应用基于Tetramer技术的致病性CD8 T细胞免疫分型探讨1型糖尿病患者 胰岛损伤异质性的研究

Pathogenic CD8 T cells have the ability to infiltrate and kill islet beta cells, and play a key role in type 1 diabetes (T1D) onset and progression. Recent studies have proved the heterogenicity of islet destruction in different T1D subjects, but it is poorly understood the mechanisms contributed to it. Our previous study indicated that CD8 T cells responses to ZnT8 107-116 (a novel HLA-A*0201 restricted CTL epitope) stimulation were significantly different between recent onset and longstanding T1D subjects, this suggested the heterogenicity of pathogenic CD8 T cells themselves might result in the heterogenicity of islet destruction in different T1D subjects. Consequently, this proposed work will utilize multi-color flow cytometry based on HLA-A*0201 restricted tetramer staining and molecular biological techniques etc, and compare HLA-A*0201 restricted recent onset T1D subjects with rapid loss of islet function to longstanding T1D subjects with residual C peptide, (1) to obtain a robust understanding of phenotype differences of various auto-antigen (including ZnT8 107-116) specific CD8 T cells, such as their activation, differentiation and cytokine secretion etc, (2) to further reveal their different capacity to infiltrate and kill islet beta cells, (3) to clarify differences of global Treg activation and differentiation and their ratio to the investigated islet-specific CD8 T cells, (4) to elucidate whether the effects of T1D risk alleles on Treg and islet-specific CD8 T cells immune phenotypes are correlated with heterogenicity of islet destruction. This project will help define potentially more precise composite biomarkers of disease progression along with providing insight into disease mechanisms and appropriate combination therapies required to prevent T1D.

致病性CD8 T细胞是浸润并杀伤胰岛，导致1型糖尿病发生的关键因素。近期研究证实该病不同患者胰岛损伤存在异质性，但其机制尚不明确。本课题组前期研究表明，该病新发及长病程患者外周血CD8 T细胞对HLA-A*0201限制性表位ZnT8 107-116的反应性差异显著，这提示致病性CD8 T细胞本身异质性可能导致患者胰岛损伤的异质性。因此本课题拟采用基于Tetramer标记的多色流式细胞术及分子生物学等技术，揭示胰岛功能快速丢失和仍有残存的两组1型糖尿病患者PBMC中，ZnT8 107-116等多种自身抗原阳性表位反应性CD8 T细胞活化分化并浸润杀伤胰岛能力的差异；阐明两组患者Treg活化分化及其与致病性CD8 T细胞比例平衡的差异；并明确该病易感基因位点对上述细胞免疫表型的影响与患者胰岛损伤异质性的关联。本课题将为1型糖尿病病程进展中免疫状态监测及其个体化免疫治疗提供新的生物学标志。

1型糖尿病作为遗传相关的自身免疫性疾病，其发病和调节性Treg细胞及致病性CD8 T细胞密切相关。但是该病和单基因糖尿病易于混淆，而中国人群该病的遗传易感性尚不清楚，并且Treg细胞亚群和胰岛抗原特异性CD8T细胞及其表型在1型糖尿病亚型中的差异，以及两者之间的关联还有待研究。基于此，本项目首先揭示自身抗体阴性临床疑似1型糖尿病患者中存在相当比例单基因糖尿病患者，尤以HNF1A和WFS1基因突变为主；基于此，我们建立了适合中国人群的单基因糖尿病规范化诊疗流程。其次，本项目首次通过GWAS筛选绘制了亚洲人群1型糖尿病遗传易感基因图谱，鉴定出GATA3和BTN3A1两个新的易感基因区域，并基于累积遗传易感基因评分，建立了1型糖尿病发病及疾病进程的预测模型，同时发现这些易感基因位点与机体调节性Treg细胞或胰岛功能水平相关。再次，本项目研究表明外周血PBMC中Helios+ Treg亚群频率虽然与1型糖尿病患者残存胰岛功能无关，但与病程呈正相关，与疾病诊断年龄呈负相关。并且在正常人群中，中央记忆性和效应记忆性Helios+ Treg细胞的负性关联，及其与调节性单核细胞的正向关联，在1型糖尿病患者中均消失。而与非特异性CD8 T细胞相比，HLA-A*0201限制性胰岛抗原特异性CD8T细胞活化（CD45RA和CD57）及功能耗竭相关（PD-1，TIGIT和KLRG1）分子表达均显著上调，但在1型糖尿病不同胰岛功能亚组中，这些特异性CD8 T细胞频率及其表型并无显著差异，并且与CD4调节性Treg细胞间也无关联。本项目从一定程度上推动了糖尿病个体化精准诊断和治疗，同样对1型糖尿病患者调节性Treg亚群及自身抗原特异性CD8 T细胞的评估也为1型糖尿病患者免疫治疗监测点提供了很好的依据。