Post on 19-Jan-2023
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
Eur. J. Immunol. 2009. 39: 3052–3065 Immunity to infection 3053
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
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.
Eur. J. Immunol. 2009. 39: 3052–3065Xuefeng Wang et al.3054
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
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).
Eur. J. Immunol. 2009. 39: 3052–3065 Immunity to infection 3055
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
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).
Eur. J. Immunol. 2009. 39: 3052–3065Xuefeng Wang et al.3056
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
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
Eur. J. Immunol. 2009. 39: 3052–3065 Immunity to infection 3057
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
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).
Eur. J. Immunol. 2009. 39: 3052–3065Xuefeng Wang et al.3058
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
(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).
Eur. J. Immunol. 2009. 39: 3052–3065 Immunity to infection 3059
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
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).
Eur. J. Immunol. 2009. 39: 3052–3065Xuefeng Wang et al.3060
& 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).
Eur. J. Immunol. 2009. 39: 3052–3065 Immunity to infection 3061
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
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
Eur. J. Immunol. 2009. 39: 3052–3065Xuefeng Wang et al.3062
& 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.
References
1 Belkaid, Y., Regulatory T cells and infection: a dangerous necessity. Nat.
Rev. Immunol. 2007. 7: 875–888.
2 Belkaid, Y., Role of Foxp3-positive regulatory T cells during infection. Eur.
J. Immunol. 2008. 38: 918–921.
3 McGuirk, P., McCann, C. and Mills, K. H., Pathogen-specific T regulatory 1
cells induced in the respiratory tract by a bacterial molecule that
stimulates interleukin 10 production by dendritic cells: a novel strategy
for evasion of protective T helper type 1 responses by Bordetella pertussis.
J. Exp. Med. 2002. 195: 221–231.
4 MacDonald, A. J., Duffy, M., Brady, M. T., McKiernan, S., Hall, W.,
Hegarty, J., Curry, M. and Mills, K. H., CD4 T helper type 1 and regulatory
T cells induced against the same epitopes on the core protein in hepatitis
C virus-infected persons. J. Infect. Dis. 2002. 185: 720–727.
5 Satoguina, J., Mempel, M., Larbi, J., Badusche, M., Loliger, C., Adjei, O.,
Gachelin, G. et al., Antigen-specific T regulatory-1 cells are associated
with immunosuppression in a chronic helminth infection (onchocercia-
sis). Microbes Infect. 2002. 4: 1291–1300.
6 Belkaid, Y. and Rouse, B. T., Natural regulatory T cells in infectious
disease. Nat. Immunol. 2005. 6: 353–360.
7 Chen, X., Zhou, B., Li, M., Deng, Q., Wu, X., Le, X., Wu, C. et al.,
CD4(1)CD25(1)FoxP3(1) regulatory T cells suppress Mycobacterium tuber-
culosis immunity in patients with active disease. Clin. Immunol. 2007. 123:
50–59.
8 Nilsson, J., Boasso, A., Velilla, P. A., Zhang, R., Vaccari, M., Franchini, G.,
Shearer, G. M. et al., HIV-1-driven regulatory T-cell accumulation in
lymphoid tissues is associated with disease progression in HIV/AIDS.
Blood 2006. 108: 3808–3817.
9 Yurchenko, E., Tritt, M., Hay, V., Shevach, E. M., Belkaid, Y. and Piccirillo,
C. A., CCR5-dependent homing of naturally occurring CD41 regulatory
T cells to sites of Leishmania major infection favors pathogen persistence.
J. Exp. Med. 2006. 203: 2451–2460.
10 Taylor, M. D., van der Werf, N., Harris, A., Graham, A. L., Bain, O., Allen,
J. E. and Maizels, R. M., Early recruitment of natural CD41Foxp31 Treg
cells by infective larvae determines the outcome of filarial infection. Eur.
J. Immunol. 2009. 39: 192–206.
Eur. J. Immunol. 2009. 39: 3052–3065 Immunity to infection 3063
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
11 Kaparakis, M., Laurie, K. L., Wijburg, O., Pedersen, J., Pearse, M., van
Driel, I. R., Gleeson, P. A. and Strugnell, R. A., CD41CD251 regulatory
T cells modulate the T-cell and antibody responses in helicobacter-
infected BALB/c mice. Infect. Immun. 2006. 74: 3519–3529.
12 Mitchell, K. M., Mutapi, F. and Woolhouse, M. E., The predicted impact of
immunosuppression upon population age-intensity profiles for schisto-
somiasis. Parasite Immunol. 2008. 30: 462–470.
