[Background] Reconstructive transplantation represents a valid therapeutic option after devastating tissue loss such as an extremity or face. Immunosuppression-free donor-specific immunological tolerance has been successfully achieved in the setting of solid organ transplantation through mixed chimerism. Routine clinical application of this approach, however, is hampered by the toxicity of the cytoreductive recipient conditioning. The current study investigated a novel non-cytoreductive immunosuppressive strategy in a murine model of hind limb transplantation.

[Methods] Fully MHC-mismatched allogeneic, orthotopic hind limb transplants were performed from Balb/C to C57BL/6 mice. Recipient animals in the experimental groups received no treatment (Group 1); 0.25mg CTLA4 Ig on postoperative days (POD) 0, and 0.25mg on POD 2, 4, and 6 (Group 2); 20mg/kg anti-T cells (anti-Thy 1.2 mAb) on POD-1 plus CTLA4-Ig and 1mg/kg Rapamycin (POD0-9) (Group 3). Flow cytometric analysis was performed to evaluate mixed chimerism and clonal deletion of alloreactive T cells.

[Results] The CTLA4 Ig treated group showed increased graft survival compared to the non-treated controls (mean survival time [MST] 17 days and 8 days, respectively, p<0.01). Interestingly, combination of T cell depletion and CTLA4 Ig plus short-course of Rapamycin increased VCA survival significantly (MST 60 days; p<0.01). Donor derived mixed chimerism was detected in recipients receiving the combined treatment protocol with 5.013 ± 1.23 % of donor derived CD11b+ cells on POD 55. Vβ – TCR staining profiles in recipients after combined treatment showed 1.570 ± 0.3700 % of νβ5+CD4+ T cells, while naïve C57BL/6 express 3.567 ± 0.3690 % of νβ5+CD4+ T cells, representing central deletion of donor-reactive T cells.

[Conclusion] This study shows that combination of T cell depletion and costimulation blockade and short-course of Rapamycin prevents VCA rejection and significantly prolongs graft survival. Also, these data show that a clinically acceptable non-cytoreductive strategy could be applied in VCA to avoid long-term maintenance immunosuppression.

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