Tumor Biology

, Volume 37, Issue 3, pp 3807–3816 | Cite as

Immunophenotyping of patients with oral squamous cell carcinoma in peripheral blood and associated tumor tissue

  • Martin Grimm
  • Oliver Feyen
  • Heiko Hofmann
  • Peter Teriete
  • Thorsten Biegner
  • Adelheid Munz
  • Siegmar Reinert
Original Article


The immune system is important for elimination of cancer cells. Tumors including oral squamous cell carcinoma (OSCC) are capable of escaping detection by host immune cells through apoptotic depletion of tumor-infiltrating lymphocytes (TILs). Circulating peripheral blood lymphocytes (PBLs) and corresponding TILs of tumor specimen were evaluated before and after curative tumor resection (n = 30) compared with PBLs of controls (n = 87). PBLs were characterized for the total number of T cells (CD3+), T helper cells (Th, CD3+/CD4+), regulatory T cells (Treg, CD4+/CD25+/CD127low), cytotoxic T cells (Tc, CD3+/CD8+), activated T cells (CD3+/HLA-DR+), and natural killer (NK) cells (CD3/CD16+/CD56+). In tumor tissue, the prevalence of CD3+, CD4+, and CD8+ TILs was assessed using immunohistochemistry, whereas the incidence of apoptosis was assessed using terminal deoxynucleotidyl transferase deoxyuridinetriphosphate nick-end labeling (TUNEL) assay. In PBLs of pretreated OSCC patients, a highly significant decrease in total number of T cells (p = 0.0001), Th cells (p < 0.0001), Treg cells (p < 0.0001), Tc cells (p < 0.0001), and NK cells (p = 0.0037) were found compared with controls. Decreased PBLs of OSCC patients were correlated with decreased numbers of corresponding TILs, which were associated with increased detection of apoptosis in the tumor tissue. Compared with the controls, the total number of T cells remained unchanged after surgery but the total number of NK cells significantly increased. Standardized immunophenotyping of OSCC may help to identify patients likely to benefit from cancer immunotherapy strategies and/or chemoradiation. Finally, future attempts to enhance an effective tumor-reactive immune response by immunotherapy or vaccination should be made by promoting tumor-specific Th and/or Tc cell/NK cell responses.


Oral squamous cell carcinoma Immunophenotyping Immunotherapy Cytotoxic T cells Regulatory T cells 



Peripheral blood lymphocytes


Tumor-infiltrating lymphocytes


Oral squamous cell carcinoma


T helper cells


Regulatory T cells


Cytotoxic T cells


Natural killer cells





We thank biovis’ Diagnostik MVZ especially Melanie Hügen and Martina Thümmler for the technical support.

Compliance with ethical standards

Authors’ contributions

MG and OF conceived the study, performed the coordination, and drafted the manuscript. OF performed flow cytometric analysis. TB and AM analyzed histopathological specimen and carried out immunohistochemistry studies. MG and PT carried out the data collection and performed the statistical analyses. SR performed surgical treatment, after care of the patients, and drafted the manuscript. All authors read and approved the final manuscript.

Conflicts of interest


Consent to participate

Written informed consent to participate was obtained prospectively from all patients (Ethics Committee Tübingen, Germany, approval number: 562-2013BO2).


