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Separation and Functional Studies of the Human Lymphokine-Activated Killer Cell Kevan Roberts, Michael T

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Separation and Functional Studies of the Human Lymphokine-Activated Killer Cell Kevan Roberts, Michael T [CANCER RESEARCH 47, 4366-4371, August 15, 1987] Separation and Functional Studies of the Human Lymphokine-activated Killer Cell Kevan Roberts, Michael T. Lotze,1 and Steven A. Rosenberg Surgery Branch, National Cancer Institute, NÃŒH,Betnesda,Maryland 20892 ABSTRACT undefined. The purification of cells responsible for LAK activity was desirable to further assess their morphology, phenotype, Cell separation studies were undertaken in an attempt to purify the and target cell binding properties prior to and following IL-2 lymphokine-activated killer (LAK) precursor cell. Null cells, prepared stimulation. by the sequential depletion of monocytes, T- and B-lymphocytes from human peripheral blood mononuclear cells, were found to be potent mediators of LAK activity. Such preparations were Leu-11* but Leu-4" MATERIALS AND METHODS and displayed high levels of natural killer activity. Incubation of these cells with recombinant interleukin 2 (11-2) for periods in excess of 24 h Media. All cell lines were maintained in suspension culture using RPMI 1640 supplemented with 10% FBS, 100 ng/m\ penicillin, 100 induced LAK lysis of fresh tumor targets which were resistant to lysis Mg/ml streptomycin, and 1% glutamine. CM consisted of RPMI 1640 by unstimulated null effectors. In contrast, lymphocytes which formed high affinity rosettes with sheep RBC (I-* lymphocytes) were poor supplemented with 10% human AB serum, antibiotics, and glutamine. Conjugation buffer, at pH 7.3, was prepared by adding 683 mg of NaCl, mediators of both natural killer and LAK activity. Interleukin 2 stimu lated null cells, retained a Leu-ll*, I.t-u-4 phenotype, and expressed 142 mg of MgCl2 6H2O, and 66 mg of CaCl2-6H2O to 450 ml of PBS supplemented with 50 ml of FBS, antibiotics, and 10 HIM4-(2-hydrox- only low levels of receptors for II,-2 and transferrin. An increase in the yethyl)-1 -piperazineethanesulfonic acid. number of binding sites, on null cells but not on T-cells, for Vicia villosa lectin with 11-2 stimulation was noted. Following II -2 stimulation, null Preparation of Lymphocyte Populations. PBMC were prepared from and T-cells were able to conjugate to K562 and fresh tumor but not to leukopheresis samples, obtained from healthy volunteer donors, by flotation on LSM (Litton Bionetics, Kensington, MD). Nonadherent autologous lymphoblast targets. PBL were subsequently isolated by sequential adherance of PBMC to plastic and nylon wool as described previously (9). PBL was resetted by mixing 4 x 10* PBL with 4 x 10'°sheep RBC INTRODUCTION suspended in 10 ml of 90% FBS-10% RPMI. The suspension was The LAK2 phenomenon represents a novel cytotoxic function centrifuged (150 x g for 7 min) and incubated on ice for 60 min. The pellet was subsequently gently resuspended by adding 30 ml of ice cold which is induced following stimulation of PBL with 112 (1). RPMI and separating rosetting (E+) from nonrosetting (E~) cells by The LAK effector cell is capable of lysing a wide selection of flotation on LSM. Briefly, 20 ml of suspended rosettes were layered fresh tumor targets (1,2), and has been applied in the adoptive over 20 ml of LSM. The gradient was centrifuged at 150 x g for 5 min immunotherapy of neoplastic disease (3). Highly purified 112 and then at 600 x g for 20 min to ensure a good separation. After has been demonstrated to be sufficient to induce LAK cytotox- washing both fractions (450 x g for 10 min) contaminant sheep RBC icity from normal donor peripheral blood mononuclear cells were removed by hypotonie lysis with the use of sterile distilled water. The T- and B cells in the E~ lymphocyte preparations were removed (4), and there appears to be no requirement for any other lymphokines or accessory cells. Furthermore, LAK cytotoxicity by treatment with monoclonal antibodies OKT3 (reactive with mature appears not to be restricted by MHC determinants since cell T-cells), HB104 (reactive with a monomorphic portion of the DR lines failing to express MHC antigens and virtually all alloge- antigen), and complement. E-lymphocytes were suspended at 20 x IO6/ ml in ice cold RPMI, both antibodies were added to the suspension neic tumors (i.e.. MHC class I disparate) are susceptible to lysis (antibodies were used as ascitic fluids at a final dilution of 1:100), and (2, 5). This raises the possibility that these cells could be used incubated for 30 min at 4 ('. After a single wash, the cells were in therapeutic protocols where the tumor fails to express class resuspended in complement (Cedarlane, Hornby, Ontario, Canada), I MHC gene products (6), a prerequisite for recognition by diluted 1:5 with the RPMI to their original volume, and incubated for cytotoxic T-cells. 