the use of trimeric isoleucine-zipper fusion proteins to study surface-receptor–ligand...

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Journal of Immunological Met

Research paper

The use of trimeric isoleucine-zipper fusion proteins to study

surface-receptor–ligand interactions in natural killer cells

Sebastian Stark, Ruediger M. Flaig, Mina Sandusky, Carsten Watzl*

Institute for Immunology, University Heidelberg, Im Neuenheimer Feld 305, 69120 Heidelberg, Germany

Received 22 July 2004; received in revised form 21 October 2004; accepted 8 November 2004

Available online 9 December 2004

Abstract

The ligands for several activating natural killer (NK) cell receptors have not been identified to date. Soluble receptor fusion

proteins can be used to stain target cells for the presence of these unidentified ligands. Here, we describe the generation and use

of soluble type I NK cell receptor isoleucine-zipper (ILZ) fusion proteins of the immunoglobulin (Ig) superfamily. ILZ-fusion

proteins are easy to produce and purify. They form trimeric complexes in solution and display a higher binding avidity than

classical immunoglobulin-fusion proteins. ILZ-fusion proteins do not interact with Fc-receptors and can therefore be used to

block receptor–ligand interactions in cellular assays. This makes ILZ-fusion proteins a valuable tool to study receptor–ligand

interactions in NK cells and other cellular systems.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Natural killer cells; Receptor–ligand interactions; Recombinant fusion proteins

1. Introduction

The activity of human natural killer (NK) cells is

regulated by different surface receptors that can

roughly be divided into activating and inhibiting

receptors (Billadeau and Leibson, 2002; Lanier,

2003). The negative signal mediated by MHC class

0022-1759/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.jim.2004.11.010

Abbreviations: Ig, immunoglobulin; ILZ, isoleucine zipper;

NCR, natural cytotoxicity receptor; NK, natural killer.

* Corresponding author. Tel.: +49 6221 564588; fax: +49 6221

565611.

E-mail address: [email protected]

(C. Watzl).

I recognizing receptors is well characterized and

protects dnormalT cells from NK cell attack (Long et

al., 2001; Leibson, 2004). Recently, much attention

has been paid to the signals and receptors leading to

NK cell activation. Several receptors that play an

essential role in the activation of human NK cells have

been identified. These include NKp30, NKp44,

NKp46 [these receptors are referred to as Natural

Cytotoxicity Receptors (NCR)], NKG2D, 2B4

(CD244), NTB-A, CS1 (CRACC), NKp80, DNAM-

1, and CD96 (Tactile) (Biassoni et al., 2001). Only

recently, some of the ligands that are recognized by

the different activating NK cell receptors have been

identified. NKG2D can recognize several different

hods 296 (2005) 149–158

S. Stark et al. / Journal of Immunological Methods 296 (2005) 149–158150

molecules. Human NKG2D ligands include MICA,

MICB, ULBP1-4 (RAET1I, H, N, and E), and

RAET1G (Jan Chalupny et al., 2003; Raulet, 2003;

Watzl, 2003; Bacon et al., 2004). 2B4 binds to CD48

while NTB-A and CS1 (CRACC) are homophilic

(Brown et al., 1998; Latchman et al., 1998; Kumar-

esan et al., 2002; Falco et al., 2004; Flaig et al., 2004;

Valdez et al., 2004). DNAM-1 recognizes Nectin-2

(CD112) and PVR (CD155) (Bottino et al., 2003;

Tahara-Hanaoka et al., 2004). CD155 is also the

ligand of CD96 (Fuchs et al., 2004). The cellular

ligands of NKp30, NKp44, NKp46, and NKp80 are

not known to date.

