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Objective:To investigate the expressions of CD200 and inducible costimulator (ICOS) protein in angioimmunoblastic T-cell lymphoma (AITL) and the relationship with prognosis as well as their significances in the differential diagnosis of AITL.Methods:A total of 39 AITL patients in the First People's Hospital of Chenzhou, the Fourth People's Hospital of Chenzhou, Xiangnan College Affiliated Hospital and Chenzhou 3rd People's Hospital from June 2012 to December 2019, and 10 patients with classic Hodgkin lymphoma (CHL) and 10 patients with peripheral T cell lymphoma, not otherwise specified (PTCL-NOS) from August 2016 to July 2019 in the First People's Hospital of Chenzhou were selected. Immunohistochemistry was used to detect the expressions of CD200, ICOS, CD10, programmed death 1 (PD-1), bcl-6 and CXC chemokine receptor-13 (CXCL13) proteins, and the correlation of CD200 and ICOS with clinicopathological features and prognosis of AITL patients was analyzed, and the diagnostic significance of both in differentiating AITL from PTCL-NOS and CHL was also analyzed.Results:The positive rates of CD200 and ICOS in 39 AITL patients were 71.79% (28/39) and 61.54% (24/39), respectively. There were 7 cases of CD200 weak and moderate positive in 10 CHL patients, and ICOS proteins were all negative. Among 10 PTCL-NOS patients, 4 patients had CD200 positive and 1 patient had ICOS positive. The differences in positive rates of ICOS protein between AITL patients and CHL, PTCL-NOS patients were statistically significant (all P < 0.05); the differences in positive rates of CD200 protein between AITL patients and CHL, PTCL-NOS patients were not statistically significant ( χ2=0.013, P=0.911; χ2=3.551, P=0.060). The positive rate of CD200 in AITL patients with elevated lactate dehydrogenase (LDH) and international prognostic index (IPI) score of 3-4 was higher than that in AITL patients with normal LDH and IPI score of 0-2 (both P < 0.05); The positive rate of ICOS in AITL patients with elevated LDH and PD-1 positive was higher than that in AITL patients with normal LDH and PD-1 negative (both P < 0.05). CD200 negative AITL patients had better 3-year overall survival (OS) rate (4.2% vs. 66.7%) and 3-year progression-free survival (PFS) rate (5.3% vs. 77.1%) compared with those in CD200 positive AITL patients, and the differences between both groups were statistically significant (both P < 0.01); there was a statistically significant difference in 3-year OS rate between ICOS positive AITL patients and ICOS negative AITL patients (15.3% vs. 38.6%, P=0.011), while there was no statistically significant difference in 3-year PFS rate of both groups (18.6% vs. 41.5%, P=0.059). Multivariate analysis showed CD200 ( HR=0.076, 95% CI 1.555-79.497, P=0.001), extranodal involvement or not ( HR=11.117, 95% CI 1.555-79.497, P=0.016) and LDH ( HR=2.147, 95% CI 0.844-5.459, P=0.109) were independent influencing factors of OS in AITL patients; CD200 ( HR=0.075, 95% CI 0.016-0.357, P=0.001) and LDH ( HR=2.335, 95% CI 0.929-5.870, P=0.071) were independent influencing factors of PFS in AITL patients. Conclusions:CD200 and ICOS can be used as immunohistochemical indicators to assist the diagnosis of AITL patients. ICOS protein helps to differentiate AITL from CHL and PTCL-NOS; CD200 can be used as indicators to judge the prognosis and deterioration of AITL patients.
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We constructed the CS1-targeted second- and third-generation CAR-T cells with genetic engineered 4-1BB or/and ICOS as a costimulatory signaling molecule by use of lentiviral platform. The CS1-targeted second-generation CAR-T cells with ICOS or 4-1BB had similar anti-neoplastic activity. When effector/target ratio was 1:1, the CAR-T cells with ICOS showed better killing effect on IM9-lucgfp cells than those with 4-1BB. However, The CS1-targeted third-generation CAR-T cells exihibited lower cytolytic capacity against IM9-lucgfp cells than the CS1-targeted second-generation CAR-T cells when the ratio of effector/target was 1:1, 2:1 or 5:1. When the ratio of effector/target was 10:1, the killing efficacy of both the second- and third-generation CAR-T cells against IM9-lucgfp cells was more than 85%, significantly higher than that of the control T cells. Taken together, both the CS1-targeted second- and third-generation CAR-T cells with ICOS or/and 4-1BB could efficiently kill CS1-positive multiple myeloma cells, but the CS1-targeted second-generation CAR-T cells had more potent killing effect on CS1-positive multiple myeloma cells than the CS1-targeted third-generation CAR-T cells.
