Thymus development, cell by cell The human thymus is the organ responsible for the maturation of many types of T cells, which are immune cells that protect us from infection. However, it is not well known how these cells develop with a full immune complement that contains the necessary variation to protect us from a variety of pathogens. By performing single-cell RNA sequencing on more than 250,000 cells, Park et al. examined the changes that occur in the thymus over the course of a human life. They found that development occurs in a coordinated manner among immune cells and with their developmental microenvironment. These data allowed for the creation of models of how T cells with different specific immune functions develop in humans. Science, this issue p. eaay3224 Single-cell RNA profiles of the human thymus over time elucidate aspects of human immunological T cell development. INTRODUCTION The thymus is the critical organ for T cell development and T cell receptor (TCR) repertoire formation, which shapes the landscape of adaptive immunity. T cell development in the thymus is spatially coordinated, and this process is orchestrated by diverse cell types constituting the thymic microenvironment. Although the thymus has been extensively studied using diverse animal models, human immunity cannot be understood without a detailed atlas of the human thymus. RATIONALE To provide a comprehensive atlas of thymic cells across human life, we performed single-cell RNA sequencing (scRNA-seq) using dissociated cells from human thymus during development, childhood, and adult life. We sampled 15 embryonic and fetal thymi spanning thymic developmental stages between 7 and 17 post-conception weeks, as well as nine postnatal thymi from pediatric and adult individuals. Diverse sorting schemes were applied to increase the coverage on underrepresented cell populations. Using the marker genes obtained from single-cell transcriptomes, we spatially localized cell states by single-molecule fluorescence in situ hybridization (smFISH). To provide a systematic comparison between human and mouse, we also generated single-cell data on postnatal mouse thymi and combined this with preexisting mouse datasets. Finally, to investigate the bias in the recombination and selection of human TCR repertoires, we enriched the TCR sequences for single-cell library generation. RESULTS We identified more than 50 different cell states in the human thymus. Human thymus cell states dynamically change in abundance and gene expression profiles across development and during pediatric and adult life. We identified novel subpopulations of human thymic fibroblasts and epithelial cells and located them in situ. We computationally predicted the trajectory of human T cell development from early progenitors in the hematopoietic fetal liver into diverse mature T cell types. Using this trajectory, we constructed a framework of putative transcription factors driving T cell fate determination. Among thymic unconventional T cells, we noted a distinct subset of CD8αα+ T cells, which is marked by GNG4 expression and located in the perimedullary region of the thymus. This subset expressed high levels of XCL1 and colocalized with XCR1+ dendritic cells. Comparison of human and mouse thymic cells revealed divergent gene expression profiles of these unconventional T cell types. Finally, we identified a strong bias in human VDJ usage shaped by recombination and multiple rounds of selection, including a TCRα V-J bias for CD8+ T cells. CONCLUSION Our single-cell transcriptome profile of the thymus across the human lifetime and across species provides a high-resolution census of T cell development within the native tissue microenvironment. Systematic comparison between the human and mouse thymus highlights human-specific cell states and gene expression signatures. Our detailed cellular network of the thymic niche for T cell development will aid the establishment of in vitro organoid culture models that faithfully recapitulate human in vivo thymic tissue. Constructing the human thymus cell atlas. We analyzed human thymic cells across development and postnatal life using scRNA-seq and spatial methods to delineate the diversity of thymic-derived T cells and the localization of cells constituting the thymus microenvironment. With T cell development trajectory reconstituted at single-cell resolution combined with TCR sequence, we investigated the bias in the VDJ recombination and selection of human TCR repertoires. Finally, we provide a systematic comparison between human and mouse thymic cell atlases. The thymus provides a nurturing environment for the differentiation and selection of T cells, a process orchestrated by their interaction with multiple thymic cell types. We used single-cell RNA sequencing to create a cell census of the human thymus across the life span and to reconstruct T cell differentiation trajectories and T cell receptor (TCR) recombination kinetics. Using this approach, we identified and located in situ CD8αα+ T cell populations, thymic fibroblast subtypes, and activated dendritic cell states. In addition, we reveal a bias in TCR recombination and selection, which is attributed to genomic position and the kinetics of lineage commitment. Taken together, our data provide a comprehensive atlas of the human thymus across the life span with new insights into human T cell development.

