Preparation of mouse anti-human B7-2 monoclonal antibody and analysis of its role in inducing Fas and FasL on the surface of 8266 cells / 中华微生物学和免疫学杂志

Objective To prepare a mouse anti-human B7-2 monoclonal antibody ( McAb) and to study its effect on the induction of death-related molecules on the surface of tumor cells. -ethods Trans-genic cells, L929-B7-2, were used as the immunogen to immunize BALB/c mice. Through cell fusion, mul-tiple screening by immunofluorescence labeling and continuous subcloning, the hybridoma secreting B7-2 McAb was obtained. Biological characteristics of the McAb were analyzed using Ig subclass identification test strip, antigenic site competition inhibition assay and specific cell membrane molecules binding test. McAb was prepared through inducing ascites in vivo and then purified by protein G affinity chromatography. The purified McAb was co-cultured with 8266 cells, naturally expressing B7-2 molecules, to observe the expres-sion of Fas and FasL on cell surface by flow cytometry ( FCM) . Results The prepared B7-2 McAb labeled as 12G4 was successfully obtained with a titer of 0. 1 μg/5×105 cells. Its heavy and light chains were IgG2b and κ, respectively. The concentration of the purified ascites-derived antibody was 1. 61 mg/ml. FCM re-sults showed that the 12G4 McAb recognized cell membrane molecules well with a positive binding rate of 89. 6％ to 8266 cells. The mean value of the Fas molecule on the cell surface increased after incubating with 20 μg/ml of 12G4 McAb for 12 h and reached the peak of 62575. 8 at 48 h, which was significantly higher than the maximum value of 57135. 4 in the IgG control group (P<0. 05). After culturing the cells with 20μg/ml of 12G4 McAb for 12 h, the expression of FasL on the cell surface also increased and reached the maximum of 7. 98％ at 48 h, which was significantly higher than the 1. 10％ in the IgG control group ( P<0. 05). Conclusions B7-2 McAb was successfully prepared. It could be used to induce the expression of some death-related molecules on the surface of tumor cells.

Effects of B7-2 gene RNAi lentiviral vector on dendritic cells and T lymphocytes proliferation induced by dendritic cells in vitro / 中国免疫学杂志

Objective: To generate mouse B7-2 gene RNAi lentivirus and study its interference effects on B7-2 expression and T lymphocytes proliferation induced by dendritic cells. Methods: Three sequences specific targeting B7-2 gene and one non-specific sequence were respectively synthesized, and inserted into lentiviral vector, then the recombinant vectors were sequencing. 293 T cells were co-transfected with lentiviral expression plasmid and packaging plasmids to produce recombinant lentivirus which titre was checked according to the expression level of green fluorescent protein ( GFP). Bone marrow cells from C57 BL/6 mice were isolated to differentiate into DCs at the present of GM-CSF, IL-4 and LPS for 48 h, then morphology and phenotypic was identified. DCs were infected by recombinant RNAi lentivirus and then the efficiency of infection and the expression of B7-2 on the surface of DCs were detected by flow cytometry. Effects on the proliferation of T cells were detected by co-culturing with DCs which were infected by B7-2 RNAi lentivirus and murine spleen T cells in vitro. Results: DNA sequencing confirmed that three B7-2 RNAi and one non-specific recombinant lentiviral transfer plasmids were successfully constructed, the titer of recombinant lentivirus was ( 2-4) × 108 TU/ml. The recombinant lentivirus could effectively infect DC and inhibit the expression of B7-2. After the B7-2 recombinant lentivirus infection, the ability of DCs to stimulate the proliferation of T cells decreased obviously ( P＜0. 05). Conclusion: The lentiviral B7-2 gene RNAi vector can effectively silence the expression of B7-2 on the surface of DCs and inhibit the proliferation effect of T cells induced by DCs.

Characterization of FSC(high) Memory B Cells from Patients with Active Systemic Lupus Erythematosus / 대한류마티스학회지

OBJECTIVE: To determine phenotypic and functional characteristics of memory B cells in patients with systemic lupus erythematosus (SLE). METHODS: The percentage of memory B cell subsets in peripheral blood mononuclear cells (PBMC) from normal control (n=11), inactive (n=15) and active (n=10) SLE patients was determined by Fluorescence Activated Cell Sorter (FACS). In addition, the activation status of memory B cells was measured by the surface expression of CD86 (B7-2). The production of antibodies to chromatin and dsDNA (IgG and IgM type) by isolated memory B cell subsets was examined by enzyme-linked immunosorbent assay (ELISA). RESULTS: In this study, we analyzed 2 subtypes of memory B cells: FSC (Forward Side Scatter)(low) and FSC(high) memory B cell. The percentage of both subtypes from active and inactive SLE patients was significantly reduced compared to that of normal controls (p<0.01). In addition, the expression of activation markers, CD86 on FSC(high) memory B cells from active SLE patients was higher than those of inactive SLE patients and normal controls (p=0.014). Upon stimulation with CpG and IL-15 in vitro for 8 days, isolated FSC(high) memory B cells from active SLE patients revealed augmented production of autoantibodies to chromatin and dsDNA. CONCLUSION: Our results suggest that abnormally activated FSC(high) memory B cells from active SLE patients might be involved in spontaneous production of autoantibodies and induce transition from inactive to active phase of the patients.

Effects of ?-interferon on proliferation and B7 expression in SPC-A-1 cell line of human lung adenocarcinoma / 中国实用内科杂志

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.

Expressions of B7.1 and B7.2 in macrophagus of rats infected by Plasmodium yoelii / 第三军医大学学报

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

Specific anti-tumor immune response induced by pEGFP-C3-B7.2-MAGE-1 fusion DNA vaccine / 中国免疫学杂志

Objective:To construct pEGFP-C3-B7.2-MAGE-1 eukaryotic expression plasmids and to study the immunological effects of esophageal cancer cell vaccine modified by human B7.2/MAGE-1 gene.Methods:The B7.2 and MAGE-1 cDNA,which was amplified by RT-PCR, and the eukaryotic expression plasmid pEGFP-C3-B7.2-MAGE-1 was constructed. Human esophageal cancer cell EC9706 was transfected with the vector of pEGFP-C3-B7.2-MAGE-1 using the technique of lipofectamine transfection.The dendritic cells(DCs)from peripheral blood mononuclear cells(PBMC)were loaded with the tumor antigen,and co-cultured with congeneric T cells derived from PBMCs for 3 days to obtain the tumor specific cytotoxicity T lymphocytes(CTL). Methy1 thiazoly1 tetrazolium (MTT) assay was used to detect inhibition effect of CTL on transfected and untransfected EC9706 cells.Results:The CTL had stronger inhibition response against the cancer cells transfected with pEGFP-C3-B7.2-MAGE-1 than to the cancer cells transfected with pEGFP-C3 and the untransfected cancer cells(P

Immunohistochemical Characterization of Dendritic Cells after Systemic Administration of Staphylococcal Enterotoxin B / 대한해부학회지

This study was performed to investigate the effects of Staphylococcal enterotoxin B (SEB) on dendritic cells (DCs) and other immune cells in the major lymphoid organs. A single dose of SEB (25 microgram/kg) was administered to BALB/c mice by intraperitoneal injection. After the mice were sacrificed in groups of three at 2 h, 6 h, 1 day, 2 days, 3 days, 1 week and 2 weeks, the spleen, lymph node and thymus were removed. The immunocytochemical characterization of the cells was carried out using various monoclonal antibodies in cryostat-cut sections. We demonstrated in this study the distribution patterns of DCs and their major costimulatory and adhesive molecules in the murine spleen, lymph node and thymus after SEB administration. We obtained the evidence for maturation of DCs in vivo in response to SEB. DCs were found in increased number in the periarterial lymphatitc sheath (PALS) of spleen, paracortex of lymph nodes and thymic medulla. CD86, ICAM-1 and MHC class II molecules were upregulated on the activated and matured DCs after SEB injection. The most salient feature of the present study was the differential expression pattern of the costimulatory and adhesive molecules on the activated DCs. In addition to DCs, T cells expressing T cell receptor Vbeta8 were increased in number after SEB treatment. In conclusion, SEB exhibited a potent and effective stimulative effect on DCs in vivo.

Fusion gene pEGFP-C3-B7.2-hTERT induces immune response in mice and restrains transplanted hepatoma / 中国肿瘤生物治疗杂志

Objective:To construct a fusion DNA vaccine pEGFP-C3-B7.2-hTERT and to observe the capability of pEGFP-C3-B7.2-hTERT DNA vaccine to induce specific anti-tumor immune responses and to inhibit growth of the hepatoma transplanted from H22 cells. Methods: The B7.2 and hTERT cDNA (amplified by RT-PCR), together with pEGFP-C3 as the vector were used to construct fusion gene plasmid pEGFP-C3-B7.2-hTERT, which was then used to vaccinated C57BL/6 mice for 3 times at a 7 d interval. Animals vaccinated with pEGFP-C3-B7.2, pEGFP-C3-hTERT, pEGFP-C3 and PBS were taken as controls. Splenocytes CTLs of immunized mice, the levels of IL-2, IFN-? in the culture supernatant, the levels of antibodies against hTERT, and the changes of the T lymphocyte subsets in the peripheral blood were all examined. The mice harboring hepatocarcinoma H22 cells were challenged with pEGFP-C3-B7.2-hTERT and the tumor forming time and the survival periods of mice were observed. Results: Agarose gel electrophoresis, nuclease digestion and sequencing confirmed the successful construction of pEGFP-C3-B7.2-hTERT. The CTLs activity of splenocytes from the mice immunized with pEGFP-C3-B7.2-hTERT fusion gene was significantly higher than those of the other groups (P

Analysis of biochemical and physical properties for a new recombinant B7-Pseudomonas exotoxin fusion protein B7-2-PE40KDEL / 中国免疫学杂志

Objective:Identification of some biochemical and physical properties for a new recombinant B7-2-PE40KDEL exotoxin fusion protein.Methods:12%SDS-PAGE separating and gel imaging analyzing,peptide mass fingerprinting,Western blot and MTT assasying were used respectively for identification of the protein.Results:Molecular weight of the recombinant B7-2-PE40KDEL was 72 628,5% of the difference to its theoretical value 69 561.The result of Western blot indicated that the purified recombinant B7-2-PE40KDEL could specifically bind with mAb anti-human B7-2 and the antibody against PEA,while the negative control did not.The recombinant B7-2-PE40KDEL digested with trypsin and then detected by MOLTI-TOF-MS.It was shown that the detected 15 peptides lied in the extracellular part of B7-2 and the truncated Pseudomonas extoxin PE40KDEL.Searching in the peptident data bank of Expasy website,we did not find any known proteins which was accordant with the above terms.The cytotoxic activity of the recombinant toxin with MTT method showed that the B7-2-PE40KDEL selectively killed Jurkat cell line which expressesed CD28 receptor well and had no killing effect on the Raji cell line unexpressing CD28 receptor.Conclusion:Recombinant B7-2-PE40KDEL exotoxin fusion protein we construct proves to be a new one with targeted killing bioactivity to B7:CD28 system.