Deep Immune Profiling of Nonchimeric Tolerance of Transplants in Nonhuman Primates

非人灵长类动物移植物非嵌合耐受性的深度免疫分析

• 批准号: 10353191
• 负责人: Bernhard Josef Hering
• 金额: $ 23.25万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-22 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10353191
• 关键词: Apoptotic; Behavior Therapy; Biological Markers; Bone Marrow; CD4 Positive T Lymphocytes; Cells; Cellular Assay; Chromatin; Chronic; Clinical Trials; Computer Analysis; Cytometry; Data Set; Epigenetic Process; Failure; Foundations; Future; Generations; Genomics; Goals; Human; Immune; Immunobiology; Immunosuppression; Infusion procedures; Investigation; Kidney Transplantation; Lead; Leukocytes; MHC Class I Genes; Macaca; Macaca mulatta; Maintenance; Memory; Minnesota; Modeling; Molecular; Monitor; Mononuclear; Peripheral; Persons; Phenotype; Population; Positioning Attribute; Protocols documentation; Public Health; Regimen; Regulatory T-Lymphocyte; Reporting; Resources; Risk; Role; Sampling; Sorting - Cell Movement; Specificity; T-Lymphocyte; Techniques; Testing; Toxic effect; Transcript; Transplant Recipients; Transplantation; Transplantation Tolerance; Transposase; Work; biomarker discovery; clinical translation; digital; exhaust; exhaustion; graft vs host disease; high dimensionality; improved; insight; islet; islet allograft; multiple omics; nonhuman primate; novel; peptide I; prevent; recruit; single cell analysis; terminally differentiated effector memory (TEM) T cells; tool; transcriptome; transcriptome sequencing; transcriptomics

Abstract Transplant (tx) tolerance would profoundly improve the lives of thousands of people who will receive tx in the years ahead. Recently, we reported safe and consistent induction of long-term (>1 year) tolerance to islet allografts in nonhuman primates (NHPs) with two peritx infusions of apoptotic donor leukocytes (ADLs) under induction immunosuppression (IIS) (in part supported by U01-AI102463). While these findings are unprecedented and suggest the possibility of nonchimeric tx tolerance in humans, further insight into the mechanisms by which tolerance is induced and maintained will be required before clinical translation of the protocol can be initiated. Owing to the progress made in the epigenetic, transcriptomic and cytometric profiling of single cells and computational analyses of high-dimensional datasets, the contributions of distinct immune cell subsets and the graft to tolerance can now be investigated with previously unimaginable explicitness. Accordingly, the goal of the proposed studies is to harness these novel tools and existing samples to delineate the critical factors and mechanisms responsible for tx tolerance induced by the ADLs+IIS regimen. To this end, we have established techniques for i) tracking and sorting allospecific CD4+ T cells, ii) single-cell ATAC- & RNA- sequencing of these sorted cells, iii) digital spatial profiling (DSP) of grafts and graft microenvironments, and iv) mass cytometry profiling of circulating mononuclear cells with four macaque-validated CyTOF panels. In Aim 1, we will dissect the molecular diversity of circulating allospecific CD4+ T cells from tolerant and nontolerant tx recipients in an unbiased manner by single-cell ATAC & RNA sequencing, testing the hypothesis that the ADLs+IIS regimen causes i) exhaustion, ii) failure to acquire a memory phenotype and iii) expansion and activation of regulatory subsets in CD4+ T cells via epigenetic and transcriptomic alterations. In Aim 2, we will leverage DSP to investigate the role of the graft in the maintenance of tx tolerance, testing the hypothesis that ADLs+IIS alters intragraft transcriptomes of co-inhibitory and protective molecules that determine immune cell recruitment to islet and kidney grafts and their survival. In Aim 3, we will utilize high-dimensional CyTOF profiling of circulating mononuclear cells (MNCs) with existing as well as novel multiomic-guided panels focused on exhausted/memory (Tex/mem) and regulatory (Treg and Tr1) T cells, testing the hypothesis that the same key heterogeneous clusters among Tex, Treg and Tr1 cell populations are associated with islet and kidney tx tolerance induced by ADLs+IIS. The studies proposed herein will be the first to leverage the power of epigenetic, transcriptomic and cytometric analyses of single cells for investigation of mechanisms of peripheral tx tolerance in NHPs. The datasets generated and analyzed will present a resource for investigating the immunobiology of nonchimeric tolerance, with implications for biomarker discovery and clinical translation of the ADLs+IIS regimen.

抽象的 移植（TX）的宽容将深刻改善成千上万的人的生活 未来几年。最近，我们报告了对胰岛的长期（> 1年）公差的安全且一致的诱导 非人类灵长类动物（NHP）中的同种异体移植物，有两个peritx输注凋亡供体白细胞（ADL） 诱导免疫抑制（IIS）（由U01-AI102463支持）。虽然这些发现是 前所未有的，并提出了人类非核TX耐受性的可能性，进一步了解 在临床翻译之前，需要诱导和维持公差的机制 可以启动协议。由于表观遗传学，转录组和细胞学分析的进展 高维数据集的单细胞和计算分析的贡献 现在可以通过以前难以想象的明确性研究子集和供耐受性的移植物。 因此，拟议的研究的目的是利用这些新颖的工具和现有样品来描述 ADL+IIS方案引起的TX公差的关键因素和机制。为此， 我们已经建立了i）跟踪和分类同种异体CD4+ T细胞的技术，ii）单细胞ATAC和RNA- 这些分类细胞的测序，iii）移植物和移植微环境的数字空间分析（DSP）以及IV） 循环单核细胞的质量细胞仪与四个猕猴的细胞图。 在AIM 1中，我们将从耐受和 通过单细胞ATAC和RNA测序以公正的方式以无偏的方式进行了非耐受性TX的接受者，检验了假设 ADLS+IIS方案导致i）疲惫，ii）未能获得记忆表型和iii）扩展 通过表观遗传和转录组改变，CD4+ T细胞中调节子集的激活。 在AIM 2中，我们将利用DSP研究移植物在维持TX公差中的作用，并测试 ADL+IIS的假设改变了确定的共抑制性和保护性分子的内部转录组 免疫细胞募集到胰岛和肾脏移植物及其生存。 在AIM 3中，我们将利用与现有的循环单核细胞（MNC）的高维细胞分析 以及专注于疲惫/记忆（TEX/MEM）和监管的新型多媒体引导面板（Treg和 TR1）T细胞，检验以下假设：Tex，Treg和Tr1细胞之间相同的关键异质簇 种群与ADL+II诱导的胰岛和肾脏TX公差有关。 本文提出的研究将是第一个利用表观遗传学，转录组和细胞图的能力的研究 单个细胞的分析，以研究NHP中外周血TX耐受性的机理。数据集 生成和分析的将提供一种资源，用于研究非核耐受性的免疫生物学， 对ADLS+IIS方案的生物标志物发现和临床翻译的影响。