Donor‐derived equine mesenchymal stem cells suppress proliferation of mismatched lymphocytes

REASONS FOR PERFORMING STUDY: 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. Howe...

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Published in:Equine veterinary journal 2016-03, Vol.48 (2), p.253-260
Main Authors: Ranera, B., Antczak, D., Miller, D., Doroshenkova, T., Ryan, A., McIlwraith, C. W., Barry, F.
Format: Article
Language:English
Subjects:
Animals
Bone marrow
Bone Marrow Cells
Cell Proliferation - physiology
Coculture Techniques
Flow cytometry
Histocompatibility Testing
horse
Horses
immunosuppression
Leukocytes, Mononuclear - drug effects
Leukocytes, Mononuclear - physiology
lymphocyte
Lymphocyte Subsets - immunology
Lymphocyte Subsets - physiology
Lymphocytes
Major Histocompatibility Complex - genetics
Major Histocompatibility Complex - immunology
major histocompatibility complex mismatch
mesenchymal stem cell
Mesenchymal Stromal Cells - physiology
Phytohemagglutinins - toxicity
Stem cells
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Summary:REASONS FOR PERFORMING STUDY: 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. OBJECTIVES: To evaluate the immunosuppressive capabilities of equine bone‐marrow‐derived MSCs (BM‐MSCs) on MHC‐mismatched lymphocytes. STUDY DESIGN: 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.
ISSN:0425-1644
2042-3306
DOI:10.1111/evj.12414