Target Auto-antigens and Pancreatic beta cell destructive T lymphocytes in Type 1 Diabetes

本研究旨在探讨1型糖尿病患者外周血T淋巴细胞对胰岛β细胞抗原的反应。迄今为止，我们已经报道了早期诊断的1型糖尿病患者中针对胰岛素肽的细胞毒性T CD 8 ^+淋巴细胞，在本研究中，我们首先尝试了利用MHC-肽四聚体的流式细胞术检测细胞毒性T CD 8 ^+淋巴细胞。但未检测到明显的阳性细胞。我们怀疑该细胞出现频率低可能是我们检测不到的原因。其次，我们利用胰岛素重叠肽建立了酶联免疫斑点（ELISPOT）检测方法。我们从A链制备了7种15个氨基酸的胰岛素肽，从B链制备了16种15个氨基酸的胰岛素肽。此外，我们还制备了8-11个氨基酸长度的11种A链和20种B链胰岛素肽，用于CD_（8 ^+）淋巴细胞。结果，19例早期诊断患者中有13例γ-INF ELISPOT阳性，而17例健康对照者中无一例阳性。在IL-4 ELISPOT试验中，未检测到阳性细胞。结论：ELISPOT检测结果表明，早期诊断的1型糖尿病患者外周血中存在胰岛素肽特异性T淋巴细胞，分泌γ-INF。这些T淋巴细胞可能在β细胞的破坏中起重要作用。

This study focused on the T lymphocytes against the pancreatic beta cell antigens in Type 1 diabetes patients. So far, we have reported the cytotoxic T CD8^+lymphocytes against the insulin peptide in the early -diagnosed Type 1 diabetes patients.In this study, the firstly, we tried the flowcytometry assay for detection of the cytotoxic T CD8^+ lymphocytes using the MHC-peptide tetramer. However, the positive cells were not significantly detected. We suspected that the low frequency of that cell might be reason why we could not detected.The secondly, we establish the enzyme-linked immunospot (ELISPOT) assay using the insulin overlapping peptides. We have made the seven kinds from A-chain and the sixteen kinds from B-chain of the insulin peptides in 15 amino acids length for CD4^+ T lymphocytes. Furthermore, we have also made the 11 kinds from A-chain and the 20 kinds of B-chain of the insulin peptides in 8-11 amino acids length for CD8^+ lymphocytes,In results, 13 patients among 19 early-diagnosed patients have showed positive in gamma-INF ELISPOT assay, but no one among 17 healthy controls. In IL-4 ELISPOT assay, no positive cell has been detected at all. Among the overlapping peptides, the positive response was detected against the peptides near portion of the peptides that have been already reported.In conclusion, ELISPOT assay have revealed that the insulin peptide specific T lymphocytes, that secrete gamma-INF, existed in the peripheral blood of the early-diagnosed human Type 1 diabetes patients. These T lymphocytes might play an important role in the destruction of the beta cells.