The Expansion of CD25 high IL-10 high FoxP3 high B Regulatory Cells Is in Association with SLE Disease Activity

Abstract

B regulatory cells (Bregs) belong to a subgroup of activated B cells tasked with maintaining self-tolerance and preventing autoimmunity. While sharing similar regulatory mechanisms such as IL-10 dependency, they also defer in exhibiting their suppressive effects by expressing Fas-Ligand, TGF-beta, and PDL-1. In this study we show, for the first time, the expansion of

CD25

high

FoxP3

high

Bregs in systemic lupus erythematosus (SLE) patients compared to healthy individuals (18.5 ± 3.052% versus 11.0 ± 1.654%,

p
<
0.001

, resp.). This expansion was also shown to correlate with SLE disease activity (

r
=
0.75

). In addition,

CD25

high

FoxP3

high

Bregs were also

IL-10

high

expressing and further expanded when stimulated with semaphorin 3A. In sum we show that

CD25

high

FoxP3

high

are an additional subtype of Bregs, involved in regulating SLE disease activity. Being IL-10 expressing, we may assume that they are one of the sources of increased serum IL-10 in SLE patients. Further studies are required in order to assess the relation between high serum IL-10 and

CD25

high

FoxP3

high

Breg cells.

Full text

Research Article
The Expansion of CD25highIL-10highFoxP3high B Regulatory Cells
Is in Association with SLE Disease Activity
Zahava Vadasz,1Regina Peri,1Nasren Eiza,1Gleb Slobodin,2
Alexandra Balbir-Gurman,3and Elias Toubi1
1Division of Allergy & Clinical Immunology, Bnai Zion Medical Center, Faculty of Medicine, Technion, 4940 Haifa, Israel
2Rheumatology Unit, Bnai Zion Medical Center, Faculty of Medicine, Technion, 4940 Haifa, Israel
3B. Shine Rheumatology Unit, Rambam Health Care Campus, Faculty of Medicine, Technion, 4940 Haifa, Israel
Correspondence should be addressed to Elias Toubi; elias.toubi@b-zion.org.il
Received  July ; Accepted  September 
Academic Editor: Carlo Perricone
Copyright ©  Zahava Vadasz et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
B regulatory cells (Bregs) belong to a subgroup of activated B cells tasked with maintaining self-tolerance and preventing
autoimmunity. While sharing similar regulatory mechanisms such as IL- dependency, they also defer in exhibiting their
suppressive eects by expressing Fas-Ligand, TGF-beta, and PDL-. In this study we show, for the rst time, the expansion of
CDhighFoxPhigh Bregs in systemic lupus erythematosus (SLE) patients compared to healthy individuals (. ±.% versus
. ±.%, 𝑝 < 0.001, resp.). is expansion was also shown to correlate with SLE disease activity (𝑟 = 0.75). In addition,
CDhighFoxPhigh Bregs were also IL-high expressing and further expanded when stimulated with semaphorin A. In sum we
show that CDhighFoxPhigh are an additional subtype of Bregs, involved in regulating SLE disease activity. Being IL- expressing,
we may assume that they are one of the sources of increased serum IL- in SLE patients. Further studies are required in order to
assess the relation between high serum IL- and CDhighFoxPhigh Breg cells.
1. Introduction
Among the many immune mediated responses involved in
systemic lupus erythematosus (SLE) is the imbalance between
T-helper cells () subsets, namely, //, and both
T and B regulatory (reg) cells [].  proinammatory
cytokine levels such as IL-, IL-, and IFNs are usually
increased in association with SLE disease activity index
(SLEDAI).  related cytokines such as IL- and IL-
 are also reported to be enhanced and contribute to
inammatory processes in SLE and other rheumatic dis-
eases such as rheumatoid arthritis (RA) and psoriasis. 
relatedcytokines,thatis,IL-andIL-,areknownfor
their ability in driving humoral immune responses, B cell
overactivation, and the production of many specic autoan-
tibodies [–]. Many studies during the last decade have
reported on the failure of Treg cells to maintain self-tolerance,
allowing the development of many autoimmune diseases.
e failure in suppressing eector  cell proliferation is
mainly considered to be IL- dependent (lower expression
and/or production of IL-) due to the altered expression of
FoxP and/or inhibitory molecules such as CTLA- in Treg
cells []. Breg cells are involved in regulating/suppressing
immune mediated inammation but act earlier than Treg
cells. ey use similar suppressive modalities, that is, IL-
, TGF-beta, and the expression of proapoptotic membrane
molecules which vary across dierent Breg subtypes [].
Among these dierent subtypes, CD19+CD24highCD38high
and CD19+CD25highCD86highCD1dhigh were both described
as being involved in suppressing autoimmune processes,
both in an IL- dependent way and with an altered
function in SLE [, ]. Breg cells have also been charac-
terized as CD5high ,FoxP3high , and Fas-Ligand expressing
cells. CD19+CD5highFoxP3high Breg cells were reported to
be involved in non-IgE-mediated food allergies, namely,
in maintaining tolerance to milk allergies []. In addi-
tion to this subtype, Breg cells were dened as being
Hindawi Publishing Corporation
Journal of Immunology Research
Volume 2015, Article ID 254245, 6 pages
http://dx.doi.org/10.1155/2015/254245

