| Type 1 diabetes mellitus (T1DM), also known as insulin dependent diabetes mellitus, results from T cell-mediated autoimmune destruction of insulin-producing β cells of the pancreas. Cytotoxic T lymphocytes (CTLs), the direct destructor of islet β cells, play an crucial role in the prime and development of T1DM.Just like other autoimmune diseases, epitope spreading also occurs in T1DM. As the prime antigen of T1DM, auto immunity against insulin is vital for exposure of downstream antigens and induction of autoimmunity against them. In nonobese diabetic (NOD) mouse, one of the most common used model animals of T1DM, the H-2Kd restricted antigenic peptide is insulin B15-23 (LYLVCGERG, abbreviated to InsB 15.23) peptide. We are interested in producing a tool to dectect InsB 15.23 specific CTLs and defining the role of InsB 15-23 peptide in T1DM.MHC (major histocompatibility complex) I multimer technique, especially tetramer technique, is often used to detect CTLs directly. But it needs to refold antigenic peptide, recombinant H-2Kd and recombinant β2m to produce MHC I monomer, and then multimerize several monomers into multimers. So these techniques require relatively high affinity between antigenic peptides and MHC I molecules. Low affinity antigenic peptides are hard to refold into stable MHC I monomer to produce multimers. Unfortunately, the side chain of the 9th glycine residue of native InsB15-23 peptide is too small to binds stably with H-2Kd, results in a big obstacle for this study. Now available approach is to mutate the 9th glycine residue of InsB15-23 peptide into valine, which has a bigger side chain, to enhance its binding ability with H-2Kd. But there have been reported that differences existed in the recognition profile of tetramer loaded with native peptide and mutant peptide. In order to maintain better affinity without changing the specificity, the disulfide-trap single-chain trimer (dtSCT) technique was selected for this study.dtSCT technique is to connect antigenic peptide, p2-microglobulin and MHC I molecule covalently by two short flexible linkers and introduced a disulfide bond between the linkerl and MHC I molecule to trap antigenic peptide into the binding groove. The easiness of rebinding to the antigen binding groove of covalently linked antigenic peptide renders improvement of stability to the whole molecule. But the covalently coupled linkerl in the C terminal will lead a 90° rotation of the last amino acid of antigenic peptide and cause negative effects. So the final net effects on the stability of dtSCT molecules still positively correlated to the affinity of antigenic peptide.Several successful works have been reported. But most of these peptides originate from viruses or bacteria and exhibit relatively higher affinity to their ligands than InsB 15-23 peptide. To determine the application value of dtSCT in T1DM of NOD mice, we construct InsB15-23 dtSCT to produce tetrmers. Besides native InsB 15.23 antigenic peptide, G9V modifided peptide was also used to assembly dtSCT. Furthermore, we introduced another Q115E mutation in H-2Kd to improve the binding ability between CD8 and H-2Kd. This Q115E mutation do not influence the specificity of dtSCT molecule. So altogether we constructed 4 types of soluble dtSCT (sdtSCT) molecules:InsB15-23 sdtSCT, InsBG9v sdtSCT, InsBQ115E sdtSCT and InsBG9V/Q115E sdtSCT to produce tetramers. We found all sdtSCT tetrmers were stable and exhibited low non-specific marking levels. When marking the peripheral blood mononuclear cells (PBMCs) of negative BALB/c mice, the non-specific marking levels were 0%,0.06%,0.02% and 0.07%, respectively. The non-specific marking levels for spleen lymphocytes of BALB/c mice were 0.01%,0.03%, 0.04% and 0.05%, respectively. The non-specific marking levels of sdtSCT tetrmers were similar to commercial conventional G9V pentamer, whose non-specific marking levels for PBMCs and spleen lymphocytes of BALB/c mice were 0.07% and 0.04%, respectively. On the other hand, all sdtSCT tetrmers were able to detect the small amount of InsB 15.23 specific CTLs existed in PBMCs of 4 weeks female NOD mice. The values were 0.29%,0.69%,0.89% and 1.03%, respectively. The detection value of spleen lymphocytes of 4 weeks female NOD mice were 0.03%,0.03%,0.15% and 0.20%, respectively. The corresponding detection value of commercial conventional G9V pentamer were 0.46% and 0.09%, respectively. It could conclude that even for low affinity InsB 15-23 antigenic peptide, the positive effect of easiness of rebinding can complement the negative effect of C terminal problem. Perhaps dtSCT approaches will help in the construction of tetramers made with lower affinity peptides.Due to its no influence on specificity and exhibition of higher detection ratio only after InsBO9V/Q115E sdtSCT tetramer, InsBQ115E sdtSCT was selected to make a membrane-expressed dtSCT (mdtSCT) molecule. After cloned into pcDNA3.1(-) eukaryotic expression vector, InsBQ115E mdtSCT was used to immunize female NOD mice subcutaneously. After 3 vaccinations, the frequency of InsB15-23 specific CTLs in PBMCs and spleen were increased to 1.93±0.55% and 0.83±0.31%, respectively. It showed highly significant difference with the empty plasmid groups, which were 0.22±0.11% and 0.37±0.20%, respectively. Thus, we affirmed the biological function of dtSCT in eukaryotic expression level.At last, we used InsBQ115E mdtSCT to intervene 3 weeks old female NOD mice. It was found that the islets infiltration of mononuclear cells were decreased and the development of T1DM delayed. The morbidity of 30 weeks female NOD mice received InsBQ115E mdtSCT vaccination was 9%, exhibited highly significant difference with the empty plasmid groups, whose morbidity was 60%. The results also showed that the immunoprotection was IGRP independent.In conclusion, with the help of dtSCT technique, we successfully produced functional tetramer of low affinity InsB15-23 antigenic peptide. It suggested that dtSCT techniques might help in construction tetramers of lower affinity peptides. The morbidity of 30 weeks female NOD mice was decreased significantly after 3 InsBQ115E mdtSCT vaccinations. This result give us a hint that dtSCT vaccine may be a new promising specific immunotherapy method. But the detailed mechanisms of its immunoprotection need to be defined in future researches. |
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