The Mucosal Adjuvant Macrophage-Activating Lipopeptide-2Directly Stimulates B Lymphocytes via the TLR2 without theNeed of Accessory Cells1
Stefan Borsutzky,* Karsten Kretschmer,2† Pablo D. Becker,* Peter F. Muhlradt,‡
Carsten J. Kirschning,§ Siegfried Weiss,† and Carlos A. Guzman3*
The macrophage-activating lipopeptide-2 (MALP-2) is an agonist of the TLR heterodimer 2/6, which exhibits potent activity asmucosal 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 fromWT mice or animals deficient in TLR2, T cells, B cells, or specific subpopulations of B cells. They demonstrated that MALP-2promotes a T cell-independent activation and maturation of B cells (mainly follicular but also B-1a and marginal zone B cells) viaTLR2. 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-mediatedstimulation of TLR2/6 was unable to circumvent the need of T cell help for efficient Ag-specific B cell activation. Immunizationof mice lacking B cells demonstrated that B cells are critical for MALP-2-dependent improvement of T cell responses. Theknowledge emerging from this work suggests that MALP-2-mediated activation of B cells through TLR2/6 is critical for adju-vanticity. B cell stimulation by pattern recognition receptors seems to be a basic mechanism that can be exploited to improve theimmunogenicity of vaccine formulations. The Journal of Immunology, 2005, 174: 6308–6313.
M ucosal tissues constitute the main portal of entry forinfectious agents. Therefore, many novel vaccinationapproaches are aimed at the stimulation of efficient
local immune responses at the mucosa. This can be achieved by theadministration of vaccine Ags via the mucosal route. On the otherhand, mucosal surfaces are not only confronted with potentiallydangerous agents, but they are also continuously exposed to harm-less entities. Thus, the responsiveness of the mucosal immune sys-tem is tightly controlled. In fact, the immune system has evolvedto recognize potentially “dangerous” rather than foreign Ags (1–3).This explains that most Ags administered via mucosal route arepoorly immunogenic, whereas those delivered in the context of aproper “danger signal” have a better chance to elicit stronger re-sponses. Therefore, one of the strategies to overcome poor immu-nogenicity is the coadministration of Ags with mucosal adjuvants(4–6). However, only a few mucosal adjuvants have been identi-fied, and their mechanisms of action, as well as the structural re-quirements for adjuvanticity are poorly understood.
We have demonstrated that a synthetic derivative (S-[2,3-bis-palmitoyloxypropyl]cysteinyl-GNNDESNISFKEK) of the macro-phage-activating lipopeptide-2 (MALP-2)4 from Mycoplasma fer-mentans is a potent mucosal adjuvant (7, 8). Intranasalcoadministration of MALP-2 with different T cell-dependent Agsstimulates strong Ag-specific T cell proliferative responses, highlevels of Ag-specific serum antibodies, and secretory IgA re-sponses both locally and at distant mucosal sites. Previous studiesrevealed that MALP-2 is an agonist of the TLR2/6 heterodimer,leading to a Toll-IL-1R domain-containing adapter protein andMyD88-dependent activation of NF-�B in macrophages (9–11).Consequently, incubation of macrophages and dendritic cells withMALP-2 results in the secretion of proinflammatory cytokines andenhances the capacity of dendritic cells to present Ags to T cells (12).Since the adjuvanticity of MALP-2 is characterized by strong humoralresponses and B cells express TLR2/6 (13), B cells could be a po-tential target for MALP-2-mediated activation in vivo. However, onlylittle is known concerning the direct effect of MALP-2 on B cells.
In the attempt to unravel the mechanism of adjuvanticity ofMALP-2, we investigated whether MALP-2 exerts a direct stim-ulatory activity on B cells. The results obtained demonstrate thatMALP-2 promotes a T cell-independent activation and maturationof B cells via TLR2. Immunization studies performed in mice lack-ing T or B cells also showed that both cell types are crucial for theadjuvanticity of MALP-2.
Materials and MethodsAnimals 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 micewere obtained from Charles River Laboratories. RAG-1�/�, CD4�/�,
*Vaccine Research Group, Division of Microbiology and †Molecular ImmunologyGroup, Division of Molecular Biotechnology, GBF-German Research Centre for Bio-technology, Braunschweig, Germany; ‡Wound Healing Research Group, BioTec-Gruenderzentrum, Braunschweig, Germany; and §Institute for Medical Microbiology,Immunology and Hygiene, Technical University of Munich, Munich, Germany
Received for publication February 23, 2004. Accepted for publication March 7, 2005.
The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by a grant from the Deutsche Forschungsgemein-schaft (to S.W.).2 Current address: Harvard Medical School, Dana-Farber Cancer Institute, 44 BinneyStreet, Smith 736, Boston, MA 02115.3 Address correspondence and reprint requests to Dr. Carlos A. Guzman, VaccineResearch Group, Division of Microbiology, GBF-German Research Centre for Bio-technology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. E-mail address:[email protected]
4 Abbreviations used in this paper: MALP-2, macrophage-activating lipopeptide-2;�-gal, �-galactosidase; WT, wild type.
L2 (14), SLP65�/� (15, 16), L2 x SLP65�/�, and Ig��/� (17) mice onBALB/c background have been bred in our animal facility.
