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Immune Homeostasis pp 147–159Cite as

Molecular Measurement of T Cell Receptor Excision Circles

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 979))

Abstract

This chapter provides protocols necessary for quantifying human, mouse, and nonhuman primate signal joint T cell receptor excision circles (sjTRECs) produced during T cell receptor alpha (TCRA) gene rearrangement. These non-replicated episomal circles of DNA are generated by the recombination process used to produce antigen-specific T cell receptors. The number of sjTRECs per mg of thymus tissue or per 100,000 lysed cells has been shown to be a molecular marker of thymopoiesis and naïve T cells. This technology is beneficial to investigators interested in quantitating the level of naïve T cell production occurring under various circumstances in a variety of systems, and complements traditional phenotypic analyses of thymopoiesis. This chapter specifically describes procedures required for rapid detection and quantitation of sjTRECs in thymus tissue or isolated cells using real-time quantitative polymerase chain reaction (PCR). The sjTREC assay system comprises species-specific forward and reverse primers for amplification of a unique site on the T cell receptor δ (TCRD) sjTREC, a fluorescently labeled (FAM/ZEN/IABkFQ) species-specific real-time probe, and a species-specific sjTREC DNA plasmid standard for quantitation.

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References

  1. Haynes BF, Denning SM, Le PT, Singer KH (1990) Human intrathymic T cell differentiation. Semin Immunol 2:67–77

    PubMed  CAS  Google Scholar 

  2. Flores KG, Li J, Sempowski GD, Haynes BF, Hale LP (1999) Analysis of the human thymic perivascular space during aging. J Clin Invest 104:1031–1039

    Article  PubMed  CAS  Google Scholar 

  3. Jamieson BD, Douek DC, Killian S, Hultin LE, Scripture-Adams DD, Giorgi JV, Marelli D, Koup RA, Zack JA (1999) Generation of functional thymocytes in the human adult. Immunity 10:569–575

    Article  PubMed  CAS  Google Scholar 

  4. Sempowski GD, Hale LP, Sundy JS, Massey JM, Koup RA, Douek DC, Patel DD, Haynes BF (2000) Leukemia inhibitory factor, oncostatin M, IL-6, and stem cell factor mRNA expression in human thymus increases with age and is associated with thymic atrophy. J Immunol 164:2180–2187

    PubMed  CAS  Google Scholar 

  5. Lynch HE, Goldberg GL, Chidgey A, Van den Brink MR, Boyd R, Sempowski GD (2009) Thymic involution and immune reconstitution. Trends Immunol 30:366–373

    Article  PubMed  CAS  Google Scholar 

  6. Douek DC, McFarland RD, Keiser PH, Gage EA, Massey JM, Haynes BF, Polis MA, Haase AT, Feinberg MB, Sullivan JL, Jamieson BD, Zack JA, Picker LJ, Koup RA (1998) Changes in thymic function with age and during the treatment of HIV infection. Nature 396:690–695

    Article  PubMed  CAS  Google Scholar 

  7. Kong F, Chen CH, Cooper MD (1998) Thymic function can be accurately monitored by the level of recent T cell emigrants in the circulation. Immunity 8:97–104

    Article  PubMed  CAS  Google Scholar 

  8. Douek DC, Vescio RA, Betts MR, Brenchley JM, Hill BJ, Zhang L, Berenson JR, Collins RH, Koup RA (2000) Assessment of thymic output in adults after haematopoietic stem-cell transplantation and prediction of T-cell reconstitution. Lancet 355:1875–1881

    Article  PubMed  CAS  Google Scholar 

  9. Davis CC, Marti LC, Sempowski GD, Jeyaraj DA, Szabolcs P (2010) Interleukin-7 permits Th1/Tc1 maturation and promotes ex vivo expansion of cord blood T cells: a critical step toward adoptive immunotherapy after cord blood transplantation. Cancer Res 70:5249–5258

