CD137 Ligand Signaling Induces Human Monocyte to Dendritic Cell Differentiation

CD137 ligand signaling induces human monocyteto dendritic cell differentiation

Shaqireen Kwajah M. M. and Herbert Schwarz

Department of Physiology and Immunology Programme, Yong Loo Lin School of Medicine,

National University of Singapore, Singapore

The ligand for CD137 (4-1BB) is expressed on peripheral human monocytes and delivers a

potent activating signal via reverse signaling. Here we show that treatment of monocytes

with a recombinant CD137 protein that induces reverse signaling through CD137 ligand

reduces typical macrophage characteristics such as phagocytosis, oxidative burst and

CD14 expression; however, typical DC characteristics including endocytosis, costimulatory

molecule expression and the ability to stimulate proliferation of naıve T cells are

induced. CD137-generated DC do not express DC-SIGN, CD1a or IL-12 and secrete less

IL-10 than classical DC. CD137-generated DC are mature, and addition of LPS1IFN-c does

not enhance their T-cell-stimulatory capacity. This indicates that CD137 as a sole factor is

sufficient to induce development to mature DC, making stimulation of CD137 ligand the

most simple protocol to generate mature DC. CD137-generated DC are more potent in

inducing T-cell proliferation than classical DC. They inhibit development of Treg cells but

induce T-cell expression of perforin, IFN-c, IL-13 and IL-17. These data demonstrate that

CD137 as a single factor is sufficient to induce differentiation of peripheral monocytes to

mature inflammatory DC that have a more potent T-cell-stimulatory capacity than clas-

sical DC.

Key words: CD137 . DC . Differentiation . Monocyte

Introduction

DC are crucial initiators of adaptive immune responses. Based on

cell lineage origin, phenotype, pathogen recognition mechan-

isms, function and tissue localization various DC subsets have

been defined [1–4]. Monocyte-derived DC are generated from

peripheral blood monocytes under the influence of inflammatory

conditions and support resident DC in initiating potent T-cell

responses [5]. Indeed, protective anti-Leishmania major immune

responses in mice were critically dependent on monocyte derived

DC while resident DC neither captured nor presented Leishmania

major antigens [6]. Also, in man, monocyte to DC differentiation

is essential for protective Th1 responses against lepromatous

leprosy [7].

Differentiation of monocytes to DC with GM-CSF1IL-4, and

subsequent maturation with LPS is the classical protocol for

generating monocyte-derived DC in vitro [8]. However, a range

of different inflammatory stimuli can induce monocyte to DC

differentiation, and not surprisingly, monocyte-derived DC

generated by different inflammatory stimuli may differ in their

functions [9].

The TNF receptor family member CD137 (TNFRSF9, 4-1BB)

has been shown to regulate several aspects of myelopoiesis.

Myeloid cells and hematopoieitic progenitor cells express CD137

ligand as a cell surface protein. Though classified as a ligand,

functionally it is a ligand as well as a receptor and it can trans-

duce signals into the cells it is expressed on, a process referred to

as reverse signaling [10]. CD137 ligand agonists induce prolif-

eration and colony formation of hematopoieitic progenitor cellsCorrespondence: Professor Herbert Schwarze-mail: [email protected]

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

DOI 10.1002/eji.200940105 Eur. J. Immunol. 2010. 40: 1938–1949Shaqireen Kwajah M. M. and Herbert Schwarz1938

and their differentiation to macrophages in man and mouse

[11, 12]. In human peripheral monocytes CD137 ligand

signaling induces adherence, activation, migration, survival and

endomitosis/proliferation [13–18]. CD137 ligand signals syner-

gize with TLR signals in inducing monocyte activation [19, 20].

Further, CD137 ligand agonists induce maturation of human

immature DC (imDC), leading to increased expression of

CD83, CD86, MHC class II, TNF and IL-12, the ability of DC to

migrate, and to induce proliferation and IFN-g secretion in T cells

[21–23].

Based on induction of monocyte activation, and macrophage

differentiation in hematopoietic progenitor cells, we suspected

that CD137 ligand signals would promote monocyte to macro-

phage differentiation. Surprisingly, we found that instead CD137

ligand signals induce monocyte to DC differentiation. Specifi-

cally, CD137 ligand signals reduce the expression of the macro-

phage marker CD14 but increase expression of CD80, CD86 and

CD83. These phenotypic changes are accompanied by functional

changes. Macrophage-typical activities such as phagoctosis and

oxidative burst are reduced while DC activities such as endocy-

tosis and the ability to induce T-cell proliferation are enhanced.

CD137-generated DC are fully mature, since they cannot be

further matured with LPS and IFN-g.

Results

CD137 ligand signaling inhibits macrophage activities

Human monocytes were purified from PBMC that had been

isolated from buffy coats. For crosslinking of CD137 ligand the

monocytes were cultured on plates that had been coated with a

fusion protein consisting of the extracellular domain of CD137

fused to the constant domain of human IgG1 (Fc). Wells coated

with an equal concentration of the Fc protein and non-coated

wells (PBS) were used as negative controls.

