Table 1.
GvHD clinical Score.
Figure 1.
BALB/c recipient mice were lethally irradiated (8,5 Gy) and received allogeneic C57BL/6 TCD-BM (5×106 cells) and Thy1.2+ T cells (5×105 cells, WT, black line, n = 17). A second group additionally received C57BL/6 Treg cells (5×105 cells, WT+WT Treg, broken line, n = 15). Survival was monitored for 100 days. Combined data from 3 independent experiments are shown. (*) indicates for P<0,05 according to a Mantel-Cox-Test.
Figure 2.
Treg cells communicate with allogeneic DC directly via cell-to-cell contact and GJIC dependent manner.
Bone marrow derived dendritic cells (BMDC from BALB/c mice, 3×104 per well) were cocultured with B6.SJL CD45.1+ T cells and C57BL/6 CD45.1− calcein-labelled pre Treg cells for 20 h in a 1∶1∶3 ratio. (A) Transfer of green fluorescent calcein after 20 h was analyzed by gating on CD45.1−CD11c+ DC, CD45.1−CD11c− Treg cells and CD45.1+CD11c− T cells, respectively. (B) Differences of median fluorescence intensity (MFI) among BMDC, CD4+ and CD8+ T cells was assessed. Where indicated Treg cells were preincubated with the gap junction inhibitor GAP27 (300 ng/ml). CFDA-SE (1 µM) labelled Treg cells were used as a control. (C) BALB/c BMDC were left untreated or cocultured with C57BL/6 pre Treg cells in an 1∶1 ratio in presence of soluble anti-CD3-mAb (3 µg/ml). Where indicated GAP27 (300 ng/ml) or rolipram (300 nM) were added. After 4 h suppressed DC (DC sup) were purified by CD11c specific MACS. Intracellular cAMP concentration was assessed by a specific ELISA after lysis of the cells. (D) Changes in cAMP levels after a 4 h coculture with BALB/c BMDC were mesured by flow cytometry in C57BL/6 preTreg. (*) indicates significant differences by Mann-Whitney U-test. The data shown are representative for 2 independent experiments in duplicate or triplicate wells.
Figure 3.
Treg cells induce a suppressive DC phenotype.
BALB/c BMDC were left untreated or stimulated with LPS (100 ng/ml) and cocultured with C57BL/6 Treg cells in an 1∶1 ratio (DC sup). For optimal Treg stimulation, a soluble anti-CD3-mAb (3 µg/ml) was added. After 4 h expression of CD80, B7-H1 and B7-DC was determined by flow cytometry gating on CD11c+ MHCII+ cells. All depicted results were assayed in triplicate wells and are representative of three independent experiments. (*) indicates significant differences by Mann-Whitney test; n.s. – no significant differences.
Figure 4.
Rolipram enhances the suppressive capacities of Treg cells in vitro.
(A, B) BALB/c splenic DC (3×104 per well) were cocultured with allogeneic C57BL/6 Thy1.2+ T cells (1×105 per well) with titrated amounts of the PDE4 inhibitor rolipram (100 to 1000 nM), PDE2 inhibitor BAY-60-7550 (100 to 1000 nM) or PDE3 inhibitor Cilostazol (1 µM to 10 µM) for 3 days. Cell proliferation was determined after 3 days by 3H-thymidine incorporation. (B) DC (3×104 per well) were cocultured with allogeneic C57BL/6 Thy1.2+ T cells (1×105 per well) as in (A), but C57BL/6 Treg cells (3×104 per well) were added. (C) DC (3×104 per well) were cocultured with allogeneic C57BL/6 Thy1.2+ T cells (1×105 per well) and C57BL/6 Treg cells in indicated ratios for 3 days. Where indicated PDE4 inhibitor rolipram (100 nM), PDE2 inhibitor BAY-60-7550 (300 nM) or PDE3 inhibitor (3 µM) was added and proliferation was assessed after 3 days by 3H-thymidine incorporation. All depicted results were assayed in triplicate wells and are representative of three independent experiments. (*) indicates significant differences by Mann-Whitney U-test; n.s. – no significant differences.
Figure 5.
Rolipram suppresses MLR directly by inhibiting T cell prolifation and indirectly by modulating DC/Treg interaction.
(A) BALB/c BMDC were cultured in absence or presence of C57BL/6 Treg cells (1∶1 ratio) and rolipram (300 nM) for 4 h. Anti-CD3 (3 µg/ml) was added to every well. After fixation cells were cocultured with C57BL/6 Thy1.2+ T cells in a 10∶1 ratio for 3 days. Proliferation was determined by 3H-thymidine incorporation. (B) BALB/c BMDC were stimulated with LPS (100 ng/ml) for 4 h or left untreated and subsequently fixed with glutaraldehyde. C57BL/6 Thy1.2+ CD25− T cells were cocultured with LPS stimulated or unstimulated fixed BMDC (1×104 per well) for 3 days in a 10∶1 ratio. All depicted results were assayed in triplicate wells and are representative of three independent experiments. (*) indicates significant differences by Mann-Whitney U-test; n.s. – no significant differences.
Figure 6.
Rolipram enhances suppressive capacities of Treg cells in vivo and ameliorates GvHD.
(A) BALB/c mice were lethally irradiated (8,5 Gy) and received either TCD bone marrow (5×106 cells) and Thy1.2+ CD25− T cells (5×105 cells) from C57BL/6 donors (n = 10, filled squares), Thy1.2+ CD25− T cells plus Treg cells (1∶1 ratio, n = 10, open squares), Thy1.2+ T cells plus rolipram (n = 10, filled circles) or Thy1.2+ T cells plus Treg cells and rolipram (n = 9, open circles). Rolipram (0.3 mg/kg) was injected i.p. on days 0 to 20 once per day. Results show the combined survival data from 2 independent experiments (* p<0.0005 by Mantel-Cox test). (B) Transplantation and rolipram treatment were performed as descibed above, but the mice were sacrificed on day 10. Representative skin sections from the back were stained with haematoxylin and eosin. The upper pictures are showing an overview (original magnification 200×), the lower pictures are showing a detailed view (original magnification 630×).