Tu151 - GAD65 Mediated Inflammatory Immune Response In HLA-DR3/DQ2+ Type 1 Diabetes Patients

AIM AND OBJECTIVE: This study was planned to define diabetogenic GAD65 peptides possibly presented by HLA-DR3/DQ2 molecules to CD4+ T-cells in type 1 diabetes patients.

METHODS: Top 30 GAD65 peptides, found to strongly bind in silico with HLA-DR3/DQ2 molecules, were selected for this study and grouped into 4 pools according to their amino acid positions. The peptides were used to stimulate CD4+T-cells of study subjects in 16 hours culture. CD4+ T-cells’ stimulation in terms of IFN-γ, TNF-α, IL-17 and IL-10 expression was analysed using flow-cytometry.

RESULTS: While, all GAD65 peptide pools resulted in significant increase in IFN-γ (p=0.003, p< 0.0001, p=0.03 and p=0.04 respectively), pool 1-3 showed significant increase in the expression IL-17 (p=0.003, p< 0.001 and p=0.004 respectively) in T1D vs. controls. However, the expression of TNF-α and IL-10 by CD4+ T-cells was comparable between patients and controls. Inter-peptide group comparison for immunogenicity revealed significant increase in IFN-γ and IL-17 producing CD4+T-cells for pool 2 as compared to other groups (p< 0.0001 in both cases) in patients but not in controls. Further, group 2 GAD65 peptides yielded significant increase in IFN-γ (p=0.002) and IL-17 (p=0.003) and significant decrease in IL-10 (p=0.04) expression by CD4+T-cells in HLA-DR3/DQ2+ patients as compared to DR3/DQ2+ healthy controls.

CONCLUSION: GAD65 peptides, particularly those belonging to group 2, induced CD4 T-cells to produce inflammatory IFN-γ and IL-17 cytokines in patients, suggesting that, group 2 GAD65 peptides possibly presented by HLA-DR3/ DQ2 molecules to CD4 T cells shift immune balance towards inflammatory phenotype in T1D patients.