Anti-insulin antibodies arise after administration of exogenous hormone and spontaneously in the autoimmune prodrome of insulin dependent diabetes (IDDM). These autoantibodies are one of the best indicators of beta cell destruction in both human and murine (NOD) diabetes. In this project, we propose to characterize the structure and molecular composition (germline genes, somatic mutation, etc.) of insulin autoantibodies. In our previous studies and preliminary data, we produced anti-insulin mAb using protocols that induce T cell dependent (TD, CFA insulin) and T cell independent (TI, Brucella-insulin) responses in BALB/c mice. Analysis of the IgG1 mAb from TD responses revels some unusual features that include utilization of underrepresented V genes and the presence of tandem prolines in CDR3 of VKs. IgG2 and IgM anti-insulins from TI immunization have characteristics of the preimmune repertoire that include germline encoded V genes, some of which are used by other autoantibodies. Many anti-insulins from both TI and TD repertoires use VK5 related L chains, and these L chains share amino acid sequence motifs with the hormone receptor for insulin. We propose to extend these observations by producing mAb from NOD mice and using reverse transcriptase-polymerase chain reaction (RT/PCR) to study the pathological anti-insulin repertoire. These data will characterize the relationship between the germline repertoire and the repertoires selected by insulin immunization and autoimmune beta cell destruction. Experiments using eukaryotic expression vectors and chain recombination will assess the effect of germline structures and specific motifs (e.g., Pro-Pro) on autoreactivity, epitope specificity, and polyreactivity. These studies will identify structural features of insulin antibodies that may be used to modify adverse immunological reactions and provide new information on genetics and structure of pathological autoantibodies for diagnosis and intervention in autoimmune diabetes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK043911-01A1
Application #
3245438
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1992-03-01
Project End
1997-02-28
Budget Start
1992-03-01
Budget End
1993-02-28
Support Year
1
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Acevedo-Suarez, Carlos A; Hulbert, Chrys; Woodward, Emily J et al. (2005) Uncoupling of anergy from developmental arrest in anti-insulin B cells supports the development of autoimmune diabetes. J Immunol 174:827-33
Kendall, Peggy L; Woodward, Emily J; Hulbert, Chrys et al. (2004) Peritoneal B cells govern the outcome of diabetes in non-obese diabetic mice. Eur J Immunol 34:2387-95
Thomas, James W; Kendall, Peggy L; Mitchell, Holly G (2002) The natural autoantibody repertoire of nonobese diabetic mice is highly active. J Immunol 169:6617-24
Hulbert, C; Riseili, B; Rojas, M et al. (2001) B cell specificity contributes to the outcome of diabetes in nonobese diabetic mice. J Immunol 167:5535-8
Rojas, M; Hulbert, C; Thomas, J W (2001) Anergy and not clonal ignorance determines the fate of B cells that recognize a physiological autoantigen. J Immunol 166:3194-200
Tikhomirov, O Y; Thomas, J W (2000) Alanine scanning mutants of rat proinsulin I show functional diversity of anti-insulin monoclonal antibodies. J Immunol 165:3876-82
Thomas, J W; Kralick, P M; Ewulonu, U K (1997) T cell-independent response to Brucella-insulin identifies a preimmune repertoire for insulin. J Immunol 159:2334-41
Tikhomirov OYu; Thomas, J W (1997) Restricted V gene repertoire in the secondary response to insulin in young BALB/c mice. J Immunol 158:4292-300
Chapman, A; Stewart, S J; Nepom, G T et al. (1996) CD11b+CD28-CD4+ human T cells: activation requirements and association with HLA-DR alleles. J Immunol 157:4771-80
Thomas, J W; Hulbert, C (1996) Somatically mutated B cell pool provides precursors for insulin antibodies. J Immunol 157:763-71

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