The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jegerlehner, A.
Right arrow Articles by Bachmann, M. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jegerlehner, A.
Right arrow Articles by Bachmann, M. F.
The Journal of Immunology, 2007, 178: 2415-2420.
Copyright © 2007 by The American Association of Immunologists, Inc.

TLR9 Signaling in B Cells Determines Class Switch Recombination to IgG2a

Andrea Jegerlehner*, Patrik Maurer*, Juliana Bessa*, Heather J. Hinton*, Manfred Kopf{dagger} and Martin F. Bachmann1,*

* Cytos Biotechnology AG, Zurich-Schlieren, Switzerland; and {dagger} Swiss Federal Institute of Technology, Institute of Integrative Biology, Molecular Biomedicine, ETH Zurich, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although IgG2a is the most potent Ab isotype in the host response to viral and bacterial infections, the regulation of class switch recombination to IgG2a in vivo is not yet well understood. Recognition of pathogen-associated molecular patterns by dendritic cells expressing TLRs, like TLR7, recognizing ssRNA, or TLR9, recognizing DNA rich in nonmethylated CG motifs (CpG), favors induction of Th1 responses. It is generally assumed that these Th1 responses are responsible for the TLR-mediated induction of IgG2a. Using virus-like particles loaded with CpGs, we show here that TLR9 ligands can directly stimulate B cells to undergo isotype switching to IgG2a. Unexpectedly, TLR9 expression in non-B cells did not affect isotype switching in the Ab response against virus-like particles. Thus, TLR9 can regulate isotype switching to IgG2a directly by interacting with B cells rather than indirectly by inducing Th1 responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Infection of mice with RNA or DNA viruses induces an antiviral Ab response that is largely restricted to the IgG2a isotype (1). The IgG2a isotype has been shown to be particularly potent in host defense against viral infections (2) due to its specific capacity to activate the complement system (3), bind to Fc receptors expressed on phagocytes (4), and induce Ab-dependent cell-mediated cytotoxicity (5). In humans, IgG1 and IgG4 correspond to IgG2a and IgG1 (6) in the mouse. Because different IgG isotypes markedly differ in their ability to opsonize bacteria and lyse infected target cells, controlled induction of the specific isotypes is of significant interest for the design of effective vaccines.

Class switch recombination of B cells from IgM to IgG is dependent on the cognate interaction of B cells with Th cells (7). Although CD40L-CD40 interaction is necessary to initiate Ab isotype switching (8, 9, 10, 11, 12, 13, 14), it is assumed that Th cell-derived cytokines determine whether the B cell switches to IgG1 or IgG2a (15). IFN-{gamma} and IL-4 are key cytokines of Th1 and Th2 cells, respectively, although the latter also typically produce IL-5, IL-10, and IL-13.

By signaling through STAT6, IL-4 drives the switch to IgG1. Indeed, IL-4 or STAT6 deficient mice have a profound defect in IgG1 responses (16, 17, 18). IFN-{gamma} has been shown to counteract IL-4 and promotes IgG2a switching in vitro (19).

However, in vivo, both IFN-{gamma}-dependent (20, 21, 22, 23, 24) and IFN-{gamma}-independent (25) IgG2a class switching have been observed, the latter mainly after infection of mice with live viruses and parasites.

Whereas IFN-{gamma}-dependent IgG2a class switching occurs as a consequence of the cognate interaction of B cells with Th1 cells, it is not yet known how IFN-{gamma}-independent IgG2a class switching occurs in vivo.

On a molecular level, T-box transcription factor T-bet seems to play a critical role in IFN-{gamma}-STAT1-mediated IgG2a class switching in B cells (26). Recently, it has been shown that in vitro T-bet expression and up-regulation of IgG2a class switching can be induced by direct stimulation of B cells with CpG DNA (27, 28, 29), suggesting that an IFN-{gamma}-independent pathway for class switch recombination to IgG2a might exist which goes via direct TLR signaling in B cells. In addition, it has recently been proposed that B cell responses are generally TLR-dependent (30); however, this has been challenged (31). Moreover, the respective roles of CpG-signaling into B cells vs induction of Th1 responses remain unclear (32).

In the present study, we dissected the importance of Th1/Th2 responses vs direct signals received by B cells for the regulation of IgG isotype switching by TLR ligands like ssRNA and CpG. Using virus-like particles (VLPs)2 loaded either with RNA, recognized by TLR7 (33), or CpG DNA, recognized by TLR9 (34), we demonstrate that the critical factor for the induction of IgG2a Abs against VLPs was not Th1 or Th2 cytokines. Rather, TLR expression in B cells was pivotal for the balance of IgG1 vs IgG2a Ab titers. Thus, direct stimulation of B cells by TLR ligands can be the driving factor for class switch recombination to IgG2a in vivo.


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

C57BL/6 and BALB/c mice were purchased from Harlan. IFN-{gamma}–/– (35), TLR9–/– (34), and µMT (36) mice on a BALB/c background have been described earlier (37). All animals were kept under specific pathogen-free conditions at BioSupport and were used at 8 to 12 wk of age. Experiments were conducted in accordance with protocols approved by the Swiss Federal Veterinary Office.

