Review Article Tolerance in Kidney Transplantation: What ...downloads.hindawi.com/journals/mi/2016/8491956.pdf · infections, malignancies, and metabolic disorders [] which may threaten

Review ArticleTolerance in Kidney Transplantation What Is on the B Side

Laura Carreras-Planella Francesc E Borragraves and Marcella Franquesa

REMAR-IVECAT Group Health Science Research Institute Germans Trias i Pujol Can Ruti Campus Badalona Spain

Correspondence should be addressed to Marcella Franquesa mfranquesaigtpcat

Received 19 August 2016 Accepted 12 October 2016

Academic Editor Margit Zeher

Copyright copy 2016 Laura Carreras-Planella et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Regulatory B cells (Breg) are in the spotlight for their role in immune homeostasis maintenance and tolerance achievement as inthe last years the correlation with functional and increased Breg numbers in autoimmune diseases and transplantation has beenextensively proven Their study is however in its infancy with still little knowledge and consensus on their origin phenotype andmechanism of action All this hampers the pursuit of an effective Breg induction method for therapeutic purposes In this reviewwe aim to summarize the studies on human Breg and their implication in kidney transplantation and to further discuss the issuessurrounding therapeutic applications of this cell subset

1 Introduction

Renal transplantation is the unique curative option forpatients suffering from end-stage renal disease but to datethe evolution of each patient after transplantation cannotbe predicted In the past decades acute graft rejectionhas decreased dramatically as a result of the introductionof immunosuppressive drugs However immunosuppres-sive drugs carry undesired and severe side effects such asinfections malignancies and metabolic disorders [1] whichmay threaten patientrsquos life Yet chronic rejection is still themain cause of long-term graft loss [2 3] The holy grail oforgan transplantation is to maintain long-term graft functionwithout immunosuppressive treatment namely operationaltolerance (OT) However OT is a rare event in kidneytransplanted patients [4] as only about 003 of cases areestimated to be in such state [5] Thus despite the effortsmade in the past there is still a clear need to find newstrategies to achieve long-term tolerance and to investigatethe immunologicalmechanisms thatmay be implicated in theprocess of OT

Among the actors implicated in the mechanisms ofthe immune response B and T lymphocytes are the maincharacters that lead to graft rejection In this play B lym-phocytes have a dual key role since they present antigensof the donor to T cells in addition to secreting antibodiesthat can lead to acute rejection or later in time chronic

rejection [6] Nevertheless a sparse B cell subset has beenattributed immune regulatory functions which conveys thatnot all B cells play on the rejection side Although it wasfirst described in 1974 [7] it was not until 2000 that thispopulation was named regulatory B cells (Breg) [8] Inthe last decade the regulatory role played by Breg hasbeen highlighted by many authors in autoimmune diseasessuch as systemic lupus erythematosus (SLE) [9] rheumatoidarthritis [10] and pathologies that promote antineutrophilcytoplasmic antibodies [11] and also in allograft tolerance inorgan transplantation [12 13] The current general consensusis that Breg develop their function mainly via the secretionof IL-10 [14 15] However a complete phenotype signaturedevelopment pathway or the immunoregulatory propertiesof Breg have not been fully discovered inmice nor in humansthus granting future research on this cell type

In this review our aim is to gather the current knowledgeabout regulatory B cells and their role in kidney transplan-tation tolerance in humans and to discuss their potentialapplication as cellular therapeutic agent

2 Regulatory B Cells Phenotype and Function

One of the darkest spots of Breg is their phenotype sincefor years researchers in the field have tried through multipleapproaches to find unique characteristic markers to definethem However there is still no consensus on it There is

Hindawi Publishing CorporationMediators of InflammationVolume 2016 Article ID 8491956 11 pageshttpdxdoiorg10115520168491956

2 Mediators of Inflammation

less discussion about their mechanism of action which isprincipally accepted to be IL-10 but the lack of knowledge onwhat triggers its secretion and the fact that other regulatorymechanisms have also been proposed leave this issue to dateunresolved

21 Does a ldquoUniquerdquo Breg Phenotype Exist As previouslyoccurred in the studies on regulatory T cells manyresearchers have prompted to identify a unique set ofmarkers transcription factors or mechanism of action thatexclusively identify Breg in all contexts In this sense geneticand surface expression studies have been conducted withpartial success to unravel a unique Breg signature [16 17]Unfortunately to date such unequivocal markers have notbeen found yet Also some hypothesis have been formulatedon Breg development pathways from a common precursor[18 19] but the results so far are not conclusive Thus mostauthors rely on the capacity to produce interleukin- (IL-) 10and on the two main phenotypical signatures used to defineBreg (1) transitional B cell phenotype CD19+CD24hiCD38hi

and (2) CD19+CD5+CD1dhi (used in both human and mice)[20 21] Nevertheless we still face a lack of specific Bregmarkers and different phenotypes for IL-10-producing Bcells with regulatory capacity have been proposed throughthe years In 2008 Yanaba and colleagues identified an IL-10-producing regulatory B cell subset in mice expressingCD1dhiCD5+ which they referred to as B10 cells [21] A fewyears later the same group characterized a similar IL-10-producing B cell subset in humans Human B10 cellsrsquo regula-tory potential was shown by their capacity to inhibit tumornecrosis factor- (TNF-) 120572 production by CD4+ T helpercells and monocytes In peripheral blood B10 cells werefound exclusively among CD24hiCD27+ cells whereas inspleen no difference was observed between IL-10-producingand nonproducing B cells regarding their surface markers[15] When testing the immunomodulatory capacity of thesame subset from patients with allergic asthma in vitrothese cells were less capable of secreting IL-10 and inducingthe secretion of IL-10 from CD4+ T compared to the samecell population from healthy subjects suggesting that thispopulation could hold immunomodulatory capability [22]Nevertheless Matsumoto et al found that CD27intCD38+immunoglobulin- (Ig-) secreting plasmablasts that arise fromnaıve and immature B cells from human blood are the majorIL-10-producing B cells after in vitro stimulation [23] Yet thetransitional B cell subset CD24hiCD38hi also seems to haveregulatory capacity since after CD40 stimulation they couldsuppress the differentiation of naıve T cells into T helper 1(Th1) andTh17 and lead CD4+CD25minus T cells conversion intoregulatory T cells (Treg) partially via IL-10 [9 24]

Due to the disparity of the results showing that differentB cell subsets can express immunomodulatory propertiesa current emerging view is that Breg are not a specific Bcell subset but rather a circumstantial B cell phenotype Inthis scenario B cells could acquire a regulatory role whenappropriate signals are generated in the environment as hasbeen already suggested by some authors [25ndash27] It wouldseem reasonable to think that depending on the type of

activated immune cells and cytokines released to the envi-ronment some B cells could shape their response towards theappropriate way to modulate the response of other immunecells The adaptability of the Breg response could explain thedifferent outcomes depending on the disease studied in vivoor the stimulation provided in vitro Therefore maybe thequest for a ldquouniquerdquo Breg marker must be reoriented to findthe right stimulation for B cells to become stable regulators ofthe immune response in a given scenario

22 IL-10 Secretion as Breg Mechanism of Action As men-tioned above IL-10 production is perhaps the principalhallmark to define regulatory B cells describing theirimmunomodulatory potential and explaining their mecha-nism of action IL-10 is a regulatory cytokine secreted byalmost all innate and adaptive immune cells that plays anessential role in maintaining immune homoeostasis [28] Itbinds as a homodimer to its receptor which is a tetramerformed of two 120572 (IL-10R1) and two 120573 (IL-10R2) chainsIL-10R1 binds to the cytokine while IL-10R2 is responsiblefor the downstream signaling activation through Jak1 andsignal transducer and activator of transcription 3 (STAT3)IL-10 is the only ligand for IL-10R1 which in turn is theunique receptor of IL-10 while IL-10R2 is shared by severalcytokines such as IL-20 IL-22 IL-24 IL-26 IL-28 and IL-29[29] Although little is known about the molecular pathwaysinvolved in IL-10 secretion in humans in mice it is medi-ated by store-operated Ca2+ influx from the endoplasmicreticulum which is further regulated by the calcium sensorsstromal interaction molecule (STIM) 1 and STIM2 [30]

Among other biological functions IL-10 promotes thedownregulation of antigen presentation by macrophages anddendritic cells and suppresses the production of proinflam-matory cytokines such as IL-1 interferon (IFN)-120574 and TNF-120572 by CD4+ T cells monocytes and macrophages [29 31](Figure 1)

Besides the functional relevance of IL-10 expression IL-10-producing CD19+CD24hiCD38hi B cells have also beenshown to promote the expansion of IL-10-producing FoxP3+Treg and to play a role in inducing their recruitment to the siteof inflammation [32] In addition human IL-10-producingB cells may block the CD28minus inducible T cell costimulator(ICOS) costimulatory pathway thus blocking T cell activa-tion via phosphorylation of Src homology region 2 domain-containing phosphatase-1 (SHP1) a downstream moleculeof the IL-10 receptor intracellular pathway [33] Moreoverthey regulate innate immune responses by reducing TNF-120572production by monocytes [15]

The key role of IL-10 released by B cells has been alsoproven in multiple sclerosis (MS) patients who have B cellswith impaired IL-10 production under CD40 stimulation[34] Similarly in SLE patients B cells fail to produce IL-10 in response to CD40 but not to CpG oligodeoxynu-cleotides (CpG) [9]These results indicate an impaired T cell-dependent Breg induction in both autoimmune diseases

23 IL-10 Independent Regulatory B Cells Not only IL-10 butalso other regulatory mechanisms like IL-35 [35] granzyme

Mediators of Inflammation 3

darr antigen presentation

darr IL-1darr IFN-120574darr TNF-120572darr IL-2

darr IL-1

darr IL-6

IL-

IDOADO CTLA-4

10

darr IFN-120574darr TNF-120572darr IL-2

darr ICOS

uarr proliferation

darr proliferation

darr proliferation

uarr recruitment

CD4+ T cell

CD4+ T cell

darr proliferationCD4+ T cell


darr proliferationCD4+ T cell CD8+ T cell

TGF-120573

GzmB

5998400-AMP

IFN-120572120573

uarr CD69

Th1

Th17

Treg

uarr proliferationTreg

BregB cell

Macrophage Dendritic cellMonocyte

Proposed inducersCD40 ligation

CpG ODNBCR engagement

IL-2IL-4IL-6

LPSIL-21IL-35BAFF

APRILMSC

Figure 1 Graphical summary of the main Breg induction methods mode of action and immunosuppressive functions that havebeen proposed in the literature ODN oligodeoxynucleotides IL interleukin BCR B cell antigen receptor IFN interferon LPSlipopolysaccharide BAFF B cell activating factor APRIL a proliferation-inducing ligand MSC Mesenchymal Stem Cells GzmB granzymeB TGF transforming growth factor ADO adenosine AMP adenosine monophosphate IDO indoleamine 23-dioxygenase CTLA-4Cytotoxic T-Lymphocyte Antigen 4 ICOS inducible T cell costimulator

B (GzmB) [36] transforming growth factor- (TGF-) 120573 andindoleamine 23-dioxygenase (IDO) [37] have been suggestedas important molecules in Breg tolerogenic function Figure 1depicts some of the different Breg inducers mechanisms ofaction and functions described in several studies In oneof them when CD4+CD25minus T cells were cocultured withCD40-CpG-stimulated B cells from either healthy controls(HC) immunosuppressive-dependent stable graft function(SI) patients or OT patients the proliferation of T cells wasinhibited When IL-10 TGF-120573 or GzmB were blocked sepa-rately only the anti-GzmB antibody hindered the inhibitoryeffect on T cell proliferation [12] However in a similarexperimental set-up where B cells were stimulated withCpG alone the blockade of TGF-120573 andor IDO activity ledto decreased antiproliferative function of Breg in coculturewith T cells [37] suggesting different immunosuppressionmechanisms depending on the stimulation

Somehow these papers entail an IL-10 alternativeimmunosuppressive mechanism of action rather than acharacterizing feature of Breg This might hold true fordetermined Breg subsets and strengthens the idea of diverseBreg phenotypes depending on the environment

