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\begin{document}

\title{IL-10-modulated dendritic cells from birch pollen- and hazelnut-allergic
patients facilitate Treg-mediated allergen-specific and cross-reactive
tolerance}



\author[1]{Patricia Vanessa Heinl}%
\author[1]{Edith Graulich}%
\author[2]{Benno Weigmann}%
\author[3]{Andrea Wangorsch}%
\author[1]{Robert Ose}%
\author[1]{Iris Bellinghausen}%
\author[3]{Stephan Scheurer}%
\author[3]{Stefan Vieths}%
\author[1]{Joachim Saloga}%
\author[1]{Kerstin Steinbrink}%
\affil[1]{Universitatsmedizin der Johannes Gutenberg-Universitat Mainz Hautklinik und Poliklinik}%
\affil[2]{Universitatsklinikum Erlangen}%
\affil[3]{Paul-Ehrlich-Institut}%


\vspace{-1em}



  
  \date{March 12, 2025}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Background} Approximately 70 \% of individuals allergic to birch
pollen (Bet v 1.01 {[}Bet{]}) develop a secondary food allergy (e.g.
hazelnut: Cor a 1.04 {[}Cor{]}), due to allergen cross-reactivity.
However, standard immunotherapy for type I allergies often does not
improve the food allergy sufficiently. We analyzed the allergen-specific
and cross-reactive suppressive capacity of primary human regulatory T
cells (Treg) induced by autologous IL-10-modulated dendritic cells
(IL-10 DC) \emph{in vitro} and \emph{in vivo}. \textbf{Methods} CD4
\textsuperscript{+} T cells of patients with birch pollen and associated
hazelnut allergies were differentiated into Bet-specific or non-specific
induced Treg (iTreg). After Bet- or Cor- specific restimulation the
phenotype, proliferation and suppressive capacity of iTreg subsets were
analyzed. iTreg function was further investigated in humanized mouse
models of airway and intestinal allergy, generated by engraftment of
peripheral blood mononuclear cells from allergic donors into
immunodeficient animals. \textbf{Results} After IL-10 DC priming and
allergen-specific restimulation (Bet or Cor) non-specific control iTreg
remained anergic, whereas Bet-specific iTreg proliferated extensively
and exhibited a regulatory phenotype (enhanced expression of CTLA-4,
PD-1, TNFR2, IL-10). Accordingly, activated Bet-specific iTreg displayed
a high capacity to suppress Bet- and Cor-induced responder T
\textsubscript{H}2 cell responses \emph{in vitro}, indicating induction
of both allergen-specific (birch) and cross-reactive tolerance
(hazelnut). \emph{In vivo}, the beneficial effect of Bet-specific iTreg
was verified in humanized mouse models of allergic airway and intestinal
inflammation, resulting in reduced allergen-induced clinical symptoms
and immune responses. \textbf{Conclusion} Human IL-10 DC-induced iTreg
facilitate allergen-specific and cross-reactive tolerance. Therefore,
they are potential candidates for regulatory cell therapy in allergic
and autoimmune diseases.%
\end{abstract}%



\sloppy


\textbf{Title Page}

\textbf{IL-10-modulated dendritic cells from birch pollen- and
hazelnut-allergic patients facilitate Treg-mediated allergen-specific
and cross-reactive tolerance}

Short title: Treg induced allergen specific- and cross-tolerance

Patricia Vanessa Heinl PhD \textsuperscript{1}, Edith
Graulich\textsuperscript{1}, Benno Weigmann PhD \textsuperscript{2},
Andrea Wangorsch PhD \textsuperscript{3}, Robert Ose MSc
\textsuperscript{4}*, Iris Bellinghausen PhD \textsuperscript{4},
Stephan Scheurer PhD\textsuperscript{3}, Stefan Vieths PhD
\textsuperscript{3}, Joachim Saloga MD \textsuperscript{4}, Kerstin
Steinbrink MD \textsuperscript{1, 5}

\textsuperscript{1} University Medical Center of the Johannes
Gutenberg-University, Department of Dermatology, Division for
Experimental and Translational Research, 55131 Mainz, Germany

\textsuperscript{2} University Hospital Erlangen, University
Erlangen-N\selectlanguage{ngerman}ürnberg, Department of Internal Medicine, 91054 Erlangen,
Germany

\textsuperscript{3} Paul-Ehrlich-Institute, Molecular Allergology, 63225
Langen, Germany\textsuperscript{4} University Medical Center of the
Johannes Gutenberg-University, Department of Dermatology, 55131 Mainz,
Germany

\textsuperscript{5} University Hospital Münster, University of Münster,
Department of Dermatology, Münster, Germany

*The author died prior to submission of this paper. This is one of his
last works.

Funding: This work was funded by the German Research Foundation (STE
791/9-1, TR156/C05-246807620, SFB1009/B11-194468054,
SFB1066/B06-213555243, SFB1450/C06-431460824 (all to KS).

Corresponding Author: Kerstin Steinbrink, MD, University Hospital
Münster, University of Münster, Von-Esmarch-Strasse 58, 48149 Münster,
Germany. Tel: +49 (0)251 / 83-56504, Fax: +49 (0)251 / 83-56522, E-mail:
kerstin.steinbrink@ukmuenster.de

\textbf{Abstract}

\textbf{Background}

Approximately 70 \% of individuals allergic to birch pollen (Bet v 1.01
{[}Bet{]}) develop a secondary food allergy (e.g. hazelnut: Cor a 1.04
{[}Cor{]}), due to allergen cross-reactivity. However, standard
immunotherapy for type I allergies often does not improve the food
allergy sufficiently. We analyzed the allergen-specific and
cross-reactive suppressive capacity of primary human regulatory T cells
(Treg) induced by autologous IL-10-modulated dendritic cells (IL-10
DC)\emph{in vitro} and \emph{in vivo} .

