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\begin{document}

\title{VitaminD3 regulates T cell immune responses in allergen and Rhinovirus
induced asthma}



\author[1]{Patricia Haag}%
\author[1]{Daria Andreev}%
\author[1]{Nina Li}%
\author[1]{Alexander Kiefer}%
\author[1]{Manfred Rauh}%
\author[1]{Luisa Gleich}%
\author[1]{Stefan Wirtz}%
\author[2]{Nikolaos Papadopoulos}%
\author[1]{Susetta Finotto}%
\affil[1]{Uniklinikum Erlangen}%
\affil[2]{University of Manchester}%


\vspace{-1em}



  
  \date{May 13, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: Serum 25(OH)-Vitamin D3 (VitD3) deficiency during infancy
has been associated with asthma. The potential therapeutic role of VitD3
given in the airways and its interference with the allergen and
Rhinovirus was the objective of this study. Methods: In two cohorts of
children with and without asthma, serum levels of the C-reactive protein
(CRP) were correlated to Serum VitD3 and in peripheral blood T cell
inhibitor marker Programmed cell death protein 1 (PD1) mRNA was
analyzed. In a murine model, VitD3 was given intranasally in vivo and in
vitro to lung cells with allergen and Rhinovirus. Results: In the
cohorts of pre-school age children without (control) asthma, CRP and
VitD3 levels inversely correlated. In preschool asthmatic children that
did not receive VitD3 supplementation as infant had more episode of
asthma exacerbation associated with high CRP serum level. In peripheral
blood cells from control but not asthmatic children with higher serum
levels of VitD3 had lower PD1 mRNA levels. In murine model, OVA
intranasal challenge induced Innate Lymphoid Cells type 2
(ILC2)-associated markers and Eosinophils in BALF and VitD3 inhibited
lung inflammation and ILC2 markers. Furthermore, VitD3 given
intranasally, induced CD4+T cells and reduced PD1, T regulatory cells in
the lung. Similarly, VitD3 had a suppressive role on CD4+PD1+ T cells
involved in T cell exhaustion in the airways in the absence of ST2 after
Rhinovirus infection. Conclusion: These data support an inhibitory role
of VitD3 on T cell exhaustion after allergen and rhinovirus infection
that is relevant for pediatric asthma.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Allergic asthma is a chronic-inflammatory disease of the airways that
affects millions of people worldwide especially
children\textsuperscript{1,2}.

Different pathological form of hyperreaction to allergen, like allergic
asthma, atopic dermatitis and food allergy are associated to
allergy-induced local inflammation driven by Th2 driven
immune-reaction\textsuperscript{3,4}. By contrast, under homeostatic
conditions, the airways are kept pathogen free by the function of cells
of the innate immunity\textsuperscript{5}. Moreover,
1,25-Dihydroxyvitamin D3 (VitD3) has been suggested as a therapeutic
compound for allergic asthma.\textsuperscript{6-8} In Germany, VitD3 is
given to the infants as supplement\textsuperscript{9} to avoid
immunological suppression especially in children which could not receive
breastfeeding. Wheezing is associated with asthma in the first years of
life.\textsuperscript{10} Moreover, RV is the factor that associated
with wheezing in infants. In this study, we wanted to better understand
the role of Vitamin D3 in pediatric asthma.

Most of the medication for asthma are given directly into the airways,
we thus thought to investigate in murine model, the role of VitD3 given
intranasally \emph{in vivo} . Recent studies described the role Innate
lymphoid cells (ILC2s) in the airways in models of allergic
asthma.\textsuperscript{11-13} When the allergen enters the airways, it
interacts with epithelial cells, first. This interaction results in
epithelial release of alarmins like IL-33, which activate ILC2 via
ST2\textsuperscript{14}. Once stimulated, ILC2s secrete IL-5, IL-9 and
IL-13, classically known as Th2 cytokines.

Here we focused on the influence of VitD3 in blood cells from two
pediatric cohorts with and without asthma. Moreover, we analysed the
effect of VitD3 given intranasally, on allergen induced airway tolerance
with focus on T regulatory cells and Innate Lymphoid Cell associated
markers. \textsuperscript{15}

The role of T regulatory immunosuppressive cells has been extensively
studied in airway tolerance\textsuperscript{16,17}, by contrast, the
role of ILC2 in airway tolerance is less understood.

Here we found that VitD3 inhibited key ILC2/ST2 markers like IL-33,
Amphiregulin (AREG) and Ror-alpha, and PD1 and induced CD4+ T
cells,\emph{in vivo} in the airways. Similarly, VitD3 had a suppressive
role on CD4+PD1+ T cells involved in T cell exhaustion in the airways in
the absence of ST2 after Rhinovirus infection.

Our findings suggest a protective role of VitD3 in antagonizing ILC2
markers in preschool children thus providing a rationale for exploring
VitD3 in immunotherapy in pediatric asthma.

