Cancer Immunology, Immunotherapy

, Volume 56, Issue 12, pp 1931–1943

Spontaneous and vaccine induced AFP-specific T cell phenotypes in subjects with AFP-positive hepatocellular cancer

  • Lisa H. Butterfield
  • Antoni Ribas
  • Douglas M. Potter
  • James S. Economou
Original Article


We are investigating the use of Alpha Fetoprotein (AFP) as a tumor rejection antigen for hepatocellular carcinoma (HCC). We recently completed vaccination of 10 AFP+/HLA-A2.1+ HCC subjects with AFP peptide-pulsed autologous dendritic cells (DC). There were increased frequencies of circulating AFP-specific T cells and of IFNγ-producing AFP-specific T cells after vaccination. In order to better understand the lack of association between immune response and clinical response, we have examined additional aspects of the AFP immune response in patients. Here, we have characterized the cell surface phenotype of circulating AFP tetramer-positive CD8 T cells and assessed AFP-specific CD4 function. Before vaccination, HCC subjects had increased frequencies of circulating AFP-specific CD8 T cells with a range of naïve, effector, central and effector memory phenotypes. Several patients had up-regulated activation markers. A subset of patients was assessed for phenotypic changes pre- and post-vaccination, and evidence for complete differentiation to effector or memory phenotype was lacking. CD8 phenotypic and cytokine responses did not correlate with level of patient serum AFP antigen (between 74 and 463,040 ng/ml). Assessment of CD4+ T cell responses by ELISPOT and multi-cytokine assay did not identify any spontaneous CD4 T cell responses to this secreted protein. These data indicate that there is an expanded pool of partially differentiated AFP-specific CD8 T cells in many of these HCC subjects, but that these cells are largely non-functional, and that a detectable CD4 T cell response to this secreted oncofetal antigen is lacking.


Alpha fetoprotein Immunotherapy T lymphocytes Immunological monitoring Cancer vaccine 



Alpha fetoprotein




Hepatocellular cancer


Dendritic cell(s)


