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\begin{document}

\title{Stromal cells and B cells orchestrate ectopic lymphoid tissue formation
in nasal polyps}



\author[1]{Zhe-Zheng  Wang}%
\author[1]{Jia Song}%
\author[1]{Hai  Wang}%
\author[1]{Jing-Xian Li}%
\author[1]{Qiao Xiao}%
\author[1]{Ze Yu}%
\author[1]{Zhi-Chao  Wang}%
\author[1]{Jin-Xin Liu}%
\author[1]{Li Pan}%
\author[2]{Yin Yao}%
\author[1]{Cai-Ling Chen}%
\author[1]{Xiang Lu}%
\author[3]{Chaohong Liu}%
\author[4]{Pei-Song Gao}%
\author[1]{Zheng Liu}%
\affil[1]{Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology}%
\affil[2]{Huazhong University of Science and Technology}%
\affil[3]{School of Basic Medicine, Huazhong University of Science and Technology}%
\affil[4]{Johns Hopkins University}%


\vspace{-1em}



  
  \date{June 07, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: Although the importance of ectopic lymphoid tissues (eLTs)
in the pathophysiology of nasal polyps (NPs) is increasingly
appreciated, the mechanisms underlying their formation remain unclear.
Objective: To study the role of IL-17A, CXCL13 and lymphotoxin (LT) in
eLT formation in NPs. Methods: The expression of CXCL13 and LT as well
as their receptors, and the phenotypes of stromal cells in NPs were
studied by flow cytometry, immunostaining, and RT-PCR. Purified nasal
stromal cells and polyp B cells were cultured and a murine model with
nasal type 17 inflammation was established for the mechanistic study.
Results: Excessive CXCL13 production was found in NPs and correlated
with enhanced IL-17A expression. Stromal cells, with an expansion of
CD31-Pdpn+ fibroblastic reticular cell (FRC) type, were the major source
of CXCL13 in NPs without eLTs. IL-17A induced FRC expansion and CXCL13
production in nasal stromal cells. In contrast, B cells were the main
source of CXCL13 and LT\selectlanguage{greek}α\selectlanguage{english}1\selectlanguage{greek}β\selectlanguage{english}2 in NPs with eLTs. CXCL13 upregulated LT\selectlanguage{greek}α\selectlanguage{english}1\selectlanguage{greek}β\selectlanguage{english}2
expression on polyp B cells, which in turn promoted CXCL13 production
from polyp B cells and nasal stromal cells. LT\selectlanguage{greek}α\selectlanguage{english}1\selectlanguage{greek}β\selectlanguage{english}2 induced expansion of
FRCs and CD31+Pdpn+ lymphoid endothelial cells, corresponding to the
phenotypic characteristic of stromal cells in NPs with eLTs. IL-17A gene
knockout, and CXCL13 and LT\selectlanguage{greek}β\selectlanguage{english}R blockage diminished nasal eLT formation in
the murine model. Conclusion: We identified an important role of
IL-17A-induced stromal cell remodeling in the initiation, and crosstalk
between B and stromal cells via CXCL13 and LT\selectlanguage{greek}α\selectlanguage{english}1\selectlanguage{greek}β\selectlanguage{english}2 in the enlargement of
eLTs in NPs.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Nasal polyps (NPs) are benign edematous outgrowth of the sinonasal
mucosa characterized by persistent and exaggerated
inflammation.\textsuperscript{1} Clinical management of NPs is largely
unsatisfying,\textsuperscript{2, 3} reflecting our limited understanding
of the pathogenesis of NPs. Emerging evidence highlight an important
role of local immunoglobulin (Ig) hyperproduction in the pathogenesis of
NPs. Local IgE, IgG, IgA and IgD may lead to the activation of mast
cells, eosinophils and classic complement pathway in NPs, and associate
with poor treatment outcome.\textsuperscript{4-7} Recently, it has been
revealed that local immunoglobulin production is supported by ectopic
lymphoid tissues (eLTs) in NPs, which are composed of T/B cell
aggregation and germinal center (GC) like
structure.\textsuperscript{8}The eLT formation associates with
refractory disease in patients with NPs as well.\textsuperscript{8, 9}
Therefore, disrupting the formation of eLTs, which likely function as
lymphoid organs in NPs, may hold significant clinical implications for
NP treatment, particularly for the refractory type. However, little is
known about what drives the appearance of eLTs in NPs.

The eLTs share morphological and functional similarities with secondary
lymphoid organs (SLOs).\textsuperscript{10} In the development of SLOs,
lymphotoxin (LT) \selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2}-expressing
lymphoid tissue inducer (LTi) cells induce the differentiation of the
LT\selectlanguage{greek}β \selectlanguage{english}receptor (LT\selectlanguage{greek}β\selectlanguage{english}R) positive stromal organizer cells and subsequent
secretion of homeostatic chemokines, such as CXCL13, CCL21 and CXCL12,
from those cells, which lead to the recruitment and compartmentalization
of T and B cells.\textsuperscript{11, 12} In contrast to the well
documented formation of SLOs, the mechanisms controlling the eLT
development are limited understood. Although homeostatic chemokines have
also been implicated in the eLT formation in inflamed tissues in
response to infection, auto-immunity, cancer and
transplantation,\textsuperscript{13-17} the involvement of specific
homeostatic chemokines is highly tissue and disease-dependent. In
addition, inflammatory cells may substitute for embryonic LTi cells to
induce the production of lymphorganogenic chemokines during ectopic
lymphoid neogenesis. For example, IL-17A has been implicated in driving
microbial infection-induced lung eLT formation by inducing CXCL12 or
CXCL13 production in murine models.\textsuperscript{18, 19}

Previous studies have demonstrated elevated IL-17A levels and increased
accumulation of IL-17A positive CD4\textsuperscript{+} and
CD8\textsuperscript{+} T cells in NPs, especially in Asian
patients.\textsuperscript{20, 21} We discovered that the presence of
eLTs associated with elevated levels of IL-17A, LT and CXCL13 in
NPs.\textsuperscript{8} IL-17A-producing cells have been identified
inside and surrounding the eLTs in NPs.\textsuperscript{8} We therefore
hypothesized that IL-17A, LT and CXCL13 may have a role in the
development of eLTs in NPs. In this study, we determined the cellular
sources of LT and CXCL13 as well as their receptors in NPs. We also
performed mechanistic studies to explore the function of IL-17A, LT and
CXCL13 in eLT formation in NPs by using nasal stromal cell and B cell
culture, and a murine model with local IL-17A inflammation.

