CC BY 4.0 · Eur J Dent
DOI: 10.1055/s-0043-1764419
Original Article

High 4-1BB Expression in PBMCs and Tumor Infiltrating Lymphocytes (TILs) in Patients with Head and Neck Squamous Cell Carcinoma

Fardeela Bin-Alee*
1   Faculty of Medicine, Princess of Naradhiwas University, Narathiwat, Thailand
Nattharee Chunthagonesupawit*
2   Master of Science Program in Medical Sciences, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Tamonwan Meesakul
2   Master of Science Program in Medical Sciences, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Areeya Diloktaweewattana
3   Division of Dengue Hemorrhagic Fever Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
4   Siriraj Center of Research Excellence in Dengue and Emerging Pathogens, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
Patnarin Mahattanasakul
5   Department of Otolaryngology, Head and Neck Surgery, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand
6   Department of Otolaryngology, Head and Neck Surgery, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Apiwat Mutirangura
7   Center of Excellence in Molecular Genetics of Cancer and Human Diseases, Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Komkrit Ruangritchankul
8   Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Somboon Keelawat
8   Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Nakarin Kitkumthorn
9   Department of Oral Biology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand
› Author Affiliations


Objective 4-1BB is a costimulatory immune-activating molecule. Increased amounts of this protein have previously been found in the plasma of patients with oropharyngeal and oral cancer. Here, we focused on this molecule that functions as part of the immune system. We investigated 4-1BB in the peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) of patients with head and neck squamous cell cancer (HNSCC).

Materials and Methods The expression level of 4-1BB in the PBMCs was determined using real-time polymerase chain reaction (PCR). The TIMER (Tumor Immune Estimation Resource) web server was utilized to approximate the 4-1BB level in HNSCC TILs. Moreover, 4-1BB immunohistochemistry (IHC) was used to validate TILs in four organs of HNSCC, including oral cancer (OC), oropharyngeal cancer (OPC), sinonasal cancer (SNC), and laryngeal cancer (LC), in both the tumor area and adjacent normal epithelium. The difference in 4-1BB expression levels in various groups was assessed using a Kruskal-Wallis test and an independent sample t-test.

Results The level of 4-1BB expression in PBMCs was highest in OPC, followed by OC and healthy controls (HC). Significant differences were discovered between HC and OPC and between OC and OPC. Bioinformatics revealed a substantial correlation between 4-1BB expression level and lymphocyte infiltration in HNSCC, including B cells, CD8+ T cells, and CD4+ T cells. IHC validation in HNSCC tissue revealed that the average number of 4-1BB positive TILs in all four HNSCC subtypes was considerably greater than the number of lymphocytes seen in adjacent normal tissue. Interestingly, the number of lymphocytes that were 4-1BB positive increased in relation to the TIL level.

Conclusion A higher number of 4-1BB expression levels were found in the PBMCs and TILs of HNSCC patients, implying that 4-1BB may be a promising approach for HNSCC patients to improve their immune function. It is important to study and create a treatment that uses 4-1BB medicine as well as existing drugs.

* Co-first author

Supplementary Material

Publication History

Article published online:
02 May 2023

© 2023. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (

Thieme Medical and Scientific Publishers Pvt. Ltd.
A-12, 2nd Floor, Sector 2, Noida-201301 UP, India

  • References

  • 1 Johnson DE, Burtness B, Leemans CR, Lui VWY, Bauman JE, Grandis JR. Head and neck squamous cell carcinoma. Nat Rev Dis Primers 2020; 6 (01) 92
  • 2 Sung H, Ferlay J, Siegel RL. et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021; 71 (03) 209-249
  • 3 Chester C, Ambulkar S, Kohrt HE. 4-1BB agonism: adding the accelerator to cancer immunotherapy. Cancer Immunol Immunother 2016; 65 (10) 1243-1248
  • 4 Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood 2018; 131 (01) 49-57
  • 5 Cheuk AT, Mufti GJ, Guinn BA. Role of 4-1BB:4-1BB ligand in cancer immunotherapy. Cancer Gene Ther 2004; 11 (03) 215-226
  • 6 Buranapraditkun S, Mahattanasakul P, Diloktaweewattana A. et al. Immune-associated plasma proteins in oral and oropharyngeal cancer patients. Heliyon 2022; 8 (09) e10753
  • 7 Doescher J, Veit JA, Hoffmann TK. The 8th edition of the AJCC Cancer Staging Manual: updates in otorhinolaryngology, head and neck surgery. HNO 2017; 65 (12) 956-961
  • 8 Li T, Fan J, Wang B. et al. TIMER: a web server for comprehensive analysis of tumor-infiltrating immune cells. Cancer Res 2017; 77 (21) e108-e110
  • 9 Li T, Fu J, Zeng Z. et al. TIMER2.0 for analysis of tumor-infiltrating immune cells. Nucleic Acids Res 2020; 48 (W1): W509-W514
  • 10 Ruangritchankul K, Sandison A, Warburton F. et al. Clinical evaluation of tumour-infiltrating lymphocytes as a prognostic factor in patients with human papillomavirus-associated oropharyngeal squamous cell carcinoma. Histopathology 2019; 75 (01) 146-150
  • 11 Zhang GB, Dong QM, Hou JQ. et al. Characterization and application of three novel monoclonal antibodies against human 4-1BB: distinct epitopes of human 4-1BB on lung tumor cells and immune cells. Tissue Antigens 2007; 70 (06) 470-479
  • 12 Glorieux C, Huang P. Regulation of CD137 expression through K-Ras signaling in pancreatic cancer cells. Cancer Commun (Lond) 2019; 39 (01) 41
  • 13 Kim AMJ, Nemeth MR, Lim S-O. 4-1BB: A promising target for cancer immunotherapy. Front Oncol 2022; 12: 968360
  • 14 Suzuki S, Ogawa T, Sano R. et al. Immune-checkpoint molecules on regulatory T-cells as a potential therapeutic target in head and neck squamous cell cancers. Cancer Sci 2020; 111 (06) 1943-1957
  • 15 Wen T, Bukczynski J, Watts TH. 4-1BB ligand-mediated costimulation of human T cells induces CD4 and CD8 T cell expansion, cytokine production, and the development of cytolytic effector function. J Immunol 2002; 168 (10) 4897-4906
  • 16 Curran MA, Kim M, Montalvo W, Al-Shamkhani A, Allison JP. Combination CTLA-4 blockade and 4-1BB activation enhances tumor rejection by increasing T-cell infiltration, proliferation, and cytokine production. PLoS One 2011; 6 (04) e19499
  • 17 Chapman CH, Yom SS. Oropharyngeal Cancer. In: Hansen EK, Roach IM. eds. Handbook of Evidence-Based Radiation Oncology. Cham: Springer International Publishing; 2018: 171-192
  • 18 Sankaranarayanan R, Ramadas K, Amarasinghe H, Subramanian S, Johnson N. Oral cancer: prevention, early detection, and treatment. In: Gelband H, Jha P, Sankaranarayanan R, Horton S. eds. Cancer: Disease Control Priorities. 3rd ed. Vol. 3. Washington, DC: The International Bank for Reconstruction and Development/The World Bank; 2015
  • 19 Llorente JL, López F, Suárez C, Hermsen MA. Sinonasal carcinoma: clinical, pathological, genetic and therapeutic advances. Nat Rev Clin Oncol 2014; 11 (08) 460-472
  • 20 Pacini L, Cabal VN, Hermsen MA, Huang PH. EGFR Exon 20 insertion mutations in sinonasal squamous cell carcinoma. Cancers (Basel) 2022; 14 (02) 394
  • 21 Liu C, Wang M, Zhang H. et al. Tumor microenvironment and immunotherapy of oral cancer. Eur J Med Res 2022; 27 (01) 198
  • 22 Locati LD, Serafini MS, Carenzo A. et al. complete response to nivolumab in recurrent/metastatic HPV-positive head and neck squamous cell carcinoma patient after progressive multifocal leukoencephalopathy: a case report. Front Oncol 2022; 11: 799453
  • 23 Chu DT, Bac ND, Nguyen KH. et al. An update on anti-CD137 antibodies in immunotherapies for cancer. Int J Mol Sci 2019; 20 (08) 1822
  • 24 Segal NH, He AR, Doi T. et al. Phase I study of single-agent Utomilumab (PF-05082566), a 4-1BB/CD137 agonist, in patients with advanced cancer. Clin Cancer Res 2018; 24 (08) 1816-1823
  • 25 Cohen EEW, Pishvaian MJ, Shepard DR. et al. A phase Ib study of utomilumab (PF-05082566) in combination with mogamulizumab in patients with advanced solid tumors. J Immunother Cancer 2019; 7 (01) 342
  • 26 Hamid O, Chiappori AA, Thompson JA. et al. First-in-human study of an OX40 (ivuxolimab) and 4-1BB (utomilumab) agonistic antibody combination in patients with advanced solid tumors. J Immunother Cancer 2022; 10 (10) e005471
  • 27 Hong DS, Gopal AK, Shoushtari AN. et al. Utomilumab in patients with immune checkpoint inhibitor-refractory melanoma and non-small-cell lung cancer. Front Immunol 2022; 13: 897991
  • 28 Gao Y, Yang T, Liu H, Song N, Dai C, Ding Y. Development and characterization of a novel human CD137 agonistic antibody with anti-tumor activity and a good safety profile in non-human primates. FEBS Open Bio 2022; 12 (12) 2166-2178
  • 29 Shindo Y, Yoshimura K, Kuramasu A. et al. Combination immunotherapy with 4-1BB activation and PD-1 blockade enhances antitumor efficacy in a mouse model of subcutaneous tumor. Anticancer Res 2015; 35 (01) 129-136