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CD80 is mainly expressed on Ag-presenting cells (APCs) as a costimulatory molecule but is also detected on T cells. However, the origin and physiological role of CD80 on CD8⁺ T cells remain unclear. In the present study, we demonstrated that effector and memory CD8⁺ T cells, but not naïve CD8⁺ T cells, displayed CD80 molecules on their surfaces after acute lymphocytic choriomeningitis virus infection. Using adoptive transfer of CD80-knockout (KO) CD8⁺ T cells into a wild type or CD80-KO recipient, we demonstrated that the effector CD8⁺ T cells displayed CD80 by both intrinsic expression and extrinsic acquisition, while memory CD8⁺ T cells displayed CD80 only by extrinsic acquisition. Interestingly, the extrinsic acquisition of CD80 by CD8⁺ T cells was observed only in the lymphoid organs but not in the periphery, indicating the trogocytosis of CD80 molecules via interaction between CD8⁺ T cells and APCs. We compared the recall immune responses by memory CD8⁺ T cells that either extrinsically acquired CD80 or were deficient in CD80, and found that CD80, presented by memory CD8⁺ T cells, played a role in limiting their expansion and IL-2 production upon exposure to secondary challenge. Our study presents the in vivo dynamics of the extrinsic acquisition of CD80 by Ag-specific CD8⁺ T cells and its role in the regulation of recall immune responses in memory CD8+ T cells.
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Adoptive Transfer , B7-1 Antigen , Interleukin-2 , Lymphocytic choriomeningitis virus , Memory , T-LymphocytesABSTRACT
Objective To observe the effect of costimulatory molecule B7-1 on cytoskeleton rearrangement in mouse podocytes induced by angiotensin Ⅱ (Ang Ⅱ),and to study the underlying molecular mechanism of B7-1 in the pathological changes of podocytes.Methods All cultivation of conditionally immortalized mouse podocytes (MPC) in vitro were divided into the following groups:normal control group,CTLA-4 group,Ang Ⅱ group (10-6 mmol/L 12 h,24 h;10-8 mmol/L 12 h,24 h) and CTLA-4 with Ang Ⅱ group.Transfect B7-1 RNA interference fragment (siRNA) to the mature podocytes,and then restimulated by Ang Ⅱ (10-6 mmol/L 12 h),the change of podocyte cytoskeleton after Ang Ⅱ stimulation were observed.The expression of B7-1 in each group was assayed by flow cytometry and Western blotting.The nephrin and p-nephrin protein levels in the four groups were also analyzed by Western blotting.At the same time,the podocyte cytoskeleton distribution as indicated by F-actin was observed by fluorescence microscopy.Results Flow cytometry and Western blotting showed that B7-1 was not expressed in the normal control group.Ang Ⅱ showed a concentration and time dependent induction of B7-1 expression in mouse podocytes (P ＜ 0.05).Western blotting indicated that Ang Ⅱ induced B7-1 protein expression (P ＜ 0.05).Expression of nephrin and p-nephrin was significantly down-regulated by Ang Ⅱ (P ＜ 0.05).Compared with the normal control group,the expression of podocyte protein nephrin and p-nephrin in Ang Ⅱ stimulation group was significantly reduced (P ＜ 0.05).Using FITC phalloidin fluorescence staining showed that CTLA-4+Ang Ⅱ stimulation group cytoskeleton rearrangement was improved significantly and F-actin recombinant score (mCFS) decreased compared with Ang Ⅱ group (P ＜ 0.05),suggesting that Ang Ⅱ led to the disorder of the podocytes cytoskeleton and the destruction of the cytoskeleton of podocytes by Ang Ⅱ could be improved after B7-1 blocking.Compared with the Ang Ⅱ stimulation,transfection of B7-1 siRNA + Ang Ⅱ stimulation group improved F-actin cytoskeletal rearrangement,and mCFS also decreased significantly (P ＜ 0.05),suggesting that transfection of B7-1 siRNA might improve the damage of Ang Ⅱ on podocytes cytoskeleton.Conclusions B7-1 participates in the process of cytoskeleton reconstruction and plays an important role in the pathological changes of podocytes.