13 Sajid, M. S., Iqbal, Z., Muhammad, G. and Iqbal, M. U., Immunomodu-
latory effect of various anti-parasitics: a review. Parasitology 2006. 132:
301–313.
14 Shen, L., Zhang, Z. S., Wu, H. W., Weir, R. E., Xie, Z. W., Hu, L. S., Chen, S.
Z. et al., Down-regulation of specific antigen-driven cytokine production
in a population with endemic Schistosoma japonicum infection. Clin. Exp.
Immunol. 2002. 129: 339–345.
15 Araujo, M. I., Hoppe, B. S., Medeiros, M, Jr. and Carvalho, E. M.,
Schistosoma mansoni infection modulates the immune response against
allergic and auto-immune diseases. Mem. Inst. Oswaldo Cruz 2004. 99:
27–32.
16 Dunne, D. W. and Cooke, A., A worm’s eye view of the immune system:
consequences for evolution of human autoimmune disease. Nat. Rev.
Immunol. 2005. 5: 420–426.
17 Layland, L. E., Rad, R., Wagner, H. and da Costa, C. U., Immunopathology
in schistosomiasis is controlled by antigen-specific regulatory T cells
primed in the presence of TLR2. Eur. J. Immunol. 2007. 37: 2174–2184.
18 Baumgart, M., Tompkins, F., Leng, J. and Hesse, M., Naturally occurring
CD41Foxp31 regulatory T cells are an essential, IL-10-independent part of
the immunoregulatory network in Schistosoma mansoni egg-induced
inflammation. J. Immunol. 2006. 176: 5374–5387.
19 Taylor, J. J., Mohrs, M. and Pearce, E. J., Regulatory T-cell responses
develop in parallel to Th responses and control the magnitude and
phenotype of the Th effector population. J. Immunol. 2006. 176: 5839–5847.
20 Wilson, M. S., Mentink-Kane, M. M., Pesce, J. T., Ramalingam, T. R.,
Thompson, R. and Wynn, T. A., Immunopathology of schistosomiasis.
Immunol. Cell Biol. 2007. 85: 148–154.
21 Yang, J., Zhao, J., Yang, Y., Zhang, L., Yang, X., Zhu, X., Ji, M. et al.,
Schistosoma japonicum egg antigens stimulate CD4 CD25 T cells and
modulate airway inflammation in a murine model of asthma. Immunology
2007. 120: 8–18.
22 Mills, K. H., Regulatory T cells: friend or foe in immunity to infection? Nat.
Rev. Immunol. 2004. 4: 841–855.
23 Hauet-Broere, F., Wieten, L., Guichelaar, T., Berlo, S., van der Zee, R. and
Van Eden, W., Heat shock proteins induce T-cell regulation of chronic
inflammation. Ann. Rheum. Dis. 2006. 65: iii65–iii68.
24 Anderton, S. M., van der Zee, R., Prakken, B., Noordzij, A. and van Eden,
W., Activation of T cells recognizing self 60-kD heat shock protein can
protect against experimental arthritis. J. Exp. Med. 1995. 181: 943–952.
25 Fontenot, J. D., Gavin, M. A. and Rudensky, A. Y., Foxp3 programs the
development and function of CD41CD251 regulatory T cells. Nat.
Immunol. 2003. 4: 330–336.
26 Walker, M. R., Carson, B. D., Nepom, G. T., Ziegler, S. F. and Buckner, J. H.,
De novo generation of antigen-specific CD41CD251 regulatory T cells from
human CD41CD25� cells. Proc. Natl. Acad. Sci. USA 2005. 102: 4103–4108.
27 Curotto de Lafaille, M. A., Lino, A. C., Kutchukhidze, N. and Lafaille, J. J.,
CD25 – T cells generate CD251Foxp31 regulatory T cells by peripheral
expansion. J. Immunol. 2004. 173: 7259–7268.
28 Zanin-Zhorov, A., Cahalon, L., Tal, G., Margalit, R., Lider, O. and Cohen,
I. R., Heat shock protein 60 enhances CD41CD251 regulatory T-cell
function via innate TLR2 signaling. J. Clin. Invest. 2006. 116: 2022–2032.
29 Vignali, D. A., Collison, L. W. and Workman, C. J., How regulatory T cells
work. Nat. Rev. Immunol. 2008. 8: 523–532.
