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Accepted Manuscript
Increased proportion of a CD38highIgD+ B cell subset in
peripheral blood is associated with clinical and immunological
features in patients with primary Sjögren's syndrome
Eriko Ishioka-Takei, Keiko Yoshimoto, Katsuya Suzuki, Ayumi
Nishikawa, Hidekata Yasuoka, Kunihiro Yamaoka, Tsutomu
Takeuchi
PII:
DOI:
Reference:
S1521-6616(17)30641-1
doi:10.1016/j.clim.2017.10.008
YCLIM 7955
To appear in:
Clinical Immunology
Received date:
Revised date:
Accepted date:
1 September 2017
13 October 2017
19 October 2017
Please cite this article as: Eriko Ishioka-Takei, Keiko Yoshimoto, Katsuya Suzuki, Ayumi
Nishikawa, Hidekata Yasuoka, Kunihiro Yamaoka, Tsutomu Takeuchi , Increased
proportion of a CD38highIgD+ B cell subset in peripheral blood is associated with clinical
and immunological features in patients with primary Sjögren's syndrome. The address
for the corresponding author was captured as affiliation for all authors. Please check if
appropriate. Yclim(2017), doi:10.1016/j.clim.2017.10.008
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ACCEPTED MANUSCRIPT
Increased proportion of a CD38 high IgD + B cell subset in periphe ral blood is
associated with clinical and immunological features in patients with primary
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Sjögren’s syndrome.
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Eriko Ishioka-Takei, MDa, Keiko Yoshimoto, PhDa,b,* , Katsuya Suzuki, MD,PhDa,
of Medicine, Tokyo, Japan
Clinical and Translational Research Center, Keio University Hospital, Tokyo, Japan
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b
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Division of Rheumatology, Department of Internal Medicine, Keio University School
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a
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PhDa and Tsutomu Takeuchi, MD, PhDa
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Ayumi Nishikawa, MD, PhDa, Hidekata Yasuoka, MD, PhDa, Kunihiro Yamaoka, MD,
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*Correspondence author,
Dr. K. Yoshimoto, Division of Rheumatology, Department of Internal Medicine, Keio
University School of Medicine, Clinical and Translational Research Center, Keio
University Hospital, 35 Shinanomachi, Shinjuku, Tokyo 160-8552, Japan.
E-mail: keikoy@a8.keio.jp Tel/FAX: +81-3-5363-3636
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ABSTRACT
We investigated the correlation between the increased proportion of peripheral
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B cell subsets and clinical and immunological features in primary Sjögren’s syndrome
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(pSS). We found that the proportion of CD19+ B cells was significantly increased in pSS
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as compared with HC and was correlated with serum IgG levels. Moreover, in vitro IgG
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production by CD19+ B cells was significantly increased in pSS and was positively and
significantly correlated with serum IgG levels. FACS analysis revealed that the
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proportions of peripherally CD38high IgD+ B cells and CD38high IgD- B cells were
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significantly increased in pSS. In addition, the proportion of CD38 high IgD+ B cells
positively correlated with ESSDAI scores and serum levels of IgG, anti- Ro/SSA and
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anti-La/SSB antibodies while that of CD38high IgD- B cells showed no correlation with
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these parameters. Our data suggest that increased proportion of CD38high IgD+ B cells in
pSS is involved in IgG overproduction including autoantibodies, and correlates with
disease progression.
Keywords: Sjögren’s syndrome, B cell, CD38, IgD, ESSDAI, IgG, autoantibodies
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1. Introduction
Primary Sjögren’s Syndrome (pSS) is an autoimmune disease, characterized by
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xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes) [1-3]. Several lines of
evidence suggest that the lesion formation and subsequent dysfunction of the glands are
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due to focal lymphocytic infiltrates in the exocrine glands, mainly T and B cells, which
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are a distinctive feature of pSS [4-5]. pSS is often accompanied by the production of
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autoantibodies and hypergammaglobulinemia [6], although hypergammaglobulinemia is
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not included in the classification criteria of pSS [7-9]. It is also well known that high
serum levels of immunoglobulin and autoantibodies, such as anti- Ro/SSA, La/SSB
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antibodies, rheumatoid factor (RF), and cryoglobulins are o ften observed in pSS
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patients [10-12]. These observations clearly indicate that abnormally activated B cells
play an important role in the pathogenesis of pSS.
