Sites-Specific Nanofiber-Coated Regulatory T Cells for the Induction of Tolerance in Vascularized Composite Allotransplants

Specific Aim 1: To determine whether nanofiber coated Tregs preferentially home to sites of inflammation. This aim is to test our hypothesis that nanofibers coated Tregs significantly enhance capacity to migrate to the sites of inflammation compared to uncoated Tregs, and that the ability to target Tregs to sites of inflammation may significantly reduce the number of Tregs required for sustained therapeutic effect. The mouse hind-limb transplants will be infused intravenously with autologous ex-vivo expanded and VCAM1b-PA-coated Tregs at various doses (times or number of Tregs), frequencies of Tregs in the transplanted limb and contralateral limb, draining lymph nodes and spleen will be determined and compared with non-coated Tregs (Aim 1.1) at earlier post transplant (d1-5). We will also test whether Tregs coated with either ICAM1 binding (ICAM1b-PA) or selectins-binding (SLb-PA) or a mixture of VCAM1b-PAs, ICAM1b-PA and SLb-PA are more effective in driving Tregs to the target sites (Aim 1.2). The infused Tregs will be tracked by using congenic mice (CD45.1 vs CD45.2), or Foxp3-EGFP transgenic or fluoresce-labeled nanofiber PAs. Frequency of Tregs recruited to the injury site will be correlated with numbers of graft infiltrating cells and tissue injury such as edema, necrosis to determine the therapeutic effects of infused Tregs. Specific Aim 2: To evaluate the functionality and efficacy of the PA-nanofiber coated Tregs in promoting peripheral nerve repair or regeneration. Recent studies have shown that Tregs promote regeneration of both central and peripheral nerves in various murine models of nerve injury and neurological disorders deceases (14, 15). We hypothesize that increased recruitment of Tregs accelerate recovery of neurological function. Mice with syngeneic hind-limb transplantation will be treated with an intravenous infusion of the optimized number of PA coated and uncoated Tregs, and will be monitored for >100 day for longitudinal evaluation will be performed to evaluate sensory and locomotor function by The Hargreaves test thermal pain sensation and DigiGait test of locomotor function, respectively, as previously described (17). In addition, to determine whether Tregs promote nerve regeneration repair, both femoral and sciatic nerves will be isolated from a group of limb transplants at post-operative days 5-14. Their nerves will be harvested and immunolabelled to observe myelin with myelin protein zero (MPZ), macrophage infiltration with cluster differentiation 68 (CD68), and axons with neurofilament (NF) (18, 19). Specific Aim 3: Determine whether PA-nanofiber coated regulatory T cells mitigate hind-limb allograft rejection and promote tolerance induction. We will test the homing capacity and tolerogenic function of the PA-nanofiber coated Tregs in our allogeneic limb-transplant model. The approach under this aim will be two-fold: 1) determine whether/which/when the nanofiber-PA coated Tregs preferentially traffic to allograft and whether/which nanofiber-PA coated Tregs significantly prevents acute allograft rejection and (Aim 3.1), and to test whether coated Tregs are more effective in synergy with currently used immunosuppression (IS) to facilitate transplant tolerance induction (Aim 3.2) with the goal of developing tolerogenic protocols for the injection of coated Tregs which will lead to controlled immunosuppressant drug minimization after limb transplantation. We will analyze the mechanisms of alloimmune reactivity and immune tolerance. Specifically, we will monitor the homing of Tregs into the VCA graft using