Investigating the Use of TGF-ß2 to Downregulate MHC Expression and Reduce the Immunogenicity of Equine Bone Marrow-derived Mesenchymal Stem Cells.

BERGLUND, ALIX KAY. Investigating the Use of TGF-2 to Downregulate MHC Expression and Reduce the Immunogenicity of Equine Bone Marrow-derived Mesenchymal Stem Cells. (Under the direction of Lauren V. Schnabel and Samuel L. Jones). Allogeneic bone marrow-derived mesenchymal stem cells (MSCs) are a promising cell therapy for effective and efficient treatment of various inflammatory and immunemediated diseases. While the prevailing dogma has been that MSCs are immune privileged, very few studies have controlled for MHC haplotype or adequately measured MSC immunogenicity in vitro or in vivo. Controlled studies have found that major histocompatibility complex (MHC)-mismatched MSCs evoke both cell-mediated and humoral immune responses in vivo. Microcytotoxicity assays were used to show that horses injected with MHC-mismatched MSCs generate cytotoxic alloantibodies capable of killing MSCs as early as 7-days post-transplantation. Rejection of MSCs likely leads to reduced therapeutic efficacy and the development of strategies to avoid allorecognition and rejection are necessary to provide safe and efficacious allogeneic therapy. Downregulation of MHC expression allows cells to avoid immune surveillance and may enhance the ability of MSCs to avoid allorecognition and rejection. Transforming growth factor-2 (TGF-2) has been shown to downregulate MHC surface expression in various cell types. In agreement with what has been demonstrated in other cell types, TGF-2 treatment significantly reduced constitutive MHC I and MHC II surface expression and partially blocked IFN--induced MHC expression on equine MSCs. TGF-2 treatment did not significantly affect the morphology, cell surface markers, viability, or secretion of TGF1 and TGF-2, but did increase the cell yield from cultures. This data indicates that TGF-2 may reduce MSC immunogenicity without altering the immunomodulatory properties of the cells. The immunomodulatory capabilities of TGF-2-treated MSCs were analyzed in modified one-way mixed leukocyte reactions and ELISAs. Naïve and TGF-2-treated MSCs both significantly reduced T cell proliferation as measured by the relative division index and relative CFSE geometric mean fluorescent intensity attenuation. Similar amounts of PGE2 and TGF-2 were also measured in the supernatant of MLRs with naïve and TGF-2-treated MSCs. This supports that TGF-2 treatment does not negatively affect the immunomodulatory properties of equine MSCs, which are critical for therapeutic function and evading immune responses in vivo. In conclusion, although MHC-mismatched equine MSCs are immunogenic in vivo, MHC I and MHC II surface expression can be manipulated by treating cells with TGF-2 in vitro. Downregulate of MHC surface expression is a promising strategy for enhancing the ability of MSCs to evade immune responses allowing for allogenic use clinically without the risk of immune rejection. The ability of TGF-2-treated MSCs to avoid immune rejection should continue to be investigated in vitro and in vivo along with the mechanism by which TGF-2 downregulates MHC expression. © Copyright 2018 Alix Kay Berglund All Rights Reserved Investigating the Use of TGF-2 to Downregulate MHC Expression and Reduce the Immunogenicity of Equine Bone Marrow-derived Mesenchymal Stem Cells. by Alix Kay Berglund A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Comparative Biomedical Sciences Raleigh, North Carolina 2018