\textbf{Materials and Methods}

HUMAN STUDIES

The present study is part of the prospective study PreDicta
(Post-infectious immune reprogramming and its association with
persistence and chronicity of respiratory allergic diseases). The
working package 1 (WP1) relates to studies in pre-school children with
and without asthma. The study WP1 in Erlangen was approved by the local
Ethics Committee of the Universit\selectlanguage{ngerman}ätsklinikum Friedrich-Alexander
Universität Erlangen-Nürnberg (Re-No 4435) and it is registered in
German Clinical Trials Register (www.germanctr.de: DRKS00004914).
Parents/ guardians of all participants gave their informed consent. Two
cohorts of pre-school children (age 4-6 years) with and without asthma
were analysed (control children n=22 and asthma children n=24). The
recruitment of the subjects, inclusion and exclusion criteria as well as
the timescale for clinical visits and data collection along with the
clinical aspects and characteristics were previously
described\textsuperscript{6, 18,19}.

\textbf{Serum 25(OH)-Vitamin-D3 measurements.}

25(OH)-Vitamin-D3 (=25(OH)VitD3) was measured in serum from baseline
visits at the Dept. of Paediatrics and Adolescent Medicine of the
Friedrich-Alexander Universität Erlangen-Nürnberg (Erlangen, Germany).
Thereby LC-MS/MS MassChrom Kit from Chromosystems was used according to
the manufacturers protocol.

\textbf{Detection of C-reactive protein (CRP).}

CRP values in serum samples of the children were analyzed on a Roche
(Basel) Integra 800 Analyzer by turbidimetry as previously described 1
(CRPL2 reagent, interday CV 1.4\% (8.1 mg/L), limit of detection
1.0mg/L).

\textbf{Human RNA Isolation from Tempus Tubes and quantitative Real-Time
PCR.}

At Baseline visit whole blood was collected in Tempus® Blood RNA Tubes
(Life Technologies\selectlanguage{english}, GmbH, Darmstadt, Germany) and RNA was extracted
with the MagMax for Stabilized Blood Tubes RNA Isolation Kit. Synthesis
of cDNA and consequently real time-PCR were performed as described for
murine cells below with the following primers andsequences: hHPRT
(5'-TGA CAC TGG CAA AAC AAT GCA-3', 5'-GGT CCT TTT CAC CAG CAA GCT-3'),
PD-1 (fw: 5'-CAG TTC CAA ACC CTG GTG GT- 3'; rev: 5'-GGC TCC TAT TGT CCC
TCG TG- 3').

\textbf{FEV1} .

The Spirometry method for the lung function analysis was performed by
using a Vyaire Bodyplethysmograph (Chicago,USA). ERS Criteria were
followed during the study. The quality control was performed by an
experienced technician and a physician and supported by the
software.FEV1 (=Forced expiratory volume in 1 second) and FVC (=Forced
vital capacity) were measured at Baseline visit (B0) by using
spirometry. After a period of normal breathing the participant should
inhale maximal, directly followed by maximal and fast exhalation. The
volume exhaled in one second is FEV1. The total exhaled volume is FVC.
The ratio FVC/FEV1 is stated as FEV1\%.

\textbf{MICE}

All mice were mantained under specific pathogen free conditions. They
had free access to food and water. The experiments were approved by the
government of Mittelfranken, Bavaria (54-2532.1-2/10).

\textbf{Balb/c Wt in vivo treatment with OVA / Vitamin D3.}

Female Balb/c wild-type mice at the age of 8-9 weeks were treated at day
0 intranasally with 25\selectlanguage{greek}μ\selectlanguage{english}l PBS, PBS + Vitamin D3 (=1\selectlanguage{greek}α,\selectlanguage{english}25
DihodroxyvitaminD3 from Sigma Aldrich, 10 ng), Ovalbumin (500 \selectlanguage{greek}μ\selectlanguage{english}g) or OVA
+ Vitamin D3 (500\selectlanguage{greek}μ\selectlanguage{english}g and 10 ng respectively, with 20 min time delay in
between) according to their group. After 48h the treatment was repeated.
After additional 48 h whole lung cells were isolated as described
previously and cultured with anti-CD3 (10\selectlanguage{greek}μ\selectlanguage{english}g/ml) respectively
anti-CD3(10\selectlanguage{greek}μ\selectlanguage{english}g/ml) and anti-CD28 (1\selectlanguage{greek}μ\selectlanguage{english}g/ml) antibodies\textsuperscript{17}.

\textbf{Collection and analysis of the BAL}

Bronchoalveolar lavage was performed 24h after the last allergen
challenge, by intratracheally injecting and aspirating 0.8 ml saline
twice. After its collection the BALF was centrifuged for 5 min at 1500
rpm.

The cell pellets were resuspended in 1 ml PBS and an aliquot was stained
with trypan blue solution and cells were counted using a Neubauer
chamber. Eosinophils and neutrophils were detected by
fluorescence-activated cell sorting (FACS) analysis. The cell surface
staining was performed with antibodies against CD3 (eBioscience,
Frankfurt, Germany), GR-1 (BD Bioscience, Heidelberg, Germany), CD45R
(eBioscience, Frankfurt, Germany) and CCR3 (BD Bioscience, Heidelberg,
Germany) for 30 min at 4\selectlanguage{ngerman}°C.

\textbf{Histological analysis}

Lung tissues were analyzed by using paraffin-embedded tissue slices for
histology. After staining with Giemsa staining, peribronchial and
perivascular inflammation was assessed by a pathologist blinded to the
experimental group assignments of the individual lungs. Inflammation was
graded by using a semi-quantitative scoring system with a range pending
between 1 (mild) and 4 (severe) as described before\textsuperscript{17}.