Instead of the expected enhancement we found that CD137

ligand signaling inhibited typical macrophage activities. Treat-

ment with CD137-Fc protein significantly reduced the phagocytic

capacity of monocytes on days 1, 2, 3 and 7, while the Fc control

protein had no effect. On days 2 and 3 the phagocytic activity was

less than one-third of that of untreated or Fc-treated monocytes.

The phagocytic activity was also reduced in immature classical

DC but not in macrophages that were differentiated in the

presence of recombinant M-CSF (Fig. 1A).

Similarly, when monocytes were activated by PMA to induce

oxidative burst, treatment with immobilized CD137 protein for 1

day was sufficient to reduce the oxidative burst to less than one-

third of untreated or Fc-treated monocytes. The oxidative burst

after CD137 treatment remained low throughout the 4-day

period of analysis (Fig. 1B). Expression of CD14, which is higher

on macrophages than on DC, was close to abolished by CD137

ligand signaling (Fig. 1C). These data indicate that CD137 not

only do not support monocyte to macrophage differentiation,

rather it inhibits it.

day 1 day 2 day 32542

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Time (days)

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Time (days)

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PBS

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ControlDHR123

C PBS Fc CD137-FcDay

1 65.9% 47.0

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7.4% 6.1

254 .9% 64.6% 1.1%16.9 19.8 1.8E

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ts

4 20.0% 18.7

28.8% 22.6

8.2%11.0

CD14

Figure 1. CD137 ligand signaling reduces macrophage functions.Monocytes were cultured on uncoated (PBS) or on plates coated with10 mg/mL of Fc or CD137-Fc protein for 1, 2, 4 or 7 days and analyzed byflow cytometry. Or monocytes were differentiated to DC or macro-phages by 80 ng/mL GM-CSF1100 ng/mL IL-4 or 100 ng/mL M-CSF,respectively. (A) Phagocytosis: FITC-labeled latex beads were added for1 h before analysis. Shown are flow cytometry histogram (top) and aquantitative comparison (bottom). Control: Autofluorescence of thecells. Numbers in histograms state mean fluorescence intensities. (B)Oxidative burst: Cells were activated by 20 mg/mL PMA, and DHR123was added for 30 min before analysis. Shown are flow cytometryhistogram (left panel) and a quantitative evaluation (right panel).Control: Autofluorescence of the cells. (C) CD14 expression: Cells wereimmunostained for CD14 and analyzed. Numbers in histogramsindicate percentages of positive cells and mean fluorescence inten-sities. �po0.05; ��po0.01 using a two-tailed unpaired Student’s t-test.These experiments have been performed three times with comparableresults.


Eur. J. Immunol. 2010. 40: 1938–1949 Cellular immune response 1939

CD137 ligand signaling induces DC characteristics

Monocytes can differentiate to macrophages or DC. Since CD137

inhibited macrophage differentiation we tested whether it

induces DC differentiation. As a positive control we included

monocyte derived DC that were generated by the classical

method of GM-CSF1IL-4 stimulation.

While the phagocytic activity decreases during monocyte to DC

differentiation the endocytic acitivity increases [24]. Exposure of

monocytes to recombinant CD137 protein enhanced the rate of

endocytosis over a 9-day period as measured by the uptake of

flourescently labeled dextran by flow cytometry. On day 7, CD137-

treated monocytes had a four times enhanced fluorescence

(120.2714.8) as compared with untreated or Fc-treated monocytes

(30.772.7) (Fig. 2A). imDC generated by GM-CSF1IL-4 had an

even higher endocytosis rate (200.775.7) but a 2-day maturation

decreased their endocytic capacity (85.872.3) to the level of CD137-

treated monocytes, suggesting that CD137 treatment induces DC

differentiation and that CD137-generated DC are already mature.

As a further criterion to assess macrophage versus DC differ-

entiation, the morphology of CD137-generated DC was compared

with that of immature and mature DC (mDC). imDC were mainly

rounded cells and maturation induced fiber-like extensions and a

spindle-shaped cell morphology as well as cells with rounded

morphology. Similar fiber-like extensions and spindle-shaped and

rounded morphologies were induced by CD137 treatment although

CD137-treated cells adhered more strongly to the culture dishes

resulting in a flatter appearance of the cells (Fig. 2B).

CD137 ligand signaling induces differentiation to mDC

DC differentiation and maturation are characterized by phenotypic

changes, such as the increase in expression of co-stimulatory

molecules. Indeed, CD137-generated DC expressed enhanced levels

of co-stimulatory molecules. After maturation with LPS1IFN-g 60%

of the cells were positive for CD80 with a MFI of 42 compared with

48% of positive cells with a MFI of 18 in the Fc control condition.

There was no significant difference between the Fc and CD137-Fc

treated monocytes in the absence of LPS1IFN-g maturation.

GM-CSF1IL-4 induced even higher levels of CD80 expression,

before as well as after maturation by LPS1IFN-g (74% positive,

MFI 5 35 versus 93% positive, MFI 5 106) (Fig. 3A).

Treatment with CD137 alone was sufficient to enhance

expression of CD86 (from 45%, MFI 5 16 in the Fc condition to

86%, MFI 5 108). Maturation with LPS1IFN-g increased CD86

expression further (97%, MFI 5 202) (Fig. 3A). imDC expressed

less CD86 than CD137-generated DC (51%, MFI 5 44), but more

after maturation by LPS1IFN-g (94%, MFI 5 203) (Fig. 3A).

