Andreatta Massimo, Gueguen Paul, Borcherding Nicholas, Carmona Santiago J
Ludwig Institute for Cancer Research, Lausanne Branch, and Department of Oncology, CHUV and University of Lausanne, Epalinges, Switzerland.
Agora Cancer Research Center, Lausanne, Switzerland.
Bio Protoc. 2023 Aug 20;13(16):e4735. doi: 10.21769/BioProtoc.4735.
T cells are endowed with T-cell antigen receptors (TCR) that give them the capacity to recognize specific antigens and mount antigen-specific adaptive immune responses. Because TCR sequences are distinct in each naïve T cell, they serve as molecular barcodes to track T cells with clonal relatedness and shared antigen specificity through proliferation, differentiation, and migration. Single-cell RNA sequencing provides coupled information of TCR sequence and transcriptional state in individual cells, enabling T-cell clonotype-specific analyses. In this protocol, we outline a computational workflow to perform T-cell states and clonal analysis from scRNA-seq data based on the R packages Seurat, ProjecTILs, and scRepertoire. Given a scRNA-seq T-cell dataset with TCR sequence information, cell states are automatically annotated by reference projection using the ProjecTILs method. TCR information is used to track individual clonotypes, assess their clonal expansion, proliferation rates, bias towards specific differentiation states, and the clonal overlap between T-cell subtypes. We provide fully reproducible R code to conduct these analyses and generate useful visualizations that can be adapted for the needs of the protocol user. Key features Computational analysis of paired scRNA-seq and scTCR-seq data Characterizing T-cell functional state by reference-based analysis using ProjecTILs Exploring T-cell clonal structure using scRepertoire Linking T-cell clonality to transcriptomic state to study relationships between clonal expansion and functional phenotype Graphical overview.
T细胞具有T细胞抗原受体（TCR），使其能够识别特定抗原并引发抗原特异性适应性免疫反应。由于每个初始T细胞中的TCR序列都是独特的，它们可作为分子条形码，通过增殖、分化和迁移来追踪具有克隆相关性和共享抗原特异性的T细胞。单细胞RNA测序提供了单个细胞中TCR序列和转录状态的耦合信息，从而能够进行T细胞克隆型特异性分析。在本方案中，我们概述了一种基于R包Seurat、ProjecTILs和scRepertoire从scRNA-seq数据进行T细胞状态和克隆分析的计算工作流程。给定一个带有TCR序列信息的scRNA-seq T细胞数据集，使用ProjecTILs方法通过参考投影自动注释细胞状态。TCR信息用于追踪单个克隆型，评估其克隆扩增、增殖率、对特定分化状态的偏向性以及T细胞亚型之间的克隆重叠。我们提供了完全可重复的R代码来进行这些分析并生成有用的可视化结果，这些结果可根据方案使用者的需求进行调整。关键特性：对配对的scRNA-seq和scTCR-seq数据进行计算分析；使用ProjecTILs通过基于参考的分析来表征T细胞功能状态；使用scRepertoire探索T细胞克隆结构；将T细胞克隆性与转录组状态联系起来，以研究克隆扩增与功能表型之间的关系；图形概述。
