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\begin{document}

\title{The proportion of CD161 on CD56+NK cells in peripheral circulation
associates with clinical features and disease activity of primary
Sj\selectlanguage{ngerman}ögren's syndrome\selectlanguage{english}}



\author[1]{Cuiping Liu}%
\author[2]{ping zhao}%
\author[1]{Yanhong Yang}%
\author[1]{Saizhe Song}%
\author[3]{wei cheng}%
\author[3]{cheng peng}%
\author[4]{Xin Chang}%
\author[3]{Jian Wu}%
\affil[1]{First Affiliated Hospital of Soochow University Jiangsu Key Laboratory of Clinical Immunology}%
\affil[2]{The First Affiliated Hospital of Bengbu Medical College Department of Rheumatology}%
\affil[3]{First Affiliated Hospital of Soochow University}%
\affil[4]{Soochow University Affiliated No 1 People's Hospital}%


\vspace{-1em}



  
  \date{May 30, 2024}


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\selectlanguage{english}
\begin{abstract}
Objectives:The purpose of this study was to examine the proportion of
CD161 on CD56+Natural Killer(NK) cells in peripheral blood of primary
Sj\selectlanguage{ngerman}ögren's syndrome(pSS) and investigate its clinical relevance of pSS.
Methods:The proportion of CD56+NK cells and CD161 on CD56+NK cells was
detected by flow cytometry in 31 pSS patients and 29 healthy controls
(HCs). The correlations between proportion of CD161+CD56+NK cells and
clinical features and disease activity of pSS were further analysed.
Meanwhile, we drew the receiver operating characteristic(ROC) curve to
evaluate the diagnostic value of CD161+CD56+NK cells in pSS. Results:
The proportion of CD56+ NK cells and CD161+CD56+NK cells decreased
markly in pSS patients compared to HCs. The correlation analysis showed
that proportion of CD161+CD56+NK cells negatively correlated with
WBC,IgA,IgM,IgG,ESSRPI and ESSDAI,and positively correlated with
complement C4.The proportion of CD161+CD56+NK cells in pSS patients with
decayed tooth, fatigue, arthralgia, skin involvement, primary biliary
cirrhosis(PBC),interstitial lung disease(ILD), anti-SSA/Ro60 positive,
anti-SSB positive and high IgG was lower than that in negative patients.
Furthermore, compared with innactive patients,the proportion of
CD161+CD56+NK cells decreased obviously in active patients. The area
under the curve(AUC)was 0.7375(P=0.0016),the results indicated that
CD161+CD56+NK cells had certain diagnostic value for pSS. Conclusion:
This study suggested that the proportion of CD56+NK cells and
CD161+CD56+NK cells decreased significantly in pSS patients,and the
proportion of CD161+CD56+NK cells negatively associated with the
clinical features and disease activity of pSS patients. The
CD161+CD56+NK cells may present as a potential target for therapy and a
biomarker of disease activity in pSS .%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{1.Introduction}

Primary Sjogren's syndrome(pSS) is a complex autoimmune disease, which
is characterized by the infiltration of lymphocytes from exocrine
glands, leading to the dysfunction of gland secretion, and then a series
of symptoms of gland injuries, such as dry mouth, dry eye and swelling
of parotid gland, as well as extraglandular manifestations such as
arthralgia, interstitial lung disease(ILD), renal tubular acidosis and
primary biliary cholangitis(PBC),and part of patients have systemic
manifestations, such as fatigue, fever and weight
loss\textsuperscript{{[}1, 2{]}}. Until now, the pathogenesis of pSS has
not been clarified.Generally speaking, the pathogenesis of pSS is
related to environmental factors,genetic susceptibility and immune
disorder.It is well known that pSS is mainly mediated by T cells and B
cells, but innate immune cells such as NK cells also play an important
role in the pathogenesis of pSS\textsuperscript{{[}3{]}}.Recent studies
have found that the expression of NK cells in peripheral blood or
salivary glands of pSS patients or model mice was abnormal, which was
related to the activity and severity of the
disease\textsuperscript{{[}4-6{]}}.

CD161 is one of a member of human NKRP1 subfamily which is called NK
cell receptor protein 1A (NKR-P1A) or killer cell lectin-like receptor
subfamily B member 1 (KLRB1),and it's also a type II transmembrane
C-type lectin glycoprotein receptor, which is called C-type lectin
domain family 5 member B (CLEC5B)\textsuperscript{{[}7{]}}. CD161 is
mostly expressed on NK cells and T cell subsets such as CD4+T cells,CD8
\selectlanguage{greek}αβ\selectlanguage{english}T cells, \selectlanguage{greek}γδ\selectlanguage{english}T cells and NKT cells\textsuperscript{{[}8{]}}.In humans,
the ligand of CD161 is called lectin-like transcript 1(LLT1),which is
mainly expressed on activated monocytes and B cells, and interacts with
CD161 to inhibit the activation of NK cells, and inhibit cytotoxic
function and cytokine secretion mediated by NK
cells\textsuperscript{{[}9, 10{]}}.

