M2表型骨髓来源巨噬细胞外泌体促进神经胶质瘤免疫逃逸和PD-1抑制剂抗性的分子机制

The molecular mechanisms of M2 phenotype bone marrow-derived macrophages’ exosomes in promoting immune escape of glioma and resistance to PD-1 inhibitors

摘要:

目的 探讨M2表型骨髓来源巨噬细胞外泌体促进神经胶质瘤免疫逃逸和PD-1抑制剂抗性的分子机制。方法 建立M0型、M1型和M2型肿瘤相关巨噬细胞(tumor associated macrophages,TAMs)与胶质瘤细胞GL261细胞或K-Luc细胞的共培养体系。细胞实验分组-1为GL261细胞M0型、M1型、M2型组以及K-Luc细胞M0型、M1型、M2型组。提取M0型、M1型和M2型TAMs细胞培养上清液中的外泌体,并分别用以处理GL261细胞或K-Luc细胞。细胞实验分组-2为GL261细胞M0-Exo组、M1-Exo组、M2-Exo组和K-Luc细胞M0-Exo组、M1-Exo组、M2-Exo组。ELISA法测定两种脑瘤细胞M0-Exo组、M1-Exo组、M2-Exo组中IFN-γ、IL-8和IL-12的水平。慢病毒介导敲低/过表达IFN-γ。C57BL/6J小鼠建立在体脑异种移植物小鼠模型,小鼠脑部立体定向注射不同预处理后的GL261细胞。96只C57BL/6J小鼠随机分入6组,每组16只：强力霉素-IgG抗体-Vector control组,强力霉素-IgG抗体-Lenti-IFN-γ-OE组和强力霉素-IgG抗体-Lenti-IFN-γ shRNA组；以及强力霉素-PD-1抗体-Vector control组,强力霉素-PD-1抗体-Lenti-IFN-γ-OE组和强力霉素-PD-1抗体-Lenti-IFN-γ shRNA组。安乐死小鼠后剥离小鼠脑瘤,采用排水法测定脑瘤体积。流式细胞术测定瘤体中浸润的CD8+ PD-1+ T细胞占比(%)。结果 与M0型TAMs和M1型TAMs相比,与M2型TAMs共培养的GL261细胞和K-Luc细胞增殖能力增强(P<0.05)。与M0型TAMs-源外泌体和M1型TAMs-源外泌体处理后的细胞相比,与M2型TAMs-源外泌体处理后的GL261细胞和K-Luc细胞增殖能力增强(P<0.05)。GL261细胞和K-Luc细胞,分别与M0-Exo组相比,M1-Exo组和M2-Exo组IL-8水平升高(P<0.05),IL-12水平降低(P<0.05)；分别与M0-Exo组相比,M1-Exo组和M2-Exo组IFN-γ水平升高(P<0.05)；特别是,与M1-Exo组相比,M2-Exo组IFN-γ水平升高(P<0.05)。与强力霉素-IgG抗体-Vector control组相比,强力霉素-IgG抗体-Lenti-IFN-γ-OE组脑瘤体积增大(P<0.05),强力霉素-IgG抗体-Lenti-IFN-γ shRNA组脑瘤体积减小(P<0.05)。与强力霉素-PD-1抗体-Vector control组相比,强力霉素-PD-1抗体-Lenti-IFN-γ-OE组脑瘤体积增大(P<0.05),脑瘤中浸润的CD8+ PD-1+ T细胞占比增多(P<0.05)；强力霉素-PD-1抗体-Lenti-IFN-γ shRNA组脑瘤体积减小(P<0.05),脑瘤中浸润的CD8+ PD-1+ T细胞占比减少(P<0.05)。结论 M2表型骨髓来源巨噬细胞外泌体中高水平IFN-γ可促进神经胶质瘤CD8+ PD-1+ T细胞浸润、形成免疫抑制微环境和获得PD-1抑制剂抗性。

Abstract:

