TLR4 promotes B cell maturation: independence and cooperation with B lymphocyte-activating factor.

Abstract

We have previously shown that TLR4 triggering promotes the generation of CD23(+)CD93(+) transitional T2-like cells in vitro from mouse B cell precursors, suggesting a possible role for this receptor in B cell maturation. In this study, we perform an extensive study of cell surface markers and functional properties of B cells matured in vitro with LPS, comparatively with the well-known B cell maturation factor B lymphocyte-activating factor (BAFF). LPS increased generation of CD23(+) transitional B cells in a TLR4-dependent way, upregulating IgD and CD21 and downregulating CD93, without inducing cell proliferation, in a manner essentially equivalent to BAFF. For both BAFF and LPS, functional maturation of the IgM(+)CD23(+)CD93(+) cells was confirmed by their higher proliferative response to anti-CD40 plus IL-4 compared with IgM(+)CD23(neg)CD93(+) cells. BAFF-R-Fc-mediated neutralization experiments showed that TLR4-induced B cell maturation was independent of BAFF. Distinct from BAFF, maturation by LPS relied on the activation of canonical NF-kappaB pathway, and the two factors together had complementary effects, leading to higher numbers of IgM(+)CD23(+)CD93(+) cells with their simultaneous addition. Importantly, BCR cross-linking abrogated the generation of CD23(+) B cells by LPS or BAFF, indicating that signals mimicking central tolerance act on both systems. Addition of cyclosporin A reverted BCR-mediated inhibition, both for BAFF and LPS, suggesting similar regulation of signaling pathways by calcineurin. Finally, LPS-injected mice showed a rapid increase of mature B cells in the bone marrow, suggesting that TLR4 signaling may effectively stimulate B cell maturation in vivo, acting as an accessory stimulus in B cell development, complementary to the BAFF physiological pathway.

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of June 8, 2010
This information is current as
doi:10.4049/jimmunol.0903253
http://www.jimmunol.org/cgi/content/full/184/9/4662
online Mar 31, 2010;
2010;184;4662-4672; originally published
J. Immunol.

Álvaro L. Bertho, Maria Bellio and Alberto Nobrega
Elize A. Hayashi, Alessandra Granato, Luciana S. Paiva,


Lymphocyte-Activating Factor
BIndependence and Cooperation with
TLR4 Promotes B Cell Maturation:


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The Journal of Immunology
TLR4 Promotes B Cell Maturation: Independence and
Cooperation with B Lymphocyte-Activating Factor
Elize A. Hayashi,* Alessandra Granato,* Luciana S. Paiva,*,†A´lvaro L. Bertho,‡
Maria Bellio,* and Alberto Nobrega*
We have previously shown that TLR4 triggering promotes the generation of CD23+CD93+transitional T2-like cells in vitro from
mouse B cell precursors, suggesting a possible role for this receptor in B cell maturation. In this study, we perform an extensive
study of cell surface markers and functional properties of B cells matured in vitro with LPS, comparatively with the well-known
B cell maturation factor B lymphocyte-activating factor (BAFF). LPS increased generation of CD23+transitional B cells in
a TLR4-dependent way, upregulating IgD and CD21 and downregulating CD93, without inducing cell proliferation, in a manner
essentially equivalent to BAFF. For both BAFF and LPS, functional maturation of the IgM+CD23+CD93+cells was confirmed by
their higher proliferative response to anti-CD40 plus IL-4 compared with IgM+CD23negCD93+cells. BAFF-R-Fc–mediated
neutralization experiments showed that TLR4-induced B cell maturation was independent of BAFF. Distinct from BAFF, mat-
uration by LPS relied on the activation of canonical NF-kB pathway, and the two factors together had complementary effects,
leading to higher numbers of IgM+CD23+CD93+cells with their simultaneous addition. Importantly, BCR cross-linking abrogated
the generation of CD23+B cells by LPS or BAFF, indicating that signals mimicking central tolerance act on both systems.
