Cancer Immunology, Immunotherapy

, Volume 63, Issue 8, pp 779–786 | Cite as

Immunologic hierarchy, class II MHC promiscuity, and epitope spreading of a melanoma helper peptide vaccine

  • Yinin HuEmail author
  • Gina R. Petroni
  • Walter C. Olson
  • Andrea Czarkowski
  • Mark E. Smolkin
  • William W. Grosh
  • Kimberly A. Chianese-Bullock
  • Craig L. SlingluffJr.
Original Article


Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4+ T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8+ T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund’s adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4+ and CD8+ T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4+ T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3281–295 (49 %) and tyrosinase386–406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8+ T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4+ and CD8+ T cell responses against melanoma antigens. CD4+ T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8+ T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.


Melanoma Peptide vaccines CD4 T cells Immunogenicity 



Combination melanoma helper peptide


Cytotoxic T lymphocyte


Peripheral blood mononuclear cell


Stimulation index


Sentinel immunized node


Conflicts of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Appay V, Zaunders JJ, Papagno L, Sutton J, Jaramillo A, Waters A et al (2002) Characterization of CD4(+) CTLs ex vivo. J Immunol 168:5954–5958PubMedCrossRefGoogle Scholar
  2. 2.
    Greenberg PD, Kern DE, Cheever MA (1985) Therapy of disseminated murine leukemia with cyclophosphamide and immune lyt-1+, 2-T cells. Tumor eradication does not require participation of cytotoxic T cells. J Exp Med 161:1122–1134PubMedCrossRefGoogle Scholar
  3. 3.
    Hunder NN, Wallen H, Cao J, Hendricks DW, Reilly JZ, Rodmyre R et al (2008) Treatment of metastatic melanoma with autologous CD4+ T cells against NY-ESO-1. N Engl J Med 358:2698–2703PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Khong HT, Yang JC, Topalian SL, Sherry RM, Mavroukakis SA, White DE et al (2004) Immunization of HLA-A*0201 and/or HLA-DPbeta1*04 patients with metastatic melanoma using epitopes from the NY-ESO-1 antigen. J Immunother 27:472–477PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Phan GQ, Touloukian CE, Yang JC, Restifo NP, Sherry RM, Hwu P et al (2003) Immunization of patients with metastatic melanoma using both class I- and class II-restricted peptides from melanoma-associated antigens. J Immunother 26:349–356PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Wong R, Lau R, Chang J, Kuus-Reichel T, Brichard V, Bruck C et al (2004) Immune responses to a class II helper peptide epitope in patients with stage III/IV resected melanoma. Clin Cancer Res 10:5004–5013PubMedCrossRefGoogle Scholar
  7. 7.
    Slingluff CL Jr, Petroni GR, Olson W, Czarkowski A, Grosh WW, Smolkin M et al (2008) Helper T-cell responses and clinical activity of a melanoma vaccine with multiple peptides from MAGE and melanocytic differentiation antigens. J Clin Oncol 26:4973–4980PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Slingluff CL Jr, Lee S, Zhao F, Chianese-Bullock KA, Olson WC, Butterfield LH et al (2013) A randomized phase II trial of multiepitope vaccination with melanoma peptides for cytotoxic T cells and helper T cells for patients with metastatic melanoma (E1602). Clin Cancer Res 19:4228–4238PubMedCrossRefGoogle Scholar
  9. 9.
    Panina-Bordignon P, Tan A, Termijtelen A, Demotz S, Corradin G, Lanzavecchia A (1989) Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur J Immunol 19:2237–2242PubMedCrossRefGoogle Scholar
  10. 10.
    Slingluff CL Jr, Yamshchikov G, Neese P, Galavotti H, Eastham S, Engelhard VH et al (2001) Phase I trial of a melanoma vaccine with gp100(280-288) peptide and tetanus helper peptide in adjuvant: immunologic and clinical outcomes. Clin Cancer Res 7:3012–3024PubMedGoogle Scholar
  11. 11.
    Johnson RP, Trocha A, Yang L, Mazzara GP, Panicali DL, Buchanan TM et al (1991) HIV-1 gag-specific cytotoxic T lymphocytes recognize multiple highly conserved epitopes. Fine specificity of the gag-specific response defined by using unstimulated peripheral blood mononuclear cells and cloned effector cells. J Immunol 147:1512–1521PubMedGoogle Scholar
