All ETDs from UAB

Advisory Committee Chair

John F Kearney

Advisory Committee Members

Peter Burrows

David D Chaplin

Christopher Klug

Harry W Schroeder

Document Type

Dissertation

Date of Award

2009

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

An understanding of the molecular mechanisms involved in the generation of protective antibody responses to polysaccharides associated with pathogenic microorganisms is of importance for improving vaccine design. The heavy chain third complementarity-determining region (HCDR3) of an antibody molecule is at the center of its antigen-binding site and plays a determinative role in antigen recognition. The goal of this dissertation was to investigate the role of HCDR3 diversity and composition on the antibody response to the polysaccharide α 1→3 Dextran (DEX). In the first study, we investigated the role of TdT, a DNA polymerase that plays a major role in generating diversity of lymphocyte antigen receptors during V(D)J recombination. We show that the DEX-specific antibody response is lower and the dominant DEX-specific J558 idiotype (Id) is not detected in TdT-/- mice when compared to wild type BALB/c (WT) mice. Complementation of TdT expression in TdT-/- mice by early forced expression of TdT restored WT expression of J558 Id+ and also abrogated the development of the minor M104E Id+ clones. These data suggest that TdT is essential for the generation of the higher affinity DEX-responsive J558 clone. In the second study, we investigated the DEX-specific antibody response of gene-targeted mice limited to use one DH gene segment in different reading frames (D-limited mice). We show that the DEX-specific antibody response is comparable in D-limited mice to that of WT mice. D-limited mice forced to use DH reading frame 2 (∆D-DμFS) showed higher antibody responses to DEX compared to other D-limited mice and higher levels of J558 Id compared to WT mice. ∆D-DμFS DEX-specific plasma cells contained greater numbers of V(D)J rearrangements that encoded for expression of J558 Id compared to other D-limited mice. D-limited mice had a higher frequency of DH-less HCDR3 regions compared to WT. These data suggest that constraints on HCDR3 diversity by limiting DH usage do not preclude the generation of a proper anti-DEX response and that altered DH reading frame usage could enhance the generation of the anti-DEX clone that is predominant in the WT.

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