T cells with multiple fine specificities are used by non-obese diabetic (NOD) mice in the response to GAD(524-543)

J Autoimmun. 1996 Jun;9(3):365-70. doi: 10.1006/jaut.1996.0049.

Abstract

Insulin-dependent diabetes mellitus (IDDM) in the non-obese diabetic (NOD) mouse results from a T lymphocyte mediated destruction of the insulin-producing beta cells of the pancreas and serves as a model for human type I diabetes. The NOD mouse develops insulitis at 4 weeks of age and diabetes later in life. It has previously been shown that a T helper 1 (Th1) response to the islet antigen, glutamic acid decarboxylase (GAD65, henceforth GAD) spontaneously develops in NOD mice concurrent with the onset of lymphocytic infiltration into the islets (insulitis). The proliferative T cell response in the spleen is initially confined to the carboxy-terminal region of GAD65 (peptides 509-528 and 524-543) followed by a progression to nearby determinants and a variety of upstream determinants. We have produced a set of overlapping synthetic peptides spanning the 509-543 region of GAD and surveyed the responses raised by immunization with peptide GAD(524-543), which is the more immunogenic of the two peptides. NOD mice immunized with GAD(524-543) demonstrate splenic proliferative responses to 524-538 and 527-541 but not to 521-535 or 530-543. Four T cell hybridomas were produced from spleen cells of GAD(524-543)-immunized NOD female mice. Each hybridoma displayed a unique cytokine profile when stimulated with peptides 524-538 and 527-541, assaying IL-2, IFN-gamma, and IL-5 production by peptide-stimulated hybridomas. To identify MHC and TCR contact residues critical for the stimulation of the hybridomas, a truncated peptide (GAD 526-538) and a panel of analogue peptides were synthesized containing single-amino acid substitutions. Hybridoma 35.13.2 was non-responsive to the truncated peptide and all of its variants. However, the four residues 530 (A), 531 (P), 536 (R), and 537 (M) were found to be critical for the activation of the three remaining hybridomas, suggesting that these positions in the GAD-524-543 determinant were MHC binding residues or conserved TCR contact sites.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Division
  • Cytokines / biosynthesis
  • Diabetes Mellitus, Type 1 / immunology
  • Epitopes
  • Glutamate Decarboxylase / immunology*
  • Hybridomas / cytology
  • Mice
  • Mice, Inbred NOD / immunology*
  • Molecular Sequence Data
  • Receptors, Antigen, T-Cell / immunology
  • Spleen / cytology
  • T-Lymphocytes / immunology*

Substances

  • Cytokines
  • Epitopes
  • Receptors, Antigen, T-Cell
  • Glutamate Decarboxylase