CD41CD251 Treg induction by an HSP60-derived peptideSJMHE1 from Schistosoma japonicum is TLR2 dependent

Xuefeng Wang1, Sha Zhou1, Ying Chi1, Xiaoyun Wen1,

Jason Hoellwarth1, Lei He1, Feng Liu1, Calvin Wu2, Shawn Dhesi2,

Jiaqing Zhao1, Wei Hu3 and Chuan Su1

1 Department of Pathogen Biology & Immunology, Department of Pharmacology, Jiangsu Key

Laboratory of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, P. R. China2 Department of Educational Affairs, Keck School of Medicine, University of Southern California,

Los Angeles, CA, USA3 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention,

Shanghai, P. R. China

Chronic schistosome infection results in the suppression of host immune responses,

allowing long-term schistosome survival and restricting pathology. Current theories

suggest that Treg play an important role in this regulation. However, the mechanism of

Treg induction during schistosome infection is still unknown. The aim of this study was to

determine the mechanism behind the induction of CD41CD251 T cells by Schistosoma

japonicum HSP60 (SjHSP60)-derived peptide SJMHE1 as well as to elucidate the cellular and

molecular basis for the induction of CD41CD251 T cells during S. japonicum infection. Mice

immunized with SJMHE1 or spleen and LN cells from naı̈ve mice pretreated with SJMHE1

in vitro all displayed an increase in CD41CD251 T-cell populations. Release of IL-10 and

TGF-b by SJMHE1 stimulation may contribute to suppression. Adoptively transferred

SJMHE1-induced CD41CD251 T cells inhibited delayed-type hypersensitivity in BALB/c

mice. Additionally, SJMHE1-treated APC were tolerogenic and induced CD41 cells to

differentiate into suppressive CD41CD251 Treg. Furthermore, our data support a role for

TLR2 in SJMHE1-mediated CD41CD251 Treg induction. These findings provide the basis

for a more complete understanding of the S. japonicum–host interactions that contribute to

host homeostatic mechanisms, preventing an excessive immune response.

Key words: CD41CD251 Treg . Immunomodulation . Schistosomes . SJMHE1 . TLR2

Supporting Information available online

Introduction

Immune regulation associated with protective immunity is

intricate and entails the effective elimination of the pathogen

without causing serious damage to the host. Conversely, effective

pathogens have developed multiple mechanisms for modulating

or suppressing host immunity as survival and dissemination

strategies. Therefore, during the course of an infection, a struggle

between host defense mechanisms and the pathogen’s immuno-

modulatory processes results in a complex interplay that may

result in pathogen eradication or damage to the host (and

persistence of the pathogen). One of the survival strategies used

by pathogens involves the induction of immunosuppressive cell

populations, e.g. Treg [1, 2].Correspondence: Professor Chuan Sue-mail: chuansu@njmu.edu.cn

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

DOI 10.1002/eji.200939335 Eur. J. Immunol. 2009. 39: 3052–3065Xuefeng Wang et al.3052

The first observations suggesting that Treg induction

occurs during infections with certain pathogens were made

in mice infected with Bordetella pertussis [3] and

in humans infected with HCV [4] or the nematode Onchocerca

volvulus [5]. More recently, Treg induction has been described

in chronic infections caused by Candida albicans [6],

Mycobacterium tuberculosis [7], HIV [8], Leishmania major

[9], Litomosoides sigmodontis [10], and Helicobacter pylori

[11]. Treg induction has also been associated with Schisto-

some infection. Schistosomiasis is a major human disease

primarily caused by one of the three species of

Schistosome endemic to parts of Asia, South America, and

Africa, i.e. S. mansoni, S. haematobium, and S. japonicum.

Mortality rates resulting from these types of parasitic

infections are second only to malaria. Chronic schistosomiasis is

characterized by a polarized Th2 response which, in the

context of many infectious agents, is highly immunosuppressive

[12, 13]. Adult schistosomes are capable of suppressing protec-

tive host responses resulting in the insufficient development of

protective immunity [12]. Our group has demonstrated that

cellular immunity was suppressed in humans living in S. japoni-

cum-endemic regions and this suppression could be reversed

following praziquantel treatment [14]. Furthermore, there is

evidence that chronic exposure to S. mansoni not only down-

regulated Th1 responses and prevented the onset of Th1-medi-

ated diseases, e.g. MS, diabetes mellitus, and Crohn’s disease,

but also down-regulated atopic diseases including some

Th2-mediated diseases [15]. The mechanism of suppression

associated with chronic schistosomiasis suggested that the

establishment of Treg, which favor parasite survival, also dim-

inishes the severity of immune-mediated pathology and may

contribute to diminishing manifestations of atopic disease [16,

17]. Treg have been described [18, 19] as having multiple roles

during schistosome infections, including either DC suppression,

or regulation of Th2-mediated responses, granuloma develop-

ment, egg antigen-induced fibrosis [19, 20], and the establish-

ment of persistent or chronic disease. Our previous work showed

that S. japonicum egg antigens stimulated the generation of Treg

[21]. However, it is still not clear how Treg are induced during

schistosome infection.

Recently, some pathogen antigens, such as filamentous

hemagglutinin, adenylate cyclase toxin (CyaA) from B. pertussis,

cholera toxin, HCV non-structural protein 4 (NS4), S. mansoni-

specific phosphatidylserine [22], HSP60, and HSP70 from

microbes [23] have been proven to have the ability to induce

Treg. In this study, we demonstrated that SjHSP60, which is

expressed in S. japonicum eggs and worms, induces Treg; this is

consistent with recent findings indicating that prokaryotic HSP60

molecule [23], a highly conserved protein, has large areas

of sequence homologies among different species and is able to

induce CD41CD251 Treg. Furthermore, we identified a

peptide from SjHSP60, SJMHE1, which contained over-

lapping human and mouse CD41 T-cell epitopes, which were

proven to induce CD41CD251 Treg with immunosuppressive

activity.

