Background and objectives: Mesenchymal stem cells (MSC) have been proposed as a way to treat graft-versus-host disease based of their immunosuppressive effect. We analyzed whether regulatory T cells can be generated in co-cultures of peripheral blood mononuclear cells (PBMC) and MSC.
Design and methods: MSC were obtained from the bone marrow of four healthy donors and nine patients with acute leukemia in complete remission following chemotherapy. Short-term (4 days) co-cultures of MSC and autologous or allogeneic PBMC were set up, the lymphocytes harvested and their regulatory activity assessed.
Results: Lymphocytes harvested from MSC-PBMC co-cultures strongly inhibit (up to 95%) mixed lymphocyte reaction (MLR), recall to alloantigen, and CD3- or PHA-induced lymphocyte proliferation. These lymphocytes, termed regulatory cells (Regc), were all CD45+CD2+ with variable proportions of CD25+ cells (range 40-75% n=10) and a minor fraction expressed CTLA4 (2-4%, n=10) or glucocorticoid-induced tumor necrosis factcor receptor-related gene (0.5-4% n=10). Both CD4+ and CD8+ Regc purified from MSC-PBMC co-cultures strongly inhibited lymphocyte proliferation at a 1:100 Regc:responder cell ratio. CD4+ Regc expressed high levels of forkhead box P3 (Foxp3) mRNA while CD8+ Regc did not. The effectiveness of Regc, whether CD4+ or CD8+, was 100-fold higher than that of CD4+CD25+high regulatory T cells. Regc were also generated from highly purified CD25- PBMC or CD4+ or CD8+ T cell subsets. Soluble factors, such as interleukin-10, transforming growth factor-b and prostaglandin E2 did not appear to be involved in the generation of Regc or in the Regc-mediated immunosuppressive effect. Furthermore, cyclosporin A did not affect Regc generation or the immunosuppression induced by Regc.
Interpretation and conclusions: These findings indicate that powerful regulatory CD4+ or CD8+ lymphocytes are generated in co-cultures of PBMC with MSC. This strongly suggests that these regulatory cells may amplify the reported MSC-mediated immunosuppressive effect.