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

Haematologica. 2009 Jul;94(7):975-83. doi: 10.3324/haematol.2008.005017. Epub 2009 Jun 8.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / pharmacology
  • Boronic Acids / pharmacology*
  • Bortezomib
  • CD28 Antigens / biosynthesis
  • CD3 Complex / biosynthesis
  • CD4-Positive T-Lymphocytes / cytology
  • Cell Proliferation
  • Coculture Techniques
  • Humans
  • Interleukin-2 / metabolism
  • Interleukin-2 Receptor alpha Subunit / biosynthesis
  • Leukocytes, Mononuclear / metabolism
  • Lymphocyte Subsets / drug effects*
  • Phenotype
  • Proteasome Inhibitors
  • Pyrazines / pharmacology*
  • T-Lymphocytes / drug effects*


  • Antineoplastic Agents
  • Boronic Acids
  • CD28 Antigens
  • CD3 Complex
  • Interleukin-2
  • Interleukin-2 Receptor alpha Subunit
  • Proteasome Inhibitors
  • Pyrazines
  • Bortezomib