title: Donor-derived equine mesenchymal stem cells suppress proliferation of mismatched lymphocytes.
abstract: BACKGROUND
  Recently, it has been shown that mesenchymal stem cells (MSCs) do not express the major histocompatibility complex (MHC) II antigen and are able to inhibit proliferation of MHC-mismatched stimulated lymphocytes, enabling their use as in vivo allogeneic transplants. However, prior to clinical application of allo-MSCs, in vitro tests are required to confirm the safety of treatment protocols.
  
  
  OBJECTIVE
  To evaluate the immunosuppressive capabilities of equine bone-marrow-derived MSCs (BM-MSCs) on MHC-mismatched lymphocytes.
  
  
  METHODS
  In vitro experiment.
  
  
  METHODS
  Phytohaemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) from 3 Thoroughbreds (recipients) were co-cultured with mismatched BM-MSCs from 3 Connemara ponies (donors). Proliferation of lymphocytes was monitored by carboxyfluorescein succinimidyl ester labelling and analysed by flow cytometry. In total, 6 horses were haplotyped using microsatellites to confirm mismatching. Optimisation of the conditions to stimulate Thoroughbred lymphocytes and titration of equine anti-CD4 and anti-CD8 antibodies were performed. Connemara pony and Thoroughbred BM-MSCs were isolated, expanded and characterised by tri-lineage differentiation. Finally, BM-MSCs from both breeds were set up in co-culture at different ratios with stimulated Thoroughbred lymphocytes. Proliferation of CD4(+) and CD8(+) cells was determined by flow cytometry.
  
  
  RESULTS
  A high proportion of CD4/CD8 double-positive lymphocytes were found in freshly isolated PBMCs, although this percentage decreased after 4 days of culture. Mismatched BM-MSCs inhibited proliferation of stimulated lymphocytes in a dose-dependent manner, with the greatest suppression occurring at a 1:10 ratio of BM-MSCs to PBMCs. Proliferation of CD4(+) and CD8(+) subpopulations decreased in 1:10 co-culture, with statistical significance in the case of CD8(+) cells, while that of the CD4/CD8 double-positive population was similar to the phytohaemagglutinin control.
  
  
  CONCLUSIONS
  The results demonstrate dose-dependent immunosuppression of stimulated lymphocytes by mismatched equine BM-MSCs, supporting their future application in allo-MSC clinical treatments.
authors.affiliation: Regenerative Medicine Institute (REMEDI), National University of Ireland Galway, Galway, Ireland.
authors.forename: B
authors.initials: B
authors.lastname: Ranera
authors.affiliation: Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.
authors.forename: D
authors.initials: D
authors.lastname: Antczak
authors.affiliation: Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.
authors.forename: D
authors.initials: D
authors.lastname: Miller
authors.affiliation: Regenerative Medicine Institute (REMEDI), National University of Ireland Galway, Galway, Ireland.
authors.forename: T
authors.initials: T
authors.lastname: Doroshenkova
authors.affiliation: Regenerative Medicine Institute (REMEDI), National University of Ireland Galway, Galway, Ireland.
authors.forename: A
authors.initials: A
authors.lastname: Ryan
authors.affiliation: Orthopaedic Research Center, Colorado State University, Fort Collins, USA.
authors.forename: C W
authors.initials: CW
authors.lastname: McIlwraith
authors.affiliation: Regenerative Medicine Institute (REMEDI), National University of Ireland Galway, Galway, Ireland.
authors.forename: F
authors.identifier: http://orcid.org/0000-0002-5535-002X
authors.initials: F
authors.lastname: Barry
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells; D049109:Cell Proliferation / Q000502:physiology*; D018920:Coculture Techniques; D006650:Histocompatibility Testing; D006736:Horses; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000502:physiology*; D016131:Lymphocyte Subsets / Q000276:immunology / Q000502:physiology*; D008285:Major Histocompatibility Complex / Q000235:genetics / Q000276:immunology; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D010835:Phytohemagglutinins / Q000633:toxicity
chemical_list: D010835:Phytohemagglutinins
keywords: horse; immunosuppression; lymphocyte; major histocompatibility complex mismatch; mesenchymal stem cell