3 Potential Role of RegulatoryB Cells in Transplantation

Due to their central role as effector cells in the immuneresponse particularly in acute organ rejection T lympho-cytes have been one of the main targets of immunosup-pressive treatments B lymphocytes also participate in acute

rejection by infiltrating allografts and presenting alloantigensto T lymphocytes promoting the production of IFN-120574 IL-4 and IL-6 among others cytokines These cells are alsocapable of differentiating into plasma cells switching fromantigen presenting cells to antibody secretory cells that maytarget MHC class I and II molecules of the graftThis processoccurs latter in time and usually leads to chronic rejection[38] To hamper this process an anti-CD20 B cell deplet-ing monoclonal antibody rituximab has been introducedas immunosuppressive treatment for transplanted patientsDespite the fact that the use of this drug has increasedpatientsrsquo survival it fails to induce chronic unresponsivenessto the graft [39 40] One of the possible reasons underneathmay be that plasmablasts and plasma cells two key players inchronic rejection do not express CD20 on their cell surfaceAn additional explanation may be that Breg are also depletedby the treatment thus hampering their tolerogenic functionIn this sense some studies have shown that preserving the Bcell compartment favors OT in renal transplantation [41]

31 Breg as a Tolerance and Good Prognostic Biomarker inKidney Transplantation Seminal papers coled by US andUKconsortia (IOT RISET and ITN) [42 43] showed a similartransitional-Breg-related gene signature corresponding toimmunosuppressant-free spontaneous OT kidney transplantpatients Using microarray analysis and real time PCR theyidentified a B cell specific gene signature and different Bcell subpopulations distribution in OT patients comparedto SI patients after transplantation The signature proposedby Newell et al relating OT patients to HC but not to


their IS counterparts includes 30 genes most of them areencoding for the 120581120582 light chains of Ig In the cross-validationexperiments three of these genes were found to be themost predictive IGkV1D-13 IGLL1 and IGkV4-1 On theother hand flow cytometry analyses revealed an increasednumber of total and naıve B cells in OT with respect toSI patients Transitional B cells (defined by the group asCD19+CD24+CD38+IgD+) were also found to be increasedin tolerant patients and that was consistent in both ITN andIOT cohorts Since then several other groups have showedsimilar traits in their OT or SI patients [13 44] Ches-neau et al reported that tolerant patients showed a higherfrequency of transitional (defined as CD20+CD24hiCD38hi)and naıve (defined as CD20+CD24loCD38lo) B cells and ahigher production of IL-10 compared to SI patients [45] Inline with this observation patients with chronic antibodymediated rejection after renal transplantation were found tohave less percentage and absolute numbers of transitional Bcells (defined as CD19+CD24hiCD38hi) when compared tothe group of SI patients [46]

A recently published update of the ITN study revealeda maintained gene signature among OT patients but sur-prisingly the gene set also increased over time in those SIpatients Flow cytometric analysis of the B cell populationshows a persistent increase in total naıve and transitional Bcell population in OT compared to SI patients [47]

Furthermore additional studies have compared transi-tionalBreg frequencies in OT SI HC and also chronicrejection patients Interestingly the last group shows lowlevels of transitional B cells comparable to the ones of SIpatients [13]

Drawing on the correlation between kidney transplanttolerance and regulatory B cells the prognostic value ofpretransplantation transitionalregulatory B cells and trans-plantation outcome has been approached [48 49] In aprospective study Shabir and colleagues show that onlyhigher transitional B cell frequencies before transplantationbut not regulatory T cells total B cells or memory B cellscorrelate with lower incidence of biopsy proven acute rejec-tion [48] Moreover patients lacking transitional B cells threemonths after transplantation are at higher risk of sufferingfrom both T cell and antibody mediated rejection [49]

Altogether these studies suggest a marked role of thetransitional B cell compartment in graft acceptance andtolerance achievement which implies that transitional B cellsand Breg are at least partially overlapping populations It stillremains unclear whether the tolerogenic effect is only createdby the ldquonaturalrdquo Breg present in the recipient or whether theycan be induced in any patient to generate a tolerance status

32 Current Immunosuppression Regime and Breg InductionSince Breg and transitional B cells have been acknowledged asa key cell type in the induction andmaintenance of toleranceseveral groups have studied the effect of different treatmentson these B cell compartments in the human setting

The study of the B cell subsets profile in patients underdifferent immunosuppressive regimes has been approached

by some groups The results reported so far have demon-strated that neither mTor nor Calcineurin inhibitors (CNI)induce transitional nor regulatory B cells [50ndash52] AlthoughmTor inhibitors have shownTreg inducing capacity this effectseems to be Breg independent Further the in vitro study ofthe effect of CNI revealed that it inhibits IL-10 expression ofB cells [50]

Other immunosuppressive agents such as the B celldepleting antibodies alemtuzumab (anti-CD52) and ritux-imab (anti-CD20) have also been tested in transplant patientsfor their capacity to induce Breg Alemtuzumab treatedpatients show a transient increase in transitional B cells alongwith a sustained increase in naıve B cells [53] Converselyrituximab has produced far more controversial results Whilea single prophylactic dose seemed to protect from develop-ing acute cellular rejection [54] and even induce a B cellrepopulation based on transitional B cells [55] a clinicaltrial using two doses of the same compound on days 0and 7 after transplantation had highly deleterious effectscausing excessive rates of acute cell rejection which forcedthe premature termination of the trial [56]These studiesmaysuggest that there is a window of time- and dose-dependenteffect of B cell depletion to induce regulatory or effector B cellsubsets in patients under these treatments

Finally next generation blockers of the B cell functionwhich are being approached in autoimmune diseases suchas belimumab (B cell activating factor (BAFF) blocker) oratacicept (transmembrane activator and CALM interactor(TACI) blocker affecting both BAFF and a proliferation-inducing ligand (APRIL)) will undoubtedly also have aneffect on the B cell profile of patients but to date there is noinformation on their effect on the Breg population

Beyond conventional immunosuppressive treatmentsother nonconventional approaches have also proven Breginduction potential Mesenchymal Stem Cells (MSC) ther-apy is one of the leading nonpharmacological therapies intransplantation Several clinical trials have approached theirtolerogenic potential and a few brought their attention toBreg induction Patients with refractory chronic graft versushost disease (cGvHD) present lower frequencies of total Bcells and CD5+IL-10+ B However after three months of MSCtreatment patients showed improvement of their symptomscorrelating with increased CD5+IL-10+ B cells Of note theplasmatic levels of IL-10 were also higher after the treatmentin these patients [57] In another phase II multicenter clinicaltrial lymphocyte subsets were analyzed in patients infusedseveral times with umbilical cord-derived MSC to treatcGvHD Although there were no differences between thecontrol and the treated groups regarding B cell numbers thenumber of particular CD27+ B cells was higher in the treatedgroup after some months of MSC infusions and the clinicalsymptoms improved [58]

An additional way to induce IL-10 and functional Bregmay rely on helminths infections [59] Individuals infectedwith Schistosoma haematobium have higher percentage of IL-10 producing B cells that are able to induce Treg and IL-10 production by T cells in coculture Moreover helminthicinfection of MS patients has shown therapeutic potentialsince those patients that were infected presented less clinical


symptoms compared to noninfected MS subjects [60] Theauthors determined that B cells from helminths-infected MSpatients produced more IL-10 than noninfected MS patientsand that these IL-10 producing cells had a phenotype similarto naıve B2 cells

Altogether these studies show the potential of severalcompounds and therapeutic approaches to induce BregHowever the significance of the increase of this particular Bcell subset and their specific role in the progression of the dis-ease or the therapeutic effect still need to be fully determinedHence a proper knowledge on Breg is mandatory to monitorthe efficacy of the treatment as well as the tolerogenic statusof the patient

4 Breg as Cell Therapy

In view of the potential of Breg many efforts have beenmade trying to find out how to effectively induce Breg invivo and to deepen into the mechanisms of action underlyingBreg induction Table 1 summarizes the principal describedmethods in human samples This vast knowledge is ofparamount importance to get more insight into the potentialmechanisms and therapeutic targets to induce Breg in vivostrategies for ex vivo induction for forthcoming cell therapy-based approaches and purification of Breg for their furtherstudy and characterization

41 Purification of B Cells for Breg Induction The cell sourceto purify B cells and to produce Breg in vitro differs fromone lab to the other While most groups use peripheral bloodmononuclear cells as the main source of B cells due to theeasy accessibility of blood other sources such as lymph nodes(ie per indication from removed tonsils) or spleen (iediscarded organ from cadaveric organ donor) may also beimportant to get even larger numbers of B cells Althoughthe levels of expression of some surface markers could varybetween B cells from different compartments [67] little isknown about how this can affect the induction of Breg in invitro experiments

The purification of the B cells may also be approachedusing different methodologies Most laboratories use positiveselection with CD19 antibodies CD19 is expressed from theearly pro-B cell stage to the B cell lymphoblast stage butthe expression is downregulated upon B cell maturation toplasma cells Aiming at minimizing B cell activation inducedby CD19 ligation many other groups use CD19 negativeselection to purify B cells CD43 is expressed on activatedB cells plasma cells CD5+ B-1a cells and non-B cells thusresulting in a good marker to isolate untouched restingmature B cells Alternatively CD22 is expressed on the surfaceof mature B cells in peripheral blood but not on plasma cellsor early stages of B cell differentiation [68] resulting in theisolation of untouched CD19+ B cells

42 In Vitro Expansion of Breg As the only current definingcharacteristic of regulatory B cells is their capacity to secreteIL-10 induction of Breg from B cells is usually measuredbased on the proportion of IL-10-producing B cells Although

the intracellular pathways are not well known yet they seemto be inducible in differentways Ligation ofCD40 B cell anti-gen receptor (BCR) andor toll-like receptors (TLR) togetherwith IL-2 or IL-4 are the most used stimulating factorsHowever a consensus regime to induce IL-10 producing Bregis still to be defined Decreased expression of TLR9 due topolymorphisms in the tlr9 gene can increase predispositionto SLE in humans [69] which suggests that this is a keyfactor in Breg induction TLR9 ligation to induce Breg canbe achieved basically with CpG type B (generally 2006)Lipopolysaccharide (LPS) which ligates to TLR4 is morefrequently used in mice since human B cells express very lowlevels of this receptor in physiological conditions Howeverstimulation with anti-IgM CD40L and IL-4 can increaseTLR4 expression by humanB cells [70]Thismay explainwhyIwata et al [15] found that both LPS and CpG induced IL-10+B cells and CD40 ligation enhanced this effect

Also APRIL has been demonstrated to promote thegeneration of IL-10 producing B cells via STAT3 induction[66] Compared to naıve B cells from peripheral blood naıveB cells from cord blood seem to have a higher capacity toproduce IL-10 after stimulation which could be related toa higher level of pSTAT3 after CD40 stimulation [65] Thisobservation reinforces the importance of the source of B cellsfor ex vivo expansion

Another strategy to induce Breg ex vivo is the use ofMSCOur group has recently demonstrated that MSC support Bcell survival and have a direct effect on their differentiationWhen B cells derived from tonsils were stimulated with BCRplus CD40 ligation in the presence of IL-2 plasmablasts wereinduced But when B cells in the same setting were cocul-tured with MSC derived from adipose tissue plasmablastformation was abrogated and Breg (CD19+CD24hiCD38hiIL-10-producing B cells) were induced [63] Similar results wereobtained using B cells fromblood as when theywere culturedtogether MSC promoted the survival and proliferation ofB cells and increased the CD5+ B cell subset which hasalso been described to have immunoregulatory capacityEven though the mechanisms underlying these effects areunknown in the same study they showed that inhibition ofthe IDO pathway partially reduced the effect of MSC on Bcells while blockade of COX-2Prostaglandin-E2 pathwayIL-6 or IL-10 did not have any effect [57]

43 Breg Based Cell Therapy Based on the studies summa-rized in this review and also in other studies that have notbeen mentioned due to space limitation it is clear that Bregmay be envisaged as an additional approach for promotingtolerance in several pathologic situations

Cell therapy is not a new concept anymore and even inthe solid organ transplantation field protocols and clinicaltrials are being set up to promote tolerance in the absenceor in a minimized immunosuppressive regime MSC therapyhas taken the lead in this area with several trials done andpublished in kidney liver and bone marrow transplantationIn parallel regulatory immune cell types such as regulatoryT cells tolerogenic DCs or regulatory macrophages are themain immune cell types being studied and used for cell


