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 construct anti-CD20scFv/CD80/CD28/zeta recombinant gene modified T cells, test its effectiveness of eradicating CD20+ lymphoma cells and provide a probably new approach to tumor adoptive immunotherapy.

METHODSCD28-zeta cDNA were amplified from vector pBULLET and inserted into pLNCX vector that contained anti-CD20scFv/CD80 gene. The recombinant vectors were transduced into PA317 cells and high titer retroviruses were obtained to infect human peripheral blood T lymphocytes. Resistant T cells were obtained by G418 selection at one week. Then transduced T lymphocytes and lymphoma cell lines Daudi Raji were cocultured. The cytotoxicity and cytokine production of transduced T cells were determined by non-radio-activation cytotoxicity assay and ELISA respectively.

RESULTSThe recombinant eukaryotic vector was constructed successfully as proved by enzyme digestion analysis and sequencing. These T cells were able to lyse CD20+ target cells and secrete high levels of IL-2 and IFN-gamma in vitro.

CONCLUSIONRecombinant gene modified T cells can be constructed successfully. It can specially kill CD20 positive lymphoma cells in vitro.

Antigens, CD20 ; genetics ; immunology ; B7-1 Antigen ; genetics ; immunology ; CD28 Antigens ; genetics ; immunology ; Cell Line ; Cytotoxicity, Immunologic ; Genetic Vectors ; Humans ; Immunotherapy, Adoptive ; Plasmids ; genetics ; T-Lymphocytes ; immunology ; metabolism ; Transfection

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

Antigens, CD ; metabolism ; B7-1 Antigen ; metabolism ; CD28 Antigens ; metabolism ; CTLA-4 Antigen ; Child ; Humans ; Interleukin-2 ; metabolism ; Mucocutaneous Lymph Node Syndrome ; immunology ; metabolism ; RNA, Messenger ; genetics ; Receptors, Immunologic ; metabolism

The V(H) and V(L) gene fragments of anti-CD28 mAb were combined to form anti-CD28 ScFv gene by using TP-PCR method. Sequence analysis showed that 6 x His tag was added to it for the ease of purification and the V(H), and V(L) gene fragments were connected by a linker containing 15 amino acids which are biased by the baculovirus promoter, ph. Then ScFv gene fragment was inserted into baculovirus transfer vector pBacPAK8. The recombinant transfer vector, pBacPAK8/CD28-ScFv was constructed successfully. The pBacPAK8/CD28-ScFv and the linear Bm-BacPAK6 were co-transfected into the cell line of Bombyx mori (BmN) with the help of Lipofectin,then the product was purified by plaque assay and identified by PCR method. The recombinant virus, Bm-BacPAK6 CD28-ScFv, was obtained successfully. The BmN cells and the larvae of Bombyx mori were infected by the recombinant baculovirus and harvested every 24h postinfection. SDS-PAGE and Western Blotting analysis confirmed the expression of ScFv with the molecular weight of about 28 kD. The expression in BmN cells was detected 24h post infection and it peaked at 72 h, while in the larvae of Bombyx mori, the expression was detected 48 h post infection and it peaked at 120 h.
Animals ; Antibodies, Anti-Idiotypic ; biosynthesis ; genetics ; Antibodies, Monoclonal ; immunology ; Baculoviridae ; genetics ; metabolism ; Bombyx ; cytology ; genetics ; metabolism ; CD28 Antigens ; genetics ; immunology ; Cell Line ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; immunology ; Larva ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

The expression of the costimulatory molecules B7/CD28 in peripheral blood mononuclear cells (PBMC) of the patients with systemic lupus erythematosus (SLE) and its relation to the pathogenesis of SLE were studied. The expression of the costimulatory molecules in PBMC in 30 patients with active SLE and 20 cases of healthy controls was detected by using the techniques of immunofluorescence and flow cytometer. The result showed that the expression percentage of CD28+, CD4+ CD28+ in T cells of PBMC from the patients with SLE decreased significantly as compared with that in healthy control group, while the expression percentage of CD80+, CD19+ CD80+ in B cells was significantly increased than that in healthy control group (P<0.01). It suggested that the abnormal expression of costimulatory molecules B7/CD28 played a role in the pathogenesis of SLE.
Adolescent ; Adult ; B7-1 Antigen ; biosynthesis ; genetics ; CD28 Antigens ; biosynthesis ; genetics ; Female ; Flow Cytometry ; Humans ; Leukocytes, Mononuclear ; metabolism ; Lupus Erythematosus, Systemic ; blood ; etiology ; immunology ; Lymphocyte Activation ; Male ; Middle Aged

CD28, a cell surface glycoprotein, predominantly expressed on T cells, belongs to the Ig superfamily and provides critical co-stimulatory signals. The data which have published indicate that the monoclonal antibody against CD28 can decrease curative effects when it was applied in vivo for a long time. In order to avoid the human-anti-mouse action, anti-CD28 mAb must be humanized before it can be used in clinical study. Chimeric antibody, consisting of variable regions of mouse antibody and the constant regions of human IgG1, is often chosen by designers in generating humanized antibody. In this study, to prepare the anti-human CD28 chimeric antibody, the genes coding variable regions of anti-CD28 mAb and the constant regions of human IgG1 were cloned by PCR method. Then, the target genes were assembled by TP-PCR, a novel method developed for fusing genes without designing endonuclease sites at the both end of the target genes, and inserted into the baculovirus transfer vector pAcUW3 respectively. Thus, the recombinant baculovirus transfer vector with two strong promoters, ph and p10 was successfully constructed, which can express two different foreign genes at the same time. The recombinant vector was identified by the methods of restriction digesting, electrophoresis, PCR amplification and further verified by DNA sequence analysis. This work will contribute to expressing the chimeric CD28 antibody in insect cells.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; immunology ; Baculoviridae ; genetics ; metabolism ; Base Sequence ; CD28 Antigens ; genetics ; immunology ; Gene Transfer Techniques ; Genetic Vectors ; Humans ; Immunoglobulin G ; biosynthesis ; genetics ; immunology ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Promoter Regions, Genetic ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Trinucleotide Repeat Expansion