Engineered IL-2 Cytokine-Cytokine Receptor Complex Enables Selective Expansion of Regulatory T Cells and Facilitates Establishment of Organ Transplantation Tolerance

Abstract

Mixed hematopoietic cell chimerism induction is a promising approach to establish transplantation tolerance, though achievement of donor cell engraftment in patients given non-myeloablative conditioning remains challenging. Adoptive transfer of regulatory T cells (Tregs) has been shown to improve alloengraftment in animal models. However, ex vivo Treg expansion for the purpose of infusing large numbers is technically challenging. An innovative approach is to engineer an orthogonal IL-2 (oIL-2) cytokine-receptor pair that selectively interacts with each other and transmits native IL-2 signals, but does not interact with the natural cytokine or receptor counterparts, thereby enabling selective stimulation of target cells in vivo. Here, we introduced this ortho cytokine-receptor combination into Treg with the goal of improving donor hematopoietic cell engraftment upon adoptive transfer in a murine mixed chimerism model to induce tolerance to a transplanted organ allograft. CD4⁺CD25^hiFoxp3^GFP+cells were isolated from Foxp3^GFP-BALB/c mice. Purified Tregs were preactivated and transduced with the construct for murine oIL-2 receptor β chain (oIL-2Rβ) through a retroviral vector. Transduction efficiency was approximately 40%. Transduced oIL-2Rβ⁺Tregs (oTreg) were injected at 1 × 10⁶ /mouse together with 15 × 10⁶ bone marrow cells (BMCs) that were obtained from luciferase transgenic C57BL/6 donor mice into WT BALB/c recipient mice that received 3.3-Gy total body irradiation and anti-CD40L mAb (0.3 mg, ip on d0) as preconditioning. Recipients were treated with PBS, WT IL-2, or oIL-2 (both 25K IU/day) for 14 days. Without oTreg transfer, donor cells were rejected by d10 as measured by bioluminescence. Improvement of engraftment by oTreg transfer was not statistically significant without IL-2 injection (P = 0.13 compared to BMC + PBS group on d30). WT IL-2 injection increased CD8⁺T cells and resulted in rapid graft rejection. On the other hand, oIL-2 injection significantly increased %Foxp3^GFP+ in CD4⁺T cells (PBS; 4.3±1.6%, oIL-2; 9.3±4.7%, each n=10, P=0.007) without increasing CD8⁺T cells. Consequently, donor cell engraftment was significantly improved in this group (P<0.0001 compared to BMC + PBS group on d30). Those recipients accepted heart allografts that were transplanted from C57BL/6 donor mice after chimerism induction, indicating the establishment of donor specific tolerance. Because ortho IL-2 did not have an impact when Tregs were transferred without oIL-2Rβ transduction, we concluded that specific interactions between oIL-2 and oIL-2Rβ resulted in improved acceptance of the heart allografts. Our data indicates that selective IL-2 stimulation improves Treg potential for facilitation of engraftment and for the induction of transplantation tolerance. Cytokine receptor engineering could be a powerful option to manipulate target cells such as Treg in vivo.