A Novel Approach to Specific Allergy Treatment: The Recombinant Allergen-S-Layer Fusion Protein rSbsC-Bet v 1 Matures Dendritic Cells That Prime Th0/Th1 and IL-10-Producing Regulatory T Cells

Abstract

An ideal vaccine for allergen-specific immunotherapy of type I allergies should display reduced mediator-releasing capacity, induce maturation of APC, and modify the disease-eliciting Th2-dominated allergen-specific response to a more physiological response. We have previously shown that rSbsC-Bet v 1, the recombinant fusion protein of a bacterial surface (S-layer) protein of Geobacillus stearothermophilus ATCC 12980 and the major birch pollen allergen Bet v 1, exhibited reduced allergenicity and induced IFN-gamma and IL-10 synthesis in Bet v 1-specific Th2 clones. In this study, we characterized the effects of rSbsC-Bet v 1 on immature monocyte-derived dendritic cells (mdDC) and the consequences for the polarization of naive CD4(+) T lymphocytes isolated from the blood of birch pollen-allergic patients. mdDC responded to rSbsC-Bet v 1 with a significant up-regulation of costimulatory molecules, functional maturation, and the synthesis of IL-10 and IL-12. mdDC matured with rSbsC-Bet v 1 induced the differentiation of naive T cells into IFN-gamma-producing cells. This effect was IL-12 dependent. In parallel, a substantial number of naive T cells developed into IL-10-producing CD25(+)Foxp3(+)CLTA-4(+) cells capable of active suppression. Thus, rSbsC-Bet v 1 showed immune stimulatory capacity on DC, which then promoted the simultaneous differentiation of Th0/Th1 cells and regulatory T cells. These data further support that the concept of conjugating allergens to bacterial agents is a promising approach to improve vaccines for specific immunotherapy of atopic allergies.

Full text

A Novel Approach to Specific Allergy Treatment: The
Recombinant Allergen-S-Layer Fusion Protein rSbsC-Bet v 1
Matures Dendritic Cells That Prime Th0/Th1 and
IL-10-Producing Regulatory T Cells
1
Marianne Gerstmayr,* Nicola Ilk,
†
Irma Schabussova,* Beatrice Jahn-Schmid,*
Eva M. Egelseer,
†
Uwe B. Sleytr,
†
Christof Ebner,
‡
and Barbara Bohle
2
*
An ideal vaccine for allergen-specific immunotherapy of type I allergies should display reduced mediator-releasing capacity,
induce maturation of APC, and modify the disease-eliciting Th2-dominated allergen-specific response to a more physiological
response. We have previously shown that rSbsC-Bet v 1, the recombinant fusion protein of a bacterial surface (S-layer) protein
of Geobacillus stearothermophilus ATCC 12980 and the major birch pollen allergen Bet v 1, exhibited reduced allergenicity and
induced IFN-
␥
and IL-10 synthesis in Bet v 1-specific Th2 clones. In this study, we characterized the effects of rSbsC-Betv1on
immature monocyte-derived dendritic cells (mdDC) and the consequences for the polarization of naive CD4
ⴙ
T lymphocytes
isolated from the blood of birch pollen-allergic patients. mdDC responded to rSbsC-Bet v 1 with a significant up-regulation of
costimulatory molecules, functional maturation, and the synthesis of IL-10 and IL-12. mdDC matured with rSbsC-Bet v 1 induced
the differentiation of naive T cells into IFN-
␥
-producing cells. This effect was IL-12 dependent. In parallel, a substantial number
of naive T cells developed into IL-10-producing CD25
ⴙ
Foxp3
ⴙ
CLTA-4
ⴙ
cells capable of active suppression. Thus, rSbsC-Bet v
1 showed immune stimulatory capacity on DC, which then promoted the simultaneous differentiation of Th0/Th1 cells and
regulatory T cells. These data further support that the concept of conjugating allergens to bacterial agents is a promising approach
to improve vaccines for specific immunotherapy of atopic allergies. The Journal of Immunology, 2007, 179: 7270 –7275.
Allergen-specific Th2 cells, i.e., CD4
⫹
T lymphocytes
that produce high levels of IL-4, IL-5, and IL-13, but
low amounts of IFN-
␥
, are pivotal for the induction and
maintenance of type I allergies (1). The only approved treatment
that counterregulates the disease-eliciting allergen-specific Th2 re-
sponse in allergic individuals is specific immunotherapy (SIT).
