Assessing the immunopotency of Toll-like receptor agonists in anin vitro tissue-engineered immunological model

Yifan Ma,1,2 Louis Poisson,1

Guzman Sanchez-Schmitz,1 Santosh

Pawar,1 Chunfeng Qu,3 Gwendalyn

J. Randolph,3 William L. Warren,1

Eric M. Mishkin1 and Russell G.

Higbee1

1VaxDesign Corporation, Orlando, FL, USA,2Shenzhen Institutes of Advanced Technology,

Chinese Academy of Sciences, Shenzhen,

China, and 3Department of Gene and Cell

Medicine, Icahn Research Institute, Mount

Sinai School of Medicine, New York, NY, USA

doi:10.1111/j.1365-2567.2009.03237.x

Received 22 September 2009; revised 9

December 2009; accepted 15 December 2009.

Correspondence: R. G. Higbee, VaxDesign

Corporation, 12612 Challenger Parkway,

Suite 365, Orlando, FL 32826, USA.

Email: rhigbee@vaxdesign.com

Senior author: Russell G. Higbee

Summary

The in vitro Peripheral Tissue Equivalent (PTE) module is a three-dimen-

sional tissue-engineered endothelial cell/collagen matrix culture system,

which has been reported to reproduce in vivo physiological conditions

and which generates dendritic cells (DC) autonomously. In the present

study, we used the PTE module to investigate the immunopotency of

Toll-like receptor (TLR) agonists, including polyinosine-polycytidylic acid,

Gardiquimod, CpG 2006 and lipopolysaccharide. Application of TLR

agonists in the PTE module induced a wide range of cytokines, including

interleukins 1a/b, 6, 8 and 10 and tumour necrosis factor-a. Compared

with traditional peripheral blood mononuclear cell (PBMC) cultures, the

PTE module produced twofold to 100-fold higher levels of cytokine secre-

tion, indicating that it can be a highly sensitive assay system. This

increased sensitivity is the result of the natural synergy between the leuco-

cytes and the endothelium. Furthermore, the application of TLR agonists,

such as lipopolysaccharide and Gardiquimod, to the PTE module

enhanced DC differentiation and promoted DC maturation, as indicated

by up-regulated expression of CD83, CD86 and CCR7(CD197). In addi-

tion, functional assays indicated PTE-derived DC treated with Gardiqui-

mod, a TLR-7 agonist, significantly augmented anti-tetanus toxoid

antibody production. Interestingly, replacing PBMC with purified myeloid

cells (CD33+) significantly reduced the responsiveness of the PTE module

to TLR stimulation. The reduced sensitivity was partly the result of the

removal of plasmacytoid DC that participated in the response to TLR

stimulation and sensitization of the PTE module. Overall, the in vitro

PTE module clearly demonstrated the effects of TLR agonists on DC gen-

eration, maturation and antigen-presenting capacity, and may serve as a

sensitive and predictive test bed for the evaluation of adjuvant candidates.

Keywords: cytokine; dendritic cell; endothelial cells; human; in vitro tissue

engineered immunological model; three-dimensional; Toll-like receptor

Abbreviations: APC, antigen-presenting cells; DC, dendritic cell; ELISPOT, enzyme-linked immunosorbent spot-forming cellassay; GM-CSF, granulocyte–macrophage colony-stimulating factor; HBSS, Hanks’ buffered salt solution; HUVEC, humanumbilical vein endothelial cell; iDC, immature DC; IFN, interferon; IgG, immunoglobulin G; IL-4, interleukin-4; IP-10,interferon inducible protein-10; LC, Langerhans cells; LPS, lipopolysaccharide; mDC, mature DC; ODN, oligonucleotides;PBMC, peripheral blood mononuclear cell; pDC, plasmacytoid DC; PE, phycoerythrin; Poly I:C, polyinosine-polycytidylic acid;PTE module, in vitro peripheral tissue equivalent module; PTE-DC, PTE-derived DC; RANTES, regulated on activation, normalT-cell expressed and secreted; RT, reverse transmigrated; TLR, Toll-like receptor; TNF, tumour necrosis factor; TT, tetanustoxoid.

374 � 2010 Blackwell Publishing Ltd, Immunology, 130, 374–387

I M M U N O L O G Y O R I G I N A L A R T I C L E

Introduction

Dendritic cells (DC) are the most potent antigen-present-

ing cells (APC), and they play an essential role in both

innate and adaptive immunity. They normally develop

from circulating bone-marrow-derived DC precursors that

distribute into the peripheral tissues and give rise to

immature DC (iDC).1 The tissue-residing iDC capture

antigens from the local environment and release cyto-

kines/chemokines, thereby participating in innate immu-

nity. Moreover, antigen capture also triggers DC

maturation and migration into draining lymph nodes. In

the T-cell region of lymph nodes, mature DC (mDC)

present antigens to naive T cells via major histocompati-

bility complex molecules, triggering the adaptive immune

response.2 Hence, DC are an important link between

innate and adaptive immunity.

In vitro DC production is an important strategy for

generating large numbers of DC. To date, the most com-

monly used method to generate human DC is to culture

blood monocytes with granulocyte–macrophage colony-

stimulating factor (GM-CSF) and interleukin-4 (IL-4) for

5–7 days.3 Although this method can produce a large

population of DC, it remains questionable whether this

method faithfully recapitulates DC development in vivo.4

Besides cytokine-derived DC, Randolph et al.5–8 have

reported a tissue-engineered in vitro immunological

model that allows for autonomous generation of DC. We

termed this system the in vitro ‘Peripheral Tissue Equiva-

lent’ (PTE) module. The PTE module is a three-dimen-

sional culture system consisting of a confluent monolayer

of unstimulated endothelial cells grown on top of an

endotoxin-free collagenous matrix (see Fig. 1). When

peripheral blood mononuclear cells (PBMC) are applied

onto the endothelial monolayer, monocytes preferentially

migrate across the endothelium into the sub-endothelial

collagen. This process probably mimics the constitutive

entry of monocytes into tissues as observed in vivo.9 After

48 hr in culture, approximately half of the migrated

monocytes differentiate into DC with varying degrees of

maturity, and migrate across the intact endothelium in

the abluminal-to-luminal direction (reverse transmigra-

tion),5 which may mimic the in vivo DC migration from

peripheral tissue into the local lymphatics.1 In the

unstimulated PTE module, the percentage of mature DC

in the reverse-transmigrated (RT) cell fraction is < 10%,

with the majority of RT cells resembling immature DC or

monocytes. Incorporation of various stimuli, such as lipo-

polysaccharide (LPS), influenza virus or zymosan, signifi-

cantly increases the percentage of mature DC in the RT

cell fraction.6 Compared with conventional cytokine-

derived DC, PTE-derived DC (PTE-DC) differentiate

more rapidly and do not require application of exogenous

cytokines. Moreover, the composition of PTE-DC is more

heterogeneous than that of cytokine-cultured DC, and

may more closely resemble the composition of the DC

populations developed in vivo.5,10 In addition, the PTE

module contains endothelial cells that can sense ‘danger

signals’ in peripheral tissues and contribute to immune

responses.11,12 Indeed, the presence of endothelial cells

has a critical role in the generation of DC in the system,5

which probably increases the sensitivity of the PTE mod-

ule in response to various stimuli.

