Dendritic Cells Engineered To Express GITRL Enhance Anti-tumor Immunity In Murine Lewis Lung Carcinoma

Lung cancer is among the most malignant tumors in human. Although a significant progress has been achieved in the treatment of lung cancer, the overall efficacy of various treatments is still limited. Thus, it has remained a major challenge in the development of more effective therapies. During recent years, dendritic cell (DC)-based immunotherapy has offered a novel promising approach for the therapeutic strategy for targeting lung cancers.Glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL), a newly discovered member of tumor necrosis factor (TNF) superfamily, are critically involved in the regulation of immune response via modulating the functions of both effector T cells and regulatory T (Treg) cells. In this study, we aimed to generate bone marrow-derived dendritic cells transfected with recombinant adenovirus expressing GITRL (pAd-GITRL-BMDC), and evaluate their therapeutic efficacy in a murine Lewis lung carcinoma model. In particular, we will determine whether pAd-GITRL-BMDC show any enhanced immune stimulatory functions and affect tumor growth by regulating the functions of both effector T cells and Treg cells in vivo. Our findings will facilitate the potential development of DC-based anti-tumor therapies.Ⅰ.Construction and verification of recombinant adenovirus expressing GITRLAim:The recombinant adenovirus expressing GITRL was produced and verified.Method:A 522bp cDNA product encoding the natural form of GITRL was cloned in an adenoviral pAdtrack-CMV vector. and then co-transfected into BJ5183 with backbone plasmid. The recombinant adenovirus were generated by transfecting the PacⅠlinearized viral genomes into the human embryonic kidney 293A cell line, which were then released from the cells by rapid freezing (-80℃) and thawing in a 37℃bath for five times. The viral titers were determined by TCID50 assay. The pAdTrack-CMV expressing eGFP was detected and verified by fluorescent microscopy after its transfection into 293A cells. The transfected cells showed marked cytopathic effect (CPE). The expression of target genes in 293A cells were further examined by qRT-PCR and Western blot analyses.Results:The target gene GITRL inserted into recombinant adenovirus was verified by restriction enzyme digestion and PCR analysis and further confirmed by sequencing analysis. The recombinant viral vectors showed high infectivity and formed viral particles in 293A cells with viral titers of 2.0 x 109 and 2.5 x 109 for pAd-GITRL and pAd-null, respectively, as detected by TCID50 assay. GFP expression was detected by fluorescence microscopy after pAd-GITRL transfection into 293A cells, and expression of GITRL transcripts was further confirmed by qRT-PCR analysis when compared to those in 293 A cells transfected with or without pAd-null vectors ( 2△ct×103:261.90±1.35 vs 0.36±0.039 vs 0). Accordingly, GITRL expression in pAd-GITRL transfected 293A cells was confirmed by Western blot analysis.Conclusion:These results have demonstrated that the constructed recombinant adenovirus can express mGITRL in 293A cells.II.Transfection of pAd-GITRL in mouse bone marrow-derived dendritic cellAim:To determine the changes of costimulatory molecules and MHC II expression in bone marrow-derived dendritic cell (BMDC) transfected with pAd-GITRL. To examine whether BMDC expressing GITRL show different effects on modulating the functions of CD4+CD25- and CD4+CD25+ T cells.Method:Bone marrow was obtained from tibia and femurs of C57BL/6 mice by flushing with the media. Marrow tissue was minced in a single-cell suspension through a nylon mesh. The cells (1.0×107) were cultured for 7 d in fresh RPMI 1640 medium containing 10% FCS, GM-CSF (10ng/mL) and IL-4 (10 ng/mL). The effect of pAd-GITRL-BMDC on anti-CD3 mAb induced CD4+CD25-T cells proliferation and the suppressive capacities of CD4+CD25+T cells was assessed by thymidine incorporation in vitro.Results:The pAd-GITRL efficiently transfected BMDC as revealed by fluorescent microscopic and flow cytometric analysis to examine eGFP expressed by the adenoviral vector. Flow cytometric analysis showed that pAd-GITRL-BMDC stably expressed high levels of GITRL in an 8-day culture. Further phenotypic analysis by flow cytometry revealed that no significant changes in the expression levels of CD40, CD80, CD86 and MHCⅡwere detected between pAd-null-BMDC and pAd-GITRL-BMDC. Mitomycin C-inactivated BMDC, pAd-null-BMDC and pAd-GITRL-BMDC were co-cultured with CD4+CD25- T cells. The effect of these DC on anti-CD3 mAb induced T cells proliferation was then assessed by thymidine incorporation. It was found that pAd-GITRL-BMDC markedly enhanced the proliferative capacity of CD4+ CD25- T cells when compared with BMDC and pAd-null-BMDC. The cpm value of pAd-GITRL-BMDC group was 6888±1304, compared with BMDC group (848±59) and pAd-null-BMDC group (1231±75) (p<0.05). The pAd-GITRL-BMDC significantly reduced the inhibitory function of regulatory T cells in suppressing CD4+ CD25- T cells proliferation. The percentage of inhibition of pAd-GITRL-BMDC group was 44.7±17.2%, compared with BMDC group (63.7±17.4%) and pAd-null-BMDC group (90.3±11.9%) (p<0.05).Conclusions:BMDC stably expressing GITRL were successfully generated. The pAd-GITRL-BMDC possess a costimulatory effect directly on CD4+CD25- T cells proliferation and can partially abrogate the immunosuppressive function exerted by Treg cells. Ⅲ. Dendritic cells engineered to express GITRL enhance therapeutic immunity in murine Lewis lung carcinomaAim:To explore whether pAd-GITRL-BMDC can enhance therapeutic efficacy response in a murine Lewis lung carcinoma model and seek its immune mechanisms.Method:To establish a tumor model, Lewis lung carcinoma cells (2.0×106) in 100μl of PBS were implanted subcutaneously (s.c.) in the midflank of C57BL/6 mice. BMDC, pAd-null-BMDC or pAd-GITRL-BMDC (1.0×106 cells) were incubated in the presence of Lewis lung carcinoma cells lysates (3.0×106 cells) in RPMI 1640 medium containing 10% FCS at 37℃and 5% CO2 for 24 h before their usage for vaccination. On days 10,12 and 14, groups of tumor-bearing mice were injected inside the tumor tissue with 1.0×106 BMDC, pAd-null-BMDC or pAd-GITRL-BMDC, respectively. As negative control, tumor-bearing mice were treated in parallel with PBS alone. The diameters of the tumors (width×length) were measured by calipers every 2-3d. The part of the mice were killed on day 20 for further analysis.FCM detected the number and proportion of CD8+IFN-γ+T, CD4+CD25+ Foxp3+ T and CD4+IL-17+ T cells in spleen of tumor-bearing mice, qRT-PCR revealed the expression of IFN-y, Foxp3, IL-17, ROR-γt and CCL20 in tumor tissues, CD8+IFN-γ+ T, CD4+Foxp3+ T and CD4+IL-17+ T cells in tumor tissues were stained by immunofluorescence.Results:Both mice survival rate and tumor growth were significantly inhibited in mice vaccinated with pAd-GITRL-BMDC. During the 70d period of observation, mice vaccinated with pAd-GITRL-BMDC exhibited a survival rate of 75% whereas mice vaccinated with pAd-null-BMDC showed a markedly reduced survival rate of 29%. In contrast, no mice survived in groups of animals vaccinated with BMDC and PBS. Flow cytometric analysis revealed that the frequency of splenic CD8+ IFN-γ+ T cells was significantly higher in mice vaccinated with pAd-GITRL-BMDC than that in the pAd-null-BMDC group (32.29±14.5% vs 24.32±6.83%)(p<0.05). The frequencies of CD4+CD25+Foxp3+ Treg cells did not show any obvious changes among various groups. However, the absolute number of splenic Treg cells was significantly decreased in pAd-GITRL-BMDC group as compared with pAd-null-BMDC group (p<0.05). Immunofluorescence characterization of T cells in tumor tissues revealed a large number of infiltrating CD8+IFN-γ+T cells and CD4+ IL-17+ T cells in pAd-GITRL-BMDC group. The number of CD4+FoxP3+ Treg cells in tumor tissues was lower in pAd-GITRL-BMDC group than that in pAd-null-BMDC group. Significantly increased levels of GITRL mRNA, IFN-γmRNA, IL-17 mRNA, ROR-γt mRNA and CCL20 mRNA were detected in tumor tissues from pAd-GITRL-BMDC group when compared with tumor tissues in mice treated with either pAd-null-BMDCs group, BMDC group or PBS group by quantitative real-time PCR analysis. Moreover, levels of Foxp3 transcripts in tumor tissues were markedly decreased in pAd-GITRL-BMDC group when compared with other experimental groups.Conclusions:Vaccination with pAd-GITRL-BMDC significantly retarded tumor growth and prolonged survival of tumor-bearing mice, which was accompanied with decreased numbers of CD4+CD25+Foxp3+ Treg cells and increased CD8+IFN-γ+T cells in peripheral lymphoid organs as well as in tumor tissues. There were also a large number of infiltrating CD4+IL-17+ T cells in tumor tissues, which is supported by markedly elevated levels of IL-17. These findings suggest pAd-GITRL-BMDC could retard tumor growth by enhancing the immune response in murine Lewis lung carcinoma.