Design and validation of a clinically applicable culture procedure for the generation of interleukin-2 activated natural killer cells in human bone marrow autografts.

Previous studies in animal models have suggested that bone marrow-derived interleukin-2 (IL-2) activated natural killer (A-NK*) cells can be used to eliminate residual leukemic cells both in vivo and in vitro. As part of initial studies to develop a clinical protocol to exploit this phenomenon in combination with culture purging of leukemic cells, we examined a number of variables that might affect either IL-2 stimulation of natural killer (NK) cell activity or maintenance of primitive hematopoietic cells in cultures suitable for manipulating human marrow autografts. Cytotoxicity of IL-2 A-NK cells was determined using K562 and Daudi target cells in a standard 4-hour 51Cr release assay. Primitive hematopoietic cells were quantitated using both direct clonogenic progenitor assays and the long-term culture-initiating cell (LTC-IC) assay. The latter measures a very primitive cell type that gives rise to clonogenic cells after > 5 weeks of culture on pre-established marrow feeder layers. Light-density (< 1.070 g/cm3) human marrow cells cultured at 10(6) cells/mL for 7 days at 37 degrees C in the presence of 1000 U of either natural or recombinant human IL-2 showed a marked generation of A-NK cell function that was not seen if IL-2 was not added. The cytotoxicity was the same whether the cells had been maintained in standard fetal calf serum (FCS)-supplemented medium or in LTC medium (which also contains horse serum and 10(-6) M hydrocortisone) or whether adherence of cells during the culture period was promoted or prevented. The level of NK activity in IL-2-stimulated cultures of marrow cells from patients with acute myeloid leukemia (AML) in remission was not significantly different from that obtained with normal marrow. The initial numbers of clonogenic cells in these samples were close to normal values (60%). For both normal and AML patient samples, these declined slightly during the 7-day culture period, regardless of whether IL-2 was present. Starting values for LTC-IC in patient marrow samples were markedly reduced to approximately 7% of normal values, but as in normal cultures, did not decrease further in culture with or without IL-2. These results indicate that activation of bone marrow-derived NK cells can be obtained in 7-day cultures containing IL-2 under conditions that preserve the most primitive hematopoietic cells currently detectable.