The Toll-like receptor (TLR) family is a group of pat-

tern-recognition receptors that play a crucial role in both

innate and adaptive immunity. TLRs can recognize con-

served microbial structures or products of microbial

metabolism called ‘pathogen-associated molecular pat-

tern’, which consequently triggers innate immunity. The

TLR signalling also promotes the activation and matura-

tion of APCs, thereby facilitating adaptive immunity. In

addition, cytokines and chemokines elicited by TLR stim-

ulation further regulate downstream T-cell and B-cell

responses.13 Currently, several TLR agonists are being

evaluated as potential adjuvants for vaccine development

against infectious diseases and cancer. For example,

polyinosine-polycytidylic acid (Poly I:C), a TLR3 agonist,

has been shown to be a potent adjuvant to enhance vac-

cine-induced protective immune responses.14 Agonists of

TLR7/8, such as Imiquimod and Resiquimod, have been

used to treat skin neoplasms and viral infections in

humans.15 CpG oligonucleotides (ODN), TLR9 agonists,

have been reported to significantly enhance the antibody

responses induced by hepatitis B and anthrax vaccines in

human clinical trials.16,17 Previous studies have demon-

strated that conventional and plasmacytoid DC (pDC) are

the major cell types mediating the adjuvant effect of TLR

agonists.18 Stimulation by TLR can promote the differen-

tiation and maturation of freshly isolated DC or cyto-

kine-cultured DC, triggering the release of cytokines.19–21

Moreover, there is evidence that TLR stimulation, such as

stimulation of TLR8 by single-stranded RNA, triggers

PTE-DC maturation.7 However, a generally broader sur-

vey as to how TLR stimulation will affect the phenotype

and function of PTE-DC has not been performed, nor is

PBMC

Remain

Monocyte/DC

48 hr

Monocytes

M φ

(a) (b)

Figure 1. The scheme of the in vitro peripheral tissue equivalent

(PTE) module. The PTE module consists of a quiescent monolayer

of human umbilical vein endothelial cells (HUVEC) grown on a col-

lagen matrix. When peripheral blood mononuclear cells (PBMC) are

applied onto HUVEC, monocytes migrate into the subendothelial

collagen and form a PTE module (a). After an additional 48 hr of

culture, about half of the monocytes reverse transmigrate across the

endothelium and develop into dendritic cells (b).

� 2010 Blackwell Publishing Ltd, Immunology, 130, 374–387 375

Assessing the immunopotency of TLR agonists in a 3D immunological model

it known how the response to TLR agonists in the PTE

module compares with traditional PBMC culture and

cytokine-cultured DC. In the present study, the immuno-

potency of TLR agonists, such as Poly I:C (TLR3), LPS

(TLR4), Gardiquimod (TLR7/8) or CpG 2006 (TLR9),

was tested in the PTE module. These TLR agonists evoked

cytokine and chemokine production, and promoted DC

generation and maturation in the PTE module. When

compared with traditional PBMC culture methods, the

PTE module was more sensitive and so was a promising

predictor of immune response. These studies support the

use of the PTE module as a reliable immunological test

bed for adjuvant and vaccine screening and development.

Materials and methods

Human PBMC preparation

Apheresis blood products were obtained from Florida’s

Blood Centers (Orlando, FL). The study was reviewed

and approved by Chesapeake Research Review Inc.

(Columbia, MD). All donors were in good health and all

blood products were negative for blood-borne pathogens

as detected by standard blood bank assays. The PBMC

were enriched by Ficoll density gradient separation

according to standard laboratory procedures. After wash-

ing, PBMC were either resuspended in culture media or

cryopreserved in dimethylsulphoxide-containing freezing

media for extended storage in liquid nitrogen vapour

phase for future assays.

In vitro PTE module preparation

The PTE module was fabricated as described previously,10

as shown in Fig. 1. Briefly, second-passage human umbili-

cal vein endothelial cells (HUVEC; Lonza, Walkersville,

MD) were cultured on a polymerized type I collagen

matrix (Inamed Biomaterials, Fremont, CA) and main-

tained in M199 media (Lonza) supplemented with 20%

fetal bovine serum until confluent. The PBMC or purified

monocytes were suspended in M199 media containing

0�1% (weight/volume) bovine serum albumin, and

applied onto the HUVEC monolayer at 500 000 cells/well

and 200 000 cells/well, respectively. In some experiments,

pDC were depleted from whole PBMC by using CD304

(BDCA-4/Neuropilin-1) MicroBeads (Miltenyi Biotec

Inc., Auburn, CA). After a 90-min incubation, non-

migrated cells were removed, and PTE modules were

incubated in culture medium (M199 medium with 20%

heat-inactivated human autologous serum) with or with-

out the following TLR agonists: 5 lg/ml CpG 2006,

25 lg/ml Poly I:C, 5 lg/ml Gardiquimod (all from Invivo-

Gen, San Diego, CA), or 10 ng/ml LPS (Sigma, St Louis,

MO). The PTE culture supernatants were collected at

24 hr or 48 hr for cytokine analysis, and the RT cells were

carefully harvested at 48 hr for fluorescence-activated cell

sorting analysis.

PBMC culture

The PBMC were resuspended at 2�5 · 106 cells/ml in

RPMI-1640 supplemented with 10% heat-inactivated

autologous human serum, and stimulated with or without

the following TLR agonists: 5 lg/ml CpG 2006, 25 lg/ml

Poly I:C, 5 lg/ml Gardiquimod (all from InvivoGen), or

10 ng/ml LPS (Sigma). PTE culture supernatants were

collected at 24 hr for cytokine analysis.

Cell isolation

The T and B cells were negatively selected using pan-

T-cell isolation kit II or B-cell isolation kit II from Miltenyi

Biotec, Inc. For isolation of CD33+ myeloid cells, PBMC

were stained with phycoerythrin (PE) -labelled anti-

human CD33 antibody (BD Biosciences, San Jose, CA).

Labelled cell populations were then enriched to > 99%

purity by selection over LS separation columns after label-

ling with anti-PE microbeads (Miltenyi Biotec Inc). For

pDC reconstitution experiments, the CD33/pDC fraction

was positively selected using CD33 and BDCA-4 micro-

beads, and the percentage of pDC in the CD33/pDC-

enriched fraction was 1�4–2%.

Cytokine-cultured DC preparation and stimulation

CD14+ monocytes were positively selected using CD14+

microbeads (Miltenyi Biotec), and cultured with GM-CSF

(100 ng/ml) and IL-4 (25 ng/ml) for 5 days to generate

iDC. The iDC were then stimulated with equivalent doses

of TLR agonists as previously described. After 48 hr cul-

ture, cells were collected for DC phenotypic analysis.

