A novel method for analysis of human T cell repertoires by real-time PCR

T lymphocyte responses to challenges with multiple pathogens depend on the diversity of their T cell receptors (TcRs) that are heteroduplexes of alpha and beta chains. The regions of alpha and beta chains that define TcR specificity are encoded by rearranged variable (V) and joining (J) genes that are separated by variable numbers of nucleotides that encode the complementarity determining region 3 (CDR3). The assumption that a "healthy" T cell compartment exhibits broad TcR and CDR3 diversity has driven development of methods to evaluate diversity of TcR beta transcripts expressed by T lymphocyte populations and subpopulations in inflammatory sites and human malignancies. To that end, we have developed the BV:BJ matrix assay that uniquely generates a single statistic that describes TcR repertoire diversity and improves identification of beta transcripts expressed by expanded T cell clonotypes. The BV:BJ matrix uses rigorously selected primers specific for individual V and J genes to amplify beta transcripts in real-time PCRs driven by 533 BV:BJ primer pairs. The quantitative control of real-time PCRs produces Shannon entropy estimates of diversity that are reproducible over a range of template amounts and amenable to statistical analyses that have been difficult to perform with existing methods of repertoire analysis.