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Recently, several clinical trials of drugs for pSS which target B cells or BAFF,
B cell activating factor belonging to the TNF family, have been conducted, including
rituximab, an anti-CD20 monoclonal antibody, and belimumab, an anti- BAFF
monoclonal antibody. Though rituximab trials failed to achieve the primary endpoint,
the antibody had some efficacy in improving fatigue in these patients [13, 14]. The
belimumab trials achieved the primary endpoint in open- label studies [15, 16]. These
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clinical trials indicate that the therapies targeting B cells or BAFF may be effective for
pSS and regulation of specific B cell subpopulations, rather than wide range of B cells,
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could provide efficacious therapy for pSS [17-19].
To date, it has been demonstrated that the distribution of peripheral blood B
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cell subsets is altered in pSS patients [20, 21]. In addition, the several comparative
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analyses by immunophenotyping showed reduced levels of CD27 + memory B cells in
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the peripheral blood [22, 23], but accumulated in the target epithelial organs [24].
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Conversely, transitional and naïve B cells are increased in the peripheral blood in pSS
[25]. It has also been reported that CD27 +CD138+ plasma cells are present in the
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peripheral blood of pSS patients [26]. Taken together, these findings imply that the
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regulation of molecular and cellular processes leading to B cell activation may be
altered in patients with pSS. In this regard, we considered that a closer look at the B cell
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lineage would be a worthwhile way of discovering better therapeutic targets for pSS.
In this study, we focused on the possible alteration of peripheral B cell subsets,
which may be associated with the clinical and immunological characteristics of pSS.
2. Materials and Methods
2.1 Patients and controls. The pSS patients enrolled in this study fulfilled at least one of
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the following criteria: 2002 American-European criteria for SS (AECG) [8], 2012
American College of Rheumatology classification criteria for SS (ACR) [9], or the
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revised Japanese Ministry of Health criteria for the diagnosis of SS [7]. Venous blood
samples were collected from pSS patients (n = 34; female, age 36–87 [mean: 59.4]) and
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HC (n = 20; females, age 29–57 [mean: 45.8]). At the time of collection, two patients
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were receiving prednisolone at a daily dose of 4 mg and 7 mg, respectively, while the
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remaining patients were free of immune- modulating medication. Informed consent was
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obtained from each participant before blood collection. pSS disease activity was
assessed using the guidelines of the European League against Rheumatism (EULAR)
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Primary Sjögren’s syndrome disease activity index (ESSDAI) [27-29]. This study was
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#20140479).
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approved by Keio University School of Medicine Ethics Committee (#20140335,
2.2 Antibodies and recombinant proteins. We used anti- human IgG antibodies for
ELISA (BD Biosciences, San Jose CA, USA) and an anti-IgM antibody for cell culture
(R&D Systems, Minneapolis MN, USA). Anti- human CD19 labeled with a
violet-excited dye (Vioblue®, Miltenyi Biotec, Bergisch-Gladbach, Germany),
anti-human CD27 labeled with a fluorochrome (PE-Cy7®, BD Biosciences),
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fluorescein isothiocyanate (FITC)- labeled anti-IgD, and fluorochrome- labeled CD38
(PE-Cy5.5®, BioLegend, San Diego CA, USA) for fluorescence-activated cell sorting
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(FACS) analysis (BD Biosciences) were used. Recombinant human CD40 ligand
(rhCD40L, Avrend®, Immunex Corp, Seattle WA, USA), recombinant human soluble
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B cell activating factor (rhsBAFF), and recombinant human interleukin-4 (rhIL-4) for
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cell culture (PEPROTECH, Rocky Hill NJ, USA) were also employed.
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2.3 Stimulation of peripheral CD19+ B cells in vitro. Peripheral CD19+ B cells were
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prepared from pSS patients (n = 16) and HC (n = 10) with anti-CD19 antibody-coated
microbeads (Miltenyi Biotec) and a cell separator (autoMACS Pro Separator®, Miltenyi
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CD19-positive.
