MATERIALS AND METHODS

CTL clones. The CTL clones HG-38, JR-2-19, and JR-2-2 were isolated from T cells of the donors HG (HLA-A2,~ 23; B40,~ 44, Cw4; DRw6, 7) and JR (HLA-A~ 23,~ 29; 87, 7; C-; DR5) as described in detail elsewhere (17, 23, 24). The clones were cultured in Iscove's modified Dulbecco's MEM (GIBCO, Glasgow, Scotland) that contained bovine serum albumin (BSA)(2.5 mgfml), transferrin (35 pg/ml), and insulin (5 pg/ml), all obtained from Sigma Chemical Co., St. Louis, MO, 2 x M ethanolamin (Merck, Darmstadt, West Germany), and 1%(v/v) of a dispersion of lipids extracted from human plasma as follows. The cells (0.5 x 10s per sample) were washed twice in PBS Phenotyping of the CTL clones. Phenotyping of the clones was carried out supplemented with 0.1% BSA and 0.1% N&N and were resuspended in an appropriately diluted sample of monoclonal antibody (OKT3, OKT4, or OKT8, all obtained from Ortho Pharmaceutical Corp.. Raritan, NJ). The cells were incubated during 30 min at OC, washed twice in PBS with 0.1% BSA and 0.1% Na, N, and incubated during 30 min with a fluorescein isothiocyanate (FITC) labeled goat anti-mouse conjugate (Nordic, Tilburg, The Netherlands). The cells were washed three times and analyzed on a fluorescence microscope and a fluorescence-activated cell sorter (FACS-IV, Becton Dickinson, Mountain View, CA).

Target cells for cytotoxic assays. The majority of the EBV-transformed lymphoblastoid cell lines used in this study were established at the Department of Haematology, University Medical Center, Leiden. LG-5, 28, 32, 38 were established by Gatti and Leibold (25) and were made available to us by the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam. Laz 467,470, and 475 were obtained from Dr. E. Yunis, Sidney Farber Cancer Institute, Boston, MA. The monocytes used as target cells were isolated as described by Feighery 8 Stastny (12) with a few modifications. Peripheral Mood lymphocytes were incubated in petri dishes (Falcon plastics, Oxnard, CA) overnight in Iscove's supplemented with 20% autologous serum at 37'C in a humidified atmosphere of 5% C02. The nonadherent cells were removed by repeatedly washing the dish with Iscove's medium. The adherent cells were then collected in Iscove's/20% serum by detaching them with a rubber policeman. These cells were more than 95% viable as judged by trypan blue exclusion, and 80% of these cells were positive for a nonspecific esterase stain (26). Cytotoxicity assay. 0.5 x 10'cells were labeled with 150 &i% r during 45 min at 37% washed three times, and used as target cells in a standard" Cr-release assay that was carried out as follows. Varied numbers of effector cells were mixed with 1000 target cells in Iscove's medium supplemented with 2.5 mg/ml BSA in a final volume of 200 pl per well of a Linbro titertek plate with Ushaped wells. The plate was centrifuged for 5 min at 50 x G and was incubated for 4 hr at 37 C in a humidified 5% Con atmosphere. One hundred microliters were harvested and counted in a gamma counter. The percentage specific lysis was calculated according to the formula: