activity of soluble ox40 ligand is enhanced by oligomerization and cell surface immobilization

Activity of soluble OX40 ligand is enhanced byoligomerization and cell surface immobilizationNicole Muller1,*, Agnes Wyzgol1,*, Sabine Munkel2, Klaus Pfizenmaier2 and Harald Wajant1

1 Department of Molecular Internal Medicine, Medical Clinic and Polyclinic II, University of Wuerzburg, Germany

2 Institute of Cell Biology and Immunology, University of Stuttgart, Germany

The ligands of the tumor necrosis factor (TNF) family

and their corresponding receptors are crucially

involved in a variety of immunoregulatory and devel-

opmental processes [1]. TNF ligands activate receptors

belonging to the TNF receptor superfamily, which can

be subdivided into death receptors and non-death

receptors. The death receptors are characterized by a

conserved protein–protein interaction domain in their

cytoplasmic tail that links these molecules to death

domain-containing adaptor proteins and proapoptotic

proteases, such as caspase-8. The non-death receptors

lack a death domain and interact with adapter proteins

of the TNF receptor-associated factor family, which

are involved in the activation of nuclear factor kappa B

(NF-jB) transcription factors and various mitogen-

activated kinases [1]. TNF receptor-associated factor

binding receptors especially include CD27, 4-1BB and

OX40, which ensure the ongoing cell division of ini-

tially T-cell receptor ⁄CD28 costimulated T-cells and

further control T-cell differentiation [2–4]. For exam-

ple, OX40 has been implicated in the generation and

reactivation of memory T-cells. Accordingly, inhibitors

of the OX40 ligand (OX40L)–OX40 interaction are

under consideration for the treatment of autoimmune

and allergic diseases, whereas OX40 agonists are

exploited as adjuvants to stimulate antitumor immu-

nity [2–4].

With the exception of lymphotoxin-a, all members

of the TNF ligand family are single spanning trans-

membrane proteins with an amino-terminal cytoplas-

mic tail (type II membrane protein). Due to

proteolytic processing or alternative splicing, naturally

Keywords

FAP; NF-jB; oligomerization, OX40L; single

chain

Correspondence

H. Wajant, Department of Molecular

Medicine, Medical Clinic and Polyclinic II,

University of Wuerzburg, Roentgenring 11,

97070 Wuerzburg, Germany

Fax: +49 931 201 71070

Tel: +49 931 201 71010

E-mail: [email protected]

*These authors contributed equally to this

work

(Received 7 December 2007, revised 25

February 2008, accepted 6 March 2008)

doi:10.1111/j.1742-4658.2008.06382.x

OX40 ligand (OX40L) and OX40 are typical members of the tumor

necrosis factor ligand family and the tumor necrosis factor receptor super-

family, respectively, and are involved in the costimulation and differentia-

tion of T cells. Like other tumor necrosis factor ligands, OX40L is a

type II transmembrane protein. Recombinant soluble human OX40L

assembles into trimers and is practically inactive despite binding to OX40.

However, oligomerization of soluble OX40L trimers by cross-linking with

antibodies or by expression as a hexameric fusion protein strongly

increased the activity of the ligand. Moreover, a fusion protein of OX40L

with a single chain fragment recognizing the tumor stroma antigen fibro-

blast activation protein showed a cell surface antigen-dependent increase

in the activity of the ligand domain of the molecule and thus mimicked

the activity of membrane OX40L upon antigen binding. Trimeric single

chain OX40L fusion proteins therefore represent a novel type of OX40L-

derived immunostimulatory molecule with potentially reduced systemic

side effects.

Abbreviations

FACS, fluorescence activated cell sorting; FAP, fibroblast activation protein; IL, interleukin; IjBa, inhibitor of jB-alpha; JNK, c-Jun N-terminal

kinase; NF-jB, nuclear factor kappa B; THD, TNF homology domain; TNC, tenascin-C; TNF-a, tumor necrosis factor-alpha; TRAIL, TNF-related

apoptosis inducing ligand.

