Bone marrow transplantation following supralethal radiochemotherapy is associated with dangerous infections due to the slow immune reconstitution during the first year post transplant. Thus, the use of reduced intensity conditioning, associated with less severe immune ablation, could have a remarkable potential in the treatment of a variety of non-malignant diseases, or for the induction of 'mixed chimerism' as a prelude for cell therapy in cancer or in organ transplantation. However, the marked level of host hematopoioetic and immune cells surviving mild preparatory regimens represents a difficult barrier for the engraftment of donor cells. In particular, the use of purified allogeneic stem cells, which do not pose any risk for GvHD and which can continuously present donor type antigens in the host thymus, thereby inducing durable tolerance to donor cells or tissues, represents one of the most desirable goals in transplantation biology. The proposed project will address this challenge by investigating the potential of veto CTLs and/or regulatory CD4+CD25+ cells, to allow engraftment of mega dose purified stem cells following reduced intensity conditioning. Initially, the optimal veto CTL numbers which can overcome CD4 and/or CD8 mediated graft rejection will be defined in a mouse model specifically developed for this purpose (aim la). Subsequently, the potential synergism of veto CTLs with rapamycin (aim 1b) and with CD4CD25 cells (aim2a, 2b) will be tested in the same model. In parallel, the minimal myeloablation required to allow durable engraftment of H-2 identical Sca-1+Lin stem cells in a congenic host, will be established (aim 1c). This minimal myeloablative conditioning will serve as a platform on which attempts to achieve engraftment of allogeneic stem cells by minimal immune suppression, will be built. The minimal rapamycin dose and the minimal number of veto CTLs and CD4CD25 cells which lead, together, to durable engraftment of allogeneic stem cells will be defined in this model (aim 1d,2c). The protocols of choice developed in 1d and subsequently in 2c will be evaluated for their contribution to the immune competence of the resulting chimera, compared to full donor type chimera achieved by stem cell transplantation in fully ablated hosts (aim 3). In particular, the immune response of chimeric mice to a challenge of murine CMV infection, at different times post transplant, will serve as an important endpoint prior to clinical application.