ABSTRACT
Objective To explore the immunopathological mechanism for the imbalance between the positive signal mediated by inducible costimulator (ICOS) and the negative signal mediated by programmed death-1 (PD-1) in patients with myasthenia gravis (MG).Methods Eighty-two patients with MG,56 healthy controls (HC) and 20 non-MG (NMG) patients,collected in the First Affiliated Hospital of Suzhou University from February 2014 to December 2016,were chosen to participate in the study.The expression of ICOS and PD-1 on peripheral blood mononuclear cells was detected by immuno-fluorescence staining and flow cytometry.The levels of soluble programmed death-1 (sPD-1),soluble programmed death ligand 1 (sPD-L1),IL-4 and other cytokines were detected by enzyme-linked immunosorbent assay.Results (1) Flow cytometry analysis:The co-expression of PD-1,ICOS on CD4 + T cells from MG group (9.64％ (8.82％)) was higher than in HC (1.81％ (2.10％),Z =-7.389,P ＜0.05) and NMG group (2.86％ (1.49％),Z =-4.636,P ＜ 0.05).The expression of ICOS on CD4 + T cells,ICOS ligand (ICOSL) on CD14+ monocytes and CD19+ B cells were increased in MG group comparing with that of the control groups.The proportion of PD-1 + CD4 + T cells (MG group 16.82％ (10.66％),HC 9.34％ (9.18％),Z =-4.345,P＜0.05;NMG group 7.07％ (3.40％),Z=-4.594,P＜0.05) and PD-1 Ligand (PD-L1) + CD14+ monocytes was higher in MG patients.All of these were detected by flow cytometry.(2) ELISA analysis:Serum sPD-1 expression significantly increased in MG group compared with that in the control groups (MG group (1.87 ± 0.64) ng/ml,NMG group (1.49 ± 0.70) ng/ml,t =2.04,P ＜ 0.05;HC (1.05 ± 0.50)ng/ml,t =2.08,P ＜ 0.05),while for serum sPD-L1,there was no significant difference between MG and control groups.(3) Serum cytokines detection:The expression of IL-4 was increased in MG patients (MG group (61.88 ±5.15) pg/ml,HC (32.03 ±1.84) pg/ml,t=2.50,P＜0.05;NMG group (42.62± 3.31) pg/ml,t =2.34,P ＜0.05),and there was a negative correlation between the expression of sPD-1 and the concentration of IL-4.Conclusions The increased expression of PD-1 + ICOS + CD4 + T cells suggested the subset involved in the pathological progress of MG.sPD-1 might disturb the ligation of PD-1 on T cells and PD-L1 on antigen presenting cells,while the ligation of ICOS and ICOSL passed positive signal,leading to over activity of the subsets and the progression of disease.
ABSTRACT
Objective To investigate the role and mechanism of B7-H4, a negative costimulatory molecule, in mediating the immunomodulatory effects of mesenchymal stem cells C3H10T1/2 (C3H10) on T cell polarization. Methods The lentiviral vectors that carried the shRNA targeting mouse B7-H4 were transfected into mouse mesenchymal stem cells (C3H10-B7-H4). The cells were co-cultured with PHA-acti-vated mice spleen lymphocytes before and after the transfection. ELISA was performed to detect the concen-trations of cytokines in supernatants of cell culture in order to elucidate the effects of B7-H4 expressed by C3H10 on T cell polarization. A mouse model of experimental allergic encephalitis (EAE) was established. Fifty C57BL/6 mice were divided into five groups including control group, EAE group, C3H10 group (injec-ting EAE mice with C3H10 cells), C3H10-NC group ( injecting EAE mice with C3H10-NC cells) and C3H10-B7-H4 group (injecting EAE mice with C3H10-B7-H4 cells). ELISA was performed to detect the soluble form of IL-2, IL-17, IFN-γ and IL-4 in plasma samples. Results Knocking down the B7-H4 gene with shRNA significantly decreased the expression of B7-H4 on C3H10 cells, which weakened the inhibitory effects of C3H10 cells on the secretion of IL-2, IL-17 and IFN-γ by spleen lymphocytes. The therapeutic effects of C3H10-B7-H4 cells on mice with EAE were weakened after silencing the B7-H4 gene expression, which was manifested as higher nerve function score and earlier onset and bring forwarded peak time of EAE than those of the C3H10 group. Treating EAE mice with C3H10-B7-H4 cells was less efficient in inhibiting the expression of IL-2, IL-17 and IFN-γin plasma. However, knocking down the B7-H4 gene had no signif-icant effect on the expression of IL-4 in terms of treating EAE with C3H10 cells. Conclusion The co-inhib-itor molecule B7-H4 expressed on C3H10 cells mediated the treatment of EAE with C3H10 cells by regula-ting Th1 and Th17 effector T cells.