RT-PCR was used to clone DNA fragment of the extracellular domain of 4-1BBL from human THP-1 cells (human monocyte), and the expression vector pAYZ4-1BBL was constructed by cloning the extracellular domain of 4-1BBL into the expression vector pAYZ. The extracellular domain of 4-1BBL was expressed in E. coli 16C9 and purified by affinity chromatography. SDS-PAGE and Western blot analysis showed that the relativae molecular weight of soluble 4-1BBL is 22kD which was consistent with the theoretically predicted value. So far as we know, it is the first time that the soluble expression of 4-1BBL in E. coli was achieved 4-1BBL induced a significant release of IL-2 in stimulated Jurkat cells after 48h incubation, especially in the presence of tumor cell. At the same time the apoptosis level of Jurkat cell reduce more than 50%. In conclusion, 4-1BBL may be useful in cancer immunotherapy.
4-1BB Ligand ; biosynthesis ; genetics ; Apoptosis ; genetics ; Cell Line ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Extracellular Space ; metabolism ; Humans ; Interleukin-2 ; biosynthesis ; Jurkat Cells ; Recombinant Proteins ; biosynthesis ; genetics

This study was purposed to investigate the molecular mechanism of 4-1BBL reverse signals in the human acute monocytic leukemia cell line of U937. The U937 cell line was used as target cells, and stimulated by the mouse anti-human 4-1BBL monoclonal antibody 1F1. The nuclear translocation of NF-κB and the co-location of 4-1BBL and CD28i molecules in U937 cells were observed with confocal laser scanning microscopy. The protein and m-RNA expression levels of 4-1BBL and CD28i were detected by flow cytometry and RT-PCR respectively. The results showed that the significant nuclear translocation of NF-κB and co-localization of 4-1BBL and CD28i on membrane of U937 cells appeared after being stimulated by mAb1F1. It is concluded that the 4-1BBL reverse signals transduction mediating the growth of U937 cells relates with the nuclear translocation of NF-κB. CD28i may be involved in intracellular 4-1BBL reverse signaling pathways.
4-1BB Ligand ; immunology ; metabolism ; Antibodies, Monoclonal ; pharmacology ; CD28 Antigens ; metabolism ; Coculture Techniques ; Humans ; NF-kappa B ; genetics ; Signal Transduction ; U937 Cells

Our previous study has demonstrated that there is a significant delay of Balb/c cardiac allograft rejection in the C57BL/6 4-1BB-deficient knockout recipient. In this study, we examined the effect of combined blockade of the 4-1BB and CD28 costimulatory pathways on cardiac allograft rejection in the C57BL/6-->Balb/c model. A long-term cardiac allograft survival was induced in CD28/4-1BB- deficient mice (>100 days survival in 3 of 4 mice), which was comparable with CD28-deficient mice (>100 days survival in 2 of 5 mice; P<0.2026). There was no long-term cardiac allograft survival in either wild-type (WT) or 4-1BB-deficient mice, even though 4-1BB-deficient recipients showed a significant delay of cardiac allograft rejection than WT mice. An in vitro mixed leukocyte reaction (MLR) assay showed that 4-1BB-deficient and WT mouse T cells had a similar responsiveness to allostimulation, whereas CD28- and CD28/4-1BB-deficient mouse T cells had a defective responsiveness to allostimulation. Furthermore, 4-1BB-deficient mice showed a similar CTL but an elevated Ab response against alloantigens as compared to WT mice, and the alloimmune responses of 4-1BB-deficient mice were abrogated in the CD28-deficient background. Overall, these results indicate that the CD28 costimulatory pathway plays a major role in the alloimmune response and that 4-1BB signals are dependent upon CD28 signals.
Transplantation, Homologous/immunology ; Signal Transduction/*immunology ; Mice, Knockout ; Mice ; Isoantigens/immunology ; Heart Transplantation/immunology ; Graft Survival/immunology ; Cytotoxicity Tests, Immunologic ; Antigens, CD28/genetics/*immunology/metabolism ; Antibodies/immunology ; Animals ; 4-1BB Ligand/deficiency/genetics/*immunology/metabolism

OBJECTIVETo study the construction of the PCI-neo mammalian expression vector system containing murine 4-1BBL gene and its stable and effective expression in rat hepatocellular carcinoma cell line CBRH7919.