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Objective:To construct lentiviral vector specific for mouse B7-1 RNA interference and study lentivirus-mediated B7-1 gene silencing effects in L929 fibroblast cells.Methods:Three candidate sequences for B7-1 RNAi selected from coding sequence of mouse B7-1 transcription were used to design short hairpin RNA ( shRNA ) templates and then cloned into lentiviral expression plasmid followed with correctness identification of inserted sequence by DNA sequencing.Recombinant lentivirus were prepared by co-transfecting lentiviral expression vector and packaging plasmids into 293T cells.Then the resulting culture supernatant containing infectious lentiviral particles was pooled and centrifuged via ultra-centrifugation.Infectious titer of the preparations was determined by detecting the expression of GFP in 293T cells after transfected by lentivirus.Cultured L929 cells were transfected with lentivirus to deter-mine transduction efficiency and silencing efficacy of B7-1 expression by flow cytometry.Transducted L929 cells were then screened using puromycin to generate stable cell clones followed by flow cytometry analysis of GFP and B7-1 expression.A mixed reaction system consisting of stable B7-1 silencing L929 cells and mouse splenic T cells was used to analyze ability of the established cell line to trigger T cells proliferation.Results: Lentiviral expression vector for mouse B7-1 RNAi was correctly constructed with inserted sequences as designed.Recombinant RNAi lentivirus were prepared with titers ranging (3-5) ×108 TU/ml and efficacy to mediate GFP transgene expression and B7-1 silencing.B7-1 expression and the ability to trigger T cells proliferation of stable L929 cells were suppressed significantly ( P<0.05 ).Conclusion: We generated lentiviral vector specific for mouse B7-1 RNAi with high performance of transduction efficiency as well as B7-1 silencing efficacy and the recombinant RNAi lentivirus can mediated stable B7-1 gene silencing in L929 cells and inhibition of T cells proliferation induced by B7-1/CD28 co-stimulatory signal.
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Objective:On the basis of the use of chemical methods to establish mouse model of lupus nephritis and its biological identification , we investigate the reverse effect of pathological lesions of B 7-1 human-mouse chimeric antibody blockade against B7/D28 signaling pathway in mice with lupus nephritis model.Methods:Pristane was injected intraperitoneally to 6-week-old female C57BL/6J mice at dose of 0.5 ml per mouse in one go,and urine protein,ANA and renal pathological changes were detected on a monthly basis.Mice whose urine protein content reached ++and ANA fluorescence intensity reached ++were randomly devided into three groups ,five each.Antibody intervention group was sequentially injected with B 7-1-mouse chimeric antibody by orbital venous , positive control group was injected with immunosuppressant CTX , negative control group was injected with isotype control IgG.Urine protein and ANA were also detected on a monthly basis.Mice were sacrificed three months after intervention was executed.Kidney was used for H&E dying , IC detection and electric microscope observation.Results: After four-month Pristane induction , urine protein content of 80%mice reached +-+++,meanwhile,serum ANA fluorescence intensity reached ++-+++.Glomerulonephritis infiltrating cells were observed Mice with urine protein and ANA , glomerular inflammatory cell infiltration , tubular epithelial cell degeneration visible edema ,vascular congestion significantly ,fibrosis.After antibody intervention ,urine protein content in antibody intervention group gradually reduced from ++-+++to ±-+++,ANA ++-+++to +-++,and were significantly different from that in the negative control group ( P<0.01 ).Analysis of kidney H&E dying showed that antibody glomerular infiltration of inflammatory cells in the intervention group and tubular congestion and other symptoms were improved significantly.Immunofluorescence staining indicated that fluorescence intensity of IC was significantly reduced in the antibody intervention group.Electron dense deposits reduction and glomerular basement membrane uniformity were observed in antibody intervention group by electric microscope when compared with the negative control group.Conclusion:B7-1 antibodies could downregulate immune response through inhibiting B 7-1/CD28 signaling pathway , reducing the production of autoantibodies and reversing pathological damage caused by autoimmune response .
