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\begin{document}

\title{The redundant role of plasmacytoid dendritic cells in Primary Sj\selectlanguage{ngerman}ögren's
syndrome\selectlanguage{english}}



\author[1]{Rongli Li}%
\author[2]{Mu Wang}%
\author[1]{Chuiwen Deng}%
\author[1]{Yu Peng}%
\author[1]{Lidan Zhao}%
\author[1]{Wen Zhang}%
\author[1]{Jiaxin Zhou}%
\author[1]{Yingying Chen}%
\author[1]{Anqi Wang}%
\author[1]{Yimeng Jia}%
\author[1]{Mengtao Li}%
\author[1]{Yan Zhao}%
\author[1]{Xiaofeng Zeng}%
\author[1]{Xunyao Wu}%
\author[1]{Yunyun Fei}%
\affil[1]{Peking Union Medical College Hospital Department of Rheumatology and Clinical Immunology}%
\affil[2]{Peking Union Medical College Hospital}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
\textbf{Objective:} The aim of our present study is to investigate the
role of plasmacytoid dendritic cells (pDCs) in the pathogenesis and type
I interferon (IFN) signatures in Primary Sj\selectlanguage{ngerman}ögren's Syndrome (pSS)
patients. \textbf{Methods:} In the present study, we compared the
percentage, activation markers, and representative cytokines secretion
of pDCs derived from treatment-naive pSS and matched healthy controls
(HCs) by flow cytometry. We performed pDC/B co-culture system to explore
the contribution of pDC to B cell functions in pSS. \textbf{Results:}
The percentage of pDC was significantly reduced in the peripheral blood
of pSS. The activation markers (CD80, CD83, and CD86) expressions,
chemokine receptors, and representative cytokines production (IFN-\selectlanguage{greek}α,
\selectlanguage{english}IL-6, and TNF-\selectlanguage{greek}α\selectlanguage{english}) of pDC were similar between pSS and HCs. Only a few
pDCs infiltration were detected in the labial gland. The percentage of
pDCs was negatively correlated with serum IgG, IgA, and anti-SSA
autoantibody levels and resting pDCs were able to efficiently promote B
cells proliferation, activation, differentiation, and antibody
production \emph{in vitro}. However, there was no difference between HC
and pSS-derived pDCs. Finally, we found that incubation of plasma from
pSS patients could significantly induce pDCs apoptosis than that from
HCs and both IgG and IgA dramatically increased the apoptotic rates of
pDCs. \textbf{Conclusion:} Our data have deciphered the redundant role
of pDC in the type I signature and disease development in pSS. Also, we
demonstrated the decreased percentage of pDC in pSS patients might
result from apoptosis induced by the excess of immunoglobulin (IgG and
IgA).%
\end{abstract}%



\sloppy


\selectlanguage{ngerman}\textbf{The redundant role of plasmacytoid dendritic cells in Primary
Sjögren's syndrome}

Rongli Li\textsuperscript{1,2}, Mu Wang\textsuperscript{3}, Chuiwen
Deng\textsuperscript{1,2}, Yu Peng\textsuperscript{1,2}, Lidan
Zhao\textsuperscript{1,2}, Wen Zhang\textsuperscript{1,2}, Jiaxin
Zhou\textsuperscript{1,2}, Yingying Chen\textsuperscript{1,2}, Anqi
Wang\textsuperscript{1,2}, Yimeng Jia\textsuperscript{1,2}, Mengtao
Li\textsuperscript{1,2}, Yan Zhao\textsuperscript{1,2}, Xiaofeng
Zeng\textsuperscript{1,2}, Xunyao Wu\textsuperscript{1,4*}, Yunyun
Fei\textsuperscript{1,2*}

\textsuperscript{1} Department of Rheumatology and Clinical Immunology,
Peking Union Medical College Hospital, Chinese Academy of Medical
Sciences, Peking Union Medical College, Beijing, China

\textsuperscript{2} National Clinical Research Center for Dermatologic
and Immunologic Diseases (NCRC-DID), The Ministry of Education Key
Laboratory, Beijing, China

\textsuperscript{3} Department of Stomatology, Peking Union Medical
College Hospital, Chinese Academy of Medical Sciences \& Peking Union
Medical College, Beijing, China.

