李浩江, 王振勇, 沈师, 高超, 张彬, 王泽昊, 眭翔, 崔雪梅, 袁玫, 刘舒云, 郭全义, 王桂琴. 小鼠多亚型热休克蛋白/肽疫苗联合PD-L1免疫检查点抑制剂的抗肿瘤实验研究[J]. 中国肿瘤临床, 2019, 46(6): 278-283. DOI: 10.3969/j.issn.1000-8179.2019.06.337
引用本文: 李浩江, 王振勇, 沈师, 高超, 张彬, 王泽昊, 眭翔, 崔雪梅, 袁玫, 刘舒云, 郭全义, 王桂琴. 小鼠多亚型热休克蛋白/肽疫苗联合PD-L1免疫检查点抑制剂的抗肿瘤实验研究[J]. 中国肿瘤临床, 2019, 46(6): 278-283. DOI: 10.3969/j.issn.1000-8179.2019.06.337
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Li Haojiang, Wang Zhenyong, Shen Shi, Chao Gao, Zhang Bin, Wang Zeha, Sui Xiang, Cui Xuemei, Yuan Mei, Liu Shuoyun, Guo Quanyi, Wang Guiqin. Anti-tumor experimental study of mouse multi-subtype heat shock protein/peptide vaccine combined with PD-L1 immunological checkpoint inhibitor[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2019, 46(6): 278-283. DOI: 10.3969/j.issn.1000-8179.2019.06.337
Citation: Li Haojiang, Wang Zhenyong, Shen Shi, Chao Gao, Zhang Bin, Wang Zeha, Sui Xiang, Cui Xuemei, Yuan Mei, Liu Shuoyun, Guo Quanyi, Wang Guiqin. Anti-tumor experimental study of mouse multi-subtype heat shock protein/peptide vaccine combined with PD-L1 immunological checkpoint inhibitor[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2019, 46(6): 278-283. DOI: 10.3969/j.issn.1000-8179.2019.06.337
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小鼠多亚型热休克蛋白/肽疫苗联合PD-L1免疫检查点抑制剂的抗肿瘤实验研究

Anti-tumor experimental study of mouse multi-subtype heat shock protein/peptide vaccine combined with PD-L1 immunological checkpoint inhibitor

    摘要
  • 摘要:
      目的  应用小鼠肉瘤组织制备混合多亚型热休克蛋白/肽疫苗（mHSP/P），联合程序性死亡配体（PD-L1）抑制剂治疗小鼠肉瘤。
      方法  免疫组织化学染色和Elisa蛋白定量鉴定肉瘤细胞MCA207中的热休克蛋白（HSP70、HSP90、Grp94）的表达。制备蛋白悬液，通过蛋白层析技术获取mHSP/P和Grp94/肽肉瘤疫苗（Grp94/P），以Western blot（WB）鉴定。流式细胞术测定细胞毒性作用。Elisa实验测定mHSP/P和Grp94/P刺激产生的干扰素（IFN-γ）、肿瘤坏死因子（TNF-α）。小鼠实验探究肉瘤疫苗对肉瘤生长以及小鼠生存状况的影响。免疫荧光染色MCA207肉瘤细胞表面PD-L1的表达，WB测定IFN-γ对PD-L1表达的影响。动物实验探究PD-L1抑制剂联合mHSP/P对肿瘤的影响。
      结果  肿瘤组织携带多种亚型的HSP（HSP70、HSP90、Grp94）；成功制备肉瘤组织来源的mHSP/P和Grp94/P，Western blot对肿瘤疫苗鉴定并且流式细胞学测定未发现细胞毒性；制备的mHSP/P较Grp94/P刺激产生更多的IFN-γ和TNF-α细胞因子（P < 0.05）。肉瘤细胞表面PD-L1的表达随着IFN-γ的介入而增高；动物实验显示PD-L1抑制剂联合mHSP/P提高了免疫反应的抗肿瘤作用（P < 0.05）。
      结论  肿瘤来源的mHSP/P和Grp94/P可以作为肿瘤疫苗在动物实验中使用。mHSP/P比Grp94/P能诱发更强的抗肿瘤免疫反应。IFN-γ刺激肉瘤细胞PD-L1的表达而导致免疫逃逸。PD-L1抑制剂联合mHSP/P提高了抗肿瘤作用。

     

    Abstract:
      Objective  To evaluate the anti-tumor activity of mouse multi-subtype heat shock protein/peptide (mHSP/P) vaccine in combination with a programmed death ligand 1 (PD-L1) inhibitor in mouse sarcoma.
      Methods  Immunohistochemical staining and enzyme-linked immunosorbent assay (Elisa) was used to quantitatively identify the expression of heat shock proteins (HSP70, HSP90, Grp94) in the sarcoma cell line MCA207. From the protein suspension prepared, mHSP/P and Grp94/peptide (Grp94/P) sarcoma vaccines were isolated using chromatography and were identified by Western blot (WB). Flow cytometry was used to determine their cytotoxic effects. The levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) produced upon mHSP/P and Grp94/P stimulation were measured by Elisa. The effect of sarcoma vaccines on the growth and survival of sarcoma was evaluated in mice. The expression of PD-L1 on the surface of MCA207 sarcoma cells was evaluated by immunofluorescent staining. The effect of IFN-γ treatment on the expression of PD-L1 was determined by WB. Animal experiments explored the effects of PD-L1 inhibitor in combination with mHSP/P treatment on tumors.
      Results  Tumor tissue carries a variety of HSP subtypes (HSP70, HSP90, Grp94). We successfully isolated sarcoma tissue-derived mHSP/P and Grp94/P tumor vaccines, which were identified by WB; flow cytometry analysis demonstrated their cytotoxicity. The levels of IFN-γ and TNF-α cytokines upon mHSP/P stimulation were significantly higher than that observed upon Grp94/ P stimulation (P < 0.05). The expression of PD-L1 on the surface of sarcoma cells increased with IFN-γ treatment. Animal experiments demonstrated that PD-L1 inhibitor in combination with mHSP/P significantly increased the immune response against tumor (P < 0.05).
      Conclusions  Tumor-derived mHSP/P and Grp94/P can be used as tumor vaccines in animal models. The mHSP/P can elicit a stronger anti-tumor immune response than Grp94/P. IFN-γ stimulates the expression of PD-L1 in sarcoma cells, which results in immune evasion. The PD-L1 inhibitor in combination with mHSP/P increased the anti-tumor effect in the tumor microenvironment.

     

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