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The Journal of Immunology, 2007, 179, 6663-6672
Copyright © 2007 by The American Association of Immunologists, Inc.
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Primary Development and Participation in a Foreign Antigen-Driven Immune Response of a Chromatin-Reactive B Cell Clonotype Are Not Influenced by TLR9 or Other MyD88-Dependent TLRs1

Francis Coffey, Xiaohe Liu and Tim Manser2

Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, 19107


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Recent findings support a central role for TLRs in both foreign Ag-driven immune responses and systemic autoimmune diseases mediated by B lymphocytes. In vitro studies have shown that the Ag receptors (BCRs) on B cells specific for nuclear autoantigens can facilitate the delivery of these autoantigens to the endocytic compartment, resulting in activation of the nucleic acid-specific TLRs present in this subcellular locale. If this pathway is operative in vivo it might promote the development, survival, or activation of such autoreactive B cells. To test this idea, we evaluated the influence of a deficiency in the CpG DNA-specific TLR, TLR9, or all MyD88-dependent TLRs on the primary development and foreign Ag-driven immune response of B cells in a line of VH knockin mice that contains a high frequency of "dual reactive" B cells specific for DNA-based autoantigens such as chromatin, as well as the hapten arsonate. We found that although development and activation of these B cells in vitro are clearly influenced by DNA-based autoantigens, TLR9 or MyD88 deficiencies had no apparent effect on the primary development and participation in the anti-arsonate response of these B cells in vivo. We discuss these results in the context of previous models for the role of TLR9 and other TLRs in the regulation of antinuclear Ag B cell development and activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The naive B cell compartment is characterized by high levels of "polyreactivity," including autospecificities (1, 2, 3, 4). How autoreactive peripheral B cell activity is regulated to prevent autoimmunity is incompletely understood. This question has become more complex with the recent discovery that the Ag receptors (BCRs) of certain autoreactive B cells can mediate autoantigen uptake and delivery to endosomal TLRs such as TLR7 and TLR9 (5, 6, 7, 8). The signaling of these TLRs can induce B cell costimulation, activation, and proliferation (5, 6, 7, 8, 9).

Such findings have led to the suggestion that production of Abs specific for intracellular autoantigens in autoimmune diseases such as systemic lupus erythematosus is facilitated by this pathway (10, 11, 12, 13, 14, 15). This idea is particularly attractive in the case of anti-DNA and antichromatin Abs, as it is now clear that mammalian DNA, particularly that released from apoptotic cells, contains sufficient levels of stimulatory CpG motifs to mediate activation of TLR9 (7, 8). Support for this idea has also been provided by recent studies of autoimmune-prone TLR9-deficient mice, which display substantially reduced levels of serum anti-DNA and antichromatin Abs (16).

The ability of TLRs to provide direct costimulation to B cells has also led to the idea that TLR activity plays an essential role in promoting anti-pathogen and other anti-foreign Ag Ab responses (9, 17). Although this is a current area of controversy (18, 19), the fact that both self and foreign ligands are capable of activating at least the endosomal TLRs raises the conundrum of how autoreactive B cell activity is regulated during an anti-pathogen immune response, particularly in the case of such B cells that are also cross-reactive with foreign Ags expressed by the pathogen. A potential solution to this paradox has been provided by a recent study indicating that TLR9 signaling is attenuated in B cells that chronically engage autoantigen via the BCR (20).

We have previously described a line of gene targeted mice termed HKIR, in which a modified form of an Ab H chain variable (VH) region gene partially encoding anti-arsonate (Ars)3 Abs is inserted into the endogenous H chain locus. This VH gene contains a mutation to arginine at position 55 in CDR2 (21). The HKIR H chain locus, in combination with a single, unmutated, endogenous {kappa} L chain gene, encodes Abs termed "canonical" that bind Ars and also have high avidity for DNA-based autoantigens and intensely stain condensed chromatin in antinuclear Ag assays (21).

B cells expressing canonical HKIR BCRs develop to mature follicular phenotype, reside in splenic and lymph node follicles and are not short-lived (22). However, these B cells express very low levels of both surface IgM and IgD (22, 23, 24), indicating that they are not "ignorant" of self-Ags. Indeed, we previously showed that modulation of BCR levels on canonical HKIR B cells developing in vitro is regulated by both endocytosis and a feedback loop in which BCR engagement by a DNA-based autoantigens is linked to transcriptional control of BCR-encoding loci (25). In addition, our previous studies demonstrated that canonical HKIR B cells residing in the follicles of the spleen do not display features of anergy in vitro and participate normally in the primary immune response to Ars in vivo (21, 22, 23, 26).

