Optimal immune-based therapies for type 1 diabetes (T1D) should restore self-tolerance without inducing chronic immunosuppression. CD4+Foxp3+ regulatory T cells (Tregs) are a key cell population capable of facilitating durable immune tolerance. However, clinical trials with expanded Tregs in T1D and solid-organ transplant recipients are limited by poor Treg engraftment without host manipulation. We showed that Treg engraftment and therapeutic benefit in nonautoimmune models required ablative host conditioning. Here, we evaluated Treg engraftment and therapeutic efficacy in the nonobese diabetic (NOD) mouse model of autoimmune diabetes using nonablative, combinatorial regimens involving the anti-CD3 (αCD3), cyclophosphamide (CyP), and IAC (IL-2/JES6–1) antibody complex. We demonstrate that αCD3 alone induced substantial T-cell depletion, impacting both conventional T cells (Tconv) and Tregs, subsequently followed by more rapid rebound of Tregs. Despite robust depletion of host Tconv and host Tregs, donor Tregs failed to engraft even with interleukin-2 (IL-2) support. A single dose of CyP after αCD3 depleted rebounding host Tregs and resulted in a 43-fold increase in donor Treg engraftment, yet polyclonal donor Tregs failed to reverse diabetes. However, infusion of autoantigen-specific Tregs after αCD3 alone resulted in robust Treg engraftment within the islets and induced remission in all mice. This novel combinatorial therapy promotes engraftment of autoantigen-specific donor Tregs and controls islet autoimmunity without long-term immunosuppression.

CD4þ Foxp3 þ T regulatory cells (T regs ) are essential for maintaining immunological tolerance, which could be harnessed for novel cell-based therapies to prevent allograft rejection and control autoimmunity. However, the use of T regs for therapy is hindered by the inability to generate sufficient cell numbers to inhibit desired immune response(s) and achieve stable engraftment of the donor-T reg cell inoculums. The present study was undertaken to investigate the in vivo requirements to promote engraftment of adoptively transferred T regs and induce tolerance. We established that not only is peripheral space required, but competition from endogenous T regs must be minimized for successful donor-T reg engraftment with IL-2 critical for driving their proliferation and survival. Moreover, these studies revealed a critical level of donor-T reg engraftment was required for tolerance induction to skin transplants. These mouse studies lay the foundation for development of novel T reg approaches for tolerance induction in the clinic involving not only organ or cellular transplantation, but also to re-establish self-tolerance in autoimmune settings.

The cytokine interleukin-2 (IL-2) is critical for the functions of regulatory T cells (T). The contribution of polymorphisms in the gene encoding the IL-2 receptor α subunit (), which are associated with type 1 diabetes, is difficult to determine because autoimmunity depends on variations in multiple genes, where the contribution of any one gene product is small. We investigated the mechanisms whereby a modest reduction in IL-2R signaling selectively in T lymphocytes influenced the development of diabetes in the NOD mouse model. The sensitivity of IL-2R signaling was reduced by about two- to threefold in T from mice that coexpressed wild-type IL-2Rβ and a mutant subunit (IL-2Rβ) with reduced signaling (designated NOD-Y3). Male and female NOD-Y3 mice exhibited accelerated diabetes onset due to intrinsic effects on multiple activities in T Bone marrow chimera and adoptive transfer experiments demonstrated that IL-2Rβ T resulted in impaired homeostasis of lymphoid-residing central T and inefficient development of highly activated effector T and that they were less suppressive. Pancreatic IL-2Rβ T showed impaired development into IL-10-secreting effector T The pancreatic lymph nodes and pancreases of NOD-Y3 mice had increased numbers of antigen-experienced CD4 effector T cells, which was largely due to impaired T, because adoptively transferred pancreatic autoantigen-specific CD4 Foxp3 T cells from NOD-Y3 mice did not accelerate diabetes in NOD.SCID recipients. Our study indicates that the primary defect associated with chronic, mildly reduced IL-2R signaling is due to impaired T that cannot effectively produce and maintain highly functional tissue-seeking effector T subsets.

There is a clear need to develop strategies to induce tolerance without the need of chronic immunosuppression in transplant recipient and in patients with autoimmunity. Adoptive T regulatory cell (Treg) therapy offers the potential of long-lasting protection. However, based on results of clinical trials so far with ex vivo expanded autologous Tregs in type 1 diabetic (T1D) patients, it seems unlikely that single immunotherapy with Treg infusion without immunomodulation regimens that promote stable donor Treg engraftment and persistence would afford truly significant clinical benefit. Combination therapies could provide improved outcomes with consideration of the fundamental factors required for Treg generation, homeostasis, and function to promote long-term donor Treg persistence to provoke beneficial therapeutic outcomes.

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