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The Journal of Immunology, 2005, 174: 6308-6313.
Copyright © 2005 by The American Association of Immunologists

The Mucosal Adjuvant Macrophage-Activating Lipopeptide-2 Directly Stimulates B Lymphocytes via the TLR2 without the Need of Accessory Cells 1

Stefan Borsutzky*, Karsten Kretschmer2,{dagger}, Pablo D. Becker*, Peter F. Mühlradt{ddagger}, Carsten J. Kirschning§, Siegfried Weiss{dagger} and Carlos A. Guzmán3,*

* Vaccine Research Group, Division of Microbiology and{dagger} Molecular Immunology Group, Division of Molecular Biotechnology, GBF-German Research Centre for Biotechnology, Braunschweig, Germany;{ddagger} Wound Healing Research Group, BioTec-Gruenderzentrum, Braunschweig, Germany; and§ Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Munich, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The macrophage-activating lipopeptide-2 (MALP-2) is an agonist of the TLR heterodimer 2/6, which exhibits potent activity as mucosal adjuvant, promoting strong humoral and cellular responses. Although B cells expressing TLR2/6 are potential targets, very little is known about the effect of MALP-2 on B cells. Studies were performed using total spleen cells or purified B cells from WT mice or animals deficient in TLR2, T cells, B cells, or specific subpopulations of B cells. They demonstrated that MALP-2 promotes a T cell-independent activation and maturation of B cells (mainly follicular but also B-1a and marginal zone B cells) via TLR2. MALP-2 also increased the frequency of IgM- and IgG-secreting cells, but bystander cells were required for IgA secretion. Activated B cells exhibited increased expression of activation markers and ligands that are critical for cross-talk with T cells (CD19, CD25, CD80, CD86, MHC I, MHC II, and CD40). Immunization of mice lacking T cells showed that MALP-2-mediated stimulation of TLR2/6 was unable to circumvent the need of T cell help for efficient Ag-specific B cell activation. Immunization of mice lacking B cells demonstrated that B cells are critical for MALP-2-dependent improvement of T cell responses. The knowledge emerging from this work suggests that MALP-2-mediated activation of B cells through TLR2/6 is critical for adjuvanticity. B cell stimulation by pattern recognition receptors seems to be a basic mechanism that can be exploited to improve the immunogenicity of vaccine formulations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mucosal tissues constitute the main portal of entry for infectious agents. Therefore, many novel vaccination approaches are aimed at the stimulation of efficient local immune responses at the mucosa. This can be achieved by the administration of vaccine Ags via the mucosal route. On the other hand, mucosal surfaces are not only confronted with potentially dangerous agents, but they are also continuously exposed to harmless entities. Thus, the responsiveness of the mucosal immune system is tightly controlled. In fact, the immune system has evolved to recognize potentially "dangerous" rather than foreign Ags (1, 2, 3). This explains that most Ags administered via mucosal route are poorly immunogenic, whereas those delivered in the context of a proper "danger signal" have a better chance to elicit stronger responses. Therefore, one of the strategies to overcome poor immunogenicity is the coadministration of Ags with mucosal adjuvants (4, 5, 6). However, only a few mucosal adjuvants have been identified, and their mechanisms of action, as well as the structural requirements for adjuvanticity are poorly understood.

We have demonstrated that a synthetic derivative (S-[2,3-bispalmitoyloxypropyl]cysteinyl-GNNDESNISFKEK) of the macrophage-activating lipopeptide-2 (MALP-2) 4 from Mycoplasma fermentans is a potent mucosal adjuvant (7, 8). Intranasal coadministration of MALP-2 with different T cell-dependent Ags stimulates strong Ag-specific T cell proliferative responses, high levels of Ag-specific serum antibodies, and secretory IgA responses both locally and at distant mucosal sites. Previous studies revealed that MALP-2 is an agonist of the TLR2/6 heterodimer, leading to a Toll-IL-1R domain-containing adapter protein and MyD88-dependent activation of NF-{kappa}B in macrophages (9, 10, 11). Consequently, incubation of macrophages and dendritic cells with MALP-2 results in the secretion of proinflammatory cytokines and enhances the capacity of dendritic cells to present Ags to T cells (12). Since the adjuvanticity of MALP-2 is characterized by strong humoral responses and B cells express TLR2/6 (13), B cells could be a potential target for MALP-2-mediated activation in vivo. However, only little is known concerning the direct effect of MALP-2 on B cells.

