Comprehensive assessment of T-cell receptor β-chain diversity in αβ T cells

The adaptive immune system uses several strategies to generate a repertoire of T- and B-cell antigen receptors with sufficient diversity to recognize the universe of potential pathogens. In alpha beta T cells, which primarily recognize peptide antigens presented by major histocompatibility complex molecules, most of this receptor diversity is contained within the third complementarity-determining region (CDR3) of the T-cell receptor (TCR) alpha and beta chains. Although it has been estimated that the adaptive immune system can generate up to 10(16) distinct alpha beta pairs, direct assessment of TCR CDR3 diversity has not proved amenable to standard capillary electrophoresis-based DNA sequencing. We developed a novel experimental and computational approach to measure TCR CDR3 diversity based on single-molecule DNA sequencing, and used this approach to determine the CDR3 sequence in millions of rearranged TCR beta genes from T cells of 2 adults. We find that total TCR beta receptor diversity is at least 4-fold higher than previous estimates, and the diversity in the subset of CD45RO(+) antigen-experienced alpha beta T cells is at least 10-fold higher than previous estimates. These methods should prove valuable for assessment of alpha beta T-cell repertoire diversity after hematopoietic cell transplantation, in states of congenital or acquired immunodeficiency, and during normal aging. (Blood. 2009; 114: 4099-4107)