cd59 is physically and functionally associated with natural cytotoxicity receptors and activates...

© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

CD59 is physically and functionally associated withnatural cytotoxicity receptors and activates humanNK cell-mediated cytotoxicity

Emanuela Marcenaro1, Raffaella Augugliaro2, Michela Falco3, Roberta Castriconi1,Silvia Parolini4, Simona Sivori1,3, Elisa Romeo2, Romano Millo1, Lorenzo Moretta1,3,5,Cristina Bottino3 and Alessandro Moretta1,5

1 Dipartimento di Medicina Sperimentale, Universita degli Studi di Genova, Genova, Italy2 Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy3 Istituto Giannina Gaslini, Genova, Italy4 Dipartimento di Scienze Biomediche e Biotecnologie, Universita di Brescia, Brescia, Italy5 Centro di Eccellenza per le Ricerche Biomediche, Universita degli Studi di Genova, Genova,

Italy

Triggering of cytotoxicity in human NK cells is induced by the combined engagement of sev-eral triggering receptors. These include primary receptors such as NKG2D and the naturalcytotoxicity receptors (NCR) NKp30, NKp46 and NKp44, while other molecules, including2B4, NTB-A and NKp80, function as co-receptors. As reported in the present study, duringan attempt to identify novel NK receptors or co-receptors, we found that CD59 functions asa co-receptor in human NK cell activation; engagement of CD59 by specific mAb deliverstriggering signals to human NK cells, resulting in enhancement of cytotoxicity. Similar toother NK co-receptors, the triggering function of CD59, a glycosylphosphatidylinositol (GPI)-linked protein, depends on the simultaneous engagement of primary receptors such asNCR. Accordingly, CD59-dependent triggering was virtually restricted to NK cells expressinghigh surface densities of NKp46, and mAb-mediated modulation of NKp46 resulted in mark-edly decreased responses to anti-CD59 mAb. Biochemical analysis revealed that CD59 isphysically associated with NKp46 and NKp30. Moreover, engagement of CD59 resulted intyrosine phosphorylation of CD3 ´ chains associated with these NCR, but not those associ-ated with CD16. Thus, CD59-mediated costimulation of NK cells requires direct physicalinteraction of this GPI-linked protein with primary triggering NK receptors.

Key words: Cytotoxicity / NK lymphocytes / Triggering receptors

Received 1/8/03Revised 30/9/03Accepted 6/10/03

[DOI 10.1002/eji.200324425]

Abbreviations: NCR: Natural cytotoxicity receptors GPI:Glycosylphosphatidylinositol ITAM: Immune receptortyrosine-based activating motif DTT: Dithiothreitol IAA:Iodoacetamide

1 Introduction

The function of human natural killer cells is regulatedthrough a balance between activating and inhibitoryreceptors. The main function of the HLA class I-specificinhibitory NK cell receptors [1–4], represented by thekiller Ig-like receptors (KIR) and the CD94/NKG2A het-erodimer, is to monitor the level and quality of HLA classI molecules expressed at the autologous cell surface.Thus, cells expressing altered levels of HLA class I, a fre-quent event during viral infections or tumor transforma-tion, become susceptible to NK-mediated cytotoxicity,

due mainly to the missing engagement of the inhibitoryreceptors for HLA class I. The fact that NK cells lyse HLAclass I-defective target cells implies the existence ofactivating receptors mediating NK cell cytotoxicity in anHLA-independent fashion. Recent studies led to theidentification and molecular characterization of threesuch receptors, NKp46, NKp30 and NKp44, collectivelytermed “natural cytotoxicity receptors” (NCR) [5]. NCRappear to play a predominant role in NK cell activationduring the process of natural cytotoxicity against mosttumor target cells [6].

The central role of NCR in NK-mediated tumor cell lysiswas underscored by the strict correlation between NCRsurface density and the magnitude of NK-mediated cyto-toxicity; only NK cells that express high NCR surfacedensities (NCRbright) kill most NK-susceptible tumor celllines efficiently, whereas NK cells characterized by anNCRdull phenotype display low cytolytic activity againstthe same panel of target cells [7].

