雷帕霉素处理的 BM-MSCs 来源的树突状细胞通过诱导 CD8CD45RCTreg 逆转大鼠肝移植模型中的移植物排斥反应。

Rapamycin treated tol-dendritic cells derived from BM-MSCs reversed graft rejection in a rat liver transplantation model by inducing CD8CD45RCTreg.

机构信息

Department of Hepatobiliary and Pancreaticosplenic Surgery, Beijing ChaoYang Hospital, Capital Medical University, Beijing, 100020, China.

Faculty of Hepato-Pancreato-Biliary Surgery, Chinese PLA General Hospital, Beijing, 100853, China.

出版信息

Mol Immunol. 2021 Sep;137:11-19. doi: 10.1016/j.molimm.2021.03.018. Epub 2021 Jun 26.

DOI:10.1016/j.molimm.2021.03.018

PMID:34182227

Abstract

OBJECTIVE

To investigate the influence of tolerance dendritic cells (tolDCs), generated from Bone marrow mesenchymal stem cells (BM-MSCs) treated with rapamycin (Rapa) on liver allograft survival in a rat acute liver transplantation model.

METHODS

Different GM-CSF induction project was used to obtain immature DCs (imDCs), mature DCs (matDCs) or tolDCs from BM-MSCs. First, MLR was performed to analyze the activity of tolDCs on polyclonaly stimulated total T cells. Then, co-cultured imDCs, matDCs and tolDCs with CD8T cells isolated by magnetic activated cell sorting to analyze the influence on its regulatory characteristic. Last, the established rat acute liver transplantation model were adoptive transfused with imDCs, matDCs or tolDCs isolated by anti-CD11c immunomagnetic beads. The phenotype of DC cells and level of CD8Treg in the culture system and in vivo, the expression of CD8 and CD45RC in the tissues were analyzed by flow cytometry and immunohistochemistry, respectively.

RESULTS

The loGM-CSF plus IL-4 decreased the costimulatory molecules of CD80/86 and MHC class II of DCs comparison with hiGM-CSF from BM-MSCs no matter whether stimulation by LPS (P<0.05). Rapa treated not only reduced the expression of CD80/86 and MHC class II but also down-regulated the expression of CD11c after LPS stimulation which was more obviously in tolDCs by loGM-CSF project (P<0.05). Moreover, tolDCs displayed a rather higher level of IL-10 and low level of IL-12p70 than others (P<0.01), which shown a rather lower stimulative effect on the proliferation of T cells comparison with matDCs and imDCs. Co-cultured with CD8Treg showed an improvement on induction of CD8TCRCD45RCT cells (CD8Treg) in ex vivo. The rats transfused with tolDCs has a delayed survival benefits with high level of CD8Tregs (P<0.01) and high expression of CD45RC in liver tissue (P<0.01) and spleen when comparison with other groups. The infused tolDCs improved a mean survival time (MST) of 32 days comparison with a MTS of 9.5 days and 15.75 days displayed by rat that per-infused with matDCs and imDCs, respectively.

CONCLUSION

Rapa modified tolDCs derived from BM-MSCs reversed graft rejection by improve tolerance characteristics of CD8CD45RCTreg in acute liver rat transplantation.

摘要

目的

研究雷帕霉素（Rapa）处理的骨髓间充质干细胞（BM-MSCs）生成的耐受树突状细胞（tolDCs）对大鼠急性肝移植模型中肝移植物存活的影响。

方法

采用不同的 GM-CSF 诱导方案，从 BM-MSCs 中获得未成熟 DC（imDCs）、成熟 DC（matDCs）或 tolDCs。首先，进行 MLR 以分析 tolDC 对多克隆刺激的总 T 细胞的活性。然后，将分离的 imDCs、matDCs 和 tolDCs 与磁激活细胞分选分离的 CD8T 细胞共培养，分析其对调节特性的影响。最后，采用抗 CD11c 免疫磁珠分离 imDCs、matDCs 或 tolDCs，对建立的大鼠急性肝移植模型进行过继性转输。通过流式细胞术和免疫组织化学分别分析细胞培养系统和体内 DC 细胞的表型以及 CD8Treg 的水平，分析组织中 CD8 和 CD45RC 的表达。

结果

与 hiGM-CSF 相比，loGM-CSF 加 IL-4 降低了 LPS 刺激时 BM-MSCs 来源的 DC 共刺激分子 CD80/86 和 MHC Ⅱ类的表达（P<0.05）。Rapa 处理不仅降低了 LPS 刺激后 CD80/86 和 MHC Ⅱ类的表达，而且还降低了 CD11c 的表达，在 loGM-CSF 方案中 tolDCs 的表达更为明显（P<0.05）。此外，tolDCs 显示出较高水平的 IL-10 和较低水平的 IL-12p70，与 matDCs 和 imDCs 相比，其对 T 细胞增殖的刺激作用较低（P<0.01）。与 CD8Treg 共培养可改善体外诱导 CD8TCRCD45RCT 细胞（CD8Treg）的效果。与其他组相比，输注 tolDCs 的大鼠具有较高的 CD8Treg 水平（P<0.01）和肝组织中 CD45RC 的高表达（P<0.01），从而获得了延迟的生存获益，且其平均生存时间（MST）为 32 天，输注 matDCs 和 imDCs 的大鼠的 MST 分别为 9.5 天和 15.75 天。