乳腺癌髓源性免疫抑制细胞 (MDSCs) 通过j激活PI3K/Akt途径诱导CTLA-4+CCR6-CD62L- B细胞发挥调节样免疫功能的研究

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of myeloid cells that suppress T-cell activity in a tumor microenvironment. However, the suppressive function of MDSCs on B cells and its underlying mechanism remain unclear. Here, we successfully constructed BALB/C mice 4T1 breast cancer model. We show that the surface molecules of B cells are remolded, with molecules such as CTLA-4, CCR6 and CD62L changing prominently in the presence of MDSCs. The subset of CTLA-4+CCR6-CD62L-B cells was obviously increased in the presence of MDSCs. MDSCs emerge as vital regulators in B-cell inhibitory immune functions such as inhibition of IFN-ɣ secretion. Further detection showed that NF-ƙB and IDO in breast cancer MDSCs increased. Less translocation of RelB was associated with IDO inhibition. Analysis by western blot demonstrated that PI3K/Akt pathway was activated in CTLA-4+CCR6-CD62L-B cells. Targeting PI3K/Akt could reverse CTLA-4 expression on B cells and further attenuate CTLA-4+CCR6-CD62L-B cells′ suppression on IFN-ɣ secretion of normal T cells. Application of the inhibitor targeting IDO could block PI3K/Akt and reverse CTLA-4 expression on B cells. Our current project is aimed to identify that MDSCs can educate normal B cells and induced a novel CTLA-4+CCR6-CD62L-B cell-subset, which can exert immunosuppressive functions via PI3K/Akt pathway. The accomplishment of this project might extend our understanding of inhibitory immune functions of B cells in tumor microenvironment and might shed new light on anti-tumor immunity and immune therapy.

MDSCs是一类与肿瘤免疫逃逸密切相关的髓系细胞，目前关于MDSCs对B细胞的免疫调控尚未阐明。前期结果表明乳腺癌MDSCs诱导CTLA-4+CCR6-CD62L-B细胞发挥免疫抑制作用，且诱导该B细胞中PI3K/Akt高表达。阻断PI3K/Akt能够逆转B细胞表面CTLA-4的表达并减弱B细胞的免疫抑制功能。进一步发现阻断MDSCs中非经典NF-ƙB途径能够下调自身IDO的表达，并减弱B细胞的免疫抑制作用。本课题将以BALB/C小鼠4T1乳腺癌为模型，阐明乳腺癌MDSCs通过非经典NF-ƙB途径调控IDO表达，激活CTLA-4+CCR6-CD62L-B细胞中PI3K/Akt通路，进而诱导其发挥免疫抑制功能。本项目的完成将有助于了解B细胞的免疫抑制作用，同时为抗肿瘤免疫效应机制以及肿瘤治疗新方法的研究提供一定的理论基础。

髓源性免疫抑制细胞（myeloid derived suppressor cell, MDSC）对多种免疫细胞如T 细胞、NK细胞等的作用已有明确报道，但关于MDSC对B细胞的影响及机制尚未阐明。因此，探讨并阐明MDSC对B细胞功能的影响可有助于更全面认识MDSC对免疫应答多细胞网络的调控功能，并且提高研究人员对B细胞免疫功能的理解，为探索肿瘤、自身免疫耐受和自身免疫性疾病等的发生、发展机制及免疫治疗的应用提供了新的研究途径。通过功能检测我们发现并初步定义了具有免疫抑制功能的CTLA-4+CCR6-CD62L-B细胞亚群。我们后续通过测序分析发现MDSCs 诱导CTLA-4+CCR6-CD62L-B细胞中PI3K/Akt通路蛋白发生改变，进一步采用western blot也证实这一结果，提示我们PI3K/Akt通路可能参与了MDSCs对CTLA-4+CCR6-CD62L-B 细胞免疫抑制功能的调控。乳腺癌MDSCs和正常B细胞共孵育体系封闭CTLA-4，能够逆转B细胞对正常T细胞分泌IFN-ɣ的抑制作用；加入PI3K抑制剂LY294002，发现阻断PI3K/Akt通路能够逆转B细胞表面CTLA-4的表达，并能够逆转B细胞对正常T细胞分泌IFN-ɣ的抑制作用，初步证实了PI3K/Akt 通路参与了MDSCs对CTLA-4+CCR6-CD62L-B细胞免疫抑制功能的调控。值得注意的是乳腺癌MDSCs与正常B细胞隔开共孵育，并不能诱导后者抑制T细胞分泌IFN-ɣ，激光共聚焦结果也初步表明MDSCs对B细胞免疫功能的调控依赖于二者直接接触。为了研究MDSCs通过何种机制激活CTLA-4+CCR6-CD62L-B细胞中PI3K/Akt通路，我们首先发现相比于正常对照，乳腺癌MDSCs中NF-ƙB通路活化，并高表达IDO，提示我们二者是否有一定关系。阻断NF-ƙB通路能够抑制非经典途径蛋白RelB的入核，并抑制IDO的表达，为后续深入研究NF-ƙB对IDO 的调控提供了较好的工作基础。此外，发现阻断IDO能够抑制B细胞内Akt的磷酸化水平，逆转B细胞表面CTLA-4的表达。最终建立了NF-ƙB/IDO对PI3K/Akt 通路的调控这样一个关系轴。