TCR and BCR deep sequencing to distinguish autoimmune recurrence from allograft rejection

项目摘要：美国国立卫生研究院估计，2400万美国人患有自身免疫性疾病，这是一个重要的 发生终末性器官衰竭的比例，器官移植将是唯一的根治方法。 原发自身免疫性疾病的复发是造成移植物丢失和患者死亡的主要原因之一。 实体器官移植(SOT)后的发病率和死亡率。管理这些患者的一个主要挑战是 正在确定免疫介导的移植物功能障碍是自身免疫还是同种异体免疫过程的结果 或者两者都有。目前的诊断涉及血清学、放射学和组织学标准的组合 明确区分急性细胞排斥反应(ACR)和RPAD。更好地理解相互作用 这两种方法之间的结合可能最终提高RPAD和ACR治疗的安全性和有效性， 针对个别患者移植物损伤的具体机制。适应性T和B细胞反应是 与自身免疫性疾病和同种异体移植排斥反应有关。我们建议使用一种新的方法来识别 同种异体反应性T细胞和自身反应性T、B细胞在同种异体肾移植后功能障碍中的作用 患有自身免疫性疾病的患者。使用T细胞受体(TCR)β的高通量测序(HTS) 链互补决定区3(CDR3)，我们发展了一种新的方法来识别和 追踪移植受者的人类同种异体反应。我们现在建议结合使用此方法 用基于HTS的方法识别和跟踪自身免疫性TCR和B细胞受体(BCR)谱系 区分同种异体移植排斥反应和RPAD在移植物功能障碍中的作用 自身免疫性疾病。我们将使用肝移植(LT)治疗自身免疫性肝病(ALD)作为一个系统 用来测试这种方法。TCRβCDR3(高变区)区域的HTS将用于识别供体- 移植前血液中反应性T细胞克隆和患者肝移植后自身反应性T细胞和B细胞克隆 活组织检查。移植后，我们将追踪这些克隆人在血液、移植物、胆汁和可能的粪便中的情况。 以及随后的同种异体移植功能障碍时期，以辨别供者特异性T细胞克隆和 自身反应性T和B细胞在导致移植物功能障碍中的作用以及评估它们对治疗的反应。我们 假设当移植物功能障碍时，血液或肝组织中的自身反应克隆将更加丰富 由复发的ALD引起，而供体反应性T细胞克隆将在ACR中占主导地位。我们还将测试 有可能在粪便中识别和跟踪这些淋巴细胞，如果呈阳性，最终可能被评估为 这是一种非侵入性的方法，可以避免将来进行肝脏活检。如果成功，一种新的工具可以 区分ACR和RADD将可用，使您能够更深入地了解以下肝功能障碍 并使改进的治疗成为可能。我们的新方法具有巨大的潜力，可以提供诊断和 SOT后同种异体免疫和自身免疫过程的预后生物标志物，并将适用于移植 自身免疫性疾病的其他类型的SOT功能障碍。

Project Summary: NIH estimates that 24 million Americans suffer from autoimmune diseases, a significant percentage of whom develop end organ failure for which organ transplant would be the only curative treatment. Recurrence of primary autoimmune diseases (RPAD) is among the leading causes of graft loss and patient morbidity and mortality following solid organ transplantation (SOT). A major challenge in managing these patients is determining whether immune-mediated graft dysfunction is the result of an autoimmune or alloimmune process or both. Current diagnostics involving a combination of serological, radiological and histological criteria do not clearly distinguish between acute cellular rejection (ACR) and RPAD. A better understanding of the interplay between these two processes may ultimately improve the safety and efficacy of RPAD and ACR treatment, targeting the specific mechanism of graft injury in individual patients. Adaptive T and B cell responses are implicated in both autoimmune disorders and allograft rejection. We propose to use a novel approach to identify the specific roles of alloreactive T cells and autoreactive T and B cells in post-transplant allograft dysfunction in patients with autoimmune disease. Using high-throughput sequencing (HTS) of the T-cell receptor (TCR) beta chain complementarity-determining region 3 (CDR3), we have developed a novel method of identifying and tracking the human alloresponse in transplant recipients. We now propose to use this method in combination with a HTS-based method of identifying and tracking the autoimmune TCR and B-cell receptor (BCR) repertoire to distinguish the roles of allograft rejection and RPAD in graft dysfunction following transplantation due to autoimmune disease. We will use liver transplantation (LT) for autoimmune liver diseases (ALD) as a system in which to test this approach. HTS of the TCR beta CDR3 (hypervariable) region will be used to identify donor- reactive T cell clones in pre-transplant blood and autoreactive T and B cell clones from patient liver explants and biopsies. Following the transplant, we will track these clones in the blood, graft, bile and possibly stool during and following periods of allograft dysfunction in order to discern the roles of donor-specific T cell clones and autoreactive T and B cells in causing graft dysfunction and to assess their responsiveness to therapy. We hypothesize that autoreactive clones will be more abundant in blood or liver tissues when graft dysfunction is caused by recurrent ALD, whereas donor-reactive T cell clones will predominate in ACR. We will also test whether it is possible to identify and track these lymphocytes in stool, which, if positive, could ultimately be evaluated as a non-invasive method that would avoid the need for liver biopsies in the future. If successful, a new tool to distinguish ACR and rALD will become available, allowing a deeper understanding of liver dysfunction following LT and enabling improved therapies. Our novel approach has enormous potential to provide a diagnostic and prognostic biomarker of alloimmune and autoimmune processes following SOT and will be applicable to graft dysfunction in other types of SOT for autoimmune diseases.