\textsuperscript{4} Department of Medical Research Center, Peking Union
Medical College Hospital.

\textsuperscript{\textbf{*}}\textbf{Correspondence:}Dr. Xunyao Wu,
e-mail: xunyaowu@mail.ustc.edu.cn and Dr. Yunyun Fei, email:
feiyunyun@pumch.cn; Department of Rheumatology and Clinical Immunology,
Peking Union Medical College Hospital, \#1 Shuai-Fu-Yuan, Dongcheng
District, Beijing, 100730, China.

\textbf{Statements and Declarations}

\textbf{Competing interests:}

The authors have declared no conflicts of interest.

\textbf{Ethics approval and consent to participate:}

The study was approved by the Ethics Committee of the Peking Union
Medical College Hospital (No: K2525) and was conducted in accordance
with the Helsinki Declaration. Informed consent of all the patients were
obtained.

\textbf{Consent for publication:}

The manuscript is approved by all authors for publication, and written
informed consent to publish were obtained.

\textbf{Funding:}

This study was supported by the National Natural Science Foundation of
China (grant numbers: 81971545, 81971544), the National High-Level
Hospital Clinical Research Funding (2022-PUMCH-C-039, 2022-PUMCH-B-013)
and the Fundamental Research Funds for the Central Universities (No.
3332022105).



\textbf{Abstract}

\textbf{Objective:} The aim of our present study is to investigate the
role of plasmacytoid dendritic cells (pDCs) in the pathogenesis and type
I interferon (IFN) signatures in Primary Sjögren's Syndrome (pSS)
patients.

\textbf{Methods:} In the present study, we compared the percentage,
activation markers, and representative cytokines secretion of pDCs
derived from treatment-naive pSS and matched healthy controls (HCs) by
flow cytometry. We performed pDC/B co-culture system to explore the
contribution of pDC to B cell functions in pSS.

\textbf{Results:} The percentage of pDC was significantly reduced in the
peripheral blood of pSS. The activation markers (CD80, CD83, and CD86)
expressions, chemokine receptors, and representative cytokines
production (IFN-\selectlanguage{greek}α, \selectlanguage{english}IL-6, and TNF-\selectlanguage{greek}α\selectlanguage{english}) of pDC were similar between pSS and
HCs. Only a few pDCs infiltration were detected in the labial gland. The
percentage of pDCs was negatively correlated with serum IgG, IgA, and
anti-SSA autoantibody levels and resting pDCs were able to efficiently
promote B cells proliferation, activation, differentiation, and antibody
production \emph{in vitro} . However, there was no difference between HC
and pSS-derived pDCs. Finally, we found that incubation of plasma from
pSS patients could significantly induce pDCs apoptosis than that from
HCs and both IgG and IgA dramatically increased the apoptotic rates of
pDCs.

\textbf{Conclusion:} Our data have deciphered the redundant role of pDC
in the type I signature and disease development in pSS. Also, we
demonstrated the decreased percentage of pDC in pSS patients might
result from apoptosis induced by the excess of immunoglobulin (IgG and
IgA).

\textbf{Key words:} Primary Sj\selectlanguage{ngerman}ögren's syndrome, Plasmacytoid dendritic
cell, Type I interferon, B cell, Hypergammaglobulinemia



\textbf{Introduction}

Primary sjögren's syndrome (pSS) is a typical autoimmune disorder
characterized by focal lymphocytic infiltrations, damage, and
dysfunction in salivary and lacrimal glands {[}1{]}. Lung, kidney, and
nervous system involvement could also occur in severe cases {[}2{]}.
Previous studies have demonstrated the type I IFN signatures in
peripheral blood mononuclear cells (PBMCs), B cells, monocytes,
neutrophils as well as the affected tissues {[}3-10{]}. Moreover, IFN-\selectlanguage{greek}α
\selectlanguage{english}inducible proteins: MDA-5, IFIT-3 {[}11{]}, and B cell-activating factor
(BAFF) {[}12{]} were found highly expressed in salivary glands. Type I
IFN could enhance T cell and B cell responses and promote the production
of autoantibodies {[}11, 13{]}, implicating their important roles in the
pathogenesis of pSS.