3
Successful SIT significantly reduces allergen-induced T cell pro-
liferation, indicating the induction of peripheral tolerance in aller-
gen-specific T cells. In parallel, the shift from Th2 toward a more
physiological Th0/Th1-like immune response, either by reduced
IL-4 production or the induction of IFN-
␥
, has been observed
(2–5). More recently, the induction of IL-10-producing allergen-
specific T cells has been associated with successful SIT (6 –9).
SIT-induced IL-10-producing CD4
⫹
CD25
⫹
T cells suppress pro-
liferation and cytokine production of allergen-specific T cells and
are thus designated T regulatory (Treg) cells (10 –13). Further-
more, the fine balance between IL-10-producing allergen-specific
Treg and Th2 cells has been considered a relevant component of
the physiological immune response to allergens (14).
One strategy to improve vaccines for SIT is the use of adjuvants
that amplify the modulation of the allergen-specific Th2 response.
In this respect, different agents from bacterial origin have been
regarded as promising candidates. Synthetic lipopeptides derived
from lipoproteins of Gram-negative bacteria have been reported to
induce IFN-
␥
and IL-10 synthesis by human T cells (15). Mono-
phosphoryl lipid A, a nontoxic derivate of LPS of Salmonella min-
nesota, mixed with grass pollen extract enhanced the production of
IFN-
␥
and reduced the production of IL-5 in PBMC from grass
pollen-allergic patients (16). Synthetic oligodeoxynucleotides con-
taining CpG motifs (CpG-ODN) derived from bacterial DNA pro-
moted Th1-like cytokine production and decreased the spontane-
ous synthesis of IgE Abs in allergic individuals in vitro (17–20).
This immunomodulatory capacity was even more pronounced
when CpG-ODN were chemically linked to a single defined aller-
gen (21–23). Both conjugated CpG-ODN and monophosphoryl
lipid A have been successfully applied in clinical trials and re-
vealed promising results regarding the treatment of type I allergies
(24 –26).
S-layer proteins forming crystalline arrays on the cell surface of
many bacteria and most archaea have already a long tradition in
vaccine development (27–29). We have proposed to engineer al-
lergy vaccines by genetic fusion of allergens with S-layer proteins:
*Department of Pathophysiology, Center for Physiology, Pathophysiology, and Im-
munology, Medical University of Vienna, Vienna, Austria; and
†
Center for Nano-
Biotechnology, University of Natural Resources and Applied Life Sciences and
‡
Al-
lergy Clinic Reumannplatz, Vienna, Austria
Received for publication July 10, 2007. Accepted for publication September 19, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Fonds zur Fo¨rderung der wissenschaftlichen For-
schung (SFB-F1807-B04); P12158 by the OeNB, Austria; P18510-B12 by the Euro-
pean Union Project NAS-SAP; and by the U.S. Air Force Office of Scientific Re-
search, Project FA9550-07-1-0313.
2
Address correspondence and reprint requests to Dr. Barbara Bohle, Department of
Pathophysiology, Center for Physiology, Pathophysiology, and Immunology, Medical
University of Vienna, Austria; AKH-3Q, Waehringer Guertel 18-20, Vienna, Austria.
E-mail address: barbara.bohle@meduniwien.ac.at
3
Abbreviations used in this paper: SIT, specific immunotherapy; DC, dendritic cell;
mdDC, monocyte-derived DC; B-DC, mdDC incubated with rBet v 1; CpG-ODN,
oligodeoxynucleotides containing CpG motifs; Ct, threshold cycle; LB-CD, mdDC
incubated with LPS and rBet v 1; SB-DC, mdDC incubated with rSbsC-Bet b 1; Treg,
T regulatory.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
www.jimmunol.org

the major birch pollen allergen Bet v 1 was fused with the S-layer
protein from the nonpathogenic bacteria Geobacillus stearother-
mophilus ATCC 12980 (30, 31). The resulting allergen-S-layer
fusion protein rSbsC-Bet v 1 induced IFN-
␥
and IL-10 production
in PBMC and Bet v 1-specific Th2 clones from birch pollen-al-
lergic donors (31). Moreover, rSbsC-Bet v 1 displayed a reduced
IgE-binding capacity, and consequently a lower risk to induce ad-
verse vaccine-related anaphylactic reactions (31). Thus, we con-
sider rSbsC-Betv1aspromising hypoallergenic molecule to skew
allergen-specific Th2 effector cells to both IFN-
␥
and IL-10 pro-
duction. In this study, we investigated whether rSbsC-Betv1af-
fected the differentiation of naive CD4
⫹
T cells from birch pollen-
allergic individuals. We first analyzed the effects of rSbsC-Bet v 1
on monocyte-derived dendritic cells (mdDC) regarding matura-
tion, functionality, and cytokine production. Next, mdDC stimu-
lated with rSbsC-Bet v 1 were used to prime naive CD4
⫹
T cells.