Bl6/C57 WT, ST2-/- in vitro treatment with Rhinovirus, OVA / Vitamin D3.

{\label{bl6c57-wt-st2---in-vitro-treatment-with-rhinovirus-ova-vitamin-d3.}}\par\null{\label{section}}

We analyzed lung cells from Wild-type and ST2-/- mice at the age of 7-9
weeks. All mice were based on a Bl6/C57 genetic background. Whole lung
cells were isolated from the mice on day 0 and partly incubated with
Rhinovirus (250\selectlanguage{greek}µ\selectlanguage{english}l RV/106 cells) for 1 hour at room temperature on a
horizontal shaker. Afterwards OVA (500 mg/ml) and/or Vitamin D3 (10nM)
were added to the cells infected with RV as well as to those not
infected. Without further stimulation, cells were then cultured for 24h
at 37\selectlanguage{ngerman}° and 5\% CO2.

\textbf{RNA isolation and quantitative real time-PCR}

To extract RNA from murine lung cells we used PeqGold RNA Pure according
to the manufacturer's protocol (PeqLab, Erlangen, Germany). For reverse
transcription of RNA (1\selectlanguage{greek}μ\selectlanguage{english}g), we used the first strand cDNA synthesis kit
for RT-PCR (MBI Fermentas, Sat. Leon-Rot, Germany) followed by the
amplification by quantitative real-time PCR (qPCR) using SoFast EvaGreen
Supermix (Bio-Rad Laboratories, M\selectlanguage{ngerman}ünchen, Germany). The qPCR itself was
performed in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad
Laboratories) with a cycle of 2 min 98°C, 50 cycles at 5 s 95°C, 10 s
60°C, followed by 5 s 65°C and 5 s 95°. The primers and sequences used
for mouse were: mHPRT (5'- GCC CCA AAA TGG TTA AGG TT-`3, 5'-TTG CGC TCA
TCT TAG GCT TT-`3), mICOS (5'- GTG CAG CTT TCG TTG TGG TA --`3, 5' - TCA
GGG GAA CTA GTC CAT GC --`3), mIL33 (5'- CCT CCC TGA GTA CAT ACA ATG
ACC- `3, 5'- GTA GTA GCA CCT GGT CTT GCT CTT --`3), mFoxp3 (5'- AGA GCC
CTC ACA ACC AGC TA --`3, 5'- CCA GAT GTT GTG GGT GAG TG --`3), mIL10
(5'- CCA AGC CTT ATC GGA AAT GA --`3, 5'- TTT TCA CAG GGG AGA AAT CG
--`3), mRora (5'-TCT CCC TGC GCT CTC CGC AC-3', 5'- TCC ACA GAT CTT GCA
TGG A --`3), mST2 (5'-GCG GAG AAT GGA ACC AAC TA-`3, 5'- AAG CAA GCT GAA
CAG GCA AT --`3), mAREGB (5'- AGC TGA GGA CAA TGC AGG GTA-`3, 5'-AGT GAC
AAC TGG GCATCT GG-`3), mPD1 (5'-TCA AGG CATGGT CAT TGG TA-`3, 5'-TAG GCC
ACA CTA GGG ACA GG-`3).

\textbf{Flow cytometric analysis and intracellular staining}

For intracellular staining total lung cells from Balb/c wild-type mice
were incubated over night with anti-CD3 antiCD28 antibodies and then
stimulated for 4h with PMA/ Ionomicin and a protein transport inhibitor
according to the manufacturer`s protocol. Cells were harvested, washed
and stained with anti-CD4, anti-CD8 and anti-CD25 antibodies for 30 min
at 4°C and then stained with anti-Foxp3, anti IFN-gamma and anti-IL10
antibodies in staining buffer (BD Biosciences, Heidelberg) for 35 min at
4° C. Afterwards cells were washed again and resuspended in stain
buffer. The following anti-mouse antibodies were used: CD4 FITC (BD
Biosciences), CD4 PerCP (BD), CD25 PerCP (BD), Foxp3 APC (Miltenyi
Biotec, Bergisch Gladbach, Germany).

Lung cell culture

{\label{lung-cell-culture}}

WT Bl6/C57 and ST2-/- total lung cells were incubated in the presence of
Vitamin D3, OVA and with or without RV infection at 37°C. After 24h
cells were harvested, washed and stained with anti-CD4 and anti PD-1
antibodies in staining buffer (BD Biosciences, Heidelberg) for 30 min at
4°C. Marked cells were acquired by using FACS-Calibur (BD Biosciences,
Heidelberg) and analyzed with FlowJo.

\textbf{Enzyme linked immunosorbent assay (ELISA).}

Murine IL-5, IL10 and IL13 were detected in cell culture supernatants by
DuoSet Elisa kit from R\&D (Hamburg,Germany), according to the
manufacturer protocol.

\textbf{Statistical analysis}

Differences were evaluated for significance (* p \selectlanguage{english}[?] 0.05; ** p \selectlanguage{english}[?] 0.01,
*** p \selectlanguage{english}[?] 0.001) by using the non-parametric one-tailed Mann-Whitney-test
(Prism version 7 for Windows; GraphPad, La Jolla, CA, USA) and a two
tailed Student t-test was used (Prism version 7 for Windows; GraphPad,
La Jolla, CA, USA). Data are shown as mean values + s.e.m. For
correlation of two parameters GraphPad Prism software (version 7 for
Windows) was used.

