A multi-step isolation scheme for obtaining CD16 + human natural killer cells

Abstract

A multi-step isolation scheme capitalizing on negative selection protocols is described for obtaining an enriched population of CD16 + human natural killer (NK) cells. The isolation scheme consists of incubating peripheral blood mononuclear cells (MNC) on nylon wool, rosetting the nylon wool non-adherent cells with sheep red blood cells (SRBCs) for 1 h at 29°C and then utilizing a ‘panning’ technique to remove CD3 +, non-rosetting cells. The final working cell population contained 70–80% CD16 + cells, 15% CD2 + cells, 1–3% CD3 + cells, 5–7% SIg + cells and no detectable MO2 + cells. In comparing the final NK cell population from the multi-step isolation protocol to NK cells obtained by the Percoll density gradient centrifugation technique, the multistep method: (1) yielded a higher percentage of CD16 + cells, (2) mediated a greater degree of cytotoxicity at a 25:1 E:T ratio, and (3) contained fewer contaminating monocytes/macrophages (none were detectable). In addition, the multi-step scheme allowed recovery of 30% of the total CD16 + cells present compared to only 7% recovered by the Percoll density gradient technique. Pretreatment of the enriched NK cells, obtained from the multi-step scheme, with interleukin-2 (3.5 and 7.0 U/ml of activity) resulted in an increase in NK cell-mediated cytotoxicity. In addition, these cells were as effective at synthesizing the cytotoxin, NKCF, at a 25:1 E:T ratio as at 50:1 and 100:1 E:T ratios. This multi-step isolation scheme consistently yields a high percentage of CD16 + NK cells and thus may greatly facilitate studies on the mechanism(s) involved in NK cell-mediated cytotoxicity and may further the study of the cytotoxins involved.