Flow cytometry

The phenotype of DC was determined using seven-colour

staining. The RT cells or iDC were first incubated with

mouse immunoglobulin G1 (IgG1; 1 lg/sample; Sigma)

at room temperature to block Fc receptor along with the

vital dye Annexin V (2�5 ll/sample; Invitrogen Corpora-

tion, Carlsbad, CA) to identify apoptotic cells. After

15 min, the cells were incubated for 40 min on ice with

the cocktail of fluorochrome-labelled monoclonal anti-

bodies. The antibody cocktail contained non-myeloid

lineage markers, allophycocyanin-conjugated CD3, CD19

and CD56, and the relevant DC surface markers, allo-

phycocyanin-Cychrome 7-conjugated CD14, peridinin

chlorophyll protein-Cychrome 5.5-conjugated human leu-

cocyte antigen (HLA) -DR, CCR7-PE-Cy7, fluorescein

isothiocyanate-conjugated CD83 and CD86-PE (BD Bio-

sciences). After incubation, the cells were washed twice

376 � 2010 Blackwell Publishing Ltd, Immunology, 130, 374–387

Y. Ma et al.

with Hanks’ buffered salt solution (HBSS, +Ca/+Mg) fol-

lowed by fixation in 2% (volume/volume) paraformalde-

hyde, also in HBSS. Viable cellular events were accrued

on a BD LSRII flow cytometer equipped with BD FACS-

DIVA software (BD Biosciences). Data analysis was with

FLOWJO software (Tree Star Inc., Ashland, OR).

Cytokine and chemokine analysis

Production of cytokines and chemokines was analysed

using a Beadlyte human 22-plex multi-cytokine detection

system (Millipore, Billerica, MA). Briefly, 50 ll appropri-

ately diluted cytokine standard or culture supernatants

was incubated with 25 ll of multi-cytokine beads in a 96-

well filter plate at room temperature for 2 hr. The beads

were then washed and incubated with 25 ll of multi-

cytokine biotin for 90 min, followed by mixing with 25 ll

of diluted Beadlyte strepavidin-PE. After 30 min, liquid

was removed, and the beads were resuspended in 125 ll

of assay buffer and analysed using the Bio-Plex 200 sys-

tem (Bio-Rad, Hercules, CA). Cytokine and chemokine

concentrations were calculated based on relevant standard

curves using the BIO-PLEX manager software (Bio-Rad).

Antigen presentation

The PTE modules were prepared as previously described.

After removing the non-migrated cells, the PTE modules

were incubated at 37� with either culture medium alone,

tetanus toxoid (TT, 1 lg/ml; Calbiochem, La Jolla, CA),

Gardiquimod (5 lg/ml), or TT plus Gardiquimod. The

RT cells were carefully collected after 48 hr and co-cul-

tured with 2�5 · 105 autologous T cells and 2�5 · 105

autologous B cells in a 24-well plate at an APC : lympho-

cyte ratio of 1 : 30. The anti-TT IgG response was

evaluated on day 7 using enzyme-linked immunosorbent

spot-forming cell assay (ELISPOT) detection.

Enumeration of anti-TT antibody-secreting cells

Numbers of TT-specific antibody-secreting cells were

assessed by ELISPOT as previously described, with minor

modifications.22 Briefly, a 96-well Multiscreen HA filtration

plate (Millipore Corporation) was coated with 1 lg/ml TT

at 4� overnight, followed by blocking with RPMI-1640 con-

taining 1% bovine serum albumin at 37� for 2 hr. Serially

diluted lymphocyte suspensions were added to the plate

and incubated at 37� for 4–5 hr. After incubation, cells

were discarded and the plate was incubated with 1 lg/ml

goat anti-human IgG-biotin (Jackson ImmunoLab, West

Grove, PA) at 4�, overnight. On the next day, the plate was

washed and incubated with 5 lg/ml horseradish peroxi-

dase-conjugated avidin (Vector Laboratories, Burlingame,

CA) for 1 hr at room temperature. The plate was washed

and incubated with 3-amino-9-ethylcarbazole substrate

solution until spots were well developed. The plate was

rinsed with deionized water to stop the reaction and spots

were counted using the AID ELISPOT reader system (Cell

Technology, Columbia, MD).

Results

TLR agonists induce more cytokines in the PTEmodule than in PBMC culture

The TLR-induced cytokine production in the PTE mod-

ule was measured first to evaluate the immunopotency of

the TLR agonists. Overall, TLR agonists induced higher

levels of cytokines in the PTE module than in conven-

tional PBMC cultures (Fig. 2). For example, both Poly

I:C and CpG 2006 induced the pro-inflammatory cyto-

kines IL-1a/b in the PTE module, which were not

observed in PBMC cultures (Fig. 2a,c). Poly I:C also trig-

gered tumour necrosis factor-a (TNF-a) production solely

in the PTE culture (Fig. 2a). Moreover, the production of

IL-6 and IL-8 induced by Poly I:C and CpG 2006 treat-

ments in the PTE module was significantly higher than in

PBMC culture (Fig. 2b,d). Although Gardiquimod and

LPS dramatically induced IL-1b, IL-6, IL-8, IL-10 and

TNF-a, both in the PTE module and in the PBMC cul-

ture, the PTE module produced approximately three- to

sixfold more IL-1b and IL-10, and 10- to 50-fold more

IL-6 and IL-8, than PBMC culture (Fig. 2e–h). Conse-

quently, the PTE module was more sensitive than conven-

tional PBMC culture in response to TLR stimulation.

The PTE module contains a confluent HUVEC mono-

layer, so it was important to determine if the higher sen-

sitivity of the PTE module to TLR agonists was

attributable to the presence of HUVEC. There was a basal

production of IL-6 (286 ± 26 pg/ml) and IL-8

(3822 ± 347 pg/ml) in unstimulated HUVEC cultures.

Although treatment with Poly I:C or LPS significantly

increased the production of IL-6 and IL-8 by about two-

fold to fourfold, Gardiquimod and CpG 2006 did not

trigger cytokine production in the HUVEC culture

(Fig. 3). These data suggest that enhanced TLR-induced

cytokine production in the PTE module is attributed to a

synergistic interaction between migrated cells and the

endothelium.