Subject(s)
Humans , 4-1BB Ligand/metabolism , Cell Line, Tumor , Genetic Engineering , Inducible T-Cell Co-Stimulator Protein/metabolism , Multiple Myeloma/therapy , Signal Transduction , T-Lymphocytes/chemistry , Xenograft Model Antitumor AssaysABSTRACT
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.
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Objective To investigate the change of circulating follicular helper T cells (cTfh) in patients with anti-neutrophil cytoplasmic myeloperoxidase antibody-associated vasculitis (MPO-AAV), and to analyze the relationship between cTfh and disease activity. Methods Thirty-eight untreated MPO-AAV patients (patient group) and thirty-eight healthy volunteers (control group) were enrolled in this study. cTfh and membrane expression of inducible co-stimulator(ICOS)and programmed cell death protein 1(PD-1) were detected by flow cytometry (FCM). Serum anti-neutrophil cytoplasmic myeloperoxidase antibody (MPO-ANCA) was measured by ELISA. Disease activity was evaluated by Birmingham vasculitis activity score (BVAS). Results Compared with those in control group, the proportions of cTfh, ICOS+Tfh and PD-1+Tfh cells in patient group were significantly higher [(25.9±3.8)％vs. (21.0±5.3)％, P<0.001;(1.8±0.8)％vs. (0.8±0.5)％, P<0.001 and (10.2±2.8)％vs. (8.2±2.2)％, P=0.001, respectively]. Meanwhile, the expression of ICOS and PD-1 on cTfh in patient group was markedly more intensive (59.6±10.0 vs.49.2±6.9, P<0.001 and 532.6±104.2 vs. 485.1±73.4, P=0.025, respectively). In patient group, the proportion of cTfh was positively correlated with the ratio of ICOS+Tfh, the expression of ICOS, the level of MPO-ANCA and BVAS (r=0.407, P=0.011; r=0.705, P<0.001; r=0.737, P<0.001 and r=0.663, P<0.001, respectively). The expression intensity of ICOS on cTfh was positively associated with ICOS+Tfh ratio, serum MPO-ANCA and BVAS (r=0.388, P=0.016; r=0.645, P<0.001 and r=0.653, P<0.001, respectively). Nevertheless, the expression of PD-1 on cTfh was only positively correlated with the ratio of PD-1+Tfh (r=0.473, P=0.003). Conclusions Enhanced cTfh in patients with MPO-AAV might produce MPO-ANCA, which is related to the aggravation of MPO-AAV. Thus, cTfh and its ICOS could be potentially targeted for the treatment of MPO-AAV.
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To detect the expression level of ICOS on Th9 cells in mice infected with Schistosoma japonicum, and investigate the relation between ICOS signaling and Th9 cell polarization.Twenty-five mice with S. japonicum infection were used as models. IL-9+cells in CD4+ T cells and ICOS+ cells in Th9 cells of the mice were detected by flow cytometry 0, 4, 7, 9 weeks and 12 weeks after the infection.Compared with that 0 week after the infection, the proportion of Th9 cells in CD4+ T cells of the mice significantly increased 4, 7, 9, 12 weeks after the infection (all P < 0.05), and the proportion of ICOS+ cells in Th9 cells also markedly improved (P < 0.05).In S. japonicum infection, the ICOS signaling may have a regulatory effect on Th9 cell polarization.