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For children suffering from primary nephrotic syndrome(PNS), podocyte is a crucial part of glomerular filtration barrier, whose injury usually causes proteinuria.The structural and functional integrity of podocyte cytoskeleton is the prerequisite of its normal physiological function.Recent studies demonstrated that under a certain pathological condition, B7-1, expressed in podocyte, can inhibit the activation of β1 integrin by competitively binding to the target site on the β1 integration, which may change the morphology and function of podocyte, consequently induced proteinuria.Other studies also have showed that abatacept, which selectively inhibit the cross-talk between B7-1 and β1-integrin,can reduce proteinuria in patients with B7-1-positive glomerular disease.These studies suggested that B7-1 may be a potential diagnostic biomarker and therapeutic target in proteinuria in PNS.This review summarizes the role of B7-1 expressed in podocyte in the pathogenesis of proteinuria and the possibility of clinical application in the future.
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This study was aimed to explore the antitumor effect of volatile oil of X ihuang pill and its immune mecha-nism in order to screen the antitumor active site of Xihuang pill. Among 70 female Wistar rats, 10 rats were random-ly selected as the blank control group; and the other 60 rats was used in the establishment of walker 256 breast can-cer cell tumor-bearing rat models. The model rats were randomly divided into the negative control group (model group), high-dose volatile oil group, middle-dose volatile oil group, low-dose volatile oil group, high-dose Xihuang pill group, and lentinan group (positive control group), with 10 rats in each group. The intragastric administration was given twice a day for 14 days. Blood was taken from the abdominal aorta. Tumor tissues was removed and weighed to calculate the tumor inhibitory rate. ELISA method was used to detect the level of IL-2, IL-6, IL-10, IFN-γ and TGF-β in peripheral blood. The flow cytometry was used to detect the content of CD3+ T cell, CD4+ T cell, CD8+ T cell, and B7-1 cell (CD80). The results showed that the tumor inhibitory rates of volatile oil of high-dose group and middle-dose group were 28.4% and 24.1%, respectively. Compared with the model group, the average level of IL-2 and IFN-γ of volatile oil of high-dose group and middle-dose group and CD3+ T cell, CD8+ T cell, B7-1 cell con-tent were obviously increased (P< 0.05). It was concluded that volatile oil of Xihuang pill had certain antitumor ef-fect, which was one of the antitumor active sites of Xihuang pill. The volatile oil of Xihuang pill upregulates the lev-els of IL-2 and IFN-γ, as well as the contents of CD3+ T cell, CD8+ T cell, B7-1 cell in order to increase the im-mune clearance function of tumor-bearing rats.
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Objective To investigate the ability of immune response of cotransfection of rat breast cancer cells with MIP-1αand B7-1 .Methods Tumor growth was observed every day after the SHZ-88 breast cancer cells were inoculated in the rats .The tumor-bearing rats were randomly divided into control group ,MIP-1αgroup ,B7-1 group and combination group ,which were injected with corresponding transgenic cells .The change of tumor volume in 1~4 weeks and the survival time were observed and compared .The peripheral blood was collected .CD4+ ,CD8+ percentage and CD4+ /CD8+ ratio and the levels of IL-2 and IFN-γof the peripheral blood were detected and compared .Results The tumorigenic rate were 100% ,20% ,30% and 0% after the SD rats were inoculated with SHZ-88 ,SHZ-88/MIP-1α,SHZ-88/B7-1 and SHZ-88/MIP-1α+B7-1 cells for 10 days .After the transgenic therapy ,the tumor volume of the combination group in 1~4 weeks were significantly smaller than the other groups .The survival time of the combina-tion group was significantly longer than the other groups .The CD4+ ,CD8+ percentage and CD4+ /CD8+ ratio and the levels of IL-2 and IFN-γof peripheral blood in the combination group were significantly higher than the other groups .The differences above were all statistically significant (P<0 .05) .Conclusion Cotransfection of MIP-1αand B7-1 could induce immune response in breast cancer rats and play anti-cancer effects .
