Mechanisms of cyclic nucleotide phosphodiesterases in modulating T cell responses in murine graft-versus-host disease

Abstract

Graft-versus-host disease (GvHD) is a key contributor to the morbidity and mortality after allogeneic hematopoetic stem cell transplantation (HSCT). Regulatory Foxp3(+) CD4(+) T cells (Treg) suppress conventional T cell activation and can control GvHD. In our previous work, we demonstrate that a basic mechanism of Treg mediated suppression occurs by the transfer of cyclic adenosine monophosphate (cAMP) to responder cells. Whether this mechanism is relevant for Treg mediated suppression of GvHD is currently unknown. To address this question, bone marrow and T cells from C57BL/6 mice were transferred into lethally irradiated BALB/c recipients, and the course of GvHD and survival were monitored. Transplanted recipients developed severe GvHD that was strongly ameliorated by the transfer of donor Treg cells. Towards the underlying mechanisms, in vitro studies revealed that Treg communicated with DCs via gap junctions, resulting in functional inactivation of DC by a metabolic pathway involving cAMP that is modulated by the phosphodiesterase (PDE) 4 inhibitor rolipram. PDE2 or PDE3 inhibitors as well as rolipram suppressed allogeneic T cell activation, indirectly by enhancing Treg mediated suppression of DC activation and directly by inhibiting responder T cell proliferation. In line with this, we observed a cooperative suppression of GvHD upon Treg transfer and additional rolipram treatment. In conclusion, we propose that an important pathway of Treg mediated control of GvHD is based on a cAMP dependent mechanism. These data provide the basis for future concepts to manipulate allogeneic T cell responses to prevent GvHD.

Figure 1. T_reg cells suppress GvHD.

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 T_reg cells (5×10⁵ cells, WT+WT T_reg, 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. T_reg 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 T_reg 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⁻ T_reg 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 T_reg cells were preincubated with the gap junction inhibitor GAP27 (300 ng/ml). CFDA-SE (1 µM) labelled T_reg cells were used as a control. (C) BALB/c BMDC were left untreated or cocultured with C57BL/6 pre T_reg 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 preT_reg. (*) indicates significant differences by Mann-Whitney U-test. The data shown are representative for 2 independent experiments in duplicate or triplicate wells.

Figure 3. T_reg cells induce a suppressive DC phenotype.

BALB/c BMDC were left untreated or stimulated with LPS (100 ng/ml) and cocultured with C57BL/6 T_reg cells in an 1∶1 ratio (DC _sup). For optimal T_reg 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 T_reg 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 T_reg 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 T_reg 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/T_reg interaction.

(A) BALB/c BMDC were cultured in absence or presence of C57BL/6 T_reg 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 T_reg 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 T_reg cells (1∶1 ratio, n = 10, open squares), Thy1.2⁺ T cells plus rolipram (n = 10, filled circles) or Thy1.2⁺ T cells plus T_reg 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×).