\textbf{METHODS}

\textbf{Subjects and specimens}

This study was approved by the Ethics Committee of Tongji Hospital and
written informed consents were obtained from all subjects. A total of 78
NP patients and 49 control subjects were recruited in this study. The
diagnosis of NPs was made according to the current
guideline.\textsuperscript{1} Patients undergoing septoplasty owing to
anatomic variations and without any other inflammatory sinonasal
disorders were enrolled as control subjects. During surgery, polyp
tissues from patients with NPs and inferior turbinate mucosal tissues
from control subjects were collected. Not all samples were included in
every experiment protocol because of limited quantity. More information
is provided in this article's Online Supplement including Table E1.

\textbf{Immunohistochemistry and immunofluorescence}

The histology, immunohistochemistry and immunofluorescence study were
conducted as described elsewhere.\textsuperscript{8} In NPs, eLTs were
defined as lymphoid aggregates with GC like structure as previously
reported.\textsuperscript{8} More information is provided in this
article's Online Supplement including Tables E2 and E3.

\textbf{Quantitative RT-PCR}

Quantitative RT-PCR was performed with specific primers (Table E4 and E5)
as previously reported.\textsuperscript{7} More information is provided
in this article's Online Supplement.

\textbf{Flow cytometry}

Nasal single cell suspensions were prepared, stained, and analyzed by
flow cytometry as previously described.\textsuperscript{8} More
information including Table E6 is provided in this article's Online
Supplement.

\textbf{Purification and culture of B cells and stromal cells}

Nasal stromal cells from control tissues and B cells from polyp tissues
with eLTs were isolated by means of immunomagnetic cell sorting, and
cultured as previously stated.\textsuperscript{18, 22, 23} More
information is provided in this article's Online Supplement.

\textbf{Mouse experiment}

Female C57BL/6 wild type (WT) mice and
\emph{il17a\textsuperscript{-/-}}mice were treated with curdlan
intranasally.\textsuperscript{24-26} In some experiment, WT mice were
given anti-CXCL13 antibody or LT\selectlanguage{greek}β\selectlanguage{english}R-Ig fusion protein intraperitoneally.
Histology and RT-PCR study were performed as mentioned
elsewhere.\textsuperscript{27} As previously
described,\textsuperscript{28} lymphoid aggregates with a dense
lymphocyte core and \textgreater{} 50 cells were defined as eLTs, and
those with fewer cells and/or dispersed lymphocyte localization were
defined as lymphocyte clusters. All mice were used following protocols
approved by the Animal Care and Use Committee of Tongji Hospital. More
information is provided in this article's Online Supplement.

\textbf{Statistics}

Statistical analysis was performed with SPSS 18.0 software (SPSS,
Chicago, IL, USA). Data derived from human tissue and animal studies are
presented in dot plots with horizontal bars representing medians and
error bars showing interquartile ranges unless specifically addressed. A
Kruskal-Wallis \emph{H} test was used to assess intergroup variability
and a Mann-Whitney \emph{U} test was used for between-group comparisons.
The Spearman rank test was used for correlations. Chi-square test or
Fisher's exact test was applied to analyze differences in proportions
between groups. Data from cell culture experiments are expressed as
means \selectlanguage{ngerman}± SEMs and were analyzed by the paired Student's \emph{t} test.
Significance was accepted at a \emph{P} value of less than 0.05.

\textbf{RESULTS}

\textbf{Distinct CXCL13 expression pattern in NPs with and without eLTs}

Although both CXCL12/CXCR4 and CXCL13/CXCR5 axis are vital for B cell
attraction and aggregation, our previous study only found an elevation
of CXCL13, but not CXCL12, in NPs with eLTs.\textsuperscript{8} In this
study, we first confirmed increase of CXCL13 production in NPs with eLTs
in comparison to NPs without eLTs and control tissues at both mRNA and
protein level (Fig 1, A and B). Compared to control tissues, NPs without
eLTs also demonstrated a mild increase of CXCL13 production (Fig 1, A
and B). We therefore explored whether there is a difference in cellular
sources of CXCL13 in NPs with and without eLTs. Immunofluorescence study
showed that CXCL13 was mainly expressed by CD20\textsuperscript{+} B
cells and vimentin\textsuperscript{+} stromal cells in NPs with and
without eLTs (Fig 1, C). Given to the difficulty in counting the number
of CXCL13 positive cells in eLTs in immunofluorescence study and the
interference of membrane-bound CXCL13 on quantification of cytoplasmic
CXCL13 in B cells by flow cytometry, we detected CXCL13 mRNA levels in
different portions of cells in NPs. We depleted stromal cells from total
polyp cells and found that CXCL13 mRNA expression was remarkably reduced
in NPs without eLTs (mean, 66\% reduction), while mildly reduced in NPs
with eLTs (mean, 36\% reduction) (Fig 1, D). On the contrary, after B
cell depletion, there was about 31\% and 60\% (mean) reduction of CXCL13
mRNA expression in relation to the expression of total cells from NPs
without and with eLTs, respectively (Fig 1, E). These results suggest
that CXCL13 is mainly produced by stromal cells in NPs without eLTs,
whereas by B cells in NPs with eLTs.

\textbf{The phentypic changes of stromal cells in NPs with or without
eLTs}

The production of CXCL13 by nasal stromal cells in NPs without eLTs may
point a role of stromal cells in the recruitment of B cells and
initiation of eLT development. We then compared the CXCL13 expression in
stromal cells from different types of sinonasal tissues. We found that
compared to those in control tissues, CXCL13 mRNA expression was
increased in stromal cells purfied from both NPs with and without eLTs,
although a more prominent upreglation was noted in NPs with eLTs than
those without eLTs (Fig 2, A).

Based on CD31 and podoplanin (Pdpn, also known as gp38)
expression,\textsuperscript{29, 30} CD45 and epithelial cell adhesion
molecule double negative stromal cells are classified into four
subpopulations including
CD31\textsuperscript{+}Pdpn\textsuperscript{-}blood endothelial cell
(BEC), CD31\textsuperscript{-}Pdpn\textsuperscript{+} \selectlanguage{english}fibroblastic
reticular cell (FRC), CD31\textsuperscript{+}Pdpn\textsuperscript{+}
lymphoid endothelial cell (LEC), and
CD31\textsuperscript{-}Pdpn\textsuperscript{-} double negative cell (DN)
(Fig 2, B), which have different functions. We studied whether specific
stromal cell types are involved in eLT formation in NPs. We discovered
that the frequencies of FRCs were increased in both NPs with and without
eLTs, and the frequencies of LECs were only increased in NPs with eLTs
compared with those in control tissues (Fig 2, C and D). In contrast,
the frequencies of DNs were reduced in NPs with eLTs and no significant
change of BECs was found in NPs in comparison to those in control
tissues (Fig 2, C and D).