At present, the few research of CD161 in pSS patients is limited to T
cells and the subsets of T cells\textsuperscript{{[}11, 12{]}}, and
there is no relevant study on the clinical relevance of CD161 expression
on NK cells in pSS patients.In this study,we detected the proportion of
CD161 in peripheral blood on CD56+NK cells of pSS patients by flow
cytometry, and analyzed the clinical correlation between the proportion
of CD161+CD56+NK cells and disease activity in pSS. This research aims
to provide new ideas and therapeutic targets for the diagnosis and
treatment of pSS.

\textbf{2.Materials and methods}

\textbf{2.1 Patients and healthy controls}

A total of 31 pSS patients were enrolled from the Rheumatology and
Immunology Department,the First Affiliated Hospital of Soochow
University in the study. All patients were diagnosed according to the
classification standard of primary Sjogren's syndrome of American
College of Rheumatology(ACR)/European League Against Rheumatism(EULAR)
in 2016\textsuperscript{{[}13{]}},and those who with malignant tumor,
chronic hepatitis C(HCV), human immunodeficiency virus infection (HIV),
sarcoidosis, amyloidosis, graft versus host disease (GVHD), IgG4-related
diseases and other rheumatic diseases were excluded.We also recruited 29
individuals (age and gender matched) without autoimmune diseases,cancer
and infectious diseases from the physical examination center as healthy
controls (HCs).This research was approved by the Ethics Committee of the
First Affiliated Hospital of Soochow University in 2020 (Ethical No.
2020105).And all participants signed the informed consent.

\textbf{2.2 Data Collection}

We recorded the detailed clinical manifestations of these pSS patients,
such as dry mouth, dry eye, decayed tooth, fatigue, arthralgia,
raynaud's phenomenon, skin involvement, gland involvement, fever, muscle
involvement, renal tubular acidosis, interstitial lung disease(ILD) and
primary biliary cirrhosis(PBC) etc.At the same time, the laboratory data
such as white blood cell count, red blood cell count, platelet count,
erythrocyte sedimentation rate (ESR), C- reactive protein (CRP),
rheumatoid factor (RF), serum globulin,
immunoglobulin(IgG,IgA,IgM),complement (C3,C4) and
autoantibodies(anti-SSA/Ro52, anti-SSA/Ro60, anti-SSB/La and
anti-centromere) were collected.The blood samples for above tests were
fasting blood from pSS patients in the morning.According to the above
clinical manifestations and laboratory parameters, the European League
Against Rheumatism Sjogren's Syndrome Disease Activity Index(ESSDAI) and
European League Against Rheumatism Sjogren's Syndrome Patient
Reported(ESSPRI) of patients were
evaluated\textsuperscript{{[}14{]}}.ESSPRI is the patient's subjective
score, which is the average of the three points including dryness, pain
and fatigue, and each evaluation score range is 0 to 10. ESSDAI is the
pSS disease activity index, which is calculated according to patients's
clinical manifestations, laboratory parameters, and corresponding weight
coefficient.At the same time,general informations of HCs such as gender
and age were collected.

\textbf{2.3 Flow cytometric analysis}

To analyze the proportion of CD56+NK cells and CD161 on the surface of
CD56+NK cells in pSS patients and HCs peripheral blood, we used the
following human fluorescent antibody markers: anti-CD56-FITC (Biolegend,
USA) and anti-CD161-PE(Biolegend, USA). The isotype control antibodies
used in our study were as follows: Mouse IgG 1\selectlanguage{greek}κ-\selectlanguage{english}FITC (Biolegend,
USA),Mouse IgG1\selectlanguage{greek}κ-\selectlanguage{english}PE (Biolegend, USA).

Fresh venous blood of pSS patients and HCs on an empty stomach were
collected with heparinized anticoagulation tubes. According to the
instructions of the manufacturer, 50\selectlanguage{greek}μ\selectlanguage{english}L whole blood samples were stained
with human CD56-FITC antibody and human CD161-PE antibody, and incubated
at 4in the dark for 30 minutes.Then, 200\selectlanguage{greek}μ\selectlanguage{english}L/test of erythrolysin(1\selectlanguage{ngerman}×) was
added into all sample tubes, and all samples vibrated by an oscillator
and placed in a thermostat at 37\selectlanguage{english} to fully lyse red blood cells. Then,
2mL/test sheath fluid was added into all samples for washing and
centrifugation(1200rpm,5min), and the waste liquid was carefully dumped
(Took and put the test tubes gently to avoid vibration!).
Next,0.5mL/test of sheath fluid was added to resuspend white blood cells
to obtain leukocyte suspension for detection. The flow cytometric
analysis was processed by flow cytometry (FC500, Beckman Coulter, USA),
and the data results were analyzed by FlowJo 7.6 software.

\textbf{2.4 Statistical analysis}

All data was performed using GraphPad Prism (version 8.0.2) software for
statistical analysis and graphic presentations. The normality of the
data was tested by the Shapiro-Wilk test(n\textless{}50). Normally
distributed data was expressed as mean+- standard deviation, and
non-normally distributed data was represented by the median (minimum
number-maximum number). The Mann-Whitney U-test was used for the data
without normal distribution. The T-test was used for normally
distributed paired samples.The correlation analysis between two
continuous variables that were non-normally distributed was analyzed
using Spearman's rank correlation. The statistical significance was
determined as \emph{P} \textless{}.05 (*\emph{P} \textless{} .05,
**\emph{P} \textless{} .01,****\emph{P} \textless{} .0001).

