TGF-β2 Reduces the Cell-Mediated Immunogenicity of Equine MHC-Mismatched Bone Marrow-Derived Mesenchymal Stem Cells Without Altering Immunomodulatory Properties

Abstract

Allogeneic mesenchymal stem cells (MSCs) are a promising cell therapy for treating numerous diseases, but major histocompatibility complex (MHC)-mismatched MSCs can be rejected by the recipient's immune system. Pre-treating MSCs with transforming growth factor-β2 (TGF-β2) to downregulate surface expression of MHC molecules may enhance the ability of allogeneic MSCs to evade immune responses. We used lymphocyte proliferation assays and ELISAs to analyze the immunomodulatory potential of TGF-β2-treated equine bone marrow-derived MSCs. T cell activation and cytotoxicity assays were then used to measure the in vitro cell-mediated immunogenicity. Similar to untreated MSCs, TGF-β2-treated MSCs inhibited T cell proliferation and did not stimulate MHC-mismatched T cells to proliferate. Additionally, similar quantities of prostaglandin E2 and TGF-β1 were detected in assays with untreated and TGF-β2-treated MSCs supporting that TGF-β2-treated MSCs retain their strong immunomodulatory properties in vitro. Compared to untreated MSCs, TGF-β2-treated MSCs induced less T cell activation and had reduced cell-mediated cytotoxicity in vitro. These results indicate that treating MSCs with TGF-β2 is a promising strategy to reduce the cell-mediated immunogenicity of MHC-mismatched MSCs and facilitate allogeneic MSC therapy.

Keywords: TGF-β2; allogeneic; cytotoxicity; immunogenicity; major histocompatibility complex; mesenchymal stem cell.

FIGURE 1

Untreated and TGF-β2-treated MSCs inhibit proliferation of MHC-mismatched T cells in vitro. (A) Representative dot plots of live, CD3⁺ cells from MLRs with MHC-mismatched PBL, untreated MSC, and TGF-β2-treated MSC stimulator cells. (B) Number of live CD3⁺ cells recovered from MLRs relative to the MHC-M MLR with PBL stimulator cells. Data shown are mean ± SD of n = 6, p = 0.0046 by ANCOVA. Superscript letters indicate significant difference between groups by Tukey’s test. (C) Representative histograms depicting CFSE attenuation in responder PBLs cultured with MHC-matched (M) and MHC-mismatched (MM) PBL, untreated, and TGF-β2-treated MSC stimulator cells. (D) Proliferation of live, CD3⁺ responder T cells shown as the average log fold change in division index relative to the MHC-M MLR with PBL stimulator cells. Data shown are mean, minimum, and maximum values of n = 6, p = 0.0025 by ANCOVA. (E) Proliferation of live, CD3⁺ responder T cells shown as the average log fold change in CFSE geometric mean fluorescent intensity relative to the MHC-M MLR with PBL stimulator cells. Data shown are mean, minimum, and maximum values for n = 6, p = 0.0001 by ANCOVA.

FIGURE 2

PGE2 and TGF-β1 are secreted in MLRs with PBL and MSC stimulator cells. (A) PGE2 concentrations in the supernatant of MLRs as measured by ELISA. Data shown are mean ± SD of n = 6, p < 0.0001 by ANCOVA. Superscript letters indicate significant difference between groups. (B) TGF-β1 concentrations in MLR supernatant as measured by ELISA. Data shown are mean, minimum, and maximum values for n = 6, non-significant by ANCOVA.

FIGURE 3

TGF-β2 treatment of MHC-mismatched MSCs prevents activation of T cells. (A) Representative histograms of CD3 expression on effector lymphocytes from one horse cultured with MHC-matched or MHC-mismatched untreated or TGF-β2-treated MSCs. (B) CD3 expression on effector lymphocytes relative to effector cells incubated without MSCs. MSC donor-paired samples are represented by connected lines for n = 5, ***p = 0.0016 by a one-sample t-test on each pair. (C) Representative histograms of CD8 expression on effector lymphocytes from one horse cultured with MHC-matched or MHC-mismatched untreated or TGF-β2-treated MSCs. (D) CD8 expression on effector lymphocytes relative to effector cells incubated without MSCs. MSC donor-paired samples are represented by connected lines for n = 5, * p = 0.0223 by a one-sample t-test on each pair.

FIGURE 4

TGF-β2 treatment decreases the cytotoxicity of MHC-mismatched MSCs. Percent cytotoxicity in a standard 6-h chromium-51 release assay for MHC-matched and MHC-mismatched untreated and TGF-β2-treated MSCs. MSC donor-paired samples are represented by connected lines for n = 4, *p = 0.0119 by a one-sample t-test on each pair.