中文翻译：

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人类胸腺发育的细胞图谱定义了 T 细胞库的形成

胸腺发育，逐个细胞 人类胸腺是负责许多类型 T 细胞成熟的器官，这些 T 细胞是保护我们免受感染的免疫细胞。然而，尚不清楚这些细胞如何发育成完整的免疫补体，其中包含保护我们免受各种病原体侵害的必要变异。Park 等人通过对超过 250,000 个细胞进行单细胞 RNA 测序。检查了人类生命过程中胸腺中发生的变化。他们发现，免疫细胞及其发育微环境以协调的方式发生发育。这些数据允许创建具有不同特定免疫功能的 T 细胞如何在人类中发育的模型。科学，本期第 3 页。eaay3224 随着时间的推移，人类胸腺的单细胞 RNA 谱阐明了人类免疫 T 细胞发育的各个方面。引言 胸腺是 T 细胞发育和 T 细胞受体 (TCR) 库形成的关键器官，它塑造了适应性免疫的格局。胸腺中的 T 细胞发育在空间上是协调的，这个过程是由构成胸腺微环境的不同细胞类型协调的。尽管已经使用多种动物模型对胸腺进行了广泛研究，但如果没有人类胸腺的详细图谱，就无法理解人类免疫。基本原理 为了提供人类生命中胸腺细胞的综合图谱，我们在发育、儿童期和成年期使用人类胸腺的解离细胞进行了单细胞 RNA 测序 (scRNA-seq)。我们从受孕后 7 到 17 周的胸腺发育阶段采集了 15 个胚胎和胎儿胸腺，以及来自儿科和成人个体的 9 个产后胸腺。应用了不同的分类方案来增加对代表性不足的细胞群的覆盖率。使用从单细胞转录组中获得的标记基因，我们通过单分子荧光原位杂交 (smFISH) 对细胞状态进行空间定位。为了提供人类和小鼠之间的系统比较，我们还生成了产后小鼠胸腺的单细胞数据，并将其与预先存在的小鼠数据集相结合。最后，为了研究人类 TCR 库的重组和选择中的偏差，我们丰富了用于单细胞文库生成的 TCR 序列。结果 我们在人类胸腺中发现了 50 多种不同的细胞状态。人类胸腺细胞状态的丰度和基因表达谱在发育过程中以及在儿科和成人生活期间会发生动态变化。我们鉴定了人类胸腺成纤维细胞和上皮细胞的新亚群，并将它们原位定位。我们通过计算预测了人类 T 细胞从造血胎肝中的早期祖细胞发育成多种成熟 T 细胞类型的轨迹。利用这一轨迹，我们构建了一个驱动 T 细胞命运决定的推定转录因子框架。在胸腺非常规 T 细胞中，我们注意到一个独特的 CD8αα+ T 细胞亚群，其以 GNG4 表达为标志，位于胸腺髓周区。该子集表达高水平的 XCL1 并与 XCR1+ 树突状细胞共定位。人类和小鼠胸腺细胞的比较揭示了这些非常规 T 细胞类型的不同基因表达谱。最后，我们确定了由重组和多轮选择形成的人类 VDJ 使用的强烈偏差，包括 CD8+ T 细胞的 TCRα VJ 偏差。结论 我们在整个人类生命周期和跨物种的胸腺单细胞转录组谱提供了天然组织微环境中 T 细胞发育的高分辨率普查。人类和小鼠胸腺之间的系统比较突出了人类特异性细胞状态和基因表达特征。我们用于 T 细胞发育的胸腺生态位的详细细胞网络将有助于建立体外类器官培养模型，该模型忠实地概括了人类体内胸腺组织。构建人类胸腺细胞图谱。我们使用 scRNA-seq 和空间方法分析了人类胸腺细胞在发育和出生后的生活，以描绘胸腺衍生 T 细胞的多样性和构成胸腺微环境的细胞的定位。通过结合 TCR 序列以单细胞分辨率重构 T 细胞发育轨迹，我们研究了 VDJ 重组和人类 TCR 库选择的偏差。最后，我们提供了人类和小鼠胸腺细胞图谱之间的系统比较。胸腺为 T 细胞的分化和选择提供了一个培育环境，这是一个由它们与多种胸腺细胞类型相互作用精心策划的过程。我们使用单细胞 RNA 测序来创建人类胸腺整个生命周期的细胞普查，并重建 T 细胞分化轨迹和 T 细胞受体 (TCR) 重组动力学。使用这种方法，我们鉴定并定位了原位 CD8αα+ T 细胞群、胸腺成纤维细胞亚型和活化的树突状细胞状态。此外，我们揭示了 TCR 重组和选择的偏差，这归因于基因组位置和谱系承诺的动力学。总之，我们的数据提供了一个全面的人类胸腺图谱，涵盖了人类 T 细胞发育的新见解。我们揭示了 TCR 重组和选择的偏差，这归因于基因组位置和谱系承诺的动力学。总之，我们的数据提供了一个全面的人类胸腺图谱，涵盖了人类 T 细胞发育的新见解。我们揭示了 TCR 重组和选择的偏差，这归因于基因组位置和谱系承诺的动力学。总之，我们的数据提供了一个全面的人类胸腺图谱，涵盖了人类 T 细胞发育的新见解。