title: Genetically-modified pig mesenchymal stromal cells: xenoantigenicity and effect on human T-cell xenoresponses.
abstract: BACKGROUND
  Mesenchymal stromal cells (MSC) are being investigated as immunomodulatory therapy in the field of transplantation, particularly islet transplantation. While MSC can regenerate across species barriers, the immunoregulatory influence of genetically modified pig MSC (pMSC) on the human and non-human primate T-cell responses has not been studied.
  
  
  METHODS
  Mesenchymal stromal cells from wild-type (WT), α1,3-galactosyltransferase gene knockout (GTKO) and GTKO pigs transgenic for the human complement-regulatory protein CD46 (GTKO/CD46) were isolated and tested for differentiation. Antibody binding and T-cell responses to WT and GTKO pMSC in comparison with GTKO pig aortic endothelial cells (pAEC) were investigated. The expression of swine leukocyte antigen (SLA) class II (SLA II) was tested. Costimulatory molecules CD80 and CD86 mRNA levels were measured. Human T-cell proliferation and the production of pro-inflammatory cytokines in response to GTKO and GTKO/CD46 pMSC in comparison with human MSC (hMSC) were evaluated.
  
  
  RESULTS
  α1,3-galactosyltransferase gene knockout and GTKO/CD46 pMSC isolation and differentiation were achieved in vitro. Binding of human antibodies and T-cell responses were lower to GTKO than those to WT pMSC. Human and baboon (naïve and sensitized) antibody binding were significantly lower to GTKO pMSC than to GTKO pAEC. Before activation, <1% of GTKO pMSC expressed SLA II, compared with 2.5% of GTKO pAEC. After pig interferon-gamma (pIFN-γ) activation, 99% of GTKO pAEC upregulated SLA II expression, compared with 49% of GTKO pMSC. Only 3% of GTKO pMSC expressed CD80 compared with 80% of GTKO pAEC without activation. After pIFN-γ activation, GTKO pAEC upregulated CD86 mRNA level stronger than GTKO pMSC. The human CD4(+) T-cell response to GTKO pMSC was significantly weaker than that to GTKO pAEC, even after pIFN-γ activation. More than 99% of GTKO/CD46 pMSC expressed hCD46. Human peripheral blood mononuclear cells and CD4(+) T-cell responses to GTKO and GTKO/CD46 pMSC were comparable with those to hMSC, and all were significantly lower than to GTKO pAEC. GTKO/CD46 pMSC downregulated human T-cell proliferation as efficiently as hMSC. The level of proinflammatory cytokines IL-2, IFN-γ, TNF-α, and sCD40L correlated with the downregulation of T-cell proliferation by all types of MSC.
  
  
  CONCLUSIONS
  Genetically modified pMSC is significantly less immunogenic than WT pMSC. GTKO/CD46 pMSC downregulates the human T-cell responses to pig antigens as efficiently as human MSC, which can be advantageous for therapeutic cell xenotransplantation.
authors.affiliation: Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, 200 Lothrop Street,Pittsburgh, PA 15261, USA. ezzelarabmb@upmc.edu
authors.forename: Mohamed
authors.initials: M
authors.lastname: Ezzelarab
authors.forename: Corin
authors.initials: C
authors.lastname: Ezzelarab
authors.forename: Tyler
authors.initials: T
authors.lastname: Wilhite
authors.forename: Goutham
authors.initials: G
authors.lastname: Kumar
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Hara
authors.forename: David
authors.initials: D
authors.lastname: Ayares
authors.forename: David K C
authors.initials: DK
authors.lastname: Cooper
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D023201:CD40 Ligand / Q000276:immunology; D002454:Cell Differentiation; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D006801:Humans; D007136:Immunoglobulins / Q000276:immunology; D007371:Interferon-gamma / Q000276:immunology; D007376:Interleukin-2 / Q000276:immunology; D051916:Membrane Cofactor Protein / Q000235:genetics / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000276:immunology* / Q000502:physiology; D010215:Papio / Q000276:immunology; D017154:Stromal Cells / Q000166:cytology / Q000276:immunology* / Q000502:physiology; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
chemical_list: C497857:CD46 protein, human; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D007136:Immunoglobulins; D007376:Interleukin-2; D051916:Membrane Cofactor Protein; D014409:Tumor Necrosis Factor-alpha; C438668:swine leukocyte antigen; D023201:CD40 Ligand; D007371:Interferon-gamma; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase