Novel Therapeutic Strategy to Overcome Skin Rejection in Composite Tissue Allotransplantation: Local intra-graft Treatment with Lymphocyte Migratory Blockers and a Kv1.3 Potassium Channel Blocker Together with Short-Term Systemic Immunosuppression

Theresa Hautz¹, Benson Pulikkottil², Johanna Grahammer¹, Rishi Jindal², Christoph Krapf¹, Vijay Gorantla², Bettina Zelger³, Gerald Brandacher^1,4, Peter Petzelbauer⁵, Michael Schoen⁶, Hartmut Glossmann⁷, Raimund Margreiter¹, Andrew Lee⁴, Johann Pratschke¹, Stefan Schneeberger^1,4, Christian Denecke¹

¹Dept. of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria; ²Div. of Plastic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA; ³Dept. of Pathology, Innsbruck Medical University, Innsbruck, Austria; ⁴Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA; ⁵Dept. of Dermatology, Vienna Medical University, Vienna, Austria; ⁶Dept. of Dermatology, University of Goettingen, Goettingen, Germany; ⁷Institute of Biomedical Pharmacology, Innsbruck Medical University, Innsbruck, Austria

Methods: ALS was given on days -1/0 and 3 after BN-to-Lewis orthotopic rat-hind-limb-transplantation. Animals were either treated with cyclosporine+IL-2-fusion-protein (21 days) followed by daily intraperitoneal or subcutaneous-intragraft BBeta15-42, or tacrolimus (50 days) together with weekly subcutaneous-intragraft efomycine-M or tacrolimus (30 days) together with subcutaneous-intragraft correolide-C twice/week. Rejection was assessed by inspection, H&E-staining and immunohistochemistry. Tacrolimus-24h-trough-blood-levels, WBC-and RBC-counts were recorded.

Results: Untreated animals rejected grafts on day 9+/-1. Treatment with Bbeta15-42, efomycine-M and correolide-C alone had no effect. Animals treated with ALS+IL-2/Fc+CyA rejected on day 50.6+/-7.2. Additional daily intraperitoneal injections with BBeta15-42 had no effect, but local-subcutaneous BBeta15-42-therapy resulted in long-term allograft survival (>150 days;p=0.0374). After discontinuation of BBeta15-42 on day 100 donor skin grafts transplanted on day 150 remained rejection-free while third-party-skin-grafts were rejected within 18+/-2 days, indicating donor-specific-tolerance. After weaning tacrolimus on day 30 or 50 limbs were rejected within 10 days+/-1. Treatment with local efomycine-M resulted in long-term (150 days) allograft survival. Histology on day 150 showed a mild lymphocytic dermal infiltrate and single vacuolized keratinocytes. Local correolide-C therapy resulted in insignificant prolongation of graft survival (day 43+/-4;p=0.24). Tacrolimus-mean-blood-levels were 2.97+/-0.98ng/ml and undetectable 5 days after weaning.

Conclusion: Local subcutaneous treatment with lymphocyte-migration-blockers together with transient immunosuppressive/immunomodulatory regimen results in long-term limb allograft survival. A Kv1.3-blocker has no effect on skin rejection.