Triggering receptors involved in natural killer cell-mediated cytotoxicity against choriocarcinoma cell lines

Triggering Receptors Involved in Natural Killer Cell-Mediated Cytotoxicity Against Choriocarcinoma Cell Lines Simona Sivori, Silvia Parolini, Emanuela Marcenaro, Romano Millo, Cristina Bottino, and Alessandro Moretta ABSTRACT: The lack of classical HLA-class I molecules on trophoblast is necessary to prevent allorecognition by maternal CTL, but may induce activation of NK cells. A protective role against NK cells equipped of suitable inhibitory receptors has been proposed for nonclassical HLA-class I molecules including HLA-E and HLA-G. In the present study we show that the NK-mediated killing of two choriocarcinoma cell lines, JAR and JEG3, is induced upon engagement of natural cytotoxicity receptors (NCR) with their specific ligands. In particular, we show that NKp44, a triggering receptor expressed at the NK cell surface only after in vitro culture in the presence of IL-2, plays a central role in triggering NK cytotoxicity against trophoblast cells. Also NKp46 appear to contribute to this function by cooperating with NKp44. On the other hand, other triggering receptors such as NKp30,

ABBREVIATIONS NK natural killer cell

INTRODUCTION Trophoblast cells in humans are in direct contact with the maternal immune system. These cells lack expression of classical HLA class I and class II molecules, with the exception of HLA-C, during the first trimester of pregnancy [1]. This is thought to represent a mechanism of escape from maternal CTL that would protect the fetus

From the Dipartimento di Medicina Sperimentale (S.S., E.M., R.M., A.M.), Universita` di Genova, Genova, Italy; the Dipartimento di Scienze Biomediche e Biotecnologie (S.P.), Universita` di Brescia, Brescia, Italy; and the Istituto Nazionale per la Ricerca sul Cancro (C.B.), Genova, Italy. Address reprint requests to: Alessandro Moretta, M.D., Dipartimento di Medicina Sperimentale, Sezione di Istologia, Via G.B. Marsano 10, 16132 Genova, Italy; Phone: ⫹39 (10) 3537868; Fax: ⫹39 (10) 512747; E-Mail: [email protected]/[email protected]. Received August 1, 2000; accepted September 15, 2000. Human Immunology 61, 1055–1058 (2000) © American Society for Histocompatibility and Immunogenetics, 2000 Published by Elsevier Science Inc.

2B4, and NKG2D are not involved in killing of choriocarcinoma. Our findings suggest that resistance of trophoblast to NK-mediated cytotoxicity is the result of insufficient activating interactions between the various triggering NK receptors and their target cell ligands. On the other hand, the interaction of nonclassical HLA class I molecules with inhibitory NK receptors appears to play only a marginal role in regulating the susceptibility of choriocarcinoma to NK mediated cytotoxicity. Human Immunology 61, 1055–1058 (2000). © American Society for Histocompatibility and Immunogenetics, 2000. Published by Elsevier Science Inc. KEYWORDS: natural cytotoxicity; triggering receptors; trophoblast

NCR

natural cytotoxicity receptors

from rejection [2]. However, lack of HLA class I expression may be expected to induce an increased susceptibility to NK cells that are particularly abundant in the decidua [3]. This does not occur because trophoblast cells are highly resistant to cytotoxicity mediated by fresh NK cells [4, 5]. A possible mechanism of protection has been evoked that involves nonclassical HLA class I molecules expressed by the trophoblast and a number of inhibiting receptors expressed by decidual NK cells [6 –12]. On the other hand, the actual role of such inhibitory interactions remains unclear because blocking of either the inhibitory receptors or of the nonclassical HLA class I molecules is not sufficient to restore lysis of choriocarcinoma cell lines by NK cells. Thus, it has been proposed that additional HLA class I independent mechanisms may be responsible for trophoblast protection [13]. One possible mechanism 0198-8859/00/$–see front matter PII S0198-8859(00)00201-9

