of April 14, 2018.This information is current as

Homeostatic and Activated Conditions Cell Population from Adult Mice inloCD45R

+Dynamics of the Splenic Innate-like CD19

Miguel Angel R. Marcos and Maria Luisa GasparAlía, Sharmili Jagtap, Natalia Serrano, Isabel Cortegano, Belén de Andrés, Carmen Prado, Beatriz Palacios, Mario

ol.1200224http://www.jimmunol.org/content/early/2012/07/25/jimmun

published online 25 July 2012J Immunol 

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The Journal of Immunology

Dynamics of the Splenic Innate-like CD19+CD45Rlo CellPopulation from Adult Mice in Homeostatic and ActivatedConditions

Belen de Andres,*,1 Carmen Prado,* Beatriz Palacios,* Mario Alıa,* Sharmili Jagtap,†

Natalia Serrano,† Isabel Cortegano,* Miguel Angel R. Marcos,† and Maria Luisa Gaspar*,1

In the adult spleen, CD19+CD45R2/lo (19+45Rlo) lymphocytes of embryonic origin exist as a distinct population to that of the

conventional B cell lineage. These cells display a plasmablast phenotype, and they spontaneously secrete IgG1 and IgA, whereas

the bone marrow population of 19+45Rlo cells contains B1 progenitors. In this study, we show that 19+45Rlo cells are also present in

Peyer’s patches and in the spleen throughout the life span of wild-type mice, beginning at postnatal day 7. Although this

population is heterogeneous, the surface phenotype of most of these cells distinguishes them from follicular, transitional, marginal

zone, and B1 cells. In CBA/CaHN mice, few 19+45Rlo cells were detected at postnatal day 7, and none was observed in the adult

spleen. Splenic 19+45Rlo cells exhibited homeostatic BrdU uptake in vivo and actively transcribed cell cycle genes. When trans-

ferred to immunodeficient RAG22/2gchain2/2 recipient mice, 19+45Rlo cells survived and differentiated into IgG1– and IgA–

plasma cells. Moreover, in vitro stimulation of splenic 19+45Rlo cells with LPS, CpG, BAFF/IL4, and CD40/IL4 induced cell

proliferation, IgG1/IgA secretion and the release of IL-10, suggesting a potential immunoregulatory role for this subset of innate-

like B cells. The Journal of Immunology, 2012, 189: 000–000.

In peripheral organs, lymphocytes shape the adaptive immunesystem responses by performing specialized functions, andthey can be categorized as either innate-like or conventional

adaptive cells. Innate-like B cells are B1 cells and marginal zone(MZ) B cells, whereas the adaptive branch mainly contains fol-licular B (FoB) cells. B-1 and MZ cells share some characteristicfeatures such as the oligoclonality of their BCRs (1, 2), Ag-independent selection (3), their distribution in particular niches,a preactivation status that is responsible for their rapid multi-specific responses (4), and their participation in maintaining ho-meostasis and in the initial phases of the immune response (5).Recently, several new innate like B lymphocytes have been de-

scribed, such as age-associated B cells (ABCs) (6, 7), gut-associatedB cells (8), and innate response activator (IRA) cells (9), which areimplicated in autoimmune disorders and the responses to microbialinfection.The peripheral B cell compartments are maintained by a series of

coordinated mechanisms. The new immature precursors compete forlimited resources, and they must pass through sequential BCRcheckpoints, which limit the number of progenitors that successfullydifferentiate (10). Furthermore, the survival and differentiationof cells are conditioned by their responses to BCR- and TLR-activating signal pathways (11) and by their access to selectedcytokines and niches (12). Soluble factors such as BAFF and aproliferation-inducing ligand (APRIL) provide survival and prolif-erative signals (13), and they induce isotype switching in mature B(MB) cells (14). Activated B cells mediate immune regulationthrough the secretion of Igs and a wide range of cytokines, all ofwhich modulate T cell activity (15–17).In the mouse embryo, studies of the development of hemato-

poietic programs revealed the first unilineage B cell progenitorto be CD19+AA4.1+Pax-5+, which lacks the CD45R/B220phosphatase (18, 19). In mouse bone marrow (BM), the CD19+

CD45R2/lo (19+45Rlo) cell population (hereafter referred to as 19+

45Rlo) is an immature population containing B1 progenitors (20–22). We previously described the presence of 19+45Rlo cells ofembryonic origin in the spleen of juvenile and adult mice (23).These cells have a singular phenotype (CD52CD11b2CD21lo

CD232IgD2) with bimodal expression of IgM and spontaneouslyrelease IgG1 and IgA.In the current study, we analyzed the homeostatic behavior and

maintenance of embryo-derived 19+45Rlo cells, as well as theircapacity for in vitro activation. We show that 19+45Rlo cells arepresent in the spleen and Peyer’s patches (PP) and as circulatingcells throughout the life span of the mouse and that the cellnumber in the spleen is tightly controlled until 1 y of age. Thesurface phenotype of this cell type distinguishes them from tran-sitional B cells, B1 cells, IRA B cells, and ABCs, although like B1

*Centro Nacional de Microbiologıa, Instituto de Salud Carlos III, Majadahonda,28220 Madrid, Spain; and †Centro de Biologıa Molecular Severo Ochoa, ConsejoSuperior de Investigaciones Cientıficas, Campus de Cantoblanco, 28049 Madrid,Spain

1B.d.A. and M.L.G. contributed equally to this study.

Received for publication January 19, 2012. Accepted for publication June 27, 2012.

This work was supported by grants from the Fondo de Investigaciones Sanitarias(MPY 1374/08), the Comunidad Autonoma de Madrid (SAL-0304-2006), and theMinisterio de Ciencia e Innovacion (SAF2007-65265 and SAF2009-12596). N.S.received a fellowship from the Centro de Biologıa Molecular Severo Ochoa, I.C.received a fellowship from the Ministerio de Ciencia e Innovacion, B.P. receiveda fellowship from the Fondo de Investigaciones Sanitarias, and S.J. received a fellow-ship from the Comunidad Autonoma de Madrid. The Centro de Biologıa Molecular,Severo Ochoa, receives institutional funding from the Fundacion Ramon Areces.

Address correspondence and reprint requests to Dr. Belen de Andres, Unidad deInmunobiologıa, Centro Nacional de Microbiologıa, Instituto de Salud Carlos III,Carretera de Majadahonda a Pozuelo km 2,5, Majadahonda, 28220 Madrid, Spain.E-mail address: bdandres@isciii.es

Abbreviations used in this article: ABC, age-associated B cell; APRIL, a prolifera-tion-inducing ligand; BM, bone marrow; btk, Bruton’s tyrosine kinase; CT, thresholdcycle; FoB, follicular B; IL12p70, p70 subunit of IL-12; IRA, innate response acti-vator; LN, lymph node; MB, mature B; MZ, marginal zone; PB, peripheral blood;PC, plasma cell; PD, postnatal day; PP, Peyer’s patches; PWC, peritoneal washedcell; 19+45Rlo, CD19+CD45R2/lo; Rag2g2/2, RAG22/2gchain2/2; RT-qPCR, quan-titative real-time PCR; T1, transitional 1.

Copyright� 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

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cells, they are Bruton’s tyrosine kinase (btk) dependent. 19+45Rlo

cells actively express cell-cycling genes and exhibit a high rate ofproliferation under homeostatic conditions in vivo. When trans-ferred to immunodeficient RAG22/2gchain2/2 (Rag2g2/2) mice,a population of IgG1/IgA-secreting 19+45Rlo cells was establishedand maintained in the spleen. Moreover, 19+45Rlo cells respondedto stimulation with TLR ligand and BAFF/IL4 by undergoingisotype class switching and secreting IL-10. We conclude that 19+

45Rlo cells constitute an activated component of the innate-likeB cell population with an exceptional ability to release IgG1 andIgA and promising immunoregulatory properties based on theirproduction of IL-10.

Materials and MethodsMice

BALB/c, C57BL/6, C57BL6.Ly5.1, CBA/CaHN (CBA/N), CBA/CaJ, andRag2g2/2 mice were bred and maintained in animal facilities at theInstituto de Salud Carlos III and the Centro de Biologıa Molecular SeveroOchoa. Animals were sacrificed by cervical dislocation, and cell suspen-sions were obtained subsequently from the lymphoid organs, peripheralblood (PB), and peritoneal exudates (peritoneal washed cells [PWC]). Allprocedures were carried in accordance with approved protocols andguidelines established by the Institutional Animal Care and Use Com-mittees of the Instituto de Salud Carlos III and the Centro de BiologıaMolecular Severo Ochoa.

Flow cytometry and cell sorting

Single-cell suspensions were prepared in staining buffer (2.5% FCS inDulbecco’s PBS; Biowhittaker, Lonza Group, Basel, Switzerland), andnonspecific binding was blocked with Fc block (BD Biosciences, San Jose,CA). Staining was performed using standard protocols with the Abs andsecondary reagents listed in Supplemental Table I. The cells were analyzedon a FACSCanto I using the FlowJo version 6.3.4 software package (TreeStar, Ashland, OR), and they were purified by FACS using a FACSAria I(BD Biosciences) apparatus with DIVA version 6.1 software. T cell de-pletion was performed by treatment with anti-Thy1.2 (J1J clone) and rabbitcomplement (Cedarlane Laboratories, Hornby, ON, Canada) prior to thesorting procedure to isolate splenic B cell subpopulations. B1 cells in PWCwere defined as IgM+CD232 populations. Samples used for adoptivetransfer and PCR array experiments were submitted to a two-round sortingprocedure to achieve 99% purity.

In vivo BrdU incorporation

Two-month-old BALB/c mice were inoculated i.p. with 0.6 mg/mouse ofBrdU (Sigma-Aldrich, St. Louis, MO) every 12 h for different periods oftime, and surface Ag expression and intracellular BrdU incorporationwere determined at the indicated time points, as described previously(24). Briefly, cells were stained with anti–CD19-PE and anti–CD45R-allophycocyanin to identify 19+45Rlo cells or with anti–CD23-biotin/streptavidin-PE and anti–CD93-allophycocyanin to discriminate betweentransitional 1 (T1) cells and MB cells (Supplemental Fig. 1). The cellswere then washed, fixed, and permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences) and subsequently incubated with DNaseI (Sigma-Aldrich). Next, the cells were stained using the Live/Dead Ex-clusion Fixable Violet Dead Cell Stain kit (Invitrogen, Carlsbad, CA) andthen with anti–BrdU-FITC or isotype FITC (BD Biosciences). Two tothree mice were analyzed for each time point.

In vivo adoptive transfer

Splenic 19+45Rlo, 19+45R+ and peritoneal B1 cells from C57BL/6.Ly5.1mice were FACS purified by double sorting and inoculated i.v. into irra-diated (1.5 Gy) 8- to 12-wk-old Rag2g2/2 hosts (2–3 3 104 cells/mouse).Three mice were sacrificed at each time point, 20, 40, 60, and 90 d aftercell transfer, and serum was collected from the mice and stored at 220˚Cfor ELISAs. The presence of Ly5.1 cells in BM, spleen, and peritonealexudate samples was measured by flow cytometry.

PCR array

RNA from purified B cell populations was extracted with the TRIzol reagent(Invitrogen), and the cDNAs prepared with the RT2 First Strand kit(SABiosciences, Qiagen, Valencia, CA) were preamplified using the RT2

Nano PreAMP cDNA cell cycle synthesis kit (SABiosciences). The ex-

pression of cell cycle genes was analyzed in samples using the mouse cellcycle RT2 Profiler PCR array (SABiosciences). The PCR array dataanalysis web portal (http://www.SABiosciences.com/pcrarraydataanalysis.php) was used to calculate the threshold cycle (CT) values and to obtainDCT values normalized to those of housekeeping genes. To determine thedifferences in relative expression between B cell populations, comparativeanalyses were performed (22DDCT) using the PCR Array Data AnalysisSoftware (SABiosciences).

Quantitative real-time PCR

Quantitative real-time PCR (RT-qPCR) was performed as previously de-scribed (23) with the following IL-10 primers: sense, 59-TCGATTTCTC-CCCTGTGA-39; and antisense, 59-GACACCTTGGTCTTGGAGCT-39.DCT values normalized with the HPRT gene were calculated.