In the attempt to unravel the mechanism of adjuvanticity of MALP-2, we investigated whether MALP-2 exerts a direct stimulatory activity on B cells. The results obtained demonstrate that MALP-2 promotes a T cell-independent activation and maturation of B cells via TLR2. Immunization studies performed in mice lacking T or B cells also showed that both cell types are crucial for the adjuvanticity of MALP-2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and cell cultures

BALB/c and C57BL/6 mice were purchased from Harlan-Winkelmann. TLR2-deficient animals were kindly provided by Tularik. BALB/c nu/nu mice were obtained from Charles River Laboratories. RAG-1–/–, CD4–/–,L2 (14), SLP65–/– (15, 16), L2 x SLP65–/–, and Ig{alpha}–/– (17) mice on BALB/c background have been bred in our animal facility.

SLP65–/– and L2 mice are characterized by a complete block of B cell ontogeny in the fetal liver (15) and the bone marrow (14), respectively. Thus, homozygous SLP65–/– mice (15, 16) lack B-1a cells in the spleen and peritoneal compartments (Fig. 1). On the other hand, L2 mice (14, 18), which are transgenic for the {lambda}2 L chain, exhibit a complete lack of follicular B cells (B-2 or conventional B cells) and a predominance of B-1a cells (Fig. 1,). In contrast to wild-type (WT) mice (i.e., SLP-65+/–), only B-1a cells and B cells of the marginal zone are found in the spleen of L2 x SLP-65+/– mice, and B-1a and B-1b cells in their peritoneum (Fig. 1). Therefore, crossing of the two deficient mouse strains led to a L2 x SLP65–/– genotype, which is characterized by a complete block of B cell ontogeny and a total lack of B cells in their peripheral lymphoid organs, as demonstrated by flow cytometric analysis of cells obtained from the spleen and the peritoneal cavity (Fig. 1).



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  		FIGURE 1. B cells are absent in L2 x SLP-65–/– mice. Flow cytometric analysis of the surface expression of CD19 (i.e., B cell marker) and CD5 (i.e., B-1a cell marker) on cells obtained from the spleen and the peritoneal cavity of SLP-65+/– (wild type), L2 x SLP-65+/– (i.e., {lambda}2 L chain transgenic), SLP-65–/–, and L2 x SLP-65–/– mice.

 
B cell purification

Small resting B cells from spleen were purified either by magnetic cell sorting using the B cell isolation kit on an auto-MACS (both obtained from Miltenyi Biotec) and biotinylated anti-CD11c (HL3; BD Pharmingen), anti-F4/80 (CI:A3-1; Serotec), anti-CD5 (53-7.3; BD Pharmingen) and anti-Mac1 (M1/70; BD Pharmingen) Abs (purity ~95%) or by FACS sorting of CD19+CD23+ and CD21+ cells (purity >98%) using a MOFLO (Cytomation).

Measurement of cellular proliferation

Proliferation assays were performed in quadruplicates, as previously described (7). Briefly, spleen cells (5 x 105 per well) were stimulated with different concentrations of MALP-2, and after 80 h of incubation [3H]thymidine (1 µCi/well) was added. Results are expressed as the mean of cpm of stimulated cells subtracted of background values from nonstimulated cells cultured in RPMI 1640 supplemented with 10% FCS.

Flow cytometry

Stimulated and nonstimulated cells were labeled with FITC-conjugated Abs against CD80 (16-10A1), CD86 (GL1), MHC I (SF1-1.1), MHC II (AMS-32.1), CD40 (HM40-3), CD5 (53-7.3), or CD25 (7D4), in combination with a PE-labeled anti-CD19 (1D3) Ab. All Abs were obtained from BD Pharmingen.

