Ex Vivo Expanded Regulatory T Cells Combined With Short-term Costimulation Blockade Prevent Rejection Of Vascularized Composite Allografts
Michael Grzelak1, Linh Vuong1, Georg Furtmüller1, Marcos Iglesias1, Madeline Fryer2, Damon Cooney1, WP Andrew Lee1, Giorgio Raimondi1, Gerald Brandacher1, Byoungchol Oh1.
1Johns Hopkins School of Medicine, Baltimore, MD, USA, 2University of Massachusetts Medical School, Worcester, MA, USA.
PURPOSE: 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 investigates 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: Fully MHC-mismatched orthotopic hind limb transplants were performed from Balb/C to C57BL/6 mice. Recipients in the experimental groups received a combination regimen consisting of 0.5mg CTLA4 Ig on day 0, 2, 4 and 6 post-transplant, 20mg/kg anti-Thy 1.2 mAb on POD-1, and 1mg/kg Rapamycin (POD 0-9), and in one group, 1 wk expanded CD4+CD25+ Treg cells. Allograft survival was monitored and flow cytometric analysis was performed to evaluate mixed chimerism and clonal deletion of alloreactive T cells. Treg activity was assessed in vitro using suppression assays in order to support and supplement in vivo data.
RESULTS: Combination of T cell depletion and CTLA4-Ig plus short course of Rapamycin increased VCA survival significantly while untreated controls rejected their allografts (MST 105 days; Untreated, MST 9 days; CTLA4 Ig only, MST 17 days, Rapamycin, MST 20 days; T cell depletion, 20 days; p<0.01). Mixed chimerism was detected in recipients receiving this combined treatment protocol with 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 νβ5+CD4+ T cells, while naïve C57BL/6 express 3.567 ± 0.3690 % of νβ5+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.
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