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 Blood, 5 November 2009, Vol. 114, No. 19, pp. 4099-4107.
Prepublished online as a Blood First Edition Paper on August 25, 2009; DOI 10.1182/blood-2009-04-217604.

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IMMUNOBIOLOGY

Comprehensive assessment of T-cell receptor β-chain diversity in {alpha}β T cells

Harlan S. Robins1,*, Paulo V. Campregher1,*, Santosh K. Srivastava1,*, Abigail Wacher1, Cameron J. Turtle1,2, Orsalem Kahsai1, Stanley R. Riddell1,2, Edus H. Warren1,2,{dagger}, and Christopher S. Carlson1,{dagger}

1 Fred Hutchinson Cancer Research Center, Seattle, WA; and 2 Department of Medicine, University of Washington, Seattle

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}β 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 β chains. Although it has been estimated that the adaptive immune system can generate up to 1016 distinct {alpha}β 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 TCRβ genes from T cells of 2 adults. We find that total TCRβ receptor diversity is at least 4-fold higher than previous estimates, and the diversity in the subset of CD45RO+ antigen-experienced {alpha}β T cells is at least 10-fold higher than previous estimates. These methods should prove valuable for assessment of {alpha}β T-cell repertoire diversity after hematopoietic cell transplantation, in states of congenital or acquired immunodeficiency, and during normal aging.


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