\textbf{Results}

\textbf{Clinical outcome of the cohorts of children analyzed in this
study}

The clinical data of these cohorts of children are reported
in\textbf{Table 1} . Further, clinical details of the cohort were
recently described\textsuperscript{6,18-20}. In Germany, Vitamin D3 is
given to the infants as supplement\textsuperscript{9} to avoid
immunological suppression especially in children which could not receive
breastfeeding. Wheezing is associated with asthma in the first years of
life\textsuperscript{10} moreover, RV is the factor that associated with
wheezing in infants. In this study, we wanted to better understand the
role of Vitamin D3 in pediatric asthma.

\textbf{Serum-C-reactive protein (CRP) and 25 (OH)-VitD3 levels
inversely correlated in control but not in asthmatic pre-school
children}

C-reactive protein (CRP) is a plasma protein, whose levels rise in
response to inflammation and infections. It is an hepatic acute-phase
protein that increases following interleukin-6 secretion by macrophages
and T cells. Its physiological role is to bind to
lysophosphatidylcholine expressed on the surface of dead or dying cells
(and some types of bacteria) promoting phagocytosis by macrophages,
which clears necrotic and apoptotic cells and bacteria. In healthy
adults, the normal concentrations of CRP varies between 0.8 mg/L to 3.0
mg/L. However, some healthy adults show elevated CRP at 10 mg/L. The
plasma half-life of CRP is 19 hours.\textsuperscript{21} We next asked
if serum CRP level at recruitment in our cohort of children correlated
with their serum level of 25(OH)VitD3.

The design of the study is reported in \textbf{Fig 1a} and the levels of
CRP of the cohorts are reported in \textbf{Table 1} . We noticed that
both control and asthmatic children group had one child with low
25(OH)VitD3 (less than 20 ng/ml) and very high CRP. We next correlated
25(OH)VitD3 and serum CRP levels without this very high CRP value in
both cohorts \textbf{(Fig 1b,c and d,e)} and next in asthma we analyzed
only data with CRP data lower than 2.5mg/ml, to be in the CRP range of
controls (\textbf{Fig 1f} ) and looked at the correlation with serum
VitD3. Here we found that, control but not asthmatic children had an
inverse correlation between their serum CRP value and serum 25(OH)VitD3
levels (\textbf{Fig1c} ). Considering the CRP value in asthma, we
noticed they were higher as compared to control children, indicating an
ongoing infection, inflammation in these asthmatic children \textbf{(Fig
1e).}

\textbf{VitD3 supplemetation in infancy is associated with reduced
asthma exacerbations}

To analyze the role of VitD3 supplementation during infancy on asthma
exacerbations, we then analyzed the number of asthma exacerbations and
asked if VitD3 supplementation during infancy would associate with less
disease exacerbations. Here we found that asthmatic children without
VitD3 supplementation had more episodes of asthma
exacerbations\textbf{(Fig 2a)} that were associated with induced CRP
\textbf{(Fig 2b),} a factor associated with increased airway
hyperresponsiveness\textbf{(Fig 2c)} . In conclusion, increased asthma
exacerbations associated with CRP in the absence of VitD3
supplementation, indicating a possible protective role of VitD3 during
asthma exacerbations.

\textbf{PD1 is decreased in blood cells of control children with higher
serum levels of 1,25 (OH) Vitamin D3}

We next wanted to analyze the role of 25(OH) VitD3 on Programmed cell
death protein 1 (PD-1) in the cohort of preschool children. PD-1 is a
protein on the surface of immune competent T cells that down-regulates T
cell inflammatory activity.\textsuperscript{22,23}Considering VitD3
serum levels lower than 20 ng/ml as low, we found that high VitD3
associated with decreased PD1 mRNA levels in blood cells of control but
not asthmatic children \textbf{(Fig 2d, e).} We also saw a trend towards
\emph{PD1 mRNA} induction in asthmatic children with higher serum VitD3
levels.

\textbf{VitD3 given intranasally reduced lung inflammation}

We next wanted to analyze the effect of intranasal treatment of Vitamin
D3 in the airways in a murine model of airway tolerance \textbf{(Fig
3a).} To this aim, we treated mice intranasally with VitD3 and looked at
airways inflammation \textbf{(Fig 3b)} . Here we found that allergen
challenge, without allergen sensitization, was accompanied by induction
of local inflammation \textbf{(Fig 3a,b).} By contrast, intranasal VitD3
treatment, reduced lung inflammation \textbf{(Fig 3b).} We then asked if
Eosinophils in bronchoalveolar lavage fluid were involved in regulating
airway tolerance after allergen challenges. Here we found that allergen
challenge, without allergen sensitization, was accompanied by induction
of local inflammation and induced eosinophils in BALF \textbf{(Fig
3c).}By contrast, intranasal VitD3 treatment, reduced lung
inflammation\textbf{(Fig 3c).} Alltogether these data indicate an
anti-inflammatory effect of VitD3 given intranasally in a model of
airway tolerance.

