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\begin{document}

\title{Increased SARS-CoV-2 reactive low avidity T cells producing inflammatory
cytokines in pediatric post-acute COVID-19 sequelae (PASC)}



\author[1]{Krystallenia Paniskaki}%
\author[1]{Sarah Goretzki}%
\author[2]{Moritz Anft}%
\author[1]{Margarethe J. Konik}%
\author[3]{Toni L. Meister}%
\author[3]{Stephanie Pfaender}%
\author[1]{Klara Lechtenberg}%
\author[1]{Melanie Vogl}%
\author[1]{Burcin Dogan}%
\author[1]{Sebastian Dolff}%
\author[4]{Timm H. Westhoff}%
\author[1]{Hana Rohn}%
\author[1]{Ursula Felderhoff-Mueser}%
\author[2]{Ulrik Stervbo}%
\author[1]{Oliver Witzke}%
\author[1]{Christian Dohna-schwake}%
\author[2]{Nina Babel}%
\affil[1]{Universitat Duisburg-Essen - Campus Essen}%
\affil[2]{Universitatsklinik Marien Hospital Herne}%
\affil[3]{Ruhr University Bochum}%
\affil[4]{Marienhospital Herne Medizinische Klinik I Innere Medizin}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Background:} A proportion of the convalescent SARS-CoV-2
pediatric population presents nonspecific symptoms, mental health
problems and a reduction in quality of life similar to myalgic
encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID-19
symptomatic. However, data regarding its clinical manifestation and
immune mechanisms are currently scarce. \textbf{Methods:} In this study,
we perform a comprehensive clinical and immunological profiling of 17
convalescent COVID-19 children with post-acute COVID-19 sequelae (PASC)
manifestation and 13 convalescent children without PASC manifestation. A
detailed medical history, blood and instrumental tests and physical
examination were obtained from all patients. SARS-CoV-2 reactive T cell
response was analyzed via multiparametric flowcytometry and the humoral
immunity was addressed via pseudovirus neutralization and ELISA assay.
\textbf{Results:} The most common PASC symptoms were shortness of
breath/exercise intolerance, paresthesia, smell/taste disturbance, chest
pain, dyspnea, headache and lack of concentration. Blood count and
clinical chemistry showed no statistical differences among the study
groups. We detected higher frequencies of spike (S) reactive CD4+ and
CD8+ T cells among the PASC study group, characterized by TNF\selectlanguage{greek}α \selectlanguage{english}and IFN\selectlanguage{greek}γ
\selectlanguage{english}production and low functional avidity. CRP levels are positively
correlated with IFN\selectlanguage{greek}γ \selectlanguage{english}producing reactive CD8+ T cells.
\textbf{Conclusions:} Our data might indicate a possible involvement of
a persistent cellular inflammatory response triggered by SARS-CoV-2 in
the development of the observed sequelae in pediatric PASC. These
results may have implications on future therapeutic and prevention
strategies.%
\end{abstract}%



\sloppy


\textbf{Increased SARS-CoV-2 reactive low avidity T cells producing
inflammatory cytokines in pediatric post-acute COVID-19 sequelae (PASC)}

Krystallenia Paniskaki \textsuperscript{1,2}*\textsuperscript{+}, Sarah
Goretzki \textsuperscript{1,3}*, Moritz Anft \textsuperscript{2},
Margarethe J. Konik \textsuperscript{1}, Toni L.
Meister\textsuperscript{4}, Stephanie Pfaender \textsuperscript{4},
Klara Lechtenberg \textsuperscript{3}, Melanie Vogl \textsuperscript{5},
Burcin Dogan \textsuperscript{3}, Sebastian Dolff \textsuperscript{1},
Timm H. Westhoff \textsuperscript{6}, Hana Rohn \textsuperscript{1},
Ursula Felderhoff-Mueser \textsuperscript{3,7}, Ulrik
Stervbo\textsuperscript{2}, Oliver Witzke \textsuperscript{1}, Christian
Dohna-Schwake \textsuperscript{1,3,7} and Nina
Babel\textsuperscript{2,8}

