One of the fundamental questions in mucosal immunology is how the intestine maintains tolerance to food antigens and commensal flora, and yet it is capable of mounting immune responses to pathogens. Peyers patches (PP) are lymphoid aggregates that are found in the small intestine and are the primary sites where adaptive immune responses are initiated in the intestine. An understanding of how PP cells regulate innate immune responses may provide information on how immune responses are regulated in the intestine. The toll-like receptors (TLRs) are a family of pattern recognition receptors (PRR) which provide a sensory mechanism for the detection of infectious threats. TLR9 recognizes bacterial DNA or synthetic CpG oligodeoxynucleotides (ODN). Cells that express TLR9 when stimulated with CpG ODN proliferate and produce Th1-like pro-inflammatory cytokines and upregulate co-stimulatory molecules. Because the intestine is constantly exposed to bacterial DNA from commensal flora, immune cells from the gut must have evolved mechanisms to modulate responses to TLR9 stimulation to prevent responses to harmless bacteria. Our hypothesis is that innate immune responses to the TLR9 agonist CpG ODN in Peyers patches (PP) are attenuated compared to other tissues such as blood or lymph nodes. This is due to local regulatory mechanisms unique to the intestinal microenvironment.<p> We conducted a number of experiments to test this hypothesis. We initially assessed the immunostimulatory activity of three available classes of CpG ODN in lymph nodes (LN), peripheral blood mononuclear cells (PBMC) and PP since this had not been done in ruminants. We found that CpG ODN induced strong IFNá, IFN-gamma, IL-12, lymphocyte proliferation and NK-like activity in LN and PBMC. In contrast, these responses were significantly less in PP stimulated with CpG ODN. We wondered whether the reduced responses of PP cells to CpG ODN were unique to the TLR9 agonist. For this reason we tested responses of cells from these tissues to poly (I:C), LPS, and single-stranded RNA, which are agonists for TLR3, TLR4, and TLR7/8 respectively. Additionally, we tested combinations of TLRs since others have reported that multiple TLR agonists may induce synergistic responses. All TLR agonists or their combinations either failed to induce detectable responses or the responses were significantly less in PP compared to other tissues. Thus we concluded that PP cells responses to TLR stimulation were attenuated. In all tissues tested, there were no synergistic responses (IFN-alpha, IFN-gamma and lymphocyte proliferation) following stimulation with combinations of agonists. However, there was inhibition of PBMC responses when TLR7/8 agonists were combined with CpG ODN (TLR9 agonist). Importantly, TLR7/8 agonists reduced the CpG-induced proliferative responses in purified blood B cells. Interestingly, ovine B cells constitutively expressed TLR7/8 and TLR9 mRNA, suggesting the potential for cross-talk between the receptors.<p> Interestingly, cell from all isolated tissues [ileal PP (IPP), jejunal PP (JPP), mesenteric LN (mLN) and PBMC] expressed similar levels of TLR9 mRNA, suggesting that the reduced responsiveness to CpG ODN stimulation in PP was not due to a lack of TLR9 expression.<p> Surprisingly, we observed that PP cells spontaneously secreted significant amounts of the immunoregulatory cytokine IL-10. Furthermore, we confirmed that CD21+ B cells were the source of the IL-10. We then examined the role of IL-10 in regulating IFN and IL-12 responses in PP. Neutralization of IL-10 resulted in a significant increase in the numbers of CpG-induced IFNá-secreting cells detected and in IFN-gamma and IL-12 production by PP cells (both follicular and interfollicular lymphocytes). Similarly, depletion of the CD21+ B cells resulted in significant increases in IFNá, IFN-gamma and IL-12 responses. These observations support the conclusion that IL-10-secreting PP CD21+ B cells suppress innate immune responses in PP. Further characterization by flow cytometry revealed that these cells were CD1b-CD5-CD11c-CD72+CD21+ IgM+ B cells. We have proposed that these IL-10-secreting PP CD21+ B cells are a novel subset of regulatory B cells (Bregs).