Human Natural Killer Cells, Activated Lymphocyte Killer Cells, And
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Proc. Nati. Acad. Sci. USA Vol. 80, pp. 7606-7610, December 1983 Immunology Human natural killer cells, activated lymphocyte killer cells, and monocytes possess similar cytotoxic mechanisms (monoclonal antibody/cytotoxic factor/inhibition of killing) GORDON F. BURNS, TONY TRIGLIA, PERRY F. BARTLETT, AND IAN R. MACKAY The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria 3050, Australia Communicated by F. M. Burnet, August 19, 1983 ABSTRACT The relationship between the killing mechanisms killing and then tested for its effect on the cytolytic activity of of human natural killer (NK) cells, mitogen- and mixed-lympho- ALK cells and, for comparison, MHC-restricted cytotoxic T cells cyte-culture-induced activated lymphocyte killer (ALK) cells, and and cultured monocytes. The results suggest that killing by ALK monocytes was investigated with a monoclonal antibody. The IgG2 cells, NK cells, and monocytes depends on similar mecha- antibody 9.1C3 was prepared from mice immunized with purified nisms. human large granular lymphocytes and selected from clones that inhibited NK cell killing. The 9.1C3 antibody bound to all mono- MATERIALS AND METHODS nuclear cells but not to granulocytes or K562 cells, and it selec- tively blocked killing of K562 targets by both NK and ALK cells Preparation of Lymphocytes and Large Granular Lympho- without affecting the binding of effector to target cells. The an- cytes (LGL). Peripheral blood mononuclear cells (MNC) were tibody blocked killing when present from time zero and-it still in- obtained from the blood of normal subjects by Ficoll/Hypaque hibited partially even when added 1 hr after initiation of the lytic centrifugation. Monocytes were removed from the MNC by reaction. Killing of Epstein-Barr virus-transformed B lympho- plastic adherence, and enrichment for LGL was carried out by blasts by classical cytotoxic T lymphocytes was not inhibited. Of centrifugation over discontinuous Percoll gradients by a mod- interest, 9.1C3 did block the killing of K562 target cells by cul- ification of a standard method (12). tured peripheral blood monocytes. Other monoclonal antibodies Preparation of Monoclonal Antibodies. BALB/c mice (main- that bound to monocytes did not block killing, and a nonspecific tained at The Walter and Eliza Hall Institute) were immunized effect of the antibody on monocytes was excluded. These data sug- by three intraperitoneal injections of 107 LGL over a period of gest that NK cells, ALK cells, and monocytes can kill tumor cell 30 days and a further intravenous boost 1 week later. After 3 targets by using similar lytic mechanisms. days spleen cells were fused with NS-1 cells by using 50% (vol/ vol) polyethylene glycol 4000 and hybrids were selected with The concept of immune surveillance over the emergence of hypoxanthine, aminopterin, and thymidine (HAT). The hybri- cancer has stimulated much investigation into the possible un- doma supernatants were tested for blocking antibodies by re- derlying mechanisms (1, 2). Since the realization that natural duction in 51Cr release in cytotoxic assays (see below). The cells killer (NK) cells may play an important role in combating virus secreting supernatant showing significant blocking of K562 lysis infections and tumor cell growth there has been considerable by NK cells were cloned at 1 cell per well, then recloned twice interest in these cells and, as a result, several mechanisms of at 0.3 cell per well on a feeder layer of thymocytes. Antibody NK-mediated lysis of tumor cells have been proposed (3-6). 9. 1C3 was purified from ascites fluid by affinity chromatog- Also of possible relevance in the prevention of tumor cell raphy with Sepharose-coupled staphylococcal protein A, and a growth in vivo is the activated (lymphocyte) killer (ALK) cell radioimmunometric assay employing "2I-labeled class-specific (7). Like NK cells, ALK cells can recognize and kill certain tu- sheep antibodies and control mouse monoclonal antibodies of mor cell targets without prior sensitization and without major known isotype (supplied by G. Morahan) was used to type 9.1C3 histocompatibility complex (MHC) restriction; however, at a as an IgG2 antibody. given effector-to-target ratio ALK cells are much more effective Induction of Cytotoxic T Cells (CTC) and ALK. CTC were killers and in addition they can rapidly lyse certain tumor cell established from MNC of normal subjects by stimulating 2 X targets, including autologous tumor cells, that are resistant to 106 MNC in 2 ml of RPMI 1640 medium containing 10% fetal freshly isolated NK cells (8). The nature and origin of ALK cells calf serum and 50 ,uM 2-mercaptoethanol with irradiated al- is disputed. They can be generated from blood mononuclear logeneic or autologous B lymphoblastoid cells at a MNC-to- cells in vitro by mixed cell culture, or with