| Type1diabetes (T1D) is an organ-specific and autoimmune disease which has a complex etiology. The final factor is the islets appear cellular infiltration. Then it causes destruction of pancreatic P-cell and other relevant cells, in the end, the patient will appear insulin deficiency and be abnormal in glucose metabolism. Antigen specific CD8+T lymphocytes via specific antigen receptors (TCR) to identify MHC-I own antigen peptide at the surface of (3cells, play cytotoxic effects by mechanisms of secrete perforation protein and particle enzyme, and destroy islet β cells. So, it is important to monitor self-reactive T cells during disease period. Among those self-reactive insulin antigens which be detected, especially InsB15-23peptide is regard as a prime trigger antigen of T1D, but the mechanism of it is still unknown. Therefore, to monitor those insulin specific CD8+T lymphocytes is significant for T1D diagnosis and treatment.MHC-I tetramer technology is a generally accepted tool of CD8+T lymphocytes quantitative assay. But the preparation of traditional MHC-I tetramer is expensive and complex. Furthermore, low affinity InsB15-23peptides are difficult to form stable MHC-I tetramer. So, we try to introduced MHC-I single-chain trimer technology (SCT) to prepare InsB15-23H-2Kd. Use two soft linkers to connect InsulinB15-23antigenic peptide, light chain β2m and MHC-I heavy chain to enhance stability of antigenic peptide and H-2Kd. Meanwhile, we mutanted tyrosine to cysteine in which residue84on peptide C terminal in the heavy chain of SCT and introduced one cysteine in linkel, then the two cysteine could form a disulfide bond, this reform can conduce the stability and combining capacity of SCT. It’s so-called dtSCT (disulfide-trap) which we used to establish a new type tetramer for quantitative assay of CD8+T cells. On account of above mentioned, our laboratory have been established soluble gene InsB15-23H-2Kd-dtSCT (sSCT) to build new tetramer as a tool for detecting CD8+T cells. In order to detect the function of the dtSCT, we try to constructed a eukaryotic expression plasmid which encodes InsB15-23H-2Kdfl-dtSCT (Kdfl is full lenth H-2Kdfl).Vaccination NOD mice with it, according to the rate of InsB15-23CTL it reduced to judge the conformation of the sSCT. We introduced a improvement named Q115E for the purpose of enhance the affinity during CD8molecular with H-2Kd and enhance the antigen presentation of the mSCT. Based on this, we obtained plnsB15-23H-2KdQ115Efl-dtSCT. Vaccination NOD(4weeks) mice partly with this plasmid for one time and three times, then we detect InsB15-23specific CD8+T lymphocytes in peripheral blood, spleen and islets by FACs. The result of immunized three times show that the rate of InsB15-23specific CD8+T in peripheral blood reach to5.3%, increasing in contrast to vaccination with control group and the group immunize with one time. But the consequence of spleen have no significant distinction regardless of one or three times, it show that the plasmid cannot induce InsB15-23specific CD8+T cells in spleen. The rate of islets which is the target cells of those specific T cells is only1.3%, the reason need to probe.The result indicates that the sSCT expression in NOD mice have correct conformation, they can induce InsB15-23specific CTLs. We establish functional sSCT successfully, thus we approach to prepare a new type tetramer as a tool for quantitative assay of CD8+T cells, and contribute to selective delete CD8+T lymphocytes pathogenicity in diabetes model NOD mice for effective treat type I diabetes. In another aspect, maybe it could provide indication of methodology in the study of other self-reactive disease. This technique has a broad prospect. |
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