Acting as a premier type I IFN producer and a key bridge between innate
and adaptive immunity, pDCs also play a nonnegligible contribution to
autoimmune disease development. pDCs in SLE patients can be activated
through various pathways (immune complexes, neutrophil extracellular
traps, mitochondrial DNA, \emph{etc} .), which in turn trigger immune
responses and promote the production of autoantibodies {[}14{]}. In
studies of systemic sclerosis (SSc) {[}15{]}, Psoriasis {[}16{]},
rheumatoid arthritis {[}17{]} and autoimmune diabetes {[}18{]}, pDCs
could infiltrate into the target tissue and exacerbate local
inflammation by releasing IFN-\selectlanguage{greek}α \selectlanguage{english}and proinflammatory factors.

Currently, studies on the role of pDCs in pSS are limited. Previous
studies have reported the reduced frequencies of pDCs in peripheral
blood {[}19, 20{]} of pSS patients. A recent study performed a
transcriptional analysis of circulating pDCs and identified the aberrant
activation of pDC in pSS patients {[}21{]}. Here, we systematically
study the percentage, phenotype, and functions of pDCs for a better
understanding of their roles in the pathogenesis and type I IFN
signatures in pSS patients.

\textbf{Methods}

\textbf{Patients and ethics}

All newly-onset pSS and SLE patients were enrolled in Peking Union
Medical College Hospital (PUMCH) and met the 2016 ACR-EULAR
classification{[}22{]} and the 1997 classification criteria of the
American College of Rheumatology{[}23{]}, respectively. Labial tissues
were obtained from patients who underwent labial gland biopsy at the
department of stomatology in PUMCH. Patients all signed informed consent
for their residual tissues after the pathological examination. This
study was approved by the Ethics Committee of PUMCH (No: K2525).

\textbf{PBMC Isolation and \emph{In vitro} stimulation}

Human PBMCs were isolated with Ficoll-Paque density (DAKEWE, China) as
previously described{[}8{]}. For the plasma stimulation, PBMCs from HCs
were seeded into 24-well plates at a density of
1\selectlanguage{ngerman}×10\textsuperscript{6}/ml and maintained in RPMI 1640 (Gibco, A10491,
USA) with 20\% mixed plasma from ten pSS or HCs in the presence of 100
ng/ml IL-3 for 4 hours.

For IgG and IgA stimulation, freshly isolated PBMCs from HCs were first
incubated with 100 mg/ml IL-3 for 2 hours, then stimulated with human
IgG (SP001, Solarbio) and human IgA (SP016, Solarbio) with indicated
concentrations. IL3 is used to assist the survival of pDCs in vitro
{[}24, 25{]}.

\emph{In vitro} \textbf{purification and stimulation of pDC}

pDC was purified using CD304 microbeads (130-090-532, Miltenyi Biotec)
and maintained in RPMI 1640 supplemented with 10\% fetal bovine serum
(FBS) (Gibco, USA), 100 U/ml penicillin and 100 \selectlanguage{greek}μ\selectlanguage{english}g/ml streptomycin
(15140122, ThermoFisher). For cytokines detection, PBMCs were stimulated
with 5 \selectlanguage{greek}μ\selectlanguage{english}g/ml R848 (tlrl-r848, InvivoGen) or 10 \selectlanguage{greek}μ\selectlanguage{english}g/ml ODN-2216
(tlrl-2216, InvivoGen) in the presence of 100 ng/ml IL-3 (Peprotech,
200-03). R848 is a ligand for Toll-like receptor 7 (TLR7) and OND-2216
is a ligand for TLR9.