SLP65�/� and L2 mice are characterized by a complete block of B cellontogeny in the fetal liver (15) and the bone marrow (14), respectively.Thus, homozygous SLP65�/� mice (15, 16) lack B-1a cells in the spleenand peritoneal compartments (Fig. 1). On the other hand, L2 mice (14, 18),which are transgenic for the �2 L chain, exhibit a complete lack of follic-ular 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-1acells and B cells of the marginal zone are found in the spleen of L2 xSLP-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 xSLP65�/� genotype, which is characterized by a complete block of B cellontogeny and a total lack of B cells in their peripheral lymphoid organs, asdemonstrated by flow cytometric analysis of cells obtained from the spleenand the peritoneal cavity (Fig. 1).
B cell purification
Small resting B cells from spleen were purified either by magnetic cellsorting using the B cell isolation kit on an auto-MACS (both obtained fromMiltenyi 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 ofCD19�CD23� and CD21� cells (purity �98%) using a MOFLO(Cytomation).
Measurement of cellular proliferation
Proliferation assays were performed in quadruplicates, as previously de-scribed (7). Briefly, spleen cells (5 � 105 per well) were stimulated withdifferent concentrations of MALP-2, and after 80 h of incubation [3H]thy-midine (1 �Ci/well) was added. Results are expressed as the mean of cpmof stimulated cells subtracted of background values from nonstimulatedcells cultured in RPMI 1640 supplemented with 10% FCS.
Flow cytometry
Stimulated and nonstimulated cells were labeled with FITC-conjugatedAbs 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 combi-nation with a PE-labeled anti-CD19 (1D3) Ab. All Abs were obtained fromBD Pharmingen.
Detection of Ab-secreting cells
The frequencies of total, IgM-, IgG-, or IgA-secreting cells were determinedby ELISPOT using PVDF plates (Millipore) coated with 100 �l/well isotype-specific capture Abs (Sigma-Aldrich) at a concentration of 5 �g/ml in 0.05 Mcarbonate buffer (pH 9.6). Serial dilutions of spleen cells in complete mediumwere incubated in triplicates for 6 h. After washing, plates were incubated with100 �l of biotinylated subclass-specific Abs (Sigma-Aldrich) overnight at 4°C.Then, plates were washed and 100 �l/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 countedusing a binocular microscope.
Immunization experiments
Groups of three mice were immunized by intranasal route on days 1, 14,and 21 with 20 �l of �-galactosidase (�-gal) (50 �g/dose; Roche) alone orcoadministered with MALP-2 (0.5 �g/dose) (19). Alternatively, animalsreceived �-gal (50 �g/dose) emulsified in Freund’s complete (priming) orincomplete (boosters) adjuvant by intraperitoneal route, according to thesame schedule. Sera were collected on days 0, 13, 20, and 31. Then, ani-mals were sacrificed and the spleens were removed for the analysis of thecellular 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 with100 �l/well of anti-IgG, anti-IgA, or anti-IgM Abs (Sigma-Aldrich),whereas Ag-specific serum IgG was determined using plates coated with�-gal (5 �g/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 ofthe double-sided Mann-Whitney U test, p � 0.05 was consideredsignificant.
ResultsProliferation of spleen cells in response to MALP-2 depends onTLR2 expression
Since it has been shown very early that bacterial lipoproteins in-duce proliferation of spleen cells (20), it was assumed that themycoplasmal lipopeptide MALP-2 acted similarly. As expected,MALP-2 stimulated [3H]thymidine incorporation in spleen cellsfrom C57BL/6 mice in a dose-dependent manner (Fig. 2A). Thisstimulatory effect of MALP-2 was evident at a concentration of 10ng/ml and reached a plateau at a concentration of 200 ng/ml. In
FIGURE 1. B cells are absent in L2 x SLP-65�/� mice. Flow cytomet-ric 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 peritonealcavity of SLP-65�/� (wild type), L2 x SLP-65�/� (i.e., �2 L chain trans-genic), SLP-65�/�, and L2 x SLP-65�/� mice.
FIGURE 2. Proliferative responses stimulated by MALP-2. A and B,Spleen cells (5 � 105) were incubated with different concentrations ofMALP-2 for 4 days. Proliferation was assessed by [3H]thymidine incor-poration. C, Flow cytometric analysis of B cells purified by sorting usinga 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 asmean cpm subtracted of background values from nonstimulated cells cul-tured in RPMI 1640 supplemented with 10% FCS; SEM of quadruplicatevalues is indicated by vertical lines.
contrast, spleen cells from TLR2�/� mice did not respond, con-firming that the proliferation induced by MALP-2 specifically de-pends on TLR2 (Fig. 2A).