Interestingly, expression of HLA-DR was reduced by GM-CSF1IL-4

PBS Fc CD137-Fc

Day 9A

B

imDC mDCAutoflourescence

FITC-dextran

250**

*

150

200

***

Fc

PBS

0

50

100

Mea

n F

luo

resc

ence

** **** GM-CSF + IL-4

GM-CSF + IL-4 + LPS

CD137-Fc

Time (days)

1 2 3 7 9

Figure 2. CD137 ligand signaling enhances DC characteristics. Monocytes were treated with immobilized Fc or CD137-Fc protein for 7 days, or with80 ng/mL GM-CSF1100 ng/mL IL-4 (imDC). In total, 1mg/mL LPS150 ng/mL IFN-g were added to the cultures for an additional 18 h. (A) The endocyticcapacity of the cells was determined by their ability to take up FITC-labeled dextran. Shown are flow cytometry histogram for day 9 (top) and aquantitative evaluation for all time points (bottom). (B) Photographs were taken on day 8 at magnifications of 630� . Scale bars: 20mm.


Eur. J. Immunol. 2010. 40: 1938–1949Shaqireen Kwajah M. M. and Herbert Schwarz1940

21%7

48%18

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γ

CD80A

88% 416

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** **

Figure 3. CD137-generated DC display markers of activated DC. (A) Cells were immunostained for the indicated surface markers and analyzed byflow cytometry. Black and grey histograms represent isotype control and indicated surface markers, respectively. Indicated are percentages ofpositive cells and the values of mean fluorescence intensities. DC: Treatment with GM-CSF1IL-4. (B) Dose response curve. Monocytes were treatedwith indicated concentrations of immobilized Fc or CD137-Fc protein, and expression of CD80 and CD86 was determined by flow cytometry onday 7. (C) Supernatants from (A) were harvested on day 8 and concentrations of IL-12p70, IL-10 and IL-23 were determined by ELISA. Depicted aremeans7standard deviations of triplicate measurements. N.d.: not detectable. �po0.05; ��po0.01 using a two-tailed unpaired Student’s t-test. Thisexperiment has been performed three times with comparable results.



treatment and even more so by CD137 treatment, especially in the

absence of maturation by LPS1IFN-g (Fig. 3A).

Both CD137 and GM-CSF1IL-4 enhaced expression of CD83, a

marker for activated DC. Similar to CD86, expression of CD83 was

higher on CD137-generated DC than on classical imDc, but LPS1

IFN-g treatment led to a stronger expression of CD83 on classical DC

(Fig. 3A). The DC markers CD1a and DC-SIGN were not expressed

on CD137-generated DC even not after maturation, whereas the

classical DC expressed considerable levels of both proteins (Fig. 3A).

The higher CD83 and CD86 expression on CD137-generated DC

than on imDC, and the pattern of endocytic activity would be

consistent with the fact that CD137-generated DC are mDC. But the

absence of CD1a and DC-SIGN expression and the reduced MHC

class II expression suggest that they are a different type of DC.

An important feature of DC is migration and migration to lymph

nodes is largely regulated via the chemokine receptor CCR7. CD137

ligand signaling upregulated CCR7 expression while it decreased

expression of CXCR4. This was especially evident when the CD137

ligand signal was combined with LPS1IFN-g (Fig. 3A). These

effects of CD137 on monocytes were dose-dependent as evidenced

by the increasing expression of CD80 and CD86 with the increasing

concentrations of CD137-Fc protein (Fig. 3B).

Another important feature of DC is the secretion of cytokines

that mediate T-cell activation and polarization. Especially IL-12 and

IL-23 are associated with the ability of DC to induce T-cell prolif-

eration [25]. Substantial levels of IL-12 could only be detected in

supernatants of classical DC after maturation by LPS1IFN-g(Fig. 3C). CD137-generated DC did not produce IL-12, even not in

the presence of LPS1IFN-g. IL-23, however, was not produced by

classical DC but was produced by CD137-generated DC in the

presence of LPS1IFN-g (Fig. 3C). Secretion of the anti-inflamma-

tory IL-10 was inhibited by CD137 treatment but was increased in

imDC by LPS1IFN-g maturation (Fig. 3C). The IL-12, IL-23 and

IL-10 profile differs between CD137-generated DC and classical DC

and so does the expression of costimulatory molecules as described

above. Both observations suggest that CD137-generated DC and

classical DC influence T-cell polarization differently.

In previous studies, it was shown that CD137 can provide the

maturation signal to monocyte-derived DC that were generated

using GM-CSF1IL-4 [21, 23]. IL-10 production of CD137-matured

classical DC cells was also measured in this study. Maturation of DC

with CD137 leads to a higher production of IL-10 as compared with

DC treated with Fc. However, IL-10 production was much lower

than in DC, which were matured in the presence of LPS1IFN-gSince IL-10 is associated with tolerogenic DC the observed decrease

in IL-10 secretion and the increase in IL-23 secretion argue for a

stimulatory rather than a tolerogenic role of CD137-generated DC.

Also, the presence of TGF-b could not be detected in these super-

natants (data not shown).

