Contributions
Abstract: S253
Type: Oral Presentation
Session title: Cellular immunotherapy and gene therapy - Experimental
Background
The use of regulatory T cells (Treg) has a positive therapeutic potential to ameliorate acute graft-versus-host disease (GvHD), which represents one of the major complications after allogeneic bone marrow transplantation (allo-BMT). One of the remaining obstacles in the clinical practice of Treg administration is the high dose of Treg cells required to prevent GvHD and the difficulty in expanding these cells ex-vivo.
Aims
In this study, we engineered murine Treg cells to express an altered IL-2 receptor β subunit that selectively interacts with an orthogonal mutant IL-2 (oIL2) without interacting with the natural cytokine or receptor counterparts, thus selectively expand engineered Treg and prevent acute -GvHD.
Methods
CD4+CD25hiFoxp3GFPcells were purified from C57Bl/6 FoxP3DRT-GFP-luc+ mice and transduced with the construct for murine oIL-2 receptor β chain (oTregs) through a retroviral vector. BALB/c mice were lethally irradiated (8.8 Gy) and transplanted with 5x106 T cell-depleted bone marrow cells (TCD-BM) from C57Bl/6 mice alone or together with fresh 1x106 C57Bl/6 FoxP3DRT-GFP-luc+ Treg cells (fTreg) or with oTreg (0.2x106 and 1x106) on day 0. On day 2, C57Bl/6 Tcon cells (1x106) were injected to induce GvHD. Recipients were treated with PBS or oIL-2 (25K IU/day) for 14 days and then 3 times a week for another 14 days.
Results
By bioluminescence assays, we observed a significant expansion of oTreg (luc+) cells treated with oIL2 at day 7 post-transplant. The expansion of oTreg with PBS and fTreg was observed on day 14 after allo-BMT. The administration of oTreg-oIL2 and fTreg (1:1 Tcon:Treg ratio) significantly increased the survival of the mice compared to Tcon group (P<0.0001 and P=0.0001, respectively). Further, the group oTreg-oIL2 (1:1) showed a significant increase in the survival curve compared to the fTreg group (P=0.03). When decreasing the number of oTreg administrated (5:1 Tcon:Treg ratio) a significant increase in the survival (Tcon vs Tcon:oTreg (5:1)+oIL2; P<0.0001) in the oIL2 group. Similar survival curves were observed between this group and fTreg group (1:1). oIL-2 injection significantly increased the percentage of Foxp3GFP+ in CD4+ T cells (PBS;14.96±2.4%, oIL-2; 27.76±23.6%, P=0.02) in the peripheral blood (PB) after 7 days post-transplant. oTreg mice group showed a decrease in the percentage of effector T cells after 7 days compared to Tcon group. However, the oIL2 group showed a significant decrease of ICOS+CD8+ cells compared to the PBS group in the PB and spleen (P=0.04 and P=0.006, respectively).
Conclusion
The administration of a reduced number of oTreg and the selective IL-2 stimulation in vivo early after transplant showed a capacity to ameliorate GvHD, representing a novel approach to the utilization of Treg in allo-BMT.
Keyword(s): Acute graft-versus-host disease, Allogeneic hematopoietic stem cell transplant, Animal model, Cellular therapy
Abstract: S253
Type: Oral Presentation
Session title: Cellular immunotherapy and gene therapy - Experimental
Background
The use of regulatory T cells (Treg) has a positive therapeutic potential to ameliorate acute graft-versus-host disease (GvHD), which represents one of the major complications after allogeneic bone marrow transplantation (allo-BMT). One of the remaining obstacles in the clinical practice of Treg administration is the high dose of Treg cells required to prevent GvHD and the difficulty in expanding these cells ex-vivo.
Aims
In this study, we engineered murine Treg cells to express an altered IL-2 receptor β subunit that selectively interacts with an orthogonal mutant IL-2 (oIL2) without interacting with the natural cytokine or receptor counterparts, thus selectively expand engineered Treg and prevent acute -GvHD.
Methods
CD4+CD25hiFoxp3GFPcells were purified from C57Bl/6 FoxP3DRT-GFP-luc+ mice and transduced with the construct for murine oIL-2 receptor β chain (oTregs) through a retroviral vector. BALB/c mice were lethally irradiated (8.8 Gy) and transplanted with 5x106 T cell-depleted bone marrow cells (TCD-BM) from C57Bl/6 mice alone or together with fresh 1x106 C57Bl/6 FoxP3DRT-GFP-luc+ Treg cells (fTreg) or with oTreg (0.2x106 and 1x106) on day 0. On day 2, C57Bl/6 Tcon cells (1x106) were injected to induce GvHD. Recipients were treated with PBS or oIL-2 (25K IU/day) for 14 days and then 3 times a week for another 14 days.
Results
By bioluminescence assays, we observed a significant expansion of oTreg (luc+) cells treated with oIL2 at day 7 post-transplant. The expansion of oTreg with PBS and fTreg was observed on day 14 after allo-BMT. The administration of oTreg-oIL2 and fTreg (1:1 Tcon:Treg ratio) significantly increased the survival of the mice compared to Tcon group (P<0.0001 and P=0.0001, respectively). Further, the group oTreg-oIL2 (1:1) showed a significant increase in the survival curve compared to the fTreg group (P=0.03). When decreasing the number of oTreg administrated (5:1 Tcon:Treg ratio) a significant increase in the survival (Tcon vs Tcon:oTreg (5:1)+oIL2; P<0.0001) in the oIL2 group. Similar survival curves were observed between this group and fTreg group (1:1). oIL-2 injection significantly increased the percentage of Foxp3GFP+ in CD4+ T cells (PBS;14.96±2.4%, oIL-2; 27.76±23.6%, P=0.02) in the peripheral blood (PB) after 7 days post-transplant. oTreg mice group showed a decrease in the percentage of effector T cells after 7 days compared to Tcon group. However, the oIL2 group showed a significant decrease of ICOS+CD8+ cells compared to the PBS group in the PB and spleen (P=0.04 and P=0.006, respectively).
Conclusion
The administration of a reduced number of oTreg and the selective IL-2 stimulation in vivo early after transplant showed a capacity to ameliorate GvHD, representing a novel approach to the utilization of Treg in allo-BMT.
Keyword(s): Acute graft-versus-host disease, Allogeneic hematopoietic stem cell transplant, Animal model, Cellular therapy