\textbf{3.Results}

\textbf{3.1 Essential informations of participants}

A total of 31 pSS patients (29 females,2 males) and 29 HCs (28 females,1
males)were recruited in this study.There was no significant difference
in age between pSS and HCs groups(47.13+-11.94 vs. 43.69+-8.79,\emph{P}
=0.212).The mean disease duration of pSS patients was 46 months, ranging
from 1 month to 122 months.The clinical manifestations and laboratory
parameters of the enrolled participants are presented in Table 1.

\textbf{3.2 The proportion of CD56+NK cells and CD161+CD56+NK cells
decreased in pSS patients}

In order to evaluate the expression of CD56+NK cells and CD161 on
surface of CD56+NK cells in peripheral blood of pSS patients and HCs,we
used flow cytometry to detect the proportion of CD56+NK cells and
CD161+CD56+NK cells.The results showed that the proportion of CD56+NK
cells decreased markedly compared with
HCs(9.16\%(3.05\%-21.80\%)vs.12.75\%(7.36\%-26.40\%),\emph{P}
=0.0052)(Fig.1C). Similarly, the proportion of CD161+CD56+NK cells was
also significantly lower than that in HCs (65.90\% (25.15\%-96.10\%) vs.
76.10\% (5.22\%-96.40\%), \emph{P} =0.0013)(Fig.1D).The gating
strategies for CD56+NK cells and CD161+CD56+NK cells are represented in
Fig.1A,and the representative FACS plots are shown in Fig.1B.

\textbf{3.3 Difference of CD161+CD56+NK cells proportion in pSS patients
with different clinical manifestations}

We compared the differences of CD161+CD56+NK cells proportion in pSS

patients with different clinical manifestations(Table 2), it was found
that the

proportion of CD161+CD56+NK cells with decayed tooth(53.60\%+-16.83\% vs.

67.00\%+-9.81\%,\emph{P} =0.013,Fig.2A) ,fatigue(55.71\%+-17.50\% vs.
67.54\%+-10.49\%,\emph{P} =0.030,Fig.2B),
arthralgia(57.38\%+-14.24\%vs.69.69\%+-12.04\%,\emph{P} =0.030,Fig.2C),
skininvolvement(49.84\%+-18.09\%vs.66.30\%+-11.94\%,\emph{P}
=0.016,Fig.2D), PBC(49.00\%+-

21.32\%vs.66.31\%+-12.64\%,\emph{P} =0.028,Fig.2E) and
ILD(53.32\%+-17.57\% vs.67.78\%+-10.23\%,\emph{P} =0.008,Fig.2F) in pSS
patients notably decreased compared with pSS

patients without above manifestations.

We also analyzed the difference in the proportion of CD161+CD56+NK

cells between pSS patients with positive and negative autoantibodies.The
resultsshowed that the proportion of CD161+CD56+NK cells in patients
with Anti-

SSA/Ro60 positive(59.59\%+-15.25\% vs.76.34\%+-13.47\%,\emph{P}
=0.021,Fig.2G) and

Anti-SSB positive (57.96\%+-15.63\% vs.74.76\%+-11.06\%,\emph{P}
=0.005,Fig.2H) was

significantly lower than that in patients with negative
autoantibodies.Compared

with pSS patients with normal IgG, the proportion of CD161+CD56+NK cells
inpatients with high IgG obviously decreased(57.22\%+-15.95\%
vs.70.85\%+-10.87\%,\emph{P} =0.018,Fig.2I).However, there was no
significant difference in the proportion of CD161+CD56+NK cells between
pSS patients with dry mouth, dry eye,

gland involvement, leukopenia and raynaud's phenomenon and patients
without

the above clinical manifestations.The proportion of CD161+CD56+NK cells

only slightly decreased in anti-SSA/Ro52 positive and anti-centromere
positive patients compared with anti-SSA/Ro52 and anti-centromere
negative patients,

but there was also no statistical difference.

\textbf{3.4 The proportion of CD161+CD56+NK cells decreased
significantly in active pSS patients}

Based on ESDDAI score, pSS patients with ESSDAI\selectlanguage{english}[?]5 were defined as
activegroup, and ESSDAI\textless{}5 were defined as inactive
group\textsuperscript{{[}15{]}}.We analyzed the

difference of proportion between CD56+NK cells and CD161+CD56+NK cells

in active and inactive pSS patients and HCs, and we observed that
CD56+NK cells proportion decreased in active and inactive pSS patients
compared with

HCs(active vs. HCs: 8.81\%(5.13\%-20.00\%)
vs.13.75\%(8.90\%-26.40\%),\emph{P} =0.002.

inactive vs. HCs: 9.50\%(3.05\%-21.80\%)
vs.13.75\%(8.90\%-26.40\%),\emph{P} =0.007)

(Fig.3A), but there was no statistical difference between active and
inactive

pSS patients(8.81\%(5.13\%-20.00\%) vs. 9.50\%(3.05\%-21.80\%),\emph{P}
=0.429)(Fig.3A).