T cell polarization was evaluated by measuring IL-4, IFN-
␥
, and
IL-10. The suppressive capacity of IL-10-secreting T cells was
tested in proliferation assays.
Materials and Methods
Patients, allergens, and reagents
Birch pollen-allergic patients with a typical case history, positive skin prick
tests, and CAP/RAST scores ⬎3 (Pharmacia) to birch were included. Sen-
sitization to Bet v 1 was assessed by means of immunoblotting. The study
was approved by the local medical ethics committee (Vienna, Austria).
RSbsC-Bet v 1 and rSbsC were produced, as described (30). Endotoxin
levels in rSbsC-Bet v 1 and rSbsC were below 0.4 EU/mg, as determined
by Limulus amebocyte lysate assay (BioWhittaker). RBet v 1 was pur-
chased from Biomay, and LPS from Sigma-Aldrich.
Generation of mdDC
Monocytes were isolated from PBMC by magnetic cell sorting using anti-
CD14 microbeads (Miltenyi Biotec). The isolated fraction contained
⬎95% CD14
⫹
cells, as determined by flow cytometry using FITC-conju-
gated anti-CD14 mAb (BD Pharmingen). CD14
⫹
cells (1 ⫻10
6
/ml) were
cultured in 24-well plates (Corning Glass) in RPMI 1640 medium (Invitro-
gen Life Technologies) supplemented with 2 mM L-glutamine, 170 mg/L
gentamycinsulfate (both Sigma-Aldrich), human rIL-4 (1000 U/ml; Strath-
mann Biotec), and GM-CSF (50 ng/ml; PeproTech) in the presence of
either 3% autologous plasma or 10% heat-inactivated FCS (PAA Labora-
tories) for 7 days. The resulting dendritic cell (DC) population was homo-
geneously CD14
⫺
, and ⬎70% of the cells reacted with PE-Cy5-conjugated
anti-CD1a mAbs (BD Pharmingen). Immature DC were either left un-
treated or stimulated with rBetv1(10
␮
g/ml), rSbsC-Betv1(59
␮
g/ml
containing 10
␮
g/ml rBet v 1), and corresponding amounts of rSbsC and
LPS (1
␮
g/ml), respectively. After 48 h, surface expression of DC matu-
ration markers on CD14
⫺
CD1a
⫹
cells was analyzed using PE-conjugated
anti-CD80, anti-CD83 mAb (both from BD Pharmingen), anti-CD86 mAb
(Caltag Laboratories), and FITC-conjugated anti-CD40 mAb (provided by
O. Majdic, Institute of Immunology, Vienna, Austria).
Mixed leukocyte reaction
Differently stimulated DC were harvested after 48 h, irradiated (60 Gy),
and added to 1 ⫻10
5
allogeneic PBMC from nonallergic donors in dif-
ferent ratios. Cells were cultured in triplicates in 96-well plates (Nunclone;
Nunc) in serum-free UC medium (BioWhittaker) supplemented with 2 mM
L-glutamine and 2 ⫻10
⫺5
M 2-ME for 6 days. Proliferation was assessed
by adding [
3
H]thymidine (0.5
␮
Ci/well) during the last 16 h of culture and
measuring the incorporated radioactivity by scintillation counting.
Real-time PCR
DC were incubated in 24-well plates (Corning Glass) with different stimuli
for 14 h. RNA was isolated using the RNeasy Mini Kit (Qiagen) and
reversely transcribed with TaqMan reverse-transcription reagents using
random hexamers (Applied Biosystems). Analysis of 18S rRNA (control
housekeeping gene), IL-10, and IL-12p40 was performed with cDNA-spe-
cific Assays-on-Demand (Applied Biosystems) using an ABI PRISM 7700
Sequence Detection System (Applied Biosystems). A negative control with-
out template and a positive control with a template of known amplification
were included in each run. All amplifications were performed in triplicates.
Relative quantification and calculation of the range of confidence were per-
formed by using the comparative threshold cycle (⌬⌬Ct) method (Applied
Biosystems). The fold induction of cytokines was calculated by the formula
2
⫺⌬⌬CT
⫽2
⫺(⌬Ct for unstimulated culture ⫺⌬Ct for stimulated culture)
.