Taken together with the data discussed, indicating that the BCR of anti-DNA and antichromatin B cells may be capable of efficient delivery of CpG containing extracellular DNA to TLR9, our results raised the possibility that this TLR plays a central role in promoting the primary or Ag-driven development and activation of canonical HKIR clonotypes. To test this idea, we generated TLR9- and MyD88-deficient versions of the HKIR line (termed HKIR.TLR9–/– and HKIR.MyD88–/–) and conducted in vivo studies of B cell development and the participation of canonical HKIR.TLR9–/– and HKIR.MyD88–/– clonotypes in the Ars-driven immune response. In addition, we investigated the influence of endogenous and exogenous TLR9 ligands on canonical HKIR B cell development and activation in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

HKIR VH knockin mice on the C57BL/6 background were generated in our laboratory and have been previously described (21, 22, 23). TLR9- or MyD88-deficient C57BL/6 mice, both obtained from S. Akira (Osaka University, Osaka, Japan), were bred to the HKIR mouse line to obtain either HKIR.TLR9–/– or HKIR.MyD88–/– mice (27, 28). To obtain the double transgenic HKIR/V{kappa}10 mouse line, HKIR mice were bred to V{kappa}10A L chain conventional transgenic mice expressing the same L chain construct as previously described Ars/A1 mice (29). C57BL/6 (CD45.2+) and C57BL/6.SJL (CD45.1+) mice were purchased from The Jackson Laboratory. Mice were housed under pathogen-free conditions and received autoclaved food and water. All mice were 8- to 12-wk-old when used in experiments. The use of mice in these studies was approved by the Animal Care and Use Committee.

Adoptive transfer

After isolation of splenocytes from age-matched donor HKIR and HKIR.TLR9–/– mice, RBCs were lysed using ACK buffer and cells were washed three times with PBS. Two adoptive transfer protocols were used. In some studies, 3–5 x 106 splenocytes were injected into the tail vein of syngeneic B6.CD45.1 recipients 1 wk after i.p. immunization with 100 µg of Ars-keyhole limpet hemocyanin (KLH) in alum. These recipient mice were i.p. injected with an additional 50 µg of Ars-KLH in PBS at the time of transfer. For studies in which in vivo CFSE dilution was analyzed, 10 x 106 splenocytes were labeled with CFSE before transfer as previously described and mice were sacrificed on day 3 to assess proliferation by flow cytometry (30). In the second protocol, 3–5 x 106 splenocytes were transferred to B6.CD45.1 recipients that were i.p. immunized 12 h later with 100 µg of Ars-KLH in alum.

Bone marrow cultures

Bone marrow cultures were generated as previously described (31) using the S17 stromal cell line, which was a gift from Dr. R. Hardy (Fox Chase Cancer Center, Philadelphia, PA), with permission from Dr. K. Dorshkind (University of California, Los Angeles, CA). Medium was supplemented with 16 ng/ml recombinant murine IL-7 (R&D Systems). These cultures were free of mature recirculating B cells as determined by flow cytometry. Cells were harvested after 12 h of treatment with DNase I (Roche), p-azophenylarsonate-L-tyrosine (Ars-Tyr) or p-aminobenzoic acid (PABA)-L-tyrosine (PABA-Tyr) (both conjugated and purified in-house as previously described) (29).

Spleen cell cultures

Splenocytes were isolated from 8- to 12-wk old age matched transgenic and nontransgenic mice and single-cell suspensions were filtered through a 70-µm strainer (Fisher Scientific). Naive splenic B cells were enriched by negative selection using MACS anti-CD43-conjugated magnetic beads (Miltenyi Biotec). The purity of cells in eluates was assessed by flow cytometry and always found to be >90% B cells. Cells were cultured in RPMI 1640, 10% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, and 5 µM 2-ME in 24-well plates (2–3 x 106 cells/well) with the indicated concentrations of chloroquine (Sigma-Aldrich), CpG ODN 1826 (InvivoGen), and/or Ars-Tyr. LPS (Difco) was added at 2.5 µg/ml. For in vitro proliferation studies, purified cells were labeled with CFSE following MACS enrichment.

Immunohistology

Spleens were snap frozen in Tissue-Tek OCT compound (Sakura Finetek), and cryosections (5–6 µm) were made as previously described (32). Three-color immunofluorescent staining was performed as previously described (33) using combinations of the following fluorochrome-conjugated Abs and reagents (from BD Pharmingen, unless otherwise indicated): E4-biotin (prepared in house); FITC-GL7, PE-anti-B220 (clone RA3-6B2), PE-anti-TCR-β (clone 104), PE-CD45.2 (clone 104; eBioscience), PE-anti-Syndecan-1 (281.2), FITC-MOMA-1 (metallophillic macrophage-1), and purified rat FDC-M2 (Serotec). Biotinylated E4 was detected with streptavidin-Alexa Fluor 633 and purified FDC-M2 was followed by goat anti-rat IgG-Alexa Fluor 633 (Molecular Probes). Images were acquired using the LSM 510 META confocal microscope (Zeiss).