<p> Finally, we examined the capacity of IL-10 secreting B cells (Bregs) to respond to CpG ODN. To achieve this, we compared CD21+ B cells from blood and JPP. Unlike blood CD21+ B cells, CD21+ B cells from JPP proliferated poorly in response to CpG ODN. Moreover, PP CD21+ B cells, unlike blood CD21+ B cells, do not secrete IgM or IL-12 in response to CpG stimulation, although both PP and blood CD21+ B cells express similar level of TLR9 mRNA. Neutralization of IL-10 did not enhance CpG-induced proliferative responses in PP CD21+ B cells. Thus IL-10 does not play a direct role in the hyporesponsiveness of PP CD21+ B cells to CpG ODN. To further explore the mechanism by which PP Bregs fail to respond to CpG ODN stimulation, we used a kinome analysis to determine whether the TLR9 pathway was functional in PP Bregs compared to blood CD21+ B cells. We observed that peptides representing critical adaptor molecules downstream of TLR9 such as IRAK1, TAK1, Casp8, p-38 MAPK, JNK, FOS, IKKá, NF-KB-p65 were not phosphorylated in JPP CD21+ B cells following CpG ODN stimulation. However, in blood CD21+ B cells stimulated with CpG ODN, the same peptides on the array were all highly phosphorylated leading to a functional TLR9 signaling pathway. Thus PP Bregs have evolved mechanisms by which the TLR9 signaling pathway is not activated following exposure to the TLR9 agonist, CpG ODN.<p> In conclusion, we clearly demonstrated that TLR9-induced responses in cells from PP are significantly attenuated. This is a consequence of PP CD21+ B cells (Bregs) that spontaneously secrete IL-10, which in turn conditions an anti-inflammatory environment in this tissue leading to poor cytokine responses to the TLR9 agonist, CpG ODN. Additionally, we show that Bregs are unresponsiveness to TLR9 stimulation. This unresponsiveness is due to regulatory mechanisms in Bregs leading to a dysfunctional TLR9 signaling pathway. These may represent strategies by which PP dampen innate responses to pathogen-associated molecular patterns (PAMPs) in intestinal immune tissues to maintain intestinal immune homeostasis. These conclusions are consistent with our hypothesis that TLR responses in PP cells are attenuated, and this is due to B cell-mediated regulatory mechanisms that are unique to the intestinal microenvironment.

One of the fundamental questions in mucosal immunology is how the intestine maintains tolerance to food antigens and commensal flora, and yet it is capable of mounting immune responses to pathogens. Peyers patches (PP) are lymphoid aggregates that are found in the small intestine and are the primary sites where adaptive immune responses are initiated in the intestine. An understanding of how PP cells regulate innate immune responses may provide information on how immune responses are regulated in the intestine. The toll-like receptors (TLRs) are a family of pattern recognition receptors (PRR) which provide a sensory mechanism for the detection of infectious threats. TLR9 recognizes bacterial DNA or synthetic CpG oligodeoxynucleotides (ODN). Cells that express TLR9 when stimulated with CpG ODN proliferate and produce Th1-like pro-inflammatory cytokines and upregulate co-stimulatory molecules. Because the intestine is constantly exposed to bacterial DNA from commensal flora, immune cells from the gut must have evolved mechanisms to modulate responses to TLR9 stimulation to prevent responses to harmless bacteria. Our hypothesis is that innate immune responses to the TLR9 agonist CpG ODN in Peyers patches (PP) are attenuated compared to other tissues such as blood or lymph nodes. This is due to local regulatory mechanisms unique to the intestinal microenvironment.<p> We conducted a number of experiments to test this hypothesis. We initially assessed the immunostimulatory activity of three available classes of CpG ODN in lymph nodes (LN), peripheral blood mononuclear cells (PBMC) and PP since this had not been done in ruminants. We found that CpG ODN induced strong IFNá, IFN-gamma, IL-12, lymphocyte proliferation and NK-like activity in LN and PBMC. In contrast, these responses were significantly less in PP stimulated with CpG ODN. We wondered whether the reduced responses of PP cells to CpG ODN were unique to the TLR9 agonist. For this reason we tested responses of cells from these tissues to poly (I:C), LPS, and single-stranded RNA, which are agonists for TLR3, TLR4, and TLR7/8 respectively. Additionally, we tested combinations of TLRs since others