Influence Of MIns_15-23H-2K^d-SCT Eukaryotic Expression Plasmid For T-lymphocyte Subsets In NOD Mice

Type 1 diabetes(T1D) is an autoimmune disease that results from a failure or breakdown of self-tolerance, and can be caused by a series of factors, including immunological abnormalities affecting lymphocytes, genevariation and microbial infections. It still remains research hightlight to interve the development of T1D before disease ongoing using autoangigen.NOD mice can spontaneously develop the disease of type 1 diabetes. In the early stage of T1D, insulitis is appeared at 3-4 weeks old and pancreatic ? cells are destroyed gradually. After 12 weeks old, clinical symptoms of T1D is appeared and the morbidity of the disease is up to 80% at 30 weeks old. Based on these characteristics, NOD mice is considered as the major animal model of T1D. In the process of type I diabetes, insulin is considered as the autoantigen that initiating disease and the antigen specific CD8+T cells play a important role in the progress. The epitope InsB 15.23 has difficulty in combining with MHC I because of its low affinity. In order to study the function of epitope, Single chain trimer is adopted which is consisted of MHC class I heavy chain, ?2m chain and epitope peptides. The recombinant plasmids mInsB15-23H-2KddtSCT constructed in our previous work advanced the affinity of InsB15-23. The disease of NOD mice immuned with combination of the recombinant plasmids and adjuvant was relieved and the morbidity was decreased.The research of the protective mechanism will provide the news ideas for immune intervention therapy of autoimmune diseases.Based on these conclusions, we try to research the protective mechanism of mInsB15-23H-2KddtSCT for NOD mice. The question that whether CTL is decreased directly by mInsB15-23H-2KddtSCT or down regulated by Th cells will be solved in the following research.In order to exclude the influence of adjuvant,we immuned NOD mice with the recombinant plasmids mInsB15-23H-2KddtSCT alone. We got the same conclusion that the disease of NOD mice was relieved and the morbidity was decreased which demonstrated that mInsB15-23H-2KddtSCT protect NOD mice from T1D.It has been well recognized that CD4+T and CD8+T cells are major immune effector cells mediating T1D. So, the relationship between the recombinant plasmids mInsB15-23 dtSCT and T cell subsets is our main research content. Insulin specific CTLs and Treg, Thl, Th2 cells in spleens of immuned NOD mice were analyzed by FACS. Insulin specific CTLs appear at 3-4 weeks old of NOD mice and are replaced by the other autoantigen specific CTLs with increase of weeks old. Detected by FACS, we found that the recombinant plasmids mlnsB 15.23 dtSCT can reduce the frequency of insulin specific CTLs in 6 weeks old. For CD4+T cell subset, m InsB15-23 dtSCT significantly decreased the number of Thl cells in 12 and 16 weeks old. The frequency of Th2 cells was increased in 16 weeks old. The number of regulatory T cells had no significant difference in the group of mInsB15-23 dtSCT and pCDNA3.1 (-).We concluded that the recombinant plasmids mInsB15-23 dtSCT can promote Th cell to differentiate to Th2 cells and had no influence on the frequency of Treg cells. The mechanism of the decrease of the frequency of CTLs and the differentiation to Th2 remains the further research.