  12. 12.
    Zarour HM, Kirkwood JM, Kierstead LS, Herr W, Brusic V, Slingluff CL Jr et al (2000) Melan-A/MART-1(51-73) represents an immunogenic HLA-DR4-restricted epitope recognized by melanoma-reactive CD4(+) T cells. Proc Natl Acad Sci USA 97:400–405PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Topalian SL, Gonzales MI, Parkhurst M, Li YF, Southwood S, Sette A et al (1996) Melanoma-specific CD4+ T cells recognize nonmutated HLA-DR-restricted tyrosinase epitopes. J Exp Med 183:1965–1971PubMedCrossRefGoogle Scholar
  14. 14.
    Kobayashi H, Kokubo T, Sato K, Kimura S, Asano K, Takahashi H et al (1998) CD4+ T cells from peripheral blood of a melanoma patient recognize peptides derived from nonmutated tyrosinase. Cancer Res 58:296–301PubMedGoogle Scholar
  15. 15.
    Manici S, Sturniolo T, Imro MA, Hammer J, Sinigaglia F, Noppen C et al (1999) Melanoma cells present a MAGE-3 epitope to CD4(+) cytotoxic T cells in association with histocompatibility leukocyte antigen DR11. J Exp Med 189:871–876PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Chaux P, Vantomme V, Stroobant V, Thielemans K, Corthals J, Luiten R et al (1999) Identification of MAGE-3 epitopes presented by HLA-DR molecules to CD4(+) T lymphocytes. J Exp Med 189:767–778PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Halder T, Pawelec G, Kirkin AF, Zeuthen J, Meyer HE, Kun L et al (1997) Isolation of novel HLA-DR restricted potential tumor-associated antigens from the melanoma cell line FM3. Cancer Res 57:3238–3244PubMedGoogle Scholar
  18. 18.
    Li K, Adibzadeh M, Halder T, Kalbacher H, Heinzel S, Muller C et al (1998) Tumour-specific MHC-class-II-restricted responses after in vitro sensitization to synthetic peptides corresponding to gp100 and annexin II eluted from melanoma cells. Cancer Immunol Immunother 47:32–38PubMedCrossRefGoogle Scholar
  19. 19.
    Consogno G, Manici S, Facchinetti V, Bachi A, Hammer J, Conti-Fine BM et al (2003) Identification of immunodominant regions among promiscuous HLA-DR-restricted CD4+ T-cell epitopes on the tumor antigen MAGE-3. Blood 101:1038–1044PubMedCrossRefGoogle Scholar
  20. 20.
    Benteyn D, Van Nuffel AM, Wilgenhof S, Corthals J, Heirman C, Neyns B et al (2013) Characterization of CD8+ T-cell responses in the peripheral blood and skin injection sites of melanoma patients treated with mRNA electroporated autologous dendritic cells (TriMixDC-MEL). Biomed Res Int 2013:976383PubMedCentralPubMedGoogle Scholar
  21. 21.
    Wilgenhof S, Van Nuffel AM, Benteyn D, Corthals J, Aerts C, Heirman C et al (2013) A phase IB study on intravenous synthetic mRNA electroporated dendritic cell immunotherapy in pretreated advanced melanoma patients. Ann Oncol 24:2686–2693PubMedCrossRefGoogle Scholar
  22. 22.
    Slingluff CL Jr, Petroni GR, Olson WC, Smolkin ME, Ross MI, Haas NB et al (2009) Effect of granulocyte/macrophage colony-stimulating factor on circulating CD8+ and CD4+ T-cell responses to a multipeptide melanoma vaccine: outcome of a multicenter randomized trial. Clin Cancer Res 15:7036–7044PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Hailemichael Y, Dai Z, Jaffarzad N, Ye Y, Medina MA, Huang XF et al (2013) Persistent antigen at vaccination sites induces tumor-specific CD8(+) T cell sequestration, dysfunction and deletion. Nat Med 19:465–472PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Salerno EP, Shea SM, Olson WC, Petroni GR, Smolkin ME, McSkimming C et al (2013) Activation, dysfunction and retention of T cells in vaccine sites after injection of incomplete freund’s adjuvant, with or without peptide. Cancer Immunol Immunother 62:1149–1159PubMedCrossRefGoogle Scholar
  25. 25.
    Bijker MS, van den Eeden SJ, Franken KL, Melief CJ, Offringa R, van der Burg SH (2007) CD8+ CTL priming by exact peptide epitopes in incomplete freund’s adjuvant induces a vanishing CTL response, whereas long peptides induce sustained CTL reactivity. J Immunol 179:5033–5040PubMedCrossRefGoogle Scholar
  26. 26.
    Chaux P, Luiten R, Demotte N, Vantomme V, Stroobant V, Traversari C et al (1999) Identification of five MAGE-A1 epitopes recognized by cytolytic T lymphocytes obtained by in vitro stimulation with dendritic cells transduced with MAGE-A1. J Immunol 163:2928–2936PubMedGoogle Scholar
