A procedure for enrichment in recombinant interleukin-2 (rIL2)-activated natural killer (NK) cells was developed and used for in vitro generation of antitumor effector cells from the peripheral blood of 20 patients with central nervous system (CNS) tumors. In comparison to the patients' unseparated mononuclear cells and nonadherent lymphocytes cultured in the presence of 1000 U/ml of rIL2 for up to 3 weeks, interleukin-2-stimulated lymphoid cells, when purified by adherence to plastic, proliferated better (up to 6,720-fold expansion) and achieved up to five times higher levels of antitumor activity against K562 cell targets and NK-resistant glioblastoma cell targets. Two-color flow cytometry analysis showed that cultures of cells purified by adherence to plastic which had the best proliferation contained 10% or less of CD3+Leu19-T-lymphocytes, while the unseparated lymphokine-activated killer cell cultures which proliferated poorly contained up to 85% of CD3+Leu19-T-cells. Cultures of adherent lymphocytes which reached the highest antitumor cytotoxicity were enriched in CD3+Leu19+ effectors (60–80%); the proportion of CD3-Leu19+ NK-cells was not greater than 25% in these cultures. Thus, using the technique of 24- or 48-h activation in rIL2 and adherence to plastic, and in contrast to the results obtained with cells from normal donors, it was not possible to enrich in activated NK cells from the blood of patients with CNS tumors. Instead of activated NK cells, a population enriched in non-major histocompatibility complex-restricted cytotoxic T-cells (CD3+Leu19+) was obtained in cultures from most but not all patients. Low NK cell activity and elevated numbers of circulating CD3+Leu11+ cells seen in the blood of these patients, previously treated by surgery/radiation/chemotherapy and maintained on steroids, could be responsible for the preferential adherence and subsequent expansion to plastic of IL2-activated non-major histocompatibility complex restricted cytotoxic T-lymphocytes.

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