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Mesenchymal stromal cells (MSCs) are attractive candidates for immunomodulatory cell therapy. However, it remains unknown how far therapeutic efficacy and potency are dependent on the dosage and activity of the MSCs. We previously observed that infusion of MSCs leads to rapid and transient changes in cytokine expression in blood, lung, and liver. In the present study, increasing doses of syngeneic adipose tissue-derived MSCs were infused in healthy mice and systemic changes in G-CSF, IL6, IL-10, and CXCL5 were detected 2 h after administration of 3 × 10 5 MSCs per animal, but not at lower doses. In lung and liver tissue, dose-dependent effects of MSCs on cytokine mRNA expression levels were detected from doses as low as 3 × 10 3 MSCs. Infusion of secretome-deficient or IFNγ-activated MSCs in healthy mice had similar effects on systemic cytokine levels as control MSCs, suggesting that in vivo at least the initial systemic effect of MSC administration is independent of the level of activity of MSCs, but depends on the response of host cells to MSCs. The results of this study reveal a rapid dose-dependent effect of MSCs and stress the important role of host cells in MSC treatment. This knowledge contributes to the design of rational MSC trials and to the quest for clinical efficacy of MSC therapy.

Mesenchymal stem or stromal cells (MSC) are under investigation as a potential immunotherapy. MSC are usually administered via intravenous infusion, after which they are trapped in the lungs and die and disappear within a day. The fate of MSC after their disappearance from the lungs is unknown and it is unclear how MSC realize their immunomodulatory effects in their short lifespan. We examined immunological mechanisms determining the fate of infused MSC and the immunomodulatory response associated with it. Tracking viable and dead human umbilical cord MSC (ucMSC) in mice using Qtracker beads (contained in viable cells) and Hoechst33342 (staining all cells) revealed that viable ucMSC were present in the lungs immediately after infusion. Twenty-four hours later, the majority of ucMSC were dead and found in the lungs and liver where they were contained in monocytic cells of predominantly non-classical Ly6C low phenotype. Monocytes containing ucMSC were also detected systemically. In vitro experiments confirmed that human CD14 ++ /CD16 - classical monocytes polarized toward a non-classical CD14 ++ CD16 + CD206 + phenotype after phagocytosis of ucMSC and expressed programmed death ligand-1 and IL-10, while TNF-α was reduced. ucMSC-primed monocytes induced Foxp3 + regulatory T cell formation in mixed lymphocyte reactions. These results demonstrate that infused MSC are rapidly phagocytosed by monocytes, which subsequently migrate from the lungs to other body sites. Phagocytosis of ucMSC induces phenotypical and functional changes in monocytes, which subsequently modulate cells of the adaptive immune system. It can be concluded that monocytes play a crucial role in mediating, distributing, and transferring the immunomodulatory effect of MSC. Stem Cells 2018;36:602-615., (© AlphaMed Press 2018.)

Mesenchymal stem cells (MSC) are studied as a cell therapeutic agent for treatment of various immune diseases. However, therapy with living culture-expanded cells comes with safety concerns. Furthermore, development of effective MSC immunotherapy is hampered by lack of knowledge of the mechanisms of action and the therapeutic components of MSC. Such knowledge allows better identification of diseases that are responsive to MSC treatment, optimization of the MSC product, and development of therapy based on functional components of MSC. To close in on the components that carry the therapeutic immunomodulatory activity of MSC, we generated MSC that were unable to respond to inflammatory signals or secrete immunomodulatory factors, but preserved their cellular integrity [heat-inactivated MSC (HI-MSC)]. Secretome-deficient HI-MSC and control MSC showed the same biodistribution and persistence after infusion in mice with ischemic kidney injury. Both control and HI-MSC induced mild inflammatory responses in healthy mice and dramatic increases in interleukin-10, and reductions in interferon gamma levels in sepsis mice. In vitro experiments showed that opposite to control MSC, HI-MSC lacked the capability to suppress T-cell proliferation or induce regulatory B-cell formation. However, both HI-MSC and control MSC modulated monocyte function in response to lipopolysaccharides. The results of this study demonstrate that, in particular disease models, the immunomodulatory effect of MSC does not depend on their secretome or active cross-talk with immune cells, but on recognition of MSC by monocytic cells. These findings provide a new view on MSC-induced immunomodulation and help identify key components of the therapeutic effects of MSC.

Mesenchymal stromal cells (MSC) are a promising therapy for inflammatory diseases. However, MSC are large and become trapped in the lungs after intravenous infusion, where they have a short survival time. To steer MSC immunoregulatory therapy beyond the lungs, we generated nm-sized particles from MSC membranes (membrane particles, MP), which have immunomodulatory properties, and investigated their internalization and mode of interaction in macrophages subtypes and human umbilical vein endothelial cells (HUVEC) under control and inflammatory conditions. We found that macrophages and HUVEC take up MP in a dose, time, and temperature-dependent manner. Specific inhibitors for endocytotic pathways revealed that MP internalization depends on heparan sulfate proteoglycan-, dynamin-, and clathrin-mediated endocytosis but does not involve caveolin-mediated endocytosis. MP uptake also involved the actin cytoskeleton and phosphoinositide 3-kinase, which are implicated in macropinocytosis and phagocytosis. Anti-inflammatory M2 macrophages take up more MP than pro-inflammatory M1 macrophages. In contrast, inflammatory conditions did not affect the MP uptake by HUVEC. Moreover, MP induced both anti- and pro-inflammatory responses in macrophages and HUVEC by affecting gene expression and cell surface proteins. Our findings on the mechanisms of uptake of MP under different conditions help the development of target-cell specific MP therapy to modulate immune responses. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are used in various clinical and preclinical models for immunomodulation. However, it remains unclear how the immunomodulatory effect of MSC is communicated. MSC-induced immunomodulation is known to be mediated through both MSC-secreted cytokines and direct cell-cell interactions. Recently, it has been demonstrated that metabolically inactive, heat-inactivated MSCs (HI-MSCs) have similar anti-inflammatory capacities in LPS-induced sepsis compared with viable MSC. To further investigate the immunomodulatory effects of MSC, we introduced MSC and HI-MSC in two animal models with different immunological causes. In the first model, allogeneic hearts were transplanted from C57BL/6 mice to BALB/c recipients. MSC in combination with mycophenolate mofetil (MMF) significantly improved graft survival compared with MMF alone, whereas the application of HI-MSC had no effect on graft survival. We revealed that control MSC dose-dependently inhibited CD3+ and CD8+ T-cell proliferation in vitro, whereas HI-MSC had no effect. In the second model, sepsis was induced in mice via cecal ligation and puncture. HI-MSC treatment significantly improved the overall survival, whereas controlMSCs had no effect. in vitro studies demonstrated that HI-MSCs are more effectively phagocytosed by monocytes than control MSCs and induced cell death in particular of activated CD16+ monocytes, which may explain the immune protective effect of HI-MSC in the sepsis model. The results of our study demonstrate that MSC-mediated immunomodulation in sepsis is dependent on a passive recognition of MSC by monocytes, whereas fully functional MSCs are required for inhibition of T-cell-mediated allograft rejection. [ABSTRACT FROM AUTHOR]

The immunomodulatory capacity of mesenchymal stem or stromal cells (MSC) makes them a promising tool for treatment of immune disease and organ transplantation. The effects of MSC on B cells are characterized by an abrogation of plasmablast formation and induction of regulatory B cells (Bregs). It is, however, unknown how MSC interact with B cells under inflammatory conditions. In this study, adipose tissue-derived MSC were pretreated with 50 ng/ml IFN-γ for 96 h (MSC-IFN-γ) to simulate inflammatory conditions. Mature B cells were obtained from spleens by CD43- selection. B cells were co-cultured with MSC and stimulated with anti-IgM, anti-CD40, and IL-2; and after 7 days, B cell proliferation, phenotype, Immunoglobulin-G (IgG), and IL-10 production were analyzed. MSC did not inhibit B cell proliferation but increased the percentage of CD38high CD24high B cells (Bregs) and IL-10 production, while MSC-IFN-γ significantly reduced B cell proliferation and inhibited IgG production by B cells in a more potent fashion but did not induce Bregs or IL-10 production. Both MSC and MSC-IFN-γ required proximity to target cells and being metabolically active to exert their effects. Indoleamine 2,3 dioxygenase expression was highly induced in MSC-IFN-γ and was responsible of the anti-proliferative and Breg reduction since addition of tryptophan (TRP) restored MSC properties. Immunological conditions dictate the effect of MSC on B cell function. Under immunological quiescent conditions, MSC stimulate Breg induction; whereas, under inflammatory conditions, MSC inhibit B cell proliferation and maturation through depletion of TRP. This knowledge is useful for customizing MSC therapy for specific purposes by appropriate pretreatment of MSC. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells ( MSCs) possess immunomodulatory properties which are of key interest for their application in autoimmunity and transplantation. In transplantation, administration of MSCs has shown promising results in preclinical models and has recently moved to clinical trials. Therefore, it is important to study the interactions between MSCs and immunosuppressive drugs currently used in transplantation. We aimed to analyze the effect of rabbit antithymocyte globulin ( rATG) MSCs. MSCs were obtained from perirenal fat of kidney donors and exposed to ranging doses of rATG (Thymoglobulin®, Genzyme; 0.5-100 μg/ml). Binding of rATG, effects on viability and susceptibility to be killed by cytotoxic lymphocytes as well as effects on their immunosuppressive potential of MSCs were tested. rATG binds dose-dependently to MSCs. This binding was associated with slightly impaired viability after 48 and 72 h when compared with nonexposed MSCs. In contrast to nontreated MSCs, rATG preexposed MSCs were susceptible to be lysed by cytokine-activated CD8+ cytotoxic cells and NKT cells. The capacity of MSCs to suppress the proliferation of anti-CD3/CD28 activated CD4 and CD8 T cells were reduced by the presence of rATG in the culture. rATG reduces the viability and antiproliferative capacity of MSCs in a dose-dependent manner and converts them into targets for CD8 T cells and NKT cell lysis. [ABSTRACT FROM AUTHOR]