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TGF-β1-Licensed Murine MSCs Show Superior Therapeutic Efficacy in Modulating Corneal Allograft Immune Rejection In Vivo.

ABSTRACT: Mesenchymal stromal cells (MSCs) are a promising therapeutic option for multiple immune diseases/disorders; however, efficacy of MSC treatments can vary significantly. We present a novel licensing strategy to improve the immunosuppressive capacity of MSCs. Licensing murine MSCs with transforming growth factor-β1 (TGF-β MSCs) 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 following co-culture assays. These TGF-β MSC-expanded regulatory T lymphocytes 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-β MSCs could significantly prolong rejection-free survival (69.2% acceptance rate compared to 21.4% for unlicensed MSC-treated recipients) in a murine corneal allograft model. Mechanistic studies revealed that (1) therapeutic efficacy of TGF-β MSCs is Smad2/3-dependent, (2) the enhanced immunosuppressive capacity of TGF-β MSCs is contact-dependent, and (3) enhanced secretion of PGE2 (via prostaglandin EP4 [E-type prostanoid 4] receptor) by TGF-β 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-β1 licensing as an unconventional strategy for enhancing MSC immunosuppressive capacity.

SUBMITTER: Lynch K

PROVIDER: S-EPMC7474271 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMesenchymal stem cells (MSCs) have been reported to promote long-term cellular and organ transplant acceptance due to their immunotherapeutic characteristics. Previous work from our lab using a rat allograft model has shown that systemic infusion of MSCs inhibited corneal allograft rejection and prolonged graft survival. Here, we further investigated the effects of local MSCs administration in the same animal model.MethodsDonor-derived MSCs were isolated and cultured while corneal grafts obtained from Wistar rats were transplanted into Lewis rat hosts. Hosts were then randomly separated into four groups and treated with previously cultured MSCs at different times and doses. Graft survival was clinically assessed using slit-lamp biomicroscopy and the median survival time (MST) was calculated. Grafts were examined histologically using hematoxylin-eosin (H-E) staining and immunohistochemically using antibodies against CD4. A comprehensive graft analysis of IL-2, IL-4, IL-10, and IFN-? expression was also conducted using both real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA).ResultsPostoperative MSCs injection prolonged graft survival time when compared with controls (MST 9.8?±?1.2 days). Injection twice of MSCs (MST 12.6?±?1.4 days) was more effective than a single injection (MST 10.8?±?1.3 days). MSCs-treated groups also showed suppression of inflammatory cell as well as CD4?+?T cell infiltration in the allograft region. IL-4 and IL-10 levels were significantly increased in grafts obtained from postoperative twice MSCs-treated rats when compared with controls. There were no significant differences in IL-2 or IFN-? expression across groups.ConclusionsSubconjunctival injection of MSCs in rats was effective in prolonging corneal allograft survival. This effect was mediated by inhibition of inflammatory and immune responses, indicating an anti-inflammatory shift in the balance of T helper (Th)1 to T helper(Th) 2.

Project description:Transforming growth factor β (TGF-β) plays a critical role in wound healing and the pathogenesis of fibrosis (scarring). Three isoforms of TGF-β have been identified in mammals. Previous studies have shown that the addition of TGF-β1 (T1) or -β2 (T2) to human corneal fibroblasts (HCF) cultured in a 3-dimensional construct resulted in a fibrotic matrix, while the addition of TGF-β3 (T3) resulted in the production of enhanced non-fibrotic matrix as compared to control (Vitamin C [VitC] only). In the current investigation, we undertook the molecular comparison of fibrosis-related gene expression in T1 or T3-treated HCF to gain further insights into the regulation and roles of these two isoforms on the fibrotic response. HCF were cultured in 100 mm dishes in basic medium (Eagles minimum essential medium [EMEM] with 10% fetal bovine serum [FBS]). At 70-80% confluency, cells were exposed to basic medium with 0.5 mM 2-O-α-d-glucopyranosyl-l-ascorbic acid (VitC) ± 2 ng/ml of T1 or T3. After 4 h or 3 days, cells were harvested, and mRNA or protein was isolated. Fibrosis related mRNA levels were assayed using a commercial qRT-PCR Array. Selected proteins were examined using Western blotting (WB). Experiments were performed 6 times for the qRT-PCR and 4 times for WB for each condition. qRT-PCR results showed that most of the fibrosis-related genes were up or downregulated in HCF exposed to T1 or T3 as compared with VitC control. At 4 h, only Smad7 expression was significantly altered in T3-treated HCF, compared to T1, and at 3 days, five genes were altered. WB confirmed that T1 significantly decreased Smad7 expression compared to T3 and control, and that the expression of thrombospondin-1 in T3-stimulated HCF was enhanced compared to T1-treated cells. Finally, both T1 and T3 decreased Smad3 expression dramatically at both time points. At early time points, T1 and T3 have similar effects on expression of fibrosis related genes; however, with a longer exposure, an increasing number of genes were differentially expressed. Interestingly, most of the differentially expressed gene products are secreted by the cells and may be related to the modulation of extracellular matrix.

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TGF-β1-Licensed Murine MSCs Show Superior Therapeutic Efficacy in Modulating Corneal Allograft Immune Rejection In Vivo.

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