IFN-α did not interfere with priming or perpetuation of T cell anergy.

<p>(A) CD4⁺ T cells were cocultured with allogeneic mDC or IL-10 DC and incubated with concentrations of IFN-α as indicated. After 5 days, T cell proliferation was assessed by [³H] thymidine incorporation, results are depicted in absolute cpm +/− SD (left panel) and in percentages +/− SD (100% = T cell proliferation induced by mDC) (right panel). One representative experiment of 3 is shown. (B) CD4⁺ T cells primed with allogeneic mDC or IL-10 DC were restimulated with anti-CD3/anti-CD28 in the presence of increasing concentrations of IFN-α as indicated. T cell proliferation was detected after 72 hours of restimulation as described. Results are shown in absolute cpm +/− SD (left panel) and in percentages +/− SD (normalized to proliferation induced by mDC = 100%) (right panel). One representative experiment of 2 is demonstrated. n.s. not significant.</p

Immunophenotype of IL-10 DC after IFN-α treatment.

<p>(A, B) Immature DC were stimulated at day 5 of culture with a maturation cocktail (mDC) or a maturation cocktail supplemented with IL-10 either with or without 10⁴ U/mL IFN-α. (A) Dot plots of one representative experiment and (B) expression of surface molecules of DC of 6 independent experiments are depicted. (C) mDC and IL-10 DC and IL-10 DC +/−10⁴ U/mL IFN-α were generated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022763#s4" target="_blank">Materials and Methods</a>. At day 7, supernatants were collected and production of IL-12 p40 was assessed by ELISA. Mean values +/− standard deviation (SD) of 4 independent experiments are shown; nd: not detected. * p<0.05; ** p<0.01; *** p<0.001, n.s. not significant.</p

IL-10 DC generated in the presence of IFN-α induced an enhanced T cell activation.

<p>(A, B) CD4⁺ or CD8⁺ T cells were stimulated in primary culture with allogeneic mDC, IL-10 DC or IL-10 DC cultured with 10⁴ U/mL IFN-α. (A) T cell proliferation after restimulation was assessed as described and pooled data of 6 (CD4⁺ T cells) or 3 (CD8⁺ T cells) independent experiments are demonstrated. Proliferation is demonstrated in % +/−SD (normalized to 100% proliferation induced by control mDC). (B) IFN-γ and IL-10 production were detected in the supernatants of CD4⁺ T cells at day 5 of primary culture by ELISA. Mean values of cytokine levels in pg/ml +/− SD of 6 (IFN-γ) or 5 (IL-10) independent experiments are shown. * p<0.05; ** p<0.01; n.s. not significant.</p

IFN-α treatment of IL-10 DC abolished suppressor function of iTregs.

<p>(A, B) CD4⁺ or CD8⁺ T cells were cultured in primary culture with allogeneic mDC, IL-10 DC or IL-10 DC treated with 10⁴ U/mL IFN-α, respectively. Restimulation experiments were performed with anti-CD3/anti-CD28. T cell proliferation is depicted in % (normalized to 100% proliferation induced by mDC). Mean values +/− SD of 4 (CD4⁺) or 3 (CD8⁺ T cells) independent experiments are shown. (B) Supernatants were obtained after 72 h and analyzed for IFN-γ production by ELISA. One representative experiment is demonstrated for CD4⁺ and CD8⁺ T cells, respectively. (C) Suppressor assay: CD4⁺ T cells primed with allogeneic mDC (iTeff), IL-10 DC (iTregs) or IL-10 DC treated with 10⁴ U/mL IFN-α (iTregs + IFN-α), respectively, or freshly isolated CD4⁺CD25^high nTregs were cocultured with CD4⁺CD25^low effector T cells and stimulated by 0.5 µg/mL anti-CD3 in the presence of irradiated PBMC. T cell proliferation was determined by [³H] thymidine incorporation. Suppressor activity is shown in proliferation of Teff + cocultured T cell population/poliferation of Teff ×100 in % +/− SD. One representative experiment of 3 is demonstrated. Results are shown in absolute cpm +/− SD for triplicates. * p<0.05; ** p<0.01; *** p<0.001, n.s. not significant.</p

γH2AX staining of Treg, Th and CTL not treated (control) and treated with mafosfamide.

