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ABSTRACT: Immune checkpoints, such as the PD-1/PD-L1 pathway, negatively interfere in the efficiency of dendritic cell (DC) vaccination in cancer immunotherapy. In this study, we demonstrated that the blockade of PD-L1 signaling could promote DC maturation, proliferation, and IL-12 secretion, augment DC primed T cell response and reverse tumor cell dampened T cell impairment. Blockade of PD-L1 signaling during DC vaccination showed better therapeutic effects than classic DC vaccination by preventing tumor growth and prolonging survival times in a breast tumor-bearing hu-SCID model. Taken together, suppressing immune checkpoints during DC vaccination might be a more efficient strategy for cancer therapy.
Cancer letters 03/2013; · 4.86 Impact Factor
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ABSTRACT: FLT3, a transmembrane molecule, was found on hematopoietic stem/progenitor cells and leukemia cells and determined to be a promising target in leukemia diagnosis and therapy. In this study a functional anti-human FLT3, monoclonal antibody (MAb) 10G6, was obtained and the specificity of this MAb was verified by flow cytometry. This MAb effectively recognized the FLT3 molecule expressed on a series of malignant cell lines. Furthermore, we demonstrated that MAb 10G6 inhibited the proliferation and migration ability and induced the apoptosis of SHI-1 cells that derived from a human monocytic leukemia. This functional anti-human FLT3 MAb provides a valuable tool for further study targeting the FLT3 on leukemia cells.
Hybridoma (2005) 02/2011; 30(1):61-7. · 0.42 Impact Factor
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ABSTRACT: CD40L is an important costimulatory molecule in the induction of the humoral and cell-mediated immune responses. 4F1, a specific murine antagonistic monoclonal antibody against human CD40L molecule, is a promising candidate biomedicine for autoimmune diseases, transplantation rejection and anti-angiogenesis therapy of cancer. To avoid the mAb induced thromboembolism as a consequence of platelet surface FcγR activation, we attempted to construct a chimeric Fab of 4F1 to minimize its side effects for potential clinical use. A chimeric version of anti-CD40L Fab was generated by transferring mouse variable regions into a human framework. Our study indicated that 4F1 could be simply and rapidly converted to chimeric Fab which could be expressed in bacteria and purified in reasonable quantities. This chimeric antibody maintained its bioreactivity to human CD40L.
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 10/2010; 65(1):52-9. · 2.24 Impact Factor
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ABSTRACT: A novel member of the human beta3-galactosyltransferase family, the beta3GalT7 gene (AY277592, EC2.4.1.-) was first isolated and cloned by our laboratory. To further study its functions, we constructed a prokaryotic expression system of beta3GalT7 and obtained anti-beta3GalT7 polyclonal antiserum by immunizing rabbit with purified beta3GalT7 protein. Using the antiserum, the expression of beta3GalT7 in various tissues and cell lines was analyzed by Western blot and immunochemical assays. Immunochemistry analysis showed the enzyme was expressed significantly higher in some tumor tissues than in normal tissues, indicating its biofunction in tumorogenesis. By immunofluorescence, the enzyme was observed highly accumulated in cytoplasm around nuclear membrane, implying that beta3GalT7 may play an important role in the assembly of galactose in RER and Golgi.
Hybridoma (2005) 04/2010; 29(2):141-6. · 0.42 Impact Factor
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ABSTRACT: Programmed death-1 (PD-1, CD279), an inhibitory co-stimulatory molecule of the CD28 superfamily, plays a critical role in immune response. In this report, four novel mouse anti-human PD-1 monoclonal antibodies (MAbs) were prepared using hybridoma technology and immunological characteristics of the MAbs were determined. The results showed that all the MAbs (clones 9H1, 4B9, 8F5, and 1F8) were IgG1(kappa) and bound specifically to human PD-1. By mutual competition, we found that the antibodies recognized three different epitopes of PD-1 antigen and 9H1 MAb could block both PD-1/PD-L1 and PD-1/PD-L2 interaction. Cross-linking of PD-1 with MAb 9H1 markedly blocked PD-1 negative signal and promoted T cell proliferation. In addition, 4B9 and 9H1 MAbs were suitable for indirect ELISA detection. Thus, the MAbs against human PD-1 with high specificity and different activity would be useful for the further study of this molecule.
Hybridoma (2005) 04/2010; 29(2):153-60. · 0.42 Impact Factor
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Fang Xie,
Qin Shi,
Qin Wang, Yan Ge,
Yongjing Chen,
Jianling Zuo,
Yongping Gu,
Haizhen Deng,
Hui Mao,
Zhenhua Hu,
Yinghui Zhou,
Xueguang Zhang
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ABSTRACT: CD40 is expressed in many tumor cells, however, its role in tumor biology is yet to be demonstrated. In the present study, we investigated the role of CD40 in gliomas. In vivo, we evaluated CD40 expression in 95 glioma tissues and 10 non-tumorous brain tissues and investigated the relationship between histopathological parameters, vascular density, and vascular endothelial growth factor (VEGF) expressions. In vitro, we aimed to understand the biological relevance of CD40 and VEGF in glioma cell lines. The results clearly demonstrated that CD40 expression, including membranous and cytoplasmic staining, was significantly higher in poorly differentiated and well differentiated gliomas than in the non-tumorous brain tissues (P=0.045 and P=0.043, respectively). In gliomas, the expression of CD40 was significantly correlated with tumor size, VEGF expressions and microvessel density (MVD) (P=0.022, P=0.023 and P=0.0316, respectively). In the in vitro study, stimulation of human glioma cells by CD40 ligation induced the expression and secretion of VEGF and was blocked by anti-CD40 monoclonal antibody. These observations provide evidence that CD40 ligation supports the expression and secretion of VEGF and may be involved in neovascularization of gliomas, they also suggest that CD40 and VEGF may be useful biomarkers for evaluating the risk of developing gliomas, and may also be used as a target for therapy.
Journal of neuroimmunology 03/2010; 222(1-2):62-9. · 2.84 Impact Factor
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ABSTRACT: Dendritic cells (DCs) pulsed by tumor antigens have been widely used as tumor vaccines to specifically trigger the cytotoxicity of CD8+ T cells. But the tumor microenviroment with enriched immunosuppressants hampered DC maturation and co-stimulation. CD40/CD40L signaling, one of the most important co-stimulatory molecules is capable of effectively skewing the immune response by promoting DCs maturation and co-stimulation. To establish a novel specific immunotherapeutic approach for the use of DC vaccine in the treatment of B lymphoma, hu-SCID mice bearing B lymphoma were vaccinated by different combination of tumor antigen pulsed DC or imDC vaccines and immune-enhancing agencies such as agonist CD40mAb and T cells. The results of immature DCs combined with agonistic CD40mAb were encouraging with achievement of tumor regression and induction of antigen-specific immune responses. These findings demonstrated the potential utility of imDC-based tumor vaccination combining with agonistic CD40mAb in the treatment of malignant lymphoma.
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 03/2010; 64(7):487-92. · 2.24 Impact Factor
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Songwen Ju,
Songguang Ju, Yan Ge,
Hongxia Qiu,
Binfeng Lu,
Yuhua Qiu,
Jingxiang Fu,
Gaoqin Liu,
Qin Wang,
Yumin Hu,
Yongqian Shu,
Xueguang Zhang
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ABSTRACT: Dendritic cells (DCs) are responsible for the initiation of immune responses. Our study demonstrates a new pathway for generating a large quantity of stimulatory monocyte-derived DCs (Mo-DCs) from human monocytes using anti-4-1BB ligand (4-1BBL) mAb to trigger reverse signaling. The anti-4-1BBL-driven Mo-DCs (DCs(alpha-4-1BBL)) not only express higher levels of CD86, CD83 and HLA-DR, when compared with the Mo-DCs matured by tumor necrosis factor alpha, but also exhibit a unique phenotype that expresses lower levels of PD-L1. High levels of GM-CSF, M-CSF and Flt3 ligand (FL) were found in the anti-4-1BBL-differentiation culture. Neutralizing M-CSF, GM-CSF and FL inhibited Mo-DC proliferation stimulated by anti-4-1BBL mAb, suggesting that M-CSF, GM-CSF and FL are involved in cell proliferation stimulated by anti-4-1BBL. Further analysis of the DCs(alpha-4-1BBL) showed increased secretion of T(h)1-type cytokines IL-12 and IFN-gamma and decreased secretion of IL-10. DCs(alpha-4-1BBL) induced much stronger proliferative responses in the mixed lymphocyte reaction assay when compared with DCs derived by GM-CSF. Moreover, DCs(alpha-4-1BBL) preferentially induced T(h)1 responses. We have further demonstrated that anti-4-1BBL antibody stimulated nuclear translocation of NF-kappaB from the cytoplasm in monocytes, suggesting that reverse signaling by 4-1BBL is likely responsible for mediating DC differentiation. Collectively, we have found that reverse signaling of 4-1BBL promotes the differentiation of potent T(h)1-inducing DCs from human monocytes.
International Immunology 09/2009; 21(10):1135-44. · 3.41 Impact Factor
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ABSTRACT: OX40L (CD252), a membrane-bound member of the tumor necrosis factor superfamily, is known to be a critical co-stimulatory molecule during immune response. Here, we describe an agonistic mouse anti-human OX40L monoclonal antibody (clone 1E7) recognizing a novel epitope of OX40L antigen. Using this antibody, we found that OX40L was transiently upregulated on CD4(+) T cells during the early stage of activation. Cross-linking of OX40L with monoclonal antibody 1E7 markedly promoted T cell proliferation and activation and enhanced cytokine production, demonstrating that OX40L transmitted a signal to T cells. Thus, OX40L may play an important role in the early phase of T cell activation and proliferation.
Hybridoma (2005) 09/2009; 28(4):269-76. · 0.42 Impact Factor
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Songwen Ju,
Hongxia Qiu,
Xiaoyue Zhou,
Biqin Zhu,
Xin Lv,
Xinen Huang,
Juan Li,
Yu Zhang,
Linxiang Liu, Yan Ge,
Daniel E Johnson,
Songguang Ju,
Yongqian Shu
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ABSTRACT: Regulatory T cells (Tregs) in peripheral blood and tumor infiltrating lymphocytes (TILs) play crucial roles in suppressing anti-tumor immune responses in cancer patients, and correlate with clinical outcomes. We identified an important subpopulation, CD13+CD4+CD25hiTreg cells, among CD4+CD25hiTreg cells in the peripheral blood of non-small cell lung cancer (NSCLC) patients. Peripheral blood mononuclear cells (PBMCs) were isolated from patients with NSCLC (n = 72) or from healthy donors (n = 30). Flow cytometric analyses were performed to study the expression of cell-surface or intracellular markers on the CD4+CD25hiTreg cells. The immune suppressive function of CD13+CD4+CD25hiTreg cells was evaluated by co-culturing with CD4+CD25-T cells that were activated by PHA. Our data showed that, compared with CD4+CD25(Low/-)T cells, CD13 expression was enriched on CD4+CD25hiTreg cells. The CD13+CD4+CD25hiTreg cells also expressed higher levels of Foxp3, CTLA-4, membrane-bound transforming growth factor beta1 (mTGFbeta1) and B7-H1, and are more suppressive to CD25 expression and proliferation of CD4+CD25-T cells. Additionally, we showed that the expression of Foxp3, CTLA-4, B7-H1, mTGFbeta1 and the secretion of TGFbeta1 and IL-10 on CD13+CD4+CD25hiTreg cells was significantly suppressed by anti-CD13 mAb (WM15), and the ability of these cells to suppress CD25 expression and proliferation of CD4+CD25-T cells was inhibited by WM15 as well. Interestingly, the percentage of CD13+CD4+CD25hiTreg cells among the CD4+CD25hiTreg population increased significantly and correlated with pathological stage in NSCLC: healthy donor (9.84% +/- 2.23%) <stage I (21.64% +/- 2.78%) <stage II (31.86% +/- 3.01%) <stage III (45.64% +/- 6.12%) <stage IV (58.78% +/- 12.89%). Moreover, the percentage of CD13+CD4+CD25hiTreg cells decreased dramatically after surgical removal of tumors. CD13 is a new surface molecule for identifying a CD4+CD25hiTreg cell subpopulation with higher suppressive ability. The percentage of CD13+CD4+CD25hiTreg cells among the CD4+CD25hiTreg cell population correlated with the pathological stage in NSCLC and tumor burden. CD13 represents a potential target to suppress Treg cells in anti-tumor therapy.
Cell cycle (Georgetown, Tex.) 08/2009; 8(16):2578-85. · 5.36 Impact Factor
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ABSTRACT: 5C11, a murine monoclonal antibody with a high specificity for human CD40 molecule, is a promising candidate for cancer targeting therapy. We have therefore attempted to construct a humanized antibody of 5C11 to minimize its immunogenicity for potential clinical use. A chimeric version of 5C11 (ch-5C11) was generated by transferring these mouse variable regions onto a human framework. This chimeric antibody retained reactivity to human CD40. In vitro, ch-5C11 could effectively inhibit B lymphoma Daudi cell proliferation, suggesting that it might have the potential to be developed for future clinical use.
Hybridoma (2005) 05/2009; 28(2):121-8. · 0.42 Impact Factor
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ABSTRACT: Dendritic cells (DCs) initiate and direct immune responses. Previous in vitro and in vivo studies have showed that DCs matured with CD40 linking signal could potentially elicit and boost antitumor immunity, however, its molecular mechanism remain elusive. This study demonstrates that expression of B7-H3 on apoptotic cell-loading DCs is up-regulated markedly by CD40 activation but not by tumor necrosis factor-alpha stimulation. There was no significant difference found with CD40, CD80, or CD86 expressions when activated by CD40 or tumor necrosis factor-alpha stimulation. In tumor-bearing mice, T cells conditioned with B7-H3-blocked on CD40-activated apoptotic tumor cell-pulsed DCs had a decreased ability to inhibit tumor growth. Therefore, it is hypothesized that high levels of B7-H3 expression contributes to the ability of CD40-activated tumor associated DCs in eliciting efficient antitumor immune response, given this fact the potentially significant clinical implications, CD40-activated DCs merit further considerations when preparing DCs for clinical application.
Journal of immunotherapy (Hagerstown, Md.: 1997) 02/2009; 32(1):29-35. · 3.20 Impact Factor
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Tao Gu,
Yi Bei Zhu,
Cheng Chen,
Min Li,
Yong Jing Chen,
Ge Hua Yu, Yan Ge,
Shi Yong Zhou,
Huan Zhou,
Yong Huang,
Yu Hua Qiu,
Xue Guang Zhang
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ABSTRACT: During maturation, murine myeloid dendritic cells (DCs) upregulated the expressions of CD11c, CD25, CD40, CD80, CD86, MHC II and programmed death 1 ligands 1 and 2 (PD-L1 and PD-L2). Differential expression patterns of PD-L1 and PD-L2 were found when DCs were triggered by CD40 ligand and TNF-alpha. PD-L1 expression was repressed and PD-L2 expression remained unchanged in mature CD40-ligated DCs, whereas TNF-alpha stimulated DCs kept high expression of PD-L1 and significantly enhanced PD-L2 expression on DCs. Proliferations of T lymphocytes stimulated by immature DCs were enhanced by blockade of the PD-1 and PD-1 ligand interaction. But inhibitive effects were found in T lymphocytes stimulated by CD40-ligated DCs. With the fine-tuned expressions of PD-L1 and PD-L2, CD40-ligated DCs could sustain a longer activation period and elicit a more efficient T lymphocyte activation.
Cellular & molecular immunology 03/2008; 5(1):33-9. · 2.99 Impact Factor
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ABSTRACT: To investigate the stable expression of a chimeric antibody against CD40 moleculeèch-5C11éin CHO and its biological activity.
Human-mouse chimeric antibody against CD40 recombinant plasmid and mock plasmid were transfected into CHO cell line through lipofectamine mediation. Human kappa chain and Fc fragment of ch-5C11 were characterized by FACS and Western blot. The concentration of ch-5C11 in cell supernatants was detected by Lowry assay and the inhibitory effect of ch-5C11 on the proliferation of Daudi cells was detected by MTT assay.
RT-PCR showed that target CHO cells integrated chimeric heavy chain and chimeric light chain gene. FACS and Western blot showed that ch-5C11 in cell supernatants maintained the binding activity and specificity to human CD40 molecule, and contained human kappa chain and Fc fragment. Cell supernatants were purified using protein G affinity chromatography. The concentration of human-mouse chimeric antibody against CD40 in cell supernatants was 0.535 mg/L. When co-cultured with B lymphoma cell line Daudi, ch-5C11 induced proliferation arrest of Daudi cells.
The human-mouse chimeric antibody against CD40 can be expressed in CHO stably and effectively, which inhibits proliferation of Daudi.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 07/2007; 23(6):565-8.
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ABSTRACT: Recent data have revealed that Ag presentation by immature dendritic cells (imDCs) plays a role in establishing and maintaining T-cell tolerance, but the mechanism remains unclear. PD-L1 and PD-L2, ligands for programmed-death receptor 1 (PD-1), members of the expanding B7 family, were highlighted for their inhibitory role in T-cell responses. Here, we show that blockade of PD-1 ligands on imDCs resulted in enhanced T-cell proliferation, which is perhaps due to the enhancement of IL-2 production from DC-stimulated T cells. PD-1 ligands blockade on mDCs did not show a significant stimulatory effect as markedly as imDCs. The inhibitory effects of PD-1 ligands would be dependent on maturation status of DCs, where attenuated positive costimulatory molecules provided the opportunity for PD-1 ligands to exert their strong capacity. Our data are consistent with the hypothesis that imDCs have an inhibitory bias, and indicate that PD-L1 and PD-L2 contribute to the poor stimulatory capacity of imDCs.
Immunobiology 02/2007; 212(3):159-65. · 3.20 Impact Factor
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ABSTRACT: OX40 ligand (CD252), a molecule originally identified as human gp34, is an important costimulatory molecule during immune response. Here, we describe a sandwich ELISA for the detection and quantification of soluble OX40L using anti-OX40L monoclonal antibodies 1G1 and 4C12 as capture and detecting antibody, respectively. With this ELISA, the existence and concentration of soluble forms of OX40L (sOX40L) was demonstrated for the first time. It was found that soluble OX40L is detectable in the sera of elderly persons (above 60 years old) and patients with Graves' disease which has the highest mean serum concentration of sOX40L, suggesting the potential diagnostic significance of sOX40L in the disease. Surprisingly, the quantitation of sOX40L was correlated with the age and among these subjects, those of 70s and 80s have much higher sOX40L concentration than those of 60s.
Cytokine 11/2006; 36(1-2):23-8. · 3.02 Impact Factor
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ABSTRACT: To explore the biofunctions of human B7-H4 generated from prokaryotic system.
The gene of human B7-H4 extracellular region (IgV-like and IgC-like domains) was obtained by PCR from human cDNA FLJ22418 and then inserted into the prokaryotic expression vector pGEX-5X-3 expressing glutathione s-transferase (GST) fusion protein. After being identified by restriction enzyme digestion and sequencing, the recombinant vector was transferred into host strain E coli BL21-RIL(DE3). A 47 kDa fusion protein (GST/hB7-H4) was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by standard methods reported in the prokaryotic system. The inhibitory effect of GST/hB7-H4 on proliferation of T cells was observed in vitro by CD3mAb activated T-cell culturing system and [(3)H]-thymidine incorporation assay. The concentrations of interleukin-2 and iterferon-g in the supernatants of T cells were determined by ELISA.
We successfully constructed the method for high-level expression and purification of the hB7-H4 extracellular domain as GST fusion protein from E coli. The GST/hB7-H4 fusion protein produced in bacteria had obvious biological activity to inhibit T-lymphocyte proliferation and IL-2 secretion.
The prokaryote expression system could be used to generate hB7-H4 protein with natural spatial conformations and biological functions, which provided an efficient and economical way for the preparation of this target protein.
