[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 a novel approach using ex vivo expanded regulatory T cells combined with a short-term immunomodulatory strategy in a murine hind limb transplantation model.

[Methods] Orthotopic hind limb transplants were performed from Balb/C to C57BL/6 mice. Recipients in the experimental groups received a combination regimen consisting of CTLA4 Ig on day 0, 2, 4 and 6 post-transplant, anti-Thy 1.2 mAb on POD-1, and 1mg/kg Rapamycin (POD 0-9), and 1 wk expanded CD4+CD25+ Treg cells. Allograft survival, suppression assays, mixed chimerism was evaluated.

[Results] Combination of T cell depletion and CTLA4-Ig plus short-course of Rapamycin increased VCA survival significantly while untreated controls rejected allografts (MST 105 days; p<0.01). Mixed chimerism was detected in recipients receiving this combined treatment protocolwith 5.013 ± 1.23 % of CD11b+ cells being donor-derived 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. In order to further prolong allograft survival, one week expanded Tregs were then included in the combination therapy. The suppressive activity of the CD4+CD25+ Tregs was confirmed with in vitro suppression assays. The addition of ex vivo expanded regulatory T cells further increased VCA survival to >200 days and induced long-term stable mixed chimerism with 16.7±1.5 % of CD11b cells being donor-derived on POD 55 after administration of expanded Treg cells.

[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 by inducing sustained mixed chimerism.

CITATION INFORMATION: Oh B., Furtmüller G., Grzelak M., Vuong L., Iglesias M., Fryer M., 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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