MALP-2 induces proliferation of B cells without the need ofaccessory cells
To identify the cellular subpopulation responding to MALP-2, weused spleen cells from BALB/c WT mice and from differentknockout animals (deficient in CD4� T cells, follicular B cells, Bcells, or T and B cells) bred onto the same genetic background. Asshown in Fig. 2B, the highest incorporation was observed whencells from BALB/c WT mice were tested, although the dose-re-sponse curve looked different from that observed with spleen cellsfrom C57BL/6 mice (Fig. 2A). Spleen cells from CD4� T cell-deficient animals were still able to proliferate. However, they ex-hibited a slightly reduced incorporation of [3H]thymidine com-pared with cells from WT mice when MALP-2 was applied at lowconcentrations (100–200 ng/ml). In contrast, no MALP-2-depen-dent stimulation was observed when cells from mice with either acombined deficiency of B and T cells (RAG�/�) or B cells alone(L2 x SLP65�/�) were tested. This suggests that B cells are themajor target subpopulation. However, this does not necessarilyrule 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 zoneB cells (L2 mice). A dose-dependent proliferation was observed(Fig. 2B), but [3H]thymidine incorporation, even at the highestconcentration tested (1 �g/ml), was lower with cells from L2 micethan with those from WT or CD4�-deficient mice. This suggeststhat follicular B cells are a major target subset for MALP-2, butthat also B-1a and/or marginal zone B cells might be able to re-spond. Interestingly, B cells from L2 mice seem to respond tolower doses of MALP-2, which underscores their function as fastreacting B cells of the first line of defense.
Additional studies were performed to assess whether the stim-ulatory effect on B cells was induced by MALP-2 directly or viathe activation of bystander cells (e.g., macrophages). To this end,the proliferative capacity of small resting B cells purified fromspleens by sorting (�98% purity) was evaluated (Fig. 2C). Stim-ulation with either 100 or 1000 ng/ml of MALP-2 resulted in asignificantly increased proliferative response, when compared witheither stimulated full-spleen cell preparations or nonstimulatedcontrol B cells (Fig. 2D). This suggests that the observed activa-tion 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 thefrequency of Ig-secreting cells, small resting B cells were enrichedfrom spleen by negative selection (�95% purity), thereby avoidinga potential nonspecific activation due to Ab binding. Then, restingB cells and total spleen cells were incubated in the presence orabsence 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-secretingcells in the samples were determined by ELISPOT. The presenceof MALP-2 increased the frequencies of IgM- and IgG-secretingcells in both total spleen cells and purified B cells (Fig. 3). Morethan 90% of Ab-secreting cells released IgM, followed by IgG andIgA. The small increment in the number of IgA-secreting cells wasonly detectable when total spleen cells were used. The number ofIgA-secreting cells was very low and maximal on day 2, whereasIgM- and IgG-expressing cells peaked on day 6. In spleen and Bcells, the absolute number of IgM-secreting cells was similar, whereasthe number of IgG-secreting cells was higher in spleen. This suggeststhat the effect of MALP-2 on Ig-producing cells was enhanced by the
FIGURE 3. Determination of the frequency of IgM-, IgG-, and IgA-se-creting cells in MALP-2-stimulated B cells. Spleen cells and highly enrichedB cells (�95%) were cultured in the presence or absence of MALP-2 (0.05�g/ml) for 2, 4, 6, and 8 days and the frequencies of Ig-secreting cells wereevaluated by ELISPOT. Results are presented as spot-forming units/105 cells.SEM of quadruplicate values are indicated by vertical lines.
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 �g/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.
presence of bystander cells. To complement these observations, theconcentrations of Ig were determined in supernatant fluids from stim-ulated and nonstimulated cells. The obtained results were consistentwith the ELISPOT data; i.e., secreted Ig was only detected in super-natants of MALP-2-stimulated cells. The supernatants of total spleencells showed significantly higher concentrations of IgG and IgA thanthose of enriched B cells (data not shown).
MALP-2 increases the expression of activation markers in B cells
The use of MALP-2 as adjuvant does not only improve humoralimmune responses, but also stimulates cellular immunity. Thus, itwas evaluated whether MALP-2 affects the expression pattern ofactivation markers and/or surface ligands that are critical for B cellinteractions with other immune cells, such as T lymphocytes.Therefore, we stimulated negatively selected small resting B cellswith MALP-2 at a concentration of 50 ng/ml. After 5 days ofincubation, 45% of the B cells were enlarged and showed an in-creased granularity (Fig. 4A). This cellular subpopulation alsoshowed higher expression levels of activation markers (CD25 andCD19), MHC I, MHC II, CD80, CD86, and CD40 (Fig. 4B).
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 with-out T cell help. Thus, the in vivo role of T cells during B cellactivation was examined by immunizing mice lacking T cells (nu/nu). High Ag-specific IgG titers (�1:300,000) were detected insera from control mice that were vaccinated with �-gal andMALP-2. In contrast, no �-gal- specific Ab responses were foundin nude mice immunized by intranasal route with �-gal, even afterMALP-2 coadministration (Fig. 5A). Thus, MALP-2-mediatedstimulation of the TLR2/6 was not able to compensate the lack ofT cell help for a T cell-dependent activation of B cells.