  1. 1.
    Huang TY, Hsu LP, Wen YH, Huang TT, Chou YF, Lee CF, et al. Predictors of locoregional recurrence in early stage oral cavity cancer with free surgical margins. Oral Oncol. 2010;46(1):49–55. doi: 10.1016/j.oraloncology.2009.10.011.CrossRefPubMedGoogle Scholar
  2. 2.
    Badoual C, Sandoval F, Pere H, Hans S, Gey A, Merillon N, et al. Better understanding tumor-host interaction in head and neck cancer to improve the design and development of immunotherapeutic strategies. Head Neck. 2010;32(7):946–58. doi: 10.1002/hed.21346.PubMedGoogle Scholar
  3. 3.
    Zamarin D, Postow MA. Immune checkpoint modulation: rational design of combination strategies. Pharmacol Ther. 2015. doi: 10.1016/j.pharmthera.2015.01.003.PubMedGoogle Scholar
  4. 4.
    Duong CP, Yong CS, Kershaw MH, Slaney CY, Darcy PK. Cancer immunotherapy utilizing gene-modified T cells: from the bench to the clinic. Mol Immunol. 2015. doi: 10.1016/j.molimm.2014.12.009.PubMedGoogle Scholar
  5. 5.
    Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013;39(1):1–10. doi: 10.1016/j.immuni.2013.07.012.CrossRefPubMedGoogle Scholar
  6. 6.
    Gildener-Leapman N, Ferris RL, Bauman JE. Promising systemic immunotherapies in head and neck squamous cell carcinoma. Oral Oncol. 2013;49(12):1089–96. doi: 10.1016/j.oraloncology.2013.09.009.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Ferrone S, Whiteside TL. Tumor microenvironment and immune escape. Surg Oncol Clin N Am. 2007;16(4):755–74. doi: 10.1016/j.soc.2007.08.004. viii.CrossRefPubMedGoogle Scholar
  8. 8.
    Tanaka H, Yoshizawa H, Yamaguchi Y, Ito K, Kagamu H, Suzuki E, et al. Successful adoptive immunotherapy of murine poorly immunogenic tumor with specific effector cells generated from gene-modified tumor-primed lymph node cells. J Immunol. 1999;162(6):3574–82.PubMedGoogle Scholar
  9. 9.
    Dobrzanski MJ. Expanding roles for CD4 T cells and their subpopulations in tumor immunity and therapy. Front Oncol. 2013;3:63. doi: 10.3389/fonc.2013.00063.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Igney FH, Krammer PH. Immune escape of tumors: apoptosis resistance and tumor counterattack. J Leukoc Biol. 2002;71(6):907–20.PubMedGoogle Scholar
  11. 11.
    Gastman BR, Atarshi Y, Reichert TE, Saito T, Balkir L, Rabinowich H, et al. Fas ligand is expressed on human squamous cell carcinomas of the head and neck, and it promotes apoptosis of T lymphocytes. Cancer Res. 1999;59(20):5356–64.PubMedGoogle Scholar
  12. 12.
    Kassouf N, Thornhill MH. Oral cancer cell lines can use multiple ligands, including Fas-L, TRAIL and TNF-alpha, to induce apoptosis in Jurkat T cells: possible mechanisms for immune escape by head and neck cancers. Oral Oncol. 2008;44(7):672–82. doi: 10.1016/j.oraloncology.2007.08.013.CrossRefPubMedGoogle Scholar
  13. 13.
    Reichert TE, Strauss L, Wagner EM, Gooding W, Whiteside TL. Signaling abnormalities, apoptosis, and reduced proliferation of circulating and tumor-infiltrating lymphocytes in patients with oral carcinoma. Clin Cancer Res. 2002;8(10):3137–45.PubMedGoogle Scholar
  14. 14.
    Kim JW, Wieckowski E, Taylor DD, Reichert TE, Watkins S, Whiteside TL. Fas ligand-positive membranous vesicles isolated from sera of patients with oral cancer induce apoptosis of activated T lymphocytes. Clin Cancer Res. 2005;11(3):1010–20.PubMedGoogle Scholar
  15. 15.
    Hoffmann TK, Dworacki G, Tsukihiro T, Meidenbauer N, Gooding W, Johnson JT, et al. Spontaneous apoptosis of circulating T lymphocytes in patients with head and neck cancer and its clinical importance. Clin Cancer Res. 2002;8(8):2553–62.PubMedGoogle Scholar
  16. 16.