TLR agonists promote differentiation and maturationof DC in the PTE module

Previous studies have shown that TLR stimulation can

induce DC maturation and enhance antigen presenta-

tion.23 The PTE module has been reported as a novel tis-

sue-engineered system to generate DC autonomously, so

we determined if TLR stimulation could also regulate the

phenotype of the PTE-DC. For this purpose, RT cells

were carefully harvested from unstimulated PTE modules

� 2010 Blackwell Publishing Ltd, Immunology, 130, 374–387 377

Assessing the immunopotency of TLR agonists in a 3D immunological model

and labelled with an antibody cocktail. Antigen-presenting

cells generated in the PTE module (PTE-APC) were

defined as Annexin) Lineage) DR+ cells, which could be

further divided into two subpopulations: DR+ CD14+

cells and DR+ CD14) cells (Fig. 4). DR+ CD14+ cells

expressed a low level of CD86 and lacked CD83 and

CCR7. In contrast, DR+ CD14) cells expressed CD83 and

CCR7, with elevated levels of CD86 and HLA-DR com-

pared with DR+ CD14+cells (Fig. 4), suggesting that these

cells were DC generated autonomously in the PTE

300

250

200

150

100

Cyt

okin

es (

pg/m

l)

Cyt

okin

es (

pg/m

l)

50

IL-1a IL-1b TNF IL-1a

PBMCPTE PBMCPTE

PBMCPTE

IL-1b IL-6 IL-8 IL-8IL-6

IL-6 IL-8 IL-8IL-6

IL-6 IL-8 IL-8IL-6

PBMCPTEIL-6 IL-8 IL-8IL-6

22,

807, 180754228,

5189 328328, 1003,

12,8058 56096720,

641,77 586

TNF

IL-1a IL-1b TNF IL-1a

PBMCPTE PBMCPTE

IL-1b TNF

IL-10IL-1b TNF IL-10

PBMCPTE

IL-1b TNF

IL-10IL-1b TNF IL-10PBMCPTE

IL-1b TNF

MediumPoly I : C

(a) (b)

(c) (d)

(e) (f)

(g) (h)

MediumPoly I : C

Medium

Medium Medium

CpG 2006

Cyt

okin

es (

pg/m

l)C

ytok

ines

(pg

/ml)

Cyt

okin

es (

pg/m

l)

Cyt

okin

es (

pg/m

l)C

ytok

ines

(pg

/ml)

Cyt

okin

es (

pg/m

l) CpG 2006

MediumGardiquimod

MediumGardiquimod

200 000

500 000

400 000

300 000

200 000

100 000

0

150 000

100 000

50 000

0

2 000 000

2 000 000

1 600 000

1 200 000

800 000

400 000

0

1 000 000

1 500 000

500 000

0

LPSMediumLPS

0

200

150

100

50

0

150 000

12 000

9000

6000

3000

0

700

600

500

400

300

200

100

0

Figure 2. Toll-like receptor (TLR) agonists induce cytokine production in the peripheral tissue equivalent (PTE) module. Peripheral blood

mononuclear cells (PBMCs) were suspended in M199-0�1% bovine serum albumin and applied onto the endothelium at 500 000 cells/well to

generate PTE modules. After a 90-min incubation period, non-migrating cells were removed and the PTE was incubated in M199 with 10%

autologous serum in the presence or absence of CpG 2006 (5 lg/ml), Poly I : C (50 lg/ml), Gardiquimod (Gar, 2�5 lg/ml), or lipopolysaccharide

(LPS; 10 ng/ml). For PBMC cultures, PBMC were suspended in RPMI-1640 with 10% autologous serum (2�5 · 106 cells/ml), and stimulated with

the same concentration of Toll-like receptor (TLR) agonists. The culture supernatants were collected after 24 hr incubation, the cytokine levels in

supernatants were analysed by Q-plex cytokine array (IL, interleukin; TNF, tumour necrosis factor). The numbers shown in the bar graphs repre-

sent the mean ± SE of four or five different donors.

378 � 2010 Blackwell Publishing Ltd, Immunology, 130, 374–387

Y. Ma et al.

module (PTE-DC). Although PTE-DC (DR+ CD14))

accounted for 20–30% of total PTE-APs in untreated PTE

modules, Gardiquimod and LPS approximately doubled

the percentage of DC recovered, a change that was statis-

tically significant (Fig. 5a). Notably, the application of

TLR agonists did not significantly impact the absolute

number of live RT cells (data not shown), which is con-

sistent with a previous report.5 Hence, selected TLR stim-

ulation enhanced the differentiation of monocytes into

DC in the PTE module. Moreover, TLR agonists pro-

moted the maturation of PTE-DC. Unstimulated PTE-DC

expressed CD86 but lacked CD83 and CCR7, a chemo-

kine receptor essential for DC migration into the lympha-

tic vessels.24 Gardiquimod and LPS significantly increased

the expression of CCR7, CD83 and CD86 on PTE-DC,

suggesting that they drive the maturation of DC (Fig. 5b).

Poly I:C and CpG 2006 promoted DC maturation to a

lesser extent, with augmented expression of CD86 and

CCR7, respectively (Fig. 5b).

In addition to promoting DC generation and matura-

tion, TLR agonists also triggered the activation of Line-

age) DR+ CD14+ APC in the PTE module. Under

unstimulated conditions, CD14+ APC expressed low levels

of CD83, CD86 and CCR7. However, administration of

Gardiquimod and LPS induced the expression of CD83

and CD86 by threefold to sixfold, and CCR7 over 10-fold

in these cells (Fig. 5c). Just as for CD14) DC, CpG 2006

and Poly I:C were less potent than LPS and Gardiquimod

in activating CD14+ APC in the PTE module (Fig. 5c).

The PTE-DC were further characterized by comparing

their phenotype with conventional cytokine-cultured DC.

In general, unstimulated PTE-DCs expressed similar levels

3000

50 000

40 000

30 000

20 000

10 000

0

2000

1000

0Med

IL-6

(pg

/ml)

IL-8

(pg

/ml)

CpG Gar LPS PIC

PICMed CpG Gar LPS

*

**

*

Figure 3. Toll-like receptor (TLR) agonists induce cytokine produc-

tion in the human umbilical vein endothelial cell (HUVEC) culture.

HUVEC were grown on a collagen matrix and maintained in M199-

10% fetal bovine serum. Once the HUVEC monolayer became con-

fluent, treatment with medium, CpG 2006 (5 lg/ml), Poly I : C

(50 lg/ml), Gardiquimod (Gar, 2�5 lg/ml), or lipopolysaccharide

(LPS; 10 ng/ml) was carried out for 48 hr. Interleukin-6 (IL-6) and

IL-8 in culture supernatants were analysed by Q-Plex cytokine array.

Values shown in the bar graphs represent the mean ± SE of four or

five different donors. Differences between unstimulated and stimu-

lated groups were analysed using the paired Student’s t-test.

*P < 0�05.

CD14

HLA

-DR

00

CD83 CD86 CCR7 HLA-DR102 103 104 105

0

0

102

102

24·2 75·8

DR+ CD14+

DR+ CD14–103

103

104

104

105

105

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105

20

40

60

80

100

Max

(%

)

Figure 4. Phenotypic analysis of unstimulated peripheral tissue equivalent antigen-presenting cells (PTE-APC). The PTE module was prepared as

described in Fig. 1, and then incubated in culture medium for 48 hr. The reverse transmigrated (RT) cells were collected and labelled with a mix-

ture of antibodies, including the lineage markers allophycocyanin-CD3, -CD19, -CD56 and the DC relevant markers, allophycocyanin-Cychrome

7-CD14, peridinin chlorophyll protein-Cychrome 5.5-HLA-DR, phycoerythrin-Cychrome 7-CCR7, fluorescein isothiocyanate-CD83 and phyco-

erythrin-CD86. Viable cells were assessed by Trypan Blue exclusion and Annexin V-Pacific Blue negativity. No significant difference between

absolute numbers of viable cells counted between different conditions was observed. PTE-APC are defined as Annexin V) Lin) DR+, and can be

further divided into two subpopulations: DR+ CD14+ and DR+ CD14). The expressions of CD83, CD86, CCR7 and HLA-DR on these two sub-

populations are shown in histograms, and the grey areas represent the staining with isotype-matched control antibodies.

