[Construction of human breast carcinoma cell line transfected with OX40L and its costimulatory effect on T cells].
OBJECTIVE
Costimulator OX40 (CD134) belongs to TNFR family, and its ligand OX40L (CD134L) belongs to TNF family. OX40-OX40L signal plays an important role in immune regulation, which can increase the production of cytokines and enhance the survival of antigen-specific T cells. This research was to transfect OX40L into human breast cancer cell line MDA-MB-435 to construct OX40L-MDA-MB-435 cells, and to investigate the costimulatory effect of OX40-OX40L signal on biological activity of T cells in vitro.


METHODS
cDNA fragment encoding OX40L was obtained from human mature dendritic cells by reverse transcription-polymerase chain reaction (RT-PCR), and inserted into eukaryotic expression vector pcDNA3.1. The recombinant plasmid pcDNA3.1-OX40L was transfected into MDA-MB-435 cells, and verified by indirect immunophenotyping and RT-PCR. The effect of OX40L-MDA-MB-435 cells to the biological activity of T cells was investigated by MTT, enzyme-linked immunosorbent assay (ELISA), and direct immunophenotyping.


RESULTS
OX40L-MDA-MB-435 cells were successfully constructed. These cells efficiently promoted the proliferation of T cells, enhanced the secretion of interleukin-2 (IL-2) (973.4 ng/ml at the 7th day) and interferon-gamma (IFN-gamma) (3,689.167 pg/ml at the 3rd day), and down-regulated the expression of Fas on T cells [(68.3+/-5.6)%, P<0.05].


CONCLUSIONS
OX40L-MDA-MB-435 cells could activate T cells in vitro, promote T cell proliferation and cytokine secretion, and suppress T cell activation-induced cell death.
Medical Biotechnology Institute, Soochow University, Suzhou, Jiangsu 215007, P. R. China.
Qin
Q
Shi
Qin
Q
Wang
Yong-Jing
YJ
Chen
Jian-Jun
JJ
Sun
Yan
Y
Ge
Xue-Guang
XG
Zhang
D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018076:DNA, Complementary / Q000235:genetics; D003713:Dendritic Cells / Q000166:cytology; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D015847:Interleukin-4 / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D053278:OX40 Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D014162:Transfection; D048069:Tumor Necrosis Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D019014:fas Receptor
D018076:DNA, Complementary; C491901:FAS protein, human; D007376:Interleukin-2; D008562:Membrane Glycoproteins; D053278:OX40 Ligand; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; C089251:TNFSF4 protein, human; D048069:Tumor Necrosis Factors; D019014:fas Receptor; D015847:Interleukin-4; D007371:Interferon-gamma