Skip to main content


Log in

Disruption of CTLA-4 expression on peripheral blood CD8 + T cell enhances anti-tumor efficacy in bladder cancer

  • Original Article
  • Published:
Cancer Chemotherapy and Pharmacology Aims and scope Submit manuscript


Activation of programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) on T cells leads to T cell exhaustion and ultimately facilitates tumor progression. Recent success of using immune cell checkpoint inhibitors offers a great promise to treat various cancers, including bladder cancer. However, the expression pattern and therapeutic value of PD-1 and CTLA-4 in peripheral blood T cells remain largely unexplored. In this study, we presume that disruption of the potential dysregulated checkpoint molecules in peripheral blood T cells may improve the anti-tumor efficacy of cytotoxic T cells in bladder cancer. We showed that both PD-1 and CTLA-4 expression were specifically elevated on CD8 + T cells but not CD4 + T cells in peripheral blood of patients with bladder cancer compared with that in healthy donors. Notably, CTLA-4 expression was significantly higher in muscle-invasive bladder cancer (MIBC) and correlated with tumor size. By blocking CTLA-4 with anti-CTLA-4 antibody and CRISPR-Cas9-mediated CTLA-4 disruption, we revealed that CTLA-4-disrupted CTLs had enhanced cellular immune response and superior cytotoxicity to the CD80/CD86-positive bladder cancer cells in vitro. Moreover, the CTLA-4-disrupted CTLs exhibited a pronounced anti-tumor effect in vivo as demonstrated by prophylactic assay and therapeutic assay in the subcutaneous xenograft model. Collectively, our findings confirm improved therapeutic efficacy of CTLA-4-disrupted CTLs and provides the potential strategy for targeting immune checkpoints to enhance the promising immunotherapy.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others


  1. Siegel RL, Miller KD, Jemal A (2016) Cancer statistics, 2016. CA Cancer J Clin 66:7–30

    Article  PubMed  Google Scholar 

  2. Miyamoto DT, Mouw KW, Feng FY, Shipley WU, Efstathiou JA (2018) Molecular biomarkers in bladder preservation therapy for muscle-invasive bladder cancer. Lancet Oncol 19:e683–e695

    Article  CAS  PubMed  Google Scholar 

  3. Zhang J, Bu X, Wang H, Zhu Y, Geng Y, Nihira NT, Tan Y, Ci Y, Wu F, Dai X, Guo J, Huang YH, Fan C, Ren S, Sun Y, Freeman GJ, Sicinski P, Wei W (2018) Cyclin D-CDK4 kinase destabilizes PD-L1 via cullin 3-SPOP to control cancer immune surveillance. Nature 553:91–95

    Article  CAS  PubMed  Google Scholar 

  4. Kim HS, Seo HK (2018) Immune checkpoint inhibitors for urothelial carcinoma. Investig Clin Urol 59:285–296

    Article  PubMed  PubMed Central  Google Scholar 

  5. Powles T, Morrison L (2018) Biomarker challenges for immune checkpoint inhibitors in urothelial carcinoma. Nat Rev Urol 15:585–587

    Article  PubMed  Google Scholar 

  6. El Rassy E, Assi T, Bakouny Z, Pavlidis N, Kattan J. Beyond first-line systemic treatment for metastatic urothelial carcinoma of the bladder. Clin Transl Oncol 2018 21:280–288

    Article  PubMed  Google Scholar 

  7. Zhao R, Song Y, Wang Y, Huang Y, Li Z, Cui Y, Yi M, Xia L, Zhuang W, Wu X (2019) Zhou Y PD-1/PD-L1 blockade rescue exhausted CD8 + T cells in gastrointestinal stromal tumours via the PI3K/Akt/mTOR signalling pathway. Cell Prolif.

