Homeostatic and Activated Conditions Cell

The Journal of Immunology

Dynamics of the Splenic Innate-like CD19+CD45Rlo Cell Population from Adult Mice in Homeostatic and Activated Conditions

Bele´n de Andre´s,*,1 Carmen Prado,* Beatriz Palacios,* Mario Alı´a,* 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 transferred 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: 2300–2308.

n peripheral organs, lymphocytes shape the adaptive immune scribed, such as age-associated B cells (ABCs) (6, 7), gut-associated system responses by performing specialized functions, and B cells (8), and innate response activator (IRA) cells (9), which are I they can be categorized as either innate-like or conventional implicated in autoimmune disorders and the responses to microbial adaptive cells. Innate-like B cells are B1 cells and marginal zone infection. (MZ) B cells, whereas the adaptive branch mainly contains fol- The peripheral B cell compartments are maintained by a series of licular B (FoB) cells. B-1 and MZ cells share some characteristic coordinated mechanisms. The new immature precursors compete for features such as the oligoclonality of their BCRs (1, 2), Ag- limited resources, and they must pass through sequential BCR independent selection (3), their distribution in particular niches, checkpoints, which limit the number of progenitors that successfully

by guest on October 1, 2021. Copyright 2012 Pageant Media Ltd. a preactivation status that is responsible for their rapid multi- differentiate (10). Furthermore, the survival and differentiation specific responses (4), and their participation in maintaining ho- of cells are conditioned by their responses to BCR- and TLR- meostasis and in the initial phases of the immune response (5). activating signal pathways (11) and by their access to selected Recently, several new innate like B lymphocytes have been de- cytokines and niches (12). Soluble factors such as BAFF and a proliferation-inducing ligand (APRIL) provide survival and proliferative signals (13), and they induce isotype switching in mature B *Centro Nacional de Microbiologıá, Instituto de Salud Carlos III, Majadahonda, (MB) cells (14). Activated B cells mediate immune regulation 28220 Madrid, Spain; and †Centro de Biologıá Molecular Severo Ochoa, Consejo through the secretion of Igs and a wide range of cytokines, all of Superior de Investigaciones Cientı´ficas, Campus de Cantoblanco, 28049 Madrid, which modulate T cell activity (15–17). Spain http://classic.jimmunol.org In the mouse embryo, studies of the development of hemato- 1B.d.A. and M.L.G. contributed equally to this study. poietic programs revealed the first unilineage B cell progenitor Received for publication January 19, 2012. Accepted for publication June 27, 2012. to be CD19+AA4.1+Pax-5+, which lacks the CD45R/B220 This work was supported by grants from the Fondo de Investigaciones Sanitarias phosphatase (18, 19). In mouse bone marrow (BM), the CD19+ (MPY 1374/08), the Comunidad Autońoma de Madrid (SAL-0304-2006), and the 2/lo + lo + Ministerio de Ciencia e Innovacioń (SAF2007-65265 and SAF2009-12596). N.S. CD45R (19 45R ) cell population (hereafter referred to as 19 received a fellowship from the Centro de Biologıá Molecular Severo Ochoa, I.C. 45Rlo) is an immature population containing B1 progenitors (20– received a fellowship from the Ministerio de Ciencia e Innovacioń, B.P. received + lo Downloaded from a fellowship from the Fondo de Investigaciones Sanitarias, and S.J. received a fellow- 22). We previously described the presence of 19 45R cells of ship from the Comunidad Autońoma de Madrid. The Centro de Biologıá Molecular, embryonic origin in the spleen of juvenile and adult mice (23). Severo Ochoa, receives institutional funding from the Fundacioń Ramoń Areces. These cells have a singular phenotype (CD52CD11b2CD21lo Address correspondence and reprint requests to Dr. Beleń de Andre´s, Unidad de CD232IgD2) with bimodal expression of IgM and spontaneously Inmunobiologıá, Centro Nacional de Microbiologıá, Instituto de Salud Carlos III, Carretera de Majadahonda a Pozuelo km 2,5, Majadahonda, 28220 Madrid, Spain. release IgG1 and IgA. E-mail address: [email protected] In the current study, we analyzed the homeostatic behavior and + lo Abbreviations used in this article: ABC, age-associated B cell; APRIL, a prolifera- maintenance of embryo-derived 19 45R cells, as well as their + lo tion-inducing ligand; BM, bone marrow; btk, Bruton’s tyrosine kinase; CT, threshold capacity for in vitro activation. We show that 19 45R cells are cycle; FoB, follicular B; IL12p70, p70 subunit of IL-12; IRA, innate response activator; LN, lymph node; MB, mature B; MZ, marginal zone; PB, peripheral blood; present in the spleen and Peyer’s patches (PP) and as circulating PC, plasma cell; PD, postnatal day; PP, Peyer’s patches; PWC, peritoneal washed cells throughout the life span of the mouse and that the cell + lo + 2/lo 2/2 2/2 2/2 cell; 19 45R , CD19 CD45R ; Rag2g ,RAG2 gchain ; RT-qPCR, quan- number in the spleen is tightly controlled until 1 y of age. The titative real-time PCR; T1, transitional 1. surface phenotype of this cell type distinguishes them from tran- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 sitional B cells, B1 cells, IRA B cells, and ABCs, although like B1

