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B-cell acute lymphoblastic leukemia (B-ALL) represents a collection of diseases that are categorized into subtypes based on the presence of recurrent cytogenetic abnormalities. These abnormalities often result in the expression of oncogenic drivers that denote a standard- or high-risk for relapse. Currently, survival rates boarder 40% for adult patients and relapses are often observed in patients lacking high-risk markers. Thus, there is an unmet need for biomarkers that can identify all high-risk leukemia, and development of novel therapies based on a better understanding of the molecular drivers of B-ALL. To address this need, I designed a multi-parameter phospho-flow cytometry platform and characterized basal and cytokine-potentiated signaling in adult B-ALL samples. I identified patterns of cytokine-responsiveness across B-ALL patients that correlated with the presence of high-risk oncogenic drivers. Furthermore, I demonstrated that small-molecule inhibitors could abrogate cytokine-induced signaling in high-risk patients suggesting these inhibitors may compliment current chemotherapeutic protocols.

B-cell acute lymphoblastic leukemia (B-ALL) represents a collection of diseases that are categorized into subtypes based on the presence of recurrent cytogenetic abnormalities. These abnormalities often result in the expression of oncogenic drivers that denote a standard- or high-risk for relapse. Currently, survival rates boarder 40% for adult patients and relapses are often observed in patients lacking high-risk markers. Thus, there is an unmet need for biomarkers that can identify all high-risk leukemia, and development of novel therapies based on a better understanding of the molecular drivers of B-ALL. To address this need, I designed a multi-parameter phospho-flow cytometry platform and characterized basal and cytokine-potentiated signaling in adult B-ALL samples. I identified patterns of cytokine-responsiveness across B-ALL patients that correlated with the presence of high-risk oncogenic drivers. Furthermore, I demonstrated that small-molecule inhibitors could abrogate cytokine-induced signaling in high-risk patients suggesting these inhibitors may compliment current chemotherapeutic protocols.

B lymphocytes constitutes an important arm of the immune system, and their response to antigen is largely dependent upon signal transduction through the B cell receptor (BCR). Such a potent receptor, however, needs to be further balanced by positive and negative regulators to prevent harmful effects that may arise from inappropriate stimulation. Src family protein tyrosine kinase Lyn is involved in both positive and negative regulation, since the both gain-of-function Lyn and loss-of-function Lyn mutations caused autoimmunity in mice. The exact signalling pathway(s) regulated by Lyn in B cells, however, are still not clear. Work presented in this thesis attempts to elucidate the biochemical mechanisms that underline the double-edged nature of Lyn in BCR signalling. (For complete abstract open document)

B lymphocytes produce a wide array of antibodies to recognize a countless number of antigens. This highly diverse repertoire is produced during B cell development in the bone marrow from the immunoglobulin heavy chain (Igh) and light chain (Igk and Igl) loci. The mouse Igh is a large (~3Mb) multigene locus that contains 195 variable (V), 10 diversity (D) and 4 joining (J) genes that undergo developmentally regulated V(D)J recombination to produce the variable region of the antibody. Gene expression depends on spatial organisation of chromatin. To ensure that all V genes have a chance to recombine, they are brought into physical proximity to the D-J region by locus contraction and DNA looping. Not all V genes recombine with equal frequencies and we aim to investigate how dynamic changes in 3D structure of the Igh locus facilitate V(D)J recombination. Chromosome conformation capture techniques have revolutionised studies of genome conformation. I have applied a novel form of enriched Hi-C to study both intra-locus (cis) and genome-wide (trans) interactions of the immunoglobulin loci in pro-B and pre-B cells. This method provides a higher resolution than Hi-C and is less biased than 4C and 5C. I have mapped all cis interactions within the Igh locus to produce a comprehensive view of the structure of the locus prior to recombination. This approach has shown that the 3’ superanchor (3’CBEs) and the Intergenic Control Region 1 (IGCR1) containing CTCF sites are the two most interacting regions in the locus making long-range contacts with all V genes. A second major conformational feature is that the distal V genes form a large tightly looped domain forming the centre of mass of the locus to which the 3’CBEs and IGCR1 loop. Thanks to a collaboration on polymer modelling, 5000 single conformations were simulated based on the ensemble Hi-C data. This showed that every structure is different, supporting a model of dynamic and flexible organisation of the locus rather than hierarchical subdomains therein. Moreover, there is only a slight trend for V genes interacting more often with the D-J region to have higher recombination scores, supporting an ‘equal opportunity for all’ model in which participation of V genes in V(D)J recombination is not constrained by linear genomic distance from the DJ region. Nevertheless, CTCF binding level does contribute to V gene recombination frequency. I have also discovered that Igh and Igk loci participate in a highly specialised network of genome-wide (trans) interactions involving genes encoding B cell-specific factors essential for activation and maintenance of B cell identity, including Pax5, Foxo1, Ebf1, and Runx1. I have validated these by 3D DNA FISH and found that at the pro-B cell stage the Igh is involved in many trans interactions, whereas Igk does not make any contacts. In contrast, Igk gains numerous trans interactions at the pre-B cell stage, many of which overlap with the interactions Igh participates in at both developmental stages. Together, these findings reveal a complex developmentally regulated orchestration of genome conformation changes that underpins B cell development.

Asthma is a debilitating lung disease that affects nearly 300 million people worldwide. Environments with high humidity and subsequent mold exposure often trigger allergic asthma. Sex differences have been reported in the incidence, prevalence, and severity of asthma. B-lymphocytes are recruited in high numbers to the allergic lung in response to the inhalation of Aspergillus fumigatus mold spores (conidia). In this work, we used a mouse model of allergic fungal asthma to assess environmental humidity, sex, and B-lymphocytes in an inhalational model of allergic fungal asthma. Our results showed that animals sensitized in low humidity conditions had no airway hyperresponsiveness (AHR), inflammation, but an increase in IgG3 antibody production. Males weighed more than females, female mice had more fibrosis and produced more IgG3 Ab, but sex showed no impact on low humidity. C19+ B-lymphocytes differentially downregulated multiple genes related to allergic asthma returning the body to homeostasis.

M.Med.(Haematology), Faculty of Health Sciences, University of the Witwatersrand, 2008 / This study aimed to identify the relevance of these prognostic features in the modern treatment era in South African children. A retrospective analysis of the presentation clinical and laboratory features and treatment outcomes of all children treated for Precursor B cell ALL at the Johannesburg Hospital was performed. Between January 1997 and May 2007, 100 children were reviewed. Clinical features (age, race and gender) emerged as significant prognostic variables. Laboratory features (white cell count and genetic features) lacked significance. Early morphologic response on day 15 identified a subgroup associated with a favourable outcome. However the presence of > 5% blasts was not significantly predictive of relapse or death at this time point. Minimal residual disease (MRD) detection by modified immunoglobulin gene rearrangement and flow cytometry techniques did not improve the predictive value of the morphological assessment. In a low resource setting, the challenge is to design cost effective MRD detection methods to improve the identification of patients at risk for relapse.

Anthrax Vaccine Adsorbed (AVA) immunization protects against anthrax disease by eliciting a neutralizing antibody response. However, antigen-specific antibody concentrations are not observed in high quantities until three immunizations have been administered over six months. Even then, humoral responses to AVA do not provide long-term immunity without an annual booster. We followed six healthy volunteers over the five-dose, 18-month AVA schedule to characterize the genetics of the immunoglobulin repertoire during the vaccination series. Two tiers of data were collected: 1) Immunoglobulin variable region genes (IgVRG) from bulk sorted naïve, memory and plasmablast (PB) B cells and 2) single cell sorted and sequenced IgVRG from plasmablasts. Samples were collected prior to and one and two weeks following each immunization. Our initial analyses indicated that technical error, the variation introduced by biological sampling and standard sample preparation, resulted in skewed output, and we developed a model to better estimate quantitative values from Ig-seq. We also utilized unique molecular identifiers to correct for nucleotide errors and PCR over-amplification. Our analysis of IgVRG following AVA administration reveals that the population of peripheral PBs following primary immunization is not distinguishable from the pre-immune peripheral PB repertoire. These PBs have more somatic mutations than expected for newly activated and differentiated naïve B cells, and are unlikely to be vaccine-elicited. In contrast, PBs observed following the 2nd dose have low mutation frequencies that increase upon subsequent vaccination. These clones are more persistent than clones first observed following any other immunization, but still make up a very small proportion of the overall repertoire. At no time is the clonal repertoire consistently dominated by a few clones, and the total and plasmablast repertoires are highly transient, even after the elicitation of vaccine-specific antibodies. AVA immunization thus results in a polyclonal B cell response which is not dominated by one or a few highly specific, strongly-elicited clones. We conclude that primary immunization by AVA is not sufficiently immunogenic to elicit vaccine-responsive, class-switched PBs to the periphery, nor is complete AVA immunization able to sustain proliferation of individual clones, providing insight into why AVA may require regular boosts.

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