Project 2 Summary Transplantation tolerance promises to facilitate long-term allograft acceptance while avoiding the need for life-long immunosuppression and its associated problems. In the past decade, operational clinical transplantation tolerance has been achievable through hematopoietic stem cell transplantation or weaning of conventional immunosuppression. However, the vast majority of transplant recipients fail to achieve allograft tolerance, and a subset of operationally tolerant transplant recipients eventually experience graft loss. High frequencies of memory alloreactive CD4+ or CD8+ T cells have been identified as potent barriers to the successful induction of transplantation tolerance. There continues to be an urgent need to identify new strategies for overcoming the barrier posed by memory T cells; Project 2 proposes to address this need. We recently showed that memory T cells resist infectious tolerance mechanisms, and cannot acquire the cell-intrinsic hypofunctional states attained by naïve T cells. Moreover, in sensitized recipients harboring memory T cells directed to just a single donor antigen, we reported that the larger repertoire of naïve donor-specific CD4+ Tconv also develops resistance to co-stimulation blockade-induced tolerance. We refer to this phenomenon as “linked sensitization” and posit that understanding the mechanism underlying this barrier to co-stimulation blockade-induced allograft tolerance is not only relevant to recipients with heterologous memory T cells, but also can lead to the identification of new targets that synergize with co-stimulation blockade to achieve transplantation tolerance. Aim 1 will focus on defining the mechanism of linked sensitization. Our observations that memory T cells are resistant to being programmed into cell-intrinsic hypofunctional states identify a gap in knowledge as to whether, and how, memory T cells may be programmed to tolerance. To this end, we leveraged the physiological