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is related to the expression on decidual NK cells of inadequate amounts of triggering receptors capable of inducing the NK cell-mediated cytotoxicity. However, the surface density of natural cytotoxicity receptors (NCR) [14], such as NKp46 and NKp30, on decidual NK cells is usually comparable with that of peripheral blood NK cells (A. Moretta, unpublished observations). A second mechanism that can be envisaged involves the ligands for NCR that may be poorly represented on the trophoblast cell population. In this context, it is noteworthy that trophoblast cells as well as trophoblast cell lines such as JAR (HLA class I-) and JEG3 (HLA-G/E⫹) are resistant to fresh NK cells but become susceptible to NK cells that have been cultured in the presence of exogenous IL-2 [4, 13]. Thus, it is possible that trophoblast cells may express surface ligands for NK triggering receptors that are lacking on fresh NK cells but become available after in vitro culture. A good candidate receptor that fits with this hypothesis is represented by the NKp44 receptor that is totally absent in fresh NK cells but become progressively expressed upon culture of NK cells in the presence of IL-2 or IL-15 [15, 16]. In this study we analyzed the involvement of various NCR (including NKp44) in the lysis of trophoblast target cells mediated by activated (IL-2 cultured) NK cells. MATERIALS AND METHODS Monoclonal Antibodies The following mAbs were produced in our lab: JT3A (IgG2a, anti-CD3); BAB281 and KL247 (IgG1 and IgM, respectively, anti-NKp46); Z231 and KS38 (IgG1 and IgM, respectively, anti-NKp44); AZ20, A76, and Z25 (all IgG1, anti-NKp30); BAT221 (IgG1, antiNKG2D); ST39 (IgG2a, anti-2B4); c127 and XA59 (IgG1 and IgM, respectively, anti-CD16); c218 and A6220 (IgG1 and IgM, respectively, anti-CD56); A6-136 (IgM, anti-HLA class I). The D1.12 (IgG2a, anti-HLADR) mAb was provided by Dr. R. S. Accolla (Pavia, Italy). The HP2.6 (IgG2a, anti-CD4) mAb was provided by Dr. P. Sanchez-Madrid (Madrid, Spain). Purification of Peripheral Blood Lymphocytes and Generation of Polyclonal or Clonal NK Cell Populations Peripheral blood lymphocytes (PBL) were derived from healthy donors by Ficoll-Hipaque gradients and depletion of plastic-adherent cells. In order to obtain enriched NK cells, PBL were incubated with anti-CD3 (JT3A), anti-CD4 (HP2.6), and anti-HLA-DR (D1.12) mAbs (30 min at 4°C) followed by goat antimouse coated Dynabeads (Dynal, Oslo, Norway) (30 min at 4 °C) and immunomagnetic depletion [15]. CD3⫺ 4⫺ DR⫺ cells

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were cultured on irradiated feeder cells in the presence of 100 U/ml rIL-2 (Proleukin; Chiron Corp., Emeryville, IL, USA) and 1.5 ng/ml PHA (Gibco Ltd, Paisley, Scotland) in order to obtain polyclonal NK cell populations, or after limiting dilution NK cell clones [7, 15]. Flow Cytofluorimetric Analysis Cells were stained with the appropriate mAb followed by PE- or FITC-conjugated isotype-specific goat antimouse second reagent (Southern Biotechnology Associated, Birmingham, AL, USA). Samples were analyzed by one- or two-color cytofluorimetric analysis (FACScan; Becton Dickinson Co., Mountain View, CA, USA) as previously described [15] Cell Lines and Cytolytic Assays Target cells used were represented by JAR [17] and JEG3 [18] (human choriocarcinoma cell lines). NK cells were tested for cytolytic activity in a 4-h 51Cr-release assay as previously described [7, 15], either in the absence or in the presence of various mAbs. The concentrations of the various mAbs were 10 ␮g/ml for the masking experiments. The E/T ratios are indicated in the text. RESULTS AND DISCUSSION In these experiments, effector cells were represented by peripheral blood NK cells cultured in the presence of IL-2 for approximately 15–20 days while target cells were represented by two choriocarcinoma cell lines termed JAR and JEG3 [17, 18]. The cytotoxicity was assessed either in the absence or in the presence of mAb directed to various NCR. As shown in Figure 1, cultured NK cells were strongly cytolytic against both types of target cells: remarkably no increase of target cell lysis could be observed in the presence of A6.136 (anti-HLA class I) mAb that reacts with both classical and nonclassical (i.e. HLA-G and HLA-E) HLA molecules [7]. This further supports the notion that the HLA-G and HLA-E molecules that are expressed by JEG3 cell line do not function as protective elements against NK-mediated cell lysis [13]. Along this line, although not shown, CD94/NKG2A⫹ and/or ILT2⫹ NK cell clones efficiently killed both trophoblast target cells. More importantly, lysis of trophoblast cell lines was sharply inhibited in the presence of anti-NKp44 (KS38, IgM) or anti-NKp46 (KL247, IgM) mAbs. On the other hand, no inhibition was detected in the presence of mAb directed to NKp30, 2B4, or NKG2D. The combined use of both anti-NKp44 and anti-NKp46 mAb resulted in enhanced inhibition of lysis while the combination of anti-NKp44 with anti-NKp30 did not increment the inhibitory effect of anti-NKp44 alone. Similarly, blocking of NKG2D or 2B4 together with NKp44 did not