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Biotech). FACS analysis of the cells demonstrated that > 98% of living cells were
The CD19+ B cells were cultured at 2.5 ×105 /mL in Roswell Park Memorial
Institute (RPMI) 1640 medium (American Type Culture Collection, Manassas, VA,
USA) supplemented with 10% fetal calf serum (FCS) upon stimulation with anti-IgM
antibody (10 g/mL), rhuCD40L (20 ng/mL), rhIL-4 100 ng/mL, and rhsBAFF (2
g/mL) in a 24-well culture plate for 96 hours. The amount of IgG in the culture
supernatants was measured by sandwich ELISA according to methods recommended by
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the manufacturer of the antibodies (BD Bioscience).
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2.4 FACS analysis. FACS and data analyses were carried out on a MACS Quant
Analyzer (MACS Quant Analyzer®, Miltenyi Biotec). FACS analysis of cells in whole
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blood was carried out according to methods recommended by the manufacturer of the
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antibodies (BD Biosciences). The proportions of B cell subsets, such as CD38 high IgD+,
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CD38high IgD- and CD27+IgD- were defined as shown in Supplementary Figure 1.
2.5 Statistical analysis. Differences between groups were examined for statistical
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significance using the Wilcoxon test for single comparisons. Pearson’s correlation
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analysis was also employed for evaluation of the linear relationship between two
continuous variables. A p value < 0.05 denoted the presence of a statistically significant
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difference.
3. Results
3.1 Clinical characteristics of pSS patients and healthy controls. Average IgG serum
levels were significantly higher in patients with pSS (1726 ± 589 mg/dL) than in HC
(1103 ± 192 mg/dL) (Table 1). The prevalence of hypergammaglobulinemia (IgG level
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> 1700 mg/dL) was 38.2% in patients. Although it has been reported that pSS patients ≤
65 years old are less likely to have hypergammagloblinemia [30], we saw no significant
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difference in prevalence between our younger (37.5%, n = 18) and older patients
(38.9%, n = 16). Average serum levels of IgA and IgM of pSS patients were 195 ± 75
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mg/dL and 140 ± 139 mg/dL, respectively (Table 1). The serum IgA level of pSS
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patients was significantly higher than that of normal controls, whereas serum IgM levels
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showed no significant difference. The proportion of patients who were positive for
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autoantibodies such as anti- nuclear, anti-Ro/SSA, anti-La/SSB, and anti-centromere
antibodies was 79.4%, 80.6%, 51.6% and 17.6%, respectively. In contrast, none of these
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autoantibodies were detected in HC except anti-nuclear antibody (15.7%).
3.2 Proportion of CD19+ B cells was elevated in peripheral blood of pSS patients and
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was positively correlated with serum IgG levels. FACS analysis revealed that the
proportion of CD19
+
B cells in peripheral blood of pSS patients was significantly
elevated compared to that of normal individuals (Figure 1A). Moreover, this proportion
was positively and significantly correlated with serum IgG levels in both pSS patients
and HC (Figure 1B). These data imply that increased number of CD19 + B cells is
associated with high titers of serum IgG and is involved in the pathogenesis of pSS
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which is often accompanied with hypergammaglobulinemia.
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3.3 Enhanced IgG production from peripheral B cells of pSS patients in vitro was
positively correlated with serum IgG levels. B cell stimulation with anti-IgM antibody,
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rhCD40L, rhIL-4, and rhsBAFF resulted in increased IgG in both normal and pSS
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CD19+ B cells. However, peripheral pSS CD19 + B cells produced a significantly higher
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amount of IgG than normal B cells when the cells were stimulated in vitro for four days
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(Figure 2A). In addition, IgG production was elevated in pSS CD19 + B cells even in the
absence of stimulation. These data indicate that the regulatory mechanism of IgG
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production is enhanced in pSS B cells. To determine the contribution of IgG
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overproduction by peripheral pSS B cells in vitro to the pathogenesis of pSS, which is
often accompanied by hypergammaglobulinemia, we evaluated the correlation between
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the amount of IgG produced by pSS CD19 + B cells in vitro and serum IgG levels of the
patients. Patient serum IgG levels were significantly and positively correlated with IgG
production from pSS CD19+ B cells in the absence of stimulation (Figure 2B r = 0.591,
p = 0.016). Moreover, when the cells were cultured with B cell stimuli, IgG production
by pSS CD19+ B cells was strongly correlated with the patients’ serum IgG levels
(Figure 2C r = 0.72, p = 0.002). In addition, IgG production by pSS CD19+ B cells with
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or without stimulation in vitro was significantly related to positive correlation (Figure
2D r = 0.732, p = 0.0013).
