Cell Maturation Via Toll-Like Receptor 2 Microbial Lipopeptides Stimulate

Microbial Lipopeptides Stimulate Dendritic Cell Maturation Via Toll-Like Receptor 2 Cheryl J. Hertz, Sylvia M. Kiertscher, Paul J. Godowski, Deborah A. Bouis, Michael V. Norgard, Michael D. Roth This information is current as and Robert L. Modlin of September 28, 2021. J Immunol 2001; 166:2444-2450; ; doi: 10.4049/jimmunol.166.4.2444 http://www.jimmunol.org/content/166/4/2444 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Microbial Lipopeptides Stimulate Dendritic Cell Maturation Via Toll-Like Receptor 21

Cheryl J. Hertz,* Sylvia M. Kiertscher,† Paul J. Godowski,‡ Deborah A. Bouis,§ Michael V. Norgard,§ Michael D. Roth,† and Robert L. Modlin2*¶

The ability of dendritic cells (DC) to initiate immune responses in naive T cells is dependent upon a maturation process that allows the cells to develop their potent Ag-presenting capacity. Although immature DC can be derived in vitro by treatment of peripheral blood monocytes with GM-CSF and IL-4, additional signals such as those provided by TNF-␣, CD40 ligand, or LPS are required for complete maturation and maximum APC function. Because we recently found that microbial lipoproteins can activate monocytes and DC through Toll-like receptor (TLR) 2, we also investigated whether lipoproteins can drive DC maturation. Immature DC were cultured with or without lipoproteins and were monitored for expression of cell surface markers indicative of maturation.

Stimulation with lipopeptides increased expression of CD83, MHC class II, CD80, CD86, CD54, and CD58, and decreased CD32 Downloaded from expression and endocytic activity; these lipopeptide-matured DC also displayed enhanced T cell stimulatory capacity in MLR, as measured by T cell proliferation and IFN-␥ secretion. The lipid moiety of the lipopeptide was found to be essential for induction of maturation. Preincubation of maturing DC with an anti-TLR2 blocking Ab before addition of lipopeptide blocked the phenotypic and functional changes associated with DC maturation. These results demonstrate that lipopeptides can stimulate DC maturation via TLR2, providing a mechanism by which products of bacteria can participate in the initiation of an immune response. The Journal of Immunology, 2001, 166: 2444–2450. http://www.jimmunol.org/

endritic cells (DC)3 are potent APCs capable of Ag up- 7). Similar changes indicative of maturation have also been re- take and presentation, as well as cytokine secretion. In ported following infection with mycoplasma, viruses, intracellular D vivo, immature DC reside in the periphery where they bacteria, and parasites (8–10). These phenotypic changes parallel serve as sentinels for foreign Ags and microbial pathogens. Inter- the functional transition of DC from Ag-capturing cells to APCs. action with microbes induces a critical maturation program during Although it is known that microbes and microbial products, par- which the DC modulate expression of cell surface molecules and ticularly LPS, induce the maturation of DC, the mechanism by migrate to lymph nodes where potent interactions with T cells which this occurs is not known. initiate the acquired immune response (1). Recent work in our laboratory has revealed that DC express by guest on September 28, 2021 In vitro studies of DC maturation have been conducted using Toll-like receptor (TLR) 2 and that this receptor mediates lipopep- cells derived from peripheral blood monocytes cultured in the tide-induced IL-12 production, but little is known of other pro- presence of GM-CSF and IL-4 (2–5). Such cells are relatively cesses mediated by TLRs (11). Although there is currently no di- immature, having a high rate of endocytosis and expressing low rect evidence that TLRs mediate DC maturation, TLRs or other levels of MHC class II (MHC-II), CD83, and costimulatory mol- pattern recognition receptors have been predicted to mediate DC ecules CD80 and CD86 (1). Upon maturation with TNF-␣, CD40 maturation events (1, 12). In this study, we examined whether ligand, or LPS, DC down-regulate mechanisms of Ag capture, in- microbial lipopeptides can induce the phenotypic and functional cluding endocytic activity and expression of Fc receptors, while changes associated with DC maturation, and whether this process increasing expression of costimulatory and adhesion molecules (6, is dependent upon TLR2.

