129 Background: Human T cell antigen receptors play a critical role in protective immune responses but are also implicated in autoimmune disease and immune-mediated adverse events during immunotherapy. The antigen specificity of the T cell receptor is determined in part by the sequence of the CDR and Framework regions encoded by the TCRB variable gene. Previous studies of population sequencing data indicate that current antigen receptor allele databases, such as IMGT, fail to capture a significant portion of human variation. Here we use long-amplicon multiplex sequencing of rearranged TCRB receptors to validate putative novel human variable gene alleles previously recovered from 1000 genomes data. Methods: TCRB rearrangements were amplified from cDNA from 85 Caucasians undergoing treatment for melanoma using AmpliSeq-based multiplex Framework 1 and Constant gene primers to produce ~330bp amplicons. Samples were sequenced using the Ion Torrent S5 530 chip to produce ~1.5M raw reads per sample. Ion Reporter was used for clonotyping and identification of variable gene sequences absent from the IMGT database. Putatively novel sequences were compared to those reported in the Lym1k database of alleles recovered from 1000 genomes data. Results: We identified 15 novel variable gene alleles that are absent from the IMGT database and result in amino acid changes to the CDR or Framework regions of the TCR. Typically, a single individual was found to be heterozygous for a novel variant, though we note two instances where multiple individuals possessed a novel variant. We also identified novel variable gene alleles that are absent from the Lym1k database, potentially due to challenges in inferring receptor alleles from short-read population sequencing studies. Conclusions: We find evidence for significant human diversity in TCRB variable gene alleles beyond what is currently represented in the IMGT database. TCRB sequencing using multiplex Framework 1 and Constant gene targeting primers is ideally suited for studying the role of TCRB polymorphism in autoimmune disease and immune-mediated adverse events during immunotherapy.
Germline DNA Retention in Murine and Human Rearranged T Cell Receptor Gene Coding Joints: Alternative Recombination Signal Sequences and V(D)J Recombinase Errors