Cancer Immunology, Immunotherapy

, Volume 55, Issue 10, pp 1198–1208 | Cite as

Telomere length of in vivo expanded CD4+CD25+ regulatory T-cells is preserved in cancer patients

  • Dominik Wolf
  • Holger Rumpold
  • Christian Koppelstätter
  • Guenther A. Gastl
  • Michael Steurer
  • Gert Mayer
  • Eberhard Gunsilius
  • Herbert Tilg
  • Anna M. Wolf
Original Article

Abstract

Purpose: CD4+CD25+ regulatory T-cells (Treg) are increased in the peripheral blood of cancer patients. It remains unclear whether this is due to redistribution or active proliferation. The latter would require the upregulation of telomerase activity, whose regulation also remains unknown for Treg. Experimental Design: Treg and CD4+CD25 T-cells were isolated from peripheral blood of cancer patients (n=23) and healthy age-matched controls (n=17) and analyzed for their content of T-cell receptor excision circles (TREC) and for telomere length using flow-FISH, real-time PCR and Southern blotting. The in vitro regulation of telomerase of Treg was studied using PCR-ELISA in bulk cultures as well as in isolated proliferating and non-proliferating Treg. Results: Treg isolated from peripheral blood of cancer patients exhibit significantly decreased levels of TREC when compared to Treg from healthy controls. Despite their in vivo proliferation, telomere length is not further shortened in Treg from cancer patients. Accordingly, telomerase activity of Treg was readily inducible in vitro. Notably, sorting of in vitro proliferating Treg revealed a significant telomere shortening in Treg with high-proliferative capacity. The latter are characterized by shortened telomeres despite high telomerase activity. Conclusions: Increased frequencies of Treg in peripheral blood of cancer patients are due to active proliferation rather than due to redistribution from other compartments (i.e., secondary lymphoid organs or bone marrow). In vivo expansion does not further shorten telomere length, probably due to induction of telomerase activity. In contrast, under conditions of strong in vitro stimulation telomerase induction seems to be insufficient to avoid progressive telomere shortening.

