Adherent lymphokine-activated killer cells suppress autologous human normal bone marrow progenitors

We have generated a homogeneous population of recombinant interleukin-2 (rIL-2)-stimulated effector cells termed adherent lymphokine-activated killer cells (A-LAK) from peripheral blood mononuclear cells (PBMNC) of 14 normal individuals and tested the effect of A-LAK cells on autologous hematopoietic bone marrow (BM) progenitor growth. Enrichment of A-LAK from PBMNC depended on the propensity of A-LAK precursors to adhere to plastic and proliferate in the presence of rIL-2. The resultant population had the morphologic appearance of large granular lymphocytes, and the majority of cells (73% ± 4%) expressed the CD56⁺/CD3^- phenotype associated with rIL-2-stimulated natural killer (NK) cells. The A-LAK population had potent lytic activity in chromium release assays against both NK-sensitive (K562) and NK-resistant (Raji) targets. When BM mononuclear cells (BMMNC) were coincubated with autologous A-LAK and rIL-2 (1,000 U/mL) added at the start of culture, dose-dependent suppression of burst-forming unit-erythroid (BFU-E) and colony-forming unit mix (CPU-MIX) colony growth was observed at effector to target ratios (E:T) ranging from 0.25:1 to 5:1 (maximal suppression BFU-E = 85% ± 6%; CFU-MIX = 95% ± 3%). This suppression was rIL-2 dose-dependent, and no suppression was seen in the absence of rIL-2. Depletion of BM monocytes and T lymphocytes did not alter A-LAK suppression of progenitors coincubated with A-LAK cells. Addition of polyclonal neutralizing antibodies against both interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) to the coincubation culture completely abrogated the suppressive effect of A-LAK on BFU-E and CPU-MIX colony growth while each neutralizing antibody used alone had intermediate effects. In contrast to coincubation studies, 36 hours of preincubation of A-LAK cells with autologous BM (E:T 2.2:1) and rIL-2 (1,000 U/mL) followed by plating of preincubated BM cells in hematopoietic progenitor culture produced significant suppression of day 14 BFU-E (47% ± 5%), but spared the more primitive CPU-MIX (7% ± 9%), suggesting a divergent effect of A-LAK cells on hematopoietic progenitors at different stages of differentiation. Addition of neutralizing antibodies against IFN-γ and TNF-α in preincubation failed to abrogate the suppressive effect of A-LAK on BFU-E colony growth, suggesting that this suppression occurs by a different mechanism than that seen in coincubation studies. Previous studies have demonstrated that the A-LAK population has cytotoxic and proliferative advantages over other killer cell populations. Here we demonstrate that A-LAK have a suppressive effect on autologous BM progenitors under some conditions that is dependent on rIL-2 stimulation, may not require accessory cells, and in coincubation may be mediated by IFN-γ and TNF-α. Suppression of more primitive, multilineage progenitors can be avoided even at high E:T ratios by preincubation with rIL-2-stimulated A-LAK over relatively short intervals. This information will be useful in developing strategies for purging of tumor-contaminated marrow or for in vivo therapy of malignant disorders with autologous A-LAK cells.