Mechanism of B-cell Delivered Tolerance in Diabetes

描述(申请人提供)：我们实验室的主要目标是利用逆转录病毒传递的基因治疗平台技术在自身免疫性疾病中诱导耐受，重点是1型糖尿病。这些研究是基于免疫球蛋白载体的耐受性和处理过的T细胞表位的B细胞呈递。在过去的十年里，我们在小鼠lgG1重链支架上设计了多个含有表位的多肽，并在B细胞中表达，通过在体内连续递呈相关表位来实现诱导和长期维持免疫耐受。在几个实验性自身免疫模型中的数据非常有希望，因为已经取得了显著的临床疗效。因此，B细胞处理和呈递Ig融合蛋白可以预防和逆转葡萄膜炎和EAE的自身免疫反应。重要的是，我们发现全长GAD65(GAD-Ig)和胰岛素B9-23-Lg(或胰岛素原-Lg)在NOD B细胞中的表达显著推迟了雌性NOD小鼠糖尿病的发生，即使在7-12周开始治疗时(即患有胰岛素周炎症的小鼠)。我们的假设是，融合的GAD-LG由NOD B细胞(包括男性和女性)处理，以产生主要和次要的GAD表位，并导致T调节细胞的产生。在这里，我们将确定B细胞激活的最佳条件和这种“耐受”的机制。我们还将直接确定这种基因治疗方法针对的是CD4和CDS T细胞，还是两者都有，因为细胞因子产生到多个表位，以及它们将糖尿病转移到NOD-SCID接受者的能力。我们将使用识别CD4或CDS表位的TCR转基因系来跟踪目标T细胞的命运。此外，我们将在直接消融和细胞混合实验中研究调节性T细胞在耐受诱导和维持中的作用和特异性，并确定抑制性细胞因子在耐受中的作用。对于未来的治疗，我们还希望为该系统在胰岛移植模型中的有效性提供原则上的证据。我们的目标是：1)优化和确定B细胞基因治疗耐受的靶点；2)确定B细胞基因治疗的细胞靶点和机制。3)研究B细胞基因治疗后致病T细胞的去向和调节性T细胞的作用；4)建立B细胞基因治疗在胰岛移植模型中的疗效。这些目标的实现将确立调节性T细胞的作用，以及B细胞传递耐受基因治疗的细胞靶点。例如，我们的目标是调节对GAD的致病反应，作为应用该方案作为补充的前奏

DESCRIPTION (provided by applicant): The major goal of our lab has been to utilize a platform technology of retrovirally-delivered gene therapy for tolerance induction in autoimmune diseases, with a focus on Type 1 diabetes. These studies are based on the tolerogenicity of Ig carriers and B-cell presentation of processed T-cell epitopes. During the last decade, we have engineered multiple epitope-containing polypeptides in frame on a murine lgG1 heavy chain scaffold, and expressed them in B cells to achieve the induction and long-term maintenance of immune tolerance through continuous in vivo presentation of relevant epitopes. Data in several experimental autoimmune models are highly promising in that significant clinical efficacy has been achieved. Thus, processing and presentation of Ig fusion proteins by B cells can both prevent and reverse autoimmune responsiveness in uveitis and EAE. Importantly, we have found that expression of full length GAD65 (GAD- Ig) and insulin B9-23-lg (or pro-insulin-lg) in NOD B cells significantly delayed the onset of diabetes in female NOD mice even when treatment was started at 7-12 weeks of age (i.e., in mice with peri-insulitis). Our hypothesis is that the fusion GAD-lg is processed by NOD B cells (both male and female) for tolerogenic presentation of both major and minor GAD epitopes, and leads to the generation of T regulatory cells. Herein, we will determine the optimal conditions for B-cell activation and the mechanism of this "tolerance". We also will directly establish whether this gene therapeutic approach targets CD4 and CDS T cells or both in terms of cytokine production to multiple epitopes, as well as their ability to transfer diabetes to NOD-scid recipients. We will follow the fate of target T cells using TCR transgenic lines recognizing CD4 or CDS epitopes. Moreover, we will examine the role and specificity of regulatory T cells may be involved in both the induction and maintenance of tolerance in direct ablation and cell mixing experiments, as well as determine the role of suppressive cytokines in tolerance. Importantly for future therapy, we also wish to provide proof of principle for the efficacy of this system in an islet transplantation model. Our aims are: 1) To optimize and determine the targets of B-cell delivered gene therapy for tolerance, 2) To identify the cellular targets and mechanism of B-cell delivered gene therapy. 3) To examine the fate of pathogenic T cells and the role of regulatory T cells after B-cell delivered gene therapy, and 4) To establish the efficacy of B-cell delivered gene therapy in an islet transplant model. The accomplishment of the aims will establish the role of regulatory T cells, as well as the cellular targets for B-cell delivered gene therapy for tolerance. Our goal is to modulate pathogenic responses to GAD, for example, as a prelude to applying this protocol as a complementary