Bone marrow transplantation (BMT) is used for the treatment of a variety of disorders, however, the acute toxicities associated with the conditioning procedure in BMT has led to the emergence of non-myeloablative conditioning therapies. Rejection of the BMC graft by host effector cells remains a significant concern. Natural killer (NK) cells are cells that spontaneously mediate the killing of transformed or virally infected cells in a MHC-unrestricted manner. We have previously shown that activated NK cells of donor-type can improve donor engraftment after allogeneic BMT in mice. We now propose to assess the effects of NK cell transfer using a non-myeloablative model to ascertain if activated NK cells, either as whole populations or through the use of purified subsets, can promote donor BMC engraftment. We hypothesize that activated NK cells of donor-type, particularly when either purified or those who have their inhibitory receptors (Ly-49 in the mouse) blocked, can eliminate or suppress host alloreactive cells that come into contact with them resulting in greater engraftment capability of subsequent donor BMC. Toward this goal we have developed 4 Specific Aims:
Specific Aim 1 will assess the effects of donor NK cell transfer on promoting donor chimerism using allogeneic strain combinations and models involving decreasing amounts of total body irradiation (TBI) as a conditioning regimen. The effect on both host NK and T cell-mediated rejection pathways will be assessed using both in vitro and in vivo assays. Extent of donor chimerism, evidence for GVHD, and specificity of tolerance induction will be assessed.
Specific Aim 2 will determine if transfer of specific Ly-49 NK cell subsets will enhance the ability of the donor NK cells to prevent subsequent marrow rejection. NK cell subsets bearing Ly-49 inhibitory receptors specific for the host MHC will be removed allowing for greater reactivity of the transferred population for the host effector cells and thus improve engraftment.
Specific Aim 3 will use F(ab?)2 fragments of various antibodies specific for Ly-49 members to block the interaction with the inhibitory receptors with MHC.
Specific Aim 4 will decipher the mechanism by which NK cells inhibit host resistance. Neutralizing antibodies to various immunosuppressive cytokines (i.e. TGF-beta) and the use of knockout mice (i.e. perforin) as a source of NK cells will be used to determine the role of cytokines versus lytic pathways in the immunosuppressive effect of NK cells. These studies should result in improving the efficacy of non-myeloablative therapies and to develop a means to induce tolerance.
