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

肾移植排斥反应中同种异体 CD8 T 细胞的单细胞分析

• 批准号: 10607410
• 负责人: Tiffany Shi
• 金额: $ 4.06万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-22 至 2026-07-21
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607410
• 关键词: Acute; Affect; Alloantigen; Allogenic; Allograft Tolerance; Allografting; Alpha Cell; Anti-CD40; Autoimmunity; Automobile Driving; Biology; Biopsy; Blood; CD8-Positive T-Lymphocytes; CTLA4-Ig; Cell Therapy; Cells; Chronic; Clonal Expansion; Clone Cells; Cross Reactions; Cytomegalovirus; Data; Development; Diabetes Mellitus; Disease; Effectiveness; End stage renal failure; Event; Frequencies; Gene Expression; Goals; Graft Rejection; Hand; Health; Home; Human; Human Herpesvirus 4; Hypertension; Immunity; Immunobiology; Immunosuppression; Individual; Infection; Infection Control; Infiltration; Jurkat Cells; Kidney Transplantation; Knowledge; Life Expectancy; Link; Literature; Lymphocyte; Lymphocytic choriomeningitis virus; Malignant Neoplasms; Mediating; Memory; Modeling; Molecular; Monoclonal Antibodies; Mus; Organ; Organ Transplantation; Pathway interactions; Patients; Peptides; Peripheral Blood Mononuclear Cell; Persons; Play; Quality of life; Reaction; Risk; Role; Sampling; Solid; Specificity; T cell infiltration; T cell receptor repertoire sequencing; T cell therapy; T memory cell; T-Lymphocyte; Tacrolimus; Testing; Therapeutic; Therapeutic immunosuppression; Time; Toxic effect; Transplant Recipients; Transplantation; Urine; Viral; Viral Antigens; Virus Diseases; Work; allograft rejection; combat; cross reactivity; experimental study; graft failure; graft function; heart allograft; human disease; insight; kidney allograft; mouse model; new therapeutic target; novel; novel therapeutics; peripheral blood; post-transplant; premature; prevent; response; screening; single cell analysis; single-cell RNA sequencing; standard of care; transcriptomics; transplant model

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.

摘要 肾移植仍然是终末期肾病（ESRD）的唯一长期解决方案， 全世界约有200万人。然而，长期接受同种异体移植需要终生 免疫抑制和目前的标准护理免疫抑制疗法具有多种毒性 直接导致患者和同种异体移植物存活率降低。针对T细胞的靶向抗排斥治疗 已经开发了共刺激阻断剂，例如贝拉西普（belatacept，CTLA 4-IG）或伊斯卡利单抗（iscalimab，抗CD 40 mAb）， 防止T细胞介导的排斥反应。尽管增强了移植物功能，但在这些治疗下的排斥反应发生率仍然很低。 目前的教条认为，这些排斥反应是由于记忆性CD 8 + T细胞， 来自先前感染的交叉反应性，其不需要共刺激。在这里，我们将检查十字架- 在来自人和小鼠的排斥活检中扩增的移植物浸润性CD 8 + T细胞的反应性。我们的新型 初步数据表明，移植物中扩增的CD 8 + T细胞克隆型是同种异体反应性的， 感染会增加同种异体反应。这些初步调查结果包括：（a）有限数量的独特 在排斥同种异体移植物中扩增克隆型;（B）在排斥同种异体移植物中扩增相同的单个扩增的CD 8 + T细胞克隆， 在持续的同种异体移植排斥反应中观察数月;（c）扩增的克隆型将其基因表达转移到 逃避免疫抑制;和（d）证实了TCR从扩增的克隆型亚克隆到Jurkat细胞中 它们的同种异体特异性。此外，我们使用已建立的病毒感染和移植小鼠模型表明： (e)先前的LCMV感染增加了对同种异体抗原的应答;和（f）没有先前的LCMV感染的小鼠排斥 移植后第14天的F1心脏移植物。最后，我们有一些病人的样本， 施用病毒特异性T细胞疗法以治疗感染。总之，我们的发现和现有文献 支持了一个非常新颖的假设，即预先存在的病毒特异性记忆T细胞的显著部分 在移植后不久，它们与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）鉴定同种异体反应性、病毒反应性和交叉反应性移植物- 在经历肾移植排斥的患者中浸润的CD 8 + T细胞（目的1）和（ii）确定 先前病毒感染对CD 8 + T细胞浸润到移植小鼠模型的同种异体移植物中的影响（目的2）。我们 这些研究将提高对肾移植排斥反应中交叉反应性CD 8 + T细胞的认识， 同种异体反应性T细胞对人类疾病的表征。考虑到器官短缺危机和有限的生命 移植患者的预期，确定肾移植中同种异体反应性CD 8 + T细胞的特异性 排斥反应将使得能够发现新的治疗靶点，以直接靶向同种异体反应性细胞，同时保留 旁观者CD 8 + T细胞负责维持对感染或癌症的免疫力。