Cancer Immunology, Immunotherapy

, Volume 62, Issue 7, pp 1249–1260 | Cite as

Ex vivo analysis of pancreatic cancer-infiltrating T lymphocytes reveals that ENO-specific Tregs accumulate in tumor tissue and inhibit Th1/Th17 effector cell functions

  • Amedeo AmedeiEmail author
  • Elena Niccolai
  • Marisa Benagiano
  • Chiara Della Bella
  • Fabio Cianchi
  • Paolo Bechi
  • Antonio Taddei
  • Lapo Bencini
  • Marco Farsi
  • Paola Cappello
  • Domenico Prisco
  • Francesco Novelli
  • Mario Milco D’Elios
Original Article


Pancreatic cancer (PC) is an aggressive disease with dismal prognosis. Surgical resection is the recommended treatment for long-term survival, but patients with resectable PC are in the minority (with a 5-year survival rate of 20 %). Therefore, development of novel therapeutic strategies, such as anti-PC immunotherapy, is crucial. α-Enolase (ENO1) is an enzyme expressed on the surface of pancreatic cancer cells and is able to promote cell migration and cancer metastasis. The capacity of ENO1 to induce an immune response in PC patients renders it a true tumor-associated antigen. In this study, we characterized the effector functions of ENO1-specific T cells isolated from PC patients, and we specifically evaluated the successful role of intra-tumoral T helper 17 (Th17) cells and the inhibitory role of regulatory T (Tregs) cells in respectively promoting or reducing the cancer-specific immune response. In this ex vivo study, we have demonstrated, for the first time, that ENO1-specific Th17 cells have a specific anti-cancer effector function in PC patients, and that there are decreased levels of these cells in cancer compared to healthy mucosa. Conversely, there are elevated levels of ENO1-specific Tregs in PC patients which lead to inhibition of the antigen-specific effector T cells, thus highlighting a possible role in promoting PC progression. These results may be relevant for the design of novel immunotherapeutic strategies in pancreatic cancer.


Pancreatic cancer Tumor-infiltrating lymphocytes (TILs) Regulatory T cells (Tregs) T helper 17 (Th17) α-Enolase (ENO1) 



This work is dedicated to the memory of Professor Gianfranco del Prete, who was a valuable researcher and teacher. We would like to thank Dr. Radhika Srinivasan for accurate editing of the manuscript. This work was supported by grants from the Italian Ministry of University and Research (PRIN 2009), the Italian Ministry of Health (Progetto Integrato Oncologia), the University of Florence, the Regione Piemonte (BIOTHER, IMMONC, Ricerca Sanitaria Finalizzata), the Associazione Italiana Ricerca sul Cancro (AIRC IG n. 11643 and 5 per 1000 n. 12182), the University of Torino-Progetti di Ateneo 2011 (grant Rethe-ORTO11RKTW), the Istituto Superiore di Sanità and European Community, the Seventh Framework Program, and the European Pancreatic Cancer-Tumor-Microenvironment Network (EPC-TM-Net, nr. 256974) and the Fondazione Internazionale di Medicina Sperimentale.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Hidalgo M (2010) Pancreatic cancer. N Engl J Med 362:1605–1617PubMedCrossRefGoogle Scholar
  2. 2.
    Goonetilleke KS, Siriwardena AK (2007) Nationwide questionnaire survey of the contemporary surgical management of pancreatic cancer in the United Kingdom & Ireland. Int J Surg 5:147–151PubMedCrossRefGoogle Scholar
  3. 3.
    Moon HJ, An JY, Heo JS, Choi SH, Joh JW, Kim YI (2006) Predicting survival after surgical resection for pancreatic ductal adenocarcinoma. Pancreas 32:37–43PubMedCrossRefGoogle Scholar
  4. 4.
