Antibodies specific for the carbohydrate antigen GaIalpha1-3Galbeta1-4GIcNAc-R (alphaGal) represent a major immunological hurdle to successful xenotransplantation of pig organs and tissues into humans, and play an important role in providing protective host immunity to various pathogens. However, in spite of the importance of antibodies specific for alphaGal , little is known about how production of these or other anticarbohydrate antibodies is regulated. In the past funding period, we successfully developed a gene therapy based approach to induce B cell tolerance to alphaGal in order to facilitate xenotransplantation, as well as develop a mouse model to study how production of alphaGal specific antibodies is regulated. Using knockout mice, which lack the enzyme that synthesizes alphaGal, and consequently produce aGal reactive natural antibodies, as do humans, we examined whether expression of a retrovirally transduced alphaGT in bone marrow-derived cells could be used to induce tolerance to alphaGal. Expression of alpha(1,3)galactosyltransferase in bone marrow-derived cells of knockout mice completely inhibited production of aGal specific antibodies, resulting in stable long-term tolerance to alphaGal. These data suggest that gene therapy approaches may be used to induce B cell tolerance, effectively reshaping the B cell repertoire. Using gene targeting in embryonic stem cells we also constructed novel immunoglobulin gene knock-in mice in which antibody variable region gene segments encoding aGal specific antibodies were introduced into the immunoglobulin heavy and light chain loci, on either an antigen deficient or antigen sufficient background. In this renewal, we will use these mice to address fundamental issues related to regulation of anti-carbohydrate antibodies.
The specific aims are to: 1) Characterize development and regulation of B cells producing aGal specific antibodies; 2) Determine the mechanisms and requirements for anti-carbohydrate B cell tolerance; and 3) Determine the B cell receptor affinity which permits development of B cells that produce antibodies that bind self antigens. These studies should advance our understanding of how production of anti-carbohydrate is regulated, provide insight into how B cells producing antibodies with specificity for self-antigens escape negative selection, and may provide information critical to designing therapeutic agents that could be used to prevent or enhance production of anti-carbohydrate antibodies for clinical applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI044268-07
Application #
7031577
Study Section
Special Emphasis Panel (ZRG1-IMM-A (01))
Program Officer
Koh, Crystal Y
Project Start
1999-02-15
Project End
2010-02-28
Budget Start
2006-03-01
Budget End
2007-02-28
Support Year
7
Fiscal Year
2006
Total Cost
$312,114
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
Benatuil, Lorenzo; Kaye, Joel; Cretin, Nathalie et al. (2008) Ig knock-in mice producing anti-carbohydrate antibodies: breakthrough of B cells producing low affinity anti-self antibodies. J Immunol 180:3839-48
Tian, Chaorui; Ansari, Mohammed Javeed I; Paez-Cortez, Jesus et al. (2007) Induction of robust diabetes resistance and prevention of recurrent type 1 diabetes following islet transplantation by gene therapy. J Immunol 179:6762-9
Bagley, Jessamyn; Tian, Chaorui; Iacomini, John (2007) New approaches to the prevention of organ allograft rejection and tolerance induction. Transplantation 84:S38-41
Benatuil, Lorenzo; Kaye, Joel; Rich, Robert F et al. (2005) The influence of natural antibody specificity on antigen immunogenicity. Eur J Immunol 35:2638-47
Tian, Chaorui; Bagley, Jessamyn; Cretin, Nathalie et al. (2004) Prevention of type 1 diabetes by gene therapy. J Clin Invest 114:969-78
Kaye, Joel; Bagley, Jessamyn; Malkowski, Denise et al. (2004) The role of complement receptors in production of antibodies specific for Galalpha1,3Gal. Transplantation 77:314-6
Tian, Chaorui; Bagley, Jessamyn; Kaye, Joel et al. (2003) Induction of T cell tolerance to a protein expressed in the cytoplasm through retroviral-mediated gene transfer. J Gene Med 5:359-65
Bagley, J; Iacomini, J (2003) Gene therapy progress and prospects: gene therapy in organ transplantation. Gene Ther 10:605-11
Cretin, Nathalie; Bracy, Jennifer; Hanson, Krista et al. (2002) The role of T cell help in the production of antibodies specific for Gal alpha 1-3Gal. J Immunol 168:1479-83
Bagley, Jessamyn; Bracy, Jennifer L; Tian, Chaouri et al. (2002) Establishing immunological tolerance through the induction of molecular chimerism. Front Biosci 7:d1331-7

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