A High-Throughput Sequencing Strategy for Clinical Repertoire Profiling of T Cell Receptor Beta Chain: Development and Reference Values Across Healthy Adults, Paediatrics, and Cord Blood Units

TL;DR

This paper introduces a reliable DNA-based sequencing method for analyzing T cell receptor beta chains in healthy individuals and patients with immune deficiencies.

Contribution

A novel, high-throughput DNA-based TCRβ sequencing strategy with reference values for healthy and diseased populations is developed.

Findings

The sequencing strategy showed high specificity, reproducibility, and sensitivity with minimal PCR bias.

CBUs and paediatrics had greater TCRβ convergence and diversity compared to adults and CMV-positive donors.

RAG-SCID/CID patients exhibited significantly shorter CDR3 length and lower repertoire diversity than healthy paediatrics.

Abstract

T cell receptor (TCR) profiling using next-generation sequencing (NGS) enables high-throughput, in-depth analysis of repertoire diversity, offering numerous clinical applications. We developed a DNA-based strategy to analyse the TCRβ-chain using NGS and established reference values for T cell repertoire characteristics in 74 healthy donors, including 44 adults, 20 paediatrics, and 10 cord blood units (CBUs). Additionally, four paediatric patients with combined immunodeficiency (CID) or severe CID (SCID) due to deleterious mutations in recombination activating genes (RAG) were analysed. The developed strategy demonstrated high specificity, reproducibility, and sensitivity, and all functional variable and joining genes were detected with minimal PCR bias. All donors had a Gaussian-like distribution of complementary-determining region 3 length, with lower presence of non-templated nucleotides and higher proportion of non-functional clonotypes in CBUs. Both CBUs and paediatrics showed greater convergence and TCRβ diversity was significantly lower in adults and donors with cytomegalovirus-positive serostatus. Finally, an analysis of paediatric patients with RAG-SCID/CID showed significantly shorter CDR3 region length and lower repertoire diversity compared to healthy paediatrics. In summary, we developed a reliable and feasible TCRβ sequencing strategy for application in the clinical setting, and established reference values that could assist in the diagnosis and monitoring of pathological conditions affecting the T cell repertoire.

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