    Laheru D, Lutz E, Burke J, Biedrzycki B, Solt S, Onners B, Tartakovsky I, Nemunaitis J, Le D, Sugar E, Hege K, Jaffee E (2008) Allogeneic granulocyte macrophage colony-stimulating factor-secreting tumor immunotherapy alone or in sequence with cyclophosphamide for metastatic pancreatic cancer: a pilot study of safety, feasibility, and immune activation. Clin Cancer Res 14:1455–1463PubMedCrossRefGoogle Scholar
  5. 5.
    Ramanathan RK, Lee KM, McKolanis J, Hitbold E, Schraut W, Moser AJ, Warnick E, Whiteside T, Osborne J, Kim H, Day R, Troetschel M, Finn OJ (2005) Phase I study of a MUC1 vaccine composed of different doses of MUC1 peptide with SB-AS2 adjuvant in resected and locally advanced pancreatic cancer. Cancer Immunol Immunother 54:254–264PubMedCrossRefGoogle Scholar
  6. 6.
    Jung S, Schluesener HJ (1991) Human T lymphocytes recognize a peptide of single point-mutated, oncogenic ras proteins. J Exp Med 173:273–276PubMedCrossRefGoogle Scholar
  7. 7.
    Nakatsura T, Senju S, Ito M, Nishimura Y, Itoh K (2002) Cellular and humoral immune responses to a human pancreatic cancer antigen, coactosin-like protein, originally defined by the SEREX method. Eur J Immunol 32:826–836PubMedCrossRefGoogle Scholar
  8. 8.
    Wobser M, Keikavoussi P, Kunzmann V, Weininger M, Andersen MH, Becker JC (2006) Complete remission of liver metastasis of pancreatic cancer under vaccination with a HLA-A2 restricted peptide derived from the universal tumor antigen survivin. Cancer Immunol Immunother 55:1294–1298PubMedCrossRefGoogle Scholar
  9. 9.
    Suso EM, Dueland S, Rasmussen AM, Vetrhus T, Aamdal S, Kvalheim G, Gaudernack G (2011) hTERT mRNA dendritic cell vaccination: complete response in a pancreatic cancer patient associated with response against several hTERT epitopes. Cancer Immunol Immunother 60:809–818PubMedCrossRefGoogle Scholar
  10. 10.
    Cappello P, Tomaino B, Chiarle R, Ceruti P, Novarino A, Castagnoli C, Migliorini P, Perconti G, Giallongo A, Milella M, Monsurrò V, Barbi S et al (2009) An integrated humoral and cellular response is elicited in pancreatic cancer by alpha-enolase, a novel pancreatic ductal adenocarcinoma-associated antigen. Int J Cancer 125:639–648PubMedCrossRefGoogle Scholar
  11. 11.
    Capello M, Ferri-Borgogno S, Cappello P, Novelli F (2011) α-Enolase: a promising therapeutic and diagnostic tumor target. FEBS J 278:1064–1074PubMedCrossRefGoogle Scholar
  12. 12.
    Tomaino B, Cappello P, Capello M, Fredolini C, Sperduti I, Migliorini P, Salacone P, Novarino A, Giacobino A, Ciuffreda L, Alessio M, Nisticò P et al (2011) Circulating autoantibodies to phosphorylated alpha-enolase are a hallmark of pancreatic cancer. J Proteome Res 10:105–112PubMedCrossRefGoogle Scholar
  13. 13.
    Cappello P, Rolla S, Chiarle R, Principe M, Cavallo F, Perconti G, Feo S, Giovarelli M, Novelli F (2013) Vaccination with ENO1 DNA prolongs survival of genetically engineered mice with pancreatic cancer. Gastroenterology. doi: 10.1053/j.gastro.2013.01.020. [Epub ahead of print]
  14. 14.
    Linehan DC, Goedegebuure PS (2005) CD25+ CD4+ regulatory T-cells in cancer. Immunol Res 32:155–168PubMedCrossRefGoogle Scholar
  15. 15.
