TCR Sequencing Core

摘要 U19提案的总体目标是将肾和胰岛移植与非清髓性移植相结合 造血细胞移植(HCT)是为了实现对供体组织的免疫耐受。我们将使用 食蟹猴模型为已故和活体捐赠者开发这种方法，使用扩展的 在项目1和2中，分别是多克隆和供体特异性受体调节性T细胞。我们假设 耐受包括供体特异性调节性T细胞的扩增和诱导，以及供体-T细胞的缺失- 特异性效应器T细胞。开发这一程序的一个关键是识别和跟踪这些问题的工具 供者特异性T细胞。我们之前在人类身上开发了这样的方法。核心B将扩展这项技术 对于食蟹猴(Cyno)，并将该检测应用于支持项目1和2。具体而言，核心B将遵循 我们用于人类和小鼠开发T细胞受体(TCR)的相同开发流程 天青素的测序分析。由于TCR在体细胞上的重新排列具有巨大的多样性，因此每个TCR几乎 独一无二。这使我们能够随着时间的推移跟踪数十万个T细胞克隆，并通过它们在组织之间 TCR序列。我们利用技术，首先通过混合淋巴细胞分离供者特异性T细胞 反应(明确刺激供体组织特异性的T细胞)。这些扩大捐赠者中的克隆人- 反应性的CD4和CD8T细胞与分类的、未刺激的功能亚群中的细胞进行比较，包括效应器 和调节性T细胞，扩增的序列被鉴定为属于供体反应亚集。 我们将在接受同种异体移植的食蟹猴身上验证这一检测方法，并优化定义 供体反应性效应器和调节性T细胞克隆。从输液树中提取的定时分类的T细胞DNA样本， 外周血和移植物浸润性淋巴细胞以及来自活检标本和尿球的DNA 项目1和项目2中移植的青色猴子将被送往核心B，在那里将进行检测。完毕 时间，我们将监测这些T细胞亚群在血液、组织和尿液中的克隆性扩张和收缩 在这一临床前模型中确定免疫耐受的机制。

Summary The overall goal of the U19 proposal is to combine kidney and islet transplantation with nonmyeloablative hematopoietic cell transplantation (HCT) to achieve immune tolerance to the donor tissues. We will use a the cynomolgus monkey model to develop this approach for deceased and living donor scenarios, using expanded polyclonal and donor-specific recipient regulatory T cells respectively, in Projects 1 and 2. We hypothesize that tolerance will involve both expansion and induction of donor-specific regulatory T cells and deletion of donor- specific effector T cells. A key to development of this procedure will be the tools to identify and track these donor-specific T cells. We previously developed such a method in humans. Core B will extend this technology to cynomolgus monkeys (cyno) and apply the assay to support Projects 1 and 2. Specifically, Core B will follow the same development pipeline we have used for human and mouse to develop a T cell receptor (TCR) sequencing assay for cyno. Since TCRs rearrange somatically with massive diversity, each TCR is nearly unique. This allows us to track hundreds of thousands of T cell clones over time and between tissues by their TCR sequence. We leverage with technology by first isolating donor-specific T cells through a mixed lymphocyte reaction (explicitly stimulating T cells that are specific to the donor tissue). The clones in these expanded donor- reactive CD4 and CD8 T cells are compared to those in sorted, unstimulated functional subsets, including effector and regulatory T cells, and the expanded sequences are identified as belonging to the donor-reactive subset. We will validate this assay in cynomolgus monkeys receiving allotransplants and optimize methods .for defining donor-reactive effector and regulatory T cell clones. Timed, sorted T cell DNA samples from infused Tregs, peripheral blood and graft-infiltrating lymphocytes as well as DNA from biopsy specimens and urine pellets from transplanted cyno monkeys in Projects 1 and 2 will be sent to Core B, where the assay will be applied. Over time, we will monitor the clonal expansion and contraction of these T cell subsets in blood, tissue, and urine pellets to identify mechanisms of immune tolerance in this preclinical model.