Analyzing CXCL13 expression in different nasal stromal cell types, we
found that CXCL13 expression was enhanced in FRCs and LECs in both types
of NPs in comparision to control tissues, with NPs with eLTs having a
higher upregulation than NPs without eLTs (Fig E1, A and B). CXCL13
expression in DNs was only upregulated in NPs with eLTs and no
upregulation of CXCL13 was found in BECs in NPs (Fig E1, A and B). We
determined the contribution of different types of stromal cells to
CXCL13 expression and found that FRCs were the major source of stromal
cell dervied CXCL13 in NPs (Fig 2, E and F).

\textbf{IL-17A induces FRC expansion and CXCL13 production in nasal
stromal cells}

Several studies have demonstrated a requirement for IL-17A signaling in
the initiation of eLT formation in animal models of chronic
inflammation.\textsuperscript{18, 19, 31-33} In this study, we confirmed
increased mRNA (Fig 3, A) and protein (Fig E2) expression of IL-17A in
NPs, especially in those with eLTs, as compared with control tissues.
Moreover, we revealed a positive correlation between IL-17A and CXCL13
mRNA expression in both NPs with and without eLTs (Fig 3, B).

Given the finding that nasal stromal cells were the major source of
CXCL13 in NPs without eLTs (Fig 1, D), we explored whether IL-17A can
regulate CXCL13 production in nasal stromal cells. We first verified
that there was IL-17RA expression on vimentin\textsuperscript{+} stromal
cells in nasal tissues by immuno\selectlanguage{english}fluorescence staining (Fig E3, A).
Additionally, we found that the mRNA expression of IL-17RA was increased
in stromal cells isolated from NPs as compared with those from control
tissues (Fig E3, B). We further found that among different types of
stromal cells, the FRC population had the highest freqencies of IL-17RA
expression in control tissues and NPs without eLTs (Fig E3, C and D).

Next, we found that IL-17A induced CXCL13 mRNA production in nasal
stromal cells purified from control tissues in a dose-dependent manner
(Fig 3, C). IL-17A also promoted CXCL13 protein production in nasal
stromal cells (Fig 3, D), in which FRCs were the major stromal cell type
producing CXCL13, accounting for 61\% (mean) of
CXCL13\textsuperscript{+} stromal cells (Fig 3, E). In addition, we
discovered that IL-17A stimulation led to a specific expansion of FRC
population and reduction of DN population in a time-dependent manner
(Fig 3, F). We did not find a change of CXCL12 mRNA expression in nasal
stromal cells after IL-17A stimulation for 12 hours (Fig E3, E).

\selectlanguage{greek}\textbf{A positive feedback loop between CXCL13 and
LTα\textsubscript{1}β\textsubscript{2} on B cells}\selectlanguage{english}

In NPs with eLTs, B cells were found to be the major producer of CXCL13
(Fig 1, E), suggesting an involvement of B cells at a later stage of eLT
formation in NPs. B cells are also known as the main source of the
membrane-bound form of LT (LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2}) in
SLOs.\textsuperscript{34, 35} We found here that the mRNA expression
levels of LT\selectlanguage{greek}α \selectlanguage{english}were upregulated in both types of NPs, whereas the levels
of LT\selectlanguage{greek}β \selectlanguage{english}were only upregulated in NPs with eLTs compared to those in
control tissues (Fig 4, A), indicating an upregulation of membrane
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} in NPs with eLTs rather than in
NPs without eLTs. Consistent with the ability of B cells to express LT,
our immunofluorescence staining demonstrated expression of LT\selectlanguage{greek}α \selectlanguage{english}and LT\selectlanguage{greek}β
\selectlanguage{english}on B cells in eLTs in NPs (Fig 4, B). Furthermore, we found that after B
cell depletion, LT\selectlanguage{greek}β \selectlanguage{english}mRNA expression was markedly decreased in patients
with NPs with eLTs (mean, 62\% reduction), but not in patients with NPs
without eLTs (mean, 32\% reduction). In contrast, a mild and comparable
reduction of LT\selectlanguage{greek}α \selectlanguage{english}mRNA expression was noted in both NPs with and without
eLTs after depleting B cells (mean, 27\% and 38\% reduction,
respectively) (Fig 4, C). These results suggest that B cells are the
main producer of LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2}, but not
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{3}, in NPs with eLTs.

Of a particular interest, it has been shown that activation of CXCR5
induced murine splenic B cells to produce membrane
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} which is required for GC
formation in SLOs.\textsuperscript{36} Here, the expression of CXCR5 on
B cells in eLTs in NPs was verified by immunofluorescence (Fig E4, A).
We further discovered that CXCL13 induced the upregulation of LT\selectlanguage{greek}β \selectlanguage{english}mRNA
expression and membrane LT\selectlanguage{greek}α \selectlanguage{english}expression in purified polyp B cells (Fig 4,
D and E). However, there was no increase of secreted
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{3} in the supernatant of B cell culture (Fig E4, B).
Collectively, these findings indicate an upregulation of membrane type,
but not secretory type of LT, in polyp B cells under CXCL13 stimulation.

In SLOs, LT\selectlanguage{greek}β\selectlanguage{english}R pathway is critical for the induction of
CXCL13.\textsuperscript{37, 38} In this study, we found that B cell were
the major source of CXCL13 in NPs with eLTs (Fig 1, E). Thereby we
tested whether the production of CXCL13 in B cells can be induced via
LT\selectlanguage{greek}β\selectlanguage{english}R pathway. We found that LT\selectlanguage{greek}β\selectlanguage{english}R was expressed by B cells in eLTs in NPs
by immunofluorescence staining (Fig E4, C). Moreover, we found that
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} treatment promoted mRNA (Fig 4,
F) and protein production of CXCL13 in polyp B cells as reflected by
CXCL13\textsuperscript{+} B cell frequencies (Fig E4, D) and CXCL13
levels in culture supernatants (Fig 4, G).

\selectlanguage{greek}\textbf{LTα\textsubscript{1}β\textsubscript{2} induces FRC and LEC
expansion and CXCL13 production in nasal stromal cells}\selectlanguage{english}

In addition to B cells, we found that LT\selectlanguage{greek}β\selectlanguage{english}R was also expressed on
vimentin\textsuperscript{+} stromal cells in eLTs in NPs by
immunofluorescence study (Fig E5, A) and the mRNA expression levels of
LT\selectlanguage{greek}β\selectlanguage{english}R was enhanced in purified stromal cells from NPs with eLTs compared
to those from NPs without eLTs and control tissues (Fig E5, B). Flow
cytometric analysis showed that LT\selectlanguage{greek}β\selectlanguage{english}R expression was upregulated in four
subsets of stromal cells in NPs with eLTs (Fig E5, C and D).

