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Generation of Lymphokine-Activated Killer Cells Proc. Nati. Acad. Sci. USA Vol. 85, pp. 6875-6879, September 1988 Immunology Generation of lymphokine-activated killer cells: Synergy between tumor necrosis factor and interleukin 2 (large granular lymphocytes/lymphokiue receptors) SALEM CHOUAIB*, JACQUES BERTOGLIO, JEAN-YVES BLAY, CARMEN MARCHIOL-FOURNIGAULT, AND DIDIER FRADELIZI Laboratoire d'Immunologie, Institut Gustave Roussy, 94805 Villejuif, France Communicated by Jean Dausset, March 28, 1988 ABSTRACT Large granular lymphocytes (LGL) can be non-Tac IL-2 binding site (p75) has been reported by several activated by interleukin 2 (IL-2) to lymphokine-activated groups, suggesting that the p75 antigen is associated with the killers (LAK). The effect of tumor necrosis factor (TNF) on p55 Tac to form the high-affinity receptor complex (12-14). LAK generation was investigated. TNF was found to act Furthermore, it was demonstrated that the p75 receptor synergistically with low concentrations of IL-2 (0.10-0.25 subunit but not the Tac subunit is expressed constitutively on ng/ml), which were ineffective by themselves in inducing LAK freshly isolated LGL (15) and is suspected to explain LAK activity, to promote the differentiation of LGL into non-major activation by IL-2. histocompatibility complex-restricted killers. When IL-2 was In the present report, we investigated the possible inter- used at concentrations optimal for LAK generation, TNF did action between IL-2 and TNF on LGL to generate LAK. We not further enhance this phenomenon. Specific binding of demonstrate that, despite their inability to induce LGL into '25I-labeled TNF to LGL was increased by IL-2 stimulation. LAK effectors, low doses of IL-2 are efficient for TNF Scatchard analysis ofTNF binding revealed the existence oftwo receptor induction on LGL and appear to be sufficient for classes of binding sites with markedly different affinities (Kd LAK activity development when used in conjunction with values of 57 and 600 pM). We also demonstrated that the exogenous TNF. We therefore provide evidence for a func- IL-2/TNF synergistic induction ofLAK activity did not involve tional interaction between TNF and IL-2 on LGL to generate either IL-1 or interferon-y. This IL-2/TNF synergistic effect LAK. was blocked by anti-Tac antibodies. Immunofluorescence anal- ysis revealed that IL-2/TNF selectively up-regulated Tac MATERIALS AND METHODS antigen expression on LAK precursors. Our results suggest a functional interaction between IL-2 and TNF on LAK precur- Cell Preparations. Human peripheral blood mononuclear sors, which results in a reduction of the IL-2 concentration cells were obtained by leukapheresis of normal blood donors required for differentiation of LGL into LAK killers. (Blood Bank, Hospital Saint Louis, Paris) and were separated on Ficoll/Hypaque. After a 1-hr adherence to plastic at 370C Tumor necrosis factor (TNF) is a cytokine produced by in 5% C02, nonadherent cells were loaded over a discontin- activated mononuclear phagocytes (1, 2). In addition to its uous Percoll (Pharmacia Fine Chemicals, Bois d'Arcy, direct cytotoxic/cytostatic effects in vitro and its antitumoral France) gradient consisting of2-ml layers of31, 34, 37, 41, 45, activity in vivo (2, 3), TNF has been shown to possess and 47% Percoll and were centrifuged 30 min at 1800 rpm (600 pleiotropic effects not on neoplastic x g). LGL were recovered from the low-density fraction and only cells but also on were further purified by removal of contaminating T cells normal cells. Recently, several studies have shown that TNF with anti-T3 antibody plus complement treatment. The re- has a broad range of biological activities, including modula- sulting LGL preparations (5% of the initial peripheral blood tory effects on growth (4), proliferation (5), and differentia- mononuclear cell preparation) contained >90%o LGL. tion (6). Like other cytokines, the action of TNF requires Source of Lymphokines and Antibodies. Highly purified specific binding to cell surface receptors, which are ex- recombinant IL-2 (rIL-2) (>95%) used in this study was pressed not only on most malignant cells but also on normal kindly provided by Sanofi (Bio-recherches, Labege, France; cells (7, 8). Lymphokines form a network of regulatory specific activity = 23.3 x 106 units/mg of protein). Highly signals, and several lines ofevidence suggest the existence of purified (>99%o) recombinant human TNF (rTNF) (specific a considerable overlap in their activities, which leads to activity = 6.63 x 106 units/mg of protein) was kindly unexpected patterns of synergism or antagonism. provided by Knoll (Ludwigshafen, F.R.G.). Recombinant The in vitro culturing of human peripheral blood lympho- interferon y(IFN-y), recombinant