Table1Summaryof

ther

ecently

used

Breg

indu

ctionmetho

dsin

human

invitro

Isolationlowast

Stim

ulation

Characteriz

ationof

indu

cedBreg

Immun

osup

pressiv

eeffect

(mechanism

ofactio

n)Re

ference

CD19

positives

election

CpG(10u

gmL)

+CD

40L(1ugm

L)for

24ho

urs

CD24

hiCD

27+IL-10+

Inhibitio

nof

TNF-120572prod

uctio

nby

antig

en-specific

CD4+

Tcells

andby

mon

ocytes

Both

CD24

hiCD

27+Bcelland

CD24

lowCD

27minusBcellinhibitT

NF-120572

prod

uctio

nby

CD4+

Tcellequally

(IL-10

independ

entp

athw

ay)

[15]

CD22

positives

electio

nCp

G(05120583M)+

BAFF

(20120583

M)+

rHIL-4

(50U

mL)

for2

4ho

urs

IL-10+IL-6+

Increasedexpressio

nof

TLR9

mdash[61]

CD19

negativ

esele

ction

Anti-IgA

+IgG

+IgM

(65120583gmL)

+IL-21

(50n

gmL)

for16ho

urs

CD38+CD

1d+IgM+CD

147+IL-10+CD

25+ID

O+

Supp

ressionof

autologous

CD4+

Tcell

proliferatio

n(G

zmBmediated)

[36]

CD19

negativ

esele

ction

CpG(1120583M)+

PPDfor4

8ho

urs

CD73minusCD

25+CD

71+IL-10+

Enric

hedinC

D5+IgM+IgDminus

IL-10+CD

27+have

lessC

D27

MFI

than

IL-10minusCD

27+

Supp

ressionof

autologous

PPD-stim

ulated

CD4+

Tcell

proliferatio

n(IL-10

mediated)

[14]

CD19

negativ

esele

ction

Ultra-CD

40L(5solutio

n)and

200I

Um

Lof

IL-4

for5

days

Increasedexpressio

nlevelsof

CD39

Dow

nregulated

CD73

Supp

ressionof

autologous

CD4+

and

CD8+

Tcellproliferatio

n(likelyto

bepartially

mediatedby

ADOand51015840-AMPprod

uctio

nby

Bcell)

[62]

CD19

positives

election

IL-2

(10n

gmL)

+IL-6

(10n

gmL)

+Cp

G2006

(1120583gmL)

+IFN-120572

orIFN-120573

(1000

Um

L)for4

days

CD27

int CD38+plasmablasts

from

naıvea

ndim

matureB

cells

mdash[23]

PBMCor

CD43

negativ

eselection

LPS(10120583

gmL)

+IL-4

(10n

gmL)

+rH

IL-35sin

gle-chainfusio

nprotein

(100

ngm

L)for4

days

IL-10+

mdash[35]

CD19

positives

election

Irradiated

CDw32Lexpressin

ganti-CD

40mAb

CD11bminusCD

5minusCD

27minusIgD+CD

1dhiIL-10+

Supp

ressionof

autologous

Tcell

proliferatio

nandIFN-120574

prod

uctio

n[60]


Table1Con

tinued

Isolationlowast

Stim

ulation

Characteriz

ationof

indu

cedBreg

Immun

osup

pressiv

eeffect

(mechanism

ofactio

n)Re

ference

CD19

negativ

esele

ction

CpG(3120583gmL)

+anti-IgA+IgG+IgM

(10120583

gmL)

for4

8h

CD11c+IL-10+

Enric

hedin

CD38+CD

27minusGCs

Depletedin

CD38+CD

27+PC

smdash

[16]

CD19

negativ

esele

ction

CpG(10120583

gmL)

+rH

CD40

Lfor4

8ho

urs

CD5+CD

27+CD

138+CD

38+IgDminusGzm

B+Inhibitio

nof

proliferatio

nof

autologous

CD4+CD

25minusTcell

(Gzm

Bmediated)

[12]

CD19

negativ

esele

ction

CpG(025120583

M)for

4days

mdash

Effectson

CD4+

autologous

Tcell

Supp

ressionof

proliferatio

nand

differentiatio

ninto

Th1cell

Redu

ctionof

TNF-120572andIFN-120574

prod

uctio

nIndu

ctionof

Foxp3+CD

4+TcellTr1

cellandTh

3cell

Upregulationof

CTLA

-4(IL-10T

GF-120573IDOand

CTLA

-4mediated)

[37]

CD43

negativ

esele

ction

from

tonsils

Adiposetissue

deriv

edMSC

(ratio

BcellM

SC5

1)+anti-IgM

(10120583

gmL)

+IL-2

(103

IU)+

anti-CD

40agon

isticmAb

(5120583gmL)

for10days

CD19+CD

27minusCD

38hiCD

24hiIL10+

mdash[63]

CD19

positives

election

CD40

L(2mgmL)

+IL-4

(200

IUm

L)for4

days

CD24

hiCD

38hiCD

25+CD

39hiCD

73hi

DecreaseinCD

69expressio

nand

proliferatio

nIncrease

ofIL-6

secretionfro

mautologous

Teffector

CD4+(A

DO-A2A

Rmechanism

)

[64]

CD19

positives

election

from

cord

bloo

dIrradiated

CD40

Ltransfe

cted

Lcells

+Cp

Gandanti-BC

Rfor4

days

IL10+foun

deither

inCD

24hiCD

38hior

CD24

int CD38

int

Supp

ressionof

proliferatio

nof

allogeneicCD

4+Tcell

Supp

ressionof

secretionof

IFN-120574

TNF-120572and

IL-2

byallogeneicCD

4+T

cell

[65]

PBMC

50cond

ition

edmediawith

APR

ILfor3

days

+Cp

G(10120583

gmL)

fora

ddition

al24

hours

IL-10+

Decreaseo

fIFN

-120574andTN

F-120572

prod

uctio

nfro

mautologous

CD4+

Tcell

[66]

PBMC

perip

heralb

lood

mon

onuclear

cellsrHrecom

binant

humanP

PDp

urified

proteinderiv

ativeADOadeno

sineCT

LA-4C

ytotoxicT-Lymph

ocyteAntigen

4A2A

RA2A

receptorlowast

Unlesso

therwise

indicatedBcells

wereisolatedfro

mPB

MC


therapy in human organ transplantation The ONE studya cooperative project that aims at developing immunoregu-latory cell therapies for organ transplanted patients [71] isthe paradigm since they compile and share the knowledgeamong the research groups devoted to that field Howeverto this moment there are no trials on the use of Breg as acell therapy The incomplete knowledge on Breg inductionstability and functional potential and the lack of a consensusBreg signature are just some of the hurdles to be bypassed togenerate a safe and efficient cell productWemight be dealingwith different subsets of Breg depending on the inductioncocktail and system used that might present different stabilityand functionality

Since most of the induction systems used at this momentpromote activation of B cells Fillatreau and colleagues pro-pose a method to induce IL-10 expression on resting B cellsto generate tolerogenic B cells which are poor immunogenicand present a lower potential risk of switching into effector Bcell [72] Another matter of concern is the antigen specificityof Breg In contrast to dendritic cells B cells cannot phagocytean antigen to present it on their surfaces but instead itneeds to be recognized by specific BCR internalized andpresented inMHC-II [73] It is unknownwhether this antigenis inducing specific tolerance but if this was the case it wouldbe necessary to find out how to generate antigen-specificBreg New technical advances in nanosciences might bringnew opportunities into that area

The effect that donor or recipient-derived Breg could havein modulating the immune reaction remains unknown if weenvision a therapy in the field of organ transplantation orthe effect of autologous or allogeneic Breg in autoimmunediseases Identifying the mechanism of action by whichone and not the other could induce allograft tolerance canshed light on the role of direct and indirect pathway ofantigen presentation and tolerance induction Moreover theage of the patient is a relevant factor in the capacity ofregulatory B cells to produce IL-10 since it is impaired inCD38hiCD24hi B cells from old individuals independentlyof the stimulating factor used (CD40L phorbol 12-myristate13-acetate (PMA)ionomycin or CpG) [74] This might bea major problem if we think of autologous cell treatmentThere are other mechanistic issues that would have to beaddressed such as the time needed to produce enough Breginfusion timing and dosage route of administration andGMP compliance

In addition to all the above mentioned issues we arefacing an added difficulty in the development of such atherapeutic strategy the model In mice models multiplestudies show that Breg can induce Treg and are capable oftransferring tolerance in allogeneic cardiac allograft [75]islet allograft [76] and arthritis [77] models These studiespoint out the central role of IL-10 in the modulation of theimmune response Also another study with islet allograft fullMCH-mismatched model suggested that Treg induction byBreg could be mediated through TGF-120573 [78] In a rat car-diac allograft model transferred Breg from tolerant animalsmigrated to the graft where they maintained their regulatory

capacity [79] Back to mice T cell Ig mucin protein-1 (TIM-1) also seems to be a key molecule for Breg since TIM-1+ B cell subset is highly enriched for IL-10 producingcells and the secretion of IL-10 increases substantially afterTIM-1 ligation [80] Nevertheless it is proven that murineand human Breg are essentially different and that their exvivo induction involves different mechanisms and molecularpathways In this scenario we will have to rely on human cellculture approaches and humanizedmousemodels in order todevelop a therapeutic strategy

Besides the generation of therapeutic protocols to induceBreg in vivo and taking into account the achievements in invitro expansion of Breg one can envision in the near future acell therapy approach using Breg to promote tolerance

5 Conclusions

Regulatory B cells are one of the newest members of theregulatory immune cells family Many researchers in the fieldof transplantation and autoimmune diseases have turnedtheir attention to this cell type for their implication inmaintaining homeostasis and achieving a tolerant state

While patients suffering from autoimmune diseases suchas SLE or MS have shown nonfunctional Breg populations[9 81] in the transplantation field higher pretransplantationBreg numbers have been associated with lower antibodymediated rejection in kidney transplant recipients [48]Further in kidney transplantation Breg have become highlyinteresting due to their association with tolerance [12 42 43]However it still remains elusive whether the increase in Bregis cause or consequence of the tolerance status [82]

Despite the increasing number of papers published aboutBreg one of the main hurdles in their study is the absenceof a Breg signature and the fact that murine Breg aresubstantially different to the human ones is hindering thisendeavor Until now the human Breg signature has beenmainly resolved by the use of transitional B cell phenotypeandor the ability to secrete IL-10 but other extracellularmarkers and released cytokines have been associated withthis cell type The variability of the Breg signature might bedue to different Breg phenotypes depending on the diseasethe activation milieu or the cell origin Unraveling a properset of markers that identify this regulatory subset will help inthe monitoring of patients and will bring new light into theirrelation with the immune homeostasis

In any case functional Breg seem to be directly involvedin graft tolerance The generation of a Breg in vitro might bethe key point to regain the lost tolerance status opening newdoors to the development of innovative therapies

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Acknowledgments

This work was supported in part by Grants from the SGRprogramme of ldquoGeneralitat de Catalunyardquo (2014SGR804


Group REMAR) ISCIII-REDinREN (160009 Feder Funds)and Fundacio Cellex Francesc E Borras is sponsored bythe ldquoResearchers Stabilization Programrdquo from the SpanishldquoSistema Nacional de Saludrdquo (SNS-ISCIII) and ldquoDirecciodrsquoEstrategia i Coordinaciordquo Catalan Health Department(CES07015) Marcella Franquesa is in receipt of a Beatriude Pinos-B contract (2014BP B00118) from the ldquoAgencia deGestio drsquoAjuts Universitaris i de Recercardquo (AGAUR) from theCatalan Government

References

[1] R F Saidi and S K Hejazii Kenari ldquoClinical transplantationand tolerance are we there yetrdquo International Journal of OrganTransplantation Medicine vol 5 no 4 pp 137ndash145 2014

[2] M M Lopez J E Valenzuela F C Alvarez M R Lopez-Alvarez G S Cecilia and P P Paricio ldquoLong-term problemsrelated to immunosuppressionrdquoTransplant Immunology vol 17no 1 pp 31ndash35 2006

