The goal of the investigations described in this proposal is to obtain detailed information on the natural history of the T cell response to islets as it develops in islet infiltrates of pre-diabetic NOD mice In this analysis we will exploit our recent finding that insulin-specific T cells are a predominant component of islet-specific T cells and focus on this insulin-specific response. The experiments are designed to test four hypotheses. i. The frequency of insulin-specific T cells within populations of T cells presents in islet infiltrates is low at the early stages of beta cell damage and these T cells are of limited TCR diversity. ii. As the degree of infiltration and beta cell damage increases the frequency of insulin-specific T cells, as a sub population of islet infiltrates, increases and the TCR diversity of the insulin-specific T cells present in islet infiltrates increases. iii. Individual pre-diabetic NOD mice at similar stages of disease progression will employ the same TCR repertoire in the response to insulin and islet antigens. iv. Islet-specific and insulin-specific T cells isolated from islet infiltrates will be of the Th 1 phenotype. Testing the above four hypotheses will require isolation and characterization of panels of islet-specific T cells clones from individuals pre-diabetic mice at successive intervals in disease progression. The preliminary results presented in this proposal demonstrate the proficiency of our laboratory in the routine isolation of islet-specific T cells from islet infiltrates of young, pre-diabetic mice. There are four specific aims. i. Isolation of islet-specific an insulin-specific T cells from islet infiltrates of pre-diabetic NOD mice at various stages of disease progression, in-vitro characterization of these cells with regard to CD4/CD8 phenotype and establishment of islet-specific and insulin- specific T cell lines and clones from these cells. ii. Determine the kinetics of appearance of insulin-specific T cells in islet infiltrates by a comprehensive limiting dilution frequency analysis of individual NOD mice in age matched groups at one week age intervals. iii. In vitro characterization of the T cells isolated from islet infiltrates for antigen-specificity, lymphokine production profiles, and sequence analysis of the TCR a/B chains by PCR techniques. iv. Assessment of the capacity of insulin-specific T cell lines and clones to: a, induce islet damage upon adoptive transfer into NOD- scid/scid mice; b, synergies with known diabetogenic T cell clones in adoptive transfer of diabetes; c, inhibit adoptive transfer of diabetes by known diabetogenic T cell clones.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK047298-01A1
Application #
2146771
Study Section
Immunological Sciences Study Section (IMS)
Project Start
1994-08-01
Project End
1997-07-31
Budget Start
1994-08-01
Budget End
1995-07-31
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Biochemistry
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
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Redondo, M J; Gottlieb, P A; Motheral, T et al. (1999) Heterophile anti-mouse immunoglobulin antibodies may interfere with cytokine measurements in patients with HLA alleles protective for type 1A diabetes. Diabetes 48:2166-70
Simone, E; Daniel, D; Schloot, N et al. (1997) T cell receptor restriction of diabetogenic autoimmune NOD T cells. Proc Natl Acad Sci U S A 94:2518-21
Schloot, N C; Roep, B O; Wegmann, D et al. (1997) Altered immune response to insulin in newly diagnosed compared to insulin-treated diabetic patients and healthy control subjects. Diabetologia 40:564-72
Simone, E A; Yu, L; Wegmann, D R et al. (1997) T cell receptor gene polymorphisms associated with anti-insulin, autoimmune T cells in diabetes-prone NOD mice. J Autoimmun 10:317-21
Daniel, D; Wegmann, D R (1996) Protection of nonobese diabetic mice from diabetes by intranasal or subcutaneous administration of insulin peptide B-(9-23). Proc Natl Acad Sci U S A 93:956-60
Schloot, N C; Daniel, D; Norbury-Glaser, M et al. (1996) Peripheral T cell clones from NOD mice specific for GAD65 peptides: lack of islet responsiveness or diabetogenicity. J Autoimmun 9:357-63
Daniel, D; Gill, R G; Schloot, N et al. (1995) Epitope specificity, cytokine production profile and diabetogenic activity of insulin-specific T cell clones isolated from NOD mice. Eur J Immunol 25:1056-62