Addition of cyclosporin A reverted BCR-mediated inhibition, both for BAFF and LPS, suggesting similar regulation of signaling
pathways by calcineurin. Finally, LPS-injected mice showed a rapid increase of mature B cells in the bone marrow, suggesting that
TLR4 signaling may effectively stimulate B cell maturation in vivo, acting as an accessory stimulus in B cell development,
complementary to the BAFF physiological pathway. The Journal of Immunology, 2010, 184: 4662–4672.
I
formed immature B lymphocytes must pass through intermediary
stages named “transitional” before reaching mature state (2–5).
Many groups have resolved the transitional B cell subsets in the BM
and spleen using different cell markers, and a standard classification
has not been clearly defined yet (2–4, 6). Transitional B lymphocytes
still express significant levels of CD93, which is absent in mature
B cells, and high levels of IgM. They are typically divided into T1
and T2, the second one bearing more mature characteristics, such as
the expression of CD23 and CD21 and higher IgD. Passage from
immature to transitional and then to mature stages is marked by
clonal selection, in which only a small fraction of the daily generated
immature B cells can survive and complete differentiation (7).
Interaction with self-Ags through the BCR is a crucial checkpoint in
B cell maturation. As part of an important mechanism for main-
n mice, as well as in humans, B lymphocytes are generated
in the bone marrow (BM) throughout postnatal life from pre-
cursors derived from hematopoietic stem cells (1). Newly
taining self-tolerance, signaling outcome in B lymphocytes upon
BCR ligation changes dramatically according to their developmental
state: whereas mature B lymphocytes tend to proliferate and secrete
Ab, immature and transitional B cells are highly susceptible to
negative selection by receptor editing or apoptosis (2, 8, 9).
InadditiontoAg-specificinteraction,contactwithcytokinesandcell
surfaceligandsonstromalcellshasalsobeenshowntoplaycriticalroles
inBcelldevelopmentanddifferentiation(1,9).Theseaccessorysignals
cancooperatepositivelyornegativelywithBCR-deliveredsignals,and
the balance among them seems to guide the fate of B lymphocytes
throughout their maturation, regulating survival, differentiation, and
proliferation. A critical factor for B cell development is B lymphocyte
activating factor (BAFF, also known as BLyS or TALL-1) (10), a cyto-
kine belonging to the TNF superfamily. Interaction of BAFF with
BAFF-R provide constitutive signals necessary for B cell survival, es-
peciallyattheT2stage,andisrequiredfornormalBcellmaturation(10,
11). The enforced expression of BAFF in transgenic mice results in
a lupus-like autoimmune disease (12). Interestingly, MyD88 has been
shown to be necessary for the outbreak of lupus-like autoimmune dis-
ease in BAFF transgenic mice, denoting a dependence on signals from
TLRs, besides the central role of BAFF signaling in this model (13).
TLR4,TLR2,andTLR9agonistssuchasLPS,bacteriallipoproteins,
and CpG-DNA are largely known as potent B cell mitogens, inducing
aT-independentpolyclonalactivationandIgsecretion(14–16),butan
increasingamountofdatahasevidencedthatTLR-derivedsignalsplay
a much more fine-tuned role in B cell activation. TLR engagement
seems to provide important costimulatory signals for optimal Ag-
specific B cell response (17, 18). Activation of autoreactive B cells
against chromatin and RNA-associated self-Ags in murine models for
systemicautoimmunediseaseshasalsobeenshowntodependonBCR-
TLR9 and BCR-TLR7 sequential engagements, respectively (19, 20).
It remains unclear, however, whether the BCR and TLR collaboration
observed in mature B cells is also valid for immature B lymphocytes
*Department of Immunology, Institute of Microbiology, Federal University of Rio de
Janeiro;†Department of Immunobiology, Institute of Biology, Fluminense Federal
University; and
Oswaldo Cruz Institute-Fiocruz, Rio de Janeiro, Brazil
‡Flow Cytometry Core Lab, Laboratory of Immunoparasitology,
Received for publication October 5, 2009. Accepted for publication February 19,
2010.
