Interferon-gamma preactivated mesenchymal stem cells (MSC-γ) are highly immunosuppressive but immunogenic in vivo due to their inherent expression of major histocompatibility (MHC) molecules. We modified human bone-marrow derived MSC with interleukin-17A (MSC-17) to enhance MSC immunosuppression on T cells. MSC-17, unlike MSC-γ, showed no induction or upregulation of MHC class I, MHC class II and T cell co-stimulatory molecule CD40, and conformed to untreated-MSC (UT-MSC) phenotype. Interestingly, in co-cultures of phytohemagglutinin (PHA) activated human T cells, MSC-17 were superior to UT:MSC and MSC-γ in enhancing suppression of T cell proliferation. MSC-17 also augmented inhibition of the CD25 T cell activation molecule similar to MSC-γ and further downregulated Th1 cytokines IFN-γ, tumor-necrosis factor-alpa (TNF-α) and IL-2 in these co-cultures when compared to UT-MSC. Enhancement of T cell suppression correlated with the increased immunoregulatory gene expression of IL-6 but not indoleamine 2,3-dioxygenase (IDO1), cyclooxygenase-1 (Cox-1), transforming growth factor-beta-1 (TGF-β1) in MSC-17. Moreover, MSC-17 but not MSC-γ consistently induced CD4+CD25highCD127lowFoxP3+ regulatory T (iTregs) cells from PHA activated human CD4+CD25- T cells (ranging from 1.47- to 7.18-fold induction relative to UT-MSC; n=10, p<0.05). MSC-induced iTregs were positive for activation and suppressive iTreg molecules CD39, CD73, CD69, OX40, CTLA-4 and GITR. Moreover, MSC-17 derived CD4+CD25highCD127low iTregs from these co-cultures were isolated by flow sorting (> 80% FoxP3 purity).These sorted MSC-17 generated iTregs were functionally suppressive as they inhibited activation induced CD154 expression on effector T cells. Thus, MSC-17 are superior T cell immunomodulators.

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