We then investigated the potential role of activated B cells asAPC, since MALP-2-treated B cells showed an increased expres-sion of MHC II and costimulatory molecules (Fig. 4). WT BALB/cmice and B cell-deficient mice in the same background (L2 xSLP65�/�) were vaccinated, and Ag-specific proliferative T cellresponses were evaluated. No proliferative response was observedin naive nonimmunized mice, as well as in BALB/c and L2 xSLP65�/� mice receiving �-gal alone (Fig. 5B). In contrast, adose-dependent proliferative response was observed when cellsfrom animals vaccinated with �-gal and MALP-2 were tested (Fig.5B). This suggests that B cells are not essential for the activationof T cells. However, the proliferative responses of spleen cellsfrom BALB/c mice vaccinated with �-gal plus MALP-2 were sig-
nificantly stronger ( p � 0.05) than those observed in L2 xSLP65�/� mice (Fig. 5B). This points to the fact that B cells maycontribute indeed to the elicitation of efficient T cell responseswhen MALP-2 is used as adjuvant. Similar results were obtainedafter performing immunization studies using a different strain of Bcell-deficient mice (i.e., Ig��/�, (17)) (Fig. 5B). To rule out thepossibility that the observed phenotype may result from crypticdefects on T cell functions of L2 x SLP65�/� mice, immunizationstudies were conducted using �-gal and Freund’s adjuvant. Theobtained results demonstrated that �-gal-specific T cell responsescan be evoked in L2 x SLP65�/� mice when the Ag is deliveredin the context of an adjuvant with a different mechanism of action(Fig. 5B). In fact, similar proliferative responses were obtained inWT BALB/c and L2 x SLP65�/� mice immunized with �-gal andFreund’s adjuvant ( p � 0.05), which were in turn comparable tothose observed in WT BALB/c mice receiving �-gal and MALP-2.
DiscussionMALP-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 Myco-plasma, is an immune dominant Ag (22). It is likely that this highimmunogenicity arises from the covalently linked lipid moiety,which acts as a danger signal (1–3) by stimulating the innate im-mune system via the pattern recognition receptor TLR 2/6. Thiscapacity is retained by both natural MALP-2 and synthetic deriv-atives, which are able to activate different cellular subtypes (12,19, 23). However, the role played by these lipopeptides duringnatural infections has not been elucidated.
We have demonstrated that MALP-2 exerts a strong adjuvanteffect after systemic and mucosal coadministration with differentAgs (7, 8). MALP-2 is not only able to activate macrophages, butalso dendritic cells (19, 23). Although its adjuvant effect is char-acterized 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, whereasspleen cells from B cell-deficient or TLR2-deficient mice wereunresponsive (Fig. 2). These findings showed that MALP-2 di-rectly stimulates proliferation of B cells in vitro via TLR2 withoutthe need of accessory cells. Interestingly, slight differences wereobserved in the proliferative responses of spleen cells fromC57BL/6 and BALB/c mice over the range of concentrations
FIGURE 5. Adjuvanticity of MALP-2 in BALB/c nu/nu, BALB/c L2 x SLP65�/�, and BALB/c Ig��/� micecompared with WT BALB/c mice. Animals were intrana-sally vaccinated with PBS, �-gal alone, or �-gal coadmin-istered with MALP-2. Additional control mice also re-ceived �-gal emulsified in Freund’s complete (prime) orincomplete (boosters) adjuvant by intraperitoneal route. A,Kinetics of �-gal-specific serum IgG responses ofBALB/c nu/nu and BALB/c mice are presented as recip-rocal geometric mean of the endpoint titers. Immuniza-tions are indicated by arrows. B, The proliferative re-sponses of �-gal-specific T cells from vaccinated micewere assessed by [3H]thymidine incorporation after re-stimulation for 4 days with �-gal. Results are expressed asthe mean cpm from triplicates subtracted of backgroundvalues from nonstimulated cells cultured in RPMI 1640supplemented with 10% FCS. SEM are indicated by ver-tical lines. Data are representative of two independent ex-periments with similar results.
tested, suggesting a certain degree of dependency on the geneticbackground. However, the general trend was exactly the same inboth mice strains.
Although the proliferative response of B cells were T cell in-dependent, it was enhanced in the presence of CD4� T cells (Fig.2B). Furthermore, the frequencies of IgG- and IgA-secretingplasma cells were higher in total spleen cells than in isolated rest-ing B cells after stimulation with MALP-2 (Fig. 3). Therefore, itseems that bystander cells provide additional costimulatory sig-nals. Considering the stimulatory activity of MALP-2 on macro-phages (19), they appear to be likely candidate cells for providingthe additional differentiation signals. This finding is in agreementwith the observation that the CD40/CD40L interaction exhibitscostimulatory properties on the activation of B cells by OspA fromBorrelia burgdorferi (24). The IgA-secreting cells observed in thepresence of MALP-2 seem to indicate maintenance of pre-existingIgA secretory cells rather than activation of resting cells in re-sponse to MALP-2 (Fig. 3). In fact, very low frequencies of IgA-secreting cells were detected on day 2 (i.e., �30 spot-formingunits/105 cells), and their number was further reduced during thecourse of the experiment (8 days).
