Anti-CD3 mAbs induce CD4+FoxP3+Regulatory T( Treg) cells and alleviate acute rejection of the pulmonary al-lografts in mice / 中华胸心血管外科杂志

Objective By using mouse orthotopic lung transplant model, we investigated the immune mechanisms of an-ti-CD3 induced lung allograft protection .Our study intends to further dissect the features of lung transplant immunology and to provide a novel therapeutic insight for the clinical application of anti-CD3 mAbs after lung transplantation.Methods Murine orthotopic allogeneic lung transplants were performed in C57BL/6 wild type(WT) mice using major histocompatibility complex (MHC) fully mismatched BALB/c donors.Syngeneic transplants were also performed in WT C57BL/6 mice using C57BL/6 donors.For immunosuppressive therapy, allograft recipients received 50g dose of anti-CD3 by intraperitoneal injection on days 2, 3, 4, 5, 6 and 9 post-operation(n=4).At day 10, histopathologic characteristics and rejection status of the pulmonary grafts were assessed.The severity of acute rejection was graded by the pathological score , and T cell and neutrophil infiltration in the pulmonary grafts was evaluated by immunohistochemical(IHC) staining for CD3 and myeloperoxidase(MPO) respective-ly.Real-time RT-PCR was performed for FoxP3, IL-17A and IFN-γexpression in the pulmonary grafts.The percentage of FoxP3+Treg in total CD4+T lymphocytes from the recipient spleens was analyzed by FACS.Results 10 days after transplan-tation, histopathologic examination demonstrated that there is no apparent acute rejection observed in the pulmonary isografts , whereas allografts from untreated recipients have marked inflammatory cell infiltration and pulmonary parenchyma lesion .IHC staining for CD3 and MPO showed that the allograft-infiltrating cells of perivascular layers are mainly T lymphocytes , and the cells around the small airways are mostly neutrophils .Anti-CD3 treatment significantly alleviated the acute rejection of pulmo-nary allografts, when compared with the untreated group.Real-time RT-PCR showed that the expression levels of IL-17A and IFN-γin allografts were markedly elevated compared to those in isografts, and anti-CD3 increased the expression of FoxP3, and reduced the expression of IL-17A and IFN-γin the pulmonary allografts.FACS analysis of splenocytes showed that the percent-age of Treg in total CD4+T lymphocytes increased significantly in the anti-CD3 treated allograft recipients, as compared with the isograft and untreated allograft recipients.Conclusion Anti-CD3 mAbs may alleviate acute rejection of the pulmonary al-lografts by promoting FoxP3 expression and Treg development.

Construção de um vetor de expressão para fragmentos de anticorpos anti-cd3 humanos com potencial imunorregulatório / Construction of an expression vector for fragments of human anti-cd3 antibodies with immunoregulatory potential

Construiu-se um novo vetor para expressão de fragmentos de anticorpos anti-CD3 humano (pMIRES FvFc R ­ Fc mutado) com potencial imunoregulatório. Foram utilizadas técnicas de clonagem no vetor pUC 57 e no vetor de expressão pMIRES FvFc R por meio de transformação bacteriana por choque térmico; seguida de preparação do DNA plasmidial em pequena e média escala; digestão dos mesmos com enzimas de restrição, e posterior análise e extração do DNA plasmidial por eletroforese. A clonagem foi confirmada pela técnica de sequenciamento de Sanger. Este trabalho resultou na construção de um novo cassete para expressão de fragmentos de anticorpos anti-CD3 humano contendo duas mutações na região variável e uma mutação na porção Fc, que se acredita que melhore a produção heteróloga do anticorpo. A construção deste novo vetor de expressão, com a adição da sequência de exportação de RNA e mutações na porção Fc, buscou contribuir para a caracterização e aprimoramento das características ligantes e efetoras dos anticorpos monoclonais anti-CD3.