    Wolf GT, Hudson JL, Peterson KA, Miller HL, McClatchey KD. Lymphocyte subpopulations infiltrating squamous carcinomas of the head and neck: correlations with extent of tumor and prognosis. Otolaryngol Head Neck Surg. 1986;95(2):142–52.CrossRefPubMedGoogle Scholar
  17. 17.
    Wolf GT, Schmaltz S, Hudson J, Robson H, Stackhouse T, Peterson KA, et al. Alterations in T-lymphocyte subpopulations in patients with head and neck cancer. Correlations with prognosis. Arch Otolaryngol Head Neck Surg. 1987;113(11):1200–6.CrossRefPubMedGoogle Scholar
  18. 18.
    Badoual C, Hans S, Rodriguez J, Peyrard S, Klein C, Agueznay Nel H, et al. Prognostic value of tumor-infiltrating CD4+ T-cell subpopulations in head and neck cancers. Clin Cancer Res. 2006;12(2):465–72. doi: 10.1158/1078-0432.CCR-05-1886.CrossRefPubMedGoogle Scholar
  19. 19.
    Balermpas P, Michel Y, Wagenblast J, Seitz O, Weiss C, Rodel F, et al. Tumour-infiltrating lymphocytes predict response to definitive chemoradiotherapy in head and neck cancer. Br J Cancer. 2014;110(2):501–9. doi: 10.1038/bjc.2013.640.CrossRefPubMedGoogle Scholar
  20. 20.
    Wolf GT, Chepeha DB, Bellile E, Nguyen A, Thomas D, McHugh J, et al. Tumor infiltrating lymphocytes (TIL) and prognosis in oral cavity squamous carcinoma: a preliminary study. Oral Oncol. 2015;51(1):90–5. doi: 10.1016/j.oraloncology.2014.09.006.CrossRefPubMedGoogle Scholar
  21. 21.
    Gaur P, Qadir GA, Upadhyay S, Singh AK, Shukla NK, Das SN. Skewed immunological balance between Th17 (CD4(+)IL17A (+)) and Treg (CD4 (+)CD25 (+)FOXP3 (+)) cells in human oral squamous cell carcinoma. Cell Oncol (Dordr). 2012;35(5):335–43. doi: 10.1007/s13402-012-0093-5.CrossRefGoogle Scholar
  22. 22.
    Gasparoto TH, de Souza Malaspina TS, Benevides L, de Melo EJ, Costa Jr MR, Damante JH, et al. Patients with oral squamous cell carcinoma are characterized by increased frequency of suppressive regulatory T cells in the blood and tumor microenvironment. Journal of the Formosan immunol, immunother: CII. 2010;59(6):819–28. doi: 10.1007/s00262-009-0803-7.CrossRefGoogle Scholar
  23. 23.
    Lim KP, Chun NA, Ismail SM, Abraham MT, Yusoff MN, Zain RB, et al. CD4 + CD25hiCD127low regulatory T cells are increased in oral squamous cell carcinoma patients. PLoS One. 2014;9(8):e103975. doi: 10.1371/journal.pone.0103975.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Liu W, Putnam AL, Xu-Yu Z, Szot GL, Lee MR, Zhu S, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med. 2006;203(7):1701–11. doi: 10.1084/jem.20060772.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Ziegler SF. FOXP3: of mice and men. Annu Rev Immunol. 2006;24:209–26. doi: 10.1146/annurev.immunol.24.021605.090547.CrossRefPubMedGoogle Scholar
  26. 26.
    Drennan S, Stafford ND, Greenman J, Green VL. Increased frequency and suppressive activity of CD127(low/-) regulatory T cells in the peripheral circulation of patients with head and neck squamous cell carcinoma are associated with advanced stage and nodal involvement. Immunology. 2013;140(3):335–43. doi: 10.1111/imm.12144.PubMedPubMedCentralGoogle Scholar
  27. 27.
    Rammensee HG, Weinschenk T, Gouttefangeas C, Stevanovic S. Towards patient-specific tumor antigen selection for vaccination. Immunol Rev. 2002;188:164–76.CrossRefPubMedGoogle Scholar
  28. 28.
    Singh-Jasuja H, Emmerich NP, Rammensee HG. The Tubingen approach: identification, selection, and validation of tumor-associated HLA peptides for cancer therapy. Cancer immunology, immunotherapy : CII. 2004;53(3):187–95. doi: 10.1007/s00262-003-0480-x.CrossRefPubMedGoogle Scholar
  29. 29.
