Expression Of OX40L And OX40L-EGFP In B16F10 Cells And Their Effects On The Function Of Mouse Lymphocytes

Objective:The immune system is subject to many levels of control, and regulatory T (T reg) cells make a major contribution to such regulation. T reg cells can inhibit anti-tumor immune response in the tumor microenvironment. Therefore, T reg cells are the major obstacle for cancer immunotherapy. OX40 and OX40L convey a late co-stimulatory signal to activated T cells. OX40-OX40L interactions have been found to block natural Treg cells activity and antagonize inducible T reg cells. Recent studies found that transfection of tumor cells or dendritic cells with OX40L had been effective in suppressing tumor growth in melanoma model, lung carcinoma, thymoma animal models. The present study aims to construct a DNA vaccine based on OX40L eukaryotic expression vectors and to construct tumor cell vaccine which can stably express OX40L. These studies will pave the way for further investigation of immunotherapy of melanoma and related tumors.Methods:The cDNA of mouse OX40L was amplified by RT-PCR from the total RNA of spleen. The eukaryotic expression vector for OX40L was constructed by inserting the DNA fragments into pVAX1 plasmid. The expression of mouse OX40L gene in B16 cells transfected with this vector was detected by fluorescence staining. Meanwhile, the proliferation and apoptosis of the activated lymphocytes in tumor cell-mixed lymphocyte culture (MLC) were analyzed by CFSE and AnnexinV-PE staining, respectively. In the in vivo tumor model, the recombinant plasmid was injected into tumor-bearing mice to detect the antitumor effects. Meanwhile, using the eukaryotic expression vector pVAX1-OX40L cDNA as a template, the OX40L gene was amplified by PCR, and then ligated with pEGFP-C3 to construct the eukaryotic expression vector. The expression of this plasmid in B16 cells transfected with it was detected by fluorescence microscope. Both pEGFP-C3 and pEGFP-C3-OX40L were transfected into B16 cells, respectively. The monoclonized sublines that stable expressed EGFP or OX40L-EGFP were selected in the presence of G418. The morphology, proliferation, migration, invasion, cell cycle of B16/EGFP was examined. The expression of EGFP in tumor cells was detected by flow cytometry.Results:1. The cDNAs encoding mouse OX40L was cloned and two eukaryotic expression vectors were constructed successfully. DNA sequencing showed that OX40L had identical sequence to that in the GenBank.2. Flow cytometry and confocal microscopy showed that OX40L was expressed on the cell surface of B16 cells transfected with these plasmids. MLC results revealed that the proliferation ratio of OX40L gene-modified group was 26.27% (Con A+) and 26.95% (Con A-) while that of empty plasmid transfected group was 18.97%(Con A+) and 13.95%(Con A-), and the apoptosis ratio of OX40L gene-modified group was 6.57% while that of empty plasmid transfected group was 17.24%. After intra-tumoral administration of plasmid DNA, the antitumor effect was not obvious.3. After transfection of B16 cells with pEGFP-C3-OX40L expression vector, OX40L expression was observed under fluorescence microscopy. The B16 cell line stably expressing EGFP was successfully obtained whereas that expressing OX40L-EGFP was not successful. There were no obvious differences in the morphology, migration, invasion and cell cycle distribution between the B16/EGFP cell line and B16 cells. Yet, the growth rate of B16/EGFP was a little slow than B16 cells. In mouse tumor model, the tumor formed from B16/EGFP had a slow growth (P<0.05), compared with control B16F10 group. Moreover, the tumor cells freshly separated from the B16/EGFP tumor expressed EGFP.Conclusion:The two eukaryotic expression vectors for OX40L were constructed successfully and could be expressed in B16 cells. The expressed OX40L had significant biological activity. Integration and expression of EGFP have some effect on cell growth, but less effect on cell phenotype and tumor formation capacity. The B16 cell line stably transfected with EGFP may be valuable for further investigation on the metastasis mechanisms of melanoma.