We characterize the natural killer (NK) cell colony-inhibiting activity (CIA) produced in supernatants from cultures of human peripheral blood lymphocytes (PBL) with NK-sensitive target cell lines, and study its relationship with NK cell-derived cytotoxic factor (NKCF). Using monoclonal antibodies (mAb) specific for NK cells or other lymphocyte populations, we unambiguously identify NK cells as the only PBL subset able to produce both NKCF and NK-CIA. We present functional and biochemical data suggesting that NKCF and NK-CIA represent the same molecule: (a) a highly significant positive correlation exists between the quantity of NKCF and NK-CIA in supernatants independently produced by different PBL subsets; (b) both NK-CIA and NKCF are induced by culture of PBL with NK-sensitive, but not with NK-insensitive cell lines, and with HLA-DR+ bone marrow cells; (c) both NKCF and NK-CIA are absorbed on the same cell lines or bone marrow cell types; (d) the two activities coelute in the same gel filtration fractions; (e) D-mannose-6-phosphate blocks both NKCF and NK-CIA activity, and prevents their absorption by K562 cells; and (f) both NKCF and NK-CIA activity are lost after 2 d at 37 degrees C. The NK-CIA-containing preparations are devoid of antiviral activity, and antiinterferon (anti-IFN) antibodies do not block the inhibitor activity of NK-CIA. The effect of NK-CIA on day 14 (early) colony-forming units of granulocytes and macrophages (CFU-GM) is synergistic with that of IFN-gamma, and this synergy is also evident on day 7 (late) CFU-GM growth. A combination of NK-CIA and IFN-gamma suppresses late CFU-GM, at concentrations of the two lymphokines that are completely ineffective when used independently. No synergy between NK-CIA and IFN-alpha or -beta was observed, due to a direct inhibitory effect of these two IFN types on late CFU-GM. Antibodies specific for tumor necrosis factor (TNF), but not those specific for lymphotoxins, inhibit both NK-CIA and NKCF activity in the NK cell-derived supernatant. Recombinant TNF, in the range of concentrations corresponding to that of the cytotoxic activity on L-929 cells present in supernatants, mediated both NKCF and NK-CIA activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Incubation of human peripheral blood lymphocytes with bone marrow cells resulted in significant inhibition of colony formation by committed myeloid and erythroid cells. Using positively selected homogeneous natural killer (NK) cell preparations and lymphocyte subpopulations depleted of or enriched for NK cells, we definitively characterize as NK cells the cells in normal peripheral blood that are responsible for inhibition of bone marrow colony growth. The inhibitory effect of NK cells on hematopoiesis can be mediated by a soluble factor that is produced only by NK cells upon culture with HLA-DR+ hematopoietic cells and with NK-sensitive cell lines. Both NK cells and the NK-produced, colony-inhibiting activity (NK-CIA) are suppressive for allogeneic and autologous bone marrow CFU-GEMM (colony-forming units, granulocyte, erythroid, monocyte, megakaryocyte), CFU-E (CFU, erythroid), and early CFU-GM (CFU, granulocyte, monocyte), but not for either BFU-E (burst-forming units, erythroid) or late CFU-GM. [3H]Thymidine incorporation was inhibited by NK-CIA-containing supernatants in HLA-DR+ but not HLA-DR- bone marrow cell populations stimulated to proliferative by colony-stimulating factor (CSF). These data suggest that the NK cell-mediated inhibitory effect on proliferation and differentiation of hematopoietic precursor cells is mediated in part or completely by the secreted NK-CIA. The concentration of NK-CIA reached in the supernatant of the mixture of NK cell-containing lymphocyte populations with bone marrow cells is sufficient to account for the inhibitory effect mediated by NK cells. Our data support the hypothesis that human NK cells play a major role in the control of hematopoiesis, down-regulating it under conditions in which the NK cells are functionally activated.

Methotrexate with leucovorin rescue (HDMTX-LV rescue), has been used to treat solid tumors and non-Hodgkin's lymphomas (NHL). We studied the use of HDMTX-LV rescue in patients with widespread NHL with histologic diagnosis of diffuse poorly differentiated lymphocytic and diffuse histiocytic including involvement of the central nervous system (CNS) and/or bone marrow. The prognosis with conventional chemotherapy is extremely poor. Three patients have bone marrow involvement, 2 patients CNS, and 2 both. These patients were unresponsive to conventional chemotherapy and had a rapid progression of their disease. Therapy with HDMTX-LV rescue induced responses in 5 patients: 2 patients achieved a complete remission and three a partial remission. Regression of CNS involvement was observed in 3 patients; bone marrow toxicity was not observed. Only 1 patient failed to respond. These data suggest that HDMTX-LV rescue may be useful as primary therapy in lymphoma patients with CNS and/or bone marrow involvement.

Bone marrow cells of a 45-year-old female with Philadelphia chromosome (Ph1)-positive, early-phase chronic myelogenous leukemia (CML), who was heterozygous for the glucose-6-phosphate dehydrogenase (G6PD) locus, were pretreated in vitro with 4-hydroperoxycyclophosphamide (4-HC) and tested for G6PD activity in several colony formation assays and for karyotypic abnormalities. All cells within the mixed (CFU-GEMM), the erythroid burst (BFU-E), and the granulocyte-macrophage (CFU-GM) colonies expressed type A and type B G6PD activity and a normal karyotype, whereas untreated cells expressed type A G6PD and the Ph1 chromosome. This reversal of G6PD activity type and the disappearance of the Ph1 chromosome in colonies grown from 4-HC-treated cells indicate that this cytotoxic agent spares a residual normal stem cell population in bone marrow cells of early-phase CML patients. This finding, in turn, suggests a therapeutic approach in CML based on in vitro chemotherapy of autologous bone marrow grafts.

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