CD137L在HBsAg DNA疫苗诱导小鼠细胞免疫应答中的佐剂效应

Adjuvant effect of co-stimulatory molecule CD137L on cellular responses to HBsAg DNA vaccination in mice

目的:研究共刺激分子CD137L重组质粒对HBsAg DNA疫苗诱导小鼠细胞免疫应答和回忆反应的佐剂作用。方法:将小鼠CD137L真核表达载体(pcD137L)体外转染NIH3T3细胞,RT-PCR、流式细胞仪和免疫荧光法分别检测CD137L mRNA和蛋白的表达;pcD137L与HBsAg DNA疫苗(pcDS)通过肌肉注射联合免疫BALB/c小鼠,LDH释放法测定体外脾细胞特异性CTL杀伤活性;流式细胞仪分析小鼠脾脏CD8+记忆T细胞数量;流式细胞仪检测CD8+T细胞及淋巴细胞中IFN-γ和IL-4的表达水平。结果:重组质粒pcD137L在NIH3T3细胞中获得有效表达。与pcDS单独免疫组比较:免疫后1周,pcDS+pcD137L免疫组能诱导更强的HBsAg特异性CTL杀伤活性(P<0.05);免疫后1周和12周,pcDS+pcD137L免疫组CD8+T细胞CD44high和CD127表达水平均显著增高(P<0.05或P<0.01);免疫后1周、12周和13周(即再次给予pcDS刺激后1周),pcDS+pcD137L免疫组分别明显上调CD8+T细胞和淋巴细胞中IFN-γ产生细胞的百分比,差异有显著意义(均P<0.05)。结论:共刺激分子CD137L能显著增强HBsAg DNA疫苗诱导的Tc1(I型)细胞免疫、特异性CTL应答及回忆反应,是诱导HBV特异性T细胞应答的有效佐剂。

Objective： To investigate the adjuvant effect of co-stimulatory molecule CD137L on cellular responses to HBsAg DNA vaccination in mice.Methods： Eukaryotic expression vector containing the full length of mouse CD137L cDNA sequence（pcD137L） was transfected into NIH3T3 cells,and then the expression of CD137L mRNA and protein in the transfected cells were detected by RT-PCR,flow cytometry and immunofluorescence method,respectively.The BALB/c mice were co-immunized with pcD137L and HBsAg DNA vaccine（pcDS） by intramuscular injection.HBsAg-specific activity of splenic cytotoxic T lymphocyte（CTL） in the immunized mice was measured by LDH release assay.The splenic memory CD8＋ T cells,and intracellular IFN-γ and IL-4 of splenic lymphocytes and CD8＋ T cells after immunization were detected by flow cytometry.Results： The NIH3T3 cells transfected with pcD137L efficiently expressed mouse CD137L mRNA and protein.HBsAg-specific CTL responses induced by the pcDS plus pcD137L group were much stronger than those induced by pcDS alone at a week after immunization（P0.05）.Compared to mice immunized with pcDS alone,CD44high and CD127（IL-7R） were all significantly up-regulated in memory CD8＋ T cells from the mice immunized with pcDS combined CD137L both at a week and 12 weeks after immunization（P0.05 and P0.01）.The pcDS plus CD137L group also elicited higher levels of IFN-γ secreted by CD8＋ T cells and splenic lymphocytes than pcDS alone at a week,12 and 13 weeks after immunization,respectively（all P0.01）.Conclusion： DNA,viral/immunol;Co-stimulatory molecule CD137L can enhance the Tc1（type I） cell-mediated immunity,HBsAg-specific CTL and memory responses induced by HBsAg DNA vaccine,and may be an efficient adjuvant in priming HBV-specific T cell response.