Maturation and Is Inhibited by TLR2 Signaling Through TLR4 Promotes

Role of TLR in B Cell Development: Signaling through TLR4 Promotes B Cell Maturation and Is Inhibited by TLR2

This information is current as Elize A. Hayashi, Shizuo Akira and Alberto Nobrega of September 28, 2021. J Immunol 2005; 174:6639-6647; ; doi: 10.4049/jimmunol.174.11.6639 http://www.jimmunol.org/content/174/11/6639 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Role of TLR in B Cell Development: Signaling through TLR4 Promotes B Cell Maturation and Is Inhibited by TLR21

Elize A. Hayashi,* Shizuo Akira,† and Alberto Nobrega2*

The role of TLR4 in mature B cell activation is well characterized. However, little is known about TLR4 role in B cell development. Here, we analyzed the effects of TLR4 and TLR2 agonists on B cell development using an in vitro model of B cell maturation. Highly puriﬁed B220؉IgM؊ B cell precursors from normal C57BL/6 mouse were cultured for 72 h, and B cell maturation in the presence of the TLR agonists was evaluated by expression of IgM, IgD, CD23, and AA4. The addition of LPS or lipid A resulted in a marked increase in the percentage of CD23؉ B cells, while Pam3Cys had no effect alone, but inhibited the increase of CD23؉ B cell population induced by lipid A or LPS. The TLR4-induced expression of CD23 is not accompanied by full activation of the lymphocyte, as suggested by the absence of activation Ag CD69. Experiments with TLR2-knockout mice conﬁrmed that the

inhibitory effects of Pam3Cys depend on the expression of TLR2. We studied the effects of TLR-agonists on early steps of B cell Downloaded from differentiation by analyzing IL-7 responsiveness and phenotype of early B cell precursors: we found that both lipid A and Pam3Cys impaired IL-7-dependent proliferation; however, while lipid A up-regulates B220 surface marker, consistent with a more mature phenotype of the IgM؊ precursors, Pam3Cys keeps the precursors on a more immature stage. Taken together, our results suggest that TLR4 signaling favors B lymphocyte maturation, whereas TLR2 arrests/retards that process, ascribing new roles for TLRs in B cell physiology. The Journal of Immunology, 2005, 174: 6639–6647. http://www.jimmunol.org/ n the adult mouse, B lymphopoiesis occurs in the bone mar- cules, the TLR family, which includes at least 10 members in the row through the commitment and differentiation of hemopoi- mouse species (15). TLRs are present in the cell types involved in I etic stem cells (1, 2). B lineage cells are characterized by cell innate immunity and are capable of recognizing several pathogen- surface expression of CD19 and B220, and the earliest B cell pre- associated molecular patterns, playing a key role in the activation cursors express several well-described surface markers such as of monocytes and granulocytes. TLR4 recognizes the LPS present AA4, c-kit, CD43, BP-1, and heat stable antigen (3–6). At a later in cell walls of Gram-negative bacteria (16, 17), while TLR2 rec- stage characterized as pre-B-II stage (6), or fraction D (4), c-kit ognizes bacterial lipoproteins and is important for triggering innate and CD43 are down-modulated, while IL-2R is expressed on cell immune response against Gram-positive bacteria (18). The activa- surface and maintained until the immature B lymphocyte stage (6). tion of mature B lymphocytes to proliferation and Ig secretion by by guest on September 28, 2021 Along B cell development, V-D-J rearrangements lead to forma- LPS and bacterial CpG-DNA has been confirmed to be mediated tion of the clonotypic BCR and surface expression of IgM mole- ϩ ϩ by TLR4 and TLR9, respectively (17, 19). Triggering of murine B cules (7). Newly formed surface IgM (sIgM ) B lymphocytes lymphocytes by bacterial cell wall lipoproteins is very likely me- are still immature cells bearing AA4, which is progressively lost as diated by TLR2 (12, 18). B lymphocytes mature (3), and must pass through transitional The final differentiation of the maturing B cell is thought to be stages before the full maturation (8–10). During the late differen- dependent on the positive selection of the lymphocyte upon entiation steps, transitional B lymphocytes acquire CD23 and IgD, gagement of the BCR with self-Ags, suggesting a role for cellular which are considered as markers of B cell maturity (8–11). activation in B cell maturation (20–22). Although TLRs are well Mature B cells can be activated to proliferation and Ig secretion, characterized as functionally triggering receptors for mature B in a BCR-independent manner, by microbial products such as LPS, lipoproteins, and CpG-enriched DNA (12–14). Recently, receptors lymphocytes, little is known about their expression and function in for such microbial ligands and their molecular signaling pathways B cell development. TLR and BCR signaling can exhibit synergy have been characterized. These receptors belong to a recently de- in activation of mature B cell (23) and it is not known whether scribed family of germline-encoded and highly conserved mole- triggering through TLRs could act in B cell development as well. Here, using an in vitro model of B cell differentiation, we have studied the role of TLR4 and TLR2 agonists, lipid A/LPS, and *Department of Immunology, Institute of Microbiology, Federal University of Rio de Pam3Cys respectively, in B cell maturation. Our results show that Janeiro, Rio de Janeiro, Brazil; †Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, and Exploratory Research for Advanced Tech- signaling through TLR4 and TLR2 modulates B cell differentiation nology, Japan Science and Technology Agency, Osaka, Japan from early precursor stage. We found that, while TLR4 promotes Received for publication September 7, 2004. Accepted for publication March maturation, TLR2 engagement arrests/retards B cell maturation. 14, 2005. Simultaneous addition of TLR2 and TLR4 ligands revealed a pre- The costs of publication of this article were defrayed in part by the payment of page viously unknown antagonism between those stimuli, suggesting an charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. inhibitory cross-talk between the respective signaling pathways in 1 This work was supported by grants from Conselho Nacional de Pesquisas, Fundaçaõ maturing B lymphocytes. Experiments with TLR2-knockout (KO) de Amparo á Pesquisa do Estado do Rio de Janeiro, and Fundaçaõ Universitária José mice showed that Pam3Cys effects depend on the expression of Bonifácio. TLR2. This study reveals a new role for TLRs in B cell maturation, 2 Address correspondence and reprint requests to Dr. Alberto Nobrega, Department of Immunology, Institute of Microbiology, Federal University of Rio de Janeiro, Rio de raising questions about their physiological role in B lymphopoiesis Janeiro 21941-590, RJ, Brazil. E-mail address: [email protected] and repertoire selection in vivo.