\textbf{Methods}

CD4\textsuperscript{+} T cells of patients with birch pollen and
associated hazelnut allergies were differentiated into Bet-specific or
non-specific induced Treg (iTreg). After Bet- or Cor- specific
restimulation the phenotype, proliferation and suppressive capacity of
iTreg subsets were analyzed. iTreg function was further investigated in
humanized mouse models of airway and intestinal allergy, generated by
engraftment of peripheral blood mononuclear cells from allergic donors
into immunodeficient animals.

\textbf{Results}

After IL-10 DC priming and allergen-specific restimulation (Bet or Cor)
non-specific control iTreg remained anergic, whereas Bet-specific iTreg
proliferated extensively and exhibited a regulatory phenotype (enhanced
expression of CTLA-4, PD-1, TNFR2, IL-10). Accordingly, activated
Bet-specific iTreg displayed a high capacity to suppress Bet- and
Cor-induced responder T\textsubscript{H}2 cell responses \emph{in vitro}
, indicating induction of both allergen-specific (birch) and
cross-reactive tolerance (hazelnut). \emph{In vivo} , the beneficial
effect of Bet-specific iTreg was verified in humanized mouse models of
allergic airway and intestinal inflammation, resulting in reduced
allergen-induced clinical symptoms and immune responses.

\textbf{Conclusion}

Human IL-10 DC-induced iTreg facilitate allergen-specific and
cross-reactive tolerance. Therefore, they are potential candidates for
regulatory cell therapy in allergic and autoimmune diseases.

\textbf{Key words}

\begin{itemize}
\tightlist
\item
  type I allergy
\item
  Bet v 1.01
\item
  tolerogenic IL-10 DC
\item
  regulatory T cells
\item
  cross-reactive tolerance
\end{itemize}

\textbf{Abbreviations}

{[}\textsuperscript{3}H{]}TdR: tritiated thymidine, AIT:
allergen-specific immunotherapy, Bet: Bet v 1.01, CFSE: Vybrant® CFDA SE
Cell Tracer Kit, Cor: Cor a 1.04, ctrl: positive control, eFluor670:
Cell Proliferation Dye eFluor\selectlanguage{english} 670, IL-10 DC: IL-10-modulated dendritic
cells, IL-10 DC\textsubscript{0}: unloaded IL-10 DC, IL-10
DC\textsubscript{Bet}: Bet-loaded IL-10 DC, iTreg: induced Treg,
iTreg\textsubscript{0}: IL-10 DC\textsubscript{0} induced iTreg,
iTreg\textsubscript{Bet}: IL-10 DC\textsubscript{Bet}-induced iTreg,
MCh: methacholine, mDC: mature dendritic cells, mDC\textsubscript{0}:
unloaded mDC, mDC\textsubscript{Bet}: Bet-loaded mDC, PBMC: peripheral
blood mononuclear cells, PFA: pollen-associated food allergy, r:
recombinant, SA: suppressor assay, SD: standard deviation, SI:
stimulation index, Teff: effector T cells, Teff\textsubscript{0}:
mDC\textsubscript{0} induced Teff, Teff\textsubscript{Bet}:
mDC\textsubscript{Bet}-induced Teff, T\textsubscript{H}2: T helper type
2, tolDC: tolerogenic dendritic cells, Treg: regulatory T cells, Tresp:
responder T cells.

\textbf{Text}

\textbf{Introduction}

Allergic diseases are becoming the focus of attention, as their
incidences as well as severity are increasing
worldwide.\textsuperscript{1--3} Among the responsible allergens, birch
pollen were identified as the third most-diagnosed allergen for
respiratory allergy,\textsuperscript{4} and around 70\% of patients
allergic to birch also develop a secondary food
allergy.\textsuperscript{5--7} The symptoms of this so-called
pollen-associated food allergy (PFA) range from nasal, ocular and oral
pruritus and angioedema to severe allergic asthma and
anaphylaxis,\textsuperscript{6,8--10} which has a comprehensive impact
on personal quality of life, work or school performance and the
socio-economic burden.\textsuperscript{4,11}

In the pathogenesis of PFA, patients are first sensitized towards a
pollen allergen, such as Bet v 1 (Bet), a birch pollen allergen
belonging to the pathogenesis-related (PR)-10 protein
family.\textsuperscript{12,13} PR-10 proteins share a high degree of
amino acid sequence identity as well as structural homology and
facilitate cross-reactivity of pollen-specific IgE antibodies and T
cells with food allergens, which initiates the development of secondary
food allergies.\textsuperscript{14--17} PR-10 proteins are found in
different plant pollen and in edible plant parts like hazelnut (Cor a 1,
Cor), apple (Mal d 1) and carrot (Dau c
1).\textsuperscript{7,18}Interestingly, the hazelnut allergen isoform
Cor a 1.04 is more similiar to the birch pollen isoallergen Bet v 1.0101
than to the hazel pollen isoallergen Cor a 1.01.\textsuperscript{19}

In contrast to the ongoing spread of allergic diseases due to
cross-reactivities, the selection of efficient and long-lasting
therapies that improve pollen and cross-reactive food allergies together
are sparse. The only disease-modifying therapy available is
allergen-specific immunotherapy (AIT), which might achieve a 20-50\%
reduction of combined symptom and medication
scores.\textsuperscript{20--22} But several years of therapy are needed
to maintain the therapeutic success\textsuperscript{23,24} and
placebo-controlled studies showed that pollen AIT have little to no
clinical effect on the associated food
allergy.\textsuperscript{25--28}Therefore, the current state of the art
regarding treatment of PFA remains unsatisfying and novel therapeutic
strategies have to be explored.