We next found that LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} induced CXCL13
mRNA production in nasal stromal cells in a dose-dependent manner (Fig
5, A). LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} also induced CXCL13
protein production in nasal stromal cells (Fig 5, B). FRCs and LECs were
found to be the major stromal cell types producing CXCL13 under
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} stimulation (Fig 5, C). Different
from the effect of IL-17A, LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2}induced
the remodeling of nasal stromal cells towards not only FRCs but also
LECs (Fig 5, D).

\textbf{IL-17A is required for \emph{de novo} nasal follicle formation
in an IL-17A high mouse model}

To further determine the role of IL-17A in eLT formation in nasal
mucosa\emph{in vivo} , we established a murine model with high local
IL-17A inflammation by nasal administration of curdlan (Fig E6,
A).\textsuperscript{24-26} Interestingly, we revealed that both 20 \selectlanguage{greek}μ\selectlanguage{english}g
and 100 \selectlanguage{greek}μ\selectlanguage{english}g curdlan treatment promoted lymphoid aggregate formation in
nasal mucosa, however, only 100 \selectlanguage{greek}μ\selectlanguage{english}g curdlan treatment induced the eLT
formation characterized by the presence of
CD21\textsuperscript{+}follicular dendritic cell networks in B cells
aggregates (Fig E6, B-D), which was supported by the elevated mRNA
expression of activation induced cytidine deaminase (AID) and CD21 in
those mice (Fig E7, A). In addition, CXCL13 and LT\selectlanguage{greek}β \selectlanguage{english}mRNA levels were
enhanced in 100 \selectlanguage{greek}μ\selectlanguage{english}g curdlan treated mice compared to other groups (Fig
E7, B). The upregulation of both Pdpn and CD31 in 100 \selectlanguage{greek}μ\selectlanguage{english}g curdlan group
suggests a remodeling of stromal cells towards FRCs and LECs during eLT
development (Fig E7, C). IL-17A and IL-17F mRNA expression in nasal
mucosa were upregulated in both 20 \selectlanguage{greek}μ\selectlanguage{english}g and 100 \selectlanguage{greek}μ\selectlanguage{english}g curdlan group compared
to control group, however, 100 \selectlanguage{greek}μ\selectlanguage{english}g curdlan group demonstrated a marked
greater upregulation than 20 \selectlanguage{greek}μ\selectlanguage{english}g curdlan group (Fig E7, D).

In contrast to WT mice, \emph{il17a\textsuperscript{-/-}} mice treated
with 100 \selectlanguage{greek}μ\selectlanguage{english}g curdlan demonstrated no lymphocyte cluster or eLT formation
(Fig 6, A and B). No upregulation of CXCL13 and LT\selectlanguage{greek}β \selectlanguage{english}(Fig 6, C), AID and
CD21 (Fig E8, A), or Pdpn and CD31 (Fig E8, B) mRNA expression was found
in \emph{il17a\textsuperscript{-/-}} mice.

\selectlanguage{greek}\textbf{Blockage of CXCL13 and LTβR inhibits \emph{de novo} nasal
follicle formation in an IL-17A high mouse model}\selectlanguage{english}

Next, we investigated the role of CXCL13 and
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} on eLT formation in the IL-17A
high mouse model induced by 100 \selectlanguage{greek}μ\selectlanguage{english}g curdlan. We found that blockage of
CXCL13 or LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} completely prevented
eLT formation, and partially diminished lymphocyte cluster development
in nasal mucosa (Fig 7). Consistently, AID and CD21 mRNA expression in
nasal mucosa were reduced in anti-CXCL13 and LT\selectlanguage{greek}β\selectlanguage{english}R-Ig treated mice (Fig
E9, A and D). The mRNA expression of CXCL13 and LT\selectlanguage{greek}β \selectlanguage{english}were decreased in
anti-CXCL13 treatment mice (Fig 7, C), while only CXCL13 mRNA expression
was significantly inhibited in mice treated with LT\selectlanguage{greek}β\selectlanguage{english}R-Ig (Fig 7, F). The
mRNA expression of CD31, but not Pdpn, was significantly inhibited by
anti-CXCL13 and LT\selectlanguage{greek}β\selectlanguage{english}R-Ig treatment (Fig E9, B and E). Nevertheless, there
was no change of IL-17A or IL-17F mRNA levels in mice treated with
anti-CXCL13 or LT\selectlanguage{greek}β\selectlanguage{english}R-Ig (Fig E9, C and F). Together with the finding of
abolished upregulation of CXCL13 and LT\selectlanguage{greek}β \selectlanguage{english}in
\emph{il17a\textsuperscript{-/-}} mice, these data suggest that CXCL13
and LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} residue downstream of IL-17A
in this model.

\textbf{DISCUSSION}

The eLTs can produce antibodies locally and therefore perpetuate
inflammation in NPs.\textsuperscript{8} However, the mechanisms
underlying the formation and expansion of eLTs in NPs remain unexplored.
In this study, for the first time, we identified a critical role for
IL-17A-induced stromal cell remodeling in the initiation, and crosstalk
between B cells and stromal cells via CXCL13 and
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} in the enlargement and
maintenance of eLTs in NPs.

Expression of homeostatic chemokines is central to the initiating events
that lead to lymphoneogenesis.\textsuperscript{13-15, 39, 40} In NPs
without eLTs, we found that stromal cells were the major cellular source
of CXCL13, indicating that stromal cells are critical for the initiation
of B cell recruitment and compartmentalization in NPs. Evidence that
IL-17A may play an important role in eLT formation by inducing the
production of lymphoid chemokine CXCL12 and CXCL13 emerge from recent
animal studies of bronchial infection and autoimmune
encephalitis.\textsuperscript{18, 32} In this study, we found that
IL-17A expression correlated with CXCL13 expression in NPs. We further
discovered that nasal stromal cells had the expression of IL-17RA and
IL-17A induced CXCL13 production in nasal stromal cells. Although IL-17A
induced the production of CXCL12, another important B cell chemokine,
during bacteria-induced lung lymphoid neogenesis,\textsuperscript{18} we
failed to find an induction of CXCL12 in nasal stromal cells by IL-17A,
which is consistent with our previous finding of no association between
CXCL12 expression and eLT formation in NPs.\textsuperscript{8}Therefore,
distinct mechanisms may underline the effect of IL-17A in promoting eLT
development in different organs and pathological conditions.