*Division of Dermatology and †Pulmonary Medicine, ¶Department of Microbiology Materials and Methods and Immunology, and Molecular Biology Institute, University of California School of Preparation and maturation of DC Medicine, Los Angeles, CA 90095; ‡Genentech Incorporated, South San Francisco, § CA 94080; and Department of Microbiology, University of Texas Southwestern Peripheral blood was collected from healthy volunteers and fractionated Medical Center, Dallas, TX 75235 over Ficoll-Paque (Amersham Pharmacia, Uppsala, Sweden) by a standard Received for publication June 22, 2000. Accepted for publication December 7, 2000. procedure. To derive DC, total PBMCs were cultured at 2 ϫ 106 cells/ml The costs of publication of this article were defrayed in part by the payment of page in complete media (RPMI 1640, 0.1 mM sodium pyruvate, 2 mM penicil- charges. This article must therefore be hereby marked advertisement in accordance lin, 50 ␮g/ml streptomycin; Life Technologies, Grand Island, NY) supple- with 18 U.S.C. Section 1734 solely to indicate this fact. mented with 1% FCS (Omega Scientiﬁc, Tarzana, CA) for 1.5 h in tissue 1 culture ﬂasks. Following incubation, nonadherent cells were removed by This work was supported in part by grants from the National Institutes of Health ϫ (AI22553, AR 40312, AI 07118) and the United Nations Development Program/ extensive washing with a 1 solution of HBSS (Life Technologies). The World Bank/World Health Organization Special Program for Research and Training remaining adherent cells were then cultured in complete media containing in Tropical Diseases (IMMLEP). C.J.H. is the recipient of a Research Fellowship 10% FCS, 200 U/ml GM-CSF (Genetics Institute, Cambridge MA), and

Award from the Dermatology Foundation sponsored by SmithKline Beecham Phar- 100 U/ml IL-4 (Schering-Plough, Madison, NJ) for 3–4 days in a CO2 maceuticals and Medicis Pharmaceutical Corporation. incubator at 37°C. The resulting cells were semi- to nonadherent and MHC ϩ ϩ Ϫ/low 2 Address correspondence and reprint requests to Dr. Robert Modlin, University of II CD14 CD83 and displayed DC morphology. California Division of Dermatology, 52-121 CHS, 10833 Le Conte Avenue, Los For further maturation, adherent and nonadherent DC were harvested Angeles, CA 90095. E-mail address: [email protected] from T-75 flasks by incubation in PBS-EDTA (1 mM) for 30 min. The cells ϫ 5 3 Abbreviations used in this paper: DC, dendritic cell(s); MFI, median fluorescence recovered were counted and recultured at 5 10 cells/ml in fresh media intensity; F-Dx, fluorescein-conjugated dextran; TLR, Toll-like receptor; MHC-II, containing GM-CSF and IL-4. Salmonella typhosa LPS (Sigma; St. Louis, MHC class II. MO), the synthetic lipopeptide Pam3CysSerLys4 (Boehringer Mannheim,