2296 FEBS Journal 275 (2008) 2296–2304 ª 2008 The Authors Journal compilation ª 2008 FEBS

occurring soluble variants also exist for most of the

TNF ligands. The structural hallmark defining the

TNF ligand family is the carboxy-terminal TNF

homology domain (THD), which is composed of two

stacked b-pleated sheets adopting a conserved jelly-

roll-like tertiary fold [5,6]. The THD mediates self

association into trimers and is necessary for receptor

binding. In accordance with the carboxy-terminal

localization of the THD, the transmembrane forms of

TNF ligands, as well as soluble ligand variants,

assemble into trimers [5,6]. Despite their similar tri-

meric organization, membrane-bound and soluble

TNF ligands can differ in their activity. With respect

to receptor binding and receptor activation by TNF

ligands, three cases can be distinguished. First, both

the transmembrane as well as the corresponding solu-

ble variant of a particular TNF ligand bind and acti-

vate their cognate TNF receptor. An example for this

case is TNF receptor-1, which becomes properly acti-

vated by soluble as well as membrane TNFa [7]. In

the second case, the transmembrane variant of a TNF

ligand interacts and stimulates the corresponding

receptor, whereas the soluble variant neither binds,

nor activates the receptor. An example of this case is

murine CD95L [8]. Third, a TNF receptor interacts

with both the transmembrane variant of its corre-

sponding ligand as well as with the derived soluble

counterpart, but is not, or only poorly, stimulated by

the later. Such a differential activity of the soluble

and transmembrane ligand form has been demon-

strated for human CD95 and human TNF receptor-2,

[7,9]. The reasons underlying the quite diverse quality

of soluble TNF ligands with respect to receptor bind-

ing and receptor activation are currently poorly

understood.

Notably, inactive soluble TNF ligands that still

bind their receptors can be converted into highly

active molecules by increasing their avidity or by

immobilization on a cell surface or the extracellular

matrix. The most prominent and best investigated

example in this respect is human CD95L. Soluble tri-

meric human CD95L binds its corresponding receptor

CD95 at physiological relevant concentrations, but

fails to stimulate it properly and even acts as an

antagonist for membrane CD95L-induced activation

of CD95 [9]. When soluble CD95L trimers were

secondarily multimerized by antibody mediated cross-

linking or genetic fusion with an oligomerizing pro-

tein domain, the increase in avidity correlated with

an up to thousand-fold enhancement of the specific

activity of CD95L [9–11]. A similar increase in activ-

ity was observed upon binding of the soluble CD95L

to components of the extracellular matrix via a bind-

ing motif located N-terminally to the THD of the

ligand [12]. Likewise, soluble trimeric single chain-

CD95L fusion proteins achieve high activity after

docking to a cell surface antigen [13,14]. Other solu-

ble ligands of the TNF family that only gain high

activity after oligomerization are TNF-a in respect to

TNF receptor-2 and CD40L, 4-1BBL and TNF-

related apoptosis inducing ligand (TRAIL) in respect

to TRAIL receptor-2 [9,15–20]. Notably, both aggre-

gated and non-aggregated TNFa and TRAIL trimers

activate comparably well with TNF receptor-1 and

TRAILR1, respectively. This indicates that the

requirement of a soluble TNF ligand for oligomeriza-

tion to gain high activity is not ligand intrinsic, but

also depends on the receptor. The previously noted

observation that trimeric soluble fusion proteins of

inactive or poorly active TNF ligands acquire high

activity after immobilization opens the possibility of

restricting the activation of their cognate receptors to

cell surface antigen positive cells and tissue

[13,14,18,21–24]. This is of potential relevance for the

therapeutic application of these molecules because it

avoids therapy restricting side effects on cell surface

antigen negative cells and tissue. Noteworthy, for sev-

eral of the TNF ligand family members, including

OX40L, it is not known whether the soluble variant

of the molecule depends on oligomerization to elicit

maximal activity.

In the present study, we report that oligomerization

of soluble OX40L trimers by cross-linking with anti-

bodies or by expression as a hexameric fusion protein

increases the activity of the molecule by more than

200-fold. Accordingly, a fusion protein of OX40L

linked to a single chain fragment recognizing the

tumor stroma antigen fibroblast activation protein

(FAP) shows a significant increase in activity after

binding to its cognate cell surface antigen, thus defin-

ing a novel type of OX40 agonist with potentially

reduced systemic side effects.

Results and Discussion

Expression, purification and molecular

organization of soluble OX40L variants

To determine the relevance of oligomerization of solu-

ble ligand trimers for OX40 activation, we generated

fusion proteins of the extracellular THD of OX40L

with a Flag-tag, with a Flag-tagged tenascin-C (TNC)

trimerization domain and a Flag-tagged immun-

globulin Fc domain (Fig. 1A). The use of a Flag-tag

facilitated purification of the various fusion proteins.