<p>A, Representative stainings 1 up to 24(1 µg/ml). Blue, nuclear staining with ToPro 3. Green, staining of γH2AX. B, Quantification for Treg, Th and CTL at different times after treatment. Mean intensities of γH2AX foci per cell of at least three independent experiments are pooled (standard error).</p

Effect of mafosfamide on the Th suppression activity of Treg.

<p>A, Th were co-incubated with the same amount of mafosfamide-pretreated Treg (1∶1), stimulated with antiCD3 antibody and allogenic high-dose irradiated PBMC, and thymidine incorporation was measured in the cell fraction. Treg were not treated or pre-treated with 0.5 and 1.5 µg/ml mafosfamide 24 h before co-cultivation with Th. Co-cultivation and stimulation occurred for 72 h, and thymidine was added to the medium 16 h before cell harvest. The thymidine incorporation in Treg was taken into account by subtracting the thymidine incorporation (cpm/cell) in mono-cultivated and stimulated Treg from the total population. Data were corrected for cytotoxicity i.e. the dead cell Treg fraction was not taken into account. Data are the mean of three independent experiments. B, Relative suppression activity of Treg. Data are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083384#pone-0083384-g004" target="_blank">Fig. 4B</a>. * p<0.05. We should note that in this assay the data were corrected as to cytotoxicity.</p

Cyctotoxicity of mafosfamide in Treg, Th and CTL.

<p>A, Time course of the induction of apoptosis, which was determined by subG1 quantification. Cells were treated with a dose of 0.5 µg/ml mafosfamide and cells were subjected to flow cytometry at the times indicated. B, Dose response of apoptosis induction in Treg, Th and CTL. The fraction of subG1 was determined 48 h after mafosfamide treatment. The asteriks label the measure points that show a significant difference to Th and CTL, except for the 1.5 µg/ml MAF level, for which significance was obtained only against Th. C, Amount of apoptosis (annexinV positive, PI negative fraction) and necrosis (annexinV positive, PI positive fraction), as determined by the annexinV/PI flow cytometry assay. Treg were treated with 0.5 µg/ml mafosfamide. D, Yield of apoptosis and necrosis in Treg, Th and CTL treated with 0.5 µg/ml. Cells were harvested 24 h after the onset of treatment. Data are the mean of at least three independent experiments.</p

Crosslink induction and repair in Treg, Th and CTL.

<p>A, Representative comets of cells not treated (control), mafosfamide (20 µg/ml) treated, gamma-ray treated (IR, 8 Gy) and IR plus mafosfamide treated. B, Quantification. ICL (%) was defined as decrease in tail moment as described in material and methods, 4 h after treatment. C, ICL (%) 24 h after treatment. D, Relative repair of ICL, which is given by the relative ICL level 24 h after treatment in relation to the level 4 h after treatment. Data are the mean of at least three independent experiments. * p<0.05; **p<0.01 significance.</p

Level of apoptosis and necrosis (determined by the annexinV/PI assay) in Treg, Th and CTL following treatment with different agents.

<p>A, apoptosis and necrosis following treatment with 50 µM (13,6 µg/ml) and 100 µM (27,3 µg/ml) nimustine (ACNU). Fixation occurred 72 h after treatment. B, apoptosis and necrosis following treatment with 100 µM (19,4 µg/ml), 200 µM (38,8 µg/ml) and 250 µM (48,5 µg/ml) temozolomide. Fixation occurred 72 h after treatment. C, apoptosis and necrosis following treatment with mitomycin C. Fixation occurred after 48 h. D, apoptosis (determined by subG1) induced by acrolein in Treg, Th and CTL after 48 h. The abscissa points to the dose of acrolein and, on the top, the equivalent dose of mafosfamide, which is in the range of 0–2 µg/ml (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083384#pone-0083384-g002" target="_blank">Fig. 2B</a>). Data are the mean of at least three independent experiments. The solvent (DMSO for temozolomide) and the aqueous controls were not toxic if given without the drug.</p

Mode of action of cyclophosphamide and mafosfamide.

<p>A, Decay into the active form, phosphoramide mustard. B, Binding to guanine and formation of guanine-guanine ICL. Structural formulas are drawn with Accelrys Draw 4.1 SP1 (Accelrys, Inc., San Diego, CA).</p