Plastic Surgery Research Council
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PSRC 60th Annual Meeting

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The Dominant Role Of Intrathymic Clone Deletion In The Mixed Chimerism Related VCA Tolerance Induction
Wensheng Zhang, M.D., Ph.D., Yong Wang, M.D., Yang Yang, M.D., Ph.D, Yang Li, M.D., Ph.D., Kia M. Washington, M.D., Vijay S. Gorantla, M.D., Mario G. Solari, M.D., Xin Xiao Zheng, M.D..
University of Pittsburgh, Pittsburgh, PA, USA.

PURPOSE: Thymic clonal deletion that ensues after donor hematopoietic cell engraftment is the major mechanism for maintaining donor-specific tolerance. We have reliably achieved stable mixed chimerism (MC) and tolerance in a mouse hind-limb transplant model using a short-term immunosuppressive regimen, and have demonstrated that the deletion of alloreactive T cells occurs in the tolerant recipients. In this study, we provide further evidence of central intrathymic deletion in MC-mediated vascularized composite allograft (VCA) tolerance.
METHODS: B6 mice received hind-limb allografts from B10.A donors and were 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 B6 nude mice. CD4+CD25- T cells (5 x 10^6) 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 grafts from B10.A (donor specific), BALB/c (third party), and B6 (syngenic control) mice. MC, TCR Vβ expression profiles were analyzed by flow cytometric analysis.
RESULTS: 1) All treated recipients permanently accepted B10.A hind-limb allografts with persist multilineage donor hematopoietic MC (2-4%) for >120 days post transplantation. Tolerance was confirmed in vivo by acceptance of the donor skin grafts and rapid rejection of third party grafts. 2) Nude mice bearing tolerant thymus showed a prolonged engraftment for donor-specific B10.A skin grafts (n=4, MST: 64.5 days) with 50% surviving infinitely (>120 days), as compared with the naïve thymus-bearing recipients (n=4, MST: 11.5 days, p12 weeks).
CONCLUSION: Our findings demonstrate thymus and T cells from tolerant VCA recipients confer donor-specific immunity to nude and RAG1-/- mice with the deletion of donor-specific Vβ clones. It suggests that intrathymic deletional mechanisms play dominant role in the induction of VCA tolerance with stable mixed chimerism.


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