[3] J Sellares D G De Freitas M Mengel et al ldquoUnderstandingthe causes of kidney transplant failure the dominant roleof antibody-mediated rejection and nonadherencerdquo AmericanJournal of Transplantation vol 12 no 2 pp 388ndash399 2012

[4] G Roussey-Kesler M Giral A Moreau et al ldquoClinical opera-tional tolerance after kidney transplantationrdquoAmerican Journalof Transplantation vol 6 no 4 pp 736ndash746 2006

[5] A Massart A Pallier J Pascual et al ldquoThe DESCARTES-Nantes survey of kidney transplant recipients displaying clinicaloperational tolerance identifies 35 new tolerant patients and 34almost tolerant patientsrdquo Nephrology Dialysis Transplantationvol 31 no 6 pp 1002ndash1013 2016

[6] A Nouel Q Simon C Jamin J-O Pers and S HillionldquoRegulatory B cells an exciting target for future therapeutics intransplantationrdquo Frontiers in Immunology vol 5 pp 1ndash8 2014

[7] R Neta and S B Salvin ldquoSpecific suppression of delayedhypersensitivity the possible presence of a suppressor B cellin the regulation of delayed hypersensitivityrdquo The Journal ofImmunology vol 113 no 6 pp 1716ndash1725 1974

[8] E Mizoguchi A Mizoguchi F I Preffer and A K BhanldquoRegulatory role of mature B cells in a murine model ofinflammatory bowel diseaserdquo International Immunology vol 12no 5 pp 597ndash605 2000

[9] P A Blair L Y Norena F Flores-Borja et alldquoCD19+CD24hiCD38hi B cells exhibit regulatory capacityin healthy individuals but are functionally impaired in systemiclupus erythematosus patientsrdquo Immunity vol 32 no 1 pp129ndash140 2010

[10] L Ma B Liu Z Jiang and Y Jiang ldquoReduced numbers ofregulatory B cells are negatively correlated with disease activ-ity in patients with new-onset rheumatoid arthritisrdquo ClinicalRheumatology vol 33 no 2 pp 187ndash195 2014

[11] L T Aybar J G Mcgregor S L Hogan et al ldquoReducedCD5+CD24hiCD38hiand interleukin-10+ regulatory B cells inactive anti-neutrophil cytoplasmic autoantibody-associatedvasculitis permit increased circulating autoantibodiesrdquo Clinicaland Experimental Immunology vol 180 no 2 pp 178ndash188 2015

[12] M Chesneau L Michel E Dugast et al ldquoTolerant kidneytransplant patients produce B cells with regulatory propertiesrdquoJournal of the American Society of Nephrology vol 26 no 10 pp2588ndash2598 2015

[13] A Cherukuri D M Rothstein B Clark et al ldquoImmunologichuman renal allograft injury associates with an altered IL-10TNF-120572 expression ratio in regulatory B cellsrdquo Journal of theAmerican Society of Nephrology vol 25 no 7 pp 1575ndash15852014

[14] W Van De Veen B Stanic G Yaman et al ldquoIgG4 productionis confined to human IL-10-producing regulatory B cells thatsuppress antigen-specific immune responsesrdquo Journal of Allergyand Clinical Immunology vol 131 no 4 pp 1204ndash1212 2013

[15] Y Iwata T Matsushita M Horikawa et al ldquoCharacterizationof a rare IL-10-competent B-cell subset in humans that parallelsmouse regulatory B10 cellsrdquo Blood vol 117 no 2 pp 530ndash5412011

[16] W Lin D Cerny E Chua et al ldquoHuman regulatory B cellscombine phenotypic and genetic hallmarks with a distinctdifferentiation faterdquo The Journal of Immunology vol 193 no 5pp 2258ndash2266 2014

[17] Q Simon J-O Pers D Cornec L Le Pottier R AMageed andS Hillion ldquoIn-depth characterization of CD24highCD38hightransitional human B reveals different regulatory profilesrdquoJournal of Allergy and Clinical Immunology vol 137 no 5 pp1577e10ndash1584e10 2016

[18] I Kalampokis A Yoshizaki and T F Tedder ldquoIL-10-producingregulatory B cells (B10 cells) in autoimmune diseaserdquo ArthritisResearch andTherapy vol 15 no 1 article S1 2013

[19] R Maddaly G Pai S Balaji et al ldquoReceptors and signal-ing mechanisms for B-lymphocyte activation proliferationand differentiationmdashinsights from both in vivo and in vitroapproachesrdquo FEBS Letters vol 584 no 24 pp 4883ndash4894 2010

[20] C Mauri and M Menon ldquoThe expanding family of regulatoryB cellsrdquo International Immunology vol 27 no 10 pp 479ndash4862015

[21] K Yanaba J-D Bouaziz K M Haas J C Poe M Fujimotoand T F Tedder ldquoA regulatory B cell subset with a uniqueCD1dhiCD5+ phenotype controls T cell-dependent inflamma-tory responsesrdquo Immunity vol 28 no 5 pp 639ndash650 2008

[22] L E PM van der Vlugt EMlejnek A Ozir-Fazalalikhan et alldquoCD24(hi)CD27(+) B cells from patients with allergic asthmahave impaired regulatory activity in response to lipopolysaccha-riderdquoClinical amp Experimental Allergy vol 44 no 4 pp 517ndash5282014

[23] M Matsumoto A Baba T Yokota et al ldquoInterleukin-10-producing plasmablasts exert regulatory function in autoim-mune inflammationrdquo Immunity vol 41 no 6 pp 1040ndash10512014

[24] F Flores-Borja A Bosma D Ng et al ldquoCD19+CD24hiCD38hi Bcells maintain regulatory T cells while limiting TH1 and TH17differentiationrdquo Science Translational Medicine vol 5 no 173Article ID 173ra23 2013

[25] M Gray and D Gray ldquoRegulatory B cells mediate tolerance toapoptotic self in health implications for diseaserdquo InternationalImmunology vol 27 no 10 pp 505ndash511 2015

[26] J M Lykken K M Candando and T F Tedder ldquoRegulatoryB10 cell development and functionrdquo International Immunologyvol 27 no 10 pp 471ndash477 2015

[27] E C Rosser and C Mauri ldquoRegulatory B cells origin pheno-type and functionrdquo Immunity vol 42 no 4 pp 607ndash612 2015

[28] L Gabrysova A Howes M Saraiva and A OrsquoGarra ldquoTheregulation of IL-10 expressionrdquo Current Topics in Microbiologyand Immunology vol 380 pp 157ndash190 2014


[29] K R Engelhardt and B Grimbacher ldquoIL-10 in humans lessonsfrom the Gut IL-10IL-10 receptor deficiencies and IL-10 poly-morphismsrdquo Current Topics in Microbiology and Immunologyvol 380 pp 1ndash18 2014

[30] M Matsumoto Y Fujii A Baba M Hikida T Kurosaki andY Baba ldquoThe calcium sensors STIM1 and STIM2 control Bcell regulatory function through interleukin-10 productionrdquoImmunity vol 34 no 5 pp 703ndash714 2011

[31] K W Moore R De Waal Malefyt R L Coffman andA OrsquoGarra ldquoInterleukin-10 and the interleukin-10 receptorrdquoAnnual Review of Immunology vol 19 pp 683ndash765 2001

[32] M K Mann K Maresz L P Shriver Y Tan and B N DittelldquoB cell regulation of CD4+CD25+ T regulatory cells and IL-10via B7 is essential for recovery from experimental autoimmuneencephalomyelitisrdquo Journal of Immunology vol 178 no 6 pp3447ndash3456 2007

[33] A Taylor M Akdis A Joss et al ldquoIL-10 inhibits CD28 andICOS costimulations of T cells via src homology 2 domainmdashcontaining protein tyrosine phosphatase 1rdquo The Journal ofAllergy and Clinical Immunology vol 120 no 1 pp 76ndash83 2007

[34] M Duddy M Niino F Adatia et al ldquoDistinct effector cytokineprofiles of memory and naive human B cell subsets andimplication in multiple sclerosisrdquo Journal of Immunology vol178 no 10 pp 6092ndash6099 2007

[35] R-X Wang C-R Yu I M Dambuza et al ldquoInterleukin-35induces regulatory B cells that suppress autoimmune diseaserdquoNature Medicine vol 20 no 6 pp 633ndash641 2014

[36] S Lindner K Dahlke K Sontheimer et al ldquoInterleukin 21-induced granzyme b-expressing b cells infiltrate tumors andregulate T cellsrdquo Cancer Research vol 73 no 8 pp 2468ndash24792013

[37] A Nouel P Pochard Q Simon et al ldquoB-Cells induce regulatoryT cells through TGF-120573IDO production in A CTLA-4 depen-dentmannerrdquo Journal of Autoimmunity vol 59 pp 53ndash60 2015

[38] A Haririan B Kiangkitiwan D Kukuruga et al ldquoThe impactof C4d pattern and donor-specific antibody on graft survival inrecipients requiring indication renal allograft biopsyrdquoAmericanJournal of Transplantation vol 9 no 12 pp 2758ndash2767 2009

[39] R Grenda W Jarmuzek J Rubik B Piątosa and S ProkuratldquoRituximab is not a lsquomagic drugrsquo in post-transplant recurrenceof nephrotic syndromerdquo European Journal of Pediatrics vol 175no 9 pp 1133ndash1137 2016

[40] M Rudnicki ldquoFSGS recurrence in adults after renal transplan-tationrdquo BioMed Research International vol 2016 Article ID3295618 7 pages 2016

[41] H M Silva M C S Takenaka P M M Moraes-Vieira etal ldquoPreserving the B-cell compartment favors operationaltolerance in human renal transplantationrdquoMolecular Medicinevol 18 no 5 pp 733ndash743 2012

[42] P Sagoo E Perucha B Sawitzki et al ldquoDevelopment of across-platform biomarker signature to detect renal transplanttolerance in humansrdquo The Journal of Clinical Investigation vol120 no 6 pp 1848ndash1861 2010

[43] K ANewell A Asare A D Kirk et al ldquoIdentification of a B cellsignature associated with renal transplant tolerance in humansrdquoJournal of Clinical Investigation vol 120 no 6 pp 1836ndash18472010

[44] D Baron G Ramstein M Chesneau et al ldquoA commongene signature across multiple studies relate biomarkers andfunctional regulation in tolerance to renal allograftrdquo KidneyInternational vol 87 no 5 pp 984ndash995 2015

[45] M Chesneau A Pallier F Braza et al ldquoUnique B cell differen-tiation profile in tolerant kidney transplant patientsrdquo AmericanJournal of Transplantation vol 14 no 1 pp 144ndash155 2014

[46] A Nouel I Segalen C Jamin et al ldquoB cells display anabnormal distribution and an impaired suppressive functionin patients with chronic antibody-mediated rejectionrdquo KidneyInternational vol 85 no 3 pp 590ndash599 2014

[47] K A Newell A Asare I Sanz et al ldquoLongitudinal studiesof a B cell-derived signature of tolerance in renal transplantrecipientsrdquo American Journal of Transplantation vol 15 no 11pp 2908ndash2920 2015

[48] S Shabir J Girdlestone D Briggs et al ldquoTransitional B lym-phocytes are associated with protection from kidney allograftrejection a prospective studyrdquo American Journal of Transplan-tation vol 15 no 5 pp 1384ndash1391 2015

[49] V Svachova A Sekerkova P Hruba et al ldquoDynamic changesof B-cell compartments in kidney transplantation lack of tran-sitional B cells is associated with allograft rejectionrdquo TransplantInternational vol 29 no 5 pp 540ndash548 2016

[50] B Tebbe B Wilde Z Ye et al ldquoRenal transplant recipientstreated with calcineurin-inhibitors lack circulating immaturetransitional CD19+CD24ℎ119894CD38ℎ119894 regulatory B-lymphocytesrdquoPLoS ONE vol 11 no 4 Article ID e0153170 2016

[51] I Latorre A Esteve-Sole D Redondo et al ldquoCalcineurinand mTOR inhibitors have opposing effects on regulatory Tcells while reducing regulatory B cell populations in kidneytransplant recipientsrdquo Transplant Immunology vol 35 pp 1ndash62016

[52] N Gong Z Chen J Wang et al ldquoImmunoregulatory effectsof sirolimus vs tacrolimus treatment in kidney allograft recipi-entsrdquo Cellular Immunology vol 297 no 2 pp 87ndash93 2015

[53] S Heidt and K J Wood ldquoBiomarkers of operational tolerancein solid organ transplantationrdquo Expert Opinion on MedicalDiagnostics vol 6 no 4 pp 281ndash293 2012

[54] G Tyden H Genberg J Tollemar et al ldquoA randomizeddoubleblind placebo-controlled study of single-dose rituximabas induction in renal transplantationrdquo Transplantation vol 87no 9 pp 1325ndash1329 2009

[55] E G Kamburova H J PM KoenenMW F VanDenHoogenM C Baas I Joosten and L B Hilbrands ldquoLongitudinal anal-ysis of T and B cell phenotype and function in renal transplantrecipients with or without rituximab induction therapyrdquo PLoSONE vol 9 no 11 Article ID e112658 pp 1ndash8 2014

[56] M R Clatworthy C J E Watson G Plotnek et al ldquoB-cell-depleting induction therapy and acute cellular rejectionrdquo TheNew England Journal of Medicine vol 360 no 25 pp 2683ndash2685 2009

[57] Y Peng X Chen Q Liu et al ldquoMesenchymal stromal cellsinfusions improve refractory chronic graft versus host diseasethrough an increase of CD5+ regulatory B cells producinginterleukin 10rdquo Leukemia vol 29 no 3 pp 636ndash646 2015

[58] L Gao Y Zhang B Hu et al ldquoPhase II multicenter ran-domized double-blind controlled study of efficacy and safetyof umbilical cord-derived mesenchymal stromal cells in theprophylaxis of chronic graft-versus-host disease after HLA-haploidentical stem-cell transplantationrdquo Journal of ClinicalOncology vol 65 pp 1ndash8 2016

[59] L Hussaarts L E P M Van Der Vlugt M Yazdanbakhshand H H Smits ldquoRegulatory B-cell induction by helminthsimplications for allergic diseaserdquo Journal of Allergy and ClinicalImmunology vol 128 no 4 pp 733ndash739 2011


[60] J Correale and T R Equiza ldquoRegulatory B cells helminths andmultiple sclerosisrdquo Methods in Molecular Biology vol 1190 pp257ndash269 2014

[61] D Yehudai A Snir R Peri et al ldquoB cell-activating factorenhances interleukin-6 and interleukin-10 production byODN-activated human B cellsrdquo Scandinavian Journal of Immunologyvol 76 no 4 pp 371ndash377 2012

[62] Z Saze P J Schuler C-S Hong D Cheng E K Jackson andT L Whiteside ldquoAdenosine production by human B cells and Bcell-mediated suppression of activated T cellsrdquo Blood vol 122no 1 pp 9ndash18 2013

[63] M F Franquesa F K Mensah R Huizinga et al ldquoHumanadipose tissue-derived mesenchymal stem cells abrogate plas-mablast formation and induce regulatory B cells independentlyof T helper cellsrdquo STEMCELLS vol 33 no 3 pp 880ndash891 2015

[64] F Figueiro L Muller S Funk E Jackson A Battastini andT Whiteside ldquoPhenotypic and functional characteristics ofCD39high human regulatory B cells (Breg)rdquo OncoImmunologyvol 5 no 2 Article ID e1082703 2016

[65] A Sarvaria R Basar R SMehta et al ldquoIL-10+ regulatory B cellsare enriched in cord blood and may protect against cGVHDafter cord blood transplantationrdquo Blood vol 128 no 10 pp1346ndash1361 2016

[66] C Hua R Audo N Yeremenko et al ldquoA proliferation inducingligand (APRIL) promotes IL-10 production and regulatoryfunctions of human B cellsrdquo Journal of Autoimmunity vol 73pp 64ndash72 2016

[67] F Medina C Segundo A Campos-Caro I Gonzalez-Garcıaand J A Brieva ldquoThe heterogeneity shown by human plasmacells from tonsil blood and bone marrow reveals graded stagesof increasing maturity but local profiles of adhesion moleculeexpressionrdquo Blood vol 99 no 6 pp 2154ndash2161 2002

[68] B Dorken G Moldenhauer A Pezzutto et al ldquoHD39 (B3)A B lineage-restricted antigen whose cell surface expression islimited to resting and activated human B lymphocytesrdquo Journalof Immunology vol 136 no 12 pp 4470ndash4479 1986

[69] K TaoM Fujii S-I Tsukumo et al ldquoGenetic variations of Toll-like receptor 9 predispose to systemic lupus erythematosus inJapanese populationrdquo Annals of the Rheumatic Diseases vol 66no 7 pp 905ndash909 2007

[70] L M Ganley-Leal Y Liang M Jagannathan-Bogdan F AFarraye and B S Nikolajczyk ldquoDifferential regulation ofTLR4 expression in human B cells and monocytesrdquo MolecularImmunology vol 48 no 1ndash3 pp 82ndash88 2010

[71] E K Geissler ldquoThe ONE Studyrdquo 2011 httpwwwonestudyorg

[72] E Calderon-Gomez and S Fillatreau ldquoUtilization of a lentiviralsystem for the generation of B cells with regulatory propertiesrdquoin Regulatory B Cells Methods and Protocols vol 1190 ofMethods in Molecular Biology pp 105ndash113 Springer BerlinGermany 2014

[73] D Rodrıguez-Pinto ldquoB cells as antigen presenting cellsrdquo Cellu-lar Immunology vol 238 no 2 pp 67ndash75 2005

[74] N A Duggal J Upton A C Phillips E Sapey and J MLord ldquoAn age-related numerical and functional deficit inCD19+CD24hiCD38hi B cells is associated with an increase insystemic autoimmunityrdquo Aging Cell vol 12 no 5 pp 873ndash8812013

[75] G Lal N Kulkarni Y Nakayama et al ldquoIL-10 from marginalzone precursor B cells controls the differentiation of Th17 Tfhand Tfr cells in transplantation tolerancerdquo Immunology Lettersvol 170 pp 52ndash63 2016

[76] K M Lee J I Kim R Stott et al ldquoAnti-CD45RBanti-TIM-1-induced tolerance requires regulatory B cellsrdquoAmerican Journalof Transplantation vol 12 no 8 pp 2072ndash2078 2012

[77] N A Carter R Vasconcellos E C Rosser et al ldquoMicelacking endogenous IL-10-producing regulatory B cells developexacerbated disease and present with an increased frequencyof Th1Th17 but a decrease in regulatory T cellsrdquo Journal ofImmunology vol 186 no 10 pp 5569ndash5579 2011

[78] K M Lee R T Stott G Zhao et al ldquoTGF-120573-producingregulatory B cells induce regulatory T cells and promotetransplantation tolerancerdquo European Journal of Immunologyvol 44 no 6 pp 1728ndash1736 2014

[79] J Durand V Huchet E Merieau et al ldquoRegulatory B cellswith a partial defect in CD40 signaling and overexpressinggranzyme B transfer allograft tolerance in rodentsrdquo Journal ofImmunology vol 195 no 10 pp 5035ndash5044 2015

[80] QGDingMYeungGCamirand et al ldquoRegulatoryB cells areidentified by expression of TIM-1 and can be induced throughTIM-1 ligation to promote tolerance in micerdquo The Journal ofClinical Investigation vol 121 no 9 pp 3645ndash3656 2011

[81] M Duddy M Niino F Adatia et al ldquoDistinct effector cytokineprofiles of memory and naive human B cell subsets andimplication in multiple sclerosisrdquo The Journal of Immunologyvol 178 no 10 pp 6092ndash6099 2007

[82] E Dugast M Chesneau J-P Soulillou and S BrouardldquoBiomarkers and possible mechanisms of operational tolerancein kidney transplant patientsrdquo Immunological Reviews vol 258no 1 pp 208ndash217 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


less discussion about their mechanism of action which isprincipally accepted to be IL-10 but the lack of knowledge onwhat triggers its secretion and the fact that other regulatorymechanisms have also been proposed leave this issue to dateunresolved

21 Does a ldquoUniquerdquo Breg Phenotype Exist As previouslyoccurred in the studies on regulatory T cells manyresearchers have prompted to identify a unique set ofmarkers transcription factors or mechanism of action thatexclusively identify Breg in all contexts In this sense geneticand surface expression studies have been conducted withpartial success to unravel a unique Breg signature [16 17]Unfortunately to date such unequivocal markers have notbeen found yet Also some hypothesis have been formulatedon Breg development pathways from a common precursor[18 19] but the results so far are not conclusive Thus mostauthors rely on the capacity to produce interleukin- (IL-) 10and on the two main phenotypical signatures used to defineBreg (1) transitional B cell phenotype CD19+CD24hiCD38hi

and (2) CD19+CD5+CD1dhi (used in both human and mice)[20 21] Nevertheless we still face a lack of specific Bregmarkers and different phenotypes for IL-10-producing Bcells with regulatory capacity have been proposed throughthe years In 2008 Yanaba and colleagues identified an IL-10-producing regulatory B cell subset in mice expressingCD1dhiCD5+ which they referred to as B10 cells [21] A fewyears later the same group characterized a similar IL-10-producing B cell subset in humans Human B10 cellsrsquo regula-tory potential was shown by their capacity to inhibit tumornecrosis factor- (TNF-) 120572 production by CD4+ T helpercells and monocytes In peripheral blood B10 cells werefound exclusively among CD24hiCD27+ cells whereas inspleen no difference was observed between IL-10-producingand nonproducing B cells regarding their surface markers[15] When testing the immunomodulatory capacity of thesame subset from patients with allergic asthma in vitrothese cells were less capable of secreting IL-10 and inducingthe secretion of IL-10 from CD4+ T compared to the samecell population from healthy subjects suggesting that thispopulation could hold immunomodulatory capability [22]Nevertheless Matsumoto et al found that CD27intCD38+immunoglobulin- (Ig-) secreting plasmablasts that arise fromnaıve and immature B cells from human blood are the majorIL-10-producing B cells after in vitro stimulation [23] Yet thetransitional B cell subset CD24hiCD38hi also seems to haveregulatory capacity since after CD40 stimulation they couldsuppress the differentiation of naıve T cells into T helper 1(Th1) andTh17 and lead CD4+CD25minus T cells conversion intoregulatory T cells (Treg) partially via IL-10 [9 24]

Due to the disparity of the results showing that differentB cell subsets can express immunomodulatory propertiesa current emerging view is that Breg are not a specific Bcell subset but rather a circumstantial B cell phenotype Inthis scenario B cells could acquire a regulatory role whenappropriate signals are generated in the environment as hasbeen already suggested by some authors [25ndash27] It wouldseem reasonable to think that depending on the type of

activated immune cells and cytokines released to the envi-ronment some B cells could shape their response towards theappropriate way to modulate the response of other immunecells The adaptability of the Breg response could explain thedifferent outcomes depending on the disease studied in vivoor the stimulation provided in vitro Therefore maybe thequest for a ldquouniquerdquo Breg marker must be reoriented to findthe right stimulation for B cells to become stable regulators ofthe immune response in a given scenario

22 IL-10 Secretion as Breg Mechanism of Action As men-tioned above IL-10 production is perhaps the principalhallmark to define regulatory B cells describing theirimmunomodulatory potential and explaining their mecha-nism of action IL-10 is a regulatory cytokine secreted byalmost all innate and adaptive immune cells that plays anessential role in maintaining immune homoeostasis [28] Itbinds as a homodimer to its receptor which is a tetramerformed of two 120572 (IL-10R1) and two 120573 (IL-10R2) chainsIL-10R1 binds to the cytokine while IL-10R2 is responsiblefor the downstream signaling activation through Jak1 andsignal transducer and activator of transcription 3 (STAT3)IL-10 is the only ligand for IL-10R1 which in turn is theunique receptor of IL-10 while IL-10R2 is shared by severalcytokines such as IL-20 IL-22 IL-24 IL-26 IL-28 and IL-29[29] Although little is known about the molecular pathwaysinvolved in IL-10 secretion in humans in mice it is medi-ated by store-operated Ca2+ influx from the endoplasmicreticulum which is further regulated by the calcium sensorsstromal interaction molecule (STIM) 1 and STIM2 [30]

Among other biological functions IL-10 promotes thedownregulation of antigen presentation by macrophages anddendritic cells and suppresses the production of proinflam-matory cytokines such as IL-1 interferon (IFN)-120574 and TNF-120572 by CD4+ T cells monocytes and macrophages [29 31](Figure 1)

Besides the functional relevance of IL-10 expression IL-10-producing CD19+CD24hiCD38hi B cells have also beenshown to promote the expansion of IL-10-producing FoxP3+Treg and to play a role in inducing their recruitment to the siteof inflammation [32] In addition human IL-10-producingB cells may block the CD28minus inducible T cell costimulator(ICOS) costimulatory pathway thus blocking T cell activa-tion via phosphorylation of Src homology region 2 domain-containing phosphatase-1 (SHP1) a downstream moleculeof the IL-10 receptor intracellular pathway [33] Moreoverthey regulate innate immune responses by reducing TNF-120572production by monocytes [15]

The key role of IL-10 released by B cells has been alsoproven in multiple sclerosis (MS) patients who have B cellswith impaired IL-10 production under CD40 stimulation[34] Similarly in SLE patients B cells fail to produce IL-10 in response to CD40 but not to CpG oligodeoxynu-cleotides (CpG) [9]These results indicate an impaired T cell-dependent Breg induction in both autoimmune diseases

23 IL-10 Independent Regulatory B Cells Not only IL-10 butalso other regulatory mechanisms like IL-35 [35] granzyme




darr IL-1

darr IL-6

IL-

IDOADO CTLA-4

10


darr ICOS

uarr proliferation

darr proliferation

darr proliferation

uarr recruitment

CD4+ T cell

CD4+ T cell




TGF-120573

GzmB

5998400-AMP

IFN-120572120573

uarr CD69

Th1

Th17

Treg


BregB cell




IL-2IL-4IL-6

LPSIL-21IL-35BAFF

APRILMSC



































Table1Summaryof

ther

ecently

used

Breg

indu

ctionmetho

dsin

human

invitro

Isolationlowast

Stim

ulation

Characteriz

ationof

indu

cedBreg

Immun

osup

pressiv

eeffect

(mechanism

ofactio

n)Re

ference

CD19

positives

election

CpG(10u

gmL)

+CD

40L(1ugm

L)for

24ho

urs

CD24

hiCD

27+IL-10+

Inhibitio

nof

TNF-120572prod

uctio

nby

antig

en-specific

CD4+

Tcells

andby

mon

ocytes

Both

CD24

hiCD

27+Bcelland

CD24

lowCD


NF-120572

prod

uctio

nby

CD4+

Tcellequally

(IL-10

independ

entp

athw

ay)

[15]

CD22

positives

electio

nCp

G(05120583M)+

BAFF

(20120583

M)+

rHIL-4

(50U

mL)

for2

4ho

urs

IL-10+IL-6+

Increasedexpressio

nof

TLR9

mdash[61]

CD19

negativ

esele

ction

Anti-IgA

+IgG

+IgM

(65120583gmL)

+IL-21

(50n

gmL)

for16ho

urs

CD38+CD

1d+IgM+CD

147+IL-10+CD

25+ID

O+

Supp

ressionof

autologous

CD4+

Tcell

proliferatio

n(G

zmBmediated)

[36]

CD19

negativ

esele

ction

CpG(1120583M)+

PPDfor4

8ho

urs

CD73minusCD

25+CD

71+IL-10+

Enric

hedinC

D5+IgM+IgDminus

IL-10+CD

27+have

lessC

D27

MFI

than

IL-10minusCD

27+

Supp

ressionof

autologous

PPD-stim

ulated

CD4+

Tcell

proliferatio

n(IL-10

mediated)

[14]

CD19

negativ

esele

ction

Ultra-CD

40L(5solutio

n)and

200I

Um

Lof

IL-4

for5

days

Increasedexpressio

nlevelsof

CD39

Dow

nregulated

CD73

Supp

ressionof

autologous

CD4+

and

CD8+

Tcellproliferatio

n(likelyto

bepartially

mediatedby


uctio

nby

Bcell)

[62]

CD19

positives

election

IL-2

(10n

gmL)

+IL-6

(10n

gmL)

+Cp

G2006

(1120583gmL)

+IFN-120572

orIFN-120573

(1000

Um

L)for4

days

CD27


from

naıvea

ndim

matureB

cells

mdash[23]

PBMCor

CD43

negativ

eselection

LPS(10120583

gmL)

+IL-4

(10n

gmL)

+rH

IL-35sin

gle-chainfusio

nprotein

(100

ngm

L)for4

days

IL-10+

mdash[35]

CD19

positives

election

Irradiated

CDw32Lexpressin

ganti-CD

40mAb

CD11bminusCD

5minusCD

27minusIgD+CD

1dhiIL-10+

Supp

ressionof

autologous

Tcell

proliferatio

nandIFN-120574

prod

uctio

n[60]


Table1Con

tinued

Isolationlowast

Stim

ulation

Characteriz

ationof

indu

cedBreg

Immun

osup

pressiv

eeffect

(mechanism

ofactio

n)Re

ference

CD19

negativ

esele

ction

CpG(3120583gmL)

+anti-IgA+IgG+IgM

(10120583

gmL)

for4

8h

CD11c+IL-10+

Enric

hedin

CD38+CD

27minusGCs

Depletedin

CD38+CD

27+PC

smdash

[16]

CD19

negativ

esele

ction

CpG(10120583

gmL)

+rH

CD40

Lfor4

8ho

urs

CD5+CD

27+CD

138+CD

38+IgDminusGzm

B+Inhibitio

nof

proliferatio

nof

autologous

CD4+CD

25minusTcell

(Gzm

Bmediated)

[12]

CD19

negativ

esele

ction

CpG(025120583

M)for

4days

mdash

Effectson

CD4+

autologous

Tcell

Supp

ressionof

proliferatio

nand

differentiatio

ninto

Th1cell

Redu

ctionof

TNF-120572andIFN-120574

prod

uctio

nIndu

ctionof

Foxp3+CD

4+TcellTr1

cellandTh

3cell

Upregulationof

CTLA

-4(IL-10T

GF-120573IDOand

CTLA

-4mediated)

[37]

CD43

negativ

esele

ction

from

tonsils

Adiposetissue

deriv

edMSC

(ratio

BcellM

SC5

1)+anti-IgM

(10120583

gmL)

+IL-2

(103

IU)+

anti-CD

40agon

isticmAb

(5120583gmL)

for10days

CD19+CD

27minusCD

38hiCD

24hiIL10+

mdash[63]

CD19

positives

election

CD40

L(2mgmL)

+IL-4

(200

IUm

L)for4

days

CD24

hiCD

38hiCD

25+CD

39hiCD

73hi

DecreaseinCD

69expressio

nand

proliferatio

nIncrease

ofIL-6

secretionfro

mautologous

Teffector

CD4+(A

DO-A2A

Rmechanism

)

[64]

CD19

positives

election

from

cord

bloo

dIrradiated

CD40

Ltransfe

cted

Lcells

+Cp

Gandanti-BC

Rfor4

days

IL10+foun

deither

inCD

24hiCD

38hior

CD24

int CD38

int

Supp

ressionof

proliferatio

nof

allogeneicCD

4+Tcell

Supp

ressionof

secretionof

IFN-120574

TNF-120572and

IL-2

byallogeneicCD

4+T

cell

[65]

PBMC

50cond

ition

edmediawith

APR

ILfor3

days

+Cp

G(10120583

gmL)

fora

ddition

al24

hours

IL-10+

Decreaseo

fIFN

-120574andTN

F-120572

prod

uctio

nfro

mautologous

CD4+

Tcell

[66]

PBMC

perip

heralb

lood

mon

onuclear

cellsrHrecom

binant

humanP

PDp

urified

proteinderiv

ativeADOadeno

sineCT

LA-4C

ytotoxicT-Lymph

ocyteAntigen

4A2A

RA2A

receptorlowast

Unlesso

therwise

indicatedBcells

wereisolatedfro

mPB

MC








5 Conclusions





Competing Interests


Acknowledgments




References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















darr IL-1

darr IL-6

IL-

IDOADO CTLA-4

10


darr ICOS

uarr proliferation

darr proliferation

darr proliferation

uarr recruitment

CD4+ T cell

CD4+ T cell




TGF-120573

GzmB

5998400-AMP

IFN-120572120573

uarr CD69

Th1

Th17

Treg


BregB cell




IL-2IL-4IL-6

LPSIL-21IL-35BAFF

APRILMSC



































Table1Summaryof

ther

ecently

used

Breg

indu

ctionmetho

dsin

human

invitro

Isolationlowast

Stim

ulation

Characteriz

ationof

indu

cedBreg

Immun

osup

pressiv

eeffect

(mechanism

ofactio

n)Re

ference

CD19

positives

election

CpG(10u

gmL)

+CD

40L(1ugm

L)for

24ho

urs

CD24

hiCD

27+IL-10+

Inhibitio

nof

TNF-120572prod

uctio

nby

antig

en-specific

CD4+

Tcells

andby

mon

ocytes

Both

CD24

hiCD

27+Bcelland

CD24

lowCD


NF-120572

prod

uctio

nby

CD4+

Tcellequally

(IL-10

independ

entp

athw

ay)

[15]

CD22

positives

electio

nCp

G(05120583M)+

BAFF

(20120583

M)+

rHIL-4

(50U

mL)

for2

4ho

urs

IL-10+IL-6+

Increasedexpressio

nof

TLR9

mdash[61]

CD19

negativ

esele

ction

Anti-IgA

+IgG

+IgM

(65120583gmL)

+IL-21

(50n

gmL)

for16ho

urs

CD38+CD

1d+IgM+CD

147+IL-10+CD

25+ID

O+

Supp

ressionof

autologous

CD4+

Tcell

proliferatio

n(G

zmBmediated)

[36]

CD19

negativ

esele

ction

CpG(1120583M)+

PPDfor4

8ho

urs

CD73minusCD

25+CD

71+IL-10+

Enric

hedinC

D5+IgM+IgDminus

IL-10+CD

27+have

lessC

D27

MFI

than

IL-10minusCD

27+

Supp

ressionof

autologous

PPD-stim

ulated

CD4+

Tcell

proliferatio

n(IL-10

mediated)

[14]

CD19

negativ

esele

ction

Ultra-CD

40L(5solutio

n)and

200I

Um

Lof

IL-4

for5

days

Increasedexpressio

nlevelsof

CD39

Dow

nregulated

CD73

Supp

ressionof

autologous

CD4+

and

CD8+

Tcellproliferatio

n(likelyto

bepartially

mediatedby


uctio

nby

Bcell)

[62]

CD19

positives

election

IL-2

(10n

gmL)

+IL-6

(10n

gmL)

+Cp

G2006

(1120583gmL)

+IFN-120572

orIFN-120573

(1000

Um

L)for4

days

CD27


from

naıvea

ndim

matureB

cells

mdash[23]

PBMCor

CD43

negativ

eselection

LPS(10120583

gmL)

+IL-4

(10n

gmL)

+rH

IL-35sin

gle-chainfusio

nprotein

(100

ngm

L)for4

days

IL-10+

mdash[35]

CD19

positives

election

Irradiated

CDw32Lexpressin

ganti-CD

40mAb

CD11bminusCD

5minusCD

27minusIgD+CD

1dhiIL-10+

Supp

ressionof

autologous

Tcell

proliferatio

nandIFN-120574

prod

uctio

n[60]


Table1Con

tinued

Isolationlowast

Stim

ulation

Characteriz

ationof

indu

cedBreg

Immun

osup

pressiv

eeffect

(mechanism

ofactio

n)Re

ference

CD19

negativ

esele

ction

CpG(3120583gmL)

+anti-IgA+IgG+IgM

(10120583

gmL)

for4

8h

CD11c+IL-10+

Enric

hedin

CD38+CD

27minusGCs

Depletedin

CD38+CD

27+PC

smdash

[16]

CD19

negativ

esele

ction

CpG(10120583

gmL)

