Introduction: We previously demonstrated stable mixed chimerism (MC) and tolerance in a murine hind-limb vascularized composite allotransplant (VCA) model using a non-myeloabative conditioning regimen, and established deletion of alloreactive T cells in tolerant recipients. In this study, we sought to further delineate the role for central intrathymic deletion in MC-mediated tolerance to hind-limb VCA.
Methods: B6 mice received hind-limb VCA from B10.A donors and treated with rapamycin, CTLA4/Fc and anti-CD40L mAb.  120 days after transplantation, thymic lobes from the tolerant recipients were isolated and secondarily transplanted into the sub-renal capsule of B6 nude mice. CD4+CD25- T cells (5X10^6) sorted from the splenocytes of same tolerant mice were i.v. injected to the B6.RAG1-/- mice. Naïve B6 thymus or CD4+CD25- T cells were used as controls in thymus transplantation and adoptive transfer. The recipient nude or Rag1-/- mice underwent skin graft challenge from B10.A (donor specific), BALB/c (third party), and B6 (syngenic control) mice. MC, TCR Vbeta expression profiles were analyzed by FACS.
Results: 1) All treated recipients permanently accepted B10.A hind-limb VCA with stable and robust multilineage donor hematopoietic MC (2-4%) for >120 days post-transplantation. Tolerance was confirmed in vivo by acceptance of donor skin grafts and rapid rejection of third party grafts. 2) Nude mice receiving tolerant thymic transplants showed prolonged engraftment of donor-specific B10.A skin grafts (n=4, MST: 64.5 days) with 50% of them surviving infinitely (>120 days), as compared with naïve thymus-bearing recipients (n=4, MST: 11.5 days, p<0.05). All nude recipients rejected BALB/c and accepted B6 skin grafts.  3) RAG1-/- mice transfused with tolerant CD4+CD25- T cells readily rejected BALB/c skin grafts (n=4, MST: 10 days), while accepting the B10.A grafts. 4) In both tolerant thymus-bearing nude mice and tolerant CD4+CD25- T cell-transfused RAG-/- mice, partial deletion of Vβ5+ and Vβ11+ CD4 T cells was observed as early as 2 weeks after skin transplantation and sustained throughout the follow-up period (>12 weeks).
Conclusion: We provide first evidence in a limb VCA model that the thymus plays an important role in stable mixed chimerism mediated allograft tolerance through a mechanism of intrathymic central deletion of allo-reactive T cell populations.