Stromal cells have a complex role at local microenvironments, which
induce immune cell migration, activation and survival, and support
lymphoid enlargement. FRCs provide homeostatic chemokines, and secrete
extracellular matrix proteins to form the structural framework for
immune cell interaction in SLOs.\textsuperscript{41} BECs and LECs
regulate lymphocyte entry into SLOs. DNs have recently been shown to
contain a novel subset of fibroblastic contractile
pericytes.\textsuperscript{42} Although the phenotype and function of
stromal cells are well documented in SLOs, little is known of their role
in eLT formation. In this study, we revealed an expansion of FRC
population of stromal cells in both NPs with and without eLTs, which was
likely induced by IL-17A and LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2}. The
expansion of FRCs in NPs without eLTs indicates a role of FRCs, but not
other types of stromal cells, in eLT formation in its infant stage given
to the findings that stromal cells were the main producer of CXCL13 in
NPs without eLTs and FRCs were the major source for CXCL13 in stromal
cells in NPs. LECs is also a fundamental compartment in controlling SLOs
organogenesis.\textsuperscript{43} Nevertheless, LECs were only expanded
in NPs with eLTs, and the expansion of LECs was induced by
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} but not IL-17A. Since
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} was only upregulated in NPs with
eLTs, LECs are more likely involved in the enlargement rather than the
initiation of eLTs in NPs by facilitating the entry of lymphocytes into
NPs.

In NPs with eLTs, we found that B cells were main producer of CXCL13.
After the B cell recruitment under the control of CXCL13 derived from
stromal cells, B cells themselves may provide a ``second wave'' of
supply of CXCL13. LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{3} and
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2}are reported to be involved in eLT
formation.\textsuperscript{44, 45}Compared with control tissues, the
mRNA expression of LT\selectlanguage{greek}β \selectlanguage{english}was only upregulated in NPs with eLTs, suggesting
an involvement of membrane form of LT in the later stage of eLT
formation in NPs. Previous studies show that CXCL13 induces murine
splenic B cells to upregulate membrane-bound LT via Grb2, and CXCL13
expression induction is dependent on LT\selectlanguage{greek}β\selectlanguage{english}R pathway in
SLOs.\textsuperscript{37, 38, 46} In this study, we demonstrated a
positive feedback loop between CXCL13 and
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} on B cells in eLTs in NPs, which
obviously exaggerates the B cell recruitment and compartmentalization.
In addition to B cells, 36\% reduction of CXCL13 expression was founded
in NPs with eLTs after depletion of stromal cells. Nasal stromal cells
also had LT\selectlanguage{greek}β\selectlanguage{english}R expression. We found that
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} reshaped stromal cells to FRC and
LEC type and promoted their CXCL13 production. Thus, the crosstalk
between stromal cells and B cells further perpetuate the eLT development
in NPs.

Using a murine model with high nasal type 17 inflammation, we confirmed
that IL-17A was able to induce eLT formation in nasal mucosa and this
process was dependent on CXCL13 and
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} \emph{in vivo}
\textsubscript{.}In the animal study, we found that only high levels of
IL-17A induced by 100 \selectlanguage{greek}μ\selectlanguage{english}g curdlan led to eLT formation. In contrast, the
comparatively lower IL-17A levels induced by curdlan at 20 \selectlanguage{greek}μ\selectlanguage{english}g only
induced lymphocyte clusters. This is in line with the finding in humans
that the IL-17A levels were more prominently elevated in NPs with eLTs
than those without eLTs.

There are several limitations in this study. We established a mouse
model by using curdlan, which elicits a high local IL-17A inflammation
but may not mirror the pathogenesis of NPs in humans. It is considered
that the type 17 response is less important for Caucasian patients with
NPs than for Chinese patients. Nevertheless, eLTs have also been
reported in Caucasian patients,\textsuperscript{47} indicating that
additional mechanisms may underlie eLT formation in Caucasian patients.

These comments notwithstanding, for the first time, we have established
a paradigm of how eLTs are formed in NPs in Chinese patients. We suspect
that targeting IL-17A, CXCL13 and
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2}may provide opportunities for the
design of therapies to manipulate eLT formation and alleviate
inflammation in patients with NPs.

\textbf{REFERENCES}

1. Fokkens WJ, Lund VJ, Hopkins C, et al. European Position Paper on
Rhinosinusitis and Nasal Polyps 2020. \emph{Rhinology.} 2020;58(Suppl
S29):1-464.

2. van der Veen J, Seys SF, Timmermans M, et al. Real-life study showing
uncontrolled rhinosinusitis after sinus surgery in a tertiary referral
centre. \emph{Allergy.} 2017;72(2):282-290.

3. Djukic V, Dudvarski Z, Arsovic N, Dimitrijevic M, Janosevic L.
Clinical outcomes and quality of life in patients with nasal polyposis
after functional endoscopic sinus surgery. \emph{European Archives of
Oto-Rhino-Laryngology.} 2015;272(1):83-89.

4. Van Zele T, Holtappels G, Gevaert P, Bachert C. Differences in
initial immunoprofiles between recurrent and nonrecurrent chronic
rhinosinusitis with nasal polyps. \emph{Am J Rhinol
Allergy.}2014;28(3):192-198.

5. De Schryver E, Calus L, Bonte H, et al. The quest for autoreactive
antibodies in nasal polyps. \emph{J Allergy Clin
Immunol.}2016;138(3):893-895 e895.

6. Tan BK, Li QZ, Suh L, et al. Evidence for intranasal antinuclear
autoantibodies in patients with chronic rhinosinusitis with nasal
polyps. \emph{J Allergy Clin Immunol.} 2011;128(6):1198-1206 e1191.

7. Cao PP, Zhang YN, Liao B, et al. Increased local IgE production
induced by common aeroallergens and phenotypic alteration of mast cells
in Chinese eosinophilic, but not non-eosinophilic, chronic
rhinosinusitis with nasal polyps. \emph{Clin Exp
Allergy.}2014;44(5):690-700.

8. Song J, Wang H, Zhang Y-N, et al. Ectopic lymphoid tissues support
local immunoglobulin production in patients with chronic rhinosinusitis
with nasal polyps. \emph{Journal of Allergy and Clinical
Immunology.}2018;141(3):927-937.

9. Lau A, Lester S, Moraitis S, et al. Tertiary lymphoid organs in
recalcitrant chronic rhinosinusitis. \emph{Journal of Allergy and
Clinical Immunology.} 2017;139(4):1371-1373.

10. Neyt K, Perros F, GeurtsvanKessel CH, Hammad H, Lambrecht BN.
Tertiary lymphoid organs in infection and autoimmunity. \emph{Trends
Immunol.} 2012;33(6):297-305.

