[Background] Reconstructive transplantation represents a valid therapeutic option after devastating tissue loss. Routine clinical application, however, is hampered by the toxicity of long-term maintenance immunosuppression. The current study investigated to a novel approach using ex vivo expanded regulatory T cells combined with short-term immunomodulatory strategy in a murine hind limb transplantation model.

[Methods] Fully MHC-mismatched orthotopic hind limb transplants were performed from Balb/C to C57BL/6 mice. Recipients in the experimental groups received various combination regimen consisting of 0.5mg CTLA4 Ig on day 0, 2, 4 and 6 post-transplant and, 20mg/kg anti-Thy 1.2 mAb on POD-1 and 1mg/kg Rapamycin (POD 0-9) and 1 wk expanded Treg cells. Allograft survival was monitored and flow cytometric analysis was performed to evaluate mixed chimerism and clonal deletion of alloreactive T cells.

[Results] Combination of T cell depletion and CTLA4-Ig plus short-course of Rapamycin increased VCA survival significantly while untreated control rejected allografts (MST 105 days vs MST 9 days; p<0.01). 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 full treatment showed 1.570 ± 0.3700 % of [nu]β5+CD4+ T cells, while naïve C57BL/6 express 3.567 ± 0.3690 % of [nu]β5+CD4+ T cells, suggesting the actuation of central deletion of developing donor-reactive T cells. Increased graft-Infiltrating Foxp3+ regulatory T cells of donor origin were detected on POD 30 and 60. The addition of ex vivo expanded regulatory T cells further significantly increased VCA survival to more than 200 days and induced long-term stable mixed chimerism (15.01 ± 2.44% of donor CD11b+ cell on POD 60) .

[Conclusion] The combination of T cell depletion, costimulation blockade, and a short-course of Rapamycin prevents VCA rejection and significantly prolongs graft survival without the need for myeloablative conditioning or maintenance therapy. Moreover, regulatory T cells added in the early post transplant period further optimize immune regulation via sustained mixed chimerism.

CITATION INFORMATION: Oh B, Furtmüller G, Iglesias M, Fryer M, Akre P, Cooney D, Lee W, Raimondi G, Brandacher G. Ex vivo Expanded Regulatory T cells Combined with Short-Term Costimulation Blockade Prevent Rejection of Vascularized Composite Allografts. Am J Transplant. 2017;17 (suppl 3).

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