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Enhanced Immunomodulation in Inflammatory Environments Favors Human Cardiac Mesenchymal Stromal-Like Cells for Allogeneic Cell Therapies.

ABSTRACT: Rising numbers of patients with cardiovascular diseases and limited availability of donor hearts require new and improved therapy strategies. Human atrial appendage-derived cells (hAACs) are promising candidates for an allogeneic cell-based treatment. In this study, we evaluated their inductive and modulatory capacity regarding immune responses and underlying key mechanisms in vitro. For this, cryopreserved hAACs were either cultured in the presence of interferon-gamma (IFN?) or left unstimulated. The expression of characteristic mesenchymal stromal cell markers (CD29, CD44, CD73, CD105, CD166) was revealed by flow cytometry that also highlighted a predominant negativity for CD90. A low immunogeneic phenotype in an inflammatory milieu was shown by lacking expression of co-stimulatory molecules and upregulation of the inhibitory ligands PD-L1 and PD-L2, despite de novo expression of HLA-DR. Co-cultures of hAACs with allogeneic peripheral blood mononuclear cells, proved their low immunogeneic state by absence of induced T cell proliferation and activation. Additionally, elevated levels of IL-1?, IL-33, and IL-10 were detectable in those cell culture supernatants. Furthermore, the immunomodulatory potential of hAACs was assessed in co-cultures with ?CD3/?CD28-activated peripheral blood mononuclear cells. Here, a strong inhibition of T cell proliferation and reduction of pro-inflammatory cytokines (IFN?, TNF?, TNF?, IL-17A, IL-2) were observable after pre-stimulation of hAACs with IFN?. Transwell experiments confirmed that mostly soluble factors are responsible for these suppressive effects. We were able to identify indolamin-2,3-dioxygenase (IDO) as a potential key player through a genome-wide gene expression analysis and could demonstrate its involvement in the observed immunological responses. While the application of blocking antibodies against both PD-1 ligands did not affect the immunomodulation by hAACs, 1-methyl-L-tryptophan as specific inhibitor of IDO was able to restore proliferation and to lower apoptosis of T cells. In conclusion, hAACs represent a cardiac-derived mesenchymal stromal-like cell type with a high potential for the application in an allogeneic setting, since they do not trigger T cell responses and even increase their immunomodulatory potential in inflammatory environments.

SUBMITTER: Diedrichs F

PROVIDER: S-EPMC6665953 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Enhanced Immunomodulation in Inflammatory Environments Favors Human Cardiac Mesenchymal Stromal-Like Cells for Allogeneic Cell Therapies.

Diedrichs Falk F Stolk Meaghan M Jürchott Karsten K Haag Marion M Sittinger Michael M Seifert Martina M

Frontiers in immunology 20190723

Rising numbers of patients with cardiovascular diseases and limited availability of donor hearts require new and improved therapy strategies. Human atrial appendage-derived cells (hAACs) are promising candidates for an allogeneic cell-based treatment. In this study, we evaluated their inductive and modulatory capacity regarding immune responses and underlying key mechanisms <i>in vitro</i>. For this, cryopreserved hAACs were either cultured in the presence of interferon-gamma (IFNγ) or left unst ...[more]