Results

SJMHE1 increased CD41CD251Foxp31 T-cellproportions in vivo and in vitro

It has been previously determined that microbial HSP60 and HSP70

[23] have the ability to induce Treg differentiation. Consistent with

these data, we also found that SjHSP60 induced Treg both in vivo

and in vitro (Supporting Information Fig. 1). Studies have

suggested that the induction of Treg by microbial HSP could be

due to the high homology of the microbial protein to its host

(mammalian) counterpart or to the induction of self-HSP-reactive

Treg [24]. Thus, the peptide SJMHE1 from SjHSP60 437-460,

VPGGGTALLRCIPVLDTLSTKNED, highly identical to host (murine

and human) HSP60 and containing overlapping human and mouse

T-cell epitopes, was selected for further study (Supporting

Information Table 1). To test whether SJMHE1 could induce CD41CD251 Treg differentiation in vivo, T cells were isolated from the

spleen and LN of mice immunized with SJMHE1, OVA323–339 or

PBS. These cells were analyzed by flow cytometry (FCM) for the

expression of CD4, CD25, and Foxp3, a regulatory function related

marker which is known to be expressed in Treg as opposed to

activated cells [25]. The proportion of CD41CD251Foxp31 T cells

in the spleens and LN of SJMHE1-immunized mice increased

significantly compared with OVA323–339-immunized or PBS-treated

mice (Fig. 1A and C). To assess the ability of SJMHE1 to induce

CD41CD251Foxp31 T cells in vitro, pooled spleen and LN cells

from naı̈ve mice were prepared and cultured for 4 days in the

presence of SJMHE1 or OVA323–339. FCM showed that only spleen

and LN cells from SJMHE1 peptide-incubated cells contained an

increased proportion of CD41CD251Foxp31 T cells (Fig. 1B and

D). No significant increases in CD41CD251Foxp31 T cells were

observed in OVA323–339 stimulated cells. Studies have shown that

CD41CD251 Treg cells can be induced from CD41CD25– T cells

[26, 27]. To further test whether CD41CD25– T cells can be

differentiated into CD251/Foxp31 Treg by SJMHE1, CD41CD25–

T cells were purified and stimulated in vitro with SJMHE1. The

results (Supporting Information Fig. 2) suggest that CD41CD25–

T cells can be induced to differentiate into CD41CD251Foxp31

Treg by SJMHE1 in an APC-dependent manner. Taken together,

these results indicated that SJMHE1 treatment increased CD41

CD251Foxp31 T-cell populations both in vivo and in vitro.

SJMHE1 enhanced the inhibitory activity of CD41

CD251 Treg

To evaluate whether SJMHE1 treatment enhanced CD41CD251

Treg-mediated suppression, CD41CD25– T cells (responder cells)

were sorted from naı̈ve mice and co-cultured with CD41CD251

T cells from SJMHE1-, OVA323–339-, or PBS-immunized mice,

respectively. The results (Fig. 2A) showed that following stimula-

tion with anti-CD3 Ab, CD41CD251 T cells from all three

immunized mouse groups were highly effective at suppressing

CD41CD25– T-cell proliferation; however, the greatest degree of

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inhibition was conferred by CD41CD251 T cells purified from

SJMHE1-immunized mice. Furthermore, following the addition of

SJMHE1 to co-cultures, the inhibitory ability of CD41CD251 T cells

harvested from SJMHE1-immunized mice was significantly

enhanced compared with the activity observed for CD41CD251

T cells purified from OVA323–339- or PBS-immunized mice or

following the addition of OVA323–339 to CD41CD251 T cells from all

immunization groups. These results suggest that SJMHE1 injection

might induce antigen-specific CD41CD251 Treg in mice. To test

whether the suppressive activity of CD41CD251 T cells could be

enhanced by SJMHE1 in vitro, CD41CD251 T cells from naı̈ve mice

were pretreated in vitro with SJMHE1, OVA323–339 or PBS and then

co-cultured with responder naı̈ve murine CD41CD25– T cells.

Results described in Fig. 2B show that in vitro pretreatment of

CD41CD251 T cells with SJMHE1 (but not OVA323–339 or PBS)

enhanced suppressive properties. This is consistent with reports that

human HSP60 pretreatment of CD41CD251 T cells enhanced

inhibition of CD41CD25– T-cell proliferation [28]. Furthermore,

our results (Supporting Information Fig. 3A) suggest that without

APC, SJMHE1 was still able to enhance CD41CD251 T-cell

suppression in a plate-bound anti-CD3 culture system. However,

SJMHE1 failed to directly induce a statistically significant expansion

of purified CD41CD251 T-cell populations (Supporting Information

Fig. 3B) in the presence or absence of APC. Taken together, these

results indicated that SJMHE1 treatment enhanced the inhibitory

activity of CD41CD251 Treg both in vivo and in vitro.

SJMHE1 triggered CD41CD251 Treg suppressivefunction in a cytokine-dependent manner

Several possible mechanisms have been identified to describe

Treg function. In contrast to naturally occurring CD41CD251

Figure 1. SJMHE1 increased CD41CD251Foxp31 T cells in vivo and in vitro. Analysis of CD41CD251Foxp31 T cells from pooled spleen and LN cells byFCM. (A) Seven days after the last immunization, spleen and LN from each mouse were pooled, single cell suspensions were prepared and redblood cells were lysed. FCM analysis for CD3, CD4, CD25, and Foxp3 was performed and the data are expressed as the mean7SD of 18 mice fromthree independent experiments. �po0.05 versus mice treated with OVA323–339 or PBS. (B) Pooled spleen and LN cells from individual naı̈ve BALB/cmice were pre-incubated with 1mg/mL SJMHE1, OVA323–339 or medium only at 371C in complete RPMI 1640. In total, 4 days later, FCM was used toidentify CD41CD251Foxp31 T cells. Data are expressed as the mean7SD of 18 mice from three independent experiments. �po0.05 versus cellstreated with OVA323–339 or medium alone. (C) In vivo injection of peptides into BALB/c mice. One representative experiment of the total data shownin (A). (D) Spleen and LN cells were treated with peptides in vitro. One representative experiment of the total data shown in (B). Double-staining forCD25 and Foxp3 expression of cells gated for CD31 and CD41 cells. Values indicate the percentage of events in the indicated quadrant. Statisticalsignificance was analyzed using an independent Student’s t-test.

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Treg that mediate suppression primarily via direct cellular

contact, antigen-induced CD41CD251 Treg function by

releasing suppressive cytokines, e.g. IL-10 and TGF-b [22, 29].

To identify soluble mediators that might be involved in SJMHE1-

triggered immunoregulation, CD41CD251 and CD41CD25–

T cells from SJMHE1-immunized mice were co-cultured in

separate Transwell chambers. The CD41CD251 T cells were not

immunosuppressive, suggesting that in the absence

of the SJMHE1 peptide and triggered only by anti-CD3,

CD41CD251 T cells harvested from SJMHE1-immunized mice

were not capable of suppressing responder cells via direct cellular

contact (Fig. 3A and B). However, when SJMHE1 was

added or SJMHE1 in vitro-pretreated CD41CD251 T cells were

added, suppression of CD41CD25– T cells was restored

(Fig. 3A and B), indicating that soluble factors mediated

SJMHE1-triggered inhibitory effects. Adding either anti-TGF-bor anti-IL-10 neutralizing Ab only slightly reversed the inhibition.