Objective Explore the molecular mechanisms of M2 phenotype bone marrow-derived macrophages’ exosomes in promoting immune escape of glioma and resistance to PD-1 inhibitors. Methods Co-culture systems of M0, M1 and M2 type tumor-associated macrophages (TAMs) with glioma cells (GL261 cells or K-Luc cells) were established. Cell experiment groups-1: GL261 cells-M0 type, M1 type, and M2 type groups, as well as K-Luc cells-M0 type, M1 type, and M2 type groups. Extracted the exosomes from the culture supernatants of M0 type, M1 type and M2 type TAMs, and used them to treat GL261 cells or K-Luc cells, respectively. Cell experiment groups-2: GL261 cells-M0-Exo group, M1-Exo group, M2-Exo groups, and the K-Luc cells-M0-Exo group, M1-Exo group, M2-Exo groups. ELISA was used to determine the levels of IFN-γ, IL-8 and IL-12 in M0-Exo group, M1-Exo group and M2-Exo group of the two types of brain tumor cells. Lentivirus-mediated knockdown/overexpression of IFN-γ were conducted. In vivo brain xenograft mice model was established by using C57BL/6J mice. Different pre-treated GL261 cells were stereotactically injected into the mice" brains. Ninety six C57BL/6J mice were randomly divided into 6 groups, with 16 mice in each group: Doxycycline-IgG antibody-Vector control group, Doxycycline-IgG antibody- Lenti-IFN-γ-OE group, and Doxycycline-IgG antibody-Lenti-IFN-γ shRNA group; as well as Doxycycline-PD-1 antibody-Vector control group, Doxycycline-PD-1 antibody-Lenti-IFN-γ-OE group, and Doxycycline-PD-1 antibody-Lenti-IFN-γ shRNA group. After euthanizing the mice, the brain tumors were removed and the volume of the tumors were measured using drainage volume method. Flow cytometry was employed to determine the percentage (%) of infiltrating CD8+ PD-1+ T cells infiltrated in the tumor. Results Compared with M0 type TAMs and M1 type TAMs, the proliferation ability of GL261 cells and K-Luc cells co-cultured with M2 type TAMs was enhanced (P<0.05). Compared with the cells treated with M0-type TAMs-derived exosomes and M1-type TAMs-derived exosomes, the proliferation ability of GL261 cells and K-Luc cells treated with M2-type TAMs-derived exosomes was enhanced (P<0.05). For GL261 cells and K-Luc cells, compared with the M0-Exo group, in the M1-Exo group and the M2-Exo group, the levels of IL-8 increased (P<0.05), and the levels of IL-12 decreased (P<0.05). Compared with the M0-Exo group, the levels of IFN-γ in the M1-Exo group and the M2-Exo group increased (P<0.05); particularly, compared with the M1-Exo group, the level of IFN-γ in the M2-Exo group increased (P<0.05). Compared with the Doxycycline-IgG antibody-Vector control group, in the Doxycycline-IgG antibody-Lenti-IFN-γ-OE group the tumor volume increased (P<0.05), while in the Doxycycline-IgG antibody-Lenti-IFN-γ shRNA group the tumor volume decreased (P<0.05). Compared with the Doxycycline-PD-1 antibody-Vector control group, the tumor volume in the Doxycycline-PD-1 antibody-Lenti-IFN-γ-OE group increased (P<0.05), and the proportion of infiltrating CD8+ PD-1+ T cells in the tumor increased (P<0.05); the tumor volume in the Doxycycline-PD-1 antibody-Lenti-IFN-γ shRNA group decreased (P<0.05), and the proportion of infiltrating CD8+ PD-1+ T cells in the tumor decreased (P<0.05). Conclusion The high level of IFN-γ in M2 phenotype bone marrow-derived macrophages’ exosomes could promote the infiltration of CD8+ PD-1+ T cells in glioma to form an immunosuppressive microenvironment, and to acquire resistance to PD-1 inhibitors.