Detection of Ab-secreting cells

The frequencies of total, IgM-, IgG-, or IgA-secreting cells were determined by ELISPOT using PVDF plates (Millipore) coated with 100 µl/well isotype-specific capture Abs (Sigma-Aldrich) at a concentration of 5 µg/ml in 0.05 M carbonate buffer (pH 9.6). Serial dilutions of spleen cells in complete medium were incubated in triplicates for 6 h. After washing, plates were incubated with 100 µl of biotinylated subclass-specific Abs (Sigma-Aldrich) overnight at 4°C. Then, plates were washed and 100 µl/well peroxidase-conjugated streptavidin (BD Pharmingen) were added for 1 h. Spots were developed using 3-amino-9-ethyl-carbazole (Sigma-Aldrich) in 0.1 M acetate buffer, pH 5.0, and 0.05% H2O2 (30%). The reaction was stopped after 60 min and spots were counted using a binocular microscope.

Immunization experiments

Groups of three mice were immunized by intranasal route on days 1, 14, and 21 with 20 µl of {beta}-galactosidase ({beta}-gal) (50 µg/dose; Roche) alone or coadministered with MALP-2 (0.5 µg/dose) (19). Alternatively, animals received {beta}-gal (50 µg/dose) emulsified in Freund’s complete (priming) or incomplete (boosters) adjuvant by intraperitoneal route, according to the same schedule. Sera were collected on days 0, 13, 20, and 31. Then, animals were sacrificed and the spleens were removed for the analysis of the cellular immune response.

Detection of Abs in sera and supernatants

The detection of Abs was performed by ELISA, as previously described (7). To measure total IgG, IgA, and IgM, 96-well plates were coated with 100 µl/well of anti-IgG, anti-IgA, or anti-IgM Abs (Sigma-Aldrich), whereas Ag-specific serum IgG was determined using plates coated with {beta}-gal (5 µg/ml). Biotinylated goat anti-mouse IgG, IgA, and IgM (Sigma-Aldrich) were used as detection antibodies.

Statistical analysis

Comparisons between experimental groups were made by application of the double-sided Mann-Whitney U test, p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Proliferation of spleen cells in response to MALP-2 depends on TLR2 expression

Since it has been shown very early that bacterial lipoproteins induce proliferation of spleen cells (20), it was assumed that the mycoplasmal lipopeptide MALP-2 acted similarly. As expected, MALP-2 stimulated [3H]thymidine incorporation in spleen cells from C57BL/6 mice in a dose-dependent manner (Fig. 2A). This stimulatory effect of MALP-2 was evident at a concentration of 10 ng/ml and reached a plateau at a concentration of 200 ng/ml. In contrast, spleen cells from TLR2–/– mice did not respond, confirming that the proliferation induced by MALP-2 specifically depends on TLR2 (Fig. 2A).



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  		FIGURE 2. Proliferative responses stimulated by MALP-2. A and B, Spleen cells (5 x 105) were incubated with different concentrations of MALP-2 for 4 days. Proliferation was assessed by [3H]thymidine incorporation. C, Flow cytometric analysis of B cells purified by sorting using a MOFLO (purity >98%). D, Total spleen cells and FACS-sorted B cells (>98% purity) were stimulated with different concentrations of MALP-2, and their proliferative capacity was then evaluated. Data are presented as mean cpm subtracted of background values from nonstimulated cells cultured in RPMI 1640 supplemented with 10% FCS; SEM of quadruplicate values is indicated by vertical lines.

 
MALP-2 induces proliferation of B cells without the need of accessory cells

To identify the cellular subpopulation responding to MALP-2, we used spleen cells from BALB/c WT mice and from different knockout animals (deficient in CD4+ T cells, follicular B cells, B cells, or T and B cells) bred onto the same genetic background. As shown in Fig. 2B, the highest incorporation was observed when cells from BALB/c WT mice were tested, although the dose-response curve looked different from that observed with spleen cells from C57BL/6 mice (Fig. 2A). Spleen cells from CD4+ T cell-deficient animals were still able to proliferate. However, they exhibited a slightly reduced incorporation of [3H]thymidine compared with cells from WT mice when MALP-2 was applied at low concentrations (100–200 ng/ml). In contrast, no MALP-2-dependent stimulation was observed when cells from mice with either a combined deficiency of B and T cells (RAG–/–) or B cells alone (L2 x SLP65–/–) were tested. This suggests that B cells are the major target subpopulation. However, this does not necessarily rule out a potential role for accessory cell subpopulations in vivo. To further define whether a specific subset of B cells is involved, we incubated MALP-2 with spleen cells from animals lacking the follicular B cell subset but still containing B-1a and marginal zone B cells (L2 mice). A dose-dependent proliferation was observed (Fig. 2B), but [3H]thymidine incorporation, even at the highest concentration tested (1 µg/ml), was lower with cells from L2 mice than with those from WT or CD4+-deficient mice. This suggests that follicular B cells are a major target subset for MALP-2, but that also B-1a and/or marginal zone B cells might be able to respond. Interestingly, B cells from L2 mice seem to respond to lower doses of MALP-2, which underscores their function as fast reacting B cells of the first line of defense.

