University of Adelaide
Gene microarray comparative analysis of Interferon-gamma and Interleukin-17A preconditioned human Mesenchymal Stem cells
Kisha Sivanathan1,2,3, Darling Rojas-Canales1,2, Christopher Hope1,2, Stan Gronthos3,5, Shane Grey6, Patrick T. Coates1,2,3,4.
1School of Medicine, Faculty of Health Sciences, , University of Adelaide, Adelaide, Australia; 2Centre for Clinical and Experimental Transplantation, Royal Adelaide Hospital, Adelaide, Australia; 3Centre for Stem Cell Research and Robinson Institute, School of Medical Sciences, University of Adelaide, Adelaide, Australia; 4Central Northern Adelaide Renal Transplantation Service, Royal Adelaide Hospital, Adelaide, Australia; 5Mesenchymal Stem Cell Group Laboratory, School of Medicine, Faculty of Health Sciences , University of Adelaide, Adelaide, Australia; 6Transplant Immunology Group, Garvin Institute of Medical Research, Sydney, Australia
Interferon-gamma (IFN-γ) preactivated mesenchymal stem cells (MSC) are highly immunosuppressive but immunogenic in vivo due to their inherent expression of MHC molecules. We have shown that human bone-marrow derived MSC pre-treated with IL-17A represents a novel immunomodulatory strategy and an alternative to IFN-γ treatment of MSC in enhancing MSC immunosuppression on T cells.
Methods: Untreated MSC (UT-MSC) or MSC treated for 5 days with IFN-γ (MSC-γ) or IL-17A (MSC-17) were assessed for their ability to suppress T cell proliferation and effector function as well as in the induction of regulatory T cells (iTregs). UT-MSC, MSC-γ and MSC-17 isolated from 3 MSC donors were also characterised for MSC phenotype by flow cytometry and gene expression profile using the Affymetrix Human Gene ST 2.0 microarrays. Significantly regulated genes (p<0.05, fold change (FC) <-2 or > 2) were identified and further analysed for their biological functions using the Database for Annotation, Visualisation and Integrated Discovery (DAVID). Results: 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. Both MSC-17 and MSC-γ inhibited T cell surface CD25 expression and suppressed the elaboration of IFN-γ, TNF-α and IL-2 compared to UT-MSC. MSC-17 in direct contact with T cells and in environments enriched of TGF-β1 and PGE2 favoured the induction of functionally suppressive CD4+CD25highCD127lowFoxP3+ iTregs. These MSC-17 induced iTregs expressed CD39, CD73, CD69, OX40, CTLA-4 and GITR. Flow sorted MSC-17 derived iTregs also inhibited activation induced CD154 expression of effector T cells. MSC-17 unlike MSC-γ showed no upregulation of MHC class I, MHC class II and T cell co-stimulatory molecule CD40, but maintained normal MSC phenotypic marker expression. Microarray analysis revealed that 1278 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 response 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.
Conclusion: MSC-17 represents a potential cell therapy to modulate T cell responses for clinical application.
11:00 - 12:30
|Mesenchymal Stromal Cells||Gene microarray comparative analysis of Interferon-gamma and Interleukin-17A preconditioned human Mesenchymal Stem cells||Room 111-112|