Project Abstract Long-term success of transplantation and achieving transplant tolerance in the clinic is hindered by recipient sensitization and immunological memory, which predominantly arise as a result of graft rejection, pregnancy and blood transfusions. Paradoxically, semi-allogeneic pregnancy provides a unique example of allogeneic tolerance spontaneously induced in adults. Using preclinical mouse models, we showed that semi-allogeneic pregnancy simultaneously induces T cell tolerance to the semi-allogeneic fetus and to subsequent offspring-matched heart grafts. However, pregnancy also induces humoral sensitization to fetal antigens, and this sensitization overrides pregnancy-induced as well as co-stimulation-induced allograft acceptance. Our research aims to define the mechanisms underlying humoral sensitization by semi-allogeneic pregnancy, and how this sensitization over- rides pregnancy-induced and co-stimulation blockade-induced T cell tolerance of offspring-matched heart grafts. In Aim 1, we will test the hypothesis that at a cellular level, pregnancy enables the differentiation of CD4+ T cells into T follicular helper (Tfh) cells providing help to fetus-specific B cells and facilitating their differentiation into post-partum B cells and antibody-secreting cells. Simultaneously, pregnancy induceds a persistent state of cell- intrinsic hypofunction in fetus-specific non-Tfh cells. In Aim 2, we will test the hypothesis that pregnancy- sensitized Tfh, B cell and antibody responses synergize to constitute a barrier to the induction of transplantation tolerance by co-stimulation blockade. Completion of this project will provide an in-depth mechanistic understanding for the basis and consequence of humoral sensitization by semi-allogeneic pregnancy, and identify treatments capable of preventing and reversing this sensitization. The long-term goal of this project is to improve access to transplantation in historically-disadvantaged multiparous women and to optimize their post- transplant outcomes through the induction of transplant tolerance.