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Objective To detect levels of C?X?C chemokine receptor type 5 (CXCR5) and inducible costimulator(ICOS)in blister fluid of patients with bullous pemphigoid(BP), and to explore their significance in the pathogenesis of BP. Methods Blister fluid samples were collected from 15 patients with BP(experimental group)and 15 patients with second?degree burns(control group). Enzyme?linked immunosorbent assay(ELISA)was performed to detect the levels of CXCR5 and ICOS in the 2 groups. Results The level of CXCR5 was significantly higher in the experimental group than in the control group(219 ± 145.31 vs. 147 ± 23.83 ng/L, t=4.577, P 0.05). Conclusion The expression of CXCR5 may be associated with the occurrence of BP, but further researches are needed to determine the relationship between ICOS and the occurrence of BP.
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Objective To investigate the changes of follicular helper T cells (Tfh cells) and Tfh cells associated molecules in the peripheral blood (PB) of patients with malignant lymphoid diseases (MLD) dynamically, and explore their roles on pathogenesis of the diseases. Methods Fifty-five patients with MLD were enrolled in this study,including 9 patients with acute lymphocyte leukemia (ALL), 30 patients with non-Hodgkin lymophoma (NHL) and 16 patients with multiple myeloma (MM), and 10 healthy controls (NC) of similar age were also enrolled. The percentage of CD4+CXCR5+cells (Tfh cells) and expression of ICOS+, PD1+among the T cells were detected by flow cytometry (FCM), while the levels of interleukin 21 (IL-21) in plasma were detected by ELISA tests. Results The percentage of Tfh cells and expression of ICOS and/or PD-1 in PB of all untreated patients were significantly higher than those of NC (all P 0.05), and apparently lower than those who achieved PR (P 0.05), and much higher than NC (P< 0.01). The concentration of IL-21 in patients were much higher than that in NC [(326.56±32.44) pg/ml] (P<0.01), and MM group
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Objective To investigate the expression of inducible co-stimulator (ICOS) and inducible co-stimulator ligand (ICOSL) on PBMCs,and the plasma concentrations of soluble forms of ICOSL and their clinical relationship with systemic lupus erythematosus (SLE) patients.Methods Peripheral blood samples were collected from 45 SLE patients and 39 healthy subjects (HC).The expressions of ICOS and ICOSL on peripheral blood mononuclear cells (PBMCs) were detected by flow cytometry.The concentrations of soluble ICOSL were assessed by measured by enzyme linked immunosorbent assay (ELISA).And the relationship between their expression levels and patients' clinical manifestations were analyzed.Levene F test was used for statistical analysis,the comparison between groups was conducted using t test,and the correlation between two variables were tested by Pearson correlation analysis.Results The expression of ICOS on CD4+ and CD8+ T cells were significantly higher than that of the HC [(19.1±1.7)％ vs (14.0±1.5)％,t=2.156,P=0.035],[(10.0± 1.0)％ vs (6.4±1.0)％,t=2.587,P=0.012].The expression of ICOSL on CD14+ mononuclear cells in SLE patients was significantly higher than that in the HC group [(2.94±0.88)％ vs (0.89 ±0.21)％,t=2.152,P=0.04].Plasma ICOSL concentrations in patients with active SLE were significantly higher than those of patients with inactive SLE [(362±25) ng/ml vs (278±15) ng/ml,t=2.356,P=0.025].We also found a significant negative correlation between the soluble ICOSL expression and the surface ICOSL expression on both mononuclear cells and B cells (r=-0.4243,P=0.022;r=-0.4099,P=0.025).MMPI induced an evident reduction in sICOSL levels released from the cells,which was statistically significant in comparison with untreated cells (P＜0.05).Conclusion The up-regulated expressions of ICOS and ICOSL on peripheral lymphocytes and the high levels of plasma concentration of soluble ICOSL are closely correlated with the severity of the disease,suggesting that ICOS/ICOSL pathway may play a critical role in the pathogenesis of SLE.