Regarding the difference of CD161+CD56+NK cells proportion in active and
inactive pSS patients and HCs, we found that not only CD161+CD56+NK
cells proportion in active and inactive pSS patients was obviously lower
than that in HCs(active vs. HCs:55.04\%+-18.73\%
vs.77.38\%+-10.53\%,\emph{P} \textless{}0.0001. inactive vs.
HCs:69.06\%+-11.39\% vs.77.38\%+-10.53\%,\emph{P} =0.014)(Fig.3B), but
also the proportion of CD161+CD56+NK cells in active pSS patients
significantly decreased compared with inactive pSS
patients(55.04\%+-18.73\% vs. 69.06\%+-11.39\%,\emph{P} =0.016)(Fig.3B).

\textbf{3.5 CD161+CD56+NK cells proportion was negatively correlated
with disease activity in pSS patients}

We subsequently analyzed the correlations between CD161+CD56+NK cells

proportion and laboratory features and disease activity index in pSS
patients

(Table 3).It was found that a positive correlation was verified between
CD161+CD56+NK cells proportion and C4(r=0.4205, \emph{P} =0.0289,Table
3, Fig.4J), and CD161+CD56+NK cells proportion was negatively correlated
with WBC(r=-0.3893,\emph{P} =0.0369,Table
3,Fig.4A),IgA(r=-0.4594,\emph{P} =0.0093,Table
3,Fig.4B),IgM(r=-0.4047,\emph{P} =0.0294,Table 3,Fig.4C),
IgG(r=-0.4968,\emph{P} =0.0159,Table 3,Fig.4D),
ESSDAI(r=-0.3624,\emph{P} =0.0451,Table 3,Fig.4E) and
ESSPRI(r=-0.4863,\emph{P} =0.0055,Table 3,Fig.4F).However,there was no
significant difference of correlations between

CD161+CD56+NK cells and ESR, serum globulin and C3(Table 3).

\textbf{3.6 The ROC curve of CD161+CD56+NK cells proportion to predict
the occurrence of pSS}

Combined with our above research results, we found that the proportion
of CD161+CD56+NK cells in pSS patients decreased significantly, and it
was related to the clinical manifestations, laboratory parameters and
disease activities of pSS.Thus, we drew receiver operating
characteristic (ROC) curve to evaluate the diagnostic value of
CD161+CD56+NK cells for predicting the occurrence of pSS disease. The
results showed that AUC(Area Under the Curve)=0.7375,\emph{P}
=0.0016(Fig 3C), and suggested that CD161+CD56+NK cells had a certain
diagnostic value for the occurrence of pSS.

\textbf{4.Discussion}

pSS is a systemic autoimmune disease.In addition to dry symptoms caused
by gland dysfunction, there are also extraglandular manifestations,
which can be life-threatening in severe cases, at the same time, these
symptoms such as dryness, fatigue and pain often adversely affect the
quality of life in patients\textsuperscript{{[}16{]}}.At present, the
therapy of pSS is mainly based on clinical experience and symptomatic
treatment, such as alleviating dryness and applying total glucosides of
paeony(TGP), hydroxychloroquine(HCQ) and leflunomide(LEF) to regulate
immunity\textsuperscript{{[}2, 17{]}}.Biological therapy of pSS has a
valuable application prospect, but its effectiveness and safety are
still controversial. Therefore, it is essential to explore effective
potential therapeutic targets for immunotherapy of pSS. NK cells are
congenital lymphocytes, which can kill their target cells through
perforin, granzyme or death-inducing receptors. At present, more and
more evidence reveals the role of NK cells in autoimmune diseases such
as systemic sclerosis(SSc),systemic lupus erythematosus(SLE), PBC and
pSS\textsuperscript{{[}6, 18-21{]}}.Although T cells and B cells are
dominant in the pathogenesis of pSS, innate immune cells such as NK
cells also play an important role in pSS.

In this study,we observed that the proportion of CD56+NK cells in
peripheral blood of pSS patients(including active and inactive patients)
was significantly lower than that of HCs. In recent years, it has also
been reported that the frequency and absolute number of CD3-CD56+NK
cells in peripheral blood of pSS patients decreased significantly, and
the ratio of CD56\textsuperscript{bright} NK to
CD56\textsuperscript{dim} NK in peripheral blood may have relatively
specific diagnostic value for pSS\textsuperscript{{[}6{]}}. It was
speculated that the reduction of NK cells in peripheral blood of pSS
patients may be due to augment homing of cytotoxic cells to exocrine
glands, which trigger and maintain tissue inflammation by producing Th1
cytokines and cytotoxic mediators\textsuperscript{{[}6{]}}.Another study
found that the absolute number of NK cells in pSS patients with renal
tubular acidosis(RTA)was significantly lower than that in patients
without RTA\textsuperscript{{[}4{]}}.The above studies indicate that NK
cells are involved in the disease development of pSS. Similarly,
previous studies found that NK cells in peripheral circulation decreased
in SSc, especially in patients with organ involvement,and speculated
that the decrease of NK cells proportion in peripheral blood may be due
to the infiltration of NK cells into the involved
tissues\textsuperscript{{[}22{]}}.However, the reasons for the decrease
of NK cells in pSS and the mechanism of NK cells affecting the
development of pSS diseases need further study.