This work was supported by grants from the Conselho Nacional de Pesquisas (Bra-
zil), Fundac ¸a ˜o de Amparo a ` Pesquisa do Estado do Rio de Janeiro, Comissao de
Aperfeic ¸oamento de Pessoal de Nival Superior, and Financiadora de Estudos e Pro-
jetos.
Address correspondence and reprint requests to Dr. Alberto Nobrega, Department of
Immunology, Institute of Microbiology, Federal University of Rio de Janeiro, Rio de
Janeiro 21941-590, Brazil. E-mail address: afnobrega@gmail.com
Abbreviations used in this paper: BAFF, B lymphocyte-activating factor; BM, bone
marrow; CsA, cyclosporin A; PI, propidium iodide.
Copyright?2010byTheAmericanAssociationofImmunologists,Inc.0022-1767/10/$16.00
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and whether signals from TLRs could influence B cell developmental
events other than cell activation. It has recently been suggested that
immatureantichromatinautoreactiveBcellscouldalsobeactivatedby
CpG-DNA (21). However, total IgM secretion and proliferation in
immature B subset were still much lower than in mature subset, even
though TLR9 is highly expressed in early immature stage. Likewise,
immature B cells poorly proliferate upon LPS stimulation (4, 5), al-
thoughtheyexpressconsiderablelevelsofTLR4(22).Incontrast,ithas
been shown that TLR4 and TLR2 agonists rapidly reach BM in
acontextofinfection,inhibitinglymphoidprecursorproliferation(23).
In addition, it has been shown that endogenous molecules with broad
distribution in the organism, such as hiauluronate and heat-shock pro-
teins,arerecognizedbyTLR4(reviewedinRef.24).Insuchacontext,
it is important to investigate the role of TLRs in B cell physiology
beyond their mitogenic property, particularly addressing their putative
roleinBcellmaturationandsurvival.OurpreviousfindingsthatTLR4
engagement is able to promote the generation of CD23+transitional
B cells with a “T2-like” phenotype fromBM B cellprecursors invitro
(25)suggestanalternativemodeofactionofLPSonimmatureBcells,
distinct from classical B cell activation and more compatible with the
progression in maturation of the developing B lymphocyte.
Inthisstudy,wehavedoneathoroughcharacterizationofinvitro
B cell maturation triggered by TLR4 signaling, comparatively with
the well-described BAFF-dependent B cell maturation process. We
foundthatdifferentiationpromotedbyLPSisessentiallyequivalent
to that induced by BAFF in everyphenotypic aspects studied in this
paper, including changes in molecular developmental markers and
functionalproperties.TLR4-drivenBcellmaturationwasfoundtobe
independent of BAFF, relying on the activation of classical NF-kB
pathway, whereas BAFF activity did not require that pathway. Both
factors together had complementary effects stimulating the gener-
ationofhighernumbersofCD23+Bcells.Importantly,high-avidity
cross-linking of BCR totally inhibited CD23+B cell generation in
the presence of LPS or BAFF, and cyclosporin A (CsA) was able to
revert that inhibition, suggesting that both pathways are similarly
regulated by BCR-mediated signals controlling central tolerance.
Finally, we found evidences in vivo that TLR4 signaling can ef-
fectivelystimulategenerationofmatureBcellsinBM.Theseresults
show consistent evidence that TLR4 can provide alternative or
complementary signals to BAFF-R along B cell maturation, re-
specting developmental control and regulation by BCR-derived
signals, raising the question of the physiological role played by this
receptor in B cell development.
