The rejection cascade in recipients sensitized to allo-Ag may trigger a form of an allograft loss that is mediated by memory T cells (""""""""accelerated"""""""" rejection;AccR). To mimic the clinical scenario of sensitized patients, we have developed a murine cardiac AccR model in which B6 mice are challenged with B/c skin, and then rested for >40 days. When B/c hearts are transplanted into such sensitized mice, they are rejected in 4-5 days, with re-activated memory CD8 T cells playing the paramount role. The central tenet of this project is that graft TLR4-dependent pro-inflammatory innate response regulates the systemic CD8 T cell memory, and precludes successful organ engraftment in sensitized hosts.
Aim 1 : Determine the role of """"""""positive"""""""" T cell costimulation in the development of alloreactive CD8 T cell memory, and its regulation by graft innate immune activation. Hypothesis: The activation of alloreactive memory CD8 T cells: 1/ proceeds via CD154- dependent direct and CD154-independent indirect mechanisms, and 2/ depends on TLR4-dependent inflammation at the graft site, which in turn critically affects the efficacy of T cell costimulation blockade. We will study costimulation requirements for memory CD8 re-activation, and dissect roles of ICOS, OX40, 4-1BB and CD27 signaling in alloreactive memory CD8 T cell generation. We will analyze the role of graft innate activation on alloreactive memory T cell activation. By utilizing mice with specific TLR4 mutations (MyD88/TRIF KO) as heart donors, we will determine: i/ how does local defective TLR signaling impact graft inflammation response; ii/ how does the absence of TLR4 affect systemic activation of alloreactive CD8+ T cells/graft infiltration by activated CD8 T cells;and iii/ the impact of deficient donor innate activation on host sensitization/efficacy of costimulation blockade. By using CD8+ TCR-tg (2C-tg) system, we will address the direct effects of T cell costimulation pathways on activation/differentiation of specific allo-CD8 T cells.
Aim 2 : Determine the role of """"""""negative"""""""" PD-1 (CD279) - PD-L1/-L2 T cell costimulation in alloreactive CD8 T cell memory generation, and its regulation by graft innate activation. Hypothesis: The PD-1-PD-L pathways negatively regulate CD8 T cell memory activation by transmitting signals that limit their proliferation or trigger cell apoptosis. Harnessing physiological mechanisms of regulation by PD-1 on alloreactive T cells with PD-L on parenchyma cells depresses local CD8 memory T cells in a mechanism regulated by graft innate activation. First, we will evaluate how PD-1 pathway influences the primary CD8 T cell activation, and the memory repertoire. Second, we will differentiate the impact of negative signaling on CD4-dependent vs. -independent CD8 T cell memory recall. Third, we will define the role of PD-L on donor endothelial vs. BM derived cells (chimera). Fourth, we will explore mechanisms by which systemic vs. local PD-1-PD-L engagement (fusion protein/gene transfer) alters cardiac parenchyma inflammation/alloimmune responses. Fifth, we will study how intragraft innate immune activation regulates the PD-1-PD-L pathway in sensitized transplant recipients.
Host sensitization remains the major problem in clinical organ transplantation. Many prospective transplant patients are sensitized following blood transfusions, pregnancies, or failed previous grafts. This project is designed to analyze cell mediated mechanisms leading to accelerated rejection of organ allografts and ultimately to design novel and much needed therapeutic approaches to ameliorate transplant rejection in sensitized patients.
