Previous studies have analyzed TCR repertoires throughout the lifespan in a cross-sectional manner ( 6, 7), but none have longitudinally assessed the temporal changes in the repertoire beginning early in life and throughout childhood. Advances in high-throughput sequencing technologies now enable deep sequencing of TCR-β chains from genomic DNA ( 3– 5). Rearrangement of TCR α and β genes results in a vast and diverse repertoire of T cells within a given individual, allowing for recognition of millions of unique antigens. The T cell receptor (TCR) on the cell surface of T lymphocytes plays a central role in immune responses by the recognition of peptides in the context of major histocompatibility complexes ( 1, 2). These results demonstrate an enrichment of preproinsulin-reactive TCR sequences during the progression to T1D, highlighting the importance of using disease-relevant TCR sequences as powerful biomarkers in autoimmune disorders. As an independent validation, we sequenced and analyzed TCR-β repertoires from a cohort of new-onset T1D patients ( n = 143), identifying the same preproinsulin-reactive TCRs. Insulin-reactive TCR-β sequences were more common and frequent in number as the disease progressed in those who developed T1D compared with genetically at risk nondiabetic children, and this was not the case for influenza-reactive sequences. We demonstrate the ability to identify specific TCR sequences, including those known to recognize influenza A and, separately, those specific for insulin and its precursor, preproinsulin. From 53 million TCR-β sequences, we show that the repertoire is extraordinarily diverse early in life and narrows with age independently of disease. Here, we deep sequenced the TCR-β chain repertoires from longitudinal peripheral blood DNA samples at 4 time points beginning early in life (median age of 1.4 years) from children who progressed to T1D ( n = 29) and age/sex-matched islet autoantibody-negative controls ( n = 25). However, a thorough investigation of longitudinal TCR repertoires throughout childhood in health and during development of a common childhood disease, type 1 diabetes (T1D), has not been undertaken. KGaA, Weinheim.T cell receptor (TCR) sequences are exceptionally diverse and can now be comprehensively measured with next-generation sequencing technologies.

T-cell receptor V gene usage phylogenetic tree polymerase chain reaction (PCR) semi-qualitative analysis.

Taken together, the new primers provide a more comprehensive coverage of the TCR gene diversity for improved TCR library generation and TCR V gene analysis studies. A semi-qualitative approach using the designed primer set was able to provide a relative view of the TCR V gene diversity in different populations. The new primer set was able to provide 93.75% and 95.63% coverage for the alpha and beta family, respectively. A total of 112 alpha and 160 beta sequences were aligned and clustered using a phylogram yielding 32 and 27 V gene primers for the alpha and beta family. The primers designed were analyzed and the PCR efficiency of each primer set was optimized. Here, we present a refined primer design strategy of the human TCR V gene by clustering V gene sequence homolog for degenerate primer design based on the data from IMGT. The successful coverage of a diverse TCR repertoire is mainly attributed to the primer design of the human TCR V genes.

The application of human TCR in cancer immunotherapy has gained momentum with developments in tumor killing strategies using endogenous adaptive immune responses.

Tcr v beta repertoire