Journal of Immunology Research
CD19+CD5highFas-Lhigh ,alsocalled“killerBcells.”Numer-
ous researchers have reported that these cells participate
in the escape of viral infections from the ecient cyto-
toxic T cell response []. e similarities and dierences
between all the above-mentioned Breg cells are not suf-
ciently understood. Are they similar in their regulatory
eects? Do they express/produce similar amounts of IL-
and TGF-beta? How do they react to various stimuli? (see
[]). In previous studies, we and others showed that Breg
cell function was enhanced when stimulated by CpG and
CDL, increasing by this autologous Treg cell properties
following their coculture [, ]. When cocultured with
semaphorin A (semaA), IL- and TGF-beta expression
was enhanced in CD19+CD25high Breg cells, suggesting that
semaA is a frontier factor in improving Breg cell function
(unpublished data). Later, we reported on the ability of
semaA in enhancing Breg cell properties by increasing CD
(a regulatory molecule) expression on B cells []. Expecting
to nd lower serum levels of IL- in some autoimmune
diseases, namely, in SLE, the opposite was found. Paradoxi-
cally, serum IL- is reported to be increased in association
with increased SLEDAI and with high titers of anti-dsDNA
antibodies. e source of increased serum IL- in SLE is
yet undened, suggested to be overproduced by  and/or
by one of the Breg subtypes. In addition, the association of
Atg rs single nucleotide polymorphism (SNP) with
SLE susceptibility and IL- serum levels was analyzed. Here,
carriage of the rs T allele was associated with IL-
upregulation and clinical features of SLE, concluding that
suchmutatedalleleinuencedbothSLEsusceptibilityandIL-
production[].Inthisstudy,weaimtoevaluatethestatus
of CD19+CD25highFoxP3high Breg cells, namely, whether they
are IL- expressing. We will also assess the status of these
cells in SLE patients when compared to healthy individuals.
We speculate on their possible contribution to increased
serum IL- in SLE patients. Finally, we will evaluate the
response of this subtype of Breg cells to semaA, to see if this
coculture increases IL- expression as it does in other Breg
cells.
2. Patients and Methods
2.1. Patients Population. is study examined  SLE patients
( females and  male; age range – years; mean
30.5 ± 9.2). All patients are routinely followed up by well-
trained rheumatologists and all fulll the ACR criteria for
the classication of SLE []. Clinical and serological data
(skin involvement; arthritis; renal involvement; full cell
blood count; serum complement levels; anti-dsDNA and
other extractable nuclear autoantibodies) were all available,
enabling the determination of SLEDAI. e serological work-
up was performed at the Bnai Zion Medical Center by a
single experienced technician to insure uniformity of all
analyses, utilizing identical kits. Patients in whom SLEDAI
was between  and  points were treated with hydroxychloro-
quine and in some patients prednisolone (.mg/daily) was
added. When SLEDAI was above  points, azathioprine was
added, but only aer analyzing specic serology and purify-
ing B cells. When SLEDAI was above  points the addition
of cyclophosphamide or MMF was considered again, only
aer performing SLE serology and purifying B cells. Twenty
healthycontrols,sexandagematched,wereassessedand
analyzed for all above-mentioned parameters. is study was
approved by both the local Helsinki Committee of the Bnai
Zion Medical Center and the Rambam Health Care Campus,
Haifa, Israel.
2.2. B Cell Purication. B cells were puried from peripheral
bloodofhealthycontrolsandSLEpatients.Todoso,
peripheral blood mononuclear cells (PBMCs) were isolated
on Lymphoprep (Axis-Shield, Oslo, Norway), and B lympho-
cytes were then twice puried by positive selection using
CD microbeads ( 𝜇L/7cells; Miltenyi Biotec, Ber-
gisch Gladbach, Germany) according to the manufacturer’s
instructions, achieving by this >% purity.
2.3. FoxP3 and IL-10 Expression in CD19+CD25ℎ𝑖𝑔ℎ BCells.
e expression of FoxP and IL- in CD19+CD25high cells
(considered as Breg cells) from healthy controls and SLE
patients was initially assessed by staining puried B cells
aer  hours of activation with ODN-CpG and CDL.
e staining was performed by using monoclonal antibodies,
human anti-CD-BUV (BD Horizon, Becton Dickinson,
NJ, USA) and human anti-CD BUV (BD Horizon,
Becton Dickinson, NJ, USA) as outer membrane antibod-
ies, and FoxP PE\CF594 and IL- APC (BD Horizon,
Becton Dickinson, NJ, USA) as intracellular staining, using
a “Fix and Perm” kit (Invitrogen, NY, USA) according to
the manufacturer’s instructions. e staining was evaluated
using ow cytometry soware (FC and CXP soware,
Beckman Coulter, Brea, CA, USA, and Becton Dickinson,
NJ, USA). CD positive cells in the puried cell culture were
determined by using monoclonal CD PerCP-Cy. antibody
(BD Pharmingen, Becton Dickinson, NJ, USA) and analyzed
by Becton Dickinson FACS-Fortessa. e results are shown
as % of CD19+CD25high Breg cells expressing FoxP or IL-,
taking into consideration that the absolute number of Breg
cells in all groups was found to be comparable. Standard
deviation(STDEV)wasusedtoquantifytheamountof
variationofasetofdatavalues(e.g.,percentageofBregcells
expressing FoxP among the patients in each indicated group
of disease or normal control).
2.4. Semaphorin 3A Enhances FoxP3 Expression. Aiming to
evaluate the eect of semaA on FoxP expression, condition-
media from HEK−cells, which were infected by NSPI-
CMV-FLAG lentivirus with or without human semaA
cDNA, a kind gi from Professor Gera Neufeld and Dr.
Ofra Kessler, Ruth and Bruce Rappaport Faculty of Medicine,
Technion, Israel, as previously described [], were added to
theabove-mentionedpuriedBcellsactivatedbyODN-CpG
and CDL and incubated for  hours. Aer incubation,
CD19+CD25high cellswereanalyzedforthepossiblechange
in FoxP expression using the above-mentioned specic
monoclonal antibodies and evaluated using an FC ow