Materials and Methods
Mice and cells
Adult C57BL/6 and C57BL/10ScCr mice were obtained from animal fa-
cilitiesofFederalUniversityofRiodeJaneiroandmaintainedunderstandard
pathogenfree-conditions.BMcellswereflushedfromfemurswithice-cooled
complete medium (OptiMEM supplemented with 10% FBS, 5 3 1025M 2-
ME, 100 mg/ml streptomycin, and 100 U/ml penicillin) (Life Technologies,
Grand Island, NY). Spleen cell suspensions were obtained by gently teasing
spleensontoacellstrainer.Aftercentrifugingcellsuspensionsat2803gfor
7 min and discarding supernatant, cells were resuspended in ammonium
chloride potassium lysis buffer (0.155 M NH4Cl, 10 mM KHCO3, and
0.1 mM sodium EDTA) to deplete erythrocytes, centrifuged for 7 min, and
resuspended in complete medium. Cells were counted using a hemacytom-
eterwithexclusionofdeadcellswithtrypanbluedye,andprocessedforflow
cytometric analysis or cell sorting. Experimental procedures were approved
by the Committee on Ethics for Animal Experimentation of the Federal
University of Rio de Janeiro.
MACS
VarioMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) system was
used for magnetic cell sorting. For depletion of IgM+B lymphocytes, BM
cellswereincubatedinMACSbuffer(PBScontaining2mMEDTA,and5%
FBS) withanti-mouseIgM MicroBeads(Miltenyi Biotec)for20 minon ice,
washed, and resuspended in MACS buffer, according to the manufacturer’s
specifications. Cells were applied into an LD depletion column (Miltenyi
Biotec), and the effluent cells were collected as the BM IgMnegfraction and
washed in complete OptiMEM medium. IgMnegfraction was incubated for
20 min on ice with anti-B220 or anti-CD19 MicroBeads (Miltenyi Biotec)
and processed as described above. Cells were applied into MS-positive sort
column, and after washing, retained cells were collected. Viable cells were
scored by trypan blue dye using a hemacytometer, and the purity of cell
preparations was verified by flow cytometry. Usually, IgM+cells corre-
sponded to ,1% of B lineage cells after depletion, and B220+cells corre-
sponded to .95% of recovered cells after positive selection.
Cell staining, flow cytometry, and cell sorting
Fresh BM cells, MACS-purified cells, and cultured cells were analyzed by
flow cytometry. The Abs used for staining were as follows: PE anti-CD23
(clone B3B4), PE anti-B220 (clone RA3-6B2), FITC rat anti-mouse IgM
(clone R6-60.2), and FITC anti-CD21 (clone 7G6) (BD Pharmingen, San
Diego, CA); Alexa 647 anti-mouse IgM (clone b-7-6) and FITC anti-B220
(provided by Dr. J. Cambier, University of Colorado Health Science Center
and NationalJewishHealthCenter,Denver,CO);allophycocyaninanti-CD23
(clone B3B4) (Caltag Laboratories, San Francisco, CA), PE-Cy7 anti-CD93
(clone AA4.1), and Alexa 647 anti–BAFF-R; PE rat IgG2a isotype control
(clone eBR2a); PE anti-CD40 (clone 1C10) (eBioscience, San Diego, CA);
biotin anti-IgD (clone 11-26) (Southern Biotechnology Associates, Bir-
mingham, AL); FITC and DyLight 649 goat F(ab9)2fragments anti-mouse
IgM (The Jackson Laboratory, Bar Harbor, ME) and biotin anti-CD93 (493
hybridoma provided by Dr. A. Rolink, Basel University, Basel, Switzerland;
the mAb was purified and biotin-conjugated according to standard proto-
cols); and Alexa 488 Annexin V (Molecular Probes, Eugene, OR). Bio-
tinylated Abs were revealed with Alexa-Fluor 680-R-PE streptavidin
(Molecular Probes) or with APC streptavidin (Caltag Laboratories). Cells
were incubated with Abs in FACS buffer (PBS, 5% FBS, and 0.05% sodium
azide) for 20 min at 4˚C and washed with FACS buffer. When biotinylated
mAbs were used, another step of incubation with Alexa 680-PE streptavidin
or APC streptavidin was performed under the same conditions as described
above. Except for the four-color–stained cell samples, propidium iodide (PI)
was added at 0.5 mg/ml to the samples immediately before data acquisition
for dead cell exclusion. Annexin V staining was performed using a calcium-
containing buffer as indicated by the manufacturer. Data were acquired by
a FACSCalibur (BD Biosciences, San Jose, CA) and analyzed using Cell-
Quest software (BD Biosciences) or Summit (DakoCytomation, Glostrup,
Denmark). For sorting of immature and transitional B cells from BM and cell
cultures, MoFlo flow cytometer (DakoCytomation) and EPICS ALTRA
(Beckman Coulter, Hialeah, FL) were used.