    Article  PubMed  Google Scholar 

  8. Tan J, Chen S, Lu Y, Yao D, Xu L, Zhang Y, Yang L, Chen J, Lai J, Yu Z, Zhu K, Li Y (2017) Higher PD-1 expression concurrent with exhausted CD8 + T cells in patients with de novo acute myeloid leukemia. Chin J Cancer Res 29:463–470

    Article  PubMed  PubMed Central  Google Scholar 

  9. Jiang X, Wang J, Deng X, Xiong F, Ge J, Xiang B, Wu X, Ma J, Zhou M, Li X, Li Y, Li G, Xiong W, Guo C, Zeng Z (2019) Role of the tumor microenvironment in PD-L1/PD-1-mediated tumor immune escape. Mol Cancer 18:10

    Article  PubMed  PubMed Central  Google Scholar 

  10. Fu C, Jiang A (2018) Dendritic cells and CD8 T cell immunity in tumor microenvironment. Front Immunol 9:3059

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Li J, Shayan G, Avery L, Jie HB, Gildener-Leapman N, Schmitt N, Lu BF, Kane LP, Ferris RL (2016) Tumor-infiltrating Tim-3(+) T cells proliferate avidly except when PD-1 is co-expressed: evidence for intracellular cross talk. Oncoimmunology 5:e1200778

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Dejaegher J, Verschuere T, Vercalsteren E, Boon L, Cremer J, Sciot R, Van Gool SW, De Vleeschouwer S (2017) Characterization of PD-1 upregulation on tumor-infiltrating lymphocytes in human and murine gliomas and preclinical therapeutic blockade. Int J Cancer 141:1891–1900

    Article  CAS  PubMed  Google Scholar 

  13. Siddiqui I, Schaeuble K, Chennupati V, Fuertes Marraco SA, Calderon-Copete S, Pais Ferreira D, Carmona SJ, Scarpellino L, Gfeller D, Pradervand S, Luther SA, Speiser DE, Held W (2019) Intratumoral Tcf1(+)PD-1(+)CD8(+) T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. Immunity 50:195–211 e110

    Article  CAS  PubMed  Google Scholar 

  14. Kurtulus S, Madi A, Escobar G, Klapholz M, Nyman J, Christian E, Pawlak M, Dionne D, Xia J, Rozenblatt-Rosen O, Kuchroo VK, Regev A, Anderson AC (2019) Checkpoint blockade immunotherapy induces dynamic changes in PD-1(-)CD8(+) tumor-infiltrating T cells. Immunity 50:181–194 e186

    Article  CAS  PubMed  Google Scholar 

  15. Felsenstein KM, Theodorescu D. Precision medicine for urothelial bladder cancer: update on tumour genomics and immunotherapy. Nat Rev Urol 2017 15:92–111

    Article  CAS  PubMed  Google Scholar 

  16. Kamphorst AO, Pillai RN, Yang S, Nasti TH, Akondy RS, Wieland A, Sica GL, Yu K, Koenig L, Patel NT, Behera M, Wu H, McCausland M, Chen Z, Zhang C, Khuri FR, Owonikoko TK, Ahmed R, Ramalingam SS (2017) Proliferation of PD-1 + CD8 T cells in peripheral blood after PD-1-targeted therapy in lung cancer patients. Proc Natl Acad Sci USA 114:4993–4998

    Article  CAS  PubMed  Google Scholar 

  17. Zhang W, Bai JF, Zuo MX, Cao XX, Chen M, Zhang Y, Han X, Zhong DR, Zhou DB (2016) PD-1 expression on the surface of peripheral blood CD4(+) T cell and its association with the prognosis of patients with diffuse large B-cell lymphoma. Cancer Med 5:3077–3084

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hultquist JF, Hiatt J, Schumann K, McGregor MJ, Roth TL, Haas P, Doudna JA, Marson A, Krogan NJ (2019) CRISPR-Cas9 genome engineering of primary CD4(+) T cells for the interrogation of HIV-host factor interactions. Nat Protoc 14:1–27

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Rupp LJ, Schumann K, Roybal KT, Gate RE, Ye CJ, Lim WA, Marson A (2017) CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Sci Rep 7:737

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Su S, Zou Z, Chen F, Ding N, Du J, Shao J, Li L, Fu Y, Hu B, Yang Y, Sha H, Meng F, Wei J, Huang X, Liu B (2017) CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer. Oncoimmunology 6:e1249558

    Article  CAS  PubMed  Google Scholar 

  21. Davarpanah NN, Yuno A, Trepel JB, Apolo AB. Immunotherapy: a new treatment paradigm in bladder cancer. Curr Opin Oncol 2017 29:184–195

    Article  CAS  PubMed Central  Google Scholar 

  22. Zhou G, Sprengers D, Boor PPC, Doukas M, Schutz H, Mancham S, Pedroza-Gonzalez A, Polak WG, de Jonge J, Gaspersz M, Dong H, Thielemans K, Pan Q, JNM IJ Bruno MJ Kwekkeboom J (2017) Antibodies against immune checkpoint molecules restore functions of tumor-infiltrating T cells in hepatocellular carcinomas. Gastroenterology 153:1107–1119

    Article  CAS  PubMed  Google Scholar 

  23. Duraiswamy J, Kaluza KM, Freeman GJ, Coukos G (2013) Dual blockade of PD-1 and CTLA-4 combined with tumor vaccine effectively restores T-cell rejection function in tumors. Cancer Res 73:3591–3603

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lussier DM, Johnson JL, Hingorani P, Blattman JN (2015) Combination immunotherapy with alpha-CTLA-4 and alpha-PD-L1 antibody blockade prevents immune escape and leads to complete control of metastatic osteosarcoma. J Immunother Cancer 3:21

    Article  PubMed  PubMed Central  Google Scholar 

  25. Di Nunno V, De Luca E, Buttigliero C, Tucci M, Vignani F, Gatto L, Zichi C, Ardizzoni A, Di Maio M, Massari F (2018) Immune-checkpoint inhibitors in previously treated patients with advanced or metastatic urothelial carcinoma: a systematic review and meta-analysis. Crit Rev Oncol Hematol 129:124–132

    Article  PubMed  Google Scholar 

  26. Tripathi A, Plimack ER (2018) Immunotherapy for urothelial carcinoma: current evidence and future directions. Curr Urol Rep 19:109

    Article  PubMed  Google Scholar 

  27. Buchbinder E, Hodi FS (2015) Cytotoxic T lymphocyte antigen-4 and immune checkpoint blockade. J Clin Investig 125:3377–3383

    Article  PubMed  Google Scholar 

  28. Sheik Ali S, Goddard AL, Luke JJ, Donahue H, Todd DJ, Werchniak A, Vleugels RA (2015) Drug-associated dermatomyositis following ipilimumab therapy: a novel immune-mediated adverse event associated with cytotoxic T-lymphocyte antigen 4 blockade. JAMA Dermatol 151:195–199

    Article  PubMed  Google Scholar 

  29. Su S, Hu B, Shao J, Shen B, Du J, Du Y, Zhou J, Yu L, Zhang L, Chen F, Sha H, Cheng L, Meng F, Zou Z, Huang X, Liu B (2016) CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients. Sci Rep 6:20070

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chapuis AG, Lee SM, Thompson JA, Roberts IM, Margolin KA, Bhatia S, Sloan HL, Lai I, Wagener F, Shibuya K, Cao J, Wolchok JD, Greenberg PD, Yee C (2016) Combined IL-21-primed polyclonal CTL plus CTLA4 blockade controls refractory metastatic melanoma in a patient. J Exp Med 213:1133–1139

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to Jianhui Cai.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, W., Shi, L., Zhao, Z. et al. Disruption of CTLA-4 expression on peripheral blood CD8 + T cell enhances anti-tumor efficacy in bladder cancer. Cancer Chemother Pharmacol 83, 911–920 (2019).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: