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ABSTRACT: To construct an eukaryotic expression vector of human muCD40Ig fusion protein, and to express it stably in Chinese hamster ovary (CHO) cells for obtaining muCD40Ig fusion protein and founding an experimental basis for investigating the soluble muCD40 molecule in vivo.
Extracellular domain of human muCD40 gene was amplified by RT-PCR from L929/muCD40-transfected cells, and the genes encoding the constant regions of human IgG1 were cloned from human splenocytes. The genes were inserted into eukaryotic expression vector pIRES2-EGFP, respectively. The recombinant vector was transfected into CHO cells by Superfectin. The transfected cells stably secreting muCD40Ig fusion protein was selected with G418 and subcloned. The serum-free culture supernatant of the selected positive clone was subjected to Western blotting and RT-PCR to confirm the expression of the fusion gene. The affinity of muCD40Ig and L929/CD40L was analyzed by flow cytometry (FCM).
The eukaryotic expression vector pIRES2-EGFP/muCD40Ig was constructed successfully. PCR and Western blotting showed that the transfected CHO cell strain was able to secret muCD40Ig fusion protein stably. FCM demonstrated a good affinity between muCD40Ig and L929/CD40L.
A transfected CHO cell strain stably expressing muCD40Ig fusion protein has been obtained, and the muCD40Ig fusion protein can bind to CD40L.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 07/2012; 28(7):673-6.
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ABSTRACT: Host anti-tumor immune responses can be attenuated by suppressor T cells of the phenotype CD8(+)CD28(-) (T(s) cells). In the present study, we investigated the presence of CD8(+)CD28(-) (T(s) cells) in the peripheral blood compartment of gastric cancer (GC) patients. Flow cytometry was used to detect the population of CD8(+)CD28(-) T(s) cells present in peripheral blood in therapy naïve patients with gastric cancer (n = 26), postoperative chemotherapy naïve gastric cancer patients (n = 23), and healthy controls (n = 27). Meanwhile, the clinical data of gastric cancer patients were analyzed. A significant difference in the percentage of T(s) cells was observed when comparing peripheral blood samples from cancer patients to healthy volunteers (27.08 ± 1.60% versus 10.86 ± 0.75%). In the patient group, the percentage of CD8(+)CD28(-) cells among lymphocytes was higher in patients with LN metastasis than those without LN metastasis. The percentage of CD8(+)CD28(-) cells was also related to tumor infiltration and size, but not with the degree of differentiation of cancer cells. Moreover, the percentage of CD8(+)CD28(-) cells was higher in preoperative gastric cancer patients (26.24 ± 1.78%) than in those of postoperation patients (15.79 ± 1.11%). These findings may reflect the possibility of tumor-induced immunosuppression, and they should be complemented with further studies.
Journal of Immunoassay and Immunochemistry 04/2012; 33(2):149-55. · 0.69 Impact Factor
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ABSTRACT: B7-homolog 4 (B7-H4), a recently identified homolog of B7.1/2 (CD80/86), has been described to exert co-stimulatory and immune regulatory functions. We investigated the expression and the functional activity of B7-H4 in lung cancer in vitro and in vivo. Although a lung cancer cell line constitutively expressed B7-H4 mRNA and protein in plasma, primary tumor cell isolated from the transplanted lung carcinoma model expressed B7-H4 on the surface. Interestingly, in transplanted lung carcinoma model, the expression of membrane-bound B7-H4 in tumor cells was increased as prolonging of tumor transformation. Exposure to tumor-associated macrophages strongly induced membrane-bound B7-H4 expression on the lung cancer cell line. To elucidate the functional significance of lung cancer-related B7-H4 expression, we performed co-culture experiments of lung cancer cell with allo-reactive T cells. Lung cancer-related B7-H4 was identified as a strong inhibitor of T-cell effect. Furthermore, B7-H4 mAb had an ability to inhibit tumor growth in vivo. B7-H4 expression may thus significantly influence the outcome of T-cell tumor cell interactions and TAM induced membrane-bound B7-H4 on the lung cancer cell represents a novel mechanism by which lung cancer cells evade immune recognition and destruction.
Cancer letters 11/2011; 317(1):99-105. · 4.86 Impact Factor
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ABSTRACT: DCs infiltrated tumors appears to be phenotypically and functionally defective. B7-H4 was highlighted for its inhibitory role in T cell responses. In this study, we showed that B7-H4 was moderately expressed in imDCs, and up-regulated by IL-10, and TNF-α could counteract the up-regulatory effects of IL-10 on expression of B7-H4 in DCs in vitro. Furthermore, tumor infiltrated DCs expressed B7-H4 at high levels. Blockade of B7-H4 expressed in DCs highly resulted in enhanced T cell proliferation and IFN-γ production significantly. Otherwise, the high level of IL-10 and TNF-α was both detected in the tumor, which suggested that TNF-α can not antagonize the effects of IL-10 on expression of B7-H4 in DCs in vivo. These data indicate that tumor environment may condition local DCs to become dysfunctional in the phenotype, and that the high expression of B7-H4 may contribute to the tumor infiltrated DCs to mediate immune invasion.
Journal of Immunoassay and Immunochemistry 10/2011; 32(4):353-64. · 0.69 Impact Factor
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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 cervical carcinoma. In vivo, we evaluated CD40 expression in 56 cervical carcinoma tissues, 43 cervicitis and 38 normal cervix, and investigated the relationship between CD40 and HPV antigen, histopathological parameters, vascular density, and vascular endothelial growth factor (VEGF) expressions. The results clearly demonstrated that CD40 expression, including membranous and cytoplasmic staining, was significantly higher in cervical carcinoma than in the cervicitis and normal cervix. The expression of CD40 was significantly correlated with HPV and VEGF expressions and microvessel density (MVD). These observations provide evidence that CD40 may be involved in neovascularization of cervical carcinoma, they also suggest that CD40 and VEGF may be useful biomarkers for evaluating the risk of developing cervical carcinoma, and may also be used as a target for therapy.
Cancer epidemiology. 01/2011; 35(4):388-92.
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ABSTRACT: CD40, a member of the tumor necrosis factor receptor superfamily, is widely expressed on various cell types. Some studies show that CD40 expression is related to several carcinomas, where its function remains largely unknown. This study investigated the expression of CD40 on cervical carcinoma and evaluated the effect of agnostic anti-CD40 mAb (5C11) on tumor cell line (SiHa). CD40 expression on the primary cervical carcinoma samples was detected by immunohistochemistry. Results showed that CD40 is commonly expressed in human cervical carcinoma, which is higher than that of normal cervix, cervical precancerous tissue and chronic cervicitis. Treatment of the SiHa cell with the agonistic anti-CD40 monoclonal antibody or Gemcitabine alone did not inhibit the proliferation of the SiHa cell in vitro, but the activation of CD40 on SiHa could enhance its sensitivity to Gemcitabine. Furthermore, CD40 activation blocked SiHa in the S phase, stimulated proapoptotic Bax and inhibited antiapoptotic Bcl-XL mRNA synthesis in the SiHa cell. Apoptosis in SiHa was associated with an increasing ratio of Bax to Bcl-XL in mRNA levels. It is concluded that use of the anti-CD40 mAb 5C11 in combination with chemotherapy may have significant therapeutic potential.
Medical Oncology 05/2010; 28(3):781-8. · 2.14 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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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: 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.
