Comprehensive and semi-quantitative TCR repertoire analysis with a novel multiplex PCR method and 454 sequencing (85.3)

Abstract CDR3 sequences, composed by V(D)J combination, form the center of the antigen binding site where they often play a critical role in defining the affinity and specificity of the receptor for individual peptide-MHC complexes of both the TCRα and TCRβ chains. The goal of our study was to produce comprehensive, unrestricted profiles of TCR diversity among key developmental and effector subsets of T cells isolated from the blood of a single, healthy donor at sequence-level resolution using a novel multiplex PCR method combined with Roche 454 Life Sciences high-throughput sequencing technology. From the sequence reads, about 1.48 million CDR3 intervals were identified, totaling 169,977 and 113,290 unique CDR3 intervals for TCRα and for TCRβ, respecitively. Our data also show numerous examples of identical CDR3 sequences shared by different T subsets. Using the compound Poisson process model, we estimated that the diversity of the TCRα and β chains expressed by our donor is around 0.47 x 106 and 0.35 x 106 unique CDR3 nucleotide sequences, respectively. Our comprehensive data demonstrates that our new method has overcome past challenges in studying the T cell immune repertoire and is highly sensitive, repeatable, and semi-quantitative. This approach provides a useful tool for assessing immune competence, tracking T cell expansion kinetics, assessing vaccine efficiency, and detecting antigen-specific T cell clones in patients with infection or cancer.