A simple method for the purification and rapid expansion of large granular lymphocytes into cells with efficient broad antitumor cytotoxicity after stimulation by human rIL-2 is described. Nylon-wool nonadherent splenic mononuclear leukocytes from Fischer 344 rats were cultured in medium containing 1,000 U/ml rIL-2. The initial response of a small subpopulation of cells (less than 2%) to rIL-2 was their adherence to the plastic surface. This response was noted as soon as 2 h after addition of rIL-2. 2-h rIL-2-activated plastic adherent lymphocytes were 90-98% LGL, expressed surface markers characteristic of rat NK cells (OX8 [CD8]+, asialo GM1, laminin+, OX19 [CD5]-, R1-3B3 [CD5]-, W3/25 [CD4]-, OX39 [CD25]-, Ia-, and Ig-), and expressed very high levels of cytotoxicity against YAC-1 target cells. In addition to the above markers, plastic-adherent LGLs obtained at 24, 48, or 72 h progressively expressed Ia surface antigens, but were not phagocytic and contained less than 1% monocytes/macrophages by morphology. When 24- or 48-h plastic-adherent LGL/NK cells were cultured over 3-4 d in rIL-2, the cells expanded between 30- and 100-fold, reaching densities between 2-3 X 10(6) cells/ml. These rapidly expanding LGL/NK cells also generated very high levels of LAK activity (including lysis of fresh NK-resistant solid tumor cells), expressed a phenotype characteristic of activated rat NK/LAK cells, and incorporated [3H]TdR into DNA. This technique not only provides a novel method for the purification of LGL/NK cells for in vitro studies but also provides a means for the rapid expansion of highly purified cells with high levels of broad antitumor (LAK) cytotoxicity.

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