Induction of cardiac allograft tolerance in a rat heart transplant model

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Abstract

Although the immunosuppressive drugs currently in use are effective in reducing the incidence of acute rejection after transplantation, they also put the patient at risk for life threatening infections and cancers. The long term administration of immunosuppressive agents results in an increase in both morbidity and mortality. In addition, the long term cost of these agents represents a financial burden. Establishment of tolerance to a well-functioning transplant without nonspecific immunosuppressive drugs is a major goal of organ transplantation therapy. The induction of recipient (host) tolerance to the histocompatibility antigens of the organ donor could eliminate the need for long term administration of these nonspecific immunosuppressive drugs. This would have a major impact on the quality and quantity of life of patients with long term surviving organ grafts by reducing the immunologic and non-immunologic complications associated with long term immunosuppressive therapy. Induction of immunotolerance in animal heart transplant models has for the most part involved donor-based tolerance. One well established model is injection of donor splenocytes plus a single injection of the T-cell suppressing agents anti-rat lymphocyte serum or RIB 5/2. Adoptive transfer (altering the hosts immune system using lymphoid cells from another individual) of ex vivo generated alloantigen-specific regulatory T cells induces immunotolerance in a rat transplant model. This method has also been used in bone marrow transplantation and can be the basis for recipient-based immunotolerance induction. In heart transplantation, recipient-based immunotolerance induction is clinically more applicable than donor-based since donor genotype is rarely known prior to transplant. To date there is limited data on recipient-based immunotolerance induction in heart transplantation, but data exists from the bone marrow transplant literature. In addition, although allograft tolerance has been achieved using a wide variety of donor-based immunologic interventions in laboratory animals, no reliable method of confirming tolerance following cardiac transplantation has been established. Standard of care in the field of solid organ transplantation remains initial treatment with triple immunosuppression followed by long term maintenance therapy with 1-3 immunosuppressive agents. One cannot withdraw drugs confidently even if the graft seems to be tolerant. This study will apply high throughput expression profiling with the goal of studying donor and recipient-based immunotolerance induction protocols and identifying protein and gene changes that could serve as possible candidate biomarkers of tolerance. Identifying laboratory methods that will permit safe and precise confirmation of immune tolerance in the transplant patient has the potential to improve outcome substantially. The protocol was approved in 2006 and a total of 403 rats were used over the duration of the protocol which was closed in 2012. The protocol was divided into 2 parts: Part 1 (recipient-based tolerance); and Part 2 (donor-based tolerance). In Part 1 (recipient-based tolerance), Stage 1 we were able to successfully generate Th2.rapa cells from recipient BN rats. In Part 1, Stage 2 these adoptively transferred ex vivo generated BN Th2 cells were tested in culture using flow and cytokine phenotype tests, and an optimal rapamycin dose was determined. In 2009, we completed studies in Part 1 Stage 3, which was designed to determine if Th2-shifted hosts (recipient-based tolerance) have reduced rejection. In Part 2 (donor-base tolerance), Stage 1 (Induction donor-based tolerance) we successfully learned the techniques to remove the spleen of the rat (DA) that will donate the heart in stage 2. We also successfully injected its processed splenocytes into the thymus of the rat (BN) that will receive the donor heart in stage 2. In 2009, we completed studies in Part 2, Stage 2 which were designed to determine if donor-based tolerance induction reduces rejection. In 2010, total RNA was prepared from peripheral blood mononuclear cells and used for high density oligonucleotide microarrays. A manuscript was published in 2011 (PloS One, 6(4): e18885, 2011) establishing the ability of host-type Th2. Rapa cell therapy given pre-transplant to shift post-transplant cytokines towards a Th2 phenotype and prolong allograft viability when used in combination with a short course of cyclosporine therapy. As mentioned above, in 2012 the protocol was closed; however, we continue to report on the project, since as new genomic techniques become available we may do further data processing and analysis of these completed studies and their stored samples.