4-1BBL Promoted the Proliferation and Migration of Human Gastric Cancer Cells through Wnt/p-catenin Signaling Pathway / 中国生物化学与分子生物学报

4-IBB and 4-IBB ligand (4-1BBL) , also known as CI) 137 and CD 137 ligand, are members of tumor necrosis factor (TNF) receptor and ligand family, respectively.The interaction of 4-1BBL and 4-IBB can activate T cell immune response.Therefore, 4-1BBL has been playing a role as a classical costimulatory molecule involving in anti-tumor immune responses.In recent decades, it was reported that 4-1BBL had multiple functions in tumor cells.However, the molecular mechanism of 4-1BBL in the progression of gastric cancer (GC) remains unclear.This study investigated the biological function and molecular mechanism of 4-1BBL in human GC cells.First, analysis from TCGA and Kaplan Meier plotter databases showed that 4-1BBL expression level in GC tissues was significantly higher than that in adjacent tissues (P<0.001) , and 4-1BBL high expression was positively associated with poor prognosis of GC (P<0.05).Knockout of 4-1BBL significantly inhibited the proliferation (P<0.05) , invasion and migration of GC cells (P<0.05 ) , and increased the apoptosis of GC cells (P<0.05).Western blot showed that 4- 1 BBL knockout decreased the protein expression levels of (3-catenin, c-Myc and Cyclin D1, and blocked Wnt/p-catenin signaling pathway.On the contrary, 4-1 BBL overexpression significantly promoted the proliferation (P<0.05) , invasion and migration of GC cells (P<0.05) , and reduced the apoptosis of GC cells (P<0.05).Moreover, 4-1 BBL overexpression increased the protein expression levels of (3- catenin, c-Myc and Cyclin D1, and activated Wnt/p-catenin signaling pathway.In summary, 4-1 BBL promoted the proliferation and migration of human GC cells through Wnt/(B-catenin signaling pathway.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6 percent, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4 percent) and CD86 (80.13 ± 2.81 percent)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

Anti-tumor effects induced by gene vaccines co-expressing truncated human prostate specific membrane antigen gene and mouse 4-1BBL / 中华泌尿外科杂志

Objective To investigate the influence of m4-1BBL on the anti-tumor effects induced by truncated human prostate specific membrane antigen (tPSMA) gene in mice. Methods A eukaryotic expression plasmid encoding tPSMA and m4-1BBL (pDC316-tPSMA-IRES-m4-1BBL), pDC316-tPSMA and pDC316 were constructed. C57BL/6 mice were vaccinated in the quadriceps femoris, respectively. The CTL activity of spleen cells from the immunized mice against prostate cancer RM-1-tPSMA was detected by CCK-8 kit in vitro. The tumor growth was then observed. Results The target cell specific cytotoxicity rate induced by pDC316-tPSMA-IRES-m4-1BBL was 42.6%, compared to 24.8% in the pDC316-tPSMA group and 10.8% in the pDC316 group. The difference was significant (P<0.05). The volume of tumor in the pDC316 group was 2657.4mm3 7 d after vaccination, compared to 1334.5 mm3 in the pDC316-tPSMA group, 9 d after vaccination. In the pDC316-tPSMA-IRES-m4-1BBL group, the tumor volume was 445.8 mm3, 12d after vaccination. The difference was significant (P<0.05). Conclusion Gene vaccines co-expressing tPSMA gene and m4-1BBL gene could significantly enhance anti-prostate cancer effects in mice.

The Change of Immunoactivity of Dendritic Cells Induced by Mouse 4-1BBL Recombinant Adenovirus