have reported that multiple TLR agonists may induce synergistic responses. All TLR agonists or their combinations either failed to induce detectable responses or the responses were significantly less in PP compared to other tissues. Thus we concluded that PP cells responses to TLR stimulation were attenuated. In all tissues tested, there were no synergistic responses (IFN-alpha, IFN-gamma and lymphocyte proliferation) following stimulation with combinations of agonists. However, there was inhibition of PBMC responses when TLR7/8 agonists were combined with CpG ODN (TLR9 agonist). Importantly, TLR7/8 agonists reduced the CpG-induced proliferative responses in purified blood B cells. Interestingly, ovine B cells constitutively expressed TLR7/8 and TLR9 mRNA, suggesting the potential for cross-talk between the receptors.<p> Interestingly, cell from all isolated tissues [ileal PP (IPP), jejunal PP (JPP), mesenteric LN (mLN) and PBMC] expressed similar levels of TLR9 mRNA, suggesting that the reduced responsiveness to CpG ODN stimulation in PP was not due to a lack of TLR9 expression.<p> Surprisingly, we observed that PP cells spontaneously secreted significant amounts of the immunoregulatory cytokine IL-10. Furthermore, we confirmed that CD21+ B cells were the source of the IL-10. We then examined the role of IL-10 in regulating IFN and IL-12 responses in PP. Neutralization of IL-10 resulted in a significant increase in the numbers of CpG-induced IFNá-secreting cells detected and in IFN-gamma and IL-12 production by PP cells (both follicular and interfollicular lymphocytes). Similarly, depletion of the CD21+ B cells resulted in significant increases in IFNá, IFN-gamma and IL-12 responses. These observations support the conclusion that IL-10-secreting PP CD21+ B cells suppress innate immune responses in PP. Further characterization by flow cytometry revealed that these cells were CD1b-CD5-CD11c-CD72+CD21+ IgM+ B cells. We have proposed that these IL-10-secreting PP CD21+ B cells are a novel subset of regulatory B cells (Bregs).<p> Finally, we examined the capacity of IL-10 secreting B cells (Bregs) to respond to CpG ODN. To achieve this, we compared CD21+ B cells from blood and JPP. Unlike blood CD21+ B cells, CD21+ B cells from JPP proliferated poorly in response to CpG ODN. Moreover, PP CD21+ B cells, unlike blood CD21+ B cells, do not secrete IgM or IL-12 in response to CpG stimulation, although both PP and blood CD21+ B cells express similar level of TLR9 mRNA. Neutralization of IL-10 did not enhance CpG-induced proliferative responses in PP CD21+ B cells. Thus IL-10 does not play a direct role in the hyporesponsiveness of PP CD21+ B cells to CpG ODN. To further explore the mechanism by which PP Bregs fail to respond to CpG ODN stimulation, we used a kinome analysis to determine whether the TLR9 pathway was functional in PP Bregs compared to blood CD21+ B cells. We observed that peptides representing critical adaptor molecules downstream of TLR9 such as IRAK1, TAK1, Casp8, p-38 MAPK, JNK, FOS, IKKá, NF-KB-p65 were not phosphorylated in JPP CD21+ B cells following CpG ODN stimulation. However, in blood CD21+ B cells stimulated with CpG ODN, the same peptides on the array were all highly phosphorylated leading to a functional TLR9 signaling pathway. Thus PP Bregs have evolved mechanisms by which the TLR9 signaling pathway is not activated following exposure to the TLR9 agonist, CpG ODN.<p> In conclusion, we clearly demonstrated that TLR9-induced responses in cells from PP are significantly attenuated. This is a consequence of PP CD21+ B cells (Bregs) that spontaneously secrete IL-10, which in turn conditions an anti-inflammatory environment in this tissue leading to poor cytokine responses to the TLR9 agonist, CpG ODN. Additionally, we show that Bregs are unresponsiveness to TLR9 stimulation. This unresponsiveness is due to regulatory mechanisms in Bregs leading to a dysfunctional TLR9 signaling pathway. These may represent strategies by which PP dampen innate responses to pathogen-associated molecular patterns (PAMPs) in intestinal immune tissues to maintain intestinal immune homeostasis. These conclusions are consistent with our hypothesis that TLR responses in PP cells are attenuated, and this is due to B cell-mediated regulatory mechanisms that are unique to the intestinal microenvironment.

