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

Abstract

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-gamma 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-gamma 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.