Interleukin-17A induced human mesenchymal stem cells are superior modulators of immunological function

Abstract

Interferon-gamma (IFN-γ) preactivated mesenchymal stem cells (MSC-γ) are highly immunosuppressive but immunogenic in vivo due to their inherent expression of major histocompatibility (MHC) molecules. This thesis presents an improved approach where human bone-marrow derived MSC were modified with IL-17A (MSC-17) to enhance T cell immunosuppression but not their immunogenicity. It was demonostrated in CHAPTER 3 that MSC-17, unlike MSC-γ, showed no induction or upregulation of MHC class I, MHC class II and T cell co-stimulatory molecule CD40, but maintained normal MSC morphology and phenotypic marker expression. When co-cultured with phytohemagglutinin (PHA) activated human T cells, MSC-17 potently suppressed T cell proliferation, inhibited surface CD25 expression and suppressed the elaboration of Th1 cytokines IFN-γ, TNF-α and IL-2 when compared to untreated MSC (UT-MSC). T cell suppression by MSC-17 correlated with increased IL-6 but not indoleamine 2,3-dioxygenase 1, cyclooxygenase-1 and TGF-β. In CHAPTER 4, it was shown that MSC-17, but not MSC-γ consistently induced CD4⁺CD25high[high superscript]CD127low[low superscript]FoxP3⁺ regulatory T cells (iTregs) from PHA activated CD4⁺CD25⁻ T cells. MSC-induced iTregs expressed the functional Treg markers CD39, CD73, CD69, OX40, CTLA-4 and GITR. Functionally, FACS-sorted MSC- 17-induced-iTregs could suppress human T cell activation (CD154 suppression assay). CHAPTER 5 was aimed at further dissecting mechanisms by which human MSC- 17 mediate their superior modulation of T cell responses. UT-MSC, MSC-γ and MSC-17 were assessed for their gene expression profile (microarray, 3 human MSC donors). Significantly regulated genes (p<0.05, fold change (FC) < -2 or >2) were identified for their biological functions (Database for Annotation, Visualisation and Integrated Discovery, DAVID). Microarray analysis revealed that 1278 differentially expressed genes (902 upregulated; 376 downregulated) were significantly regulated between MSC-γ and UT-MSC and only 67 genes (39 upregulated; 28 downregulated) between MSC-17 and UT-MSC. Gene ontology analysis of upregulated MSC-γ genes uncovered significant enrichment of genes involved in immune response, antigen processing and presentation, humoral responses and complement activation (eg. HLA genes, complement components and CIITA). This data is consistent with the upregulation of MHC molecules and studies showing increased MSC-γ immunogenicity. MSC-17 upregulated genes were mainly associated with chemotaxis response. This may be essential for T cell recruitment for MSC-17 immunosuppression. MMP13 was highly expressed only in MSC-17 as determined by microarray (FC 15.6) and validated by real-time PCR, hence the potential involvement of MMP13 in the superior immunomodulatory function of MSC-17. The final results CHAPTER 6 was to translate the findings with human MSC-17 to compact bone-derived mouse MSC (CB mMSC). Unexpectedly, CB mMSC-17 unlike human MSC-17 showed no enhancement of in vitro immunosuppression of allogeneically induced CD4⁺ and CD8⁺ T cell proliferation. Nevertheless, CB mMSC differ from bone marrow-derived mMSC and may represent a new source to isolate mouse MSC with high purity and potent immunosuppression properties that would be beneficial in a context of allotransplantation rejection. CB mMSC, without IL-17A preconditioning mediated potent in vitro suppression of T cells even when used at low doses. In conclusion, human MSC-17 are superior modulators of T cells and can engender Tregs to potently suppress T cell activation with minimal immunogenicity. MSC-17 represent a potential cell therapy to modulate T cell responses for clinical application.