CD137-differentiated monocytes are functional DC

To functionally assess CD137-generated DC their ability to induce

T-cell proliferation in an allogeneic MLR was tested. Monocytes

treated with CD137-Fc protein for 8 days had a five-fold

stronger capacity than cells treated with the Fc control protein

to enhance proliferation of total T cells in an allogeneic MLR

(2483572768.9 versus 4812.771064.5 cpm), (Fig. 4). Addition

of LPS during the final 48 h did not further increase the T–cell-

stimulatory capacity of CD137-generated DC, demonstrating that

CD137 as a sole factor is able to generate mDC. CD137 was also

more potent than LPS in inducing maturation of classical imDC

that were generated by culturing monocytes for 7 days in GM-

CSF1IL-4 (20924.371836.7 versus 9282.77384.6 cpm).

However, their capacity to stimulate T-cell proliferation was still

lower than that of CD137-generated DC (Fig. 4A).

These results based on 3H-thymidine incorporation assays

were confirmed by the decrease in fluorescence intensities of

CFSE-labeled T cells during coculture with CD137-generated DC.

CD137-generated DC induced proliferation in 51.9%

of the T cells (MFI 5 861.6) whereas Fc-treated control mono-

cytes only induced proliferation in 31.9% of the T cells

(MFI 5 1170.2), (Fig. 4B). Here too maturation of CD137-

generated DC had no effect on the ability of the cells to induce

T-cell proliferation.

Proliferation of memory Tells can be induced by either

macrophages or DC while proliferation of naıve T cells can only

be induced by DC [26]. Therefore, we tested the ability of CD137-

generated DC to induce proliferation of CD45RA1 T cells. The

overall proliferative response pattern of CD45RA1 T cells

resembled that of total T cells (Fig. 4C). However, CD137-

generated DC increased proliferation of CD45RA1 T cells by

around eight-fold (12256.771396.8 versus 1480.77653.7 cpm),

compared with five-fold of total Tells. Again, maturation did not

increase the ability to induce T-cell proliferation any further, and

CD137-generated DC were more potent than classical DC. The

stronger induction of CD45RA1 T-cell proliferation was also

evident from the CFSE dilution. The number of proliferating T

cells was increased from 24.1 to 52.5% when the cells were

stimulated by CD137-generated DC rather than by Fc-treated

monocytes (Fig. 4D). The ability of CD137-treated monocytes to

induce proliferation of naıve T cells can be regarded as the final

proof that CD137 treatment induces monocyte to DC differ-

entiation.

While the maturation of classical DC by LPS or LPS1IFN-g is

the most frequently used method the combination of IL-6,

IL-1b, TNF, and PGE2 is often being used for maturation of DC

that are to be used in therapeutic settings. Maturation by this

cocktail of factors resulted indeed in a significantly higher

potency to stimulate allogeneic T cells, and the potency of these

DC were comparable to that of CD137-generated DC (Fig. 4E).

T-cell polarization induced by CD137-generated DC

DC can induce different types of immune responses, reflected by

the activation and/or generation of different T-cell subsets. One

distinguishing feature of these subsets is their cytokine pattern

[27, 28].



Levels of IL-10 were enhanced more than five-fold

(128.8713.4 versus 661.27164.5 pg/mL) in supernatants of

MLR cultures that contained CD137-generated DC. Since CD137

treatment of monocytes reduces their IL-10 secretion (Fig. 3B)

the source of the increased IL-10 levels in the MLR supernatants

should be the T cells. Maturation by LPS1IFN-g reduced

IL-10 levels to 237.7723.6 pg/mL. Supernatants from classical

imDC and mDC contained intermediate levels of IL-10

(521.1712.0 versus 487.274.1 pg/mL) but here it could have

come from the DC and/or the T cells. Maturation of imDC by

CD137 instead of LPS1IFN-g induced highest IL-10 secretion

(878.6789.1 pg/mL).

IL-10 is associated with Treg cells and tolerogenic DC but even

classical DC produce IL-10 (Fig. 5A). Therefore, the increase in

IL-10 does not necessarily implicate a Treg polarization. Indeed,

expression of the Treg-specific transcription factor FOXP3 was

almost completely abolished in T cells that were stimulated by

CD137-generated DC (Fig. 5B).

IL-4, IL-17 and IFN-g are indicative of Th2, Th17 and Th1 cells,

respectively. IL-4 could neither be detected by ELISA nor by

intracellular cytokine staining in T cells cocultured with CD137-

generated DC (data not shown). Secretion of IL-17 was increased

by an order of magnitude in T cells cocultured with CD137-

generated DC (3235.47186.0 versus 359.1710.5 pg/mL).

Maturation of CD137-generated DC by LPS1IFN-g did not change

IL-17 levels. Classical imDC induced only low levels of

IL-17 release in T cells (1028.9767.6 pg/mL) and classical mDC

induced even less (674.1716.7 pg/mL). However, classical imDC,

matured with CD137 released by far the highest concentration of

IL-17 (5625.2713.6 pg/mL) (Fig. 5A). These data together with

the release of IL-23 from CD137-generated DC indicate that CD137

ligand signaling enables DC to activate Th17 cells.

