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

本研究主要针对1型糖尿病患者抗胰岛β细胞抗原的T淋巴细胞进行研究。到目前为止，我们已经报道了在早期诊断的1型糖尿病患者中存在针对胰岛素肽的细胞毒性T CD8~+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来检测细胞毒性T CD8~+淋巴细胞。但未见明显的阳性细胞。我们怀疑该细胞的低频率可能是我们无法检测到的原因。其次，我们建立了利用胰岛素重叠多肽的酶联免疫斑点(ELISPOT)方法。我们从15个氨基酸长度的胰岛素多肽A链制备了7种胰岛素多肽，从B链制备了16种胰岛素肽，用于CD4~+T淋巴细胞。此外，我们还从8-11个氨基酸长度的11种胰岛素多肽A链和20种B链制备了CD8~+淋巴细胞。结果19例早期诊断的患者中有13例γ-INF-ELISPOT试验阳性，而17例健康对照中无一例阳性。在IL-4 ELISPOT检测中，未检测到阳性细胞。在重叠的多肽中，与部分已报道的多肽有阳性反应。综上所述，ELISPOT分析表明，在早期诊断的人类1型糖尿病患者的外周血中存在胰岛素多肽特异性T淋巴细胞，分泌伽马-干扰素。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.