    Liyanage UK, Moore TT, Joo HG, Tanaka Y, Herrmann V, Doherty G, Drebin JA, Strasberg SM, Eberlein TJ, Goedegebuure PS, Linehan DC (2002) Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J Immunol 169:2756–2761PubMedGoogle Scholar
  16. 16.
    Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK (2006) Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441:235–238PubMedCrossRefGoogle Scholar
  17. 17.
    Sharma MD, Hou DY, Liu Y, Koni PA, Metz R, Chandler P, Mellor AL, He Y, Munn DH (2009) Indoleamine 2,3-dioxygenase controls conversion of Foxp3+ Tregs to TH17-like cells in tumor-draining lymph nodes. Blood 113:6102–6111PubMedCrossRefGoogle Scholar
  18. 18.
    Bettelli E, Oukka M, Kuchroo VK (2007) T(H)-17 cells in the circle of immunity and autoimmunity. Nat Immunol 8:345–350PubMedCrossRefGoogle Scholar
  19. 19.
    Kryczek I, Wei S, Zou L, Altuwaijri S, Szeliga W, Kolls J, Chang A, Zou W (2007) Cutting edge: Th17 and regulatory T cell dynamics and the regulation by IL-2 in the tumor microenvironment. J Immunol 178:6730–6733PubMedGoogle Scholar
  20. 20.
    Zhang B, Rong G, Wei H, Zhang M, Bi J, Ma L, Xue X, Wei G, Liu X, Fang G (2008) The prevalence of Th17 cells in patients with gastric cancer. Biochem Biophys Res Commun 374:533–537PubMedCrossRefGoogle Scholar
  21. 21.
    Koyama K, Kagamu H, Miura S, Hiura T, Miyabayashi T, Itoh R, Kuriyama H, Tanaka H, Tanaka J, Yoshizawa H, Nakata K, Gejyo F (2008) Reciprocal CD4+ T-cell balance of effector CD62Llow CD4+ and CD62LhighCD25+ CD4+ regulatory T cells in small cell lung cancer reflects disease stage. Clin Cancer Res 14:6770–6779PubMedCrossRefGoogle Scholar
  22. 22.
    Takahashi T, Kuniyasu Y, Toda M, Sakaguchi N, Itoh M, Iwata M, Shimizu J, Sakaguchi S (1998) Immunologic self-tolerance maintained by CD25+ CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int Immunol 10:1969–1980PubMedCrossRefGoogle Scholar
  23. 23.
    Shevach EM (2001) Certified professionals: CD4(+)CD25(+) suppressor T cells. J Exp Med 193:F41–F46PubMedCrossRefGoogle Scholar
  24. 24.
    Ikemoto T, Yamaguchi T, Morine Y, Imura S, Soejima Y, Fujii M, Maekawa Y, Yasutomo K, Shimada M (2006) Clinical roles of increased populations of Foxp3+ CD4+ T cells in peripheral blood from advanced pancreatic cancer patients. Pancreas 33:386–390PubMedCrossRefGoogle Scholar
  25. 25.
    Viehl CT, Moore TT, Liyanage UK, Frey DM, Ehlers JP, Eberlein TJ, Goedegebuure PS, Linehan DC (2006) Depletion of CD4+ CD25+ regulatory T cells promotes a tumor-specific immune response in pancreas cancer bearing mice. Ann Surg Oncol 13:1252–1258PubMedCrossRefGoogle Scholar
  26. 26.
    Warshaw AL, Fernández-del Castillo CN (1992) Pancreatic carcinoma. N Engl J Med 26:455–465CrossRefGoogle Scholar
  27. 27.
    Amedei A, Della Bella C, Niccolai E, Stanflin N, Benagiano M, Duranti R, Del Prete G, Murphy TF, D’Elios MM (2009) Moraxella catarrhalis-specific Th1 cells in BAL fluids of chronic obstructive pulmonary disease patients. Int J Immunopathol Pharmacol 22:979–990PubMedGoogle Scholar
  28. 28.