32000** **

A B

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Figure 4. CD137-activated monocytes induce proliferation of T cells. Monocytes were treated for 8 days with immobilized Fc or CD137-Fc protein,or with GM-CSF1IL-4 for 7 days (imDC) and matured for 16 h with (A and B) LPS or (C and D) LPS1IFN-g (mDC) or (E) IL-6, IL-1b, TNF, PGE2 or theywere matured with Fc (mDC-Fc) or CD137-Fc (mDC-CD137). These cells were then cocultured for another 7 days with (A, B and E) total or (C and D)naıve T cells at a ratio of 1:10. Proliferation was quantified by (A, C and E) 3H-thymidine incorporation and (B and D) CFSE dilution. For3H-thymidine incorporation means and standard deviations of triplicate measurements are depicted. Numbers in the flow cytometry histogramsrepresent percentages of positive cells and mean fluorescence. �po0.05; ��po0.01 using a two-tailed unpaired Student’s t-test. This experiment hasbeen performed three times with comparable results.



IFN-g was not detectable in T cells cocultured with

Fc-treated control monocytes, but was secreted at high levels

(5575.7720.8 pg/mL) by T cells cocultured with CD137-Fc-

treated monocytes (Fig. 5A). Maturation of CD137-generated DC

by LPS1IFN-g made no difference to IFN-g secretion.

Classical imDC induced little IFN-g in T cells but maturation by

LPS1IFN-g or by CD137 profoundly increased IFN-g secretion

(490.0721.4 and 7297.57155.5 pg/mL, respectively). This

increase in IFN-g was observed in both CD41 and CD81 T cells

but to a greater extent in CD81 than in CD41 T cells as assessed

by intracellular cytokine staining (Fig. 5C). The production of

IFN-g by T cells during coculture with CD137-generated DC

suggests that CD137 ligand signaling induces or enhances Th1

responses.

Despite the absence of IL-4, another Th2 cytokine, IL-13

was present in supernatants from the MLR. Its profile was similar

to that of IFN-g. IL-13 was not detectable in supernatants

from MLR with control monocytes but was present at high

levels in supernatants of T-cell cocultures with CD137-generated

DC (1610.1742.0 pg/mL). Maturation by LPS1IFN-g increased

IL-13 levels further (to 2266.5735.7 pg/mL). Classical imDC and

mDC induced little IL-13 but maturation of imDC by CD137

resulted in intermediate IL-13 levels (1080.3712.6 pg/mL).

IL-13 could not be detected in supernatant of monocytes

**3000

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AB

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**

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(pg

/ml)

0

1000

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8000 **

**

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2000

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44.5%33.7

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γ

LPS + IFN-γ

Figure 5. T-cell polarization induced by CD137-generated DC. (A) Cytokine secretion. Supernatants from the T cells in Fig. 4A were collected andcytokine levels of IFN-g, IL-13, IL-10 and IL-17 were determined by ELISA. Scales are in pg/mL. Depicted are means7standard deviations oftriplicate measurements. �po0.05; ��po0.01 using a two-tailed unpaired Student’s t-test. (B) Foxp3 expression: Monocytes were treated as indicatedfor 8 days and then cocultured for another 7 days with T cells at a ratio of 1:10. The T cells were immunostained for CD4, CD25 and FOXP3 andanalyzed by flow cytometry. Open and filled histograms represent isotype control and FOXP3 staining, respectively. FOXP3 expression is shown forCD4, CD25 double positive T cells. (C) Intracellular IFN-g staining: T cells from Fig. 4A were immunostained for CD4 and IFN-g and analyzed by flowcytometry. Numbers in quadrants indicate percentages of cells. These experiments have been performed three times with comparable results.



indicating that it was secreted by T cells (Fig. 5A). TGF-b could

not be detected in these supernatants (data not shown).

Cytolytic T-cell activity induced by CD137-generatedDC

Expression of perforin in CD81 T cells is indicative of

their cytolytic potential as release of perforin is one of the

mechanism by which CD81 T cells kill their target cells.

Intracellular perforin production was higher in CD81 T cells,

which were co-cultured with CD137-generated DC than those co-

cultured with Fc-treated monocytes (Fig. 6). Maturation by LPS1

IFN-g only slightly increased perforin expression. Perforin

expression in T cells cocultured with CD137-generated DC was

higher than in those T cells that were cocultured with classical

DC, even after maturation by LPS1IFN-g (Fig. 6). This suggests

that CD137-generated DC might be more potent than the classical

DC in inducing cytolytic activities in T cells.

The activation status of CD137-generated DC differsfrom that of classical DC

IL-12 is a key cytokine mediating T-cell activation by classical DC.

Since CD137-generated DC did not secrete IL-12 we tested

whether they may be employing the related cytokine IL-23 for

induction of T-cell proliferation. When IL-23 was neutralized by

specific antibodies induction of IL-17 in T cells by CD137-

generated DC was blocked whereas an isotype control antibody

had no effect (Fig. 7A).

The difference between CD137-generated DC and

classical DC is also reflected by intracellular signal transduction.

GM-CSF1IL-4 induce activation of NF-kB during monocyte to

classical DC differentiation [29, 30]. However, during CD137-

induced DC differentiation activation of NF-kB is inhibited

(Fig. 7B).