Soluble recombinant NK cell receptor proteins

have been very useful for studying the function of NK

cells. They have successfully been used to identify the

ligands of several NK cell receptors and are the only

tools to test for the expression of the unknown ligands

for NKp30, NKp44, NKp46, and NKp80. In the case

of NKG2D or DNAM-1 that recognize several

ligands, soluble receptors are especially useful to

determine the expression of NKG2D or DNAM-1

ligands on the surface of target cells with a single

reagent. So far, most studies have used the extrac-

ellular domain of surface receptors fused to the Fc

portion of human IgG1 [immunoglobulin (Ig)-fusion

proteins]. These Ig-fusion proteins are relatively stable

in solution and form disulfide-linked dimers, enhanc-

ing the avidity towards their ligands. However, the Fc

part of human IgG1 can interact with Fc receptors on

cells, resulting in nonspecific staining or unwanted

effects when used in in vivo or in vitro assays.

The naturally occurring leucine-zipper motif con-

sists of a characteristic seven amino acid residue

repeat with hydrophobic residues at positions 1 and 4

and functions to dimerize bZIP transcription factors

(Landschulz et al., 1988). The isoleucine-zipper (ILZ)

sequence is derived from the yeast transcription factor

GCN4 leucine-zipper dimerization domain by iso-

leucine substitutions at positions 1 and 4 (Harbury et

al., 1993). This 31 amino acid sequence has been

shown to fold into a parallel three-stranded, alpha-

helical coiled-coil (Harbury et al., 1994).

Here, we describe the generation and use of

isoleucine-zipper (ILZ) fusion proteins of type I

activating NK cell receptors and ligands of the Ig

superfamily. These soluble fusion proteins are easy to

produce and to purify and form trimeric molecules in

solution, thereby enhancing their binding avidity. ILZ-

fusion proteins do not interact with Fc receptors,

which makes it possible to use these reagents to block

receptor–ligand interactions in vitro or in vivo without

inducing unwanted effects in Fc-receptor positive

cells.

2. Materials and methods

2.1. Cells and antibodies

The cells used in this study were 293T, MEL1106

(both cultured in DMEM, 10% FCS, Pen/Strep), BaF3

(cultured in RPMI1640, 10% FCS, 50 AM 2-ME, Pen/

Strep), YTS (cultured in IMDM, 12.5% FCS, 50 AM2-ME, Pen/Strep), and 721.221 (cultured in IMDM,

10% FCS, Pen/Strep). The antibodies used were: anti-

NKp30, anti-NKp44, anti-NKp46, anti-2B4 (C1.7; all

Beckman Coulter, Krefeld, Germany), anti-CD4 (BD

Bioscience, Heidelberg, Germany), anti-CD48 (Santa

Cruz Biotechnology, Heidelberg, Germany), anti-

NKG2D (R&D Systems, Wiesbaden, Germany), goat

anti-mouse IgG HRPO-conjugated, goat anti-mouse

IgG PE-conjugated, goat anti-human IgG biotin-

conjugated, and Streptavidin PE-conjugated (all Jack-

son ImmunoResearch, West Grove, PA). The mono-

clonal anti-NTB-A antibody (NT-7) has been

described (Flaig et al., 2004). The monoclonal anti-

ILZ (ILZ-11) and CS1 (CS1.4) antibodies were

generated as described below.

2.2. Plasmid construction and receptor cloning

Using standard PCR and cloning techniques, we

constructed the pZipH vector shown in Fig. 1A. The

expression cassette consisting of the Ign-leader, a

multiple cloning site (BamHI, EcoRI, EcoRV, NotI,

SpeI), the ILZ sequence (generous gift of Dr.

Henning Walczak, Apogenix Biotechnology, Heidel-

berg, Germany), followed by a 6xHis tag and a stop-

codon was cloned between the HindIII and XbaI

sites of the pEF1 expression vector (Invitrogen,

Karlsruhe, Germany).

The following primers were used to amplify the

extracellular domains of the different receptors used in

this study (restriction sites are underlined): CS1: GGA

TCC CCT CTG GAC CCG TGA AAG and ACT

Fig. 1. Expression and purification of ILZ-fusion proteins. (A)

Schematic representation of the ILZ-fusion protein expression

vector pZipH. EF-1a, elongation factor-1a promotor; MCS, multi-

ple cloning site; ILZ, isoleucine-zipper sequence. (B) The indicated

ILZ-fusion proteins were expressed and purified as described in the

Materials and methods section. Two micrograms of each fusion

protein was analyzed by 10% SDS-PAGE and coomassie blue

staining.

