The study on the cross talk between B and T lymphocytes in type 1 diabetes with different insulinoma-associated protein 2 epitope conformation peptides

Abstract

Objective To explore the interaction of T cell and insulinoma-associated protein 2(IA-2) conformation and peptide primary sequence on B and T lymphocyte. Methods We designed and synthesized four types of IA-2 epitope peptides, parental β-fold structure IA-2843-855 peptide, α-helical IA-2843-855 peptide (conformation variability), random coil IA-2843-855 peptide (sequence variance) and further β-fold structure IA-2 843-855 peptide which incorporated three hydrophobic amino acid. Patients who were diagnosed as type 1 diabetes mellitus (T1DM, n=12) within 1 year and with high titer IA-2 autoantibodies, T2DM (n=12) and healthy control(n=12) were recruited from the second hospital of Jilin university. Their peripheral blood mononuclear cell (PBMC) cells were stimulated with four types of IA-2843-855 antigens (10 μg/ml）and nonspecific mitogen PHA (5 μg/ml) was added as positive control. Cellular proliferation was expressed as the stimulation index(SI). The suspensions collected from culture medium after centrifuged were used to test the levels of cytokines interleukin (IL)-2 and IL-4. T-test was used to compare the means between groups. Results The SI of T1DM stimulated by parental β-fold structure IA-2843-855 peptide, the α-helical IA-2843-855 peptide, the random coil IA-2843-855 peptide and the further β-fold structure IA-2843-855 peptide were significantly higher than those in T2DM(1.33 ± 0.05 vs 1.13 ± 0.06, 1.27 ± 0.06 vs 1.19 ± 0.05, 1.31 ± 0.07 vs 1.21 ± 0.05, 1.20 ± 0.07 vs 1.11 ± 0.05, respectively,t=-11.6--2.71,P<0.05), and also significantly higher than that in the healthy group (t=-12.6--2.12,P<0.05). The IL-4 level of T1DM stimulated by the four types of IA-2843-855 peptide were significantly lower than that in T2DM and the healthy group(t=2.43-11.75, all P<0.05). The IL-2 level of T1DM stimulated by the four types of IA-2843-855 peptide were significantly higher than that in T2DM and the healthy group(t=-7.20--2.14, all P<0.05).The IL-2 level stimulated by the further β-fold structure of IA-2843-855 peptide and IL-4 cytokines level was lower than the other three peptides(t=-11.9-4.91, all P<0.01). Conclusions The four types of IA-2843-855 peptides we designed and synthesized can specifically make the T lymphocyte proliferate, the further β-fold structure IA-2843-855 peptide is different from the parental peptide in effect on proliferation and cytokines secretion. The level of amino acid sequence plays an important role between T and B lymphocyte cross talk. Key words: Diabetes mellitus , type 1; Insulinoma associated protein-2; Autoimmune; B lymphocyte; T lymphocyte