Combined multidimensional single-cell protein and RNA profiling dissects the cellular and functional heterogeneity of thymic epithelial cells

The network of thymic stromal cells provides essential niches with unique molecular cues controlling T-cell development and selection. Recent single-cell RNA-sequencing studies uncovered a large transcriptional heterogeneity among thymic epithelial cells (TEC) demonstrating a previously unappreciated complexity. However, there are only very few cell markers that allow a comparable phenotypic identification of TEC. Here we deconvoluted by massively parallel flow cytometry and machine learning known and novel TEC phenotypes into novel subpopulations and related these by CITEseq to the corresponding TEC subtypes defined by the cells' individual RNA profiles. This approach phenotypically identified perinatal cTEC, physically located these cells within the cortical stromal scaffold, displayed their dynamic change during the life course and revealed their exceptional efficiency in positively selecting immature thymocytes. Collectively, we have identified novel markers that allow for an unprecedented dissection of the thymus stromal complexity, the cells physical isolation and assignment of specific functions to individual TEC subpopulations. Cells were isolated from 1- and 16-week-old mice and depleted of CD45+ cells by autoMACS. Subsequently cells were stained for CD45, EpCAM1, Ly51, Ter119 and with PI. In addition cells were stained with antibodies coupled to oligonucleotides directed against CD9, CD40, CD49a, CD54, CD63, CD73, CD83, CD117, CD146 (human with cross reactivity to mouse), CD200, CD274, HVEM, Ly6D, Ly6C/Ly6G (Gr1), MadCAM1, Podoplanin, CD80, CD86, MHCII, Sca1, CD31, EpCAM1, CD36, CD133, CD157, CD300LG and the Ulex europaeus agglutinin I (UEA1) lectin labeled with biotin, followed by secondary staining with streptavidin-PE coupled to an oligonucleotid. CD45-Ter119-EpCAM1+ and CD45-Ter119-EpCAM1- cells were sorted in a 70% to 30% ratio into a 1.5 mL tube containing FACS buffer for the 1-week-old and 16-week-old samples, respectively. For both timepoints an estimate of 28000 total cells were loaded on two wells of a 10x Genomics Chromium Single Cell Controller.胸腺基质细胞网络可提供具备独特分子信号的必需微环境，调控T细胞的发育与选择。近期的单细胞RNA测序（single-cell RNA-sequencing）研究揭示了胸腺上皮细胞（thymic epithelial cells, TEC）中存在广泛的转录异质性，展现出此前未被认知的复杂性。然而，目前仅存在极少数细胞标志物可实现对TEC的同等表型鉴定。 本研究通过大规模流式细胞术（massively parallel flow cytometry）与机器学习（machine learning），将已知及新型TEC表型解卷积为全新的细胞亚群，并借助CITE-seq（CITEseq）将这些亚群与基于细胞个体RNA表达谱定义的对应TEC亚型进行关联。该方法从表型层面鉴定出了围产期皮质胸腺上皮细胞（perinatal cTEC），在皮质基质支架中实现了这些细胞的物理定位，展示了其在生命周期中的动态变化，并揭示了它们在阳性选择未成熟胸腺细胞方面的卓越效率。 综上，本研究鉴定出了全新的细胞标志物，可实现对胸腺基质复杂性的前所未有的解析、细胞的物理分离，并可将特定功能赋予单个TEC亚群。 我们从1周龄和16周龄的小鼠体内分离细胞，并通过autoMACS去除CD45+细胞。随后对细胞进行染色：首先标记CD45、EpCAM1、Ly51、Ter119以及碘化丙啶（propidium iodide, PI）；此外，还使用偶联寡核苷酸（oligonucleotides）的抗体进行染色，所针对的靶点包括CD9、CD40、CD49a、CD54、CD63、CD73、CD83、CD117、CD146（与人源靶点具有交叉反应，可用于小鼠样本）、CD200、CD274、HVEM、Ly6D、Ly6C/Ly6G（Gr1）、MadCAM1、Podoplanin、CD80、CD86、MHCII、Sca1、CD31、EpCAM1、CD36、CD133、CD157、CD300LG，以及生物素标记的荆豆凝集素I（Ulex europaeus agglutinin I, UEA1）；之后再使用偶联寡核苷酸的链霉亲和素-PE（streptavidin-PE）进行二次染色。 对于1周龄和16周龄的样本，我们分别以70%:30%的比例将CD45-Ter119-EpCAM1+与CD45-Ter119-EpCAM1-细胞分选至含有流式缓冲液（FACS buffer）的1.5 mL离心管中。两个时间点的样本均预计加载约28000个总细胞至10x Genomics Chromium单细胞控制器（10x Genomics Chromium Single Cell Controller）的两个孔中。