Additional studies were performed to assess whether the stimulatory effect on B cells was induced by MALP-2 directly or via the activation of bystander cells (e.g., macrophages). To this end, the proliferative capacity of small resting B cells purified from spleens by sorting (>98% purity) was evaluated (Fig. 2C). Stimulation with either 100 or 1000 ng/ml of MALP-2 resulted in a significantly increased proliferative response, when compared with either stimulated full-spleen cell preparations or nonstimulated control B cells (Fig. 2D). This suggests that the observed activation is mediated by the direct effect of MALP-2 on B cells.

MALP-2 treatment promotes B cell differentiation into Ig-secreting cells

To further characterize the stimulatory activity of MALP-2 on the frequency of Ig-secreting cells, small resting B cells were enriched from spleen by negative selection (~95% purity), thereby avoiding a potential nonspecific activation due to Ab binding. Then, resting B cells and total spleen cells were incubated in the presence or absence of MALP-2 at its half-maximal concentration of 50 ng/ml (Fig. 2A). After 2, 4, 6, and 8 days, the frequencies of Ig-secreting cells in the samples were determined by ELISPOT. The presence of MALP-2 increased the frequencies of IgM- and IgG-secreting cells in both total spleen cells and purified B cells (Fig. 3). More than 90% of Ab-secreting cells released IgM, followed by IgG and IgA. The small increment in the number of IgA-secreting cells was only detectable when total spleen cells were used. The number of IgA-secreting cells was very low and maximal on day 2, whereas IgM- and IgG-expressing cells peaked on day 6. In spleen and B cells, the absolute number of IgM-secreting cells was similar, whereas the number of IgG-secreting cells was higher in spleen. This suggests that the effect of MALP-2 on Ig-producing cells was enhanced by the presence of bystander cells. To complement these observations, the concentrations of Ig were determined in supernatant fluids from stimulated and nonstimulated cells. The obtained results were consistent with the ELISPOT data; i.e., secreted Ig was only detected in supernatants of MALP-2-stimulated cells. The supernatants of total spleen cells showed significantly higher concentrations of IgG and IgA than those of enriched B cells (data not shown).



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  		FIGURE 3. Determination of the frequency of IgM-, IgG-, and IgA-secreting cells in MALP-2-stimulated B cells. Spleen cells and highly enriched B cells (~95%) were cultured in the presence or absence of MALP-2 (0.05 µg/ml) for 2, 4, 6, and 8 days and the frequencies of Ig-secreting cells were evaluated by ELISPOT. Results are presented as spot-forming units/105 cells. SEM of quadruplicate values are indicated by vertical lines.

 
MALP-2 increases the expression of activation markers in B cells

The use of MALP-2 as adjuvant does not only improve humoral immune responses, but also stimulates cellular immunity. Thus, it was evaluated whether MALP-2 affects the expression pattern of activation markers and/or surface ligands that are critical for B cell interactions with other immune cells, such as T lymphocytes. Therefore, we stimulated negatively selected small resting B cells with MALP-2 at a concentration of 50 ng/ml. After 5 days of incubation, 45% of the B cells were enlarged and showed an increased granularity (Fig. 4A). This cellular subpopulation also showed higher expression levels of activation markers (CD25 and CD19), MHC I, MHC II, CD80, CD86, and CD40 (Fig. 4B).



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  		FIGURE 4. Flow cytometric analysis of MALP-2-stimulated B cells. Enriched small resting B cells (~95%) were incubated with or without MALP-2 (0.05 µg/ml). A, Forward scatter and sideward scatter were determined, and gates G1 and G2 were established. B, The surface expression of CD25, CD19, CD40, MHC I, MHC II, CD80, and CD86 in MALP-2-stimulated G1 and G2 (filled) cells was evaluated.