\textbf{pDC and B cell coculture}

B cells were purified using B cell isolation kit II (130-091-151,
Miltenyi Biotec) following the manufacturer's protocol. B cells were
activated with 5 \selectlanguage{greek}μ\selectlanguage{english}g/mL anti-IgM (314502, BioLegend), 500 ng/mL sCD40L
(310-02, Peprotech), 100 ng/ml IL-4 (200-04, Peprotech), and 50 ng/ml
IL21 (200-21, Peprotech). For pDCs and B cells coculture, purified
CD19\textsuperscript{+} B cells (5\selectlanguage{ngerman}×10\textsuperscript{5}/ml) and pDC
from allogeneic HCs were added at the ratio of 10:1. The supernatants
were harvested at day 7.

\textbf{Flow cytometry}

Cells were harvested and resuspended at the concentration of
5×10\textsuperscript{6} cells/ml. Fluorochrome-conjugated monoclonal
antibodies for cell surface staining were as follows: anti-human CD303,
CD123, Lineage, HLA-DR, CD80, CD83, CD86, CD19, CD40, CD74, CD27, IgD,
CCR2, CCR4, CCR5, CCR7, CCR10, CD38, CD138, CD24.

The Intracellular Fixation \& Permeabilization Buffer Set (eBioscience)
was used for intracellular staining following manufacturer's protocol.
Fluorochrome-conjugated monoclonal antibodies for intracellular staining
are as follows: anti-human Ki-67, TLR7, TLR9, IFN-\selectlanguage{greek}α, \selectlanguage{english}TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-6.

Detailed information about conjugated fluorescence and manufacturers for
antibodies is shown in Supplementary Table 1.

\textbf{Apoptosis assay}

Cell apoptosis was detected by PE Annexin V Apoptosis Detection Kit I
(559763, BD Pharmingen). Briefly, cells were washed with 1ml 1\selectlanguage{ngerman}× Annexin
V binding buffer after regular surface staining, then incubated with
PE-conjugated Annexin V and 7-AAD for 15 minutes at room temperature,
all the samples were analyzed by BD Accuri C6 flow cytometer (Becton
Dickinson, USA).

\textbf{Immunofluorescence}

Freshly labial tissue was embedded in O.C.T. (Tissue-Tek) and 8\selectlanguage{greek}μ\selectlanguage{english}m frozen
sections were prepared. Sections were immersed in 95\% ethanol for 15
min, blocked in normal goat serum for 3h at room temperature, followed
by incubating with 25 \selectlanguage{greek}μ\selectlanguage{english}g/ml mouse anti-human BDCA-2 (CD303) monoclonal
antibody (MAB62991, R\&D), 1:50 diluted mouse anti-human IFN-\selectlanguage{greek}α
\selectlanguage{english}monoclonal antibody (sc-373757, SANTA CRUZ BIOTECHNOLOGY, INC.) or 1:500
diluted rabbit anti-human EpCAM monoclonal antibody (also known as
CD326, ab223582, abcam) at 4\selectlanguage{ngerman}°C overnight. CoraLite594-conjugated goat
anti-mouse secondary antibody (proteintech, China) or
CoraLite488-conjugated goat anti-rabbit secondary antibody (proteintech,
China) was incubated at a dilution of 1:500 for 1h at room temperature.
Further, the slides were mounted by an antifading mounting medium with
DAPI (S2110, Solarbio) and scanned by Pannoramic~MIDI (3DHISTECH,
Hungary).

\textbf{ELISA}

The levels of IgG, IgM, and IgA in culture supernatants were measured
with Human IgG/IgM/IgA Precoated ELISA Kit (1128162, 1128182, 1128172
DAKEWE, China) respectively. The level of IFN\selectlanguage{greek}α \selectlanguage{english}in plasma from pSS
patients and matched HCs was determined by Human IFN-\selectlanguage{greek}α \selectlanguage{english}Precoated ELISA
Kit (1110012, DAKEWE, China) following the manufacturer's instructions.

\textbf{Statistical analysis}

All the data analyses were conducted by IBM SPSS statistics (Version
25.0, IBM, Armonk, NY, USA) and GraphPad Prism 8 (GraphPad Software,
Inc., La Jolla, CA, USA) software. The data were first performed with
the normality distribution. Student's t-test was used for variables in a
normal distribution, otherwise, the Mann-Whitney test was used. Linear
regression and Pearson's correlation were applied to explore the
correlation analysis. A\emph{p} -value \textless{} 0.05 was defined as
statistically significant.