However, adding both anti-IL-10 and anti-TGF-b blocked

inhibition completely (Fig. 3A and B). These results suggest

that both IL-10 and TGF-b contribute to SJMHE1-mediated

inhibition.

CD41CD251 T cells from SJMHE1-immunized miceinhibited the delayed-type hypersensitivity response

To assess the in vivo regulatory activity of CD41CD251 Treg

generated following SJMHE1 immunization, purified CD41

CD251 or CD41CD25– T cells from SJMHE1- or OVA323–339-

immunized mice were injected i.v. into BALB/c mice. One day

later, BALB/c mice were immunized in the footpad with OVA/

CFA. Delayed-type hypersensitivity (DTH) responses and in vitro

proliferation to OVA were measured on day 14 post-adoptive

transfer. As shown in Fig. 4A, DTH responses were significantly

suppressed in mice that received CD41CD251 T cells from

either SJMHE1- or OVA323–339-immunized mice. In addition,

adoptive transfer of CD41CD251 T cells from SJMHE1-

immunized mice caused a stronger suppression of the DTH

response compared with similarly purified CD41CD251 T cells

from OVA323–339-immunized mice. There were no

significant effects in mice that received CD41CD25– T cells from

SJMHE1- or OVA323–339-immunized mice. As shown in Fig. 4B,

proliferation of pooled spleen and LN cells from mice receiving

adoptively transferred CD41CD251 T cells from SJMHE1-

immunized mice following in vitro OVA-stimulation was also

significantly suppressed. However, the expansion of pooled

spleen and LN cells in response to OVA from mice immunized

with OVA323–339 was suppressed less potently. Alternatively,

proliferation increased in pooled spleen and LN cells from mice

injected with CD41CD25– T cells from either SJMHE1- or

OVA323–339-immunized mice (Fig. 4B). Furthermore, when

CD41CD25– T cells from OVA-immunized mice were used as

responder cells, CD41CD251 T cells purified from SJMHE1-

immunized mice showed stronger inhibitory properties than

CD41CD251 T cells harvested from OVA323–339-immunized mice

(Fig. 4C). These results suggested that the enhanced suppressive

activity of CD41CD251 T cells following SJMHE1 immunization

could be the result of an SJMHE1-induced increase in CD41

CD251 Treg activity in vivo.

Figure 2. SJMHE1 enhanced the inhibitory activity of CD41CD251 Treg.(A) Responder CD41CD25– T cells (1�105/well) from naı̈ve mice werecultured with naı̈ve, irradiated APC (1�105 cells/well) and CD41CD251

T cells (5� 104 cells/well) harvested from either SJMHE1-, OVA323–339- orPBS-treated mice in the presence or absence of SJMHE1 or OVA323–339

(0.1 mg/mL). (B) CD41CD251 T cells were incubated with SJMHE1,OVA323–339 (0.1 mg/mL) or PBS for 30 min at 371C in complete RPMI1640 medium, washed, mixed with CD41CD25– T cells at the celldensities indicated in (A) and irradiated APC and 1mg/mL anti-CD3.Proliferation was determined by measuring 3H-thymidine incorpora-tion for the last 16 h of the experiment. Data are expressed as themean7SD (n 5 6 per group) and are representative of three indepen-dent experiments. Statistical significance was analyzed using anindependent Student’s t-test. Asterisks indicate significant differences(�po0.05).

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BM-derived DC and M/ are involved in the generationof SJMHE1-specific CD41CD251 T cells

To investigate the role of APC in SJMHE1-induced CD41

CD251 Treg, BM-derived DC (BMDC) from BALB/c mice

and the RAW264.7 Mf cell line were pulsed in vitro with

SJMHE1, OVA323–339, LPS or medium, respectively, prior to

incubation with CD41 T cells from naı̈ve mice. FCM analysis

showed that BMDC or Mf pulsed with SJMHE1 (but not with

OVA323–339, LPS or medium) stimulated a marked increase in

CD41CD251Foxp31 T cells (Fig. 5A and B). Parallel to the

increase in CD41CD251Foxp31 T cells, the proliferation of CD41

T cells co-cultured with SJMHE1-pulsed BMDC or Mfproliferated poorly compared with CD41 T cell proliferation

induced by BMDC or Mf co-cultured with either OVA323–339 or

LPS (Fig. 5C and D).

SJMHE1 failed to induce BMDC and M/ maturation

Previous reports indicated that APC could trigger generation of

Treg as a function of their activation state, e.g. immature APC

may induce differentiation of CD41 and CD81 Treg and mediate

Ag-specific peripheral tolerance [30, 31]. Considering that

SJMHE1-treated BMDC/Mf (but not OVA323–339-treated

BMDC/Mf) induced CD41CD251 Treg, we investigated BMDC/

Mf maturation following an SJMHE1 pulse. Figure 6A shows that

untreated BMDC or Mf (BMDCmedium/Mfmedium) retained an

immature morphology (more rounded in shape), possessing only

small microvillous structures on their cell surface. In contrast,

LPS treatment (BMDCLPS/MfLPS) induced BMDC/Mf maturation

evidenced by structural changes represented by the formation of

uropods and ruffles. However, similar to the untreated BMDC/

Mf, SJMHE1-treated BMDCSJMHE1/MfSJMHE1 also exhibited an

immature morphology.

The effects of SJMHE1 on BMDC and Mf (BMDCSJMHE1/

MfSJMHE1) maturation were further determined by analyzing

surface marker expression and cytokine production.

BMDCmedium/Mfmedium and BMDCLPS/MfLPS were used as

negative and positive controls, respectively. As shown in Fig. 6B,

BMDCLPS/MfLPS expressed high levels of co-stimulatory

(e.g. CD40, CD80, and CD86) and MHCII molecules following

LPS stimulation. However, the levels of surface markers

on BMDCSJMHE1/MfSJMHE1 were consistently lower and

showed an immature phenotype similar to BMDCmedium/

Mfmedium, which has been shown in the reports describing

tolerogenic DC/Mf [32, 33]. With LPS stimulation, BMDC or

Mf produced high levels of inflammatory cytokines. In

contrast to BMDCLPS/MfLPS, BMDCSJMHE1/MfSJMHE1 produced

lower amounts of proinflammatory cytokines (TNF-a and IL-12)

but released significant levels of the anti-inflammatory

cytokines IL-10 and TGF-b1 (Fig. 6C). Taken together,

these results indicate that BMDC and Mf cultured in the presence

of SJMHE1 showed a phenotype resembling that of tolerogenic

DC/Mf.