ELISAs

ELISAs were performed using sera collected from transferred mice andin vitro culture supernatants, as described previously (25). Standard curvesfor each isotype were generated using the following purified myelomaproteins or Abs: IgM (clone G155-228; BD Biosciences), IgG1 (clone4.19) (26), and IgA (clone M18-254; BD Biosciences). GraphPad Prism4.0 software was used to calculate Ig concentrations.

CFSE labeling

Purified 19+45Rlo and 19+45R+ cells were incubated in darkness at a den-sity of 105 cells/100 ml in 5 mM CFSE dissolved in PBS/0.1% BSA. After15 min at room temperature, the cells were washed twice before being usedfor in vitro stimulation.

B cell cultures

CFSE-labeled cells (40,000 cells/well) were plated in flat-bottom 96-wellculture plates (BD Biosciences) in RPMI 1640 supplemented with 10%heat-inactivated FCS, 2 mM L-glutamine, 1 mM pyruvate, 50 mM 2-ME,10 mM HEPES, and antibiotics (complete RPMI 1640). Cells were incu-bated at 37˚C and 5% CO2 in a humidified atmosphere for 96 h in thepresence or absence of 10 mg/ml polyclonal goat anti-mouse IgM (JacksonImmunoResearch Laboratories, West Grove, PA), 25 mg/ml LPS (Difco,Detroit, MI), 1 mg/ml BAFF (R&D Systems, Minneapolis, MN), 1 mg/mlAPRIL (PeproTech, Rocky Hill, NJ), 150 ng/ml IL-4 (PeproTech), 10 mg/ml hamster anti-mouse CD40 (HM40-3; eBioscience, San Diego, CA), or10 ng/ml IL-10 (PeproTech). In 72-h cultures, oligodeoxynucleotides S-ODN-1826 (59-TCCATGACGTTCCTGACGTT-39) and S-ODN-1911 (59-TCCAGGACTTTCCTCAGGTT-39; Sigma-Aldrich) were used at 1 mM asCpG DNA and non-CpG control DNA, respectively (27). For intracellularIL-10 staining, B-enriched cells from T cell-depleted spleens were culturedat 2 3 106 cells/ml in complete RPMI 1640, and they were stimulated asindicated above for 72 h. Brefeldin A (10 mg/ml) was added to thesecultures for the final 3.5 h. At the time points indicated, the culturesupernatants were collected for quantification of soluble Ig and cytokines,and cultured cells were recovered for flow cytometry studies.

Cytokine detection

Supernatants from cultured B cells were used to measure IL-6, IL-10, andthe p70 subunit of IL-12 (IL12p70) using the Milliplex MAP mousecytokine/chemokine kit (Merck, Darmstadt, Germany). Murine cytokines ofknown concentrations were used to generate standard curves to calculate thecytokine concentrations in the samples. Supernatants from cultures of themurine Th2 clone D10.G4.1 were used as positive controls for IL-6 and IL-10 (28). To detect cytoplasmic IL-10 in individual cells, LPS and BAFF/IL4-stimulated B cells were washed and stained for surface CD19 andCD45R expression, before they were incubated with the Live/Dead Ex-clusion kit. Stained cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences), prior to intracellular cytokine stainingwith anti–IL-10-PE, and the isotype-PE control (eBioscience), as describedpreviously (28). For in vivo GM-CSF detection, injections of LPS (10 mg/ml) were performed with four daily i.p. injections of LPS as describedpreviously (9), and the cytoplasmic detection was as indicated above forIL-10.

Statistical analysis

The data are presented as the means 6 SEM and all statistical analyseswere performed using GraphPad Prism 4.0 software. The p values werecalculated with the two-tailed Student t test (*p, 0.05, **p, 0.01, ***p,0.001).

2 CELL DYNAMICS OF SPLENIC CD19+CD45Rlo CELLS

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ResultsLymphoid organ distribution and postnatal progression of 19+

45Rlo cells in normal mice

We previously reported that 19+45Rlo cells of embryonic origin arepresent in the spleen of infant mice (23). In this study, we tracedthese cells to different lymphoid locations beginning at postnatalday (PD) 7. We detected these cells in the spleen, PB, and PP ofnormal, unmanipulated BALB/c, C57BL/6, and CBA/CaJ adultmice but not in the thymus or lymph nodes (LN; Fig. 1A, 1B).CBA/N mice bearing the btk mutation exhibited a 30–50% re-duction in FO cells and a severe reduction of B1a and B1b sub-populations. We confirmed that btk-deficient CBA/N micecontained very few 19+45Rlo cells in these structures, suggestinga strong btk dependency of these cells, as reported elsewhere(23). However, 19+45Rlo cells did not express CD11b or CD5 (Fig.1C). Spleens from BALB/c mice were colonized early after birthby 19+45Rlo cells (Fig. 1D), which accounted for up to 11.2 6 2%(n = 8) of the total splenic B cells at PD7. In absolute terms, thenumber of 19+45Rlo cells was maintained constant until adulthood(Fig. 1D). Surprisingly, young CBA/N spleen samples had more19+45Rlo cells than those from PD30 or adult CBA/N mice, al-though this number was 3-fold less than those detected in controlage-matched mice. The adult PP contained a significant number of19+45Rlo cells, which were first detected at PD30.We examined the relationship between adult 19+45Rlo cells and

transitional B cell subsets in the spleen by analyzing the expres-sion of CD24, CD21, and CD93, as described previously (29). The19+45Rlo cells were mapped outside the transitional CD21/CD24B cell subsets, although CD93 was expressed at similar levels in19+45Rlo and 19+45R+ cells (Fig. 1E). Splenic 19+45Rlo cellsdiffered also from the recently described ABCs (6, 7), which areB220/CD45R+, CD11b/CD11c+, CD80+, and CD86+, whereas 19+

45Rlo cells were B220/CD45R2/lo and negative for CD11b,CD11c, CD80, and CD86 (Fig. 1A, 1C, 1F). Finally, althoughexpression of the preplasmablast marker CD138 (30, 31) wasminimal in splenic CD19+CD45R+ cells (0.9 6 0.4%; n = 8), itwas more abundant in 19+45Rlo cells (6.8 6 1.7%; n = 8), both inthe IgM2 and IgM+ subsets of this population from the spleen andPP samples (Fig. 1E, 1G). Taken together, these findings suggestthat these cells are in a natural preactivation state.These phenotypic data show that 19+45Rlo cells are distributed

in the spleen, PP, and PB from infant stages. In the spleen, thispopulation could be clearly distinguished from the Transitionalcell subsets, conventional MB cells, ABC, and B1 cells. Theseobservations, together with our previous confocal microscopyfindings demonstrating a perifollicular distribution of 19+45Rlo

cells (23), suggest that these cells represent a B cell subset ofembryonic origin.

