![]() Genomic (g) DNA-based methods use a primer set designed to cover all V or J regions. (A) Exemplary workflow of TCR library preparation methods. In addition, the impact of each TCRseq method on the repertoire data should be considered when comparing TCR repertoire data obtained by different methods.įigure 1 Summary of TCRseq and TCR repertoire analysis. Because each method has its advantages and disadvantages ( Table 1) ( 11, 12), it is preferable to adopt the most appropriate TCRseq method for a given research purpose based on these characteristics. There are several TCRseq methods, which can be classified based on (i) whether genomic DNA (gDNA) or messenger RNA (mRNA) is used as a template, and (ii) whether multiple V region-specific primers are used to amplify the TCR sequence (multiplex PCR) or universal primers for the adapter sequence are used (5′ rapid amplification of cDNA ends ) ( 10) ( Figure 1A). In TCRseq, the TCR gene, including complementary determining region 3 (CDR3), which is the most variable region in the TCR and a significant contributor to antigen specificity, is amplified and then sequenced by NGS. TCRs are produced through rearrangement of the variable (V), diversity (D), joining (J), and constant (C) gene segments as well as insertions and deletions, resulting in a vast diversity ( 9). Additionally, we introduce two novel approaches in TCR repertoire analysis that facilitate the immunological interpretation of TCR repertoire data: inter-organ clone tracking analysis, which identifies and analyzes T-cell clones present in the tumor and other tissues, and single-cell (sc) TCRseq, which identifies TCR sequences and their gene expression profiles at a single cell resolution. In this review, we provide an overview of the TCRseq methods, and summarize the findings and limitations of current TCR repertoire analysis in the field of cancer immunotherapy. TCR repertoire analysis is currently being used for studies in various medical fields, including infectious diseases ( 6), transplantation immunity ( 7), and tumor immunity ( 8). Recent advances in next-generation sequencing (NGS) technology have enabled TCR sequencing (TCRseq), which allows a comprehensive determination of the TCR sequences in individuals ( 5). The collection of TCRs in vivo, termed as the TCR repertoire, is considered to be a new indicator to evaluate T-cell responses based on antigen specificity. However, it is becoming increasingly clear that antitumor T-cell responses are driven by a wide variety of T-cell clones ( 3, 4). Conventional preclinical and clinical studies on antitumor T-cell responses have analyzed a limited number of tumor-specific T-cell clones using TCR transgenic mice ( 1) or peptide-major histocompatibility complex multimer technology ( 2). These activated T cells proliferate in the periphery, eliciting responses against the corresponding antigen, including cancer antigens. T cells specifically recognize their cognate antigens via the TCRs and are activated. TCR is composed of alpha and beta chains, whose specificity is determined by the gene rearrangement that occurs in the thymus. These approaches for TCR repertoire analysis will provide a more accurate understanding of the response of tumor-specific T cells in the tumor microenvironment.Įach T cell has a unique antigen receptor (T-cell receptor TCR). In this review, we introduce two approaches that facilitate an immunological interpretation of the TCR repertoire data: inter-organ clone tracking analysis and single-cell TCR sequencing. However, the immunopathological significance of TCR repertoire indices is still undefined. With the recent advancement in next-generation DNA sequencers, comprehensive analysis of the collection of TCR genes (TCR repertoire analysis) is feasible and has been used to investigate the clonal responses of antitumor T cells. Thus, elucidating the overall antitumor responses of diverse T-cell clones is an emerging challenge in tumor immunology. They comprise various T-cell clones with diverse antigen-specific T-cell receptors (TCRs). 2Department of Hygiene, Graduate School of Medicine, The University of Tokyo, Tokyo, JapanĬD8 + T cells are the key effector cells that contribute to the antitumor immune response.1Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba, Japan.Hiroyasu Aoki 1,2 Shigeyuki Shichino 1 Kouji Matsushima 1 Satoshi Ueha 1*
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