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200224 The Journal of Immunology 2301

cells, they are Bruton’s tyrosine kinase (btk) dependent. 19+45Rlo pression of cell cycle genes was analyzed in samples using the mouse cell 2 cells actively express cell-cycling genes and exhibit a high rate of cycle RT Profiler PCR array (SABiosciences). The PCR array data proliferation under homeostatic conditions in vivo. When trans- analysis web portal (http://www.SABiosciences.com/pcrarraydataanalysis. php) was used to calculate the threshold cycle (C ) values and to obtain ferred to immunodeficient RAG22/2gchain2/2 (Rag2g2/2) mice, T DCT values normalized to those of housekeeping genes. To determine the a population of IgG1/IgA-secreting 19+45Rlo cells was established differences in relative expression between B cell populations, comparative 2 and maintained in the spleen. Moreover, 19+45Rlo cells responded analyses were performed (2 DDCT) using the PCR Array Data Analysis to stimulation with TLR ligand and BAFF/IL4 by undergoing Software (SABiosciences). + isotype class switching and secreting IL-10. We conclude that 19 Quantitative real-time PCR 45Rlo cells constitute an activated component of the innate-like B cell population with an exceptional ability to release IgG1 and Quantitative real-time PCR (RT-qPCR) was performed as previously de- IgA and promising immunoregulatory properties based on their scribed (23) with the following IL-10 primers: sense, 59-TCGATTTCTC- CCCTGTGA-39; and antisense, 59-GACACCTTGGTCTTGGAGCT-39. production of IL-10. DCT values normalized with the HPRT gene were calculated. Materials and Methods ELISAs Mice ELISAs were performed using sera collected from transferred mice and in vitro culture supernatants, as described previously (25). Standard curves BALB/c, C57BL/6, C57BL6.Ly5.1, CBA/CaHN (CBA/N), CBA/CaJ, and 2 2 for each isotype were generated using the following purified myeloma Rag2g / mice were bred and maintained in animal facilities at the proteins or Abs: IgM (clone G155-228; BD Biosciences), IgG1 (clone Instituto de Salud Carlos III and the Centro de Biologıá Molecular Severo 4.19) (26), and IgA (clone M18-254; BD Biosciences). GraphPad Prism Ochoa. Animals were sacrificed by cervical dislocation, and cell suspen- 4.0 software was used to calculate Ig concentrations. sions were obtained subsequently from the lymphoid organs, peripheral blood (PB), and peritoneal exudates (peritoneal washed cells [PWC]). All CFSE labeling procedures were carried in accordance with approved protocols and guidelines established by the Institutional Animal Care and Use Com- Purified 19+45Rlo and 19+45R+ cells were incubated in darkness at a den- mittees of the Instituto de Salud Carlos III and the Centro de Biologıá sity of 105 cells/100 mlin5mM CFSE dissolved in PBS/0.1% BSA. After Molecular Severo Ochoa. 15 min at room temperature, the cells were washed twice before being used for in vitro stimulation. Flow cytometry and cell sorting B cell cultures Single-cell suspensions were prepared in staining buffer (2.5% FCS in Dulbecco’s PBS; Biowhittaker, Lonza Group, Basel, Switzerland), and CFSE-labeled cells (40,000 cells/well) were plated in flat-bottom 96-well nonspecific binding was blocked with Fc block (BD Biosciences, San Jose, culture plates (BD Biosciences) in RPMI 1640 supplemented with 10% CA). Staining was performed using standard protocols with the Abs and heat-inactivated FCS, 2 mM L-glutamine, 1 mM pyruvate, 50 mM 2-ME, secondary reagents listed in Supplemental Table I. The cells were analyzed 10 mM HEPES, and antibiotics (complete RPMI 1640). Cells were incu- on a FACSCanto I using the FlowJo version 6.3.4 software package (Tree bated at 37˚C and 5% CO2 in a humidified atmosphere for 96 h in the Star, Ashland, OR), and they were purified by FACS using a FACSAria I presence or absence of 10 mg/ml polyclonal goat anti-mouse IgM (Jackson (BD Biosciences) apparatus with DIVA version 6.1 software. T cell de- ImmunoResearch Laboratories, West Grove, PA), 25 mg/ml LPS (Difco, pletion was performed by treatment with anti-Thy1.2 (J1J clone) and rabbit Detroit, MI), 1 mg/ml BAFF (R&D Systems, Minneapolis, MN), 1 mg/ml complement (Cedarlane Laboratories, Hornby, ON, Canada) prior to the APRIL (PeproTech, Rocky Hill, NJ), 150 ng/ml IL-4 (PeproTech), 10 mg/

by guest on October 1, 2021. Copyright 2012 Pageant Media Ltd. sorting procedure to isolate splenic B cell subpopulations. B1 cells in PWC ml hamster anti-mouse CD40 (HM40-3; eBioscience, San Diego, CA), or + 2 were defined as IgM CD23 populations. Samples used for adoptive 10 ng/ml IL-10 (PeproTech). In 72-h cultures, oligodeoxynucleotides S- transfer and PCR array experiments were submitted to a two-round sorting ODN-1826 (59-TCCATGACGTTCCTGACGTT-39) and S-ODN-1911 (59- procedure to achieve 99% purity. TCCAGGACTTTCCTCAGGTT-39; Sigma-Aldrich) were used at 1 mMas CpG DNA and non-CpG control DNA, respectively (27). For intracellular In vivo BrdU incorporation IL-10 staining, B-enriched cells from T cell-depleted spleens were cultured 3 6 Two-month-old BALB/c mice were inoculated i.p. with 0.6 mg/mouse of at 2 10 cells/ml in complete RPMI 1640, and they were stimulated as BrdU (Sigma-Aldrich, St. Louis, MO) every 12 h for different periods of indicated above for 72 h. Brefeldin A (10 mg/ml) was added to these time, and surface Ag expression and intracellular BrdU incorporation cultures for the final 3.5 h. At the time points indicated, the culture were determined at the indicated time points, as described previously supernatants were collected for quantification of soluble Ig and cytokines, (24). Briefly, cells were stained with anti–CD19-PE and anti–CD45R- and cultured cells were recovered for flow cytometry studies. http://classic.jimmunol.org allophycocyanin to identify 19+45Rlo cells or with anti–CD23-biotin/ streptavidin-PE and anti–CD93-allophycocyanin to discriminate between Cytokine detection transitional 1 (T1) cells and MB cells (Supplemental Fig. 1). The cells Supernatants from cultured B cells were used to measure IL-6, IL-10, and were then washed, fixed, and permeabilized using the BD Cytofix/ the p70 subunit of IL-12 (IL12p70) using the Milliplex MAP mouse Cytoperm kit (BD Biosciences) and subsequently incubated with DNase cytokine/chemokine kit (Merck, Darmstadt, Germany). Murine cytokines of I (Sigma-Aldrich). Next, the cells were stained using the Live/Dead Ex- known concentrations were used to generate standard curves to calculate the clusion Fixable Violet Dead Cell Stain kit (Invitrogen, Carlsbad, CA) and cytokine concentrations in the samples. Supernatants from cultures of the Downloaded from then with anti–BrdU-FITC or isotype FITC (BD Biosciences). Two to murine Th2 clone D10.G4.1 were used as positive controls for IL-6 and IL- three mice were analyzed for each time point. 10 (28). To detect cytoplasmic IL-10 in individual cells, LPS and BAFF/ In vivo adoptive transfer IL4-stimulated B cells were washed and stained for surface CD19 and CD45R expression, before they were incubated with the Live/Dead Ex- Splenic 19+45Rlo,19+45R+ and peritoneal B1 cells from C57BL/6.Ly5.1 clusion kit. Stained cells were fixed and permeabilized using the Cytofix/ mice were FACS purified by double sorting and inoculated i.v. into irra- Cytoperm kit (BD Biosciences), prior to intracellular cytokine staining diated (1.5 Gy) 8- to 12-wk-old Rag2g2/2 hosts (2–3 3 104 cells/mouse). with anti–IL-10-PE, and the isotype-PE control (eBioscience), as described Three mice were sacrificed at each time point, 20, 40, 60, and 90 d after previously (28). For in vivo GM-CSF detection, injections of LPS (10 mg/ cell transfer, and serum was collected from the mice and stored at 220˚C ml) were performed with four daily i.p. injections of LPS as described for ELISAs. The presence of Ly5.1 cells in BM, spleen, and peritoneal previously (9), and the cytoplasmic detection was as indicated above for exudate samples was measured by flow cytometry. IL-10. PCR array Statistical analysis RNA from purified B cell populations was extracted with the TRIzol reagent The data are presented as the means 6 SEM and all statistical analyses (Invitrogen), and the cDNAs prepared with the RT2 First Strand kit were performed using GraphPad Prism 4.0 software. The p values were (SABiosciences, Qiagen, Valencia, CA) were preamplified using the RT2 calculated with the two-tailed Student t test (*p , 0.05, **p , 0.01, ***p , Nano PreAMP cDNA cell cycle synthesis kit (SABiosciences). The ex- 0.001). 2302 CELL DYNAMICS OF SPLENIC CD19+CD45Rlo CELLS

