| Objective:To observe and discuss the immunological significance of T follicular helper cells,inducible co-stimulator molecule and its ligand(ICOS/ICOSL),programmed apoptosis factor-1 and its ligands(PD-1/PD-L1)expressed in peripheral blood from myasthenia gravis(MG)patients on different stages.Methods:From May 2016 to December 2017,72 untreated stage myasthenia gravis patients(USMG),29 remission stage myasthenia gravis patients(RSMG),22 other autoimmune disease of non-myasthenia gravis patients(NMG),and 29 cerrbrovascular disease patients(CVD)treatet at the Neurology Department of the Fist Affiliated Hospital of Soochow university and 25 health controls(HC)were enrolled,thier whole blood were collected.Immuno-flourenscence staining and flow cytometry(FCM)were applied to detect the proportion of T follicular helper cells(Tfh),and the expression of ICOS/ICOSL and PD-1/PD-L1 on the surface of peripheral blood mononuclear cells,Enzyme-linked immunosorbent assay was performed to detect the level of IL-4,IL-21,and the soluble form of ICOS/ICOSL,PD-1/PD-L1 in plasma samples from different groups respectively.Results:1.There was no significant difference of IL-4 levels in the peripheral blood between the USMG and the other experimental groups,P>0.05.2.The expression of ICOS~+PD-1~+on CD4~+T cells from USMG patient PBMC was higher than that from RSMG,CVD and HC,[19.53(9.25,27.06)%vs13.3(6.40,17.49)%,6.24(3.23,9.00)%,5.17(2.94,8.66)%,P=0.003,0.001,0.000].The percentage of PD-1~+ICOS~+CD4~+T cells was higher in generalized myasthenia gravis(GMG)patients than that in Ocular myasthenia gravis(OMG)patients,[23.24(15.29,31.77)%vs 10.38(6.70,20.03)%,P=0.000].The percentage of PD-1~+ICOS~+CD4~+T cells was higher in MG patients with thymoma than that in MG patients with normal thymus,[(32.03(14.61,35.01)%vs 17.96(7.92,23.44)%,P=0.005].The percentage of PD-1~+ICOS~+CD4~+T cells was higher in late-onset of MG patients than that in early-onset of MG patients,[20.73(11.06,28.12)%vs 9.44(5.25,22.39)%,P=0.016].There were no differences between differnt genders MG patients.The proportion of CD4~+ICOS~+PD-1~+CXCR5~+T cells(Tfh)on the USMG patients’peripheral blood was significantly higher than that of RSMG,CVD and HC.[12.54(7.62,19.48)%vs6.39(4.21,8.57)%.3.76(2.80,4.23)%.3.99(3.09,4.75)%,P=0.000.0.000.0.000].The percentage of CXCR5~+PD-1~+ICOS~+CD4~+T cells was higher in GMG patients than that in OMG patients[12.54(7.62,19.48)%vs 11.05(5.97,15.44)%,P=0.022].Which was higher in MG patients with thymoma than that in MG patients with normal thymus,[23.20(10.66,27.47)%vs11.85(7.60,14.62)%,P=0.002].There were no differences between different genders or onset ages type of MG patients.There was a positive correlation(r=0.318)between the proportion of Tfh cells and the quantitive MG score.The concentration of IL-21 in USMG patients was increased compared with those in RSMG,CVD and HC[(390.5±53.90)ng/ml vs(106.7±8.08)ng/ml,(138.0±17.87)ng/ml,(135.5±7.76)ng/ml,P<0.001].It was higher in MG patients with thymoma than that in MG patients with normal thymus[(717.7±25.02)ng/ml vs(390.5±53.90)ng/ml,P=0.035],and was higher in GMG patients than that in OMG patients,[(579.0±46.68)ng/ml vs(224.8±64.70)ng/ml,P=0.002].There were no differences between different genders or onset ages in MG patients.3.The expression of CD4~+ICOS~+T cells,ICOSL~+CD14~+monocytes and CD19~+B cells from USMG patient PBMC was higher than those from the RSMG,CVD,and HC(P<0.05).CD4~+ICOS~+T cells and ICOSL~+CD19~+B cells and from GMG patients was higer than that from the other groups,and were increased in MG patients with thymoma than that in MG patients with normal thymus,(P<0.05).There were no differences between different genders or onset ages in MG patients.The expression of ICOSL~+CD14~+monocytes from USMG patient PBMC was higher than those from the RSMG,CVD,and HC(P<0.05).ICOSL~+CD14~+B monocytes were increased in MG patients with thymoma than that in MG patients with normal thymus,(P<0.05).There were no differences between different clinical type,genders or onset ages in MG patients.The soluble form of ICOSL(s ICOS)in USMG is higer than that from the RSMG,CVD,and HC[(12.19±0.79)ng/ml vs(6.40±0.27)ng/ml,(2.76±0.55)ng/ml,(3.00±0.30)ng/ml,P<0.0001].It was higher in GMG patients than that in OMG patients[(15.40±1.33)ng/ml vs(10.44±1.06)ng/ml,P=0.02],and was higher in MG patients with thymoma than that in MG patients with normal thymus,[(23.02±1.95)ng/ml vs(12.19±0.79)ng/ml,P<0.0001].There was no significant difference of s ICOS levels in the peripheral blood among the five experimental groups(P>0.05).4.The expression of CD4~+PD-1~+T cells and PD-L1~+CD14~+monocytes and CD19~+B cells from USMG group were higher than those from the RSMG,CVD and HC groups(P<0.05).The expression of CD4~+PD-1~+T cells and PD-L1~+CD19~+B cells from GMG group were higher than those in the other groups,and were increased in MG patients with thymoma than in MG patients with normal thymus,P<0.05.The expression of PD-L1~+CD14~+monocytes from patients with thymoma were higher than that in MG patients with normal thymus,P<0.05.The soluble form of PD-1 in USMG are higer than that from the other groups[(54.35±6.00)ng/ml vs(22.91±2.12)ng/ml,(23.84±3.12)ng/ml,(26.85±2.76)ng/ml,P<0.001],andwas also higher in GMG than OMG[(63.32±7.18)ng/ml vs(28.73±2.30)ng/ml,P=0.0015].There was no differences between different genders,as well as in different onset age groups,or with or without mymoma MG patients.The concentration of s PD-L1 in the USMG group was higher than that from the other experimental groups[(15.35±1.04)ng/ml vs(7.00±0.35)ng/ml,(5.76±1.18)ng/ml,(4.71±0.57)ng/ml,P<0.0001].It was also higher in GMG than OMG[(18.55±1.00)ng/ml vs(13.42±1.34)ng/ml,P=0.014].and was higher in MG patients with thymoma than that in MG patients with normal thymus[(16.17±1.07)ng/ml vs(9.55±0.28)ng/ml,P=0.032]There was no differences between different genders,as well as in different onset age groups,The expression of s PD-1 was increased about six times than s PD-L1 in GMG patients[(63.32±7.18)ng/ml vs(15.35±1.04)ng/ml,P<0.001].Conclusion:The Tfh cells and ICOS/ICOSL,PD-1/PD-L1 pathway took part in the pathological process of MG,and these molecular might be potential useful immune-biomarkers for monitor MG immune response and clinical stages. |
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