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Objective:To investigate the role of mTOR in regulation of ICOS expression in human blood regulatory T cells. Methods:Isolation of Treg cells from human PBMC using MACS beads. We detected the ICOS expression on purified Treg cells and Treg cells viability using flow cytometry in anti-CD3 plus anti-CD28 ( antibody or beads) or anti-CD3 plus ICOSL-Fc for 3 days and 7 days. CFSE labeling human PBMC cells and in vitro cultured Treg mixed, Treg contact inhibition activity was detected by flow analysis. Results:After in vitro stimulation of Treg cells in the presence of anti-CD3+anti-CD28 for 3 days, there was no significant statistic difference in viability between ICOS+(92. 00±2. 69)% and ICOS-(90. 30±3. 53)% Treg-cells. After cultured for 7 days,the decreased ICOS+ Treg cells percentage within total Treg cells from ( 40. 20 ± 1. 83 )% to ( 11. 60 ± 1. 10 )% compared with that of 3 days. Further more,the ICOS expression level between stimulated with anti-CD28 or ICOSL-Fc condition group,compared with the ICOS MFI in the condition of anti-CD3 plus anti-CD28 treatment for 3 days was (2410. 0±746. 4) obviously higher than (403. 30±74. 42), that of the group treated with anti-CD3 plus ICOSL-FC. Rapamycin could partially suppress Treg cells ICOS expression,but unaffected the Treg suppression ability. Conclusion:ICOS expression level may not important for in vitro cultured human PBMC Treg cells survival although mTOR signling is important for regulation ICOS expression on in-vitro cultured Treg cells,but the ICOS expression on Treg regulated by multiply signaling pathways. CD28 signaling is the key stimulation factor for ICOS upregulation on in-vitro cultured Treg cells compared to ICOSL signaling.
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Objective:To study the pathological mechanism of the inducible co-stimulator molecular and ligand ( ICOS/ICOSL) in Graves disease animal.Methods:45 out-bred BALB/c mice were randomly divided into three groups with 15 rats in each group;using gene gun to deliver different plasmid injection.Group A was delivered with pCDNA3.0-mICOSL and pCDNA3.0-hTSHR, Group B with pCDNA3.0-hTSHR and null pCDNA3.0 with Group C for immunization as the control group.The concentration of serum free thyroxine immunization was deter mined with immunoassay and serum thyrotropin receptor antibody ( TRAb ) with ELISA, supernatant of IFN-γconcentration in mouse spleen cells was measured with radioimmunoassay,and hTSHR transected CHO cells were incubated to detect the concentration of cAMP to deter mine autoantibody TRAb activity.Results: After plasmid injection serum FT4 level in Group A (0.49±0.25) pg/ml ( q=6.571,P=0.023) was higher than that in Group C,the standard rate was higher than Group B and C (χ2=14.47,P=0.005).IFN-γconcentration of mice spleen cultured supernatant in Group A (1.88±0.41) pmol/L was significantly higher than the other two groups.The activity of autoantibody TRAb in Group A 188.3 (179.7-260.2) %was higher than that in the other two groups ( P=0.027 ) .Conclusion: Exogenous delivery of pCDNA3.0-mICOSL plasmid in GD mice could stimulate the spleen lymphocytes to secrete more IFN-γ,increase the activity of TRAb autoantibodies and might lead to upregulation of immune response in Graves animal model in vivo.