In vivo BrdU uptake in splenic 19+45Rlo cells

On the basis of the conservation of 19+45Rlo cell number observedthroughout life, we investigated their maintenance in vivo. Wepreviously found that these cells were of embryonic origin,spontaneously incorporated thymidine, and expressed the nuclearAg Ki67 (23). In this study, we determined their proliferativestatus in vivo by analyzing BrdU incorporation after continuousi.p. injection over 17 d, which allowed us to study the kinetics ofBrdU labeling in distinct peripheral B cell populations bycytometry. Electronically gated 19+45Rlo cells, T1 stage cells(CD232CD93+), and MB cells (CD23+CD932) were analyzed(Fig. 2A) and as described previously, maximal BrdU incorpora-tion was observed in the T1 cell subset (Fig. 2B) (30, 32). Bycontrast, only 11.6 6 1.2% (n = 3) of MB cells were labeled as

a resting population. Splenic 19+45Rlo cells incorporated BrdUcontinuously, with 43 6 1.3% (n = 3) of cells labeled on day 17.No significant differences in BrdU labeling were detected betweensubsets of 19+45Rlo cells, regardless of the expression of IgM,IgG, or CD24 (Fig. 2B, Supplemental Fig. 1). However, 19+45Rlo

FIGURE 1. Lymphoid organ distribution and postnatal development of

19+45Rlo cells. (A) Cells from the organs indicated of BALB/c and CBA/N

mice were stained with anti-CD19 and anti-CD45R and analyzed by flow

cytometry. Representative dot plots are shown in which the numbers

represent the frequencies as percentages (mean6 SEM; n = 8) of 19+45Rlo

cells (ellipse in the left dot plot) of the lymphoid gate. (B) Bar graphs show

the number of 19+45Rlo cells/organ (mean6 SEM; n = 6) in the spleen and

PP of different strains of mice at 2 mo of age. Absolute numbers were

calculated using the frequencies obtained by cytometry and the total

number of lymphoid cells counted in each sample. **p , 0.01; ***p ,0.001. (C) Representative examples of electronically gated 19+45Rlo (left),

19+45R+ (middle), and of total spleen cells (right) from BALB/c mice,

showing staining with anti-CD11b and anti-CD5. (D) The dot diagrams

represent the number of 19+45Rlo cells/organ at the ages indicated. Indi-

vidual values are represented by dots, and the mean values are represented

by horizontal bars. Comparisons between BALB/c and CBA/N mice of the

same ages were performed, with significance values shown in the boxes.

The values obtained at #PD15 versus $PD30 were also compared. **p ,0.01; ***p , 0.001. (E) Staining with anti-CD24, anti-CD21, and anti-

CD93 defined subsets of cells (upper and middle dot plots) in electroni-

cally gated 19+45Rlo and 19+45R+ spleen cells: T1, T2, and MZ precursor

(MZP)/MZ. Lower dot plots show CD138 and IgM expression in both

populations. Numbers represent the frequency of the cells in the boxes

(mean 6 SEM; n = 5 and 8 for CD93/CD24 and CD138/IgM staining,

respectively). (F) Expression of CD11c, CD80, and CD86 on electronically

gated 19+45Rlo cells. (G) Representative histograms of cells from PP

stained with anti-CD19, anti-CD45R, and anti-CD138 (thick line) or iso-

type (dotted line). The scale in the cytometry plots is logarithmic.

The Journal of Immunology 3

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cells that were CD93+ and CD432 incorporated significantly moreBrdU than 19+45RloCD932/CD43+ cells (p, 0.001 and p, 0.05,respectively), suggesting that these CD93+CD19+CD45Rlo cellsmay be newly formed B cells and emphasizing the heterogeneouscomponent of the 19+45Rlo cell subset. We also analyzed thepersistence of BrdU when its administration was ceased after8 d of continuous injections (Fig. 2C). BrdU labeling was notevident 21 d later in T1 cells, whereas BrdU+19+45Rlo andMB cells persisted at this time.In summary, our data demonstrate the heterogeneity of splenic

19+45Rlo cells, with ∼50% of these cells proliferating spontane-ously and ∼25% of the 19+45Rlo cells that divide being main-tained for extended periods.

Transcriptional cell cycle signature of 19+45Rlo cells

The spontaneous proliferative status of 19+45Rlo cells was studiedin more detail by analyzing the expression of a broad range of cellcycle-related genes in PCR arrays. Using the 22DDCT method andusing the values obtained from splenic 19+45R+ cells as a refer-ence, we performed a comparative analysis between FACS-purified splenic 19+45Rlo, 19+45R+, and peritoneal B1 cells(Fig. 3). Several genes were differentially expressed in 19+45Rlo

versus 19+45R+ cells, showing differences of .2-fold. In partic-ular, 17 genes were more strongly (3-fold) expressed in 19+45Rlo

cells. These included genes involved in regulating G1 and G1-S(Camk2a, Itgb1, c-myb, and Nfatc1), DNA replication (Ki67), Mphase (Ccna1, Ccnb1, Nek2, and Wee1), cell cycle checkpointcontrol (Ak1, Cdkn1a, Cdkn2a, Dst, and Pmp22), and positive/negative cell cycle progression (Ccna2, E2f2, and Trp63, respec-tively). Only two genes (Notch2 and Slnf1) were expressed moreweakly in 19+45Rlo than 19+45R+ cells. Comparison of B1 and19+45R+ cells revealed changes in the expression of several genes;seven genes were more strongly expressed in B1 cells, whereastwo genes were expressed more weakly. Direct comparison of 19+

45Rlo and peritoneal B1 cells (the latter used as a reference)highlighted the profound differences between the two subsets ofcells. Changes in expression of up to 3-fold were observed for 16genes, including c-myb, Ki67, Ccnb1, Cdc25a, Npm2, Prm1,Cks1b, Dst, Gadd45a, Inha, Ccna2, and Trp63 (stronger expres-

FIGURE 2. Peripheral BrdU turnover in different B cell compartments.

