Expression Of Extracellular Domain Of Co-inhibition Molecular VISTA And The Study On Its Treatment Of EAE In Mice

Research Background: Co-stimulate molecules play an important role in the regulation of T cell activation/apoptosis. Negatively co-stimulate molecules, also known as co-inhibition molecules, could activate negative signals to weak, inhibite, and even terminate the immune response. It can induce immune tolerance; maintain peripheral tolerance and finally prevent autoimmunity.It has been shown that co-inhibition molecules involved in the pathogenesis of MS/EAE, so these molecules are considered to be potential targets for the treatment of MS/EAE. VISTA(V-domain Ig suppressor of T cell activation), also known as PD-1H(Programmed death-1 homolog), is a novel co-inhibition molecule. It belongs to the immunoglobulin superfamily. VISTA widely expressed on the surface of hematopoietic cells, T cells, as well as antigen presenting cells(APCs) such as macrophages, dendritic cells and monocytes. The expression of VISTA always exhibited inhibition of T cell responses, regardless of expressed on APCs or T cells. Studies have proved that VISTAKO 2D2 mice showed significant higher morbidity and much serious in EAE, which suggest that VISTA is closely related with MS/EAE, and could increase the sensitivity of spontaneous EAE. To investigate the efficacy of VISTA treatment in EAE mice, in this study, extracellular domain of VISTA protein were successfully expressed in different expression systems. The EAE model was successfully established and the expression of VISTA m RNAs were examined in different courses of EAE mice. Finally, we studied the effect of extracellular domain of VISTA protein on EAE therapy.Research methods: 1. Prokaryotic expression vector p ET22b-VISTA was constructed and transformed into E. coli. Then the extracellular domain of VISTA protein was induced and purified. CCK8 was used to verify the extracellular domain of VISTA protein’s effect on proliferation of spleen cells.2. The yeast expression vector p PICZa A-VISTA was constructed and recombinant protein was induced by methanol. Mammalian expression vector PFUSE-VISTA-m Ig G2 a was also constructed and transfected into 293 T cells. The expression of recombinant protein was verified by RT-PCR and Western Blot. 3. MOG35-55 peptide was used as antigen to immunize C57BL/6 mice to establish the EAE model. The expression of VISTA m RNAs in different tissues during different courses of EAE mice were detected by Real-time PCR. 4. The extracellular domain of VISTA protein was used as treatment on EAE mice. The curative effects were verified by clinical score and pathological examination. And Real-time PCR assay was used to detect the expression of IFN-γ in brain and spleen in EAE mice.Research result: 1. The prokaryotic expression vector p ET22b-VISTA was successfully constructed, and the extracellular domain of VISTA protein was collected and purified. Result showed that extracellular domain of VISTA protein could significantly inhibited the proliferation of Con A-stimulated spleen cells; 2. We successfully constructed the yeast expression vector p PICZa A-VISTA, after induced by methanol, the host cells were successfully expressed the extracellular domain of VISTA protein; Mammalian expression vector p FUSE-VISTA-m Ig G2 a was also constructed and verified, the expression of Extracellular domain of VISTA protein was detected by western blot and Real-time PCR; 3. EAE model in mice was successfully established, and the incidence rate was 100%(proved by clinical score and pathological examination).The expression of VISTA m RNAs in the spleen and brain during different courses of EAE mice were detected by Real-time PCR, and we’ve found that the expression of VISTA correlated with different courses of EAE. 4. After injection of the extracellular domain of VISTA protein, the EAE mice showed the following results: the incidence of EAE mice was 100%, compared with 71% incidence in treatment group; onset time of treatment group were 14.40 ± 0.60 days, which significantly later than the EAE group(12.14 ± 0.40 days, ** p <0.01);the average clinical score of treatment group was much lower than the same period of EAE mice; H&E/LFB stained brain and spinal cord showed that the disease severity of treatment group were better than EAE group. The expression of IFN-γ gene in the brain and spleen of treatment group were significantly lower than EAE group.Conclusion: In this study we successfully constructed the recombinant vector p ET22b-VISTA by means of genetic engineering. Extracellular domain of VISTA protein with biological activity was obtained and purified. EAE model mice were successfully induced after immunization of MOG35-55 peptide. The expression of VISTA m RNA in the spleen and brain during different courses of EAE mice were detected by Real-time PCR, and this result proved that VISTA involved in the development of EAE courses. Injection of extracellular domain of VISTA protein in EAE mice could delay and suppress the onset of EAE, and could also reduce the severity of EAE mice. Extracellular domain of VISTA protein was able to decrease the expression IFN-γ in spleen and brain of EAE mice. This study provides a theoretical basis for the therapeutic effect of VISTA on MS/EAE diseases.