PURPOSE: The purpose of this study is to construct a recombinant adenovirus vector carrying mouse 4-1BBL and observe its effects in dendritic cells. MATERIALS AND METHODS: Mouse 4-1BBL cDNA was taken from the plasmid pcDNA3-m4-1BBL and subcloned into adenovirus shuttle plasmid pAdTrack-CMV, and then transformed into competent BJ5183 with plasmid pAdEasy-1. After recombination in E. coli, Ad-4-1BBL was packaged and amplified in HEK 293 cells. The expression of 4-1BBL in Ad-4-1BBL-transfected mouse prostate cancer cell line RM-1 was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. After the co-culture of dendritic cells (DCs) with Ad-4-1BBL-transfected RM-1 cells, interleukin (IL)-6 and IL-12 production were assessed by enzyme-linked immunosorbent assay (ELISA) and co-stimulatary moleculs (CD80 and CD86) on DCs were analyzed by flow cytometry. RESULTS: The levels of IL-6 (3,960 pg/mL) and IL-12 (249 pg/mL) production in Ad-m4-1BBL-pulsed DCs were more than those in none-pulsed DCs. The differences were statistically significant (p < 0.05). The expression of co-stimulatary molecules (CD80 and CD86) was up-regulated in Ad-m4-1BBL-pulsed DCs. CONCLUSION: The results indicated the recombinant mouse 4-1BBL can effectively activate DCs.

Location of extracellular cysteine-rich domains of 4-1BB binding to murine 4-1BB ligand and analysis of its possible structure / 中华微生物学和免疫学杂志

ObJective To locate the cysteine-rich domains(CRD) of murine 4-1BB binding to its natural ligand. Methods A serial soluble extracellular CRDs of routine 4-1BB and 4-1BBL fusion proteins was constructed and prepared. The binding of purified 4-1BB-Igs to 4-1BBL and 4-1BB monoclonal antibody were tested using ELISA assay and Western blot analysis. Blocking experiment with 4-1BBL and 4-1BB mon-oclonal antibody was performed by ELISA assay. Results All truncated overlapped proteins containing ex-tracellular CRD Ⅱ of murine 4-1BB were able to bind to 4-1BBL by ELISA assay, excepting the CRD Ⅰ do-main alone. A 4-1BB monoclonal antibody proved to block the interaction of 4-1BB and 4-1BBI, was also able to bind to CRD Ⅱ. Conclusion Murine 4-1BBL whose specificity was mapped to CRD Ⅱ of 4-1BB ex-tracellular region with a possible conformational structure.

Expression of 4-1BB and 4-1BBL in Graves'Disease / 대한내분비학회지

BACKGROUND: 4-1BB mediated costimulatory signal is a recently identified immunotherapeutic strategy for treating autoimmune diseases without depressing the immune response. In this study, we investigated the expression of 4-1BB and 4-1BBL on the peripheral blood mononuclear cells (PBMC) and we assessed whether the serum levels of soluble (s) 4-1BB and s4-1BBL in the patients with Graves' disease (GD) and compared them with normal subjects. METHODS: Expression of 4-1BB and 4-1BBL on PBMC of GD patients was determined by flow cytometry. The concentrations of s4-1BB and s4-1BBL were assessed in the sera of GD patients by performing ELISA. RESULTS: 4-1BB was constitutively expressed on naive CD4+ and CD8+ T cells of the GD patients and this was increased by stimulation. 4-1BBL was also expressed on the antigen-presenting cells such as CD19+ B cells, monocytes and dendritic cells in GD patients. The serum levels of s4-1BB and s4-1BBL were significantly higher in GD patients than those in controls, and these levels were significantly correlated with the serum levels of thyroid-binding inhibitory immunoglobulin and free T4. CONCLUSION: These results indicate that effector T cells of GD patients can be activated through the 4-1BB-mediated costimulatory signal. Elevated s4-1BB and s4-1BBL levels in the sera of GD patients may affect modulation of the clinical course in GD patients.

Study on the relationship between high expression of co-stimulatory molecule 4-1BBL on tumor cell lines and immune escape / 中国免疫学杂志

0.05),but the proliferation rate(P

Expression of Recombinant Human Soluble 4-1BBL in Yeast Pichia Pastoris and It′s Costimulating Activity on T Cells / 中国肿瘤生物治疗杂志

