Numerous reports have shown that MSCs yielded a favorable therapeutic benefit for GVHD, MSCs have been widely used in several clinical trials [5, 13]. Nevertheless, controversy about their safety and efficacy remains. Although bone marrow (BM) is the main source of MSCs, bone marrow MSCs (BM-MSCs) are not always used because of the invasive harvesting procedure; the number of BM-MSCs declines with increasing age [14, 15]. Placenta tissue-derived MSCs are readily available and easy to collect from a waste product, which has been reported to contain a population of multipotent stem cells exhibiting characteristics of MSCs . Therefore, MSCs from amniotic membranes might be a better option than BM-MSCs. In the present study, we established a human allogeneic acute GVHD model in humanized mice by adoptive transfer of allogeneic hPBMCs into immunodeficient NOD-PrkdcscidIL2rγnull (NPG) mice [17, 18]. The humanized aGVHD model is mediated mainly by donor T cells and characterized by disease appearance (hunching, activity, ruffling, and diarrhea), recruitment of alloreactive cells in target organs, and dysregulation of proinflammatory cytokines . Although important differences remain between GVHD in humanized NPG mouse models and humans, key mechanisms of GVHD pathogenesis are shared in human and xenogeneic aGVHD. The pathogenesis of xeno-aGVHD shares important features with human GVHD such as TCR/co-stimulatory-mediated expansion of selected T cell clones that acquire mainly a Th1/Tc1 profile . Furthermore, significantly increased human cell reconstitution and better immune responses, including immunoglobulin class switching and elevated human IgG responses, have been observed in NPG mice . Thus, the human allogeneic acute GVHD model established here may provide a more relevant approach for studies of human immunopathogenesis and therapeutics for aGVHD after BMT. Our data showed that the murine inflammatory environment was capable of activating human T cells to produce acute GVHD pathology regardless of whether human APCs are co-transplanted. We elected to infuse 3 × 106 PBMCs from donors following 200 cGy irradiation, because we previously found that infusion of 3 × 106 PBMCs from donors induced a moderate GVHD in that model while administration of 7–9 × 106 PBMCs resulted in severe GVHD (unpublished data). In addition, the irradiation dose is proportional to the degree of tissue damage and the subsequent cytokine storm, and thus is directly proportional to aGVHD-related mortality in the mouse [21, 22]. Our results showed that low dose irradiation before PBMCs translation was necessary for mouse antigen exposure and to successfully induce a xeno-aGVHD model.
The hAMSCs are a novel source of stem cells that can be obtained in large quantities. Different from BM-MSCs, MSCs isolated from placenta exhibit greater proliferative and differentiation potential than BM-MSCs, most likely because of the early embryologic origin of amniotic membrane and placenta MSCs compared with BMMSCs [23, 24]. The hAMSCs have intermediate levels of HLA MHC class I molecules, but do not have HLA class II antigens, FAS ligand, and the co-stimulatory molecules, and therefore, do not activate alloreactive T cells [25, 26, 27]. The hAMSCs exhibited a fibroblast-shaped morphology and adherence to plastic, our data show that the cell-surface markers of the hAMSCs were positive for CD90, CD73 and CD105 while negative for CD45, CD11b, HLA-DR and CD34. These findings met the criteria for MSCs identification by The Association of International Cell Therapy. Our observations further demonstrated GFP-labeled hAMSCs had better growth for tracers in the mouse, with a labeling rate of 84%. The hAMSCs labeled with GFP fluorescence could be reproducibly and noninvasively detected by immunofluorescence in the lung, liver, spleen, and gut at day one and day three, respectively, after cell infusion via the mouse tail vein. This phenomenon may be caused by secreted cytokines such as fibroblast growth factor, chemokine receptors, and stem cell homing factors [28, 29, 30]. secreted by hAMSCs and rich marginal blood flow in that region, which are beneficial for cell migration. We also found that from day one to day three, GFP labeled cells increased gradually and were observed in the lung but also can track to the liver, spleen, and gut. However, the number of hAMSCs gradually decreased over time in those organs. It has been reported that BM-MSCs were largely trapped in the lungs, liver, and spleen with abundant capillaries after intravenous transplantation . Our data were consistent with this research; we also found that hAMSCs reached target organs such as the small intestine and liver early. Therefore, hAMSCs exhibited homing ability to target damaged tissues, relieving the severity of inflammation and accelerating tissue repair. Homing of MSCs may also be associated with local microvascular changes, increased capillary permeability, hemostasis, and passive retention.