Small resting B cells showed an increased size after MALP-2stimulation (Fig. 4A). They also exhibited a higher expression ofthe differentiation marker CD25 (Fig. 4B), which is only found onactivated B cells (25, 26). In addition, the expression of CD19 wasalso up-regulated, which was demonstrated to correlate with alower threshold for Ag receptor stimulation (27, 28), suggestingthat MALP-2 sensitizes B cells for Ag. Moreover, MALP-2 mayfacilitate the interaction of B cells with other immune cells, suchas 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 cellsalone or the enhanced interaction with T cells is mainly responsiblefor the strong humoral responses observed using MALP-2 as mucosaladjuvant (7, 8), mice lacking T cells (nu/nu) were immunized. Theresults showed that MALP-2 was unable to suffice as second signal toB cells to circumvent the need for T cell help. This highlights theimportance of the observed up-regulation of surface molecules on Bcells, which are critical for the interaction between T and B cells.
The increased expression of MHC I, MHC II, CD80, CD86, andCD40 suggests an enhanced capacity of MALP-2-treated B cells topresent Ags to T cells (29–31). Therefore, we investigated Ag-specific T cell proliferation in spleen cells of mice lacking B cells(L2 � SLP65�/�, Fig. 1) after intranasal immunization with �-galand MALP-2. The use of MALP-2 as adjuvant stimulated an Ag-specific proliferative response in B cell-deficient mice, demonstrat-ing the role of other APC (e.g., macrophages and dendritic cells)in the observed T cell activation (Fig. 5B). However, the cellularresponse detected in B cell-deficient mice was strongly impaired( p � 0.05) with respect to that observed in WT BALB/c mice. Thespecificity of these results was further supported by the fact thatsimilar results were obtained when a different strain of B cell-deficient mice was used (i.e., Ig��/�). In addition, similar T cellresponses were observed in BALB/c and L2 x SLP65�/� miceimmunized with �-gal and Freund’s adjuvant ( p � 0.05), whichwere in turn comparable to those observed in BALB/c mice re-ceiving �-gal with MALP-2 (Fig. 5B). This demonstrates that thereare no cryptic defects on T cell functions affecting the responsesagainst �-gal in the L2 x SLP65�/� mice. In conclusion, B cellengagement plays indeed an important role for T cell activationwhen MALP-2 is used as mucosal adjuvant.
A two-phase model was suggested for B cell activation (32) inwhich, upon Ag contact or stimulation, B cells are initially primedand proliferate, thereby increasing the chances for B-T cell con-
tact. In the second phase, the interaction between Ag-specific Tand B cells leads to B cell differentiation, affinity maturation andmemory B cell development. B cell activation often results in thesecretion of IgM, which seems to play an important role in theelicitation and modulation of the immune response (32). SecretedIgM would provide positive feedback to Ag-specific B cells, lead-ing to positive selection. In fact, mice with deficient secretion ofIgM show delayed and impaired serum IgG responses, which canbe rescued by coadministration of soluble IgM with the Ag (33–37). Serum IgM is also a potent complement activator (38). Thus,complement-containing immune complexes can be trapped bycomplement receptors on follicular dendritic cells, thereby leadingto efficient germinal center reactions during the T cell-dependentactivation of B cells (39–41). Immune complexes also exhibit astrong stimulatory activity on B cells, by lowering the threshold forAg receptor stimulation via binding to the CD19/CD21 complex(27, 28, 42). The importance of this interaction is underlined by thefact that the expression of complement receptors is crucial for Tcell-dependent B cell responses (43–45). A murine Fc�� receptorhas also been characterized, which is expressed on B cells andmacrophages but not on granulocytes, T cells, and NK cells. Thisreceptor mediates the endocytosis of immune complexes into mu-rine B cells, thereby facilitating Ag processing and presentation toTh cells (46).
Thus, the improvement of cellular responses upon application ofMALP-2 as mucosal adjuvant could be explained, at least in part,by an enhanced activity of B cells as APC. The stimulation of IgMsecretion can also favor Ag trapping and internalization by follic-ular dendritic cells. On the other hand, IgM binding to Ags mightfacilitate their uptake and transport across the mucosal barrier,since IgM is transported through epithelia by polymeric Ig recep-tors (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 foradjuvanticity. B cells seem to be a common target for moleculesacting on different pattern recognition receptors. In fact, ligandsspecific 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 forTLR2 (50), R-848 for TLR7 (51–53) and CpG motifs for TLR9(54, 55). It has been also demonstrated that these agonists canenhance specific B cell responses against coadministered Ags invivo (56–60). Thus, B cell stimulation by TLR ligands seems to bea basic mechanism, which can be exploited to improve the immu-nogenicity of vaccine formulations.
AcknowledgmentsWe thank Jana Stopkowicz and Susanne zur Lage for technical assistanceand M. Morr for the production of MALP-2. We are deeply indebted toHassan Jumaa (MPI for Immunobiology, Freiburg) for providing us theSLP65�/� mouse strain.
DisclosuresThe authors have no financial conflict of interest.
References1. Matzinger, P. 2002. The danger model: a renewed sense of self. Science 296:
301–305.2. Matzinger, P. 1994. Tolerance, danger, and the extended family. Annu. Rev.
Immunol. 12: 991–1045.3. Gallucci, S., and P. Matzinger. 2001. Danger signals: SOS to the immune system.