Materials and Methods of 10%. Harvested cells were submitted to MACS for the depletion of IgMϩ cells. The recovered B220ϩIgMϪ B cell precursors at the end of the Mice and cells ϩ fifth day represented ϳ85% of the total cells after elimination of IgM C57BL/6 and C57BL/10ScCr mice were from Fluminense Federal Uni- cells. Cells were recultured in 96-well flat-bottom plates at 2 ϫ 105 cells versity and from the Oswaldo Cruz Foundation. TLR2-KO mice were ob- per well with rIL-7 for 48 h, without or with either Pam3Cys (1 ␮g/ml), tained from Centro de Pesquisas Rene´Rachou from breeding stock orig- lipid A (1 ␮g/ml), or both at 1 ␮g/ml. Living cells were counted excluding inally provided by Dr. Akira (Osaka University). Bone marrow (BM)3 cells dead cells with trypan blue dye and processed for FACS analyses. from 2- to 3-mo-old mice were flushed out of femurs with ice-cooled OptiMEM plus 10% FCS (Invitrogen Life Technologies) by using a 10-ml Proliferation assay syringe with a 21-gauge needle. Spleen cell suspensions were obtained by gently teasing the spleen. Erythrocytes were lysed with NH4Cl lysis buffer Spleen cells from C57BL/6 or TLR2-KO mice were cultured with different and BM and spleen cells were washed with OptiMEM plus 10% FCS. concentrations of Pam3Cys or lipid A at 2 ϫ 105 cells per well in 96-well flat-bottom plates in 200 ␮l/well of OptiMEM supplemented with 10%

MACS FCS in a humidified atmosphere of 7% CO2 at 37°C. After 48 h, cultures 3 ϩ were pulsed with 1 ␮Ci of [ H]thymidine and incubated for further 24 h. For depletion of IgM B lymphocytes, BM cells were incubated in MACS Cells were harvested, and the incorporation of [3H]thymidine was mea- buffer (PBS containing 2 mM EDTA, 5% FCS) with anti-mouse IgM Mi- sured by scintillation counting. croBeads (Miltenyi Biotec) for 20 min on ice, washed, and resuspended in MACS buffer, according to specifications of the manufacturer. Cells were applied onto the magnetic column (Miltenyi Biotec) and the effluent was Results collected, and washed in OptiMEM plus 10% FCS. For purification of B220ϩIgMϪ B precursors, IgMϩ-depleted cell preparations were incu- In vitro generation of immature and transitional B lymphocytes bated for 20 min on ice with anti-B220 MicroBeads (Miltenyi Biotec) and In vitro experimental systems for studying B cell differentiation Downloaded from processed as described above. Cells were applied to the magnetic column (Miltenyi Biotec), and after washing, retained cells were collected as the have long been established as useful models for dissecting B cell B220ϩ fraction. Viable cells were scored by trypan blue dye, using a maturation process (4, 24–26). To study the effects of TLR ligands Neubauer hemacytometer, and the purity of the cell preparations were ver- on B cell development, we used a system of B cell differentiation ϩ ified by FACS analyses. IgM cells corresponded to Ͻ1% of B lineage in vitro from highly purified B220ϩIgMϪ B cell precursors ϩ Ͼ cells after depletion, and B220 cells corresponded to 99% of recovered (Ͼ99% purity). This culture system allowed B220ϩIgMϪ B cell cells after positive selection. ϩ