  27. 27.
    Kittlesen DJ, Thompson LW, Gulden PH, Skipper JC, Colella TA, Shabanowitz J et al (1998) Human melanoma patients recognize an HLA-A1-restricted CTL epitope from tyrosinase containing two cysteine residues: implications for tumor vaccine development. J Immunol 160:2099–2106PubMedGoogle Scholar
  28. 28.
    Kawakami Y, Robbins PF, Wang X, Tupesis JP, Parkhurst MR, Kang X et al (1998) Identification of new melanoma epitopes on melanosomal proteins recognized by tumor infiltrating T lymphocytes restricted by HLA-A1, -A2, and -A3 alleles. J Immunol 161:6985–6992PubMedGoogle Scholar
  29. 29.
    Traversari C, van der Bruggen P, Luescher IF, Lurquin C, Chomez P, Van Pel A et al (1992) A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E. J Exp Med 176:1453–1457PubMedCrossRefGoogle Scholar
  30. 30.
    Gaugler B, Van den Eynde B, van der Bruggen P, Romero P, Gaforio JJ, De Plaen E et al (1994) Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes. J Exp Med 179:921–930PubMedCrossRefGoogle Scholar
  31. 31.
    Celis E, Tsai V, Crimi C, DeMars R, Wentworth PA, Chesnut RW et al (1994) Induction of anti-tumor cytotoxic T lymphocytes in normal humans using primary cultures and synthetic peptide epitopes. Proc Natl Acad Sci USA 91:2105–2109PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Skipper JC, Hendrickson RC, Gulden PH, Brichard V, Van Pel A, Chen Y et al (1996) An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins. J Exp Med 183:527–534PubMedCrossRefGoogle Scholar
  33. 33.
    Kawakami Y, Eliyahu S, Jennings C, Sakaguchi K, Kang X, Southwood S et al (1995) Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression. J Immunol 154:3961–3968PubMedGoogle Scholar
  34. 34.
    Cox AL, Skipper J, Chen Y, Henderson RA, Darrow TL, Shabanowitz J et al (1994) Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines. Science 264:716–719PubMedCrossRefGoogle Scholar
  35. 35.
    Huang LQ, Brasseur F, Serrano A, De Plaen E, van der Bruggen P, Boon T et al (1999) Cytolytic T lymphocytes recognize an antigen encoded by MAGE-A10 on a human melanoma. J Immunol 162:6849–6854PubMedGoogle Scholar
  36. 36.
    Kawakami Y, Eliyahu S, Sakaguchi K, Robbins PF, Rivoltini L, Yannelli JR et al (1994) Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes. J Exp Med 180:347–352PubMedCrossRefGoogle Scholar
  37. 37.
    Romero P, Dutoit V, Rubio-Godoy V, Lienard D, Speiser D, Guillaume P et al (2001) CD8+ T-cell response to NY-ESO-1: relative antigenicity and in vitro immunogenicity of natural and analogue sequences. Clin Cancer Res 7:766s–772sPubMedGoogle Scholar
  38. 38.
    Skipper JC, Kittlesen DJ, Hendrickson RC, Deacon DD, Harthun NL, Wagner SN et al (1996) Shared epitopes for HLA-A3-restricted melanoma-reactive human CTL include a naturally processed epitope from pmel-17/gp100. J Immunol 157:5027–5033PubMedGoogle Scholar
  39. 39.
    Hogan KT, Sutton JN, Chu KU, Busby JA, Shabanowitz J, Hunt DF et al (2005) Use of selected reaction monitoring mass spectrometry for the detection of specific MHC class I peptide antigens on A3 supertype family members. Cancer Immunol Immunother 54:359–371PubMedCrossRefGoogle Scholar
  40. 40.
    Wang RF, Johnston SL, Zeng G, Topalian SL, Schwartzentruber DJ, Rosenberg SA (1998) A breast and melanoma-shared tumor antigen: T cell responses to antigenic peptides translated from different open reading frames. J Immunol 161:3598–3606PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Yinin Hu
    • 1
    Email author
  • Gina R. Petroni
    • 2
  • Walter C. Olson
    • 1
  • Andrea Czarkowski
    • 3
  • Mark E. Smolkin
    • 2
  • William W. Grosh
    • 4
  • Kimberly A. Chianese-Bullock
    • 1
  • Craig L. SlingluffJr.
    • 1
  1. 1.Department of Surgery/Division of Surgical OncologyUniversity of Virginia Health SystemCharlottesvilleUSA
  2. 2.Department of Public Health SciencesUniversity of Virginia Health SystemCharlottesvilleUSA
  3. 3.Cancer CenterUniversity of Virginia Health SystemCharlottesvilleUSA
  4. 4.Department of Medicine/Division of Hematology-OncologyUniversity of Virginia Health SystemCharlottesvilleUSA

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