\textbf{Results}

\textbf{The frequency and phenotype analysis of peripheral pDC in pSS
patients}

We first analyzed the frequency of pDC in PBMCs from treatment-na\selectlanguage{ngerman}ïve pSS
(n=31), HCQ-treated pSS (n=17), SLE (n=9), and HCs (n=29) by flow
cytometry. The demographic characteristics of all the included pSS
patients were demonstrated in Supplementary Table 2 and SLE patients
were demonstrated in Supplementary Table 3. The gating strategy of pDCs
was shown in Figure S1a. In accordance with previous studies {[}20,
26{]}, the percentage of pDCs was significantly lower in PBMCs from
treatment naïve SLE and pSS patients compared with HCs. Interestingly,
the reduction of pDCs rebounded in pSS patients after Hydroxychloroquine
(HCQ) therapy (Fig 1a). However, we did not observe the hyperactivation
status of pDCs in pSS as the activation markers CD80, CD83, and CD86
expressions were equal to HCs (Fig 1b, Fig S1c).

Detection of TLR7/9 expressions showed that unstimulated pDCs from pSS
patients showed higher TLR7 but not TLR9 expressions compared with HCs
(Fig 1c, d). However, the difference vanished after TLR7 (R848) or TLR9
ligand (ODN2216) stimulation (Fig 1e, f).

\textbf{pDCs did not contribute to peripheral and affected tissue type I
IFN signature in pSS patients}

To determine whether pDC from pSS patients displays the enhanced
capacity of IFN-\selectlanguage{greek}α \selectlanguage{english}secretion, we stimulated freshly isolated PBMCs with
TLR7 (R848) or TLR9 ligand (ODN-2216) and determined representative
cytokines production by flow cytometry. However, upon stimulation, there
was no difference in the IFN-\selectlanguage{greek}α \selectlanguage{english}secretion of pDCs between HC and pSS
patients (Fig 2a-b). The same was observed in the IL-6 and TNF-\selectlanguage{greek}α
\selectlanguage{english}production (Fig 2c-f).

We next detected the infiltration of pDCs in labial glands by
immunofluorescence to explore whether they contribute to tissue type I
IFN signature in pSS patients. Only one patient had a few pDCs
infiltration, while the other three hardly had any pDCs in the labial
gland (Fig 3a). Recent researches highlighted the role of salivary
epithelial cells in producing type I IFNs {[}27-29{]}, consistently, our
immunofluorescent results indicated that epithelial cells are
responsible for the production of tissular IFN-\selectlanguage{greek}α \selectlanguage{english}in patients with or
without pDC infiltration (Fig 3b). We also detected the main chemokine
receptors, including CCR2, CCR4, CCR5, CCR7, and CCR10 on the pDC
surface showed no difference between pSS patients and HCs (Fig 3c, Fig
S2j).

\textbf{pDCs promote B cell class-switching and antibody production}

Correlation analysis between the frequency of peripheral pDCs and
clinical parameters in pSS patients showed that the percentage of pDCs
was negatively correlated with serum IgG, IgA, and anti-SSA autoantibody
levels (Fig S2a-i, Fig 3d). We, therefore, established the pDC/B cells
co-culture system to explore the potential role of pDC in promoting B
cell responses and antibody production in pSS patients.

We found that resting or TLR-7-activated pDCs were able to efficiently
promote the proliferation, activation, and differentiation into
plasmablast/plasma cell of B cells (Fig 4a-e, Fig S3a-e). Also, we
explored the effects of pDCs on antibody production and found that even
resting pDCs could promote IgG, and IgA production by B cells (Fig 4f).
However, there was no difference between HC and pSS-derived pDCs.
Similarly, no difference was found in the effects of HC or pSS derived
pDCs on naive or memory B cells (Fig S4).