\textsuperscript{1} Department of Infectious Diseases, West German
Centre of Infectious Diseases, University Hospital Essen, University
Duisburg-Essen, Essen, Germany

\textsuperscript{2} Center for Translational Medicine and Immune
Diagnostics Laboratory, Medical Department I, Marien Hospital Herne,
University Hospital of the Ruhr-University Bochum, Bochum, Germany

\textsuperscript{3} Department of Pediatrics I, University of
Duisburg-Essen, Children\selectlanguage{ngerman}´s Hospital Essen, Germany

\textsuperscript{4} Department of Molecular and Medical Virology,
Ruhr-University Bochum, Bochum, Germany

\textsuperscript{5} Department of Pediatrics III, Pediatric Pulmonology
and Sleep Medicine, University of Duisburg-Essen, Children´s Hospital
Essen, Germany

\textsuperscript{6} Medical Department I, Marien Hospital Herne,
University Hospital of the Ruhr-University Bochum, Herne, Germany

\textsuperscript{7} Center for Translational Neuro- and Behavioral
Sciences C-TNBS, University Duisburg-Essen, Essen, Germany

\textsuperscript{8} Berlin Institute of Health at Charité -- University
Clinic Berlin, BIH Center for Regenerative Therapies (BCRT) Berlin,
Berlin, Germany

* equal contribution

\textsuperscript{+} Correspondence:

Dr. Krystallenia Paniskaki

email: kpaniskaki@gmail.com

\textbf{Running title:} Cellular inflammation in pediatric PASC

Word count: 2623

Tables: 2

Figures: 3

\textbf{Competing Interests}

All authors declare no competing interests.

\textbf{Financial support}

This work was supported by grants of Mercator Foundation, EFRE grant for
COVID.DataNet. NRW, AiF grant for EpiCov, and BMBF for NoChro (FKZ
13GW0338B).

\textbf{Abstract}

\textbf{Background:} A proportion of the convalescent SARS-CoV-2
pediatric population presents nonspecific symptoms, mental health
problems and a reduction in quality of life similar to myalgic
encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID-19
symptomatic. However, data regarding its clinical manifestation and
immune mechanisms are currently scarce.

\textbf{Methods:} In this study, we perform a comprehensive clinical and
immunological profiling of 17 convalescent COVID-19 children with
post-acute COVID-19 sequelae (PASC) manifestation and 13 convalescent
children without PASC manifestation. A detailed medical history, blood
and instrumental tests and physical examination were obtained from all
patients. SARS-CoV-2 reactive T cell response was analyzed via
multiparametric flowcytometry and the humoral immunity was addressed via
pseudovirus neutralization and ELISA assay.

\textbf{Results:} The most common PASC symptoms were shortness of
breath/exercise intolerance, paresthesia, smell/taste disturbance, chest
pain, dyspnea, headache and lack of concentration. Blood count and
clinical chemistry showed no statistical differences among the study
groups. We detected higher frequencies of spike (S) reactive CD4+ and
CD8+ T cells among the PASC study group, characterized by TNF\selectlanguage{greek}α \selectlanguage{english}and IFN\selectlanguage{greek}γ
\selectlanguage{english}production and low functional avidity. CRP levels are positively
correlated with IFN\selectlanguage{greek}γ \selectlanguage{english}producing reactive CD8+ T cells.

\textbf{Conclusions:} Our data might indicate a possible involvement of
a persistent cellular inflammatory response triggered by SARS-CoV-2 in
the development of the observed sequelae in pediatric PASC. These
results may have implications on future therapeutic and prevention
strategies.

\textbf{Keywords:} pediatric PASC, long COVID, T cells, neutralizing
antibodies

\textbf{Main text}

\textbf{Introduction}

Already from the first pandemic months, it became evident that clinical
manifestations of SARS-COV-2 infection in pediatric patients were
strongly differentiated compared to adults(1). Very few children
progress to any significant respiratory distress(2,3), however a small
percentage of pediatric patients presents multisystem inflammatory
syndrome(4,5) as a post infectious complication. Similar to adults, a
part of the convalescent COVID-19 pediatric population presents
multisystemic nonspecific symptoms, mental health problems and a
reduction in quality of life similar to ME/CFS and long COVID-19
symptomatic(6). However, current reports are conflicting regarding its
direct or indirect connection to SARS-CoV-2 prevalence, duration and
impact on daily life(1). Sorg et al and Borch et al report ME/CFS
similar symptomatic in seronegative and seropositive SARS-CoV-2 children
and suggest that containment measures and reduction of social contacts
during 2020 and 2021 reflect on these results(6,7).

