Therapeutic Use of Regulatory T Cells for Tolerance Induction: Mechanisms and Specificity in a Murine Mixed Chimerism Model

Background: Mixed chimerism is an effective strategy for the induction of transplantation tolerance but widespread clinical application is impeded by the toxicities of current bone marrow (BM) transplantation (BMT) protocols. We combined the therapeutic use of regulatory T cells (Tregs) with the mixed chimerism approach to achieve a protocol that is both effective and safe. Previous data demonstrate that Treg therapy allows engraftment of conventional doses of fully mismatched BM without the need for irradiation, leading to robust skin and humoral tolerance, preventing acute and chronic rejection. Here we investigated regulatory mechanisms and specificity of Tregs in this novel Treg-chimerism protocol. Methods: B6 recipients received 20×106 fully mismatched Balb/c BM cells, under costimulation blockade and short-course rapamycin together with 0.5-4×106 polyclonal WT Tregs from either recipient, donor or third-party strains. Multilineage chimerism was followed by flow-cytometric analysis, tolerance was assessed by heart and skingrafts and analysis of anti-donor Abs. Persistence of Tregs was assessed by PCR in lymphoid organs. Regulatory mechanisms were investigated by analysis of donor-reactive T cells and MLRs. Results: Therapeutic application of as few as 0.5×106 (25x106/kg) polyclonal recipient Tregs reliably induces mixed chimerism and tolerance in a non-cytotoxic BMT model. MLR data and analysis of donor-reactive T cells revealed donor-specific tolerance in the absence of peripheral deletion early after BMT, which was also confirmed by early skingrafts. Treg treated chimeras lack anti-donor antibodies and absence of chronic rejection was confirmed by histologic analysis of heart allografts. Donor heats remained functional for the follow up of >100d. Pathohistologic examination showed no signs of cardiac allograft vasculopathy. Hearts were devoid of interstitial fibrosis, vasculitis and myointimal thickening. Donor-specific tolerance was robust and sustained despite repeated challenge with secondary donor and third-party grafts. PCR analysis revealed preserved homing abilities and limited life-span of transferred Tregs. Tregs home to lymph nodes and spleen, but not to thymus or BM early after transplantation (7 days), no transferred Tregs could be detected in lymphoid organs or skingrafts late after BMT (30 weeks). Tregs derived from donor or third-party strains led to transient chimerism in some mice, however, longterm donor-specific tolerance could not be achieved. Conclusions: Therapeutic Treg treatment promotes engraftment of allogeneic BM and reliably induces mixed chimerism with clinical relevant Treg numbers. Although central tolerance is established, peripheral deletion progresses only slowly. Regulatory mechanisms are sufficient to maintain donor-specific tolerance before peripheral deletion takes place and seem to be more important than in BMT protocols relying on myelosuppression. Transferred Tregs are suggested to be short-lived, suggesting an infectious-like tolerance mechanism in Treg treated chimeras. Recipient derived Tregs seem to be superior over donor or third-party Tregs in the promotion of BM engraftment. Due to positive selection for recipient MHC, recipient Tregs might be superior over donor and third-party Tregs in being activated by the indirect pathway of antigen presentation.