全葡聚糖颗粒逆转小鼠调节性树突状细胞免疫抑制功能的机制研究

Our recent clinical study showed that, DCs therapy could significantly improve the 3-year progression free survival in ER/PR double-negative breast cancer patients, while the overall survival rates were not different. In vivo tumor microenvironment potentially drived mature immunostimulatory DCs induced in vitro to re-differentiate into regulatory DCs, which resulted in only a minority of cancer patients could have clinical benefits from DCs vaccine. Our present results showed that, whole glucan particles (WGP) could improve DCs recruitment in tumor issues, directly induce DCs maturation depended on Dectin-1 pathway, and inhibit the growth of breast cancer in mouse. Importantly, WGP had similar effects on human DCs. We used LLC lung cancer cells culture supernatant or IL-10 combined TGF-β to mimic tumor microenvironment to induce regulatory DCs in vitro, and found that WGP could significantly decrease antigen specific CD4+/CD8+ Foxp3+ T cells generation induced by regulatory DCs..Based on our previous results, the current study is aiming at investigate the converting effects of WGP on immunosuppressive functions of regulatory DCs in vitro and in vivo. Then we will further study about the process of signaling complex formation and intracellular down signal transduction pathway in regulatory DCs boosted by WGP stimulation to clarify the mechanisms. Our study may provide a new strategy of cancer therapeutic DCs vaccine application.

我们的临床研究显示，树突状细胞（DC）治疗乳腺癌能改善患者3年无进展生存期，但总生存期尚无差异。肿瘤免疫抑制微环境下，体外诱导成熟的免疫激发性DC可能再次分化为调节性DC，限制了它的临床疗效。我们的研究发现，全葡聚糖颗粒（WGP）口服会引起CD11c+CD8+DC在小鼠肿瘤组织中的浸润，抑制肿瘤生长，在体外可直接诱导小鼠DC成熟，对人DC也有相似的免疫调节作用，这些效应呈Dectin-1依赖性。我们模拟肿瘤微环境，以两种方式体外诱导小鼠骨髓细胞制备调节性DC。结果表明，WGP可以逆转调节性DC的免疫抑制效应，显著降低其诱导的抗原特异性CD4+/CD8+Foxp3+调节性T细胞分化。本项目以肺癌为模型体内外研究WGP对调节性DC免疫抑制功能的影响，进一步通过分析WGP激发后调节性DC中Dectin-1下游信号分子的募集和传导途径，阐明逆转效应的作用机制，为肿瘤DC疫苗临床应用策略提供新思路。

肿瘤微环境会诱导DC免疫耐受，促进肿瘤发展。我们使用小鼠肺癌细胞培养上清（或其外泌体）或细胞因子组合（IL-10+TGF-β）与DC共培养，模拟肿瘤微环境驯化诱导免疫抑制性DC（TEDC），发现TEDC会上调表达CD11b、PD-L1，主要诱导Treg分化，促进荷瘤小鼠肿瘤生长。我们前期的研究发现，全颗粒β-葡聚糖（WGP）可诱导小鼠骨髓来源或人外周血单核细胞来源的DC成熟。本项目研究WGP对TEDC免疫抑制功能的影响及其机制。流式显示，WGP激发后，TEDC表面会上调CD40、CD80、CD86、MHC-Ⅱ，而下调PD-L1、TIM-3；Real-time PCR和ELISA显示，TEDC对TNF-α、IL-12p40的表达、分泌增加，而Arginase、TGF-β降低。TEDC与OT-Ⅱ小鼠CD4+T细胞共培养后，WGP可改善TEDC诱导OVA特异性T细胞的增殖，且Treg降低，而IFN-γ+T细胞增加。同时，WGP可提高TEDC诱导OT-Ⅰ小鼠CD8+T细胞向CTL分化。此外，WGP会上调TEDC表面CCR7的表达，增强CCL19/CCL21对TEDC的趋化，促进其向淋巴结的迁移，诱导OVA特异性T细胞增殖。小鼠模型中，联合注射WGP可以显著抑制TEDC促瘤效应。流式显示，WGP可以增加CD11b+F4/80+、CD11b+Gr-1+细胞在肿瘤中的浸润，而降低CD11c+TIM-3+DC的数量；肿瘤和引流淋巴结中CD11c+CD8+DC增加，而PD-1+CD4+T细胞减少。Real-time PCR显示，WGP组肿瘤中TNF-α、IL-12p40、IFN-γ、iNOS、Gzmb上调，Arginase、TGF-β、IL-17、Foxp3下降。体外T细胞诱导实验显示，WGP组小鼠肿瘤和淋巴结中分选出的DC显著提高诱导OVA抗原特异性CD8+IFN-γ+T的能力，而减少对Treg的诱导。综上，WGP可调节TEDC的免疫抑制功能，上调共刺激分子和Th1细胞因子的表达，而降低共抑制分子的表达，促进TEDC向淋巴结迁移，并抑制其诱导抗原特异性T细胞向Treg分化，而转向Th1分化；荷瘤小鼠体内，WGP可调节肿瘤浸润TEDC免疫抑制效应，改变肿瘤微环境中免疫细胞分布，增强机体抗肿瘤效应，延缓肿瘤生长。本项目研究结果可为β-葡聚糖作为免疫佐剂的应用转化提供理论基础。