In addition, it allowed oligomerization of the various

N. Muller et al. Soluble OX40L variants with different activities

FEBS Journal 275 (2008) 2296–2304 ª 2008 The Authors Journal compilation ª 2008 FEBS 2297

OX40L variants by treatment with Flag-specific

monoclonal antibody. The Flag-TNC-OX40L fusion

protein was included in the study due to our recent

finding that a disulfide-bonded TNC trimerization

domain can enhance receptor binding by some mem-

bers of the TNF ligand family (e.g. murine TRAIL

and murine or human CD95L) without changing the

principle trimeric assembly of these molecules [8]. The

dimerizing Fc domain of human immunoglobulin G1

was used with the intention to drive the formation of

hexameric ligands, as recently shown for a Fc-CD95L

fusion protein [10]. All fusion proteins were produced

in Hek293 cells and were purified by affinity chroma-

tography on monoclonal M2 anti-Flag agarose

(Fig. 1B,C). The migration pattern of Flag-OX40L in

nonreducing SDS ⁄PAGE analysis revealed three

bands with molecular masses of 20, 23 and 27 kDa,

respectively. Flag-TNC-OX40L showed a band of

approximately 100 kDa and a broad fuzzy band of

60–80 kDa. Fc-Flag-OX40L migrated as single band

with a molecular mass of 90 kDa. Under reducing

conditions, Flag-Ox40L ran as a triplet with 23, 27

and 30 kDa, Flag-TNC-OX40L migrated with 27 and

31 kDa and Fc-Flag-OX40L revealed a single band of

60 kDa. The data obtained for Flag-TNC-OX40L are

in accordance with the expected disulfide-bonded

formation of Flag-TNC-OX40L trimers. The values

observed for Fc-Flag-OX40L suggest that this mole-

TNF homology domain

Flag-OX40L (52–183)Flag OX40L aa 137-281Flag OX40L aa 52–183

Flag-TNC-OX40L (52–183)TNCFlag OX40L aa 137-281OX40L aa 52–183

Fc-Flag-OX40L (52–183)Flag OX40L aa 137-281Flag OX40L aa 52–183hIgG1 (hinge + Fc)

A

Flag-OX40L

Fc-Flag-OX40L

Flag-TNC-OX40L

83

6248

33

25

17

C

+ – + – + –dithiothreitol dithiothreitol

Flag-OX40L

Fc-Flag-OX40L

Flag-TNC-OX40L

836248

33

25

17

175

B

+ – + – + –

D

Elution volume

Fc-Flag-OX40LFlag-OX40L TNC-Flag-OX40LMW standards

HMW670

150

4417

Abs

orba

nce

Fig. 1. Characterization of recombinant variants of soluble OX40L. (A) Scheme of the OX40L variants used in the present study. Fc, human

immunoglobulin G1 Fc fragment; F, Flag tag; OX40L, human OX40L (amino acids 52–183); TNC, chicken tenascin-C (amino acids 110–139).

(B) The indicated Flag-tagged variants of soluble OX40L containing the THD were produced in Hek293 cells, purified by M2 affinity chroma-

tography, separated by SDS ⁄ PAGE under reducing and nonreducing conditions and finally visualized by silver staining. (C) Western blot anal-

ysis of the various OX40L variants using the monoclonal M2 anti-Flag serum. (D) The OX40L variants were separated by gel filtration on a

BioSep-Sec-S3000 column. Arrows indicate the elution volume of the molecular weight standards thyroglobulin (670 kDa), IgG (150 kDa),

ovalbumin (44 kDa) and myoglobulin (17 kDa).

Soluble OX40L variants with different activities N. Muller et al.


cule assembles into hexamers composed of three

noncovalently linked, disulfide bonded Fc-Flag-

OX40L dimers. The observed molecular masses signif-

icantly exceed the calculated mass of 17, 21 and

47 kDa for the various OX40L fusion proteins, but

are in good accordance with glycosylation of OX40L

on three asparagine residues in the THD because one

N-linked carbohydrate accounts for 2–5 kDa by

SDS ⁄PAGE. Based on gel filtration using a BioSep-

Sec-S3000 column, molecular masses of 80, 130 and

580 kDa were calculated for native Flag-OX40L,

Flag-TNC-OX40L and Flag-Fc-OX40L (Fig. 1D).

The molecular mass standards used for calibration in

the gel filtration analysis experiments were all globular

shaped proteins, whereas, from X-ray crystallography,

it is known that ligand trimers of the TNF family

form elongated bell-shaped structures. Thus, these val-

ues, despite deviating somewhat from the expected

calculated molecular masses, are in reasonable agree-

ment with the expected trimeric organization of Flag-

OX40L and Flag-TNC-OX40L and a hexameric orga-

nization of Flag-Fc-OX40L. A hexameric organization

has recently also been shown for an immunoglobulin

OX40L fusion protein that was linked with an isoleu-

cine zipper domain [25].