METHODSThe murine full-length 4-1BBL cDNA was obtained and subcloned into the PCI-neo mammalian expression vector. The recombinant named as PCI-neo-4-1BBL was identificated by restriction enzyme digestion and sequencing. Subsequently, PCI-neo-4-1BBL was transfected into CBRH7919 with lipofectamine reagent, then G418-resistance clone was acquired and named as PCI-neo-4-1BBL-CBRH7919+. The stable expression of 4-1BBL mRNA in PCI-neo-4-1BBL-CBRH7919+ was detected by RT-PCR.

RESULTSThe fragment of 980 bp (4-1BBL) and 5.4 kb (PCI-neo) was shown after PCI-neo-4-1BBL had been digested by EcoR I and Not I and agarose gel electrophoresis. The DNA sequencing of 4-1BBL, proved to be identical to the data of 4-1BBL in Genebank, showed stable and effective expression in PCI-neo-4-1BBL-CBRH7919+.

CONCLUSIONThe PCI-neo mammalian expression vector system containing murine 4-1BBL has been constructed successfully, which shows stable and effective expression in CBRH7919.

4-1BB Ligand ; Animals ; Cell Line, Tumor ; Cloning, Molecular ; DNA, Complementary ; genetics ; Genetic Vectors ; Liver Neoplasms, Experimental ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Recombination, Genetic ; Transfection ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

OBJECTIVETo explore the anti-tumor immunity in vitro induced by prostate cancer cell vaccine transfected with recombinant adenovirus encoding 4-1BBL in mice.

METHODSThe replication-deficient adenovirus AdEasy-1 system was used to construct recombinant adenovirus Ad-m4-1BBL and Ad-eGFP. The prostate cancer cell RM-1 of mice was transfected with Ad-m4-1BBL and Ad-eGFP, and treated with mitomycin (MMC) to produce TCV, TCV-Ad-eGFP and TCV-Ad-m4-1BBL, followed by co-culture with syngeneic murine spleen cells. Then the cytotoxic activity of the lymphocytes against RM-1 cells was analyzed with CCK-8 solution, and IL-2 and INF-gamma were detected by ELISA.

RESULTSThe 4-1BBL protein was highly expressed in the TCV-Ad-m4-1BBL of the 4-1BBL-transfected mice. TCV-Ad-m4-1BBL significantly increased the expressions of IL-2 ([180.24 +/- 2.22] pg/ml) and INF-gamma ([1512.46 +/- 23.64] pg/ml) as compared with TCV and TCV-Ad-eGFP (P < 0.05), and induced higher RM-1 cell specific cytotoxicity ([34.24 +/- 2.64]%) than the latter two ([9.82 +/- 1.48]%) and ([14.65 +/- 3. 21]%), (P < 0.05). But none of them exhibited significant cytotoxicity against hepatocellular carcinoma Hepal-6.

CONCLUSIONThe m4-1BBL-expressing prostate cancer cell vaccine can effectively induce anti-tumor immune responses.

4-1BB Ligand ; genetics ; immunology ; Animals ; Cancer Vaccines ; genetics ; immunology ; Cell Line, Tumor ; Coculture Techniques ; Cytotoxicity, Immunologic ; genetics ; Female ; Interleukin-2 ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Prostatic Neoplasms ; Transfection