We then analyzed the function of hAMSCs in xeno-aGVHD using the humanized mice. We revealed that hAMSCs therapy reduced villous blunting and lymphocyte infiltration into the lamina propria of the gut while reducing vascular endothelialitis and lymphocyte infiltration into the parenchyma of the liver and lung. In addition, hAMSCs suppressed xenogenesis of the CD3 + CD4 + T and CD3 + CD8 + T cell proportions and increased the expression of Treg cells in target organs. The induction of immune tolerance involves a precise balance between activation and inhibition of T cell responses, which is important in the development of aGVHD . Tobin et al  found that BM-MSCs blocked TNF-α secretion by dendritic cells via promotion of IL-10 and IL-4 secretion, which impeded T cell differentiation into Th1 cells, directing differentiation of these cells into Treg and Th2 cells, respectively . A recent study showed that AMSCs reduced the activity of human CD8 + T cells and TNF-α in the peripheral blood of xeno-GVHD mice. They also compared the immunomodulatory effects of AMSCs and BM-MSCs in vitro, and found that both AMSCs and BM-MSCs reduced the concentration of TNF-α and IFN-γ expressed by PBMCs . However, this study did not detect T cell and inflammatory cytokine levels in the target organs of aGVHD mice.
We examined CD3 + CD4 + T and CD3 + CD8 + T cells in aGVHD target organs, and found a significant decrease in the proportion of CD3 + CD4 + T and CD3 + CD8 + T cells in these tissues of mice treated with hAMSCs compared to PBS. MSCs further favor Treg expansion in vitro indirectly by inhibiting dendritic cell maturation, and CD8 + T cell and NK cell expansion [36, 37]. Our observations further demonstrated that the proportion of CD4 + CD25 + Foxp3 + Treg cells was increased after hAMSCs treatment in the liver, spleen, and gut. Our data is consistent with the group that showed that murine CD4+CD25+Foxp3+ Tregs were induced during GVHD after allogeneic BMT, and the induction of these Tregs was positively correlated with the protection of GVHD in mice . GVHD involves a pathophysiology that includes host tissue damage, increased secretion of proinflammatory cytokines (TNF, IFN-γ, IL-1, IL-2, and IL-12), and activation of dendritic cells, macrophages, NK cells, and cytotoxic T cells . Inhibition of proinflammatory cytokines has been shown to be beneficial in resolution of the severity and incidence of GVHD [40, 41]. Although some in vitro studies have suggested that IL-10 or TGF-β may be involved in the suppression of MSCs [42, 43]. it remains unknown whether these molecules participate in the suppression mediated by hAMSCs in vivo. Here, we found that the amounts of IL-17A, IFN-γ, TNF, and IL-2 in target organs such as the liver, lung, and gut decreased after hAMSCs treatment. IL-17A was initially reported to be produced by T helper 17 (Th17) cells . In general, pro-inflammatory cytokines such as IFN-γ, TNF-α, IL-1α, or IL-1β have been extensively reported in MSCs activation in vitro . However, there are few studies on inflammatory cytokines by hAMSCs in vivo. Our data also showed that hAMSCs treatment inhibited IL-17A, INF-γ, TNF, and IL-2, which are involved in the pathogenesis of GVHD target organs. Th17 cells and Th17-associated cytokines play a central role in the occurrence of aGVHD [46, 47]. IL-17 contributed to the development of aGVHD in recipient mice by recruiting or priming Th1 cells during the early stages of the disease, reflecting a shift from Th1 to Th17 cells in the physiopathology of aGVHD . A subset of Th17 cells in the gut has been described as having regulatory properties with high levels of IL-10 and low TNF and IL-2 production . We found that IL-17A, IFN-γ and IL-2 were increased in the gut of the aGVHD model; after hAMSCs treatment, the levels of IL-17A, IFN-γand IL-2 were significantly decreased, which means hAMSCs can suppressed Th1 and Th17 cells in aGVHD mouse model. It has been well-established from both murine studies and immune reconstitution data in the clinic that the production of IFN-γ was an adverse effect of GVHD-associated cytokines [49, 50, 51]. Aggarwal et al. suggested that MSCs inhibited IFN-γ and increased IL-4 secretion, and may orchestrate a shift from the prominence of proinflammatory Th1 cells toward an increase in anti-inflammatory Th2 cells, beneficial for GVHD management . Other studies have also shown that a high level of IL-2 might favored the exacerbation of T cell-mediated inflammation rather than the survival of Treg cells under proinflammatory conditions . Furthermore, hAMSCs decreased the level of IFN-γ in the liver, lung, gut, and blood and decreased the level of IL-2 in the gut and blood simultaneously. The hAMSCs possessed potent immunomodulatory properties capable of suppressing allogeneic T cell responses in vivo. The immune suppressive activity of hAMSCs in vivo was associated with a significant decrease in Th1 and Th17 cytokines, including IFN-γ, TNF, IL-17A, and IL-2.
In summary, using humanized mice with a complete human immune system, we successfully established a human allogeneic acute GVHD model. Using this model, we demonstrated that hAMSCs could control acute GVHD by regulating the balance of Treg and T effector cells. Our study provided a proof of concept of hAMSCs treatment to control GVHD after BMT. This strategy could be readily extended to human clinical trials using hAMSCs alone or in combination with minimal conventional immunosuppression to control GVHD. Furthermore, our data also demonstrated that the pathogenesis of aGVHD shared important features with human GVHD and that NPG mice could serve as a better model to study GVHD.