title: Comprehensive assessment of T-cell receptor beta-chain diversity in alphabeta T cells.
abstract: 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 alphabeta 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 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 measure 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 2 adults. We find that total TCRbeta receptor diversity is at least 4-fold higher than previous estimates, and the diversity in the subset of CD45RO(+) antigen-experienced alphabeta T cells is at least 10-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.
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA. hrobins@fhcrc.org
authors.forename: Harlan S
authors.initials: HS
authors.lastname: Robins
authors.forename: Paulo V
authors.initials: PV
authors.lastname: Campregher
authors.forename: Santosh K
authors.initials: SK
authors.lastname: Srivastava
authors.forename: Abigail
authors.initials: A
authors.lastname: Wacher
authors.forename: Cameron J
authors.initials: CJ
authors.lastname: Turtle
authors.forename: Orsalem
authors.initials: O
authors.lastname: Kahsai
authors.forename: Stanley R
authors.initials: SR
authors.lastname: Riddell
authors.forename: Edus H
authors.initials: EH
authors.lastname: Warren
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Carlson
mesh_terms: D000328:Adult; D001483:Base Sequence; D022801:Complementarity Determining Regions; D004247:DNA / Q000235:genetics; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; D019674:Genes, T-Cell Receptor beta; D014644:Genetic Variation; D006801:Humans; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D016176:T-Lymphocyte Subsets / Q000276:immunology*
chemical_list: D022801:Complementarity Determining Regions; D016693:Receptors, Antigen, T-Cell, alpha-beta; D004247:DNA; D017493:Leukocyte Common Antigens
references.citation: Nature. 1988 Aug 4;334(6181):395-402
references.pmid: 3043226
references.citation: Nucleic Acids Res. 2006 Jan 1;34(Database issue):D781-4
references.pmid: 16381979
references.citation: Res Immunol. 1995 Feb;146(2):65-80
references.pmid: 7481075
references.citation: Nat Biotechnol. 2008 Oct;26(10):1135-45
references.pmid: 18846087
references.citation: Blood. 2000 Jun 15;95(12):3990-5
references.pmid: 10845939
references.citation: J Immunol. 1993 Nov 15;151(10):5762-9
references.pmid: 8228260
references.citation: Scand J Immunol. 1997 Nov;46(5):520-6
references.pmid: 9393636
references.citation: Immunol Today. 1995 Apr;16(4):176-81
references.pmid: 7734044
references.citation: Exp Gerontol. 2007 May;42(5):400-6
references.pmid: 17218073
references.citation: J Immunol. 1998 Mar 15;160(6):2842-52
references.pmid: 9510187
references.citation: Proc Natl Acad Sci U S A. 2009 Mar 31;106(13):5008-13
references.pmid: 19276111
references.citation: Annu Rev Immunol. 2006;24:419-66
references.pmid: 16551255
references.citation: Hum Immunol. 1996 Jun-Jul;48(1-2):125-34
references.pmid: 8824581
references.citation: J Exp Med. 2008 Jul 7;205(7):1701-14
references.pmid: 18573906
references.citation: Tissue Antigens. 1999 Feb;53(2):122-34
references.pmid: 10090612
references.citation: Proc Natl Acad Sci U S A. 1982 Jul;79(13):4118-22
references.pmid: 6287467
references.citation: Nat Rev Immunol. 2008 Mar;8(3):231-8
references.pmid: 18301425
references.citation: J Immunol. 2005 Jun 1;174(11):7446-52
references.pmid: 15905594
references.citation: Blood. 1998 Jan 1;91(1):340-6
references.pmid: 9414303
references.citation: Science. 1999 Oct 29;286(5441):958-61
references.pmid: 10542151
references.citation: J Exp Med. 2001 Nov 5;194(9):1385-90
references.pmid: 11696602
references.citation: Proc Natl Acad Sci U S A. 1993 May 1;90(9):4319-23
references.pmid: 8483950
references.citation: Hum Immunol. 1999 Nov;60(11):1090-100
references.pmid: 10600007
references.citation: Bioinformatics. 2004 Aug 4;20 Suppl 1:i379-85
references.pmid: 15262823
references.citation: Bone Marrow Transplant. 2003 Nov;32(10):1001-14
references.pmid: 14595388
references.citation: J Immunol. 1994 May 15;152(10):5109-19
references.pmid: 8176227
references.citation: Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):274-8
references.pmid: 10618408
references.citation: J Immunol. 2008 Dec 1;181(11):7853-62
references.pmid: 19017975