precursors to differentiate into newly formed sIgM B cells, as http://www.jimmunol.org/ Cell staining and FACS analysis previously established (4, 26). The first IgMϩ B lymphocytes arise at 24 h, and continue to accumulate during cell culture, represent- Fresh BM cells, MACS-purified cells, and cultured cells were monitored ϩ by flow cytometry. The Abs used for staining were as follows: PE anti- ing 40–50% of B220 cells after 72 h (Fig. 1A). Beyond the 72-h ϩ CD23, PE anti-c-kit, FITC anti-B220, biotin anti-CD69 (BD Pharmingen), culture period, the absolute number of recovered IgM B lympho- allophycocyanin goat anti-mouse IgM (Caltag Laboratories), biotin anti- cytes decreased while cell mortality increased (data not shown). IgD, FITC goat anti-mouse anti-IgM (Southern Biotechnology Associates), The percentage of IgMϩCD23ϩ cells, which represent a more ma- and biotin anti-AA4 (493 hybridoma provided by Dr. A. Rolink, Basel ture developmental stage of the B lymphocyte, augments during Institute, Basel, Switzerland; the mAb was purified and biotin-conjugated ϩ according to standard protocols). Biotinylated mAbs were revealed with the culture, up to 10% of the total IgM B cells at 72 h (Fig. 1A). Alexa-Fluor 680-R-PE streptavidin (Molecular Probes) or with allophyco- In adult BM, three major subsets of B lymphocytes have been by guest on September 28, 2021 cyanin streptavidin (Caltag Laboratories). Cells were incubated with the characterized according to B220 and IgM expression patterns (8): mAbs in FACS buffer (PBS, 1% FCS, 0.05% sodium-azide) for 20 min on the B220lowIgMlow immature B cells, which correspond to the ear- ice and washed twice with FACS buffer. When biotinylated mAbs were ϩ dull/high high used, another step of incubation with Alexa 680-PE streptavidin or allo- liest stage of the IgM B lymphocyte, the B220 IgM phycocyanin streptavidin were performed under the same conditions as transitional B cells, a more advanced stage of B cell maturation, described above. Except for the four-color-stained cell samples, propidium and the B220highIgMϩ follicular recirculating mature B lympho- iodide was added to the samples immediately before data acquisition at 0.5 cytes. Fig. 1B shows the IgM/B220 flow cytometry plot represen- ␮g/ml, for dead cell exclusion. Data were acquired on a FACSCalibur (BD Biosciences) and analyzed using CellQuest software (BD Biosciences). tative of fresh BM cells from young mice (8–12 wk of age), indicating the gates we used to define the three different B cell Cultures for B cell differentiation subsets: I, immature; T, transitional; and M, mature. The gates Purified B220ϩIgMϪ cells were cultured in 96-well flat-bottom plates were defined according to the original description of Carsetti et al. (Corning) at 2 ϫ 105 cells per well in OptiMEM plus 10% FCS, 5 ϫ 10Ϫ5 (8). For each of these subsets we studied in more detail the phe- M 2-ME, 100 ␮g/ml streptomycin, and 100 U/ml penicillin (Invitrogen notype of the B cell generated in vivo and in vitro. We analyzed Life Technologies) in a humidified atmosphere of 7% CO at 37°C. Stimuli 2 the up-regulation of maturation markers acquired as the B cell added to cultures included highly purified, lipoprotein-free LPS preparations (provided by Dr. D. Golenbock, University of Massachusetts Medical differentiates, CD23 and IgD, and the down-modulation of AA4, School, Worcester, MA and Dr. P. Tobias, Scripps Research Institute, La an immature B cell marker that is progressively lost during differ- Jolla, CA) at 10 ␮g/ml, lipid A from Salmonella minnesota Re595 (Cal- entiation, from the transitional B cell population to the mature biochem) at 1 ␮g/ml, and Pam3Cys-Ser-(Lys)4 (EMC Microcollections) at stage. Down-regulation of AA4 is considered as the hallmark dis- 1 ␮g/ml. At 24, 48, or 72 h of culture, cells were harvested and counted; living cells were determined excluding dead cells with trypan blue dye and criminating immature and transitional from mature B cell (3, 9). processed for FACS analyses. Statistical analysis of the data (one-sample As can be seen in Fig. 1B, in fresh BM cells, while immature B Ϫ Ϫ analysis, Student’s t test) was performed using StatView software (Abacus cells are mostly CD23 IgD , a significant part of the transitional Concepts); results were considered statistically significant if p Ͻ 0.05. B cells are already CD23ϩ and IgDϩ and mature B cells, instead, Ϫ ϩ ϩ Ϫ ϳ Cultures for early B cell precursors expansion are all AA4 CD23 IgD . AA4 cells, which represented 10 and 30% of immature and transitional subsets, respectively, were In experiments performed to study early stages of the B cell precursors, probably mature B cells that could not be completely separated BM cells were cultured at 5 ϫ 105 cells/ml in OptiMEM with the supple- from these populations with the gating used in the B220/IgM plot. ments described above plus rIL-7 for 5 days to obtain pro-B cell-enriched Ϫ population. The source of rIL-7 was the culture supernatant of J558 cells If AA4 cells are excluded from immature and transitional gates, ϩ transfected with IL-7 cDNA, added to the culture at a final concentration then the percentage of CD23 cells drops to ϳ10% in transitional B subset, and 0% in immature B. 3 Abbreviations used in this paper: BM, bone marrow; sIgM, surface IgM; KO, B lymphocytes generated in vitro bear essentially a typical im- knockout. mature/transitional phenotype (Fig. 1C), mostly expressing AA4. The Journal of Immunology 6641 Downloaded from http://www.jimmunol.org/

FIGURE 2. B lymphocyte differentiation in vitro in the presence of TLR-4 ligands. B220ϩIgMϪ B cell precursors purified from mouse BM were cultured for 72 h in the absence or presence of either highly purified LPS (10 ␮g/ml) or lipid A (1 ␮g/ml), and stained with anti-B220-FITC, -IgM-APC, and -CD23-PE Abs for FACS analysis, as in Fig. 1A. Dead cells were excluded from analyses by propidium iodide staining. A, The IgM vs B220 profiles of total cultured cells (upper plots) and the CD23 vs B220 profiles of the B220ϩIgMϩ population (lower plots); B, the IgM vs CD23 profiles for total cultured cells. by guest on September 28, 2021

