Objective:To test in liver transplant recipients identified as non-tolerant whether discontinuation of calcineurin inhibitors followed by 6-month treatment with sirolimus modifies the pattern of expression of the set of genes associated with tolerance~Background: Sirolimus is an immunosuppressive drug used to counter autoimmunity and to prevent acute graft rejection in human and has remarkable tolerance-promoting properties in animal transplant models.~Hypothesis/Specific Aims:We hypothesize that sirolimus promotes tolerogenic pathways in human liver transplantation.~To test in liver transplant recipients identified as non-tolerant whether discontinuation of calcineurin inhibitors followed by 6-month treatment with sirolimus modifies the pattern of expression of the set of genes associated with tolerance~To test in liver transplant recipients identified as non-tolerant whether conversion from calcineurin inhibitors to sirolimus modifies memory type immune responses.~To test in liver transplant recipients identified as non-tolerant whether conversion from calcineurin inhibitor monotherapy to sirolimus affects the frequency, phenotype and function of potentially immunoregulatory peripheral blood lymphocyte subsets~To test in liver transplant recipients identified as non-tolerant whether conversion from calcineurin inhibitors to sirolimus promotes epigenetic changes related to immunoregulation and cancer development/progression~Proposed Methods:Gene expression experiments: we will quantify the expression in peripheral blood of a set of genes previously identified as predictive of successful immunosuppression withdrawal in stable liver transplant recipients. Blood samples will be obtained before and 6 months after conversion to sirolimus treatment. Measurement of gene expression levels will be conducted employing real-time TaqMan PCR. Classification of patients in the tolerant/non-tolerant categories will be conducted utilizing thresholds and predictive algorithms developed in our laboratory.~Immunophenotyping studies: we will quantify in peripheral blood various mononuclear cell subsets implicated in immunoregulatory pathways before and 6 months after conversion to sirolimus treatment. Measurements will be conducted employing flow cytometry.~Functional assays: we will isolate CD4+CD25+ regulatory T cells (Treg) from peripheral blood by Sorter before and 6 months after conversion to sirolimus treatment. Serial dilution experiments will be conduct in an antigen non-specific assay to assess the relative suppressive properties of Tregs. IFNg ELISpot assays will be conducted in parallel employing peripheral blood mononuclear cells as responder cells to measure donor-specific alloimmune responses.~Measurement of DNA-methylation: recipient DNA will be extracted from peripheral blood samples before and after 6-month sirolimus treatment and used to conduct whole-genome methylation studies employing the ILLUMINA array platform.~Expected results: We expect to precisely define the effects of sirolimus on previously identified tolerogenic pathways in humans and, to assess the capacity of this drug to enhance the proportion of liver recipients undergoing successful immunosuppression weaning.
In contrast to calcineurin inhibitors, sirolimus is known to exert remarkable tolerance-promoting properties in multiple animal transplant models. Whether sirolimus is capable of enhancing tolerance-related pathways and/or promoting complete withdrawal of immunosuppressive drugs in human transplant recipients has not been previously addressed. The goal of the investigators study is to evaluate the effects of sirolimus on previously identified tolerogenic pathways in humans and, indirectly, to assess the capacity of this drug to enhance the proportion of liver recipients undergoing successful immunosuppression weaning.