IMMUNE REACTIVITY OF DIABETES-ASSOCIATED HUMAN MONOCLONAL AUTOANTIBODIES DEFINES MULTIPLE EPITOPES AND DETECTS 2 DOMAIN BOUNDARIES IN GLUTAMATE-DECARBOXYLASE

Autoreactive islet cell Abs (ICA) accompany the pathogenic destruction of pancreatic beta cells in insulin-dependent diabetes mellitus (IDDM ). Human monoclonal ICA (MICA 1-6) previously derived from a DR1/DR7-p ositive newly diagnosed diabetic patient, recognized the islet cell au toantigen glutamate decarboxylase 65 (GAD65) and defined two distinct conformational (MICA 1/3 and MICA 4/6) and one linear (MICA 2) autoimm une epitopes in this molecule. We have isolated 4 new ICA-reactive B c ell lines, one from a DR4/DR11-positive newly diagnosed IDDM patient ( MICA 7) and three from a DR3 homozygous patient with both IDDM and Cra ves' disease (MICA 8-10). Like MICA 1-6, MICA 7-10 are specific for GA D65, suggesting that GAD65-reactive B cells dominate the ICA response in IDDM. Comparative analysis of MICA 1-6 and MICA 7-10, using GAD65 m utants and blocking experiments, showed that MICA 7-10 define three no vel conformational autoimmune epitopes in GAD65. Further structural an alysis of the MICA 1-10 epitopes revealed two distinct and one overlap ping region of epitope clusters. Thus, the C-terminal region, defined by amino acids 450 to 570, harbors the conformational MICA 4/6 and MIC A 7 epitopes as well as the linear epitope of MICA 2 (amino acids 506- 531). The MICA 4/6 and MICA 10 epitopes are located in the middle regi on of the molecule defined by aminoacids 245 to 449, whereas the N-ter minal region contributes only to the MICA 8/9 epitopes (encompassed in amino acids 39-585). MICA 1-6, 7, and 8-10, derived from three IDDM p atients of different HLA haplotypes, define six different epitopes in GAD65 and represent tools to determine the spectrum, possible HLA asso ciation, and temporal order of epitope recognition in IDDM.