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

## TL;DR

This paper introduces HTGTS-TCR-seq, a new method for efficiently profiling T-cell receptor diversity in mice and humans, revealing insights into development, aging, and immune function.

## Contribution

HTGTS-TCR-seq is a novel, cost-effective method for profiling TCR rearrangements with minimal primer bias.

## Key findings

- HTGTS-TCR-seq captures both productive and nonproductive TCR rearrangements in mice and humans.
- The method reveals stage-specific V and J usage in murine thymocytes and age-related repertoire changes.
- Analysis shows conserved CDR3 features and subset-specific Vβ usage in human T cells.

## 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.

HTGTS‐TCR‐seq is an efficient and cost‐effective method for profiling TCRα and TCRβ rearrangements using only a few primers. It captures both productive and nonproductive recombination events, minimizes primer bias, and reveals stage‐specific and age‐related repertoire changes in mice, as well as conserved features in human peripheral T cells.