Skip to main content


Log in

CD4+ T-cell recognition of human 5T4 oncofoetal antigen: implications for initial depletion of CD25+ T cells

  • Original Article
  • Published:
Cancer Immunology, Immunotherapy Aims and scope Submit manuscript



The human 5T4 (h5T4) oncofoetal antigen is expressed by a wide variety of human carcinomas including colorectal, ovarian, gastric and renal, but rarely on normal tissues. Its restricted expression on tumour tissues as well as its association with tumour progression and bad prognosis has driven the development of a MVA-based vaccine (TroVax) which has been tested in several early phase clinical trials and these studies have led to the start of a phase III trial in renal cell carcinoma patients. We have recently shown that CD8+ T cells recognizing h5T4 can be generated in the absence of CD4+ T cells from peripheral blood lymphocytes of human healthy individuals.


We report the existence and expansion of human CD4+ T cells against h5T4 by stimulation with autologous monocyte-derived dendritic cells infected with a replication defective adenovirus encoding the h5T4 cDNA (Ad-h5T4). The h5T4-specific T-cell responses in normal individuals are enhanced by initial depletion of CD25+ cells (putative T regulatory cells) prior to the in vitro stimulation. We have identified a novel h5T4-derived 15-mer peptide recognized by CD4+ T cells in HLA-DR4 positive healthy individuals. Interestingly, CD4+ T cells spontaneously recognizing a different 5T4 epitope restricted by HLA-DR were identified in tumour-infiltrating lymphocytes isolated from a regressing renal cell carcinoma lung metastasis.


Our data show that CD4+ T cells recognizing h5T4 can be expanded and detected in healthy individuals and a renal cell carcinoma patient. Such h5T4-specific CD4+ T cells boosted or induced by vaccination could act to modulate both cell or antibody mediated anti-tumour responses.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others





Cytotoxic T lymphocyte


Dendritic cells


Dimethyl sulfoxide


Enzyme linked immunosorbent assay


Enzyme linked immunospot


Green fluorescent protein


Granulocyte-macrophage colony stimulating factor


Human 5T4


Healthy donor


Interferon gamma




Major histocompatibility complex


Modified vaccinia Ankara


Peripheral blood lymphocytes


Peripheral blood mononuclear cells


Plaque forming unit


Peptide pool


Recombinant human interleukin


Staphylococcal enterotoxin B


Tumour-associated antigen


T cell receptor


Tumour-infiltrating lymphocytes


T regulatory cells


  1. van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den Eynde B, Knuth A, Boon T (1991) A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 254:1643–1647

    Article  PubMed  Google Scholar 

  2. Kawakami Y, Eliyahu S, Delgado CH, Robbins PF, Sakaguchi K, Appella E, Yannelli JR, Adema GJ, Miki T, Rosenberg SA (1994) Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection. Proc Natl Acad Sci USA 91:6458–6462

    Article  PubMed  CAS  Google Scholar 

  3. Schultze JL, Vonderheide RH (2001) From cancer genomics to cancer immunotherapy: toward second-generation tumor antigens. Trends Immunol 22:516–523

    Article  PubMed  CAS  Google Scholar 

  4. Greenberg PD (1991) Adoptive T cell therapy of tumors: mechanisms operative in the recognition and elimination of tumor cells. Adv Immunol 49:281–355

    Article  PubMed  CAS  Google Scholar 

  5. Chikamatsu K, Albers A, Stanson J, Kwok WW, Appella E, Whiteside TL, DeLeo AB (2003) P53(110–124)-specific human CD4+ T-helper cells enhance in vitro generation and antitumor function of tumor-reactive CD8+ T cells. Cancer Res 63:3675–3681

    PubMed  CAS  Google Scholar 

  6. Hole N, Stern PL (1990) Isolation and characterization of 5T4, a tumour-associated antigen. Int J Cancer 45:179–184

    Article  PubMed  CAS  Google Scholar 

  7. Myers KA, Rahi-Saund V, Davison MD, Young JA, Cheater AJ, Stern PL (1994) Isolation of a cDNA encoding 5T4 oncofetal trophoblast glycoprotein. An antigen associated with metastasis contains leucine-rich repeats. J Biol Chem 269:9319–9324

    PubMed  CAS  Google Scholar 

  8. Southall PJ, Boxer GM, Bagshawe KD, Hole N, Bromley M, Stern PL (1990) Immunohistological distribution of 5T4 antigen in normal and malignant tissues. Br J Cancer 61:89–95

    PubMed  CAS  Google Scholar 

  9. Mulder WM, Stern PL, Stukart MJ, de Windt E, Butzelaar RM, Meijer S, Ader HJ, Claessen AM, Vermorken JB, Meijer CJ, Wagstaff J, Scheper RJ, Bloemena E (1997) Low intercellular adhesion molecule 1 and high 5T4 expression on tumor cells correlate with reduced disease-free survival in colorectal carcinoma patients. Clin Cancer Res 3:1923–1930