(A) Flow cytometry analysis to define splenic 19+45Rlo cells (ellipse in the

left dot plot) was performed using anti-CD19 and anti-CD45R. Anti-IgM

was also used to identify IgM2 (m2) and IgM (m+) 19+45Rlo cells. Anti-

CD93 and anti-CD23 mAbs were used to identify T1 cells (CD93+CD232,

square) and MB cells (CD932CD23+, rectangle). Dead cells were excluded

using the Live/Dead Exclusion kit. The incorporation of BrdU over time

was determined using anti-BrdU and isotype nonspecific incorporation.

(B) Schematic design of the BrdU i.p. injection protocol. Left graph, Kinetics

of BrdU incorporation by 19+45Rlo cells (n), T1 (;), and MB (s). Results

represent mean 6 SEM of one of three experiments (three mice per point).

The histograms on the right show representative profiles of BrdU incorpo-

ration on day 17 (thick line), with the isotype signal overlaid (dotted line). In

the 19+45Rlo cell population, m and m2 cells were electronically gated for

analysis. (C) Schematic outline of BrdU decay experiments. BrdU was

injected i.p. for 8 d, and BrdU-labeled cells were monitored for different time

periods after ceasing BrdU administration. Left graph, Kinetics of BrdU

incorporation. Right overlaid histograms, Profile of BrdU (thick line) and

isotype (dotted line) obtained in the cells indicated at 5 and 21 d after ceasing

BrdU administration. Results are represented as in (B).

FIGURE 3. RT-qPCR array results comparing cell cycle genes in B cell

populations. Anti-CD19 and anti-CD45R were used to purify splenic 19+

45Rlo and 19+45R+ populations. Anti-CD23 and anti-IgM Abs were used

to purify B1 cell populations from PWC. RT-qPCR was performed as

described in Materials and Methods. Data were from three different

preparations of 19+45Rlo, 19+45R+, and B1 cell samples. Results were

normalized by the 22DDCT method, relative to 19+45R+ cell preparations

(left and middle columns) or to B1 cell preparations (right column), and

shown as the fold change. Transcripts with changes .2-fold are displayed

grouped in five different gene clusters: 1, G1 and G1-S phases; 2, S-phase

and DNA replication; 3, M phase; 4, cell cycle checkpoint and arrest; and

5, cell cycle regulation. *p , 0.05, **p , 0.01, ***p , 0.001.

4 CELL DYNAMICS OF SPLENIC CD19+CD45Rlo CELLS

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sion in 19+45Rlo versus B1 cells) and Nek2, Notch2, Pmp22, andSlnf1 (weaker expression versus B1 cells), whereas less dramaticchanges were observed for several other genes. Confirmatory RT-qPCR studies were performed for c-myb, Notch2, and Ki67transcripts in 19+45Rlo, 19+45R+, and peritoneal B1-sorted cells(Supplemental Fig. 2). Taken together, these data reveal remark-able differences in the expression of components of the cell cyclemachinery used by the B cell subpopulations analyzed in thisstudy, reflecting the high proliferative status of 19+45Rlo cells.

Homeostatic proliferation of the 19+45Rlo cell population

To eliminate any possible contribution from the BM in the homeostaticcontrol and life span of 19+45Rlo cell subset, we performed adoptivetransfer experiments into Rag2g2/2 hosts, taking advantage of thefact that a lymphopenic environment maximizes the homeostaticcell proliferation in vivo under physiological conditions (33).Splenic 19+45Rlo, 19+45R+, and peritoneal B1 cells were purifiedby double sorting from C57BL/6.Ly5.1 mice prior to their adop-tive transfer to Rag2g2/2 recipients (Fig. 4A). The repopulation ofdonor-derived Ly5.1+ cells was determined at 20-d intervals invarious organs (Fig. 4B, 4C). Only CD19+ cells were detected inthe Ly5.1+ population of these animals. After 20 d, recipients of19+45Rlo cells exhibited Ly5.1+CD19+ cells in the spleen and inthe peritoneal exudates, with the maximal number reached at 40–60 d. Recipients of B1 and 19+45R+ cells reconstituted a smallerLy5.1+CD19+ cell population in the spleen (1.9- and 5.5-fold less,respectively). In PWC, similar numbers of Ly5.1+CD19+ cellswere observed in recipients of B1 cells as those receiving 19+

45Rlo cells, whereas no Ly5.1+ cells were detected in recipientsof 19+45R+ cells. Neither 19+45Rlo nor 19+45R+ cells repopulatedthe BM. Cells recovered from the spleen exhibited similar CD45Rlevels to those detected in the original transferred cells (Fig. 4D).Moreover, the recovered cells were CD11b2 and displayed min-imal levels of CD5 (range, 3–20%; mean, 10%; n = 6). CD11bexpression was detected in cells from PWC (range, 30–70%; n =6), although no CD5 expression was observed (n = 6). Similarresults were obtained in competitive adoptive transfer experimentsusing adult C57BL/6 recipients after sublethal irradiation (Sup-plemental Fig. 3). After BM efflux arrest, B cells in the peripheryadopt an activated state and a differentiated Ig secretion profile,similar to MZ, B1, or plasma cells (PCs), as shown on IL72/2

mice (34). Analysis of the serum Ig levels in transferred mice (Fig.4E) revealed similar levels of IgM in recipients of 19+45Rlo, 19+

45R+, and B1 cells. By contrast, levels of both IgG1 and IgAweresignificantly higher in mice repopulated with 19+45Rlo cells,further demonstrating a marked differentiation bias toward IgG1and IgA in this homeostatic proliferative context.Taken together with the findings regarding BrdU incorporation,

the reconstitution potential observed and the persistence of the 19+

45Rlo cells demonstrate the innate activation status of the 19+

45Rlo cell population in vivo. Accordingly, these cells can bemaintained in mice under homeostatic conditions while preservingtheir preferential differentiation bias toward IgG1- and IgA-secreting PCs.