Objective: Methylotropic yeast pichia pastoris system was used to express recombinant human soluble 4 1BBL protein with biological activity. Methods: According to the nuclear acid sequence coding human soluble 4 1BBL, we cloned the genes with PCR from XG 4 1BBL transfection cell line,then the gene fragment for extracellar domain was subcloned into the PUCm T vector and sequence of s4 1BBL cDNA was confirmed by sequencing. The s4 1BBL gene was inserted into the pPICZ?A , which was transformed into Pichia pastoris GS115 by linearized electroportion.The recombinant protein was identified by the assay of SDS PAGE and Western blot. Costimulating activity of rhs4 1BBL on T cell proliferation in vitro was evidenced by 3 H TdR incorporation assay. Results: The s4 1BBL cDNA was successfully obtained and insected into pPICZ?A. The protein molecular weight of hs4 1BBL in the yeast supernamant was about 21 kD by SDS PAGE analyses,and the specificitity was identified by western blot. Finally, rhs4 1BBL protein could costimulate the proliferation of T cells in vitro. Conclusion: The rhs4 1BBL protein was efficiently expressed in Pichia pastoris (GS115)and showed natural biological activities. And it may provide a valuable materials for further study of 4 1BB/4 1BBL.

Construction and indentification of the recombinant vaccine of Attenuated Salmonella typhimurium containing pIRES2-EGFP-4-1BBL vector / 中国免疫学杂志

0.05).The transfected HepG2 cells by infection of SL3261 containing the vector were shown with GFP and the 930 bp target expression by RT-PCR.Conclusion:Attenuated Salmonella typhimurium SL3261 containing recombined eukaryotic expressing plasmid pIRES2-EGFP-4-1BBL is successfully constructed which can deliver recombinant plasmid into HepG2 cells.

Preparation of a functional monoclonal antibody against human 4-1BBL molecule and analysis of its biological characteristics / 中国免疫学杂志

Objective:To prepare functional monoclonal antibodies against human 4-1BBL molecule and analysis of their biological characteristics.Methods:Female BALB/c mice of 6-8 weeks old were immunized with 4-1BBL transfectant (L929/4-1BBL) as immunogen.The spleen B cells of the mice were fused with sp2/0 and hybridoma cells were screened with 4-1BBL transfectant (L929/4-1BBL) by FCM.The biological characteristics of antibody were investigated by rapid isotyping analysis,karyotype analysis,competitive inhibition test etc.Furthermore,the growth of monocytes in vitro was determined by cell number counting and the cytokine concentration in the supernatants was assayed by ELISA.Results:One hybridoma cell line named 3E7 was obtained,which had the property of secreting anti-human 4-1BBL monoclonal antibody continuously and steadily.This mAb specifically recognized human 4-1BBL molecules.The experimental results manifested that mAb 3E7 could effectively enhance the growth of monocytes and the high level IL-6 and TNF-? secretion.Conclusion:One hybridoma cell line which can secret a functional mouse anti-human 4-1BBL mAb has been developed successfully.This mAb can specifically recognize human 4-1BBL and regulate growth and functions of monocytes in vitro.

The costimulatory effect of 4-1BBL and B7-1 molecules on human peripheral blood T lymphocyte / 中国免疫学杂志

Objective:To explore the role and mechanism of 4-1BBL and B7-1 molecules in the activation and proliferation of human peripheral blood T lymphocyte. Methods:T lymphocyte was purified by magnetic beads negative selecting. The proliferation of T cells in primary allogenic MLR was evaluated by using 3H-TdR incorporation; The immunophenotype of T cells was analyzed by FACS and the quantitative measurement of IL-2 in culture supernatant were performed by ELISA.Results:After negative selection, the purity of CD3+ T cells was over 90%; 4-1BBL and B7-1 molecules stably expressed on XG cells after 4-1BBL and B7-1 cDNA transfection; 4-1BBL or B7-1 gene transfected XG cells could more effectively mediate the activation, proliferation and IL-2 secretion of T cells than XG cells did, and the results also showed that the costimulatory molecules had a co-activating effect on T cells.Conclusion:Human multiple myeloma cells failed to induced antitumor immunoresponse because of the lack or weak expression of costimulatory molecules. The costimulatory molecules of 4-1BBL and B7-1 has a synthetic effect on endowing myeloma cells with enhancing the activation、proliferation and IL-2 secretion of T cells in vitro. [