Independent observational studies conclude that the main
symptoms/diagnoses of PASC among children are exertional dyspnea, cough
and exercise intolerance, loss of taste/smell, fatigue and to a lesser
extend muscle weakness and chest pain (8--14).

Studies in adult subjects with PASC manifestation suggest chronic
stimulation of the immune system as a result of antigen persistence,
autoimmunity or microbiota-gut-brain axis dysregulation(15--17).
However, data regarding the pathology and immune mechanisms behind
pediatric PASC is scarce. To address these knowledge gaps and the
contribution of immunity, including humoral and cellular response in
pediatric PASC pathogenesis, we performed a comprehensive immune
profiling of 17 pediatric patients with PASC. As control we used 13
healthy COVID-19 convalescent children.

\textbf{Materials and methods}

\textbf{Study participants}

We included 17 pediatric patients with PASC (further referred as PASC)
and 13 convalescent COVID-19 pediatric subjects without PASC symptomatic
(further referred as control) treated in the Pediatric Infectious
Diseases, Outpatient Clinic at the Department of Pediatrics I,
University Hospital Essen from April 2021 to December 2021. Seventeen
patients, who met the criteria of long COVID according to the German
Association of the Scientific Medical Societies (AWMF) and NICE
guidelines (18,19) were recruited as PASC cohort. Patients with at least
two symptoms, which could not be explained by an alternative diagnosis
were included. The study was approved by the Ethics Committee of the
University Hospital Essen (21-9998\_1-KOBO \& 22-10581-BO). Written
informed consent was obtained from all participants/caretakers.
Demographic and clinical characteristics are provided in tables 1 and 2.

\textbf{Clinical Diagnostic Workup}

As previously described(20), a detailed medical history, blood tests,
physical examination, oxygen saturation and blood pressure were obtained
from all patients. Body mass indices (BMI; kg/m\selectlanguage{ngerman}²) were assessed using
BMI-centiles adapted to age, sex, and race. All further consultations
with other medical specialists were conducted based on clinical
presentation. Electroencephalogram (EEG), electrocardiogram (ECG),
echocardiography, ultrasound of the abdomen and thyroid, chest-X-rays,
lung function with lung clearance index (LCI) measurement, cranial or
spinal magnetic resonance imaging, and Nerve Conduction Velocity (NCV)
were performed, if necessary, based on clinical presentation.
Demographic variables, preexisting diagnoses, and newly detected
underlying diseases as well as long COVID-associated symptoms were
documented. PASC associated symptoms were assessed through a
questionnaire (see supplementary file) and/or medical history. Shortness
of breath/exercise intolerance were associated via the six-minute
walking test. Patients were systematically queried as to symptoms
associated with long COVID or other somatic symptoms were included in
the evaluation. An improvement was defined by less reported intensity,
quantity of symptoms, and/or alleviation of restrictions in daily
routines, and numbers of symptoms or less restrictions in daily routines
caused by long COVID symptoms.

\textbf{Preparation of PBMCs}

As previously described (21), peripheral blood was collected in
S-Monovette K3 EDTA blood collection tubes (Sarstedt). Collected blood
was prediluted in PBS/BSA (Gibco) at a 1:1 ratio and underlaid with 15
mL of Ficoll-Paque Plus (GE Healthcare). Tubes were centrifuged at 800g
for 20 min at room temperature. Isolated PBMCs were washed twice with
PBS/BSA and stored at -80 °C until use. The cryopreserved PBMCs were
thawed by incubating cryovials 2-3 minutes at 37 °C in bead bath, washed
twice in 37°C RPMI 1640 media (Life Technologies) supplemented with 1\%
penicillin-streptomycin-glutamine (Sigma-Aldrich), and 10\% fetal calf
serum (FCS) (PAN-Biotech) medium, and incubated overnight at 37 °C.