Journal of Immunology Research 
103
102
101
100
CD25 PC5
FoxP3Pe
28%48%
100101102103
(a)
100102
101103104105
0
SSC-A
Comp-PerCP-Cy5.5-A::CD3
250 K
200 K
150 K
100 K
50 K
CD3+0.33%
(b)
F : (a) A representative FACS analysis of puried B cells (expressing CD25high FoxP) following CpG-ODN and CDL activation. Of
note is that CD25dim-low B cells (upper le quadrant) do not express FoxP. However, CD25high B cells coexpress signicant amount of FoxP
(upper right quadrant). (b) A representative FACS analysis of puried activated B cells, showing that CD+T cell contamination (gated CD3+
T cells) is less than .%.
cytometer and Becton Dickinson FACS-Fortessa. e results
are shown as % of Breg cells expressing FoxP, taking into
consideration that the absolute number of Breg cells in all
groups was found to be comparable.
2.5. Clinical Correlation and Statistical Analysis. Comparison
of FoxP expression in B cells from SLE patients and healthy
controls was done using the unpaired Student 𝑡-test. e
correlation coecient (𝑟)ofclinicalcorrelationbetween
SLEDAI score and % of Breg cells expressing FoxP was
determined using the Pearson correlation test. A two-tailed
𝑝value of . or less was considered to be statistically
signicant.
3. Results
3.1. CD19+CD25ℎ𝑖𝑔ℎ Activated B Cells Are FoxP3ℎ𝑖𝑔ℎ.First, we
examined whether CD19+CD25high B regulatory cells are also
FoxP expressing cells. Puried resting B cells (immediately
following purication) were FoxP3dim (weakly detectable)
(data not shown). However, following their stimulation with
CpG-ODN and CDL for  h, CD19+CD25high Bcells
turned to become FoxP3high (Figure (a)). As also seen, there
are less than .% gated CD T cells and therefore B cell
contamination with CD is unlikely and FoxP expression in
CD25high B cells is very prominent (Figure (b)).
3.2. Activated CD19+CD25ℎ𝑖𝑔ℎ FoxP3ℎ𝑖𝑔ℎ Are Also IL-10ℎ𝑖𝑔ℎ.
Gating on activated CD25highFoxP3high one can see that most
Comp-APC-A::IL-10
Comp-PE-CF594-A::FoxP3
Q1
6.84%
Q4
2.91%
Q3
4.06%
Q2
86.2%
100102
101103104105
105
104
103
101
102
100
F : A demonstrative FACS analysis of puried B cells,
showing that activated CD19+CD25highFoxP3high are also IL-10high .
Of note is that CD25dim-low \FoxP3dim-low B cells express very little
IL-.
of these cells (>% of these cells) are IL-10high (Figure ) in
contrast to B cells that are FoxP3dim being also IL-10dim.
3.3. CD19+CD25ℎ𝑖𝑔ℎFoxP3ℎ𝑖𝑔ℎ in SLE. e percentage of Breg
cells (CD19+CD25high cells) in peripheral blood (highly