CFSE staining
For coculture experiments and cell proliferation analysis, cells were stained
with CFSE prior to the culture. Briefly, BM-purified B cell precursors and
sorted immature and transitional B cells were incubated at 5 3 106cells/ml
with 0.5 mM CFSE (Molecular Probes) in prewarmed PBS for 5 min at
37˚C and washed twice with complete OptiMEM medium.
Cultures for B cell differentiation
PurifiedB220+IgMnegBcellprecursorcellswereculturedfor72hin96-wellflat-
bottom plates (Corning Glass, Corning, NY) at 2 3105cells/well in 200 ml/well
in OptiMEM supplemented with 10% FCS, 5 3 1025M 2-ME, 100 mg/ml
streptomycin, and 100 U/ml penicillin (Life Technologies) in humidified atmo-
sphere of 7% CO2at 37˚C. In mixed cocultures of C57BL/6 and C57BL10/
ScCrBcellprecursors,13105Bcellprecursorsfromeachstrainwereaddedper
well.Typically,stimuliwereadded16–20hbeforetheendofcultureandincluded
12.5 mg/ml ultrapure LPS from Escherichia coli 0111:B4 strain, 1 mg/ml mono-
phosphoryllipidAfromSalmonellaminnesotaR595LPS(InvivoGen,SanDiego,
CA), or 100 ng/ml rBAFF (R&D Systems, Minneapolis, MN). In some experi-
ments, we added simultaneously to stimuli: 10 mg/ml decoy BAFF-R (BAFF-
R-Fc) (R&D Systems), 200 ng/ml CsA (Sandimmun; Novartis Pharmaceuticals,
East Hanover, NJ), or F(ab9)2fragments anti-IgM at concentrations ranging from
0.005to5mg/ml.InculturesforinhibitionofclassicalNF-kBpathwayactivation,
we added 50 mg/ml SN50 peptide (Marligen Biosciences, Ijamsville, MD) to the
culture 2 h before stimulation with BAFF or LPS. Recovered live cells were
counted using hemacytometer with exclusion of dead cells with trypan blue dye
and processed for flow cytometric analysis or cell sorting. Statistical analysis
(unpairedStudentttest)wasdoneusingPrismsoftware(GraphPadSoftware,San
Diego, CA); results were considered statistically significant if p , 0.05.
In vitro proliferation assay
Sorted cells were cultured in flat-bottom 96-well plates at 50,000 cells/well in
RPMI1640mediumsupplementedwith2mMglutamine,1mMsodiumpyruvate,
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10%FCS,531025M2-ME,100mg/mlstreptomycin,and100U/mlpenicillin
(LifeTechnologies).Anti-CD40(clone1C10)(eBioscience)at5mg/mlandIL-4
(0.1% supernatant from cultures of XR63-4 cells) were added to the indicated
cultures.After48h,cultureswerepulsedwith1mCi(0.037MBq)[3H]thymidine
for 20 h before cells were harvested onto glass microfiber filters (Whatman,
Maidstone, U.K.) and analyzed on a scintillation counter (Beckman Coulter).