Keywords

Human Regulatory T-cell Tumor immunity Telomere length 

References

  1. 1.
    Adkins B, Charyulu V, Sun QL, Lobo D, Lopez DM (2000) Early block in maturation is associated with thymic involution in mammary tumor-bearing mice. J Immunol 164:5635PubMedGoogle Scholar
  2. 2.
    Allsopp RC, Cheshier S, Weissman IL (2002) Telomerase activation and rejuvenation of telomere length in stimulated T cells derived from serially transplanted hematopoietic stem cells. J Exp Med 196:1427CrossRefPubMedGoogle Scholar
  3. 3.
    Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher AB, Greider CW, Harley CB (1992) Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci USA 89:10114PubMedCrossRefGoogle Scholar
  4. 4.
    Blasco MA, Lee HW, Hande MP, Samper E, Lansdorp PM, DePinho RA, Greider CW (1997) Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91:25CrossRefPubMedGoogle Scholar
  5. 5.
    Boyer O, Saadoun D, Abriol J, Dodille M, Piette JC, Cacoub P, Klatzmann D (2004) CD4+CD25+ regulatory T cells deficiency in patients with hepatitis C-mixed cryoglobulinemia vasculitis. Blood 103:3428CrossRefPubMedGoogle Scholar
  6. 6.
    Buchkovich KJ, Greider CW (1996) Telomerase regulation during entry into the cell cycle in normal human T cells. Mol Biol Cell 7:1443PubMedGoogle Scholar
  7. 7.
    Cawthon RM (2002) Telomere measurement by quantitative PCR. Nucleic Acids Res 30:e47CrossRefPubMedGoogle Scholar
  8. 8.
    Counter CM, Avilion AA, LeFeuvre CE, Stewart NG, Greider CW, Harley CB, Bacchetti S (1992) Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J 11:1921PubMedGoogle Scholar
  9. 9.
    Dieckmann D, Plottner H, Berchtold S, Berger T, Schuler G (2001) Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood. J Exp Med 193:1303CrossRefPubMedGoogle Scholar
  10. 10.
    Hoffmann P, Eder R, Kunz-Schughart LA, Andreesen R, Edinger M (2004) Large scale in vitro expansion of polyclonal human CD4+CD25high regulatory T cells. Blood 104:895CrossRefPubMedGoogle Scholar
  11. 11.
    Kasow KA, Chen X, Knowles J, Wichlan D, Handgretinger R, Riberdy JM (2004) Human CD4(+)CD25(+) regulatory T cells share equally complex and comparable repertoires with CD4(+)CD25(-) counterparts. J Immunol 172:6123PubMedGoogle Scholar
  12. 12.
    Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW (1994) Specific association of human telomerase activity with immortal cells and cancer. Science 266:2011PubMedCrossRefGoogle Scholar
  13. 13.
    Liu K, Hodes RJ, Weng N (2001) Cutting edge: telomerase activation in human T lymphocytes does not require increase in telomerase reverse transcriptase (hTERT) protein but is associated with hTERT phosphorylation and nuclear translocation. J Immunol 166:4826PubMedGoogle Scholar
  14. 14.
    Luiten RM, Pene J, Yssel H, Spits H (2003) Ectopic hTERT expression extends the life span of human CD4+ helper and regulatory T-cell clones and confers resistance to oxidative stress-induced apoptosis. Blood 101:4512CrossRefPubMedGoogle Scholar
  15. 15.
    Meyne J, Ratliff RL, Moyzis RK (1989) Conservation of the human telomere sequence (TTAGGG)n among vertebrates. Proc Natl Acad Sci USA 86:7049PubMedCrossRefGoogle Scholar
  16. 16.
    Morin GB (1989) The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 59:521CrossRefPubMedGoogle Scholar
  17. 17.
    Ogoshi M, Takashima A, Taylor RS (1997) Mechanisms regulating telomerase activity in murine T cells. J Immunol 158:622PubMedGoogle Scholar
  18. 18.
    Onizuka S, Tawara I, Shimizu J, Sakaguchi S, Fujita T, Nakayama E (1999) Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor alpha) monoclonal antibody. Cancer Res 59:3128PubMedGoogle Scholar
  19. 19.
    Plunkett FJ, Soares MV, Annels N, Hislop A, Ivory K, Lowdell M, Salmon M, Rickinson A, Akbar AN (2001) The flow cytometric analysis of telomere length in antigen-specific CD8+ T cells during acute Epstein-Barr virus infection. Blood 97:700CrossRefPubMedGoogle Scholar
  20. 20.
    Roth A, Yssel H, Pene J, Chavez EA, Schertzer M, Lansdorp PM, Spits H, Luiten RM (2003) Telomerase levels control the lifespan of human T lymphocytes. Blood 102:849CrossRefPubMedGoogle Scholar
  21. 21.
    Roth A, Baerlocher GM, Schertzer M, Chavez E, Duhrsen U, Lansdorp PM (2005) Telomere loss, senescence, and genetic instability in CD4+ T lymphocytes overexpressing hTERT. Blood 106:42CrossRefGoogle Scholar
  22. 22.
    Rufer N, Migliaccio M, Antonchuk J, Humphries RK, Roosnek E, Lansdorp PM (2001) Transfer of the human telomerase reverse transcriptase (TERT) gene into T lymphocytes results in extension of replicative potential. Blood 98:597CrossRefPubMedGoogle Scholar
  23. 23.
    Sakaguchi S (2004) Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 22:531CrossRefPubMedGoogle Scholar
  24. 24.
    Sasada T, Kimura M, Yoshida Y, Kanai M, Takabayashi A (2003) CD4+CD25+ regulatory T cells in patients with gastrointestinal malignancies: possible involvement of regulatory T cells in disease progression. Cancer 98:1089CrossRefPubMedGoogle Scholar
  25. 25.
    Shimizu J, Yamazaki S, Sakaguchi S (1999) Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity. J Immunol 163:5211PubMedGoogle Scholar
  26. 26.
    Somasundaram R, Jacob L, Swoboda R, Caputo L, Song H, Basak S, Monos D, Peritt D, Marincola F, Cai D, Birebent B, Bloome E, Kim J, Berencsi K, Mastrangelo M, Herlyn D (2002) Inhibition of cytolytic T lymphocyte proliferation by autologous CD4+/CD25+ regulatory T cells in a colorectal carcinoma patient is mediated by transforming growth factor-beta. Cancer Res 62:5267PubMedGoogle Scholar
  27. 27.
    Taams LS, Vukmanovic-Stejic M, Smith J, Dunne PJ, Fletcher JM, Plunkett FJ, Ebeling SB, Lombardi G, Rustin MH, Bijlsma JW, Lafeber FP, Salmon M, Akbar AN (2002) Antigen-specific T cell suppression by human CD4+CD25+ regulatory T cells. Eur J Immunol 32:1621CrossRefPubMedGoogle Scholar
  28. 28.
    Thornton AM, Shevach EM (2000) Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J Immunol 164:183PubMedGoogle Scholar
  29. 29.
    Viglietta V, Baecher-Allan C, Weiner HL, Hafler DA (2004) Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J Exp Med 199:971CrossRefPubMedGoogle Scholar
  30. 30.
    Wang HY, Lee DA, Peng G, Guo Z, Li Y, Kiniwa Y, Shevach EM, Wang RF (2004) Tumor-specific human CD4+ regulatory T cells and their ligands: implications for immunotherapy. Immunity 20:107CrossRefPubMedGoogle Scholar
  31. 31.
    Weng NP, Hathcock KS, Hodes RJ (1998) Regulation of telomere length and telomerase in T and B cells: a mechanism for maintaining replicative potential. Immunity 9:151CrossRefPubMedGoogle Scholar
  32. 32.
    Weng NP, Levine BL, June CH, Hodes RJ (1995) Human naive and memory T lymphocytes differ in telomeric length and replicative potential. Proc Natl Acad Sci USA 92:11091PubMedCrossRefGoogle Scholar
  33. 33.
    Wolf AM, Wolf D, Steurer M, Gastl G, Gunsilius E, Grubeck-Loebenstein B (2003) Increase of regulatory T cells in the peripheral blood of cancer patients. Clin Cancer Res 9:606PubMedGoogle Scholar
  34. 34.
    Woo EY, Yeh H, Chu CS, Schlienger K, Carroll RG, Riley JL, Kaiser LR, June CH (2002) Cutting edge: regulatory T cells from lung cancer patients directly inhibit autologous T cell proliferation. J Immunol 168:4272PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Dominik Wolf
    • 1
  • Holger Rumpold
    • 1
  • Christian Koppelstätter
    • 3
  • Guenther A. Gastl
    • 1
  • Michael Steurer
    • 1
  • Gert Mayer
    • 3
  • Eberhard Gunsilius
    • 1
  • Herbert Tilg
    • 2
  • Anna M. Wolf
    • 1
  1. 1.Department of Hematology and OncologyInnsbruck Medical UniversityInnsbruckAustria
  2. 2.Department of Gastroenterology and HepatologyInnsbruck Medical UniversityInnsbruckAustria
  3. 3.Department of Nephrology, Internal MedicineInnsbruck Medical UniversityInnsbruckAustria

Personalised recommendations