Mechanism of B-cell Delivered Tolerance in Diabetes

描述（由申请人提供）：我们实验室的主要目标是利用逆转录病毒递送基因治疗的平台技术，用于诱导自身免疫性疾病的耐受性，重点是1型糖尿病。这些研究基于IG载体的耐受性和加工的T细胞表位的B细胞呈递。在过去的十年中，我们已经在鼠IgG 1重链支架上框内工程化了含有多个表位的多肽，并在B细胞中表达它们，以通过相关表位的连续体内呈递来实现免疫耐受的诱导和长期维持。在几个实验性自身免疫模型中的数据是非常有希望的，因为已经实现了显著的临床疗效。因此，B细胞对IG融合蛋白的加工和呈递可以预防和逆转葡萄膜炎和EAE中的自身免疫应答。重要的是，我们已经发现，全长GAD 65（GAD- IG）和胰岛素B 9 -23-Ig（或胰岛素原-Ig）在NOD B细胞中的表达显著延迟了雌性NOD小鼠中糖尿病的发作，即使在7-12周龄时开始治疗（即，在患有胰岛周围炎的小鼠中）。我们的假设是，融合GAD-Ig被NOD B细胞（雄性和雌性）加工，用于主要和次要GAD表位的致耐受性呈递，并导致调节性T细胞的产生。在此，我们将确定B细胞活化的最佳条件和这种“耐受性”的机制。我们还将直接确定这种基因治疗方法是否靶向CD 4和CDS T细胞或两者，以产生多个表位的细胞因子，以及它们将糖尿病转移到NOD-scid受体的能力。我们将使用识别CD 4或CDS表位的TCR转基因系来跟踪靶T细胞的命运。此外，我们将研究调节性T细胞的作用和特异性可能参与直接消融和细胞混合实验中耐受的诱导和维持，以及确定抑制性细胞因子在耐受中的作用。对于未来的治疗，重要的是，我们还希望提供该系统在胰岛移植模型中的功效的原理证明。我们的目标是：1）优化和确定B细胞介导的耐受性基因治疗的靶点; 2）确定B细胞介导的耐受性基因治疗的细胞靶点和机制。3)检测B细胞递送基因治疗后致病性T细胞的命运和调节性T细胞的作用，以及4）在胰岛移植模型中建立B细胞递送基因治疗的功效。这些目标的实现将确立调节性T细胞的作用，以及B细胞递送的耐受性基因治疗的细胞靶点。我们的目标是调节对GAD的致病反应，例如，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