Figure 3. SJMHE1 triggered CD41CD251 Treg-mediated suppression ina cytokine-dependent manner. (A) In a Transwell system, CD41CD25�

T cells (5� 105 cells/well) and irradiated APC (2.5� 105 cells/well) fromnaı̈ve mice were cultured in the bottom chambers. CD41CD251 T cells(2.5�105 cells/well) from SJMHE1-immunized mice plus 2.5� 105 APC/well from naı̈ve mice were cultured in the upper chambers andstimulated with anti-CD3 (1 mg/mL) in the presence or absence ofSJMHE1 (0.1mg/mL). Certain wells contained anti-IL-10 (3 mg/mL), anti-TGF-b (0.5 mg/mL), Ab or isotype control Ab. (B) CD41CD251 T cells(2.5�105 cells/well) from naı̈ve mice were incubated with SJMHE1(0.1 mg/mL) or PBS for 30 min, washed with medium, then mixed with2.5� 105 APC/well from naı̈ve mice in the upper chambers. CD41CD25–

T cells (5� 105 cells/well) and irradiated APC (2.5� 105 cells/well) fromnaı̈ve mice were cultured in the bottom chambers and stimulated withanti-CD3 (1 mg/mL) in Transwells. As indicated above, cells werecultured in the presence of anti-IL-10 (3 mg/mL), anti-TGF-b (0.5 mg/mL), both Ab or isotype control Ab. Data are expressed as themean7SD (n 5 6 per group) and are representative of three indepen-dent experiments. Statistical significance was analyzed using anindependent Student’s t-test. Asterisks indicate significant differences(�po0.05).

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Induction of CD41CD251 Treg by SJMHE1 is abrogatedin TLR2�/� mice

The above results showed that exposure to SJMHE1 elevated the

number and efficacy of CD41CD251 T cells in WT BALB/c mice

both in vivo and in vitro. Recent studies have shown that TLR2

and TLR4 signaling promoted CD41CD251 Treg proliferation

[34, 35]. To further investigate the mechanism through which

SJMHE1 triggered CD41CD251 Treg induction, TLR2�/�,

TLR4�/�, and WT control mice were used. As shown in Fig. 7A

and B, pooled spleen and LN cells harvested from immunized

TLR4�/� and WT control mice or SJMHE1 in vitro pretreated

pooled spleen and LN cells from naı̈ve TLR4�/� and WT control

mice showed an increase in CD41CD251Foxp31 T-cell popula-

tions. However, an increase in CD41CD251Foxp31 T cells was

not observed in TLR2�/� mice treated in a similar fashion, while

WT controls still showed an increase. Furthermore, CD41CD251

T cells from SJMHE1-immunized TLR4�/� and WT control mice

were highly effective at suppressing CD41CD25– T-cell prolifera-

tion (Fig. 7C). Adding SJMHE1 to co-cultures enhanced

suppression further, consistent with the results shown in Fig. 2.

In addition, in SJMHE1-pretreated TLR4�/� and WT control

mice, CD41CD251 T cells had also enhanced suppressive activity

(Fig. 7D). In contrast, addition of SJMHE1 to spleen and LN cells

harvested from TLR2�/� mice not only failed to enhance

suppression but also slightly increased the proliferation of

CD41CD25– responder T cells for unknown reasons at this time.

Taken together, these results indicate that TLR2 but not TLR4

plays an important role in specifically promoting the develop-

ment of CD41CD251 Treg induced by SJMHE1.

Induction of CD41CD251 T cells by SJMHE1-pulsedBMDC and BMM/ was abrogated in TLR2�/� mice

The above results demonstrated that SJMHE1-pulsed BMDC and

Mf induced CD41CD251 Treg in WT BALB/c mice and that TLR2

was involved in CD41CD251 T-cell induction. To further

investigate whether SJMHE1 acted as an inducer of CD41

CD251 Treg via TLR2 on APC, BMDC, and BMMf from

TLR2�/�, TLR4�/�, and WT control mice primed in vivo or

pulsed in vitro with SJMHE1 were used to induce CD41CD251

Treg. TLR4�/� and WT control BMDC (prepared from either

SJMHE1-immunized mice or from an in vitro SJMHE1 pulse)

increased the number of CD41CD251Foxp31 T cells, whereas

BMMf from TLR4�/� mice did not significantly increase the

Figure 4. DTH and cell proliferation were suppressed by CD41CD251 Tcells from SJMHE1-immunized mice. (A) CD41CD251 and CD41CD25– Tcells from SJMHE1- or OVA323–339-treated mice were purified andimmediately injected i.v. into BALB/c mice (1�106 cells/mouse). Micewere then sensitized and challenged with OVA and ear thickness wasmeasured as described in Materials and Methods. Change in earthickness (Dear thickness) 5 (thickness of left ear)–(thickness of rightear). The data are expressed as the mean7SD of six mice/group fromtwo independent experiments. (B) Pooled spleen and LN cells fromBALB/c mice receiving CD41CD25– T cells from SJMHE1-, OVA323–339-immunized or medium only-treated mice or CD41CD251

T cells from SJMHE1- or OVA323–339-immunized mice were prepared1 day after OVA challenge. The cells were then cultured at 5� 105 cells/well in complete RPMI 1640 with OVA for 3 days and proliferation wasmeasured. Data are expressed as the mean values of two experimentswith three mice per group. (C) BALB/c mice were sensitized andchallenged with OVA and CD41CD25– T cells (1� 105/well) were sortedand co-cultured with purified CD41CD251 T cells (1� 105/well) fromSJMHE1- or OVA323–339-immunized mice in the presence of 100 mg/mLOVA. Data are expressed as the mean7SD of two experiments with sixmice per group. Statistical significance was analyzed using anindependent Student’s t-test. Asterisks indicate significant differences(�po0.05; ��po0.01).

b

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number of CD41CD251Foxp31 T cells (Fig. 8). In contrast,

neither BMDC nor BMMf from TLR2�/� mice increased the

CD41CD251 T-cell counts regardless of whether they were

immunized in vivo or pulsed in vitro with SJMHE1 (Fig. 8).