Immature and transitional B cell subsets obtained in culture express levels and percentages of CD23 and IgD similar to the respective B cell subsets generated in vivo. In vitro-generated transitional B cells display higher levels of AA4ϩ compared with their in vivo equivalent, but the level of expression of this Ag is dimin- ished when compared with immature B cells, suggesting that this population obtained in vitro indeed represents a more advanced stage of B cell maturation. Overall, the in vitro maturation culture generated immature and transitional B lymphocytes with a phenotype very similar to the lymphocytes differentiated in vivo. A very small percentage of phenotypically mature B cells could be generated in vitro, conﬁrming that cell culture systems of differentiation do not generate mature B lymphocytes (27).

TLR4 and TLR2 ligands have distinct effects on B lymphocyte FIGURE 1. Representative FACS analyses of B cells differentiated in ϩ Ϫ differentiation vivo and in vitro. A, Kinetic of B cell differentiation in vitro. B220 IgM B cell precursors purified from mouse BM were cultured (2 ϫ 105 cells per The addition of TLR4 agonists, lipid A, or highly purified LPS, to well) for 24, 48, or 72 h, and stained with anti-B220-FITC, -IgM-APC, and the cultures of B220ϩIgMϪ B cell precursors promoted an in- -CD23-PE. Percentages of IgMϩ cells in total B220ϩ population are indi- ϩ ϩ ϩ crease in the absolute number and percentage of IgM CD23 B cated in the upper panel, and in the lower panel, the percentage of CD23 lymphocytes, resulting in a 2- to 4-fold increase in the percentage cells in B220ϩIgMϩ population is indicated. Cells from fresh BM (B)or 72-h cultures of purified B220ϩIgMϪ B cell precursors (C) were stained with anti-B220-FITC, -IgM-APC, -CD23-PE and -AA4- or -IgD-biotin/ Alexa 680-PE-SA. Left plots, B and C, B cell subsets defined according to AA4 in T subset compared with I subset. Dead cells were excluded from Numbers under I, T, and M ,ء .literature: B220lowIgMlow immature (I), B220dull/highIgMhigh transitional analyses by propidium iodide staining ϩ ϩ ϩ (T), and B220highIgM mature (M). Percentages of IgM cells in B220 indicate the percentages of the subsets in IgMϩ population. Due to the population were 42.8 (fresh BM) and 49.0 (72-h cultures). In the right narrow gates defined to avoid mutual contaminations, and get more rep- panels, B and C, CD23, IgD, and AA4 expression patterns were determined resentative patterns for each B cell subset, ϳ15% of IgMϩ cells were not for I, T, and M subsets. Lower right histogram in C, Down-regulation of included in any of the subset gates. 6642 ROLE OF TLR IN B CELL DEVELOPMENT

Table I. Cell numbers of different B-lineage cell subsets along differentiation in vitroa

B220ϩ (%) B220ϩIgMϩ (%) B220ϩIgMϩCD23ϩ (%) Percentage of CD23ϩ In B220ϩIgMϩ

Expt. 1 Control 114,540 (100)b 61,359 (53.6) 8,038 (7.0) 13.1c Lipid A 150,726 (100) 87,919 (58.3) 22,454 (14.9) 25.5 Expt. 2 Control 104,216 (100) 47,564 (45.6) 3,368 (3.2) 7.1 Lipid A 100,989 (100) 51,818 (51.3) 13,105 (13.0) 25.3 Expt. 3 Control 97,439 (100) 47,472 (48.7) 3,556 (3.6) 7.5 Lipid A 139,962 (100) 72,444 (51.8) 15,568 (11.1) 21.5 Expt. 4 Control 166,554 (100) 49,733 (29.9) 5,560 (3.3) 11.2 LPS 165,744 (100) 68,784 (41.5) 18,070 (10.9) 28.3 Expt. 5 Control 144,864 (100) 65,870 (45.5) 6,148 (4.2) 9.3 LPS 164,110 (100) 75,326 (45.9) 17,739 (10.8) 23.6 Expt. 6 Control 160,904 (100) 75,303 (46.6) 4,255 (2.6) 5.7 LPS 130,800 (100) 75,681 (57.9) 14,924 (11.4) 19.7 Downloaded from a Analyses were performed after 72 h of culture of purified B220ϩIgMϪ B cell precursors under the indicated conditions (control, LPS at 10 ␮g/ml or lipid A at 1 ␮g/ml). Viable cell recovery was scored, excluding dead cells with trypan blue dye, and FACS profile analysis was performed as described in Fig. 1A. b Absolute cell numbers per well (96-well plates) for the indicated subsets. Numbers in parentheses indicate percentage relative to total B220ϩ cell population. c Percentage of IgMϩCD23ϩ cells relative to total IgMϩ cells, obtained as in Fig. 1A. of CD23ϩ B lymphocytes (Fig. 2A and Table I). The percentage of showed a limited up-regulation of CD69 Ag in 24 h of culture and http://www.jimmunol.org/ IgMϩCD23ϩ cells augments during the 72-h culture period, which there was not a significant increase of the marker even at 72 h, in could suggest the proliferation of a small number of IgMϩCD23ϩ contrast to the massive and sustained expression of CD69 in ma- cells. However, this seems unlikely because the addition of LPS in ture B lymphocytes (Fig. 3A). Moreover, four-color staining for the last 24 h of culture had the same result as the addition of LPS B220, IgM, CD69, and CD23 revealed that the limited expression in the beginning of the culture (Table II). Indeed, the effect of LPS is of CD69 following the addition of LPS bears no correlation with already observed even if the addition occurred 12 h before the end of the expression of CD23 (Fig. 3B). the culture (data not shown). Absolute cell counts are also shown in We next investigated if the agonist specific for the TLR2, Tables I and II, allowing the conclusion that the percent increase of Pam3Cys, would have similar effects as TLR4 agonists in B cell ϩ ϩ

