Donor-derived equine mesenchymal stem cells suppress proliferation of mismatched lymphocytes.

Abstract

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.