CD161 is a type C lectin-like type II transmembrane protein, which is
mainly expressed on the surface of most natural killer cells and
circulating memory T cells\textsuperscript{{[}23, 24{]}}.Human CD161
binds to its ligand LLT1, and inhibits the activity and function of NK
cells, but it is still controversial whether it inhibits or activates T
cells\textsuperscript{{[}25{]}}.In pSS patients, there are few
researches on CD161.There were two studies on CD161 expression on T
cells in peripheral blood of pSS and its clinical relevance with
diseases. Zhao et al. found that the expression of CD161 on CD4+T cells
of pSS patients was higher than that in HCs, and the retinoic acid
receptor-related orphan nuclear receptor (ROR)-\selectlanguage{greek}γ \selectlanguage{english}frequency on CD161
CD4+T cells in peripheral blood increased, which was positively
correlated with anti-SSA/SSB autoantibodies and
hypergammaglobulinemia\textsuperscript{{[}11{]}}.Another study reported
that compared with HCs, CD4+CD25+CD161+T cell subsets significantly
increased in the peripheral blood of pSS patients, and the proportion of
IL-17-producing cells in CD161+ T cell was higher than that in CD161-T
cell, and CD4+CD161+T cells in peripheral circulation were related to
the activity and severity of pSS disease\textsuperscript{{[}12{]}}.These
studies indicated that CD161 played an important role in the
pathogenesis of pSS and may be a potential therapeutic target for pSS.

In this study, we found that the proportion of CD161 on the surface of
CD56+NK cells in peripheral blood decreased significantly compared with
HCs.Similar results were also reflected in SLE.Two studies were reported
that the expression of CD161 decreased on the surface of NK cells in
peripheral circulation of SLE patients, and suggested that CD161+NK
cells were involved in the pathogenesis of SLE\textsuperscript{{[}26,
27{]}}. The receptors on the surface of NK cells, including activated
receptors and inhibitory receptors, regulate the function of NK cells
through balancing signal transmission. CD161, as an inhibitory receptor
on the surface of NK cells, inhibits the transmission of cytotoxic
functional signals of NK cells\textsuperscript{{[}9{]}}.Therefore, we
speculated that the decease of CD161 expression on NK cells in
peripheral blood of pSS patients to weaken the inhibition of function on
NK cells, which led to the enhancement of cytotoxicity and the increase
release of cytokine.Likewise, it was reported that the frequency of
circulating CD56+CD161+ NK Cells decreased in human visceral
leishmaniasis\textsuperscript{{[}28{]}}.All the above studies reflected
that CD161 involved in the pathogenesis of autoimmune diseases and
infectious diseases by mediating the function of NK cells.

We further observed that the proportion of CD161+CD56+NK cells was
associated with the clinical characteristics and laboratory parameters
in pSS.The CD161+CD56+NK cells proportion was significantly lower in pSS
patients with decayed tooth, fatigue, arthralgia,skin involvement, PBC
and ILD than that in patients without above features.Furthermore, we
found that the proportion of CD161+CD56+NK cells in peripheral blood of
active patients (ESSDAI\textgreater{}5) reduced obviously compared with
that in inactive pSS patients.Further clinical correlation analysis
showed that the proportion of CD161+CD56+NK cells was negatively
correlated with disease activity and severity of pSS.These results
suggested that the decrease of CD161+CD56+NK cells may contribute to the
progression of pSS.Lenart M. et al. found that activation of the
LLT1-CD161 axis can inhibit granzyme B and interferon-\selectlanguage{greek}γ\selectlanguage{english}(IFN-\selectlanguage{greek}γ\selectlanguage{english})
production by NK cells and hamper the function of NK
cells\textsuperscript{{[}29{]}}.CD161 is expressed in the early stage of
NK cell development,and in the peripheral circulation, the crosslinking
of CD161 leads to upregulate the expression of IFN-\selectlanguage{greek}γ \selectlanguage{english}and inhibits the
cytotoxicity of NK cells\textsuperscript{{[}30, 31{]}}.Another study
showed that CD161 on NK cells combined with its ligand on target cells
and inhibited NK cytotoxicity by activating acidic
sphingomyelinase\textsuperscript{{[}32{]}}.Thus, we speculated that the
decrease of CD161 may affect the function of CD56+NK cells through some
mechanism, leading to enhanced cytotoxicity and increased secretion of
inflammatory cytokines,and aggravating the progress of pSS disease.

