T-cell activation is a key step in the amplification of an immune response. Over the course of an immune response, cells may be chronically stimulated, with some proportion becoming exhausted; an enormous number of molecules are involved in this process. There remain a number of questions about the process, namely: (1) what degree of heterogeneity and plasticity do T-cells exhibit during stimulation? (2) how many unique cell states define chronic stimulation? and (3) what markers discriminate activated from exhausted cells? We addressed these questions by performing single-cell multiomic analysis to simultaneously measure expression of 38 proteins and 399 genes in human T cells expanded in vitro. This approach allowed us to study –with unprecedented depth–how T cells change over the course of chronic stimulation. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states and the identification of a donor-specific subset of terminally differentiated T-cells that would have been otherwise overlooked using canonical cell classification schema. As expected, activation downregulated naïve-cell markers and upregulated effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation, defined by markers temporarily expressed upon 3 days of stimulation (PD-1, CD69, LTA), markers constitutively expressed throughout chronic activation (CD25, GITR, LGALS1), and markers uniquely up-regulated upon 14 days of stimulation (CD39, ENTPD1, TNFDF10); expression of these markers could be associated with the emergence of short-lived cell types. Notably, different ratios of cells expressing activation or exhaustion markers were measured at each time point. These data reveal the high heterogeneity and plasticity of chronically stimulated T cells. Our study demonstrates the power of a single-cell multiomic approach to comprehensively characterize T-cells and to precisely monitor changes in differentiation, activation, and exhaustion signatures during cell stimulation.

中文翻译：

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体外 T 细胞耗竭的单细胞多组学分析

T细胞活化是免疫反应放大的关键步骤。在免疫反应过程中，细胞可能会受到长期刺激，部分细胞会耗尽；这个过程涉及大量的分子。关于该过程仍有许多问题，即：（1）T细胞在刺激过程中表现出何种程度的异质性和可塑性？(2) 有多少独特的细胞状态定义了慢性刺激？(3) 哪些标记可以区分激活的细胞和耗尽的细胞？我们通过进行单细胞多组学分析来解决这些问题，以同时测量体外扩增的人类 T 细胞中 38 种蛋白质和 399 种基因的表达。这种方法使我们能够以前所未有的深度研究 T 细胞如何在慢性刺激过程中发生变化。在第 0 天进行全面的免疫表型和转录组学分析，可以对 T 细胞成熟状态进行精确表征，并识别出终末分化 T 细胞的供体特异性亚群，否则这些细胞会被典型的细胞分类模式所忽视。正如预期的那样，激活下调幼稚细胞标志物并上调效应分子、增殖调节剂、共抑制和共刺激受体。我们的深度动力学分析进一步揭示了识别独特激活状态的蛋白质和基因簇，这些激活状态由刺激 3 天后临时表达的标记物（PD-1、CD69、共抑制和共刺激受体。我们的深度动力学分析进一步揭示了识别独特激活状态的蛋白质和基因簇，这些激活状态由刺激 3 天后临时表达的标记物（PD-1、CD69、共抑制和共刺激受体。我们的深度动力学分析进一步揭示了识别独特激活状态的蛋白质和基因簇，这些激活状态由刺激 3 天后临时表达的标记物（PD-1、CD69、LTA)、在慢性激活过程中组成型表达的标志物（CD25、GITR、LGALS1），以及在刺激 14 天后唯一上调的标志物（CD39、ENTPD1、TNFDF10）；这些标记的表达可能与短寿命细胞类型的出现有关。值得注意的是，在每个时间点测量了表达激活或衰竭标记的细胞的不同比例。这些数据揭示了长期刺激的 T 细胞的高异质性和可塑性。我们的研究证明了单细胞多组学方法在全面表征 T 细胞并精确监测细胞刺激过程中分化、激活和耗竭特征的变化方面的能力。