Curr. Opin. Immunol. 13: 114–119.4. Holmgren, J., N. Lycke, and C. Czerkinsky. 1993. Cholera toxin and cholera B
subunit as oral-mucosal adjuvant and antigen vector systems. Vaccine 11:1179–1184.
5. Yamamoto, S., Y. Takeda, M. Yamamoto, H. Kurazono, K. Imaoka, K. Fujihashi,M. Noda, H. Kiyono, and J. R. McGhee. 1997. Mutants in the ADP-ribosyltrans-ferase cleft of cholera toxin lack diarrheagenicity but retain adjuvanticity. J. Exp.Med. 185: 1203–1210.
6. Roberts, M., A. Bacon, R. Rappuoli, M. Pizza, I. Cropley, G. Douce, G. Dougan,M. Marinaro, J. McGhee, and S. Chatfield. 1995. A mutant pertussis toxin moleculethat lacks ADP-ribosyltransferase activity, PT-9K/129G, is an effective mucosal ad-juvant for intranasally delivered proteins. Infect. Immun. 63: 2100–2108.
7. Rharbaoui, F., B. Drabner, S. Borsutzky, U. Winckler, M. Morr, B. Ensoli, P. F.Muhlradt, and C. A. Guzman. 2002. The Mycoplasma-derived lipopeptideMALP-2 is a potent mucosal adjuvant. Eur. J. Immunol. 32: 2857–2865.
8. Borsutzky, S., V. Fiorelli, T. Ebensen, A. Tripiciano, F. Rharbaoui, A. Scoglio,C. Link, F. Nappi, M. Morr, S. Butto, et al. 2003. Efficient mucosal delivery ofthe HIV-1 Tat protein using the synthetic lipopeptide MALP-2 as adjuvant. Eur.J. Immunol. 33: 1548–1556.
9. Takeuchi, O., A. Kaufmann, K. Grote, T. Kawai, K. Hoshino, M. Morr, P. F.Muhlradt, and S. Akira. 2000. Cutting edge: preferentially the R-stereoisomer ofthe mycoplasmal lipopeptide macrophage-activating lipopeptide-2 activates im-mune cells through a toll-like receptor 2- and MyD88-dependent signaling path-way. J. Immunol. 164: 554–557.
10. Takeuchi, O., T. Kawai, P. F. Muhlradt, M. Morr, J. D. Radolf, A. Zychlinsky,K. Takeda, and S. Akira. 2001. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int. Immunol. 13: 933–940.
11. Yamamoto, M., S. Sato, H. Hemmi, H. Sanjo, S. Uematsu, T. Kaisho,K. Hoshino, O. Takeuchi, M. Kobayashi, T. Fujita, K. Takeda, and S. Akira.2002. Essential role for TIRAP in activation of the signalling cascade shared byTLR2 and TLR4. Nature 420: 324–329.
12. Link, C., R. Gavioli, T. Ebensen, A. Canella, E. Reinhard, and C. A. Guzman.2004. The Toll-like receptor ligand MALP-2 stimulates dendritic cell maturationand modulates proteasome composition and activity. Eur. J. Immunol. 34:899–907.
13. Caramalho, I., T. Lopes-Carvalho, D. Ostler, S. Zelenay, M. Haury, andJ. Demengeot. 2003. Regulatory T cells selectively express toll-like receptors andare activated by lipopolysaccharide. J. Exp. Med. 197: 403–411.
14. Engel, H., B. Bogen, U. Muller, J. Andersson, A. Rolink, and S. Weiss. 1998.Expression level of a transgenic �2 chain results in isotype exclusion and com-mitment to B1 cells. Eur. J. Immunol. 28: 2289–2299.
15. Jumaa, H., B. Wollscheid, M. Mitterer, J. Wienands, M. Reth, and P. J. Nielsen.1999. Abnormal development and function of B lymphocytes in mice deficientfor the signaling adaptor protein SLP-65. Immunity 11: 547–554.
16. Flemming, A., T. Brummer, M. Reth, and H. Jumaa. 2003. The adaptor proteinSLP-65 acts as a tumor suppressor that limits pre-B cell expansion. Nat. Immunol.4: 38–43.
17. Pelanda, R., U. Braun, E. Hobeika, M. C. Nussenzweig, and M. Reth. 2002. Bcell progenitors are arrested in maturation but have intact VDJ recombination inthe absence of Ig-� and Ig-�. J. Immunol. 169: 865–872.
18. Kretschmer, K., A. Jungebloud, J. Stopkowicz, B. Stoermann, R. Hoffmann, andS. Weiss. 2003. Antibody repertoire and gene expression profile: implications fordifferent developmental and functional traits of splenic and peritoneal B-1 lym-phocytes. J. Immunol. 171: 1192–1201.
19. Muhlradt, P. F., M. Kiess, H. Meyer, R. Sussmuth, and G. Jung. 1997. Isolation,structure elucidation, and synthesis of a macrophage stimulatory lipopeptide fromMycoplasma fermentans acting at picomolar concentration. J. Exp. Med. 185:1951–1958.