Results Lymphoid organ distribution and postnatal progression of 19+45Rlo cells in normal mice We previously reported that 19+45Rlo cells of embryonic origin are present in the spleen of infant mice (23). In this study, we traced these cells to different lymphoid locations beginning at postnatal day (PD) 7. We detected these cells in the spleen, PB, and PP of normal, unmanipulated BALB/c, C57BL/6, and CBA/CaJ adult mice but not in the thymus or lymph nodes (LN; Fig. 1A, 1B). CBA/N mice bearing the btk mutation exhibited a 30–50% reduction in FO cells and a severe reduction of B1a and B1b subpopulations. We confirmed that btk-deficient CBA/N mice contained very few 19+45Rlo cells in these structures, suggesting a 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 birth by 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, the number of 19+45Rlo cells was maintained constant until adulthood (Fig. 1D). Surprisingly, young CBA/N spleen samples had more 19+45Rlo cells than those from PD30 or adult CBA/N mice, although this number was 3-fold less than those detected in control age-matched mice. The adult PP contained a significant number of 19+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 expression of CD24, CD21, and CD93, as described previously (29). The 19+45Rlo cells were mapped outside the transitional CD21/CD24 B cell subsets, although CD93 was expressed at similar levels in 19+45Rlo and 19+45R+ cells(Fig.1E).Splenic19+45Rlo cells differed also from the recently described ABCs (6, 7), which are B220/CD45R+, CD11b/CD11c+, CD80+, and CD86+, whereas 19+ lo 2/lo FIGURE 1. Lymphoid organ distribution and postnatal development of 45R cells were B220/CD45R and negative for CD11b, 19+45Rlo cells. (A) Cells from the organs indicated of BALB/c and CBA/N CD11c, CD80, and CD86 (Fig. 1A, 1C, 1F). Finally, although mice were stained with anti-CD19 and anti-CD45R and analyzed by flow by guest on October 1, 2021. Copyright 2012 Pageant Media Ltd. expression of the preplasmablast marker CD138 (30, 31) was cytometry. Representative dot plots are shown in which the numbers minimal in splenic CD19+CD45R+ cells (0.9 6 0.4%; n = 8), it represent the frequencies as percentages (mean 6 SEM; n =8)of19+45Rlo was more abundant in 19+45Rlo cells (6.8 6 1.7%; n = 8), both in cells (ellipse in the left dot plot) of the lymphoid gate. (B) Bar graphs show the IgM2 and IgM+ subsets of this population from the spleen and the number of 19+45Rlo cells/organ (mean 6 SEM; n = 6) in the spleen and PP samples (Fig. 1E, 1G). Taken together, these findings suggest PP of different strains of mice at 2 mo of age. Absolute numbers were that these cells are in a natural preactivation state. calculated using the frequencies obtained by cytometry and the total , , These phenotypic data show that 19+45Rlo cells are distributed number of lymphoid cells counted in each sample. **p 0.01; ***p 0.001. (C) Representative examples of electronically gated 19+45Rlo (left), in the spleen, PP, and PB from infant stages. In the spleen, this 19+45R+ (middle), and of total spleen cells (right) from BALB/c mice, population could be clearly distinguished from the Transitional http://classic.jimmunol.org showing staining with anti-CD11b and anti-CD5. (D) The dot diagrams cell subsets, conventional MB cells, ABC, and B1 cells. These represent the number of 19+45Rlo cells/organ at the ages indicated. Indi- observations, together with our previous confocal microscopy vidual values are represented by dots, and the mean values are represented findings demonstrating a perifollicular distribution of 19+45Rlo by horizontal bars. Comparisons between BALB/c and CBA/N mice of the cells (23), suggest that these cells represent a B cell subset of same ages were performed, with significance values shown in the boxes. embryonic origin. 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-

Downloaded from CD93 deﬁned subsets of cells (upper and middle dot plots) in electroni- In vivo BrdU uptake in splenic 19+45Rlo cells cally gated 19+45Rlo and 19+45R+ spleen cells: T1, T2, and MZ precursor On the basis of the conservation of 19+45Rlo cell number observed (MZP)/MZ. Lower dot plots show CD138 and IgM expression in both throughout life, we investigated their maintenance in vivo. We populations. Numbers represent the frequency of the cells in the boxes previously found that these cells were of embryonic origin, (mean 6 SEM; n = 5 and 8 for CD93/CD24 and CD138/IgM staining, F spontaneously incorporated thymidine, and expressed the nuclear respectively). ( ) Expression of CD11c, CD80, and CD86 on electronically + lo G Ag Ki67 (23). In this study, we determined their proliferative gated 19 45R cells. ( ) Representative histograms of cells from PP stained with anti-CD19, anti-CD45R, and anti-CD138 (thick line) or iso- status in vivo by analyzing BrdU incorporation after continuous type (dotted line). The scale in the cytometry plots is logarithmic. i.p. injection over 17 d, which allowed us to study the kinetics of BrdU labeling in distinct peripheral B cell populations by cytometry. Electronically gated 19+45Rlo cells, T1 stage cells a resting population. Splenic 19+45Rlo cells incorporated BrdU (CD232CD93+), and MB cells (CD23+CD932) were analyzed continuously, with 43 6 1.3% (n = 3) of cells labeled on day 17. (Fig. 2A) and as described previously, maximal BrdU incorpora- No signiﬁcant differences in BrdU labeling were detected between tion was observed in the T1 cell subset (Fig. 2B) (30, 32). By subsets of 19+45Rlo cells, regardless of the expression of IgM, contrast, only 11.6 6 1.2% (n = 3) of MB cells were labeled as IgG, or CD24 (Fig. 2B, Supplemental Fig. 1). However, 19+45Rlo The Journal of Immunology 2303

