Abstract

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.

Highlights

  • The interaction of the immune system with foreign antigens initiates inflammation and allorecognition

  • Human studies have repeatedly shown that Mesenchymal stromal cell (MSC) have immunosuppressive effects via T regulatory (Treg) and B regulatory cell upregulation leading to decreased activation of T lymphocytes and B cells, respectively [13, 63]

  • MSC haplotyping The Connemara pony was of a different equine leukocyte antigen (ELA) haplotype than all of the other horses utilized in this study (Supplemental information)

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Summary

Introduction

The interaction of the immune system with foreign antigens initiates inflammation and allorecognition. When the foreign source of antigenic stimulation is a therapy such as allogeneic mesenchymal stem cells (MSCs), the immune reaction can be detrimental to the survival of the. Human studies have repeatedly shown that MSCs have immunosuppressive effects via T regulatory (Treg) and B regulatory cell upregulation leading to decreased activation of T lymphocytes and B cells, respectively [13, 63]. Immunosuppression within the recipient site by MSCs is necessary as allogeneic MSCs may be rejected due to their expression of foreign surface antigens. Mismatched ELA haplotype donor MSCs have been shown to induce greater lymphocyte activation in vitro as compared to matched donor MSCs [57]. Mesenchymal stromal cells (MSCs) are believed to be hypoimmunogeneic with potential use for allogeneic administration

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