Combinational Regimens for Antigen-specific Treg-based Immunotherapy for Autoimmune Diabetes and Transplantation Tolerance

Immune-based therapies for T1D need to focus on restoring self-tolerance without the need for chronic immunosuppression. CD4⁺Foxp3⁺ regulatory T-cells (T_regs) maintain immunological tolerance and can be harnessed to control autoimmunity and prevent transplant rejection. However, clinical trials with expanded polyclonal T_regs in T1D and solid-organ transplant patients demonstrate limitations of this therapy in the absence of recipient’s manipulation to promote T_reg engraftment and lack antigen-specific T_regs. Here, we studied a novel, non-ablative combinatorial immunomodulatory regimens to improve T_reg engraftment and therapeutic efficacy in NOD mice. We tested aCD3, cyclophosphamide (CyP), and IAC (IL-2/JES6-1 complex). aCD3 induced substantial T-cell depletion, including T_regs in early stages. But T_regs returned to normal within 7 days of 1^st dose, much faster than conventional T-cells, which recover around day 32-39 leading to increased T_reg:T_eff ratio consistent with earlier reports. Despite robust depletion, donor NOD T_regs failed to significantly engraft even with IAC, which expands T_regs in vivo. However, a single dose of CyP after aCD3, which depletes rebounding host T_regs resulted in a 43-fold increase in donor T_reg engraftment; IAC boosted several more fold. Importantly, infusion of islet-specific T_regs isolated from BDC2.5 TCR NOD mice following immunomodulation resulted in 100% diabetes remission and islet-specific T_regs trafficked to the pancreatic islets. In a skin allograft model, the combinational regimen with aCD3, CyP, and short-course rapamycin together with donor-specific C57BL/6 T_regs resulted in tolerance induction to C57BL/6 skin allografts, while 3^rd party C3H allografts were rejected. Collectively, we have identified novel therapeutic synergies between αCD3 and CyP, which together promote donor T_reg engraftment and therapeutic efficacy with antigen-specific T_regs in resetting the autoimmune b-cell destruction and inducing transplantation tolerance without the need for chronic immunosuppression.