以Ad SIINFEKL—Luc为抗原研究免疫反应的量效关系

Dose-effect relationship between Ad SIINFEKL-Luc immunization and file induced imnmne response

目的定量地探讨腺病毒载体编码的已知抗原与其诱发的抗原特异性细胞免疫反应的量效关系。方法用不同MOI的编码鸡卵清蛋白MHCI多肽SIINFEKL（OVAp257-264）与荧光素（luciferase，Luc）融合蛋白的腺病毒（Ad SIINFEKL-Luc）感染小鼠骨髓来源的树突状细胞（DC），在不同的时间点定量检测DC中Luc的表达；相应地，用表达不同水平SIINFEKL-Luc融合蛋白的DC皮下免疫C57BL／6小鼠，通过体内CTL杀伤试验和细胞内γ干扰素（IFN-γ）染色（ICS）定量分析CTL杀伤活性及其产生IFN-γ的能力。结果Luc在DC中的表达水平与Ad SIINFEKL-Luc所用剂量具有剂量效应关系，AdSIINFEKL-Luc一次感染后在DC中持续4d仍然检测得到Luc的表达。分别用0．1、1、10和100MOI的Ad SIINFEKL-Luc／5×10^5 DC免疫小鼠1周，体内CTL杀伤试验分析小鼠脾细胞4hCTL杀伤率分别是14．4％±2．3％、32．6％±4．5％、86．7％±3．8％和99．8％±0．3％。ICS分析脾脏IFN-γ产生的CD8^＋T细胞占总CD8^＋T细胞比率分别是0．49％±0．12％、1．74％±0．21％、4．12％±0．34％和9．53％±0．58％。结论Ad SIINFEKL-Luc表达抗原水平的高低与其诱发特异性细胞免疫反应的强弱明显相关。在一定范围内，抗原表达高低与其诱发的抗原特异性细胞免疫反应的强弱呈正相关。

Objective To quantitatively investigate the relationship between a known antigen encoded by adenoviral vector（Ad SIINFEKDLuc） and the induced immune response. Methods Murine bone marrow-derived dendritic cells（DC） were infected by Ad SIINFEKL-Luc encoding a fusion protein consisting of chicken ovalbumin MHC class Ⅰ poptide SIINFEKL（OVAp257-264 ） and luciferase in different MOI and the expression levels of luciferase（Luc） in DC were quantitatively checked. C57BL/6 mice were subcutaneously immunized with the Ad SIINFEKL-Luc-infected DC, and then the killing activity of antigen-specific CTL in spleen were analyzed by in vivo CTL assay and intracellular cytokine staining（ICS） assay for IFN-T produced by antigen-specific CD8^＋ T cells were performed. Results A dose-dependent relationship between the levels of Luc expression and doses of Ad SIIN- FEKL-Luc was observed, and the expression of Luc conld be still detected in DC 4 d after Ad SIINFEKD-Luc infection. One week after the subcutaneous immunization of C57BL/6 mice with 0.1, 1, 10 and 100 MOI Ad SIIN- FEKL-Luc × 10^5 DC, the four-hour CTL killing rate were 14.4% ± 2.3%, 32.6% ± 4.5%, 86.7% ± 3.8% and 99.8 % ± 0.3 %, respectively, and the ratio of IFN-γ-producing CD8^＋ T cells to total CD8^＋ T cells in spleen were 0.49% ±0.12%, 1.74% ±0.21%, 4.12% ±0.34% and 9.53% ±0.58%, respectively. Conclusion There is an obvious relationship between levels of the antigen expressed by Ad SIINFEKL-Luc and antigen-specific cellular immune responses elicited by the antigen. In a certain range, a positive correlation exists.