Le virus d’Epstein-Barr (EBV) est le premier virus transformant à avoir été identifié chez l’Homme. Il infecte plus de 90% de la population adulte mondiale, persistant sous forme épisomale dans le compartiment B mémoire tout au long de la vie de l’hôte. Lors de la primo-infection et lors de phases de réactivation du virus, les cellules B immortalisées sont en programme de latence III, aussi appelée phase de prolifération, où l’ensemble des protéines de latence sont exprimées. Lorsque les hôtes sont immunocompétents, un équilibre entre hôte et virus s’établit et la plupart des cellules B infectées sont éliminées par le système immunitaire de l’hôte, principalement par les lymphocytes T cytotoxiques. En cas de déficit immunitaire, il peut y avoir émergence de lymphomes, tels que les désordres lymphoprolifératifs des patients immunodéprimés (PTLDs), les lymphomes non-Hodgkiniens (LNH) et Hodgkiniens (LH). Les travaux antérieurs du laboratoire ont permis de révéler que l’immuno-inhibiteur PD-L1/B7-H1/CD274 est surexprimé à la surface des lymphocytes B en latence III de l’EBV. L’interleukine-10 (IL-10) est également sécrétée par ces cellules. Ces caractéristiques sont communes aux cellules B régulatrices (Bregs). Le but de ma thèse était d’interroger les caractéristiques immuno-modulatrices des cellules B en latence III de l’EBV, dans le cadre des propriétés des Bregs. Nous montrons que les cellules B en latence III de l’EBV possèdent les déterminants antigéniques communs aux Bregs immatures (CD24High CD38High PD-L1High), associée à une surexpression des cytokines immunosuppressives cardinales des Bregs (IL-10, TGF-β1 et IL-35). Nous montrons que les cellules B en latence III de l’EBV peuvent conduire à la mort des cellules T CD4 autologues, ainsi qu’à l’inhibition de la prolifération des lymphocytes T CD4 et CD8, au profit de l’expansion de lymphocytes T régulateurs (Tregs). Nous avons trouvé que cette expansion est médiée par l’axe PD-1/PD-L1. Ces travaux mettent en évidence un nouveau mécanisme de l’EBV concernant le détournement du système immunitaire de l’hôte, augmentant ses capacités oncogéniques. / The Epstein-Barr virus (EBV) is the first transforming virus discovered in humans. It infects more than 90% of the global adult population, persisting in an episomal form in the memory B-cell compartment throughout the life of the host. During primo-infection and during phases of viral reactivation, immortalized B-cells are in latency III, also called the proliferation program, in which the full range of latency proteins are expressed. In immunocompetent subjects, a balance between virus and host is established, and most infected B-cells are eliminated by the host’s immune system, mainly by cytotoxic T lymphocytes. Deficit of the immune system may lead to lymphomagenesis, such as post-transplantation lymphoproliferative disorders (PTLDs), non-Hodgkin’s (NHL) or Hodgkin’s lymphomas (HL). Previous studies in the lab revealed that the immuno-inhibitor PD-L1/B7-H1/CD274 was overexpressed on the surface of EBV latency III B-cells. Interleukin-10 (IL-10was also secreted by these cells. These features are shared with regulatory B-cells (Bregs). Our objective was to examine the immunomodulatory features of EBV latency III B-cells, in the frame of Bregproperties. We found that EBV latency III B-cells possessed the antigenic determinants common to immature Bregs (CD24High CD38High PD-L1High), associated with overexpression of Breg immunosuppressive cytokines (IL-10, TGF-β1 and IL-35). EBV latency III B-cells led to death of autologous CD4 T-cells, as well as inhibition of CD4 and CD8 T-lymphocyte proliferation, favoring regulatory T-cell (Treg) expansion. We found that this expansion was mediated by the PD-1/PD-L1 axis. This study highlights a new mechanism of EBV for t diversion of the host immune system thereby increasing its oncogenic properties.

Toll-like receptors (TLRs) recognize microbial components as danger signals and induce immune responses. TLR’s are expressed in many tissues of the host that are involved in immune responses including the intestines where they are abundantly expressed. This situation presents a challenge in the gastrointestinal tract which is constantly exposed to a wide variety of commensal organisms. Therefore, innate immune recognition in the intestine must be tightly regulated to prevent unwanted inflammation against harmless commensal micro-organisms and yet allow for the induction of protective immunity to invading pathogens. A dysregulation of this balance can result in intestinal inflammation. Peyer’s patches (PP) are the primary site for the induction of immune responses in the intestine and abundantly express TLRs. It is not known how PP regulate microbial signals from commensal bacteria and yet mount vigorous immune responses against dangerous pathogens. CpG DNA, an agonist for TLR9, can strongly activate immune cells in blood, lymph nodes and spleen. However, CpG very poorly activates immune cells from Peyer’s patches, although these cells express TLR9 [1, 2]. Understanding how TLR responses are regulated in PP cells will unveil important information on how immune responses are regulated in the intestine. Investigations from our laboratory have revealed a B cell population (CD5-CD11c-CD21+) in PP that spontaneously secrete high levels of IL-10 which in turn down