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Objective To study the effects of IFN-?on expression of B7 molecules in human lung adenocarcinoma cell line cultured in vitro.Methods Human lung adenocarcinoma SPC-A-1 cells were incubated in the medium with IFN-?respectively.Cell proliferation effect was measured by MTT assay;apoptosis was detected with flow cytometry assay;expression of B7-1 and B7-2 mRNA was determined by RT-PCR.Results Compared with those of control group,MTT shows that IFN-?could reduce the SPC-A-1 cell proliferation.FCM shows the apoptosis rate in the IFN-? group was significantly higher,but there is no different changes in each concentration group.Compared with the control group,expression of B7-1 and B7-2 significantly increased in the IFN-?group,and the expression was not correlated with the concentration of IFN-?in each control group.Conclusion IFN-?can inhibit cell proliferation and induce apoptosis in SPC-A-1 cell line.IFN-?markedly enhance the expression of B7-1 and B7-2 in SPC-A-1 line.The apoptosis may be mediated by up-expression of gene B7-1 and B7-2.
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Objective To eukaryotically exp ress B7.1 extracellular domian and investigate its biological activities. Methods Recombinant expression vector pDisplay/B7.1(V+C) wa s transfected into Hela cells by electroporation method. The expression of B7.1( V+C) was identified by immunohistochemistry and in situ hybridization, respectiv ely. In vitro T lymphocyte activation activity was detected by 3H-TdR incoporation method and the aroused cytotoxicity after mixed culture of pDispla y/B7.1(V+C) transfected Hela cell-T cell was observed by MTT method. R esults B7.1(V+C) was expressed successfully on the surface of Hela c ells. The expressed B7.1(V+C) was able to activate T lymphocytes in the pres ence of anti-CD3 monoclonal antibody. Cytotoxicity could be observed when pDisp lay/B7.1(V+C) transfected Hela cells were mixed and co-cultured with T lymphocy tes. Conclusion Eukaryotically expressed B7.1(V+C) main tains its T lymphocyte costimulatory activity, and B7.1(V+C) can be adopted to construct bifunctional molecules for tumor immunotherapy.
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BACKGROUND/AIMS: Immunogene therapy is extensively studied for a therapeutic modality of various cancers. This study was conducted to investigate the efficacy of immunogene therapy using the T-cell costimulatory molecule and human B7-1 (CD80, hB7-1) in an in vivo human hepatocellular carcinoma (HCC) model. METHODS: The stable HCC cell line expressing hB7-1 gene was established using retroviral vector (Huh-7/hB7-1). Of fourteen BALB/c nude mice, 7 were subcutaneously injected with 2 X 10(6) Huh-7/hB7-1 cells, while the other 7 were injected with 2 X 10(6) Huh-7/mock cells as a control group. After the injection, the mice were observed weekly for three months for subcutaneous tumor formation. Assay for natural killer (NK) cell cytotoxicity and serum IFN-gamma was performed at 1 and 2 weeks after inoculation. RESULTS: In BALB/c nude mice inoculated with Huh-7/hB7-1 cells, no tumor growth was observed. BALB/c nude mice inoculated with Huh-7/hB7-1 cells showed significantly increased NK cell activities of splenocytes compared with those with Huh-7/mock cells. Serum IFN-gamma was not measurable at 1 week, but significantly increased at 2 weeks after inoculation to the level of 470 pg/ml in BALB/c nude mice with Huh-7/mock cells and 521 pg/ml in BALB/c nude mice with Huh-7/hB7-1. CONCLUSIONS: Our results demonstrate the in vivo anti-tumor immunity and NK cell activation by transfer of hB7-1 gene into human HCC in xenogeneic BALB/c nude mice model. This approach may provide a tool for the development of immunogene therapies against human malignant tumors.
