HTGTS‐TCR‐Seq for Profiling of Mouse and Human T‐Cell Receptor α and β Gene Rearrangements and Diversity

Abstract Developing αβ T lymphocytes generate T‐cell receptor (TCR) diversity through V(D)J recombination, which assembles Tcra and Tcrb genes from germline variable (V), diversity (D), and joining (J) segments. Approaches to characterize TCR rearrangements and diversity are critical for studying T‐cell development and immune function. Several existing methods, such as multiplex PCR and 5′RACE, have advanced the field; however, each carries inherent technical limitations. Here, high‐throughput Genome‐wide translocation sequencing‐based TCR sequencing (HTGTS‐TCR‐seq), a complementary and cost‐effective strategy for quantitative profiling of Tcra and Tcrb gene rearrangements, is presented. HTGTS‐TCR‐seq employs a limited set of 3–5 J or V region primers to enrich for V(D)J recombination products, allowing detection of both productive and nonproductive rearrangements. Application to wild‐type murine thymocytes at defined developmental stages, as well as young and aged T cells, reveals stage‐specific V and J usage and age‐associated repertoire alterations. Analysis of Wapl ‐knockout preselection double‐positive thymocytes uncovers a cell division‐independent role for the cohesin‐unloading factor WAPL in Tcra rearrangement. Moreover, analysis of human peripheral T cells demonstrates conserved complementarity‐determining region 3 (CDR3) features and subset‐specific Vβ usage across species. Collectively, HTGTS‐TCR‐seq provides an efficient and accessible approach for quantifying TCR rearrangements and diversity across development, aging, and immune‐related conditions.