Purification and stimulation of naive CD4
⫹
T cells
Peripheral CD4
⫹
T cells were purified using the CD4 Negative Isolation
Kit from Dynal Biotech. From the resulting CD4
⫹
population, CD45RA
⫹
cells were isolated by magnetic cell sorting using anti-CD45RA mi-
crobeads (Miltenyi Biotec). A purity of ⬎92% CD4
⫹
CD45RA
⫹
T cells
**
*
Mean fluorescence intensity
200
150
100
50
0
CD40
Percent positive cells
100
80
60
40
20
0
CD80
100
80
60
40
20
0
CD83
100
80
60
40
20
0
CD86
**
Medium
rBet v1
rSbsC-Bet v1
rSbsC
LPS
Medium
rBet v1
rSbsC-Bet v1
rSbsC
LPS
Medium
rBet v1
rSbsC-Bet v1
rSbsC
LPS
Medium
rBet v1
rSbsC-Bet v1
rSbsC
LPS
**
**
**
*
**
*
*
**
*
*
*
**
**
***
**
Mean fluorescence intensity
200
150
100
50
0
CD40
Percent positive cells
100
80
60
40
20
0
CD80
100
80
60
40
20
0
CD83
100
80
60
40
20
0
CD86
** **
Medium
rBet v1
rSbsC-Bet v1
rSbsC
LPS
Medium
rBet v1
rSbsC-Bet v1
rSbsC
LPS
Medium
rBet v1
rSbsC-Bet v1
rSbsC
LPS
Medium
rBet v1
rSbsC-Bet v1
rSbsC
LPS
Medium
rBet v1
rSbsC-Bet v1
rSbsC
LPS
Medium
rBet v1
rSbsC-Bet v1
rSbsC
LPS
Medium
rBet v1
rSbsC-Bet v1
rSbsC
LPS
Medium
rBet v1
rSbsC-Bet v1
rSbsC
LPS
****
*
***
** *
*
**
*
***
**
**
**
**
***
**
*
*
**
FIGURE 1. rSbsC-Bet v 1 induces
DC maturation. Immature mdDC were
left untreated or stimulated with rBet v
1, rSbsC-Bet v 1, rSbsC, or LPS. The
percentage of DC expressing CD80,
CD83, and CD86 surface markers and
the mean fluorescence intensity of
CD40 are shown. Data from seven
different birch pollen-allergic donors
were summarized in box plots. Each
box represents the interquartile range
containing 50% of the data. The line
across the box indicates the median.
The significances of differences to rBet
v 1 were calculated using the Wilcoxon
signed ranks test (ⴱ,p⬍0.05).
0
30
60
90
120
150
180
2510
x10³ DC/well
Proliferation (cpm x103)
Medium rBet v 1
rSbsC-Bet v1 rSbsC
LPS
0
30
60
90
120
150
180
2510
x10³ DC/well
Proliferation (cpm x103)
Medium rBet v 1
rSbsC-Bet v1 rSbsC
LPS
FIGURE 2. rSbsC-Bet v 1 induces functional DC. Increasing numbers
of untreated mdDC or mdDC incubated with rBet v 1, rSbsC-Bet v 1,
rSbsC, or LPS were cultured with 1 ⫻10
5
allogeneic PBMC. Mean cpm
of triplicates of one experiment of three are shown.
7271The Journal of Immunology

was achieved. Autologous immature DC were stimulated with either rBet
v 1 (10
␮
g/ml), rSbsC-Betv1(59
␮
g/ml), or LPS (100 ng/ml) plus rBet
v 1 (10
␮
g/ml). After 24 h, 1 ⫻10
6
CD4
⫹
CD45RA
⫹
T cells were added
(day 0). At day 7, T cells were restimulated with the respective DC pop-
ulation and analyzed for cytokine production and proliferation at day 14. In
some experiments, a neutralizing anti-IL-12 mAb (50
␮
g/ml; R&D Sys-
tems) was added at days 0 and 7.
