国家重大科学研究计划项目“生物医学纳米材料对血细胞作用的研究”工作进展与讨论

纳米材料对血液免疫功能细胞的作用及其在白血病免疫治疗中的新方法研究

中国医学科学院基础医学研究所 许海燕 教授


PLGA微球包裹WT1和CpG对DC介导的抗白血病免疫治疗作用研究
张亮,孙钊,段金虹,胡艳,顾宁,许海燕,杨先达
中国医学科学院,北京协和医学院,基础医学研究所

WT1抗原(Wilms’肿瘤蛋白)特异性高表达于急性髓系白血病和淋巴系白血病原始细胞,在正常原始造血细胞中表达水平较低,有望作为白血病免疫治疗的特异性靶标。但是WT1多肽抗原的免疫原性较低,很难激发起有效的免疫响应。为探索基于纳米技术的白血病免疫治疗新策略,我们课题研究了承载WT1抗原的PLGA 微球对树突细胞(DC)的刺激作用,分析了PLGA 微球对DC 细胞介导的特异性抗白血病免疫反应的影响,探索其是否能够改善现有WT1-DC 疫苗的抗白血病免疫效应。

利用常规的W/O/W方法制备了PLGA纳米微球,激光共聚焦观察显示微球为典型球状颗粒(Fig.1A)。DLS检测分析表明微球的直径分布范围为0.84-1.60μm,平均直径为1.22μm(Fig.1B)。与DC细胞孵育后,PLGA微球可以有效的被DC细胞吞噬并进入到DC细胞内(Fig.1C)。我们研究了负载不同形式抗原复合物的PLGA微球的免疫刺激作用。将PLGA分别包裹WT1抗原(简称MP(WT1))以及WT1与佐剂CpG复合物(简称为MP(WT1+CpG)),继而与DC细胞共孵育,形成DC疫苗。进一步用DC疫苗刺激同源人外周血单个核细胞(PBMC),用CFSE 荧光标记法标记NB4,检测PBMC对NB4的特异性杀伤作用。同时用分泌型人IFN-γ酶联免疫吸附方法检测PBMC中IFN-γ的表达,对细胞杀伤的实验结果进行验证。免疫杀伤实验显示承载WT1 抗原的MP(WT1)微球可诱发更强的针对靶细胞的免疫杀伤效应(Fig.1D);而同时承载WT1抗原和佐剂CpG 的PLGA微球MP(WT1+CpG),又可进一步地提升针对靶细胞的免疫杀伤效应(Fig.1e);MP(WT1+ CPG)中抗原和CpG的剂量和比例是决定免疫杀伤效果的重要因素。WT1 和CpG 的浓度分别为1 μg/ml 和2 μg/ml 时,杀伤效果最好; ELISA 实验结果显示,经过MP(WT1+CpG)刺激后,淋巴细胞分泌IFN-γ 的分泌量为17.8 ng/ml,显著高于MP(WT1)组(Fig.1F);特异性免疫杀伤实验结果显示,MP(WT1+CpG)微球所提升的抗肿瘤免疫仅对WT1 阳性/HLA-A2 阳性的NB4 白血病细胞具有特异性杀伤作用,对WT1 阴性/HLA-A2 阳性的U973 细胞以及WT1 阳性/HLA-A2 阴性的K562 细胞的杀伤无显著增强 (Fig.1G)。由实验结果推测 PLGA 微球共包裹WT1 多肽抗原和CpG 佐剂后,能够显著提升DC所介导的对人早幼粒白血病细胞NB4 的特异性免疫杀伤作用。上述结果提示,该技术可能在改善现有WT1-DC 疫苗的抗白血病免疫效应方面具有潜在的应用价值。相应文章发表在Protein & Cell.0 2013 4(3): 163–167上。


Figure 1. Effects of encapsulation of WT1 peptide by PLGA MP on DC-mediated anti-leukemic immunity. (A) Confocal microscopic image of PLGA particles. (B) Size distribution of the particles per DLS analysis. (C) Confocal microscopy images of the DCs incubated with FITC-loaded MPs. Left panel: DCs incubated with PLGA-MPs containing FITC-labeled protein (green). Middle panel: the nuclei of the DCs were stained by DAPI (blue). Right panel: superimposed images of A and B are shown. (D) Effects of MP, WT1, or MP (WT1) on anti-leukemic response. Tumor inhibition produced by lymphocytes mixed with DCs that had been exposed to empty MP, WT1 of 1 μg/mL, or MP (WT1) containing 1 μg/mL peptide. The DC group was treated by saline and used as the control. The stimulated DCs were co-cultured with lymphocytes, which were subsequently mixed with the target cells (NB4 leukemic cells) to induce immune cytotoxicity. The tumor inhibition was measured using the 5- or 6-(N-Succinimidyloxycarbonyl)- 3’,6’-O,O’-diacetyl-fl uorescein (CFSE) fl uorescence-based cytotoxicity assay (n = 6, ± SD). (E) Comparison of tumor inhibition induced by various forms of MPs, using NB4 as the target cells. The WT1 concentration of 1μg/mL was used in experiment groups of WT1, MP (WT1), MP (WT1) + MP (CpG), and MP (WT1 + CpG). The CpG concentration of 2 μg/mL was used in experiment groups of MP (CpG), MP (WT1) + MP (CpG), and MP (WT1 + CpG) (n = 6, ± SD). (F) Production of IFN-γ by lymphocytes stimulated with various immunogenic treatments. Secreted IFN-γ in supernatant of culture medium was measured by ELISA (n = 6, ± SD). (G) Immune cytotoxicity triggered by MP or MP(WT1 + CpG) on target cells of NB4, K562, and U937 (n = 6, ± SD, E:T = 20:1). The star indicates a statistically significant difference.

国家重大计划研究项目课题(2011CB933504)资助研究。