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In partial fulfillment of the requirements for the degree of
Doctor of Philosophy in Bioinformatics
in the School of Biological Sciences
Meixue Duan
Defends her thesis:
investigation of maturation and survival of human Long-lived plasma cells using integrated single-cell analysis
Wednesday, August 10
12:00 PM Eastern Time
Whitaker-BME, classroom 1232
Thesis Advisors:
Dr. Greg Gibson
School of Biological Sciences
Georgia Institute of Technology
Dr. Frances Eun-Hyung Lee
Department of Medicine
Emory University
Committee Members:
Dr. I. King Jordan
School of Biological Sciences
Georgia Institute of Technology
Dr. Peng Qiu
Department of Biomedical Engineering
Georgia Institute of Technology
Dr. Julia Babensee
Department of Biomedical Engineering
Georgia Institute of Technology
Abstract:
Single-cell transcriptomic sequencing (scRNA-seq), first introduces in 2009, provides an unprecedented opportunity to profile gene expression at the level of individual cells. This approach has rapidly become the favored technique to address fundamental cell biological phenomena such as the pathways of differentiation and maturation of cell types, as well as to explore and elucidate the pathology of disease. Long-lived plasma cells (LLPC), which are quiescent and terminally differentiated B lineage cells, primarily function as antibody (Ab) manufacturing factories that secrete of Abs, which are the primary contributor to durable humoral-mediated immunity.
The Lee Lab at Emory University has been working on elucidating of heterogeneity of plasma cells. Using cell surface protein levels of CD19, CD38 and CD138 to define subtypes, they have demonstrated transcriptional and functional differences between short-lived plasma cell (SLPC) and LLPC using bulk RNA-seq techniques. They have also devised a human in vitro antibody-secreting cell (ASC) culture media that supports experimental manipulations. The first aim of the thesis utilized scRNA-seq combined with single cell BCR repertoire sequencing (scVDJ-seq) to characterize the heterogeneity, maturationmaturation, and survival of human bone marrow plasma cells (BMPC). These analyses first demonstrated that there are two maturation stages and four maturation trajectories associated with 15 robust human BMPC subgroups. High resolution bioinformatics analysis of the data provided by scRNA-seq identified critical pathways associated with maturation, and notably, manual curation of all annotated apoptosis/apoptotic related pathways led to discovery of a novel ASC pro-survival factor, MDK. Therefore, the second aim of my thesis was to explore the genes and pathways that MDK regulates so as to infer the intracellular downstream signaling it activates by gene regulatory network analysis. Integration with experimental manipulation validated the identities of active receptors for MDK, and these results are expected to support engineering of ASC in future research.
Previous studies from the Sanz lab at Emory University observed that a subgroup of circulating pathogenic autoreactive ASCs expand during systemic lupus erythematosus (SLE) patient flares. A distinct subset of class-switched B cells lacking the memory B cell markers CD27, IgD and CXCR5, termed DN2 (CXCR5-CD21-CD11c+) was also reported to be expanded in the peripheral blood of SLE patients. Considering the success of applying scRNA-seq and scVDJ-seq to elucidate fundamental biology with respect to human BMPC, the third aim of my thesis utilized my analytical pipeline to explore the potential pathogenesis of SLE by comparing B cell heterogeneity in human BM between healthy controls and SLE patients. Unfortunately, the results reported here were compromised due to a sample mixing issue that was only detected late in the analysis. However, the enhanced methodology I constructed for the study appears to have improved good performance by discovering that 1) the general genes and functions that shift during B cell development between HD and SLE; 2) most SLE B cells prior to the immature B stage are less differentiated from normal B cells, except for a subgroup of Pro B and immature B cells; 3) SNP-heritability of SLE disease is enriched at a central tolerance checkpoint in the immature B stage; and 4) multiple key transcription factors exhibit turnover in gene expression that differs between SLE and controls at the central tolerance checkpoint.
