Treatment with bortezomib of human CD4+ T cells preserves natural regulatory T cells and allows the emergence of a distinct suppressor T-cell population

• Published in: Haematologica (Roma) 2009-07, Vol.94 (7), p.975-983
• Main Authors: Blanco, Belen, Perez-Simon, Jose A, Sanchez-Abarca, Luis I, Caballero-Velazquez, Teresa, Gutierrez-Cossio, Silvia, Hernandez-Campo, Pilar, Diez-Campelo, Maria, Herrero-Sanchez, Carmen, Rodriguez-Serrano, Concepcion, Santamaria, Carlos, Sanchez-Guijo, Fermin M, Canizo, Consuelo del, San Miguel, Jesus F
• Format: Article
• Language: English
• Subjects: Antineoplastic Agents - pharmacology; Biological and medical sciences; Boronic Acids - pharmacology; Bortezomib; CD28 Antigens - biosynthesis; CD3 Complex - biosynthesis; CD4-Positive T-Lymphocytes - cytology; Cell Proliferation; Coculture Techniques; Hematologic and hematopoietic diseases; Humans; Interleukin-2 - metabolism; Interleukin-2 Receptor alpha Subunit - biosynthesis; Leukocytes, Mononuclear - metabolism; Lymphocyte Subsets - drug effects; Medical sciences; Original; Phenotype; Proteasome Inhibitors; Pyrazines - pharmacology; T-Lymphocytes - drug effects
• ISSN: 0390-6078; 1592-8721
• DOI: 10.3324/haematol.2008.005017

Servicio de Hematología, Hospital Universitario de Salamanca and Centro de Investigación del Cáncer (CIC/CSIC) Salamanca, Centro en Red de Medicina Regenerativa y Terapia celular de Castilla y León, Spain Correspondence: José A Pérez Simón, Servicio de Hematología y CIC Salamanca, Paseo de San Vicente, s/n 37007, Salamanca, Spain. E-mail: pesimo{at}usal.es Background: In vitro depletion of alloreactive T cells using the proteasome inhibitor bortezomib is a promising approach to prevent graft-versus-host disease after allogeneic stem cell transplantation. We have previously described the ability of bortezomib to selectively eliminate alloreactive T cells in a mixed leukocyte culture, preserving non-activated T cells. Due to the role of regulatory T cells in the control of graft versus host disease, in the current manuscript we have analyzed the effect of bortezomib in regulatory T cells. Design and Methods: Conventional or regulatory CD4 + T cells were isolated with immunomagnetic microbeads based on the expression of CD4 and CD25. The effect of bortezomib on T-cell viability was analyzed by flow cytometry using 7-amino-actinomycin D staining. To investigate the possibility of obtaining an enriched regulatory T-cell population in vitro with the use of bortezomib, CD4 + T cells were cultured during four weeks in the presence of anti-CD3 and anti-CD28 antibodies, IL-2 and bortezomib. The phenotype of these long-term cultured cells was studied, analyzing the expression of CD25, CD127 and FOXP3 by flow cytometry, and mRNA levels were determined by RT-PCR. Their suppressive capacity was assessed in co-culture experiments, analyzing proliferation and IFN- and CD40L expression of stimulated responder T cells by flow cytometry. Results: We observed that naturally occurring CD4 + CD25 + regulatory T cells are resistant to the pro-apoptotic effect of bortezomib. Furthermore, we found that long-term culture of CD4 + T cells in the presence of bortezomib promotes the emergence of a regulatory T-cell population that significantly inhibits proliferation, IFN- production and CD40L expression among stimulated effector T cells. Conclusions: These results reinforce the proposal of using bortezomib in the prevention of graft versus host disease and, moreover, in the generation of regulatory T-cell populations, that could be used in the treatment of multiple T-cell mediated diseases. Key words: bortezomib, regulatory T cells, graft-versus-host disease, prevention.