Solid organ transplantation, the best available treatment for end-stage kidney, liver, lung and heart failure, may fail due to chronic immunological rejection, often taking the form of allograft vasculopathy. Allograft vasculopathy results from recruitment to and activation within the graft vessel wall of IFN-γ-producing graft- reactive host T cells by graft ECs. The development of donor specific antibodies (DSA) that recognize non-self alleles of MHC molecules expressed by graft endothelial cells (ECs) and that fix complement is a major risk for allograft vasculopathy. Complement enhances this process by activating immune functions of the ECs that mediate both recruitment and activation of alloreactive T cells. Binding of high titer panel reactive antibody (PRA) from allo-sensitized transplant candidates, used to model DSA, deposits complement membrane attack complex (MAC) on human ECs, both in culture or in vessel grafts in immunodeficient mouse hosts, and induces gene expression of adhesion molecules and chemokines in a manner dependent upon MAC internalization and non-canonical NF-κB signaling. The mechanism(s) by which MAC potentiates T cell activation, measured as effector memory T cell proliferation and cytokine production in vitro or augmented vasculopathic changes in vivo is unknown. My preliminary data show that MAC induces formation of an active inflammasome in ECs, a previously undescribed phenomenon. Activated caspase-1 in the inflammasome processes pro-IL-1β to active IL-1β and mediates its release. Blocking caspase-1 activity or inhibiting IL-1 signaling with IL-1 receptor antagonist (IL-1Ra) blocks both downstream inflammatory gene expression in ECs secondary to canonical NF-κB activation and also blocks the augmentation of allogeneic T cell response in vitro. I hypothesize that complement activation of the inflammasome in ECs intensifies the host T cell response to graft arterial ECs by increasing local production of IL-1, potentiating allograft vasculopathy. In Specific Aim 1, I will characterize the MAC-induced inflammasome and determine the mechanisms linking MAC to inflammasome assembly. I will examine if kinases of non-canonical NF-κB signaling are linked to inflammasome assembly or IL-1 mRNA and protein synthesis. In Specific Aim 2, I will investigate the biological consequences of alloantibody-induced inflammasome activation in ECs on the alloreactive T cell response that drives allograft vasculopathy. I will determine if this response is also MAC-dependent and if mature IL-1 released from MAC-induced inflammasomes acts on ECs, T cells or both. I will characterize IL-1 dependent changes of the clonal repertoire and subsets of activated alloreactive effector memory T cells using TCR deep sequencing and FACS-analytic phenotyping and the effect of IL-1Ra or caspase-1 inhibition on the alloreactive T cell response in vitro using cultured human ECs. I will use well-developed humanized mouse models of allograft vasculopathy to assess the role of the EC inflammasome in vivo. Successful completion of this project may reveal novel targets for therapies to improve patient outcomes in transplant medicine.

Project Narrative Chronic immunological rejection in the form of allograft vasculopathy is a major cause for the failure of live- saving transplantation therapy. Complement-activating donor specific antibodies (DSA) enhance allograft vasculopathy pathogenesis by activating immune functions of the ECs and thereby intensify host T cell responses, but the mechanism(s) underlying how complement potentiates the immunogenicity of endothelial cells, measured as effector memory T cell proliferation and cytokine production, is unknown. In this project, I will elucidate both mechanisms of how antibody-mediated complement activation induces formation of an inflammasome in ECs, a phenomenon that has not been previously described, and the biological consequences of the subsequent pro-inflammatory IL-1 cytokine release on the host T cell responses, findings that may lead to new, vessel-directed therapies to prevent or treat chronic rejection.