11. Drayton DL, Liao S, Mounzer RH, Ruddle NH. Lymphoid organ
development: from ontogeny to neogenesis. \emph{Nat
Immunol.}2006;7(4):344-353.

12. Cupedo T, Kraal G, Mebius RE. The role of CD45+CD4+CD3- cells in
lymphoid organ development. \emph{Immunol Rev.} 2002;189:41-50.

13. Bracke KR, Verhamme FM, Seys LJ, et al. Role of CXCL13 in cigarette
smoke-induced lymphoid follicle formation and chronic obstructive
pulmonary disease. \emph{Am J Respir Crit Care Med.}2013;188(3):343-355.

14. de Chaisemartin L, Goc J, Damotte D, et al. Characterization of
chemokines and adhesion molecules associated with T cell presence in
tertiary lymphoid structures in human lung cancer. \emph{Cancer
Res.}2011;71(20):6391-6399.

15. Salomonsson S, Larsson P, Tengner P, Mellquist E, Hjelmstrom P,
Wahren-Herlenius M. Expression of the B cell-attracting chemokine CXCL13
in the target organ and autoantibody production in ectopic lymphoid
tissue in the chronic inflammatory disease Sjogren's
syndrome.\emph{Scand J Immunol.} 2002;55(4):336-342.

16. Di Carlo E, D'Antuono T, Contento S, Di Nicola M, Ballone E,
Sorrentino C. Quilty effect has the features of lymphoid neogenesis and
shares CXCL13-CXCR5 pathway with recurrent acute cardiac
rejections.\emph{Am J Transplant.} 2007;7(1):201-210.

17. Yamamoto K, Nishiumi S, Yang L, et al. Anti-CXCL13 antibody can
inhibit the formation of gastric lymphoid follicles induced by
Helicobacter infection. \emph{Mucosal Immunol.} 2014;7(5):1244-1254.

18. Fleige H, Ravens S, Moschovakis GL, et al. IL-17-induced CXCL12
recruits B cells and induces follicle formation in BALT in the absence
of differentiated FDCs. \emph{Journal of Experimental
Medicine.}2014;211(4):643-651.

19. Rangel-Moreno J, Carragher DM, de la Luz Garcia-Hernandez M, et al.
The development of inducible bronchus-associated lymphoid tissue depends
on IL-17. \emph{Nat Immunol.} 2011;12(7):639-646.

20. Miljkovic D, Psaltis AJ, Wormald PJ, Vreugde S. Chronic
Rhinosinusitis with Polyps Is Characterized by Increased Mucosal and
Blood Th17 Effector Cytokine Producing Cells. \emph{Front
Physiol.}2017;8:898.

21. Ma J, Shi LL, Deng YK, et al. CD8(+) T cells with distinct
cytokine-producing features and low cytotoxic activity in eosinophilic
and non-eosinophilic chronic rhinosinusitis with nasal polyps.\emph{Clin
Exp Allergy.} 2016;46(9):1162-1175.

22. Zhai GT, Wang H, Li JX, et al. IgD-activated mast cells induce IgE
synthesis in B cells in nasal polyps. \emph{J Allergy Clin
Immunol.}2018;142(5):1489-1499 e1423.

23. Barone F, Nayar S, Campos J, et al. IL-22 regulates lymphoid
chemokine production and assembly of tertiary lymphoid organs.\emph{Proc
Natl Acad Sci U S A.} 2015;112(35):11024-11029.

24. Osorio F, LeibundGut-Landmann S, Lochner M, et al. DC activated via
dectin-1 convert Treg into IL-17 producers. \emph{Eur J
Immunol.}2008;38(12):3274-3281.

25. Zhang Z, Biagini Myers JM, Brandt EB, et al. beta-Glucan exacerbates
allergic asthma independent of fungal sensitization and promotes
steroid-resistant TH2/TH17 responses. \emph{J Allergy Clin
Immunol.}2017;139(1):54-65 e58.

26. Ryu JH, Yoo JY, Kim MJ, et al. Distinct TLR-mediated pathways
regulate house dust mite-induced allergic disease in the upper and lower
airways. \emph{J Allergy Clin Immunol.} 2013;131(2):549-561.

27. Zhang XH, Zhang YN, Li HB, et al. Overexpression of miR-125b, a
novel regulator of innate immunity, in eosinophilic chronic
rhinosinusitis with nasal polyps. \emph{Am J Respir Crit Care
Med.}2012;185(2):140-151.

28. Seys LJ, Verhamme FM, Schinwald A, et al. Role of B Cell-Activating
Factor in Chronic Obstructive Pulmonary Disease. \emph{Am J Respir Crit
Care Med.} 2015;192(6):706-718.

29. Hahnlein JS, Nadafi R, de Jong T, et al. Impaired lymph node stromal
cell function during the earliest phases of rheumatoid
arthritis.\emph{Arthritis Res Ther.} 2018;20(1):35.

30. Fletcher AL, Malhotra D, Acton SE, et al. Reproducible isolation of
lymph node stromal cells reveals site-dependent differences in
fibroblastic reticular cells. \emph{Front Immunol.} 2011;2:35.

31. Deteix C, Attuil-Audenis V, Duthey A, et al. Intragraft Th17
infiltrate promotes lymphoid neogenesis and hastens clinical chronic
rejection. \emph{J Immunol.} 2010;184(9):5344-5351.

32. Peters A, Pitcher LA, Sullivan JM, et al. Th17 cells induce ectopic
lymphoid follicles in central nervous system tissue
inflammation.\emph{Immunity.} 2011;35(6):986-996.

33. Libbey JE, Tsunoda I, Fujinami RS. Possible role of interleukin-17
in a prime/challenge model of multiple sclerosis. \emph{Journal of
Neurovirology.} 2012;18(6):471-478.

34. Endres R, Alimzhanov MB, Plitz T, et al. Mature follicular dendritic
cell networks depend on expression of lymphotoxin beta receptor by
radioresistant stromal cells and of lymphotoxin beta and tumor necrosis
factor by B cells. \emph{J Exp Med.} 1999;189(1):159-168.

35. Tumanov A, Kuprash D, Lagarkova M, et al. Distinct role of surface
lymphotoxin expressed by B cells in the organization of secondary
lymphoid tissues. \emph{Immunity.} 2002;17(3):239-250.

36. Ansel KM, Ngo VN, Hyman PL, et al. A chemokine-driven positive
feedback loop organizes lymphoid follicles.
\emph{Nature.}2000;406(6793):309-314.