Eur. J. Immunol. 2003. 33: 3367–3376 NK cell costimulation via CD59 3367


Another receptor that plays an important role in NK-mediated cytolytic activity against certain target cells isNKG2D [8, 9]. Expressed by NK cells and most cytolyticT lymphocytes, NKG2D is specific for the stress-inducible MICA/B molecules as well as for ULBP pro-teins expressed on cell tumors of different histotypes. Anadditional group of stimulating surface molecules maycontribute to the induction of NK cell triggering, butbecause their ability to induce NK cell activationdepends on the simultaneous co-engagement of othertriggering receptors, such as NKp46, they are consid-ered to be co-receptors rather than “primary” receptors[6]. Accordingly, analysis revealed that the function ofsuch co-receptors is mainly restricted to NK cell clonescharacterized by an NCRbright phenotype. Three such co-receptor molecules are 2B4 [10], NTB-A [11] and NKp80[12].

In an attempt to reveal novel surface molecules capableof triggering NK-mediated killing, we selected a mAbspecific for CD59. In this study, we show that mAb-mediated cross-linking of CD59 induces triggering ofcytotoxicity in most, but not all, human NK cells. Similarto other NK co-receptors, CD59 function is dependenton the expression of high levels of NCR, in particularNKp46. Finally, we provide direct evidence that the func-tion of CD59 is due to its physical and functional associ-ation with the NKp46 and NKp30 receptors.

2 Results

2.1 Isolation of an mAb that activates the NKcell-mediated cytotoxicity

Mice were immunized with the human NK cell cloneSE192 (surface phenotype p58–, p70–, p140–, CD94/NKG2A+). After cell fusion, mAb ON251 was selected onthe basis of its ability to trigger NK cells in a redirectedkilling assay against Fc + R+ P815 murine target cells. Toassess the cell surface distribution of the ON251-reactive molecules, peripheral blood mononuclear cells(PBMC) from normal donors or tumor cell lines of differ-ent histotypes were analyzed by indirect immunofluores-cence and flow cytometry. ON251 mAb brightly stainedNK cells, T and B lymphocytes, monocytes, granulo-cytes and most human tumors cell lines analyzed, withthe exception of NK.L, NK92 and Raji (Table 1). Celllysates derived from different polyclonal NK cell popula-tions were immunoprecipitated with ON251 mAb, andsamples were analyzed by SDS-PAGE. Under non-reducing conditions, the ON251 mAb recognized mole-cules characterized by molecular masses of approxi-mately 15 and 60 kDa (Fig. 1, upper panel). Under strongreducing conditions (Dithiothreitol [DTT]-mediated

Table 1. Surface expression of ON251 mAb-reactive mole-culesa)

Cells Histotype Anti-CD59mAb

Resting NK cells +Activated NK cells +Resting T cells +PHA Blasts +Resting B cells +Monocytes +Granulocytes +

YT NK cell line +/–NK.L NK cell line –NK3.3 NK cell line +NK92 NK cell line –NKYS NK cell line –

JA3 T leukemia +H9 T leukemia +HSB2 T leukemia +

Raji Burkitt lymphoma –DAUDI Burkitt lymphoma +LCL 721.221 EBV-cell line +C1R EBV-cell line +

K562 Proeritrocytic leukemia +HL60 Promyelocytic leukemia +TF1 Promyelocytic leukemia +MM6 Promyelocytic leukemia +Eo/A3 Eosinophilic leukemia –MEL15392 Melanoma +M14 Melanoma +FO-1 Melanoma +1074 mel Melanoma +SK-NEP-1 Renal cell

adenocarcinoma+

A172 Glioblastoma cell line +

YAC-1 Murine thymoma –BW1502 Murine thymoma –P815 Murine mastocytoma –COS-7 Monkey kidney fibroblast –a) Normal cells and tumor cell lines of different histotypes

were analyzed by immunofluorescence and flowcytometry for reactivity with the ON251 mAb (used incombination with PE-conjugated goat anti-mouse IgG1).Cells were of human origin unless otherwise specified.

reduction followed by Iodoacetamide [IAA]-inducedalkylation), however, only the 15 kDa protein could bedetected (Fig. 1, lower panel). This pattern of proteinmigration suggests that the ON251 mAb specifically rec-ognizes a 15 kDa molecule characterized by a high num-ber of cysteine residues responsible for the formation ofhigher molecular weight aggregates under denaturatingconditions. Phosphatidylinositol-specific phospholipaseC treatment of NK cells strongly affected the expression