\textbf{Flow cytometry - Measurement of SARS-CoV-2 reactive T cells}

As previously described, PBMCs were plated in 96-U-Well plates in RPMI
1640 media (Life Technologies) (22,23). Each well was stimulated with
wildtype (WT) S-protein (Miltenyi Biotec) or left untreated as a control
for 16 h. The proteins were dissolved per manufacturer's directions. As
a positive control, cells were stimulated with staphylococcal
enterotoxin B (1 \selectlanguage{greek}μ\selectlanguage{english}g/mL, Sigma-Aldrich). After 2 h, brefeldin A (1 \selectlanguage{greek}μ\selectlanguage{english}g/mL,
Sigma-Aldrich) was added. Detailed listing of the antibody panels for
general phenotyping and T cell activation \emph{ex vivo} is in Table S2.
The PBMCs stimulated overnight were stained with optimal concentrations
of antibodies for 10 min at room temperature in the dark. Stained cells
were washed twice with PBS/BSA before preparation for intracellular
staining using the Intracellular Fixation \& Permeabilization Buffer Set
(Thermo Fisher Scientific) as per the manufacturer's instructions. Fixed
and permeabilized cells were stained for 30 min at room temperature in
the dark with an optimal dilution of antibodies against the
intracellular antigen. All samples were immediately acquired on a
CytoFLEX flow cytometer (Beckman Coulter). Quality control was performed
daily using the recommended CytoFLEX daily QC fluorospheres (Beckman
Coulter). No modification to the compensation matrices was required
throughout the study. Antigen-reactive responses were considered
positive after the non-reactive background was subtracted, and more than
0.01\% were detectable. Negative values were set to zero.

\textbf{SARS-CoV-2 neutralization assay}

As previously described(22), for the virus neutralization assay, sera
were incubated for 30 min at 56\selectlanguage{ngerman}°C in order to inactivate complement
factors. Single cycle VSV\selectlanguage{english}*\selectlanguage{greek}Δ\selectlanguage{english}G(FLuc) pseudoviruses bearing the SARS-CoV-2
WT S (D614G) protein in the envelope were incubated with quadruplicates
of two-fold serial dilutions of immune sera in 96-well plates prior to
infection of Vero E6 cells (1x104 cells / well) in DMEM + 10\% FBS (Life
Technologies). At 18 hours post infection, firefly luciferase (FLuc)
reporter activity was determined as previously described S5 using a
CentroXS LB960 (Berthold) and the reciprocal antibody dilution causing
50\% inhibition of the luciferase reporter was calculated (PVND50).

\textbf{Statistics}

Flow cytometry data were analyzed using FlowJo version 10.6.2 (BD
Biosciences); gating strategy is presented in figures S1. For the
analysis of SARS-CoV-2 reactive T cells, a threshold of 0.01\% was
employed to define a detectable response. Single stains and
fluorescence-minus-one controls were used for gating. Gates of each
study participant were adjusted according to the negative control. CD4+
T cells expressing CD154 and CD137 and CD8+ T cells expressing CD137
were defined as reactive T cells. Statistical analysis was performed
using GraphPad Prism v7. Categorical variables are summarized as numbers
and frequencies; quantitative variables are reported as median and
interquartile range. Normality tests were performed with the
Shapiro-Wilk Test. All applied statistical tests are two-sided.
Kruskal-Wallis Test and Mann-Whitney-Test were applied to perform
comparisons. The age between the two cohorts was compared using unpaired
two-tailed t-test, and gender was compared using two-tailed Fisher's
exact test. Correlational relationships were explored with Spearman\selectlanguage{ngerman}´s
test. p values below 0.05 were considered significant; only significant
p values are reported in the figures. p values were not corrected for
multiple testing, as this study was of an exploratory nature.

