Browsing by Subject "B cell"

Now showing 1 - 5 of 5
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    B cell receptor signaling in receptor editing and leukemia.
    (2009-07) Ramsey, Laura Bailey
    There are many unanswered questions in the B cell field, but one of paramount importance is how B cells are tolerized to avoid autoimmunity. The majority of developing B cells are probably self-reactive, and many that make it through B cell development to the periphery show evidence of receptor editing. The question remains as to how the B cells signal to re-induce the editing machinery, whether it is a positive reactivation signal or the release of an inhibitory tonic signal. We propose that it is the release of a tonic signal through the B cell receptor that inhibits recombination when the BCR is present on the surface, but when the BCR binds self-antigen it is internalized, the inhibitory signal is abrogated, and the recombination machinery is reactivated. We tested this hypothesis using several transgenic models, including RAG2-GFP mice, HEL Ig mice, and anti-κ mice. We also used several mice deficient in BCR signaling to test this hypothesis, and found that reduced BCR signaling induces more receptor editing. When we tested mice with excessive BCR signaling or pharmacologically mimicked the BCR signal, we found a decrease in receptor editing. These experiments provide compelling evidence for the inhibitory tonic signaling hypothesis and against the activation signaling hypothesis. Another question unanswered in the B cell field is how B cells are transformed into leukemia and lymphoma. There have been whole genome analysis studies to show that genes involved in the BCR signaling pathway are involved in many B cell malignancies, including leukemia, lymphoma and myeloma. Our studies in mice provide evidence that in combination with constitutive STAT5 activation, a loss of genes involved in B cell development ( ebf1 and pax5 ) or pre-BCR signaling ( blnk, PKCβ, and btk ) results in leukemia. These are genes that have been shown to be deleted in B cell acute lymphoblastic leukemia (ALL). We also demonstrate an increase the phospho-STAT5 levels in adult BCR-ABL + ALL patients. Herein we have developed a new mouse model for B-ALL that may be useful in testing and perfecting treatment protocols used on ALL patients.
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    Characterization of Broadly Neutralizing Antibodies to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)
    (2020-01) Young, Jordan
    Porcine reproductive and respiratory syndrome (PRRS) is an all too common disease with a devastating impact on pork producers in the US and worldwide. The disease is caused by the PRRS virus (PRRSV) which leads to abortions and other forms of reproductive failure in sows and severe respiratory disease in adults and neonates. Unfortunately, current vaccines against PRRSV provide limited protection and often only protects against closely related viruses. Efficacious vaccine-based prevention of infectious disease is based on antigen-specific long-lived memory B and T cells. Protective, or neutralizing, antibodies produced by memory B cells, that are activated in response to virulent pathogen challenge, are critical to this process. Thus, it is of utmost importance to investigate antibodies with neutralizing potential especially broadly neutralizing antibodies (bnAbs), which possess the ability to neutralize distantly related strains. In this manuscript, I take a reverse approach to the classic neutralization study, starting first with pigs we know are broadly neutralizing and then investigating the identity of the responsible B cells, using a novel technique for the isolation of PRRSV neutralizing antibodies. PBMCs were harvested from pigs sequentially exposed to divergent PRRSV isolates and vaccine. Memory B cells were then transduced, with a proprietary retroviral vector developed by AIMM Therapeutics, containing genes highly expressed by germinal center B cells, creating an immortalized B-cell population. B-cells were then sorted by FACS and five PRRSV-specific B cells were isolated. All identified PRRSV-specific antibodies were found to be broadly binding to all PRRSV-2 isolates tested, but not PRRSV-1. Antibodies against GP5 protein, commonly thought to be the PRRSV neutralizing epitope, were found to be highly abundant, as four out five clonal B cells were GP5 specific. Next, an isolated GP5 clone was discovered to be neutralizing against homologous, but not heterologous PRRSV. Sequencing of this clone’s heavy chain variable region and CDR3 revealed a gene that was heavily mutated compared to germline sequence, suggesting somatic hypermutation playing an essential role in generation of broadly neutralizing antibodies. Further investigation of these antibodies, and others, may lead to the elucidation of conserved neutralizing epitopes that can be exploited for improved vaccine design and lays the groundwork for the study of bnAbs against other porcine pathogens.
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    Immunopathogenesis of avian metapneumovirus in the Turkeys
    (2009-09) Cha, Ra Mi
    Avian metapneumovirus subtype C (aMPV/C) causes a severe upper respiratory tract (URT) disease in turkeys. The disease is characterized by viral replication and extensive lymphoid cell infiltrations in the URT. The identity of infiltrating cells and their possible involvement in the immunopathogenesis of the disease are not known. The role of local mucosal immunity in viral defense has not been examined for aMPV/C. The overall objective of the study was to examine the immunopathogenesis of aMPV/C in ovo and hatched turkeys, with emphasis on the involvement of local mucosal immunity in viral defense. Three specific objectives were pursued. First, the immune cells, especially mucosal T cells that infiltrate the URT of turkeys following aMPV/C exposure were characterized. Two-week-old aMPV/C antibody-free turkeys were inoculated oculonasally (O/N) with live aMPV/C. At 5 and 7 days post inoculation (DPI), lymphoid cells infiltrating the mucosal lining of the turbinates of the virus-exposed and untreated control turkeys were isolated by enzymatic treatment. In the URT, aMPV/C exposure increased the proportion of CD8+ T cells but not of CD4+ T cells. In addition, CD8 gene expression was upregulated after virus exposure whereas CD4 gene expression remained unchanged. At 5 and 7 DPI, aMPV/C-exposed turkeys showed upregulated gene expression of IFN-gamma and IL-10 in the turbinate tissue. These results suggested that aMPV/C modulated local cellular immunity in the URT of turkeys. Secondly, the ability of an adjuvanted inactivated aMPV/C (Ad-iaMPV/C) inoculated by the respiratory route to induce protective mucosal immunity in the URT was examined. aMPV/C antibody-free turkeys were inoculated via the O/N route with inactivated virus adjuvanted with synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid (Poly IC). Ad-iaMPV/C immunized turkeys showed an increased number of mucosal IgA+ cells in the URT and increased levels of virus-specific IgG and IgA in the lachrymal fluid and serum. After 7 or 21 days post immunization, turkeys were challenged with pathogenic aMPV/C via the O/N route. Turkeys immunized with Ad-iaMPV/C were protected against microscopic lesions and the replication of the challenge virus in the URT. These observations revealed that inactivated aMPV/C administered by the respiratory route induced protective immunity against challenge with the pathogenic virus. As the last objective, we studied the immunopathogensis and protective immunity of aMPV/C in turkeys following in ovo exposure. aMPV/C was inoculated into commercial aMPV/C antibody-free turkey eggs via the amniotic route at embyronation day (ED) 24. Hatchability of eggs was not affected by the virus inoculation. At the day of hatch (ED 28) (4DPI) and 5 days post hatch (9DPI), the virus genome was detected by qRT-PCR in the turbinate, trachea and lung but not in the thymus or the spleen. Turbinate mucosa had mild lymphoid cell infiltration, and there were no detectable lesions in the lung. Spleen cells and thymus cells from virus-exposed turkeys responded poorly to T cell mitogens. In addition, IFN-gamma and IL-10 gene expression was increased in the turbinate tissue of virus-exposed turkeys. In ovo virus exposure increased the levels of aMPV/C-specific IgG in the serum and the lachrymal fluid. At 3 weeks of age, the in ovo immunized turkeys were protected against a challenge with pathogenic aMPV/C. These data indicated that in ovo vaccination may be used in turkeys to control aMPV/C.
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    The porcine memory B cell in conferring long term adaptive immunity to viral pathogens
    (2017-06) Rahe, Michael
    Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important pathogen of swine health and well-being worldwide. Discovered nearly thirty years ago, there is still no vaccine capable of producing a broadly protective immune response against the virus. This deficiency is due in large part to a failure to understand how the adaptive immune system responds to vaccination or infection. Specifically, there is little to no knowledge regarding the all-important memory immune response to PRRSV. The objective of this dissertation was to fill in this significant gap in knowledge by identifying and characterizing the memory B cell response to PRRSV vaccination. First, we identified the presence of memory B cells against PRRSV non-structural protein 7 (nsp7) through the use of a novel in vitro B cell culture system. Next, we created and validated a novel reagent, a B cell tetramer, against nsp7 to enhance the speed and sensitivity of memory cell identification. Finally, through the utilization of the nsp7 tetramer, we evaluated the regional specificity and dynamic nsp7 specific B cell response to PRRSV MLV vaccination within select secondary lymphoid organs. These results constitute the first evidence of regional specialization of the B cell response to vaccination in an outbred animal species. Furthermore, the presented methods are a blueprint for the study of antigen specific cellular responses to any significant pathogen of animals important for food or fiber.
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    Protective B cell responses during Salmonella infection
    (2013-07) Nanton, Minelva Romelia
    In this thesis, we aim to further define the protective role of B cells during Salmonella infection. Understanding how B cells contribute to protective immunity is essential for designing more efficacious next generation vaccines against Typhoid and new vaccines against Paratyphoid and Non-Typhoid Salmonella strains. In the second chapter we test the hypothesis that systemic IgG production is an essential mechanism by which B cells protect against virulent infection. Surprisingly, we found that unlike B cells deficient mice, mice that lack the ability to secrete IgG or all antibodies are still largely protected from virulent Salmonella following resolution of infection with a vaccine strain. We further show that B cells, in the absence of antibody, are required for establishing robust memory Th1 responses. That B cells are important for protective immunity during secondary infection, suggests that we should better understand how this protection develops during a primary infection. In the third chapter, we aimed to define the kinetics and characteristics of the primary endogenous ovalbumin-specific B cell response during live vaccination with attenuated Salmonella expressing ovalbumin and OVA-specific B cell tetramers. We show that unlike immunization with OVA and adjuvant alone, there was a delay in the OVA-specific B cell responses until the infection had cleared. Additionally, we observed a delay in germinal center formation in these OVA-specific B cells, also in contrast to immunization with OVA and adjuvant alone. Upon co-administration of OVA with Salmonella infection, there was a decrease in the expansion and germinal center formation of OVA-specific B cells compared to mice that received OVA alone. We further discovered that, unlike culling of T cells during Salmonella infection, this inhibition of the B cell might not only be linked to expression of Salmonella Pathogenicity Island II proteins, but to effectors that modify the intraphagosomal load of bacteria.