In vivo LPS injection
Adult C57BL/6 micewere inoculated i.p. with 0.1 mg/g weight LPS or with
PBS only and were killed 20 h later for spleen and BM viable cell counting
and flow cytometric analysis of B cell compartments. Cells were processed
for counting and flow cytometric analysis as described above. In some
experiments, BrdU was injected simultaneously to LPS and control PBS
for analysis of in vivo proliferation.
BrdU incorporation
Adult C57BL/6 mice were inoculated i.p. with 38 mg/kg BrdU (Sigma-
Aldrich, St. Louis, MO) in PBS with or without LPS. Mice were killed 20 h
later, and BM and spleen cells were cell surface stained in standard FACS
buffer, washed once with protein-free PBS, resuspended in 0.5 ml plus
0.5 ml 2% paraformaldehyde 0.02% Tween 20 in PBS, and incubated for
18 h at 4˚C. Subsequently, cells were washed, incubated with DNase I
(150 mM NaCl, 5 mM MgCl2, 10 mM HCl, and 100 KU/ml DNAse I) for
20 min at room temperature, washed, then stained with FITC anti-BrdU
(eBioscience) before analysis by flow cytometry.
Results
Generation of CD23+B cells in vitro in the presence of LPS
does not involve cell proliferation and respects developmental
program
Our group has previously shown that TLR4 agonists promote the
generation of CD23+B cells with a transitional T2-like phenotype
in a system of B cell differentiation in vitro from purified B cell
precursors (25), suggesting a role for TLR4 signal in B cell
maturation. We have obtained convincing evidence that such ef-
fect is due to differentiation of CD93+CD232immature B cells
into CD93+CD23+T2-like B cells and does not involve B cell
proliferation. We considered it important to confirm these results
with a more straightforward approach, investigating whether LPS
was able to induce short-term differentiation of immature B cells
directly obtained ex vivo from fresh BM. To better characterize
the effect of LPS on distinct steps of B cell maturation, we tested
purified immature (IgMlowCD23neg) and transitional (IgMhigh
CD23neg) B subsets separately. Immature and CD23negtransitional
T1-type B cells were sorted from adult mouse BM with sort pu-
rities of .95% (Fig. 1A), stained with CFSE, and cultured for 16 h
with or without LPS. LPS stimulation was able to promote a sig-
nificantly higher generation of CD23+cells from transitional
B cells than in immature B cell, both in percentage (Fig. 1B) and
cell numbers (Fig. 1C), clearly showing that the responsiveness to
LPS is under developmental control. When the CFSE staining was
analyzed in CD23+B cells generated in vitro for the last 16 h, we
observed a uniform CFSEhighpopulation both in LPS-treated and
untreated control cultures (Fig. 1B), providing direct proof that
LPS was not inducing proliferation of CD23+cells, as suggested
by our previous studies (25). Total viable cell recovery after cul-
ture was always less than input (70–90%) of transitional B cells,
both in control and LPS-stimulated cultures (Fig. 1C and data not
shown), in contrast to a large increase in CD23+cell numbers in
LPS-stimulated cultures only, showing that TLR4 agonists stim-
ulate the differentiation of immature B cells to CD23+B cells.
These data obtained with B cells ex vivo confirmed and extended
the results obtained previously with a culture system of B cell
differentiation in vitro from purified CD19+IgMnegBM B cell
precursors (25). Hereafter, we used the later system as our basal
approach for the experiments performed in vitro, because of its
simplicity and much higher yield of cells. Complementary ex-
periments were also done with sorted BM B cells.
LPS and BAFF induce similar alterations in surface markers
and functional pattern along B lymphocyte maturation in vitro
BAFFisthemostwellcharacterizedmaturationandsurvivalfactorfor
BlymphocytesandhasbeenshowntoinduceBcellsmaturationinvitro
aswell(11,26).Wethusdecidedtoperformacomparativeanalysisof
the effect of LPS and BAFF on B cell maturation. Purified B cell
precursorswereculturedfor72h,inthepresenceorabsenceofLPSor
BAFFforthelast20h.TheexpressionofCD23andothermaturation
markers were followed by flow cytometry. We could clearly distin-
guish three main stages of B cell maturation in our system: IgMlow
CD23negimmature B cells, IgMhighCD23negtransitional B cells, and
IgMhighCD23+transitional B cells (Fig. 2A). Both BAFF and LPS
promoted, very similarly, the increase of the more advanced transi-
tionalCD23+B cellpopulation,both inpercentage (Fig. 2A) and cell
numbers(Fig.2C),andadecreaseinthepercentageofmoreimmature
B cell subset (IgMlowCD23neg). Immature CD23negtransitional and
CD23+transitionalBcellsubsetgeneratedbythedifferenttreatments
were further analyzed for the expression of the maturation markers
CD21, IgD,and CD93 (Fig. 2B). Weverifiedthat CD23+transitional
BcellsgeneratedinthepresenceofeitherBAFForLPScorresponded
phenotypically to a more advanced maturation state, with higher
expressionofCD21andIgDandlowerCD93,resemblingtheCD23+
fraction E subset in the BM (27) or its splenic counterpart, the tran-
sitional T2 subset (3–5). These results indicate that the expression of
CD23+molecule in maturing B cells promoted by LPS, similarly to
BAFF, is not due to a simple isolated induction of CD23 expression
but to the stimulation of a coherent set of phenotypic maturational
changes on B lymphocytes. LPS and BAFF had a similar effect on
culturesofsortedimmatureandtransitionalBcellsaswell(Fig.2D),
withamassiveincreaseinCD23+BcellnumbersinLPS-andBAFF-
treated transitional B cells and a lesspronounced increaseinduced in
immature B cells.
GiventheclosesimilaritiesbetweenBAFFandLPSeffectsinBcell
maturation in vitro and the presence of low amounts of B220neg-
contaminatingcellsthatcouldbeproducingBAFFinourcultures,we
aimed to formally exclude the possibility that the LPS-induced mat-
uration would need the presence BAFF in some extent. We then
treated B cell maturation cultures with Fc fusion decoy BAFF-R
(BAFF-R-Fc) (Fig. 2E). Addition of BAFF-R-Fc, which block the
interaction of BAFF with BAFF-R (28), completely abrogated the
maturation promoted by BAFF but did not inhibit the maturation
promoted by LPS (Fig. 2E), discarding a role of BAFF-R on LPS-
induced maturation.
We next asked whether maturation observed in this study for both
BAFF and LPS with the analysis of cell surface markers also corre-
sponded to a functional maturation. It has been reported that B lym-
phocytesacquireproliferativeresponsiveness to anti-CD40 plusIL-4
stimulation at CD23+Fraction E stage and that CD23negstage is still
poorlyresponsivetothisstimulus(27).Thus,wetestedthefunctional
stateofIgMlowCD23neg,IgMhighCD23neg,and IgMhighCD23+B cells
differentiated in the presence of LPS and BAFF in vitro, measuring
invitroproliferationuponstimulationwithanti-CD40plusIL-4(Fig.
3A).WeverifiedthatsortedtransitionalCD23+Bcellsobtainedfrom
bothBAFF-andLPS-differentiatedcultureshadasignificantlyhigher
proliferation upon stimulation, compared with the CD23negsubsets
(Fig. 3A), showing that the CD23+subsets generated upon LPS and
BAFF stimulation in vitro behave similar to their counterparts gen-
erated in vivo. We further investigated whether the acquisition of
proliferative responsiveness corresponds to a higher expression of
CD40 (Fig. 3B). We observed an increase in CD40 expression along
the passage through maturation stages in vitro. That increase is evi-
dentcomparingIgMlowCD23negtoIgMhighCD23negbutonlymarginal
from the IgMhighCD23negto IgMhighCD23+subsets (Fig. 3B).
