PD-L1修饰的间充质干细胞联合iCasp9自杀基因在噬血细胞性淋巴组织细胞增生症治疗作用中的研究

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome with a high lethality, and one of the key processes of pathogenesis is the over-activation of NK and T lymphocytes. Mesenchymal stem cells (MSCs) have the property of targeted migration to the inflammation sites and multiple immunosuppressive function, which makes MSCs a promising therapy strategy for HLH. However, as HLH is an acute and life-threatening disease, it is crucial to control the excessive activation of lymphocytes in time for HLH treatment. The PD-1 / PD-L1 immune checkpoint played an important role in the inhibitory pathway that limited lymphocytes activation; furthermore, we found that PD-1 molecules were highly expressed in the peripheral blood lymphocytes of HLH children in our previous work. Therefore, we designed to induce MSC cells overexpress PD-L1 molecules and established MSC.PD-L1 treatment system. First, MSCs acted as the PD-L1 carriers in vivo and quickly initiated the immune suppression effect depending on PD-1/PD-L1 checkpoint mechanism; subsequently, the immunomodulatory effects of MSCs were induced, which further suppressed the lymphocytes activation. In order to prevent the potential over-immunosuppression，we combined the MSC.PD-L1 treatment system with iCasp9 suicide gene to remove MSCs from the body in time. In summary, we established an effective and precise cell therapy strategy for HLH, which could provide support for the development of new HLH treatment strategy.

噬血细胞性淋巴组织细胞增生症(HLH)是一种临床上严重威胁患儿生命的血液疾病，NK及T淋巴细胞的过度激活是发病的关键环节。间充质干细胞（MSC）具有向炎症部位定向迁移及多重免疫抑制作用，因而在HLH治疗中成为一种可能。然而，HLH是急危重症，及时快速控制免疫激活是治疗的关键。PD-1/PD-L1免疫检查点在限制淋巴细胞过度活化中发挥重要作用；申请人在前期工作中发现PD-1分子在HLH患儿外周血淋巴细胞中高表达，因此设计使MSC高表达PD-L1分子，建立MSC.PD-L1双重治疗体系。首先利用PD-1/PD-L1调控机制，快速启动免疫抑制效应；随后MSC免疫调节作用被诱导，进一步强化免疫抑制。为防止对机体产生持续过度免疫抑制，我们联合iCasp9自杀基因凋亡机制，及时将MSC从体内清除，从而建立一种安全高效HLH细胞治疗策略，为开发新的HLH治疗手段提供思路及必要的临床前基础研究支持。

噬血细胞性淋巴组织细胞增生症(HLH)是一种临床上严重威胁患儿生命的血液疾病，NK及T淋巴细胞的过度激活是发病的关键环节。本研究以HLH发病关键机制充分联系间充质干细胞（MSC）生物学特性，构建PD-L1修饰的MSC联合iCasp9/AP1903 自杀基因治疗系统，建立一种具有安全高效、双重免疫抑制作用的HLH细胞治疗策略。首先检测难治性HLH患儿外周血样本，并利用健康儿童外周血样本作对照，证明HLH患儿外周血淋巴细胞高表达PD-1分子。随后，分离培养人脐带来源的MSC细胞（HUMSC），并以介导HLH发病的关键细胞因子IFN-γ和IL-1β刺激HUMSC细胞，证明IFN-γ和IL-1β可诱导HUMSC表达PD-L1分子，且二者发挥协同诱导作用，进而建立PD-L1分子修饰的HUMSC细胞（HUMSC.PD-L1）。在此基础上，进一步探索HUMSC.PD-L1的双重免疫抑制功能。首先证明IFN-γ和IL-1β炎症因子协同刺激下，HUMSC可表达具有酶活性的IDO，发挥自身免疫抑制功能。随后，以HUMSC.PD-L1与人外周血PBMCs细胞共培养，证明MSC.PD-L1体系可高效抑制活化PBMC的增殖和细胞因子分泌，同时PBMC的凋亡比例增加，提示MSC.PD-L1体系具有快速免疫抑制功能。另一方面，构建pLenti-iCasp9.fLuc.eGFP载体，分选iCasp9阳性的MSC细胞，AP1903小分子化合物诱导MSC细胞凋亡，16小时后 MSC细胞凋亡比例达74.3%，证明iCasp9/AP1903系统具有高效快速的MSC凋亡控制能力。综上，本研究通过体外实验证明MSC.PD-L1治疗体系联合 iCasp9自杀基因治疗HLH的可行性，从而为开发新的HLH治疗手段提供了必要的临床前基础研究支持。