interleukin la (IL-la), and cytes with interleukin 2 (IL-2) results in the generation of IL-1p8 were supplied by Biogen (Geneva, Switzerland). Rab- lymphokine-activated killers (LAK) that are able to lyse a bit antisera directed against IL-la and IL-1p were kindly wide range of target cells in a non-major histocompatibility provided by A. Shaw (Biogen). The monoclonal antibody complex-restricted manner. These cells have been shown to against IFN-y was kindly provided by M. A. Cousin (Rous- be therapeutically effective in eliminating neoplastic cells in sel-Uclaf, Romainville, France). vivo (9, 10). The LAK precursor cells have been described Indirect Immunofluorescence and Flow Cytofluorometric predominantly as CD16+ large granular lymphocytes (LGL) Analysis. Cells were stained by incubation with the mono- (11). The LAK activation by IL-2 most probably results from at followed a ligand-receptor interaction. There are at least two subunits clonal antibodies the appropriate dilution by the ofthe cellular receptors for IL-2. One is the Tac protein (p55) addition of fluorescein-labeled goat F(ab')2 anti-mouse im- defined by the anti-Tac monoclonal antibody. Recently, a Abbreviations: IL-1, -2, interleukin 1 and 2; TNF, tumor necrosis factor; LGL, large granular lymphocnytes; LAK, lymphokine- The publication costs of this article were defrayed in part by page charge activated killer; IFN-y, interferon y; 12 I-TNF, 1251I-labeled TNF; payment. This article must therefore be hereby marked "advertisement" rIL-2 and rTNF, recombinant IL-2 and TNF. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 6875 Downloaded by guest on October 1, 2021 6876 Immunology: Chouaib et al. Proc. Natl. Acad. Sci. USA 85 (1988) munoglobulin as previously described (16). The following Table 1. Synergistic effect of TNF with low doses of IL-2 on antibodies were used in this study: The NKH1 monoclonal LAK generation antibody, a mouse immunoglobulin (IgM) reactive with LGL Cytotoxic activity, lytic units per 106 effector cells (17), was a kind gift from T. Hercend (Institut Gustave Roussy). OKT3 antibody was purchased from Ortho- TNF, IL-2, ng/ml Diagnostic (Aubervilliers, France). M232, a monoclonal Exp. ng/ml 0 0.25 0.5 1.25 2.5 5 antibody that reacts with the chain of the LFA-1 molecule, 1 0 2 2 8 27 79 102 and BW209/2 (anti-CD16), which reacts with the Fc receptor 250 3 36 42 66 92 106 for IgG expressed on natural killer cells, were a kind gift from 500 3 38 46 69 89 101 A. Bernard (Institut Gustave Roussy). Anti-Tac antibody, 2 0 3 3 9 24 52 63 which reacts with the human IL-2 receptor, was provided by 250 2 22 33 43 59 62 T. Waldmann (National Institutes of Health, Bethesda, MD). 500 3 29 37 38 58 65 LAK Activation and Cytotoxicity Assay. LGL (106 per ml) 3 0 2 3 6 21 63 71 were cultured at 370C for 3 days in complete medium (RPMI 250 3 18 24 31 68 74 1640/10%o normal human serum) supplemented with rIL-2. 500 2 24 27 34 65 76 were times, and used The cells then harvested, washed three LGL were cultured with the indicated concentrations ofrIL-2 and as effector cells. The 51Cr release cytolysis assay was rTNF for 3 days and then were assayed for LAK-mediated cytotox- performed as previously described (18). The natural killer- icity against Daudi cells in a 4-hr 51Cr release assay. resistant Burkitt lymphoma-derived B lymphoblastoid cell line, Daudi, was used as the target. Data are reported as otherwise ineffective doses ofIL-2 when TNF is added to the either specific lysis or the number of lytic units. The percent culture. specific cytotoxicity was calculated as follows: TNF and IL-2 Act by Means of a Direct Mechanism on LGL for LAK Generation. Because TNF has been shown to induce cpm - cpm released experimental released spontaneous x 100 the production of IL-1 in some cells and because IL-2 has maximum cpm released - spontaneous cpm released been shown to induce the production of IFN-'y (21, 22), one A lytic unit is the reciprocal of the number of effector cells possible indirect mechanism could be the induction of these that cause 30% lysis of 104 51Cr-labeled target cells. two factors following IL-2/TNF treatment of LGL. To rule Radiolabeling of TNF and Binding Assay. TNF was radio- out this possibility, we examined the possible involvement of labeled with 125I by the method of Bolton and Hunter (19). endogenously produced IL-la, IL-1f, and IFN-y in the Briefly, 10 ,ug of rTNF in 100 ,ul of borate buffer (pH 8.5) was process ofLAK generation by LGL in response to IL-2/TNF incubated for 3-5 hr on ice with 1 mCi of diiodinated Bolton- stimulation. Hunter reagent (New England Nuclear). Iodinated TNF was LGL were cultured in the presence of neutralizing doses of purified by gel filtration on a Sephadex G-25 column.
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