IgM CD23 B cells cannot be accounted by cell death or prolifera- maturation, inasmuch as those receptors share most of their sig- by guest on September 28, 2021 tion, and represents the transition of a subset of B cells in culture, from naling transduction pathways (15). However, the addition of CD23Ϫ to CD23ϩ. It is interesting to note that only IgMhigh B cells Pam3Cys to the cultures of B220ϩIgMϪ B cell precursors had no express CD23, both in control and LPS-treated cultures (Fig. 2B). The effects on the percentage of CD23ϩ B cells (Fig. 4A). Interestingly, effect of LPS on B cell maturation was not observed in cultures with when Pam3Cys and lipid A were simultaneously added to the cul- B cell precursors obtained from C57BL/10ScCr mice, a natural null- ture, the increase in the percentage of CD23ϩ cells promoted by mutant for TLR4 (data not shown). LPS/lipid A was completely abolished by Pam3Cys (Fig. 4A). Expression of CD23 induced by LPS could depend on the ac- Pam3Cys alone inhibited the expected down-modulation of AA4 tivation of the B lymphocyte. We have addressed this point com- expression on transitional B cells (Fig. 4B), when compared with paring the expression of CD69 and CD23 following the addition of control. In contrast, the presence of LPS/lipid A did not interfere LPS to cultures of mature splenic B cells or B cells generated in in the down-modulation of AA4 expression (Fig. 4B). The expres- vitro (Fig. 3A). In the presence of LPS, B cells generated in vitro sion of IgD was also inhibited in the presence of Pam3Cys alone

Table II. Kinetic of B cell differentiation in vitro in the presence of LPSa

Hours B220ϩ (%) B220ϩIgMϩ (%) B220ϩIgMϩCD23ϩ (%) Percentage of CD23ϩ in B220ϩIgMϩ

0 200,000 (100)b 1,800 (0.9) 0 0c 24 Control 196,184 (100) 38,472 (19.6) 850 (0.4) 2.2 LPS 218,949 (100) 42,848 (19.6) 1,842 (0.8) 4.3 48 Control 198,323 (100) 80,856 (40.4) 4,447 (2.2) 5.5 LPS 222,616 (100) 91,562 (41.1) 13,835 (6.2) 15.1 72 Control 160,904 (100) 75,303 (46.8) 4,255 (2.6) 5.7 LPS 130,800 (100) 75,681 (57.9) 14,924 (11.4) 19.7 LPS last 48 h 144,005 (100) 72,247 (50.2) 12,289 (8.5) 17.0 LPS last 24 h 180,013 (100) 85,956 (47.8) 16,022 (8.9) 18.6

a Analyses were performed after 24, 48, and 72 h of culture of B220ϩIgMϪ BϪ cell precursors under the indicated conditions (control or LPS at 10 ␮g/ml). In 72-h cultures, LPS was added either at 0 h (LPS), at 24 h (LPS last 48 h) or at 48 h (LPS last 24 h) of culture. Viable cell recovery was scored, excluding dead cells with trypan blue dye, and FACS proﬁle analysis was performed as described in Fig 1A. Results are representative of two independent experiments. b Absolute cell numbers per well (96-well plates) for the indicated subsets. Numbers in parentheses indicate percentage relative to total B220ϩ cell population. c Percentage of IgMϩCD23ϩ cells relative to total IgMϩ cells, obtained as in Fig. 1A. The Journal of Immunology 6643 Downloaded from http://www.jimmunol.org/

