Date of Award

Summer 2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Eline T. Luning Prak

Abstract

Antibody diversity is generated through the random recombination of immunoglobulin gene segments. As a consequence of this stochastic process, antibodies recognizing self-antigens can also be produced. Continued antibody gene rearrangement, termed receptor editing, is an important mechanism of central B cell tolerance that serves to alter the antibody specificity of developing B cells. Although it has been demonstrated to be critical for normal B cell development, the role of receptor editing in the context of autoimmune disease and the forces that stimulate receptor editing are not fully understood. To address these issues, a novel quantitative assay based upon Recombining Sequence (RS) recombination was developed to measure receptor editing rearrangements in B cell populations. RS rearrangement is a recombination of a non-coding gene segment in the immunoglobulin κ light chain locus. Unlike other markers of receptor editing, the RS rearrangement assay does not rely upon expressed components of the B cell receptor and therefore is not restricted to specific autoantigens.

As receptor editing may be defective in some autoimmune individuals, the RS assay was applied to autoimmune mouse models of systemic lupus erythematosus (SLE) and type 1 diabetes (T1D), which demonstrated decreased receptor editing levels relative to wild-type mice. Low RS rearrangement levels were also observed in human subjects with SLE or T1D, suggesting that defects in receptor editing may contribute to disease susceptibility. Additionally, RS rearrangement was used to assess the role of self-antigen mediated B cell signaling in the stimulation of receptor editing. B cells expressing an anti-DNA heavy chain were found to undergo more extensive receptor editing and functional precursors of editing were found to be biased towards autoreactivity, indicating that self-reactivity may promote receptor editing. To begin to examine whether positive signaling is required for receptor editing, the roles of the inhibitory kinase Lyn and a disease associated variant of the lymphoid phosphatase PTPN22 were analyzed. The results indicate that aberrant activity of these signaling components alone does not influence receptor editing. Collectively the findings described in this dissertation demonstrate that RS rearrangement is an effective means of estimating receptor editing that may be used to monitor B cell central tolerance and further evaluate the regulation of editing rearrangement.

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