\textbf{1,25(OH)VitD3 inhibited IL33 and Ror-alpha in the airways}

We then asked if ILC2 were upregulated in our model of airway tolerance
after intranasal treatment with VitD3 and allergen challenge\textbf{(Fig
4a).} \textsuperscript{24}

Here we found that, intranasal delivery of VitD3, reduced Amphiregulin
(AREG), which is a member of the epidermal growth factor
family\textbf{(Fig 4b),} as well as IL-33, an alarmin and ligand of ST2,
a receptor on Innate lymphoid cells\textsuperscript{6} \textbf{(Fig 4c}
). Intranasal delivery of VitD3 did not regulate the ILC2 markers ST2
nor ICOS \textbf{(Fig 4 d, e)} but reduced Ror-alpha, an ILC2 signature
transcription factor \textbf{(Fig 4f)}\textsuperscript{25}. Thus airway
tolerance induced by VitD3 and allergen given intranasally did not
suppress completely ILC2 as confirmed by residual ILC2 cytokine IL-5 and
IL-13 in the airways of treated mice \textbf{(Fig 4 g,h)} .

\textbf{Airway tolerance induced T regulatory cells and with 1,25
(OH)VitD3 reduced PD1 in the airways of OVA challenged mice}

To further analyze the underlined mechanism of VitD3 in airway
tolerance, in the same model \textbf{(Fig 5a)} , VitD3 induced CD4+ T
cells and reduced CD4+CD25+Foxp-3+ T regulatory cells in the
airways\textbf{(Fig 5b-d)} \textsuperscript{16}. Moreover, allergen
challenge induced of IL-10 production, was kept upregulated by VitD3
treatment after anti-CD3 antibody challenge\textbf{(Fig 5e).} Finally,
treatment of Vitamin D3 with allergen, reduced significantly PD1 mRNA in
the airways (\textbf{Fig 5f).}

\textbf{Vit D3} \textbf{rescued CD4+ T cells after RV infection \emph{in
vitro} in the absence of ST2 in lung cells}

Murine IL-C2 in the lung were described in a model of influenza, where
they promote a pathologic effect on airway hyperresponsivness as well as
a protective role in epithelial repair. To understand the effect of ILC2
in RV infection present during allergen challenge and the protective
effect of VitD3 on this axis, we challenged \emph{in vitro} lung cells
from wt and ST2-/- mice with OVA and infected them with RV and
challenged them with VitD3 \textbf{(Fig. 6a)} . Here we found that, Vit
D3 induced the number of CD4+ T cells in RV infected lung cells in the
absence of ST2 \textbf{(Fig 6b)} . These experiments further support a
CD4+ T cells inducing role of VitD3 in the airways during allergen
challenge only in the absence of IL-33/ST2. Thus, IL33/ST2 axis is
involved in T cell death induced by rhinovirus.

\textbf{Vit D3 reduced lung CD4+PD1+ CD4+ T lymphocytes independently
from ST2 deficient cells}

We next asked if, in this in vitro system ViTD3 would rescue T cells
from cell death by inhibiting PD1 a marker of T cell
exhaustion.\textsuperscript{26} In addition, we wanted to better
understand the role of ST2 in T cell survival. Here we found that, in
the same experimental conditions depicted in Fig 7, CD4+PD1+ T cells
were inhibited by VitD3 in the presence of allergen challenge
dependently from ST2 \textbf{(Fig 6c)} . In summary, VitD3, has a
suppressive role on CD4+PD1+ T cells involved in T cell exhaustion in
the airways in the absence of ST2.

\textbf{Discussion}

VitD3 influences immune system in asthma.\textsuperscript{6-8} Here we
found that in healthy, but not in asthmatic preschool children serum
25(OH)VitD3 inversely correlated with CRP, a protein upregulated during
ongoing inflammation and infections.\textsuperscript{27}Furthermore, we
found that, asthmatic children had higher CRP serum level, indicating
induced ongoing inflammation. Finally, asthmatic pre-school children
which did not receive VitD3 supplementation in infancy had more episodes
of asthma exacerbations associated with high serum CRP levels indicating
a limiting effect of VitD3 on infection also in preschool children with
asthma.

Next, we wanted to see how Vitamin D3 affects T cells activation\emph{in
vivo} in asthma. Therefore we decided to look at PD1, a marker for
downregulation of T cell mediated immune responses and also a marker for
T cell exhaustion\textsuperscript{22}. We found that control children
with high Vitamin D3 serum levels have less PD1 mRNA in their blood
cells than those with low VitD3 levels. This was not seen in asthmatic
children, indicating the presence of a PD1 inducing pathway in asthma.

To look for a new molecular therapy for allergic asthma, we treated mice
intranasally with VitD3 within 20 minutes after OVA allergen challenge.
Here, VitD3 induced CD4+T cells and down-regulated Foxp-3 T regulatory
cells and PD1 in the lung.

We further demonstrated that VitD3 induced CD4+ T cells after OVA
challenge. CD25, Foxp3 and IL10, all markers for T regulatory
cells\textsuperscript{28,29}, were increased by OVA and downregulated by
VitD3.

Furthermore, some ILC2 inducing markers like IL-33 were reduced by
Vitamin D3 combined with allergen, indicating an homeostatic function in
the airways of treated mice\textsuperscript{30}. It would be very
interesting to know if Vitamin D3 could show a direct prophylactic
effect.