+rH

CD40

Lfor4

8ho

urs

CD5+CD

27+CD

138+CD

38+IgDminusGzm

B+Inhibitio

nof

proliferatio

nof

autologous

CD4+CD

25minusTcell

(Gzm

Bmediated)

[12]

CD19

negativ

esele

ction

CpG(025120583

M)for

4days

mdash

Effectson

CD4+

autologous

Tcell

Supp

ressionof

proliferatio

nand

differentiatio

ninto

Th1cell

Redu

ctionof

TNF-120572andIFN-120574

prod

uctio

nIndu

ctionof

Foxp3+CD

4+TcellTr1

cellandTh

3cell

Upregulationof

CTLA

-4(IL-10T

GF-120573IDOand

CTLA

-4mediated)

[37]

CD43

negativ

esele

ction

from

tonsils

Adiposetissue

deriv

edMSC

(ratio

BcellM

SC5

1)+anti-IgM

(10120583

gmL)

+IL-2

(103

IU)+

anti-CD

40agon

isticmAb

(5120583gmL)

for10days

CD19+CD

27minusCD

38hiCD

24hiIL10+

mdash[63]

CD19

positives

election

CD40

L(2mgmL)

+IL-4

(200

IUm

L)for4

days

CD24

hiCD

38hiCD

25+CD

39hiCD

73hi

DecreaseinCD

69expressio

nand

proliferatio

nIncrease

ofIL-6

secretionfro

mautologous

Teffector

CD4+(A

DO-A2A

Rmechanism

)

[64]

CD19

positives

election

from

cord

bloo

dIrradiated

CD40

Ltransfe

cted

Lcells

+Cp

Gandanti-BC

Rfor4

days

IL10+foun

deither

inCD

24hiCD

38hior

CD24

int CD38

int

Supp

ressionof

proliferatio

nof

allogeneicCD

4+Tcell

Supp

ressionof

secretionof

IFN-120574

TNF-120572and

IL-2

byallogeneicCD

4+T

cell

[65]

PBMC

50cond

ition

edmediawith

APR

ILfor3

days

+Cp

G(10120583

gmL)

fora

ddition

al24

hours

IL-10+

Decreaseo

fIFN

-120574andTN

F-120572

prod

uctio

nfro

mautologous

CD4+

Tcell

[66]

PBMC

perip

heralb

lood

mon

onuclear

cellsrHrecom

binant

humanP

PDp

urified

proteinderiv

ativeADOadeno

sineCT

LA-4C

ytotoxicT-Lymph

ocyteAntigen

4A2A

RA2A

receptorlowast

Unlesso

therwise

indicatedBcells

wereisolatedfro

mPB

MC








5 Conclusions





Competing Interests


Acknowledgments




References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































Table1Summaryof

ther

ecently

used

Breg

indu

ctionmetho

dsin

human

invitro

Isolationlowast

Stim

ulation

Characteriz

ationof

indu

cedBreg

Immun

osup

pressiv

eeffect

(mechanism

ofactio

n)Re

ference

CD19

positives

election

CpG(10u

gmL)

+CD

40L(1ugm

L)for

24ho

urs

CD24

hiCD

27+IL-10+

Inhibitio

nof

TNF-120572prod

uctio

nby

antig

en-specific

CD4+

Tcells

andby

mon

ocytes

Both

CD24

hiCD

27+Bcelland

CD24

lowCD


NF-120572

prod

uctio

nby

CD4+

Tcellequally

(IL-10

independ

entp

athw

ay)

[15]

CD22

positives

electio

nCp

G(05120583M)+

BAFF

(20120583

M)+

rHIL-4

(50U

mL)

for2

4ho

urs

IL-10+IL-6+

Increasedexpressio

nof

TLR9

mdash[61]

CD19

negativ

esele

ction

Anti-IgA

+IgG

+IgM

(65120583gmL)

+IL-21

(50n

gmL)

for16ho

urs

CD38+CD

1d+IgM+CD

147+IL-10+CD

25+ID

O+

Supp

ressionof

autologous

CD4+

Tcell

proliferatio

n(G

zmBmediated)

[36]

CD19

negativ

esele

ction

CpG(1120583M)+

PPDfor4

8ho

urs

CD73minusCD

25+CD

71+IL-10+

Enric

hedinC

D5+IgM+IgDminus

IL-10+CD

27+have

lessC

D27

MFI

than

IL-10minusCD

27+

Supp

ressionof

autologous

PPD-stim

ulated

CD4+

Tcell

proliferatio

n(IL-10

mediated)

[14]

CD19

negativ

esele

ction

Ultra-CD

40L(5solutio

n)and

200I

Um

Lof

IL-4

for5

days

Increasedexpressio

nlevelsof

CD39

Dow

nregulated

CD73

Supp

ressionof

autologous

CD4+

and

CD8+

Tcellproliferatio

n(likelyto

bepartially

mediatedby


uctio

nby

Bcell)

[62]

CD19

positives

election

IL-2

(10n

gmL)

+IL-6

(10n

gmL)

+Cp

G2006

(1120583gmL)

+IFN-120572

orIFN-120573

(1000

Um

L)for4

days

CD27


from

naıvea

ndim

matureB

cells

mdash[23]

PBMCor

CD43

negativ

eselection

LPS(10120583

gmL)

+IL-4

(10n

gmL)

+rH

IL-35sin

gle-chainfusio

nprotein

(100

ngm

L)for4

days

IL-10+

mdash[35]

CD19

positives

election

Irradiated

CDw32Lexpressin

ganti-CD

40mAb

CD11bminusCD

5minusCD

27minusIgD+CD

1dhiIL-10+

Supp

ressionof

autologous

Tcell

proliferatio

nandIFN-120574

prod

uctio

n[60]


Table1Con

tinued

Isolationlowast

Stim

ulation

Characteriz

ationof

indu

cedBreg

Immun

osup

pressiv

eeffect

(mechanism

ofactio

n)Re

ference

CD19

negativ

esele

ction

CpG(3120583gmL)

+anti-IgA+IgG+IgM

(10120583

gmL)

for4

8h

CD11c+IL-10+

Enric

hedin

CD38+CD

27minusGCs

Depletedin

CD38+CD

27+PC

smdash

[16]

CD19

negativ

esele

ction

CpG(10120583

gmL)

+rH

CD40

Lfor4

8ho

urs

CD5+CD

27+CD

138+CD

38+IgDminusGzm

B+Inhibitio

nof

proliferatio

nof

autologous

CD4+CD

25minusTcell

(Gzm

Bmediated)

[12]

CD19

negativ

esele

ction

CpG(025120583

M)for

4days

mdash

Effectson

CD4+

autologous

Tcell

Supp

ressionof

proliferatio

nand

differentiatio

ninto

Th1cell

Redu

ctionof

TNF-120572andIFN-120574

prod

uctio

nIndu

ctionof

Foxp3+CD

4+TcellTr1

cellandTh

3cell

Upregulationof

CTLA

-4(IL-10T

GF-120573IDOand

CTLA

-4mediated)

[37]

CD43

negativ

esele

ction

from

tonsils

Adiposetissue

deriv

edMSC

(ratio

BcellM

SC5

1)+anti-IgM

(10120583

gmL)

+IL-2

(103

IU)+

anti-CD

40agon

isticmAb

(5120583gmL)

for10days

CD19+CD

27minusCD

38hiCD

24hiIL10+

mdash[63]

CD19

positives

election

CD40

L(2mgmL)

+IL-4

(200

IUm

L)for4

days

CD24

hiCD

38hiCD

25+CD

39hiCD

73hi

DecreaseinCD

69expressio

nand

proliferatio

nIncrease

ofIL-6

secretionfro

mautologous

Teffector

CD4+(A

DO-A2A

Rmechanism

)

[64]

CD19

positives

election

from

cord

bloo

dIrradiated

CD40

Ltransfe

cted

Lcells

+Cp

Gandanti-BC

Rfor4

days

IL10+foun

deither

inCD

24hiCD

38hior

CD24

int CD38

int

Supp

ressionof

proliferatio

nof

allogeneicCD

4+Tcell

Supp

ressionof

secretionof

IFN-120574

TNF-120572and

IL-2

byallogeneicCD

4+T

cell

[65]

PBMC

50cond

ition

edmediawith

APR

ILfor3

days

+Cp

G(10120583

gmL)

fora

ddition

al24

hours

IL-10+

Decreaseo

fIFN

-120574andTN

F-120572

prod

uctio

nfro

mautologous

CD4+

Tcell

[66]

PBMC

perip

heralb

lood

mon

onuclear

cellsrHrecom

binant

humanP

PDp

urified

proteinderiv

ativeADOadeno

sineCT

LA-4C

ytotoxicT-Lymph

ocyteAntigen

4A2A

RA2A

receptorlowast

Unlesso

therwise

indicatedBcells

wereisolatedfro

mPB

MC








5 Conclusions





Competing Interests


Acknowledgments




References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























Table1Summaryof

ther

ecently

used

Breg

indu

ctionmetho

dsin

human

invitro

Isolationlowast

Stim

ulation

Characteriz

ationof

indu

cedBreg

Immun

osup

pressiv

eeffect

(mechanism

ofactio

n)Re

ference

CD19

positives

election

CpG(10u

gmL)

+CD

40L(1ugm

L)for

24ho

urs

CD24

hiCD

27+IL-10+

Inhibitio

nof

TNF-120572prod

uctio

nby

antig

en-specific

CD4+

Tcells

andby

mon

ocytes

Both

CD24

hiCD

27+Bcelland

CD24

lowCD


NF-120572

prod

uctio

nby

CD4+

Tcellequally

(IL-10

independ

entp

athw

ay)

[15]

CD22

positives

electio

nCp

G(05120583M)+

BAFF

(20120583

M)+

rHIL-4

(50U

mL)

for2

4ho

urs

IL-10+IL-6+

Increasedexpressio

nof

TLR9

mdash[61]

CD19

negativ

esele

ction

Anti-IgA

+IgG

+IgM

(65120583gmL)

+IL-21

(50n

gmL)

for16ho

urs

CD38+CD

1d+IgM+CD

147+IL-10+CD

25+ID

O+

Supp

ressionof

autologous

CD4+

Tcell

proliferatio

n(G

zmBmediated)

[36]

CD19

negativ

esele

ction

CpG(1120583M)+

PPDfor4

8ho

urs

CD73minusCD

25+CD

71+IL-10+

Enric

hedinC

D5+IgM+IgDminus

IL-10+CD

27+have

lessC

D27

MFI

than

IL-10minusCD

27+

Supp

ressionof

autologous

PPD-stim

ulated

CD4+

Tcell

proliferatio

n(IL-10

mediated)

[14]

CD19

negativ

esele

ction

Ultra-CD

40L(5solutio

n)and

200I

Um

Lof

IL-4

for5

days

Increasedexpressio

nlevelsof

CD39

Dow

nregulated

CD73

Supp

ressionof

autologous

CD4+

and

CD8+

Tcellproliferatio

n(likelyto

bepartially

mediatedby


uctio

nby

Bcell)

[62]

CD19

positives

election

IL-2

(10n

gmL)

+IL-6

(10n

gmL)

+Cp

G2006

(1120583gmL)

+IFN-120572

orIFN-120573

(1000

Um

L)for4

days

CD27


from

naıvea

ndim

matureB

cells

mdash[23]

PBMCor

CD43

negativ

eselection

LPS(10120583

gmL)

+IL-4

(10n

gmL)

+rH

IL-35sin

gle-chainfusio

nprotein

(100

ngm

L)for4

days

IL-10+

mdash[35]

CD19

positives

election

Irradiated

CDw32Lexpressin

ganti-CD

40mAb

CD11bminusCD

5minusCD

27minusIgD+CD

1dhiIL-10+

Supp

ressionof

autologous

Tcell

proliferatio

nandIFN-120574

prod

uctio

n[60]


Table1Con

tinued

Isolationlowast

Stim

ulation

Characteriz

ationof

indu

cedBreg

Immun

osup

pressiv

eeffect

(mechanism

ofactio

n)Re

ference

CD19

negativ

esele

ction

CpG(3120583gmL)

+anti-IgA+IgG+IgM

(10120583

gmL)

for4

8h

CD11c+IL-10+

Enric

hedin

CD38+CD

27minusGCs

Depletedin

CD38+CD

27+PC

smdash

[16]