PMID: 31396228

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Project description:Rising numbers of patients with cardiovascular diseases and limited availability of donor hearts require new and improved therapy strategies. Human atrial appendage-derived cells (hAACs) are promising candidates for an allogeneic cell-based treatment. In this study, their inductive and modulatory capacity regarding immune responses and underlying key mechanisms were evaluated in vitro. For this, cryopreserved hAACs were analyzed either unstimulated or after stimulation with interferon-gamma by flow cytometry. This revealed the expression of characteristic mesenchymal stromal cell markers (CD29, CD44, CD73, CD166), but also highlighted a predominant negativity for CD90. A low immunogenic phenotype in an inflammatory milieu was shown by lacking expression of co-stimulatory molecules and upregulation of the inhibitory ligands PD-L1 and PD-L2, despite of de novo expression of HLA-DR. Co-cultures of hAACs with allogeneic peripheral blood mononuclear cells, proved the low immunogenic state by absence of induced CD4+ and CD8+ T cell proliferation and activation. However, elevated levels of IL-1β, IL-33 and IL-10 were detectable in those cell culture supernatants. In a second step, the immunomodulatory potential of hAACs was assessed in co-cultures with αCD3/αCD28-activated peripheral blood mononuclear cells. A strong inhibition of T cell proliferation and lowering of pro-inflammatory cytokines (IFNγ, TNFα, TNFβ, IL-17A, IL-2) were observable after pre-stimulation of hAACs with interferon-gamma. Transwell experiments confirmed that mostly soluble factors are responsible for these suppressive effects. We were able to identify indolamin-2,3-dioxygenase (IDO) as a potential key player through a genome-wide gene expression analysis and could demonstrate its involvement in lowered proliferation and induction of apoptosis in T cells. While the application of blocking antibodies against both PD-1 ligands did not affect the immunomodulation by hAACs, 1-Methyl-L-Tryptophan as specific inhibitor of IDO was able to restore proliferation and to lower apoptosis of T cells. In conclusion, hAACs represent a cardiac-derived mesenchymal stromal-like cell type with a high potential for application in allogeneic settings, since they do not trigger T cell responses and even increase their immunomodulatory potential in inflammatory environments.

Project description:BackgroundMesenchymal stromal cells (MSCs) are believed to be hypoimmunogeneic with potential use for allogeneic administration.MethodsBone marrow was harvested from Connemara (n = 1), Standardbred (n = 6), and Thoroughbred (n = 3) horses. MSCs were grouped by their level of expression of major histocompatibility factor II (MHC II). MSCs were then sub-grouped by those MSCs derived from universal blood donor horses. MSCs were isolated and cultured using media containing fetal bovine serum until adequate numbers were acquired. The MSCs were cultured in xenogen-free media for 48 h prior to use and during all assays. Autologous and allogeneic MSCs were then directly co-cultured with responder leukocytes from the Connemara horse in varying concentrations of MSCs to leukocytes (1:1, 1:10, and 1:100). MSCs were also cultured with complement present and heat-inactivated complement to determine whether complement alone would decrease MSC viability. MSCs underwent haplotyping of their equine leukocyte antigen (ELA) to determine whether the MHC factors were matched or mismatched between the donor MSCs and the responder leukocytes.ResultsAll allogeneic MSCs were found to be ELA mismatched with the responder leukocytes. MHC II-low and universal blood donor MSCs caused no peripheral blood mononuclear cell (PBMC) proliferation, no increase in B cells, and no activation of CD8 lymphocytes. Universal blood donor MSCs stimulated a significant increase in the number of T regulatory cells. Neutrophil interaction with MSCs showed that universal blood donor and MHC II-high allogeneic MSCs at the 6 h time point in co-culture caused greater neutrophil activation than the other co-culture groups. Complement-mediated cytotoxicity did not consistently cause MSC death in cultures with active complement as compared to those with inactivated complement. Gene expression assays revealed that the universal blood donor group and the MHC II-low MSCs were more metabolically active both in the anabolic and catabolic gene categories when cultured with allogeneic lymphocytes as compared to the other co-cultures. These upregulated genes included CD59, FGF-2, HGF, IDO, IL-10, IL-RA, IL-2, SOX2, TGF-β1, ADAMSTS-4, ADAMSTS-5, CCL2, CXCLB/IL-8, IFNγ, IL-1β, and TNFα.ConclusionsMHC II-low MSCs are the most appropriate type of allogeneic MSC to prevent activation of the innate and cell-mediated component of the adaptive immune systems and have increased gene expression as compared to other allogeneic MSCs.

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Enhanced Immunomodulation in Inflammatory Environments Favors Human Cardiac Mesenchymal Stromal-Like Cells for Allogeneic Cell Therapies.

Publications

Enhanced Immunomodulation in Inflammatory Environments Favors Human Cardiac Mesenchymal Stromal-Like Cells for Allogeneic Cell Therapies.

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