These data further confirmed that the induction of CD41CD251

Treg by SJMHE1 was dependent on TLR2.

Discussion

Reports have suggested that Treg can generate conditions that may

be favorable for the persistence of pathogens. Therefore, manip-

ulation of immune-regulatory pathways by pathogens facilitating

the development of Treg is not surprising. This process may be

expedited by antigens that elicit Treg differentiation and activa-

tion, and thus enabling pathogen survival [1, 2]. Human

schistosomes can survive for decades in immunocompetent hosts

with long-term parasite survival linked to suppression of host

protective immune responses. Evidence has suggested that Treg

are generated during schistosomal infections (and other helminth

infections) that down-regulate immune responsiveness and thus

promoting parasite survival [20, 36]. However, the mechanism(s)

resulting in Treg development are at this time undefined.

In this study, we demonstrated that the SjHSP60 and more

importantly, the SjHSP60-derived peptide SJMHE1, which is

highly identical to murine and human HSP60 and composed of

overlapping T-cell epitopes in mice and humans, induced CD41

CD251Foxp31 Treg both in vivo and in vitro. It remains unclear

precisely how CD41CD251Foxp31 Treg can be expanded in an

antigen-specific manner in vivo and in vitro and what conditions

are required to establish the proliferation of Treg. Recent studies

have shown that CD41CD251 T cells can be expanded in vivo and

in vitro upon antigen stimulation [37, 38]. Moreover, CD41

CD251 Treg can be induced from CD41CD25– T cells [26, 27]. To

further test whether CD41CD25– T cells can be differentiated into

CD41CD251Foxp31 Treg by this peptide, we purified CD41CD25–

T cells from mice and treated with SJMHE1 or OVA323–339 in vitro.

The results suggested that CD41CD25– T cells can be induced into

CD41CD251Foxp31 Treg by SJMHE1 in an APC-dependent

manner. However, either in the presence or in the absence of APC,

SJMHE1 failed to induce a statistically significant expansion of

purified CD41CD251 T-cell populations. In contrast to Tr1/Tr2

Figure 5. SJMHE1-pulsed BMDC and Mf (RAW264.7 cells) induced CD41CD251 Treg. BALB/c mouse BMDC were generated as described in Materialsand Methods. BMDC or Mf (5�104 cells/well) were pulsed with either SJMHE1 (DCSJMHE1, MfSJMHE1), OVA323–339 (DCOVA323-339, MfOVA323-339), LPS(DCLPS, MfLPS) or medium (DCmedium, Mfmedium) as described. CD41 T cells (2�105/well) were purified from naı̈ve mice and cultured for 3 days withirradiated BMDC (A) or Mf (B). CD41CD251Foxp31 T cells were sorted using FCM following double staining for CD25 and Foxp3 expression of cellsgated for CD31 and CD41 expression. The data are representative of three similar experiments. After 3 days of co-culture with irradiated DC (C) orMf (D), CD41 T-cell proliferation was determined by measuring 3H-thymidine incorporation. The data are expressed as the mean7SD of threeexperiments performed in triplicate. Statistical significance was analyzed using an independent Student’s t-test. Asterisks indicate significantdifferences (�po0.05; ��po0.01; ���po0.01).

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(Th3) that produce immunosuppressive cytokines, e.g. IL-10 or

TGF-b, or naturally occurring CD41CD251 Treg, which induce

suppression primarily via direct cellular contact [22, 29], our study

suggested that secreting inhibitory cytokines might contribute to

SJMHE1-induced CD41CD251 Treg-mediated suppression. These

data were consistent with the finding that SJMHE1 administration

markedly augmented IL-10 and TGF-b production by CD41CD251

T cells both in vivo and in vitro (data not shown). IL-10 and TGF-bare speculated to be generated by immune cells in response to egg-

and worm-derived antigens, and dampen the host immune

response during chronic schistosome infection [16]. Production of

IL-10 and TGF-b might also result in the development of Treg [16].

Our studies support the view that CD41CD251 Treg constitute

heterogeneous populations. Natural CD41CD251 Treg have a

cytokine-independent mechanism of action, but CD41CD251 Treg

induced in the periphery may have either cytokine-dependent

effects or cytokine-independent effects [39]. Furthermore,

SJMHE1-induced CD41CD251 Treg had regulatory properties in

vivo, i.e. inhibition of DTH in a murine model.

Few immunosuppressive peptides have been characterized

that elicit Treg development in the periphery and were also

shown to be protective against autoimmune diseases such as

collagen-induced arthritis, inflammatory bowel disease [40–42],

or experimental autoimmune encephalomyelitis [43]. Therefore,

SJMHE1’s homology to other Treg-inducing-peptides was

analyzed. SJMHE1 is 66% identical to P277, a fragment of the

human HSP60, which can arrest the spontaneous diabetogenic

process in NOD mice [28]. Additionally, a sequence in SJMHE1

contained a random polymer of four amino acids (GLAT) also

present in myelin basic protein that has been proven to be an

effective treatment for MS [38, 44]. Whether SJMHE1 can be

used as a peptide-based therapy for allergic and autoimmune

disease treatment requires further analysis.

The mechanisms involved in the generation and activation of

Treg by SJMHE1 are not fully understood. Naturally occurring

CD41CD251 Treg are generated mainly in the thymus due to

high-affinity TCR interactions with self-ligands [45, 46].

However, Treg can also be generated in the periphery. For

example, transgenic expression of an agonist peptide by non-

activated hematopoietic cells elicited CD41CD251 Treg induction

in the periphery even in the absence of a functioning thymus

[47]. One of the mechanisms of Treg generation is antigen

stimulation by a tolerizing APC [30]. Consistent with our find-

ings, the role of APC in the induction of peripheral tolerance and

of Treg is supported by multiple studies [48, 49]. In our inves-

tigation, Treg were also induced in vitro by SJMHE1-pulsed

mouse BMDC and Mf. These cells displayed an immature or non-

activated phenotype with down-regulated MHCII and co-stimu-

latory molecule expression (i.e. CD40, CD80, and CD86) on their

surface, as well as increased production of suppressive cytokines

such as IL-10 and TGF-b and decreasing production of inflam-

matory cytokines such as TNF-a and IL-12. When cultured with

allogeneic CD41 T cells harvested from naı̈ve mice, SJMHE1-

pulsed DC/Mf were less effective in stimulating T-cell prolif-

eration and inducing CD41CD251 Treg. These data strongly

Figure 6. SJMHE1 failed to induce BMDC and Mf maturation. MouseBMDC were generated as described in Materials and Methods. BMDC and Mf(RAW264.7 cells) were separately pulsed with either SJMHE1 (BMDCSJMHE1,MfSJMHE1), LPS (BMDCLPS, MfLPS) or medium (BMDCmedium, Mfmedium).(A) Morphological analysis of BMDC or Mf was carried out microscopically.(B) Cell surface and co-stimulatory BMDC or Mf markers were analyzed byFCM. Numbers represent the MFI and the percent expression of eachmarker. Histograms are representative of three independent experiments.(C) The cytokines in culture supernatants were analyzed by ELISA. Barsshow the mean7SD from two experiments performed in triplicate.Statistical significance was analyzed using an independent Student’st-test. Asterisks indicate significant differences (��po0.01; ���po0.001).

