iPS细胞由来调节性DC的分化诱导及其免疫调节作用机制的解析

Dendritic cells (DCs) have several functions in innate and adaptive immunity as the most potent antigen-presenting cells. In addition, there is increasing evidence that DCs in situ induce antigen specific unresponsiveness or tolerance in immune system,central lymphoid organs and in the periphery, which were designated as regulatory DCs (rDCs). The mechanisms of rDCs to negative regulate the immune response were proved to inducing regulatory T cells or inducing T cells anergy or secreting immuregulation cytokines. Thus, the usage of rDCs was considered as a promising cell therapeutic strategy for autoimmune diseases and transplantation. . Now, DCs for transfer therapy are generated by in vitro differentiation of peripheral blood monocytes of the patients. However, there is a limitation in the number of available monocytes, and the potential of DC-differentiation in monocytes varies depending on the blood donor. Induced pluripotent stem (iPS) cells possess both pluripotency and infinite propagation capacity. Thus we consider iPS cells to be an ideal source for rDCs to be used in immunotherapy. . Generation and characterization of conventional DCs (cDCs) derived from mouse and human iPS cells were reported by Senju etc. The iPS cell derived cDCs (iPS-cDCs) possessed the characteristics of cDCs including the capacity of T cell-stimulation, antigen-processing and presentation, and cytokine production. Whereas, there are no any reports of the generation rDCs from iPS cells, which we consider is potential useful for the treatment of autoimmune diseases. . Therefore, in the present study, we plan to generate rDCs from mouse iPS cells, using the protocol that iPS cells cultured on the bone marrow stromal cells (OP9 cells) or spleen stromal cells layer, content with granulocyte/macrophage colony stimulating factor (GM-CSF),interleukin (IL)-10 and transforming growth factor(TGF)-beta. And then, we plan to analyze how the generated iPS-rDCs play the immune regulatory functions and it's mechanisms in vitro and in vivo experiments.

调节性树突状细胞(Regulatory Dendritic Cell,rDC)因其在维持免疫耐受方面的重要作用，被认为可以用来治疗自身免疫性疾病及移植免疫排斥。体外成功诱导rDC的研究已有报道,但是用骨髓或血液单核细胞诱导rDC的不足在于：获得的rDC数量少且性质不稳定，限制了rDC用于临床治疗的研究。为突破这个瓶颈，并根据不同组织来源、不同条件下诱导的rDC是通过不同的机制来调节免疫应答，抑制器官炎性损害的理论，我们设想是否可以利用诱导性多能干细胞（Induced Pluripotent Stem，iPS）作为源细胞，诱导得到大量、稳定、高效的rDC？我们前期研究已知iPS由来的经典DC其纯化程度远高于骨髓细胞由来的经典DC。因此，我们计划采用iPS细胞+不同基质细胞+不同诱导刺激引物的组合来探索iPS细胞向rDC的分化诱导，并通过一系列体内、外实验来检验其免疫调节功能，解明其作用机制。

调节性树突状细胞(Regulatory Dendritic Cell,rDC)因维持免疫耐受方面的重要作用，被认为可以用来治疗自身免疫性疾病等。用骨髓或血液单核细胞诱导rDC的不足在于来源受限获得的rDC数量少，限制了其应用。为突破瓶颈，我们设想利用iPS细胞作为源细胞，探索iPS细胞向rDC分化诱导的方法，并通过体内、外实验来检验其免疫调节功能，解明其作用机制。在本项目中我们利用三段式方法成功诱导了iPS 细胞分化成调节性DC。我们发现经过15天左右，绝大部分的悬浮细胞都呈现出树突状细胞的形态，这些细胞高表达CD11b 和 CD11c，低表达CD80、 CD40 和MHC-II 分子。我们发现在整个培养过程中细胞数量增加了约160倍。同时发现作为干细胞标志的Nanog和 Oct3/4随着培养逐渐减少直至消失，这预示了分化后的细胞不再具有干细胞的多能性，是稳定的分化终末细胞，也证明了iPS-DCreg的安全性。在功能测定方面，我们通过抗原吞噬试验发现iPS-DCregs无论经LPS刺激与否都保持着很高的抗原吞噬功能，与BM-DCregs一致，远远高于BM-DCcon，后者在经LPS刺激后吞噬能力降低。两种MLR实验显示iPS-DCregs作为stimulator不能或极弱地刺激CD4和CD8阳性T细胞增殖，却能作为regulator具有很强的降低CD4+和CD8+T细胞在DCcon刺激下增殖的能力。我们发现无论作为stimulator还是regulator，iPS-DCregs都能诱导CD4+/Foxp3+调节性T细胞，是其发挥免疫调节作用的主要机制。体内实验中，我们进行了小鼠腘窝淋巴结实验，我们发现与对照组BM-DCcons相比，iPS-DCregs作为stimulator组仅引起淋巴结少许增重，与BM-DCregs组结果近似，均显著低于对照组。淋巴细胞分析结果显示，与对照组BM-DCcons相比，iPS-DCregs作为stimulator组引起淋巴细胞增殖不明显，与BM-DCregs组结果近似，均显著低于对照组。因此，我们判断iPS-DCreg具备了调节性DC应有的性状与功能。本课题的成果填补了iPS细胞向调节性DC定向分化诱导的研究空白；为体外获得大量的调节性DC找到了合适的细胞源；为应用调节性DC治疗自身免疫性疾病提供了数据支持。