Single Cell Analysis of Alloreactive CD8+ T Cells in Kidney Transplant Rejection

摘要 肾移植仍然是终末期肾病(ESRD)的唯一长期解决方案，这种疾病会影响 全球约有200万人。然而，长期接受同种异体移植物需要终生接受。 免疫抑制和目前的标准护理免疫抑制疗法有多种毒性。 直接导致患者和同种异体移植物存活率下降。以T细胞为靶向的抗排斥治疗 共刺激阻断剂，如拉他赛普(CTLA4-Ig)或iscalimab(抗CD40单抗)已被开发为 防止T细胞介导的排斥反应。尽管移植物功能得到了增强，但在这些疗法下发生的排斥反应 更频繁也更难治疗。目前的教义认为这些排斥反应是由于记忆中的CD8+T细胞 与先前感染的交叉反应，不需要共刺激。在这里，我们将检查十字架- 人和小鼠的排斥活检中扩增的、移植物渗入的CD8+T细胞的反应性。我们的小说 初步数据表明，移植物中扩增的CD8+T细胞克隆型是同种异体反应性的，而在病毒 感染会增加同种异体反应。这些初步调查结果包括：(A)数量有限的独一无二 克隆型在排斥同种异体移植物中扩大；(B)相同的单个扩增的CD8+T细胞克隆 在持续的同种异体移植排斥反应中观察了几个月；(C)扩大的克隆型将其基因表达转移到 逃避免疫抑制；以及(D)证实将TCRs从扩增克隆型亚克隆到Jurkat细胞 他们的同种异体专一性。此外，我们还表明，使用已建立的病毒感染和移植小鼠模型： (E)先前感染LCMV增加了对同种异体抗原的反应；和(F)未感染LCMV的小鼠发生排斥反应 移植后第14天进行F1同种异体心脏移植。最后，我们有接受排斥反应的患者的样本 应用病毒特异性T细胞疗法治疗感染。总而言之，我们的发现和现有文献 支持一种非常新颖的假设，即相当一部分先前存在的病毒特异性记忆T细胞 移植后不久回到同种异体移植物，它们对同种异体抗原的交叉反应导致排斥反应 事件。我们建议通过以下方法来检验这一假设：(I)识别同种异体反应、病毒反应和交叉反应移植物- 肾移植排斥反应患者CD8+T细胞的渗入(目标1)和(Ii)确定CD8+T细胞的影响 既往病毒感染对CD8+T细胞向同种异体移植小鼠模型的侵袭作用(目标2)。我们的 研究将加强对CD8+T细胞在肾移植排斥反应中的交叉反应的认识 人类疾病中同种异体反应性T细胞的特征。鉴于器官短缺危机和有限的生命 移植患者的期望值，确定肾移植中同种异体反应性CD8+T细胞的特异性 排斥反应将使发现新的治疗靶点直接针对同种异体反应细胞，同时节省 旁观者CD8+T细胞，负责维持对感染或癌症的免疫力。

ABSTRACT Renal transplantation remains the only long-term solution to end-stage renal disease (ESRD), which affects approximately 2 million people worldwide. However, long term allograft acceptance requires lifelong immunosuppression, and current standard-of-care immunosuppressive therapies have a multitude of toxicities directly leading to decreased patient and allograft survival. Targeted anti-rejection therapies focusing on T cell co-stimulatory blockade, such as belatacept (CTLA4-Ig) or iscalimab (anti-CD40 mAb) have been developed to prevent T cell mediated rejection. Despite enhanced graft function, rejection episodes under these therapies are more frequent and difficult to treat.Current dogma suggests that these rejections are due to memory CD8+ T cell cross-reactivity from prior infections, which do not require co-stimulation. Here we will examine the cross- reactivity of expanded, graft-infiltrating CD8+ T cells in rejection biopsies from both humans and mice. Our novel preliminary data suggest that expanded CD8+ T cell clonotypes in the graft are alloreactive, and prior viral infection increases the alloresponse. These preliminary findings include: (a) a limited number of unique clonotypes are expanded in the rejecting allograft; (b) the same individual expanded CD8+ T cell clones are observed for months in persistent allograft rejection; (c) expanded clonotypes shift their gene expression to escape immunosuppression; and (d) subcloning of TCRs from expanded clonotypes into Jurkat cells confirmed their allospecificity. Further, we show that using established mouse models of viral infection and transplantation: (e) prior LCMV infection increases the response to alloantigen; and (f) mice without prior LCMV infection reject F1 heart allografts by day 14 post-transplant. Finally, we have samples from patients undergoing rejection after administration of viral-specific T cell therapy to treat infection. Together, our findings and existing literature support a highly novel hypothesis that a significant fraction of pre-existing viral-specific memory T cells home to the allograft shortly after transplant and their cross-reactivity to alloantigens drives rejection events. We propose to test this hypothesis by: (i) identifying alloreactive, viral-reactive, and cross-reactive graft- infiltrating CD8+ T cells in patients undergoing renal allograft rejection (Aim 1) and (ii) determining the impact of prior viral infection on CD8+ T cell infiltration into the allograft of a mouse model of transplantation (Aim 2). Our studies will enhance the knowledge of cross-reactive CD8+ T cells in renal allograft rejection and the characterization of alloreactive T cells on human disease. Given the organ shortage crisis and the limited life expectancies of transplant patients, defining the specificity of alloreactive CD8+ T cells in renal transplant rejection will enable discovery of novel therapeutic targets to directly target alloreactive cells while sparing bystander CD8+ T cells responsible for maintaining immunity to infections or cancers.