Study on HL-60 cell apoptosis induced by CD3 McAb-activated killer cells in serum-free culture system / 中国免疫学杂志

Objective:To investigate whether CD3McAb activated killer(CD3AK)cells could induce apoptosis in HL 60 cells in serum free culture system.Methods:HL 60 cells were coincubated with CD3AK cells expanded in serum free medium,then apoptosis was assessed by means of morphological studies,DNA agarose gel electrophoresis and flow cytomether(FCM) analysis.Results:After 7 h of coincubation with CD3AK cells in serum free medium,HL 60 cells showed morphological features of apoptosis and typical DNA ladder on gel electrophoresis.FCM analysis showed that 24.59% cells were found to undergo apoptosis.Conclusion:CD3AK cells can induce apoptosis in HL 60 cells under serum free culture conditions.

A Case of Common Variable Immunodeficiency with Autoimmune Hemolytic Anemia

Common variable immunodeficiency (CVID) is a heterogeneous collection of disorders with hypogammaglobulinemia with recurrent bacterial infections and high incidence of autoimmune disorders as its hallmark. We report a 7-year-old girl suffering from CVID with Coombs' test positive hemolytic anemia. She had been relatively well until 23-months old when she was admitted to Taejon St. Mary's Hospital with pneumonia 5 years ago. Afterwards, she had suffered from recurrent otitis media, paranasal sinusitis, bronchitis and pneumonia, experiencing 13 admissions. She was diagnosed as autoimmune hemolytic anemia at 4-years old and had been treated with prednisolone. Laboratory finidings showed hypogammaglobulinemia(gamma-globulin in immunoelectrophoresis 0.04g/dL, IgG 170mg/dL, IgA 31mg/dL, IgM 27.5mg/dL) which was previously within normal limits checked at the age of 3- and 5-years old. Isohemmagglutinins (Anti-A,-B IgM and IgG) and anti-measles IgG, anti-mumps IgG, anti-rubella IgG and anti-HBs antibody along with PPD skin test were all negative. Peripheral lymphocyte subsets revealed as follows : pan T cells (CD3+) 48.6% (normal values : 60-85%), pan B cells (CD19+) 36.7% (8-20%), CD4+ T cells 24.4% (28+/-8%), CD8+ T cells 15.3% (5+/-10%), and CD4/CD8 ratio of 1.6 (0.6-2.8). Proliferations of peripheral blood mononuclear cells induced by various T cell stimulants were all markedly depressed. Chronic paranasal sinusitis and lung parenchymal damages were revealed on computerized tomography and lung scan, and a monthly intravenous immunoglobulin therapy was started.

The Enhancing IL-2R alpha mRNA Expression induces A Marked T Cel Proliferation with Interleukin-2 and Anti-CD3 mAb / 대한면역학회지

Culture of human peripheral T lymphocytes with irnmobilized anti-CD3 rnAb plus IL-2 resulted in a marked proliferation and the enhancing IL-2Ra mRNA expression. The process of the T cell activation involves a series of biochemical events which ultimately lead to the proliferation and IL-2Ra mRNA expression. Although the above results have been observed, the celluar signal mechanisms between the proliferative response and the IL-2Ra mRNA expression through T cell receptor and IL-2 receptor remains unresolved. In the present study, We have used genistein (the selective PTK inhibitor) or chronic PMA treatment (depletion of intracelluar PKC activity), to investigate the role of PTK or PKC both in a synergistic proliferation and in the enhancing IL-2Ra mRNA expression by IL-2/anti-CD3. Genistein (30 ug/ml) completely blocked IL-2 induced T cell proliferation, and inhibited anti-CD3 induced T cell proliferation (93.4%). But genistein downregulated the IL-2Ra mRNA expression by IL-2, anti-CD3 and IL-2/anti-CD3. The chronic PMA treatment failed to inhibit the proliferation and the IL-2R#u mRNA expression by IL-2 alone. But PKC depleted T cells stimulated with anti-CD3 mAb showed the decrease of the proliferation (68.6%) and IL-2Ra mRNA expression. In activated with IL-2/anti- CD3, the proliferative response showed a half of reduction, but the IL-2Ra mRNA expression were not regulated. These results demonstrate that proliferative response to IL-2 appears to be dependent on PTKs activity and independent of PKC involvement, but the IL-2Ra mRNA expression may be required another signals. PTKs and PKC activity may be important in TCR/CD3 signaling. But IL-2/anti-CD3 are coupled up different signal transduction pathways responsible for the synergistic T cell proliferation and the enhancing IL-2Ru mRNA expression.

