IL-2 cytokine/antibody mediated transplantation tolerance

The induction of donor-specific tolerance is considered as the Holy Grail in the field of transplantation medicine. The elimination of chronic immunosuppressive therapy would decrease morbidityand mortalityby diminishingside effects andsimultaneously sustaining immunocompetence towards infectious agents. Moreover, immunological tolerance would prevent the risk of chronic rejection which has not improved during the last decade and remains the leading cause for late graft loss. Regulatory T cells (Tregs) appear to play a critical role in the maintenance of self-tolerance and have also been shown to be extremely potent in suppressing alloresponses. Therapeutic Treg cellular therapy has been shown to prolong allograft survival in preclinical animal models, however large numbers are required, selective markers for isolation are still lacking and optimization of large scale in vitro expansion would be an essential requirement for their effective clinical intervention. This project builds on the guest institutes expertise in manipulating and expanding Tregs in vivo using IL2 cytokine/antibody complexes. Prof. Sprents group pioneered an approach to selectively and potently stimulate Tregs in vivo with IL-2/mAb complexes in order to maximize the benefits of Treg therapy while sparing the challenges and risks of cell-based therapy. Using IL2 cytokine/antibody complexes, long-term survival of fully MHC mismatched islet allografts was achieved in over 80% of mice without the need for immunosuppressive therapy. Specifically, we will address the role of Tregs in the suppression of acute allograft rejection and the requirements for cytokines and Treg/dendritic cell interactions. The role of Tregs in longterm graft survival will be investigated by using a unique genetic model of timed Treg ablation. Moreover, we aim to develop a IL2 cytokine/antibody therapy-based skin allograft model in order to investigate the role of intragraft regulatory mechanisms and graft resident Tregs. These data are essential to reveal the underlying tolerance mechanisms in this model and moreover have impact on the mechanisms of long-term graft survival in clinical transplantation. The necessity for novel approaches for the treatment of chronic allograft rejection are beyond dispute and the elucidation of Treg based tolerance mechanisms would be a substantial progress in the field of transplantation immunology.

Solid organ transplantation is the treatment of choice for numerous end-stage organ diseases. Unspecific suppression of immune responses by pharmacological drugs enables graft survival, yet chronic graft dysfunction and subsequent graft loss is still a common clinical problem. Induction of tolerance would prevent chronic rejection and decrease morbidity and mortality by diminishing side effects of chronic immunosuppressive therapy. Regulatory T cells (Tregs) appear to play a critical role in the maintenance of self-tolerance and have also been shown to be extremely potent in suppressing alloresponses. Therapeutic cellular therapy has shown huge potency in prolonging allograft survival in pre-clinical animal models and first clinical phase trials are ongoing, however there are several hurdles hampering clinical translation. Even 20 years after the discovery of Tregs as ideal candidate for tolerance induction, there are several unanswered questions concerning sources, isolation strategy, doses, timing of infusion, best immunosuppressive regimen, and cell fate post- infusion. This project was built on the expertise of Prof. Sprent at the Garvan Institute, who pioneered an approach to selectively and potently stimulate Tregs in vivo with specific Cytokine/antibody complexes (IL-2-complexes). In this project we investigated the potency of IL2 complex based therapy to prolong skin allograft survival and the mechanisms of tolerance therein. Mechanisms of tolerance were investigated by fully mismatched skingrafts, analysis of anti-donor- reactive antibodies, flow-cytometric analysis of leucocyte subsets in peripheral blood, lymphoid organs and the graft itself and assays for measuring proliferative capacity of recipient immune cells in response to antigen. We could show that Treg expansion via IL2-complexes synergizes with low-dose rapamycin and anti-inflammatory treatment with anti-IL6, leading to significantly prolonged skin allograft survival and prevention of acute rejection even in the absence of ongoing treatment. This is remarkable as we used a stringent mouse strain combination and skingrafts are considered the most immunogeneic transplant model. Tolerogeneic therapy was given for 4 weeks with skingrafts remaining viable for another 8-10 weeks without any immunosuppressive intervention. Further analysis revealed the complete absence of humoral response indicated by a lack of anti-donor antibodies and deficiency in memory cell formation. Importantly mice remained fully immunocompetent and no adverse events were noted.Further experiments to find an optimal dosing regimen leading to indefinite survival and a deeper understanding of the underlying mechanisms are warranted. We think that these encouraging results will have significant impact on the development of new protocols for tolerance induction in transplantation.