TCR sequencing in cancer immunology and immunotherapy: what, when, where, why, and how

T-cell receptors (TCRs) are generated through somatic recombination of variable (V), diversity (D), and joining (J) gene segments, resulting in an extraordinarily diverse receptor repertoire that is essential for immune surveillance and host defense. TCR sequencing (TCR-seq) has emerged as a powerful tool for comprehensive characterization of the adaptive immune repertoire, offering deep insights into T-cell diversity, antigen specificity, and clonal dynamics. TCR-seq enables the tracking of T-cell clones across both temporal and spatial dimensions. From a longitudinal perspective, it allows for the monitoring of clonal dynamics before and after therapeutic interventions or over the course of disease progression. Temporal shifts in clonal composition can reveal the persistence, contraction, or expansion of specific T-cell populations, thereby providing valuable information on the durability of immune responses and the efficacy of treatments. From a spatial standpoint, TCR-seq facilitates comparative analyses of repertoires across distinct anatomical compartments, including tumors, blood, and lymph nodes. Such analyses yield insights into tissue-specific immune responses, T-cell trafficking, and infiltration patterns. Moreover, the ability to track antigen-specific T-cell clones enables the visualization and quantification of tumor-specific immune responses. Advances in spatial TCR-seq now integrate spatial context with clonal identity and repertoire diversity, further illuminating complex immune architecture within tissue microenvironments. Nonetheless, despite the development of various approaches for antigen specificity prediction, further advances are needed to improve their accuracy and generalizability. A wide range of TCR-seq platforms are currently available, including DNA-based and RNA-based protocols, short-read and long-read sequencing technologies, and bulk and single-cell approaches. Each method presents unique advantages in terms of resolution, throughput, cost, and biological relevance. For instance, DNA-based TCR-seq is well suited for longitudinal tracking of clonal populations, whereas RNA-based approaches are advantageous for detecting actively transcribed, antigen-responsive clones. Short-read sequencing offers high-throughput capabilities, while long-read and paired-chain sequencing provide comprehensive structural and functional information on TCRs. Additionally, computational methods, including machine learning algorithms and motif-based clustering, are increasingly employed to infer antigen specificity directly from TCR-seq data. In this review, we examine the current landscape of TCR-seq through the lenses of what, when, where, why, and how, highlighting recent technological developments and emerging applications that are shaping the field of immune repertoire analysis.