\textbf{Results}

\textbf{1} .\textbf{Demographic and clinical characterization of the
study groups}

Our study group comprised 17 convalescent COVID-19 children with PASC
manifestation (further referred as PASC) and 13 convalescent COVID-19
children without PASC manifestation (further referred as control). All
study participants had a negative SARS-CoV-2 nasal swab tested via
RT-PCR on recruitment. During the acute phase of COVID-19 disease 100\%
of both study groups presented moderate/asymptomatic COVID-19 disease
severity without need for hospitalization. The median COVID-19
convalescence time was 5 and 3 months for the PASC and the control study
group respectively (PASC range 2-11 months, control 2-8 months)
(unpaired t test PASC vs control p=0.89). All children participated did
not receive a COVID-19 vaccine. The median age of PASC study group was
11 years (range 3-18 years) and of the control cohort 12 years (range
6-15 years) (unpaired t test PASC vs control p=0.39). The PASC and
control cohorts comprised of 53\% (n=9) and 15\% (n=2) female
participants, respectively and showed no significant gender difference
(Fisher´s exact test, p=0.0575). The demographic characteristics of the
study cohorts are presented in Table 1.

The most common PASC symptoms among the PASC study group were shortness
of breath/exercise intolerance and paresthesia with 71\% (n=12) and 59\%
(n=59) respectively, while smell/taste disturbance, chest pain, dyspnea,
headache and concentration disturbance were also quite common (29 to
41\% of the PASC study cohort) (table 2). The blood count (hemoglobin,
leukocytes \& thrombocytes) and the clinical chemistry (AST/GOT,
GPT/ALP, \selectlanguage{greek}γ-\selectlanguage{english}GT, creatinine, D-dimers, CRP) excluding CK showed no
statistical differences among the study groups (table 2). However, the
control study group showed significantly higher CK-levels compared to
PASC subjects (p=0.011, Mann Whitney Test) (median 153, range 113-253,
normal\textless{}170U/l). This result may reflect more intense
physical/athletic activity among the control study group due to the
absence of PASC symptomatic.

\textbf{2. Persistent SARS-CoV-2 reactive T cell response characterized
by low avidity among the PASC subjects}

In order to assess the contribution of SARS-CoV-2 S triggered persistent
inflammation in PASC pathogenesis, we analyzed SARS-CoV-2 reactive T
cell immunity. As the majority of the study participants were infected
with either the WT or the alpha variant, we addressed the WT S reactive
CD4+ and CD8+ T cell response. CD4+CD154+CD137+ and CD8+CD137+ T cells
are defined as reactive CD4+ and CD8+ T cells, respectively. PASC
subjects showed significantly higher frequencies of WT reactive CD4+ T
cells compared to controls (Mann Whitney test p=0.008) (fig. 1A). WT
reactive CD8+ T cell frequencies among the PASC study group were higher
compared to controls, however, without statistical significance (fig.
1D).

As previously described and applied (22,24,25), we performed
additionally an analysis of the functional avidity among reactive CD4+
and CD8+ T cells (gating strategy,~fig. S1) using CD3low (high avidity)
and CD3high (low avidity) expression as discrimination marker. We
detected similar frequencies of WT reactive CD4+CD3low reactive T cells
among PASC and controls (fig. 1B). However, the PASC study group
presented higher frequencies of low avidity WT reactive CD4+CD3high T
cells (Mann Whitney test p=0.003) (fig. 1C). Furthermore, we detected
significantly higher frequencies of high avidity WT reactive CD8+CD3low
T cells among the controls compared to PASC study group (Mann Whitney
test p=0.007) (fig.1E), while low avidity WT reactive CD8+CD3high T cell
frequencies were significantly higher among PASC subjects compared to
controls (Mann Whitney test p=0.015) (fig.1F).

As next, we assessed the functionality of WT specific T cells regarding
their cytokine production. PASC subjects showed significantly higher
frequencies of WT reactive CD4 T cells producing TNF\selectlanguage{greek}α \selectlanguage{english}compared to
controls (Mann Whitney test p=0.016) (fig. 2B). Additionally, WT
reactive CD8+ T cells producing IFN\selectlanguage{greek}γ \selectlanguage{english}were significantly higher among the
PASC study group (Mann Whitney test p=0.0045) (fig. 2G). Other subsets
did not show any statistical difference in the cytokine-producing T
cells (Fig. 2 A, C-F, H).