FIGURE 4. Effects of TLR-4 and TLR-2 ligands on B lymphocyte dif- FIGURE 3. ϩ Ϫ by guest on September 28, 2021 Comparative analyses of the effect of LPS on splenic B ferentiation in vitro. A, B220 IgM cells purified from BM were cultured A Left panel cells and B cells generated in vitro. , , spleen cells were cultured for 72 h without or with either lipid A (1 ␮g/ml), Pam3Cys (1 ␮g/ml), lipid ␮ right panel ϩ Ϫ for 24 and 72 h with or without LPS (10 g/ml); , B220 IgM A plus Pam3Cys (1 ␮g/ml each), LPS (10 ␮g/ml), or LPS (10 ␮g/ml) plus cells purified from BM were cultured for 72 h, and allowed to differentiate Pam3Cys (1 ␮g/ml). Cells were stained with anti-B220-FITC, -IgM-APC, ␮ without or with LPS (10 g/ml) added either at 0 h (LPS 72 h) or at 48 h and -CD23-PE, analyzed by FACS as in Fig. 1A, and the fold-increase in (LPS last 24 h). Cells were stained with anti-B220-FITC, -IgM-APC, and percentage of CD23ϩ cells in treated cultures over control (normalized to -CD69-PE or -CD23-PE for FACS analysis. Plots were gated to show Ϯ ϩ ϩ ϩ Ϫ 1) was calculated. Bars represent mean of three independent experiments B ϩ Ϫ B220 IgM B cells only. , B220 IgM cells purified from BM were SD. B, B220 IgM cells were cultured under the same conditions as de- cultured under the same conditions described above and stained with anti- scribed in A and stained with anti-B220-FITC, -IgM-APC, -AA4-biotin/ CD23-PE, -CD69-biotin/APC-SA, and IgM-FITC. CD69 ϫ CD23 profiles low low ϩ Alexa 680-PE-SA. Histograms show AA4 expression by B220 IgM are shown for IgM cells, and the percentages in each quadrant are indi- immature and B220dull/highIgMhigh transitional B cell populations generated top cated on the of the plots. Results are representative of two independent upon each treatment. Median values of transitional B population for AA4 experiments. Dead cells were excluded from analyses by propidium iodide are as follow: control ϭ 93.9, lipid A ϭ 79.2, Pam3Cys ϭ 138.2, LPS ϭ staining 82.8. C, The same experiment as described in B showing staining with anti-IgM-APC, -B220-FITC, -CD23-PE, and –IgD-biotin/Alexa 680-PE- SA. Plots show IgD expression profiles of B220ϩIgMϩCD23ϩ B cells Statistical significance from control: p ϭ 0.0347 ,ء .Fig. 4C). Altogether, these results suggest that, besides inhibiting from each treatment) TLR4 induction of CD23, engagement of TLR2 alone can interfere (lipid A) and p ϭ 0.0129 (one-sample analysis, Student’s t test). and arrest the differentiation in a late step of B cell maturation.

The effect of Pam3Cys is abrogated in TLR2-KO B220ϩIgMϪ cells from TLR2-KO mice (Fig. 5B). We verified that Pam3Cys has been characterized as a specific TLR2 ligand; how- lipid A and highly purified lipoprotein-free LPS, induced a 2-fold ϩ ϩ ever, to rule out the possibility that Pam3Cys would be acting increase in the percentage of IgM CD23 transitional B cells rel- through an alternative receptor, we also studied the effects of ative to control. Pam3Cys addition alone did not show any effect. Pam3Cys in the TLR2-KO mice to confirm its dependence on When Pam3Cys was added simultaneously with LPS/lipid A no TLR2. As shown in Fig. 5A, splenocytes from normal C57BL/6 inhibition of their activity was observed, demonstrating that the mice responded with intense proliferation in response both to effect of Pam3Cys is directly dependent upon the activation Pam3Cys and lipid A. By contrast, TLR2-KO mouse splenocytes via TLR2. did not proliferate with Pam3Cys, while the response to lipid A was not affected, showing that the proliferative response of mature TLR2 and TLR4 ligand effects on early B cell precursors B lymphocytes to Pam3Cys is TLR2-dependent. We then tested The experiments described above characterize the effects of TLR4 the effects of TLR2 and TLR4 ligands in cultures of purified and TLR2 in late steps of B cell maturation. We then investigated 6644 ROLE OF TLR IN B CELL DEVELOPMENT

FIGURE 5. Effect of Pam3Cys on B cell maturation is TLR2-dependent. A, Splenocytes from C57BL/6 (left) and TLR2-KO (right) mice were cultured with the indicated concentrations of Pam3Cys or lipid A for 48 h and for further 24 h after addition of [3H]thymidine. Results shown are mean values of duplicate cultures Ϯ SD. B, B220ϩIgMϪ B cell precursors puriﬁed from BM of TLR2-KO mice were cultured for 72 h without or with either highly puriﬁed LPS (10 ␮g/ml), lipid A (1 ␮g/ml), Pam3Cys (1 ␮g/ml), or lipid A plus Pam3Cys (1 ␮g/ml each). Harvested cells were stained for B220, IgM, and CD23. Graphics show CD23 expression on Downloaded from B220ϩIgMϩ cells. Results shown are representative of two independent experiments. http://www.jimmunol.org/