Production of Qbeta VLP (VLP/ssRNA), RNA-free VLP (VLP), and CpG oligomer packaging into VLP (VLP/CpG)

Qbeta VLPs were expressed in Escherichia coli using the vector pQbeta10 and purified as described previously (38). During the self-assembly of the Qbeta coat proteins into VLPs, E. coli-derived mRNA (ssRNA) is packaged into the VLPs; the amount of packaged ssRNA is 300 µg of ssRNA per milligram of Qbeta.

RNA-free VLP can be obtained by digestion of VLP/ssRNA with RNase A. Shortly, 300 µg of RNase A was added to 1 mg of VLP/ssRNA (1 mg/ml in 20 mM HEPES, pH 7.4), the mixture was incubated for 1 h at 37°C in a thermoshaker (650 rpm), and to ensure complete digestion of RNA, the treatment was repeated three times. The digested VLP was dialyzed overnight against 20 mM HEPES (pH 7.4) with a 300 kDa m.w. cut-off membrane (Spectrum Medical Industries) to remove RNase A. Removal of RNase A was analyzed on a 12% SDS PAGE under reducing conditions. To analyze RNA digestion equal amounts (10 µg) of untreated and treated VLP was loaded on a 1% agarose Tris/acetic acid/EDTA (TAE)/ethidium bromide (EtBr) gel. Based on the disappearance of the fluorescent signal, success of RNA digestion could be determined.

Packaging of the CpG oligonucleotide 1668 (5'-TCCATGACGTTCCTGAATAAT-3', thioester stabilized; Microsynth) into the VLPs was performed as described previously (39). Shortly, the CpG oligonucleotides was added to RNase-treated VLPs to a final concentration of 100 nmol/mg VLP and incubated at 37°C for 3 h. To remove CpG oligonucleotides that were not packaged into the VLPs, the VLPs were dialyzed three times against 20 mM HEPES (pH 7.4) with a 300 kDa m.w. cut-off membrane (Spectrum Medical Industries). To analyze packaging of the CpG oligonucleotides, equal amounts (10 µg) of untreated and treated VLP were loaded on a 1% agarose TAE/EtBr gel. Based on the appearance of the fluorescent signal, success of CpG packaging could be determined. The amount of packaged CpGs was analyzed by Tris borate-EDTA/urea gel electrophoresis. First, the Qbeta/CpG VLP was digested with proteinase K overnight at 37°C to remove protein. An equivalent of 0.5 µg of digested VLP was applied on a 10% Tris borate-EDTA/urea gel. As a standard, 5, 10, and 20 pmol of the CpG oligonucleotides packaged into the VLPs were loaded onto the gel. One milligram of packaged Qbeta contains 40 nmol of CpG oligonucleotides.

Chemical coupling of OVA peptide to Qbeta VLP

Chemical coupling of peptides to Qbeta VLPs was described in detail previously (40). OVA peptide was produced in a modified version with an additional cysteine added at the N terminus (NH2-CSSAESLKISQAVHAAHAEINEAGR-COOH; Peter Henklein, Charité) to allow coupling to Qbeta VLPs.

ELISA

ELISA was performed as described previously (40) using HRP-conjugated secondary Abs (HRP goat anti-mouse IgG (H+L), Jackson ImmunoResearch; HRP rat anti-mouse IgG1, BD Pharmingen; HRP rat anti-mouse IgG2a, BD Pharmingen; HRP rat anti-mouse IgG2b, Southern Biotech; HRP rat anti-mouse IgG3, Southern Biotech).

The dilutions at which secondary Abs were used were standardized as follows: ELISA plates were coated with a defined concentration of mouse IgG1, IgG2a, IgG2b, and IgG3 Abs. Serial dilutions of secondary Abs that were prediluted 1/100 to 1/5000 were done, and titers at which half-maximal OD was reached were defined. For each secondary Ab, the predilution at which half-maximal OD was 8'000 was chosen as working dilution for ELISAs which were the following: anti-mouse IgG1, 1/500; anti-mouse IgG2a, 1/2000; anti-mouse IgG2b, 1/500; anti-mouse IgG3, 1/500. Secondary Abs were also tested for stability over time by repeating standardization over time.

IFN-{gamma}/IL-13 ELISPOT assay

Specific IFN-{gamma} and IL-13 secreting CD4+ T cells were quantified using single-cell suspensions of spleens and inguinal lymph nodes from immunized mice. Briefly, for IFN-{gamma} ELISPOT assay, single-cell suspensions at concentrations of 3 x 106 cells/well in duplicates were stimulated for 2 days with 10 µg/ml VLPs in 96-well round-bottom plates at 37°C, 5% CO2. After 2 days, cells were transferred to multiscreen plates (Millipore Corporation) precoated with 10 µg/ml anti-IFN-{gamma} Ab (BD Pharmingen) and blocked with RPMI 1640 (10% FCS). In the IL-13 ELISPOT assay, single-cell suspensions at concentrations of 3 x 106 cells/well in duplicate were distributed directly to the multiscreen plates precoated with 10 µg/ml anti-mouse IL-13 Ab (BD Pharmingen) and blocked with RPMI 1640 (10% FCS) and stimulated with 10 µg/ml VLPs. Cells were incubated on multiscreen plates for 24 h at 37°C, 5% CO2. For the detection, cells were removed (PBS, 0.05% Tween 20), and plates were incubated with a biotinylated anti-IFN-{gamma} Ab (BD Pharmingen (4 µg/ml)) or a biotinylated anti-mouse IL-13 Ab (R&D Systems (4 µg/ml)). Subsequently streptavidin-conjugated alkaline phosphatase (Roche) was added, and alkaline phosphatase substrate was finally used to obtain coloration of the spots (AP substrate kit; Bio-Rad).