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Objective:To investigate the expression of inducible costimulatory ( ICOS) and inducible costimulatory ligand ( ICOSL) on peripheral blood mononuclear cells ( PBMCs ) and their clinical relationship with rheumatoid arthritis ( RA ) patients.Methods:Peripheral blood samples were collected from 85 RA patients and 50 HC in this study.Expression of ICOS and ICOSL on PBMC from the subjects were detected by flow cytometry and real-time polymerase chain reaction( RT-PCR).The alteration of ICOS and ICOSL were observed after hormone therapy in 15 patients with RA and the relationship between their expression level and patients′clinical manifestations were analysed.Results:The ICOS and ICOSL mRNA level of RA patients′PBMCs were significantly higher than that in HC.The expression level of ICOS on CD4+T cells was higher than than that in HC[(7.08±4.72)% vs (3.01+1.39)%,P<0.0001].The expression of ICOSL on monocytes[(5.77±3.45)%vs (3.64±1.43)%,P<0.05] and B cells [(5.78± 4.52)%vs (3.97±1.63)%,P<0.05] were significantly elevated in RA patients.In RA patients with active disease,however,ICOSL expression on monocytes and B cells were increased as compared with those in inactive RA patients [ ( 5.45 ±3.50 )% vs ( 4.04 ± 1.55)%,P=0.036],[(6.59 ±5.74)%vs (5.63±4.30)%,P=0.016].Furthermore,after receiving immunosuppressive therapy, the expressions of ICOS and ICOSL were notably reduced as compared with pre-therapy levels on PBMCs from patients [ ( 5.45 ±3.50)%vs (4.04±1.55)%,P=0.036],[(6.59 ±5.74)%vs (5.63±4.30)%,P=0.016].Conclusion:The high levels of ICOS and ICOSL expression were closely correlated with the degree of disease and therapeutic response,suggesting that ICOS/ICOSL pathway may play a critical role in pathogenesis of RA.
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Objective To analyze the effect of inducible costmulator (ICOS)/inducible costmulator ligand (ICOSL) signaling pathway on hepatic fibrosis in mice infected with Schistosoma japonicum.Methods Seventy-eight ICOSL knockout (ICOSL-KO) mice and 77 wild type C57BL/6J mice were used as experimental schistosomiasis model infected with Schistosoma japonicum.The sera of mice were collected on the day before infection (0 week),and at 4,7,12,16 and 20 weeks post infection.Then,the concentrations of hyaluronic acid (HA) and hydroxyproline (HYP) in mice sera were measured by sandwich enzyme linked immunosorbent assay (ELISA) kits.The expressions of transforming growth factor β1 (TGF-β1),α-smooth muscle actin (a-SMA) and Collagen-Ⅰ in livers from ICOSL-KO/wild type mice were assessed by immunohistochemical staining.The granulomatous pathology and fibrosis level in mice liver were dynamically observed by hematoxylin and eosin (HE) staining and Masson's staining,respectively.The difference between groups was detected by t test or x2 test when appropriate.Results After infection with Schistosoma japonicum,the levels of HA and HYP were gradually increased.In ICOSL-KO mice,the levels of HA at 7,12,16 and 20 weeks post infection were all significantly lower than those in wild type mice [(161.32±15.44) vs (186.01±21.24) ng/mL,t=2.528 2,P＜0.05; (166.73±18.18) vs (231.39±20.12) ng/mL,t=4.342 4,P＜0.05; (193.58±21.06) vs (252.51±25.29) ng/mL,t=4.003 9,P＜0.05; (253.98±24.53) vs (310.88±23.86) ng/mL,t=3.718 0,P＜0.05].Similarly,HYP levels in ICOSL-KO mice at 12,16 and 20 weeks post infection were all significantly lower than those in wild type mice (all P＜0.05).Immunohistochemical staining showed that TGF-β1,α-SMA and Collagen-Ⅰ expressions in liver of ICOSL-KO mice from 7 to 20 weeks post infection were all significantly lower than those of wild type mice (all P＜0.05).HE staining showed,the volume of liver egg granulomas of ICOSL-KO mice was significantly smaller than that of wild type C57BL/6J mice (P＜0.01).Furthermore,Masson's staining showed that the level of hepatic fibrosis in ICOSL-KO mice was lower than that in wild type mice and the fibrosis scores were statistically different between two groups (all P＜0.05).The mortality rate of the wilde type C57BL/6J mice was higher than that of ICOSL-KO mice.After 20 weeks of infection,the difference was statistically significant (55.84 ％ vs 37.18 ％,x2 =5.427,P＜0.05).Conclusions The degree of hepatic fibrosis and related indicators are obviously down-regulated in ICOSL-KO mice infected with Schistosoma japonicum.These findings suggest that ICOS/ICOSL signaling pathway has an important impact on the process of hepatic fibrosis caused by Schistosoma japonicum.
