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Objective: Natural killer (NK) cells kill allogeneic cells that lack a class I MHC ligand for clonally distributed killer inhibitory receptors (KIR). Following HLA-mismatched hematopoietic cell transplantation (HCT), donor NK cells might mediate graft-vs-host (GVH) reactions that promote donor chimerism and mediate anti-tumor effects. Additionally, recipient NK cells might mediate donor marrow rejection. We have developed a nonmyeloablative approach to haploidentical HCT involving recipient treatment with a T cell-depleting mAb, Medi-507, that can achieve donor engraftment and mixed hematopoietic chimerism without graft-vs-host disease (GVHD). Donor lymphocyte infusions (DLI) are later administered in an effort to achieve graft-vs-leukemia/lymphoma (GVL) effects without GVHD. It is unknown whether NK cell "tolerance" develops in human mixed chimeras., Methods: We have addressed these issues in 12 patients receiving Medi-507-based nonmyeloablative haploidentical HCT., Results: NK cells recovered relatively early, despite the presence of circulating anti-CD2 mAb, but the majority of initially recovering cells lacked CD2 expression. These NK cells showed a reduced capacity, compared to those from normal donors, to kill class I-deficient targets. No association was detected between KIR mismatches in the host-vs-graft (HVG) or GVH direction and graft or tumor outcomes in this small series. NK cell chimerism did not correlate with chimerism in other lineages in mixed chimeras. NK cell tolerance to the host was not observed in a patient with full donor chimerism. One patient developed NK cell reactivity against donor-derived lymphoblast targets after loss of chimerism, despite the absence of an HVG KIR mismatch., Conclusion: Our results do not show an impact of NK cells on the outcome of nonmyeloablative, even T cell-depleted, HCT across haplotype barriers using an anti-CD2 mAb. Our data also raise questions about the applicability of observations made with NK cell clones to the bulk NK cell repertoire in humans.

Objective: We developed a nonmyeloablative conditioning regimen for allogeneic bone marrow transplantation (BMT) followed by donor lymphocyte infusions (DLI) for treatment of chemotherapy refractory malignancies. Although the majority of patients who receive this regimen achieve lasting mixed or full allogeneic chimerism, approximately 30% show initial mixed chimerism followed by loss of the donor graft. These patients recover host hematopoiesis without significant cytopenias. To assess the role of immunologic rejection in graft loss, we compared T-cell recovery and in vitro alloresponses in six patients who lost their marrow graft to that in 16 concurrent patients with sustained donor chimerism., Patients and Methods: Conditioning included pretransplant cyclophosphamide (150-200 mg/kg), thymic irradiation (700 cGy), and pre- and post-transplant equine antithymocyte globulin (ATG; ATGAM). HLA-identical related donor BMT was followed by DLI at approximately day 35 in patients without graft-vs-host disease., Results: The group with transient chimerism showed significantly increased circulating host T-cell (median 416 cells/mm(3) vs 10 cells/mm(3), p<0.05) and CD8 T-cell numbers (354 cells/mm(3) vs 71 cells/mm(3), p<0.05) compared to the group with stable mixed or full donor chimerism within the first 100 days post-BMT. All DLI recipients who lost chimerism following DLI had greater than 80% recipient T cells at the time of DLI, whereas those with persistent chimerism had <60% host T cells. Graft rejection was associated with the development of a sensitized anti-donor bulk cytotoxic T-lymphocyte (CTL) response in 4 of 6 evaluated patients, compared to only 1 of 10 evaluated patients with sustained chimerism (p<0.05). Additionally, 3 of 5 evaluated transient chimeras showed high anti-donor CTL precursor frequencies in limiting dilution assays, and 3 of 4 evaluated transient chimeras showed high anti-donor interleukin-2 (IL-2)-producing T-helper (T(H)) cell frequencies. High anti-donor T(H) or cytotoxic T-lymphocyte precursors were not detected in sustained chimeras., Conclusion: These data indicate that loss of chimerism in patients receiving this nonmyeloablative regimen is due to immune-mediated rejection. This rejection appears to bemediated by recovering recipient cytolytic CD8(+) cells as well as IL-2-producing recipient T(H) cells. These data are the first to demonstrate sensitization of recipient anti-donor IL-2-producing cells in association with human marrow allograft rejection.