    Feyen O, Coy JF, Prasad V, Schierl R, Saenger J, Baum RP. EDIM-TKTL1 blood test: a noninvasive method to detect upregulated glucose metabolism in patients with malignancies. Future Oncol. 2012;8(10):1349–59. doi: 10.2217/fon.12.98.CrossRefPubMedGoogle Scholar
  30. 30.
    Grimm M, Cetindis M, Lehmann M, Biegner T, Munz A, Teriete P, et al. Association of cancer metabolism-related proteins with oral carcinogenesis—indications for chemoprevention and metabolic sensitizing of oral squamous cell carcinoma? J Transl Med. 2014;12:208. doi: 10.1186/1479-5876-12-208.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Zweig MH, Campbell G. Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clin Chem. 1993;39(4):561–77.PubMedGoogle Scholar
  32. 32.
    Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–74. doi: 10.1016/j.cell.2011.02.013.CrossRefPubMedGoogle Scholar
  33. 33.
    Miyazaki A, Kobayashi J, Torigoe T, Hirohashi Y, Yamamoto T, Yamaguchi A, et al. Phase I clinical trial of survivin-derived peptide vaccine therapy for patients with advanced or recurrent oral cancer. Cancer Sci. 2011;102(2):324–9. doi: 10.1111/j.1349-7006.2010.01789.x.CrossRefPubMedGoogle Scholar
  34. 34.
    Devarapu SK, Sharma SC, Das SN. Triggering of T cell-mediated immune responses by allogenic tumor cell vaccine in patients with oral cancer. Immunopharmacol Immunotoxicol. 2006;28(3):387–95. doi: 10.1080/08923970600927348.CrossRefPubMedGoogle Scholar
  35. 35.
    Ostrand-Rosenberg S. CD4+ T lymphocytes: a critical component of antitumor immunity. Cancer Invest. 2005;23(5):413–9.PubMedGoogle Scholar
  36. 36.
    Agarwal A, Mohanti BK, Das SN. Ex vivo triggering of T-cell-mediated immune responses by autologous tumor cell vaccine in oral cancer patients. Immunopharmacol Immunotoxicol. 2007;29(1):95–104. doi: 10.1080/08923970701282742.CrossRefPubMedGoogle Scholar
  37. 37.
    Timar J, Ladanyi A, Forster-Horvath C, Lukits J, Dome B, Remenar E, et al. Neoadjuvant immunotherapy of oral squamous cell carcinoma modulates intratumoral CD4/CD8 ratio and tumor microenvironment: a multicenter phase II clinical trial. J Clin Oncol. 2005;23(15):3421–32. doi: 10.1200/JCO.2005.06.005.CrossRefPubMedGoogle Scholar
  38. 38.
    Yeh CY, Lin CL, Chang MC, Chen HM, Kok SH, Chang SH, et al. Differences in oral habit and lymphocyte subpopulation affect malignant transformation of patients with oral precancer. J Formos Med Assoc=Taiwan yi zhi. 2015. doi: 10.1016/j.jfma.2015.07.017.Google Scholar
  39. 39.
    Tabata T, Hazama S, Yoshino S, Oka M. Th2 subset dominance among peripheral blood T lymphocytes in patients with digestive cancers. Am J Surg. 1999;177(3):203–8.CrossRefPubMedGoogle Scholar
  40. 40.
    Young M. Immunological phenotypes of premalignant oral lesions and the immune shifts with the development of head and neck cancer. Austin J Otolaryngol. 2014;1(2):7.Google Scholar
  41. 41.
    Ma Y, Zhang Z, Tang L, Xu YC, Xie ZM, Gu XF, et al. Cytokine-induced killer cells in the treatment of patients with solid carcinomas: a systematic review and pooled analysis. Cytotherapy. 2012;14(4):483–93. doi: 10.3109/14653249.2011.649185.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Martin Grimm
    • 1
  • Oliver Feyen
    • 2
  • Heiko Hofmann
    • 3
  • Peter Teriete
    • 4
  • Thorsten Biegner
    • 5
  • Adelheid Munz
    • 1
  • Siegmar Reinert
    • 1
  1. 1.Department of Oral and Maxillofacial SurgeryUniversity Hospital TübingenTübingenGermany
  2. 2.Zyagnum AG, Reißstraße 1aPfungstadtGermany
  3. 3.biovis’ Diagnostik MVZLimburg an der LahnGermany
  4. 4.Cancer Research CenterSanford-Burnham Medical Research InstituteLa JollaUSA
  5. 5.Department of PathologyUniversity Hospital TübingenTübingenGermany

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