Anti-Third Party Veto Cells Facilitate Allogeneic Hematopoietic Cell Engraftment in MHC-Mismatched Dogs.

Donor-derived veto cells have the potential to facilitate engraftment of major histocompatibility complex (MHC)-mismatched marrow by neutralizing radioresistant host T and NK cells that mediate rejection of the donor graft. Anti-3^rd party cytotoxic T-lymphocyte (CTL) veto activity is CD8 and Fas ligand dependent (Reich-Zeliger et al., Immunity, 2000) and studies in murine models of hematopoietic cell transplantation (HCT) showed that CTL veto cells facilitate engraftment across MHC barriers (Bachar-Lustig et al., Blood, 2003). To translate these results to the well-established pre-clinical dog model of HCT, we had previously demonstrated that donor-derived, anti-3^rd party CTL that are not alloreactive against host cells could function as veto cells and block the emergence of host-derived, anti-donor CTL in vitro. Previous studies showed that DLA-nonidentical, unrelated bone marrow grafts were uniformly rejected by host T and NK cells following 920 cGy total body irradiation (TBI) without postgrafting immunosuppression (90% of dogs with graft rejection). In the current study we asked if infusion of donor derived, anti-3^rd party CTL veto cells could facilitate engraftment of MHC-mismatched marrow. To date, six recipient dogs have been transplanted. For each hematopoietic cell transplant, four DLA-nonidentical, unrelated dogs were used: one marrow donor, one marrow recipient and two 3^rd party dogs. Dendritic cells (DC) were cultured from bone marrow CD34+ cells obtained from 3^rd party dogs. Ten days before transplant, donor lymphocytes were collected by leukapheresis and cultured separately with 3^rd party DCs to generate anti-3^rd party CTL veto cells. On day 0, recipients were irradiated with 920 cGy TBI and transplanted with donor bone marrow (2.3–5.4x10⁸ TNC/kg; 4–12x10⁶ CD34⁺/kg) and donor derived anti-3^rd party CTL veto cells (30–57 x10⁶ cells/kg). Three of six (50%) dogs engrafted 7 to 18 days after HCT (as determined by bone marrow biopsy, complete donor hematopoietic chimerism and recovery of absolute neutrophil count >500/μL) and developed 3-system graft-versus-host disease (GVHD). The other 3 dogs had transient donor engraftment with clinical and histopathological evidence of GVHD. Dogs were euthanized between days 14 and 19 after HCT. In contrast, none of five historical control dogs engrafted after transplantation with DLA-nonidentical, unrelated donor marrow and 30-40x10⁶/kg freshly isolated donor peripheral blood mononuclear cells, p<0.05 (Panse JP et al., Transplantation, 2003). These preliminary data suggest that donor derived, anti-3^rd party CTL veto cells eliminate host-anti-donor T and NK cell responses and facilitate engraftment of MHC-mismatched marrow. Future studies will aim to transplant veto cells with T-cell-depleted marrow and administer post-grafting immunosuppression to decrease the intensity of the conditioning regimen needed for MHC-mismatched HCT engraftment without risking graft rejection or GVHD.