    Article  PubMed  CAS  Google Scholar 

  10. Page KM, Mendizabal AM, Prasad VK, Martin PL, Parikh S, Wood S, Sempowski GD, Szabolcs P, Kurtzberg J (2008) Posttransplant autoimmune hemolytic anemia and other autoimmune cytopenias are increased in very young infants undergoing unrelated donor umbilical cord blood transplantation. Biol Blood Marrow Transplant 14:1108–1117

    Article  PubMed  Google Scholar 

  11. Liu C, Chen BJ, Deoliveira D, Sempowski GD, Chao NJ, Storms RW (2010) Progenitor cell dose determines the pace and completeness of engraftment in a xenograft model for cord blood transplantation. Blood 116:5518–5527

    Article  PubMed  CAS  Google Scholar 

  12. Sarzotti-Kelsoe M, Win CM, Parrott RE, Cooney M, Moser BK, Roberts JL, Sempowski GD, Buckley RH (2009) Thymic output, T-cell diversity, and T-cell function in long-term human SCID chimeras. Blood 114:1445–1453

    Article  PubMed  CAS  Google Scholar 

  13. Sempowski GD, Gooding ME, Liao HX, Le PT, Haynes BF (2002) T cell receptor excision circle assessment of thymopoiesis in aging mice. Mol Immunol 38:841–848

    Article  PubMed  CAS  Google Scholar 

  14. Hockett RD Jr, Nunez G, Korsmeyer SJ (1989) Evolutionary comparison of murine and human delta T-cell receptor deleting elements. The New Biologist 1:266–274

    PubMed  CAS  Google Scholar 

  15. de Villartay JP, Hockett RD, Coran D, Korsmeyer SJ, Cohen DI (1988) Deletion of the human T-cell receptor delta-gene by a site-specific recombination. Nature 335:170–174

    Article  PubMed  Google Scholar 

  16. Toda M, Fujimoto S, Iwasato T, Takeshita S, Tezuka K, Ohbayashi T, Yamagishi H (1988) Structure of extrachromosomal circular DNAs excised from T-cell antigen receptor alpha and delta-chain loci. J Mol Biol 202:219–231

    Article  PubMed  CAS  Google Scholar 

  17. Takeshita S, Toda M, Yamagishi H (1989) Excision products of the T cell receptor gene support a progressive rearrangement model of the alpha/delta locus. EMBO J 8:3261–3270

    PubMed  CAS  Google Scholar 

  18. Elliott JF, Rock EP, Patten PA, Davis MM, Chien YH (1988) The adult T-cell receptor delta-chain is diverse and distinct from that of fetal thymocytes. Nature 331:627–631

    Article  PubMed  CAS  Google Scholar 

  19. Min B, McHugh R, Sempowski GD, Mackall C, Foucras G, Paul WE (2003) Neonates support lymphopenia-induced proliferation. Immunity 18:131–140

    Article  PubMed  CAS  Google Scholar 

  20. Chen BJ, Cui X, Sempowski GD, Gooding ME, Liu C, Haynes BF, Chao NJ (2002) A comparison of murine T-cell-depleted adult bone marrow and full-term fetal blood cells in hematopoietic engraftment and immune reconstitution. Blood 99:364–371

    Article  PubMed  CAS  Google Scholar 

  21. Billard MJ, Gruver AL, Sempowski GD (2011) Acute endotoxin-induced thymic atrophy is characterized by intrathymic inflammatory and wound healing responses. PloS One 6:e17940

    Article  PubMed  CAS  Google Scholar 

  22. Shi X, Zhang P, Sempowski GD, Shellito JE (2011) Thymopoietic and bone marrow response to murine Pneumocystis pneumonia. Infect Immun 79:2031–2042

    Article  PubMed  CAS  Google Scholar 

  23. Chen BJ, Deoliveira D, Spasojevic I, Sempowski GD, Jiang C, Owzar K, Wang X, Gesty-Palmer D, Cline JM, Bourland JD, Dugan G, Meadows SK, Daher P, Muramoto G, Chute JP, Chao NJ (2010) Growth hormone mitigates against lethal irradiation and enhances hematologic and immune recovery in mice and nonhuman primates. PloS One 5:e11056

