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


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.
Blanco
Belén
B
Servicio de Hematología y CIC Salamanca, Hospital Universitario de Salamanca and Centro de Investigación del Cáncer Salamanca, Centro en Red de Medicina Regenerativa y Terapia celular de Castilla y León, Spain.
Pérez-Simón
José A
JA
Sánchez-Abarca
Luis I
LI
Caballero-Velazquez
Teresa
T
Gutierrez-Cossío
Silvia
S
Hernández-Campo
Pilar
P
Díez-Campelo
María
M
Herrero-Sanchez
Carmen
C
Rodriguez-Serrano
Concepción
C
Santamaría
Carlos
C
Sánchez-Guijo
Fermín M
FM
Del Cañizo
Consuelo
C
San Miguel
Jesús F
JF
D000970:Antineoplastic Agents / Q000494:pharmacology; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D018106:CD28 Antigens / Q000096:biosynthesis; D017252:CD3 Complex / Q000096:biosynthesis; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D049109:Cell Proliferation; D018920:Coculture Techniques; D006801:Humans; D007376:Interleukin-2 / Q000378:metabolism; D053645:Interleukin-2 Receptor alpha Subunit / Q000096:biosynthesis; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D016131:Lymphocyte Subsets / Q000187:drug effects*; D010641:Phenotype; D061988:Proteasome Inhibitors; D011719:Pyrazines / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000187:drug effects*
D000970:Antineoplastic Agents; D001897:Boronic Acids; D018106:CD28 Antigens; D017252:CD3 Complex; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D061988:Proteasome Inhibitors; D011719:Pyrazines; D000069286:Bortezomib