Vascularized composite allotransplantation has developed as a specialty at the interface of reconstructive and transplant surgery, offering restoration of function and form in scenarios where options for autologous reconstruction are limited, and for which the burden of donor-site morbidity may be high. Over the past 15 years some 28 patients have received face, and 85 patients upper extremity transplants. Results have been encouraging, with good functional outcomes, and the majority of patients reporting return to independence, employment and improved quality of life. However, the immunological management of these patients remains a significant challenge. While conventional immunosuppressive regimens have proven effective in preventing graft loss to rejection, they have failed to protect patients from acute rejection episodes, which have been reported in 85% during the first year post-transplant. When considered alongside the burden of comorbidity associated with life-long immunosuppression, the impetus for development of novel approaches to the prevention of rejection is clear. Induction of hematopoietic mixed chimerism has successfully achieved transplant tolerance, defined as specific unresponsiveness to donor antigens permitting life-long acceptance of transplanted tissues without maintenance immunosuppression, in numerous animal models and recently, of renal allografts in clinical trials. The work presented in this thesis investigates mixed chimerism for induction of tolerance of vascularized composite allografts (VCAs) across class I and II major histocompatibility (MHC) barriers in the Massachusetts General Hospital miniature swine model; a large animal model with defined MHC immunogenetics, and skin closely analogous to that of humans. The data presented demonstrate development of a reproducible model of VCA tolerance and stable hematopoietic mixed chimerism in a preclinical model. Importantly, tolerance extended to all components of VCAs including the epidermis and dermis, a previously un-reproducible finding. In vitro analysis demonstrated no evidence for either IL-2 reversible anergy or cellular regulation as mechanisms of donor-specific unresponsiveness, suggesting that at a systemic level, tolerance in this model may be primarily mediated by clonal deletion. In contrast, characterization of the cutaneous immune system demonstrated rapid infiltration of VCAs with recipient T cells and Langerhans’ cells, which in chimeric recipients did not cause rejection but rather established stable chimerism in all tissue-resident populations including dermal T cells with the phenotype of Tregs (CD25+FoxP3+); findings which suggest tissue-specific, and regulatory, mechanisms may play important roles. These data support the hypothesis that mixed chimerism is sufficient for whole-skin tolerance of VCAs, but further work is required to demonstrate the necessity of stable, rather than transient, chimerism and to confirm the necessity of other systemic or tissue-specific factors for prevention of epidermal rejection.

Unsere Untersuchungen belegen, dass nichtmyeloablative Stammzelltransplantationen (NST) bei Patienten mit Hochrisiko-ALL oder -AML eine neue therapeutische Option darstellen. Die NST ermöglichte eine allogene Stammzelltransplantation bei Transplantationskandidaten mit Kontraindikationen gegen eine hochdosierte Strahlen- und Chemotherapie (Standardtransplantation). Nach NST kam es häufig zur Entwicklung von Infektionen und einer spät auftretenden akuten Transplantat-gegen-Wirt Reaktion (GvHD), diese waren aber mit einer geringeren Mortalität verbunden. Das erkrankungsfreie Überleben und das Gesamt-Überleben nach NST und nach Standardtransplantation waren fast gleich. Der höheren Rezidivrate nach NST stand eine höhere transplantationsassoziierte Mortalität nach Standardtransplantation gegenüber. Das Konditionierungsregime selber war ohne Einfluss auf das Überleben der Patienten. Sequenzielle Chimärismusuntersuchungen von Leukozytensubpopulationen erlaubten eine frühe Diagnose eines gemischten Chimärismus, der einen prädiktiven Wert für das Auftreten eines Rezidivs hatte. Ein stabiler gemischter Chimärismus wurde nicht beobachtet. Durch eine adoptive Immuntherapie mit Spenderlymphozyten (DLI) konnte bei der Mehrzahl der transplantierten Patienten mit gemischtem Chimärismus eine Chimärismuskonversion und eine lang anhaltende komplette Remission induziert werden, ein wichtiger Hinweis auf einen Transplantat-gegen-Leukämie Effekt (GvL) der Spenderzellen. Durch die NST rückt der immunologische Effekt der Transplantation gegenüber der Zytoreduktion bei der Standardtransplantation stärker in den Vordergrund. Unsere Ergebnisse sprechen für die Wirksamkeit eines GvL-Effektes bei akuten Leukämien auch nach NST. Die Erfahrungen mit der NST bei akuten Leukämien haben zu einer Anwendung bei refraktären Nierenzellkarzinomen geführt. Eine Tumorregression wurde erst nach Chimärismuskonversion und/oder Entwicklung einer GvHD beobachtet; diese Befunde sind vereinbar mit einem Transplantat-gegen-Tumor Effekt (GvT). Die transplantationsassoziierte Morbidität war allerdings bei diesen meist älteren Patienten erheblich. Die Therapie sollte ausschließlich im Rahmen klinischer Studien erfolgen, da es sich nach wie vor um ein experimentelles Therapieverfahren handelt. Die Analyse minimal residueller Erkrankung und der Einsatz hochauflösender Chimärismusuntersuchungen von Leukozytensubpopulationen sollte die Bedeutung des gemischten Chimärismus weiter klären und zu einem gezielteren Einsatz von DLI führen. Prospektive vergleichende Studien müssen in naher Zukunft den Stellenwert der NST untersuchen. / Our clinical investigations demonstrate, that nonmyeloablative stem cell transplantation (NST) is a novel therapeutic option in patients with high-risk ALL or AML. NST can be administered to patients eligible for allogeneic stem cell transplantation with contraindications against high-dose radio- and chemotherapy (standard SCT). After NST, infections and late onset acute graft-versus-host disease (GvHD) frequently occurred, but transplant-related mortality was low in contrast to standard SCT. Leukemia-free survival and overall survival were similar after NST and standard SCT. A higher relapse rate after NST was balanced by a higher transplant-related mortality after standard SCT. The conditioning regimen itself had no relevant impact on survival. Sequential chimerism analyses of leukocyte subpopulations resulted in early diagnosis of mixed chimerism, which proved to be predictive for later relapses. Stable mixed chimerism was not established in these patients. Adoptive immunotherapy in patients, who were in hematologic remission but had mixed chimerism after transplantation, induced conversion to complete donor chimerism and long-lasting complete remissions in the majority of patients, a strong hint to a graft-versus-leukemia-effect (GvL) of donor T-lymphocyte infusions. A change in the character of stem cell transplantation was achieved by NST with a shift from the predominantly cytoreductive effect of standard SCT towards an emphasis on the immunologic GvL-effect. Our results demonstrated the efficacy of a GvL-effect in acute leukemias after NST. The experiences with NST in acute leukemias have prompted studies in patients with refractory advanced renal cell cancer. Tumor regressions were observed only after chimerism conversion and / or development of GvHD, these results being compatible with a graft-versus-tumor effect (GvT). However, transplant-related morbidity was substantial in these mostly elderly patients. Therapy within clinical studies is mandatory, because allogeneic stem cell transplantation still has to be regarded an experimental procedure in these patients. The analysis of minimal residual disease and application of high-resolution chimerism analysis of leukocyte subpopulations by microarrays could lead to a more profound understanding of mixed chimerism and to a more rational use of DLI in the near future. Prospective randomized trials should be conducted to evaluate the role of NST.