In vitro activation of 19+45Rlo cells

To analyze the proliferative and differentiation responses of 19+

45Rlo cells to in vitro stimulation, sorted purified 19+45Rlo and19+45R+ cells were labeled with the cell tracer CFSE prior tostimulation in vitro. The TLR4 agonist LPS, which inducesa proliferative response in all B cell compartments and the dif-ferentiation of MZ and B1 cells to mature PCs (35), induceda potent proliferative response in both 19+45Rlo and 19+45R+

cells. A similar effect was observed when TLR9 was stimulated

with CpG (Fig. 5A). In addition, a marked induction of CD138was detected in LPS-responding cells (Fig. 5B). B cell signalingvia the BAFF and APRIL receptors is associated with survival andPC generation (36). Our results revealed greater proliferativeresponses in both B cell subsets following stimulation with BAFF/IL4 versus APRIL/IL4, although neither of these stimuli resultedin CD138 upregulation. BCR activation with anti-IgM promoteda significant proliferative response in 19+45R+ cells but had noeffect in 19+45Rlo cells. These findings indicate that although IgMwas expressed by 40–50% of 19+45Rlo cells, BCR activation was

FIGURE 4. Adoptive transfer of 19+45Rlo cells repopulates the spleen

and peritoneum. Purified 19+45Rlo and 19+45R+ splenic B cell populations

from C57BL/6.Ly5.1 donors were inoculated into irradiated Rag2g2/2

hosts. Donor-derived cells were identified by the expression of Ly5.1 in

cell suspensions from the spleen, BM, and PWC of the recipients using

anti-Ly5.1, anti-CD19, and anti-CD45R Abs. Cytometry data are from one

representative experiment out of three with similar results. (A) Schematic

outline of the experimental design. (B) Representative reconstitution ex-

periment showing the expression of Ly5.1 and CD19 at 40 d posttransfer.

Numbers are as in Fig. 1A. (C) Total numbers of donor Ly5.1+ cells at

different times in the spleens and PWC of recipients of 19+45Rlo (n), 19+

45R+ (s), and peritoneal B1 (:) cells calculated as in Fig. 1B. Data

represent the mean 6 SEM of three independent experiments performed

with two mice per point. (D) Preparations from spleen and PWC of

recipients of Ly5.1+19+45Rlo and of Ly5.1+19+45R+ cells, analyzed 60 d

posttransfer. For spleen preparations, anti-CD19 and anti-CD45R staining

in electronically gated donor-derived Ly5.1+ cells detected as in Fig. 3B is

shown in the upper zebra plots. Histograms show staining with anti-CD5

or anti-CD11b (thick line), overlaid with the isotype-control staining

(dotted line). For PWC preparations, the upper zebra plots are as described

for the spleen cells, and the bottom zebra plots show staining with Ly5.1,

anti-CD5, and anti-CD11b. (E) Dot plots (logarithmic scale) represent the

serum levels of IgM, IgG1, and IgA in recipients of Ly5.1+19+45Rlo (n),

Ly5.1+19+45R+(s), and B1 cells (:) that were quantified by specific

ELISAs using sera collected from mice between 20 and 60 d posttransfer.

Data are shown as in Fig. 1D. *p , 0.05, **p , 0.01.

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unable to induce proliferative signals or CD138 induction. Acti-vation mediated by anti-CD40/IL4 induced the proliferation ofboth B cell types. Moreover, exposing 19+45Rlo cells to LPS andBAFF/IL4 provoked the release of IgM and IgG1, whereas onlyLPS induced the secretion of IgA (Fig. 5C). Collectively, thesein vitro results demonstrate the potent effect of TLR4, TLR9, andBAFF/IL4 ligation on the final differentiation of 19+45Rlo cells toIgG1- and IgA-secreting PCs.

Cytokine production by activated 19+45Rlo cells

Several reports have described the role of activated B cells incytokine secretion, suggesting that different subsets of cells fulfill

distinct regulatory roles (16, 17). Thus, we analyzed the secretionof several cytokines following in vitro activation of 19+45Rlo cells.Supernatants were collected after in vitro stimulation, performedas described above, and the amount of secreted IL-6, IL-10, andIL12p70 was determined (Fig. 6). LPS stimulation resulted in thesecretion of large amounts of IL6 from purified 19+45R+ lym-phocytes, whereas only minimal release of this cytokine wasdetected in 19+45Rlo cells. By contrast, stimulation of 19+45Rlo

cells with LPS or BAFF/IL4 resulted in a 10-fold increase in IL-10release as compared with 19+45R+ cells. APRIL/IL4 and anti-

FIGURE 5. Differential in vitro responses of 19+45Rlo and conventional

19+45R+ cells. Purified 19+45Rlo and CD19+CD45R+ cells were labeled

with CFSE and cultured for 96 h (LPS, BAFF/IL4, APRIL/IL4, anti-IgM,

and anti-CD40/IL4 and IL-10) or 72 h (CpG or control DNA), and then,

they were subsequently analyzed by flow cytometry to monitor changes in

size (detected by the forward scatter, FCS-A) and CFSE signal. Contour

plots and histograms represent one of three experiments, all of which

revealed similar results. Culture supernatants were stored to determine Ig

secretion. (A) Activation and proliferation of 19+45Rlo and 19+45R+ cells in

response to different stimuli. The box in the contour plots indicates cells

with a high FCS-A that displayed decreasing levels of CFSE. Numbers

represent the frequency of the boxed populations (mean6 SEM; n = 3). (B)

Induction of CD138 after stimulation with LPS and BAFF/IL4 or in control

cells is shown in the histograms. The numbers represent the frequency of

the cells that proliferated and exhibited upregulated CD138 expression

(mean 6 SEM; n = 3). (C) IgM-, IgG1-, and IgA-specific quantification in

the supernatants collected was performed by isotype-specific ELISA (mean6SEM; n = 6). Comparisons for each isotype were performed using data ob-

tained for supernatants from the cultures of 19+45Rlo and 19+45R+ cells

(boxed asterisks). *p , 0.05, **p , 0.01, ***p , 0.001.