    PubMed  CAS  Google Scholar 

  10. Starzynska T, Marsh PJ, Schofield PF, Roberts SA, Myers KA, Stern PL (1994) Prognostic significance of 5T4 oncofetal antigen expression in colorectal carcinoma. Br J Cancer 69:899–902

    PubMed  CAS  Google Scholar 

  11. Starzynska T, Rahi V, Stern PL (1992) The expression of 5T4 antigen in colorectal and gastric carcinoma. Br J Cancer 66:867–869

    PubMed  CAS  Google Scholar 

  12. Starzynska T, Wiechowska-Kozlowska A, Marlicz K, Bromley M, Roberts SA, Lawniczak M, Kolodziej B, Zyluk A, Stern PL (1998) 5T4 oncofetal antigen in gastric carcinoma and its clinical significance. Eur J Gastroenterol Hepatol 10:479–484

    Article  PubMed  CAS  Google Scholar 

  13. Wrigley E, McGown AT, Rennison J, Swindell R, Crowther D, Starzynska T, Stern PL (1995) 5T4 oncofetal antigen expression in ovarian carcinoma. Int J Gynecol Cancer 5:269–274

    Article  PubMed  Google Scholar 

  14. Griffiths RW, Gilham DE, Dangoor A, Ramani V, Clarke NW, Stern PL, Hawkins RE (2005) Expression of the 5T4 oncofoetal antigen in renal cell carcinoma: a potential target for T-cell-based immunotherapy. Br J Cancer 93:670–677

    Article  PubMed  CAS  Google Scholar 

  15. Harrop R, Connolly N, Redchenko I, Valle J, Saunders M, Ryan MG, Myers KA, Drury N, Kingsman SM, Hawkins RE, Carroll MW (2006) Vaccination of colorectal cancer patients with modified vaccinia Ankara delivering the tumor antigen 5T4 (TroVax) induces immune responses which correlate with disease control: a phase I/II trial. Clin Cancer Res 12:3416–3424

    Article  PubMed  CAS  Google Scholar 

  16. Mulryan K, Ryan MG, Myers KA, Shaw D, Wang W, Kingsman SM, Stern PL, Carroll MW (2002) Attenuated recombinant vaccinia virus expressing oncofetal antigen (tumor-associated antigen) 5T4 induces active therapy of established tumors. Mol Cancer Ther 1:1129–1137

    PubMed  CAS  Google Scholar 

  17. Forsberg G, Ohlsson L, Brodin T, Bjork P, Lando PA, Shaw D, Stern PL, Dohlsten M (2001) Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen. Br J Cancer 85:129–136

    Article  PubMed  CAS  Google Scholar 

  18. Shaw DM, Connolly NB, Patel PM, Kilany S, Hedlund G, Nordle O, Forsberg G, Zweit J, Stern PL, Hawkins RE (2007) A phase II study of a 5T4 oncofoetal antigen tumour-targeted superantigen (ABR-214936) therapy in patients with advanced renal cell carcinoma. Br J Cancer 96:567–574

    Article  PubMed  CAS  Google Scholar 

  19. Dermime S, Gilham DE, Shaw DM, Davidson EJ, Meziane el K, Armstrong A, Hawkins RE, Stern PL (2004) Vaccine and antibody-directed T cell tumour immunotherapy. Biochim Biophys Acta 1704:11–35

    PubMed  CAS  Google Scholar 

  20. Guest RD, Hawkins RE, Kirillova N, Cheadle EJ, Arnold J, O’Neill A, Irlam J, Chester KA, Kemshead JT, Shaw DM, Embleton MJ, Stern PL, Gilham DE (2005) The role of extracellular spacer regions in the optimal design of chimeric immune receptors: evaluation of four different scFvs and antigens. J Immunother 28:203–211

    Article  PubMed  CAS  Google Scholar 

  21. Sakaguchi S, Sakaguchi N, Shimizu J, Yamazaki S, Sakihama T, Itoh M, Kuniyasu Y, Nomura T, Toda M, Takahashi T (2001) Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol Rev 182:18–32

    Article  PubMed  CAS  Google Scholar 

  22. Shevach EM (2002) CD4+ CD25+ suppressor T cells: more questions than answers. Nat Rev Immunol 2:389–400

    PubMed  CAS  Google Scholar 

  23. Takahashi T, Sakaguchi S (2003) The role of regulatory T cells in controlling immunologic self-tolerance. Int Rev Cytol 225:1–32