Transcriptional cell cycle signature of 19+45Rlo cells The spontaneous proliferative status of 19+45Rlo cells was studied in more detail by analyzing the expression of a broad range of cell cycle-related genes in PCR arrays. Using the 22DDCT method and using the values obtained from splenic 19+45R+ cells as a reference, 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 particular, 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), M phase (Ccna1, Ccnb1, Nek2, and Wee1), cell cycle checkpoint control (Ak1, Cdkn1a, Cdkn2a, Dst, and Pmp22), and positive/ negative cell cycle progression (Ccna2, E2f2, and Trp63, respectively). Only two genes (Notch2 and Slnf1) were expressed more weakly in 19+45Rlo than 19+45R+ cells. Comparison of B1 and 19+45R+ cells revealed changes in the expression of several genes; seven genes were more strongly expressed in B1 cells, whereas two 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 of cells. Changes in expression of up to 3-fold were observed for 16 genes, 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 2 2 + + lo

by guest on October 1, 2021. Copyright 2012 Pageant Media Ltd. was also used to identify IgM (m )andIgM(m )1945R 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 incorporation on day 17 (thick line), with the isotype signal overlaid (dotted line). In + lo 2 http://classic.jimmunol.org the 19 45R cell population, m and m 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). Downloaded from

cells that were CD93+ and CD432 incorporated signiﬁcantly more BrdU than 19+45RloCD932/CD43+ cells (p , 0.001 and p , 0.05, respectively), suggesting that these CD93+CD19+CD45Rlo cells FIGURE 3. RT-qPCR array results comparing cell cycle genes in B cell may be newly formed B cells and emphasizing the heterogeneous populations. Anti-CD19 and anti-CD45R were used to purify splenic 19+ component of the 19+45Rlo cell subset. We also analyzed the 45Rlo and 19+45R+ populations. Anti-CD23 and anti-IgM Abs were used persistence of BrdU when its administration was ceased after to purify B1 cell populations from PWC. RT-qPCR was performed as described in Materials and Methods. Data were from three different 8 d of continuous injections (Fig. 2C). BrdU labeling was not + lo + + + + lo preparations of 19 45R ,1945R , and B1 cell samples. Results were evident 21 d later in T1 cells, whereas BrdU 19 45R and 2 normalized by the 2 DDCT method, relative to 19+45R+ cell preparations MB cells persisted at this time. (left and middle columns) or to B1 cell preparations (right column), and In summary, our data demonstrate the heterogeneity of splenic shown as the fold change. Transcripts with changes .2-fold are displayed + lo ∼ 19 45R cells, with 50% of these cells proliferating spontane- grouped in ﬁve different gene clusters: 1, G1 and G1-S phases; 2, S-phase + lo ously and ∼25% of the 19 45R cells that divide being main- and DNA replication; 3, M phase; 4, cell cycle checkpoint and arrest; and tained for extended periods. 5, cell cycle regulation. *p , 0.05, **p , 0.01, ***p , 0.001. 2304 CELL DYNAMICS OF SPLENIC CD19+CD45Rlo CELLS

sion in 19+45Rlo versus B1 cells) and Nek2, Notch2, Pmp22, and Slnf1 (weaker expression versus B1 cells), whereas less dramatic changes were observed for several other genes. Confirmatory RT- qPCR studies were performed for c-myb, Notch2, and Ki67 transcripts 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 cycle machinery used by the B cell subpopulations analyzed in this study, 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 homeostatic control and life span of 19+45Rlo cell subset, we performed adoptive transfer experiments into Rag2g2/2 hosts, taking advantage of the fact that a lymphopenic environment maximizes the homeostatic cell proliferation in vivo under physiological conditions (33). Splenic 19+45Rlo,19+45R+, and peritoneal B1 cells were purified by double sorting from C57BL/6.Ly5.1 mice prior to their adoptive transfer to Rag2g2/2 recipients (Fig. 4A). The repopulation of donor-derived Ly5.1+ cells was determined at 20-d intervals in various organs (Fig. 4B, 4C). Only CD19+ cells were detected in the Ly5.1+ population of these animals. After 20 d, recipients of 19+45Rlo cells exhibited Ly5.1+CD19+ cells in the spleen and in the peritoneal exudates, with the maximal number reached at 40– 60 d. Recipients of B1 and 19+45R+ cells reconstituted a smaller Ly5.1+CD19+ cell population in the spleen (1.9- and 5.5-fold less, respectively). In PWC, similar numbers of Ly5.1+CD19+ cells were observed in recipients of B1 cells as those receiving 19+ 45Rlo cells, whereas no Ly5.1+ cells were detected in recipients + lo of 19+45R+ cells. Neither 19+45Rlo nor 19+45R+ cells repopulated FIGURE 4. Adoptive transfer of 19 45R cells repopulates the spleen + lo + + the BM. Cells recovered from the spleen exhibited similar CD45R and peritoneum. Purified 19 45R and 19 45R splenic B cell populations g2/2 levels to those detected in the original transferred cells (Fig. 4D). from C57BL/6.Ly5.1 donors were inoculated into irradiated Rag2 2 hosts. Donor-derived cells were identified by the expression of Ly5.1 in Moreover, the recovered cells were CD11b and displayed min- cell suspensions from the spleen, BM, and PWC of the recipients using imal levels of CD5 (range, 3–20%; mean, 10%; n = 6). CD11b anti-Ly5.1, anti-CD19, and anti-CD45R Abs. Cytometry data are from one

