| Background and ObjectiveCD20-positive B-cell lymphoma is the most common type of malignant lymphomas. Immunotherapy plays an important role in the treatment of lymphoma. Rituximab has improved the prognosis of the CD20~+ B cell lymphoma after it was used as a target therapy drug. However, rituximab can only produce passive immunity, active cell immunity is needed in antitumor immunity. Eliciting cellular immune response, or both humoral and cell immunity against lymphomas is an important strategy to treatment of lymphomas.Following surgical operation, radiotherapy and chemotherapy, biotherapy has become an important approach of tumor treatment. Dendritic cells (DC), which are the strongest antigen presenting cells (APC) in immune system in vivo, play a vital role in inducing specific anti-tumor response. DC-based tumor vaccines represent an effective strategy of immunotherapy for cancers. In other hand, Adenovirus as a vector delivering the gene encoding a tumor-associated antigen into DC may enhance expression of the antigen in DC infected by Ad carrying the gene and improve the ability of DC antigen presentation,therefore inducing strong antigen-specific T cell response.In this research, we used the DCs infected by adenovirus of 5/35 chimeric type encoding the mouse CD20 gene(mCD20)as a vaccine to investigate the antigen-specific immune response to CD20 elicited by DCs infected by Ad5/35 mCD20, and evaluated the immune protective effects produced by them, providing a new strategy and experimental evidence for immunotherapy on CD20-positive lymphomas in a clinical setting.Methods1. Establishment of stably expression of mCD20 tumor cell line: After the plasmid pcDNA3.1 mCD20/EGFP was transfected into B16.F10 cells by liposome-mediated method, B16.F10 cell were cultured and selected in RPMI-1640 medium with 10% calf Serum and 1 mg/ml G418. Using EGFP as a report gene, the EGFP-positive cells were isolated by flow cytometer and the B16.F10 cells expressing mCD20 were reserved.2. Cytotoxicity assay in vitro. Two groups were divided in experiments in vitro in which three mice were included in each group: control group (dl 70-3/DCs) and experimental group Ad5/35 mCD20. Co-culture of DCs on day 7 days with Ad5/35 mCD20 or dl 70-3 at the doses of 100 pfu/per DC for 4 hours were harvested, counted and adjusted to the number of DCs at 5×10~5/200μl. Then 5×10~5 DCs were subcutaneously injected into per allogenetic mouse, and the same doses of DCs infected by adenovirus dl 70-3 were used as control. Using B16.F10/ mCD20/EGFP cells as target cells, the splenocytes of the immunized mice as effectors, cytotoxicity assay in vitro for killing activity of CTL was carried out via the lactic acid dehydrogenase release method to detect the antigen specific cell immunity according to three ratio of effectors:targets (E:T)to 100:1, 50:1, and 25:1, respectively.3. Tumor-bearing experiments in vivo. 20 C57BL/6 female mice at the age of weeks weighting 18±2 g were randomly divided into two groups, the experimental group containing 10 mice immunizate once with Ad5/35 mCD20/CDs and the control group containing the same mice with dl 70-3/DCs. 7 days after immunization, each mouse was subcutaneously inoculated with 2×105 B16.F10/ mCD20/EGFP cells at a side of back of the mice, and the immune protection of mice from the tumors was observed in two month, including the tumor-bearing time and the tumor's size.Reuslts1. B16.F10 tumor cell line Stably expressing mCD20 (B16.F10 mCD20/EGFP) was successfully established by combination of G418-selected and FACS-isolated techniques, 99.1% B16.F10 mCD20/EGFP cells have the expression of EGFP detected and 99.7% B16.F10 mCD20/EGFP cells have the expression of mCD20 detected by flow cytometer。2. Cytotoxicity assay in vitro showed that the DCs with Ad5/35 mCD20 induced the production of antigen- specific CTL. The killing activity of CTL was positively related to the ratio of E:T. At the ratio of 100:1, 50:1 and 25:1, the killing activities of CTL were 63.83±7.72% (t=15.33, P<0.01),47.70±5.31% (t=14.23, P<0.01), and 22.73±3.11%(t=8.74, P<0.01) in the experiment group, whereas 15.37±0.80%,12.83±2.84%, and 11.10±1.01% in the control group, respectively. The results have statistically significance. 3. Tumor-bearing experiments indicated that all of the mice in the control group had the tumor growth in 16 to 18 days after the inoculation with 2×10~5 mCD20~+ B16.F10 cells and just 40% of the mice in the experimental group had tumor growth in 18 days (one mouse), 20 days (two mice), 24 days (one mouse), respectively. Moreover, the size of the tumors in the experiment group was smaller than that of the control group. The rest of mice without tumors in the experimental group continued to be observed up to two months, no tumor growth was still found. The rate of tumor growth was 100% in the control group and 40% in the experimental group, which had a significant difference between the two group (x2=8.571, P<0.01). These results suggested that the DCs with Ad5/35 mCD20 could induce the antigen-specific immunity against mouse CD20-positive lymphoma.ConclusionsDCs infected by Ad5/35 mCD20 can induce the production of antigen-specific CTL, the killing activity of CTL are related to the ratio of E:T. Specific cell immunity by Ad5/35 mCD20/DCs can effectively make the immune protection of mice from mouse lymphoma expressing CD20, which might provide a new strategy and experimental evidence for immunotherapy for CD20~+ lymphoma in a clinical setting.
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