    Article  PubMed  CAS  Google Scholar 

  24. 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 59:3128–3133

    PubMed  CAS  Google Scholar 

  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 163:5211–5218

    PubMed  CAS  Google Scholar 

  26. Sutmuller RP, van Duivenvoorde LM, van Elsas A, Schumacher TN, Wildenberg ME, Allison JP, Toes RE, Offringa R, Melief CJ (2001) Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25(+) regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses. J Exp Med 194:823–832

    Article  PubMed  CAS  Google Scholar 

  27. 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 193:1303–1310

    Article  PubMed  CAS  Google Scholar 

  28. Fontenot JD, Gavin MA, Rudensky AY (2003) Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 4:330–336

    Article  PubMed  CAS  Google Scholar 

  29. Yagi H, Nomura T, Nakamura K, Yamazaki S, Kitawaki T, Hori S, Maeda M, Onodera M, Uchiyama T, Fujii S, Sakaguchi S (2004) Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells. Int Immunol 16:1643–1656

    Article  PubMed  CAS  Google Scholar 

  30. Smyth LJ, Elkord E, Taher TE, Jiang HR, Burt DJ, Clayton A, van Veelen PA, de Ru A, Ossendorp F, Melief CJ, Drijfhout JW, Dermime S, Hawkins RE, Stern PL (2006) CD8 T-cell recognition of human 5T4 oncofetal antigen. Int J Cancer 119:1638–1647

    Article  PubMed  CAS  Google Scholar 

  31. Hole N, Stern PL (1988) A 72 kD trophoblast glycoprotein defined by a monoclonal antibody. Br J Cancer 57:239–246

    PubMed  CAS  Google Scholar 

  32. Antony PA, Restifo NP (2005) CD4+CD25+ T regulatory cells, immunotherapy of cancer, and interleukin-2. J Immunother 28:120–128

    Article  PubMed  CAS  Google Scholar 

  33. Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanovic S (1999) SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50:213–219

    Article  PubMed  CAS  Google Scholar 

  34. D’Amaro J, Houbiers JG, Drijfhout JW, Brandt RM, Schipper R, Bavinck JN, Melief CJ, Kast WM (1995) A computer program for predicting possible cytotoxic T lymphocyte epitopes based on HLA class I peptide-binding motifs. Hum Immunol 43:13–18

    Article  PubMed  CAS  Google Scholar 

  35. Ito D, Albers A, Zhao YX, Visus C, Appella E, Whiteside TL, DeLeo AB (2006) The wild-type sequence (wt) p53(25–35) peptide induces HLA-DR7 and HLA-DR11-restricted CD4+ Th cells capable of enhancing the ex vivo expansion and function of anti-wt p53(264–272) peptide CD8+ T cells. J Immunol 177:6795–6803

    PubMed  CAS  Google Scholar 

  36. Mesel-Lemoine M, Cherai M, Le Gouvello S, Guillot M, Leclercq V, Klatzmann D, Thomas-Vaslin V, Lemoine FM (2006) Initial depletion of regulatory T cells: the missing solution to preserve the immune functions of T lymphocytes designed for cell therapy. Blood 107:381–388

    Article  PubMed  CAS  Google Scholar 

  37. Hori S, Nomura T, Sakaguchi S (2003) Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057–1061

    Article  PubMed  CAS  Google Scholar 

  38. Knutson KL, Disis ML, Salazar LG (2007) CD4 regulatory T cells in human cancer pathogenesis. Cancer Immunol Immunother 56:271–285

    Article  PubMed  Google Scholar 

  39. Walker MR, Kasprowicz DJ, Gersuk VH, Benard A, Van Landeghen M, Buckner JH, Ziegler SF (2003) Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25- T cells. J Clin Invest 112:1437–1443

    PubMed  CAS  Google Scholar 

  40. Fantini MC, Becker C, Monteleone G, Pallone F, Galle PR, Neurath MF (2004) Cutting edge: TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7. J Immunol 172:5149–5153

    PubMed  CAS  Google Scholar 

  41. Allan SE, Crome SQ, Crellin NK, Passerini L, Steiner TS, Bacchetta R, Roncarolo MG, Levings MK (2007) Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production. Int Immunol 19:345–354

    Article  PubMed  CAS  Google Scholar 

  42. Morgan ME, van Bilsen JH, Bakker AM, Heemskerk B, Schilham MW, Hartgers FC, Elferink BG, van der Zanden L, de Vries RR, Huizinga TW, Ottenhoff TH, Toes RE (2005) Expression of FOXP3 mRNA is not confined to CD4+CD25+ T regulatory cells in humans. Hum Immunol 66:13–20

    Article  PubMed  CAS  Google Scholar 

  43. Tran DQ, Ramsey H, Shevach EM (2007) Induction of FOXP3 expression in naive human CD4+FOXP3- T cells by T cell receptor stimulation is TGF{beta}-dependent but does not confer a regulatory phenotype. Blood 110(8):2983–2990

