Browsing by Subject "Antibody repertoire"
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Item Elucidation of the human B cell immune repertoire by high-throughput sequencing and computational simulation(2016-12) Wu, Gabriel Chi Sun; Marcotte, Edward M.; Wilke, Claus O; Georgiou, George; Tucker, Haley; Daniels, Michael JThe human immune system carefully balances the need to maintain stable responses to familiar stimuli with the need for agile responses to an ever changing array of potential dangers. Classic techniques allow for detailed evaluation of parts of the immune system, while emerging technologies allow for more systems-level analysis of the immune system as a whole. In this dissertation, I use high-throughput techniques and computational analysis to advance our understanding of the human bone marrow B cell repertoire. First, I describe the variation in composition of human bone marrow plasma cells from the same individual over time. I show that the frequency of gene and gene combination usage, assayed by high-throughput sequencing, is temporally stable over 6.5 years. Next, I describe a computational model that simulates the process of high-throughput sequencing of immune cells and identify the major sources of error in these experiments. Specifically, this simulation demonstrates that the typical shape of the experimental distribution of antibodies may be in large part be due to error generated in the experimental process and not a biologically relevant observation. I go on to demonstrate the current limits in understanding the initial distribution of the immune repertoire due to accumulated noise in the experimental process. The work presented here represents the longest longitudinal study to date of high-throughput sequencing techniques used to study the repertoire of human B cells. In addition, the computational model frames the technical challenges of immunological repertoire analysis. This knowledge will provide the basis of future studies to understand the nature of B cells in human bone marrow. It will be relevant for both academic and clinical researchers studying the immune system at basal state as well as at an active defense state. Ultimately, it provides guidance to the community at large with the intent of improving immunology and human health.Item Global survey of the immunoglobulin repertoire using next generation sequencing technology(2014-12) Hoi, Kam Hon; Georgiou, GeorgeSpecific and sensitive recognition of foreign agents is a critical attribute of the overall effective immune system required for maintaining host protection against challenge from pathogenic cells. In the humoral arm of the immune system, this recognition attribute is carried out by the cell surface bound immunoglobulin-like receptors (BCR) and its soluble forms i.e. antibodies. Over several million years of evolution, the immune system has adopted several strategies for diversifying the antibody sequence and thus its ability to recognize an astronomical variety of molecules through the combinatorial assembly of a small number of DNA segments or genes. Among these immunoglobulin gene diversification strategies, antibody somatic VDJ recombination and junctional diversity are the fundamental mechanisms in generating a broad range of antibody specificities. Understanding how the genetic diversity of antibodies is affected in health and disease is critical for a wide range of medical applications, from vaccine evaluation to diagnostics and therapeutics discovery. Because of the very large number of distinct antibodies encoded by the more than 100 billion B cells in humans, it is essential to use high throughput next generation sequencing technologies in order to obtain an adequate sampling of the sequences and relative abundance of different antibodies expressed by B cells in clinical samples. The process requires rigorous methods for first, experimentally determining the sequences of antibodies in a sample and for second, informatics tools designed for distilling this information for practical purposes. This dissertation describes a variety of experimental approaches and informatics tools developed for the determination and mining of the antibody repertoire. The information from this work has led to major conclusions regarding the nature of the antibody repertoire in healthy individuals, in volunteers following vaccination, and in HIV-1 patients.Item Methods for proteomic characterization of antibody repertoires and de novo peptide sequencing(2016-05) Horton, Andrew Pitchford; Marcotte, Edward M.; Georgiou, George; Brodbelt, Jennifer S; Jiang, Ning; Ippolito, Gregory CDriven by the increased performance and availability of protein mass spectrometry and next generation sequencing technologies, research in proteomics and systems biology has expanded far beyond the study of model organisms. This heralds a deeper understanding of biology, the world, and human health. However, it also brings significant new challenges to the interpretation of sequencing and mass spectrometry data, the current software tools ill-suited for many modern studies. The first half of this dissertation explores some of these challenges and solutions in the context of a particularly demanding domain – that of serological antibody proteomics. Our team has developed a combined sequencing and proteomics approach for profiling the human serum antibody repertoire. This opens an unprecedented view into the nature of the adaptive immune system and provides insight on antibody repertoire dynamics in both health and disease. The platform also provides effective means to evaluate vaccine efficacy and identify potential antibody therapeutics. Chapter 1 reviews recent advances in and results from such molecular level characterization of the serum antibody repertoire. Detailed in the second chapter, challenges specific to antibody repertoire proteomics preclude the use of standard analysis methods and motivated our development of novel tools and approaches for interpreting serum repertoire proteomic data. I will shift focus in chapters 3 and 4 to present an experimental and computational workflow for accurate and full-length de novo peptide sequencing. We applied 351 nm ultraviolet photodissociation (UVPD) on chromophore-tagged peptides and developed software for sequencing the resultant UVPD mass spectra. Improvements described in chapter 4 enable the software to automatically learn from and interpret new types and combinations of spectra from the same precursor peptide. We demonstrate the effectiveness of this machine learning framework on CID/UVPD spectral pairs and obtain results, from low resolution spectra, comparable to current state of the art. Continued development of these de novo interpretation and sequencing methods, in part or in whole, may sidestep many of the remaining challenges facing repertoire proteomics, and successful application of these efforts promises further advancement in antibody repertoire characterization and understanding.