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suggest that APC are often targeted by schistosomes during

chronic infections and may play a role in inducing Treg [1]. The

induction of Treg by these mechanisms might be an important

way of controlling potent immune responses during the course of

a chronic infection. This enables the establishment of persistent

infections, a situation beneficial to both host and parasite [16].

Additionally, during infection, pathogens produce a range of

conserved molecules that interact with PRR such as TLR that may

drive the differentiation of naı̈ve T cells into distinct CD41 T-cell

subtypes, including T-regulatory phenotypes [1]. For example,

bacterial LPS acts through TLR4 and has been shown to enhance

murine CD41CD251 Treg proliferation and suppression [35].

The S. mansoni-specific phosphatidylserine is recognized by TLR2

on the surface of DC and may promote Treg induction [50]. In

this study, SJMHE1-immunization induced increases in the

number and function of CD41CD251 T cells. This increase was

blocked when the experiments were carried out in TLR2�/�mice.

Additionally, in vitro induction of CD41CD251 T cells was also

eliminated when using SJMHE1-pulsed DC and Mf from

TLR2�/� mice. These observations suggest that induction of

CD41CD251 Treg by SJMHE1 occurs through TLR2-mediated

signals. However, our present data are not sufficient to confirm

whether the induction of Treg by SJMHE1 is due to stimulation of

TLR2 on APC or direct stimulation of TLR2-expressing Treg,

which will require further analysis.

Our data support the views that HSP might be potential

candidates for molecular mimicry and that the induction of self-

HSP-reactive Treg is essential for an organism to prevent exces-

sive inflammation and subsequent organ damage [23]. Our

findings that parasite HSP60-derived peptide induced CD41

CD251 Treg through TLR signals point to the existence of a

feedback-inhibition mechanism regulating TLR-dependent

inflammatory processes during infection. This suggests that in the

early stages of inflammatory responses following infections with

certain pathogens, APC are activated via TLR and deliver a

stimulatory signal to CD41 T cells, initiating adaptive immune

responses. Simultaneously, CD41CD251 Treg are amplified and

their suppressive potential could be enhanced following TLR

Figure 7. Induction of CD41CD251 Treg by SJMHE1 is abrogated in TLR2�/� mice. (A) TLR2�/� and TLR4�/� mice were immunized s.c. with 10mg ofSJMHE1 or PBS. Seven days after the last immunization, pooled spleen, and LN cells from each mouse were isolated and analyzed for CD3, CD4,CD25, and Foxp3 expression. Data are expressed as the mean7SD of 12 mice from two independent experiments. �po0.05 versus mice treated withPBS. (B) Pooled spleen and LN cells from individual naı̈ve TLR2�/� and TLR4�/� mice were incubated with SJMHE1 (1 mg/mL) for 4 days and FCM wasused to identify CD41CD251Foxp31 T cells. Double staining for CD25 and Foxp3 expression was determined on CD31 and CD41 gated cells. Dataare expressed as the mean7SD of 12 mice from two independent experiments. �po0.05 versus mice treated with medium alone. (C) Purified CD41

CD251 (5� 104/well) and CD41CD25– T cells (1� 105/well) from SJMHE1-immunized TLR2�/� or TLR4�/� mice were co-cultured for 3 days with APC(1� 105/well), SJMHE1 (0.1 mg/mL), and anti-CD3 (1 mg/mL). Proliferation was determined by measuring 3H-thymidine incorporation. (D) CD41CD251

T cells (5� 104/well) from naı̈ve TLR2�/� or TLR4�/�mice were incubated with SJMHE1 (0.1mg/mL) for 30 min, washed and mixed with CD41CD25� Tcells (1� 105/well), APC (1� 105/well) and anti-CD3 (1 mg/mL). Proliferation was determined using 3H-thymidine incorporation. The data areexpressed as the mean7SD of 12 mice from two independent experiments. Statistical significance was analyzed using an independent Student’s t-test. Asterisks indicate significant differences (�po0.05).

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& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

activation [51]. The increase in the numbers and function of

CD41CD251 Treg induced via TLR-mediated processes indicates

a mechanism that could reduce potentially harmful side effects to

the host. These side effects are a result of uncontrolled inflam-

mation, although this type of response may also favor the

establishment of chronic infections. We hypothesize that

the development of Treg populations (which are induced by the

interaction of TLR ligands and antigens from pathogens such as

S. japonicum) is an evolutionary phenomenon that provides

protection for the host against potentially damaging anti-parasite

immune responses. It also concomitantly provides a favorable

environment to the pathogen. Targeting TLR-signaling processes

represents a novel approach toward designing anti-parasite

therapies associated with boosting host immunity (either natu-

rally or in the context of vaccination) and blocking or deleting the

activity of immunosuppressive-inducing molecules, e.g. SJMHE1

(which would also facilitate the development of an effector

response instead of suppression). Furthermore, identification of

the immunosuppressive antigenic epitopes, e.g. SEA of S. japo-

nicum, may have potential therapeutic applications for allergic

and autoimmune diseases.

Materials and methods

Mice and cell line

Eight-week-old female BALB/c mice were purchased from the

SLAC Laboratory (Shanghai, China). Eight-week-old female WT

C57BL/6, C57BL/10, TLR2�/� mice (B6.129-Tlr2tm1Kir/J) and

TLR4�/� mice (C57BL/10ScNJ) were provided by the Center for

Experimental Animals (Nanjing University, Nanjing, China). All

animal experiments were performed in accordance with Chinese

animal protection laws and with permission from the Institu-

tional Review Board.