Flow cytometry analysis

Three- and four-color flow cytometry analysis was done on single-cell suspensions (106 cells/sample) prepared from spleen and bone marrow cells obtained from naive and immunized mice or in vitro cultures. Samples were stained with combinations of the following Abs and reagents (from BD Pharmingen, unless otherwise indicated): streptavidin-CyChrome to detect biotinylated Abs, FITC-, PE-, or biotin-anti-B220 (RA3-6B2; eBioscience), FITC-anti-IgM (Jackson ImmunoResearch Laboratories), PE-anti-IgD (11-26; Southern Biotechnology Associates), PE-anti-C1qRp (AA4.1; eBioscience), FITC-anti-CD21/CD35 (7G6), biotin- or PE-anti-CD23 (B3B4), FITC-anti-CD43 (S7, Ly48), FITC-anti-CD86 (GL-1), PE-anti-CD69 (H1.2F3), FITC-anti-I-A/I-E (2G9), PE- or biotin-anti-CD45.2 (clone 104), FITC- or biotin-peanut agglutinin (PNA; Vector Laboratories), biotin-PNA, PE-, FITC-, and biotin-anti-B220 (clone RA3-6B2), and biotin-E4 (in house). Where indicated, propidium iodide was added at 2.5 µg/ml to cell suspensions for subsequent exclusion of dead cells during analysis. Flow cytometry analysis was conducted on a Coulter EPICS XL-MCL, and data were analyzed using the FlowJo software (Tree Star).

ELISPOT assay

Multiscreen 96-well plates (Millipore) were coated with 50 µl of 10 µg/ml goat anti-mouse IgM, IgG, IgG1, IgG2a, IgG2b, or IgG3 (Caltag Laboratories) at 4°C overnight. Splenocytes from immunized recipient mice were prepared as described and added in serial dilution to the coated plates in RPMI 1640 containing 10% FCS. Following incubation for 6 h at 37°C, plates were blocked with 5% normal mouse serum and biotinylated E4 (prepared in house) was added. Streptavidin conjugated to alkaline phosphatase (Vector Laboratories) was added at 1/500 dilution, and plates were developed using the alkaline phophatase substrate III kit (Vector Laboratories) to detect E4+ Abs. Images of plates were acquired using Image Acquisition and spots counted using ImmunoSpot 3 software (Cellular Technology).

ELISA

Ars-specific total serum IgM or IgG and E4 was measured by ELISA on 96-well plates (Immunolon-4; Thermo Electron) as previously described (34).

Statistical analyses

Statistical significance was determined using two-tailed, unpaired Student’s t test in the Microsoft Excel program.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
As previously described (25), the precursor frequency of canonical B cells in HKIR mice can be dramatically increased by crossing HKIR mice with a line of conventional Ig transgenic mice expressing the canonical {kappa} L chain gene (V{kappa}10A). We refer to the resulting double transgenic mice as HKIR/V{kappa}10. In HKIR single transgenic mice, canonical HKIR B cells are present at a frequency of ~5% of splenic B cells, due to the expression of endogenous L chains (22). Canonical B cells in these mice can be directly detected using the anti-clonotypical mAb E4 (21, 35).

Down-regulation of the BCR on canonical HKIR B cells developing in vitro is influenced by a DNA-based autoantigen

We previously showed that the down-regulation of BCR levels characteristic of canonical HKIR clonotype development could be inhibited in bone marrow cultures by the monovalent form of Ars, Ars-Tyr (25). Our finding that Ars-Tyr treatment also decreased the level of binding of a FITC-labeled form of DNase I to the surface of canonical HKIR B cells supported the idea that down-regulation was due to reduction of binding of canonical HKIR BCRs to a DNA-based autoantigen present in the cultures. Ars-Tyr treatment also lowered the characteristically elevated intracellular Ca2+ levels in immature canonical HKIR B cells to baseline values. To further investigate the nature of the autoantigen influencing canonical HKIR B cell BCR levels, cultures of HKIR/V{kappa}10 bone marrow were set up that included Ars-Tyr, the control hapten PABA-Tyr, for which canonical HKIR BCRs have no measurable affinity, or DNase I. The levels of surface IgM on these cells were measured 12 h later by flow cytometry. Fig. 1 illustrates that DNase I treatment, like Ars-Tyr, resulted in substantial increases in canonical surface IgM levels. In total, these data suggest that a DNA-based autoantigen is responsible for inducing BCR signaling and down-regulation in developing canonical HKIR B cells.


Figure 1
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  		FIGURE 1. DNase I treatment of HKIR/V{kappa}10 bone marrow cultures results in elevation of surface IgM levels on developing B cells. Cultures from HKIR/V{kappa}10 bone marrow were set up as described in Materials and Methods and after 3–4 days nothing (medium), PABA-Tyr (final concentration 0.1 mM), Ars-Tyr (0.1 mM), or DNase I (300 U/ml) was added. E4 and surface IgM levels were evaluated 12 h later by flow cytometry. Data shown are representative of two independent experiments.