Discussion

Based on the results from this study we propose that CD137

ligand signaling induces monocyte to DC differentiation. This

claim may seem questionable considering that CD137-generated

DC have a reduced MHC class II expression, secrete no IL-12 and

do not express CD1a and DC-SIGN.

However, DC are a heterogenous population with no single

surface marker that unequivocally identifies them [31]. Thus

their characterization usually relies on the functional abilities of

these cells and the expression of molecules involved in these

functions. CD137-generated DC show an up-regulation of the DC

maturation marker CD83, and the co-stimulatory molecules

CD80 and CD86. But most importantly, CD137-generated DC can

induce T-cell proliferation of total T cells as well as of naıve

T cells.

In human and murine hematopoietic progenitor cells CD137

has been shown to induce proliferation and differentiation to the

myeloid lineage and specifically to macrophages, with no

evidence of DC differentiation [11, 12]. It is therefore surprising

that CD137 inhibits macrophage and induces DC differentiation

when using peripheral monocytes instead of hematopoietic

progenitor cells as a starting population. A likely explanation is

the different maturation states of these two cell populations.

LPS + IFN-γ

24.7% 20.0

33.5% 24.4

55.8% 32.2

50.6% 31.9

43.1% 24.7

33.2% 21.3

Fc

CD

137-

Fc

GM

-CS

F +

IL-4

38.1% 22.1

39.2% 25.3

Fc CD137-Fc

Perforin

Figure 6. CD137-activated monocytes induce cytolytic T-cell activity.Monocytes were treated as indicated for 8 days and then cocultured foranother 7 days with T cells at a ratio of 1:10. The T cells wereimmunostained for perforin and CD8, and analyzed by flow cytometry.Perforin expression is shown for CD81 T cells. Black and greyhistograms represent isotype control and perforin staining, respec-tively.

20

25

A B

Fc

5

10

15CD137-Fc

0No

antibodyIsotypecontrol

1 μg/ml 5 μg/mlα-IL-23 antibody

IL-1

7 (n

g/m

l)

1.2

1.4 Fc CD137-Fc

**

*

0.6

0.8

1

0

0.2

0.4

NF

-κB

act

ivit

y (r

el. u

nit

s)

5 min 30 min 1 h 18 h

Figure 7. Activation status of CD137-activated DC. (A) Isotype controlor neutralizing anti-IL-23 antibodies were added to an allogeneic MLRwith CD137-generated DC. IL-17 release was measured by ELISA on day5. (B) NF-kB activity was measured in monocytes that were cultured onimmobilized Fc or CD137-Fc protein for the indicated times. �po0.05using a two-tailed unpaired Student’s t-test. These experiments havebeen performed three times with comparable results.



Peripheral monocytes have been exposed to myeloid growth

factors during their differentiation from hematopoietic progeni-

tor cells, and therefore the CD137 ligand signal is sufficient to

induce DC differentiation. It is plausible to assume that mono-

cytes/macrophages that are generated from HSC by CD137

ligand signals can be converted to DC by additional signals such

as LPS and/or IFN-g. That would mean that multiple signals are

necessary, which is in line with the current knowledge of DC

development, and places CD137 as one among several factors

that can promote progenitor cell to DC development.

But are the activities of CD137 entirely redundant or is there

anything specific about CD137? First, expression of CD137 is

induced during immune responses and expression is strictly acti-

vation dependent [32]. Therefore, CD137 would have a role in the

development of inflammatory DC, and indeed that is the phenotype

of CD137-generated DC, evidenced by the increased expression of

CD80, CD86 and CD83 and the reduced secretion of IL-10. Also,

T cells stimulated by CD137-generated DC lose expression of the

Treg-specific transcription factor FOXP3. Another interesting

feature of CD137-generated DC is that they supported all T-cell

polarizations, aside from the inhibition of Treg. T cells stimulated

by CD137-generated DC secrete the Th1 cytokine IFN-g, the Th2

cytokine IL-13 and the Th17 cytokine IL-17. That distinguishes

CD137-generated DC from other types of DC.

Differences in surface marker expression, cytokine production

and T–cell-stimulatory capacity also suggest that CD137-gener-

ated DC are different from the classic DC. Classic DC express high

levels of CD1a and DC-SIGN while no expression of these mole-

cules was observed on CD137-generated DC. Different subtypes

of DC are known to exist in man but unlike in the murine system,

these subtypes have not been well characterized [31]. The cyto-

kine profile also suggests that CD137-generated DC and classic

DC induce different types of T-cell development. CD137 treat-

ment significantly decreases IL-10 while classic DC produce high

concentrations of IL-10. This decrease of IL-10 may explain the

higher potency of CD137-generated DC in inducing T-cell

proliferation despite the lower MHC class II expression compared

with classical DC. The low IL-10 levels could also explain why

CD137-generated DC do not support Treg polarization even

though they support polarization to Th1, Th2 and Th17 T cells.

IFN-g production by T cells is thought to be induced by DC-

released IL-12. Therefore, the high production of IFN-g by T cells

co-cultured with CD137-generated DC was unexpected since no

IL-12 was secreted by CD137-generated DC. Potentially, IL-23

released by CD137-generated DC could substitute IL-12. Indeed,

neutralization of IL-23 inhibited IL-17 release by T cells stimu-

lated by CD137-generated DC.