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These data clearly indicate that the regulatory mechanisms of IgG production
in pSS B cells are spontaneously enhanced and highly accelerated once the cells
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undergo stimulation.
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3.4 Altered proportion of peripheral B cell subsets in pSS patients. Then, we
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investigated which B cell subpopulations might be responsible for the pathogenesis of
pSS. We analyzed the proportions of CD27-IgD+, CD27+IgD+, CD27+IgD-,
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CD38high IgD+ and CD38high IgD- among CD19+ B cells. Statistical analysis indicated that
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the proportion of CD38high IgD+ and CD38high IgD- B cells among CD19+ B cells was
significantly elevated in pSS patients (Figure 3D, 3E), whereas that of CD27+IgD- B
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cells was decreased (Figure 3C). There was no significant difference of the proportion
of CD27-IgD+, CD27+ IgD+ B cells between pSS and HC (Figure 3A, 3B). We then
focused on increased populations of B cell subsets that were possibly involved in the
overproduction of IgG, such as CD38 high IgD+ and CD38high IgD- B cells.
3.5 Proportion of CD38high IgD+ B cells was positively and significantly correlated with
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clinical parameters in pSS patients. We investigated the relationship between
significantly elevated B cell subsets, such as CD38 high IgD+ and CD38high IgD- B cells,
and clinical parameters to clarify the possible involvement of altered B cell subsets in
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the pathogenesis of pSS. As shown in Figure 4, the proportion of CD38 high IgD+ B cells
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was positively and significantly correlated with serum levels of IgG (Figure 4A r =
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0.881, p < 0.0001), an anti-SSA antibody (Figure 4D r = 0.508, p = 0.003) and an
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anti-SSB antibody (Figure 4E r = 0.478, p = 0.006) of the patients. In contrast,
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CD38high IgD- B cells (serum IgG: r = 0.0001, p = 0.994, anti-SSA antibody: r = 0.253, p
= 0.163, anti-SSB antibody: r = 0.055, p = 0.765) and CD27+IgD- B cells, which was
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significantly decreased in pSS patients (serum IgG: r = 0.256, p = 0.143, anti-SSA
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antibody: r = 0.263, p = 0.146, anti-SSB antibody: r = 0.231, p = 0.203) did not
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significantly correlate with these parameters.
3.6 Proportion of CD38high IgD+ B cells and serum IgG level were elevated in pSS
patients with anti-Ro/SSA and/or La/SSB antibodies positive.
To investigate the contribution of CD38high IgD+ B cells to B cell activation, we further
examined the relationship between CD38high IgD+ B cells and serum IgG level or titers of
autoantibodies, such as anti-Ro/SSA and anti- La/SSB antibodies. As shown in Figure 5,
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remarkable elevated proportion of CD38high IgD+ B cells was observed in anti-Ro/SSA
and/or anti-La/SSB positive patients (Figure 5A and 5B). Moreover, serum level of IgG
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was also significantly elevated in patients with anti-Ro/SSA and/or anti-La/SSB
antibodies positive (Figure 5C and 5D). In addition, the titers of anti- Ro/SSA and
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La/SSB antibodies were positively and significantly correlated with serum IgG level of
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the patients (anti- Ro/SSA: r = 0.523, p = 0.0025, anti- La/SSB: r = 497, p = 0.0044).
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These results indicated that CD38high IgD+ B cells are involved in B cell hyperfunction,
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such as IgG overproduction and autoantibodies production which is often accompanied
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with pSS.
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3.7 Proportion of CD38high IgD+ B cells was positively and significantly correlated with
disease activity of pSS measured by ESSDAI. ESSDAI, the EULAR Sjögren syndrome
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(SS) disease activity index, is a systemic disease activity index that was designed to
measure disease activity in patients with pSS. As shown in Figure 6, the proportion of
CD38high IgD+ B cells positively and significantly correlated with the ESSDAI of the
patients (r = 0.596, p = 0.0002; Figure 6A) whereas that of CD38high IgD- (r = 0.102, p =
0.564; Figure 6B) and CD27+IgD- B cells (r = 0.279, p = 0.110; Figure 6C) did not
correlate with the ESSDAI. Then we further investigated the association between
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clinical variables and peripheral B cell subsets and found that only the proportion of
CD38high IgD+ B cells was significantly correlated with ESSDAI and biological domain
of ESSDAI as shown in Table 2. These data strongly indicate that the proportion of
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CD38high IgD+ B cells is reflective of the disease activity of pSS and that elevation of
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this B cell subset may be involved in the pathogenesis of pSS.
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4. Discussion
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In this study, we found that the proportion of CD19+ B cells in peripheral blood was
significantly elevated in pSS patients compared with that of HC and that IgG production
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by peripheral pSS CD19+ B cells was significantly increased in vitro compared with
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normal CD19+ B cells. We further demonstrated that the proportion of CD38high IgD+ B
cells is increased in pSS and correlates with disease activity, such as serum levels of IgG,
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and anti-SS-A/anti-SS-B antibodies and ESSDAI. Our findings may be a consequence
of hyperactivation of B cells and help elucidate the pathogenesis of pSS and provide
pathways to new therapies.
IgG overproduction was observed not only in stimulated pSS CD19+ B cells
but also in the unstimulated cells of individual patients, and positively and significantly
correlated with the serum levels of IgG. Moreover, enhanced IgG production by
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unstimulated CD19+ B cells was positively correlated with that of stimulated cells in
pSS patients. These data strongly indicate that pSS B cells are spontaneously activated
and overreact to the stimulation. Therefore, our data collectively suggest that not only
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the proportion of CD19+ B cells but also the ability of IgG production of the cells are
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elevated in pSS patients and IgG overproduction is possibly attributable to the
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hyperactivity of B cells.
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FACS analysis revealed that the proportions of CD38 high IgD+ and CD38high IgD-
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subsets among CD19+ B cells were significantly higher than in HC, whereas the
proportions of CD27-IgD+ naïve B cells and CD27+IgD+ memory B cells among CD19+
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B cells did not significantly differ between pSS patients and HC. To elucidate the
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contribution of these subsets to the pathogenesis of pSS, we analyzed the correlation
between their respective proportions and the clinical parameters and disease activity of
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the patients. Then we found that only the proportion of CD38high IgD + B cells was
positively and significantly correlated with clinical parameters, such as serum levels of
IgG and anti-SS-A/anti-SS-B antibodies, and the ESSDAI scores of the patients.
It has been reported that double staining for CD38 and IgD can discriminate
between naïve and memory B cells [31]. CD38high IgD+ B cells have been classified as
activated naïve B cells and migrate to bone marrow and/or secondary lymphoid organs,
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such as lymph nodes and tonsils, and differentiate into plasmablasts and then plasma
cells to produce antibodies [32-34]. This process may explain the strong correlation of
the proportion of peripheral CD38 high IgD+ B cells with serum IgG levels and ESSDAI
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scores of the patients. Although the proportion of CD38high IgD- B cells was also
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elevated in pSS patients, this population was not significantly correlated with serum IgG
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levels or ESSDAI score. These findings might be explained by the differences in B cell
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differentiation pathways into plasmablasts and plasma cells in the peripheral blood and
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in target organs. To support this hypothesis, it has been reported that the proportion of
long- lived plasma cells that produce higher amounts of IgG is elevated in pSS patients
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with higher lymphocytic infiltration focus sco res in their salivary glands [33] and large
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portion of pSS patients has ectopic development of B cell proliferation, which are
genuine germinal centers in salivary glands that include autoreactive B cells [35, 36]. In
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addition, a phenotyping study of B cells infiltrating the salivary glands of pSS patients
identified the presence of a possibly more developed subset of transitional B cells. This
subset had a lower expression level of CD38, which is exp ressed on activated naïve
mature B cells differentiated from transitional B cells [37]. In other words, activated
naïve mature B cells, CD38high IgD+ cells migrate into target organs such as salivary
glands and bone marrow or secondary lymphoid organs, which finally become
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antibody-producing cells, whereas plasmablasts, CD38high IgD- B cells in peripheral
blood might not migrate into other organs. Our findings suggest that elevated proportion
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of CD38high IgD+ B cells in peripheral blood may indicate increasing precursor of
antibody producing cells in the organs, which is distinctive features of pSS.