    Article  PubMed  CAS  Google Scholar 

  44. Wang J, Ioan-Facsinay A, van der Voort EI, Huizinga TW, Toes RE (2007) Transient expression of FOXP3 in human activated nonregulatory CD4+ T cells. Eur J Immunol 37:129–138

    Article  PubMed  CAS  Google Scholar 

  45. Mincheff M, Zoubak S, Altankova I, Tchakarov S, Pogribnyy P, Makogonenko Y, Botev C, Meryman HT (2005) Depletion of CD25+ cells from human T-cell enriched fraction eliminates immunodominance during priming with dendritic cells genetically modified to express a secreted protein. Cancer Gene Ther 12:185–197

    Article  PubMed  CAS  Google Scholar 

  46. Fehervari Z, Sakaguchi S (2004) Control of Foxp3+ CD25+CD4+ regulatory cell activation and function by dendritic cells. Int Immunol 16:1769–1780

    Article  PubMed  CAS  Google Scholar 

  47. Yang Y, Huang CT, Huang X, Pardoll DM (2004) Persistent Toll-like receptor signals are required for reversal of regulatory T cell-mediated CD8 tolerance. Nat Immunol 5:508–515

    Article  PubMed  CAS  Google Scholar 

  48. Baecher-Allan C, Viglietta V, Hafler DA (2004) Human CD4+CD25+ regulatory T cells. Semin Immunol 16:89–98

    Article  PubMed  CAS  Google Scholar 

  49. Chattopadhyay S, Mehrotra S, Chhabra A, Hegde U, Mukherji B, Chakraborty NG (2006) Effect of CD4+CD25+ and CD4+CD25- T regulatory cells on the generation of cytolytic T cell response to a self but human tumor-associated epitope in vitro. J Immunol 176:984–990

    PubMed  CAS  Google Scholar 

  50. Hsieh CS, Liang Y, Tyznik AJ, Self SG, Liggitt D, Rudensky AY (2004) Recognition of the peripheral self by naturally arising CD25+ CD4+ T cell receptors. Immunity 21:267–277

    Article  PubMed  CAS  Google Scholar 

  51. Danke NA, Koelle DM, Yee C, Beheray S, Kwok WW (2004) Autoreactive T cells in healthy individuals. J Immunol 172:5967–5972

    PubMed  CAS  Google Scholar 

  52. Nishikawa H, Jager E, Ritter G, Old LJ, Gnjatic S (2005) CD4+ CD25+ regulatory T cells control the induction of antigen-specific CD4+ helper T cell responses in cancer patients. Blood 106:1008–1011

    Article  PubMed  CAS  Google Scholar 

  53. Clarke SL, Betts GJ, Plant A, Wright KL, El-Shanawany TM, Harrop R, Torkington J, Rees BI, Williams GT, Gallimore AM, Godkin AJ (2006) CD4CD25FOXP3 regulatory T cells suppress anti-tumor immune responses in patients with colorectal cancer. PLoS ONE 1:e129

    Article  PubMed  CAS  Google Scholar 

  54. Thistlethwaite FC, Elkord E, Griffiths RW, Burt DJ, Shablak AM, Campbell JD, Gilham DE, Austin EB, Stern PL, Hawkins RE (2007) Adoptive transfer of T(reg) depleted autologous T cells in advanced renal cell carcinoma. Cancer Immunol Immunother. doi:10.1007/s00262-007-0400-6

  55. van der Burg SH, Piersma SJ, de Jong A, van der Hulst JM, Kwappenberg KM, van den Hende M, Welters MJ, Van Rood JJ, Fleuren GJ, Melief CJ, Kenter GG, Offringa R (2007) Association of cervical cancer with the presence of CD4+ regulatory T cells specific for human papillomavirus antigens. Proc Natl Acad Sci USA 104:12087–12092

    Article  PubMed  CAS  Google Scholar 

  56. Singh H, Raghava GP (2001) ProPred: prediction of HLA-DR binding sites. Bioinformatics 17:1236–1237

    Article  PubMed  CAS  Google Scholar 

  57. Reche PA, Glutting JP, Zhang H, Reinherz EL (2004) Enhancement to the RANKPEP resource for the prediction of peptide binding to MHC molecules using profiles. Immunogenetics 56:405–419

    Article  PubMed  CAS  Google Scholar 

Download references


This work was funded by Cancer Research UK. We are very grateful to all the volunteers who donated blood to this study. We are also grateful to Dr Alaaeldin Shablak for providing the tumour sample from which TILs were isolated.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Eyad Elkord.

Additional information

This work was supported by Cancer Research UK.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Elkord, E., Burt, D.J., Drijfhout, J.W. et al. CD4+ T-cell recognition of human 5T4 oncofoetal antigen: implications for initial depletion of CD25+ T cells. Cancer Immunol Immunother 57, 833–847 (2008).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: