TGF-ß1 licensed murine mesenchymal stromal cells show superior therapeutic efficacy in modulating corneal allograft immune rejection in vivo

We report a novel licensing strategy to improve the immunosuppressive capacity of MSCs. Licensing murine MSCs with TGF-ß1 (TGF-ß MSC) significantly improved their ability to modulate both the phenotype and secretome of inflammatory bone marrow-derived macrophages and significantly increased the numbers of regulatory T lymphocytes (Tregs) following co-culture assays. These TGF-ß MSC-expanded Tregs also expressed significantly higher levels of PD-L1 and CD73, indicating enhanced suppressive potential. Detailed analysis of T lymphocyte co-cultures revealed modulation of secreted factors, most notably, elevated prostaglandin E2 (PGE2). Furthermore, TGF-ß MSCs could significantly prolong rejection-free survival (69.2% acceptance rate compared to 21.4% for un-licensed MSC treated recipients) in a murine corneal allograft model. Mechanistic studies revealed that (i) therapeutic efficacy of TGF-ß MSCs is Smad2/3-dependent; (ii) TGF-ß MSC's enhanced immunosuppressive capacity is contact-dependent and (iii) enhanced secretion of PGE2 (via prostaglandin EP4 receptor) by TGF-ß MSCs is the predominant mediator of Treg expansion and T cell activation and is associated with corneal allograft survival. Collectively, we provide compelling evidence for the use of TGF-ß1 licensing as an unconventional strategy for enhancing MSC immunosuppressive capacity. Overall design: RNA sequencing of untreated murine mesenchyaml stromal cells (MSCs) (n=3) compared to TGF-ß1 licensed MSCs (n=3)

本研究报道了一种可提升间充质干细胞（mesenchymal stromal cells, MSCs）免疫抑制能力的新型预激活策略。采用转化生长因子-β1（transforming growth factor-β1, TGF-β1）对小鼠间充质干细胞进行预激活（下称TGF-β1预激活MSC），可显著增强其调节炎性骨髓来源巨噬细胞表型与分泌组的能力，并在共培养实验后显著增加调节性T淋巴细胞（regulatory T lymphocytes, Tregs）的数量。经TGF-β1预激活MSC扩增的Tregs还可高表达PD-L1与CD73，提示其免疫抑制潜能得到增强。对T淋巴细胞共培养体系的详细分析显示，培养上清中的分泌因子发生显著调控，其中尤以前列腺素E2（prostaglandin E2, PGE2）水平升高最为突出。此外，在小鼠角膜异体移植模型中，TGF-β1预激活MSC可显著延长移植物无排斥存活期（接受率达69.2%，未预激活MSC处理组受体的接受率仅为21.4%）。机制研究揭示：① TGF-β1预激活MSC的治疗效果依赖Smad2/3通路；② 其增强的免疫抑制能力具有接触依赖性；③ TGF-β1预激活MSC通过前列腺素EP4受体（prostaglandin EP4 receptor）提升PGE2分泌，这是介导Treg扩增与T细胞活化调控的核心机制，同时与角膜异体移植物存活密切相关。综上，本研究提供了有力证据，证明采用TGF-β1预激活是一种非常规的增强MSC免疫抑制能力的策略。实验整体设计：对未处理的小鼠间充质干细胞（n=3）与TGF-β1预激活的间充质干细胞（n=3）进行RNA测序分析。