PROJECT I - SUMMARY/ABSTRACT Ischemia-reperfusion injury (IRI) remains the primary obstacle limiting the success of orthotopic liver transplantation (OLT) in patients with end-stage liver disease and those with tumors of hepatic origin. Our group has pioneered the concept that hepatic IRI requires activated CD4+ T cells to facilitate liver tissue damage. T cell immunoglobulin-3 (TIM-3; encoded by Havcr2 gene) is the central negative regulator of T cell activation. CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1; encoded by CC1 gene) has been identified as a new cellular ligand determining TIM-3 function. First, we found that compared with CEACAM1 proficient (WT) livers, CEACAM1 null-mutation (CC1-/-) exacerbated IRI in OLT. Second, we discovered that the benefit of recipient CD4+TIM-3+ signaling in IR-stressed OLT (WT→TIM-3Tg) was completely lost when CEACAM1 KO mice served as organ donors (CC1-/-→TIM-3Tg). These preliminary data have led us to central hypothesis that 1/ TIM-3 – CEACAM1 negative regulation is essential to control IRI by imposing exhaustion-like dysfunction in OLT-infiltrating CD4+ T cells; and 2/ CEACAM1 in the donor liver promotes hepatoprotection. Project I will test this hypothesis through two interlocked specific aims: Aim 1: Define mechanisms of TIM-3 – CEACAM1 negative T cell regulation in IR-stressed iso-OLT. A panel of mice available to us for Aim 1 experiments include CD4+ T cell mutants, which are: i/ CEACAM1Tg; ii/ double TIM-3Tg and CEACAM1-/-; as well as: iii/ TIM-3Tg and TIM-3-/- mice. Aim 1.1. Hypothesis: CEACAM1 - TIM-3 signaling on host circulating CD4+ T cells promotes exhaustion- type phenotype in IRI–OLT. Aim 1.2. Hypothesis: Under dominant CAECAM1 signaling, TIM-3+CD4+ exhausted T cells inhibit the development and progression of IRI in OLT. Aim 2: Define mechanisms by which hepatocellular CEACAM1 in donor liver regulates IRI in iso-OLT. Gene- targeted strains for Aim 2 studies include: i/ hepatic CEACAM1 inactivation (loss-of- function; L-CC1-/-); or ii/ forced hepatic CEACAM1 overexpression (gain-of-function; L-CC1Tg). Aim 2.1. Hypothesis: Enhancement of hepatocyte-specific CEACAM1 – β-catenin regenerative functions facilitates hepatoprotection. Aim 2.2. Hypothesis: TIM-3 – CECACAM1 signaling enhances hepatocyte autophagy program. Integration with PPG: By providing novel insights into TIM-3 – CEACAM1 checkpoint regulation at the innate – adaptive immune interface in IR-stressed iso-OLT, Project I naturally informs/precedes studies assessing how host rejection regulates innate immune activation/IRI sequel in allo-OLT (Project II). Direct application of approaches blunting inflammation while promoting hepatoprotection in mouse OLT models will accelerate assessments of immune phenotypes in human liver transplants (Project III).