3368 E. Marcenaro et al. Eur. J. Immunol. 2003. 33: 3367–3376


Fig. 1. Immunoprecipitation of ON251 mAb-reactive surfacemolecules. Normal polyclonal NK cell populations fromdonors EM (lanes A, D, G), CB (lanes B, E) and AM (lane C)and the NK92 NK cell line (lane F, negative control) werelabeled with 125I, lysed with 1% NP-40 and immunoprecipi-tated with ON251 or anti-CD69 (positive control) mAb. Sam-ples were analyzed by 11.5% SDS-PAGE under non-reducing (upper panel) or reducing (DTT and IAA, lowerpanel) conditions. Molecular weight markers (kDa) are indi-cated.

Fig. 2. ON251 mAb recognizes the human CD59 surfacemolecule. COS-7 cells transfected with the VR1012/ME1construct were stained with ON251 or ECM1 mAb and PE-conjugated goat anti-mouse second reagents, followed byflow cytometric analysis. White profiles indicate cells incu-bated with the second reagent alone.

of ON251 mAb-reactive molecules, suggesting that theymay be glycosylphosphatidylinositol (GPI)-anchored sur-face structures (data not shown).

2.2 ON251 mAb recognizes CD59 surfacemolecules

An expression cloning strategy [13] was used to identifythe cDNA encoding the molecule recognized by ON251mAb. A cDNA library prepared from activated NK cellswas transiently transfected into COS-7 cells, and theselection of positive pools was performed by immunocy-tochemical staining using the ON251 mAb and sib-selection. This approach allowed isolation of the con-struct VR1012/ME1 containing a cDNA of 1,141 bp char-

acterized by a 387-bp ORF and encoding a 128 aminoacid type I GPI-anchored protein. Comparison of theidentified sequence with the EMBL/GenBank/DDBJdatabase revealed identity of the cloned cDNA with thegene coding for CD59 [14]. COS-7 cells transiently trans-fected with this construct stained brightly with theON251 mAb (Fig 2). A second Ab, ECM1 (IgM), display-ing a surface reactivity analogous to ON251, also reactswith CD59.

2.3 Clonal heterogeneity in response to anti-CD59 mAb-mediated cross-linking

It was previously shown that a strict correlation existsbetween NCR surface density and the ability of differentNK cells to kill a panel of susceptible target cells [7]. Inparticular, when NK cell clones were assessed for cyto-toxicity against the P815 murine cell line, a target cellcommonly used in redirected killing assays, a direct cor-relation was found between the surface density ofNKp46 and the ability of the clone to kill P815 targetcells. Since killing was abrogated following mAb-mediated masking of NKp46, it was concluded that thekilling is NKp46-dependent and that the differentialbehavior of NCRdull and NCRbright NK cells reflects differ-ences in NKp46 surface densities [7].

Importantly, NKp46 surface density was also found tocorrelate with the triggering capabilities of other surfacereceptors, including 2B4, NTB-A and NKp80; mAb spe-cific for such molecules could efficiently trigger cytotox-icity in redirected killing assays only for NKp46bright NKcells [10–12]. As previously shown, these molecules actas co-receptors, as their induction of NK cell activationrequires a first signal provided by the engagement ofNKp46 by a still undefined ligand expressed at the P815cell surface [5, 6]. In preliminary experiments somedegree of heterogeneity was observed among differentNK cell clones in the ability to respond to the ON251



Fig. 3. Different cytolytic responses of NKp46bright andNKp46dull NK clones to anti-CD59 mAb. RepresentativeNKp46bright (A54) or NKp46dull (B37) NK cell clones wereassessed for cytotoxicity in a redirected killing assay againstP815 murine target cells either in the absence or the pres-ence of 0.5 ? g/ml mAb specific for the indicated surfacemolecules. The E/T ratio used was 5:1. Data are representa-tive of five independent experiments. The SD of the mean ofthe triplicates was X 5%.