Another difference to classical DC is that CD137-generated DC

are already mature. Though addition of LPS1IFN-g increased

expression of CD80, CD86, MHC class II and CCR7 on CD137-

generated DC and was necessary for IL-23 release it had no effect

on the ability of CD137-generated DC to induce T-cell prolifera-

tion. The molecular basis for this potency may be that CD137

initiates two signals, one through CD137 ligand and the other

through TLR [20]. This may explain why CD137 as a single factor

is able to generate mDC whereas in the classical method the

combination of GM-CSF1IL-4 and the subsequent maturation by

a TLR signal or IFN-g are required. The CD137 ligand signal

cascade in human monocytes has only partly been analyzed but

so far an involvement of protein tyrosine kinases, p38 MAPK,

ERK1,2, MAP/ERK kinase, Phosphoinositide-3-kinase and protein

kinase A but not by protein kinase C have been identified [33].

Since CD137 ligand signaling employs signaling molecules well

known for myeloid cell activation it was very surprising to find

that NF-kB was not activated but rather inhibited especially since

NF-kB activation has been shown during differentiation of clas-

sical DC [29, 30]. Future studies will need to determine the full

mechanisms of CD137-induced DC generation.

DC migrate from the periphery to lymph nodes where they

interact with T cells, and this migration is mediated by CCR7. We

have recently shown that maturation of immature classical DC by

CD137 ligand signaling induces expression of CCR7, which in turn

mediates the migration of these DC in vivo [23]. Similarly, expres-

sion of CCR7 is also induced on CD137-generated DC, which is

expected to mediate migration also for CD137-generated DC.

Our results are in agreement with reports that CD137 ligand

signaling induces maturation of imDC and with recent findings

that CD137 ligand signals in combination with IL-4 induce DC

differentiation [21–23, 34]. However, in all these studies hema-

topoietic progenitor cells or monocytes were differentiated with a

cocktail of cytokines before stimulation with CD137 or other

factors were present during stimulation with CD137. Our study

shows that CD137 as a single factor alone is sufficient to induce

the differentiation and maturation of DC, making the induction of

CD137 ligand signaling the most simple protocol to generate

mDC. In addition CD137-generated DC are more potent than

classical DC, generated by GM-CSF1IL-4 and matured by LPS1

IFN-g. This in vitro potency and the easiness to generate them

recommend a further characterization of CD137-generated DC to

evaluate their potential for human immunotherapy.

Materials and methods

Recombinant protein and antibodies

CD137-Fc protein was purified from supernatants of stable

transfected CHO cells by protein G sepharose, as described

previously [35]. Human IgG1 Fc protein was purchased from

Chemicon International (Temecula, CA, USA). Recombinant

human GM-CSF, M-CSF, IL-4, IL-6, IL-1b and TNF were

purchased from Peprotech (NJ, USA). PGE2 was purchased from

Sigma. Antibodies against human HLA-DR (clone LN3), CD14

(clone 61D3), CD80 (clone 2D10.4), CD83 (clone HB15e), CD86

(clone IT2.2), CD209 (clone eB-h209), CD3 (clone Okt3), CD4

(clone SK-3), CD8 (clone SK-1), IFN-g (clone 4S.B3), IL-4 (clone

MP4-25D2), CCR7 (clone 3D12) and CXCR4 (clone 12G5) were

purchased from eBioscience (San Diego, CA, USA). PE-conju-

gated anti-human perforin (clone B-D48) and anti-human IL-23



(sub-unit p19) (clone B-Z23) was purchased from abcam

(Cambridge, UK). APC-conjugated anti-human FoxP3 (clone

3G3) and PE-conjugated anti-human CD25 were purchased from

Miltenyi Biotec (Bergisch Gladbach, Germany) PE-conjugated

anti-human CD1a (clone HI149) was obtained from BD Bios-

ciences Pharmigen (San Diego, CA, USA).

Cells and cell culture

Buffy coat of healthy donors were obtained from the National

University Hospital Blood Donation Centre under IRB approved

protocol 07-005E. Human peripheral blood mononuclear cells were

prepared by Ficoll-Hypaque density (1.077 g/dL) (Sigma) gradient

centrifugation. Monocyte population was then isolated from PBMC

using the Monocyte isolation kit II (Miltenyi Biotech) according to

the manufacturer’s instructions. Isolated monocytes were more than

95% pure as determined by flow cytometric analysis after CD14

staining. Cells were cultured in polystyrene dishes (Becton

Dickinson, Franklin Lakes, NJ, USA) that were untreated or coated

with 10mg/mL Fc or CD137-Fc protein, in RPMI 1640 supplemented

with 10% FBS, 50mg/mL streptomycin and 50 IU/mL penicillin.

For the generation of DC, isolated monocytes were cultured in

the presence of 80 ng/mL recombinant human GM-CSF1100 ng/

mL recombinant human IL-4 for 7 days followed by maturation with

1mg/mL LPS (Sigma) or 1mg/mL LPS150 ng/mL IFN-g for a further

1 day. Where indicated maturation was induced by IL-6 (100 U/

mL), IL-1b (10 ng/mL), TNF (10 ng/mL), PGE2 (1mg/mL).