20. Melchers, F., V. Braun, and C. Galanos. 1975. The lipoprotein of the outer mem-brane of Escherichia coli: a B-lymphocyte mitogen. J. Exp. Med. 142: 473–482.
21. Davis, K. L., and K. S. Wise. 2002. Site-specific proteolysis of the MALP-404lipoprotein determines the release of a soluble selective lipoprotein-associatedmotif-containing fragment and alteration of the surface phenotype of Myco-plasma fermentans. Infect. Immun. 70: 1129–1135.
22. Yogev, D., G. F. Browning, and K. Wise. 2002. Genetic mechanisms of surfacevariation. In Molecular Biology and Pathogenesis of Mycoplasmas. R. Herrmann,ed. Kluwer Academic/Plenum, New York, Boston, Dordrecht, London, Moscow,p. 417–444.
23. Weigt, H., P. F. Muhlradt, A. Emmendorffer, N. Krug, and A. Braun. 2003.Synthetic mycoplasma-derived lipopeptide MALP-2 induces maturation andfunction of dendritic cells. Immunobiology 207: 223–233.
24. Snapper, C. M., F. R. Rosas, L. Jin, C. Wortham, M. R. Kehry, and J. J. Mond.1995. Bacterial lipoproteins may substitute for cytokines in the humoral immuneresponse to T cell-independent type II antigens. J. Immunol. 155: 5582–5589.
25. Lowenthal, J. W., R. H. Zubler, M. Nabholz, and H. R. MacDonald. 1985. Sim-ilarities between interleukin-2 receptor number and affinity on activated B and Tlymphocytes. Nature 315: 669–672.
26. Taniguchi, T., and Y. Minami. 1993. The IL-2/IL-2 receptor system: a currentoverview. Cell 73: 5–8.
27. Carter, R. H., and D. T. Fearon. 1992. CD19: lowering the threshold for antigenreceptor stimulation of B lymphocytes. Science 256: 105–107.
28. Sato, S., D. A. Steeber, and T. F. Tedder. 1995. The CD19 signal transductionmolecule is a response regulator of B-lymphocyte differentiation. Proc. Natl.Acad. Sci. USA 92: 11558–11562.
29. Kurt-Jones, E. A., D. Liano, K. A. HayGlass, B. Benacerraf, M. S. Sy, andA. K. Abbas. 1988. The role of antigen-presenting B cells in T cell priming invivo. Studies of B cell-deficient mice. J. Immunol. 140: 3773–3778.
30. Morris, S. C., A. Lees, and F. D. Finkelman. 1994. In vivo activation of naive Tcells by antigen-presenting B cells. J. Immunol. 152: 3777–3785.
31. Constant, S. L. 1999. B lymphocytes as antigen-presenting cells for CD4� T cellpriming in vivo. J. Immunol. 162: 5695–5703.
32. Baumgarth, N. 2000. A two-phase model of B-cell activation. Immunol. Rev. 176:171–180.
33. Boes, M., C. Esau, M. B. Fischer, T. Schmidt, M. Carroll, and J. Chen. 1998.Enhanced B-1 cell development, but impaired IgG antibody responses in micedeficient in secreted IgM. J. Immunol. 160: 4776–4787.
34. Ehrenstein, M. R., T. L. O’Keefe, S. L. Davies, and M. S. Neuberger. 1998. Targetedgene disruption reveals a role for natural secretory IgM in the maturation of theprimary immune response. Proc. Natl. Acad. Sci. USA 95: 10089–10093.
35. Henry, C., and N. K. Jerne. 1968. Competition of 19S and 7S antigen receptorsin the regulation of the primary immune response. J. Exp. Med. 128: 133–152.
36. Heyman, B., G. Andrighetto, and H. Wigzell. 1982. Antigen-dependent IgM-mediated enhancement of the sheep erythrocyte response in mice: evidence forinduction of B cells with specificities other than that of the injected antibodies.J. Exp. Med. 155: 994–1009.
37. Lehner, P., P. Hutchings, P. M. Lydyard, and A. Cooke. 1983. II. IgM-mediatedenhancement: dependency on antigen dose, T-cell requirement and lack of evi-dence for an idiotype-related mechanism. Immunology 50: 503–509.
38. Cooper, N. R. 1985. The classical complement pathway: activation and regulationof the first complement component. Adv. Immunol. 37: 151–216.
39. Papamichail, M., C. Gutierrez, P. Embling, P. Johnson, E. J. Holborow, andM. B. Pepys. 1975. Complement dependence of localisation of aggregated IgG ingerminal centres. Scand. J. Immunol. 4: 343–347.
40. Chen, L. L., A. M. Frank, J. C. Adams, and R. M. Steinman. 1978. Distributionof horseradish peroxidase (HRP)-anti-HRP immune complexes in mouse spleenwith special reference to follicular dendritic cells. J. Cell Biol. 79: 184–189.
41. Enriquez-Rincon, F., and G. G. Klaus. 1984. Follicular trapping of hapten-eryth-rocyte-antibody complexes in mouse spleen. Immunology 52: 107–116.
