Figure Legends
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+ 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 (iTreg0/iTregBet) and Teff
(Teff0/TeffBet, as controls).(A) After 3 days of iTreg induction, the coculture was pulsed
with [3H]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 mDC0 (SI = 1). The
data are pooled from 34 independent experiments. (B-F) Cytokine
concentrations (B IL-5, C IL-9, D IL-13,E IL-2 and 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 < 0.0001, ** p < 0.01, * p < 0.05, ns =
not significant (p > 0.05). PC: primary culture (induction)
Figure 2: Bet-specific iTreg lost their anergic phenotype after
Bet- and Cor-induced restimulation. In primary culture (PC),
Teff0, TeffBet, iTreg0and iTregBet were primed for five days by coculture of
CD4+ 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 [3H]TdR for
16-18 h on day 3. T cell proliferation is presented as SI (mean ± SD)
normalized to Teff0 stimulated with mDC0(SI = 1). (A) Bet- and Cor-stimulated T cell proliferation of
Teff0, TeffBet and
iTregBet was pooled from 8 independent experiments.(B) Data of 9 independent experiments were pooled demonstrating
T cell proliferation of iTregBet and
iTreg0 restimulated with mDCBet or
mDCCor, respectively. P values calculated with paired
student’s t-test are depicted as asterisks: ** p < 0.01, * p
< 0.05, ns = not significant (p > 0.05). PC:
primary culture (induction), RS: restimulation
Figure 3: Bet-specific iTreg inhibited allergen-specific and
cross-reactive responder T cell responses in vitro. Bet- and
Cor-specific proliferation of autologous
CD4+CD25low responder T cells
(Tresp) obtained from birch-pollen allergic donors with associated
hazelnut allergy was induced by stimulation with mDCBet(green) or mDCCor (brown), respectively. For analysis of
the suppressive activity iTreg (iTregBet or
iTreg0) 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). (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). (A)Percentage (mean ± SD) of proliferating Bet- (upper panel, green) and
Cor-stimulated (lower panel, brown) Tresp, cocultured with
iTregBet in the ratios 1: 1 and 1:2 , respectively, are
demonstrated (Pooled data: Bet-stimulation: n= 15; Cor-stimulation:
n=14). (B) Function of iTregBet was compared to
iTreg0 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). (C) IL-13 (n=5-9) and (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 < 0.0001,
*** p < 0.001, ** p < 0.01, * p < 0.05, ns
= not significant (p > 0.05).
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. (A)Activation and (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). (C) Five
independent experiments were pooled to show the percentage (mean ± SD)
of CD49b+LAG3+ T cells. P values
calculated with paired student’s t-test are depicted as asterisks: ****
p < 0.0001, *** p < 0.001, ** p < 0.01, *
p < 0.05, ns = not significant (p > 0.05).
Figure 5: Bet-specific iTreg abrogated birch-specific allergic
symptoms in humanized mouse models of allergen-induced intestinal and
airway inflammation. (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. (B) The allergic intestinal
inflammation was monitored by mini-endoscopy and scoring of colitis
activity. One representative set of pictures is shown. (C)Quantitative endoscopic assessment of colitis activity in all groups is
shown as mean ± SD from 8 independent experiments. (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). (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 < 0.0001,
** p < 0.01, * p < 0.05. Figure 5A was created using
Servier Medical Art (http://smart.servier.com/).
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. (A) The endoscopic score of the
allergic intestinal inflammation of four independent experiments is
depicted as pooled data (mean ± SD). (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 < 0.001, ** p < 0.01, ns = not significant (p
> 0.05)