Autologous tolerogenic dendritic cells (tolDC) are promising candidates
for cell-based immunomodulatory therapies, as they can be differentiated
from peripheral blood cells of patients \emph{ex vivo} based on
standardized protocols.\textsuperscript{29--31} After loading with the
desired antigen, they are reintroduced into the patients, where they are
generally well tolerated without any severe side
effects.\textsuperscript{32--34} In our group, we found that Interleukin
(IL) 10-modulated DC (IL-10 DC) are exceptionally suited for
tolerance-inducing therapies, as they displayed a stable, tolerogenic
phenotype, with strong migratory and suppressive
functions.\textsuperscript{30,35--37} Induced Treg (iTreg) primed by
these IL-10 DC exhibited a suppressive phenotype resulting in highly
efficient antigen-specific regulatory
functions.\textsuperscript{38,39}In this study, we combined our
expertise in IL-10 DC and iTreg biology demonstrating the capacity of
IL-10 DC as inducers of iTreg which facilitated allergen-specific and
cross-reactive tolerance in birch pollen allergic patients with
associated hazelnut allergy \emph{in vitro} and in a humanized mouse
model of allergy \emph{in vivo} .

\textbf{Methods}

All experiments with human samples were conducted according to approval
of the local ethics committee of Rhineland Palatinate (authorization no.
837.303.13 and 837.054.17 (10888)) and written informed consent was
obtained from all participants. Murine experiments were in accordance
with current federal, state, and institutional guidelines (authorisation
no. 23 177-07/G 18-1-015).

Methods are described in brief, for further details see Supplementary
Methods.

\textbf{Induction of iTreg and effector T cells (Teff)}

Immature dendritic cells (DC) from donors with birch pollen and hazelnut
allergies were generated by culture of plastic-adherent peripheral blood
mononuclear cells (PBMC) with IL-4 and GM-CSF for 6 days and were then
differentiated into mature DC (mDC) by addition of a maturation cocktail
or into IL-10 DC by addition of the maturation cocktail and IL-10
(Supplementary Figure 1). Bet-loaded mDC (mDCBet) or IL-10 DC (IL-10
DCBet) were generated by addition of rBet v 1.0101 during DC
differentiation. Autologous CD4\textsuperscript{+} T cells were
cocultured with mDC or IL-10 DC to prime effector T cells (Teff) or
iTreg, respectively.

\textbf{Suppressor assay (SA)}

CD4\textsuperscript{+}CD25\textsuperscript{low} responder T cells
(Tresp) from donors with birch pollen and associated hazelnut allergy
were stimulated with autologous Bet- or Cor- loaded mDC (control, ctrl).
iTreg primed by IL-10 DC\textsubscript{Bet} (iTreg\textsubscript{Bet})
or IL-10 DC0 (iTreg\textsubscript{0}) were added in 1:1 or 1:2
Tresp:iTreg ratios (SA 1:1 / SA 1:2), respectively. Tresp, mDC and iTreg
were stained with different cell proliferation dyes for identification
and assessment of T cell proliferation.

\textbf{Humanized mouse model of type I allergy}

As described previously\textsuperscript{40,41},
NOD.CB17-Prkdc\textsuperscript{scid}/J\selectlanguage{greek}γ\selectlanguage{english}c\textsuperscript{-/-} mice were
engrafted with PBMC from donors with birch pollen and associated
hazelnut allergy and were simultaneously injected with birch pollen
extract (=PBMC + birch, allergic positive control) or without (=PBMC,
negative control). iTreg\textsubscript{Bet} were co-injected at day 0
into animals treated like the allergic positive control. After three
weeks, blood samples for human IgE analysis were taken, the mice were
challenged rectally with birch pollen or hazelnut extract and the
allergen-induced intestinal inflammation was scored endoscopically. On
the next day the mice were challenged intranasally with birch pollen
extract and allergen-induced airway inflammation was evaluated by
measurement of methacholine (MCh)-provoked airway resistance.

\textbf{Results}

\textbf{Induction of Bet-specific iTreg from birch-pollen allergic
patients with associated hazelnut allergy by autologous IL-10 DC}

As previously demonstrated by us, human IL-10 DC induce iTreg with a
high capacity to suppress T cell responses in an antigen-specific
manner.\textsuperscript{35,38,42,43} In this study, we wanted to
investigate whether these tolerogenic IL-10 DC are able to induce an
allergen-specific as well as cross-reactive tolerance in patients
suffering from a pollen (birch, Bet v 1) and associated food (hazelnut,
Cor a 1) allergy (for patients\selectlanguage{ngerman}´ details see Supplementary Table 1). For
this purpose, we used our previously established \emph{in vitro} model
with human DC to analyze allergen-specific and cross-reactive T cell
responses.\textsuperscript{12}

IL-10 DC obtained from birch pollen allergic patients with associated
hazelnut allergy were loaded with Bet or left unloaded (IL-10
DC\textsubscript{Bet}/IL-10 DC\textsubscript{0}) and cocultured with
autologous CD4\textsuperscript{+} T cells to induce Bet-specific or
control iTreg, respectively
(iTreg\textsubscript{Bet}/iTreg\textsubscript{0}). In addition, mDC
(mDC\textsubscript{Bet}/mDC\textsubscript{0}) were used to generate
Bet-specific and non-specific Teff
(Teff\textsubscript{Bet}/Teff\textsubscript{0}) as controls (see
Methods, Supplementary Methods and Supplementary Figure 1).

After primary culture, Teff\textsubscript{Bet} exhibited a significantly
increased proliferative capacity compared to Teff\textsubscript{0},
demonstrating an allergen (Bet)-specific T cell response (Figure 1A). In
contrast, stimulation with IL-10 DC\textsubscript{0} or
IL-10DC\textsubscript{Bet} resulted in a significantly reduced T cell
proliferation of both iTreg\textsubscript{0} and
iTreg\textsubscript{Bet}.

