Single-cell RNA sequencing (scRNA-seq) has enabled high-resolution characterization of molecular signatures of tumor-infiltrating lymphocytes. However, analyses at the transcript isoform level are rarely reported. As alternative splicing is critical to T-cell differentiation and activation, here, we proposed a computational method named IDEA (Isoform Detection, Enrichment, and functional Annotation) to comprehensively detect and annotate differentially used isoforms across cell subtypes. We applied IDEA on a scRNA-seq data set of 12,346 T cells from non-small-cell lung cancer (NSCLC). We found that most genes tend to dominantly express one isoform in single T cells, enabling typing T cells based on the isotypes, given a gene. Isotype analysis suggested that tumor-infiltrating T cells significantly preferred specific isotypes for 245 genes in CD8⁺ T cells and 456 genes in CD4⁺ T cells. Functional annotation suggests that the preferred isoforms involved in coding/noncoding switches, transcription start site changes, gains/losses of domains, and subcellular translocation. Clonal analysis revealed that isoform switching occurred during T-cell activation/differentiation. Our analysis provides precise characterization of the molecular events in tumor-infiltrating T cells and sheds new light on the regulatory mechanisms of tumor-infiltrating T cells.

单细胞RNA测序（scRNA-seq）已实现了肿瘤浸润淋巴细胞分子标记的高分辨率表征。但是，很少有关于转录亚型水平的分析的报道。由于替代剪接对于T细胞的分化和激活至关重要，因此，我们提出了一种称为IDEA（异构体检测，富集和功能注释）的计算方法，以全面检测和注释跨细胞亚型的差异使用的异构体。我们将IDEA应用于来自非小细胞肺癌（NSCLC）的12,346个T细胞的scRNA-seq数据集。我们发现，大多数基因倾向于在单个T细胞中主要表达一种同种型，从而在给定基因的情况下，根据同种型分型T细胞。同种型分析表明，肿瘤浸润性T细胞明显偏爱CD8中245个基因的特定同种型⁺ T细胞和CD4 ⁺ T细胞中的456个基因。功能注释表明，参与编码/非编码开关，转录起始位点变化，结构域的获得/缺失和亚细胞易位的优选同工型。克隆分析显示，同种型转换发生在T细胞活化/分化过程中。我们的分析提供了肿瘤浸润T细胞中分子事件的精确表征，为肿瘤浸润T细胞的调控机制提供了新的思路。