\textbf{pDCs are more sensitized to pSS plasma-induced apoptosis}

Finally, we sought to explore the potential mechanism of reduced pDC
frequency in the peripheral blood of pSS patients. Incubation of plasma
from pSS patients could significantly reduce pDCs viability than that
from HCs (Fig 5a). Considering that pSS is characterized by high
autoantibodies in serum and pDC could promote antibody production as we
demonstrated above, we inferred that plasma-derived IgG and IgA might be
important inducers of pDC apoptosis. As we expected, both IgG and IgA
dramatically increased the apoptotic rates of pDCs in a dose-dependent
manner (Fig 5b-e). The viability of freshly isolated pDCs showed no
significance between pSS and HCs (Fig S5a). Although pSS had higher
serum IFN-\selectlanguage{greek}α \selectlanguage{english}levels (Fig S5b), they had little effect on the pDC
apoptosis (Fig S5c-e). These results indicate that hyper immunoglobulin
in the serum of pSS patients contributes to the reduction of pDC in the
peripheral blood (Fig 6).

\textbf{Discussion}

The critical role of the type I IFN pathway in the pathogenesis of pSS
has been widely addressed {[}30, 31{]}. Acting as the premier producer
of IFN-\selectlanguage{greek}α, \selectlanguage{english}the role of pDC in the pathogenesis of pSS still remains
inclusive. In the present study, we have deciphered the redundant role
of pDC in the type I signature and disease development in pSS.

Consistent with previous studies, we have detected a peripheral
reduction in pSS {[}20, 26{]}. We have demonstrated a negative feedback
loop, in which, pDC promoted antibody production and in turn, high
immunoglobulin could induce pDC apoptosis. It has been reported that
IgG-complexed adenoviruses induce the apoptosis of human pDCs {[}32{]}.
Hyperactivated B cells, characteristic autoantibodies (SSA, SSB), and
hypergammaglobulinemia are hallmarks of pSS {[}33{]}. Previous studies
have found that pDCs could efficiently promote the differentiation of B
cells into plasmablasts and plasma cells through type I IFNs and IL-6
{[}34, 35{]}. Moreover, pDCs could enhance the autoreactivity of B cells
via type I IFNs in a T cell-independent manner {[}36{]}. Although our
data showed that, pSS-derived pDC did not display a stronger capacity
for promoting plasma cell differentiation and antibody production, we
gave a good explanation that the excess immunoglobulin (IgG and IgA)
produced by hyperactivated B cells might result in the apoptosis and
reduction of pDC in peripheral blood of pSS. In addition,
Hydroxychloroquine (HCQ) effectively inhibits B cell activation {[}37{]}
and a recent systematic review revealed that serum IgA in patients with
pSS decreased significantly after using HCQ {[}38{]}, which is
consistent with our results that pDC frequency rebounded after HCQ
therapy.

Nonetheless, pDC is noted for its unique ability in producing type I
IFNs, our data suggest that it might not be the culprit of hyperactive
type I IFN signaling in pSS. Recent studies in pSS have emphasized the
critical role of dysregulated epithelial cells {[}29, 39{]}, especially
the active IFN signaling in salivary gland epithelial cells (SEGCs)
{[}29{]}. SEGCs from pSS were found to be sensitive to the stimulation
of TLR agonists and produced type I IFNs as a response to the
stimulation {[}27, 28, 40{]}. Type I IFNs released by SEGCs could
further promote the secretion of BAFF in an NF-\selectlanguage{greek}κ\selectlanguage{english}B dependent way {[}12,
27{]}. These results imply that gland epithelial cells are not purely
innocent victims, but can also be the trigger of type I IFN signaling in
pSS.