Journal of Immunology Research
0
5
10
15
20
25
30
Controls SLE
W/O sema3A
With sema3A
p < 0.002
p < 0.005
p < 0.001
Average % of CD19+CD25high expressing FoxP3
F : e percentage of CD19+CD25high FoxP3high Breg cells in
normal controls (𝑛=20)andinpatientssueringfromSLE(𝑛=
21). One can see that this subtype of B cells is signicantly increased
in SLE patients. In addition, the addition of semaA to these cells
increased signicantly the percentage of these cells.
expressing FoxP) was signicantly higher in SLE patients
when compared to that of healthy individuals (18.5%±3.052
versus 11.0 ± 1.654%, resp., 𝑝 < 0.005) (Figure ).
3.4. Semaphorin 3A Increases FoxP3 Expression in Breg Cells.
We then sought to determine if semaA increases the
expression of FoxP in these Breg cells. As is demonstrated
in Figure , semaA increases the percentage of Breg cells
(CD19+CD25high cells) in peripheral blood expressing FoxP,
in normal controls and to a higher extent in SLE patients
(in normal controls up to 13.6 ± 1.806%frombaseline,𝑝<
0.002, and in SLE patients up to 28.5 ± 3.506%, 𝑝 < 0.0001)
(Figure ).
3.5. FoxP3 Expression in B Cells Is Correlated with SLEDAI.
Figure  demonstrates the correlation between the percentage
of CD19+CD25highFoxP3high cells of SLE patients and the
SLEDAI score of these patients. As can be seen, there is a
positive correlation with an “𝑟” Pearson coecient of ..
isresultisinlinewiththeknowncorrelationbetweenIL-
 level in SLE patients and their SLEDAI.
4. Discussion
In most autoimmune diseases, immune mediated inamma-
torydamageisalwaystheresultofanetbalancebetween
the overactivity of self-reactive cells (T and B eector cells)
and immune regulatory mechanisms (T and B regulatory
cells). Most B regulatory cells are dened as being IL-
 expressing/producing cells; however, they have dierent
subtypes, are heterogeneous, and have dierent mechanisms
in diseases in which they are involved. eir homology
to Treg subtypes, namely, Br cells (expressing IL-), Br
05 10152025
0
20
40
60
80
CD19+CD25highFoxP3high cells (%)
r = 0.75
SLEDAI
F : Clinical correlation between the percentage of
CD19+CD25highFoxP3high cells of SLE patients and the SLEDAI
score of these patients. e correlation was done using the Pearson
correlation test.
cells (mainly expressing TGF-beta), and B-FoxP positive
cells, was recently mentioned. In this case, Breg cells were
shown to initiate immune regulatory responses by facilitating
the recruitment of Tregs and then disappearing once Tregs
become dominant in the immune response []. As men-
tioned above, when CD19+CD24highCD38high Bcellswere
evaluatedinSLE,theyhadbothareducedabilitytoproduce
IL- and a reduced ability to suppress T cell cytokine pro-
duction, although it is unclear if this latter defect is a cause or
a consequence of SLE. In contrast to this nding, human IL-
 producing CD24highCD27high Breg cells (found to suppress
monocytes in an IL- dependent manner) were increased
in patients with rheumatoid arthritis, SLE, and multiple
sclerosis when compared to healthy individuals, suggesting
this increase to be compensatory, aiming (with little success)
to maintain self-tolerance []. e role of CD5highFasLhigh
“killer B cells” was assessed in lupus susceptible MRL/lpr
mice. Being cytotoxic to T cells they were found to be
increased, probably in attempt to suppress autoreactive T cells
in these mice []. Focusing on CD19+CD25highFoxP3high
Breg cells we rst assessed their status in healthy individuals.
Here,weshowforthersttimethatbothIL-andFoxP
expressions were noticed mainly in activated CD25high B
cells (activated with CpG and ODN) and that this expression
was enhanced when these B cells were stimulated with
add-onsemaA.InthiscaseCD25high FoxP3high Breg cells
were characterized by being IL-10high whereas FoxP3dim B
cells were IL-10dim aswell.WhenanalyzedinSLEpatients,
we found CD19+CD25high FoxP3high cells to be signicantly
increased as compared to healthy individuals. is was found
to be in positive correlation with increased SLEDAI and in
association with lupus nephritis. In a recent study and in line
with our nding, CD19+CD25highFoxP3high Bregulatorycells
were found to be increased in the cerebrospinal uid of active
patients suering from relapsing-remitting multiple sclerosis
(MS) when compared to that of nonclinically active MS. is