S. Stark et al. / Journal of Immunological Methods 296 (2005) 149–158 151

AGT GGA GGA ATC TGG GTC ATC; 2B4: GGA

TCC AGG GCA AAG GAT GCC AGG G and ACT

AGT TCT GAATTC CTG ATG GGC; NTB-A: GGA

TCC GCT TTG GCC CAG GG AAT G and ACT

AGT TTT GGT ATC TGT ATA TTG; CD48: GGA

TCC GTC ACT TGG TAC ATA TGA CC and ACT

AGT GGA CCG GGC CAG GGTACA GG; NKp30:

GAATTC CTC TCT GGG TGT CCC AGC and ACT

AGT TGTACC AGC CCC TAG CTG; NKp44: GGA

TCC CAC AAT CCA AGG CTC AGG and ACT

AGT GGG GGC TGC AGG GCC AGG; NKp46:

GGA TCC CCC AGC AGC AGA CTC TCC and

ACT AGT AAC CAG GAA CCA CAC TAG AGC;

CD4: GGG GGA TCC CAA TGA ACC GGG GAG

TCC CTT TTA GGC and GGG CCG AAT TCC CGG

GGT GGA CCA TGT GGG CAG. PCR products

were cloned in the pZipH vector and sequence

verified.

2.3. ILZ-fusion protein production and purification

When almost confluent, five 175 cm2 flasks of

293T cells were transfected with 125 Ag pZipH vector

using the CaPO4 precipitation method (Pear et al.,

1993). The following day, medium was replaced by

20 ml of fresh culture medium per flask. Supernatants

were harvested on days 3 and 6, adjusted to 20 mM

imidazole and pH 8.0, and incubated with 750 Alpacked Ni2+-NTA agarose beads (Qiagen, Hilden,

Germany) per 100 ml supernatant. After rotating for 2

h at RT, beads were spun down and washed three

times with washing buffer (10 mM imidazole, 50 mM

NaH2PO4, 300 mM NaCl, 0.05% Tween 20, pH 8.0).

Beads were transferred to a column and ILZ-fusion

proteins were eluted in 1-ml fractions using elution

buffer (250 mM imidazole, 50 mM NaH2PO4, 300

mM NaCl, 0.05% Tween 20, pH 8.0). Eluted proteins

were analyzed by SDS-PAGE and coomassie staining.

Peak fractions were pooled and dialyzed against PBS.

ILZ-fusion proteins were concentrated by ultra-filtra-

tion using 10,000 MWCO PES centrifugation devices

(Vivascience, Hannover, Germany). Quantification of

the protein yield was done using the BCA protein

assay kit (Pierce, Rockford, IL). The typical yield of

pure ILZ-fusion proteins was 0.2–1 mg per 100 ml of

culture supernatant.

2.4. mAb production

Female BALB/c mice (10 weeks old) were injected

s.c. with 40 Ag of ILZ-CS1 in complete Antibody-

Multiplier (ABM-S, Linaris, Wertheim-Bettingen,

Germany) followed by additional i.p. injections of

40 Ag of ILZ-CS1 in incomplete Antibody-Multiplier

(ABM-N) at days 21, 35, and 49 after the initial

immunization. Three days after the last injection, the

animals were sacrificed, the spleen was removed and

fused with the myeloma cell line Ag8. Two weeks

after fusion, culture supernatants from wells positive

for growth were tested in an enzyme-linked immu-

noadsorbent assay (ELISA) with ILZ-CS1 or ILZ-

CD48 as coated antigens to distinguish between

antibodies directed against the receptor and the ILZ

portion of the fusion protein. Hybridomas that

produced anti-CS1 or anti-ILZ mAbs were subcloned

several times by limited dilution.