 
Primary development of canonical HKIR B cells is not altered by a TLR9 deficiency

We previously reported that the rate of BCR internalization in immature HKIR/V{kappa}10 B cells was higher than in control B cells in bone marrow cultures, but that these differences disappeared when Ars-Tyr was added to the HKIR/V{kappa}10 cultures (25). Combined with the data presented, these findings suggested that the canonical HKIR BCR might mediate efficient uptake of a DNA-based autoantigen, allowing delivery to endosomes and activation of TLR9. However, preliminary results using bone marrow cultures generated from TLR9-deficient HKIR mice (HKIR.TLR9–/–) did not support the idea that either development of canonical HKIR clonotypes or down-regulation of their BCRs is dependent on TLR9 (25).

To address this question in vivo, detailed flow cytometric studies of B cell developmental stages in HKIR.TLR9–/– bone marrow and spleen were conducted using multiparameter flow cytometry. Fig. 2A illustrates that no major quantitative or qualitative differences in either bulk or E4+ B cell development in the bone marrow of HKIR and HKIR.TLR9–/– mice could be detected. Analogous results were obtained from analysis of the spleen (Fig. 2B). In particular, the number of transitional B cells (AA4.1+) was not influenced by the TLR9 deficiency, and nearly all canonical (E4+) B cells in both lines of mice have a mature follicular phenotype. In addition, levels of surface IgM and IgD were similarly reduced as compared with control B cells in HKIR mice that were TLR9 sufficient or deficient (Fig. 2C).


Figure 2
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  		FIGURE 2. B cell development of HKIR.TLR9–/– mice in bone marrow and spleen. A, Bone marrow cells from mice of the indicated genotypes were stained with anti-B220, anti-CD43, and anti-IgM and analyzed by flow cytometry. Gates were set to show the percentage of pro early pre-B cells (B220+CD43+) and late pre-B cell immature B cells (CD43–B220low) (top). Gates were set to show recirculating mature B cells (B220highIgM+), pre-B cells (B220lowIgM–), and immature B cells (B220lowIgMhigh). Gates show same fractions as in A of noncirculating, immature CD23–E4+ B cells analyzed by four-color flow cytometry (bottom). B, Splenocyte suspensions from mice of the indicated genotype were stained with the indicated Abs and analyzed by flow cytometry. Gates were set to show levels of surface IgM and IgD, frequencies of E4+ B cells, follicular B cells (CD23highCD21+), marginal zone B cells (CD23lowCD21high), and transitional B cells (B220lowAA4.1+). C, Splenic B cells from mice of the indicated genotypes were analyzed for levels of surface IgM and IgD by flow cytometry. All data are representative of at least three independent experiments from multiple mice.

 
TLR9 deficiency does not alter the participation of HKIR B cells in the anti-Ars response in vivo

To evaluate participation of B cells in HKIR mice in the immune response to Ars in vivo, a previously developed adoptive transfer protocol was used (36, 37). Donor HKIR or HKIR.TLR9–/– splenocytes were transferred to unirradiated CD45.1+ congenic C57BL/6 mice (5 x 106 donor B cells per recipient) that had been preimmunized with Ars-KLH in alum. In some experiments, these splenocytes were labeled with the cell division tracking dye CFSE before transfer. The immune responses mounted by the donor cells were then monitored via flow cytometry and histology.

Fig. 3A shows that similar percentages of CD45.2+ donor B cells entered the PNA+ germinal center (GC) compartment in both types of chimeric mice, as assessed by flow cytometry. This finding was corroborated by qualitative histological examination of GCs (Fig. 3B). Flow cytometric analysis of CFSE dilution levels in various subpopulations revealed that nearly all donor B cells (CD45.2+CFSE+) that were becoming PNA+ had undergone multiple cell divisions, and the number of these divisions was not influenced by the presence or absence of TLR9 (Fig. 3C). Moreover, the majority of E4+ B cells had proliferated and entered the PNA+ GC compartment in both types of mice. Such E4+ B cells made up ~50% of the proliferating CD45.2+ donor B cell pool in both cases (data not shown).


Figure 3
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  		FIGURE 3. Anti-Ars GC responses by HKIR B cells are not altered by a TLR9 deficiency. A and B, Recipient B6.CD45.1 mice were immunized 1 wk before transfer of HKIR or HKIR.TLR9–/– splenocytes. Mice were sacrificed on day 5 after transfer and boost. A, Recipient splenocytes were stained for three-color flow cytometry analysis. Gated percentages represent B220highPNA+CD45.2+ GC B cells. B, Spleen sections were stained with GL7 (green), FDC-M2 (blue), and CD45.2 (red). Data are representative of three independent experiments with three to five mice of each genotype. C, CFSE-labeled HKIR or HKIR.TLR9–/– splenocytes were transferred to immunized B6.CD45.1 as in A and B. On day 3 posttransfer, mice were sacrificed and splenocytes were stained for flow cytometry analysis. B220high cells were gated and donor (B220highCD45.2+) (left), canonical (B220highE4+) (center), and total (B220highE4+) (right) B cell proliferation was assessed by dilution of CFSE. Data are representative of two independent experiments with two mice of each genotype.

 
We next analyzed the magnitude and isotype profile of the Ab-forming cell (AFC) response mounted in the spleens by both types of chimeric mice using a different transfer protocol in which mice were immunized 12 h after transfer of donor splenocytes. Fig. 4A illustrates the results of ELISPOT analysis showing that total IgM and IgG E4+ AFC responses produced by these mice were similar. In addition, the number (Fig. 4A) and size (data not shown) of ELISPOTs produced by E4+ AFCs of different IgG subtypes did not significantly differ between the two strains. These results were corroborated by ELISA of serum anti-Ars and E4+ Ab (Fig. 4B). Histological analysis of spleens showed that in all mice these E4+ and other AFCs were largely confined to the bridging channels, a characteristic location of AFCs during the primary immune response (Fig. 4C).


Figure 4
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  		FIGURE 4. TLR9 deficiency does not alter primary AFC responses of canonical HKIR B cells. HKIR or HKIR.TLR9–/– spleen cells were transferred to B6.CD45.1 recipients 1 day before immunization, and mice were sacrificed on day 7 after transfer. A, Recipient splenocytes were harvested and E4+ AFCs were measured by ELISPOT assay. Each circle represents the number of E4+ IgM, IgG, or individual IgG subclass secreting AFCs per 1 x 106 splenocytes obtained from an individual recipient mouse. Horizontal bar represents the average number of E4+ AFCs. All p values indicated differences that were not significant between HKIR and HKIR.TLR9–/– mice. p > 0.5 for IgG, IgG2a, IgG2b, IgG3, and IgM. p > 0.4 for IgG1. B, Recipient serum was collected to evaluate total anti-Ars IgG or IgM and clonotypic (E4+) Ab titers by ELISA. Each circle represents the serum dilution factor for an individual mouse at an appropriate OD450 value: 0.7 for anti-Ars IgG and 0.3 for anti-Ars IgM or 0.35 for E4. Horizontal bar indicates the average value of dilution factors. Statistical analysis was performed using Student’s t test. C, Adjacent spleen sections from recipient mice were stained for E4 (blue), syndecan (red), metallophillic macrophage-1 (MOMA-1) (green) (left); E4 (blue), TCR-β (red), metallophillic macrophage-1 (green) (center); and E4 (blue), B220 (red), metallophillic macrophage-1 (green) (right). All data are representative of three independent experiments with at least five mice of each genotype.

 
In vitro activation of HKIR B cells by exogenous and endogenous TLR9 ligands

In total, these data did not support a role for TLR9 in either the primary or Ag-driven development of HKIR B cells or the canonical subset. Potential explanations for these results included that, despite allowing BCR cross-linking and internalization, the affinity of the BCR-autoantigen interaction on these cells was too low to result in efficient receptor mediated endocytosis and delivery of DNA to TLR9, or that TLR9 signaling was attenuated in these B cells. Alternatively, the engaged autoantigen might lack the required agonistic CpG motifs to trigger TLR9. To first investigate these issues in the case of canonical HKIR B cells, we used HKIR/V{kappa}10 B cells.

In agreement with previous studies indicating that autoreactive B cells can be spontaneously activated by TLR ligands present in cell culture (6, 8), control studies on the activation status of HKIR/V{kappa}10 splenic B cells after in vitro culture showed that levels of CD69, CD86, and class II MHC increased substantially over a 12-h period (Fig. 5A). Additionally, short-term culture of B cells in serum-free medium resulted activation, indicating that the autoantigen is not serum-derived. Incubation of CFSE-labeled B cells under these conditions for 2 days revealed that although B6 B cells had not proliferated measurably, most HKIR/V{kappa}10 B cells had divided one to two times. Addition of a CpG containing oligodeoxynucleotide TLR9 agonist to the cultures resulted in induction of cell division by B6 B cells, and enhanced proliferation of HKIR/V{kappa}10 B cells (Fig. 5B). Combined with the results shown in Fig. 1, these data suggested that a DNA-based autoantigen was responsible for spontaneous HKIR/V{kappa}10 B cell activation and proliferation in vitro. To test this idea, and to determine whether this activation was dependent on canonical HKIR BCR engagement and endosomal TLRs, additional experiments were performed.


Figure 5
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  		FIGURE 5. In vitro spontaneous activation and proliferation of canonical HKIR/V{kappa}10 B cells. A, CD43– MACS purified HKIR/V{kappa}10 or control B6 B cells were analyzed for the expression of activation markers ex vivo (top), after 4 h in culture without serum (middle), or after 12 h in culture with serum (bottom). Cells were harvested and stained for flow cytometry to assess expression of CD69, CD86, and class II MHC I-A/I-E on B220+ gated lymphocytes. Data are representative of three independent experiments. B, CFSE-labeled purified HKIR/V{kappa}10 or control B6 B cells were left untreated or stimulated with CpG ODN (1 µM). Following 48 h in culture, the extent of CFSE dilution was assessed by flow cytometry. Plots represent live cells gated according to forward and side scatter profiles and propidium iodide exclusion. Data are representative of two independent experiments.