\selectlanguage{ngerman}\textbf{3. PASC subjects´ neutralizing potential is slightly inferior
compared to controls}

High neutralizing antibodies titers are considered to protect
effectively against SARS-CoV-2 infection, and their waning is related
with high risk of reinfection or vaccine breakthrough infection(26).
Currently it is unclear, whether the neutralizing antibodies (NAb) play
a role in PASC pathogenesis, therefore we measured the titers of WT NAb
in PASC and control individuals. We found slightly higher titers of WT
NAb among the controls compared to PASC subjects, however without
reaching a statistical significance (fig. 3).

\selectlanguage{greek}\textbf{CRP levels are positively correlated with IFNγ producing
reactive CD8+ T cells}\selectlanguage{english}

At last, we explored potential correlations between clinical diagnostic
parameters and S-reactive CD4+ or CD8+ T cell frequencies among the PASC
study group. We found moderate positive correlation with statistical
significance between GOT, GPT and IFN\selectlanguage{greek}γ \selectlanguage{english}producing reactive CD4 T cells as
well as between creatinine and TNF\selectlanguage{greek}α \selectlanguage{english}producing reactive CD4+ T cells
(table S2). Of interest, we demonstrate a statistically significant
positive correlation between CRP and IFN\selectlanguage{greek}γ \selectlanguage{english}producing reactive CD8+ T
cells (table S2).

\textbf{Discussion}

In this study, we performed an immune profiling of the cellular and
humoral immune response in 17 pediatric patients with non-specific PASC.
We demonstrate an increased SARS-CoV-2 CD4+ and CD8+ T cell response
characterized by low avidity, TNF\selectlanguage{greek}α \selectlanguage{english}and IFN\selectlanguage{greek}γ \selectlanguage{english}production among PASC
patients. These findings might indicate a possible role of
SARS-CoV-2-specific cellular immunity that triggers inflammation as
possible immune mechanism of pediatric PASC. While immunological data
among pediatric PASC patients are scarce, accumulating data regarding
the pathogenesis of PASC in adult cohorts indicate expansion of
cytotoxic CD8+ T cells to be an important pathogenic component of PASC
(15,27--30). Bacher et al have already demonstrated the relevance of low
avidity S-reactive T cells in immunopathogenesis of acute SARS-CoV-2
infection(31), while our group recently demonstrated increased number of
SARS-CoV-2 reactive CD8+ T cells with a low TCR avidity associated with
adult PASC(32).

Although the clinical laboratory workout showed comparable creatinine
and liver parameters between the studied cohorts, we found association
between GOT, GPT and IFN\selectlanguage{greek}γ \selectlanguage{english}producing reactive CD4 T cells as well as
between creatinine and TNF\selectlanguage{greek}α \selectlanguage{english}producing reactive CD4+ T cells. These
results may suggest a possible role of SARS-CoV-2-reactive T cells
producing inflammatory cytokines in the frame of pediatric PASC with
hepatic and renal involvement. In agreement with these findings,
post-COVID-19~cholangiopathy~has been increasingly reported in
adults(33,34), while Cooper et al described recently PASC liver
manifestation in 5 pediatric patients(35).

Of interest, we demonstrated a strong positive correlation between CRP
and IFN\selectlanguage{greek}γ \selectlanguage{english}producing reactive CD8+ T cells. The inflammatory immune
response and cytokine levels have been associated with both depression
and fatigue in a large body of literature across different
disorders(36--40). In the frame of adult PASC, elevated pro-inflammatory
proteins in cerebrospinal fluid, microglia activation markers and
persistent loss of oligodendrocytes and myelinated axons in PASC
patients and mice with neurological PASC manifestation have been
demonstrated (41,42), while Kiho et al showed TNF-\selectlanguage{greek}α \selectlanguage{english}and C-reactive
protein predicted strong association with PASC symptoms at 12 months
(43).