Acta Pharmacologica Sinica 07/2006; 27(6):741-6. · 1.95 Impact Factor
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ABSTRACT: Aim: To explore the biofunctions of human B7-H4 generated from prokaryotic system. Methods: The gene of human B7-H4 extracellular region (IgV-like and IgC-like domains) was obtained by PCR from human cDNA FLJ22418 and then inserted into the prokaryotic expression vector pGEX-5X-3 expressing glutathione s-transferase (GST) fusion protein. After being identified by restriction enzyme digestion and sequencing, the recombinant vector was transferred into host strain E coli BL21-RIL(DE3). A 47 kDa fusion protein (GST/hB7-H4) was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by standard methods reported in the prokaryotic system. The inhibitory effect of GST/hB7-H4 on proliferation of T cells was observed in vitro by CD3mAb activated T-cell culturing system and [3H]-thymidine incorporation assay. The concentrations of interleukin-2 and iterferon-g in the supernatants of T cells were determined by ELISA. Results: We successfully constructed the method for high-level expression and purification of the hB7-H4 extracellular domain as GST fusion protein from E coli. The GST/hB7-H4 fusion protein produced in bacteria had obvious biological activity to inhibit T-lymphocyte proliferation and IL-2 secretion. Conclusion: The prokaryote expression system could be used to generate hB7-H4 protein with natural spatial conformations and biological functions, which provided an efficient and economical way for the preparation of this target protein.
Acta Pharmacologica Sinica 06/2006; 27(6):741 - 746. · 1.95 Impact Factor
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ABSTRACT: To construct and express a chimeric antibody against human CD40 molecule by genetic engineering antibody transformation.
Total RNA was extracted from the murine hybridoma cell 5C11 which secreted anti-CD40 monoclonal antibody (mAb). The genes encoding V(H) and V(L) of mAb 5C11 were amplified by RT-PCR. According to sequence analysis, the primer was designed to amplify signal peptide sequences relative to V(H) and V(L) genes. The V(H) and V(L) genes of mAb 5C11 and relative signal peptide sequences were spliced with C(H) and C(kappa) genes of human IgG1 to construct expression plasmid pIRES/h5C11 of human-mouse chimeric antibody gene and the plasmid was transfected into 293T cells under Lipofectamine mediation for transient expression. Expressed product was analyzed by flow cytometry.
The result of NCBI gene data bank blast revealed that cloned gene sequence accorded with mice' V(H) and V(L) genes and feature of signal peptide sequence. The constructed plasmid pIRES/h5C11 was correct. The restriction endonuclease digestion analysis and PCR showed that the recombinant genes were subsequently cloned into vector pIRES. FACS analysis showed that human-mouse chimeric antibody against human CD40 maintained the binding activity and specificity to human CD40 molecule.
The gene encoding variable region of human-mouse chimeric antibody against CD40 is cloned successfully and the human-mouse chimeric antibody against CD40 is expressed transiently in 293T cells.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 04/2006; 22(2):189-92.
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ABSTRACT: Costimulator OX40 (CD134) belongs to TNFR family, and its ligand OX40L (CD134L) belongs to TNF family. OX40-OX40L signal plays an important role in immune regulation, which can increase the production of cytokines and enhance the survival of antigen-specific T cells. This research was to transfect OX40L into human breast cancer cell line MDA-MB-435 to construct OX40L-MDA-MB-435 cells, and to investigate the costimulatory effect of OX40-OX40L signal on biological activity of T cells in vitro.
cDNA fragment encoding OX40L was obtained from human mature dendritic cells by reverse transcription-polymerase chain reaction (RT-PCR), and inserted into eukaryotic expression vector pcDNA3.1. The recombinant plasmid pcDNA3.1-OX40L was transfected into MDA-MB-435 cells, and verified by indirect immunophenotyping and RT-PCR. The effect of OX40L-MDA-MB-435 cells to the biological activity of T cells was investigated by MTT, enzyme-linked immunosorbent assay (ELISA), and direct immunophenotyping.
OX40L-MDA-MB-435 cells were successfully constructed. These cells efficiently promoted the proliferation of T cells, enhanced the secretion of interleukin-2 (IL-2) (973.4 ng/ml at the 7th day) and interferon-gamma (IFN-gamma) (3,689.167 pg/ml at the 3rd day), and down-regulated the expression of Fas on T cells [(68.3+/-5.6)%, P<0.05].
OX40L-MDA-MB-435 cells could activate T cells in vitro, promote T cell proliferation and cytokine secretion, and suppress T cell activation-induced cell death.
Ai zheng = Aizheng = Chinese journal of cancer 03/2006; 25(2):148-52.