Journal of Immunology Research 
expansion of B regulatory cells was attributed to the compen-
satory attempt of these cells to maintain immune regulatory
processes []. In contrast to this study, rheumatoid arthritis
patients had signicantly lower proportions of peripheral
blood CD19+FoxP3+B cells as compared to healthy controls,
particularly in patients with interstitial lung disease. is
nding suggests that Breg phenotypes may have dierent
functions in the pathogenesis of dierent rheumatic diseases
[]. e fact that serum IL- is increased in SLE and in
association with SLE disease activity has been established in
many previous studies. In one, increased IL- was shown to
exhibit a modulatory eect by suppressing the dierentiation
and function of monocyte-derived dendritic cells []. In
a recent study, increased IL- in the sera of SLE patients
was capable of inducing Fas and FasL expression on CD+
T cell surfaces, promoting apoptosis of this cell subset, thus
contributing to many other mechanisms of self-tolerance
[].However,westillneedtoexplainthemechanismsby
which serum IL- is increased in SLE. In this regard, the
expansion of IL- producing B cells was shown to be in
parttheresultofincreasedBcellactivatingfactor(BAFF).
Enhanced serum BAFF in SLE was described in many studies
as being associated with increased expression of TLR- and
other markers of B cell activation [, ]. is may explain
ourndingofincreasedIL-10high FoxP3high Bregcellsaswell
as increased serum IL- in SLE. Another signicance of
FoxP3high B cells being increased in SLE is the possibility
that by multiplying they also increase their IL- production
improving by this their regulatory function. When B cells
were cocultured with semaA they responded by increasing
their FoxP expression. is raises the possibility that if
provided with the proper stimulation Bregs may develop
higher regulatory properties and that by increasing their IL-
production they may induce a better regulatory mechanism
in SLE.
5. Conclusion
CD25highFoxP3high Bregs (highly expressing IL-) are sig-
nicantly increased in SLE, in correlation with SLEDAI.
Semaphorin A increases FoxP expression in Breg cells
improving by this their regulatory properties. We assume that
the expansion of these cells is the attempt of our regulatory
immune responses to maintain self-tolerance and to suppress
as much as possible SLE disease activity. Further studies are
required in order to better understand the role of this subset
of B cells in autoimmunity.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
References
[]R.M.Talaat,S.F.Mohamed,I.H.Bassyouni,andA.A.
Raouf, “///Treg cytokine imbalance in systemic
lupus erythematosus (SLE) patients: correlation with disease
activity,” Cytokine, vol. , no. , pp. –,  (Chinese).
[] K.Shah,W.-W.Lee,S.-H.Leeetal.,“Dysregulatedbalanceof