NK Cell-Mediated Cytotoxicity Against Choriocarcinoma Cells

FIGURE 1 Killing of trophoblast cell lines by cultured NK cells is inhibited by blocking of NKp44 and NKp46 receptors but is not modified by blocking of HLA-class I. The polyclonal-activated NK cells from donor D.P. were tested for cytotoxicity against the indicated target cells either in the absence of mAb (●) or in the presence of anti-HLA class I mAb (E) anti-NKp44 (■) or anti-NKp46 (䊐). The effector/target ratios (E/T) are indicated.

result in incremented inhibition of cytotoxicity (Figure 2) even when the three molecules were blocked simultaneously (not shown). These data indicate that killing of trophoblast cell lines by activated NK cells is dependent upon triggering of NKp44 and NKp46 while no accessory role is played by other activating receptors that are known to regulate NK-mediated cytotoxicity against NK-susceptible targets [14]. Importantly, in some of the donors analyzed (see donor CT) the cytolytic activity against trophoblast cell

FIGURE 2 NKp44 and NKp46 but not other triggering receptors cooperate in the induction of cytotoxicity against trophoblast cells. Polyclonal NK cell lines derived from the indicated donors were assessed for cytotoxicity against JAR target cells either in the absence or in the presence of mAb against the indicated triggering receptor. The effector/target (E:T) ratio in this representative experiment was 10:1.

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lines was mostly dependent upon the function of NKp44 while only a marginal role could be assigned to NKp46. Thus, in this donor anti-NKp46 mAb alone was not inhibiting cell lysis whereas anti-NKp44 mAb was strongly inhibitory. That NKp46 could cooperate, at least in part, with NKp44 was indicated by experiments in which both molecules were simultaneously blocked. Indeed, under these experimental conditions the inhibition mediated by anti-NKp44 mAb could be incremented by the simultaneous blocking of NKp46. Altogether these data provide evidence for a central role of NKp44 in NK-mediated killing of trophoblast cell lines. Because NKp44 is expressed by human NK cells only upon in vitro culture in the presence of exogenous IL-2 [15, 16], the present observation may explain the trophoblast resistance to fresh NK cells. Regarding the other known triggering NK receptors, only NKp46 appears at least in part involved in killing of trophoblast cells. Because NK-mediated killing of NK-susceptible target cells is normally dependent upon the concerted function of several triggering receptors [14], our data also suggest that efficient killing of trophoblast cells can be achieved only when both NKp44 and NKp46 are simultaneously engaged by their respective ligands. This also implies that trophoblast cells may escape NK-mediated killing because of the lack of surface ligands for most triggering NK receptors including those for NKp30, NKG2D, and 2B4. Thus, as previously proposed by others [13, 19], the basis of trophoblast resistance to NK-mediated killing appears to be independent from mechanisms involving either classical or nonclassical HLA class I molecules. On the contrary, resistance to lysis is achieved by a mechanism of downregulation of the ligands for the triggering receptors that are involved in NK cell activation.

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ACKNOWLEDGMENTS

This work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), Istituto Superiore di Sanita` (I.S.S.), Ministero della Sanita`, Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (M.U.R.S.T.), and Consiglio Nazionale delle Ricerche, Progetto Finalizzato Biotecnologie. The financial support of Telethon-Italy (grants E.0892 and E.668) is gratefully acknowledged.

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