by guest on October 1, 2021. Copyright 2012 Pageant Media Ltd. expression was detected in cells from PWC (range, 30–70%; n = representative experiment out of three with similar results. (A) Schematic 6), although no CD5 expression was observed (n = 6). Similar outline of the experimental design. (B) Representative reconstitution ex- results were obtained in competitive adoptive transfer experiments periment showing the expression of Ly5.1 and CD19 at 40 d posttransfer. using adult C57BL/6 recipients after sublethal irradiation (Sup- Numbers are as in Fig. 1A. (C) Total numbers of donor Ly5.1+ cells at plemental Fig. 3). After BM efflux arrest, B cells in the periphery different times in the spleens and PWC of recipients of 19+45Rlo (n), 19+ + adopt an activated state and a differentiated Ig secretion profile, 45R (s), and peritoneal B1 (:) cells calculated as in Fig. 1B. Data 2 2 6 similar to MZ, B1, or plasma cells (PCs), as shown on IL7 / represent the mean SEM of three independent experiments performed D mice (34). Analysis of the serum Ig levels in transferred mice (Fig. with two mice per point. ( ) Preparations from spleen and PWC of recipients of Ly5.1+19+45Rlo and of Ly5.1+19+45R+ cells, analyzed 60 d 4E) revealed similar levels of IgM in recipients of 19+45Rlo,19+ + posttransfer. For spleen preparations, anti-CD19 and anti-CD45R staining http://classic.jimmunol.org 45R , and B1 cells. By contrast, levels of both IgG1 and IgA were in electronically gated donor-derived Ly5.1+ cells detected as in Fig. 3B is + lo significantly higher in mice repopulated with 19 45R cells, shown in the upper zebra plots. Histograms show staining with anti-CD5 further demonstrating a marked differentiation bias toward IgG1 or anti-CD11b (thick line), overlaid with the isotype-control staining and IgA in this homeostatic proliferative context. (dotted line). For PWC preparations, the upper zebra plots are as described Taken together with the findings regarding BrdU incorporation, for the spleen cells, and the bottom zebra plots show staining with Ly5.1, the reconstitution potential observed and the persistence of the 19+ anti-CD5, and anti-CD11b. (E) Dot plots (logarithmic scale) represent the lo + serum levels of IgM, IgG1, and IgA in recipients of Ly5.1+19+45Rlo (n), Downloaded from 45R cells demonstrate the innate activation status of the 19 + + + s : 45Rlo cell population in vivo. Accordingly, these cells can be Ly5.1 19 45R ( ), and B1 cells ( ) that were quantified by specific maintained in mice under homeostatic conditions while preserving 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. their preferential differentiation bias toward IgG1- and IgA- secreting PCs.

+ lo with CpG (Fig. 5A). In addition, a marked induction of CD138 In vitro activation of 19 45R cells was detected in LPS-responding cells (Fig. 5B). B cell signaling To analyze the proliferative and differentiation responses of 19+ via the BAFF and APRIL receptors is associated with survival and 45Rlo cells to in vitro stimulation, sorted purified 19+45Rlo and PC generation (36). Our results revealed greater proliferative 19+45R+ cells were labeled with the cell tracer CFSE prior to responses in both B cell subsets following stimulation with BAFF/ stimulation in vitro. The TLR4 agonist LPS, which induces IL4 versus APRIL/IL4, although neither of these stimuli resulted a proliferative response in all B cell compartments and the dif- in CD138 upregulation. BCR activation with anti-IgM promoted ferentiation of MZ and B1 cells to mature PCs (35), induced a significant proliferative response in 19+45R+ cells but had no a potent proliferative response in both 19+45Rlo and 19+45R+ effect in 19+45Rlo cells. These findings indicate that although IgM cells. A similar effect was observed when TLR9 was stimulated was expressed by 40–50% of 19+45Rlo cells, BCR activation was The Journal of Immunology 2305

distinct regulatory roles (16, 17). Thus, we analyzed the secretion of several cytokines following in vitro activation of 19+45Rlo cells. Supernatants were collected after in vitro stimulation, performed as described above, and the amount of secreted IL-6, IL-10, and IL12p70 was determined (Fig. 6). LPS stimulation resulted in the secretion of large amounts of IL6 from puriﬁed 19+45R+ lymphocytes, whereas only minimal release of this cytokine was detected in 19+45Rlo cells. By contrast, stimulation of 19+45Rlo cells with LPS or BAFF/IL4 resulted in a 10-fold increase in IL-10 release 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

by guest on October 1, 2021. Copyright 2012 Pageant Media Ltd. 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 FIGURE 6. Cytokine production by activated B cells. (A) Determination + with a high FCS-A that displayed decreasing levels of CFSE. Numbers of IL-6, IL-10, and IL12p70 in supernatants from cultures of purified 19 lo + + represent the frequency of the boxed populations (mean 6 SEM; n =3).(B) 45R (n)and19 45R (N) cells, stimulated as indicated in Fig. 5C. Control; http://classic.jimmunol.org Induction of CD138 after stimulation with LPS and BAFF/IL4 or in control D10.G4.1, IL-6–, and IL-10–producing clone. Numbers indicate the amount cells is shown in the histograms. The numbers represent the frequency of of cytokine detected in the D10.G4.1 supernatants. The figure shows the the cells that proliferated and exhibited upregulated CD138 expression results from three independent experiments performed in duplicate. *p , (mean 6 SEM; n =3).(C) IgM-, IgG1-, and IgA-specific quantification in 0.05, **p , 0.01. (B) Intracellular determination of IL-10 by flow cytom- the supernatants collected was performed by isotype-specific ELISA (mean 6 etry. Enriched spleen B cells were incubated with LPS and BAFF/IL4 before SEM; n = 6). Comparisons for each isotype were performed using data ob- performing surface and intracellular staining. Contour plots show data from tained for supernatants from the cultures of 19+45Rlo and 19+45R+ cells one representative experiment out of five with similar results. Dead cells