    Amedei A, Niccolai E, Della Bella C, Cianchi F, Trallori G, Benagiano M, Bencini L, Bernini M, Farsi M, Moretti R, Del Prete G, D’Elios MM (2009) Characterization of tumor antigen peptide-specific T cells isolated from the neoplastic tissue of patients with gastric adenocarcinoma. Cancer Immunol Immunother 58:1819–1830PubMedCrossRefGoogle Scholar
  29. 29.
    Yu P, Haymaker CL, Divekar RD, Ellis JS, Hardaway J, Jain R, Tartar DM, Hoeman CM, Cascio JA, Ostermeier A, Zaghouani H (2008) Fetal exposure to high-avidity TCR ligand enhances expansion of peripheral T regulatory cells. J Immunol 181:73–80PubMedGoogle Scholar
  30. 30.
    De Monte L, Reni M, Tassi E, Clavenna D, Papa I, Recalde H, Braga M, Di Carlo V, Doglioni C, Protti MP (2011) Intratumor T helper type 2 cell infiltrate correlates with cancer-associated fibroblast thymic stromal lymphopoietin production and reduced survival in pancreatic cancer. J Exp Med 208:469–478PubMedCrossRefGoogle Scholar
  31. 31.
    von Bernstorff W, Voss M, Freichel S, Schmid A, Vogel I, Jöhnk C, Henne-Bruns D, Kremer B, Kalthoff H (2001) Systemic and local immunosuppression in pancreatic cancer patients. Clin Cancer Res 7:925s–932sGoogle Scholar
  32. 32.
    Miyahara Y, Odunsi K, Chen W, Peng G, Matsuzaki J, Wang RF (2008) Generation and regulation of human CD4+ IL-17-producing T cells in ovarian cancer. Proc Natl Acad Sci USA 105:15505–15510PubMedCrossRefGoogle Scholar
  33. 33.
    Su X, Ye J, Hsueh EC, Zhang Y, Hoft DF, Peng G (2010) Tumor microenvironments direct the recruitment and expansion of human Th17 cells. J Immunology 184:1630–1641CrossRefGoogle Scholar
  34. 34.
    Kryczek I, Banerjee M, Cheng P, Vatan L, Szeliga W, Wei S, Huang E, Finlayson E, Simeone D, Welling TH, Chang A, Coukos G et al (2009) Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments. Blood 114:1141–1149PubMedCrossRefGoogle Scholar
  35. 35.
    Horlock C, Stott B, Dyson PJ, Morishita M, Coombes RC, Savage P, Stebbing J (2009) The effects of trastuzumab on the CD4+ CD25+ FoxP3+ and CD4+ IL17A+ T-cell axis in patients with breast cancer. Br J Cancer 100:1061–1067PubMedCrossRefGoogle Scholar
  36. 36.
    Benchetrit F, Ciree A, Vives V, Warnier G, Gey A, Sautès-Fridman C, Fossiez F, Haicheur N, Fridman WH, Tartour E (2002) Interleukin-17 inhibits tumor cell growth by means of a T-cell-dependent mechanism. Blood 99:2114–2121PubMedCrossRefGoogle Scholar
  37. 37.
    Sfanos KS, Bruno TC, Maris CH, Xu L, Thoburn CJ, DeMarzo AM, Meeker AK, Isaacs WB, Drake CG (2008) Phenotypic analysis of prostate-infiltrating lymphocytes reveals TH17 and Treg skewing. Clin Cancer Res 4:3254–3261CrossRefGoogle Scholar
  38. 38.
    Tartour E, Fossiez F, Joyeux I, Galinha A, Gey A, Claret E, Sastre-Garau X, Couturier J, Mosseri V, Vives V, Banchereau J, Fridman WH et al (1999) Interleukin 17, a T-cell-derived cytokine, promotes tumorigenicity of human cervical tumors in nude mice. Cancer Res 59:3698–3704PubMedGoogle Scholar
  39. 39.
