title: 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 degrees 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.
authors.affiliation: Department of Immunology and Cancer, Cleveland Clinic Foundation, OH.
authors.forename: L M
authors.initials: LM
authors.lastname: Cosentino
authors.forename: M K
authors.initials: MK
authors.lastname: Cathcart
mesh_terms: D000954:Antigens, Surface / Q000032:analysis*; D002469:Cell Separation / Q000379:methods; D002499:Centrifugation, Density Gradient; D003602:Cytotoxicity, Immunologic; D005434:Flow Cytometry; D006801:Humans; D007111:Immunity, Cellular; D007113:Immunity, Innate; D007376:Interleukin-2 / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000166:cytology*; D055646:Killer Factors, Yeast; D016169:Lymphocyte Function-Associated Antigen-1; D014176:Protein Biosynthesis; D011506:Proteins; D012397:Rosette Formation
chemical_list: D000954:Antigens, Surface; D007376:Interleukin-2; D055646:Killer Factors, Yeast; D016169:Lymphocyte Function-Associated Antigen-1; D011506:Proteins