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Objective To study the expression levels of inducible costimulator (ICOS) on CD4+ T cells in peripheral blood in patients with chronic hepatitis B and its change after treated with interferon.Methods All 56 patients were divided into two groups (interferon group has 28 cases,lamivudine group has 28 cases),and interferon group treated by PEG-IFN-α 2a,lamivudine group treated by lamivudine,respectively.There were 20 healthy individuals as control group.The levels of ICOS on CD4+ T cells of patients with chronic hepatitis B were kinetically detected by flow cytometry.The copies of HBV DNA in sera were dynamically detected by real-time PCR.Results The levels of ICOS on CD4+ T cells in patients with chronic hepatitis B was higher than that of normal controls(P＜0.001 ).However,the expressions of ICOS on CD4+ T cells in patients could be deduced by PEG-IFN-α 2a and obviously decreased after treatment.There was significant difference between interferon group and lamivudine group (P＜0.05 in 24 weeks,and P＜0.01 in 48 weeks).After treatment,the change values of ICOS in interferon group was positively correlated with the change copies of HBV DNA (r =0.972,P＜0.001 ).However,the change values mentioned above that did not find correlation in lamivudine group(r=-0.101,P=0.608).Conclusion The study shows the patients with chronic hepatitis B have disordered in cellular immunity and increased with the expression of ICOS on CD4+ T cells.PEG-IFN-α 2a could decrease the expression of ICOS on CD4+ T cells in patients,and correct the Th2 type immune polarization to some extent,and that plays a positive role in antiHBV.
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Objective To analyze whether human-derived ICOSIg can bind specifically to ICOSL on mouse immature dendritic celis(DCs) and to explore its biological funciions. Methods The binding of ICOSIg to immature DCs was observed by FCM. The cytotoxic effect of ICOSIg on DCs was examined by Annexin V /PI, CFSE staining, and CCK-8 kit. [3 H] thymine incorporation was used to analyze the blocking effect ICOSIg on mixed lymphocyte reaction (MLR). Results Human-derived soluble fusion protein ICOSIg could bind to ICOSL on mouse bone marrow-derived immature DCs and inhibited the MLR, but it neither induced early or late apoptosis of DCs nor affected their proliferation. Concluiion Human-derived ICOSIg constructed in this study has a potent biological function; it has no toxic effect against mouse immature DCs. It is demonstrated that human- derived ICOSIg can sepecifically bind to ICOSL on mouse immature DCs.
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Objective: To construct a recombinant retroviral vector carrying human inducible costimulator (ICOS) gene, screen for CHO cell line stably expressing ICOS protein and to study its biological activity. Methods: ICOS cDNA was obtained from human peripheral blood mononuclear cells (PBMC) through RT-PCR and was cloned into retroviral vector to construct retroviral recombinant pMSCV-ICOS; the latter was then packed and the high-titer virus producing cells were screened. Then CHO cell was infected by this high-titer virus and the stable cell line was screened. CHO-ICOS cells were co-cultured with PBMC (the ratio of CHO-ICOS to PBMC being 1:1,1:2,1:5, and 1:10) in presence of substimulating dose of anti-human CD3 antibody. The proliferation of PBMC and the CD25 expression on T cells were examined by 3H-TdR incorporation method and flow cytometry,respectively. CHO-pMSCV cells co-cultured with PBMC (1:1) served as the negative control and PBMC served as blank control. Results: We successfully constructed the retroviral recombinant pMSCV-ICOS and obtained CHO cell line stably expressing ICOS protein. 3H-TdR incorporation method and flow cytometry showed that, compared with the negative control group and the blank control group, co-culture with CHO-ICOS cells significantly inhibited the anti-CD3 antibody-induced activation and proliferation of PBMC(P<0.05);the maximal inhibitory rates of activation and proliferation were obtained when the ratio of CHO-ICOS to PBMC was 1:1, being (68±5.9)% and (44.08±3.26)%, respectively. Conclusion: CHO cell line stably expressing ICOS protein has been successfully established, which lays a foundation for future study.