In line with the data of allergen-specific T cell proliferation,
analysis of cytokine production after primary culture revealed
significantly increased levels of T\textsubscript{H}2 cytokines (IL-5,
IL-9 and IL-13) and of IL-2 (T cell activation) in supernatants of
mDC\textsubscript{Bet} activated Teff\textsubscript{Bet} compared to
control Teff\textsubscript{0}, demonstrating highly stimulated
Bet-specific Teff (Figure 1B-E). In contrast, supernatants of iTreg
primed by IL-10 DC\textsubscript{0} or IL-10 DC\textsubscript{Bet},
respectively, exhibited reduced amounts of T\textsubscript{H}2 cytokines
in comparison to Bet-specific control Teff, confirming the diminished
activity of iTreg after primary culture. However, we observed very high
IL-10 concentrations produced by iTreg\textsubscript{Bet} compared to
control Teff\textsubscript{Bet}/Teff\textsubscript{0} as well as to
iTreg\textsubscript{0}, suggesting the immunosuppressive cytokine IL-10
as mediator of allergen-specific iTreg suppressor function (Figure 1F).

\textbf{Bet-specific iTreg induced by IL-10 DC exhibited an activated
phenotype after Bet-and Cor-specific restimulation}

Several studies revealed an anergic phenotype of
CD4\textsuperscript{+}CD25\textsuperscript{+}FOXP3\textsuperscript{+}natural
Treg and iTreg, in particular of iTreg induced by IL-10
DC.\textsuperscript{35,38,42,44} In order to prove the allergen-specific
and cross-reactive induction of T cell anergy in our system, we have
performed restimulation experiments with autologous
mDC\textsubscript{0}, mDC\textsubscript{Bet} or mDC\textsubscript{Cor},
respectively (see Supplementary Methods). Compared to control T cells
(Teff\textsubscript{0}), restimulation of Teff\textsubscript{Bet} with
mDC\textsubscript{Bet} led to a significantly increased and in the case
of mDC\textsubscript{Cor} slightly enhanced Teff proliferation,
demonstrating the induction of both a Bet-specific and cross-reactive,
Cor-specific T cell response (Figure 2A). However, also restimulation of
iTreg\textsubscript{Bet} with mDC\textsubscript{Bet} or
mDC\textsubscript{Cor} resulted in a very pronounced and significantly
enhanced T cell proliferation, compared to Teff\textsubscript{0}.
iTreg\textsubscript{Bet} proliferated even stronger compared to
Teff\textsubscript{Bet,} indicating that allergen-specific iTreg did not
display an anergic but activated phenotype after specific (Bet) or cross
(Cor)-specific restimulation (Figure 2A). In contrast to
iTreg\textsubscript{Bet}, iTreg\textsubscript{0} exhibited a
significantly lower proliferation and remained anergic regardless of the
allergen-specific or cross-specific restimulation with
mDC\textsubscript{Bet} or mDC\textsubscript{Cor} (Figure 2B).

\textbf{Profound capacity of Bet-specific iTreg to suppress
allergen-specific and cross-reactive T cell responses \emph{in vitro}}

In order to analyze the function of iTreg with regard to their
suppressive capacity on allergen-specific and cross-reactive responses,
we investigated the suppressive capacity of iTreg\textsubscript{Bet} and
iTreg\textsubscript{0} on Bet- or Cor-stimulated responder T cells
(Tresp) obtained from birch-pollen allergic patients with associated
hazelnut allergy in a flow cytometry-based suppressor
assay.\textsuperscript{35} (see Methods and Supplementary Methods;
gating strategy in Supplementary Figure 2).

Coculture with iTreg\textsubscript{Bet} resulted in a significantly
reduced proliferation of allergen (Bet) -specific, and cross-reactive,
Cor- specific Tresp, respectively, indicating the induction of
allergen-specific (birch) and cross-reactive (hazelnut) tolerance
(Figure 3A). Further analysis revealed that higher numbers of iTreg
(ratio 1:2) led to a significantly more impaired Tresp proliferation
regardless of the allergen-specificity, demonstrating a dose-dependency
of the iTreg suppressive capacity (Figure 3A). We also listed the
results from each individual allergic donor (Supplementary Figure 3A).

We further compared the suppressive activity of
iTreg\textsubscript{Bet}with non-specific iTreg\textsubscript{0} on
antigen-specific and cross-reactive T cell immune responses (Figure 3B).
The experiments revealed a moderately impaired Tresp proliferation after
coculture with iTreg\textsubscript{0}. However, iTreg\textsubscript{Bet}
exhibit a significantly higher suppressive potential for both Bet- and
Cor-specific T cell responses, indicating the strong potential of
antigen-specific stimulated iTreg\textsubscript{Bet} as inducers of
allergen-specific and cross-reactive tolerance in birch pollen- and
hazelnut-allergic patients. Further evidence is depicted in
Supplementary Figure 3B, which presents the corresponding data of each
individual donor. In 8 out of 10 (80\%) experiments with
Bet-stimulation, and 6 out of 8 (75\%) with Cor-stimulation,
iTreg\textsubscript{Bet} showed a stronger capacity to reduce Tresp
proliferation compared to iTreg\textsubscript{0,} suggesting their
specific and cross-reactive suppressive capacity as superior to
non-specific iTreg. Thus, the data demonstrated the high, specific and
dose-dependent suppressive capacity of iTreg\textsubscript{Bet} to
suppress allergen-specific (birch) as well as cross-reactive (hazelnut)
T cell responses, highlighting the importance of allergen-specific iTreg
induction.

To support our findings, we harvested the supernatants of suppressor
assay samples after Bet- or Cor-specific mDC-stimulation and analyzed
the T cell cytokine profile (Figure 3C,D). The presence of
iTreg\textsubscript{Bet} -- but not non-specific iTreg\textsubscript{0}-
resulted in significantly reduced levels of the T\textsubscript{H}2
cytokine IL-13 (Figure 3C) compared to pronounced cytokine levels
produced by control allergen-specific (both, Bet or Cor-stimulated)
responder T cells. These results strongly confirmed our T cell
proliferation data (Figure 3A,B) and indicated an allergen-specific and
cross\textbackslash{}sout-reactive downregulation of T\textsubscript{H}2
immunity mediated by Bet-specific iTreg (iTreg\textsubscript{Bet}).