Phenotyping of primed CD4
⫹
T cells
T cells were stained with PE-conjugated anti-CD25, PE-Cy5-conjugated
anti-CD152 mAb (both from BD Pharmingen), and a FITC-conjugated
anti-human Foxp3 (eBioscience). Intracellular cytokine detection in T cells
was performed using FITC-, PE-, PerCP-labeled anti-IFN-
␥
, anti-IL-4, anti-
CD3 mAbs, and respective isotype control Abs, as described (31). To quan-
tify cytokine levels, 1 ⫻10
5
T cells were stimulated in triplicates with
anti-CD3 (1
␮
g/ml OKT3) and anti-CD28 (1
␮
g/ml; Sanquin) in the pres-
ence of irradiated PBMC (60 Gy) in 96-well plates for 48 h. Levels of
IL-10, IFN-
␥
, and TGF-
␤
in supernatants were analyzed by ELISA using
matched Ab pairs (Pierce) and a TGF-
␤
1 ELISA kit (IBT) (31).
Evaluation of suppressive capacity of IL-10-secreting T cells
Purified CD4
⫹
T cells (1 ⫻10
6
) were stimulated with immobilized anti-
CD3 (0.5
␮
g/ml) and soluble anti-CD28 (1
␮
g/ml) in 48-well plates. After
18 h, IL-10-secreting cells were isolated using the IL-10 Secretion Assay-
Cell Enrichment and Detection Kit (Milteny Biotec), according to the man-
ufacturer⬘s protocol. Autologous CD4
⫹
CD25
⫺
cells were cocultured with
IL-10-secreting cells in different ratios in the presence of anti-CD3 (OKT3
0.5
␮
g/ml) or without any stimulus. After 72 h, proliferation was assessed.
Statistical analysis
Statistical significance of differences was determined using the Wilcoxon
signed ranks test. Differences were considered statistically significant for
p⬍0.05.
Results
rSbsC-Bet v 1 induces phenotypic and functional maturation
of DC
Immature mdDC from seven different birch pollen-allergic patients
were left untreated or incubated with rBet v 1, rSbsC-Bet v 1, and
rSbsC for 48 h (Fig. 1). As positive control, LPS, a well-charac-
terized agent to mature mdDC, was used. Incubation of mdDC
with rBet v 1 showed no effects as compared with medium alone.
Incubation with rSbsC-Bet v 1 and rSbsC induced a significant
up-regulation of CD80 ( p⫽0.018, p⫽0.018), CD83 (p⫽0.018,
p⫽0.018), CD86 ( p⫽0.018, p⫽0.018), and CD40 ( p⫽0.018,
p⫽0.028) as compared with rBet v 1 (Fig. 1). RSbsC-Bet v 1
induced a significantly higher expression of CD83 than rSbsC
(p⫽0.043) (Fig. 1). LPS-stimulated mdDC displayed signifi-
cantly higher levels of all surface markers as compared with
rSbsC-Bet v 1 (CD80, p⫽0.018; CD83, p⫽0.043; CD86, p⫽
0.018; CD40, p⫽0.018). To test whether the differently matured
mdDC were capable of activating T cells, MLR were performed.
The allostimulatory capacity of mdDC reflected the observed
changes of surface marker expression (Fig. 2). In comparison with
unstimulated or rBet v 1-stimulated mdDC, mdDC incubated with
rSbsC-Betv1orrSbsC induced enhanced proliferative responses
in allogeneic T cells. LPS-stimulated mdDC induced a more pro-
nounced T cell proliferation as compared with rSbsC-Bet v 1
(Fig. 2).
rSbsC-Bet v 1 induces IL-10 and IL-12 production in DC
IL-10 and IL-12 mRNA expression levels in response to the dif-
ferent stimuli were assessed by real-time PCR in mdDC generated
from six different birch pollen-allergic donors (Fig. 3). The me-
dium control was used as baseline for calculating the fold induc-
tion of IL-10 and IL-12, respectively. The major birch pollen al-
lergen marginally enhanced IL-10 and IL-12 mRNA expression in
mdDC. Compared with rBet v 1, rSbsC-Bet v 1 induced a signif-
icantly higher mRNA expression for both cytokines ( p⫽0.028,
p⫽0.028) as did LPS ( p⫽0.046, p⫽0.028; Fig. 3), and stim-
ulation with rSbsC resulted in a significant increase of IL-12
mRNA expression ( p⫽0.046). These results obtained at the
mRNA level were confirmed by ELISA of supernatants derived
from mdDC stimulated for 48 h (data not shown).
IL-10
rBet v1
rSbsC-Betv1
rSbsC
LPS
0
2
4
6
8
10
12
14
16 *
***
IL-10 mRNA (fold induction)
IL-12
rBet v1
rSbsC-Bet v 1
rSbsC
LPS
0
10
20
30
40
50
60 *
*
**
IL-12 mRNA (fold induction)
IL-10
rBet v1
rSbsC-Betv1
rSbsC
LPS
0
2
4
6
8
10
12
14
16 *
***
IL-10 mRNA (fold induction)
IL-12
rBet v1
rSbsC-Bet v 1
rSbsC
LPS
0
10
20
30
40
50
60 *
*
**
IL-12 mRNA (fold induction)
FIGURE 3. rSbsC-Bet v 1 induces IL-10 and IL-12
in DC. Immature mdDC from six patients were left un-
treated or stimulated with rBet v 1, rSbsC-Bet v 1,
rSbsC, or LPS. The relative expression of IL-10 and
IL-12 mRNA was individually calculated as compared
with the mRNA expression in unstimulated cells. Lines
indicate the median. ⴱ,p⬍0.05 (Wilcoxon signed ranks
test).
