Summary Prolonged cold ischemia storage and recipient sensitization remain among the leading risks for poor transplant outcome. There is a continual need to investigate novel molecular pathways required for activation and functions of various immune cell types and to identify targets for potential therapeutic intervention. Using clinically relevant mouse models of heart transplantation, we have recently identified water channel Aquaporin (AQP)-4 as a promising target in mouse models of prolonged CIS and allosensitized recipients. AQPs are expressed in all tissues and cells and play key roles in various physiologic functions and disease processes. Until our studies, the expression and role of AQP4 in immune cells have not been previously considered. We found that short-term peritransplant treatment with a novel specific AQP4 inhibitor diminishes alloimmune T cell responses and significantly prolongs allograft survival of mouse heart allografts subjected to 8 h cold ischemia storage and of heart allografts transplanted into sensitized recipients. Our preliminary studies indicate that AQP4 is expressed on naïve and memory T lymphocytes and regulates T cell receptor (TCR)-mediated signaling by facilitating actin cytoskeleton rearrangement. In addition, AQP4 is expressed on antigen presenting cells (APCs) and AQP4-/- APCs have diminished antigen presenting capacity. Based on these findings, we hypothesize that AQP4 is required for optimal immune cell functions, in particular responses by both naïve and memory T cells and antigen presentation by antigen presenting cells (APCs). We further propose that pharmacological inhibition or genetic deficiency in AQP4 will decrease alloimmune responses following transplantation and facilitate long-term allograft survival. This hypothesis will be tested in the following aims using novel reagents and a unique combination of advanced in vivo and in vitro approaches: Aim 1: To investigate the mechanisms underlying impaired T cell activation caused by the deficiency or inhibition of AQP4. Aim 2: To test efficacy of AQP4 blockade in inhibiting donor-reactive memory T cell functions and heart allograft rejection by sensitized recipients. Aim 3: To test the role of AQP4 in antigen presenting cell functions in the context of transplantation. Impact: The proposed studies will reveal previously unknown molecular mechanisms leading to immune cell activation in transplant recipients and explore the possibility of targeting AQP4 as a novel component of graft- prolonging strategies.