37. Suto H, Katakai T, Sugai M, Kinashi T, Shimizu A. CXCL13 production
by an established lymph node stromal cell line via lymphotoxin-beta
receptor engagement involves the cooperation of multiple signaling
pathways. \emph{Int Immunol.} 2009;21(4):467-476.

38. Yu P, Wang Y, Chin RK, et al. B cells control the migration of a
subset of dendritic cells into B cell follicles via CXC chemokine ligand
13 in a lymphotoxin-dependent fashion. \emph{J
Immunol.}2002;168(10):5117-5123.

39. van de Pavert SA, Olivier BJ, Goverse G, et al. Chemokine CXCL13 is
essential for lymph node initiation and is induced by retinoic acid and
neuronal stimulation. \emph{Nat Immunol.} 2009;10(11):1193-1199.

40. Ansel KM, Cyster JG. Chemokines in lymphopoiesis and lymphoid organ
development. \emph{Curr Opin Immunol.} 2001;13(2):172-179.

41. Fletcher AL, Acton SE, Knoblich K. Lymph node fibroblastic reticular
cells in health and disease. \emph{Nat Rev Immunol.} 2015;15(6):350-361.

42. Malhotra D, Fletcher AL, Astarita J, et al. Transcriptional
profiling of stroma from inflamed and resting lymph nodes defines
immunological hallmarks. \emph{Nat Immunol.} 2012;13(5):499-510.

43. Onder L, Danuser R, Scandella E, et al. Endothelial cell-specific
lymphotoxin-beta receptor signaling is critical for lymph node and high
endothelial venule formation. \emph{J Exp Med.} 2013;210(3):465-473.

44. Motallebzadeh R, Rehakova S, Conlon TM, et al. Blocking lymphotoxin
signaling abrogates the development of ectopic lymphoid tissue within
cardiac allografts and inhibits effector antibody responses. \emph{FASEB
J.} 2012;26(1):51-62.

45. Ruddle NH. Lymphotoxin and TNF: how it all began-a tribute to the
travelers. \emph{Cytokine Growth Factor Rev.} 2014;25(2):83-89.

46. Jang IK, Cronshaw DG, Xie LK, et al. Growth-factor receptor-bound
protein-2 (Grb2) signaling in B cells controls lymphoid follicle
organization and germinal center reaction. \emph{Proc Natl Acad} Sci U S
A. 2011;108(19):7926-7931.

47. Gevaert P, Holtappels G, Johansson SG, Cuvelier C, Cauwenberge P,
Bachert C. Organization of secondary lymphoid tissue and local IgE
formation to Staphylococcus aureus enterotoxins in nasal polyp tissue.
Allergy. 2005;60(1):71-79

\textbf{FIGURE LEGENDS}

\textbf{Fig 1. CXCL13 expression in NPs with and without eLT formation.
A,} The mRNA expression levels of CXCL13 in different study groups as
detected by quantitative RT-PCR. \textbf{B,} The protein levels of
CXCL13 in different study groups as detected by ELISA.
\textbf{C,}Representative photomicrographs showing immunofluorescence
staining of CXCL13 on vimentin\textsuperscript{+} stromal cells and
CD20\textsuperscript{+} B cells in polyp tissues with and without eLTs.
Original magnification \selectlanguage{ngerman}×200. Insets show a higher magni\selectlanguage{english}fication of the
outlined area, and arrows denote representative positive
cells.\textbf{D,} Reduction of CXCL13 mRNA expression after depletion of
stromal cells in relation to total polyp cells from NPs with and without
eLTs. Data are expressed as means +- SEMs. \selectlanguage{english}\textbf{E,} Reduction of
CXCL13 mRNA expression after depletion of B cells in relation to total
polyp cells from NPs with and without eLTs. Data are expressed as means
+- SEMs. NPs, nasal polyps; eLTs\textsuperscript{+}, with ectopic
lymphoid tissues; eLTs\textsuperscript{-}, without ectopic lymphoid
tissues; DAPI, 4, 6-Diamidino-2-phenylindole dihydrochloride.
*\emph{P}\textless{} 0.05; ***\emph{P} \textless{} 0.001.

\textbf{Fig 2. Phenotypic changes of stromal cells in NPs A,} The mRNA
expression levels of CXCL13 in nasal stromal cells purified from
different types of tissues as detected by quantitative
RT-PCR.\textbf{B,} The gating strategy and representative \selectlanguage{english}flow plots
showing nasal stromal cells in tissues. \textbf{C,} The frequencies of
different stromal cell subpopulations in different study groups as
detected by flow cytometry. \textbf{D,} The composition of stromal cell
subsets in different study groups. Mean percentages are shown.
\textbf{E,} The gating strategy of CXCL13\textsuperscript{+} stromal
cells. \textbf{F,}The composition of different types of stromal cells
for CXCL13\textsuperscript{+} stromal cells in different types of
tissues as detected by flow cytometry. Mean percentages of different
stromal cell types are shown. NPs, nasal polyps;
eLTs\textsuperscript{+}, with ectopic lymphoid tissues;
eLTs\textsuperscript{-}, without ectopic lymphoid tissues; EpCAM,
epithelial cell adhesion molecule; Pdpn, podoplanin; FRCs, \selectlanguage{english}fibroblastic
reticular cells; LECs, lymphoid endothelial cells; BECs, blood
endothelial cells; DNs, double negative cells. **\emph{P} \textless{}
0.01; ***\emph{P} \textless{} 0.001.

\textbf{Fig 3. IL-17A induces CXCL13 production and FRC expansion in
nasal stromal cells. A,} The mRNA expression levels of IL-17A in
different study groups as detected by quantitative RT-PCR.
\textbf{B,}The correlation between IL-17A and CXCL13 mRNA expression
levels in NPs with and without eLTs. \textbf{C,} Purified nasal stromal
cells from control tissues were stimulated with various doses of IL-17A
for 12 hours and mRNA expression of CXCL13 were measured by quantitative
RT-PCR (n = 6). \textbf{D,} Purified nasal stromal cells from control
tissues were stimulated with IL-17A at 100 ng/mL for 36 hours and CXCL13
positive cells were quantified by flow cytometry (n = 6). Representative
flow plots are shown. \textbf{E,} Purified nasal stromal cells from
control tissues were stimulated with IL-17A at 100 ng/mL for 36 hours,
and CXCL13 positive cells were analyzed for the composition of different
types of stromal cells by flow cytometry (n = 6). Mean percentages of
different stromal cell types are shown. \textbf{F,} After treatment with
IL-17A at 100 ng/mL for different time points, the frequencies of
different types of stromal cells were detected by flow cytometry (n =
6). NPs, nasal polyps; eLTs\textsuperscript{+}, with ectopic lymphoid
tissues; eLTs\textsuperscript{-}, without ectopic lymphoid tissues;
Pdpn, podoplanin; FRCs, \selectlanguage{english}fibroblastic reticular cells; LECs, lymphoid
endothelial cells; BECs, blood endothelial cells; DNs, double negative
cells. **\emph{P} \textless{} 0.01; ***\emph{P} \textless{} 0.001.