Our data show a protective homeostatic effect of VitD3 \emph{in vivo}and
\emph{in vitro} . Further, considering its limiting effect on lung ILC2,
a cell type persistent in the lung of asthmatics, VitD3 given
intranasally represent a new avenue for the treatment of asthma
especially in asthmatic children.

Here we further found that, Vit D3 induced the number of CD4+ T cells in
RV infected lung cells in the absence of ST2. These experiments further
support a CD4+ T cells inducing role of VitD3 in the airways during
allergen challenge only in the absence of IL-33/ST2. Thus, IL33/ST2 axis
is involved in T cell death induced by rhinovirus.

In addition we found that VitD3, has a suppressive role on CD4+PD1+ T
cells involved in T cell exhaustion in the airways in the absence of
ST2. These data support an interactive role of ViD3 and ILC2 in the lung
that can be used for the treatment of allergic diseases and rhinovirus
exacerbations seen in pediatric asthma.\textsuperscript{6}

\textbf{Author Contributions.}

PH generated the results for Figure1,2,6. and analysed all the data
present in this manuscript KA is the paediatricians responsible of this
study in Erlangen, MR measured the VitD3 and CRP levels in the serum of
the children, NL further analyzed the results for Figure 1. NGP is the
coordinator of Predicta. SF supervised the whole work and wrote the
manuscript with PH.

\textbf{References}

1 Saglani, S., Fleming, L., Sonnappa, S. \& Bush, A. Advances in the
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14 Kouzaki, H., Tojima, I., Kita, H. \& Shimizu, T. Transcription of
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22 Cheung, A. K. L. \emph{et al.}Gut-homing Delta42PD1(+)Vdelta2 T cells
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24 Maazi, H. \emph{et al.} Activated plasmacytoid dendritic cells
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25 De Grove, K. C. \emph{et al.}Dysregulation of type 2 innate lymphoid
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26 Velu, V. \emph{et al.} Enhancing SIV-specific immunity in vivo by
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27 Wu, Y., Potempa, L. A., El Kebir, D. \& Filep, J. G. C-reactive
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28 Ziegler, S. F. FOXP3: of mice and men. \emph{Annual review of
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29 Fantini, M. C., Dominitzki, S., Rizzo, A., Neurath, M. F. \& Becker,
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30 Koch, S. \emph{et al.} Targeted deletion of
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\textbf{Ethical Statement:} All human and murine studies performed in
this study received ethical approval from local institutions.