mitogens, and can stimulator-cell ratio of 50:1 for 10 days (13). Previous studies be maintained in culture with lymphokines, probably T-cell have demonstrated that this latter protocol results in the recall growth factor (7, 8). ALK cells express the T-cell membrane of memory CTC specific for Epstein-Barr virus (EBV)-trans- antigens T3 and T8 and can be generated in vitro from NK-de- formed lymphoblasts (13, 14). The definition of ALK cells is pleted mononuclear cells (7-9). Because of this they have been essentially operational, and the cultures that were used to in- described as activated T lymphocytes (10). However, studies on duce CTC also generated high levels of ALK cells; CTC were precursor cell depletion suggest that ALK cells are derived from identified by their killing of the stimulating B lymphoblasts and NK cells (11) or from a cell lineage distinct from both T lym- ALK cells were measured by the killing of K562 target cells. phocytes and NK cells (8). Mitogen-activated MNC were also used to generate ALK cells To investigate the mechanism of tumor cell killing by ALK in the absence of specific CTC by stimulating MNC with con- cells we prepared a monoclonal antibody that blocked NK cell Abbreviations: ALK, activated lymphocyte killer; CTC, cytotoxic T cell(s); The publication costs of this article were defrayed in part by page charge EBV, Epstein-Barr virus; LGL, large granular lymphocytes; MHC, ma- payment. This article must therefore be hereby marked "advertise- jor histocompatibility complex; MNC, mononuclear cells; NK, natural ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. killer. 7606 Downloaded by guest on September 28, 2021 Immunology: Bums et al. Proc. Nati. Acad. Sci. USA 80 (1983) 7607 ditioned medium containing trace amounts of phytohemagglu- tinin and maintaining the cells for 14 days with phytohemag- glutinin-free T-cell growth factor; such ALK cells have a T-cell phenotype and there are no identifiable cells with the markers of NK cells (15). 70 --------------------------- Cytotoxicity Assay. A 51Cr release assay was employed (14). Target cells were labeled with 51Cr, 100 j1l at 105 cells per ml 601- was placed into the wells of V-bottomed microtiter plates, and different concentrations of effector cells were added in 100 ul. 50 F Plates were centrifuged (400 X g) for 1 min and incubated at 370C for 3-4 hr before recentrifugation. One hundred microli- ters of supernatant was removed from each well and the re- 30 leased radioactivity was measured in a gamma counter, and the 0O results of triplicate tests were calculated as mean percent spe- CL 0 30 cific lysis (14). In antibody blocking tests the effector cells were (n added to the wells in 50 S4 together with 50 .l of test or control 20V antibody before the addition of labeled target cells. Cell Lines. The cell lines used as target cells were K562, an erythroleukemic cell line, the melanoma cell line LiBr, and B 10 lymphoblastoid cells grown from the peripheral blood of nor- mal subjects by infection with EBV. All of the cell lines were 5x105 5x104 5x103 500 50 5 0.5 demonstrated to be free of mycoplasma (16). Antibody Concentration (ng/ml) Antibodies. The broad specificities of the monoclonal anti- bodies used are given later. The OK reagents were purchased FIG. 1. Inhibition of NK cell-mediated killing of K562 targt cells from Ortho Pharmaceutical (Raritan, NJ) and the Leu reagents by antibody 9.1C3. Shown is the mean specific lysis (±SD) of 'Cr-la- from Becton Dickinson (Sunnyvale, CA). The NIMP-R10 and beled target cells by 5 x 10' effector MNC at an effector-to-target ratio of 50:1 in the presence of dilutions of purified 9.1C3 antibody to the MAC-1 antibodies were a gift from A. L6pez (Walter and Eliza final concentration given. The broken line is the control in the absence Hall Institute), 3A1 from B. F. Haynes (Duke University, Dur- of antibody. The dose-response experiment was repeated with tissue ham, NC) and A. S. Fauci (National Institutes of Health, Be- culture supernatant and with ascites fluid containing 9.1C3, and the thesda, MD), FMC 17 from H. Zola (Flinders Medical Centre, results were similar. Adelaide, Australia), A2 and T200 from I. F. C. McKenzie (University of Melbourne, Melbourne, Australia), and UCHT1 treated with 9.1C3 and washed before assaying in the standard and UCHT3 from P. C. L. Beverley (University College Hos- cytotoxicity test, but it did block when the effector cells were pital, London). allowed to react with antibody and washed prior to testing. When purified LGL that contained most of the NK cell activity of RESULTS MNC were tested for conjugate formation, visual examination Blocking of NK Cell Function. Purified 9. 1C3 antibody in showed that the presence of 9.1C3 did not inhibit binding to the absence of complement was tested for its ability to block the targets; indeed, in one experiment, enhanced effector-tar- killing of K562 cells by freshly isolated MNC.