One of the features in pSS is the production of autoantibodies and
increase of immunoglobulins in patients after overactivation of B
cells.\textsuperscript{{[}33{]}}.In our study, we also found that the
proportion of CD161+CD56+NK cells in peripheral circulation decreased
significantly in pSS patients with anti-SSA/Ro60 positive,anti-SSB
positive and high IgG.It has been shown that cytokines produced by NK
cells, such as IFN-\selectlanguage{greek}γ \selectlanguage{english}can promote the activation of B cells and enhance
the production of immunoglobulin\textsuperscript{{[}34{]}}.Rosen DB et
al. reported that CD161 interacted with LLT1 expressed on activated B
cells, regulating the crosstalk between NK cells and B
cells\textsuperscript{{[}30{]}}.Early studies have also confirmed that
NK cells can enhance the proliferation of B
cells\textsuperscript{{[}35{]}}.Another research showed that human
invariant NKT cell could directly help autologous B lymphocytes, induce
the proliferation of naive and memory B cells,produce immunoglobulin and
antibodies in vitro\textsuperscript{{[}36{]}}.Therefore, we speculated
that CD161, as an inhibitory receptor of NK cells, decreased on the
surface of CD56+NK cells in pSS patients,which weakened the inhibition
on NK cell function, and led to the increased secretion of cytokines
such as INF-\selectlanguage{greek}γ, \selectlanguage{english}promoted the activation and proliferation of B cells, and
produced more autoantibodies and immunoglobulins in pSS patients.

However, there still exist some limitations in our research.According to
the expression of CD56, human NK cells can be divided into
CD56\textsuperscript{bright} and CD56\textsuperscript{dim} subsets.Our
study didn't deeply analyze the difference of CD161 expression on
CD56\textsuperscript{bright} and CD56\textsuperscript{dim} subsets.On
the other hand,this research was a cross-sectional and observational
study, and the number of participants recruited was small.We only
analyzed the clinical correlation between the proportion of
CD161+CD56+NK cells and pSS, and it was not clear how CD161 mediated the
function of NK cells to participate in the pathogenesis of pSS.It needs
our further study in the later stage.Finally,it is generally believed
that CD56+ NK cell subsets in salivary glands of pSS patients are more
appropriate to reflect the lesions in glands,but,this study was lack of
the histopathological verification of target tissues such as salivary
gland tissues.Next, we will further explore how CD161 mediates the
function of NK cell to involve in the pathogenesis of pSS from the above
aspects.

\textbf{5.Conclusion}

In conclusion, in this study, we revealed that the proportion of CD56+NK
cells and CD161 on CD56+NK cells in peripheral blood of pSS patients
significantly decreased compared to HCs. The proportion of CD161+CD56+NK
cells was significantly correlated with the clinical features and
laboratory parameters including autoantibodies and immunoglobulins in
pSS patients, and negatively associated with disease activity and
severity of pSS. The ROC curve showed that CD161+CD56+NK cells had
certain reference value for the diagnosis of pSS.In short, our findings
suggest that CD161+CD56+ NK cells may influence the progression of pSS
and serve as a biomarker of disease activity and potential targets for
therapy of pSS.

\textbf{Fundings:} The study was funded by Science and Technology
Programme of Suzhou(SLJ2021009), Natural Science Key Project of Bengbu
Medical College (2022byzd070),Gusu Talent Project of Suzhou
(GSWS2020011,GSWS2020018),Science and Technology Programme of

Suzhou (SKY2023138).

\textbf{Author contributions:}

Ping Zhao:Data curation, Writing original draft, Writing

review \& editing. Cuiping Liu, and Jian Wu designed the study and
provided resources and fundings.Ping Zhao, YanHong Yang and Saizhe Song
conducted the experiment, performed data analysis, and wrote the
manuscript. Wei Cheng, Cheng Peng, and Xin Chang participated in the
sample and clinical data collection. Jian Wu and Cuiping Liu helped
optimize the research and proofread the paper and revised the
manuscript. All authors helped the final approval of the version.

\textbf{Acknowledgements:} We sincerely thank the platform provided by
Jiangsu Institute of Clinical Immunology \& Jiangsu Key Laboratory of
Clinical Immunology. We are grateful for the help provided by all
department of rheumatology in the First Afffliated Hospital of Soochow
University.

\textbf{Data Availability Statement:} The data sets generated during
and/or analysed during the current study are available from the
corresponding author on reasonable request.