    Article  PubMed  Google Scholar 

  24. Gruver AL, Ventevogel MS, Sempowski GD (2009) Leptin receptor is expressed in thymus medulla and leptin protects against thymic remodeling during endotoxemia-induced thymus involution. J Endocrinol 203:75–85

    Article  PubMed  CAS  Google Scholar 

  25. Sodora DL, Douek DC, Silvestri G, Montgomery L, Rosenzweig M, Igarashi T, Bernacky B, Johnson RP, Feinberg MB, Martin MA, Koup RA (2000) Quantification of thymic function by measuring T cell receptor excision circles within peripheral blood and lymphoid tissues in monkeys. Eur J Immunol 30:1145–1153

    Article  PubMed  CAS  Google Scholar 

  26. Bona R, Macchia I, Baroncelli S, Negri DR, Leone P, Pavone-Cossut MR, Catone S, Buffa V, Ciccozzi M, Heeney J, Fagrouch Z, Titti F, Cara A (2007) T cell receptor excision circles (TRECs) analysis during acute intrarectal infection of cynomolgus monkeys with pathogenic chimeric simian human immunodeficiency virus. Virus Res 126:86–95

    Article  PubMed  CAS  Google Scholar 

  27. Aspinall R, Pido-Lopez J, Imami N, Henson SM, Ngom PT, Morre M, Niphuis H, Remarque E, Rosenwirth B, Heeney JL (2007) Old rhesus macaques treated with interleukin-7 show increased TREC levels and respond well to influenza vaccination. Rejuvenation Res 10:5–17

    Article  PubMed  CAS  Google Scholar 

  28. Muthukumar A, Zhou D, Paiardini M, Barry AP, Cole KS, McClure HM, Staprans SI, Silvestri G, Sodora DL (2005) Timely triggering of homeostatic mechanisms involved in the regulation of T-cell levels in SIVsm-infected sooty mangabeys. Blood 106:3839–3845

    Article  PubMed  CAS  Google Scholar 

  29. Sodora DL, Milush JM, Ware F, Wozniakowski A, Montgomery L, McClure HM, Lackner AA, Marthas M, Hirsch V, Johnson RP, Douek DC, Koup RA (2002) Decreased levels of recent thymic emigrants in peripheral blood of simian immunodeficiency virus-infected macaques correlate with alterations within the thymus. J Virol 76:9981–9990

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by NIH grants AG-025150 and AI-067798 and was performed in the Regional Biocontainment Laboratory at Duke which received partial support for construction from NIH/NIAID (UC6-AI-058607).

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Authors and Affiliations

  1. Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC, USA

    Heather E. Lynch & Gregory D. Sempowski

  2. Department of Medicine, Duke University Medical Center, Durham, NC, USA

    Heather E. Lynch & Gregory D. Sempowski

Authors
  1. Heather E. Lynch
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  2. Gregory D. Sempowski
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Corresponding author

Correspondence to Gregory D. Sempowski .

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Editors and Affiliations

  1. of the Health Sciences, Department of Pharmacology, Uniformed Services University, Jones Bridge Road 4301, Bethesda, 20814, Maryland, USA

    Andrew L. Snow

  2. NIAID, Laboratory of Immunology, National Institutes of Health, Rockville Pike 9000, Bethesda, 20892, Maryland, USA

    Michael J. Lenardo

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Lynch, H.E., Sempowski, G.D. (2013). Molecular Measurement of T Cell Receptor Excision Circles. In: Snow, A., Lenardo, M. (eds) Immune Homeostasis. Methods in Molecular Biology, vol 979. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-290-2_12

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  • DOI: https://doi.org/10.1007/978-1-62703-290-2_12

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-289-6

  • Online ISBN: 978-1-62703-290-2

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