Fig. 4. Effect of mAb-mediated NKp46 modulation on NKcell responsiveness to anti-CD59 mAb. Both untreated andNKp46-modulated NK cells were assessed in a redirectedkilling assay against P815 target cells in the absence or inthe presence of the indicated mAb (0.5 ? g/ml). The E/T ratioused in this experiment, representative of four independentexperiments, was 2:1. The SD of the mean of the triplicateswas X 5%.

mAb. We therefore analyzed the abilities of NKp46bright

and NKp46dull NK cell clones to lyse P815 target cells inthe absence or presence of ON251 mAb in a redirectedkilling assay. All NK cell clones analyzed, independent oftheir NKp46 phenotype, expressed comparable surfacedensities of CD59 (data not shown). While NKp46bright

clones responded to both anti-NKp46 and anti-CD59mAb, NKp46dull clones were poorly responsive to bothmAb. However, the NKp46dull clones responded withequal efficiency to anti-CD16 mAb [7, 10], indicating thatthey possess a cytolytic potential comparable to that ofNKp46bright clones (Fig. 3).

2.4 Effect of mAb-mediated modulation ofNKp46

In order to demonstrate the existence of a correlationbetween the surface density of NKp46 and the function(or lack thereof) of CD59, we analyzed the effect of mod-ulation of NKp46 expression [10]. NKp46bright NK cellclones were incubated overnight in the presence of puri-fied immobilized anti-NKp46 mAb (KL247, IgM). Thistreatment led to an almost complete disappearance ofNKp46 from the cell surface, while the expression ofCD59 was only minimally affected (data not shown).Untreated (NKp46bright) or NKp46-modulated NK cellclones were then used as effector cells in a redirectedkilling assay against P815. NKp46-modulated cells dis-played markedly reduced spontaneous (in the absenceof added mAb) cytotoxicity against P815. Addition of

anti-NKp46 or anti-2B4 mAb (both IgG1 isotype) did notsignificantly increase cytolytic activity [10]. Remarkably,mAb-mediated modulation of NKp46 also affectedresponses to anti-CD59 mAb, while cytolytic responsesto anti-CD16 mAb were unmodified (Fig. 4). The require-ment of NKp46 co-engagement for CD59-mediated trig-gering further suggests that this molecule, similar to 2B4,NTB-A and NKp80, may function as a co-receptor in NKcell activation.

2.5 NK cell activation via CD59 is not dependenton CD16 expression

Human NK cells display clonal heterogeneity in expres-sion of both NCR and the triggering Fc + receptor CD16;NK cell clones may be either CD16+ or CD16– [10]. Inorder to assess whether CD59 is involved in triggering ofboth CD16+ and CD16– NK clones, we compared suchclones (derived from the same donor) in a redirected kill-ing assay against P815. CD16+ and CD16– NK cell clonesexpressed similar levels of NKp46 and CD59 (data notshown) and displayed comparable cytolytic responses toanti-NKp46 or anti-CD59 mAb (Fig 5). As expected, anti-CD16 mAb triggered cytotoxicity only in CD16+ NK cellclones. These results indicate that, unlike NKp46, CD16molecules are not required for induction of CD59-dependent NK cytotoxicity.



Fig. 5. Both CD16+ and CD16– NK clones are responsive toanti-CD59 mAb. The cytolytic response of representativeCD16+ (EM11) and CD16– (EM72) NK clones to anti-CD59mAb was assessed in a redirected killing assay against P815target cells. In this experiment, which is representative offive independent experiments, the E/T ratio used was 5:1.The SD of the mean of the triplicates was X 5%.

2.6 Physical and functional association of CD59with the NCR molecular complex

It is well known that NCR transduce activating signalsthrough their association with the immune receptortyrosine-based activating motif (ITAM)-containing poly-peptides CD3 ´ , Fc 4 RI + and DAP12 [5, 6]. Importantly, itwas recently shown that NCR form a functionally coordi-nated molecular complex. Indeed, mAb-mediatedengagement of an individual NCR leads to activation ofthe signal transduction machinery used by the otherNCR [15]. This effect is restricted to NCR; other activat-ing receptors (e.g. CD16), are excluded from this molec-ular interplay although they use similar (or identical) sig-nal transducing polypeptides [15].

