We previously demonstrated that autologous natural killer (NK)-cell therapy after hematopoietic cell transplantation (HCT) is safe but does not provide an antitumor effect. We hypothesize that this is due to a lack of NK-cell inhibitory receptor mismatching with autologous tumor cells, which may be overcome by allogeneic NK-cell infusions. Here, we test haploidentical, related-donor NK-cell infusions in a nontransplantation setting to determine safety and in vivo NK-cell expansion. Two lower intensity outpatient immune suppressive regimens were tested: (1) low-dose cyclophosphamide and methylprednisolone and (2) fludarabine. A higher intensity inpatient regimen of high-dose cyclophosphamide and fludarabine (Hi-Cy/Flu) was tested in patients with poor-prognosis acute myeloid leukemia (AML). All patients received subcutaneous interleukin 2 (IL-2) after infusions. Patients who received lower intensity regimens showed transient persistence but no in vivo expansion of donor cells. In contrast, infusions after the more intense Hi-Cy/Flu resulted in a marked rise in endogenous IL-15, expansion of donor NK cells, and induction of complete hematologic remission in 5 of 19 poor-prognosis patients with AML. These findings suggest that haploidentical NK cells can persist and expand in vivo and may have a role in the treatment of selected malignancies used alone or as an adjunct to HCT.

Marrow is cryopreserved for use in autologous bone marrow transplants, but little is known of the incidence of reactions in patients transfused with these cryopreserved marrows. Reactions in patients transfused during a 4-year period with 134 autologous marrows cryopreserved in dimethyl sulfoxide (DMSO) were compared with those in patients transfused with marrow that had been collected from HLA-compatible donors and that had not been cryopreserved. Patients transfused with cryopreserved marrow had significantly more nausea (44.8 vs. 14.1%; p less than 0.0005), vomiting (23.9 vs. 8.5%; p less than 0.01), chills (31.3 vs. 1.4%; p less than 0.0005), and fever (17.9 vs. 0%; p less than 0.005) than patients transfused with fresh allogeneic marrow. The incidence of emesis correlated with the dose of DMSO received, but that of nausea did not. All cryopreserved marrows were cultured for bacteria at the time of transfusion and 17 (12.7%) were found to be positive. Only 1 of the 17 patients transfused with culture-positive marrow developed sepsis during the transplant course with the same organism that was present in the transfused marrow. Although the reactions in donors transfused with cryopreserved marrow were readily treated, this study suggests that the incidence of some reactions might be decreased by reducing the dose of DMSO transfused. Bacterial contamination of transfused marrow was a worrisome complication, and efforts should be made to improve marrow collection and processing techniques to minimize that risk.

Clinical-scale production of NK cells is efficient and can be performed under GMPs. The purified NK cell product results in high NK cell purity with minimal contamination by T cells, monocytes, and B cells, but it requires more time for processing and results in a lower NK cell recovery when compared to NK cell enrichment with CD3 cell depletion alone. Additional laboratory studies and results from clinical trials will identify the best source and type of NK cell product.

Donor lymphocyte infusions (DLIs) can produce lasting remissions in patients with relapsed chronic myeloid leukemia (CML), but are less effective in non-CML diseases. We hypothesized that lymphodepletion, achieved with cyclophosphamide (Cy) and fludarabine (Flu), would promote in vivo expansion of the infused lymphocytes enhancing their immunologic effects. Fifteen patients with relapsed non-CML disease who received Cy/Flu/DLI were compared with 63 controls who received DLI without chemotherapy. Only the patients receiving Cy/Flu/DLI became lymphopenic at the time of DLI. Compared with controls, patients who received Cy/Flu/DLI developed significantly more grades II to IV (60% vs 24%, P ‫؍‬ .01) and grades III to IV acute graft-versus-host disease (GVHD) (47% vs 14%, P ‫؍‬ .01) with greater GVHD lethality. In Cy/Flu/DLI patients, T-cell proliferation was elevated at 14 days after DLI. Although these data suggest that chemotherapy-induced lymphodepletion enhances activation of donor lymphocytes, the toxicity needs to be managed before testing whether better disease control can be achieved. IntroductionThe success of allogeneic hematopoietic cell transplantation (HCT) to treat leukemia depends upon both lymphohematopoietic reconstitution and also the induction of a graft-versusleukemia immunologic response. The importance of the lymphocyte antitumor effect was shown by the ability of donor lymphocyte infusion (DLI) to induce durable remissions in patients with chronic myeloid leukemia (CML) relapse after transplantation. [1][2][3][4][5] However, for patients with non-CML malignancies, this approach remains inadequate. 6,7 One strategy to potentiate the immune effects of donor lymphocytes is to manipulate the host milieu to promote in vivo expansion of donor T cells from the DLI product. Adoptive transfer experiments have shown that lymphodepletion improves in vivo lymphocyte expansion by (1) providing lymphoid space, (2) eliminating host antidonor immune reactivity, (3) decreasing competition for growth factors that promote lymphocyte expansion, or (4) elimination of suppressive regulatory T cells. [8][9][10][11][12][13][14] Based on these observations, we designed a clinical trial to test whether lymphodepleting chemotherapy could enhance the immune effects of DLI. Patients, materials, and methodsPatients were given a lymphodepleting regimen of intravenous cyclophosphamide (Cy) 50 mg/kg once on day Ϫ6 and fludarabine (Flu) 25 mg/m 2 for 5 consecutive days (Ϫ6 to Ϫ2), a regimen that included one less dose of Cy than that initially reported by the NIH group. 15 , 1 multiple myeloma, 1 juvenile CML, and 1 myelofibrosis) received Cy/Flu/DLI as their first treatment of relapse after HCT. The control group included CML patients who received DLIs containing 10 8 T cells /kg (n ϭ 28) and non-CML patients (n ϭ 35) who received DLIs with higher T-cell doses (3 daily lymphapheresis products) from 1993 to 2003. The CML and non-CML patients were analyzed as a single control group because there was no difference in their rat...