Peripheral blood mononuclear cells


  1. 1.
    Butterfield LH et al (2003) T-cell responses to HLA-A*0201 immunodominant peptides derived from alpha-fetoprotein in patients with hepatocellular cancer. Clin Cancer Res 9(16 Pt 1):5902–5908PubMedGoogle Scholar
  2. 2.
    Butterfield LH et al (2006) A phase I/II trial testing immunization of hepatocellular carcinoma patients with dendritic cells pulsed with four alpha-fetoprotein peptides. Clin Cancer Res 12(9):2817–2825PubMedCrossRefGoogle Scholar
  3. 3.
    Ruoslahti E (1979) Alpha-fetoprotein in cancer and fetal development. Adv Cancer Res 29:275–346PubMedCrossRefGoogle Scholar
  4. 4.
    Alisa A et al (2005) Analysis of CD4+ T-Cell responses to a novel alpha-fetoprotein-derived epitope in hepatocellular carcinoma patients. Clin Cancer Res 11(18):6686–6694PubMedCrossRefGoogle Scholar
  5. 5.
    Butterfield LH et al (1999) Generation of human T-cell responses to an HLA-A2.1-restricted peptide epitope derived from alpha-fetoprotein. Cancer Res 59(13):3134–3142PubMedGoogle Scholar
  6. 6.
    Butterfield LH et al (2001) T cell responses to HLA-A*0201-restricted peptides derived from human alpha fetoprotein. J Immunol 166(8):5300–5308PubMedGoogle Scholar
  7. 7.
    Hanke P et al (2002) Cirrhotic patients with or without hepatocellular carcinoma harbour AFP-specific T-lymphocytes that can be activated in vitro by human alpha-fetoprotein. Scand J Gastroenterol 37(8):949–955PubMedCrossRefGoogle Scholar
  8. 8.
    Liu Y et al (2006) Hierarchy of AFP-specific T cell responses in subjects with AFP-positive hepatocellular cancer. J Immunol 177(1):712–721PubMedGoogle Scholar
  9. 9.
    Hamann D et al (1997) Phenotypic and functional separation of memory and effector human CD8+ T cells. J Exp Med 186(9):1407–1418PubMedCrossRefGoogle Scholar
  10. 10.
    Sallusto F, Geginat J, Lanzavecchia A (2004), Central memory and effector memory T cell subsets: function, generation, and maintenance. Annu Rev Immunol 22:745–763PubMedCrossRefGoogle Scholar
  11. 11.
    Sallusto F et al (1999) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401(6754):708–712PubMedCrossRefGoogle Scholar
  12. 12.
    Lee PP (1999) Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat Med 5(6):677–685PubMedCrossRefGoogle Scholar
  13. 13.
    Dunbar PR et al (2000) A shift in the phenotype of melan-A-specific CTL identifies melanoma patients with an active tumor-specific immune response. J Immunol 165(11):6644–6652PubMedGoogle Scholar
  14. 14.
    Pittet MJ et al (1999) High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals. J Exp Med 190(5):705–715PubMedCrossRefGoogle Scholar
  15. 15.
    Speiser DE, Cerottini JC, Romero P (2002) Can hTERT peptide (540–548)-specific CD8 T cells recognize and kill tumor cells? Cancer Immun 2:14PubMedGoogle Scholar
  16. 16.
    Pittet MJ et al (2001) Expansion and functional maturation of human tumor antigen-specific CD8+ T cells after vaccination with antigenic peptide. Clin Cancer Res 7(3 Suppl):796s–803sPubMedGoogle Scholar
  17. 17.
    Kim JW et al (2004) Expression of pro- and antiapoptotic proteins in circulating CD8+ T cells of patients with squamous cell carcinoma of the head and neck. Clin Cancer Res 10(15):5101–5110PubMedCrossRefGoogle Scholar
  18. 18.
    Kim JW, Ferris RL, Whiteside TL (2005) Chemokine C receptor 7 expression and protection of circulating CD8+ T lymphocytes from apoptosis. Clin Cancer Res 11(21):7901–7910PubMedCrossRefGoogle Scholar
  19. 19.
    Meng WS et al (2001) alpha-Fetoprotein-specific tumor immunity induced by plasmid prime-adenovirus boost genetic vaccination. Cancer Res 61(24):8782–8786PubMedGoogle Scholar
  20. 20.
    Herr W et al (1996) Detection and quantification of blood-derived CD8+ T lymphocytes secreting tumor necrosis factor alpha in response to HLA-A2.1-binding melanoma and viral peptide antigens. J Immunol Meth 191(2):131–142CrossRefGoogle Scholar
  21. 21.
    Mayer S et al (1996) A sensitive proliferation assay to determine the specific T cell response against HLA-A2.1-binding peptides. J Immunol Meth 197(1–2):131–137CrossRefGoogle Scholar
  22. 22.
    Huang J et al (2005) Survival, persistence, and progressive differentiation of adoptively transferred tumor-reactive T cells associated with tumor regression. J Immunother 28(3):258–267PubMedCrossRefGoogle Scholar
  23. 23.
    Appay V et al (2002) Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat Med 8(4):379–385PubMedCrossRefGoogle Scholar
  24. 24.
    Smith CL et al (2005) Immunodominance of poxviral-specific CTL in a human trial of recombinant-modified vaccinia Ankara. J Immunol 175(12):8431–8437PubMedGoogle Scholar