� 2010 Blackwell Publishing Ltd, Immunology, 130, 374–387 379

Assessing the immunopotency of TLR agonists in a 3D immunological model

of CCR7, CD83 and CD86 as cytokine-cultured iDC

(Fig. 5d). Lipopolysaccharide significantly promoted mat-

uration of cytokine-cultured DC by inducing CCR7,

CD83 and CD86, which was consistent with observations

made in the PTE module. However, Gardiquimod signifi-

cantly promoted generation and maturation of PTE-DC,

but failed to regulate cytokine-cultured DC maturation.

Conversely, Poly I : C moderately enhanced the matura-

tion of PTE-DC, yet dramatically induced expression of

CCR7 and CD86 on cytokine-cultured DC (Fig. 5d).

Hence, cytokine-cultured DC and PTE-DC respond

differently to TLR stimulations.

Non-monocytic cell populations contribute to TLR-induced cytokine production in the PTE module

Previous studies6–8 and our unpublished observations

have indicated that the unstimulated RT fraction con-

tained about 10–15% non-monocytic cells, including lym-

phocytes and pDC. The PTE-DC and cytokine-derived

DC showed differential responsiveness to TLR agonists,

especially Gardiquimod and Poly I:C, so it was of interest

to determine if a small population of non-monocytic cells

was involved in the response of the PTE module to TLR

stimulation. For this purpose, we established the PTE

Med (a)

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iD

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P(D

PIC

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-DR

Figure 5. Toll-like receptor (TLR) agonists promote the generation and maturation of peripheral tissue equivalent dendritic cells (PTE-DC). The

PTE modules were prepared and treated as described in Fig. 1. The reverse transmigrated (RT) cells were carefully harvested at 48 hr and stained

with fluorochrome labelled antibodies as described in Fig. 2. PTE-antigen-presenting cells (APC) are defined as Annexin V) Lin) DR+ shown in

(a), and can be further divided into two subpopulations: PTE-DC (DR+ CD14)) and CD14+ APC (DR+ CD14+). The percentage of PTE-DC is

shown in (a), and the expression of CD83, CD86 and CCR7 on PTE-DC and CD14+ APC are shown in (b) and (c), respectively. CD14+ cells

positively selected from peripheral blood mononuclear cells (PBMC) were cultured with granulocyte–macrophage colony-stimulating factor

(100 ng/ml) and interleukin-4 (25 ng/ml) for 5 days to generate immature cytokine-cultured DCs (iDC). The iDC were then cultured in the pres-

ence or absence of CpG 2006 (5 lg/ml), Poly I : C (50 lg/ml), Gardiquimod (Gar, 2�5 lg/ml), or lipopolysaccharide (LPS; 10 ng/ml). The cells

were harvested at 48 hr and stained with fluorochrome labelled antibodies as previously described, and the expression of CD83, CD86 and CCR7

on DC are shown in (d). The numbers shown in the bar graphs represent the mean ± SE of four or five different donors, and differences

between the stimulated and unstimulated PTE modules were analysed using the Student’s t-test. *P < 0�05; **P < 0�01.

380 � 2010 Blackwell Publishing Ltd, Immunology, 130, 374–387

Y. Ma et al.

module with PBMC (PTE-PBMC) or monocytes (PTE-

Mo) by applying PBMC or purified CD33+ monocytes

onto the HUVEC monolayer, respectively. Although the

PBMC populations used in these studies were typically

comprised of � 20% monocytes, the enriched myeloid

cell populations for the PTE-Mo module were devoid of

non-monocytic cell populations and consistently exhibited

purities in excess of 99%.

Cytokine analysis indicated that TLR agonists evoked

different cytokine profiles in the PTE-PBMC and PTE-

Mo modules. The production of IL-6, IL-8 and IL-10 was

not significantly different between the PTE-PBMC and

PTE-Mo modules (data not shown), yet several cytokines

were significantly reduced only in the PTE-Mo module.

For example, levels of IL-12 p70 and TNF-a were signifi-

cantly increased by TLR stimulation in the PTE-PBMC

module. However, in the PTE-Mo module, the produc-

tion of IL-12 p70 and TNF-a was reduced by between

five- and 20-fold (Fig. 6a,b). Similarly, production of

interferon (IFN) also declined in the PTE-Mo module.

Interferon-a, a type I IFN, is a potent stimulus for DC

maturation.3,25 In the PTE-PBMC module, IFN-a was

induced by Poly I:C and Gardiquimod, whereas in the

PTE-Mo module its production was reduced or com-

pletely abolished (Fig. 6c). In addition to the cytokines,

the production of regulated on activation, normal T-cell

expressed and secreted (RANTES) and interferon-induc-

ible protein-10 (IP-10) was reduced in the PTE-Mo

module. Although TLR agonists induced RANTES about

10- to 200-fold in the PTE-PBMC module, RANTES pro-

duction was partially or completely ablated in the PTE-

Mo module (Fig. 6d). Another chemokine, IP-10, was

dramatically reduced in the PTE-Mo, particularly in those

treated with LPS, Gardiquimod and CpG 2006 (Fig. 6e).

These data suggest that the relatively few non-monocytic

cells in the PTE module may play a complementary role

in TLR-triggered cytokine and chemokine production.

Non-monocytic cell populations contribute to TLR-induced DC generation and non-DC APC activationin the PTE module

As TLR agonists were observed to evoke different cyto-

kine profiles in the PTE-PBMC and PTE-Mo modules, we

next wished to determine if myeloid APC and DC derived

from PTE-Mo and PTE-PBMC modules would also

respond differently to TLR stimulation. The percentage of

DC derived from the PTE-Mo module was less than half

that in the PTE-PBMC module in unstimulated condi-

tions. The TLR-induced DC generation was significantly

reduced by 30–60% in the PTE-Mo module compared

with the PTE-PBMC module (Fig. 7a). Surprisingly,

despite decreased DC generation, the induction of CD83,

CD86 and CCR7 on DC by TLR stimulation remained

similar in the PTE-Mo module (Fig. 7b). These data sug-

gest that the small population of non-monocytic cells in

1000Monocyte

IL-12p70 TNF-a IFN-a

PBMC

1 000 000 RANTES IP-10

100 000

10 000

1000

100

10

1Med CpG Gar LPS PIC Med CpG Gar LPS PIC

Med CpG Gar LPS PIC Med CpG Gar LPS PICMed CpG Gar LPS PIC

10 000 000

1 000 000

100 000

10 000

10 000

1000

1000

800

600

400

200

0

100

10

1

1000

100

10

1

100

10

pg/m

l

pg/m

l

1

(a)

(d)(e)

(b) (c)

Figure 6. Purified monocytes diminish the effect of Toll-like receptor (TLR) agonists on cytokine/chemokine production in the peripheral tissue

equivalent (PTE) module. Peripheral blood mononuclear cells (PBMC) or purified monocytes were applied onto the human umbilical vein endo-

thelial cell (HUVEC) monolayer to establish the PTE-PBMC module and the PTE-monocyte (Mo) module, respectively. After removal of the

non-migrated cell fraction, the PTE modules were incubated in the presence or absence of CpG 2006 (5 lg/ml), Poly I : C (50 lg/ml), Gardiqui-

mod (Gar, 5 lg/ml), or lipopolysaccharide (LPS; 10 ng/ml) for 48 hr. The cytokine levels in culture supernatants were analysed by the Bio-plex

cytokine array. The numbers shown in the bar graphs (a–e) represent the mean ± SE of four or five different donors.