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\textbf{Table 1 Clinical characteristics and laboratory parameters of
included participants}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
& pSS & HC\tabularnewline
Number & 31 & 29\tabularnewline
Age(years) & 47.13\selectlanguage{ngerman}±11.94 & 43.69\selectlanguage{ngerman}±8.79\tabularnewline
Female\selectlanguage{english},n(\%) & 29(93.55\%) & 28(96.55\%)\tabularnewline
Male,n(\%) & 2(6.45\%) & 1(3.45\%)\tabularnewline
Disease duration(months) & 46(1-122) & NA\tabularnewline
\textbf{Major clinical features} & &\tabularnewline
Dry mouth,n(\%) & 24( 77.42\%) & NA\tabularnewline
Dry eye,n(\%) & 15(48.39\%) & NA\tabularnewline
Decayed tooth,n(\%) & 11(35.48\%) & NA\tabularnewline
Glandular swelling,n(\%) & 5(16.13\%) & NA\tabularnewline
Raynaud's phenomenon,n(\%) & 3(9.68\%) & NA\tabularnewline
Fatigue,n(\%) & 15(48.39\%) & NA\tabularnewline
Weigh loss,n(\%) & 1(3.23\%) & NA\tabularnewline
Arthralgia,n(\%) & 9(29.03\%) & NA\tabularnewline
Skin involvement,n(\%) & 6(19.35\%) & NA\tabularnewline
Primary biliary cirrhosis,n(\%) & 5(16.13\%) & NA\tabularnewline
Renal involvement,n(\%) & 4(12.90\%) & NA\tabularnewline
Interstitial lung disease,n(\%) & 11(35.48\%) & NA\tabularnewline
Fever,n(\%) & 1(3.23\%) & NA\tabularnewline
Muscle involvement,n(\%) & 1(3.23\%) & NA\tabularnewline
Nervous system involvement,n(\%) & 1(3.23\%) & NA\tabularnewline
High-IgG & 20(64.52\%) & NA\tabularnewline
Leukocytopenia & 5(16.13\%) & NA\tabularnewline
\textbf{Major laboratory features} & &\tabularnewline
ESR(mm/h) & 14.50(2.00-62.00) & NA\tabularnewline
CRP(mg/L) & 1.76(0.20-8.91) & NA\tabularnewline
RF(IU/ml) & 46.20(20.00-777.00) & NA\tabularnewline
WBC(\selectlanguage{ngerman}×10\textsuperscript{9}/L) & 4.72(2.79-11.78) & NA\tabularnewline
Lymphocyte(\selectlanguage{ngerman}×10\textsuperscript{9}/L) & 1.55\selectlanguage{ngerman}±0.60 & NA\tabularnewline
NC(\selectlanguage{ngerman}×10\textsuperscript{9}/L) & 2.69(1.44-8.74) & NA\tabularnewline
RBC(\selectlanguage{ngerman}×10\textsuperscript{12}/L) & 4.23\selectlanguage{ngerman}±0.38 & NA\tabularnewline
Hb(g/L) & 126\selectlanguage{ngerman}±11.26 & NA\tabularnewline
Plt(\selectlanguage{ngerman}×10\textsuperscript{9}/L) & 203.80\selectlanguage{ngerman}±49.31 & NA\tabularnewline
Serum globulin(g/L) & 31.70\selectlanguage{ngerman}±6.13 & NA\tabularnewline
IgG(g/L) & 17.84\selectlanguage{ngerman}±5.01 & NA\tabularnewline
IgA(g/L) & 2.77(1.40-10.60) & NA\tabularnewline
IgM(g/L) & 1.16(0.41-7.35) & NA\tabularnewline
C3(g/L) & 0.79 (0.62-1.52) & NA\tabularnewline
C4(g/L) & 0.19\selectlanguage{ngerman}±0.06 & NA\tabularnewline
Anti-SSA/Ro52 (+) ,n(\%) & 28(90.32\%) & NA\tabularnewline
Anti-SSA/Ro60 (+),n(\%) & 24(77.42\%) & NA\tabularnewline
Anti-SSB(+),n(\%) & 20(64.52\%) & NA\tabularnewline
Anti-centromere(+),n(\%) & 3(9.68\%) & NA\tabularnewline
ESSDAI & 3.00(1.00-8.00) & NA\tabularnewline
ESSPRI & 3.00(1.33-5.00) & NA\tabularnewline
ILD: Interstitial lung disease , PBC:Primary biliary cirrhosis
ESR:Erythrocyte Sedimentation Rate, & ILD: Interstitial lung disease ,
PBC:Primary biliary cirrhosis ESR:Erythrocyte Sedimentation Rate, &
ILD: Interstitial lung disease , PBC:Primary biliary cirrhosis
ESR:Erythrocyte Sedimentation Rate,\tabularnewline
RF: Rheumatoid Factor, CRP:C-Reactive Protein, WBC: White Blood Cell, &
RF: Rheumatoid Factor, CRP:C-Reactive Protein, WBC: White Blood Cell, &
RF: Rheumatoid Factor, CRP:C-Reactive Protein, WBC: White Blood
Cell,\tabularnewline
RBC: Red Blood Cell , Hb: Hemoglobin, Plt :Platelet, NC: Neutrophil
Cell, & RBC: Red Blood Cell , Hb: Hemoglobin, Plt :Platelet, NC:
Neutrophil Cell, & RBC: Red Blood Cell , Hb: Hemoglobin, Plt :Platelet,
NC: Neutrophil Cell,\tabularnewline
IgG: Immunoglobulin G, IgA: Immunoglobulin A, IgM: Immunoglobulin M, &
IgG: Immunoglobulin G, IgA: Immunoglobulin A, IgM: Immunoglobulin M, &
IgG: Immunoglobulin G, IgA: Immunoglobulin A, IgM: Immunoglobulin
M,\tabularnewline
C3: Complement 3, C4: Complement 4, NA: Not Applicable, & C3: Complement