In SSc, pDCs directionally migrated to target organs and secreted IFN-\selectlanguage{greek}α
\selectlanguage{english}and CXCL4, thereby accelerated tissue fibrosis {[}15, 41{]}. In another
autoimmune disease with fibrosis signature, IgG4-related disease, active
pDCs and relevant IFN-\selectlanguage{greek}α \selectlanguage{english}signaling were also observed in related
pancreas, pDC also enhanced the production of IgG4 by B cells {[}42{]}.
Dual role of pDCs on IFN-\selectlanguage{greek}α \selectlanguage{english}production and promoting activation of
pathogenic T cells in psoriasis has been revealed in psoriasis {[}43{]}.
Study in RA mouse model suggested that pDC aggravated joint inflammation
and bone erosion via TLR7 dependent type I IFNs {[}17{]}. Reduction of
pDC in the peripheral blood as well as the concomitant infiltration and
activation in related tissue were reported in SLE {[}44, 45{]}. A recent
randomized controlled trial in SLE clarified that Litifilimab
(anti-BDCA2 antibody, BDCA2 is an exclusive marker of pDC) is effective
in disease remission {[}46{]}, which indicated the feasibility of pDC
purge strategy in SLE treatment. Whereas, newly published research
revealed that SLE-derived pDC had senescence and inert phenotype, active
keratinocytes should be responsible for the tupe I IFN signaling rather
than pDC {[}26{]}. In general, pDCs contributed to the pathogenesis of
autoimmune diseases through type I IFNs or interplay with other immune
cells {[}47{]}. However, further studies are needed to elucidate the
potential mechanism about pDC's contribution to SLE and pSS.

Our available evidence does not support an overactive phenotype (resting
and activating status) of pDCs from pSS patients, particularly in the
production of IFN-\selectlanguage{greek}α. \selectlanguage{english}However, a previous transcriptional study reported
that pDCs from patients with pSS secreted more type I IFNs after TLR7
stimulation {[}21{]}. Antonios et al. 's work that pDCs from patients
with SLE or pSS did not show stronger secretion ability for inflammatory
cytokines, especially IFN\selectlanguage{greek}α\selectlanguage{english} {[}26{]}, which is consistent with our
findings. These divergent results may be caused by the difference of
detection methods, further study will help to elucidate underlying
mechanism.

In conclusion, we demonstrated the decreased percentage of pDC in pSS
patients, which might result from the excess immunoglobulin (IgG and
IgA) induced apoptosis. Moreover, we showed that pDC might not be the
major contributor to the hyperactivation of type I interferon signaling
in pSS patients. Our research provides a good addition to pSS
pathogenesis and gives implications that targeting pDC might not be a
good strategy for clinical pSS treatment.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Fig 1. Frequency and phenotype analysis of peripheral pDCs in
pSS patients.}

\textbf{a:} The frequency of peripheral pDCs in untreated patients with
pSS or SLE, pSS patients treated with HCQ and matched HC (HC: n=29;
untreated pSS: n=31; pSS treated with HCQ: n=17; untreated SLE:
n=9).\textbf{b:} Comparison of activation markers (Mean Fluorescence
Intensity, MFI) on the surface of pDCs between pSS (n=12) and HCs
(n=12). Comparison of TLR7 (n=8) and TLR9 (n=6) expression levels
between pDCs from pSS and HC without stimulation \textbf{(c, d)} and
activated by TLR7 agonist \textbf{(e)} or TLR9 agonist \textbf{(f)} for
6 hours with 100ng/ml IL3 (n=6). Data are shown as mean \selectlanguage{ngerman}± SEM. Unpaired
two-tailed Student's t-test was applied. *P-value \textless{} 0.05,
**P-value \textless{} 0.01, ***P-value \textless{}0.001, **** P-value
\textless{}0.0001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig 2. Detection of characteristic cytokines in pDCs with
different activation states.}