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LPS does not depend on induction of secondary factors to
generate CD23+B cells
Our previous results with cultures of purified B cell precursors from
LPS-unresponsivemicehaveshownthatgenerationofCD23+Bcells
is strictly dependent on TLR4 signaling on B lymphocytes. As
showed above (Fig. 2), BAFF is not involved in this process, and we
sought to investigatewhether TLR4 signaling would be inducing the
secretionofothersolublefactorsorupregulationofmembrane-bound
factors,whichwouldthendriveB cellmaturation.Weaddressedthis
issuewithamixedcocultureofCD19+IgMnegB cellprecursorsfrom
C57BL/6 mice and precursors from LPS-unresponsive C57BL10/
ScCr mice (Fig. 4). To distinguish those two populations, we stained
the LPS-unresponsive population with CFSE prior to culture. Only
C57BL/6 LPS-responsive cells exhibited a significant augmented
percentageofCD23+cellsuponadditionofLPS,andthepresenceof
C57BL/6 cells could not promote responsiveness of C57BL10/ScCr
cells to LPS. This result indicates that no other soluble factor or cell
surface ligand potentially produced by LPS-stimulated C57BL/6
cellscanactonC57BL10/ScCrimmatureBcells,stronglysuggesting
thatTLR4signalingisdirectlystimulatingBlymphocytematuration.
BlockingofmaturationuponBCRcross-linkingismaintainedin
B lymphocytes differentiating in the presence of LPS or BAFF
Immature and transitional B lymphocytes are highly susceptible to
negative selection upon BCR cross-linking, which is considered an
important mechanism to eliminate autoreactive B lymphocytes (2,
8, 9). To address whether the presence of LPS or BAFF could lead
to a change in such a crucial checkpoint of B lymphocyte
development, we used F(ab9)2fragments anti-IgM to mimic Ag-
specific interaction and induce BCR cross-linking on B lympho-
cytes generated in vitro from purified precursors (Fig. 5A–C). As
expected, in control cultures, increasing dose of anti-IgM led to
a decrease in CD23+B cell percentages (Fig. 5A). Importantly,
cells differentiated in the presence of LPS or BAFF were also
dose-responsive to anti-IgM inhibition, and at a saturating con-
centration of 5 mg/ml anti-IgM, the percentages of CD23+B cells
dramatically dropped, similarly to unstimulated culture with the
same dose of anti-IgM, indicating that negative selection mecha-
nisms mediated by BCR are active in the presence of both factors.
In this study, we could not distinguish whether the decrease of
CD23+B cells upon BCR ligation was mainly due to induction of
FIGURE 1.
(CD93+B220lowIgMlowCD23neg) and CD23negtransitional (CD93+B220dullIgMhighCD23neg) B lymphocytes were sorted by FACS from mouse BM, stained
with CFSE, and cultured for 16 h without or with LPS at 12.5 mg/ml. A, Purity of sorted subsets are shown. Numbers indicate percentage of CD23+B cells
relative to total BM cells (presorting) and to the sorted subsets. B, After culture under the indicated conditions, cells were restained for CD23, IgM, and B220
expression analysis by flow cytometry. Numbers indicate percentage of CD23+B cells (upper panel) or background staining with isotype control Ab (lower
panel) in the recovered population. C, Numbers of viable total (left panel) and CD23+B (right panel) cells per well in 0 h and after 16 h of culture of sorted
transitionalandimmatureBcells,withorwithoutLPS.D,HistogramsshowCFSEstainingofCD23+Bcellsgeneratedintheabsence(gray)orinthepresence
(thickline)ofLPS,fromsortedimmature(upperpanel)andtransitional(lowerpanel)populations.Resultsarerepresentativeofthreeindependentexperiments.
Analysis of CD23+B cells generated in vitro in the presence of LPS from FACS-sorted BM immature and transitional B subsets. Immature
The Journal of Immunology4665
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