Trimeric variants of soluble OX40L are practically

inactive, but gain high activity after cross-linking

To analyse OX40 binding of the various OX40L vari-

ants, fluorescence activated cell sorting (FACS) analy-

sis were performed with OX40 negative HT1080 cells

and HT1080 transfectants expressing OX40 (Fig. 2A).

Although concentrations up to 10 lgÆmL)1 failed to

show significant binding to HT1080 cells, 1 lgÆmL)1

of Flag-OX40L or Flag-TNC-OX40L and

0.01 lgÆmL)1 of Fc-Flag-OX40L already were suffi-

cient to reveal significant and specific binding to

OX40 transfected cells (Fig. 2B). Next, we analysed

OX40 activation by the various OX40L variants, with

and without cross-linking (Fig. 3A–D). Fc-Flag-

OX40L induced significant interleukin (IL)-8 produc-

tion in HT1080-OX40 at concentrations below

100 ngÆmL)1 and showed only a modest increase in

activity after cross-linking with monoclonal M2 anti-

Flag serum (Fig. 3C). The high cross-linking indepen-

dent activity of Fc-Flag-OX40L are in accordance

with other studies, where non-oligomerized OX40L-Fc

fusion proteins have been successfully used to enhance

in animal models immunity against Leishmania

donovani, Cryptococcus neoformans, methylcholanthrene-

induced sarcomas and various tumor cell lines [26–

28]. In contrast to Fc-Flag-OX40L, Flag-OX40L and

Flag-TNC-OX40L triggered only marginal IL-8

production even at concentrations exceeding

1000 ngÆmL)1 (Fig. 3A,B). Notably, after oligomeriza-

tion with monoclonal M2 anti-Flag serum, significant

IL-8 induction by the latter two reagents occurred

with concentrations of approximately 10 ngÆmL)1

(Fig. 3A,B). Cytokine induced IL-8 production typi-

cally involves stimulation of the p38, extracellular sig-

nal-regulated kinase, c-Jun N-terminal kinase (JNK)

and NF-jB signaling pathways. The activity of the

latter two was analyzed by determination of phos-

phorylation of inhibitor of jB-alpha (IjBa) and JNK.

In accordance with the requirement of oligomerization

of soluble OX40L trimers for robust OX40-induced

IL-8 production, phosphorylation of JNK and IjBawas evident with 5 ngÆmL)1 of cross-linked Flag-

OX40L, whereas even 200 ngÆmL)1 of non-aggregated

Flag-OX40L failed to trigger significant phosphoryla-

tion of these proteins (Fig. 3D). Based on these data

the OX40L–OX40 interaction can be included in the

growing list of TNF–TNF receptor interactions where

oligomerization, and thus an increase in avidity,

is necessary to ensure robust receptor activation by

soluble ligand variants.

Cell surface antigen-mediated ‘immobilization’

confers high activity towards a trimeric single

chain-OX40L fusion protein

Single chain fusion proteins of TNF ligands that

were practically inactive as soluble trimers gain high

activity compared to their corresponding membrane

bound form after binding to a cognate cell surface

antigen [13,14,18,21–24]. To determine whether tri-

meric fusion proteins of soluble OX40L can be acti-

vated in a similar manner, we generated and

analysed an OX40L fusion protein with a N-terminal

single chain antibody fragment recognizing the cell

surface tumor stroma antigen FAP (Fig. 4A). The

anti-FAP fusion protein contained an internal Flag-

tag, which was used to purify the protein from cell

culture supernatants of transiently transfected Hek293

cells by M2-agarose affinity chromatography. Gel

filtration analysis of the purified single chain OX40L

fusion protein (sc40-Flag-OX40L) confirmed the

expected trimeric organization of the molecule (data

not shown). Specific binding of the sc40-Flag-OX40L

fusion protein to HT1080-OX40 and HT1080-

FAP transfectants, but not HT1080 cells, indicated

that both domains of the fusion proteins retained

their binding capabilities (Fig. 4C). Similar to soluble

Flag-OX40L, the sc40-Flag-OX40L fusion protein

showed only weak activity on OX40 expressing cells



at high concentrations (Fig. 4D). After cross-linking

with monoclonal M2 anti-Flag serum, the single

chain OX40 fusion protein was comparably active as

cross-linked Flag-OX40L or Fc-Flag-OX40L (data

not shown). More importantly, in the presence of

FAP expressing cells, sc40-Flag-OX40L showed an

approximately 200-fold higher activity in inducing

IL-8 production in cocultured HT1080-OX40 cells

(Fig. 4D). Using agonistic antibodies or OX40L

immunoglobulin fusion proteins, it has been demon-

strated that OX40 activation, either alone or in com-

bination with other immunstimuli, can enhance an

anti-tumoral immune response in mouse tumor

models with melanoma, colon cancer, glioma and

breast cancer [28–32]. Potential problems that might

arise from clinical applications of OX40 activating

reagents are the induction of autoimmunity and

inflammatory side effects [4]. Trimeric OX40L fusion

proteins that show cell surface antigen-restricted acti-

vation of OX40L ⁄OX40 thus appear to be promising

Rel

ativ

e ce

ll nu

mbe

r

log fluorescence intensity

con.

HT1080-OX40 IgG1


Pos

itive

cel

ls (

%) con.

Flag-OX40L

Flag-CD27L

0

80

20

60

40

101 10–1 10–2 10 µg·mL–1

CD27L-F

0

20

40

60

80

100

A

Rel

ativ

e ce

ll nu

mbe

rR

elat

ive

cell

num

ber

Rel

ativ

e ce

ll nu

mbe

r


B

con.

Flag-TNC-OX40L

Flag-CD27L

HT1080-OX40 aOX40

Flag-OX40L (µg·mL–1)

Flag-TNC-OX40L (µg·mL–1)

10–1 10–2 100

Fc-Flag-OX40L (µg·mL–1)

10–3 10–4

Pos

itive

cel

ls (

%)

0

80

20

60

40


con.

Fc-Flag-OX40L

Fc-Flag-4-1BBL

101 10–1 10–2 100 10 µg·mL–1

CD27L-F

10 µg·mL–1

Fc-F-41BBL

Fig. 2. OX40 binding of OX40L variants.

(A) OX40 cell surface expression of HT1080

and HT1080-OX40 cells was determined by

FACS analysis with PE-labelled anti-OX40.

(B) HT1080 and HT1080-OX40 cells were

incubated with increasing concentrations of

Flag-OX40L, Flag-TNC-OX40L and Flag-

Fc-OX40L on ice and, after repeated

washes, bound proteins were detected

using monoclonal M2 anti-Flag serum and

PE-labelled anti-mouse IgG. Amino-termi-

nally Flag-tagged soluble human CD27L was

used as a control.



for making therapy concepts that rely on OX40 acti-

vation safer.

Experimental procedures

Production and purification of recombinant

proteins

The various OX40L fusion proteins were produced in

Hek293 cells. In brief, cells were electroporated

(10–20 · 106 cellsÆmL)1; 4 mm cuvette; 250 V, 1800 lF,maximum resistance) in culture medium with 10% fetal

bovine serum and 40 lg of the corresponding expression

plasmid using an Easyject Plus electroporator (PeqLab,

Erlangen, Germany). Transfected cells were cultured over-

night and, the next day, medium was changed to RPMI

containing 0.5% fetal bovine serum. Supernatants were col-

lected 3 days post transfection and recombinant proteins

were purified by affinity chromatography using anti-Flag

M2 agarose columns. After elution with 100 lgÆmL)1 Flag

peptide (Sigma, Steinheim, Germany) in NaCl ⁄Pi, fractions

containing the OX40L variants were dialyzed against

NaCl ⁄Pi and analyzed by SDS ⁄PAGE.

Gelfiltration chromatography on

BioSep-SEC-S3000

Purified OX40L variants (25 lL) were applied to a BioSep-

SEC-S3000 (300 · 7.8) column (Phenomenex, Aschaffen-

burg, Germany) equilibrated in NaCl ⁄Pi and eluted at a

flow rate of 0.5 mLÆmin)1. The columns were calibrated

with thyroglobulin (670 kDa), IgG (150 kDa), ovalbumin

(44 kDa) and myoglobulin (17 kDa).

Flow cytometry with OX40L variants

Cells were incubated for 2 h at 4 �C with the indicated con-

centration of the OX40L variant of interest and, after three

cycles of washing with NaCl ⁄Pi, 0.2% fetal bovine serum

and 0.02% sodium azide, bound proteins were detected

by monoclonal M2 anti-Flag serum (1 lgÆmL)1; Sigma)

and phycoerythrin (PE)-labelled, mouse IgG-specific rabbit

C

A B

P-I B

I B

P-JNK

+ Flag mAb M2

JNK

1000

405 5000

200– Flag-OX40L (ng·mL–1)1000

405 5000

200–

0

1

2

3

Fc-Flag-OX40L (ng·mL–1)

+M2

4

5

6

0

2

4

6

8

Flag-TNC-OX40L (ng·mL–1)

+ M2

102100 101 1030

D

0

1

2

3

4

IL8

(ng·

mL–

1 )

IL8

(ng·

mL–

1 )

Flag-OX40L (ng·mL–1)102100 101 1030

–+ M2

102100 101 1030

IL8

(ng·

mL–

1 )

–

–

Fig. 3. Activity of OX40L trimers is

enhanced by oligomerization. (A–C) HT1080-

OX40 cells were stimulated in triplicates

with the indicated concentrations of Flag-

OX40L (A), Flag-TNC-OX40L (B) and Fc-Flag-

OX40L (C) with and without anti-Flag

cross-linking. After 6 h, supernatants were

removed and their relative IL-8 content was

determined by ELISA. Cell culture medium

was changed before stimulation to minimize

the contribution of OX40-independent IL-8

production due to constitutive IL-8 expres-

sion. (D) To determine JNK and NF-jB acti-

vation, lysates of HT1080-OX40 cells

stimulated for 10 min with the indicated

concentrations of cross-linked and non-

cross-linked Flag-OX40L were analyzed by

SDS ⁄ PAGE and western blotting with anti-

JNK, anti-phospho-JNK, anti-IjBa, anti-phos-

pho-IjBa and anti-tubulin.



antibodies (1 lgÆmL)1; Sigma). Analyses were performed

using FACSCalibur (BD Biosciences, Heidelberg, Germany)

according to standard procedures.

IL-8 ELISA

Cells (2 · 104 well)1) were seeded in 96-well tissue culture

plates and grown overnight. The next day, the medium was

changed to minimize the contribution of basal IL-8 produc-

tion and cells were stimulated in triplicates with varying

concentrations of the indicated OX40L variants. After 6 h,

supernatants were collected and analyzed for IL-8 produc-

tion using a commercially available ELISA kit (BD

Biosciences) according to the manufacturer’s instructions.

Western blot detection of phosphorylated

proteins

For western blot analysis of phosphorylated proteins, cells

were scraped into ice-cold NaCl ⁄Pi with a rubber policeman,

collected by centrifugation and lysed after sonification (ten

pulses) by boiling (5 min, 96 �C) in 4 · Laemmli sample buf-

fer (8% SDS, 0.1 m dithiothreitol, 40% glycerol, 0.2 m Tris,

pH 8.0) supplemented with phosphatase inhibitor cocktails

I and II (Sigma). Proteins were separated by SDS ⁄PAGE

and transferred to nitrocellulose membranes. After blocking

of nonspecific binding sites by incubation in Tris-buffered

saline containing 0.1% Tween 20 and 5% dry milk, immuno-

blotting was performed with primary antibodies recognizing

JNK, phospho-JNK, phospho-IjBa (Cell Signalling, Frank-

furt, Germany) and IjBa (Santa Cruz Biotechnologies Inc.,

Heidelberg, Germany), horseradish peroxidase-conjugated

secondary antibodies (Dako, Hamburg, Germany) and the

ECL western blotting detection reagents and analysis system.

Acknowledgements

This work was supported by Deutsche Krebshilfe

[Grants 106222 (H.W.) and 106235 (K.P.)] and Deut-

sche Forschungsgemeinschaft (SFB 487 project B7).

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HT1080-FAP

CHT1080

log fluorescence intensity log fluorescence intensity

HT1080

HT1080-OX40

0

1

2

3

4

sc40-Flag-OX40L (ng·mL–1)102100 101 1030

HT1080 + HT1080-OX40HT1080-FAP + HT1080-OX40

D

B175

83

62

48

33

25

+ –

SC40-Flag-OX40L (52–183)Flag OX40Laa137-281Flag OX40L aa 52–183FAP-specific single chain fragment “sc40”A

Rel

ativ

e ce

ll nu

mbe

r

Rel

ativ

e ce

ll nu

mbe

rIL

8 (n

g·m

L–1)

dithiothreitol

Fig. 4. A single chain-OX40L fusion protein

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activity of soluble ox40 ligand is enhanced by oligomerization and cell surface immobilization

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