R848联合Poly(I:C)促进DC抗原呈递和增强T细胞杀伤效果的研究

目的:研究R848联合聚肌胞苷酸[Poly(I:C)]对树突状细胞(DC)成熟的影响及DC诱导的细胞毒性T淋巴细胞(CTL)对人肺腺癌A549细胞的杀伤作用。方法:分离人外周血单个核细胞,诱导其成为DC,用A549细胞裂解物即肿瘤细胞裂解物(TCL)作为抗原,R848(Toll样受体7/8激动剂)联合Poly(I:C)(Toll样受体3激动剂)作用于DC,流式细胞术分析DC表面共刺激分子;将抗原刺激活化后的DC与T淋巴细胞混合培养7 d,收集培养上清液和CTL,检测上清液中白细胞介素12(IL-12)p70、干扰素γ(IFN-γ)和肿瘤坏死因子α(TNF-α)的含量;CTL与肺腺癌A549细胞共培养16 h,观察其杀伤效果。结果:DC-R848+Poly(I:C)组DC表面CD83和CD80的表达较DC-TCL组显著提高(P0.01);同时可明显刺激DC分泌IL-12 p70(P0.01);此外,DC-R848+Poly(I:C)组较DC-TCL组对肺腺癌A549细胞杀伤效果亦显著提高(P0.01);DC-R848+Poly(I:C)组IFN-γ和TNF-α的分泌水平较DC-TCL组均显著提高(P0.01)。结论:R848联合Poly(I:C)作用于DC可显著促进其成熟、活化,并增强其抗原呈递作用及CTL对肺腺癌细胞的特异性杀伤作用。

Combination of R848 and Poly(I: C) promotes antigen presentation by DC and enhances killing effect of CTL on lung adenocarcinoma A549 cells

AIM: To investigate the effect of R848 (a Toll-like receptor 7/8 agonist) combined with poly-inosinic:polycytidylic acid[Poly(I:C), a Toll-like receptor 3 agonist] on dendritic cell (DC) maturation, and the killing effect of DC-induced cytotoxic T-lymphocytes (CTL) on human lung adenocarcinoma A549 cells. METHODS: Mononuclear cells were isolated from human peripheral blood and induced to differentiate into DC. The whole-cell lysate of A549 cells, namely tumor cell lysate (TCL), was used as antigen. R848 combined with Poly(I:C) was used as adjuvant to stimulate the DC. DC surface markers were analyzed by flow cytometry. The DC stimulated by antigen was co-cultured with T-lymphocytes for 7 d to induce CTL. The culture supernatant and CTL were collected. The levels of interleukin-12 (IL-12) p70, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the supernatant were measured by ELISA. The CTL and A549 cells were co-cultured for 16 h, and the cytotoxicity was observed by LDH assay.RESULTS: The expression of CD83 and CD80 on the DC surface, and the secretion of IL-12 p70 in DC-R848+Poly(I:C) group were significantly increased compared with DC-TCL group (P<0.01). In addition, the cytotoxicity of CTL for A549 cells in DC-R848+Poly(I:C) group was significantly enhanced compared with DC-TCL group (P<0.01). The secretion levels of IFN-γ and TNF-α in DC-R848+Poly(I:C) group were significantly elevated compared with DC-TCL group (P<0.01). CONCLUSION: R848 combined with Poly(I:C) significantly promotes DC maturation and activation, and enhances the antigen-presenting effect of DC and the cytotoxicity of DC-induced CTL.