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Animals , Humans , Mice , B7-1 Antigen/genetics , Cytotoxicity, Immunologic , Gene Transfer Techniques , Interferon-gamma/metabolism , Killer Cells, Natural/immunology , Liver Neoplasms, Experimental/genetics , Mice, Inbred BALB C , Mice, Nude , Neoplasm TransplantationABSTRACT
Objective To study the expression levels of B7.1 and B7.2 in the macrophagus of rats infected with the Plasmodium yoelii sporozoites and investigate the roles of these costimulators in parasite infection immunity. Methods The mammal model was established by infecting rats with the sporozoites, then macrophagus was separated from the rat abdominal cavity 2, 12, 24, 48, 72 h after infection. The expressions were quantitative analyzed by immunofluorescence staining and laser scanning confocal microscopy. Result The expressions of B7.1 and B7.2 were upregulated after infected with the sporozoites. The expression of B7.1 was slowly induced and then descended at 72 h. B7.2 was rapidly induced and maximally expressed at 48 h and downregulation at 72 h. The expression of B7.2 was significantly higher than that of B7.1 at all time points assayed after stimulation (P
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Objective:To explore the action of the second signal system in the activatied and proliferating T cells and the induction of apoptosis of the hepatoma cells.Methods:The T cells were costimulated by anti CD28 and anti CD80(B7.1)McAb and acted on the hepatoma cells(BEL 7402),then testing the concentration of cAMP?cGMP and Ca 2+ in the T cells and the apoptosis of the hepatoma cells.Results:The concentration of cAMP was increased temporarily at first,then decreased rapidly,and increased 1 2 times again when acted on the hepatoma cells.The concentration of cGMP was increased 6 8 times fast and the concentration of Ca 2+ obviously increased 2 3 times too.The peak of them was at the fourth day and positive related to apoptosis of the hepatoma cells.Conclusion:The level of the second signal system of cAMP?cGMP and Ca 2+ were significant correlated with the T cells activated and porliferating and the cytotoxic effect.
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Objectives To evaluate the relationship between the expression of B7-1 in liver tissue and the effect of interferon-alpha(?-IFN) treatment in patients with chronic hepatitis B(CHB).Methods The expression of B7-1 in liver biopsy specimens from 68 CHB patients was studied with immunohistochemistry before ?-IFN treatment.Results B7-1 was expressed in 45(66 2%) liver tissues among 68 patients with CHB,but none in 5 normal controls.The total response ratio of ?-IFN in patients with B7-1 positive was 66 7%(30/45),which was significantly higher than 39 1%(9/23)in the patients with B7-1 negative (? 2=7 20,P
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Objective:Study the relationship of HLA I and B7 1 to elucidate the role of costimulatory molecule on antigen presentation in immunological rejection.Methods:FCM was applied to detect the expression of B7 1 Then the cytotoxicity of PBML was observed before and after blocking the HLA I Results:The level of HLA I expression increased greatly(P
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Objective:To obtain a monoclonal antibody(mAb) against human CD80(B7 1) and to study of its biological effects.Methods:The hybridoma cell line was obtained by using the B lymphoma hybridoma technique after immunization of Balb/c mice with XG7 B7 the cells.Ascites were induced to produce the mAb.The specificity and affinity of mAb were verified B7 competition and FACS.Expression of B7 1 in PBLs,DCs,Raji and Daudi were studied by indirect immunofluorescence.Using counting and trypan blue staining,inhibitory effects of mAb on Raji and Daudi cells were analyzed.The neutralization activity of the 4E5 determined by MTT assay using PBLs as response cells.Results:The anti CD80(B7 1) was obtained.The expression of B7 1 in PBLs、DC、Raji ad Daudi was 10 2%,95 1%,96 7% and 89 2%,respectived 4E5 can inhibit the growth in Raji and Daudi cells and block the costimulatory signals of B7/CD28.Conclusion:4E5 is a specific and functional anti CD80(B7 1) and has high affinity for its ligand.It may be of significant value in basic studies and find clinical applications.