The present study has limitations. The number of included patients was
small so that we were unable to differentiate immune response according
to symptom clusters. Rao et al in a large-scale exploratory study found
that the burden of pediatric PASC on the health system was low(10).
However, despite its phenomenal benignity in most of the cases,
long-term psychological and somatic effects of pediatric PASC are
currently unknown. Our study group underlined the risk for the
development of restrictive eating disorders in children and adolescents
with long-COVID-associated smell and taste dysfunction(20). Pediatric
PASC is a public health and social issue requiring interdisciplinary
vigilant attention.

Taken together, our results suggest that cellular inflammatory response
triggered by SARS-CoV-2 may be responsible for the observed sequelae in
pediatric PASC. These results may have implications on future
therapeutic and prevention strategies.

\textbf{Study Approval}

The study was approved by the Ethics Committee of University Hospital
Essen (21-9998\_1-KOBO, 20-9791-BO \& 22-10581-BO). Written informed
consent was obtained from all participants.

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\end{enumerate}

\textbf{Tables}

\textbf{Table 1:} Demographic characteristics of the study cohorts\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& \textbf{PASC (N=17)} & \textbf{Control (N=13)} & \textbf{p
value}\tabularnewline
\midrule
\endhead
Age years -median (range) & 11 (3-18) & 12 (6-15) & 0.39\tabularnewline
Female gender N (\%) & 9 (53) & 2 (15) & 0.0575\tabularnewline
Time since COVID-19 Diagnosis (months) & 5 (2-11) & 3 (2-8) &
0.89\tabularnewline
Duration of PASC symptoms (months)* & 5 (2-11) & N/A &
N/A\tabularnewline
COVID-19 Severity N (\%) & COVID-19 Severity N (\%) & COVID-19 Severity
N (\%) &\tabularnewline
\emph{Moderate} & 17(100) & 13(100) & N/A\tabularnewline
SARS-CoV-2 vaccination & 0 & 0 & N/A\tabularnewline
Variant of concern N (\%) & Variant of concern N (\%) & Variant of
concern N (\%) &\tabularnewline
\emph{WT} & 10 (59) & 7(54) & 1\tabularnewline
\emph{Alpha} & 7 (41) & 6 (46) & 0.8167\tabularnewline
\textbf{PASC Type} & \textbf{PASC Type} & \textbf{PASC Type}
&\tabularnewline
Ongoing COVID-19 & 0 & N/A & N/A\tabularnewline
Post-acute COVID-19 Syndrome & 17 (100) & N/A & N/A\tabularnewline
\bottomrule
\end{longtable}

*Months from SARS-CoV-2 Diagnosis (positive PCR) till the time point of
study recruitment

