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

• Published in: Haematologica (Roma) 2007-07, Vol.92 (7), p.881-888
• Main Authors: Prevosto, Claudia, Zancolli, Marta, Canevali, Paolo, Zocchi, Maria Raffaella, Poggi, Alessandro
• Format: Article
• Language: English
• Subjects: Acute Disease; Biological and medical sciences; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Proliferation; Coculture Techniques; Hematologic and hematopoietic diseases; Humans; Leukemia - immunology; Leukemia - pathology; Leukocytes, Mononuclear - cytology; Lymphocyte Culture Test, Mixed; Medical sciences; Mesenchymal Stem Cells - cytology; T-Lymphocytes, Regulatory - cytology
• ISSN: 0390-6078; 1592-8721
• DOI: 10.3324/haematol.11240

From the Laboratory of Experimental Oncology D, National Cancer Research Institute, 16132-Genoa, Italy (CP, PC, AP); Laboratory of Tumor Immunology, San Raffaele Scientific Institute, 20132, Milan, Italy (MZ, MRZ) Correspondence: Alessandro Poggi, PhD, MD, National Cancer Research Institute (IST) Genoa, Laboratory of Experimental Oncology D, Department of Translational Oncogenesis, Largo R. Benzi 10, 16132 Genoa, Italy. E-mail: alessandro.poggi{at}istge.it 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-ß and prostaglandin E 2 did not appear to be involved in the generation of Reg c or in the Reg c -mediated immuno-suppressive 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 regula