| Objective: In the past decades, the vaccine research and development has made made great achievements. The classical vaccine development strategy such as live attenuated vaccines, inactivated vaccine, toxoid and subunit vaccines were successful in a variety of bacterial and viral vaccines. But for many types of intracellular parasitic pathogen, the development of effective vaccines was still difficult. With many advantages, such as inducing both humoral immune response as well as cellular immune responses; encoding a variety of antigens, which constitute a multivalent vaccine; without the risk of virulence recovery; no risks of heterologous immune response that occurred in subunit vaccine due to insufficient protein purification; simple to design and easy to preparation, low cost; relatively stable, resistant to 50℃temperature, which made it can be delivered and stored without refrigeration system, etc, the development of DNA vaccines provides a new way for the development of the vaccine. However, after more than 10 years of research, there is not yet DNA vaccine available in clinical for disease prevention and control. The most important reason is that immune protection induced by DNA vaccine is not strong enough. to overcome the poor protection of DNA vaccine. our laboratory and other researchers have explored a variety of methods, including the use of different inoculation methods to improve the efficiency of transfection of host cell; optimizing the genetic code, or the transcriptional regulatory element to improve the expression of plasmid encoded antigen; co-administration of dendritic cell-specific growth factor FLT3l, GM-CSF, MIP-1αencoding plasmids with the DNA vaccine, to enhance the antigen-presenting effect; application cytokines encoding plasmid such as IL-2, IL-12, IFN-γ, etc, to increase the expansion of T lymphocytes and the application of different vectors, to enhancing the effect of immune protection of DNA vaccine, but failed to meet the expected results.The mechanism of DNA vaccines inducing the immune response was not fully understood. It is generally believed that, when the DNA vaccine was inoculated, dendritic cells and other professional antigen presenting cells can be transfected directly, or through the uptake of antigen expression in somatic cells and dead antigen expression cells, the antigen encoded by the plasmid can be produced, processing, and presented by the way of MHC-I, MHC-II antigen complex or cross presentation to the immune active cells to induce immune responses, was the primary mechanism. Muscle tissue, as the commonly used site of inoculation of DNA vaccine, can efficiently express target antigen, but as a non-professional antigen presenting cells, the muscle cells lack the expression of B7.1, B7.2 and other costimulatory molecules, therefore its role in the inducing the immune response in DNA vaccination was controversial. It is reported that the muscle cells, as a non-professional antigen presenting cells, can induce immune responses in the process of DNA vaccine, but more research shows that muscle cells can leads to immune tolerance rather than immune stimulation in this process, the mechanism may involve the activation of PD-L1/PD-1 negative immune regulation pathway. As the attempts to improve the immunogenicity of DNA vaccines was failed to reach a satisfactory immune effects as mentioned above, We believe that from another perspective, blocking negative regulator signal pathway may be a feasible way to solve the problem of DNA vaccine.Based on the above considerations, the mice NOR-10 cells were treated with different cytokines to mimic the microenvironment encounted activated T cell and muscle cell after DNA vaccine inoculation, the expression of cellular immune related molecule MHC-I, costimulatory molecules B7 and negative immune regulator molecules PD-L1 were evaluated by RT-PCR and flow cytometry to investigate the possible role of muscle cells in the induction of immune response and the possible of improving the effect of DNA vaccine by blocking the negative regulator of signal pathway mentioned PD-L1/PD-1.Methods:⑴Stimulating cells in vitro: NOR-10 cells were cultured at the density of 5×105 /ml in a 100ml flask, 24 hours later, the cells were treated with interferon-γ(500U/ml), interleukin-4 (IL-4, 50g/ml), interferon-β(IFN-β, 500U/ml), untreated cells with fresh medium as a normal control group.⑵Trizol extraction of total cellular RNA: After treated with different cytokines, total cellular RNA were extracted, at 3 hour, 6 hour, 12 hour, 24 hour, 48 hour time points, respectively, untreated normal cells were used as control, RT-PCR were performed using different primers.⑶Detection cell surface proteins by flow cytometry: Cells treated with different cytokines were harvested at 12 hour, 24 hour, 48 hour, respectively, 1×106 cells / each sample were stained with different antibodies and the surface proteins were detected by flow cytometry, untreated cells were used as normal control group.Result: (1) The expression of PD-L1 coinhibiting molecules: We confirmed that PD-L1 mRNA was expressed wakely but not PD-L1 protein was expressed in normal NOR-10 cell. IFN-γ(500U/ml) and IFN-β(500U/ml) stimulation could increase PD-L1 expression both at mRNA and protein levels. (2) The expression of molecules MHC-I: We found that the normal NOR-10 cells had a small amount of MHC-I mRNA expression, IFN-γand IFN-βtreatment increased the expression of MHC-I at mRNA level. There was no MHC-I protein expression on untreated cells. IFN-βcould induce expression of MHC-I molecules, but IFN-γnot. (3) Expression of costimulatory molecule B7:Under normal circumstances, cultured NOR-10 cells expressed a small amount of B7.1 mRNA, but no expression of B7.2 mRNA. Given stimulation by IFN-γand IFN-β, B7.1 mRNA had no significant change in expression and B7.2 mRNA still negative. (4) When cells were stimulated with IL-4, both PD-L1 and MHCI were not affected.Conlusion: (1) Normal NOR-10 cells constitutively expressed PD-L1, MHC-I and B7.1 mRNA, but no B7.2 mRNA expression; (2) There was no MHC-I, co-stimulatory molecules B7.1 protein expression in normal NOR-10 cells, as well as co-suppression molecule PD-L1; (3) IFN-γcould up-regulate PD-L1 mRNA and protein expression, but had no effect on B7.1 and B7.2 mRNA; (4)MHC-I mRNA other than protain could be up-regulated by IFN-γ; (5) IFN-βcould increase the mRNA and protein expression of PD-L1 and MHC-I,but have no impact on the B7.1 and B7.2; (6) IL-4 had no effect on PD-L1 and MHC-I, B7.1expression. |
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