Purification and adoptive transfer of B cells

Splenocytes were incubated with biotinylated Abs (BD Pharmingen) to CD43 (S7) and Ly-76 (TER-119), and B cells were negatively selected using streptavidin MACS beads (Miltenyi Biotech) and LD MACS separation columns according to the manufacturer’s instructions; purity of B220+ cells was >95%. Cells were subsequently washed with PBS at 4°C; 2 x 107 B cells were resuspended in 200 µl of PBS and injected into the tail vein of a sex-matched recipient. After 2 h, recipients were immunized s.c. with 200 µg of VLP. When DO11.10 transgenic CD4+ T cells were cotransferred, recipients were immunized with 50 µg of VLPs after transfer of B cells.

CFSE labeling and adoptive transfer of transgenic T cells

DO11.10 transgenic CD4+ T cells with a purity of ≥90% were obtained by positive MACS MicroBeads isolation, according to the manufacturer’s instructions (Miltenyi Biotech). Cells were labeled with 2.5 µM CFSE (Molecular Probes) for 7 min at room temperature. The reaction was stopped by addition of FCS to a final concentration of 10%, and cells were subsequently washed with PBS at 4°C; 5 x 106 labeled T cells were resuspended in 200 µl of PBS and injected into the tail vein of a sex-matched recipient. After 14 h, recipients were s.c. immunized with 50 µg of VLP.

Intracellular cytokine staining and flow cytometry

To analyze cell proliferation and cytokine release of adoptively transferred transgenic OVA-specific T cells, single-cell suspensions were prepared from inguinal lymph nodes of treated mice, lymph node cells were resuspended in RPMI 1640 plus 10% FCS and restimulated with 10–7 M PMA (Sigma-Aldrich) and 1 µg/ml ionomycin (Sigma-Aldrich) in vitro for 4 h. The cultures were supplemented with 10 µg/ml brefeldin A (Sigma-Aldrich) during the last 2 h of incubation. Restimulated cells were then fixed in PBS/2% formaldehyde for 20 min and permeabilized in PBS/0.5% saponin (Sigma-Aldrich) for 40 min on ice. During permeabilization, cells were incubated with allophycocyanin-labeled anti-IFN-{gamma} and PE-labeled anti-DO11.10 Abs (BD Biosciences). Events were acquired on a FACSCalibur and analyzed using CellQuest software (BD Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
ssRNA and CpGs packaged in VLPs determine IgG isotype pattern

We have previously shown that VLPs derived from the coat protein of the bacteriophage Qbeta contain E. coli-derived ssRNA when recombinantly produced in E. coli. This ssRNA can be digested by RNase and replaced by CpGs (39). To test the influence of ssRNA and CpGs on the Ab response, we immunized C57BL/6 mice subcutaneously with VLPs devoid of TLR ligands and VLPs containing ssRNA or CpGs in absence of additional adjuvants. All three forms induced potent IgG responses (Fig. 1A). Thus, as shown earlier, highly repetitive Ags, such as viruses and VLPs are able to induce potent IgG responses in the absence of additional signals (41, 42). In marked contrast, when the IgG subclasses were measured, the VLPs behaved qualitatively different: VLPs devoid of TLR ligands induced a response dominated by IgG1 (Fig. 1C), whereas the VLPs filled with RNA or CpGs induced mostly IgG2a (Fig. 1, B and D). The same observations were made in BALB/c mice (not shown).


Figure 1
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 1. Immunogenicity and Ab isotype profile of VLP-containing ssRNA (VLP/ssRNA) or CpG (VLP/CpG) compared with VLP devoid of TLR ligands (VLP). C57BL/6 mice were immunized s.c. with 50 µg of VLP/ssRNA, VLP, or VLP/CpG. Mean VLP-specific Ab titers in sera of twelve mice per group with SDs are shown. A, Time course of VLP-specific IgG titers; Ab isotype profile of mice immunized with VLP/ssRNA (B), with VLP (C), or VLP/CpG (D), sera of day 14 were analyzed. Differences in the IgG1 and IgG2a titer between the VLP/ssRNA and the VLP and the VLP/CpG and the VLP group as well as differences in the IgG2b titer between the VLP/ssRNA and the VLP group are statistically significant (*, p < 0.05). One representative experiment of three similar experiments is shown.

 
IgG isotype pattern is regulated by TLR expression

CpGs are well described ligands of TLR9. To test whether CpGs influence IgG subclasses via TLR9, we immunized TLR9-deficient mice with Qbeta/CpG. In TLR9-deficient mice, induction of IgG2a was abrogated indicating that CpG-induced TLR9 signaling is required for IgG2a class switching (Fig. 2).


Figure 2
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 2. Ab isotype switching to IgG2a is dependent on TLR expression. A, Groups of TLR9+/+ or TLR9–/– mice (n = 3) were immunized with 50 µg of VLP/CpG and IgG subclasses were determined on day 15. Differences in the IgG1, IgG2a, and IgG3 titer between the two groups are statistically significant (*, p = 0.035; **, p = 6.9 x 10–7; ***, p = 0.014). IgG subclasses were also determined on day 28, and the isotype pattern corresponded to the isotype pattern of day 15. One representative experiment of three similar experiments is shown.

