The adaptive immune system is founded on lymphocytes expressing a vast array of antigen receptors that provide specific recognition in responding to pathogen. Receptor diversity is generated by V(D)J rearrangement and, in immunoglobulin (Ig) genes, post-rearrangement somatic hypermutation. In higher vertebrates mechanisms involving not only hierarchical activation of the 3 Ig loci but also feedback by a successful rearrangement are thought to restrict expression to one allele and one isotype (allelic and isotype exclusion). This process, which ensures primarily one receptor species per cell, is not well understood. Elucidating shared and divergent regulatory pathways in mammals and in sharks, representatives of the earliest vertebrates with a RAG recombinase-based immune system, will allow us to gain insight into the underlying principles upon which adaptive immune systems are constructed. Preliminary data suggest that shark B cells expressing one light (L) chain type at the cell surface also transcribe other L chain types. However, it is not clear how the more than 70 L chain miniloci in nurse shark are regulated.
Specific Aim 1 is to examine L chain expression in single shark B cells and determine whether there is clonal expression of the antigen receptor. Some heavy (H) chain cDNA sequences contain V(D)J in combination with C regions different from what is anticipated from the established genomic organization. This genetic exchange occurs somatically and between distantly located IgH genes;it resembles the H chain switch recombination that exists in mammals.
Specific Aim 2 is to determine the linkage relationship among the 15 IgH genes and investigate the mechanistic basis for the IgH chain switching process in sharks. Since the switching occurs at the same time as somatic hypermutation, it is hypothesized that both are initiated activation- induced cytidine deaminase. Hypermutation in shark Ig uniquely includes tandem changes of 2-5 bp stretches and sometimes non-templated insertions. To understand how these changes are generated, we will in Specific Aim 3 isolate activated B cells in G2/M phase of the cell cycle, looking for those carrying both mutated and parental V(D)J sequence. The proposed research is part of long-term studies on Ig gene expression in early vertebrates. Understanding the nature of B lymphocyte diversification mechanisms involving DNA breakage and repair will provide insight into the pathogenesis of disease states that include autoimmunity and lymphoid malignancies predisposed by aberrant translocations.

Public Health Relevance

Our shark model system carries about 85 immunoglobulin genes that are targeted by enzymes introducing nicks or lesions in the course of generating antibody diversity. We have observed genetic exchange taking place between the distantly located loci. Studying these phenomena will help determine parameters for genetic exchange between non-allelic genes and give insight into the origins of somatic translocation events leading to disease states.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068095-08
Application #
8299117
Study Section
Cellular and Molecular Immunology - B Study Section (CMIB)
Program Officer
Marino, Pamela
Project Start
2004-01-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
8
Fiscal Year
2012
Total Cost
$343,917
Indirect Cost
$128,295
Name
Suny Downstate Medical Center
Department
Physiology
Type
Schools of Medicine
DUNS #
040796328
City
Brooklyn
State
NY
Country
United States
Zip Code
11203
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Hsu, Ellen (2011) The invention of lymphocytes. Curr Opin Immunol 23:156-62
Zhu, Catherine; Feng, Wendy; Weedon, Jeremy et al. (2011) The multiple shark Ig H chain genes rearrange and hypermutate autonomously. J Immunol 187:2492-501
Zhu, Catherine; Hsu, Ellen (2010) Error-prone DNA repair activity during somatic hypermutation in shark B lymphocytes. J Immunol 185:5336-47
Lee, Victor; Huang, Jing Li; Lui, Ming Fai et al. (2008) The evolution of multiple isotypic IgM heavy chain genes in the shark. J Immunol 180:7461-70
Malecek, Karolina; Lee, Victor; Feng, Wendy et al. (2008) Immunoglobulin heavy chain exclusion in the shark. PLoS Biol 6:e157
Hsu, Ellen; Pulham, Nicolas; Rumfelt, Lynn L et al. (2006) The plasticity of immunoglobulin gene systems in evolution. Immunol Rev 210:8-26
Hsu, Ellen; Criscitiello, Michael F (2006) Diverse immunoglobulin light chain organizations in fish retain potential to revise B cell receptor specificities. J Immunol 177:2452-62
Malecek, Karolina; Brandman, Julie; Brodsky, Jennie E et al. (2005) Somatic hypermutation and junctional diversification at Ig heavy chain loci in the nurse shark. J Immunol 175:8105-15

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