Generation of CD4+ or CD8+ regulatory T cells upon mesenchymal stem cell-lymphocyte interaction

Background and Objectives Mesenchymal stem cells (MSC) have been proposed as a way to treat graft-versus-host disease based on 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 phytohemagglutinin-induced lymphocyte proliferation. These lymphocytes, termed regulatory cells (Reg(c)), 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(+) Reg(c), purified from MSC-PBMC co-cultures strongly inhibited lymphocyte proliferation at a 1:100 Reg(c):responder cell ratio. CD4(+) Reg(c) expressed high levels of forkhead box P3 (Foxp3) mRNA while CD8(+) Reg(c) did not. The effectiveness of Reg(c), whether CD4(+) or CD8(+), was 100-fold higher than that of CD4(+)CD25(+high) regulatory T cells. Reg(c) were also generated from highly purified CD25(-) PBMC or CD4(+) or CD8(+) T cell subsets. Soluble factors, such as interleukin-10, transforming growth factor-beta and prostaglandin E(2 )did not appear to be involved in the generation of Reg(c) or in the Reg(c)-mediated immunosuppressive effect. Furthermore, cyclosporine A did not affect Reg(c) generation or the immunosuppression induced by Reg(c). 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.