Downloaded from 5 (boxed asterisks). *p , 0.05, **p , 0.01, ***p , 0.001. were excluded using the Live/Dead cell exclusion kit. At least 3 3 10 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 unable to induce proliferative signals or CD138 induction. Acti- dotted boxes. The bar graph (right) represents the frequency of specific IL- vation mediated by anti-CD40/IL4 induced the proliferation of 10–producing cells in unstimulated (control), LPS-, and BAFF/IL4-activated + both B cell types. Moreover, exposing 19+45Rlo cells to LPS and cells after subtracting the isotype-matched Ab background from purified 19 lo + + BAFF/IL4 provoked the release of IgM and IgG1, whereas only 45R (n)and19 45R (N) cells (mean 6 SEM; n =3).**p , 0.01. (C)RT- + lo n LPS induced the secretion of IgA (Fig. 5C). Collectively, these qPCR analysis of constitutive IL-10 mRNA expression in 19 45R cells ( ), 19+45R+ cells (s), peritoneal B1 cells (:), and LPS-stimulated (25 mg/ml) in vitro results demonstrate the potent effect of TLR4, TLR9, and ♦ + lo splenocytes after 48 h in culture (LPS blasts, ). The DCT values (loga- BAFF/IL4 ligation on the final differentiation of 19 45R cells to rithmic scale) of each transcript over HPRT were calculated as described in IgG1- and IgA-secreting PCs. Supplemental Fig. 2 (**p , 0.01). (D) Intracellular determination of GM- Cytokine production by activated 19+45Rlo cells 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- Several reports have described the role of activated B cells in sentative experiment out of three with similar results. Dead cells were ex- cytokine secretion, suggesting that different subsets of cells fulfill cluded as in (B), and .5 3 105 live cells are analyzed. 2306 CELL DYNAMICS OF SPLENIC CD19+CD45Rlo CELLS

CD40/IL4 also induced low levels of IL10 secretion by 19+45Rlo dependent activation (ABCs are unresponsive to LPS). The re- cells, whereas no IL12p70 was detected in any of the in vitro cently described IRA B cells (9) constitute a novel innate cell conditions assayed. We also quantified IL-10 secretion in indi- subset, yet they have a distinct phenotype, secretion profile, and vidual 19+45Rlo cells stimulated with LPS or BAFF/IL4, mea- ontogeny to most 19+45Rlo cells. IRA B cells express CD45R/ suring intracellular IL-10 by cytometry (Fig. 6B). Cytoplasmic B220, they are CD138+CD21loCD93+CD23lo, they secrete IgM IL10 was detected in higher numbers of 19+45Rlo cells stimulated and GM-CSF (not IgG, IgA, and IL-10), and they are produced in with LPS and BAFF/IL4 than in stimulated 19+45R+ cells. In the spleen from mature circulating B1a peritoneal cells upon TLR addition, constitutive expression of IL10 mRNA was stronger in stimulation. 19+45Rlo cells than in CD19+CD45R+ or B1 cells in vivo (Fig. In vivo BrdU labeling demonstrated considerable turnover of 19+ 6C). However, the addition of IL-10 to purified 19+45Rlo cells 45Rlo cells under homeostatic conditions, which was significantly revealed no proliferative effect of this cytokine in these cell sub- higher than MB but did not reach the level observed in T1 cells. A sets (Fig. 5A). Finally, TLR4 activation, which promotes the se- small subset of 19+45Rlo cells shared some features with newly cretion of GM-CSF by IRA cells (9), failed to induce cytoplasmic formed transitional stage T1 cells (CD212CD232CD93+BrdU+). expression of GM-CSF by 19+45Rlo cells (Fig. 6D). In summary, However, the differential expression of CD24 and CD43 (Fig 1E, these data demonstrate that in vitro activation of 19+45Rlo cells Supplemental Fig. 1) and the responsiveness to BAFF distin- results in their proliferation and differentiation to IgM-, IgG1-, and guished 19+45Rlo cells from these populations. Maintaining the IgA-secreting PCs and in active production of the immunoregu- mature B cell compartments requires an adequate balance between latory cytokine IL-10, which is constitutively expressed in vivo. proliferation and apoptosis and tight regulation of differentiation programs. BrdU decay experiments revealed that this analog Discussion persisted in 19+45RloBrdU+ cells for ∼1 m after cessation the In the current study, we demonstrate the presence of 19+45Rlo cells injections, which indicates that following one or several rounds of in the spleen, PB, and PP of normal unmanipulated mice. In the division, these cells remain in G0 phase, favoring their final dif- spleen, the number of 19+45Rlo cells is maintained from infant ferentiation. In support of this hypothesis, we observed strong c- stages to adulthood as cells different from transitional, MZ, FoB, myb and weak Notch2 transcription, genes implicated in the and B1 cells. Moreover, these cells are in an innate activated transcriptional profile of highly differentiated B cells (42). proliferative state in which cyclin-dependent cascades are in- Adoptive transfer experiments using immunodeficient animals duced. This homeostatic proliferation is also witnessed by their further supported the proposed innate-like nature of 19+45Rlo prolonged survival in immunodeficient animals, indicating that cells, which are capable of self-maintenance while preserving they require no influx of progenitors from the BM in which 19+ their preferential differentiation status in a homeostatic context. 45Rlo cells maintain their initial 19+45Rlo phenotype and differ- To ascertain the functional role of this cell population, we an- entiate to IgG1- and IgA-switched PCs. We also present in vitro alyzed the proliferative and differentiation responses of 19+45Rlo evidence demonstrating the proliferative and differentiation cells to different stimuli, including TLR and BCR ligation, T- responses of 19+45Rlo cells to T-dependent and -independent dependent signals, and soluble survival factors. TLR4 ligation by stimuli, and in the case of TLR and BAFF/IL4 stimulation, the LPS stimulation induced a potent proliferative reaction and the