2.5. Protein analysis

For immunoblot analysis, ILZ-fusion proteins were

incubated in reducing or nonreducing SDS sample


buffer and separated using 10% Bis-Tris or 4–8%

Tris–Acetate NuPAGE gels (Invitrogen, Carlsbad,

CA). Gels were blotted onto a PVDF membrane

(Millipore, Bedford, MA) and membranes were

blocked with 5% milk in TPBS (PBS containing

0.05% Tween-20) for 1 h at RT. After washing three

times with TPBS, the membranes were incubated at 4

8C overnight with ILZ-11 mAb (0.5 Ag/ml in TPBS/

5% BSA). Membranes were washed three times in

TPBS containing 0.5 M NaCl and incubated with

HRP-conjugated goat anti-mouse antibody (1/20,000

in TPBS) for 1 h at RT. After washing three times in

TPBS, the blots were developed using Super Signal

West Pico (Pierce).

2.6. ELISA and FACS measurements

ILZ-fusion proteins (0.5 Ag/ml in PBS) were

absorbed onto a 96-well microtiter plate (Maxi-Sorb,

Nunc, Rochester, NY) by incubating at 4 8C for 16 h.

After washing three times with TPBS, the plates were

incubated with an isotype control antibody or specific

antibodies (1 Ag/ml in TPBS) for 1 h at RT and

washed three times with TPBS. Bound antibodies

were detected by 1-h incubation at RT with HRP-

coupled goat anti-mouse IgG antibodies and devel-

oped with o-Phenylenediamine Dihydrochloride per-

oxidase substrate (Sigma).

For surface staining, the cells were incubated with

Ig or ILZ-fusion proteins in 50 Al FACS buffer (PBS,

2% FCS) for 20 min on ice. After washing once in

cold FACS buffer, the cells were incubated in 50 AlFACS buffer with an anti-ILZ monoclonal antibody

(ILZ-11; 5 Ag/ml) or biotin-conjugated goat anti-

human IgG (1/200) for 20 min on ice. After washing

once in cold FACS buffer, cells were incubated in 50

Al FACS buffer with a PE-conjugated goat anti-mouse

antibody or PE-conjugated Streptavidin (both 1/200)

for 20 min on ice. Cells were washed once in FACS

buffer and analyzed by flow cytometry (Becton

Dickinson, Heidelberg, Germany).

2.7. Cytotoxicity assay

Target cells (721.221) were grown to mid-log

phase and 5�105 cells were labeled in 100 Al CTLmedium (IMDM with 10% FCS and Pen/Strep) with

100 ACi 51Cr for 1 h at 37 8C. Cells were washed

twice in CTL medium and resuspended at 5�104

cells/ml in CTL medium. Five thousand target cells

per well were used in the assay. Effector cells (YTS)

were resuspended in CTL medium and preincubated

with ILZ-fusion proteins (5 Ag/ml final concentration)

at 25 8C for 15 min. After preincubation, the effector

cells were mixed with labeled target cells in a V-

bottom 96-well plate. Maximum release was deter-

mined by incubating target cells in 1% Triton X-

100. For spontaneous release, targets were incubated

without effectors in CTL medium alone. All

samples were analyzed in triplicate. After a 1-min

centrifugation at 1000 rpm, plates were incubated

for 3 h at 37 8C. Supernatant was harvested and51Cr release was measured in a gamma counter.

Percent specific release was calculated as ((exper-

imental release�spontaneous release)/(maximum

release�spontaneous release))�100.

3. Results

3.1. Production and purification of ILZ-fusion

proteins

To generate fusion proteins between the extra

cellular domains of type I activating human NK cell

receptors and an isoleucine-zipper sequence, we

constructed the expression vector pZipH shown in

Fig. 1A. The strong EF-1a promotor ensures high

expression in mammalian cells. The leader sequence

of the immunoglobulin kappa light chain (Ign) resultsin the secretion of the fusion protein into the culture

medium for easy purification. The cDNA encoding

the extracellular portion of cell surface receptors

lacking the signal sequence can be cloned into the

multiple cloning site in frame with the ILZ sequence.