We also found a profound and significant upregulation of the
immunosuppressive cytokine IL-10 in suppressor assays with cocultured
Bet-specific, but not with non-specific iTreg, when compared to control
(Figure 3D). Production of additional T\textsubscript{H}2 (IL-5, IL-9)
and T\textsubscript{H}1 (IFN-y, TNF-\selectlanguage{greek}α\selectlanguage{english}) cytokines were mostly unchanged
(see Supplementary Figure 4).

In addition to T cell proliferation and cytokine production, we analyzed
the phenotype of Tresp after presence or absence of
iTreg\textsubscript{Bet} in suppressor assays (Supplementary Figure 5A).
Coculture of Bet-stimulated Tresp with iTreg\textsubscript{Bet} in
suppressor assays resulted in a significantly impaired activation
(reduction of CD25, HLA-DR) and differentiation (reduced CD45RO /
increased CD45RA expression) of Bet-stimulated Tresp. Similar results
were observed for Cor-stimulated Tresp (Supplementary Figure 5B),
confirming the data of the allergen-specific as well as cross-reactive
suppressive capacity of iTreg\textsubscript{Bet} on Tresp proliferation
and IL-13 production.

\textbf{iTreg\textsubscript{Bet} displayed an activated and suppressive
phenotype after allergen-specific and cross-reactive stimulation}

In order to identify the phenotype of allergen-specific iTreg in more
detail, we performed a flow cytometric analysis of
iTreg\textsubscript{Bet}, thereby gating on proliferating (activated)
and non-proliferating iTreg\textsubscript{Bet} in the setting of
suppressor assays after Bet- and Cor-specific stimulation (by
allergen-loaded mDC) (Figure 4, gating strategy see Supplementary Figure
6). Allergen (Bet or Cor)-specifically restimulated proliferating
iTreg\textsubscript{Bet} populations exhibited significantly higher
percentages of CD45RO\textsuperscript{+} (enhanced differentiation into
a memory phenotype) and activated CD25\textsuperscript{+} and
HLA-DR\textsuperscript{+} cells compared to Bet- or Cor-specifically
stimulated non-proliferating iTreg\textsubscript{Bet} or Tresp,
respectively (Figure 4A). Compared to non-proliferating iTreg and to
Tresp, the proliferating iTreg\textsubscript{Bet} population was
characterized by a significantly enhanced expression of CTLA-4, TNFR2,
PD-1, IL-10 and ICOS, molecules known to be involved in the
immunosuppressive capacity of regulatory T cells, confirming the
regulatory phenotype of allergen-stimulated iTreg (Figure 4B). These
results emphasized the activation state induced by allergen-specific
(mDC\textsubscript{Bet}) or cross-reactive (mDC\textsubscript{Cor})
stimulation as prerequisite for iTreg-mediated suppressive activity. For
further characterization, the expression of CD49b and LAG3 as parameters
for Tr1 differentiation and Treg function were investigated (Figure
4C).\textsuperscript{45--48} Compared to Tresp,
iTreg\textsubscript{Bet}showed an increase in
CD49b\textsuperscript{+}LAG3\textsuperscript{+}cells after
mDC\textsubscript{Bet} stimulation, although this was not significant
under mDC\textsubscript{Cor} stimulation.

\textbf{Bet-specific iTreg ameliorated allergic symptoms in humanized
mouse models of allergen-induced airway and intestinal inflammation}

Humanized mice are a remarkable investigative tool and preclinical study
system, which close the gap between exclusively murine and human
studies.\textsuperscript{49} Here, we used well-established and
standardized humanized mouse models of type I allergy to analyze the
function of iTreg\textsubscript{Bet} on allergic symptoms in
allergen-induced intestinal and airway
inflammation.\textsuperscript{41,50,51} An overview of the
reconstitution and challenge protocol is visualized in Figure 5A (see
also Methods and Supplementary Methods). Briefly, immunodeficient mice
(NOD.CB17-Prkdc\textsuperscript{scid}/J\selectlanguage{greek}γ\selectlanguage{english}c\textsuperscript{-/-}) were
engrafted with human PBMC from birch pollen allergic donors with
associated hazelnut allergy and injected with birch pollen extract +/-
iTreg\textsubscript{Bet} as indicated.\textsuperscript{41,52} After
allergen-specific (birch) rectal challenge the intestinal inflammation
was evaluated by a clinical score, that revealed a significantly
increased inflammatory reaction of animals serving as allergic positive
controls (PBMC + birch) compared to negative controls (PBMC), thereby
demonstrating the validity of the humanized model and the development of
an allergic immune reaction (Figure 5B, C). Intriguingly, injection of
iTreg\textsubscript{Bet} resulted in a significant abrogation of the
allergen-specific immune reaction as shown by a reduced intestinal
inflammation (Figure 5B, C).

For induction of allergic asthma, the animals were engrafted and boosted
as described above and were challenged intranasally with birch pollen
extract as published previously.\textsuperscript{40,51} Subsequently,
the airway resistance was assessed as outcome of the allergic immune
reaction.\textsuperscript{40,51} In these experiments, allergic positive
controls (PBMC + birch) showed a significantly enhanced airway
hyperreactivity in contrast to negative control groups (PBMC) (Figure
5D). Importantly, co-injection of iTreg\textsubscript{Bet} curtailed the
development of allergic asthma symptoms, as the airway resistance was
significantly decreased compared to allergic asthma positive control
animals (Figure 5D).

We also analyzed the serum concentrations of human birch-specific IgE
(Figure 5E) as typical immunological parameter of type I allergic
reactions prior to allergen challenge. Compared to enhanced amounts of
birch-specific IgE in allergic positive controls (PMBC + birch),
treatment with iTreg\textsubscript{Bet} significantly reduced
birch-specific IgE concentrations \emph{in vivo} , confirming our data
of significantly reduced clinical symptoms of allergen-induced
intestinal and airway inflammation after iTreg\textsubscript{Bet}
application.

In order to investigate the induction of cross-reactive
tolerance\emph{in vivo} , the immunodeficient mice were engrafted and
boosted as described above, but were challenged rectally with hazelnut
extract prior to assessment of the intestinal inflammation (Figure 6).
In these experiments, we found a less severe allergic immune response in
the hazelnut-challenged compared to the birch-challenged control group
(Figure 5C), likely due to lack of \emph{in vivo} booster with hazelnut
extract and/or to the donors' less severe sensitization towards the food
allergen. However, even after hazelnut challenge co-injection of
iTreg\textsubscript{Bet} resulted in a pronounced inhibition of
intestinal allergic symptoms compared to control animals, which was
shown with data of individual experiments (Figure 6A). In addition, we
observed reduced human hazelnut-specific IgE levels after
iTreg\textsubscript{Bet} application and hazelnut challenge compared to
the allergic positive control (Figure 6B). These data indicate the
induction of allergen-specific (birch) as well as cross-reactive
tolerance (hazelnut) \emph{in vivo} through IL-10 DC-induced
iTreg\textsubscript{Bet}.

\textbf{Discussion}

Although PFA is a widespread allergic disorder, the only available
disease-modifying therapy for pollen allergy has a rather limited effect
on the associated food allergy.\textsuperscript{25--27} Therefore in
this study, human monocyte-derived IL-10 DC were investigated with
regard to their potential to induce allergen-specific (birch) and
cross-reactive (hazelnut) tolerance in birch pollen allergic patients
with associated hazelnut allergy \emph{in vitro} and \emph{in vivo} . We
found that IL-10 DC induce Bet-specific iTreg which show a regulatory
phenotype and strong suppressive capacities to inhibit allergen-specific
and cross-reactive immune responses \emph{in vitro} . In addition,
Bet-specific iTreg were able to ameliorate allergic symptoms \emph{in
vivo} in a humanized mouse model of allergic intestinal and airway
inflammation.

PFA results from highly conserved protein structures of pollen (e.g.
Bet) and food allergens (e.g. Cor), which was shown to facilitate
cross-reactions on IgE level and in T cell
clones.\textsuperscript{12,53,54} In a previous study, we confirmed the
data of cross-reactivity between pollen and food allergens in primary T
cells directly obtained from patients with allergies to birch pollen and
associated food allergens.\textsuperscript{12} Here, we focused on the
induction of allergen-specific and cross-reactive iTreg to modulate the
primary and secondary allergic immune response in patients suffering
from PFA.

Bet-specifically stimulated iTreg but not non-specific iTreg underwent
vigorous proliferation towards Bet- and Cor-induced restimulation,
suggesting activation as prerequisite for suppressive activity. In this
context, \emph{Pellerin et al.} investigated peanut-specific Tr1 cells
induced \emph{in vitro} by IL-10 DC from allergic
subjects.\textsuperscript{55} They found a highly proliferative
phenotype with T\textsubscript{H}2-cytokine profile upon peanut-specific
restimulation and, in contrast to our data, suggested a functional
impairment of the peanut-specific Tr1 subset.\textsuperscript{55} Our
experiments revealed that IL-10 DC-induced Bet-specific iTreg did have
the ability to suppress allergen-specific responder T cell
proliferation, displayed an activated and suppressive phenotype, even
though they were highly proliferative. This discrepancy might be due to
(1) different protocols for IL-10 DC culture and Treg generation, (2)
different allergen-specific immune responses and/or (3) lack of
functional assays in the study by \emph{Pellerin et al}
.\textsuperscript{55} Anergy has been initially described as a
fundamental characteristic of functional Treg, but this idea has hence
been revised: although breaking Treg anergy can be accompanied by loss
of suppressive function, this is not always the
case.\textsuperscript{56} In fact, it was shown that proliferating Treg
can suppress T cell responses \emph{in vivo} \textsuperscript{56,57} and
Treg that have been stimulated to proliferate can even display an
enhanced suppressive capacity.\textsuperscript{57--59}

One very crucial aspect of therapeutic tolerance induction is the
allergen-specificity. We are therefore thrilled to report that
Bet-specific iTreg showed significantly greater abilities to suppress
allergen-specific responder T cell proliferation than non-specific
iTreg, as was seen in \emph{in vitro} suppressor assays from up to 80\%
of allergic donors. These results were strongly supported by the T cell
cytokine profile in suppressor assays. Here, we found that Bet-specific
iTreg significantly decreased levels of the T\textsubscript{H}2 cytokine
IL-13, which was not achieved with non-specific iTreg. IL-13 is an
IgE-promoting T\textsubscript{H}2 cytokine, which contributes to airway
inflammation and food-induced anaphylaxis in asthma and type 1
allergies.\textsuperscript{60,61} In line with these data, amounts of
the immunosuppressive cytokine IL-10 were significantly increased in the
presence of Bet-specifically primed iTreg, which was not the case for
non-specific iTreg. Accordingly, IL-10 is well known for its suppressive
function in regulatory T cell activity, and particularly in control of
T\textsubscript{H}2-driven allergic diseases.\textsuperscript{62} As the
described cytokine shift was observed for both, Bet- and Cor-specific
responder T cells after coculture with Bet-stimulated iTreg - but not
with non-specific iTreg\textsubscript{0}- these data underlined the
induction of an allergen-specific (birch) and cross-reactive tolerance
(hazelnut) through IL-10 DC-induced Bet-specific iTreg priming.

