Purpose: Induction of donor-specific tolerance in composite tissue allografts (CTA) remains one of the most challenging goals in transplant surgery. Recently we have developed a protocol (abTCR mAb/ CsA) for tolerance induction in the rat hind limb transplantation model (LBN; RT1^n+l;F1® Lewis; LEW; RT1^l). In this study we present tolerance induction in the fully MHC mismatched allograft recipients under 7 days, combined (abTCR/CSA protocol. Methods: Thirty transplantations across strong MHC barrier were performed between Brown-Norway donors (RT1ⁿ) and LEW recipients. Isograft and allograft rejection control groups received no treatment. Experimental groups received either rat abTCR or CsA or combination of abTCR and CsA at the day of transplantation and for 7 days thereafter. The efficacy of immunosuppressive treatment and chimerism were monitored by flow cytometry (FC). Donor specific tolerance and immunocompetence of the limb recipients were determined in vivo by secondary skin graft from the recipient (LEW), the donor (BN) and the third party (ACI) graft. Mixed lymphocyte reaction (MLR) was performed for the assessment of donor specific tolerance in vitro. Results: Only fully mismatched allograft recipients under abTCR/CsA protocol survived indefinite (over 250 days). Three color FC analysis at day 120 post-transplant demonstrated stable, multilineage, donor specific chimerism in the periphery of the tolerated recipients (CD4+^PE/RT1^n+APC 7.6% and 1.3% of CD8+^PE/RT1^n+APC positive T cell subpopulations and CD45RA+^PE/RT1^n+APC 16.5% B cell population). Donor specific tolerance and immunocompetence in vivo was confirmed by acceptance of the secondary skin graft from the donor and rejection of the third-party (ACI) grafts. MLR revealed no reactivity to host (LEW) and donor (BN) antigens but strong reactivity to the third- party (ACI) alloantigens. Conclusions: To the best our knowledge, for the first time donor specific tolerance was induced under 7 days protocol of abTCR/CsA therapy in the fully mismatched limb allograft transplanrs. Stable mixed chimerism was achieved without the need for the myeloablative bone marrow modification of the recipients and without the need for chronic immunosuppression. This protocol may have direct impact on transplantation of composite tissue allografts in the clinical scenario.
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