Duane R. Wesemann, Ph.D. - US grants

This proposal details a 5 year training program for the development of an academic career in the study of B cell developmental biology and immune tolerance. The PI has joined the laboratory of Fred Alt to study B cell receptor editing and the regulation of immunoglobulin light chain rearrangement, and plans to utilize the rigorous scientific environment in the Alt to develop expertise in cutting edge molecular tools. The PI plans to then utilize these tools as he becomes an independent investigator to dissect the mechanistic and molecular underpinnings of clinically relevant problems in B cell immunology. The PI is currently a Clinical Fellow in Medicine conducting research under the direction of Dr. Frederick Alt (mentor) and Dr. Frank Austen (co-mentor). His research focus involves the characterization of a subpopulation of B cells that he recently found to rearrange the immunoglobulin lambda light chain (Ig() locus in mesenteric lymph node B cells. In the proposed plan, the PI will characterize this population of peripheral editing cells (Aim 1) and develop mouse models to investigate this Ig( locus rearrangement activity with respect to immunological tolerance to the gut environment (Aim 2). In Aim 3, he will pursue studies to elucidate the molecular mechanisms involved in regulation of immunoglobulin light chain gene rearrangements. Together, results from this proposal will advance our understanding of the mechanisms of B cell development, antibody specificity modulation and regulation of DNA accessibility to V(D)J rearrangement machinery. Moreover, these studies may aid the understanding of peripheral B cell receptor editing in allergic and/or inflammatory disease processes. The mentor is an eminent scientist in the field with over 100 established trainees and is fully focused on his current laboratory program. The co-mentor is also an eminent scientist with expertise in mouse models of hypersensitivity diseases who will aid in the candidates career development. Advisory committee members are exceptionally outstanding, respected immunologists who will also oversee the progress of this proposal and provide critique on the methods and interpretation of results at scheduled meetings and informally. The research will take place within the highly collaborative environment of the Immune Disease Institute/Program for Cellular and Molecular Medicine at the Children's Hospital Boston. The PI will also benefit from the environment of his home institution, the Inflammation and Allergic Diseases Research Section at BWH. Both of these institutions are committed to providing the necessary resources and training to help the primary investigator establish an independent career. Failure of our immune system to develop tolerance food and "good" bacteria leads to disease. Studies to determine the mechanism of tolerance to these entities will be critical to designing new therapies for prevention and treatment of food allergy and inflammatory bowel disease.

DESCRIPTION (provided by applicant): A fundamental goal in modern immunology is to understand the factors that contribute to the homeostatic balance between commensal microbes and the host immune system. Although a great deal has been done to understand the role of commensals in shaping T cell responses, a large gap remains in understanding how commensals impact B lymphocyte development and associated antibody diversification. Continued existence of this gap is an important problem because until filled, our understanding of regulatory principles underlying humoral immune fitness as well as vulnerability to allergic and inflammatory diseases will remain incomplete. The overall objective of this proposal is to determine how commensal microbes affect primary B cell development and pre-immune immunoglobulin (Ig) diversification. Based on recent findings from the applicant, the central hypothesis is that commensal microbes provide inputs through host sensory instruments to regulate early B cell developmental activities in the host gut mucosa, thus empowering luminal antigens with the capacity to influence the primary Ig repertoire locally. This is consistent with observations that several other species, such as rabbits and sheep, utilize the intestine as a significant site for primary Ig diversification early in post-natal life. The rationale for the proosed research is that, once accomplished, the field will move vertically towards a greater understanding of the role of commensals in immune homeostasis and will open the door to further exploration of modifiable environmental factors that could be targeted, resulting in new and innovative approaches to influence a variety of health issues relevant to antibody production. Using both new as well as established technologies, this proposal's three aims will build upon the applicant's recent work. With the deployment of ex vivo co-culture as well as established in vivo labeling methods, aim 1 will test the notion that commensals influence both gut-resident as well as bone marrow support systems to stimulate early B lineage cell development and is expected to identify the gut-resident support mechanisms enabling early B lineage cells to survive and respond to microbial input. With the use of germ-free and gnotobiotic facilities, aim 2 will determine the extent to which microbe composition has specific effects to shape the primary Ig repertoire. With sophisticated genetically modified mouse models as well as powerful imaging technologies, aim 3 will evaluate the degree to which luminal antigens gain access to the intestinal lamina propria and interact with freshly-produced, cell-bound Ig in young mice. The proposed research is significant for the following three reasons - first, it promises to provide a more complete picture of the general principles of host:microbe interactions in immune regulation; second, it will be a first step to identify the extent to which primary Ig repertoires can be influenced by modifiable environmental exposures early in life; and third, it provides an essential component to establish a platform for testing future hypotheses regarding the effect of specific commensal microbe ecologies on downstream antibody responses to pathogens, allergens and vaccines.