For easy purification, we included a 6-histidine tag

downstream of the ILZ sequence followed by a stop

codon. The cDNAs encoding the extracellular portion

of 2B4, CD48, NTB-A, CS1 (CRACC), NKp30,

NKp44, NKp46, and CD4 were cloned in frame into

the pZipH vector and sequence verified. The resulting

expression vectors were transiently transfected into

the human embryonic kidney cell line 293T and

supernatant was collected on days 3 and 6 after

transfection. The ILZ-fusion proteins were purified

from the supernatant using nickel beads and purity


was confirmed by SDS-PAGE and coomassie staining

(Fig. 1B). The apparent size of the ILZ-fusion proteins

was greater than the calculated molecular weight of

the recombinant protein, indicating that the fusion

proteins were glycosylated.

3.2. Characterization of ILZ-fusion proteins

To confirm the identity of the different ILZ-

fusion proteins, we performed an ELISA. The

purified ILZ-fusion proteins were all recognized by

specific antibodies that also bind the native proteins

on cells (Fig. 2A). This confirmed the identity of

the ILZ-fusion proteins and suggests their correct

folding. We used some of the ILZ-fusion proteins to

immunize mice for the production of monoclonal

antibodies against the different NK cell receptors.

The resulting antibodies also recognized the native

protein on NK cells. This is another indication that

the ILZ-fusion proteins were correctly folded. As a

Fig. 2. Characterization of the ILZ-fusion proteins. (A) The indicated ILZ-f

ELISA using specific monoclonal antibodies (open bars) or an isotype cont

Ag/ml) was analyzed by gel filtration (Superdex200 column) using HPLC

protein standard. Higher-order multimers were eluted at a size of over 1 M

weight of 167 kDa. (C) Fifty nanograms of the indicated ILZ-fusion protei

(0.1% SDS) and separated by 3–8% Tris–Acetate gel electrophoresis. Samp

antibody (ILZ-11).

result of immunizing with ILZ-fusion proteins, we

also generated a monoclonal antibody against the

ILZ portion common to all ILZ-fusion proteins. This

antibody (ILZ-11) enabled us to detect the ILZ-

fusion proteins in immunoblot analysis (Fig. 2C)

and in cell surface staining (Fig. 3).

The ILZ sequence has been shown to form trimers

in solution (Harbury et al., 1994). To investigate

whether the ILZ-fusion proteins would also form

trimers, we analyzed their size in solution by gel

filtration. This analysis confirmed that the ILZ-fusion

proteins form trimers in solution, but also revealed

that a large part of the material formed higher-order

multimers (Fig. 2B and data not shown). Interest-

ingly, the oligomerization of the ILZ-fusion proteins

could also be visualized by SDS-PAGE and immu-

noblotting. Some protein complexes were stable

enough to survive heating in the presence of 0.1%

SDS and formed distinct bands corresponding to the

molecular weight of monomers, dimers, or trimers

usion proteins were coated onto a microtiter plate and detected in an

rol antibody (black bars). (B) Twenty microliters of ILZ-NKp44 (500

. Molecular weights were determined by comparison to a known

Da whereas trimeric molecules were found to possess a molecular

ns was incubated for 10 min at 70 8C in nonreducing sample buffer

les were analyzed by immunoblotting using an anti-ILZ monoclonal

Fig. 3. Surface staining using ILZ- and Ig-fusion proteins. (A) 293T cells were mock transfected (control) or transfected with plasmids encoding

CD48 or 2B4. Transfected cells were stained with the indicated ILZ-fusion proteins (0.5 Ag/ml for ILZ-CD48 and ILZ-2B4 and 2 Ag/ml for ILZ-

CD4) followed by anti-ILZ (ILZ-11) and PE-labeled goat anti-mouse antibodies. As a control, cells were stained with ILZ-11 (control) or with

the indicated antibodies followed by PE-labeled goat anti-mouse antibodies. (B) BaF3 or MEL1106 cells were incubated with (open plots) or

without (filled plots) the indicated ILZ-fusion proteins (0.5 Ag/ml) followed by anti-ILZ (ILZ-11) and PE-labeled goat anti-mouse antibodies.