\textbf{Table 1: Clinical data of children at baseline visit}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{Control} & \textbf{Age} & \textbf{Gender} & \textbf{Skin Prick
Test} & \textbf{FEV1\%} & \textbf{VitD Supplement*}\tabularnewline
\midrule
\endhead
208 & 6 & m & / & 77 & No\tabularnewline
211 & 6 & f & / & 121 & No\tabularnewline
214 & 5 & m & / & 110 & No\tabularnewline
215 & 4 & m & / & 118 & No\tabularnewline
218 & 4 & f & / & 111 & Yes\tabularnewline
219 & 5 & f & / & 107 & Yes\tabularnewline
220 & 5 & f & - & 84 & No\tabularnewline
221 & 3 & m & / & / & Yes\tabularnewline
222 & 6 & m & / & 105 & Yes\tabularnewline
226 & 4 & f & / & 109 & No\tabularnewline
227 & 6 & m & - & 87 & no\tabularnewline
232 & 4 & m & - & 100 & no\tabularnewline
233 & 5 & f & / & 112 & no\tabularnewline
234 & 5 & f & + & 119 & yes\tabularnewline
235 & 4 & m & / & 113 & no\tabularnewline
236 & 5 & m & - & 111 & yes\tabularnewline
237 & 4 & m & - & 109 & yes\tabularnewline
240 & 4 & f & - & 92 & no\tabularnewline
241 & 5 & m & - & 123 & no\tabularnewline
244 & 5 & f & - & 107 & no\tabularnewline
245 & 4 & m & - & 121 & yes\tabularnewline
246 & 5 & m & / & 109 & no\tabularnewline
\textbf{Mean} & \textbf{4,73} & \textbf{f=41\%};\textbf{m=59\%} &
\textbf{neg=90\%} \textbf{pos=10\%} & \textbf{106,9} &
\textbf{Yes=36,4\%;} \textbf{No=63,6\%}\tabularnewline
\textbf{SEM} & \textbf{0,18} & & & \textbf{2,67} &\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllll@{}}
\toprule
\begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Asthma}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Age}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Gender}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Asthma* Severity}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Phenotype**}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Skin Prick Test}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{FEV1\%}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Treatment}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{VitD3*** Prophylaxis}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Level of VitD3}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Number of exacerbations in the last 3 month}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Number of exacerbations in the last 12 month}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
201\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
m\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
V\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
126\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
9,69\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
202\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
m\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
II\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
U\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
111\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
12\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
203\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
f\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
II\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
U\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
95\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
6,72\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
204\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
m\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
II\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
A\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
128\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
9,85\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
205\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
m\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
U\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
/\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
102\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
11\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
206\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
f\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
U\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
129\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
16,8\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
207\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
m\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
V\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
143\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
17,6\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
10\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
209\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
f\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
II\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
v,a\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
115\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
yes\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
3-4\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
7-8\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
210\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
f\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
V\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
98\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Non-Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
11,3\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
10\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
212\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
m\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
II\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
e,v\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
96\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
12\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
213\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
m\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
III\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
e\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
115\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
17,8\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
12\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
216\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
f\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
III\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
a,v\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
92\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
19,9\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
15\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
20\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
217\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
f\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
a,e,v\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
111\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
15,8\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
223\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
m\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
V\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
99\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
37,8\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
224\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
f\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
V\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
135\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
yes\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
24,3\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
225\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
m\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
V\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
/\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
99\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
yes\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
33,5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
10\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
228\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
m\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
V\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
88\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Non-Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
yes\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
33,5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
229\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
m\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
V\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
87\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Non-Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
230\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
m\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
V\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
101\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Non-Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
28,7\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
231\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
m\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
V\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
71\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
11,8\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
8\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
238\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
m\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
V\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
77\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
8,5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
9\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
239\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
f\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
I\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
E\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
/\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
98\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Non-Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
yes\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
13,1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textasciitilde{}200\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
242\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
m\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
II\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
a,e,v\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
/\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
81\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
19,2\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
3-5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
12\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
243\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
f\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
II\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
V\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
69\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Steroid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
no\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
30,6\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
120\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{Mean}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{4,92}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{f=37,5\%; m=62,5\%}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{I=62,5\%;} \textbf{II=29,2\%}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{v=50\%} \textbf{u=16,7\%}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{neg=15\%} \textbf{pos=85\%}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{102,75}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{Non Steroid=20,9\%} \textbf{Steroid 79,1\%}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{Yes 20,8} \textbf{No=79,2}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{SEM}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{0,15}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{4,04}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2. Analysed data of children at baseline visit (B0)}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\textbf{Control} & \textbf{Virus Swab**} &
\textbf{25(OH)VitD3***}\tabularnewline
\midrule
\endhead
208 & - & /\tabularnewline
211 & RV ++ & 16,9\tabularnewline
214 & RV ++ & 13,4\tabularnewline
215 & - & 26\tabularnewline
218 & RV +++ & 17,4\tabularnewline
219 & RV + & 16,4\tabularnewline
220 & - & 22,3\tabularnewline
221 & RV ++ & 25,5\tabularnewline
222 & - & 34,1\tabularnewline
226 & - & 20,3\tabularnewline
227 & - & 16,5\tabularnewline
232 & RV +++ & 13,7\tabularnewline
233 & RV +, Other & 25,9\tabularnewline
234 & RV ++, Other & 12,6\tabularnewline
235 & RV ++ & 14,2\tabularnewline
236 & - & /\tabularnewline
237 & Other & 10,7\tabularnewline
240 & RV+, Other & 18,3\tabularnewline
241 & RV ++ & 25,9\tabularnewline
244 & / & /\tabularnewline
245 & - & 28,4\tabularnewline
246 & RV+ & 24,4\tabularnewline
\textbf{Mean} & & \textbf{20,15}\tabularnewline
\textbf{SEM} & & \textbf{1,46}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure legends}

\textbf{Figure 1: Higher serum levels of 1,25(OH) VitaminD3(=VitD3) are
associated with lower CRP levels in the blood of control pre-school
children. a:} Experimental design of the study. \textbf{b,c} Linear
regression analysis of serum levels of CRP and 25(OH)VitD3 in control
and asthmatic children \textbf{(d-f).}

\textbf{Fig 2: VitD3 supplementation is correlated with less
exacerbations in preschool asthma children} \textbf{and PD1 is decreased
in control children with higher serum levels of 1,25 (OH) Vitamin
D3.(a)} Exacerbations in asthmatic children during last 6 months (B0)
depending on Vitamin D3 supplement intake (0=no Vitamin D3 supplement,
1= VitaminD3 supplement) as infant (No VitaminD3 supplement n=16,Vitamin
D3 supplement n=5). \textbf{(b)} C-Reactive protein (CRP) measured in
serum of asthmatic children at B0, in relation to number of
exacerbations during the last 6 months retrospective from B0 (n= 8 in
both groups). \textbf{(c)} CRP in same children related to their FEV1\%,
investigated at B0 (n=3-16 per group). p=0,0059. Two-tailed Student's T
Test. \textbf{d.} Experimental design. \textbf{b.} Total blood cells
(tempus tube) RNA isolation for qPCR from healthy and asthmatic
children. \textbf{e} .PD1/HPRT mRNA expression in control children (CN)
and asthmatic children (A), considering the serum level of 25 (OH) VitD3
lower than 20 ng/ml (low) and higher than 20 ng/ml (high). (N=6-6-7-5;
CN low 25(OH)VitD3 versus CN high 25(OH)VitD3: p=0.001; CN low
25(OH)VitD3 versus A low 25(OH)VitD3: p= 0.0033. Two-tailed Students
T-test)\textbf{.}

\textbf{Figure 3. VitD3 given intranasally limits airways inflammation.
a.} Experimental design. \textbf{b.} Representative histological
sections of the lung after Giemsa staining (n=4 per group) and lung
inflammation score (PBS versus 1x and 2xOVA p=0.079;1xOVA versus
VitD3+1xOVA p= 0.028 after Mann Whithney)\textbf{.c.} BALF was collected
and Eosinophils (into the red square) were stained by FACS staining (n=4
or 3 per group as indicated) and analysed by using Two tailed T test
analysis (PBS versus 2xVitD3 p= 0.029 ; PBS versus 2xOVA p=0.066).