 
To evaluate whether blocking the ability of the canonical HKIR BCR on mature splenic B cells to interact with endogenous autoantigens would inhibit spontaneous activation in vitro, Ars-Tyr was added to the medium in which these cells and control C57BL/6 B cells were incubated, and CD69 levels were monitored by flow cytometry 12 h later. Fig. 6A (left panels) shows that Ars-Tyr had no effect on CD69 levels on B6 B cells, but that it completely inhibited the up-regulation of CD69 on HKIR/V{kappa}10 B cells. These data strongly indicate that BCR engagement of an endogenous autoantigen is required for this up-regulation. In contrast, addition of the direct CpG TLR9 agonist resulted in up-regulation and enhanced up-regulation of CD69 on B6 and HKIR/V{kappa}10 B cells, respectively, and neither effect was influenced by the presence of Ars-Tyr.


Figure 6
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  		FIGURE 6. In vitro spontaneous activation of HKIR B cells is reduced by inhibitors of BCR binding and endosomal TLRs. A, CD43– MACS purified HKIR/V{kappa}10 or control B6 B cells were left untreated or treated with chloroquine (10 µM) or Ars-Tyr (10–4 M) alone, or for 30 min before the addition of CpG ODN (1 µM). Cells were harvested after overnight culture and stained for flow cytometry analysis of CD69 expression on B220high lymphocytes. B, Splenocytes from HKIR.TLR9–/–, HKIR, and control B6 mice were left untreated or treated with chloroquine or CpG ODN. Cells were harvested and stained for flow cytometry to assess CD69 expression on whole B220+ (top row), B220+E4– (middle row), or B220+E4+ (bottom row) gated lymphocytes. (C) CD43– MACS purified HKIR, HKIR.MyD88–/–, or control B6 B cells were left untreated or stimulated with CpG oligonucleotide and harvested after overnight culture to assess CD69 expression on B220+ lymphocytes by flow cytometry. All data are representative of at least three independent experiments.

 
To determine whether delivery of the putative autoantigen to endosomal TLRs was required for CD69 up-regulation, cultures of B6 or HKIR/V{kappa}10 B cells were treated with chloroquine, a compound which has been shown to block the ability of such TLRs to productively interact with their endocytosed ligands. Fig. 6A (right panels) shows this treatment only partially inhibited the up-regulation of CD69 on HKIR/V{kappa}10 B cells. As expected, however, this treatment neutralized the effect of the CpG TLR9 agonist on CD69 levels.

To evaluate the specific role of TLR9 in this in vitro activation, TLR9-deficient HKIR B cells were used in these assays. Fig. 6B, left panels, illustrates that total and E4– splenic B cells from HKIR.TLR9–/– mice displayed only very low levels of CD69 expression after 12 h in culture, as compared with HKIR B cells. However, this dependence on TLR9 for CD69 up-regulation was far less pronounced for E4+ (canonical HKIR) B cells in the cultures. Addition of the CpG TLR9 agonist to the cultures resulted in dramatic and equivalent up-regulation of CD69 levels on TLR9-sufficient total E4– and E4+ splenic B cells that were at least 10-fold higher than obtained with medium alone (Fig. 6B, right panels). As expected, TLR9-deficient B cells showed no further increase in CD69 upon CpG addition. This indicated that TLR9 is expressed at equivalent functional levels in E4– and E4+ HKIR B cells. However, the addition of chloroquine substantially, but only slightly reduced induction of CD69 on E4–, and E4+ B cells in HKIR and HKIR.TLR9–/– cultures, respectively (Fig. 6B, middle panels). In the analogous HKIR.TLR9–/– cultures, no discernible effect of chloroquine on CD69 levels were observed. In total, these data indicate that TLR9 plays a predominate role in the in vitro activation of most E4– HKIR B cells. In contrast, all endosomal TLRs, including TLR9, play minor roles in the spontaneous activation of canonical HKIR B cells.

An intrinsic MyD88 deficiency prevents the spontaneous activation of canonical HKIR clonotypes in vitro

The data presented suggested that either autoantigen-mediated cross-linking of the BCR on canonical HKIR B cells is sufficient to result in the elevated levels of CD69 observed on these cells after in vitro culture or that another, rather chloroquine-insensitive TLR was involved in this activation. To initially investigate this issue, MyD88-deficient versions of the HKIR line were generated. Splenic B cells enriched from these mice, as well as from HKIR.TLR9–/– and control B6 mice, were incubated in either medium alone or medium plus the CpG oligonucleotide, and levels of CD69 on these cells were assessed 24 h later. Fig. 6C shows that a MyD88 deficiency completely ablated the spontaneous and CpG-induced activation of all HKIR B cells.