The mouse Mf cell line RAW264.7 was purchased from the

American Type Culture Collection (Manassas, VA, USA).

Preparation of recombinant SjHSP60

SjHSP60 was expressed as a GST fusion protein in the Escherichia

coli strain DH5a and purified by glutathione-Sepharose 4B

(Amersham Biosciences, Piscataway, NJ, USA) affinity chromato-

graphy. The GST moiety was removed with PreScission Protease

(Amersham Bioscience) according to the manufacturer’s instruc-

tions. The purity of the prepared SjHSP60 was 495% as

determined by SDS-PAGE followed by Coomassie Blue staining

(SDS-PAGE/densitometry) and N-terminal protein sequencing.

Furthermore, the recombinant SjHSP60 was treated with poly-

myxin B-agarose (Sigma, St. Louis, MO, USA) to reduce residual

endotoxin. The purified protein contained o0.001 FU/mL

(0.1 pg/mL) of bacterial endotoxin, as determined by a Limulus

amebocyte lysate assay (Associates of Cape Cod, Woods Hole,

Ma, USA) used according to the manufacturer’s instructions. Full

information on the GST construct used can be found in the

Supporting Information.

Prediction of epitopes and synthesis of peptides

To predict and further identify the possible regions within

SjHSP60 that might have the ability to induce Treg, the software

Figure 8. Induction of CD41CD251 T cells by SJMHE1-pulsed BMDC andBMMf was abrogated in TLR2�/� mice. (A) TLR2�/� and TLR4�/� micewere immunized s.c. with 10 mg of SJMHE1. Seven days after the lastimmunization, BMDC/BMMf (5� 104 cells/well) were harvested, irra-diated and co-cultured with naı̈ve mice CD41 T cells (2� 105/well) for 3days. FCM was used to identify CD41CD251Foxp31 T cells. Data areexpressed as the mean7SD of 12 mice from two independentexperiments. (B) BMDC/BMMf from naı̈ve TLR2�/� and TLR4�/� mice(5� 104 cells/well) were pulsed with (BMDCSJMHE1, BMMfSJMHE1) orwithout (BMDCmedium, BMMfmedium) SJMHE1 and then irradiated andco-cultured for 3 days with CD41 T cells (2�105/well). FCM was used toidentify CD41CD251Foxp31 T cells, gating for CD31 and CD41 cells.Data are expressed as the mean7SD of 12 mice from two independentexperiments. Statistical significance was analyzed using an indepen-dent Student’s t-test. Asterisks indicate significant differences(�po0.05).

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GUATIF, TEPITOPE, and ANTHIWHIN were used. Epitope

prediction was carried out as described previously [52–54].

Briefly, the amino acid sequences of SjHSP60 (GenBank accession

no. AAW24883) were entered into the software to predict T-cell

epitopes. Candidate peptides were chosen according to prediction

scores and identity to murine and human HSP60. SjHSP60 437-

460 (SJMHE1) and the control OVA peptide 323–339

(OVA323–339) (ISQAVHAAHAEINEAGR), which has not been

shown to induce CD41CD251 cells in BALB/c mice [55, 56],

were synthesized and purified by Songong (Shanghai, China).

The purity of the peptides was determined by mass spectrometry

and was greater than 99%. LPS has been reported to activate

various types of lymphocytes via TLR2 signaling [57]. To exclude

possible LPS contamination, we pre-treated the SjHSP60,

SJMHE1, and OVA323–339 with polymyxin B-agarose as described

previously [58].

Vaccinations

BALB/c, C57BL/6, C57BL/10, TLR2�/�, and TLR4�/� mice in

each of four experimental groups (six mice/group) were

immunized with 20 mg SjHSP60, 10 mg of either SJMHE1,

OVA323–339 or PBS emulsified in 100 mL of incomplete Freund’s

adjuvant (Sigma) respectively and boosted 2 wk later, with the

described above antigens.

Generation of BMDC and BMM/

BM cells were isolated by flushing the marrow cavities of mouse

femurs with pre-cooled RPMI 1640 medium and gently refluxing

the expelled cell plug through a 25-gauge needle to form a single-

cell suspension. The BMMf were derived by culturing BM cells in

complete DMEM containing 2 mM glutamine, 50 U/mL penicillin,

50mg/mL streptomycin, 10% FCS (Gibco, Gaithersburg, MD, USA)

and 1 ng/mL recombinant macrophage colony stimulating factor

(Peprotech, Rocky Hill, NJ, USA). BMDC were derived by culturing

in complete RPMI 1640 supplemented with 1 ng/mL recombinant

granulocyte/monocyte-colony-stimulating factor and 1 ng/mL rIL-

4 (Peprotech), as described previously [59, 60]. Differentiation to

DC and Mf was assessed by morphologic observation and

detection of specific surface markers by FCM.

Cell isolation

Single cell suspensions were prepared by teasing apart spleens

and inguinal and mesenteric LN from six mice/group in PBS

containing 1% FCS and 1% EDTA followed by RBC lysis with Tris

ammonium chloride buffer. CD41 T cells were purified from

single cell suspensions with a CD41 T-cell negative-isolation kit

(Miltenyi Biotec, Auburn, CA, USA) and a MACS according to the

manufacturer’s recommendations (497% CD41 T cells by FCM

analysis).

CD41CD251 and CD41CD25– cell populations were separated

from purified CD41 T cells using a mouse Treg isolation kit

(Miltenyi Biotec) following the manufacturer’s protocol. The

CD251 populations were 495% CD41CD251, and the CD41

CD25– populations were 98% pure, as determined by FCM.

APC were obtained from single-cell suspensions by depleting

T cells using a mixture of magnetic beads conjugated with either

anti-CD8 or anti-CD4 mAb (Miltenyi Biotec) and then irradiated

(30Gy).

Cell culture

For suppression assays, 1�105 CD41CD25� T cells/well, 5�104

CD41CD251 T cells/well or both populations were cultured in

96-well U-bottom plates with 1�105 APC/well in triplicate for

72 h at 371C in complete RPMI 1640 medium (0.2 mL/well).

Cultures were stimulated with 1mg/mL soluble anti-CD3 (BD

PharMingen, San Diego, CA) in the presence or absence of

0.1 mg/mL SJMHE1 or 0.1 mg/mL OVA323–339. In certain experi-

ments, CD41CD251 T cells were pre-incubated with 0.1 mg/mL

SJMHE1 or OVA323–339 for 30 min at 371C in complete RPMI

1640 medium, washed, and co-cultured with CD41CD25– T cells

as per the above cell proportions plus 1mg/mL anti-CD3.