    Numasaki M, Fukushi J, Ono M, Narula SK, Zavodny PJ, Kudo T, Robbins PD, Tahara H, Lotze MT (2003) Interleukin-17 promotes angiogenesis and tumor growth. Blood 101:2620–2627PubMedCrossRefGoogle Scholar
  40. 40.
    Alvarez E, Moga E, Barquinero J, Sierra J, Briones J (2009) Dendritic and tumor cell fusions transduced with adenovirus encoding CD40L eradicate B-cell lymphoma and induce a Th17-type response. Gene Ther 17:469–477PubMedCrossRefGoogle Scholar
  41. 41.
    Kuang DM, Peng C, Zhao Q, Wu Y, Chen MS, Zheng L (2010) Activated monocytes in peritumoral stroma of hepatocellular carcinoma promote expansion of memory T helper 17 cells. Hepatology 51:154–164PubMedCrossRefGoogle Scholar
  42. 42.
    Kyte JA, Trachsel S, Risberg B, Thor Straten P, Lislerud K, Gaudernack G (2009) Unconventional cytokine profiles and development of T cell memory in long-term survivors after cancer vaccination. Cancer Immunol Immunother 58:1609–1626PubMedCrossRefGoogle Scholar
  43. 43.
    Martin-Orozco N, Muranski P, Chung Y, Yang XO, Yamazaki T, Lu S, Hwu P, Restifo NP, Overwijk WW, Dong C (2009) T helper 17 cells promote cytotoxic T cell activation in tumor immunity. Immunity 31:787–798PubMedCrossRefGoogle Scholar
  44. 44.
    von Euw E, Chodon T, Attar N, Jalil J, Koya RC, Comin-Anduix B, Ribas A (2009) CTLA4 blockade increases Th17 cells in patients with metastatic melanoma. J Transl Med 7:35CrossRefGoogle Scholar
  45. 45.
    Derhovanessian E, Adams V, Hähnel K, Groeger A, Pandha H, Ward S, Pawelec G (2009) Pretreatment frequency of circulating IL-17+ CD4+ T-cells, but not Tregs, correlates with clinical response to whole-cell vaccination in prostate cancer patients. Int J Cancer 125:1372–1379PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Amedeo Amedei
    • 1
    • 2
    Email author
  • Elena Niccolai
    • 1
    • 2
  • Marisa Benagiano
    • 1
    • 2
  • Chiara Della Bella
    • 1
    • 2
  • Fabio Cianchi
    • 3
  • Paolo Bechi
    • 2
    • 4
  • Antonio Taddei
    • 2
    • 4
  • Lapo Bencini
    • 5
  • Marco Farsi
    • 5
  • Paola Cappello
    • 6
    • 7
  • Domenico Prisco
    • 2
    • 3
  • Francesco Novelli
    • 6
    • 7
  • Mario Milco D’Elios
    • 1
    • 2
  1. 1.Division of Internal Medicine, Department of Experimental and Clinical MedicineUniversity of FlorenceFlorenceItaly
  2. 2.Department of BiomedicineAzienda Ospedaliera Universitaria Careggi (AOUC)FlorenceItaly
  3. 3.Department of Medical and Surgical Critical CareUniversity of FlorenceFlorenceItaly
  4. 4.Department of Surgery and Translational MedicineUniversity of FlorenceFlorenceItaly
  5. 5.Division of General and Oncologic Surgery, Department of OncologyAOUCFlorenceItaly
  6. 6.Centre for Experimental Research and Medical Studies (CERMS)Azienda Ospedaliera Città della Salute e della Scienza di TorinoTurinItaly
  7. 7.Department of Molecular Biotechnology and Health SciencesUniversity of TurinTurinItaly

Personalised recommendations