In addition, Bet-specific iTreg were able to ameliorate asthmatic and
intestinal allergic symptoms provoked by challenge with birch extract
and reduced birch-specific IgE in allergic mice \emph{in vivo} . These
combined pieces of evidence strongly suggest an allergen-specific
tolerance induction \emph{in vitro} and \emph{in vivo} by iTreg which
have been stimulated by allergen-loaded tolerogenic IL-10 DC.
Intriguingly, Bet-specific iTreg also reduced the allergic gut
inflammation and hazelnut-specific IgE levels \emph{in vivo} after
challenge with the hazelnut extract in mice engrafted with PBMC from
birch pollen allergic patients with associated hazelnut allergy,
facilitating cross-reactive tolerance.

Aiming to replace general immunosuppressive therapies, tolerogenic DC
(tolDC) have been applied as antigen-specific immune-suppressors in
numerous phase 1 clinical trials for multiple sclerosis, type I
diabetes, rheumatoid arthritis and organ
transplantation.\textsuperscript{32,34,63--65} In all studies, tolDC had
negligible adverse effects and did not worsen disease symptoms. Clinical
outcomes were only investigated in a few trials so far but preliminary
evidence for antigen-specific tolerance induction was found.

In a comparative study by \emph{Boks et al.} IL-10 modulation for human
tolDC generation was identified as the protocol most suited for tolDC
vaccination.\textsuperscript{66} We developed a protocol for IL-10 DC
that resulted in a subpopulation of tolerogenic
CD83\textsuperscript{high}CCR7\textsuperscript{+} IL-10 DC that exhibit
a high migratory activity, stability to pro-inflammatory stimuli and
profound capacity to induce iTreg with a strong suppressive
function.\textsuperscript{35} In our current study, we did show that
human IL-10 DC through priming of allergen-stimulated iTreg are able to
induce specific- and cross-reactive tolerance \emph{in vitro}
and\emph{in vivo} and, therefore are promising candidates to modulate
pollen as well as associated food allergies.

Combined with previous findings by us and other
groups,\textsuperscript{35,51,66--68} our study results might further
support the development of DC-based tolerance-inducing therapies for
allergic and autoimmune diseases.

\textbf{Acknowledgements}

Our gratitude is owed to all patients and donors who contributed to this
work.

We thank Elsbeth Sellenies-Huber, Sandra Wagner-Urban, Anja Bertsch and
Kevin L\selectlanguage{ngerman}öscher (all Department of Dermatology, University Medical Center
of the Johannes Gutenberg-Universität, Mainz, Germany) for the blood
sample collections, Annette Jamin (Paul-Ehrlich-Institut, Langen,
Germany) for technical support and Toni Anusic (Institute of Medical
Biometry, Epidemiology and Informatics (IMBEI) of the Johannes
Gutenberg-Universität, Mainz, Germany) for statistical guidance.

This work was supported by the German Research Foundation (DFG): STE
791/9-1, TR156/A4/C05-246807620, SFB1009/B11-194468054,
SFB1066/B06-213555243 and SFB1450/C06-431460824 (all to KS)

\textbf{Figure Legends}

\textbf{Figure 1: iTreg induced by unloaded or Bet-loaded IL-10 DC
displayed an anergic phenotype after primary culture.} PBMC for DC
generation and T cells were obtained from birch-pollen allergic patients
with associated hazelnut allergy. CD4\textsuperscript{+} T cells were
primed with autologous unloaded IL-10 DC (IL-10 DC0) and Bet-loaded
IL-10 DC (IL-10 DCBet) or mDC (mDC0/mDCBet), respectively, to induce
non-specific and Bet-specific iTreg
(iTreg\textsubscript{0}/iTreg\textsubscript{Bet}) and Teff
(Teff\textsubscript{0}/Teff\textsubscript{Bet}, as
controls).\textbf{(A)} After 3 days of iTreg induction, the coculture
was pulsed with {[}\textsuperscript{3}H{]}TdR for 16-18 h to assess T
cell proliferation which is shown as stimulation index (SI, mean ± SD)
normalized to T cells stimulated with mDC\textsubscript{0} (SI = 1). The
data are pooled from 34 independent experiments. \textbf{(B-F)} Cytokine
concentrations (\textbf{B} IL-5, \textbf{C} IL-9, \textbf{D}
IL-13,\textbf{E} IL-2 and \textbf{F} IL-10) in the supernatants of
primary cultures are depicted as mean ± SD normalized to ctrl (=1). P
values calculated with paired student's t-test are depicted as
asterisks: **** p \textless{} 0.0001, ** p \textless{} 0.01, * p
\textless{} 0.05, ns = not significant (p \textgreater{} 0.05). PC:
primary culture (induction)

\textbf{Figure 2: Bet-specific iTreg lost their anergic phenotype after
Bet- and Cor-induced restimulation.} In primary culture (PC),
Teff\textsubscript{0}, Teff\textsubscript{Bet},
iTreg\textsubscript{0}and iTreg\textsubscript{Bet} were primed for five
days by coculture of CD4\textsuperscript{+} T cells with autologous
unloaded and Bet-loaded mDC or IL-10 DC, respectively. After a
subsequent resting phase of 3 days, T cells were restimulated (RS) with
unloaded, Bet-loaded or Cor-loaded mDC and were pulsed with
{[}\textsuperscript{3}H{]}TdR for 16-18 h on day 3. T cell proliferation
is presented as SI (mean ± SD) normalized to Teff\textsubscript{0}
stimulated with mDC\textsubscript{0}(SI = 1). \textbf{(A)} Bet- and
Cor-stimulated T cell proliferation of Teff\textsubscript{0},
Teff\textsubscript{Bet} and iTreg\textsubscript{Bet} was pooled from 8
independent experiments.\textbf{(B)} Data of 9 independent experiments
were pooled demonstrating T cell proliferation of
iTreg\textsubscript{Bet} and iTreg\textsubscript{0} restimulated with
mDC\textsubscript{Bet} or mDC\textsubscript{Cor,} respectively. P values
calculated with paired student's t-test are depicted as asterisks: ** p
\textless{} 0.01, * p \textless{} 0.05, ns = not significant (p
\textgreater{} 0.05). PC: primary culture (induction), RS: restimulation