\textbf{a:} Freshly isolated PBMCs from pSS or HC were cultured in the
presence of IL3, R848 (TLR7 agonist) or ODN2216 (TLR9 agonist) was added
at the 6th hour, GolgiPlug was added at the 9th hour. secretion of IFN-\selectlanguage{greek}α
\selectlanguage{english}by pDCs was examined through intracellular staining at the 12th hour.
pDCs were gated as CD303+CD123+HLA-DR+LIN- cells; \textbf{b:} Comparison
of IFN-\selectlanguage{greek}α \selectlanguage{english}secretion between HC (n=7) and pSS (n=7). \textbf{c:} Freshly
isolated PBMCs from pSS or HC were cultured in the presence of IL3 and
GolgiPlug, stimulated by R848 or ODN2216 for 12 hours, intracellular
staining was used to detect the secretion of IL-6; \textbf{c:}Comparison
of IL-6 secretion between HC (n=7) and pSS (n=7); Representative flow
cytometric graphs \textbf{(d)} and statistical graph\textbf{(f)} for
TNF-\selectlanguage{greek}α \selectlanguage{english}secretion (detected by the same method as IL-6). Data are shown as
mean \selectlanguage{ngerman}± SEM. Unpaired two-tailed Student's t-test was applied.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Fig 3. Few infiltrations of pDCs in labial gland and theclinical
correlation analysis. a:} Immunofluorescent results of 4 pSS patients
who had labial pathological report with typical focal lymphocytic
infiltrations.\textbf{b:} Immunofluorescent~colocalization of epithelial
cells (CD326) and IFN-\selectlanguage{greek}α \selectlanguage{english}in salivary glands of pSS patients (n=2).
\textbf{c:}Comparison of surface chemotactic receptors (MFI) in pDCs
between pSS and HCs, CCR2 (HC: n=11; pSS: n=11), CCR4 (HC: n=11; pSS:
n=10), CCR5 (HC: n=12; pSS: n=12), CCR7 (HC: n=10; pSS: n=12), CCR10
(HC: n=14; pSS: n=12). \textbf{d:} Correlation analysis of pDC
percentage in PBMC and serum IgG (n=30), IgA (n=29), anti-SSA IgG
(n=23). Data are shown as mean \selectlanguage{ngerman}± SEM. Unpaired two-tailed Student's
t-test, Linear regression and Pearson's correlation were performed for
IgG and IgA, Spearman rank correlation were performed for anti-SSA IgG.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Fig 4. Co-culture of unstimulated pDCs and B cells.} Purified
CD19+ B cells from allogeneic healthy donors cultured alone or with pDCs
(without extra activation) from pSS and HC in the presence of 100ng/ml
IL3 for 7 days, the ratio of pDC to B cell was 1:10.
\textbf{a:}Representative flow cytometric graph of ki-67 in B cells
cultured alone or cultured with pDCs. \textbf{b:} The effect of pDCs on
B cell proliferation (n=12) and the effect comparison between HC (n=6)
and pSS (n=6) derived pDC on B cells.\textbf{c:} Expression of
activation markers on B cells cultured alone or cultured with
pDCs.\textbf{d:} Representative flow cytometric graph of B cells
differentiation into plasma cells/plasmablasts. \textbf{e:} The effect
of pDCs on B cell differentiation (n=12) and the effects comparison
between HC (n=6) and pSS (n=6) derived pDCs on B cell
differentiation.\textbf{f:} Secretion of IgG, IgA and IgM by B cells
cultured alone or cultured with pDCs (n=12). Data are shown as mean \selectlanguage{ngerman}±
SEM. Paired and unpaired two-tailed Student's t-test was performed.
*P-value \textless{} 0.05, **P-value \textless{} 0.01, ***P-value
\textless{}0.001, **** P-value \textless{}0.0001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Fig 5. pSS plasma, IgG and IgA down-regulate the viability of
pDCs. }

\textbf{a.} Representative flow cytometric graph and summary graph of
pDCs viability. pDCs were stimulated by HC or pSS derived plasma (n=5)
for 4 hours with 100ng/ml IL3. Representative flow cytometric graph of
apoptosis detection, pDCs were stimulated by increasing concentration of
IgG \textbf{(b)} or IgA \textbf{(c)} for 2 hours with 100ng/ml IL3
(n=4). Statistical graph of pDCs viability when stimulated by different
concentrations of human IgG \textbf{(d)} and IgA \textbf{(e)} . Unpaired
two-tailed Student's t-test was performed. *P-value \textless{} 0.05,
**P-value \textless{} 0.01, ***P-value \textless{}0.001, **** P-value
\textless{}0.0001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\selectlanguage{ngerman}\textbf{Fig 6. The interplay between pDC and B cell contributed to the
pathogenesis of primary Sjögren's syndrome.}

pDCs promote the proliferation, activation, and differentiation into
plasmablast/plasma cell of B cells, hyperactive B cells in pSS induce
the apoptosis of pDC via excess IgG, IgA and autoantibodies.

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