In light of the data shown above, we analyzed whetherCD59 also participates in the NCR-complex signalingcascade. Cell lysates were obtained from polyclonal NKcells left untreated or stimulated with an anti-CD59 mAbof IgM isotype (ECM1), and CD59 immunoprecipitateswere analyzed by Western blot with antisera specific fordifferent signal transducing polypeptides, includingCD3 ´ , Fc 4 RI + and DAP12, or with anti-phosphotyrosineAb. Controls included immunoprecipitates using mAbspecific for CD56 (negative control), CD16 or differentNCR. CD3 ´ and Fc 4 RI + , but not DAP12, molecules couldbe detected in CD59 immunoprecipitates (Fig. 6a).Moreover, mAb-mediated engagement of CD59 mole-cules resulted in tyrosine phosphorylation of the CD3 ´polypeptides present not only in CD59 immunoprecipi-

tates, but also in NKp46 and NKp30 immunoprecipitates(Fig. 6b). In contrast, CD59 engagement did not result intyrosine phosphorylation of CD3 ´ molecules associatedwith CD16.

It is notable that CD59 lacks the transmembrane portionrequired for the association between activating recep-tors and signal transducing polypeptides [14]. It is there-fore likely that CD59 is not directly associated with thesepolypeptides, but rather with the activating receptorsthemselves, in particular NKp46 and NKp30. Thishypothesis was confirmed by experiments in which NCRwere immunoprecipitated from NK cells left untreated ortriggered via NKp46 or NKp30. Immunoprecipitates werethen probed with anti-CD59 mAb. CD59 molecules werespecifically detected in NKp46 and NKp30 immunopre-cipitates (Fig. 6c). The CD59/NKp46 association wasevident in both untreated and anti-NKp46 mAb-stimulated NK cells, whereas the CD59/NKp30 associa-tion occurred only upon NK cell triggering with anti-NKp30 mAb. In contrast, CD59 did not associate withthe triggering receptors NKp44 or CD16, even after NKcell activation with anti-NKp44 or anti-CD16 mAb.

2.7 CD59 cooperates with NKp46 and NKp30 inNK cell triggering

Since CD59 appears to be physically and functionallyassociated with NKp46 and NKp30, we further evaluatedwhether engagement of CD59 could amplify theresponse of NK cells to these receptors. Polyclonal NKcells were analyzed in redirected killing assays in thepresence of anti-NKp46 or anti-NKp30 mAb used aloneor in combination with anti-CD59 mAb. Sub-optimaldoses of anti-NKp46 or anti-NKp30 mAb induced a lowdegree of NK-mediated cytotoxicity against P815 cells[10], and the addition of anti-CD59 mAb resulted in astrong enhancement of cytolysis (Fig. 7). These data pro-vide further experimental evidence that CD59 cooper-ates with NKp46 and NKp30 in the induction of NK-mediated cytotoxicity by amplifying the triggeringresponses to sub-optimal engagement by their respec-tive cellular ligands.

3 Discussion

In this study we identified CD59 as an additional trigger-ing co-receptor involved in the induction of human NKcell-mediated cytotoxicity. The triggering function ofCD59 was particularly evident in NK cells expressinghigh surface densities of NCR. Moreover, the engage-ment of CD59 resulted in amplification of the NKp46-mediated cytolytic responses, as indicated by experi-



P Fig. 6. Functional and physical association between CD59and NKp46 or NK p30. Cells from a polyclonal NK cell popu-lation were lysed in 1% digitonin and (a) immunoprecipitatedwith mAb specific for the indicated molecules. Sampleswere analyzed by 14% SDS-PAGE under reducing condi-tions, and membranes were probed with anti-CD3 ´ , anti-Fc 4 RI + or anti-DAP12 Ab after blotting. (b) Cell lysates wereleft untreated or stimulated with anti-CD59 or anti-CD16mAb, followed by immunoprecipitation with mAb specific forthe indicated molecules. Samples were analyzed in 14%SDS-PAGE under reducing conditions, and membraneswere probed with anti-phosphotyrosine (anti-P-Tyr) Ab afterblotting. (c) Cell lysates were left untreated or stimulated asindicated and were immunoprecipitated with mAb specificfor the indicated molecules. Samples were analyzed by11.5% SDS-PAGE under non-reducing conditions, andmembranes were probed with the anti-CD59 mAb after blot-ting. Molecular weight markers (kDa) are indicated. All mAbused for NK cell stimulation were of IgM isotype.