 and  cells in systemic lupus erythematosus,” Arthritis
Research & erapy,vol.,no.,articleR,.
[] D.Li,B.Guo,H.Wu,L.Tan,C.Chang,andQ.Lu,“Interleukin-
 in systemic lupus erythematosus: a comprehensive review,”
Autoimmunity,vol.,no.,pp.–,.
[] B. Terrier, N. Costedoat-Chalumeau, M. Garrido et al., “Inter-
leukin  correlates with T cell and B cell subset alterations in
systemic lupus erythematosus,” e Journal of Rheumatology,
vol.,no.,pp.–,.
[]S.Futatsugi-Yumikura,K.Matsushita,A.Fukuokaetal.,
“Pathogenic -type follicular helper T cells contribute to
the development of lupus in Fas-decient mice,” International
Immunology,vol.,no.,pp.–,.
[]B.Yan,S.Ye,G.Chen,M.Kuang,N.Shen,andS.Chen,
“Dysfunctional CD+,CD+ regulatory T cells in untreated
active systemic lupus erythematosus secondary to interferon-𝛼-
producing antigen-presenting cells,” Arthritis and Rheumatism,
vol.,no.,pp.–,.
[]A.Ray,L.Wang,andB.N.Dittel,“IL--independentregu-
latory B-cell subsets and mechanisms of action,” International
Immunology,.
[]P.A.Blair,L.Y.Nore
˜
na, F. Flores-Borja et al.,
“CD+CDhiCDhi B cells exhibit regulatory capacity
in healthy individuals but are functionally impaired in systemic
lupus erythematosus,” Immunity,vol.,no.,pp.–,
.
[] A. Kessel, T. Haj, R. Peri et al., “Human CD+CDhigh B
regulatory cells suppress proliferation of CD+Tcellsand
enhanceFoxpandCTLA-expressioninT-regulatorycells,”
Autoimmunity Reviews, vol. , no. , pp. –, .
[] J. Noh and G. Noh, “Allergen specic responses of CDhigh
and CDlow B cells in non-IgE mediated food allergy of late
eczematous reactions in atopic dermatitis: presence of IL- and
IL- producing regulatory B cells (Br & Br),” Inammation
&Allergy-DrugTargets,vol.,no.,pp.–,.
[] S. K. Lundy, “Killer B lymphocytes: the evidence and the
potential,” Inammation Research,vol.,no.,pp.–,
.
[] F. Mion, S. Topnon, B. Tooletto, D. Cesselli, C. E. Pucillo, and
G. Vitale, “IL- production by B cells is dierentially regulated
by immune-mediated and infectious stimuli and requires p
activation,” Molecular Immunology,vol.,no.,pp.–,
.
[] K.Yanaba,J.-D.Bouaziz,T.Matsushita,T.Tsubata,andT.F.
Tedder, “e development and function of regulatory B cells
expressing IL- (B cells) requires antigen receptor diversity
and TLR signals,” Journal of Immunology,vol.,no.,pp.
– , .
[]Z.Vadasz,T.Haj,A.Balbiretal.,“Aregulatoryrolefor
CD expression on B cells in systemic lupus erythematosus,”
Seminars in Arthritis & Rheumatism,vol.,no.,pp.–,
.
[] P. L´
opez, E. Alonso-P´
erez, J. Rodr´
ıguez-Carrio, and A. Su´
arez,
“Inuence of Atg mutation in SLE depends on functional IL-
genotype,” PLoS ONE,vol.,no.,ArticleIDe,.
[] C. Bombardier, D. D. Gladman, M. B. Urovitz, D. Caron, and C.
H. Chang, “Derivation of the SLEDAI. A disease activity index
for lupus patients. e committee on prognosis studies in SLE,”
Arthritis and Rheumatism,vol.,no.,pp.–,.