MyD88 deficiency does not alter primary or Ars-driven development of canonical HKIR clonotypes in vivo

These data indicated that a MyD88-dependent TLR other than TLR9 was required for the spontaneous activation of canonical HKIR B cells in vitro, raising the possibility that this putative TLR influenced primary or Ag-driven development of these clonotypes in vivo. To test this idea, experiments analogous to those described for TLR9-deficient versions of HKIR clonotypes were performed on MyD88-deficient HKIR B cells. Fig. 7 shows that, as was found for TLR9-deficient HKIR mice, no major defects in E4+ primary B cell development in the bone marrow or spleen were apparent in HKIR.MyD88–/– mice. As in HKIR and HKIR.TLR9–/– mice, canonical B cell clonotypes developed nearly exclusively to a mature, follicular phenotype.


Figure 7
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  		FIGURE 7. B cell development in TLR9- and MyD88-deficient HKIR mice in the bone marrow and spleen. A, Bone marrow cells from mice of the indicated genotypes were stained with anti-B220, anti-CD43, and anti-IgM and analyzed by flow cytometry (top panels). Gates were set to show the percentage of pro early pre-B cells (B220+CD43+) and late pre-B cell immature B cells (CD43–B220low) or to show recirculating mature B cells (B220highIgM+), pre-B cells (B220lowIgM–), and immature B cells (B220lowIgMhigh). Noncirculating, immature CD23–E4+ B cells were analyzed by four-color flow cytometry (bottom panels). B, Splenocyte suspensions from mice of the indicated genotype were stained with the indicated Abs and analyzed by flow cytometry. Gates were set to show levels of surface IgM and IgD, frequencies of E4+ B cells, follicular B cells (CD23highCD21+), marginal zone B cells (CD23lowCD21high), and transitional B cells (B220lowAA4.1+). C, Splenic B cells from mice of the indicated genotypes were analyzed for levels of surface IgM and IgD by flow cytometry. All data are representative of at least three independent experiments with multiple mice of each genotype.

 
Fig. 8, A and D, illustrate that whether using either of the two splenic B cell adoptive transfer/Ars immunization protocols described, canonical HKIR.MyD88–/– B cells participated in the GC response at levels similar to canonical HKIR and HKIR.TLR9–/– B cells. Histological examination revealed that CD45.2+ HKIR.MyD88–/– B cells efficiently entered the GL7+ GC compartment (Fig. 8E). CFSE dilution studies showed that HKIR.MyD88–/– B cells also proliferated with the same kinetics and extents as control B cells (data not shown). Fig. 8, B and C, show that the AFC response and isotype profile produced by HKIR.MyD88–/– B cells was comparable to those produced by control B cells.


Figure 8
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  		FIGURE 8. A MyD88 deficiency does not alter the primary AFC response of canonical HKIR B cells or their participation in the anti-Ars GC response. A–C, HKIR, HKIR.TLR9–/–, or HKIR.MyD88–/– spleen cells were transferred to B6.CD45.1 recipients 1 day before immunization and mice were sacrificed on day 7 after transfer. B, E4+ AFCs were measured by ELISPOT assay. Each circle represents the number of E4+ secreting AFCs per 1 x 106 splenocytes obtained from an individual recipient mouse and bars indicate averages. C, Recipient sera were collected to evaluate total anti-Ars IgG or IgM and clonotypic (E4+) Ab titers by ELISA. Each circle represents the serum dilution factor for an individual mouse at an appropriate OD450 value: 0.85 for anti-Ars IgG and 0.375 for anti-Ars IgM or 0.35 for E4. The horizontal bar indicates the average value of dilution factors. All p values indicating differences between genotypes were not significant. D and E, HKIR, HKIR.TLR9–/–, or HKIR.MyD88–/– spleen cells were transferred to B6.CD45.1 recipients 1 wk after immunization. Recipient mice were boosted at the time of transfer and sacrificed 5 days later. D, Spleen cells harvested from recipient mice were stained for three-color flow cytometry analysis. Gated populations represent B220highPNA+CD45.2+ (top row) and B220highPNA+E4+ (bottom row) GC B cells. E, Spleen sections from Ars-KLH preimmunized mice receiving HKIR.MyD88–/– spleen cells were stained with GL7 (green), FDC-M2 (blue), and CD45.2 (red) as in Fig. 3B. Data are representative of three independent experiments with three to four mice of each genotype.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous studies have ascribed a central role for TLR9 expressed by B cells in the regulation of immunity and autoimmunity (38, 39, 40). During immune responses to pathogens, uptake of foreign Ag-DNA complexes by the BCR may facilitate the delivery of CpG ligands to the endocytic compartment where engagement of TLR9 results in augmentation of the response (7, 8, 41). In autoimmunity, studies of TLR9-deficient mice have indicated that production of anti-DNA serum autoantibodies, particularly of the IgG2 isotypes, is dependent on this TLR (42). In contrast, we find no influence of a TLR9 deficiency on the primary development and Ars-driven immune responses of HKIR canonical and noncanonical B cells. These responses included vigorous GC reactions and AFC development, resulting in production of IgG2 isotypes. This finding is despite the influence of an endogenous DNA-based autoantigen on the BCR levels on these B cells in vitro, and presumably in vivo. Moreover, both canonical and noncanonical HKIR B cells clearly express functional active TLR9, and the spontaneous activation of noncanonical HKIR B cells in vitro is largely TLR9-dependent. The latter observation does not support previous suggestions that TLR9 signaling is attenuated in anti-DNA and antichromatin B cells (20).

