Introduction: We have achieved stable mixed chimerism (MC) and tolerance in mice hind-limb transplant model using a non-myeloabative conditioning regimen, and demonstrated that the deletion of specific Vbeta-bearing T cells occurs in the periphery of mixed chimeras. In this study, we sought further evidence of intrathymic deletion and to determine its role in the MC-mediated allograft tolerance.

Methods: B6 mice received hind-limb allografts from B10.A donors and treated with rapamycin, CTLA4/Fc and anti-CD40L mAb. 120 day after transplantation, thymus lobes from the tolerant mice were isolated and transplanted into the subrenal capsule of the 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. Recipient nude or RAG1-/- mice were further challenged with skin allografts from B10.A (donor specific), BALB/c (third party), and B6 (syngenic control) mice. MC, TCR Vbeta expression were analyzed by FACS.

Results: 1)All recipients permanently accepted B10.A limb allografts with persist multilineage donor hematopoietic MC (2-4%) for >120 days post transplantation. Tolerance was confirmed by acceptance of the donor skin grafts and rejection of third party grafts. 2)B6 nude mice bearing tolerant thymus showed a prolonged engraftment for B10.A skin grafts (n=4, MST: 64.5 days) with 50% of them survived infinitely (>120 days), as compared with the 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 B10.A grafts. 4) In both tolerant thymus-bearing nude mice and tolerant CD4+CD25- T cells transfused RAG-/- mice, partial deletion of VΒ5+ and VΒ11+ CD4 T cells was observed 2 weeks after skin transplantation and sustained throughout the follow-up period (>12 weeks).

Conclusion: Thymus and T cells from tolerant chimeras conferring donor-specific immunity to nude and RAG1 deficient mice with the deletion of donor-specific Vbeta clones suggest that the dominant role of intrathymic deletional mechanisms in allograft tolerance with stable mixed chimerism.

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