Proliferation was measured by incubating with 0.5mCi/well 3H-

thymidine and measuring incorporation during the final 16 h of a

3-day culturing period.

Co-cultures in Transwells were performed in 24-well plates

(Millipore, Bedford, MA, USA) in the presence of cytokine

blocking Ab (or isotype controls) in triplicate: 3mg/mL rat IgG1

anti-mouse IL-10 (Biolegend, San Diego, CA, USA), 0.5mg/mL rat

IgG1 anti-mouse TGF-b1 (US Biological, Swampscott, MA, USA)

or 3mg/mL rat IgG1 (Biolegend). In total, 5� 105 murine CD41

CD25� T cells and 2.5� 105 APC/well were cultured in the

bottom chamber and 2.5� 105 CD41CD251 T cells plus 2.5�105

APC/well were cultured in the upper chamber and stimulated

with 1mg/mL anti-CD3 in the presence or absence of 0.1 mg/mL

SJMHE1 for 3 days. In total, 16 h before cell harvesting, 0.5 mCi/

well of 3H-thymidine was added. The upper chamber was

removed and proliferation was measured. In some experiments,

CD41CD251 T cells in the upper chamber were pre-incubated

with 0.1 mg/mL SJMHE1 for 30 min as above and stimulated with

only 1mg/mL anti-CD3.

For in vitro antigen stimulation assays, 2� 105 BMMf or

BMDC/well were cultured in 24-well plates in triplicate and

pulsed with 0.1 mg/mL SJMHE1 (DCSJMHE1, MfSJMHE1), 0.1 mg/

mL OVA323–339 (DCOVA323–339, MfOVA323–339) or medium alone

(DCmedium, Mfmedium) for 8 days. Some samples were also

cultured in the presence of 1mg/mL LPS from E. coli 055:B5

(Sigma) (DCLPS, MfLPS) for the last 48 h of an 8-day culture.

Additionally, 2� 105 RAW264.7 cells/well were pulsed with

either 0.1 mg/mL SJMHE1, 0.1 mg/mL OVA323–339, or with 1mg/

mL E. coli LPS or medium alone for 24 h.

For induction of CD41CD251 T cells in vitro, 2�105 allo-

geneic CD41 T cells/well were purified from naı̈ve mice and

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& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

cultured with or without 5�104 DCmedium/Mfmedium cells,

DCSJMHE1/MfSJMHE1 cells, DCOVA323–339/MfOVA323-339 cells, or

DCLPS/MfLPS cells/well, respectively. These experiments were

performed in 96-well U-bottom plates for 72 h at 371C in

complete RPMI 1640 medium (0.2 mL/well). The CD41CD251

population was assessed by FCM or cell proliferation was eval-

uated by measuring 3H-thymidine incorporation.

Cytokine quantitation

TNF-a, IL-12, IL-10, and TGF-b1 in the supernatants of BMDC or

Mf stimulated by antigens were quantified using an ELISA kit

(Bender Med Systems, Vienna, Austria), according to the

manufacturer’s protocol.

FCM

For the analysis of CD41CD251Foxp31 T-cell induction, the

Mouse Regulatory T Cell Staining Kit (eBioscience, San Diego, CA,

USA) was used. Pooled spleen and LN cells from immunized

mice or from co-cultures were surface-stained with PerCP

anti-CD3 mAb (eBioscience), FITC anti-CD4 mAb and APC

anti-CD25 mAb followed by fixation and permeabilization

with Cytofix/Cytoperm and then stained intracellularly

with PE mouse anti-Foxp3 or PE IgG2a rat Ig control Ab, according

to the manufacturer’s protocol. Surface markers expressed by Mfand DC were determined by FCM using the following mAb: FITC-

conjugated anti-CD80, PE-conjugated anti-CD86, PE-conjugated

anti-CD40, and FITC-conjugated anti-MHC II (eBioscience).

Staining was done according to the manufacturer’s protocol.

Adoptive transfer of CD41CD251 or CD41CD25� T cellsand induction of the DTH response

CD41CD251 or CD41CD25– T cells from SJMHE1 or OVA323–339-

immunized mice were purified by MACS and immediately

injected into the caudal vein of 8-wk-old BALB/c mice at a dose

of 1� 106 cells/mouse. One day after transfer, mice were

sensitized in the footpad with 100 mg of OVA (fraction V; Sigma,

Poole, UK) in 100 mL of CFA (Sigma). Thirteen days after

sensitization, mice were s.c. challenged with 20 mL of OVA

(1 mg/mL in PBS) in the left ear and 20 mL of PBS in the right ear.

Ear thickness was measured at 0 and 24 h in a blind fashion using

a micrometer (Mitutoyo, Osaka, Japan). Results are reported as

mean7SD difference between the left and right ear thickness (six

mice per group) [55].

Statistical analysis

The statistical analysis was performed using SPSS version 10.1

(Statistical Package for Social Sciences, Chicago, IL statistical

software). Statistical significance was determined by Student’s t-

test with po0.05 considered statistically significant.

Acknowledgements: The authors gratefully acknowledge

assistance from Raychel Chambers (Department of Microbiology

and Immunology, University of Rochester School of Medicine and

Dentistry) for review of the manuscript. This work was supported

by grants from the National Basic Research Program of P. R.

China (973 Program) (No.2007CB513106), the National Natural

Science Foundation of P. R. China (No. 30571629 and No.

30872206), and the grants BK2007533 and 07KJA31023 from

Jiangsu Province to Chuan Su.

Conflict of interest: The authors declare no financial or

commercial conflict of interest.

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Abbreviations: BMDC: BM-derived DC � BMMf: BM-derived

macrophages � DTH: delayed-type hypersensitivity � FCM: flow

cytometry

Full correspondence: Professor Chuan Su, Department of Pathogen

Biology & Immunology, Department of Pharmacology, Jiangsu Key

Laboratory of Pathogen Biology, Nanjing Medical University, 140

Hanzhong Road, Nanjing, Jiangsu 210029, P. R. China

Fax: 186-25-86862774

e-mail: chuansu@njmu.edu.cn

Supporting Information for this article is available at

www.wiley-vch.de/contents/jc-2040/2009/39335_s.pdf

Received: 14/2/2009

Revised: 5/8/2009

Accepted: 24/8/2009

Eur. J. Immunol. 2009. 39: 3052–3065 Immunity to infection 3065

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu