Toll样受体通路联合骨髓基质细胞体外扩增髓系抑制细胞治疗急性肝衰竭

Adoptive transfer of immunosuppressor cells is a novel therapy for relieving the T lymphocyte-mediated liver failure. Recently, we observed that ConA-induced liver injury could be relieved by adoptive transfer of myeloid-derived suppressor cells (MDSCs) purified from inflammatory mice models. Due to limited number of in vivo MDSCs, in vitro expansion of MDSCs is necessary for adoptive transplantation. However, our previous study indicated that the activation of TLRs pathway alone was not sufficient for MDSC expansion in vitro, which was quite different from the in vivo study. Thus, we hypothesized that differentiation and proliferation signals are both critical for MDSC expansion from hemopoietic stem cells and myeloid precursor cells. TLR signalling pathway only provides signals for differentiation, while hematopoietic microenvironments are crucial for support cell proliferation. Thus, we intend to: 1) in vitro expand MDSCs from bone marrow hemopoietic stem cells and myeloid precursor cells in the presence of bone marrow stromal cells supporting and TLR ligand; 2) evaluate the expansion efficiency and suppressive ability of MDSCs, and adoptive transfer MDSCs to acute liver failure mouse models to access their therapeutic potential in vivo; 3) explore the mechanism of MDSC expansion with neutrolizing antibodies. Our data will provide theoretical and experimental evidence of cell therapy for acute liver failure.

免疫抑制细胞过继转移是治疗T淋巴细胞介导的急性肝衰竭（ALF）的新思路。我们采用感染模型小鼠，体内扩增髓系抑制细胞（MDSC），并过继治疗ConA诱导的急性肝损伤模型小鼠取得成功。但是，内源性MDSC数量有限，体外扩增MDSC是解决移植细胞来源的重要手段。申请人在前期工作中发现，与体内研究不同，体外培养条件下，Toll样受体（TLR）激动剂不能有效扩增MDSC。由此，我们提出：分化信号和增殖信号是体外扩增MDSC的两个重要条件。TLR只能提供分化信号，MDSC的体外有效扩增需微环境提供增殖信号。本研究拟1）分离造血干细胞和髓系祖细胞，联合运用TLR配体和骨髓基质细胞模拟炎症造血微环境，体外扩增MDSC；2）评价MDSC扩增效率和免疫抑制功能，并过继转移MDSC治疗急性肝衰竭模型小鼠，检测其在体效应；3）采用中和抗体阻断实验探讨MDSC体外扩增机制，为急性肝衰竭的临床治疗提供理论和实验基础。

免疫抑制细胞过继转移是治疗T淋巴细胞介导的急性肝衰竭（ALF）的新思路。我们采用感染模型小鼠，体内扩增髓系抑制细胞（MDSC），并过继治疗ConA诱导的急性肝损伤模型小鼠取得成功。但是，内源性MDSC数量有限，体外扩增MDSC是解决移植细胞来源的重要手段。本研究利用不同Toll样受体激动剂联合骨髓基质细胞，模拟炎症造血微环境，为造血干、祖细胞同时提供分化和增殖的双重信号，在体外成功诱导出具有不同潜能的MDSC亚群。骨髓造血干细胞发育重要的细胞因子GM-CSF和TLR4激动剂LPS均可使培养体系中的G-MDSC和M-MDSC细胞大量扩增，且G-MDSC亚群以具有较强免疫抑制功能的幼稚粒细胞为主，上述培养体系可诱导抑制性分子PD-L1的高表达，并有效抑制脾脏来源的CD4+T淋巴细胞的增殖。有趣的是，TLR1/2激动剂Pam3CSK4虽然也可体外高效扩增具有免疫抑制活性的MDSC细胞，但诱导所得细胞低表达PD-L1而高表达TNF-a，该结果提示，Pam3CSK4诱导体系在肿瘤治疗领域也具有潜在的应用价值。过继移植上述培养体系体外诱导分化获得的MDSC细胞，治疗ConA诱导的急性肝衰竭小鼠模型，与对照组相比，MDSC细胞移植组小鼠模型的肝功能得到明显改善，模型小鼠的成活率显著提高。本研究结果表明：TLR激动剂联合骨髓基质细胞可模拟炎症造血微环境，体外扩增大量具有免疫抑制功能的MDSC细胞，并介导过继转移后急性肝衰竭小鼠的肝脏保护作用，为急性肝衰竭的临床治疗提供理论和实验基础。