 
B and T cells are both critical for the adjuvant activity of MALP-2

As shown above, MALP-2 is able to activate B cells in vitro without T cell help. Thus, the in vivo role of T cells during B cell activation was examined by immunizing mice lacking T cells (nu/nu). High Ag-specific IgG titers (>1:300,000) were detected in sera from control mice that were vaccinated with {beta}-gal and MALP-2. In contrast, no {beta}-gal- specific Ab responses were found in nude mice immunized by intranasal route with {beta}-gal, even after MALP-2 coadministration (Fig. 5A). Thus, MALP-2-mediated stimulation of the TLR2/6 was not able to compensate the lack of T cell help for a T cell-dependent activation of B cells.



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  		FIGURE 5. Adjuvanticity of MALP-2 in BALB/c nu/nu, BALB/c L2 x SLP65–/–, and BALB/c Ig{alpha}–/– mice compared with WT BALB/c mice. Animals were intranasally vaccinated with PBS, {beta}-gal alone, or {beta}-gal coadministered with MALP-2. Additional control mice also received {beta}-gal emulsified in Freund’s complete (prime) or incomplete (boosters) adjuvant by intraperitoneal route. A, Kinetics of {beta}-gal-specific serum IgG responses of BALB/c nu/nu and BALB/c mice are presented as reciprocal geometric mean of the endpoint titers. Immunizations are indicated by arrows. B, The proliferative responses of {beta}-gal-specific T cells from vaccinated mice were assessed by [3H]thymidine incorporation after restimulation for 4 days with {beta}-gal. Results are expressed as the mean cpm from triplicates subtracted of background values from nonstimulated cells cultured in RPMI 1640 supplemented with 10% FCS. SEM are indicated by vertical lines. Data are representative of two independent experiments with similar results.

 
We then investigated the potential role of activated B cells as APC, since MALP-2-treated B cells showed an increased expression of MHC II and costimulatory molecules (Fig. 4). WT BALB/c mice and B cell-deficient mice in the same background (L2 x SLP65–/–) were vaccinated, and Ag-specific proliferative T cell responses were evaluated. No proliferative response was observed in naive nonimmunized mice, as well as in BALB/c and L2 x SLP65–/– mice receiving {beta}-gal alone (Fig. 5B). In contrast, a dose-dependent proliferative response was observed when cells from animals vaccinated with {beta}-gal and MALP-2 were tested (Fig. 5B). This suggests that B cells are not essential for the activation of T cells. However, the proliferative responses of spleen cells from BALB/c mice vaccinated with {beta}-gal plus MALP-2 were significantly stronger (p < 0.05) than those observed in L2 x SLP65–/– mice (Fig. 5B). This points to the fact that B cells may contribute indeed to the elicitation of efficient T cell responses when MALP-2 is used as adjuvant. Similar results were obtained after performing immunization studies using a different strain of B cell-deficient mice (i.e., Ig{alpha}–/–, (17)) (Fig. 5B). To rule out the possibility that the observed phenotype may result from cryptic defects on T cell functions of L2 x SLP65–/– mice, immunization studies were conducted using {beta}-gal and Freund’s adjuvant. The obtained results demonstrated that {beta}-gal-specific T cell responses can be evoked in L2 x SLP65–/– mice when the Ag is delivered in the context of an adjuvant with a different mechanism of action (Fig. 5B). In fact, similar proliferative responses were obtained in WT BALB/c and L2 x SLP65–/– mice immunized with {beta}-gal and Freund’s adjuvant (p > 0.05), which were in turn comparable to those observed in WT BALB/c mice receiving {beta}-gal and MALP-2.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
MALP-2 represents a natural cleavage product released by site-specific proteolysis from a larger lipoprotein (MALP-404) from M. fermentans (21). MALP-404, like other lipoproteins from Mycoplasma, is an immune dominant Ag (22). It is likely that this high immunogenicity arises from the covalently linked lipid moiety, which acts as a danger signal (1, 2, 3) by stimulating the innate immune system via the pattern recognition receptor TLR 2/6. This capacity is retained by both natural MALP-2 and synthetic derivatives, which are able to activate different cellular subtypes (12, 19, 23). However, the role played by these lipopeptides during natural infections has not been elucidated.

