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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
RESEARCH ARTCILE |
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
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Abstract  

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,Email:fengzhg@public.wh.hb.cn   
Issue Date: 05 March 2009
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-009-0006-9     OR     http://academic.hep.com.cn/fmd/EN/Y2009/V3/I1/20
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
pg/mL
groupcytotoxicity to H22 cellscytotoxicity to B16 cells
10∶120∶110∶120∶1
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.
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