(C) 293T cells were incubated with (open plots) or without (filled plots) the indicated fusion proteins. ILZ-fusion proteins were detected as

described in (A). Ig-fusion proteins were detected using biotin-conjugated goat anti-human antibodies followed by PE-conjugated Streptavidin.


(Fig. 2C). The ratio between monomers, dimers, and

trimers was dependent on the fusion protein. While in

this assay, ILZ-CD4 was mostly detected as mono-

mers, ILZ-CS1 was predominately detected as a

trimeric complex (Fig. 2C, note that the monomeric

ILZ-CS1 migrates at around 40 kDa). As all ILZ-

fusion proteins form trimers in solution as demon-

strated by the gel filtration analysis, the different

formation of SDS-stable oligomers must be a result

of the receptor part within the fusion protein. For

some ILZ-fusion proteins, bands migrating at a

higher molecular weight were observed (Fig. 2C),

again indicating that some of the fusion proteins also

form higher-order multimers. In the case of NTB-A

and CS1, these multimers may be a result of the

homophilic interaction of these receptors (Kumaresan

et al., 2002; Falco et al., 2004; Flaig et al., 2004;

Valdez et al., 2004).


Soluble receptor fusion proteins can be used to

stain their ligands on the surface of cells. This is

particularly useful in cases where the ligand is

unknown or where the receptor can bind to multiple

ligands. To test whether the ILZ-fusion proteins

could bind to their known ligands, we transfected

293T cells with CD48, 2B4, CS1, or NTB-A, the

ligands of 2B4, CD48, CS1, and NTB-A, respec-

tively, and stained the transfected cells with the

different ILZ-fusion proteins. We observed binding

of the ILZ-fusion proteins only to cells that

expressed the ligand for the respective soluble

receptor, indicating the specificity of this interaction

and the usefulness of ILZ-fusion proteins in this

assay (Fig. 3A, Flaig et al., 2004, and data not

shown).

The detection of known ligands on the surface of

cells using ILZ-fusion proteins of the corresponding

receptor has only a slight advantage over using

specific antibodies against the ligands for their

detection. An ILZ-fusion protein interacts with the

same epitope of the ligand as the cellular receptor

does and can therefore detect the presence of a

dfunctionalT ligand. Also in the case where one

receptor can bind to multiple ligands, such as

NKG2D, the use of soluble receptor constructs can

be advantageous as they assess the presence of

different ligands in a single staining. The real

application of soluble receptor constructs, however,

is to detect the presence of unknown ligands. To test

whether the ILZ-NKp30, NKp44, and NKp46 fusion

proteins can bind to their unknown ligands on the

surface of cells, we stained the human melanoma cell

line MEL1106. We detected clear binding of ILZ-

NKp30, NKp44, and NKp46 to MEL1106 cells,

indicating the presence of their ligands on the

surface of these cells (Fig. 3B). This binding was

specific as the fusion proteins did not stain the

mouse B cell line BaF3 (Fig. 3B) and as ILZ-CD4

did not stain the MEL1106 cells (data not shown).

Interestingly, the MEL1106 cells were stained most

strongly by ILZ-NKp30 and ILZ-NKp46. This is in

line with earlier reports showing that the killing of

melanoma cell lines by NK cells depends mostly on

NKp30 and NKp46 (Pende et al., 1999) and suggests

that the amount of staining with the ILZ-fusion

protein may be a direct measure of the amount of

ligand present on the cells.

3.3. Comparison between ILZ- and Ig-fusion proteins

The standard approach for generating soluble

receptors is to fuse the extracellular part with the Fc

portion of human IgG1. To compare such Ig-fusion

proteins with ILZ-fusion proteins, we used NKp30 as

an Ig or an ILZ-fusion protein for staining 293T cells

that express the ligand for NKp30 (Flaig et al., 2004).

