This is an R01 renewal application to study the mechanisms and genetics of immune tolerance in mice expressing B cell receptor-reactive superantigens. In the previous funding period, we generated and studied a large series of transgenic mice carrying custom designed superantigens reactive to immunoglobulin constant regions. Single chain antibodies reactive to Ig C regions were generated by engineering monoclonal antibody genes from hybridomas. These combining sites were expressed as chimeric membrane proteins, called macroself antigens (Ags), in transgenic mice. Macroself Ag- expressing mice were then analyzed for perturbations of lymphocyte survival, function, and receptor gene usage. Macroself Ag transgenics with specificity for IgM, Ig-kappa, IgD, and IgG2a allowed analysis of immune tolerance in different cell compartments and developmental stages in a non-clonal immune system. The utility of using macroself Ag mice as recipients of bone marrow to rapidly study the phenotypes of mouse mutants was also demonstrated. In this grant we propose to follow up these findings by focusing on two novel strengths of the experimental system-- the ability to conveniently study tolerance in B cells that have undergone an IgG class switch and the ability to impose a stringent in vivo screen for cells manifesting defective immune tolerance. In the first Aim we propose to characterize how IgG2a B cells are rendered tolerant in IgG2a-macroself Tg mice and to generate and analyze tolerance in IgG1-macroself Tgs. Candidate mouse mutants will be screened for defects in immune tolerance in the IgG compartment. In the second Aim, we will take advantage macroself Tg mice to impose selections in order to reveal tolerance defects in B cells with candidate or random mutations. We will take advantage of a random insertional mutagenesis screen to identify genes regulating tolerance. The long term goal of these studies is to understand the genetic basis of immune tolerance and autoimmunity.

Public Health Relevance

During the antibody response, antibody binding to microbes improves, owing to an intentional mutation process in B cells, the precursors of antibody forming cells. Because this natural mutation process can lead to self reactivity, it is important to understand how self reactive antibody formation is avoided. It is suspected that the regulation of B cells may go awry in autoimmune diseases such as lupus erythematosus. The goal of this project is to determine how immune tolerance normally suppresses the generation of self reactive antibodies and to identify genes involved in the process.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI059714-10
Application #
8418759
Study Section
Transplantation, Tolerance, and Tumor Immunology (TTT)
Program Officer
Ferguson, Stacy E
Project Start
2004-02-01
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2015-01-31
Support Year
10
Fiscal Year
2013
Total Cost
$393,648
Indirect Cost
$186,356
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Doyle-Cooper, Colleen; Hudson, Krystalyn E; Cooper, Anthony B et al. (2013) Immune tolerance negatively regulates B cells in knock-in mice expressing broadly neutralizing HIV antibody 4E10. J Immunol 191:3186-91
Ota, Takayuki; Doyle-Cooper, Colleen; Cooper, Anthony B et al. (2013) B cells from knock-in mice expressing broadly neutralizing HIV antibody b12 carry an innocuous B cell receptor responsive to HIV vaccine candidates. J Immunol 191:3179-85
Aoki-Ota, Miyo; Torkamani, Ali; Ota, Takayuki et al. (2012) Skewed primary Ig* repertoire and V-J joining in C57BL/6 mice: implications for recombination accessibility and receptor editing. J Immunol 188:2305-15
Duong, Bao Hoa; Ota, Takayuki; Aoki-Ota, Miyo et al. (2011) Negative selection by IgM superantigen defines a B cell central tolerance compartment and reveals mutations allowing escape. J Immunol 187:5596-605
Ota, Takayuki; Ota, Miyo; Duong, Bao Hoa et al. (2011) Liver-expressed Igkappa superantigen induces tolerance of polyclonal B cells by clonal deletion not kappa to lambda receptor editing. J Exp Med 208:617-29
Duong, Bao Hoa; Ota, Takayuki; Ait-Azzouzene, Djemel et al. (2010) Peripheral B cell tolerance and function in transgenic mice expressing an IgD superantigen. J Immunol 184:4143-58
Ait-Azzouzene, Djemel; Kono, Dwight H; Gonzalez-Quintial, Rosana et al. (2010) Deletion of IgG-switched autoreactive B cells and defects in Fas(lpr) lupus mice. J Immunol 185:1015-27
Beck, Kristina; Peak, Mandy M; Ota, Takayuki et al. (2009) Distinct roles for E12 and E47 in B cell specification and the sequential rearrangement of immunoglobulin light chain loci. J Exp Med 206:2271-84
Vela, Jose Luis; Ait-Azzouzene, Djemel; Duong, Bao Hoa et al. (2008) Rearrangement of mouse immunoglobulin kappa deleting element recombining sequence promotes immune tolerance and lambda B cell production. Immunity 28:161-70
Ait-Azzouzene, Djemel; Verkoczy, Laurent; Duong, Bao et al. (2006) Split tolerance in peripheral B cell subsets in mice expressing a low level of Igkappa-reactive ligand. J Immunol 176:939-48

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