3, C4: Complement 4, NA: Not Applicable, & C3: Complement 3, C4:
Complement 4, NA: Not Applicable,\tabularnewline
ESSDAI:European League Against Rheumatism Sjogren's Syndrome Disease
Activity Index, & ESSDAI:European League Against Rheumatism Sjogren's
Syndrome Disease Activity Index, & ESSDAI:European League Against
Rheumatism Sjogren's Syndrome Disease Activity Index,\tabularnewline
ESSPRI:European League Against Rheumatism Sjogren's Syndrome Patient
Reported Index. & ESSPRI:European League Against Rheumatism Sjogren's
Syndrome Patient Reported Index. & ESSPRI:European League Against
Rheumatism Sjogren's Syndrome Patient Reported Index.\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2 The proportion of CD161+CD56+NK cells in different
clinical manifestations,autoantibodies and IgG in pSS patients}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& with & without & t & \emph{P}\tabularnewline
Dry mouth & 62.15\%\selectlanguage{ngerman}±14.11\% & 68.50\%\selectlanguage{ngerman}±15.09\% & 0.970 &
0.341\tabularnewline
Dry eye & 64.73\%\selectlanguage{ngerman}±10.86\% & 66.74\%\selectlanguage{ngerman}±15.50\% & 0.407 &
0.687\tabularnewline
Decayed tooth & 53.60\%\selectlanguage{ngerman}±16.83\% & 67.00\%\selectlanguage{ngerman}±9.81\% & 2.671 &
0.013*\tabularnewline
Fatigue & 55.71\%\selectlanguage{ngerman}±17.50\% & 67.54\%\selectlanguage{ngerman}±10.49\% & 2.280 &
0.030*\tabularnewline
Arthralgia & 57.38\%\selectlanguage{ngerman}±14.24\% & 69.69\%\selectlanguage{ngerman}±12.04\% & 2.300 &
0.030*\tabularnewline
Skin involvement & 49.84\%\selectlanguage{ngerman}±18.09\% & 66.30\%\selectlanguage{ngerman}±11.94\% & 2.570 &
0.016*\tabularnewline
Gland involvement & 64.74\%\selectlanguage{ngerman}±11.84\% & 65.90\%\selectlanguage{ngerman}±13.58\% & 0.177 &
0.861\tabularnewline
Leukocytopenia & 67.25\%\selectlanguage{ngerman}±11.54\% & 64.18\%\selectlanguage{ngerman}±12.19\% & 0.470 &
0.643\tabularnewline
Raynaud's phenomenon & 67.04\%\selectlanguage{ngerman}±10.45\% & 64.05\%\selectlanguage{ngerman}±15.38\% & 0.326 &
0.747\tabularnewline
PBC & 49.00\%\selectlanguage{ngerman}±21.32\% & 66.31\%\selectlanguage{ngerman}±12.64\% & 2.316 & 0.028*\tabularnewline
ILD & 53.32\%\selectlanguage{ngerman}±17.57\% & 67.78\%\selectlanguage{ngerman}±10.23\% & 2.860 & 0.008**\tabularnewline
High IgG & 57.22\%\selectlanguage{ngerman}±15.95\% & 70.85\%\selectlanguage{ngerman}±10.87\% & 2.508 &
0.018*\tabularnewline
Anti-SSA/Ro52\textbf{+} & 60.84\%\selectlanguage{ngerman}±15.21\% & 62.89\%\selectlanguage{ngerman}±6.00\% & 0.228 &
0.821\tabularnewline
Anti-SSA/Ro60\textbf{+} & 59.59\%\selectlanguage{ngerman}±15.25\% & 76.34\%\selectlanguage{ngerman}±13.47\% & 2.445 &
0.021*\tabularnewline
Anti-SSB\textbf{+} & 57.96\%\selectlanguage{ngerman}±15.63\% & 74.76\%\selectlanguage{ngerman}±11.06\% & 3.029 &
0.005**\tabularnewline
Anti-centromere\textbf{+} & 59.94\%\selectlanguage{ngerman}±14.83\% & 70.72\%\selectlanguage{ngerman}±5.30\% & 1.233 &
0.228\tabularnewline
\emph{P}\textless{} 0.05, **\emph{P} \textless{} 0.01 &
*\emph{P}\textless{} 0.05, **\emph{P} \textless{} 0.01 &
*\emph{P}\textless{} 0.05, **\emph{P} \textless{} 0.01 &
*\emph{P}\textless{} 0.05, **\emph{P} \textless{} 0.01 &
*\emph{P}\textless{} 0.05, **\emph{P} \textless{} 0.01\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Laboratory parameters and disease activity index & CD161+CD56+NK cells
\% & CD161+CD56+NK cells \%\tabularnewline
& r & \emph{P}\tabularnewline
ESR & -0.3884 & 0.0607\tabularnewline
WBC & -0.3893 & 0.0369\tabularnewline
Serum globulin & -0.1095 & 0.5867\tabularnewline
IgA & -0.4594 & 0.0093\tabularnewline
IgM & -0.4047 & 0.0294\tabularnewline
IgG & -0.4968 & 0.0159\tabularnewline
C3 & 0.0934 & 0.6236\tabularnewline
C4 & 0.4205 & 0.0289\tabularnewline
ESSDAI & -0.3624 & 0.0451\tabularnewline
ESSPRI & -0.4863 & 0.0055\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 3 Correlations between CD161+CD56+NK cells proportion and
laboratory features and disease activity index in pSS patients}

\textbf{Hosted file}

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