We have demonstrated that MALP-2 exerts a strong adjuvant effect after systemic and mucosal coadministration with different Ags (7, 8). MALP-2 is not only able to activate macrophages, but also dendritic cells (19, 23). Although its adjuvant effect is characterized by the stimulation of strong humoral immune responses, only little is known about MALP-2 activity on B cells. Therefore, in the present study we evaluated the effect of MALP-2 on B cells. The results obtained demonstrated that MALP-2 leads to a T cell-independent activation of B cells through TLR2.

Purified B cells proliferate upon MALP-2 stimulation, whereas spleen cells from B cell-deficient or TLR2-deficient mice were unresponsive (Fig. 2). These findings showed that MALP-2 directly stimulates proliferation of B cells in vitro via TLR2 without the need of accessory cells. Interestingly, slight differences were observed in the proliferative responses of spleen cells from C57BL/6 and BALB/c mice over the range of concentrations tested, suggesting a certain degree of dependency on the genetic background. However, the general trend was exactly the same in both mice strains.

Although the proliferative response of B cells were T cell independent, it was enhanced in the presence of CD4+ T cells (Fig. 2B). Furthermore, the frequencies of IgG- and IgA-secreting plasma cells were higher in total spleen cells than in isolated resting B cells after stimulation with MALP-2 (Fig. 3). Therefore, it seems that bystander cells provide additional costimulatory signals. Considering the stimulatory activity of MALP-2 on macrophages (19), they appear to be likely candidate cells for providing the additional differentiation signals. This finding is in agreement with the observation that the CD40/CD40L interaction exhibits costimulatory properties on the activation of B cells by OspA from Borrelia burgdorferi (24). The IgA-secreting cells observed in the presence of MALP-2 seem to indicate maintenance of pre-existing IgA secretory cells rather than activation of resting cells in response to MALP-2 (Fig. 3). In fact, very low frequencies of IgA-secreting cells were detected on day 2 (i.e., <30 spot-forming units/105 cells), and their number was further reduced during the course of the experiment (8 days).

Small resting B cells showed an increased size after MALP-2 stimulation (Fig. 4A). They also exhibited a higher expression of the differentiation marker CD25 (Fig. 4B), which is only found on activated B cells (25, 26). In addition, the expression of CD19 was also up-regulated, which was demonstrated to correlate with a lower threshold for Ag receptor stimulation (27, 28), suggesting that MALP-2 sensitizes B cells for Ag. Moreover, MALP-2 may facilitate the interaction of B cells with other immune cells, such as T cells, by up-regulating the expression of MHC I, MHC II, CD80, CD86, and CD40.

To assess whether the T cell-independent activation of B cells alone or the enhanced interaction with T cells is mainly responsible for the strong humoral responses observed using MALP-2 as mucosal adjuvant (7, 8), mice lacking T cells (nu/nu) were immunized. The results showed that MALP-2 was unable to suffice as second signal to B cells to circumvent the need for T cell help. This highlights the importance of the observed up-regulation of surface molecules on B cells, which are critical for the interaction between T and B cells.

The increased expression of MHC I, MHC II, CD80, CD86, and CD40 suggests an enhanced capacity of MALP-2-treated B cells to present Ags to T cells (29, 30, 31). Therefore, we investigated Ag-specific T cell proliferation in spleen cells of mice lacking B cells (L2 x SLP65–/–, Fig. 1) after intranasal immunization with {beta}-gal and MALP-2. The use of MALP-2 as adjuvant stimulated an Ag-specific proliferative response in B cell-deficient mice, demonstrating the role of other APC (e.g., macrophages and dendritic cells) in the observed T cell activation (Fig. 5B). However, the cellular response detected in B cell-deficient mice was strongly impaired (p < 0.05) with respect to that observed in WT BALB/c mice. The specificity of these results was further supported by the fact that similar results were obtained when a different strain of B cell-deficient mice was used (i.e., Ig{alpha}–/–). In addition, similar T cell responses were observed in BALB/c and L2 x SLP65–/– mice immunized with {beta}-gal and Freund’s adjuvant (p > 0.05), which were in turn comparable to those observed in BALB/c mice receiving {beta}-gal with MALP-2 (Fig. 5B). This demonstrates that there are no cryptic defects on T cell functions affecting the responses against {beta}-gal in the L2 x SLP65–/– mice. In conclusion, B cell engagement plays indeed an important role for T cell activation when MALP-2 is used as mucosal adjuvant.