\textbf{Figure 3: Bet-specific iTreg inhibited allergen-specific and
cross-reactive responder T cell responses \emph{in vitro}.} Bet- and
Cor-specific proliferation of autologous
CD4\textsuperscript{+}CD25\textsuperscript{low} responder T cells
(Tresp) obtained from birch-pollen allergic donors with associated
hazelnut allergy was induced by stimulation with
mDC\textsubscript{Bet}(green) or mDC\textsubscript{Cor} (brown),
respectively. For analysis of the suppressive activity iTreg
(iTreg\textsubscript{Bet} or iTreg\textsubscript{0}) were added in a
Tresp:iTreg ratio of 1:1 or 1:2, respectively. DC, Tresp and iTreg were
stained with different proliferation dyes for cell identification and
assessment of proliferation by flow cytometry (see Supplementary Figure
2 for gating strategy). \textbf{(A, B)} Percentage of proliferating
Tresp are shown from one representative experiment (top) and as pooled
data (mean ± SD) relative to control ( = 100\%) from independent
experiments (bottom, number of independent experiments indicated below).
\textbf{(A)}Percentage (mean ± SD) of proliferating Bet- (upper panel,
green) and Cor-stimulated (lower panel, brown) Tresp, cocultured with
iTreg\textsubscript{Bet} in the ratios 1: 1 and 1:2 , respectively, are
demonstrated (Pooled data: Bet-stimulation: n= 15; Cor-stimulation:
n=14). \textbf{(B)} Function of iTreg\textsubscript{Bet} was compared to
iTreg\textsubscript{0} in suppressor assays with Tresp:iTreg = 1:2 and
percentages (mean ± SD) of proliferating Bet- and Cor-stimulated Tresp
are depicted as pooled data (Bet-stimulation: n= 10; Cor-stimulation:
n=8). \textbf{(C)} IL-13 (n=5-9) and \textbf{(D)} IL-10 concentrations
(n=5-10) in the supernatants of suppressor assay samples are depicted as
mean ± SD normalized to ctrl (=1). P values calculated with paired
student's t-test are depicted as asterisks: **** p \textless{} 0.0001,
*** p \textless{} 0.001, ** p \textless{} 0.01, * p \textless{} 0.05, ns
= not significant (p \textgreater{} 0.05).

\textbf{Figure 4: Bet- and Cor-specifically stimulated iTreg showed an
activated and suppressive phenotype.} Prior to flow cytometry analysis
of suppressor assays, T cell populations were stained for expression of
extra- and intracellular markers and with different cell proliferation
dyes (see supplementary Figure 5 for gating strategy) to distinguish
between proliferating and non-proliferating T cells.
\textbf{(A)}Activation and \textbf{(B)} immunosuppressive parameters are
pooled from independent experiments as indicated (CD45RO n=5, CD25 n=9,
HLA-DR n=10, CTLA-4 n=10, TNFR2 n=9, PD-1 n=10, IL-10 n=5, ICOS n=5) and
expression is shown as percentage (mean ± SD), except for ICOS which is
presented as the mean fluorescence intensity (mean ± SD). \textbf{(C)}
Five independent experiments were pooled to show the percentage (mean ±
SD) of CD49b\textsuperscript{+}LAG3\textsuperscript{+} T cells. P values
calculated with paired student's t-test are depicted as asterisks: ****
p \textless{} 0.0001, *** p \textless{} 0.001, ** p \textless{} 0.01, *
p \textless{} 0.05, ns = not significant (p \textgreater{} 0.05).

\textbf{Figure 5: Bet-specific iTreg abrogated birch-specific allergic
symptoms in humanized mouse models of allergen-induced intestinal and
airway inflammation.} \textbf{(A)} Immunodeficient mice were engrafted
with human PBMC from allergic donors suffering from birch pollen and
associated hazelnut allergy and were boosted twice (d0, d8) with birch
pollen extract to induce allergic immune responses. iTreg were
co-injected to analyze their impact on allergy development. After three
weeks, blood samples for IgE analysis were collected and subsequently
the mice were challenged with birch pollen extract either rectally for
induction of allergic intestinal inflammation or intranasally for
allergic asthma induction. \textbf{(B)} The allergic intestinal
inflammation was monitored by mini-endoscopy and scoring of colitis
activity. One representative set of pictures is shown.
\textbf{(C)}Quantitative endoscopic assessment of colitis activity in
all groups is shown as mean ± SD from 8 independent experiments.
\textbf{(D)} Results of airway resistance as parameter of allergic
asthma were pooled from four independent experiments and are depicted as
relative changes to baseline in \% (mean ± SD). \textbf{(E)} The
concentration of birch-specific IgE was obtained from 7 independent
experiments as indicated and are presented in kU/L. P values calculated
with paired student's t-test are depicted as asterisks: **** p
\textless{} 0.0001, ** p \textless{} 0.01, * p \textless{} 0.05. Figure
5A was created using Servier Medical Art (http://smart.servier.com/).

\textbf{Figure 6: Bet-specific iTreg facilitated a cross-tolerance to
hazelnut in a humanized mouse model of allergic gut
inflammation.}Immunodeficient mice were treated as described in Figure
5A. After three weeks, the mice were challenged rectally with either
birch pollen or hazelnut extract, respectively, 2 h prior to scoring of
the intestinal inflammation by mini-endoscopy. \textbf{(A)} The
endoscopic score of the allergic intestinal inflammation of four
independent experiments is depicted as pooled data (mean ± SD).
\textbf{(B)} Blood samples were collected and human hazelnut-specific
IgE was analyzed and is shown as mean value ± SD. Lines connect values
from individual experiments. P values calculated with paired student's
t-test are shown as asterisks: *** p \textless{} 0.001, ** p \textless{}
0.01, ns = not significant (p \textgreater{} 0.05)

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