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Some limitations of this study warrant mention. First, it was not possible to
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demonstrate IgG production in vitro by CD19 + B cells from all pSS patients (16/34)
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because of the limitation of quantity of blood collection. Second, since the degree of
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disease activity of the patients enrolled in this study was relatively moderate (ESSDAI:
0-4), further demonstration in patients with higher disease activity may be required.
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In conclusion, our results indicate that CD38 high IgD+ B cells play a pivotal role
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in the pathogenesis of pSS, especially in regulatory mechanisms in the activation of B
for pSS.
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cells. In addition, these findings may guide the development of new therapeutic targets
Conflict of interests
The authors declare that they have no conflict of interests for this study.
Authors’ contributions
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ET and KYo designed and performed the experiments and prepared manuscript. KYo,
KS, AN and HY helped with the acquisition of data. ET, KS, AN and HY collected
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clinical data of the patients. KYa participated in the interpretation of the study results,
and TT is the principal investigator. All authors have read and approved the final
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manuscript.
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Acknowledgements
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We are very grateful to Ms. Tsukasa Satoh and Ms. Yumi Ikeda for their technical
supports. This study was supported in part by a Grant-in-Aid for Scientific Research
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from the Ministry of Education, Culture, Sports, Science and Technology of Japan
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(#13243518).
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Figure Legends
Figure 1: Proportion of CD19+ B cells in peripheral blood and correlation with
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serum IgG levels.
Whole blood samples collected from healthy controls (HC n = 20) and primary
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Sjögren’s Syndrome (pSS n = 34) patients were incubated with violet-excited
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dye- labeled anti-CD19 antibody. The proportion of CD19+ B cells was analyzed by
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FACS (A). Serum IgG level was plotted against the proportion of CD19+ B cells for
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each pSS patient and HC (B). Wilcoxon test and Pearson’s correlation analysis were
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considered significant.
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examined for statistical significance between the groups. A p value < 0.05 was
Figure 2: IgG production from peripheral CD19 + B cells and correlation with
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serum IgG levels.
Peripheral CD19+ B cells (2.5 ×105 /mL) prepared from pSS patients (n = 16) and HC (n
= 10) were cultured with or without B cell stimulation in a 24-well plate for 96 hours.
The amount of IgG in the culture supernatants was measured by ELISA (A). Serum IgG
level of the patients was plotted against IgG production by CD19 + B cells with (C) or
without (B) B cell stimulation for each patient. Correlation between IgG production by
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pSS CD19+ B cells with stimulation and without stimulation in vitro was shown (D).
Wilcoxon test and Pearson’s correlation analysis were examined for statistical
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significance between the groups. A p value < 0.05 was considered to be significant.
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Figure 3: Comparative analysis of the proportion of peripheral B cell subsets
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between pSS patients and HC.
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Whole blood samples collected from healthy individuals (HC n = 20) and primary
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Sjögren Syndrome patients (pSS n = 34) were incubated with violet-excited dye-labeled
anti-CD19, FITC-labeled anti-IgD, PE-Cy5- labeled anti-CD38 and PE-Cy7-labeled
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anti-CD27 antibodies. The proportion of CD27-IgD+ (A), CD27+IgD+ (B), CD27+IgD-
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(C), CD38high IgD+ (D) and CD38high IgD- (E) cells in CD19+ B cells were analyzed by
FACS. The quantitative analysis of peripheral B cell subsets are shown. Wilcoxon test
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was examined for statistical significance between the groups. A p value < 0.05 was
considered to be significant.
Figure 4: Correlation between proportion of B cell subsets and clinical parameters
in pSS patients.
The proportion of CD38high IgD+ B cells in pSS patients (n = 34) were plotted against
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clinical parameters, such as serum levels of IgG (A), IgM (B), IgA (C), anti-SS-A
antibody (D) and anti-SS-B antibody (E) for each patient. Pearson’s correlation analysis
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was examined for statistical significance between the groups. A p value < 0.05 was
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considered to be significant.
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IgG level and autoantibodies of the patients.
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Figure 5: The relationship between the proportion of CD38high IgD+ B cells, serum
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The proportion of CD38high IgD+ B cells was defined by FACS analysis as described in
Materials and Methods. Comparative analysis of the proportion of CD38 high IgD+ B cells
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(A, B) and serum level of IgG (C, D) between the patients with anti- Ro/SSA and
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La/SSB antibodies positive and negative. Wilcoxon test was examined for statistical
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significance between the groups. A p value < 0.05 was considered to be significant.
Figure 6: Correlation between the proportion of periphe ral B cell subsets in pSS
patients and ESSDAI.
The proportion of CD38high IgD+ (A), CD38high IgD- (B) and CD27+IgD- (C) cells in
CD19+ B cells in pSS patients (n = 34) were plotted against ESSDAI score for each
patient. Pearson’s correlation analysis was examined for statistical significance between
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the groups. A p value < 0.05 was considered to be significant.
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Suppleme ntary Figure 1: The definition of pe ripheral B cell subsets by FACS
analysis.
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B cell subsets were defined by FACS as described in Materials and Methods.
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Representative data of peripheral B cell subsets, such as CD19 + B cells (A), CD27+IgD-
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B cells (B), CD38high IgD+ and CD38high IgD- B cells (C) in HC are shown.
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Table 1: Clinical characteristics of pSS patients and healthy controls (HC).
pSS (n=34)
p value
Age, years (mean ± SD)
45.8 ± 4.0
59.4 ± 14.9
< 0.001
Female (%)
100
100
1
Disease duration, median (years, range)
-
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HC (n=20)
4.8
±
4.0
-
Ocular symptoms, n (%)
-
31/34 (91.2)
-
32/34 (94.1)
-
4/34 (11.8)
-
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Oral symptoms, n (%)
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(0.5-12.2)
-
Extraglandular manifestation, n (%)
3/19 (15.7)
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Anti-nuclear antibody positive, n (%)
27/34 (79.4)
< 0.001
0/19 (0)
25/31 (80.6)
< 0.001
Anti-La/SSB antibody positive, n (%)
0/19 (0)
16/31 (51.6)
< 0.001
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Anti-Ro/SSA antibody positive, n (%)
-
IgM rheumatoid factor positive, n (%)
0/19 (0)
19/29 (65.5)
< 0.001
Serum IgG (mg/dL)
1103 ± 192
1726 ± 589
< 0.001
Serum IgM (mg/dL)
108 ± 54
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Anti-centromere antibody positive, n (%)
6/34 (17.6)
140 ± 139
0.321
Serum IgA (mg/dL)
195 ± 75
ESSPRI
-
4.5 ± 2.3
-
-
1.1 ± 1.4
-
-
2/34 (5.9)
-
ESSDAI
Corticosteroid administration, n (%)
250 ± 82
-
ESSPRI: EULAR Sjögren’s Syndrome Patient Reported Index
ESSDAI: EULAR Primary Sjögren’s syndrome disease activity index
0.025
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Table 2. Clinical variables associated with B cell subsets
CD38 highIgD - B cells/
CD27 +IgD - B cells/
CD19 + B cells (% )
CD19 + B cells (% )
CD19 + B cells (% )
r
p
r
r
0.596
0.0002
0.102
0.564
0.279
0.11
Constitutional
NA
NA
NA
NA
NA
NA
Lymphadenopathy
NA
NA
NA
NA
NA
NA
Glandular
NA
NA
NA
NA
NA
NA
Articular
NA
NA
NA
NA
NA
NA
Cutaneous
0.152
0.391
0.071
0.69
0.136
0.442
Pulmonary
NA
NA
NA
NA
NA
NA
Renal
NA
NA
NA
NA
NA
NA
Muscular
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
0.399
0.02
0.318
0.067
0.174
0.324
0.771
<0.0001
0.021
0.907
0.205
0.244
34
0.881
<0.0001
<0.0001
0.994
0.256
0.143
34
NA
NA
NA
NA
NA
NA
C4
34
0.261
0.136
0.169
0.34
0.034
0.848
CH50
33
0.039
0.83
0.103
0.567
0.21
0.241
34
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Peripheral nervous system
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ESSDAI score
Central nervous system
Hematological
IgG
C3
AC
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Biological
Not applicable: NA
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p
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CD38 highIgD+ B cells/
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N
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Variable
p
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Fig 5
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Fig 6
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Highlights

The proportion of CD19+ B cells in lymphocytes was increased in pSS.

IgG production by pSS CD19+ B cells was increased and correlated with serum

IgG.
The proportion of CD38high IgD+ B cells in CD19+ B cells was increased in pSS.
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The proportion of CD38high IgD+ B cells was correlated with clinical parameters of
pSS.
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
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