4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a major costimulatory receptor that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. The interaction of 4-1BB with 4-1BBL regulates immunity and promotes the survival and expansion of activated T cells. In this study, the expression of 4-1BB and 4-1BBL was examined during regeneration of the murine thymus following acute cyclophosphamide-induced involution. Four-color flow cytometry showed that 4-1BB and 4-1BBL were present in the normal thymus and were preferentially expressed in the regenerating thymus, mainly in CD4+CD8+ double-positive (DP) thymocytes. Furthermore, the CD4loCD8lo, CD4+CD8lo and CD4loCD8+ thymocyte subsets, representing stages of thymocyte differentiation intermediate between DP and single-positive (SP) thymocytes, also expressed 4-1BB and 4-1BBL during thymus regeneration but to a lesser degree. Interestingly, the 4-1BB and 4-1BBL positive cells among the CD4+CD8+ DP thymocytes present during thymus regeneration were TCR(hi) and CD69+ unlike the corresponding controls. Moreover, the 4-1BB and 4-1BBL positive cells among the intermediate subsets present during thymus regeneration also exhibited TCRhi/int and CD69+/int phenotypes, indicating that 4-1BB and 4-1BBL are predominantly expressed by the positively selected population of the CD4+CD8+ DP and the intermediate thymocytes during thymus regeneration. RT-PCR and Western blot analyses confirmed the presence and elevated levels of 4-1BB and 4-1BBL mRNA and protein in thymocytes during thymus regeneration. We also found that the interaction of 4-1BB with 4-1BBL promoted thymocyte adhesion to thymic epithelial cells. Our results suggest that 4-1BB and 4-1BBL participate in T lymphopoiesis associated with positive selection during recovery from acute thymic involution.
4-1BB Ligand/genetics/*metabolism ; Animals ; Antigens, CD137/genetics/*metabolism ; CD4-Positive T-Lymphocytes/cytology/metabolism ; CD8-Positive T-Lymphocytes/cytology/metabolism ; Cell Adhesion ; Cell Differentiation ; Cell Line ; Cells, Cultured ; Cyclophosphamide/pharmacology ; Epithelial Cells/cytology ; Gene Expression Regulation ; Immunosuppressive Agents/pharmacology ; Male ; Mice ; Mice, Inbred C57BL ; RNA, Messenger/genetics ; *Regeneration ; T-Lymphocytes/*cytology/metabolism ; Thymus Gland/*cytology/drug effects/*physiology

OBJECTIVETo understand the influence of co-expression of CD80, CD86 and CD137L genes on tumor immunogenicity in hepatocellular carcinoma H22-BAL B/c mouse models.

METHODSThe mice were randomly divided into five groups, named A, B, C, D and E, and control groups A and B, 20 mice in each group. Hepatocellular carcinoma H22-BAL B/c mouse model was established by subcutaneous injection of cells H22-Wt, H22-neo, H22-CD80/CD86+, H22-CD137L+ and H22-CD80/CD86/CD137L+, respectively. The rate and incubation period of tumor development, survival rate, and the tumor growth in vivo were observed and recorded. The effects of gene transduction on immunogenicity of the tumor and antitumor immunity of the animals were assessed by re-innoculation of wild type H22 cells.

RESULTSThe rate of tumor development in group E was only 50%, much lower than that in other four groups (P < 0. 01). The tumor growth in group C was reduced with complete tumor regression in two hosts (20%, 2/10). In group E, there was more pronounced reduction of tumor size. The maximal tumor sizes were remarkably smaller than those of group C, and there was complete tumor regression in three mice (60%, 3/5). No tumor regression was found in the other three groups. Survival rates of group C and E were significantly higher than that of animals in groups A, B and D (P < 0. 01), but no significant difference was seen between group C and E. The results of re-inoculation test showed that tumor formation rate was 40% (4/10) in group C, 100% (8/8) in group D, and 0 (0/5) in group E. There were significant differences between groups C and E and control group, between group E and C, but not between C and D. After the third time of re-inoculation with H22-Wt cells at the 56th day, tumor occurred in 6/6 mice (100%) of group C, but 0 (0/5) in group E. The difference was very significant. Five animals without tumor formation after the first inoculation in group E, were re-inoculated with H22-Wt cells on the 21st day and the third re-inoculation on the 56th day, no tumor was found (0/5).

CONCLUSIONBoth co-expression and solo-expression of CD80+ CD86 and CD137L genes reduce the tumorigenicity of wild type H22 cells, but co-expression of CD80, CD86 and CD137L genes can more significantly improve the immunogenicity of H22-CD80/CD86/CD137L+ cells.

4-1BB Ligand ; genetics ; immunology ; metabolism ; Animals ; B7-1 Antigen ; genetics ; immunology ; metabolism ; B7-2 Antigen ; genetics ; immunology ; metabolism ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Liver Neoplasms, Experimental ; genetics ; immunology ; pathology ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Random Allocation ; Survival Analysis