FIGURE 4. DC matured with rSbsC-Bet v 1 promote a Th0/Th1-like
response. Naive CD4
⫹
T cells were primed with autologous B-DC, LB-
DC, and SB-DC in the absence or presence of anti-IL-12. Intracellular
synthesis of IFN-
␥
and IL-4 was determined. Three experiments (patients
I–III) of 10 are shown.
7272 EFFECTS OF rSbsC-Betv1ONDCANDNAIVE CD4
⫹
T CELLS

rSbsC-Bet v 1-matured DC promote IL-12-dependent Th0/Th1
differentiation of naive CD4
⫹
T cells
CD4
⫹
CD45RA
⫹
cells derived from 10 birch pollen-allergic pa-
tients were stimulated twice with autologous mdDC incubated
with either rBet v 1 (B-DC), rSbsC-Bet v 1 (SB-DC), or simulta-
neous addition of LPS and rBet v 1 (LB-DC). T cell polarization
was determined by intracellular cytokine detection (Fig. 4). Com-
pared with B-DC, both SB-DC and LB-DC induced significantly
higher numbers of IFN-
␥
-producing T cells ( p⫽0.005; p⫽
0.013) and enhanced numbers of IL-4-producing T cells. Similar to
LB-DC, SB-DC induced significantly more IFN-
␥
⫹
than IL-4
⫹
T
cells ( p⫽0.005), reflecting a Th1-like response. Interestingly,
significantly higher numbers of IL-4
⫹
IFN-
␥
⫹
double-positive T
cells ( p⫽0.017) were detected in SB-DC-primed T cells as com-
pared with LB-DC-stimulated cultures. The simultaneous addition
of neutralizing anti-IL-12 mAb and SB-DC to CD4
⫹
CD45RA
⫹
cells drastically reduced the number of IFN-
␥
-producing CD4
⫹
T
cells (Fig. 4).
rSbsC-Bet v 1-matured DC promote the differentiation of
regulatory T cells
To investigate whether the different DC maturation induced by
rSbsC-Bet v 1 and LPS plus rBet v 1 translated into different Th
responses, the production of IFN-
␥
, IL-10, and TGF-
␤
was quan-
tified in supernatants of anti-CD3-stimulated T cells primed with
the differently stimulated mdDC by ELISA (Fig. 5). Compared
with B-DC-primed T cells, SB-DC- as well as LB-DC-primed T
cells produced significantly higher levels of IFN-
␥
(p⫽0.037;
p⫽0.047) and IL-10 ( p⫽0.005; p⫽0.009). SB-DC-primed T
cells synthesized comparable levels of IFN-
␥
and significantly
higher amounts of IL-10 ( p⫽0.022) and TGF-
␤
(p⫽0.028) than
LB-DC-primed T cells. Because these cytokines are typically syn-
thesized by Treg cells, T cells from six individuals were stained for
CD25 and intracellular expression of Foxp3 7 days after the sec-
ond addition of DC. SB-DC-primed T cell cultures contained sig-
nificantly higher numbers of CD25
⫹
Foxp3
⫹
cells than LB-DC-
primed T cells ( p⫽0.028). One representative experiment is
shown in Fig. 6A. Parallel staining for CTLA-4 revealed that
CD25
⫹
Foxp3
⫹
cells were all CTLA-4 positive (data not shown).
To analyze whether IL-10-producing cells expressed these Treg
markers, T cells needed to be stimulated before intracellular cy-
tokine detection. Most T cells became Foxp3 positive in response
to PMA/ionomycin, whereas the expression of CTLA-4 was hardly
affected (data not shown). Therefore, we costained T cells for
IL-10 and CTLA-4 and observed that all IL-10
⫹
cells were CTLA-
4
⫹
. One representative example is shown in Fig. 6B. Next, T cells
from four individuals were stained for their anti-CD3-induced se-
cretion of IL-10 and IFN-
␥
. The majority of IL-10-secreting cells
constituted a population different from IFN-
␥
-secreting cells, but
also cells secreting both cytokines were detected. One example is
depicted in Fig. 6C. Subsequently, we isolated IL-10-secreting
cells from SB-DC-primed T cell cultures from two patients. Pro-
liferative responses of these cells were markedly lower as com-
pared with autologous CD4
⫹
CD25
⫺
cells (Fig. 6D). Coculturing
IL-10-secreting T cells with autologous CD4
⫹
CD25
⫺
cells in the
presence of irradiated autologous PBMC and anti-CD3 Abs re-
sulted in the suppression of the proliferation of CD4
⫹
CD25
⫺
cells
in a dose-dependent manner (Fig. 6D). In two independent exper-
iments, a mean suppression of 83% was observed at a suppressor:
responder cell ratio of 1:1 and 74% at a ratio of 1:3, respectively.