Fig. 7. CD59 enhances NK cell responses to sub-optimaldoses of anti-NKp46 or anti-NKp30 mAb. Polyclonal NKcells were analyzed in a redirected killing assay against P815murine target cells in the presence of sub-optimal doses ofanti-NKp46 (a) or anti-NKp30 (b). The mAb were used eitheralone ( Æ , | ) or in combination with an optimal dose (0.5 ? g/ml) of anti-CD59 mAb ( 1 , ß ). The E/T ratio used was 6:1.

ments in which sub-optimal doses of anti-NKp46 mAbwere used to activate NK cells. These data suggested apossible link between NCR and CD59 and, indeed, wecould provide evidence that CD59 is physically associ-ated with NKp46 in human NK cells and associates with

NKp30 following mAb-mediated cross-linking of thisNCR. Importantly, mAb-mediated engagement of CD59results in tyrosine phosphorylation of CD3 ´ chains asso-ciated with NKp46 or NKp30. This physical/functionalassociation provides a likely explanation for the function-ing of CD59. A direct association of CD59 with ITAM-containing polypeptides is unlikely, as CD59 in NK cellsis a GPI-anchored molecule [15] lacking the positivelycharged transmembrane portion required for associationwith the signal transducing polypeptides [14]. On theother hand, our results strongly suggest that CD59 co-receptor activity requires direct interaction with theNKp46 and NKp30 signal transduction machinery.



NKp44 has also been shown to be part of the NCRmolecular complex in activated NK cells [16]. Under ourexperimental conditions, however, NKp44 does notappear to directly interact with CD59; neither co-precipitation of the two molecules nor CD59-mediatedtyrosine phosphorylation of the NKp44-associated DAP-12 chains upon CD59 cross-linking could be detected.

Further evidence of the functional link between CD59and NKp46 was provided by the finding that mAb-induced surface modulation of NKp46 rendered NK cellsunresponsive to anti-CD59 mAb in redirected killingassays; NKp46-modulated cells display a functionalbehavior similar to that of NCRdull NK cells (low levels ofNKp46 are not sufficient to induce significant triggeringof NK cells upon interaction with murine ligandsexpressed on P815) [7]. Although conventional cytofluo-rimetric analysis did not reveal comodulation of CD59following mAb-mediated modulation of NKp46, we can-not exclude the occurrence of a partial comodulation ofCD59 molecules due to their association with NKp46.Such an effect may not be easily detectable becauseCD59 is expressed at a higher surface density thanNKp46 in NK cells. Experiments of mAb-induced CD59modulation did not clarify this issue since, in our hands,no significant downmodulation of CD59 expressioncould be induced.

With regard to the cellular ligand(s) of CD59, no definitivedata are currently available, although previous reportsimplicated CD2 as a surface molecule capable of inter-acting with CD59 [17, 18]. It is well known that CD59(also termed membrane inhibitor of reactive lysis, MIRL),like CD35 (complement receptor type 1, CR1) [19], CD55(decay-accelerating factor, DAF) [20] and CD46 (mem-brane cofactor protein, MCP) [21], is involved in the con-trol of complement activation during immune responses[22–25]. In particular, CD59 protects normal cells fromcomplement attack by binding to C8a and/or C9 in theassembling the membrane attack complex (MAC) andinterfering with C9 membrane insertion and polymeriza-tion. In this context, recent studies have reported thatcomplement inhibitors that are expressed or even up-regulated in tumor cells of different histotypes may playa role in tumor escape from complement-dependentcytotoxicity [26, 27]. Moreover, in xenogeneic transplan-tation, the use of transgenic animals expressing humanCD59 protects transplanted organs from MAC-mediatedhyperacute rejection [28–30]. Finally, it is notable that inparoxysmal nocturnal hemoglobinuria (PNH), the geneticdefect in GPI anchoring results in no detectable surfaceexpression of GPI-anchored molecules such as CD59and CD55, leading to an unusually high susceptibility oferythrocytes to the lytic action of complement [31]. It willbe interesting to analyze whether the CD59 defect in

PNH patients causes alterations in the process of NK cellactivation against target cells.