� 2010 Blackwell Publishing Ltd, Immunology, 130, 374–387 381

Assessing the immunopotency of TLR agonists in a 3D immunological model

the PTE module participate in DC generation but may

not be essential for DC maturation.

Furthermore, the PTE-Mo module dampened the acti-

vation of CD14+ APC. The expression of CD83, CD86

and CCR7 on CD14+ APC in unstimulated PTE-Mo

modules was approximately half that observed in PTE-

PBMC modules. Although Gardiquimod was the most

potent inducer of CD14+ APC activation, the enhance-

ment of CD83, CD86 and CCR7 on APC was reduced by

over 50% in PTE-Mo modules compared with PTE-

PBMC modules (Fig. 7c). In contrast, LPS-induced

expression of activation markers on CD14+ APC

remained similar in both PTE-PBMC and PTE-Mo mod-

ules except for CD83, which was significantly reduced in

the PTE-Mo module (Fig. 7c). CpG 2006 and Poly I:C

both moderately activated non-DC APC by inducing

CD86 in the PTE-PBMC module; however, their effect

was partially or completely abolished in the PTE-Mo

module. These data indicated that the relatively few non-

monocytic cells in the PTE module may contribute to

TLR-induced DC generation and APC activation.

Plasmacytoid DC recovers the immunoregulatoryeffect of Gardiquimod in the PTE module

Plasmacytoid DC are the key cell population for produc-

ing type I IFN and mediating TLR7/TLR9-triggered

immune responses.26 Considering that Gardiquimod is a

particularly potently TLR7 agonist,27 we wanted to deter-

mine if the impaired effects of Gardiquimod on DC

development and cytokine production in the PTE-Mo

module were the result of the lack of a pDC population,

which are always depleted by CD33-positive selection. We

first tested whether the removal of pDC alone was suffi-

cient to change Gardiquimod-induced DC maturation in

the PTE module. The up-regulation of CCR7 and CD86

on PTE-APC induced by Gardiquimod in the PTE-PBMC

module were markedly diminished when pDC were selec-

tively depleted from the PBMC fraction (Fig. 8a). How-

ever, when pDC were added back into the PTE-Mo

module, Gardiquimod-induced DC generation and the

expression of CD86 and CCR7 were effectively rescued

(Fig. 8b,c). These data indicated that the small number of

80

60

40

20

%%

0

40

20

0

% %%

%%

0Med CpG Gar LPS PIC Med CpG Gar LPS PIC Med CpG Gar LPS PIC

Med CpG Gar LPS PIC

PTE-DC

CD14+ APC(DR+ CD14+)

(DR+ CD14–)

Med CpG Gar LPS PICMed CpG Gar LPS PIC

Med

Monocyte

DCs

CCR7

CCR7 CD83

CD83 CD86

CD86

PBMC

*

*

*

*

**

*

*

*

**

CpG Gar LPS PIC

2

4

6

8

8

4

0

12

1610

10

30

50 25 120

100

80

60

40

20

0

100

80

60

40

20

0

20

15

10

5

0

(a)

(b)

(c)

Figure 7. Purified monocytes diminish the Toll-like receptor (TLR) agonist-induced dendritic cell (DC) generation and antigen-presenting cell

(APC) activation in the peripheral tissue equivalent (PTE) module. PTE-peripheral blood mononuclear cells (PBMC) and PTE-monocytes (Mo)

were established and treated as described in Fig. 4. After 48 hr, the reverse transmigrated (RT) cells were carefully harvested and stained with

fluorochrome labelled antibodies. PTE-DC and CD14+ APC are defined as described in Fig. 3. The percentage of PTE-DC in total PTE-APC are

shown in (a), and the expression of CCR7, CD83 and CD86 on PTE-DC and CD14+ APC are shown in (b) and (c), respectively. The numbers

shown in the bar graphs represent the mean ± SE of four or five different donors, and differences between PTE-Mo and PTE-PBMC were analy-

sed using the paired Student’s t-test. *P < 0�05; **P < 0�01.

382 � 2010 Blackwell Publishing Ltd, Immunology, 130, 374–387

Y. Ma et al.

pDC remaining in the PTE module crucially contribute

to Gardiquimod-induced PTE-DC differentiation and

APC activation.

Gardiquimod enhances TT-specific IgG responsesin vitro

The major function of APC is to present antigen to T

cells and initiate adaptive immunity. As TLR agonists

could enhance the generation and/or maturation of APC

and DC, we next investigated if adjuvant-treated PTE-

APC could facilitate antigen-specific immune responses.

For these studies, we used Gardiquimod because it is a

novel TLR7 ligand and has been shown to potently

enhance the maturation of PTE-APC. The RT cells were

collected from the PTE module treated with either culture

medium alone or with TT ± Gardiquimod for 48 hr.

Consistent with previous results, Gardiquimod alone sig-

nificantly enhanced DC generation and maturation; TT

alone also markedly increased the DC generation and

promoted the expression of CD86 and CCR7 on DC. The

combination of TT and Gardiquimod resulted in the

highest percentage of CD86+ CCR7+ DC (Fig. 9a),

indicating that TT synergized with Gardiquimod in the

generation and maturation of PTE-DC. When co-cultured

with autologous T cells and B cells, PTE-DC treated with

Gardiquimod and/or TT did not impact TT-specific

T-cell and B-cell proliferation (data not shown). However,

TT-primed PTE-DC significantly induced the anti-TT IgG

response, suggesting that these DC were competent APC.

Notably, the PTE-DC treated with TT + Gardiquimod

elicited about a twofold increase in the number of anti-TT

IgG-secreting cells (Fig. 9b). Clearly, administration of

TLR agonists in the PTE module not only promoted the

generation and maturation of PTE-DCs, but also facilitated

a more pronounced antigen-specific B-cell response.

Discussion

Vaccination is currently the most cost-effective and suc-

cessful approach to prevent infectious disease in the

world. Adjuvants are an important part of vaccines and

are frequently used to enhance vaccine immunogenicity.

