I型主要组织相容性复合体参与1型糖尿病异常自身抗原呈递的机制研究

Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease, in which auto-reactive CD8+ T cells de-stroy pancreatic β cells. Diagnosed with which, patients cannot produce insulin and eventually cannot control blood glucose levels. Managing auto-reactivity of CD8+ T cells at the early stage of pancreas destruction could be a viable therapeutic strategy. In T1DM CD8+ T cells auto-reactivity is caused by a self-antigen presentation by certain MHC class I molecules. MHC class I molecules are hyper-expressed at the surface of pancreatic β cells, and there is a genetic correlation of MHC class I polymorphism to T1DM. .We hypothesized that the stability of MHC class I molecules determines susceptibility to T1DM. We compared two closely-related MHC class I molecules - one T1DM-predisposing - HLA-B*44:05, the other T1D-protective - HLA-B*44:02 in a cell line lacking MHC I chaperone protein tapasin and/or with inhibited antigen delivery. We found that B*44:05 is stable and does not require tapasin for its intracellular transport nor antigen presentation, while B*44:02 is unable to bind and present antigen without tapasin. We observed that MHC I stability is confined to the region where C-terminus of antigen binds and designed a novel mutant Y84F that indeed showed reduced stability. .We will study the stability of eight other MHC class I molecules associated with T1DM in pancreatic β cell lines with knockout of TAPASIN gene by CRISPR/Cas9. We will identify possible diabetogenic antigens pre-sented by these MHC I molecules. This will allow us to develop two strategies to modify MHC class I antigen presentation in T1DM - first one by reducing the stability of T1DM-predisposing molecules (Y84F mutation) and the second by peptide therapy - exchanging diabetogenic peptides for an antigen that may confer tolerance. These are two strategies when proven effective ex vivo could be further developed into treatment options for T1D patients.

1型糖尿病（T1DM）由CD8+T细胞对胰腺β细胞破坏引起，表现为胰岛素绝对缺乏，血糖水平增高。基于MHC I类分子稳定性决定T1DM易感性的假说，我们前期在分子伴侣tapasin敲除及抗原呈递抑制细胞系中，配对比较易感（HLA-B*44:05）和保护型（HLA-B*44:02）MHC I类分子的差异，发现前者稳定较好，无需tapasin帮助即可实现胞内运输和抗原递呈，而后者相反。此外，MHC I类分子的稳定性由其羧基端决定，诱导Y84F突变可降低其稳定性。在此基础上本项目在tapasin敲除的胰岛β细胞系中研究T1DM相关的8种MHC I类分子的稳定性，识别其致病抗原肽，同时提出治疗该病的两种新策略：诱导Y84F突变降低易感型MHC I类分子稳定性；肽治疗－置换致病抗原肽而诱导免疫耐受。我们将开展离体实验验证上述，并为T1DM的临床治疗提供新思路。

1型糖尿病（T1DM）是一种由CD8+ T细胞过度活化引起胰腺β细胞损伤，而导致胰岛素缺乏及血糖升高的慢性自身免疫性疾病。在T1DM中，CD8+ T细胞会识别特定主要组织相容性复合体I（MHC I）所呈递的自身抗原。了解MHC I和T1DM的相关性可以为T1DM提供更多的治疗方式。.我们认为MHC I分子的稳定性决定了其对T1DM的易感型。应用细胞系实验及电脑模拟技术比较了T1DM易感型和保护型MHC I分子的不同特点，发现相比保护型MHC I，易感型MHC I在细胞表面的表达水平更高，其定位于细胞表面和胞吞作用通路中，而保护型MHC I在细胞表面的表达水平则较低且定位于细胞内。这些数据表明T1DM易感型MHC I基因的稳定性强于保护型MHC I基因。T1DM易感型和保护型MHC I在C端环状结构（氨基酸66-90位点）和116位点处有差异，因此在以上位点设计突变体可以降低HLA-A*24:02的稳定性从而使其趋于保护型。为了将以上发现应用于干扰T1DM相关的抗原呈递，我们构建并实验筛选出HLA-A*24:02 H114D Y116D突变结构来模拟保护型MHC I。我们将含有HLA-A*24:02 WT及HLA-A*24:02 H114D Y116D两种基因的质粒分别转染入胰腺β细胞系1.1.B.4中，构建T1DM细胞模型，后与HLA-A*24:02阳性T1DM患者的外周血单个核细胞进行共培养以验证是否可以通过降低T1DM易感型HLA-A*24:02的稳定性来降低CD8+ T细胞的活化水平的猜想。如果这种猜想在体外实验中得到证实，则会为T1DM提供新的临床治疗方式。.我们从已知的T1DM自身抗原中比对出所有可能的抗原肽并用电脑模拟技术评估了它们和相应MHC I分子的结合情况。通过对比联系紧密的T1DM易感型和保护型MHC I分子的抗原肽组，发现T1DM中19种被HLA-B*39:06呈递和11种被HLA-B*44:05呈递的抗原肽。我们将会进一步采用已经获得专利的温度可置换多聚体技术在患者血液中检测它们是否为T1DM中新的CD8+ T细胞表位（抗原肽）。

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