One explanation for these findings is that, despite the DNA reactivity of HKIR-derived canonical and many noncanonical BCRs in in vitro assays (21, 43), the relevant costimulatory autoantigen in vivo is not DNA, but a ligand for another TLR. In this regard, recent data obtained from a line of Ig transgenic mice expressing a BCR that binds ssDNA, ssRNA, and nucleosomes showed that spontaneous serum Ab production by the B cells in these mice was dependent on the ssRNA-specific TLR, TLR7 (10). However, the data we obtained from the analysis of MyD88-deficient versions of HKIR mice renders this explanation unlikely. Although a MyD88 deficiency completely ablated the spontaneous in vitro activation of canonical HKIR B cells, it had no major influence on the primary and Ars-driven secondary development of these B cells in vivo. We cannot rule out that a compensatory effect of the ablation of the function of multiple TLRs that conveys either positive or negative signals to canonical HKIR B cells is responsible for these results. Nonetheless, these data suggest that none of the MyD88-dependent TLRs are required for the normal primary development and participation in the foreign Ag-driven immune response of this type of antichromatin B cell. However, it remains possible that the DNA-based autoantigen is influencing canonical HKIR B cell development via a nucleic acid-specific pattern receptor that is not MyD88-dependent, such as those described recently (40, 44, 45, 46).

Our data confirm and extend the studies of Nemazee and colleagues (18) who showed that T cell dependent immune responses to hapten-protein conjugates in alum did not require MyD88 or Trif, effectively ruling out a requisite role for most TLRs in this type of response. Our studies show that even when the BCRs expressed by B cells responding to such Ags also have affinity for molecules containing TLR9 and perhaps other TLR ligands, that the activity of TLR9 and other MyD88 TLRs is not required for the efficient participation of these B cells in the foreign Ag-driven response. However, we have yet to investigate whether TLRs alter the participation of canonical HKIR B cells in the memory response.

In the context of speculations regarding the role of TLRs in the induction or loss of B cell tolerance, previous studies have suggested that in autoimmune-prone mice, spontaneous production of the type of autoantibodies that stain condensed chromatin is completely dependent on TLR9 (16). The canonical HKIR Ab, in the form of an IgG, intensely stains condensed chromatin in antinuclear Ag assays (21). Nonetheless, our data do not indicate a role for TLR9 in the BCR down-regulation and primary development of B cells expressing this type of BCR. This does not support the idea that TLR9 is involved in the induction of tolerance of B cells with this type of specificity, but does not rule out a potential role for this TLR in contributing to spontaneous autoantibody production from such B cells in an autoimmune environment. We are currently testing this idea by crossing the HKIR TLR9-deficient and -sufficient lines to strains with genetic backgrounds that promote autoimmune disease. In this regard, defects in the regulation of apoptotic pathways and the clearance of apoptotic debris conferred by such genetic backgrounds (40, 47, 48, 49, 50) may be particularly relevant as these would result in altered extracellular availability of intracellular autoantigens such as CpG-containing chromatin. Indeed, such an environment may exist in our in vitro cultures, explaining the "spontaneous" activation of HKIR B cells these cultures.

Finally, we should hasten to point out the results reported in this study were confined to the influence of TLRs expressed by HKIR B cells themselves on the Ag-driven development and functional capabilities of this class of antichromatin B cell. TLRs are expressed by a variety of other cell types (46, 51), whose functions could clearly quantitatively and qualitatively influence the nature of anti-DNA and antichromatin B cell development and activity. For this reason, it will be important to conduct future studies that investigate the potential influence of TLRs expressed by accessory cells on the regulation of canonical HKIR B cell behavior.


   Acknowledgments
 
We thank Dr. Shizuo Akira for TLR9–/– and MyD88–/– mice, the Kimmel Cancer Center flow cytometry and laboratory animal facilities, Scot Fenn for technical assistance, and all other members of the Manser laboratory for indirect contributions to this work.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the National Institutes of Health (AI038965) to T.M. F.C. received support from National Research Service Award Training Grants T32-AI07492 and T32-CA09683. Back

2 Address correspondence and reprint requests to Tim Manser, BLSB Room 708, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107. E-mail address: manser{at}mail.jci.tju.edu Back

3 Abbreviations used in this paper: Ars, arsonate; AFC, Ab-forming cell; GC, germinal center; KLH, keyhole limpet hemocyanin; PABA, p-aminobenzoic acid; PNA, peanut lectin agglutinin; Tyr, tyrosine. Back

Received for publication August 15, 2007. Accepted for publication August 29, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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X. Liu, S. Shen, and T. Manser
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