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Objective To investigate the potential role of IC0s-Ig and CsA in inducing transplantation tolerance and the mechanisms thereof. Methods ICOS-Ig was a fusion protein of human ICOS extracellular region and IgG Fc fragment.Cardiac allograft from BALB/c mouse was transplanted to C57BL/6 mouse Animals were randomly divided into 5 groups:(1) un-treated group;(2)IgG-treated group,250g,i.p.day 2,4,6;(3)ICOS Ig-treated group,250g,i.P.day 2,4,6;(4) CsA-treated group,10 mg/kg,i.p.day 0-6;(5)ICOS-Ig+CsA-treated group.The survival time and pathological changes of the cardiac allografts were monitored.The mixed lymphoeyte reaction (MLR) and the alloantibody level of the recipients were also detected.Results The median survival time (MST) of the cardiac allografts was (8.5±1.5) days in un-treated group,(8.0±0.8) days in IgG-treated group,(29.5±7.7) days in ICOS-Ig-treated group,and(21.0±5.0) days in CsA-treated group.respectively.In ICOS-Ig + CsA-treated group,the MST was prolonged to longer than 100 days,which was significantly longer than other four groups(P<0.01).Allogeneic hearts from ICOS-Ig and/or CsA immunized recipients revealeel milder histological changes than control groups(P<0.05).Mechanical ahalysis revealed that splenic T cells from recipients also exhibited depressed MLR activities.The alloantibody level in ICOS-Ig-treated group and/or CsA-treated group was lower than in control groups(P<0.05),suggesting ICOS-Ig not only inhibited cell immunity,but also depressed humoral response.Conclusion ICOS-Ig combined with CsA leads to a long-term survival of mouse cardiac allografts.The induced tolerance is donor-specific and the mechanisms may be associated with T cell anergy.
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Objective To explore the effects of inducible co-stimulator (ICOS) gene on the cytotoxic activity of cytokine-induced killer (CIK) cells against cholangiocarcinoma cells. Methods CIK-ICOS cells were obtained by stable transfecting ICOS genes into CIK cells through the adenovirus vector whereas untransfected and EGFP-transfected CIK cells were treated as controls. The proliferation and apoptosis of different CIK cells, as well as their cytotoxicity against cholangiocarcinoma cells in the three groups were detected. The expressions of IFN-T, IL-2 and TNF-α in the supernatant of different CIK cells were measured by ELISA. SCID mice with cholangiocarcinoma were randomly divided into CIK group, CIK-EGFP group, CIK-ICOS group and normal saline group. The cytotoxic activity of CIK-ICOS cells against cholangiocarcinoma cells in vivo was observed. Results CIK-ICOS cells displayed better proliferation than CIK cells and CIK-EGFP cells. At day 20 and 23 of culture, the apoptosis rate of CIK-ICOS cells was 0.69% and 0.89%, respectively, while that of the CIK cells was 2.90% and 4.92%. The cytotoxic effect of CIK-ICOS cells at different E: T ratio against cholangiocarcinoma cells was significantly stronger than that of CIK cells and CIK-EGFP cells (F=13.37, 6.46, 25.51, P<0.05). The concentration of IFN-γ in CIK-ICOS cultured supernatant was (49.50±4.73)μg/L, which was significantly higher than that in the cultured supernatant of CIK cells [(30.53±3.73)μg/L] and CIK-EGFP cells [(30.12±2.64)μg/L](F=38.89, P<0.05). The growth of cholangiocarcinoma was significantly slower in CIK-ICOS group than that in CIK group and CIK-EGFP group, whereas the necrosis area of tumor was larger and the CIK cells in CIK-ICOS group was more than those in the other two groups. Conclusions CIK cells had the function of killing cholangiocarcinoma cells in vitro and in vivo. After ICOS genes were transfected into CIK cells, the survival time of CIK cells in vitro was prolonged and the proliferation of CIK cells was enhanced, as well as the secretion of IFN-γ was increased so that the cytotoxicity of CIK cells against cholangiocarcinoma cells in vitro and in vivo was enhanced.
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Objective To assessed the expression of inducible costimulator(ICOS)on peripheral blood and joint fluid CD4,CDS,CD45RO T cells and B cells in rheumatoid arthritis(RA).Methods Expression of ICOS and ICOS/CD45RO on peripheral blood and joint fluid CD4~+CD8~+T cells and ICOS ligand(ICOSL)on CD19 B cells from RA patients and healthy volunteers were determind by three-color flow cytometry.Compar- ision with active and inactive RA,initial and relapsed RA had been done.Results Joint fluid CD4 and CD8 T cells expressing ICOS,ICOS/CD45RO were significantly increased than peripheral blood in RA patients and healthy subjects.Joint fluid B cells expressing ICOSL were significantly reduced than peripheral blood in RA patients.Meanwhile,peripheral blood B cells expressing ICOSL were significantly reduced in active RA than inactive RA patients.Conclusion Hyperexpression of ICOS and ICOS/CD45RO on joint fluid CD4 and CD8 T cells and lowexpression of ICOSL in B cells from RA patients,expecially in active RA may contribute to the local immunopathological roles and joint destructions in the pathogenesis of RA.
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Objective:To produce a fusion protein(ICOS-Ig)between extracellular portion of human ICOS and Fc portion of mouse IgG 2a and study on its biological activity.Methods:cDNA encoding the extracellular domain of human ICOS was prepared by RT-PCR from the RNA of the stimulated human peripherial blood mononuclear cells. The Fc portion of mouse IgG2a was cloned by PCR from the vector that contains the sequence-encoding Fc portion of mouse IgG2a.Two resulting amplified PCR products were ligated into a secrection mammalian expression vector, pSecTag2/Hygro A. The recombined vector was transfected into CHO cells by lipofectamine2000.Results:RT-PCR demonstrated the integration and mRNA synthesis of fusion gene. ELISA and Western blot analysis showed the expression of ICOS-Ig and its molecular weight was between 43-66 kD, its concentration was 5-25 ?g/ml. FACS analysis assured that ICOS-Ig had ligand specific binding activity. Addition of ICOS-Ig to MLR resulted in inhibition of T-cell proliferation and IL-2,IFN-? secretion.Conclusion:The fused gene ICOS-Ig was constructed and expressed successfully. It had the bioactivity of inhibition of T cell proliferation and cytokine secretion.
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Objective:To construct a recombinant retroviral vector carrying human inducible costimulator (ICOS) gene,screen for CHO cell line stably expressing ICOS protein and to study its biological activity.Methods: ICOS cDNA was obtained from human peripheral blood mononuclear cells (PBMC) through RT-PCR and was cloned into retroviral vector to construct retroviral recombinant pMSCV-ICOS; the latter was then packed and the high-titer virus producing cells were screened.Then CHO cell was infected by this high-titer virus and the stable cell line was screened.CHO-ICOS cells were co-cultured with PBMC (the ratio of CHO-ICOS to PBMC being 11,12,15, and 110) in presence of substimulating dose of anti-human CD3 antibody.The proliferation of PBMC and the CD25 expression on T cells were examined by 3H-TdR incorporation method and flow cytometry,respectively.CHO-pMSCV cells co-cultured with PBMC (11) served as the negative control and PBMC served as blank control.Results: We successfully constructed the retroviral recombinant pMSCV-ICOS and obtained CHO cell line stably expressing ICOS protein.3H-TdR incorporation method and flow cytometry showed that,compared with the negative control group and the blank control group,co-culture with CHO-ICOS cells significantly inhibited the anti-CD3 antibody-induced activation and proliferation of PBMC(P
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Objective:To study the bioactivity of inducible costimulator Ig(ICOS-Ig) as an inhibitor of ICOS-B7RP-1 costimulatory pathway in vitro. Methods: cDNA encoding the extracellular domain of human ICOS was prepared by RT-PCR from the RNA of the stimulated human peripheral blood mononuclear cells. The Fc portion of mouse IgG2a was cloned by PCR from the vector containing the sequence-encoding Fc portion of mouse IgG2a.The above 2 PCR products were ligated into a clone vector: pGL-3-Basic. The fusion gene was then cloned and ligated into a mammalian expression vector: pcDNA4/HisMAX A. The recombined vector was transfected into CHO cells by Lipofectamine2000 and the expression of the fusion protein was identified by Western blot. The mixture lymphoproliferation reaction(MLR) of the lymphocytes derived from BALB/c and C57BL mice was used to detect the fusion protein function in vitro. Results: Western blot analysis showed the expression of fusion protein, with the molecular weight being 43 000-66 000. FACS analysis assured that expression products had ligand specific binding activity. MLR was inhibited by the fusion protein. Conclusion: The constructed recombinant fusion protein has ligand specific binding activity and can inhibit the lymphoproliferation.