Aim: Using murine models, we sought to identify the mechanisms of responsible for vascularized composite allotransplant (VCA) rejection, and to thereafter, using clinically relevant peri-transplant strategies that do not require maintenance immunosuppression, seek to achieve long-term allograft survival.

Methods: Using a heterotopic murine hindlimb VCA model (BALB/c->C57BL/6) and 6-10 allografts/group for screening, (i) we tested the ability of various CD40/CD40L and/or CD28/B7 costimulation blockade-based strategies to promote VCA survival, and (ii) thereafter analyzed how the most effective of these strategies altered the key pathogenic mechanisms underlying VCA rejection.

Results: Whereas control untreated VCA recipients rejected their grafts by 10 days (with 50% rejected by 7 days), surprisingly, given published efficacy in skin allograft models, recipients treated with 6 doses of MR-1 mAb, directed against CD40L (CD154), plus murine CTLA4Ig, using 500 μg of each, qod from transplantation, resulted in only a doubling of allograft survival (p<0.05). However, VCA recipients treated with MR-1 (CD154) mAb (200 μg) plus donor splenocyte transfusion (DST, 5 x 106 cells, i.v.) at the time of transplantation, had a tripling of allograft survival (p<0.01), and addition of 2 weeks of rapamycin (RPM, 2 mg/kg/d, i.p.) to the CD40L/DST protocol led to long-term VCA survival (>100 days). qPCR, histologic and flow cytometric studies at 3, 5 or 7 days post-Tx showed that, compared to untreated recipients, CD40L/DST-based therapy suppressed intragraft expression of IL-2 and IFN-g cytokines, CXCR3 and its chemokine ligands, and infiltration by CD4 and CD8 T cells, whereas circulating immune cells were largely unaffected. In addition, we noted that optimal effects were associated with preservation of infiltrating Foxp3+ Treg cells, and with the presence of donor VCA bone and/or bone marrow components.

Conclusion: Potent beneficial effects on VCA survival are seen using CD40L/DST/RPM in the early post-Tx period. While studies of donor-specific allograft tolerance induction, and both the role of Tregs and requirement for donor bone for optimal efficacy are ongoing, it is clear that this overall approach leads to permanent VCA survival with excellent preservation of bone, muscle and skin histology long-term, showing that limb engraftment may be achieved without maintenance immunosuppression in murine models.

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