B淋巴细胞亚群失衡在1型糖尿病发生发展中对效应性T细胞的调控作用及机制研究

B cells play a crucial role in the autoimmune destruction of type 1 diabetes. However, little is known about the role of B-lymphocyte subpopulations with distinct immune function, eapecially regulatory B cells, in the onset and progression of patients with T1D. Our previous study showed altered frequencies of various B-cell subsets associated with autoimmune diabetes. We also found that B cell depletion delayed and prevented spontaneous diabetes, and regenerated B cells expressed immune regulatory function. We hypothesized that patients with T1D have an altered B-lymphocyte subpopulations towards pathogenic B cell subsets, with decreased and/or impaired Breg. This study will firstly investigate the number and frequency of different B-lymphocyte subsets in the diabetes spectrum of patients with new-onset T1D, first degree relatives at high risk for T1D, and healthy controls with normal glucose tolerance using flow cytometry. We next assess phenotypic characterization and immunological suppression of Breg. T-cell proliferation and cytokine production will be evaluated through CFSE and intracellular cytokine staining after coculture with specified B-cell subsets, which are isolated by fluorescence-activated cell sorting. Furthermore, we evaluate the potential effector mechanism by which Bregs suppress proinflammatory cytokine production by CD4+ T cells. Magnetic-bead-purified CD4+CD25- T cells are cultured with Breg, stimulated with plate-bound CD3 mAb in the presence of blocking IL-10, TGF-β, CD80, or CD86 mAb, to assess cytokine or cell-cell contact dependent pathway. The frequency of Th cells and cytokine production will be determined through flow cytometry and ELISA. This study will provide important new data characterizing the number and frequency of different B-cell subsets in the spectrum of new-onset T1D, FDR and healthy controls, and defining the phenotype, suppressive activity and the effector mechanism of Breg, which could be used as a biomarker and potential target for immunological intervention selectively deleting pathogenic B cells while preserving Breg.

B淋巴细胞在T1D中发挥多重作用。不同B细胞亚群，尤其Breg的表型及免疫调节功能在T1D发生发展中的作用尚不清楚。申请人前期研究发现长病程T1D患者存在B细胞亚群谱改变，去除B细胞降低小鼠糖尿病发病率，且新生的B细胞具有免疫调节功能。据此提出假设T1D及高危一级亲属存在B细胞亚群失衡即致病性B细胞增多，Bregs细胞数量减少及免疫调节功能受损。本课题通过流式细胞术探索T1D、高危一级亲属及健康对照外周血B细胞亚群的数量及比例。Breg细胞与效应性T细胞共培养，流式细胞仪和ELISA评估Breg抑制CFSE标记的T细胞增殖、Th细胞比例及培养上清液炎症因子浓度。加入不同单抗阻断细胞因子或细胞直接接触依赖途径，阐明Breg调控效应性T细胞的机制。本研究将为T1D早期诊断筛选B细胞标志物，优化临床免疫治疗开展选择性去除致病性B细胞，同时保留Breg细胞提供临床依据。

B淋巴细胞在T1D中发挥多重作用。Breg的表型及免疫调节功能在T1D发生发展中的作用尚不清楚。本课题通过流式细胞术探索T1D外周血B淋巴细胞亚群的数量及比例，评估Breg细胞的表型与免疫调节功能，探索T1D患者B细胞受体谱的多样性与复杂性。课题组发现人外周血Breg细胞高表达CD28hiCD38hi，流式分选CD19+CD24hiCD38hi B细胞与T细胞进行混合共培养，有效抑制效应性T细胞炎症因子的分泌。T1D患者Breg细胞数量降低和功能受损。研究还发现Tim-1+ Breg和IL-10+ Breg的比例低于健康对照组，且与胰岛功能（空腹C肽）呈正相关。IL-10+ Breg的频率也与糖化血红蛋白（HbA1c）呈负相关。进一步通过磁珠分选技术分选出B淋巴细胞，应用Illumina HiSeq 2500平台高通量免疫测序技术检测B细胞免疫球蛋白重链（IGH）互补决定区3（CDR3）的基因序列，评估克隆组成多样性和复杂性。课题组发现NGT对照组常见CDR3克隆型频率相对较低，T1D与LADA患者存在不同的氨基酸克隆扩增偏好，以及一些共同升高的氨基酸克隆型。IGH谱克隆多样性在各组之间尚未达到统计学差异，但LADA多样性相比NGT和T2D有降低趋势。各组间CDR3长度分布模式相似。这些数据表明B淋巴细胞失衡和功能紊乱可能参与胰岛β细胞破坏。这些发现将为理解自身免疫糖尿病抗原特异性免疫谱的进展带来机遇，有助于改善T1D的适应性免疫治疗策略。

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