The Study For The Mechanism Of The Negative Co-stimulatory Molecule B7-H4 In The Treatment Of EAE By C3H10 T1/2 Cells

Objective: To explore the mechanism of the negative co-stimulatory molecule B7-H4 in the treatment of EAE using mesenchymal stem cell line C3H10 T1/2(C3H10) cells,by investigating the role and mechanism of B7-H4 of C3H10 on T cell immune modulating,and by studying the proliferation, migration effect of MSC mediated by B7-H4.Methods: 1、The lentiviral vectors with mouse B7-H4 target sh RNA were transfected into mouse mesenchymal stem cell(C3H10-B7-H4); the expression of Ki-67 on C3H10 cell by B7-H4 knocked down was detected by flow cytometry. The proliferation abilities of different C3H10 Cells were observed by drawing growth curves. To study the migration abilities of these C3H10 cells with or without trasfection, the expression of chemokine receptor CXCR4 were detected by flow cytometry(FCM). Cell wound scratch assay was used to detect migration of these cells. C3H10 cell, C3H10-B7-H4 cell were co-cultured with the PHA activated mice spleen lymphocytes; ELISA was used to detecte the concentration of s B7-H4, IL-2, IL-17, IFN-γ and IL-4 in the supernatant of the co-culture system to explore the effect of C3H10 expressed B7-H4 on T cell polarizing.2 、 Experimental allergic encephalitis(EAE) mice models were immunized with peptide MOG35-55 in complete Freund’s adjuvant(CFA); C57BL/6 mice(n=50) were divided into five groups including: control group(n=10), EAE group(n=10), C3H10group(implanting C3H10 cells)(n=10), C3H10-NC group(implanting C3H10-NC cells)(n=10), and C3H10-B7-H4 group(implanting C3H10-B7-H4 cells)(n=10). The neurological impairment function score was measured everyday. 6 days after the immunization, about 1×106 C3H10 cells, C3H10-NC cells and C3H10-B7-H4 cells were injected to each EAE mouse respectively. In the acute and chronic aggravating stage of the disease, the expression of B7-H4 on peripheral blood cells was detected by flow cytometry.ELISA was used to detect the expression of s B7-H4、IL-2、IL-17、IFN-γ、IL-4 on plasma.Flow cytometry was used to detect the expression of B7-H4 on spleenocytes. Combining with the neural function defect score, the effect of transplantation of mice with different cells was evaluated.Result: 1、By knocking down B7-H4 on C3H10, the expression of B7-H4 on C3H10 were decreased significantly; the expression of Ki-67 and chemokine receptor CXCR4 was also decreased; the logarithmic growth phase prolonged; even more, wound healing time of C3H10-B7-H4 cells were significantly slower than that of the control cells. These result suggested the proliferation and migration capacity were diminished.The inhibitory effect of C3H10 on spleen lymphocyte secreting B7-H4、IL-2、IL-17、IL-4、IFN-γ was partly revised.2、The onset time of EAE group was earlier than that of the other groups; and the nerve function score was higher than that of the other groups. Transplantation of C3H10 cells into EAE mice can decrease and delay the attack. The onset time and nerve function score of C3H10-B7-H4 group were between C3H10 group and EAE group; the percentage of CD19+B7-H4+ B cells in peripheral blood and spleen from EAE group were increased; the number of cells in C3H10-B7-H4 group was between C3H10 group and EAE group. The concentration of soluble B7-H4 in EAE group mice plasma was lower than that of the normal group; the difference of IL-4 expression in the five groups is not obviously. The expression of IL-2, IL-17, IFN-γin EAE mice was significantly higher than those in the normal group. The expression of IL-2, IL-17 was inhibited by C3H10 cell transplantation. The inhibitory effect of IL-2、IL-17 was partly revised in C3H10-B7-H4 cells transplanting group.Conclusions:1、The B7-H4 molecule involved in the effect of immunoregulationof C3H10 cells for proliferation and activation of T cell, which might mediate by affecting the expression of soluble molecules, such as s B7-H4, IL-2, IL-17 and IFN-γ,which suggested the possible mechanism might be modulating T cells’ polarization.2、B7-H4 molecule played an important role in the treatment of EAE by C3H10 cells transplantation,which might mediate by affecting the proliferation, migration,immune-modulating and other biologic characteristics of C3H10 cells.3、The co-inhibitor molecule B7-H4 expressed on C3H10 cell involved in the effect of the treatment of EAE by C3H10 transplantation,which might mediate by affecting theexpression of IL-2, IL-17 and other related cytokines; in other words, C3H10 cells expressed B7-H4 might regulate T cell polarizing.