While both fusion proteins specifically stained 293T

cells, the concentration necessary for optimal staining

was drastically different (Fig. 3C). The NKp30-Ig

fusion protein needed to be used at a concentration of

10 Ag/ml in order to achieve optimal staining (data not

shown). At such high concentrations, we observed

nonspecific binding of the ILZ-fusion protein. Opti-

mal binding of ILZ-NKp30 was observed at about 10-

fold lower concentrations (ca. 1 Ag/ml). Titration of

ILZ-NKp30 and NKp30-Ig showed that the ILZ-

fusion protein still stained the 293T cells at concen-

trations of 0.15 Ag/ml whereas the staining of NKp30-

Ig was lost at concentrations below 0.5 Ag/ml (Fig.

3C). This demonstrates that the trimeric ILZ-fusion

proteins have a higher avidity than dimeric Ig-fusion

proteins. Not surprisingly, we did not detect any

difference in binding affinity between the ILZ- and Ig-

fusion proteins since the staining of 293T cells was

comparable when using the optimal concentration of

both fusion proteins.

3.4. Blocking receptor–ligand interactions with

ILZ-fusion proteins


block the receptor–ligand interaction between differ-

ent cells. To test whether ILZ-fusion proteins can also

be used in such an application, we evaluated the

killing of the MHC-class I negative target cell

721.221 by the NK cell line YTS in the presence of

different ILZ-fusion proteins. The killing of 721.221

by YTS cells is partly dependent on the interaction

between 2B4 and CD48 (Watzl et al., 2000). The

presence of ILZ-CD48 or ILZ-2B4 could almost

completely inhibit the killing of 721.221 cells while

ILZ-CD4 as a control had little effect (Fig. 4). This

demonstrates that ILZ-fusion proteins can be used

successfully to block the interaction between activat-

ing NK cell receptors and their ligands. As ILZ-fusion

proteins do not interact with Fc receptors, no

Fig. 4. Blocking NK cell cytotoxicity using ILZ-fusion proteins.

The killing of 721.221 cells by the NK cell line YTS was analyzed

in a 3-h 51Cr release assay in the absence or presence of 5 Ag/ml

ILZ-CD4, ILZ-2B4, or ILZ-CD48. The use of 10 Ag/ml of the ILZ-

fusion proteins yielded identical results. All samples were done in

triplicates. Mean and standard deviation are shown.


unwanted effects are to be expected when using ILZ-

fusion proteins to interfere with receptor–ligand

interactions either in vitro or in vivo.

4. Discussion

The natural cytotoxicity receptors NKp30, NKp44,

and NKp46 play a major role in the activation of

human NK cells against a variety of different target

cells (Biassoni et al., 2001). The functional inves-

tigation of these important receptors is hampered by

the fact that their cellular ligands are unknown at the

moment. Soluble receptor constructs enable us to

identify the unknown ligands on the surface of target

cells and are therefore valuable tools to study such

orphan receptors. Here, we have described the use of a

novel form of recombinant receptor fusion proteins

with an ILZ sequence. ILZ-fusion proteins display a

higher avidity than classical Ig-fusion proteins, most

likely because of their trimeric structure. We also

observed higher-order multimers of ILZ-fusion pro-

teins in our analysis. We can only speculate about the

structure and activity of such multimers. On the one

hand, the avidity of such complexes may be very high,

which could contribute to the efficient staining of the

ILZ-fusion proteins at low concentrations. On the

other hand, some of the activity may be lost by

burying receptor molecules inside such complexes,

making them unavailable for ligand binding. Future

modification of the ILZ sequence may reduce the

amount of multimer formation, possibly increasing the

activity of ILZ-fusion proteins.

The approach for the construction of ILZ-fusion

proteins described here is only applicable to type I

transmembrane proteins. However, we have recently

adapted our expression vector for type II transmem-

brane proteins. In this vector, the Ign leader is

followed by a 6xHis tag, the ILZ sequence, and a

multiple cloning site into which the extracellular

portion of a receptor can be cloned. We have

successfully produced an ILZ-fusion protein of the

type II NK cell receptor NKG2D (Fig. 2A). However,

the binding activity of ILZ-NKG2D was weaker than

that of an NKG2D-Ig fusion protein (data not shown).

