Deep Immune Profiling of Nonchimeric Tolerance of Transplants in Nonhuman Primates

摘要 移植（tx）耐受性将大大改善成千上万的人谁将接受tx在世界各地的生活。 几年前最近，我们报道了安全和一致的诱导长期（>1年）的耐受性， 非人灵长类动物（NHP）中的同种异体移植物，在非人灵长类动物（NHP）中， 诱导免疫抑制（IIS）（部分由U 01-AI 102463支持）。虽然这些发现是 这是前所未有的，并表明在人类中存在非嵌合的TX耐受性的可能性， 在临床翻译之前，需要诱导和维持耐受性的机制。 可以启动协议。由于在表观遗传学、转录组学和细胞计量学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 现在可以用以前无法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工具和现有样本来描绘 ADLs+IIS方案诱导耐受的关键因素和机制。为此目的， 我们已经建立了用于i）追踪和分选同种异体特异性CD 4 + T细胞，ii）单细胞ATAC- & RNA- 这些分选的细胞的测序，iii）移植物和移植物微环境的数字空间分析（DSP），和iv） 使用四个macaque验证的CyTOF板对循环单核细胞进行质谱细胞术分析。 在目标1中，我们将剖析循环同种异体特异性CD 4 + T细胞的分子多样性， 通过单细胞ATAC和RNA测序，以无偏的方式检测非耐受性tx受体，检验假设 ADL +IIS方案导致i）耗竭，ii）不能获得记忆表型，和iii）扩增 以及通过表观遗传学和转录组学改变激活CD 4 + T细胞中的调节亚群。 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ADL +IIS改变了共抑制和保护分子的移植物内转录组， 免疫细胞向胰岛和肾移植物的募集及其存活。 在目标3中，我们将利用循环单核细胞（MNCs）的高维CyTOF分析， 以及新的多组学指导小组，集中在耗尽/记忆（Tex/Tex）和调节（Treg和 Tr 1）T细胞，检验Tex、Treg和Tr 1细胞之间相同的关键异质簇的假设 群体与由ADL +IIS诱导的胰岛和肾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。 分析单细胞以研究NHP中外周tx耐受性的机制。数据集 产生和分析将为研究非嵌合耐受性的免疫生物学提供资源， 对生物标志物的发现和ADL +IIS方案的临床转化具有意义。

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.