外泌体PD-L1激活Treg细胞-M2型巨噬细胞促进喉癌免疫逃逸的机制研究

Immune escape is an important reason for the development of cancer. Our study demonstrated that activated Treg cells (aTreg) and M2 macrophages (M2) promote the generation of each other, forming a positive feedback loop which aggravates their immunosuppression in laryngeal cancer. However, the initial factor is not clear. Further study showed that laryngeal cancer-derived exosomes could induce the positive feedback loop between aTreg and M2. Levels of PD-L1 carried by exosomes correlated with the expression of aTreg and M2 in laryngeal carcinoma. Blockade of exosomal PD-L1 / PD-1 signaling reduced the generation of aTreg and M2. Based on this, we hypothesized that exosomal PD-L1 may promote the activation and generation of aTreg and M2 and enhance their interactions, forming the positive feedback loop which leads to immune escape. This study aims to investigate the mechanism of aTreg-M2 loop mediated by laryngeal cancer-derived exosomal PD-L1 in vitro and verify their positive feedback relationship in vivo. This study will help to reveal the inhibitory effect and mechanism of exosomal PD-L1 in tumor microenvironment, and provide a new way of thinking for the immunotherapy of laryngeal cancer.

免疫逃逸是肿瘤发展的重要原因。我们已证实：喉癌中活化型调节性T细胞（aTreg）诱导产生M2型巨噬细胞（M2），后者可促进aTreg的生成，形成正反馈环，导致免疫逃逸。但其始动因素尚不清楚。预实验发现：喉癌来源外泌体能够促使aTreg-M2正反馈环形成；外泌体含有PD-L1，其在喉癌组织中的表达水平与aTreg、M2的浸润程度有关；阻断外泌体PD-L1与受体PD-1结合能够减少aTreg、M2的生成。我们提出：外泌体PD-L1促进aTreg及M2活化生成，二者形成正向反馈环，导致免疫逃逸。本项目拟通过体外实验阐明喉癌外泌体PD-L1激活aTreg-M2正反馈环的作用机制，体内实验探讨靶向外泌体PD-L1--aTreg--M2正反馈环通路抑制喉癌免疫逃逸的可行性。本研究有助于揭示外泌体PD-L1在肿瘤微环境中发挥免疫负调控的作用和机制，为喉癌的免疫治疗提供新的思路和策略。

活化的调节性T细胞(aTregs)和M2巨噬细胞(M2)的正反馈环在促进头颈部鳞状细胞癌(HNSCC)肿瘤免疫抑制微环境中起着至关重要的作用。然而，调节正反馈回路的关键因素尚不清楚。在此，我们研究了肿瘤细胞外泌体(TEXs)携带PD-L1对aTreg-M2正反馈环和介导免疫抑制的影响。在该研究中，我们将携带以及不携带PD-L1的外泌体 (TEX-PD-L1或TEX-PD-L1KO)与CD4+CD25- T细胞和M0巨噬细胞处理， 并评估其对aTregs、M2分化以及aTregs -M2正反馈环的影响。体内实验通过构建SCC-VII-PD-L1WT（野生型）及SCC-VII-PD-L1KO小鼠移植瘤模型。在SCC-VII-PD-L1KO荷瘤小鼠中，随机分三组，分别予尾静脉注射PBS、TEX-PD-L1WT和TEX-PD-L1KO，观察小鼠肿瘤的生长状况。光谱流式分析小鼠脾脏、引流淋巴结以及肿瘤组织的免疫细胞浸润特点。研究结果表明，TEXs比肿瘤细胞携带更多的PD-L1，不仅促进aTregs和M2细胞的分化，而且最重要的是增强aTregs -M2的正反馈环路，抑制CD4+CD25- T细胞的增殖，进而导致肿瘤免疫逃逸。此外，体内研究表明TEX-PD-L1KO在外周和肿瘤微环境中均能抑制肿瘤生长，显著提高抗肿瘤疗效。综上所述，本研究揭示了TEX-PD-L1在负性免疫调节中的作用和机制，靶向TEX-PD-L1可能是HNSCC肿瘤免疫治疗的新思路和新策略。