the activity of lipid A and synthetic Pam3Cys on early B cell 50% of B220ϩ cells after 72 h (Fig. 1A and Table I); the percent precursors. For that purpose, B cell precursors were assayed for the enrichment for B cells is true differentiation and cannot be attrib- effects of Pam3Cys and lipid A on the expression of B220, c-kit, uted to selective loss of viability of B cell precursors (Tables I and IgM, and IL-7 responsiveness (Fig. 6). BM cells were cultured for II). Comparison of the phenotype of in vitro with in vivo matura- by guest on September 28, 2021 5 days with IL-7 to obtain a B cell precursor population highly tion supports the conclusion that differentiation in vitro generates enriched for IL-7 responsiveness, and then recultured in the pres- mostly immature and transitional B lymphocytes, with a phenotype ence of IL-7 without or with Pam3Cys and/or lipid A for further very similar to their homologous populations generated in vivo 48 h. While in cultures with IL-7 alone, the number of B cell (Fig. 1, B and C). In line with previously described results, few precursors recovered was 2-fold the input number, in cultures with mature B lymphocytes could be generated in vitro (26, 27). Our Pam3Cys, lipid A or both, no changes in the B cell precursor culture systems consisted of purified B220ϩIgMϪ cells, and numbers occurred, showing that the presence of the TLR ligands support B cell maturation until the T1 (IgMhighAA4ϩCD23Ϫ) impaired the proliferative expansion of B cell precursors in re- and T2 (IgMhighAA4ϩCD23ϩ) transitional stages, while T3 sponse to IL-7 (Fig. 6A). The addition of lipid A resulted in higher low ϩ ϩ (IgM AA4 CD23 ) is poorly represented (9). expression of B220 on B cell precursors when compared with con- Addition of TLR4 agonists—lipoprotein-free LPS or lipid trol and Pam3Cys-treated cultures (Fig. 6B). Interestingly, we ϩ Ϫ A—to purified B220 IgM cell cultures promoted a 2- to 4-fold found that upon the addition of both lipid A and Pam3Cys, the ϩ increase in the generation of CD23 B cells (Fig. 2 and Table I). effect of lipid A prevailed over the Pam3Cys (Fig. 6B), in contrast ϩ ϩ Again, the percent enrichment for CD23 B cells is true differen- to the results found in the study of CD23 population, where tiation and cannot be attributed to selective loss of viability of TLR2 predominated, suggesting a developmental control on dom- Ϫ ϩ inance of the TLR4/TLR2 signaling. CD23 cells or proliferation of CD23 cells (Tables I and II). This is particularly evident in cultures where LPS was added in the last ϩ ϩ Discussion 24 h, resulting in the same increase in IgM CD23 population as for cells cultured in the continuous presence of LPS for 72 h (Table There is little information about the direct effects of LPS, as well ϩ as of other TLR agonists, on B cell development. Indeed, most II). The percent increase of CD23 B cells induced by LPS is studies investigate the response to LPS of the maturing B lympho- dependent on the expression of TLR4, as indicated by results with cyte without addressing the effect of LPS in the maturation process cultures of B cell precursors from C57BL10.ScCr mice, a natural itself (3, 9, 24, 25). In vitro studies with transformed 70Z/3 pre-B TLR4-deficient mutant (data not shown). CD23 is a low-affinity cell line showed that LPS promotes expression of IgM on cell receptor for Fc⑀ (Fc⑀RII) highly expressed on mature follicular B surface, indicating that LPS can provide stimulus for differentia- lymphocytes (29). It is considered as a maturation marker due to tion (28). To study direct effects of TLR2 and TLR4 agonists in B the close correlation of arising of this marker on B lymphocyte cell differentiation, we used an in vitro culture system of B cell surface with progression in maturation process, as has been char- maturation from normal, highly purified B220ϩIgMϪ cells. In acterized for the transitional B cell subsets (9). The data obtained vitro culture of B220ϩIgMϪ cells allows for the differentiation of with cultures of purified B220ϩIgMϪ cells in the presence of lipid a significant number of sIgMϩ B lymphocytes that make up near A or LPS showed that TLR4 can stimulate B cell maturation as The Journal of Immunology 6645

maturation steps. LPS is a potent mitogen for mature B cells, promoting extensive proliferation and differentiation of the lymphocytes into Ig-secreting plasma cells. It is interesting to speculate whether the action of LPS in B cell differentiation in vitro would depend on the full activation of the B lymphocyte, or would follow a pattern of partial activation suggested by the positive/negative selection through the BCR. We have investigated this point analyzing the expression of the very early activation Ag CD69. CD69 is readily and massively expressed by B and T cells a few hours following activation through the clonotypic receptors or mitogens (33). CD69 is expressed in conditions of suboptimal stimulation, being a very sensitive indicator of lymphocyte activation. Strik- ingly, the addition of LPS to B cells generated from puriﬁed B220ϩIgMϪ cells in vitro resulted in little expression of CD69 (Fig. 3A). In contrast, mature splenic B cells show a massive and sustained expression of CD69 upon stimulation with LPS (Fig. 3A). Moreover, there is no correlation between the expression of CD23 and CD69, as assessed by four-color ﬂow cytometry anal-

ysis (Fig. 3B). These results are in agreement with the interpreta- Downloaded from tion that stimuli inducing the expression of CD23 upon triggering of TLR4 do not promote a full cell activation, analogous to BCR- derived intracelullar signaling involved in B cell maturation (34). Further evidence of the differential activation of LPS in maturing B cells was obtained analyzing the expression of AA4 Ag, which

