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Immunization with Dendritic Cells Infected by Adenovirus Vector Encoding Mouse CD20 Effectively Elicits Immunity Against Mouse Lymphoma

Author: WangYouChen
Tutor: TanXiaoHua
School: Third Military Medical University
Course: Oncology
Keywords: Dendritic cells Adenovirus vector CD20 Lymphoma Immunotherapy
CLC: R392
Type: Master's thesis
Year: 2008
Downloads: 38
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Abstract


The background and purpose of the CD20-positive B-cell lymphoma is the most common type of malignant lymphoma in clinical immunotherapy occupies a significant position in the treatment of lymphoma. Rituximab (Rituximab, MabThera) in the clinical application of a significant improvement in the prognosis of patients with CD20 ~ B-cell lymphoma. However, of rituximab monoclonal antibodies produced in the body is passive immunization in anti-tumor immunity, mainly cellular immune-based initiative. Stimulate the patient's body to produce one of the important research direction lymphoma immunotherapy for lymphoma cells immune response or stimulate the production of cellular and humoral immune response. Following surgery, radiotherapy and chemotherapy, biological therapy has become one of the important means of cancer treatment. Dendritic cells (Dendritic cells, DCs) is the body's immune system is currently the most powerful antigen-presenting cells (antigen presenting cell, APC), DC-based tumor vaccine has become an important strategy of tumor immunotherapy. Plays a critical role in induced tumor-specific cellular immune responses, dendritic cells (DC), adenovirus vectors can greatly enhance the purpose of antigen in turn sent to the DC of DC antigen presentation and induce the efficiency of the antigen-specific immune response. In this study, the adenovirus vector encoding the mouse CD20 Ad5/35 modified DC-induced tumor-specific immune mouse CD20 lymphoma, to evaluate the immune protective effect of CD20 lymphoma cells, the the clinical immunotherapy CD20 lymphoma a new ideas and experimental evidence. Method 1. Establish stable expression of murine CD20 tumor cell lines: the the pcDNA3.1 mCD20/EGFP plasmid with liposome transfected into the B16.F10 mouse tumor cells by G418, EGFP (enhanced immunofluorescence protein) as a reporter gene, the sorting by flow cytometry, and final B16.F10 mCD20/EGFP cell lines. Cytotoxicity assay: The experiment was divided into two groups, each group of three mice: experimental group (Ad5/35 mCD20 infected DCs group); control group (no-load adenovirus dl 70-3 infected DCs group). DC culture in normal mice, day 7, Ad5/35 mCD20 and DL 70-3 to 100 pfu / DC concentration and DC co-incubated for 4 hours, collect DCs were counted, adjusted DCs number of 5 × 10 5/200 ul. Per mice 5 to the mice left rear leg Ministry dorsal immune mice injected intradermally × 105/200μl of DCs, a week after the killing mice spleen CTL cell cytotoxicity hurt experiment. B16.F10mCD20/EGFP cells as target cells after immunization of mouse spleen cells as effector cells, using three effector to target ratio (E: T) 100:1, 50:1 and 25:1, by lactate dehydrogenase release assay CTL killing rate. Vivo tumor-bearing experiment: Take C57BL / 6 mice 20, 4-6 weeks (18 ± 2) g, female, were randomly divided into two groups, the experimental group (Ad5/35 mCD20 1 infected DCs immunization, 10 ), the control group of (dl 70-3 1 infection of DCs immunization, 10). Specific immune method with the cytotoxicity assay. 1 week after immunization with saline adjusted concentration of B16.F10 mCD20/EGFP cells for 2 × 10 5/200 μl, per 2 × 10 ~ 5 B16.F10 mCD20/EGFP cell mouse right hind upper mice The dorsal subcutaneous inoculation. Observed 14 days before the next day after inoculation, after 14 days of daily observation of tumor formation in mice, not yet tumorigenicity in mice continue to observe to two months. Results. G418 selection and flow cytometry sorting technology combined to obtain a stable expression the mCD20 of B16.F10 mCD20/EGFP cancer cell lines, flow cytometry 99.1% cell expression of EGFP, 99.7% express mCD20 2. vitro cytotoxic T lymphocyte killing experiments: effector to target ratio of 100:1, 50:1 and 25:1, the destruction rate of the experimental group were 63.83 ± 7.72% (t = 15.33, P lt ; 0.01), 47.70 ± 5.31% (t = 14.23, P lt; 0.01) and 22.73 ± 3.11% (t = 8.74, P lt; 0.01), the destruction rate of the control group were 15.37 ± 0.80%, 12.83 ± 2.84% and 11.10 ± 1.01%. The experimental results are statistically significant. 3 after the experiment: control group of tumor-bearing mice inoculated with 2 × 10 ~ 5 cells B16.F10 mCD20/EGFP cells within 16 to 18d all tumor growth, the experimental group at 18d (1 only), 20d (2 4 mice), 24d (1 only) into the tumor, and the tumor volume is less than the control group. The remaining mice continued observation of the experimental group to two months, and still can not see the growth of tumors. Control the composition of tumor was 100% and 40% of the experimental group, significant difference (x ~~ 2 = 8.57, P lt; 0.01) between the two groups of tumor formation rate. Tip Ad5/35 mCD20 anti-mouse lymphoma specific immune immune protective effect of the CD20 ~ lymphomas in mice. Conclusion Ad5/35 mCD20 immunized mice were able to induce the generation of specific cytotoxic T lymphocytes (CTL), CTL killing rate of effector to target ratios related; Ad5/35 mCD20 anti-mouse lymphoma specific immune CD20 lymphoma in mice immune protective effect.

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