42. Dempsey, P. W., M. E. Allison, S. Akkaraju, C. C. Goodnow, and D. T. Fearon.1996. C3d of complement as a molecular adjuvant: bridging innate and acquiredimmunity. Science 271: 348–350.
43. Rickert, R. C., K. Rajewsky, and J. Roes. 1995. Impairment of T-cell-dependentB-cell responses and B-1 cell development in CD19-deficient mice. Nature 376:352–355.
44. Croix, D. A., J. M. Ahearn, A. M. Rosengard, S. Han, G. Kelsoe, M. Ma, andM. C. Carroll. 1996. Antibody response to a T-dependent antigen requires B cellexpression of complement receptors. J. Exp. Med. 183: 1857–1864.
45. Ahearn, J. M., M. B. Fischer, D. Croix, S. Goerg, M. Ma, J. Xia, X. Zhou,R. G. Howard, T. L. Rothstein, and M. C. Carroll. 1996. Disruption of the Cr2locus results in a reduction in B-1a cells and in an impaired B cell response toT-dependent antigen. Immunity 4: 251–262.
46. Shibuya, A., N. Sakamoto, Y. Shimizu, K. Shibuya, M. Osawa, T. Hiroyama,H. J. Eyre, G. R. Sutherland, Y. Endo, T. Fujita, et al. 2000. Fc �/� receptormediates endocytosis of IgM-coated microbes. Nat. Immunol. 1: 441–446.
47. Brandtzaeg, P., and H. Prydz. 1984. Direct evidence for an integrated function ofJ chain and secretory component in epithelial transport of immunoglobulins. Na-ture 311: 71–73.
48. Alexopoulou, L., V. Thomas, M. Schnare, Y. Lobet, J. Anguita, R. T. Schoen,R. Medzhitov, E. Fikrig, and R. A. Flavell. 2002. Hyporesponsiveness to vacci-nation with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice. Nat. Med. 8: 878–884.
49. Takeuchi, O., S. Sato, T. Horiuchi, K. Hoshino, K. Takeda, Z. Dong,R. L. Modlin, and S. Akira. 2002. Cutting edge: role of Toll-like receptor 1 inmediating immune response to microbial lipoproteins. J. Immunol. 169: 10–14.
50. Massari, P., P. Henneke, Y. Ho, E. Latz, D. T. Golenbock, and L. M. Wetzler.2002. Cutting edge: Immune stimulation by neisserial porins is toll-like receptor2 and MyD88 dependent. J. Immunol. 168: 1533–1537.
51. Bishop, G. A., Y. Hsing, B. S. Hostager, S. V. Jalukar, L. M. Ramirez, andM. A. Tomai. 2000. Molecular mechanisms of B lymphocyte activation by theimmune response modifier R-848. J. Immunol. 165: 5552–5557.
52. Bishop, G. A., L. M. Ramirez, M. Baccam, L. K. Busch, L. K. Pederson, andM. A. Tomai. 2001. The immune response modifier resiquimod mimics CD40-induced B cell activation. Cell. Immunol. 208: 9–17.
53. Tomai, M. A., L. M. Imbertson, T. L. Stanczak, L. T. Tygrett, andT. J. Waldschmidt. 2000. The immune response modifiers imiquimod and R-848are potent activators of B lymphocytes. Cell Immunol. 203: 55–65.
54. Krieg, A. M., A. K. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale,G. A. Koretzky, and D. M. Klinman. 1995. CpG motifs in bacterial DNA triggerdirect B-cell activation. Nature 374: 546–549.
55. Chen, W., Y. Yu, C. Shao, M. Zhang, W. Wang, L. Zhang, and X. Cao. 2001.Enhancement of antigen-presenting ability of B lymphoma cells by immunostimu-latory CpG-oligonucleotides and anti-CD40 antibody. Immunol. Lett. 77: 17–23.
56. Erdile, L. F., and B. Guy. 1997. OspA lipoprotein of Borrelia burgdorferi is amucosal immunogen and adjuvant. Vaccine 15: 988–996.
57. Schlecht, S., K. H. Wiesmuller, G. Jung, and W. G. Bessler. 1989. Enhancementof protection against Salmonella infection in mice mediated by a synthetic li-popeptide analogue of bacterial lipoprotein in S. typhimurium vaccines. ZentralblBakteriol. 271: 493–500.
58. Vasilakos, J. P., R. M. Smith, S. J. Gibson, J. M. Lindh, L. K. Pederson, M. J. Reiter,M. H. Smith, and M. A. Tomai. 2000. Adjuvant activities of immune response mod-ifier R-848: comparison with CpG ODN. Cell. Immunol. 204: 64–74.
59. Fusco, P. C., F. Michon, M. Laude-Sharp, C. A. Minetti, C. H. Huang, I. Heron,and M. S. Blake. 1998. Preclinical studies on a recombinant group B meningo-coccal porin as a carrier for a novel Haemophilus influenzae type b conjugatevaccine. Vaccine 16: 1842–1849.
60. Moldoveanu, Z., L. Love-Homan, W. Q. Huang, and A. M. Krieg. 1998. CpGDNA, a novel immune enhancer for systemic and mucosal immunization withinfluenza virus. Vaccine 16: 1216–1224.