Over the past few decades, numerous adjuvants have been

discovered, but only a few are licensed for human use.28

The limited application of new vaccine adjuvants can be

0 102 103 104 105

0102 103 104 105 0 102 103 104 105

0 102 103 104 105 102 103 104 101 100 102 103 104 101 100

102 103 104101100 102 103 104101100

102 103 104101100 102 103 104101100

Gardiquimod

Gardiquimod

Medium

Isotype

Medium

Isotype

Cel

l num

ber

Cel

l num

ber

Who

le P

BM

Cs

Cel

l num

ber

pDC

dep

lete

d C

ell n

umbe

r

Unstained control Medium

Isotype control Gardiquimod

PBMCs CD33 CD33/pDC

Who

le P

BM

Cs

Cel

l num

ber

Pur

ified

CD

33

Cel

l num

ber

CD

33+

pD

Cs

Cel

l num

ber

CCR7 CD86

Med Gar 0

10203040506070

DC

(%

)

Unstained

Unstained

CCR7 CD86

(a) (b) (c)

Figure 8. Plasmacytoid dendritic cells (pDC) are involved in Gariquimod-induced DC development and antigen-presenting cell (APC) activation

in peripheral tissue equivalent (PTE) modules. Different PTE modules were established either using peripheral blood mononuclear cells (PBMC)

versus PTE. PBMC depleted with pDC (a), or using purified monocytes (CD33+ cells) versus purified monocytes with pDC (CD33/pDC) (b, c),

as described in the Materials and methods. The PTE modules were treated with medium only or Gardiquimod for 48 hr. The reverse transmi-

grated (RT) cells were carefully harvested and labelled with a mixture of antibodies, including the lineage markers allophycocyanin-CD3, -CD19,

-CD56 and the DC relevant markers, allophycocyanin-Cychrome 7-CD14, peridinin chlorophyll protein-Cychrome 5.5-HLA-DR, phycoerythrin-

Cychrome 7-CCR7, fluorescein isothiocyanate-CD83 and phycoerythrin-CD86. The expression of CCR7, CD83 and CD86 on PTE-APC are

shown in (a) and (c) (a representative of three independent experiments, using different donors) and the percentage of PTE-DC is shown in (b).

Filled area: unstained control; dash line: isotype control; thin line: medium-treated group; thick line: Gardiquimod-treated group.

� 2010 Blackwell Publishing Ltd, Immunology, 130, 374–387 383

Assessing the immunopotency of TLR agonists in a 3D immunological model

attributable to the paucity of reliable evaluation systems.

Although human clinical trials are the most reliable way

to evaluate vaccine/adjuvant safety and efficiency, the cost

and sample sizes required for validation are daunting.29

Some in vitro assessments, such as PBMC culture assays

and/or cytokine-derived DC culture assays, are used for

screening and validating adjuvants. Unfortunately, results

from these in vitro studies do not provide a complete

immunological picture and as such have been poor pre-

dictors of response because of functional limitations.

Therefore, it is desirable to have a new reliable test bed to

evaluate adjuvants. In the present study, the immuno-

potency of TLR agonists using the PTE module, a tissue

engineered in vitro immunological model, was tested. The

PTE module has been reported as a novel autonomous

DC-generating system that more closely resembles in vivo

physiological conditions5,10 than other models. Results

reported herein show that the PTE module is highly sen-

sitive to TLR stimulation and may be a reliable test bed

for adjuvant evaluation.

Compared with conventional PBMC cultures, the PTE

module produced significantly higher levels of cytokines

in response to TLR stimulation, suggesting that it has a

better sensitivity. The increased sensitivity of the PTE

module may be partly the result of the enrichment of

APC (monocytes and DC), which are the major cell pop-

ulation expressing TLR. Muller et al.30 demonstrated that

over 97% of migrated cells in a similar PTE module were

monocytes. We report that about 80% of live RT cells

were DR+ APC. Enriched APC in the PTE module can

amplify the stimulatory signals of TLR, and therefore will

help to assess the immunoregulatory potency of TLR

agonists and other adjuvant candidates. Moreover, endothe-

lial cells (HUVEC) play a key role in TLR-triggered DC

differentiation and cytokine production in the PTE mod-

ule. Although endothelial cells are classically regarded as

non-immune cells, they can produce a variety of inflam-

matory mediators (chemokines and adhesion molecules)

upon stimulation, and mediate local recruitment and

migration of leucocytes. Previous studies have shown that

endothelial cells express certain levels of TLR2, -3, -4, -5

and -9, and can be stimulated by TLR agonists to release

cytokines and chemokines.10,30 More importantly, the

presence of endothelial cells synergized with monocytes to

produce cytokines, and enhanced monocyte activation in

a co-culture system.31 In addition, the cell–cell contact

between endothelial cells and monocytes was found to be

critical for the rapid DC differentiation and antigen-

induced DC maturation in vitro.5,6 In the present study,

Poly I:C (TLR3) and LPS (TLR4) induced production of

0

0102

103

104

105

102 103 104 105 0

0102

103

104

105

102 103 104 105 0

0102

103

104

105

102 103 104 105 0

0102

103

104

105

102 103 104 105

0

0102

103

104

105

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0102

103

104

105

102 103 104 105 0

0102

103

104

105

102 103 104 105 0

0102

103

104

105

102 103 104 105

200

CCR7

CD14

Med(a)

(b)

20·5

20·3 3·26 59·8 37

3·1 0·13

48·8 44·5

6·43 0·35

33·3 58·2

7·57 0·9876·2 0·3

50·6 30·8 60·8

HLA

-DR

CD

86Gar TT TT + Gar

160

120

80

Ant

i-TT

IgG

spo

ts (

per

106

cells

)

40

0Med

*

*

Gar TT TT + Gar

Figure 9. Co-administration of Gardiquimod

with tetanus toxoid (TT) in the peripheral tis-

sue equivalent (PTE) module enhances the

anti-TT immunoglobulin G (IgG) response.

The PTE module was cultured in medium

alone or in the presence of TT (1 lg/

ml) ± Gardiquimod (Gar, 5 lg/ml). After

48 hr, the reverse transmigrated (RT) cells

were carefully harvested and stained with fluo-

rochrome labelled antibodies. The percentage

of PTE-dendritic cells (DC) in the total PTE-

antigen-presenting cell (APC) fraction and the

expression of CCR7 and CD86 on DC are

shown in (a). RT cells were co-cultured with

2�5 · 105 autologous CD3 T cells and

2�5 · 105 autologous B cells at an APC : T cell

ratio of 1 : 30 in a 96-well plate. After 7 days,

the cells were harvested and the anti-TT IgG

response was evaluated using an enzyme-linked

immunosorbent spot-forming cell assay. The

numbers shown in (b) represents the mean ±

SE of three or four different donors, and dif-

ferences between the unstimulated and stimu-

lated groups were analysed using the Student’s

t-test. *P < 0�05.