In conclusion, our study revealed another moleculeinvolved in the process of NK cell activation. Previousreports suggested that cross-linking of CD59 by specificmAb could induce neutrophils to release Ca2+ from inter-nal stores [32], and a costimulatory role for CD59 in Tcells has also been proposed [33, 34]. At least in NKcells, CD59-mediated costimulation appears to bedependent upon association with certain triggeringreceptors, and to selectively contribute to the activationof their downstream signaling pathways.

4 Materials and methods

4.1 Monoclonal antibodies

ON251 and ECM1 (IgG1 and IgM, respectively, anti-CD59)were obtained by immunizing a 5-week-old BALB/c mousewith the NK clone SE192 (surface phenotype: p58–, p70–,p140–, CD94/NKG2A+) as previously described [11, 12]. Thefollowing mAb, produced in our lab, were used in this study:JT3A (IgG2a, anti-CD3), BAB281 and KL247 (IgG1 and IgM,respectively, anti-NKp46), Z231 and KS38 (IgG1 and IgM,respectively, anti-NKp44), Z25 and F252 (IgG1 and IgM,respectively, anti-NKp30), PP35 and S39 (IgG1 and IgG2a,respectively, anti-2B4), XA59 and c127 (IgM and IgG1,respectively, anti-CD16), c218 and A6/220 (IgG1 and IgM,respectively, anti-CD56), c227 (IgG1, anti-CD69) and MA152(IgG1, anti-NKp80).

D1.12 (IgG2a, anti-HLA-DR) mAb was provided by Dr. R. S.Accolla (Universita di Insubria, Varese, Italy). HP2.6 (IgG2a,anti-CD4) mAb was provided by Dr. P. Sanchez-Madrid (Uni-versidad Autonoma de Madrid, Madrid, Spain). The anti-NKp30 rabbit antiserum was previously described [16].

4.2 Biochemical characterization of CD59

Polyclonal NK cells (20×106) were labeled with 125I (NEN,Boston, MA), lysed in 1% NP-40 and immunoprecipitatedwith Sepharose-CNBr- (Pharmacia Biotech Inc., Piscataway,NJ) coupled mAb. Samples were analyzed by discontinuousSDS-PAGE under non-reducing or strong reducing (20 mMDTT, 30 min at room temperature followed by 60 mM IAA,1 h at room temperature) conditions [11, 16].

For Western blotting experiments, 90×106 NK cells wereused either untreated or stimulated with 150 ? g IgM mAb(30 min, 4°C) followed by 60 ? g F(ab’)2 rabbit anti-mouseIgM (3 min, 37°C) (Histoline Laboratories, Milan, Italy). Cellswere lysed in 1% digitonin and immunoprecipitated withSepharose-CNBr-coupled Ab. Samples were analyzed indiscontinuous SDS-PAGE, transferred to Immobilon P (Milli-



pore Corp, Bedform, MA), and detected with the Renais-sance Chemiluminescence Kit (NEN) after probing [16]. Thefollowing Ab were used in this study: anti-phosphotyrosinemAb (PY20-HRPO, Becton Dickinson Transduction Labora-tories, Franklin Lakes, NY); anti-CD3 ´ mAb (2H2, Coulter/Immunotech, Marseille, France) and anti-DAP12 (SI-28) [16]rabbit antisera. The anti-Fc 4 RI + rabbit antiserum was pro-duced by Eurogentec S. A. (Herstal, Belgium) using the 15amino acid KLH-conjugated C-NQETYETLKHEKPPQ pep-tide. HRPO-conjugated donkey anti-rabbit (Amersham,Buckingamshire, GB) or rabbit anti-mouse (DAKO A/S, Den-mark) Ab were used as second reagents [16]. For Westernblotting experiments, the anti-CD59 mAb, purified usingAKTA PRIME (Amersham), was labeled with biotin (Pierce,Rockford, IL), and Neutravidin (Pierce) was used as secondreagent.