CD19

negativ

esele

ction

CpG(10120583

gmL)

+rH

CD40

Lfor4

8ho

urs

CD5+CD

27+CD

138+CD

38+IgDminusGzm

B+Inhibitio

nof

proliferatio

nof

autologous

CD4+CD

25minusTcell

(Gzm

Bmediated)

[12]

CD19

negativ

esele

ction

CpG(025120583

M)for

4days

mdash

Effectson

CD4+

autologous

Tcell

Supp

ressionof

proliferatio

nand

differentiatio

ninto

Th1cell

Redu

ctionof

TNF-120572andIFN-120574

prod

uctio

nIndu

ctionof

Foxp3+CD

4+TcellTr1

cellandTh

3cell

Upregulationof

CTLA

-4(IL-10T

GF-120573IDOand

CTLA

-4mediated)

[37]

CD43

negativ

esele

ction

from

tonsils

Adiposetissue

deriv

edMSC

(ratio

BcellM

SC5

1)+anti-IgM

(10120583

gmL)

+IL-2

(103

IU)+

anti-CD

40agon

isticmAb

(5120583gmL)

for10days

CD19+CD

27minusCD

38hiCD

24hiIL10+

mdash[63]

CD19

positives

election

CD40

L(2mgmL)

+IL-4

(200

IUm

L)for4

days

CD24

hiCD

38hiCD

25+CD

39hiCD

73hi

DecreaseinCD

69expressio

nand

proliferatio

nIncrease

ofIL-6

secretionfro

mautologous

Teffector

CD4+(A

DO-A2A

Rmechanism

)

[64]

CD19

positives

election

from

cord

bloo

dIrradiated

CD40

Ltransfe

cted

Lcells

+Cp

Gandanti-BC

Rfor4

days

IL10+foun

deither

inCD

24hiCD

38hior

CD24

int CD38

int

Supp

ressionof

proliferatio

nof

allogeneicCD

4+Tcell

Supp

ressionof

secretionof

IFN-120574

TNF-120572and

IL-2

byallogeneicCD

4+T

cell

[65]

PBMC

50cond

ition

edmediawith

APR

ILfor3

days

+Cp

G(10120583

gmL)

fora

ddition

al24

hours

IL-10+

Decreaseo

fIFN

-120574andTN

F-120572

prod

uctio

nfro

mautologous

CD4+

Tcell

[66]

PBMC

perip

heralb

lood

mon

onuclear

cellsrHrecom

binant

humanP

PDp

urified

proteinderiv

ativeADOadeno

sineCT

LA-4C

ytotoxicT-Lymph

ocyteAntigen

4A2A

RA2A

receptorlowast

Unlesso

therwise

indicatedBcells

wereisolatedfro

mPB

MC








5 Conclusions





Competing Interests


Acknowledgments




References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Summaryof

ther

ecently

used

Breg

indu

ctionmetho

dsin

human

invitro

Isolationlowast

Stim

ulation

Characteriz

ationof

indu

cedBreg

Immun

osup

pressiv

eeffect

(mechanism

ofactio

n)Re

ference

CD19

positives

election

CpG(10u

gmL)

+CD

40L(1ugm

L)for

24ho

urs

CD24

hiCD

27+IL-10+

Inhibitio

nof

TNF-120572prod

uctio

nby

antig

en-specific

CD4+

Tcells

andby

mon

ocytes

Both

CD24

hiCD

27+Bcelland

CD24

lowCD


NF-120572

prod

uctio

nby

CD4+

Tcellequally

(IL-10

independ

entp

athw

ay)

[15]

CD22

positives

electio

nCp

G(05120583M)+

BAFF

(20120583

M)+

rHIL-4

(50U

mL)

for2

4ho

urs

IL-10+IL-6+

Increasedexpressio

nof

TLR9

mdash[61]

CD19

negativ

esele

ction

Anti-IgA

+IgG

+IgM

(65120583gmL)

+IL-21

(50n

gmL)

for16ho

urs

CD38+CD

1d+IgM+CD

147+IL-10+CD

25+ID

O+

Supp

ressionof

autologous

CD4+

Tcell

proliferatio

n(G

zmBmediated)

[36]

CD19

negativ

esele

ction

CpG(1120583M)+

PPDfor4

8ho

urs

CD73minusCD

25+CD

71+IL-10+

Enric

hedinC

D5+IgM+IgDminus

IL-10+CD

27+have

lessC

D27

MFI

than

IL-10minusCD

27+

Supp

ressionof

autologous

PPD-stim

ulated

CD4+

Tcell

proliferatio

n(IL-10

mediated)

[14]

CD19

negativ

esele

ction

Ultra-CD

40L(5solutio

n)and

200I

Um

Lof

IL-4

for5

days

Increasedexpressio

nlevelsof

CD39

Dow

nregulated

CD73

Supp

ressionof

autologous

CD4+

and

CD8+

Tcellproliferatio

n(likelyto

bepartially

mediatedby


uctio

nby

Bcell)

[62]

CD19

positives

election

IL-2

(10n

gmL)

+IL-6

(10n

gmL)

+Cp

G2006

(1120583gmL)

+IFN-120572

orIFN-120573

(1000

Um

L)for4

days

CD27


from

naıvea

ndim

matureB

cells

mdash[23]

PBMCor

CD43

negativ

eselection

LPS(10120583

gmL)

+IL-4

(10n

gmL)

+rH

IL-35sin

gle-chainfusio

nprotein

(100

ngm

L)for4

days

IL-10+

mdash[35]

CD19

positives

election

Irradiated

CDw32Lexpressin

ganti-CD

40mAb

CD11bminusCD

5minusCD

27minusIgD+CD

1dhiIL-10+

Supp

ressionof

autologous

Tcell

proliferatio

nandIFN-120574

prod

uctio

n[60]


Table1Con

tinued

Isolationlowast

Stim

ulation

Characteriz

ationof

indu

cedBreg

Immun

osup

pressiv

eeffect

(mechanism

ofactio

n)Re

ference

CD19

negativ

esele

ction

CpG(3120583gmL)

+anti-IgA+IgG+IgM

(10120583

gmL)

for4

8h

CD11c+IL-10+

Enric

hedin

CD38+CD

27minusGCs

Depletedin

CD38+CD

27+PC

smdash

[16]

CD19

negativ

esele

ction

CpG(10120583

gmL)

+rH

CD40

Lfor4

8ho

urs

CD5+CD

27+CD

138+CD

38+IgDminusGzm

B+Inhibitio

nof

proliferatio

nof

autologous

CD4+CD

25minusTcell

(Gzm

Bmediated)

[12]

CD19

negativ

esele

ction

CpG(025120583

M)for

4days

mdash

Effectson

CD4+

autologous

Tcell

Supp

ressionof

proliferatio

nand

differentiatio

ninto

Th1cell

Redu

ctionof

TNF-120572andIFN-120574

prod

uctio

nIndu

ctionof

Foxp3+CD

4+TcellTr1

cellandTh

3cell

Upregulationof

CTLA

-4(IL-10T

GF-120573IDOand

CTLA

-4mediated)

[37]

CD43

negativ

esele

ction

from

tonsils

Adiposetissue

deriv

edMSC

(ratio

BcellM

SC5

1)+anti-IgM

(10120583

gmL)

+IL-2

(103

IU)+

anti-CD

40agon

isticmAb

(5120583gmL)

for10days

CD19+CD

27minusCD

38hiCD

24hiIL10+

mdash[63]

CD19

positives

election

CD40

L(2mgmL)

+IL-4

(200

IUm

L)for4

days

CD24

hiCD

38hiCD

25+CD

39hiCD

73hi

DecreaseinCD

69expressio

nand

proliferatio

nIncrease

ofIL-6

secretionfro

mautologous

Teffector

CD4+(A

DO-A2A

Rmechanism

)

[64]

CD19

positives

election

from

cord

bloo

dIrradiated

CD40

Ltransfe

cted

Lcells

+Cp

Gandanti-BC

Rfor4

days

IL10+foun

deither

inCD

24hiCD

38hior

CD24

int CD38

int

Supp

ressionof

proliferatio

nof

allogeneicCD

4+Tcell

Supp

ressionof

secretionof

IFN-120574

TNF-120572and

IL-2

byallogeneicCD

4+T

cell

[65]

PBMC

50cond

ition

edmediawith

APR

ILfor3

days

+Cp

G(10120583

gmL)

fora

ddition

al24

hours

IL-10+

Decreaseo

fIFN

-120574andTN

F-120572

prod

uctio

nfro

mautologous

CD4+

Tcell

[66]

PBMC

perip

heralb

lood

mon

onuclear

cellsrHrecom

binant

humanP

PDp

urified

proteinderiv

ativeADOadeno

sineCT

LA-4C

ytotoxicT-Lymph

ocyteAntigen

4A2A

RA2A

receptorlowast

Unlesso

therwise

indicatedBcells

wereisolatedfro

mPB

MC








5 Conclusions





Competing Interests


Acknowledgments




References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Con

tinued

Isolationlowast

Stim

ulation

Characteriz

ationof

indu

cedBreg

Immun

osup

pressiv

eeffect

(mechanism

ofactio

n)Re

ference

CD19

negativ

esele

ction

CpG(3120583gmL)

+anti-IgA+IgG+IgM

(10120583

gmL)

for4

8h

CD11c+IL-10+

Enric

hedin

CD38+CD

27minusGCs

Depletedin

CD38+CD

27+PC

smdash

[16]

CD19

negativ

esele

ction

CpG(10120583

gmL)

+rH

CD40

Lfor4

8ho

urs

CD5+CD

27+CD

138+CD

38+IgDminusGzm

B+Inhibitio

nof

proliferatio

nof

autologous

CD4+CD

25minusTcell

(Gzm

Bmediated)

[12]

CD19

negativ

esele

ction

CpG(025120583

M)for

4days

mdash

Effectson

CD4+

autologous

Tcell

Supp

ressionof

proliferatio

nand

differentiatio

ninto

Th1cell

Redu

ctionof

TNF-120572andIFN-120574

prod

uctio

nIndu

ctionof

Foxp3+CD

4+TcellTr1

cellandTh

3cell

Upregulationof

CTLA

-4(IL-10T

GF-120573IDOand

CTLA

-4mediated)

[37]

CD43

negativ

esele

ction

from

tonsils

Adiposetissue

deriv

edMSC

(ratio

BcellM

SC5

1)+anti-IgM

(10120583

gmL)

+IL-2

(103

IU)+

anti-CD

40agon

isticmAb

(5120583gmL)

for10days

CD19+CD

27minusCD

38hiCD

24hiIL10+

mdash[63]

CD19

positives

election

CD40

L(2mgmL)

+IL-4

(200

IUm

L)for4

days

CD24

hiCD

38hiCD

25+CD

39hiCD

73hi

DecreaseinCD

69expressio

nand

proliferatio

nIncrease

ofIL-6

secretionfro

mautologous

Teffector

CD4+(A

DO-A2A

Rmechanism

)

[64]

CD19

positives

election

from

cord

bloo

dIrradiated

CD40

Ltransfe

cted

Lcells

+Cp

Gandanti-BC

Rfor4

days

IL10+foun

deither

inCD

24hiCD

38hior

CD24

int CD38

int

Supp

ressionof

proliferatio

nof

allogeneicCD

4+Tcell

Supp

ressionof

secretionof

IFN-120574

TNF-120572and

IL-2

byallogeneicCD

4+T

cell

[65]

PBMC

50cond

ition

edmediawith

APR

ILfor3

days

+Cp

G(10120583

gmL)

fora

ddition

al24

hours

IL-10+

Decreaseo

fIFN

-120574andTN

F-120572

prod

uctio

nfro

mautologous

CD4+

Tcell

[66]

PBMC

perip

heralb

lood

mon

onuclear

cellsrHrecom

binant

humanP

PDp

urified

proteinderiv

ativeADOadeno

sineCT

LA-4C

ytotoxicT-Lymph

ocyteAntigen

4A2A

RA2A

receptorlowast

Unlesso

therwise

indicatedBcells

wereisolatedfro

mPB

MC








5 Conclusions





Competing Interests


Acknowledgments




References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























5 Conclusions





Competing Interests


Acknowledgments




References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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