Discussion
We have previously shown that genetic fusion of the major birch
pollen allergen and a bacterial S layer resulted in a recombinant
protein exhibiting reduced mediator-releasing capacity, retained
pg/ml
3000
2500
2000
1500
1000
500
0
*
*
IFN-gamma IL-10 TGF-beta
600
500
400
300
200
100
0
*
*
*
160
140
120
100
80
60
40
20
0
B-DC
LB-DC
SB-DC
B-DC
LB-DC
SB-DC
B-DC
LB-DC
SB-DC
*
pg/ml
3000
2500
2000
1500
1000
500
0
**
**
IFN-gamma IL-10 TGF-beta
600
500
400
300
200
100
0
**
**
**
160
140
120
100
80
60
40
20
0
B-DC
LB-DC
SB-DC
B-DC
LB-DC
SB-DC
B-DC
LB-DC
SB-DC
B-DC
LB-DC
SB-DC
B-DC
LB-DC
SB-DC
B-DC
LB-DC
SB-DC
**
FIGURE 5. T cells primed with mdDC matured with rSbsC-Bet v 1
produce IFN-
␥
, IL-10, and TGF-
␤
. Naive CD4
⫹
T cells were primed with
autologous B-DC, LB-DC, and SB-DC. After stimulation with anti-CD3
plus anti-CD28, supernatants were assessed for cytokines by ELISA. Data
from 10 different donors were summarized in box plots. ⴱ,p⬍0.05 (Wil-
coxon signed ranks test).
FIGURE 6. rSbsC-Bet v 1-matured mdDC prime IL-
10-producing Treg cells. Naive CD4
⫹
T cells were
primed with autologous LB-DC or SB-DC and stained
for A, CD25 and intracellular Foxp3; B, intracellular
IL-10 and CTLA-4; and C, secretion of IL-10 and
IFN-
␥
assessed by cytokine secretion assays. One rep-
resentative experiment of each staining is shown. D,
SB-DC-induced IL-10-secreting cells were isolated
from two donors and cocultured with autologous
CD4
⫹
CD25
⫺
cells in the presence of irradiated autol-
ogous PBMC and anti-CD3. Mean cpm and SD of trip-
licates are depicted.
7273The Journal of Immunology

Bet v 1-T cell epitopes, and the potency to induce IFN-
␥
and IL-10
production in Bet v 1-specific Th2 clones (31). In this study, we
analyzed the effects of rSbsC-Betv1onimmature DC and naive
CD4
⫹
T cells isolated from the blood of birch pollen-allergic pa-
tients. We demonstrate that DC matured with rSbsC-Bet v 1 pro-
mote the simultaneous differentiation of Th0/Th1 cells and IL-10-
producing Treg cells. These data further support the concept that
recombinant fusion of allergens and S-layer proteins is a promising
approach to improve vaccines for SIT of atopic allergies.
In a first step, the effects of rSbsC-Bet v 1 and rSbsC on im-
mature mdDC were analyzed. Stimulation with these proteins re-
sulted in DC maturation, increased T cell stimulatory property, and
the production of IL-10 and IL-12 (Figs. 1–3). Thus, the recom-
binant S-layer protein from G. stearothermophilus ATCC 12980
stimulates the innate immune system of allergic patients. These
findings are in line with the effects of natural S layers isolated from
Clostridium difficile on monocytes and mdDC of nonallergic indi-
viduals (32). Stimulation of DC with rBet v 1 alone resulted nei-
ther in activation nor maturation, indicating that an additional stim-
ulus is required to induce strong immune responses to the
recombinant allergen. This activity was introduced by recombinant
fusion of rBetv1totheS-layer protein. The fact that rSbsC-Bet
v 1 displayed stronger effects than rSbsC may be referred to the
involvement of Ab receptor-mediated uptake of rSbsC-Bet v 1. We
have previously shown that the fusion protein contains relevant
IgE-binding epitopes of Bet v 1 (31). It may well be possible that
rSbsC-Bet v 1 was recognized by Bet v 1-specific IgE Abs bound
to the high affinity receptor Fc␧RI on APC, resulting in IgE-me-
diated uptake in addition to phagocytosis (33).