\selectlanguage{greek}\textbf{Fig 4. A positive feedback loop between CXCL13 and LTα1β2 on B
cells. A,}\selectlanguage{english} The mRNA expression levels of LT\selectlanguage{greek}α \selectlanguage{english}and LT\selectlanguage{greek}β \selectlanguage{english}in different study
groups as detected by quantitative RT-PCR. \textbf{B,} Representative
photomicrographs showing immunofluorescence staining of LT\selectlanguage{greek}α \selectlanguage{english}and LT\selectlanguage{greek}β \selectlanguage{english}on
CD20\textsuperscript{+} B cells in eLTs in NPs. Original magnification
\selectlanguage{ngerman}×200. Insets show a higher magni\selectlanguage{english}fication of the outlined area, and arrows
denote representative positive cells. \textbf{C,} Reduction of LT\selectlanguage{greek}α \selectlanguage{english}and
LT\selectlanguage{greek}β \selectlanguage{english}mRNA expression after depletion of B cells in relation to total
polyp cells from NP with and without eLTs. Data are expressed as means \selectlanguage{ngerman}±
SEMs. \textbf{D,} B cells purified from polyp tissues with eLTs were
stimulated with CXCL13 at various doses for 12 hours, and the mRNA
expression of LT\selectlanguage{greek}β \selectlanguage{english}in B cells was detected by RT-PCR (n = 6).
\textbf{E,}B cells purified from poly tissues with eLTs were stimulated
with CXCL13 at 1000 ng/mL for 36 hours, the membrane expression of LT\selectlanguage{greek}α
\selectlanguage{english}on B cells was detected by flow cytometry (n = 6). The representative
flow plots are shown. \textbf{F,} B cells purified from polyp tissues
with eLTs were stimulated with LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} at
various doses for 12 hours, and the mRNA expression of CXCL13 in B cells
was detected by RT-PCR (n = 6). \textbf{G,} B cells purified from polyp
tissues with eLTs were stimulated with
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} at 1000 ng/mL for 48 hours, the
protein levels of CXCL13 in culture supernatants were detected by ELISA
(n = 6). NPs, nasal polyps; eLTs\textsuperscript{+}, with ectopic
lymphoid tissues; eLTs\textsuperscript{-}, without ectopic lymphoid
tissues; LT, lymphotoxin. **\emph{P} \textless{} 0.01;
***\emph{P}\textless{} 0.001.

\selectlanguage{greek}\textbf{Fig 5. LTα\textsubscript{1}β\textsubscript{2} induces FRC and
LEC expansion and CXCL13 production in nasal stromal cells. A,}\selectlanguage{english} Nasal
stromal cells purified from control tissues were treated with
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} at various doses for 12 hours,
and the mRNA expression of CXCL13 in stromal cells were detected by
RT-PCR (n = 6). \textbf{B,} After stimulation with
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} at 100 ng/mL for 36 hours, CXCL13
positive nasal stromal cells were analyzed by flow cytometry (n = 6).
Representative flow plots are shown. \textbf{C,} After stimulation with
LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} at 100 ng/mL for 36 hours, CXCL13
positive stromal cells were analyzed for the composition of different
stromal cell types by flow cytometry (n = 6). Mean percentages of
different types of stromal cells are shown. \textbf{D,} After
stimulation with LT\selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{2} at 100 ng/mL for
different time points, the frequencies of different stromal cell
populations were detected by flow cytometry (n = 6). NPs, nasal polyps;
eLTs\textsuperscript{+}, with ectopic lymphoid tissues;
eLTs\textsuperscript{-}, without ectopic lymphoid tissues; LT,
lymphotoxin; Pdpn, podoplanin; FRCs, fibroblastic reticular cells; LECs,
lymphoid endothelial cells; BECs, blood endothelial cells; DNs, double
negative cells. *\emph{P} \textless{} 0.05; **\emph{P} \textless{} 0.01;
***\emph{P} \textless{} 0.001.

\textbf{Fig 6. IL-17A is required for \emph{de novo} nasal follicle
formation in an IL-17A high mouse model. A,} Histologic studies showed
lymphoid aggregates in WT and \emph{il17a\textsuperscript{-/-}} mice
challenged by curdlan at 100 \selectlanguage{greek}μ\selectlanguage{english}g or PBS (control). \textbf{B,} The number
of eLTs and lymphocyte clusters in different study groups.
\textbf{C,}The mRNA levels of CXCL13 and LT\selectlanguage{greek}β \selectlanguage{english}in nasal mucosa in
different study groups. WT, wild type; eLTs, ectopic lymphoid tissues;
LT, lymphotoxin. *\emph{P} \textless{} 0.05; **\emph{P} \textless{}
0.01.

\selectlanguage{greek}\textbf{Fig 7. Nasal eLT formation is diminished by CXCL13 and LTβR
blockage in the mouse model. A,}\selectlanguage{english} Histologic studies showed lymphoid
aggregates in WT mice challenged by curdlan at 100 \selectlanguage{greek}μ\selectlanguage{english}g and treated with
anti-CXCL13 or rat control IgG1. \textbf{B,} The number of eLTs and
lymphocyte clusters in WT mice with and without anti-CXCL13
treatment.\textbf{C,} The mRNA levels of CXCL13 and LT\selectlanguage{greek}β \selectlanguage{english}in nasal mucosa
in WT mice with and without anti-CXCL13 treatment. \textbf{D,}
Histologic studies showed lymphoid aggregates in WT mice challenged by
curdlan at 100 \selectlanguage{greek}μ\selectlanguage{english}g and treated with LT\selectlanguage{greek}β\selectlanguage{english}R-Ig or mouse control IgG1.
\textbf{E,} The number of eLTs and lymphocyte clusters in WT mice with
and without LT\selectlanguage{greek}β\selectlanguage{english}R-Ig treatment. \textbf{F,} The mRNA levels of CXCL13 and
LT\selectlanguage{greek}β \selectlanguage{english}in WT mice with and without LT\selectlanguage{greek}β\selectlanguage{english}R-Ig treatment. WT, wild type; eLTs,
ectopic lymphoid tissues; LT, lymphotoxin. *\emph{P} \textless{} 0.05;
**\emph{P}\textless{} 0.01.

\par\null

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