B cells to IgE+ B cells, as well as differentiates naïve T cells into Th2 cells, thus propagating the allergic cascade. We found that IL-4 promoted higher proportions of IL-10+ and FoxP3+ Bregs, which demonstrates that Bregs may have a role in dampening IgE-mediated inflammation in a type 2 environment. However, further functional studies are warranted. Taken together, the findings of this dissertation highlight the local compartmental changes in IgE+ B cells and Bregs following allergen challenge of allergic airways. Better understanding the temporal and compartmental shifts in B cell subpopulations, particularly IgE+ B cells and Bregs, may aid in future development of therapeutics. / Thesis / Doctor of Philosophy (PhD)