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.

Purpose The increasing application of virus-specific T cell therapy for treating BK virus infections in immunocompromised patients highlights the necessity for rapid identification of compatible cell donors with optimal BK-specific T cell response. This study aims to characterize the BK virus-specific T cell response in relation to demographic factors, blood group, serological status, and HLA genotypes using samples from a cell donor registry. Methods Peripheral blood mononuclear cells from cell donors were stimulated with peptide pools derived from VP1 and LTA proteins, and the IFN-γ production was analyzed using ELISpot and validated by flow cytometry. Results Our findings provide an overview of the T cell response to BK virus proteins in healthy donors, revealing associations with demographic characteristics, RhD status, CMV or EBV serological status, and HLA alleles. Remarkably, RhD-negative, CMV-seronegative, and EBV-seronegative donors showed a major T cell response against BK virus proteins. Notably, certain HLA alleles were associated with either enhanced or diminished T cell response. Furthermore, our results suggest that HLA-B leader dimorphism, specifically the presence of threonine at position 2, influences the VP1-specific immune response, resulting in enhanced T cell activation. Conclusion This study, beyond advancing our understanding of the relationship between donor characteristics and BK virus-specific T cell response, has significant implications for improving the selection of optimal cell donors for patient-specific adoptive therapy.