is up-regulated in mature lymphocytes upon LPS activation (data http://www.jimmunol.org/ not shown and Ref. 35), but is down-regulated in CD23ϩ B cells generated in vitro (Fig. 1C). Altogether these data show that signaling through TLR4 can promote B cell maturation, a finding that FIGURE 6. Effects of TLR-4 and TLR-2 ligands on early B cell pre- has not been previously described. cursors. BM cells were cultured for 5 days with saturating concentrations ϩ ϩ Ϫ Pam3Cys inhibited the increased generation of CD23 cells in of IL-7. At the end of the culture, ϳ85% of the cells were B220 IgM B cell precursors. After depletion of IgMϩ cells, cells were recultured with the presence of lipid A (Fig. 4), suggesting that engagement of IL-7 for additional 48 h without or with either lipid A (1 ␮g/ml), Pam3Cys TLR2 blocked TLR4-induced maturation. We sought further evi- (1 ␮g/ml), or lipid A plus Pam3Cys (1 ␮g/ml each). Cells were stained dences that engagement of TLR2 alone could be interpreted as with anti-IgM-APC and -B220-FITC. A, Numbers of total viable cell re- arrest in B cell maturation. Interestingly, while transitional B cells by guest on September 28, 2021 covery. B, Histograms show levels of B220 expression on B220ϩIgMϪ (B220dull/highIgMhigh) down-modulate AA4 in the presence of lipid cells from control (....), and the indicated treatment ( ). Results shown A, the equivalent population in the presence of Pam3Cys kept a are representative of two independent experiments. high expression of AA4 (Fig. 4B). This result confirms that TLR2 and TLR4 agonists have opposite effects on B cell maturation. The finding that Pam3Cys completely inhibits lipid A-mediated in- suggested by the increase in the more mature CD23ϩ B cell pop- crease of the generation of CD23ϩ cells suggests a new cross-talk ulation. It is interesting to note that in control cultures, the expres- interaction between those TLR signaling pathways. Experiments sion of CD23 only begins in B lymphocytes with transitional with TLR2 KO mice (Fig. 5) showed that the inhibition mediated phenotype B220dull/highIgMhigh (Fig. 2B), which have down-mod- by Pam3Cys cannot be attributed to competition for the TLR4, ulated the expression of AA4 relative to immature B lymphocytes, with Pam3Cys behaving as a hypothetical antagonist for that re- as expected accordingly with the in vivo maturation process (Fig. ceptor; the presence of TLR2 is necessary for the inhibition to 1C). We found that the CD23ϩ B lymphocytes generated in the occur. These experiments also rule out putative molecular inter- presence of TLR4 agonists also arise at transitional stage express- actions between Pam3Cys and lipid A. In most cell culture systems ing low levels of AA4, just like the control cultures, thus suggest- studied in vitro, TLR2 and TLR4 share many of their biological ing that CD23 expression induced by TLR4 engagement obeys the activity promoting the synthesis of a common set of cytokines, differentiation program, and cannot be expressed out of its timing although TLR2 seems to be less comprehensive than TLR4, but in maturation (Figs. 2B and 4B). Little is known about the stimuli both stimuli act to promote the synthesis of inflammatory media- necessary to promote the expression of CD23 in maturing B cells. tors (15). It has also been demonstrated that simultaneous addition A recent study has shown that incubation of immature B cells with of agonists for different TLRs can result in a synergistic effect on BAFF, characterized as an important factor for survival and mat- transcription factors and cytokine production (36). Surprisingly, uration of transitional B lymphocytes (30, 31), is able to induce our results show a clear antagonism between TLR4 and TLR2 expression of CD23 (32). Our results indicate that TLR4 signaling engagement in B cell maturation. To our knowledge, this is the can also provide stimuli for directing the expression of CD23 on first report of a direct biological antagonism between these TLRs. late steps of B lymphocyte maturation. Cross-tolerance between TLR2 and TLR4 has been shown to re- The final maturation stages of B lymphocyte, in which immature quire previous stimulation of the first receptor to inhibit the other B cells progress along the transitional stages, are not associated (36, 37), and it has been explained by inhibition of downstream with cell proliferation (9, 10). However, evidences for negative or signaling pathways or down-regulation of receptors upon preincu- positive selection of B cell clones depending on the engagement of bation with heterologous TLR agonists. However, here the ligands the clonotypic BCR (20–22) suggest that intracellular signaling were added simultaneously, and our results indicate a competition linked to B cell activation by surface Ig receptor is directing these between two concurrent activation pathways each one leading to 6646 ROLE OF TLR IN B CELL DEVELOPMENT distinct cell fates, rather than a desensitization phenomenon. TLR4 simultaneous alteration of BCR and TLR4 signaling in immature signal transduction involves both MyD88-dependent and indepen- B, as TLR4 is normally associated with lipid rafts. Further studies dent pathways, whereas TLR2 signals only through MyD88 (38), on the functional maturation of BCR signaling vs TLR signaling suggesting a possible explanation for the different effects of TLR4 would be necessary to understand how those receptor pathways are and TLR2 agonists. Studies with others TLRs restricted to the integrated in the same cell type and how they affect the generation MyD88-dependent (TLR7/9) or MyD88-independent (TLR3) sig- of the emerging B cell repertoire. nal transduction pathways are necessary to test this hypothesis. Alternatively, LPS may also act through RP105 besides TLR4, Acknowledgments involving different signaling cascades (39). We thank Dr. G. Dos Reis, A. Coutinho, M. Bozza, and M. Bellio for We also investigated the effects of TLR4 and TLR2 agonists in suggestions and critical reading of the manuscript, Dr. Golenbock and early stages of B cell development analyzing cultures of IL-7- Dr. P. Tobias for providing highly purified LPS preparations, Dr. A. Rolink responsive B220ϩIgMϪ cells. In the presence of Pam3Cys or lipid for providing mAb 493 hibridoma, and Dr. L. Zingali and A. L. de A, the expansion of B cell precursors in response to IL-7 is almost Oliveira-Carvalho for purifying 493 mAb. totally inhibited, showing that these early B cell precursors are also Disclosures direct targets of those TLR agonists (Fig. 6A). The increased B220 The authors have no financial conflict of interest. expression in cultures that received lipid A is again suggestive of a maturation effect of TLR4 signaling for early B cell precursors References (Fig. 6B). In contrast, B cell precursors in Pam3Cys-added cultures 1. Osmond, D., and G. Nossal. 1974. Differentiation of lymphocytes in mouse bone did not show up-regulation of B220. Therefore, despite the similar marrow. I. 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