Construction of Anti-CD3 Human/Murine Chimeric Antibody Genes for Expression in Mammalian Cells / 中国肿瘤生物治疗杂志

For construction of anti-CD3 human/murine chimeric antibody genes, a selective first-strand cDNA synthesis from mRNA or RNA of murine McAb HIT3a was performed using murine Ig constant region primers, and then cDNA of heavy and light chain variable domains of murine immunoglobulin were amplified by PCR using a set of degenerated oligonucleotide primers. Using these cDNA fragments as probes, the L and H chain V region exons encoding the murine McAb anti-CD3 were isolated from the gene library of HIT3a DNA and inserted into mammalian expression vectors containing the human ? and yl constant region exons for construction of human/murine chimeric antibody genes.

Expression and Characterization of Human/Murine Chimeric Antibodies with Specificity for CD3 Antigen / 中国肿瘤生物治疗杂志

Exons encoding the variable regions of the light and heavy chain of the murine McAb HIT3a against CD3 antigen were isolated from HIT3a gene library and inserted into mammalian expression vectors containing the human K and y1 region exons. The chimeric genes were transfected into murine SP2/0 hybridoma cells by Lipofectin. Antibody levels of culture supernatants from transfectomas ranged 21~32 ?g/ml. The chimeric antibodies bound specifically to human T cells and competed effectivelly with the parental anti-CD3 murine McAb for binding to CD3 antigen on human T cells. The ability to promote antibody-dependent cell-mediated cytolysis was significantly enhanced with the chimeric antibodies as compared with anti-CD3 murine McAb. The mitogenic activity of human T cells can be suppressed and enhanced by the anti-CD3 chimeric antibodies. The cytotoxicity to tumor cells and proliferation of human T cells mediated by the anti-CD3 chimeric antibodies were much higher than IL-2 alone. Chimeric HIT3a antibody is a clinically relevant, genetically engineering antibody with potential use in treatment of graft-versus-host disease in tranplantation, autoimmune diseases and some tumors.

A New Approach to Enhance the Induction and Activation of PHA-LAK Cells / 中国肿瘤生物治疗杂志

A new type of killer cells, named PHA-?CD3LAK, was induced by means of costimulating the peripheral blood mononuclear cells (PBMC) with anti-CD3McAb (?CD3) and rIL-2 after PHA-priming for 48 hours. Some biological characteristics of PHA-?CD3LAK, PHA-LAK and CD3AK were compared. The results showed that PHA-?CD3LAK exhibited some advantages over CD3AK and PHA-LAK in proliferation, cytotoxicity, the expression level of mIL-2R, as well as the utilizing of IL-2, suggesting the synergistic enhancing role of PHA, ?CD3 and IL-2. All three groups of effector cells were heterogeneous populations, predominantly CD3 + CD8 + T cells. The CD8 ~(+) cell percentage of PHA-?CD3LAK was higher than that of the other two groups. The application of PHA-?CD3LAK might open a new prospect to clinical therapeutic approach.

The preparation and the killer function on Hep-2 clone of bifunction antibody of anti-human laryngocarcinoma/anti-CD_3 / 中国免疫学杂志

Objective:To prepare bifunction antibody of anti-human laryngocarcinoma/anti-CD 3 for the active immune therapy of laryngocarcinoma.Methods:To corss link monoclonal antibody of anti-human laryngocarcinoma and anti-CD 3 into bifunction antibody by chemical method,which can guide the killer function on carcinoma cells.Results:The killer rate of effector cells guided by bifunction antibody is higher than that of anti-CD 3 which can arise with the elevation of effect target ratio.Conclusion:Bifunction antibody of anti-human laryngocarcinoma/anti-CD 3 prepared by chemical cross linking method has the potential value for clinical application. [