The adaptive immune system employs several strategies to generate a repertoire of T- and B-cell antigen receptors with sufficient diversity to recognize the universe of potential pathogens. In alphabeta T cells, which primarily recognize peptide antigens presented by MHC 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 alphabeta 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 measurement of TCR CDR3 diversity based on single-molecule DNA sequencing, and used this approach to determine the CDR3 sequence in millions of rearranged TCRbeta genes from T cells of two adults. We find that total TCRbeta receptor diversity is at least four-fold higher than previous estimates, and the diversity in the subset of CD45RO(+) antigen-experienced alphabeta T cells is at least ten-fold higher than previous estimates. These methods should prove valuable for assessment of alphabeta T cell repertoire diversity after hematopoietic cell transplantation, in states of congenital or acquired immunodeficiency, and during normal aging.
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