Deep Immune Profiling of Nonchimeric Tolerance of Transplants in Nonhuman Primates

非人类灵长类动物移植物非嵌合耐受性的深度免疫分析

基本信息

批准号：
10612925
负责人：
Bernhard Josef Hering
金额：
$ 19.38万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-22 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10612925
关键词：
Apoptotic Behavior Therapy Biological Markers Bone Marrow CD4 Positive T Lymphocytes Cell Separation 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 Leukocytes MHC Class I Genes MHC Class II 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 Specificity T-Lymphocyte Techniques Technology Testing Toxic effect Transcript Transplant Recipients Transplantation Transplantation Tolerance Transposase Work biomarker discovery biomarker validation clinical translation digital exhaust exhaustion graft vs host disease high dimensionality improved insight islet islet allograft multidimensional data multiple omics nonhuman primate novel peptide I prevent recruit single cell analysis tool transcriptome transcriptome sequencing transcriptomics

项目摘要

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）耐受性将深刻改善成千上万将在未来接受移植的人的生活未来几年。最近，我们报告了安全一致的胰岛长期（> 1 年）耐受诱导非人灵长类动物 (NHP) 的同种异体移植物，在以下条件下进行两次 Peritx 输注凋亡供体白细胞 (ADL) 诱导免疫抑制 (IIS)（部分由 U01-AI102463 支持）。虽然这些发现是史无前例的，并表明人类非嵌合 tx 耐受的可能性，进一步深入了解在临床转化之前需要诱导和维持耐受性的机制可以启动协议。由于表观遗传学、转录组学和细胞计数分析方面取得的进展单细胞和高维数据集的计算分析，不同免疫细胞的贡献现在可以以以前难以想象的明确性来研究子集和移植到耐受性。因此，拟议研究的目标是利用这些新颖的工具和现有样本来描绘 ADLs+IIS 方案诱导 tx 耐受的关键因素和机制。为此，我们已经建立了 i) 跟踪和分选同种异体特异性 CD4+ T 细胞，ii) 单细胞 ATAC- 和 RNA- 的技术对这些分选的细胞进行测序，iii) 移植物和移植物微环境的数字空间分析 (DSP)，以及 iv) 使用四个经过猕猴验证的 CyTOF 面板对循环单核细胞进行质谱流式分析。在目标 1 中，我们将剖析循环同种异体特异性 CD4+ T 细胞的分子多样性，这些细胞来自耐受性和通过单细胞 ATAC 和 RNA 测序以公正的方式对不耐受的 tx 接受者进行测试，检验假设 ADLs+IIS 方案会导致 i) 耗尽，ii) 无法获得记忆表型，以及 iii) 扩展以及通过表观遗传和转录组改变激活 CD4+ T 细胞中的调节子集。在目标 2 中，我们将利用 DSP 来研究移植物在维持 tx 耐受性中的作用，测试假设 ADLs+IIS 改变了共抑制分子和保护分子的移植物内转录组，这些分子决定了免疫细胞招募到胰岛和肾移植物及其存活。在目标 3 中，我们将利用现有的循环单核细胞 (MNC) 的高维 CyTOF 分析以及专注于耗尽/记忆（Tex/mem）和监管（Treg 和 Tr1) T 细胞，检验 Tex、Treg 和 Tr1 细胞之间具有相同关键异质簇的假设人群与 ADL+IIS 诱导的胰岛和肾脏 tx 耐受性相关。本文提出的研究将是第一个利用表观遗传学、转录组学和细胞计数的力量的研究分析单细胞以研究 NHP 外周 tx 耐受机制。数据集生成和分析将提供用于研究非嵌合耐受的免疫生物学的资源，对 ADL+IIS 方案的生物标志物发现和临床转化具有影响。