  25. 25.
    Lake RA, Robinson BW (2005) Immunotherapy and chemotherapy–a practical partnership. Nat Rev Cancer 5(5):397–405PubMedCrossRefGoogle Scholar
  26. 26.
    Ajuebor MN, Carey JA, Swain MG (2006) CCR5 in T cell-mediated liver diseases: what’s going on? J Immunol 177(4):2039–2045PubMedGoogle Scholar
  27. 27.
    Tatsumi T et al (2003) Disease stage variation in CD4+ and CD8+ T-cell reactivity to the receptor tyrosine kinase EphA2 in patients with renal cell carcinoma. Cancer Res 63(15):4481–4489PubMedGoogle Scholar
  28. 28.
    Butterfield LH et al (2003) Determinant spreading associated with clinical response in dendritic cell-based immunotherapy for malignant melanoma. Clin Cancer Res 9(3):998–1008PubMedGoogle Scholar
  29. 29.
    Ribas A et al (2004) Role of dendritic cell phenotype, determinant spreading, and negative costimulatory blockade in dendritic cell-based melanoma immunotherapy. J Immunother 27(5):354–367PubMedCrossRefGoogle Scholar
  30. 30.
    Evdokimova VN et al (2007) AFP specific CD4+ helper T cell responses in healthy donors and HCC patients. J Immunother (in press)Google Scholar
  31. 31.
    Seregni E, Botti C, Bombardieri E (1995) Biochemical characteristics and clinical applications of alpha-fetoprotein isoforms. Anticancer Res 15(4):1491–1499PubMedGoogle Scholar
  32. 32.
    Heydtmann M et al (2006) Detailed analysis of intrahepatic CD8 T cells in the normal and hepatitis C-infected liver reveals differences in specific populations of memory cells with distinct homing phenotypes. J Immunol 177(1):729–738PubMedGoogle Scholar
  33. 33.
    Dumortier H et al (2005) Antigen presentation by an immature myeloid dendritic cell line does not cause CTL deletion in vivo, but generates CD8+ central memory-like T cells that can be rescued for full effector function. J Immunol 175(2):855–863PubMedGoogle Scholar
  34. 34.
    Speiser DE et al (2002) In vivo activation of melanoma-specific CD8(+) T cells by endogenous tumor antigen and peptide vaccines. A comparison to virus-specific T cells. Eur J Immunol 32(3):731–741PubMedCrossRefGoogle Scholar
  35. 35.
    Harari A et al (2005) Functional heterogeneity of memory CD4 T cell responses in different conditions of antigen exposure and persistence. J Immunol 174(2):1037–1045PubMedGoogle Scholar
  36. 36.
    Beckebaum S, et al (2002) Reduction in the circulating pDC1/pDC2 ratio and impaired function of ex vivo-generated DC1 in chronic hepatitis B infection. Clin Immunol 104(2):138–150PubMedCrossRefGoogle Scholar
  37. 37.
    Ninomiya T et al (1999) Dendritic cells with immature phenotype and defective function in the peripheral blood from patients with hepatocellular carcinoma. J Hepatol 31(2):323–331PubMedCrossRefGoogle Scholar
  38. 38.
    Piccioli D et al (2005) Comparable functions of plasmacytoid and monocyte-derived dendritic cells in chronic hepatitis C patients and healthy donors. J Hepatol 42(1):61–67PubMedCrossRefGoogle Scholar
  39. 39.
    Bei R et al (1999) Cryptic epitopes on alpha-fetoprotein induce spontaneous immune responses in hepatocellular carcinoma, liver cirrhosis, and chronic hepatitis patients. Cancer Res 59(21):5471–5474PubMedGoogle Scholar
  40. 40.
    Curtsinger JM, (2005) Signal 3 tolerant CD8 T cells degranulate in response to antigen but lack granzyme B to mediate cytolysis. J Immunol 175(7):4392–4399PubMedGoogle Scholar
  41. 41.
    Curtsinger JM et al (2005) Type I IFNs provide a third signal to CD8 T cells to stimulate clonal expansion and differentiation. J Immunol 174(8):4465–4469PubMedGoogle Scholar
  42. 42.
    Schumacher L et al (2004) Human dendritic cell maturation by adenovirus transduction enhances tumor antigen-specific T-cell responses. J Immunother 27(3):191–200PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Lisa H. Butterfield
    • 1
    • 6
  • Antoni Ribas
    • 2
    • 3
  • Douglas M. Potter
    • 4
  • James S. Economou
    • 2
    • 5
  1. 1.Department of Medicine, Surgery and Immunology University of Pittsburgh Cancer InstituteUniversity of PittsburghPittsburghUSA
  2. 2.Divisions of Surgical OncologyUniversity of CaliforniaLos AngelesUSA
  3. 3.Hematology/OncologyUniversity of CaliforniaLos AngelesUSA
  4. 4.UPCI BiostatisticsUniversity of PittsburghPittsburghUSA
  5. 5.Department of Microbiology, Immunology and Molecular GeneticsUniversity of CaliforniaLos AngelesUSA
  6. 6.Hillman Cancer Center, Research PavilionUniversity of PittsburghPittsburghUSA

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