TCR Sequencing Core

总结 U19提案的总体目标是将联合收割机肾和胰岛移植与非清髓性移植结合起来。 造血细胞移植（HCT）以实现对供体组织的免疫耐受。我们将使用一个 食蟹猴模型开发这种方法的死者和活体供体的情况下，使用扩大 多克隆和供体特异性受体调节性T细胞，分别在项目1和2中。我们假设 耐受性将涉及供体特异性调节性T细胞的扩增和诱导以及供体特异性调节性T细胞的缺失。 特异性效应T细胞。制定这一程序的一个关键是确定和跟踪这些问题的工具。 供体特异性T细胞我们以前在人类身上开发了这样的方法。核心B将扩展此技术 食蟹猴（cyno），并应用该试验支持项目1和2。具体而言，核心B将遵循 与我们用于人类和小鼠开发T细胞受体（TCR）的开发管道相同， 食蟹猴的测序测定。由于TCR以巨大的多样性进行体细胞重排，每个TCR几乎都是 独特.这使我们能够跟踪数十万个T细胞克隆随着时间的推移和组织之间的通过他们的 TCR序列。我们利用技术首先通过混合淋巴细胞分离供体特异性T细胞， 反应（明确刺激对供体组织特异性的T细胞）。在这些扩增的供体中的克隆- 将反应性CD 4和CD 8 T细胞与分选的、未刺激的功能亚群中的那些进行比较，包括效应子T细胞。 和调节性T细胞，并且扩增的序列被鉴定为属于供体反应性亚群。 我们将在接受同种异体移植的食蟹猴中验证该测定并优化方法。 供体反应性效应子和调节性T细胞克隆。来自输注的TcR的定时分选的T细胞DNA样品， 外周血和移植物浸润淋巴细胞以及来自活组织检查标本的DNA和来自 项目1和2中的移植食蟹猴将被送往核心B，在那里进行试验。超过 同时，我们将监测这些T细胞亚群在血液、组织和尿液中的克隆扩增和收缩 颗粒，以确定在这个临床前模型的免疫耐受机制。

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.