\textbf{Figure 4. Airway challenge with 1,25 (OH) Vitamin D3 inhibited
IL-33, Ror-alpha in the airways. a.} Experimental design of airway
tolerance. \textbf{b.} AREGB/HPRT mRNA expression in \selectlanguage{greek}α\selectlanguage{english}CD3/CD28
stimulated lung cells. p=0,0038. Two-tailed T-test.\textbf{c.}
IL-33/HPRT mRNA expression in \selectlanguage{greek}α\selectlanguage{english}CD3/CD28 stimulated lung cells. P=0,035.
Two-tailed T-test\textbf{. d.} ST2/HPRT mRNA expression in \selectlanguage{greek}α\selectlanguage{english}CD3/CD28
stimulated lung cells. \textbf{e.} ICOS/HPRT mRNA expression in
\selectlanguage{greek}α\selectlanguage{english}CD3/CD28 stimulated lung cells. p=0,042. p=0,015.\textbf{f.} RORa/HPRT
mRNA expression in \selectlanguage{greek}α\selectlanguage{english}CD3/CD28 stimulated lung cells. PBS vs. 2xVitD3:
p=0,0005. 2xVitD3 vs 2xOVA: p=0,011. 2xOVA vs 2xVitD3: p=0,026.
\textbf{g.} IL-5 ELISA; p=0.047. Two-Tailed T-test.\textbf{h.} IL-13
ELISA. PBS vs. VitD3: p=0.0188; PBS vs. 2xOVA+VitD3: p=0.0109. Both
two-tailed T-test.

\textbf{Figure 5: Analysis of the mechanism of airway tolerance: OVA
intranasal challenge induced T regulatory cells associated markers and
VitD3 reduced PD1mRNA} . \textbf{a.} Experimental design. Balb/c n=3 per
group; VitD3=1a,25 DihodroxyvitaminD3 (Sigma Aldrich); 10 ng
intranasal/mouse; OVA = OVA Texas Red; 500 ug/mouse. \textbf{b.}Analysis
of CD4+ lymphocytes in unstimulated, 1,25(OH)-Vitamin-D3 (=VitD3) and/or
OVA in vivo treated wild-type mice. p=0,049. Two-tailed T-test
\textbf{c.} CD25+ Foxp3+CD4+: n per group=3; PBS vs 2xVitD3
p=0,025,p=0,0504; PBS vs 2xOVA p=0,003; PBS vs 2xOVA+VitD3, p= 0,034;
2xOVA vs 2xOVA+VitD3, p=0,032; 2x VitD3 vs 2xOVA, p=0,008.
\textbf{d.}Foxp3/HPRT mRNA expression in total lung cells by qPCR: PBS
vs 2xOVA \textgreater{}p= 0,00069; 2x VitD3 vs 2xOVA p=0,0015; 2xOVA vs
2xOVA+VitD3 p= 0,0073.Two-tailed T test. \textbf{e.} IL10 measured by
Elisa in the supernatants of \selectlanguage{greek}α\selectlanguage{english}CD3 antibodies stimulated lung cells. mIL
10 Elisa: PBS vs 2xOVA p=0,035; PBS vs 2xVitD3+OVA p=0,0149; 2xVitD3 vs
2x OVA p=0,0207 ; 2x OVA vs 2xOVA+VitD3 p=0,0042; two-tailed T
test.\textbf{f.} Lung PD1/HPRT p=0.0099 n=3,3,2,3, respectively. Two
tailed T test. Balb/c n=3 per group; VitD3=1\selectlanguage{greek}α,\selectlanguage{english}25 DihodroxyvitaminD3
(Sigma Aldrich)10 ng intranasal/mouse; OVA = OVA Texas Red
\textgreater{} 500 ug/mouse 20ug/ul -\textgreater{} 25 ul. Two-tailed
Student's T test.

\textbf{Figure 6: Vit D3 rescued CD4+ T cells inhibited PD1+CD4+ T cells
in the absence of ST2 after RV infection of lung cells}

(a) Experimental design. WT B6 n=4. ST2-/- n=2. IL33-/- n=4. (b)
Analysis of CD4+ lymphocytes from wild-type and ST2-/- mice, infected
with RV1b and afterwards treated with 1a,25 DihodroxyvitaminD3 (=VitD3)
and/or OVA in vitro. p=0,029. p=0,009. p=0,026. Two-tailed Student's T
test. p= 0,00028. p=0,022. p=0,028. Two-tailed Student's T test.

(c) PD1+CD4+ cells gated on CD4+lymphocytes, from wild-type (n=4) and
ST2-/- (n=2) mice, infected with RV1b and afterwards treated with 1a,25
DihodroxyvitaminD3 (=VitD3) and/or OVA in vitro. p=0,009. p=0,048.
p=0,045. Two-tailed Student's T test.\selectlanguage{english}
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