30 Vlad, G., Cortesini, R. and Suciu-Foca, N., License to heal: bidirectional
interaction of antigen-specific regulatory T cells and tolerogenic APC.
J. Immunol. 2005. 174: 5907–5914.
31 Vigouroux, S., Yvon, E., Biagi, E. and Brenner, M. K., Antigen-induced
regulatory T cells. Blood 2004. 104: 26–33.
32 Gonzalez-Rey, E., Chorny, A., Fernandez-Martin, A., Ganea, D. and
Delgado, M., Vasoactive intestinal peptide generates human tolerogenic
dendritic cells that induce CD4 and CD8 regulatory T cells. Blood 2006.
107: 3632–3638.
33 Kosiewicz, M. M. and Alard, P., Tolerogenic antigen-presenting cells:
regulation of the immune response by TGF-beta-treated antigen-present-
ing cells. Immunol. Res. 2004. 30: 155–170.
34 Liu, H., Komai-Koma, M., Xu, D. and Liew, F. Y., Toll-like receptor 2
signaling modulates the functions of CD41CD251 regulatory T cells. Proc.
Natl. Acad. Sci. USA 2006. 103: 7048–7053.
35 Caramalho, I., Lopes-Carvalho, T., Ostler, D., Zelenay, S., Haury, M. and
Demengeot, J., Regulatory T cells selectively express toll-like receptors
and are activated by lipopolysaccharide. J. Exp. Med. 2003. 197: 403–411.
36 Hesse, M., Piccirillo, C. A., Belkaid, Y., Prufer, J., Mentink-Kane, M.,
Leusink, M., Cheever, A. W. et al., The pathogenesis of schistosomiasis is
controlled by cooperating IL-10-producing innate effector and regulatory
T cells. J. Immunol. 2004. 172: 3157–3166.
37 Walker, L. S., Chodos, A., Eggena, M., Dooms, H. and Abbas, A. K.,
Antigen-dependent proliferation of CD41CD251 regulatory T cells in vivo.
J. Exp. Med. 2003. 198: 249–258.
38 Hong, J., Li, N., Zhang, X., Zheng, B. and Zhang, J. Z., Induction of CD41
CD251 regulatory T cells by copolymer-I through activation of transcrip-
tion factor Foxp3. Proc. Natl. Acad. Sci. USA 2005. 102: 6449–6454.
39 Zheng, S. G., Wang, J. H., Gray, J. D., Soucier, H. and Horwitz, D. A.,
Natural and induced CD41CD251 cells educate CD41CD25� cells to
develop suppressive activity: the role of IL-2, TGF-beta, and IL-10.
J. Immunol. 2004. 172: 5213–5221.
40 Delgado, M., Abad, C., Martinez, C., Leceta, J. and Gomariz, R. P.,
Vasoactive intestinal peptide prevents experimental arthritis by down-
regulating both autoimmune and inflammatory components of the
disease. Nat. Med. 2001. 7: 563–568.
41 Abad, C., Martinez, C., Juarranz, M. G., Arranz, A., Leceta, J., Delgado, M.
and Gomariz, R. P., Therapeutic effects of vasoactive intestinal peptide in
the trinitrobenzene sulfonic acid mice model of Crohn’s disease.
Gastroenterology 2003. 124: 961–971.
42 Keino, H., Kezuka, T., Takeuchi, M., Yamakawa, N., Hattori, T. and Usui,
M., Prevention of experimental autoimmune uveoretinitis by vasoactive
intestinal peptide. Arch. Ophthalmol. 2004. 122: 1179–1184.
43 Gonzalez-Rey, E., Fernandez-Martin, A., Chorny, A., Martin, J., Pozo, D.,
Ganea, D. and Delgado, M., Therapeutic effect of vasoactive intestinal
peptide on experimental autoimmune encephalomyelitis: down-regula-
tion of inflammatory and autoimmune responses. Am. J. Pathol. 2006. 168:
1179–1188.
44 Sela, M. and Mozes, E., Therapeutic vaccines in autoimmunity. Proc. Natl.
Acad. Sci. USA 2004. 101: 14586–14592.
45 Jordan, M. S., Boesteanu, A., Reed, A. J., Petrone, A. L., Holenbeck, A. E.,
Lerman, M. A., Naji, A. and Caton, A. J., Thymic selection of CD41CD251
regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2001.
2: 301–306.
46 Bensinger, S. J., Bandeira, A., Jordan, M. S., Caton, A. J. and Laufer, T. M.,
Major histocompatibility complex class II-positive cortical epithelium
Eur. J. Immunol. 2009. 39: 3052–3065Xuefeng Wang et al.3064
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
mediates the selection of CD4(1)25(1) immunoregulatory T cells. J. Exp.
Med. 2001. 194: 427–438.
47 Apostolou, I., Sarukhan, A., Klein, L. and von Boehmer, H., Origin of
regulatory T cells with known specificity for antigen. Nat. Immunol. 2002.
3: 756–763.
48 Hoves, S., Krause, S. W., Schutz, C., Halbritter, D., Scholmerich, J.,
Herfarth, H. and Fleck, M., Monocyte-derived human macrophages
mediate anergy in allogeneic T cells and induce regulatory T cells.
J. Immunol. 2006. 177: 2691–2698.
49 Steinbrink, K., Graulich, E., Kubsch, S., Knop, J. and Enk, A. H., CD4(1)
and CD8(1) anergic T cells induced by interleukin-10-treated human
dendritic cells display antigen-specific suppressor activity. Blood 2002. 99:
2468–2476.
50 van der Kleij, D., Latz, E., Brouwers, J. F., Kruize, Y. C., Schmitz, M., Kurt-
Jones, E. A., Espevik, T. et al., A novel host-parasite lipid cross-talk.
Schistosomal lyso-phosphatidylserine activates toll-like receptor 2 and
affects immune polarization. J. Biol. Chem. 2002. 277: 48122–48129.
51 Crellin, N. K., Garcia, R. V., Hadisfar, O., Allan, S. E., Steiner, T. S. and
Levings, M. K., Human CD41 T cells express TLR5 and its ligand flagellin
enhances the suppressive capacity and expression of FOXP3 in CD41
CD251 T regulatory cells. J. Immunol. 2005. 175: 8051–8059.
52 Zhang, L., Yang, Y., Yang, X., Zhao, J., Yang, J., Liu, F., Zhang, Z. et al.,
T cell epitope-based peptide-DNA dual vaccine induces protective
immunity against Schistosoma japonicum infection in C57BL/6J mice.
Microbes Infect. 2008. 10: 251–259.
53 Fonseca, C. T., Cunha-Neto, E., Goldberg, A. C., Kalil, J., de Jesus, A. R.,
Carvalho, E. M., Correa-Oliveira, R. et al., Identification of paramyosin
T-cell epitopes associated with human resistance to Schistosoma mansoni
reinfection. Clin. Exp. Immunol. 2005. 142: 539–547.
54 Li, G. F., Wang, Y., Zhang, Z. S., Wang, X. J., Ji, M. J., Zhu, X., Liu, F. et al.,
Identification of immunodominant Th1-type T cell epitopes from
Schistosoma japonicum 28 kDa glutathione-S-transferase, a vaccine candi-
date. Acta. Biochim. Biophys. Sin. (Shanghai) 2005. 37: 751–758.
55 Zhang, X., Izikson, L., Liu, L. and Weiner, H. L., Activation of CD25(1)
CD4(1) regulatory T cells by oral antigen administration. J. Immunol. 2001.
167: 4245–4253.
56 Thorstenson, K. M. and Khoruts, A., Generation of anergic and
potentially immunoregulatory CD251CD4 T cells in vivo after induction
of peripheral tolerance with intravenous or oral antigen. J. Immunol. 2001.
167: 188–195.
57 Yang, R. B., Mark, M. R., Gray, A., Huang, A., Xie, M. H., Zhang, M.,
Goddard, A. et al., Toll-like receptor-2 mediates lipopolysaccharide-
induced cellular signalling. Nature 1998. 395: 284–288.
58 Gao, B. and Tsan, M. F., Endotoxin contamination in recombinant human
heat shock protein 70 (Hsp70) preparation is responsible for the induction
of tumor necrosis factor alpha release by murine macrophages. J. Biol.
Chem. 2003. 278: 174–179.
59 Racoosin, E. L. and Swanson, J. A., Macrophage colony-stimulating factor
(rM-CSF) stimulates pinocytosis in bone marrow-derived macrophages.
J. Exp. Med. 1989. 170: 1635–1648.
60 Qureshi, M. H., Empey, K. M. and Garvy, B. A., Modulation of
proinflammatory responses to Pneumocystis carinii f. sp. muris in neonatal
mice by granulocyte-macrophage colony-stimulating factor and IL-4: role
of APCs. J. Immunol. 2005. 174: 441–448.
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