 
Th1/Th2 dichotomy has only a minor influence on IgG1 vs IgG2a isotype switching

To test the role of IFN-{gamma} in driving IgG2a responses, IFN-{gamma}-deficient mice were immunized with VLPs devoid of TLR ligands or VLPs loaded with ssRNA or CpGs. Both wild-type and IFN-{gamma}-deficient mice mounted strong IgG2a responses upon immunization with VLPs containing TLR ligands, while VLPs devoid of TLR ligands predominantly induced IgG1 (Fig. 3). Thus, the absence of IFN-{gamma} did not affect the induction of IgG2a.


Figure 3
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 3. Isotype switch to IgG2a does not require IFN-{gamma}. IFN-{gamma}–/– or IFN-{gamma}+/+ mice were immunized s.c. with 50 µg of VLP/ssRNA (A), VLP (B), or VLP/CpG (C). Mean VLP-specific Ab titers in sera of three mice per group at day 19 with SDs are shown.

 
To further study the link between IgG isotype patterns and Th1/Th2 responses, we assessed the number of Qbeta-specific Th cells producing IFN-{gamma} or IL-13 in C57BL/6 and BALB/c mice. VLP/ssRNA and VLP/CpG induced more IFN-{gamma}-producing Th cells than IL-13-producing Th cells in both mouse strains. Surprisingly, however, VLPs devoid of TLR ligands only induced slightly higher numbers of IL-13-producing Th cells than IFN-{gamma}-producing Th cells (Fig. 4, A and B). Thus, despite very similar Th cell responses (Fig. 4A), there was a clear difference in the IgG isotype pattern induced by VLPs with or without TLR ligands (Fig. 1).


Figure 4
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 4. Th1/Th2 differentiation has a minor influence on IgG isotype pattern. C57BL/6 or BALB/c mice were immunized with 200 µg of VLP/ssRNA, VLP, or VLP/CpG. After 10 days, spleen and inguinal lymph nodes were collected and restimulated with VLP at a final concentration of 10 µg/ml for 3 days in vitro. Numbers of specific T cells expressing IFN-{gamma} or IL-13 in individual C57BL/6 mice (n = 3) (A) or BALB/c mice (n = 3) (B) with mean values are shown. Restimulated cells of naive control mice or unstimulated cells of immunized mice did not induce measurable numbers of IFN-{gamma}- or IL-13-producing T cells (data not shown). Ab isotype profile in sera of immunized C57BL/6 or BALB/c mice were similar to those shown in Fig. 1 (data not shown). One of two similar experiments is shown. C, VLP and VLP/CpG induce robust Th1 responses in vivo. CFSE-labeled OVA-specific CD4+ T cells from DO11.10 transgenic mice were adoptively transferred into C57BL/6 mice. Mice were immunized 14 h later s.c. with 50 µg of OVA-VLP or OVA-VLP/CpG; single-cell suspensions were obtained from inguinal lymph nodes of recipient mice immunized 3 or 6 days previously and stimulated in vitro for 2 h with PMA/ionomycin. KJ+ CD4+ T cells were analyzed for cell division and intracellular IFN-{gamma} release. No IFN-{gamma} release was measured in unstimulated KJ+CD4+ T cells (data not shown). One of two similar experiments is shown.

 
To independently confirm that a Th1 response could be induced in the absence of TLR stimulation, CFSE-labeled DO11.10 TCR-transgenic T cells were transferred into naive recipient mice. These mice were then immunized with VLPs devoid of TLR ligands or VLP/CpG, which had been coupled to the MHC class II-restricted OVA peptide that is recognized by the transferred DO11.10 T cells. Proliferation and production of IFN-{gamma} was assessed 3 and 6 days later by intracellular cytokine staining. As observed in the ELISPOT experiments, a robust Th1 response was induced in the absence of a TLR9 ligand (Fig. 4C).

Th is needed for the isotype switching toward IgG

The results shown above established that the cytokines released by Th cells were not a key factor for the switching toward IgG2a. In a next set of experiments, we assessed whether cognate interaction between B and Th cells through CD40-CD40L interaction was needed for isotype switching toward IgG induced by CpG-loaded VLPs. To do this, CD40-deficient mice were immunized with VLP/CpG. As shown in Fig. 5, CD40-deficient mice failed to efficiently produce IgG Abs upon immunization with VLP/CpG, indicating that TLR ligands may modulate the IgG isotype pattern, however, that CD40-CD40L interaction and Th cells were still essential for isotype switching, confirming data by Pasare and Medzhitov (30).


Figure 5
View larger version (10K):
[in this window]
[in a new window]

  		FIGURE 5. CD40-CD40L interaction needed for IgG response against VLP/CpG. Groups of CD40–/– mice (n = 3) and C57BL/6 mice (n = 3) were immunized s.c. with 50 µg of VLP/CpG. Mean VLP-specific Ab titer was determined with SDs in sera of day 10.

 
TLR expression in B cells rather than dendritic cells (DCs) drives production of specific IgG2a antibodies

To analyze the influence of TLR9 expression in DC and B cells on Ab isotype switching, we transferred either TLR9-deficient B cells or control B cells into B cell-deficient mice. In B cell-deficient mice reconstituted with TLR9-deficient B cells, all professional APCs express TLR9 whereas all B cells are TLR9 deficient. First, we wanted to confirm that the absence of TLR9 in B cells would not affect the Th1/Th2 balance. To this end, CFSE-labeled DO11.10 TCR-transgenic T cells were transferred into B cell-deficient mice reconstituted with TLR9-deficient B cells. These mice were then immunized with VLP/CpG coupled to the MHC class II-restricted OVA peptide, which is recognized by the transferred T cells. As expected, no difference in the production of IFN-{gamma} could be detected in mice harboring TLR9-deficient or control B cells (Fig. 6A). To assess the role of TLR9 expression in B cells in regulating isotype switching to IgG2a, B cell reconstituted mice were immunized with VLP/CpG and IgG2a was assessed. Although B cell-deficient mice reconstituted with normal B cells mounted strong IgG2a responses, mice reconstituted with TLR9-deficient B cells had a greatly reduced IgG2a response (Fig. 6B). This demonstrates that TLR9 expression in B cells and not other APCs is critical for the regulation IgG isotype patterns.


Figure 6
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 6. TLR9 expression in B cells dictates the pattern of IgG isotypes. A, CFSE-labeled OVA-specific CD4+ T cells from DO11.10 transgenic mice were either adoptively transferred into B cell deficient (µMT) mice together with B cells from TLR9–/– mice or TLR9+/+ mice. Mice were immunized 14 h later s.c. with 50 µg of OVA-VLP or OVA-VLP/CpG. Single-cell suspensions were obtained from lymph nodes of recipient mice immunized 6 days previously and stimulated in vitro for 4 h with OVA peptide or 2 h with PMA/ionomycin. KJ+ CD4+ T cells were analyzed for cell division and intracellular IFN-{gamma} release. One of two similar experiments is shown. B, B cells from TLR9–/– or TLR9+/+ mice were adoptively transferred into B cell-deficient (µMT) mice, and mice were immunized with 50 µg of VLP/CpG 2 h later. Mean VLP-specific Ab titers per group (n = 3) with SDs are shown. Sera of day 11 were analyzed. The difference in the IgG2a titer between the two groups is statistically significant (*, p < 0.05), whereas the difference in the IgG1 titer is not statistically different (p > 0.05). Also the sera of the mice of experiment Fig. 6A were analyzed, and the Ab isotype pattern was the same as shown in Fig. 6B. One of two similar experiments is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Most pathogens induce potent Ab responses. There are several features which account for their high immunogenicity including the high density and repetitiveness of their epitopes (Ag organization) as well as their association with TLR ligands. Here we dissect the two parameters. We identified Ag organization as an important feature of pathogens in regulating the overall strength of the IgG response in the past (41, 42). However, TLR ligands further increase the magnitude of the IgG response and in particular determine the IgG isotype pattern. It is generally assumed that TLR ligands drive expression of IgG2a through induction of Th1 responses and IFN-{gamma}. Here we show that this is only partly the case. Rather than stimulating the production of IgG2a Abs indirectly through induction of Th1 responses, the induction of IgG2a class switching is mainly induced via CpG-mediated engagement of TLR9 in B cells. These data help to explain why pathogens generally induce strong IgG2a responses, because most of these pathogens have TLR ligands that are able to directly activate B cells.

In a previous study it was shown that TLR9/MyD88 signaling is required for class switching to pathogenic IgG2a and 2b autoantibodies in a murine model of systemic lupus erythematosus (SLE). Fc{gamma}RIIB–/– mice or 56R+Fc{gamma}RIIB–/– mice, two mouse strains which show spontaneous development of IgG2a and IgG2b autoantibodies were crossed with MyD88–/– and TLR9–/– mice and it was shown that MyD88 and TLR9 signaling was required for class switching to pathogenic IgG2a and IgG2b autoantibodies. The development of immune pathology and mortality but not the development of anti-self IgM+ B cells was dramatically reduced in the absence of TLR9 or MyD88 signaling. These findings are consistent with our results. Interestingly, however, this previous study reported that generation of non-autoreactive IgG2a and 2b Abs by vaccination were not impaired in TLR9-deficient mice, which might seem inconsistent with our data (43). A possible explanation for the difference may be the different nature of the Ags used and the different questions asked. VLPs induce an IgG response that is dominated by IgG1 unless a TLR ligand is added that deviates the IgG response toward IgG2a. However, even without a TLR ligand some isotype switching toward IgG2a occurs. We have not addressed the question whether this IgG2a switching is reduced in TLR9-deficient mice. However, Ehlers et al. (43) by immunizing mice with NP-CGG or NP-Ficoll have compared the Ab isotype pattern against these Ags in TLR9+/– and TLR9–/– mice and found no difference.

In another study, it was shown that activation of autoreactive B cells specific for host IgG is mediated by IgG2a-chromatin immune complexes and requires the synergistic engagement of the Ag receptor and a member of the MyD88-dependent TLR family (44). Further studies have shown that the role of the BCR was mainly delivery of the chromatin ligand to TLR9 and revealed hypomethylated CpG motifs, which are part of mammalian chromatin, as the critical ligand for TLR9 and the initiation of systemic autoimmune disease (45). Similarly it was shown that RNA-associated autoantigens activate autoreactive B cells by BCR/TLR7 activation (46). In summary, these studies identify TLR signaling as an essential component for induction of isotype-switched autoantibodies. In marked contrast, using CpG-loaded VLPs, we found a key role for TLR9 signaling for the isotype pattern (IgG1 vs IgG2a) but not for the IgG response per se. Furthermore, we demonstrate that TLR9 expression in B cells rather than DCs is pivotal. A similar pathway has recently been described for LPS-mediated induction of IgG1 and IgG2a class switching in mice via direct engagement of TLR4 in B cells (30).

Our findings offer a rationale for selective induction of this isotype by combined administration of TLR ligands linked to B cell Ags. This is particularly interesting as the only accepted adjuvant in humans is alum, which induces Th2 responses. By combined administration of TLR ligands with B cell Ags, Ag-specific IgG2a can be induced irrespective of the type of Th response.


   Acknowledgments
 
We thank N. Schmitz, E. Meijerink, J. Shamshiev, V. Manolova, S. Martin, and P. Saudan for helpful and stimulating discussions, M. Maudrich and A. Flace for excellent technical support, and A. Fulurija for critical review of the manuscript.


   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 Address correspondence and reprint requests to Dr. Martin F. Bachmann, Cytos Biotechnology AG, Wagistrasse 25, 8952 Zurich-Schlieren, Switzerland. E-mail address: martin.bachmann{at}cytos.com Back

2 Abbreviations used in this paper: VLP, virus-like particle; DC, dendritic cell. Back

Received for publication June 12, 2006. Accepted for publication November 15, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Coutelier, J. P., J. T. van der Logt, F. W. Heessen, G. Warnier, J. Van Snick. 1987. IgG2a restriction of murine antibodies elicited by viral infections. J. Exp. Med. 165: 64-69. [Abstract/Free Full Text]
  2. Nimmerjahn, F., J. V. Ravetch. 2006. Fc{gamma} receptors: old friends and new family members. Immunity 24: 19-28. [Medline]
  3. Klaus, G. G., M. B. Pepys, K. Kitajima, B. A. Askonas. 1979. Activation of mouse complement by different classes of mouse antibody. Immunology 38: 687-695. [Medline]
  4. Heusser, C. H., C. L. Anderson, H. M. Grey. 1977. Receptors for IgG: subclass specificity of receptors on different mouse cell types and the definition of two distinct receptors on a macrophage cell line. J. Exp. Med. 145: 1316-1327. [Abstract/Free Full Text]
  5. Kipps, T. J., P. Parham, J. Punt, L. A. Herzenberg. 1985. Importance of immunoglobulin isotype in human antibody-dependent, cell-mediated cytotoxicity directed by murine monoclonal antibodies. J. Exp. Med. 161: 1-17. [Abstract/Free Full Text]
  6. Lundgren, M., U. Persson, P. Larsson, C. Magnusson, C. I. Smith, L. Hammarstrom, E. Severinson. 1989. Interleukin 4 induces synthesis of IgE and IgG4 in human B cells. Eur. J. Immunol. 19: 1311-1315. [Medline]
  7. McHeyzer-Williams, L. J., M. G. McHeyzer-Williams. 2005. Antigen-specific memory B cell development. Annu. Rev. Immunol. 23: 487-513. [Medline]
  8. Foy, T. M., J. D. Laman, J. A. Ledbetter, A. Aruffo, E. Claassen, R. J. Noelle. 1994. gp39-CD40 interactions are essential for germinal center formation and the development of B cell memory. J. Exp. Med. 180: 157-163. [Abstract/Free Full Text]
  9. Foy, T. M., D. M. Shepherd, F. H. Durie, A. Aruffo, J. A. Ledbetter, R. J. Noelle. 1993. In vivo CD40-gp39 interactions are essential for thymus-dependent humoral immunity. II. Prolonged suppression of the humoral immune response by an antibody to the ligand for CD40, gp39. J. Exp. Med. 178: 1567-1575. [Abstract/Free Full Text]
  10. Van den Eertwegh, A. J., R. J. Noelle, M. Roy, D. M. Shepherd, A. Aruffo, J. A. Ledbetter, W. J. Boersma, E. Claassen. 1993. In vivo CD40-gp39 interactions are essential for thymus-dependent humoral immunity. I. In vivo expression of CD40 ligand, cytokines, and antibody production delineates sites of cognate T-B cell interactions. J. Exp. Med. 178: 1555-1565. [Abstract/Free Full Text]
  11. Gray, D., P. Dullforce, S. Jainandunsing. 1994. Memory B cell development but not germinal center formation is impaired by in vivo blockade of CD40-CD40 ligand interaction. J. Exp. Med. 180: 141-155. [Abstract/Free Full Text]
  12. Lane, P., T. Brocker, S. Hubele, E. Padovan, A. Lanzavecchia, F. McConnell. 1993. Soluble CD40 ligand can replace the normal T cell-derived CD40 ligand signal to B cells in T cell-dependent activation. J. Exp. Med. 177: 1209-1213. [Abstract/Free Full Text]
  13. Renshaw, B. R., W. C. Fanslow, 3rd, R. J. Armitage, K. A. Campbell, D. Liggitt, B. Wright, B. L. Davison, C. R. Maliszewski. 1994. Humoral immune responses in CD40 ligand-deficient mice. J. Exp. Med. 180: 1889-1900. [Abstract/Free Full Text]
  14. Kawabe, T., T. Naka, K. Yoshida, T. Tanaka, H. Fujiwara, S. Suematsu, N. Yoshida, T. Kishimoto, H. Kikutani. 1994. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. Immunity 1: 167-178. [Medline]
  15. Coffman, R. L., B. W. Seymour, D. A. Lebman, D. D. Hiraki, J. A. Christiansen, B. Shrader, H. M. Cherwinski, H. F. Savelkoul, F. D. Finkelman, M. W. Bond, et al 1988. The role of helper T cell products in mouse B cell differentiation and isotype regulation. Immunol. Rev. 102: 5-28. [Medline]
  16. Kuhn, R., K. Rajewsky, W. Muller. 1991. Generation and analysis of interleukin-4 deficient mice. Science 254: 707-710. [Abstract/Free Full Text]
  17. Takeda, K., T. Tanaka, W. Shi, M. Matsumoto, M. Minami, S. Kashiwamura, K. Nakanishi, N. Yoshida, T. Kishimoto, S. Akira. 1996. Essential role of Stat6 in IL-4 signalling. Nature 380: 627-630. [Medline]
  18. Kopf, M., H. Baumann, G. Freer, M. Freudenberg, M. Lamers, T. Kishimoto, R. Zinkernagel, H. Bluethmann, G. Kohler. 1994. Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature 368: 339-342. [Medline]
  19. Snapper, C. M., W. E. Paul. 1987. Interferon-{gamma} and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236: 944-947. [Abstract/Free Full Text]
  20. Finkelman, F. D., I. M. Katona, T. R. Mosmann, R. L. Coffman. 1988. IFN-{gamma} regulates the isotypes of Ig secreted during in vivo humoral immune responses. J. Immunol. 140: 1022-1027. [Abstract]
  21. Coutelier, J. P., P. G. Coulie, P. Wauters, H. Heremans, J. T. van der Logt. 1990. In vivo polyclonal B-lymphocyte activation elicited by murine viruses. J. Virol. 64: 5383-5388. [Abstract/Free Full Text]
  22. Graham, M. B., D. K. Dalton, D. Giltinan, V. L. Braciale, T. A. Stewart, T. J. Braciale. 1993. Response to influenza infection in mice with a targeted disruption in the interferon {gamma} gene. J. Exp. Med. 178: 1725-1732. [Abstract/Free Full Text]
  23. van den Broek, M. F., U. Muller, S. Huang, M. Aguet, R. M. Zinkernagel. 1995. Antiviral defense in mice lacking both {alpha}/beta and {gamma} interferon receptors. J. Virol. 69: 4792-4796. [Abstract]
  24. Maloy, K. J., B. Odermatt, H. Hengartner, R. M. Zinkernagel. 1998. Interferon {gamma}-producing {gamma}{delta} T cell-dependent antibody isotype switching in the absence of germinal center formation during virus infection. Proc. Natl. Acad. Sci. USA 95: 1160-1165. [Abstract/Free Full Text]
  25. Markine-Goriaynoff, D., J. T. van der Logt, C. Truyens, T. D. Nguyen, F. W. Heessen, G. Bigaignon, Y. Carlier, J. P. Coutelier. 2000. IFN-{gamma}-independent IgG2a production in mice infected with viruses and parasites. Int. Immunol. 12: 223-230. [Abstract/Free Full Text]
  26. Peng, S. L., S. J. Szabo, L. H. Glimcher. 2002. T-bet regulates IgG class switching and pathogenic autoantibody production. Proc. Natl. Acad. Sci. USA 99: 5545-5550. [Abstract/Free Full Text]
  27. Liu, N., N. Ohnishi, L. Ni, S. Akira, K. B. Bacon. 2003. CpG directly induces T-bet expression and inhibits IgG1 and IgE switching in B cells. Nat. Immunol. 4: 687-693. [Medline]
  28. Peng, S. L., J. Li, L. Lin, A. Gerth. 2003. The role of T-bet in B cells. Nat. Immunol. 4: 1041 author reply 1041.. [Medline]
  29. Lin, L., A. J. Gerth, S. L. Peng. 2004. CpG DNA redirects class-switching towards "Th1-like" Ig isotype production via TLR9 and MyD88. Eur. J. Immunol. 34: 1483-1487. [Medline]
  30. Pasare, C., R. Medzhitov. 2005. Control of B-cell responses by Toll-like receptors. Nature 438: 364-368. [Medline]
  31. Nemazee, D., A. Gavin, K. Hoebe, B. Beutler. 2006. Immunology: Toll-like receptors and antibody responses. Nature 441: E4 discussion E4. [Medline]
  32. Chu, R. S., O. S. Targoni, A. M. Krieg, P. V. Lehmann, C. V. Harding. 1997. CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Th1) immunity. J. Exp. Med. 186: 1623-1631. [Abstract/Free Full Text]
  33. Heil, F., H. Hemmi, H. Hochrein, F. Ampenberger, C. Kirschning, S. Akira, G. Lipford, H. Wagner, S. Bauer. 2004. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303: 1526-1529. [Abstract/Free Full Text]
  34. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, S. Akira. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408: 740-745. [Medline]
  35. Dalton, D. K., S. Pitts-Meek, S. Keshav, I. S. Figari, A. Bradley, T. A. Stewart. 1993. Multiple defects of immune cell function in mice with disrupted interferon-{gamma} genes. Science 259: 1739-1742. [Abstract/Free Full Text]
  36. Kitamura, D., J. Roes, R. Kuhn, K. Rajewsky. 1991. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature 350: 423-426. [Medline]
  37. Kopf, M., F. Brombacher, M. F. Bachmann. 2002. Role of IgM antibodies versus B cells in influenza virus-specific immunity. Eur. J. Immunol. 32: 2229-2236. [Medline]
  38. Cielens, I., V. Ose, I. Petrovskis, A. Strelnikova, R. Renhofa, T. Kozlovska, P. Pumpens. 2000. Mutilation of RNA phage Qbeta virus-like particles: from icosahedrons to rods. FEBS Lett 482: 261-264. [Medline]
  39. Storni, T., C. Ruedl, K. Schwarz, R. A. Schwendener, W. A. Renner, M. F. Bachmann. 2004. Nonmethylated CG motifs packaged into virus-like particles induce protective cytotoxic T cell responses in the absence of systemic side effects. J. Immunol. 172: 1777-1785. [Abstract/Free Full Text]
  40. Jegerlehner, A., T. Storni, G. Lipowsky, M. Schmid, P. Pumpens, M. F. Bachmann. 2002. Regulation of IgG antibody responses by epitope density and CD21-mediated costimulation. Eur. J. Immunol. 32: 3305-3314. [Medline]
  41. Bachmann, M. F., U. H. Rohrer, T. M. Kundig, K. Burki, H. Hengartner, R. M. Zinkernagel. 1993. The influence of antigen organization on B cell responsiveness. Science 262: 1448-1451. [Abstract/Free Full Text]
  42. Bachmann, M. F., R. M. Zinkernagel. 1997. Neutralizing antiviral B cell responses. Annu. Rev. Immunol. 15: 235-270. [Medline]
  43. Ehlers, M., H. Fukuyama, T. L. McGaha, A. Aderem, J. V. Ravetch. 2006. TLR9/MyD88 signaling is required for class switching to pathogenic IgG2a and 2b autoantibodies in SLE. J. Exp. Med. 203: 553-561. [Abstract/Free Full Text]
  44. Leadbetter, E. A., I. R. Rifkin, A. M. Hohlbaum, B. C. Beaudette, M. J. Shlomchik, A. Marshak-Rothstein. 2002. Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 416: 603-607. [Medline]
  45. Viglianti, G. A., C. M. Lau, T. M. Hanley, B. A. Miko, M. J. Shlomchik, A. Marshak-Rothstein. 2003. Activation of autoreactive B cells by CpG dsDNA. Immunity 19: 837-847. [Medline]
  46. Lau, C. M., C. Broughton, A. S. Tabor, S. Akira, R. A. Flavell, M. J. Mamula, S. R. Christensen, M. J. Shlomchik, G. A. Viglianti, I. R. Rifkin, A. Marshak-Rothstein. 2005. RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement. J. Exp. Med. 202: 1171-1177. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
W. Xu, P. A. Santini, A. J. Matthews, A. Chiu, A. Plebani, B. He, K. Chen, and A. Cerutti
Viral Double-Stranded RNA Triggers Ig Class Switching by Activating Upper Respiratory Mucosa B Cells through an Innate TLR3 Pathway Involving BAFF
J. Immunol., July 1, 2008; 181(1): 276 - 287.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. Lembo, M. Pelletier, R. Iyer, M. Timko, J. C. Dudda, T. E. West, C. B. Wilson, A. M. Hajjar, and S. J. Skerrett
Administration of a Synthetic TLR4 Agonist Protects Mice from Pneumonic Tularemia
J. Immunol., June 1, 2008; 180(11): 7574 - 7581.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
N. Gao, P. Jennings, and D. Yuan
Requirements for the natural killer cell-mediated induction of IgG1 and IgG2a expression in B lymphocytes
Int. Immunol., May 1, 2008; 20(5): 645 - 657.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
L. M. Gargano, J. M. Moser, and S. H. Speck
Role for MyD88 Signaling in Murine Gammaherpesvirus 68 Latency
J. Virol., April 15, 2008; 82(8): 3853 - 3863.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. V. Rubtsov, C. L. Swanson, S. Troy, P. Strauch, R. Pelanda, and R. M. Torres
TLR Agonists Promote Marginal Zone B Cell Activation and Facilitate T-Dependent IgM Responses
J. Immunol., March 15, 2008; 180(6): 3882 - 3888.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
A. Meyer-Bahlburg, S. Khim, and D. J. Rawlings
B cell intrinsic TLR signals amplify but are not required for humoral immunity
J. Exp. Med., December 24, 2007; 204(13): 3095 - 3101.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. F. Cunningham, F. Gaspal, K. Serre, E. Mohr, I. R. Henderson, A. Scott-Tucker, S. M. Kenny, M. Khan, K.-M. Toellner, P. J. L. Lane, et al.
Salmonella Induces a Switched Antibody Response without Germinal Centers That Impedes the Extracellular Spread of Infection
J. Immunol., May 15, 2007; 178(10): 6200 - 6207.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jegerlehner, A.
Right arrow Articles by Bachmann, M. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jegerlehner, A.
Right arrow Articles by Bachmann, M. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS