Please wait a minute...
Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front Med Chin    2009, Vol. 3 Issue (1) : 20-25     DOI: 10.1007/s11684-009-0006-9
4-1BBL expressed by eukaryotic cells activates immune cells and suppresses the progression of murine tumor
Hui QIU1, Hui ZHANG2, Zuohua FENG2()
1. Department of Oncology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China; 2. Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
Download: PDF(182 KB)   HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks

The interaction by co-stimulatory molecules 4-1BB and 4-1BB ligand (4-1BBL) plays an important role in the activation, proliferation and differentiation of T lymphocytes. The function of 4-1BB/4-1BBL expressed by the immune cells has been the focus for many tumor immunotherapy efforts. In this study, 4-1BBL was expressed in non-immune cells and non-tumor cells, and the role of 4-1BBL in lymphocyte activation and tumor suppression was investigated. The plasmid p4-1BBL containing the full length of mouse 4-1BBL cDNA sequence was constructed, and the plasmid was transfected into baby hamster kidney (BHK) cells and murine muscle cells by means of lipofectin-mediated or naked plasmid DNA injection into the muscle directly. The study demonstrated that the molecule 4-1BBL expressed by BHK cells in vitro could enhance the proliferation and cytotoxicity of lymphocytes, and it could increase the expression level of IL-2 and IFN-γ. The treatment with plasmid p4-1BBL in vivo revealed that the number of CD8+ T cells in the peri-tumoral tissue increased markedly, and the growth rate of the tumor was significantly lower than that of control group. These findings suggest that expression of 4-1BBL by normal cells in the tumor microenvironment can enhance the proliferation and other functions of T lymphocytes. This therapeutic method may provide a promising approach for tumor immunotherapy.

Keywords 4-1BB ligand      tumor immunotherapy      tumor microenvironment     
Corresponding Authors: FENG Zuohua,   
Issue Date: 05 March 2009
URL:     OR
Fig.1  Electrophoresis map of plasmid p4-1BBL digested by restriction enzymes. M: DNA marker; 1: p4-1BBL/d III+R I; 2: p4-1BBL/d III; 3: p4-1BBL.
Fig.2  Detection of expression of 4-1BBL on BHK cells by RT-PCR. M: DNA marker; 1: p4-1BBL transfected BHK cells; 2: pcDNA3.1 transfected BHK cells; 3: BHK cells control.
Fig.3  Detection of expression of 4-1BBL on BHK cells by western blot. 1: BHK cell control; 2: pcDNA3.1 transfected BHK cells; 3: p4-1BBL transfected BHK cells.
Fig.4  Detection of expression of 4-1BBL on BHK cells by immunocytochemistry (× 200)
Fig.5  Effect of 4-1BBL on the proliferation of spleen lymphocytes
groupconcentration of IL2concentration of IFN-γ
BHK cells49.68±12.65156.84±15.62
pcDNA3.1/BHK cells54.37±15.97178.64±18.47
P4-1BBL/BHK cells204.51±21.87**1249.55±25.69**
Tab.1  Concentration of IL2 and IFN-γ in supernatants of spleen lymphocytes detected by ELISA
groupcytotoxicity to H22 cellscytotoxicity to B16 cells
BHK cells42.59±2.5639.67±4.2513.21±2.5415.37±1.90
pcDNA3.1/BHK cells45.64±4.3141.56±6.8918.63±3.6716.38±2.37
p4-1BBL/BHK cells76.86±6.97**72.59±6.31**32.21±2.96?31.89±4.32?
Tab.2  Cytotoxicity effect of spleen lymphocytes to H22 cells and B16 cells by MTT
Fig.6  Detection of expression of 4-1BBL in muscle cells by immunohistochemistry (× 200). (a) p4-1BBL plasmid transfected muscle cells; (b) pcDNA3.1 plasmid transfected muscle cells.
Fig.7  Growth of murine tumor after muscle injection with plasmid
Fig.8  Distribution of CD8 T lymphocytes in tumor tissue by Immunohistochemical analysis(× 400). (a) The pcDNA3.1 plasmid control group; (b) the p4-1BBL plasmid treat group.
1 Goodwin R G, Din W S, Davis-Smith T, Anderson D M, Gimpel S D, Sato T A, Maliszewski C R, Brannan C I, Copeland N G, Jenkins N A. Molecular cloning of a ligand for the inducible T cell gene 4-1BB: a member of an emerging family of cytokines with homology to tumor necrosis factor. Eur J Immuno , 1993, 23(10): 2631-2641
doi: 10.1002/eji.1830231037
2 Pollok K E, Kim Y J, Zhou Z, Hurtado J, Kim K K, Pickard R T, Kwon B S. Inducible T cell antigen 4-1BB. Analysis of expression and function. J Immunol , 1993, 150(3): 771-781
3 Wilcox R A, Chapoval A I, Gorski K S, Otsuji M, Shin T, Flies D B, Tamada K, Mittler R S, Tsuchiya H, Pardoll D M, Chen L. Cutting edge: expression of functional CD137 receptor by dendritic cells. J Immunol , 2002, 168(9): 4262-4267
4 DeBenedette M A, Shahinian A, Mak T W, Watts T H. Costimulation of CD28-T lymphocytes by 4-1BB ligand. J Immunol , 1997, 158(2): 551-559
5 Cannons J L, Lau P, Ghumman B, DeBenedette M A, Yagita H, Okumura K, Watts T H. 4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy. J Immunol , 2001, 167(3): 1313-1324
6 Shuford W W, Klussman K, Tritchler D D, Loo D T, Chalupny J, Siadak A W, Brown T J, Emswiler J, Raecho H, Larsen C P, Pearson T C, Ledbetter J A, Aruffo A, Mittler R S. 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J Exp Med , 1997, 186(1): 47-55
doi: 10.1084/jem.186.1.47
7 Saoulli K, Lee S Y, Cannons J L, Yeh W C, Santana A, Goldstein M D, Bangia N, DeBenedette M A, Mak T W, Choi Y, Watts T H. CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand. J Exp Med , 1998, 187(11): 849-862
doi: 10.1084/jem.187.11.1849
8 Strome S E, Martin B, Flies D, Tamada K, Chapoval A I, Sargent DJ, Shu S, Chen L. Enhanced therapeutic potential of adoptive immunotherapy by in vitro CD28/4-1BB costimulation of tumor-reactive T cells against a poorly immunogenic, major histocompatibility complex class I-negative A9P melanoma. J Immunother , 2000, 23(4): 430-437
doi: 10.1097/00002371-200007000-00006
9 Xiang J. Expression of co-stimulatory 4-1BB ligand induces significant tumor regression and protective immunity. Cancer Biother Radiopharm , 1999, 14(5): 353-361
doi: 10.1089/cbr.1999.14.353
10 O'Driscoll L, Daly C, Saleh M, Clynes M. The use of reverse transcriptase-polymerase chain reaction (RT-PCR) to investigate specific gene expression in multidrug-resistant cells. Cytotechnology , 1993, 12(1-3): 289-314
doi: 10.1007/BF00744669
11 Lee N, Zhang S Q, Testa D. A rapid multicolor Western blot. J Immunol Methods , 1988, 106(1): 27-30
doi: 10.1016/0022-1759(88)90267-0
12 Schmidt-Kastner R, Meller D, Bellander B M, Str?mberg I, Olson L, Ingvar M. A one-step immunohistochemical method for detection of blood-brain barrier disturbances for immunoglobulins in lesioned rat brain with special reference to false-positive labeling in immunohistochemistry. J Neurosci Methods , 1993, 46(2): 121-132
doi: 10.1016/0165-0270(93)90147-J
13 Feng Z, Huang B, Zhang G, Li D, Wang H. Investigation on the effect of peptides mixture from tumor cells inducing anti-tumor specific immune response. Sci China C Life Sci , 2002, 45(4): 361-369
doi: 10.1360/02yc9040
14 Blachere N E, Li Z, Chandawarkar R Y, Suto R, Jaikaria N S, Basu S, Udono H, Srivastava P K. Heat shock protein-peptide complexes reconstituted in vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity. J Exp Med , 1997, 186(8): 1315-1322
doi: 10.1084/jem.186.8.1315
15 Huang B, Feng Z, Zhang G, Li D, Wang H. Hsp70-H22 tumor antigen peptide complex activated dendritic cell in the induction of antitumor immunity. Zhonghua Zhong Liu Za Zhi , 2002, 24(5): 421-425 (in Chinese)
16 Ferrari M, Fornasiero M C, Isetta A M. MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. J Immunol Methods , 1990, 131(2): 165-172
doi: 10.1016/0022-1759(90)90187-Z
17 Feng Z, Zhang G, Huang B, Li D, Wang H. Inducement of specific CTLs by antigen-peptides from human leukemia cells and their cytotoxicity to leukemia cells. J Huazhong Univ Sci Technol Med Sci , 2002, 22(4): 265-269
18 Kubota A, Lian R H, Lohwasser S, Salcedo M, Takei F. IFN-gamma production and cytotoxicity of IL-2-activated murine NK cells are differentially regulated by MHC class I molecules. J Immunol , 1999, 163(12): 6488-6493
19 Puliaev R, Nguyen P, Finkelman F D, Via C S. Differential requirement for IFN-gamma in CTL maturation in acute murine graft-versus-host disease. J Immunol , 2004, 173(2): 910-919
20 Langstein J, Becke F M, S?llner L, Krause G, Brockhoff G, Kreutz M, Andreesen R, Schwarz H. Comparative analysis of CD137 and LPS effects on monocyte activation, survival, and proliferation. Biochem Biophys Res Commun , 2000, 273(1): 117-122
doi: 10.1006/bbrc.2000.2889
21 Nam K O, Shin S M, Lee H W. Cross-linking of 4-1BB up-regulates IL-13 expression in CD8(+) T lymphocytes. Cytokine , 2006, 33(2): 87-94
doi: 10.1016/j.cyto.2005.12.003
22 Ju S A, Lee S C, Kwon T H, Heo S K, Park S M, Paek H N, Suh J H, Cho H R, Kwon B, Kwon B S, Kim B S. Immunity to melanoma mediated by 4-1BB is associated with enhanced activity of tumour-infiltrating lymphocytes. Immunol Cell Biol , 2005, 83(4): 344-351
doi: 10.1111/j.1440-1711.2005.01330.x
23 Lee H W, Park S J, Choi B K, Kim H H, Nam K O, Kwon B S. 4-1BB promotes the survival of CD8+ T lymphocytes by increasing expression of Bcl-xL and Bfl-1. J Immunol 2002, 169(9): 4882-4888
[1] Qiang Gao, Yinghong Shi, Xiaoying Wang, Jian Zhou, Shuangjian Qiu, Jia Fan. Translational medicine in hepatocellular carcinoma[J]. Front Med, 2012, 6(2): 122-133.
[2] Min Cheng, Jian Zhang, Wen Jiang, Yongyan Chen, Zhigang Tian. Natural killer cell lines in tumor immunotherapy[J]. Front Med, 2012, 6(1): 56-66.
[3] XU Qingwen, CHEN Weifeng. Developing effective tumor vaccines: basis, challenges and perspectives[J]. Front. Med., 2007, 1(1): 11-19.
Full text