FIGURE 6. Cytokine production by activated B cells. (A) Determination

of IL-6, IL-10, and IL12p70 in supernatants from cultures of purified 19+

45Rlo (n) and 19+45R+ (N) cells, stimulated as indicated in Fig. 5C. Control;

D10.G4.1, IL-6–, and IL-10–producing clone. Numbers indicate the amount

of cytokine detected in the D10.G4.1 supernatants. The figure shows the

results from three independent experiments performed in duplicate. *p ,0.05, **p , 0.01. (B) Intracellular determination of IL-10 by flow cytom-

etry. Enriched spleen B cells were incubated with LPS and BAFF/IL4 before

performing surface and intracellular staining. Contour plots show data from

one representative experiment out of five with similar results. Dead cells

were excluded using the Live/Dead cell exclusion kit. At least 3 3 105 live

cells were analyzed and a minimum of 3 3 103 events is shown in the 19+

45Rlo plots. The numbers shown represent the frequency of the cells in the

dotted boxes. The bar graph (right) represents the frequency of specific IL-

10–producing cells in unstimulated (control), LPS-, and BAFF/IL4-activated

cells after subtracting the isotype-matched Ab background from purified 19+

45Rlo (n) and 19+45R+ (N) cells (mean 6 SEM; n = 3). **p , 0.01. (C) RT-

qPCR analysis of constitutive IL-10 mRNA expression in 19+45Rlo cells (n),

19+45R+ cells (s), peritoneal B1 cells (:), and LPS-stimulated (25 mg/ml)

splenocytes after 48 h in culture (LPS blasts, ♦). The DCT values (loga-

rithmic scale) of each transcript over HPRT were calculated as described in

Supplemental Fig. 2 (**p , 0.01). (D) Intracellular determination of GM-

CSF by flow cytometry in B cell splenocytes after four daily i.p. injections

of LPS. The results are displayed as in (B), showing data from one repre-

sentative experiment out of three with similar results. Dead cells were ex-

cluded as in (B), and .5 3 105 live cells are analyzed.

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CD40/IL4 also induced low levels of IL10 secretion by 19+45Rlo

cells, whereas no IL12p70 was detected in any of the in vitroconditions assayed. We also quantified IL-10 secretion in indi-vidual 19+45Rlo cells stimulated with LPS or BAFF/IL4, mea-suring intracellular IL-10 by cytometry (Fig. 6B). CytoplasmicIL10 was detected in higher numbers of 19+45Rlo cells stimulatedwith LPS and BAFF/IL4 than in stimulated 19+45R+ cells. Inaddition, constitutive expression of IL10 mRNA was stronger in19+45Rlo cells than in CD19+CD45R+ or B1 cells in vivo (Fig.6C). However, the addition of IL-10 to purified 19+45Rlo cellsrevealed no proliferative effect of this cytokine in these cell sub-sets (Fig. 5A). Finally, TLR4 activation, which promotes the se-cretion of GM-CSF by IRA cells (9), failed to induce cytoplasmicexpression of GM-CSF by 19+45Rlo cells (Fig. 6D). In summary,these data demonstrate that in vitro activation of 19+45Rlo cellsresults in their proliferation and differentiation to IgM-, IgG1-, andIgA-secreting PCs and in active production of the immunoregu-latory cytokine IL-10, which is constitutively expressed in vivo.

DiscussionIn the current study, we demonstrate the presence of 19+45Rlo cellsin the spleen, PB, and PP of normal unmanipulated mice. In thespleen, the number of 19+45Rlo cells is maintained from infantstages to adulthood as cells different from transitional, MZ, FoB,and B1 cells. Moreover, these cells are in an innate activatedproliferative state in which cyclin-dependent cascades are in-duced. This homeostatic proliferation is also witnessed by theirprolonged survival in immunodeficient animals, indicating thatthey require no influx of progenitors from the BM in which 19+

45Rlo cells maintain their initial 19+45Rlo phenotype and differ-entiate to IgG1- and IgA-switched PCs. We also present in vitroevidence demonstrating the proliferative and differentiationresponses of 19+45Rlo cells to T-dependent and -independentstimuli, and in the case of TLR and BAFF/IL4 stimulation, therelease of the immunoregulatory cytokine IL-10.The rapid neonatal colonization of the spleen by 19+45Rlo cells,

their dramatic btk dependency in juvenile CBA/N mice, and theirability to release IgG and IgA in 7-d-old mice (23) suggest aprotective or regulatory role in primary/neonatal Ag encountersinvolving btk-dependent mechanisms. Btk-dependent B1 cellshave a major embryonic origin whereas B2 cells are BM derived.Furthermore, B1 cells can largely persist by self-replenishmentthroughout adult life (37). Therefore, the embryonic origin of19+45Rlo cells (23), their absence from the spleen of adult CBA/Nmice and their role in the BM as progenitors of peritoneal B1 cells(20–22) situate splenic 19+45Rlo cells close to the B1 lineage. Thisproposal is further supported by the observed induction of CD11bin the peritoneal cavity of immunodeficient mice repopulated withthese cells. These results are supported by the observed presenceof splenic B1 progenitors with a 19+45Rlo phenotype (38). Inaddition, 19+45Rlo cells constitutively express IL-10 transcripts,and they exhibit spontaneous ERK phosphorylation (data notshown), as previously described for B1 cells (39, 40). 19+45Rlo

cells exhibit no CD5 or CD11b expression in the adult spleen, and19+45Rlo cells are present in the infant CBA/N spleen, as well asthey are found in PP and PB, which are conventional B2 organs(41). The transcriptional fingerprint, which involves higher con-stitutive Blimp-1 expression (Supplemental Fig. 2) (23) and thespontaneous release of isotype-switched Igs, further distinguishesthe 19+45Rlo cell population from B1 cells. As compared withABCs, which are quiescent cells derived from FoB cells that ac-cumulate slowly with age (6, 7), 19+45Rlo cells did not increase inaged mice and exhibited major differences in their surface phe-notype, developmental origin, proliferative status, and TLR-

dependent activation (ABCs are unresponsive to LPS). The re-cently described IRA B cells (9) constitute a novel innate cellsubset, yet they have a distinct phenotype, secretion profile, andontogeny to most 19+45Rlo cells. IRA B cells express CD45R/B220, they are CD138+CD21loCD93+CD23lo, they secrete IgMand GM-CSF (not IgG, IgA, and IL-10), and they are produced inthe spleen from mature circulating B1a peritoneal cells upon TLRstimulation.In vivo BrdU labeling demonstrated considerable turnover of 19+