? DESCRIPTION (provided by applicant): With increasing incidence, allergic disease is a significant source of morbidity in adults and children. The pathogenesis of allergic disease requires that immunoglobulin (Ig) E (IgE) molecules be produced against otherwise innocuous substances. Despite the essential role of IgE in allergic disease, there is a fundamental gap in understanding how IgE is produced and regulated from its source-namely, IgE B cells. Continued existence of this gap represents an important problem because, until it is filled, the pathogenesis of IgE- mediated diseases such as asthma, food allergies and allergic rhinoconjunctivitis will remain largely incomprehensible. The long-term goal is to uncover regulatory mechanisms of allergic disease and other environmental intolerances so as to identify new targets that can be manipulated for therapeutic purposes. The objective in this particular application is to deploy unique mouse models to determine how B cell function and fate is influenced by IgE expression in the context of the B cell receptor (BCR) from its natural genomic context-and to begin to translate the findings to human IgE B cells. The central hypothesis is that IgE as a BCR directs a program for B cell survival and function that is distinct from the other IgH isotypes due to its unique signaling as a BCR. This hypothesis has been formulated on the basis of preliminary data using an IgE mouse model cloned from an IgH isotype-switched B cell. The rationale for the proposed research is that once it is known how IgE functions as a BCR, new targets for manipulation of the IgE response may be generated. This hypothesis will be tested by pursuing three specific aims: 1) Discover how BCR signaling is influenced in the context of IgE; 2) Determine the consequences of IgE expression on B cell fate and survival; and 3) Determine the role of antigen-specific IgE on cognate interactions and downstream developmental potential compared to other IgH isotypes specific to the same antigen. Under the first aim, unique mouse models producing B cells with pre-switched IgH isotypes, as well as IgE, IgM, IgA and IgG1 B cell lines, will be deployed to uncover the unique molecular, biochemical, and cellular pathways downstream of antigen engagement of IgE as a BCR. Under the second aim, these tools will be utilized to examine the extent to which IgE B cells can particulate in somatic hypermutation and Ig class switch recombination (CSR). In the third aim, novel antigen-specific IgE mice will be used to identify the degree to which IgE B cells may process and provide antigen to cognate T cells and recruit limiting T cell help. The proposed work is innovative, because it represents a departure from the status quo by providing unique models to examine IgE B cell biology wherein membrane and secreted IgE are produced from natural genomic contexts. The proposed work is significant because it is a key step in a continuum of research that is expected to uncover new targets for preventive and therapeutic interventions at the IgE B cell stage. Ultimately, such knowledge has the potential to inform the development of new strategies that will help to reduce the growing problem of allergic disease in the U.S.

Project Summary How host-environment homeostasis is established and maintained at mucosal surfaces are fundamental concepts in immunology. Due to the abundance of IgA specifically at mucosal surfaces, it is thought to be a key regulator at these barriers. However, the relevance of IgA in shaping microbial communities in humans and the role IgA plays in human gut barrier integrity is not well defined. These gaps in knowledge are an important problem because until they are filled, a full understanding of host-environment homeostasis will remain incomplete. The long-term goal is to understand the role of B lineage cells in host-environment homeostasis and immune fitness. In pursuit of this goal, the objective of this particular application is to explore the role that IgA plays influencing gut barrier function and in managing microbial membership and diversity in humans. The central hypothesis is that IgA can regulate host-microbe interactions and gut barrier integrity at mucosal surfaces. This hypothesis was formulated based on mouse model findings of commensal microbe-focused IgA repertoires in small intestine and on findings that IgA in mice may regulate the microbial membership and diversity. The rationale for the proposed research is that, once accomplished, the role of IgA in regulating microbial membership and gut barrier function in humans will be clarified. The central hypothesis will be tested by way of three specific aims: 1) Explore the effect of IgA deficiency on the human microbiome; 2) Explore the extent of symbiont-coated IgM compensation in IgA deficiency; and 3) Explore the role of IgA-deficiency on barrier integrity. Aim 1 is expected to reveal the role of IgA in regulating microbial membership and diversity. Aim 2 is expected to reveal the role of IgM in potentially compensating for lack of IgA. It is also expected to uncover the degree to which lack of IgA in the serum corresponds to lack of IgA coating of bacteria in the intestinal lumen. Aim 3 is expected to uncover the degree to which lack of IgA may be associated with barrier dysfunction. Together, these anticipated results and new approaches are expected to have an important positive impact, as they break new ground into human IgA function. Results promise to deepen the understanding of host-microbe homeostasis and fundamentally advance the field of human immunology. The proposed work is innovative, because it is the first study to directly assess the role of human IgA in human microbial ecology and intestinal barrier integrity. The proposed work is significant because it is a key step in a continuum of research that is required to achieve a full understanding of IgA's role in the regulation of host-microbe homeostasis in humans.

PROJECT SUMMARY Antibodies diversify through two distinct pathways. The first involves combinatorial assembly of Immunoglobulin (Ig) variable region (V) exons during B cell development. The second involves V exon somatic hypermutation (SHM) and affinity-based selection in germinal centers (GCs). There are fundamental gaps in understanding how these systems collaborate to recognize, adapt, and neutralize diverse pathogenic threats. The long-term goal is to shed light onto fundamental GC B cell biology and elucidate underlying mechanisms of protective antibody development. The objective for this proposal is to elucidate mechanisms underlying GC plasticity?in particular, the extent of GC diversification, and the parameters that allow B cell GC entry and continued antibody evolution. The central hypothesis is that the GC system provides dual function with regard to antibody development. On one hand, the GC reaction intensifies affinities readily available from the primary repertoire. On the other hand, GC plasticity is flexible enough to allow for the recruitment of extremely low affinity/non-cognate B cell clones whose BCR is not initially of sufficient affinity to compete well in the GC, but which may have unique potential (given a few needed mutations) to recognize critical epitopes not otherwise targeted well by the primary repertoire. A deeper understanding of how these GC functions are regulated promises to reveal new insights into how to more effectively recruit low frequency/low affinity B cells with potential to become broadly neutralizing, and shepherd them toward protective efficacy. This hypothesis will be explored with two specific aims: 1) Characterize the capacity and limitations of diversity mediated by the GC SHM diversification system; and 2) Define features that regulate B cell participation in germinal centers. Under the first aim, the flexibility of affinity development and specificity potential by the GC diversification system will be examined using an extremely low BCR/antigen affinity (Ka<102 M-1), and BCR-negative model systems in the physiologic context of competitive settings. Under the second aim, modifiable factors that regulate the flexibility of SHM-mediated Ig evolution in physiologic contexts will be defined. The approach is innovative, because the applicant's recent published work and preliminary data indicate that GC-mediated diversification can provide specificities to new epitopes not otherwise present in the primary Ig repertoire within a physiologic, competitive environment of a diverse primary Ig repertoire. Innovative mouse models will be used to probe the parameters and mechanistic aspects of the roles of affinity and Ig frequency on participation in GC maturation and contribution to protective antibody responses in the context of an animal model expressing a diverse human Ig repertoire. Discovering how extremely low affinity B cell clones gain access to the GC and continue to mature in the highly competitive GC environment?often accumulating extremely high levels of SHM not yet replicated with vaccines?would be a significant contribution to fundamental B cell knowledge. Such knowledge also has the potential to inform strategies for vaccination as well as antibody bioengineering.

Project Summary IgE-mediated allergic disease is a growing problem. The pathogenesis of allergic disease requires that immunoglobulin (Ig) E (IgE) molecules be produced against what are otherwise usually innocuous substances. Upon activation in the setting of cytokines such as IL-4 or IL-13, B cells can undergo IgH CSR to IgE. IgE secreted from B lineage cells can, in the presence of cognate antigen, activate mast cells and basophils to release potent inflammatory mediators. While IgE responses can lead to protective immunity as a part of a specialized responses to multicellular pathogens or other noxious threats, they also underlie allergic disease. Allergic disease can be manifest by localized inflammation, or by multiorgan involvement, including deadly systemic anaphylactic reactions via IgE-sensitized mast cell degranulation. Thus, the production and dissemination of IgE play a significant role in dictating the strength and extent of tissue mast cell sensitization. It is therefore critical to understand not only how B cell IgE production is controlled, but also the principles underlying distribution of IgE from point of origin to distal sites throughout the body. The overall objective of this application is to understand biological aspects of IgE production and dissemination and to gain insights into how this is influenced in allergic disease. Emerging literature and preliminary data from the applicant suggest a general hypothesis that biological constraints cooperate to restrict IgE dissemination under homeostatic conditions, and that accumulation of bone marrow IgE LLPCs is an aberrancy underlying systemic manifestations of allergic disease. This hypothesis will be tested by pursuing three specific aims, which are: 1) to determine the mechanisms of IgE expression dynamics on IgE B cell function; 2) to elucidate mechanisms underlying IgE distribution from point of origin to effector sites; and 3) to characterize IgE plasma cells in allergic patients. Under the first aim, IgE mRNA splicing and IgE surface density will be genetically perturbed to examine the hypothesis that mRNA production away from the mIg splice variant (i.e. the BCR variant) restricts the number and longevity IgE expressing cells, thus constraining IgE secretion largely to the insult site. Under the second aim, models of anatomic location-specific allergic challenge will be deployed to examine the degree to which IgE distribution is locally biased, and the role of B cells in this process. Under the third aim, bone marrow aspirations from healthy and allergic individuals will be obtained for IgH isotype-resolved deep sequencing as well as single cell transcriptomics to elucidate the cellular sources and biological properties of IgE in patients with long-standing severe allergies. This contribution is significant because it is expected to elucidate a more complete picture of how IgE responses are regulated. Ultimately, such knowledge has the potential to inform the development of new strategies that will help to reduce the growing problem of allergic disease.