by guest on October 1, 2021. Copyright 2012 Pageant Media Ltd. release of the immunoregulatory cytokine IL-10. strongest differentiation response in these cells. Indeed, pro- The rapid neonatal colonization of the spleen by 19+45Rlo cells, nounced CD138 expression was induced in CFSE low-responding their dramatic btk dependency in juvenile CBA/N mice, and their cells and CFSE high nonproliferating 19+45Rlo cells, as well as ability to release IgG and IgA in 7-d-old mice (23) suggest a the release of IgM and isotype-switched Igs (IgG1 and IgA). protective or regulatory role in primary/neonatal Ag encounters Similar findings were obtained following TLR9 stimulation (CpG) involving btk-dependent mechanisms. Btk-dependent B1 cells of 19+45Rlo cells, suggesting the relevance of TLR-dependent have a major embryonic origin whereas B2 cells are BM derived. activation of these cells. Activation mediated by anti-CD40 plus Furthermore, B1 cells can largely persist by self-replenishment IL-4 induces a potent proliferative and differentiation signaling throughout adult life (37). Therefore, the embryonic origin of cascade in B lymphocytes (43). A similar effect was observed in + lo + lo http://classic.jimmunol.org 19 45R cells (23), their absence from the spleen of adult CBA/N 19 45R cells, with the proliferative response indicating that T- mice and their role in the BM as progenitors of peritoneal B1 cells dependent signals can regulate the functional response of 19+ (20–22) situate splenic 19+45Rlo cells close to the B1 lineage. This 45Rlo cells. Indeed, a cell population defined as CD19+B2202 proposal is further supported by the observed induction of CD11b BST-2+ (specific for viral Ag) mediated Ag presentation functions in the peritoneal cavity of immunodeficient mice repopulated with and was described in the periphery of mice postinfection with these cells. These results are supported by the observed presence West Nile encephalitis virus (44). BCR ligation with anti-IgM had + lo + lo

Downloaded from of splenic B1 progenitors with a 19 45R phenotype (38). In no effect on 19 45R cells, despite their expression of IgM. Ef- addition, 19+45Rlo cells constitutively express IL-10 transcripts, fective BCR–cross-linking stimulation depends both on the qual- and they exhibit spontaneous ERK phosphorylation (data not ity of the interaction (45) and the concomitant coligation of shown), as previously described for B1 cells (39, 40). 19+45Rlo coreceptors that modulate receptor engagement by enhancing cells exhibit no CD5 or CD11b expression in the adult spleen, and BCR signaling (CD19, CD21, and FcgRIIb) (46). BCR activation 19+45Rlo cells are present in the infant CBA/N spleen, as well as in early T1CD212 cells induces apoptosis, and thus, late transi- they are found in PP and PB, which are conventional B2 organs tional cells become resistant to BCR-induced death (47). The low (41). The transcriptional ﬁngerprint, which involves higher con- levels of CD21 found in 19+45Rlo cells may contribute to their stitutive Blimp-1 expression (Supplemental Fig. 2) (23) and the refractory response after BCR ligation. Additional mechanisms, spontaneous release of isotype-switched Igs, further distinguishes such as the differential expression of other coreceptors, inhibitory the 19+45Rlo cell population from B1 cells. As compared with transduction mechanisms, or the presence of CD93+ immature ABCs, which are quiescent cells derived from FoB cells that ac- preplasmablasts, may also operate in these cells. cumulate slowly with age (6, 7), 19+45Rlo cells did not increase in The survival factor BAFF exerts its action by activating three aged mice and exhibited major differences in their surface phe- receptors, B cell maturation Ag, transmembrane activator calcium notype, developmental origin, proliferative status, and TLR- modulator and cyclophilin-ligand interactor, and BAFF receptor, The Journal of Immunology 2307

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