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.

Figures

Figure 1.

(A) High doses of bortezomib (1000 nM) did not decrease viability among Treg cells (PKH-67+CD25+ population) or resting T cells (PKH-67−CD25− population) in either un-stimulated (control), MLC or αCD3/αCD28 stimulated CD4+ T-cell cultures. Conversely, percentages of viable activated T cells (PKH-67−CD25+ population) significantly decreased in a dose dependent manner in stimulated cultures. (B) Viability of isolated CD4+CD25− cells versus CD4+CD25+ cells after exposure to increasing concentrations of bortezomib.

Figure 2.

Phenotypic analysis of long-term cultured CD4+ T cells in the presence or in the absence of 500nM bortezomib. (A) Histograms representing the expression of CD25, FOXP3 and CD127; one of five similar experiments is shown. Mean fluorescence intensity ratio (MFIR) (MFI cells cultured with 500nM bortezomib/MFI cells cultured with 0nM bortezomib) were 4.9 for CD25, 1.4 for FoxP3 and 0.95 for CD127 expression. (B) Mean (SD) of FOXP3 mRNA level was 0.45 (0.17), 1.95 (1.1) and 28.5 (36.1) for bortezomib untreated and bortezomib treated long-term cultured T cells, and for freshly isolated CD4+CD25+ T cells, respectively.

Figure 3

CD4+ T-cell sub-populations obtained after long-term culture in the presence of bortezomib. (A) Immunophenotypic analysis allowed the identification of CD25−FOXP3−CD127+/− cells, CD25+FOXP3+CD127+/− cells and CD25−FOXP3−CD127−cells among freshly isolated or long-term cultured (bortezomib 0 or 500 nM) CD4+ T cells. One representative experiment of five is presented. (B) Percentages of each of the three subpopulations obtained after CD4+ T-cell long-term culture in the presence or in the absence of borteozmib 500 nM.

Figure 4.

Suppression assays: proliferation. (A) proliferation, monitored by PKH-67 dilution of control or αCD3/αCD28 stimulated responder T cells, co-incubated or not with cells long-term cultured in the presence or in the absence of 500 nM bortezomib (1:1 ratio); one of five similar experiments is shown. (B) Mean (SD) number of non-proliferating cells among responding T lymphocytes were 92 (18.3), 25 (8.4), 27.3 (7.3), and 68.7 (9.6) for (a) unstimulated T cells (negative control), (b) T cells stimulated with aCD3/aCD28 (positive control) and T cells incubated with αCD3/αCD28 plus T lymphocytes long-term cultured (c) without bortezomib or (d) with 500nM bortezomib. (C) Titration assays showing the suppressive capacity of T cells long-term cultured at 500nM bortezomib (bTreg) at increasing ratios of long-term cultured versus effector T cells. Naturally occurring CD4+CD25+ Treg as well as CD4+ T cells long-term cultured in the absence of bortezomib (shown as 0nM) were used as controls.

Figure 5.

Suppression assays: IFN-γ and CD40L intracytoplasmic production after co-culture of responder plus long-term cultured T cells; expression by both responder and long-term cultured T cells is shown.

Figure 6.

Analysis of long-term cultured CD4+CD25− T cells in the presence or in the absence of 500 nM bortezomib. (A) Dot plots representing the subpopulations obtained; one of five similar experiments is shown. (B) Box-plots showing cell percentages of the three subpopulations obtained: C D 2 5 − F O X P 3 − C D 1 2 7 +/−, CD25+FOXP3+/−CD127+/− and CD25+FOXP3+CD127−. (C) Suppression of proliferation of responder T cells by long-term cultured CD4+CD25− T cells. The mean (SD) number of non-proliferating cells among responding T lymphocytes was 98.4 (0.7), 13.4 (1.63), 17.9 (5.9), and 72 (9.3) for (a) un-stimulated T cells (negative control), (b) T cells stimulated with αCD3/αCD28 (positive control) and T cells incubated with αCD3/αCD28 plus (c) untreated versus (d) 500nM bortezomib-treated long-term cultured T lymphocytes.