Flow cytometry

To determine surface marker expression, cells were stained with

antibodies in PBS containing 0.5% BSA and 0.1% sodium azide

(FACS buffer) for 1 h at 41C in the dark. Cells were then washed

and resuspended in 400mL of FACS buffer. Non-specific staining

was controlled by isotype-matched antibodies. Flow cytometry was

performed on a CyAn FACS machine (Dako, Glostrup, Denmark).

For intracellular cytokine and perforin staining, CD31 cells

after stimulation with allogeneic DC were re-activated with

50 ng/mL PMA and 100 nM calcium ionophore in the presence of

Brefeldin A (eBioscience) for 5–6 h. After which, cells were

harvested and stained for CD4 or CD8. Following which, cells

were fixed and permeabilized using the BD Cytofix/Cytoperm

fixation/permeabilization kit (BD Biosciences Pharmigen) and

stained with FITC-conjugated anti-IFN-g antibody or PE-conju-

gated anti-perforin antibody.

Phagocytosis and endocytosis assays

In the phagocytosis assays, monocytes were cultured under no

treatment, Fc or CD137-Fc treatment. At the desired time points

50 fluorescent latex beads (Invitrogen, CA, USA) per cell were

added, and cells were incubated at 371C for 1 h. After which,

phagocytosis was stopped by the addition of ice-cold PBS and

cells were washed and treated with trypsin to dislodge any

surface adherent latex beads. Cells were then resuspended in

400mL PBS and flow cytometry was performed to determine the

percentage of cells that had phagocytosed beads.

For the endocytosis assays, pre-treated monocytes were

incubated at 371C for 30 min with FITC-labeled dextran (1 mg/

mL) (Sigma) at the desired time points. Endocytosis was stopped

by adding ice-cold PBS containing 1% FBS. Endocytosis was then

analyzed by flow cytometry to determine the mean fluorescence

intensity of the cells.

Respiratory burst

Pre-treated monocytes were activated with 20mg/mL of PMA 10 min

prior to the addition of 0.1mg/mL dihydrorhodamine 123 (DHR123)

(Invitrogen). Cells were incubated for 30min at 371C. After which

cells were harvested and washed to remove excess DHR123. Mean

fluorescence intensity of cells was determined by flow cytometry.

ELISA

The concentrations of IL-12p70, TGF-b, IL-10, IL-4, IFN-g, IL-13,

IL-23 and IL-17 in cell supernatants were determined by human

IL-12p70 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA),

human TGF-b, human IL-10, human IL-4, human IFN-g, human

IL-13, human IL-23 and human IL-17 ELISA kits (eBioscience),

respectively, according to the manufacturers’ instructions. Each

sample was assayed in triplicate within each experiment.

Allogeneic mixed lymphocyte reaction

Pre-treated monocytes or DC were harvested and cultured with

allogeneic CD31 or naıve CD41 T cells at a ratio of 1:10.

Co-cultures were maintained in RPMI 1640 supplemented with

10% FBS, 50mg/mL streptomycin and 50 IU/mL penicillin for 5–7

days. Cells were pulsed with 0.5mCi of 3H-thymidine (PerkinElmer,

Boston, MA, USA) for the last 24 h of the culture period. The cells

were then harvested onto a Packard Unifilter Plate using a

MicroMate 196 Cell Harvester and counted using a TopCount

(Perkin Elmer, Waltham, MA, USA). Each condition was assayed in

triplicate. Alternatively, T cells were stained with CFSE (Invitrogen)

prior to co-culture with pre-treated monocytes. Cell proliferation is

determined by dilution of CFSE, using flow cytometry.

NF-jB assay

Monocytes were treated with immobilized Fc or CD137-Fc for

5 min, 30 min, 1 h or overnight. Nuclear protein was extracted

using the Nuclear Extract Kit (Active Motif, USA), and protein

concentration was determined using the Bio-Rad Protein Assay



(Bio-Rad, USA). In total, 5mg of extracted nuclear protein

were used in a TransAM NF-kB Family Transcription Factor

Assay kit (Active Motif, USA) according to the manufacturer’s

instruction.

Photographs

Morphological changes of cells were documented using the Zeiss

Axiovert 40 inverted microscope (Zeiss, Gottingen, Germany)

and Canon PowerShot G6 digital camera.

Statistics

Statistical significance was determined by a two-tailed unpaired

Student’s t-test.

Acknowledgements: This study was supported by grant

06/1/21/19/453 from the Biomedical Research Council,

Singapore to H.S. We thank Dr. Paul Macary, Department of

Microbiology, National University of Singapore, for valuable

discussions.

Conflict of interest: The authors declared no financial or

commercial conflict of interest.

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Abbreviations: DHR123: dihydrorhodamine 123 � imDC: immature DC �mDC: mature DC

Full correspondence: Professor Herbert Schwarz, Department of

Physiology and Immunology Programme, Yong Loo Lin School of

Medicine, Centre for Life Sciences ]03-05, National University of

Singapore, 28 Medical Drive, 117456 Singapore

Fax: 165-6778-2684

e-mail: [email protected]

Received: 29/10/2009

Revised: 25/3/2010

Accepted: 20/4/2010

Accepted article online: 28/4/2010



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CD137 Ligand Signaling Induces Human Monocyte to Dendritic Cell Differentiation

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