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Interferon y (IFN-?) could enhance the metastatic potential of a variety of tumors via increasing expression of MHC class I molecules or other unidentified mechanisms. It has been demonstrated that transfection of T-cell costimulatory molecule B7 gene into tumor cells could increase tumor immunogenicity and render them susceptible to immune attack. In this study, we investigate whether transfection of B7-1 gene could antagonize IFN-?-induced tumor metastasis. Lipofectamine-mediated transfection was used to introduce murine B7-1 (mB7-1) gene or neo gene into a low-metastatic variant of B16 melanoma cell line. The transfectants and parent B16 cells were treated with 100U/ml of IFN-? for 36 h before being subjected to experimental metastasis assay. The expression of MHC class I and class II molecules on the cells was analysed by flow cytometry. Pre-treatment of parent B16 cells and mock gene-transfected B16 cells (B16-neo) with IFN-? significantly increased their lung-metastizing capacity, while the same IFN-? treatment of B16 cells transfected with mB7-1 gene (B16-B7-1) showed no increase in the number of lung metastatic nodules as compared with control cells. Almost equally elevated expression of MHC class I (H-2Kb and H-2Db) molecules was found on the surface of B16, B16-neo and B16-B7-1 cells. However, a much higher expression of class II (I-Ab) molecule on B16-B7-1 cells than that on B16 and B16-neo cells was observed. These results demonstrate that transfection of B7-1 gene into B16 melanoma could reduce its IFN-?-induced metastasis and that the elevated MHC class II expression on B7-1-expressing cells might play a role in the B7-1 -associated reduction of metastasis.
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Objective: To study the vaccine potency of gene-modified tumor cells, we have constructed IL-12, H7-1 and GM-CSF express vector using retrovirus. Methods: It was transfected into EL-4 thymic lylmphoma cells respectively and the effect of gene transduction on anti-tumor immunity were investigated. Results: The tumorigenicity of EL-4/IL-12 transfectant in C57BI/6 syn- ergistical mice was decreased significantly after implanted with EL-4/IL-12 transfectant compaired with EL-4/Wt or EL-4/Neo groups (P
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We have studied the effects of B7-1 on antitumor immunity induced in vivo by murine B7-1 gene transfected EL-4 lymphoma. At a lethal dose of wild - type(wt) or plasmid controlled (pc) B7-1 negative tumor cells. B7-1 gone transfected EL-4 cells inoculated in syngeneic mice were regressed. In contrast to the mice immunized with B7-1 tumor cells, immunization of mice with B7-l~EL-4 tumor cells showed significant protective effects against the sequence rechallenge of wt EL-4 tumor. Vaccination with X-irradiated B7-1 positive tumor cells at the early stage ( 7 days) after inoculation of wt EL-4 tumor cells showed some therapeutically effects but not at the late stage (14 days after inoculation). Vaccination with B7-l~+ EL-4 tumor cells delayed the occurrence of wt EL-4 tumors and prolonged the survival period of tumor-bearing mice. Our results indicate that the transfcction of B7-1 into tumor cells can increase the immunogenicity of EL-4 tumor and improve host response to wt EL-4 lymphoma. Immunization with B7-1 positive tumors can elicit effective antitumor immunity.
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Objective To study the cotransfection mB7-1 and mCD1D gene into pancreatic cancer cells of rats and to observe its anti-tmor responses.Methods Recombinant retroviral vectors expressing mB7-1and mCD1D gene were packaged into GP2-293 cell lines and transfected.The expressions of mB7-1 and mCD1D were detected with PCR and Western blot.The positive cells of mB7-1 and mCD1D were used to induce the anti-tumor immunity in vitro.Results Anti-tumor immunity was induced after B7-1 and CD1D positive cells were coinoculated in syngeneic mice.Furthermore,the growth of tumor was inhibited.Conclusions Cotransfection of B7-1 and CD1D could induce anti-tumor effect.This study provide a foundation for the application of B7-1 and CD1D gene therapy in tumor.