60 min at 37°C.After washing, the cells were retreated with antibody The cell responsible for LAK activity appears distinct from as above prior to a final wash and suspension with complement. monocytes and mature T-lymphocytes insofar as the precursor Following a 30-min incubation at room temperature the cells were is nonadherent, T3~, and does not readily form E-rosettes (5). washed and viable cells were isolated by flotation on LSM. LAK activity was generated from separated lymphocyte populations Although capable of lysing tumor cells not normally sensitive by incubating cells at llC'/ml in complete medium supplemented with to NK lysis, LAK precursors do have characteristics in common 1000 units/ml RIL-2 (Cetus Corp., Emeryville, CA). Incubations were with NK cells. We have observed only low levels of lysis (and performed in 24-well cluster plates for 3 days. usually no lysis) of fresh tumors by highly enriched populations Cytotoxicity Assays. Cytolytic activity of separated lymphocytes was of LAK precursors prior to IL-2 stimulation. Most significantly, evaluated against fresh tumor [prepared as detailed previously (2)], and both activities are associated with cells which express receptors cultured targets by 4-h 5lCr release assays over a range of effectontarget for the Fc portion of IgG of the type found on neutrophils (7, ratios. Labeled target cells (5 x IO3)were mixed with a variable number 8). Despite such close associations the exact relationship be of effector cells (final volume, 200 i<I) in round bottom 96-well micro- tween the two activities and the cells mediating them remains liter plates (Dynatech Laboratories, Alexandria, VA). Following cen- trifugation (80 x g for 5 min) the plates were incubated for 4 h at 37*C Received 2/13/87; revised 5/21/87; accepted 5/25/87. prior to harvesting supernates by using the Skatron harvesting system The costs of publication of this article were defrayed in part by the payment (Flow Laboratories, Rockville, MD). Spontaneous and maximum 5lCr of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. releases were determined by incubating targets in medium or 1% sodium 1To whom requests for reprints should be addressed. dodecyl sulfate, respectively. Each assay was set up in triplicate, and 2The abbreviations used are: LAK, lymphokine-activated killer; 112. interleu cytotoxicity values were calculated by using the formula: kin 2; RIL-2, recombinant IL-2; FBS, fetal bovine serum; HE, hydroethidine; IL- 2R, interleukin 2 receptor, LGL, large granular lymphocyte, LSM, lymphocyte Test 51Cr release - spontaneous s'Cr release separation medium; PBL. peripheral blood lymphocytes; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; SFDA, sulfofluorescein di- Maximum "Cr release - spontaneous 5lCr release acetate; MHC, major histocompatibility complex; NK, natural killer; E* cells, rosetting E cells; E~ cells, nonrosetting E cells; CM, complete medium. from which means and standard deviations were derived. 4366 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1987 American Association for Cancer Research. HUMAN LAK CELLS Membrane Antigen Phenotype. Lymphocytes were phenotyped by E+ fraction, which formed 81-92% of the cells, displayed only direct immunofluorescence with the use of a variety of fluorescein- low levels of LAK activity. Consequently, the depletion of E+ labeled monoclonal antibodies (Becton Dickinson, Mountain View, cells rosetting at 4°Cformed a simple and efficient means of CA). Lymphocytes which had been treated with monoclonal antibodies enriching for the LAK precursor. and complement were cultured for 18 h in CM prior to phenotypie- Phenotypic analysis of the E~ fractions revealed a high level analysis. Such preparations were shown not to have identifiable murine immunoglobulin on their plasma membranes by staining with a flu- of contamination since both DR-positive cells (presumably B- oresceinated goat anti-mouse IgG and IgM (Boehringer Mannheim lymphocytes and contaminant monocytes and Leu-4-positive Biochemicals, Indianapolis, IN). Cells (10*) were incubated first with cells (T-lymphocytes) were demonstrated (30-60%). Contami heat-inactivated human AB serum at room temperature for 10 min, nant cells were removed from E-lymphocytes by using specific and following a single wash, were stained at 4'C with the appropriate antibodies (OKT3 and HB104) and complement. The remain amount of antibody for 30 min prior to 3 washes and subsequent ing cells, designated null cells, mediated 2 to 7 times the LAK analysis. Fluoresceinated lectins used to stain lymphocytes (Sigma activity of the E-lymphocytes (Table 1) and contained little T- Chemical Co., St. Louis, MO) were used at saturating concentrations. or B cell contamination. Cytofluorographic analysis was performed by analysis of IO4cells by Prior to IL-2 incubation, E~ and null cells mediated higher using a fluorescence-activated cell sorter 440 (Becton Dickinson) as levels of NK activity (i.e., lysis of K562 targets) than the PBL previously described (10). from which they were derived (Fig. 1). In contrast, E+ lympho Conjugation Assay.
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