Preclinical Studies of Living and Deceased Donor Liver Allograft Tolerance

PROJECT SUMMARY Acute rejection following liver transplantation (LTx) occurs in up to 30% of recipients and is associated with significantly increased risk of graft failure, graft failure-related death, and all-cause mortality. Induction of liver tolerance would decrease these risks and the toxicities associated with immunosuppressive medications. Although rare, spontaneous tolerance after LTx can be achieved in immunosuppression withdrawal studies. Broad application of this approach is limited by the lack of knowledge about the mechanism(s) of tolerance, markers for tolerance, and therefore the lack of rational targets for immune induction therapy to increase the rate of tolerance. To address these gaps, we developed a nonhuman primate LTx model to test induction strategies for liver tolerance. Preliminary data in our model demonstrated that transient donor chimerism failed to lead to liver tolerance. LTx without bone marrow transplantation but with memory T cell depletion also failed to induce tolerance. These results demonstrate that alternate strategies are required to induce tolerance to livers. We now propose to achieve liver tolerance by inducing durable donor mixed chimerism with conditioning protocols designed for use in living and deceased donor settings. Our long-term goals are 1) to develop clinically applicable protocols for tolerance induction after living or deceased donor LTx; and 2) to define the mechanism by which tolerance develops. Our central hypothesis is that induction of durable mixed chimerism without GVHD will result in tolerance. The proposed research is innovative because it utilizes a translational animal model to develop a clinically applicable tolerance induction protocol. It will also use innovative techniques to characterize the donor- specific immune response to liver transplants, and the interaction of graft-infiltrating lymphocytes and recipient antigen-presenting cells. In Aim 1, we will test the ability of Bcl-2 inhibitors to promote durable chimerism for simultaneous bone marrow and liver transplants while reducing the toxicity of the induction regimen. We will also determine the effect of immunosuppression duration on the subsequent rates of tolerance and rejection. In Aim 2, we will attempt to induce durable mixed chimerism with delayed immune conditioning and donor bone marrow transplant for 4 months after the LTx. This approach would be applicable for frail recipients or recipients of deceased donors. In Aim 3, we will adapt our laboratory’s high-throughput alloreactive TCR sequencing assay to the cynomolgus model to track and analyze the activation of donor-reactive effector and regulatory T-cell clones after transplant. We will also characterize the transcriptome of graft-infiltrating T cells and APCs from liver biopsies to determine if (and how) the liver promotes tolerance. These mechanistic studies will have a positive impact by revealing potential biomarkers of tolerance. They will also allow us to refine our induction protocol to minimize toxicities. Upon successful completion of the proposed research, we expect to develop tolerance induction regimens for living and deceased donor LTx. These achievements would be significant as withdrawal of immunosuppression early after LTx would result in improved patient survival and quality of life.