4.3 Purification of polyclonal or clonal NK cellpopulations

In order to obtain peripheral blood lymphocytes (PBL),PBMC were isolated on Ficoll-Hypaque gradients anddepleted of plastic-adherent cells [11]. Enriched NK cellswere isolated by incubating PBL with anti-CD3 (JT3A), anti-CD4 (HP2.6) and anti-HLA-DR (D1.12) mAb (30 min at 4°C),followed by incubation with goat anti-mouse-coated Dyna-beads (Dynal, Oslo, Norway) for 30 min at 4°C and subse-quent immunomagnetic depletion [12]. CD3–4–DR– cellswere cultured on irradiated feeder cells in the presence of100 U/ml rIL-2 (Proleukin, Chiron Corp., Emeryville, CA) and1.5 ng/ml PHA (Gibco Ltd., Paisley, Scotland) in order toobtain polyclonal NK cell populations or, after limiting dilu-tion, NK cell clones [12].

4.4 Cytolytic activity and flow cytofluorimetric analysis

NK cells were tested for cytolytic activity against the (Fc + R+)P815 murine mastocytoma cell line in a 4-h 51Cr-releaseassay as previously described [7, 10–12]. For the redirectedkilling assay, 0.5 ? g/ml of the various mAb were added.

For one- or two-color cytofluorimetric analysis (FACScan,Becton Dickinson, Mountain View, CA), cells were stainedwith the appropriate mAb followed by a PE- or FITC-conjugated isotype-specific goat anti-mouse secondreagent (Southern Biotechnology Associated, Birmingham,AL) [10–12].

To test whether the molecules recognized by ON251 mAbwere represented by GPI-linked surface structures, NK cellswere washed twice in RPMI and incubated for 1 h at 37°C inRPMI containing 10 mM Hepes and 1 U/ml phosphat-idylinositol-specific phospholipase C (PI-PLC, Sigma)before staining with specific mAb [35].

4.5 Modulation of surface molecules

Flat-bottom 24-well Costar plates were coated overnight at4°C with 2 ? g/ml (1 ml) KL247 (IgM, anti-NKp46) mAb [10].NK cells (106) were added to coated or uncoated wells andincubated for 18 h at 37°C. Cells were then washed twicebefore the cytofluorimetric analyses or cytolytic activityassays.

4.6 cDNA library screening and sib-selection

The cDNA library used in this study was prepared from acti-vated human NK cells derived from healthy donors. Briefly,the cDNA library, fractionated into ten different pools, wastransiently transfected into COS-7 cells using non-liposomalFuGene-6 reagent (Roche, Monza, Italy) following the manu-facturer’s instructions. Selection of positive pools was per-formed by immunocytochemical staining using the ON251mAb and sib-selection [13]. The screening yielded a positivefraction, and the plasmid DNA of this pool was amplified inEscherichia coli, fractionated into smaller sub-pools andtransfected into COS-7 cells. This approach was performeduntil a single construct, termed VR1012/ME1, was identified.

4.7 Transient transfection and DNA Sequencing

COS-7 cells (5×105/plate) were transfected with VR1012/ME1 construct using FuGene-6 reagent. After 48 h, trans-fected cells were analyzed by sib-selection or stained withthe mAb ON251 and ECM1 followed by a PE-conjugatedisotype-matched goat anti-mouse second reagent (IgG1and IgM, respectively) and flow cytometric analysis using aFACSort (Becton Dickinson) [13, 36].

DNA sequencing was performed using the d-RhodamineTerminator Cycle Sequencing kit and a 3100 ABI automaticsequencer (Perkin Elmer-Applied Biosystems).

Acknowledgements: This work was supported by grantsawarded by Associazione Italiana per la Ricerca sul Cancro(A.I.R.C.), Istituto Superiore di Sanita (I.S.S.), Ministero dellaSanita, Ministero dell’Universita e della Ricerca Scientifica eTecnologica (M.I.U.R.) and Consiglio Nazionale delle Ricer-che, Progetto Finalizzato Biotecnologie and FunctionalGenomics. The financial support of Fondazione Compagniadi San Paolo, Torino, Italy, is also gratefully acknowledged.We thank Ms. Tiziana Baffi for secretarial assistance. R.Castriconi is the recipient of a fellowship awarded by Fonda-zione Italiana per la Ricerca sul Cancro.

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Correspondence: Alessandro Moretta, Dipartimento diMedicina Sperimentale, Sezione di Istologia, Via G. B. Mar-sano 10, I-16132 Genova, ItalyFax: +39-010-512747e-mail: alemoret — unige.it



cd59 is physically and functionally associated with natural cytotoxicity receptors and activates...

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