regulates TLR9 induced IFN and IL-12 production. These IL-10-secreting PP B cells represent a novel subset of the recently proposed regulatory B cells (Bregs) in the intestine [1, 3]. Bregs may have a role in maintaining tolerance to commensal bacteria thereby achieving intestinal homeostasis. The overall goal of the work described in this thesis was to improve our understanding of the immunobiology of Bregs. We performed several experiments to achieve this goal. First, we studied the development of regulatory B cells in lambs of different ages. Jejunal PP were collected from 3-4 month old, neonatal and fetal lambs and the production of IL-10 (the immunoregulatory cytokine secreted by Bregs) was assayed. We found that IL-10 was secreted by CD21+ B cells from the PP in all the three age groups, confirming that Bregs develop prior to birth. We then wondered whether our CD21+ B cells might be contaminated with other cells or activated when using MACS to enrich B cells. To address this issue, we prepared very highly purified CD21+ B cell population using high speed cell sorting to negatively enrich for B cells. We also sorted DCs and assayed IL-10 production in both cell populations. Only the PP B cells spontaneously secreted IL-10. In contrast, dendritic cells, T cells, macrophages, neutrophils and NK cells did not secrete detectable IL-10. Since B cells exist as regulatory and effector populations in mice, we wondered whether an effector B cell population also existed in ovine PP that secreted the pro-inflammatory cytokines IFN-, IFN- and IL-12. Therefore, ovine PP B cells were fractionated into CD72+CD21+and CD72+CD21- subpopulations to assess their capacity to secrete pro-inflammatory cytokines. Interestingly, the CD72+CD21- B cell population secreted the cytokines IFN-, IFN- and IL-12 suggesting there was an effector population. We then surveyed for Bregs in different mucosal and peripheral tissues in sheep. We observed the Bregs frequency varied among the different lymphoid tissues. Finally, we investigated whether Bregs were present in PP of other ruminant species. We identified Bregs exist in PP of neonatal calves. In conclusion, our investigations reveal that ovine Bregs develop in utero prior to antigen exposure, and are present in a variety of mucosal and peripheral tissues. We also report the novel observation that two distinct B cell sub-populations are present in ovine jejunal PP’s: Regulatory and effector B cells.

<p>Inflammatory bowel disease (IBD) is chronic, relapsing, intestinal inflammation characterized by periods of remission and exacerbation. Pathogenesis of IBD is a complex process and the exact aetiology is unclear yet. Studies have provided evidences that IBD is a result of a genetic predisposition that leads to a mucosal immune regulatory cell defect, barrier defects, and susceptibility to environmental triggers like luminal bacteria and specific antigens. Innate and adaptive immune responses to commensal bacteria are balanced by the multiple regulatory cells like T regs and B regs. Monoclonal-antibody-based therapies have been identified for the treatment of IBD but, they have side effects and the efficacy is not stable. Hence, great expectations lie towards finding a successful cure for IBD.</p> <p>Recent studies have proved the involvement of several different genes in the pathogenesis of IBD and there expressions were highly reduced in IBD. Homeodomain only protein- Hopx is a nuclear protein required to modulate heart growth and function. Expression of Hopx has been reported in various types of normal tissues but not in malignant tissues. Recent study on Hopx revealed that Hopx is needed for the function of regulatory T cells induced by DCs. However the correlation between Hopx gene and IBD remains unanswered. In this study, we examined the role of Hopx expressing regulatory B cell in IBD.</p> <p>The aim of this study is to investigate whether the expression of Hopx in regulatory B cell play a key role in suppressing colitis in murine model.</p> <p>The expression of Hopx was studied using cellular and molecular techniques including flowcytometry, immunohistochemistry, western blotting and real time RT-PCR.</p> <p>As a first step, we investigated the Hopx expression in colitis and control murine model. After we found the possible involvement of Hopx gene in regulating intestinal inflammation, we further our study to investigate whether Hopx is expressed by regulatory B cell and function together to inhibit intestinal inflammation. After establishing Hopx and B regs association, we used probiotics to modulate Hopx expression in regulatory B cells in order to prevent/reduce intestinal inflammation. Finally we elucidate the importance of Hopx expression by injecting neutralizing anti-Hopx antibody to block Hopx.</p> <p>Together, studies on human intestinal tissue and murine model revealed that Hopx expression is suppressed during inflammation condition. Probiotic administration helps to increase Hopx expressing Breg cell thereby, prevent IBD. Hopx deficient group expressed high frequency of proinflammatory cytokines and reduced immune regulatory cells. This particular study proposes that the down regulation of Hopx contributes to the development of intestinal inflammation.</p> / Master of Health Sciences (MSc)

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)