384 � 2010 Blackwell Publishing Ltd, Immunology, 130, 374–387

Y. Ma et al.

IL-6 and IL-8 about two- to threefold in the HUVEC cul-

tures, indicating a direct stimulation of HUVEC by TLR

agonists. More importantly, compared with the conven-

tional PBMC culture, the PTE module produces signifi-

cantly higher levels of cytokines after TLR stimulation,

confirming that HUVEC and migrated monocytic cells

synergistically contributed to TLR-induced cytokine pro-

duction in the PTE module.

Furthermore, the PTE module is capable of elucidating

the effect of TLR agonists on both DC differentiation and

maturation. Both LPS and Poly I : C have been shown to

fully induce the maturation and the activation of cyto-

kine-cultured DC.32,33 However, their effect on DC gener-

ation remains unclear. The present study shows that LPS

up-regulates the expression of CD83, CD86 and CCR7 on

PTE-DC, consistent with observations in cytokine-

cultured DC. Moreover, LPS significantly increases the

percentage of DC in the PTE module. Hence, LPS pro-

motes both DC generation and maturation in the PTE

module. On the other hand, although Poly I : C induces

CD86 expression on PTE-DC, it does not significantly

regulate the percentage of DC produced. These data sug-

gest that Poly I:C can moderately induce PTE-DC matu-

ration without impacting DC generation. Surprisingly,

our results showed a weaker effect of Poly I : C on

PTE-DC maturation in comparison with that shown in

cytokine-cultured DC, which may be to the result of the

heterogeneity of the PTE-APC population, consisting of

� 20% PTE-DC and 80% monocyte-like APC. As human

TLR3 has been shown to be restricted to freshly isolated

mature DC and cytokine-derived DC,34 the presence of

monocyte-like APCs may diminish the stimulatory effect

of Poly I:C in the PTE module. In fact, in vivo DC have

been shown as a heterogeneous population. Morelli

et al.10 reported that migrating DC from skin tissue were

heterogeneous and contained both dermal DC and Lan-

gerhans cells (LC) as well as a subpopulation (� 20%) of

CD14+ LC precursors. Additionally, cells in healthy

human superficial lymph are known to contain three- to

fourfold more monocytes than DC.10 Therefore, although

the heterogeneity of PTE-APC diminished the extent of

Poly I:C-induced DC maturation, it may more closely

resemble in vivo physiological conditions.

The CpG ODNs are synthetic oligonucleotides contain-

ing unmethylated CpG motifs, which can mimic bacterial

DNA and stimulate both innate and adaptive immunity.16

Latest clinical studies also indicated that co-administra-

tion of CpG ODN with vaccine was safe and effectively

increased vaccine-specific immune responses in humans.35

The immunostimulatory effects of CpG ODN are medi-

ated by intracellular TLR9 expression predominantly in

pDC and B cells.36 The recognition of CpG ODN by

TLR9 can directly activate B cells and pDC, up-regulating

type I IFN production.37 Moreover, CpG ODN are

known to induce the differentiation of monocytes into

DC via pDC-derived type I IFN.38 Interestingly, although

cytokine-cultured DC did not respond to CpG ODN,

CpG significantly induces differentiation and maturation

of freshly isolated peripheral blood DC.37 The different

responses of cytokine-cultured DC compared with in vivo

DC implies that there are also functional differences. In

the present study, the application of CpG 2006 increased

PTE-DC generation by about 50%, and moderately

enhanced PTE-DC maturation by inducing CCR7 expres-

sion. These data are consistent with observations made in

fresh-blood DC.37 On the other hand, CpG 2006 failed to

augment DC generation in the PTE-Mo modules, suggest-

ing that CpG-induced PTE-DC generation should require

the involvement of pDC, which account for 1–2% of live

migrated cells in the PTE-PBMC module but were absent

in the PTE-Mo module. Compared with conventional

PBMC cultures and cytokine-derived DC, the PTE mod-

ule shows better sensitivity in response to CpG 2006, and

therefore could serve as a promising test bed for evaluat-

ing the immunopotency of different CpG motifs.

Gardiquimod, as a novel TLR7 ligand, has been shown

to enhance vaccine-induced immune responses in animal

models.39 However, the role of TLR7 in DC differentia-

tion and maturation remains controversial. Ito et al.40

reported that TLR7 is expressed on freshly isolated mDC

and pDC, and the treatments with TLR7 ligands, such as

Imiquimod and Resiquimod, significantly enhances

expression of CD80 and CD86 on freshly isolated DC. In

contrast, cytokine-induced iDC neither express TLR7 nor

respond to TLR7 agonists.41,42 Furthermore, co-adminis-

tration of TLR7 agonists with GM-CSF suppressed differ-

entiation and maturation of cytokine-derived DC.43 These

conflicting observations between cytokine-cultured DC

and freshly isolated DC further supports the theory that

functional differences exist between cytokine-derived and

in vivo DC. Interestingly, although cytokine-induced iDC

did not respond to TLR7 agonist, priming iDC with type

I IFN induces TLR7 expression and sensitizes iDC to

TLR7 stimulation.42

In the present study, Gardiquimod did not affect the

maturation of traditional cytokine-cultured DC; how-

ever, it did enhance PTE-DC generation and matura-

tion as well as cytokine/chemokine production. Notably,

the immunoregulatory effects of Gardiquimod were sig-

nificantly reduced when PBMC were replaced with

purified monocytes in the PTE module. Analysis of

whole PBMC selectively depleted of pDC indicates that

reduced sensitivity of the PTE module in response to

Gardiquimod could be the result of the absence of

pDC in the PTE module. Furthermore, the reconstitu-

tion of pDC effectively rescued the immunoregulatory

effect of Gardiquimod in the PTE module. Taken

together, our studies indicate that pDC are indispens-

able for Gardiquimod-triggered immune responses in

the PTE module.

� 2010 Blackwell Publishing Ltd, Immunology, 130, 374–387 385

Assessing the immunopotency of TLR agonists in a 3D immunological model

In addition to promoting PTE-DC development and

inducing cytokine production, Gardiquimod co-adminis-

tered with TT in the PTE module also significantly

increases the production of anti-TT IgG antibodies, which

is consistent with the results in animal models.39 The cor-

relation between the PTE-DC development and antibody

production suggests that adjuvant-triggered immune

responses in the PTE module could be used to predict

their modulatory effects on the downstream antigen-

specific immune response.

Overall, the PTE module shows marked sensitivity to

TLR stimulation. Application of TLR agonists into the

PTE module not only induces cytokine and chemokine

production, but also promotes DC differentiation and

maturation. Moreover, the PTE module mimics the gen-

eration of DC that functionally resembles in vivo DC.

Hence, the tissue-engineered PTE module may serve as a

sensitive and reliable in vitro immunological model for

adjuvant assessment.

Disclosures

G.J.R. has received stock options from VaxDesign. G.J.R.

and Mount Sinai School of Medicine (MSSM) have

applied for a patent with VaxDesign for technology in

which vascular and connective tissue is reconstructed

from human cells for the purposes of vaccine testing and

selection. If this technology were licensed to a commercial

entity, then G.J.R. and MSSM would benefit financially.

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