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Objective To investigate the role of T follicular helper ( Tfh) cells in the immuno-pathogenesis of persistent immune thrombocytopenic purpura ( pITP) .Methods Twenty children with pITP and twenty healthy controls were enrolled in this study .The proportion of CD4+CXCR5+ICOShigh PD-1high T ( cTfh) cells and the expression of ICOSL on CD 19+B cells in peripheral blood of the patients and healthy subjects were analyzed by flow cytometry .The expressions of Bcl-6, c-Maf, IL-21 and ICOSL at mRNA level were detected by real-time PCR.The plasma concentrations of IL-2, IL-6 and IL-21 were determined by ELISA.Results (1) Compared with the healthy controls , the proportions of cTfh cells increased signifi-cantly in patients with pITP [(17.45±9.04) %vs.(7.57±2.57) %, P0.05).(4) The plasma concentration of IL-21 was remarkably elevated in patients with pITP , regardless of DEX treatment (P0.05).Con-clusion The immunopathogenesis of persistent immune thrombocytopenic purpura might be associated with the hyper-activation of Tfh cells caused by excessive expression of ICOSL and IL-21.The persistent high expression of ICOSL and IL-21 might be one of the important factors resulting in the recurrence of pITP in children .
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To determine immune responses and immunopathology in ICOSL knockout (ICOSL KO) mice infected with Schistosoma japonicum,ICOSL- KO mice and wild-type C57BL/6J mice were used as experimental models for Schistosoma japonicum infection.The splenic lymphocytes were isolated from the mice the day before infection (0 week) as well as 4,7,12,16 and 20 weeks post infection,and stimulated with SEA for 72 hours in culture.The concentrations of Th1 cytokines (IFN-γand IL- 12) and Th2 cytokines (IL- 4,IL-10 and IL-13) in the culture supernatants were measured by sandwich ELISA.The levels of SEA-specific IgG antibody and its subtypes (IgG1 and IgG2a) were measured in mouse sera by ELISA.Pathological changes of hepatic granuloma in mice were determined by hematoxylin and eosin (H&E) staining.After the infection,the levels of Th1 cytokines,IFN- γ and IL 12,in ICOSL- KO mice were higher than those in wild-type C57BL/6J mice.However,the levels of Th2 cytokines (IL- 4,IL- 10 and IL- 13) were significantly decreased in ICOSL-KO mice compared to those in wild-type C57BL/6J mice.The levels of SEA-specific IgG antibody and its subtypes (IgG1 and IgG2a) in the sera of ICOSL- KO mice were also significantly lower than those of wild -type C57BL/6J mice.Moreover,the Th2 differentiation index was lower in ICOSL- KO mice than in wild-type C57BL/6J mice at 4,7,12,16 and 20 weeks post-infection.Similarly,the ratio of IgG1/IgG2a in ICOSL-KO mice was significantly lower than that in wild- type C57BL/6J mice at 7,12 and 16 weeks post- infection.Furthermore,throughout the course of disease progression,the volume of hepatic egg granuloma in ICOSL- KO mice was significantly smaller than that in wild-type C57BL/6J mice.In conclusions,there is a substantially down-regulated Th2 immune response in ICOSL- KO mice infected with Schistosoma japonicum,thus results in an attenuated hepatic lesion caused by egg granulomas.The findings indicate that the ICOS ICOSL co-stimulatory pathway plays an important role in the hepatic egg granuloma formation of schistosomiasis.
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The CD28-B7 are members of immunoglobulin superfamily.When B7 is engated by the CD28 ligand, a costimulatory signal occurs and transfers to T cell and it is important in the activation, proliferation, anti-apoptosis and promoting cytokine secretion of T cell.The CD28-B7 family is associated with tumor and autoimmune disease.