\textbf{Table 2:} Clinical characteristics of the study cohorts\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
PASC (N=17) median (range)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Controls (N=13) median (range)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
p value\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Blood parameters\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Blood parameters\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Blood parameters\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Blood parameters\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Blood parameters\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
BMI (kg/m\textsuperscript{2})\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
18 (14.3-23.7)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
19 (13-26)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.669\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Leukocytes/nl\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
6.44 (4.13-11.42)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
5.73 (4.73-9.29)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.426\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Hb (g/dl)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
13 (11-14.5)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
13.4 (11-15.4)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.836\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Thrombocytes/nl\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
291 (189-465)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
270.5 (226-351)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.967\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Creatinine (\selectlanguage{greek}μ\selectlanguage{english}mol/l)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
55 (18-78)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
48 (35-72)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.095\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
AST/GOT (U/l)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
23 (12-34)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
27 (17-36)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.094\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
ALT/GPT (U/l)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
18 (9-30)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
19 (9-36)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.503\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{greek}γ-\selectlanguage{english}GT (U/l)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
13 (7-24)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
14 (12-19)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.205\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Creatine Kinase (U/l)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
101 (15-230)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
153 (113-253)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{p=0.011}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Ddimers (mg/l)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.22 (0.19-0.54)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.2 (0.2-0.6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.802\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
CRP (mg/dl)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.4 (0.02-0.5)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.1 (0.0-0.5)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.342\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
PASC Symptoms N(\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
PASC Symptoms N(\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
PASC Symptoms N(\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
PASC Symptoms N(\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
PASC Symptoms N(\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Shortness of breath/exercise intolerance\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
12 (71)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Paresthesia\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
10 (59)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Smell/taste disturbance\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
7 (41)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Chest pain\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
6 (35)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Dyspnea\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
5 (29)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Headache\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
5 (29)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Concentration disturbance (brain fog)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
5 (29)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Vertigo\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4 (24)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Fatique\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2 (12)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Diarrhea\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2 (12)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Sleep disturbances\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Sensory disorder\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Palpitations\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Abdominal pain\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
New diagnosis since COVID-19\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
New diagnosis since COVID-19\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
New diagnosis since COVID-19\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
New diagnosis since COVID-19\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
New diagnosis since COVID-19\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Eating disorder\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Depressive disorder\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Asthma exacerbation\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Increased susceptibility to infections\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Instrumental Diagnostics N (\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Instrumental Diagnostics N (\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Instrumental Diagnostics N (\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Instrumental Diagnostics N (\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Instrumental Diagnostics N (\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
ECG\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11 (65)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{normal}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11 (65)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
NLG\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11 (65)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{normal}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
10 (65)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{pathological}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Echocardiogram\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11 (65)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11 (85)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{EF normal}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11 (65)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11 (85)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{mild TI}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
8 (47)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
3 (27)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.0861\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{mild AI}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{mild MI}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2 (15)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.476\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{Not relevant pericardial effusion}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (8)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Lung function test\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
9 (53)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{Ventilatory disorder}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{Bronchial hyperreactivity}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\emph{Hyperinflation}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Comorbidities\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Comorbidities\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Comorbidities\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Comorbidities\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Comorbidities\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Asthma bronchiale\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure legends}

\textbf{Figure legends}

\textbf{Figure 1: SARS-CoV-2 CD4+ driven T cell response characterized
by low avidity among the PASC subjects.} Characterization of SARS-CoV-2
S-reactive T cells in PASC and control subjects. Blood samples of 17
pediatric PASC patients and 13 convalescent controls were stimulated
with SARS-CoV-2 S-WT and analyzed by flow cytometry. (A) Frequencies of
WT-reactive CD4+ T cells among PASC and controls. (D) Frequencies of
WT-reactive CD8+ T cells among PASC and controls. (B \& E) Frequencies
of WT reactive CD4+CD3low+ and CD8+CD3low+ T cells. A high functional
avidity of SARS-CoV-2 reactive T cells was approached by determining the
CD3low+ cells among CD4+CD154+CD137+ and CD8+CD137+ cells. (C \& F)
Frequencies of WT reactive CD4+CD3high+ and CD8+CD3high+ T cells. A low
functional avidity capacity of SAR-CoV-2 reactive T cells was determined
by detecting CD3high+ cells among CD4+CD154+CD137+ and CD8+CD137+ cells.
Unpaired data were compared with Mann-Whitney-test. P\textless{}0.05 was
considered significant, only significant p values are documented in the
figures.

\textbf{Figure 2: Analysis of cytokine-producing SARS-CoV-2 reactive T
cells.} The frequencies of IL2, IFN\selectlanguage{greek}γ, \selectlanguage{english}TNF\selectlanguage{greek}α \selectlanguage{english}or GrB producing WT-reactive
T cells were analyzed among PASC subjects and controls. (A \& E) IL2
producing SARS-CoV-2 reactive CD4+ and CD8+ T cells. (B \& F) TNF\selectlanguage{greek}α
\selectlanguage{english}producing SARS-CoV-2 reactive CD4+ and CD8+ T cells. (C \& G) IFN\selectlanguage{greek}γ
\selectlanguage{english}producing SARS-CoV-2 reactive CD4+ and CD8+ T cells. (D \& H) GrB
producing SARS-CoV-2 reactive CD4+ and CD8+ T cells.

\textbf{Figure 3: Analysis of neutralizing capacity of humoral immunity
in pediatric PASC compared to controls.} Analysis of WT S NAbs titers of
both study groups. Scatterplots show line at median. Unpaired data were
compared with Mann-Whitney-test. P\textless{}0.05 was considered
significant, only significant p values are documented in the figures.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

