T cell response pattern to glutamic acid decarboxylase 65 (GAD65) peptides of newly diagnosed type 1 diabetic patients sharing susceptible HLA haplotypes

Abstract

Autoantibodies and autoreactive T lymphocytes directed against several pancreatic beta cell proteins such as GAD65 have been identified in the circulation before and at the onset of clinical type 1 (insulin-dependent) diabetes. Using GAD65 synthetic peptides, we studied the proliferative response of peripheral blood mononuclear cells (PBMC) either from recently diagnosed type 1 diabetic patients, of whom the majority share the disease-associated HLA class II haplotype (DR4-DQB1*0201 or DR3-DQB1*0302), or from HLA-matched control subjects. We found that 67% (14/21) of the type 1 diabetic patients and 39% (9/23) of the control subjects exhibited a positive proliferative response. Compared with control subjects, however, PBMC from diabetic patients proliferated more frequently (P < 0.05) in the presence of peptide pools from the C-terminal region of GAD65 (amino acids 379-585). Diabetic patients with the same HLA-DQ or HLA-DR alleles showed partially identical T cell reactivity, but no clear correlation could be made between MHC class II specificity and T cell epitopes because of multiple combinations of class II alleles. In addition, by flow cytometry, we studied the direct binding of GAD65 peptides to MHC class II molecules of Epstein-Barr virus (EBV)-transformed B (EBV-B) cells obtained from a diabetic patient. We found that 11 GAD peptides were able to bind to the highly susceptible haplotype DRB1*0301/0401-DQA1*0301/0501-DQB1*0302/0201 on the surface of EBV-B cells in partial correlation with the results obtained in the proliferation assays.

Fig. 1

Proliferative response of peripheral blood mononuclear cells (PBMC) obtained from one diabetic patient in the presence of tetanus toxoid alone (TT) or in combination with each of the nine GAD65 peptide pools (1–9). Peptide pools numbered 1–9 correspond to GAD65 regions [1–66], [61–132], [127–192], [187–264], [259–324], [319–384], [379–450], [445–522] and [517–585], respectively. The basal ³H-thymidine incorporation was obtained without antigen stimulation. The values are the mean of quadruplicate from a single representative experiment.

Fig. 2

Percentage of individuals demonstrating T cell proliferation (stimulation index (SI) > 3) in the presence of GAD65 peptide pools (1–9) among the 21 newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients (▪) and 23 control subjects (□). For each peptide pool (1–9), the proportion of positive responses among the patients was compared with that among the controls. A significant difference between the response of peripheral blood mononuclear cells (PBMC) from patients and controls was only observed with peptide pool 7 and 9 (*P < 0.05, Fisher's exact test).

Fig. 3

Peripheral blood mononuclear cell (PBMC) proliferation of five type 1 diabetic patients in the presence of each individual peptide forming pool 7 (GAD65 residues 379–450).

Fig. 4

Typical example of flow cytometry histograms of binding of biotinylated GAD65 peptides to DPC cells or HLA class II-deficient RJ 2.2.5 cells.