title: Increased proliferation, lytic activity, and purity of human natural killer cells cocultured with mitogen-activated feeder cells.
abstract: The addition of mitogen-prestimulated periferal blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) cultures to enriched populations of natural killer (NK) cells obtained from PBL of normal donors in the presence of rIL-2 resulted in highly significant increases in proliferation, purity, and cytolytic activity of cultured NK cells. Two sources of enriched NK cell preparations were used: (i) Adherent-lymphokine activated killer (A-LAK) cells obtained by adherence to plastic during 24 hr activation with 10(3) Cetus U/ml rIL-2; and (ii) NK cells negatively selected from PBL by removal of high-affinity rosette-forming cells and CD3+ lymphocytes. Coculture of A-LAK cells for 14 days with autologous or allogeneic Con A-activated PBL (10(6) cells/ml) or selected EBV-transformed LCL (2 x 10(5) cells/ml) as feeder cells increased fold expansion by a mean +/- SEM of 629 fold +/- 275 (P less than 0.019) and 267 fold +/- 54 (P less than 0.0001), respectively, compared to 55 +/- 20 in A-LAK cultures without feeder cells. The addition of either activated PBL or EBV lines to A-LAK cultures also led to a significant increase in the percentage of NK cells (CD3- CD56+) (84 +/- 2.4 and 84 +/- 2.6%, respectively, P less than 0.0001 for both), compared to 53 +/- 7.2% in cultures without feeders. The presence of feeder cells in cultures of A-LAK cells also led to significantly higher anti-tumor cytolytic activity compared to control cultures, as measured against NK-sensitive (K562) and NK-resistant (Daudi) target cells. Mitogen-stimulated CD4+ PBL purified by positive selection on antibody-coated flasks were better feeders than CD8+ or unseparated PBL. In the presence of feeder cells, it was possible to generate up to 6 x 10(9) activated NK cells from 2 x 10(8) fresh PBL by Day 13 of culture. Enhanced NK cell proliferation in the presence of feeder cells was not attributable to a detectable soluble factor. The improved method for generating A-LAK or activated-NK cells should facilitate cellular adoptive immunotherapy by providing sufficient numbers of highly enriched CD3- CD56+ effector cells with high anti-tumor activity.
authors.affiliation: Pittsburgh Cancer Institute, Pennsylvania.
authors.forename: H
authors.initials: H
authors.lastname: Rabinowich
authors.forename: P
authors.initials: P
authors.lastname: Sedlmayr
authors.forename: R B
authors.initials: RB
authors.lastname: Herberman
authors.forename: T L
authors.initials: TL
authors.lastname: Whiteside
mesh_terms: D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis; D017252:CD3 Complex; D015704:CD4 Antigens / Q000032:analysis; D019002:CD56 Antigen; D016827:CD8 Antigens; D002478:Cells, Cultured; D003208:Concanavalin A / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000276:immunology*; D008213:Lymphocyte Activation; D011948:Receptors, Antigen, T-Cell / Q000032:analysis
chemical_list: D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D015704:CD4 Antigens; D019002:CD56 Antigen; D016827:CD8 Antigens; D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell; D003208:Concanavalin A