The adaptation of this method for type II trans-

membrane proteins may therefore require further

modifications. The NK cell receptors used for the

generation of soluble ILZ-fusion proteins are mono-

meric proteins in their native state on the cell surface.

Trimerization of these receptors by the ILZ sequence

did not interfere with their ability to bind to their

respective ligands. NKG2D forms homodimers in its

native state. The trimerization of such dimeric

proteins may interfere with their ligand binding which

could be an explanation for the weaker binding of the

ILZ-NKG2D compared to a dimeric Ig-fusion protein.

The successful use of ILZ-fusion proteins for naturally

trimeric proteins, such as members of the TNF

superfamily, has already been demonstrated (Walczak

et al., 1999). Therefore, the approach described here

seems best suitable for molecules that are either

monomeric or trimeric in their native state.

When compared to the standard method of

creating soluble receptor constructs by fusing them

to the Fc part of human IgG1, the ILZ-fusion

proteins have several advantages. Both fusion

proteins can easily be produced in human cell lines

and are secreted into the culture medium. The

purification of the ILZ-fusion proteins by Nickel-

chelate chromatography is cheap, very easy and


effective compared to the purification of Ig-fusion

proteins using protein-A beads. ILZ-fusion proteins

display a higher avidity than Ig-fusion proteins and

do not bind to Fc-receptor positive cells, thereby

avoiding unwanted effects when used for surface

staining or as blocking reagents in cellular assays.

The production of ILZ-fusion proteins in human cell

lines ensures their proper glycosylation, which may

be essential for the function of the soluble receptor.

This may be an advantage of the trimeric ILZ-fusion

proteins when compared to the novel tools of

tetramers, which are produced in bacteria and there-

fore lack any glycosylation (Altman et al., 1996).


block the interaction of a receptor with its ligand. We

have shown that blocking the interaction between 2B4

and CD48 can inhibit the activation of NK cells and

reduce the killing of 721.221 cells by the NK cell line

YTS (Fig. 4). However, the binding affinity between

the soluble receptor and the ligand on target cells is

likely to be the same as for the membrane-bound

ligand. Therefore, such blocking experiments need to

use an excess of soluble receptor in order to work

effectively. Blocking by specific antibodies may be

advantageous as antibody–antigen interactions usually

display a higher affinity, but the availability of such

specific reagents may be limited. In particular, when a

ligand is unknown, the use of soluble receptor

constructs may be the only way to effectively block

the ligand.

When using soluble ligands to engage a receptor,

this interaction can either block the receptor function

or lead to receptor stimulation. In the case of 2B4, we

observed that the binding of ILZ-CD48 is antagonistic

and can block 2B4-mediated NK cell activation (Fig.

4). We observed similar effects for ILZ-NTB-A (Flaig

et al., 2004). However, the binding of ILZ-fusion

proteins of TNF-receptor family ligands to TNF-

receptor family members can effectively stimulate the

receptor by inducing its trimerization (Walczak et al.,

1999). The effect of ILZ-fusion protein binding to a

receptor may therefore differ depending on the nature

of the receptor.

The use of ILZ-fusion proteins is not limited to the

study of receptor–ligand interactions on NK cells.

This method may be a valuable tool to further enhance

our knowledge about receptor–ligand interaction in

many different areas of research.

Acknowledgements

The authors would like to thank Dr. Frank

Momburg and Dr. Adelheid Cerwenka (German

Cancer Research Center, Heidelberg, Germany) for

their gifts of NKp30-Ig fusion protein, MEL1106

and BaF3 cells. We also thank Dr. Henning

Walczak (Apogenix Biotechnology, Heidelberg,

Germany) for the ILZ sequence. This work was

supported by the Deutsche Forschungsgemeinschaft

(SFB405, A9).

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