乳腺癌MDSCs通过CyclinD-CDK4/6-pRb轴调控PD-1-PD-L1+B细胞免疫抑制功能的研究

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 checkpoint-related molecules such as PD-1 and PD-L1 changing prominently in the presence of MDSCs. The subset of PD-1-PD-L1+B cells was obviously increased in the presence of MDSCs. MDSCs emerge as vital regulators in B-cell inhibitory immune functions such as proliferation inhibition on normal T cells and transformation of Th1 to Th2. Further detection by flow cytometry showed that TGF-β in breast cancer MDSCs increased. Analysis by western blot demonstrated that molecules of CyclinD1, CyclinD3, CDK4/6, pRb in PD-1-PD-L1+B cells increased. Application of inhibitors targeting TGF-β or CDK4/6 could reverse PD-L1 expression on B cells and further attenuate PD-1-PD-L1+B cells′ suppression on proliferation of normal T cells. Blockage of TGF-β conferred to decreased p-Smad2/3 expression and also suppressed translocation of Smad4 into nuclus of B cells. Our current project is aimed to identify that MDSCs can educate normal B cells and induced a novel PD-1-PD-L1+B cell-subset, which can exert immunosuppressive functions via CyclinD-CDK4/6-pRb axis. 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，以往的研究主要集中于它们对T细胞及NK细胞等细胞的免疫调控作用，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激活 PD-1-PD-L1+B细胞CyclinD-CDK4/6-pRb轴，并证实该B细胞具有免疫抑制功能。阻断CDK能够逆转B细胞表面PD-L1的表达并减弱B细胞的免疫抑制功能。进一步发现阻断TGF-β能够抑制B细胞下游Smad2/3磷酸化及Smad4入核，并抑制B细胞中CDK通路的激活。本课题将以BALB/C小鼠4T1乳腺癌为模型，阐明乳腺癌MDSCs通过TGF-β-Smad途径调控CDK通路，继而影响PD-1-PD-L1+B细胞的免疫抑制功能。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。

髓源性抑制细胞（Myeloid-derived suppressor cells, MDSCs）是一群与肿瘤免疫逃逸密切相关的异质性细胞的集合，其对 B 细胞的免疫调控机制尚未阐明。我们之前的结果表明，乳腺癌衍生的MDSCs可以诱导一群具有免疫抑制功能的PD-1-PD-L1+Bregs。而在本研究中，我们发现阻断MDSCs和B细胞之间的 PD-1/PD-L1 轴的相互作用可以逆转PD-1-PD-L1+Bregs 对T细胞免疫应答的抑制效应。同时，本研究还证实了PI3K/AKT/NF-κB信号通路的激活对于PD-1-PD-L1+Bregs 发挥免疫抑制功能也至关重要。并且，MDSCs是通过其表面受体 PD-1 与 B 细胞上的PD-L1 配体相互结合，从而激活了B细胞中的PI3K/AKT/NF-κB路，以此发挥对 T细胞的免疫抑制作用。此外，在体内阻断PD-1/PD-L1 轴的结合或抑制PI3K/AKT信号通路的激活可抑制荷瘤小鼠的肿瘤生长和PD-1-PD-L1+Bregs的免疫抑制功能。PD-1/PD-L1轴和 PI3K/AKT通路的双重抑制发挥了更好的抗肿瘤作用。MDSCs和PD-1-PD-L1+Bregs 被证实共定位于乳腺癌患者的肿瘤组织样本中；MDSCs和PD-1-PD-L1+Bregs 均与乳腺癌患者的不良预后呈正相关。因此，我们的研究提出了乳腺癌来源的MDSC介导的 B 细胞免疫调节的新机制，这可能为肿瘤免疫治疗提供新的思路。