In a second step, we used rSbsC-Bet v 1-matured DC (SB-DC)
to prime naive CD4
⫹
T cells from birch pollen-allergic donors and
investigated the polarization of the T cell response. To induce an
allergen-specific Th1 response, mdDC were primed with rBet v 1
plus LPS (LB-DC). Similar to this positive control, priming of T
cells with SB-DC resulted in significantly more IFN-
␥
⫹
than
IL-4
⫹
cells (Fig. 4). In line with our previous data showing that
S-layer-induced IFN-
␥
synthesis in effector T cells was mediated
by IL-12 (34), SB-DC-induced T cell polarization toward IFN-
␥
production also depended on the presence of this cytokine (Fig. 4).
Interestingly, significantly higher numbers of IL-4
⫹
IFN-
␥
⫹
dou-
ble-positive T cells were detected in SB-DC-primed T cells as
compared with LB-DC-primed T cells. These cells may reflect a
Th0 phenotype, simultaneously synthesizing Th1 and Th2 cyto-
kines. Thus, rSbsC-Bet v 1 may have the advantage of not pro-
moting exclusive Th1-like immune responses. Nevertheless, po-
tential risks of in vivo administration of rSbsC-Bet v 1 need to be
carefully evaluated.
SB-DC primed naive CD4
⫹
T cells to synthesize significantly
more IL-10 and TGF-
␤
as compared with LB-DC (Fig. 5). Fur-
thermore, higher numbers of IL-10
⫹
CTLA-4
⫹
Foxp3
⫹
CD25
⫹
cells were detected in SB-DC-primed cultures (Fig. 6, A–C).
Coculturing IL-10-secreting T cells isolated from SB-DC-primed
T cells suppressed proliferative responses of autologous
CD4
⫹
CD25
⫺
T cells (Fig. 6D). Together, these data provide
strong evidence that SB-DC primed naive T cells toward a popu-
lation of IL-10-producing Treg cells in addition to Th0/Th1 cells.
Preliminary studies in our laboratory indicated that rSbsC-Bet v 1
targets TLR2, because HEK293 cells stably transfected with TLR2
produced IL-8 in response to rSbsC-Bet v 1, whereas HEK293
cells transfected with TLR1 or TLR3–9 did not (data not shown).
The involvement of TLR2 may promote Treg cells through the
induction of IL-10 production in DC (35, 36). However, to date,
we observed comparable IL-10 levels produced by DC stimulated
with rSbsC-Betv1orLPSatthemRNA (Fig. 3) and protein level.
In contrast, the significantly lower expression of CD40, CD80, and
CD86 on the surface of SB-DC as compared with LB-DC may
indicate that rSbsC-Bet v 1 does not induce a fully matured DC
phenotype (Fig. 1). Immature and semimature DC have been dem-
onstrated to induce IL-10-producing Treg cells (37–39). Whether
other potential influences, e.g., enhanced expression of inhibitory
molecules of the B7 family or DC-derived TGF-
␤
, are involved in
rSbsC-Bet v 1-induced development of Treg cells is currently un-
der investigation.
Our previous (31) and present data strongly suggest that rSbsC-
Bet v 1 meets several desired requirements for an ideal allergy
vaccine: less IgE-binding and reduced mediator-releasing capac-
ity; immune stimulatory effects on DC; immune modulatory effects
on naive and allergen-specific effector T cells; and a constant ratio
between allergen and adjuvant that should improve consistency of
the product used for SIT. In addition to the induction of a Th0/
Th1-like T cell response, rSbsC-Bet v 1 also promoted IL-10-pro-
ducing Treg cells. Both immune deviation and increased numbers
of circulating IL-10-secreting CD4
⫹
CD25
⫹
cells have been asso-
ciated with successful allergen SIT. Our in vitro data indicate that
rSbsC-Bet v 1 will simultaneously support these two immune
mechanisms. Due to its hypoallergenic features (31), this fusion
molecule may also reduce the risk of IgE-mediated side effects.
Therefore, allergens genetically fused to S-layer proteins are prom-
ising future vaccines for SIT of atopic allergies.
Acknowledgments
We thank Astrid Radakovics for excellent technical assistance. This work
is dedicated to Margit Sa´ra.
Disclosures
The authors have no financial conflict of interest.
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