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The process of generating the vast diversity of immunoglobulin receptors and secreted antibodies begins with the recombination of the joining (JH), diversity (DH) and variable (VH) genes in the immunoglobulin heavy chain locus. The ability to produce antibodies is restricted to the B cell lineage and is tightly regulated, starting with the temporal separation of the recombination process, in which DH-JH precedes VH-DHJH recombination. Successful recombination of both heavy and light chain loci results in the expression of an antigen receptor on the cell surface. Subsequent selection stages remove non‑functional and autoreactivity receptors from the final pool of antigen responding B cells that ultimately give rise to antibody secreting plasma cells. Understanding the complexity of the recombination processes and the diversity of the resulting antibody repertoire has been a major focus of academic and industrial research alike. Therapeutic monoclonal antibodies have seen many successful applications within the clinic and they constitute a billion-dollar industry. However, limitations therein have resulted in the emergence of antibody engineering approaches and the use of natural sources of alternative heavy chain only antibodies (HCAbs/nanobodies). The biotechnology company Crescendo Biologics has taken the highly desired characteristics of HCAbs a step further with the creation of a mouse platform capable of producing fully humanized HCAbs. The Crescendo platform presents a unique opportunity to expand our understanding of how mouse B cell development functions by exploiting the features of heavy chain only antibody production. Furthermore, the platform enables the expansion of our limited knowledge of the epigenetic mechanisms involved in the recombination of the human immunoglobulin heavy chain locus. Using flow cytometry, with dimensionality reduction analysis approaches, I investigated B cell development in the context of HCAbs. These studies revealed a previously uncharacterised developmentally intermediate B cell population. Due to ethical and availability limitations to studies of human bone marrow, the primary pre-selection human B cell repertoire has not been studied in detail. The isolation of several B cell developmental stages and the use of our novel DNA-based high-throughput unbiased repertoire quantification technique, VDJ-seq, allowed me to study recombination of the human IGH locus sequence and observe HCAb repertoire selection within the mouse environment. The adaptation of next generation sequencing techniques to antigen receptor repertoire quantification has provided an unprecedented insight into repertoire diversity and the alterations it undergoes during infection or ageing. Our VDJ-seq assay is unique in its ability to interrogate DNA recombinants. To expand its capabilities, I investigated several limitations of the technique, including mispriming and PCR/sequencing errors, and implemented experimental and bioinformatics solutions to overcome them, which included the creation of a comprehensive analysis workflow. Finally, I have developed and applied a novel network visualisation method for genome-wide promoter interaction data generated by promoter capture Hi-C. The availability of high quality human pluripotent stem cell datasets allowed me to utilise the new techniques to further our understanding of the dynamics of genome organisation during early human embryonic development. This visualisation approach will be directly applicable to understanding B cell development. |