45Rlo cells under homeostatic conditions, which was significantlyhigher than MB but did not reach the level observed in T1 cells. Asmall subset of 19+45Rlo cells shared some features with newlyformed transitional stage T1 cells (CD212CD232CD93+BrdU+).However, the differential expression of CD24 and CD43 (Fig 1E,Supplemental Fig. 1) and the responsiveness to BAFF distin-guished 19+45Rlo cells from these populations. Maintaining themature B cell compartments requires an adequate balance betweenproliferation and apoptosis and tight regulation of differentiationprograms. BrdU decay experiments revealed that this analogpersisted in 19+45RloBrdU+ cells for ∼1 m after cessation theinjections, which indicates that following one or several rounds ofdivision, these cells remain in G0 phase, favoring their final dif-ferentiation. In support of this hypothesis, we observed strong c-myb and weak Notch2 transcription, genes implicated in thetranscriptional profile of highly differentiated B cells (42).Adoptive transfer experiments using immunodeficient animalsfurther supported the proposed innate-like nature of 19+45Rlo

cells, which are capable of self-maintenance while preservingtheir preferential differentiation status in a homeostatic context.To ascertain the functional role of this cell population, we an-

alyzed the proliferative and differentiation responses of 19+45Rlo

cells to different stimuli, including TLR and BCR ligation, T-dependent signals, and soluble survival factors. TLR4 ligation byLPS stimulation induced a potent proliferative reaction and thestrongest differentiation response in these cells. Indeed, pro-nounced CD138 expression was induced in CFSE low-respondingcells and CFSE high nonproliferating 19+45Rlo cells, as well asthe release of IgM and isotype-switched Igs (IgG1 and IgA).Similar findings were obtained following TLR9 stimulation (CpG)of 19+45Rlo cells, suggesting the relevance of TLR-dependentactivation of these cells. Activation mediated by anti-CD40 plusIL-4 induces a potent proliferative and differentiation signalingcascade in B lymphocytes (43). A similar effect was observed in19+45Rlo cells, with the proliferative response indicating that T-dependent signals can regulate the functional response of 19+

45Rlo cells. Indeed, a cell population defined as CD19+B2202

BST-2+ (specific for viral Ag) mediated Ag presentation functionsand was described in the periphery of mice postinfection withWest Nile encephalitis virus (44). BCR ligation with anti-IgM hadno effect on 19+45Rlo cells, despite their expression of IgM. Ef-fective BCR–cross-linking stimulation depends both on the qual-ity of the interaction (45) and the concomitant coligation ofcoreceptors that modulate receptor engagement by enhancingBCR signaling (CD19, CD21, and FcgRIIb) (46). BCR activationin early T1CD212 cells induces apoptosis, and thus, late transi-tional cells become resistant to BCR-induced death (47). The lowlevels of CD21 found in 19+45Rlo cells may contribute to theirrefractory response after BCR ligation. Additional mechanisms,such as the differential expression of other coreceptors, inhibitorytransduction mechanisms, or the presence of CD93+ immaturepreplasmablasts, may also operate in these cells.The survival factor BAFF exerts its action by activating three

receptors, B cell maturation Ag, transmembrane activator calciummodulator and cyclophilin-ligand interactor, and BAFF receptor,

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whereas APRIL binds only to B cell maturation Ag and trans-membrane activator calcium modulator and cyclophilin-ligandinteractor. Our results point to BAFF receptor as the main par-ticipant in BAFF-mediated activation of 19+45Rlo cells, becauseactivation with APRIL/IL4 induced only a weak response. Bycontrast, activation by BAFF/IL4 triggered an important prolif-erative and differentiation response. Activated B cells mediateimmune regulation by secreting cytokines (16, 17), which con-tribute to the polarization of subsets of T cells and the formationof cell clusters with other cell types (15). We found that LPS andBAFF/IL4 activation of 19+45Rlo cells induced significant IL-10release. IL-10 induces the downregulation of the inflammatoryresponse in various models of infectious, autoimmune and allergicdiseases (48).The following points summarize our findings: embryo-derived

Pax-5+CD19+CD45RloCD93+IgM2 cells migrate to the postna-tal spleen and PP, displaying a strict dependency on btk activa-tion, and are maintained at these sites throughout the life span ofthe organism. In the adult, splenic 19+45Rlo cells are heteroge-neous, containing IgM2 cells and IgM+ cells, and these latterones may include newly formed B cells, IRA and activated B1cells. These adult splenic cells have a spontaneous proliferativeprofile that involves a network of cyclin-dependent pathwaysand the expression of genes related to their differentiation, whichenables their renewal under homeostatic conditions of prolifer-ation. In vitro LPS stimulation studies revealed the importanceof TLR-dependent activation of 19+45Rlo cells, linking thispopulation with those that recognize T-independent stimuli suchas MZ, B1, and gut-associated B cells, although T-dependentsignals can also activate 19+45Rlo cells. These findings alsodefine a regulatory role for cytokines from activated 19+45Rlo

cells in the immune response to common bacterial/microbialchallenge. In this scenario, these innate-like B cells may rec-ognize highly conserved motifs expressed by common patho-gens, commensal bacteria, or self-Ags, requiring a minimalcontribution of BCR-dependent mechanisms. Although we haveyet no formal proof of a protective role of 19+45Rlo cells inresponses to pathogens, ongoing in vivo studies using severalmodels of activation will further clarify the role of 19+45Rlo cellsin these situations.The characterization of 19+45Rlo cells as an innate-like splenic

B cell subpopulation helps us to understand the organizationalcomplexity of the multiple subsets of B cells, each mediatingdistinct responses and functions, including Ab secretion, cytokineimmunoregulation, and Ag presentation. Furthering our knowl-edge of these innate-like mechanisms is critical to design in-novative strategies to vaccinate against common widespreadpathogens that affect large segments of human newborn and infantpopulations.

AcknowledgmentsWe thank Dr. M.L. Toribio and Dr. M. Garcıa-Peydro for assistance with the

in vivo reconstitution experiments involving the RAG2g2/2 and C57BL/6.

Ly5.1 mice. We also acknowledge the assistance of Dr. P. Portoles with the

detection of cytoplasmic IL-10 and thank Mark Sefton for editorial assis-

tance.

DisclosuresThe authors have no financial conflicts of interest.

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