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Transplanted donor Treg cells can stably engraft leading to tolerance to allogeneic bone marrow cells in NOD mice

Cecilia Cabello², Jackeline Chirinos¹, Robert Levy¹, Thomas Malek¹, Allison L. Bayer^1,2

¹Department of Microbiology and Immunology; ²Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, United States

There is increasing interest in the use of naturally occurring CD4⁺Foxp3⁺ regulatory T cells (T_reg) in their application for adoptive immunotherapy in the treatment of autoimmune diseases, including type 1 diabetes (T1D). Studies have shown T_reg cells have therapeutic effects on the course of autoimmunity in NOD mouse, a model of human T1D. However, T_reg immunotherapy is hindered by the inability to generate sufficient cell number to inhibit the desired immune response(s) and achieve stable donor T_reg engraftment. In the present study, we examined the in vivo biological environment necessary to promote stable donor T_reg engraftment and induce tolerance to allogeneic (allo) BM cells. We describe that not only is peripheral space required, but competition from endogenous T_reg cells needs to be overcome for successful engraftment of transplanted donor T_reg cells together with IL-2, which is critically important for driving their proliferation and survival. We found that conditioning prediabetic NOD mice with varying amounts of total body irradiation resulted in a dose-dependent level of donor Thy1.1⁺ NOD T_reg engraftment. Furthermore, treatment with busulfan and cyclophosphamide (Bus+CyP) leads to significant engraftment of transplanted donor T_reg cells compared to unmanipulated mice or mice treated with Bus or CyP alone. Importantly, adoptive transfer of syngeneic or allo-T_reg cells together with short course of anti-IL-2 (clone JES6)/IL-2 complex and rapamycin, which favors T_reg expansion, induces tolerance to small numbers (10x10⁶) of allo-BM cells resulting in mixed chimerism and prevention of diabetes in these recipients. Importantly, donor T_reg cells were readily detected 48 weeks post-transfer. Collectively, our findings indicate that some level of manipulation of the recipient’s immune system greatly facilitated long-term donor T_reg cell engraftment and therapeutic efficacy. Thus, T_reg immunotherapy can lead to alloantigen tolerance even in NOD mice, which are highly resistant to tolerance induction (Supported by ADA Junior Faculty Award 7-09-JF-06 and Diabetes Research Institute Foundation)