Journal of Immunology Research
[] B. Kigel, A. Varshavsky, O. Kessler, and G. Neufeld, “Suc-
cessful inhibition of tumor development by specic class-
 semaphorins is associated with expression of appropriate
semaphorin receptors by tumor cells,” PLoS ONE,vol.,no.,
article e, .
[] J.-M. Berthelot, C. Jamin, K. Amrouche, B. Le Go, Y. Maugars,
and P. Youinou, “Regulatory B cells play a key role in immune
system balance,” Joint Bone Spine,vol.,no.,pp.–,.
[] Y. Iwata, T. Matsushita, M. Horikawa et al., “Characterization
of a rare IL--competent B-cell subset in humans that parallels
mouse regulatory B cells,” Blood,vol.,no.,pp.–,
.
[] D. Bonardelle, K. Benihoud, N. Kiger, and P. Bob´
e, “B lym-
phocytes mediate Fas-dependent cytotoxicityin MRL/lpr mice,”
JournalofLeukocyteBiology,vol.,no.,pp.–,.
[] C. de Andr´
es, M. Tejera-Alhambra, B. Alonso et al., “New
regulatory CD+CD+B-cell subset in clinically isolated
syndrome and multiple sclerosis relapse. Changes aer gluco-
corticoids,” Journal of Neuroimmunology,vol.,no.-,pp.
–, .
[] Y. Guo, X. Zhang, M. Qin, and X. Wang, “Changes in peripheral
CD+Foxp+and CD+TGF-𝛽+regulatory B cell populations
in rheumatoid arthritis patients with interstitial lung disease ,”
Journal of oracic Disease,vol.,no.,pp.–,.
[] Z. Sun, R. Zhang, H. Wang et al., “Serum IL- from systemic
lupus erythematosus patients suppresses the dierentiation
and function of monocyte-derived dendritic cells,” Journal of
Biomedical Research, vol. , no. , pp. –, .
[] X. Yang, B. Sun, H. Wang, C. Yin, X. Wang, and X. Ji, “Increased
serum IL- in lupus patients promotes apoptosis of T cell
subsets via the caspase  pathway initiated by Fas signaling,”
JournalofBiomedicalResearch,vol.,no.,pp.–,.
[] M. Yang, L. Sun, S. Wang et al., “Cutting edge: novel function
of B cell-activating factor in the induction of IL--producing
regulatory B cells,” e Journal of Immunology,vol.,no.,
pp. –, .
[] D. Yehudai, A. Snir, R. Peri et al., “B cell-activating factor
enhances interleukin- and interleukin- productionby ODN-
activated human B cells,” Scandinavian Journal of Immunology,
vol. , no. , pp. –, .