Deep Immune Profiling of Nonchimeric Tolerance of Transplants in Nonhuman Primates

摘要 移植(TX)耐受性将深刻改善成千上万将在未来接受TX治疗的人的生活 未来几年。最近，我们报告了对胰岛长期(1年)耐受的安全和一致的诱导 非人灵长类动物两次经腹输注凋亡供者白细胞的同种异体移植 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，并暗示了人类对非嵌合体TX耐受性的可能性，进一步深入了解 在临床翻译之前，将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、转录学和细胞学方面取得的进展 单细胞和高维数据集的计算分析，不同免疫细胞的贡献 子集和移植到耐受性的研究现在可以用以前无法想象的清晰程度来研究。 因此，拟议研究的目标是利用这些新的工具和现有的样本来描绘 ADLS-IIS方案诱导TX耐受的关键因素及机制。为此， 我们已经建立了用于i)跟踪和分选同种异体CD4T细胞的技术，ii)单细胞ATAC-&RNA- 这些分选细胞的测序，iii)移植物和移植物微环境的数字空间轮廓(DSP)，以及iv) 用四个猕猴验证的CyTOF板对循环单核细胞进行质量细胞术分析。 在目标1中，我们将剖析免疫耐受和免疫缺陷患者外周血中同种异体CD4T细胞的分子多样性。 通过单细胞ATAC和RNA测序以无偏见的方式对非耐受性TX受体进行测序，验证假设 ADLS IIS方案导致i)衰竭，ii)未能获得记忆表型和iii)扩展 以及通过表观遗传和转录改变激活CD4T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 假说ADLS IIS改变共抑制分子和保护性分子的移植物内转录，从而决定 免疫细胞在胰岛和移植肾中的募集及其存活。 在目标3中，我们将利用现有的循环单个核细胞(MNC)的高维CyTOF图谱 以及专注于耗尽/记忆(tex/mem)和调节(Treg和 Tr1)T细胞，验证了TeX、Treg和TR1细胞之间相同的关键异质簇的假设 群体与ADLS IIS诱导的胰岛和肾脏TX耐受有关。 这里提出的研究将是第一次利用表观遗传学、转录学和细胞学的力量。 单细胞分析用于研究NHP的外周TX耐受机制。数据集 产生和分析将为研究非嵌合耐受的免疫生物学提供资源， 对ADLS IIS方案的生物标记物的发现和临床翻译具有重要意义。

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.