A two-phase model was suggested for B cell activation (32) in which, upon Ag contact or stimulation, B cells are initially primed and proliferate, thereby increasing the chances for B-T cell contact. In the second phase, the interaction between Ag-specific T and B cells leads to B cell differentiation, affinity maturation and memory B cell development. B cell activation often results in the secretion of IgM, which seems to play an important role in the elicitation and modulation of the immune response (32). Secreted IgM would provide positive feedback to Ag-specific B cells, leading to positive selection. In fact, mice with deficient secretion of IgM show delayed and impaired serum IgG responses, which can be rescued by coadministration of soluble IgM with the Ag (33, 34, 35, 36, 37). Serum IgM is also a potent complement activator (38). Thus, complement-containing immune complexes can be trapped by complement receptors on follicular dendritic cells, thereby leading to efficient germinal center reactions during the T cell-dependent activation of B cells (39, 40, 41). Immune complexes also exhibit a strong stimulatory activity on B cells, by lowering the threshold for Ag receptor stimulation via binding to the CD19/CD21 complex (27, 28, 42). The importance of this interaction is underlined by the fact that the expression of complement receptors is crucial for T cell-dependent B cell responses (43, 44, 45). A murine Fc{alpha}µ receptor has also been characterized, which is expressed on B cells and macrophages but not on granulocytes, T cells, and NK cells. This receptor mediates the endocytosis of immune complexes into murine B cells, thereby facilitating Ag processing and presentation to Th cells (46).

Thus, the improvement of cellular responses upon application of MALP-2 as mucosal adjuvant could be explained, at least in part, by an enhanced activity of B cells as APC. The stimulation of IgM secretion can also favor Ag trapping and internalization by follicular dendritic cells. On the other hand, IgM binding to Ags might facilitate their uptake and transport across the mucosal barrier, since IgM is transported through epithelia by polymeric Ig receptors (47). This would prevent their rapid degradation in the lumen, thereby promoting strong mucosal immune responses.

The knowledge emerging from this work suggests that MALP-2-mediated activation of B cells through TLR2/6 is critical for adjuvanticity. B cells seem to be a common target for molecules acting on different pattern recognition receptors. In fact, ligands specific for other TLR combinations can activate B cells in vitro, such as OspA and S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-N-palmitoyl-(R)-Cys for TLR2/1 (24, 48, 49), neisserial porins for TLR2 (50), R-848 for TLR7 (51, 52, 53) and CpG motifs for TLR9 (54, 55). It has been also demonstrated that these agonists can enhance specific B cell responses against coadministered Ags in vivo (56, 57, 58, 59, 60). Thus, B cell stimulation by TLR ligands seems to be a basic mechanism, which can be exploited to improve the immunogenicity of vaccine formulations.


   Acknowledgments
 
We thank Jana Stopkowicz and Susanne zur Lage for technical assistance and M. Morr for the production of MALP-2. We are deeply indebted to Hassan Jumaa (MPI for Immunobiology, Freiburg) for providing us the SLP65–/– mouse strain.


   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 in part by a grant from the Deutsche Forschungsgemeinschaft (to S.W.). Back

2 Current address: Harvard Medical School, Dana-Farber Cancer Institute, 44 Binney Street, Smith 736, Boston, MA 02115. Back

3 Address correspondence and reprint requests to Dr. Carlos A. Guzmán, Vaccine Research Group, Division of Microbiology, GBF-German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. E-mail address: cag{at}gbf.de Back

4 Abbreviations used in this paper: MALP-2, macrophage-activating lipopeptide-2; {beta}-gal, {beta}-galactosidase; WT, wild type. Back

Received for publication February 23, 2004. Accepted for publication March 7, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Matzinger, P.. 2002. The danger model: a renewed sense of self. Science 296: 301-305.[Abstract/Free Full Text]
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