All ETDs from UAB

Advisory Committee Chair

Peter D Burrows

Advisory Committee Members

Louis B Justement

John D Mountz

Harry W Schroeder

Document Type

Dissertation

Date of Award

2009

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

VH replacement occurs through RAG-mediated secondary recombination between an upstream VH gene and an existing VHDJH joint. In the first part of this dissertation, we focused on the potential regulation of VH replacement in the human B lineage EU12 cells muHC+ population, which underwent spontaneous VH replacement during in vitro culture. We cloned one of the dominant IgH genes, A19H, expressed in the EU12 muHC+ cells, and generated recombinant antibodies. We found that these recombinant antibodies recognize SM antigens. The anti-SM reactivity of A19H can not be efficiently neutralized by a randomly selected group of fourteen IgL chains from healthy controls, suggesting that the SM reactivity is mainly contributed by the A19H heavy chain. Indeed, the anti-SM reactivity was attenuated in the subsequent VH replacement product, A21H, cloned from the EU12 muHC+ cells. These results indicated that VH replacement is essential to edit IgH genes encoding anti-SM antibodies. In the second part of this dissertation, we investigated the potential contribution of VH replacement in systematic lupus erythematosus (SLE). We performed single cell PCR assay and cloned 447 IgH genes with paired Igkappa or Iglambda genes from plasma cells (CD19lowCD27high) and naïve B cells (CD19+CD27-) of three SLE patients and two healthy controls. Analyses of the IgL genes from SLE patients showed signs of excessive receptor editing of IgL genes, leading to elevated usages of downstream Jkappa5 and Jlambda3 genes. Strikingly, the frequency of VH replacement products in the IgH genes from all three SLE patients is significantly higher than in the control clones, especially in plasma cells. The VH replacement `footprints' preferentially encoded charged amino acids in the IgH CDR3 regions. Analyses of 12 recombinant antibodies derived from SLE plasma cells showed that 75% of recombinant antibodies recognize with cytoplasmic or nuclear antigens, in which more than half of them are encoded by VH replacement products. Taken together, the works presented in this dissertation suggested that VH replacement is normally employed to delete unwanted IgH genes; however, excessive VH replacement might generate IgH genes with extremely long CDR3 regions, which encode autoreactive antibodies.

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