Indianapolis, IN), the 19-kDa lipoprotein from Mycobacterium tuberculo- were analyzed using WinMDI 2.8 (Joseph Trotter, Scripps Research Insti- sis (13) (courtesy of John Belisle, Colorado State University, Ft. Collins, tute, San Diego, CA). Histograms were drawn from and median fluores- CO), a synthetic 19-kDa lipopeptide, a synthetic lipopeptide based on the cence intensity (MFI) values were determined on the gated population. In sequence of the 47-kDa lipoprotein from Treponema pallidum (14), or some experiments, the percentage of cells positive for a particular marker unlipidated forms of the synthetic lipopeptides were added to some DC was determined. cultures for 24–48 h (15). All lipopeptides contained Ͻ40 pg/␮g of LPS, as determined by the Limulus Amoebocyte Assay (BioWhittaker, Walk- Endocytic activity ersville, MD). Cells were cultured for an additional 24–48 h before analysis by flow cytometry. Endocytic activity of DC was measured by the uptake of fluorescein-conjugated dextran (F-Dx; m.w. 40,000; Molecular Probes, Eugene, OR) as Blocking of TLR2 previously described (2). Briefly, DC at various states of maturation were incubated in complete media plus 10% FCS plus 1 mg/ml F-Dx for1hat Blocking experiments with anti-TLR2 mAb were performed on DC 3 days 4°C to measure nonspecific binding, or at 37°C to measure specific uptake. after the initiation of the culture from PBMCs. Anti-TLR2 Ab (16) or IgG1 Cells were then washed extensively and analyzed by flow cytometry as isotype control Ab (10 ␮g/ml) was added to the cells 30 min before the described above. addition of the lipopeptides or LPS. Cells were harvested with PBS-EDTA 40–48 h later and analyzed for expression of cell surface molecules by Results flow cytometry, or used directly in a MLR. Phenotype of immature DC Mixed leukocyte reactions It has previously been shown that both LPS and lipopeptides in- DC for use in MLRs were harvested from T-75 flasks following the initial duce cytokine secretion in monocytes via TLRs (11, 13, 17, 18), 3-day culture period, and recultured in the same media in 96-well round- and that LPS induces maturation of DC (1). Hence, we asked 4 3 3 ϫ ϫ ϫ Downloaded from bottom plates (Costar, Corning, NY) at 1 10 ,4 10 ,or2 10 whether lipopeptides can also induce DC maturation and whether cells/well. Lipopeptides were added to some cultures, and the DC were incubated for an additional 2 days in a total volume of 100 ␮l. On day 5 TLR2 mediates this process. To this end, we used an in vitro cul- after initiation of the cells from PBMC, the DC were irradiated (3000 rad ture system for the derivation of DC from adherent PBMCs cul- from a 137Cs source) and cocultured with purified T cells. Blocking of tured with GM-CSF and IL-4 (2). As shown in Fig. 1, cells cul- TLR2 was performed as described above. tured in this manner for 3 days stably expressed MHC-II. The T cells from an unrelated donor were prepared from total PBMC by negative selection using Ab depletion and magnetic beads. Briefly, total monocyte marker CD14 was also expressed, whereas there was PBMCs were prepared as described above and diluted to 5 ϫ 106 cells/ml little or no expression of CD83, a marker expressed on DC. With http://www.jimmunol.org/ in RPMI 1640 plus 10% human serum (Omega Scientific). The cells were additional days in culture, cells became more differentiated and then cultured for 30 min in tissue culture flasks to remove the adherent acquired a DC morphology. After 5 days in culture, the MFI of cells. The nonadherent cells were collected and incubated for 20 min at 4°C cells stained with anti-CD14 expression decreased 5-fold relative with anti-CD14, anti-CD16, and anti-CD19 mAb (no azide, low endotoxin (NA/LE); PharMingen, San Diego, CA) at a concentration of 0.4 ␮g Ab/ to cells cultured for 3 days (Fig. 1); in some donors, we observed 106 nonadherent cells. Following two washes with PBS plus 2% serum, a concomitant increase in CD83 expression (data not shown). cells were incubated with sheep anti-mouse IgG-conjugated Dynabeads These cells also expressed TLR2, as we have previously shown (10:1 bead:cell ratio; Dynal, Lake Success, NY) for 20 min at 4°C. The (Fig. 1) (11). nonmagnetic fraction was collected and contained Ͼ95% CD3ϩ T cells, as assessed by flow cytometry. Synthetic and microbial lipopeptides drive DC maturation by guest on September 28, 2021 Purified T cells were added to the lipopeptide-matured DC at 2 ϫ 105 cells/well to give final ratios of 1:100, 1:50, or 1:20 DC:T cells and incu- Final maturation of DC has been shown to occur upon treatment with bated for 5–6 days. Culture supernatant fluids were collected from some TNF-␣, CD40 ligand, or LPS (6). This maturation results in increased cultures for use in an IFN-␥ ELISA. To measure T cell-proliferative responses, [3H]thymidine was added at 1 ␮Ci/well and incubated for an expression of CD83. To determine whether lipopeptides also mediate additional 18 h. The assay was then harvested, and the incorporation of DC maturation, immature DC were cultured with various concentra- 3 [ H]thymidine was measured in a liquid scintillation counter. tions of the synthetic lipopeptide PAM3CysSerLys4. As shown in Fig. 2, immature DC cultured with increasing doses of lipopeptide had IFN-␥ ELISA increasing levels of CD83 expression, as assessed by flow cytometry. IFN-␥ in culture supernatant fluids was assessed by a standard sandwich At the maximum dose of 10,000 ng/ml the MFI of cells stained with ELISA. Microtiter plates (Costar) were coated with an unconjugated anti- anti-CD83 Ab was 3.5 times greater than unstimulated cells. A similar IFN-␥ capture Ab (clone NIB42, 5 ␮g/ml), and detection was achieved using a biotinylated Ab (clone 4s.B3, 2 ␮g/ml; PharMingen). The plate was dose-dependent effect was also observed with a 19-kDa lipoprotein developed using Immunopure HRP-conjugated streptavidin (Pierce, Rock- from M. tuberculosis and a synthetic 19-kDa lipopeptide (data not ford, IL) and an ABTS Microwell Peroxidase Substrate System (Kirkeg- aard & Perry Laboratories, Gaithersburg, MD). The absorbance at 405 nm was read using a microtiter plate reader, and concentrations of IFN-␥ were calculated from a standard curve of recombinant human IFN-␥ (PharMingen). Flow cytometry Standard flow cytometric analysis was used to assess surface expression of various markers. Surface expression of TLR2 was determined using a mAb (clone 2392, IgG1) and a PE-conjugated goat anti-mouse IgG secondary Ab. The following mAbs directly conjugated with either PE or FITC were used in single-color flow cytometric analysis: PE-CD14 (clone TUK4, IgG2a), PE-CD54 (clone MEM111, IgG2a), PE-CD58 (clone 1C3, IgG2a), PE-CD80 (clone L3007.4, IgG1), PE-CD83 (clone HB15e, IgG1), PE- CD86 (clone IT2.2, IgG1), FITC-HLA-DR (clone TU36, IgG2b), and FIGURE 1. Phenotype of immature DC. Adherent cells from peripheral FITC-CD32 (clone FLI8.26, IgG2b). Isotype control Abs (mouse IgG1, blood were cultured in vitro with 200 U/ml GM-CSF and 100 U/ml IL-4. PE-IgG2a, PE-IgG1, FITC-IgG2b) were used in all experiments. All conjugated Abs were purchased from Caltag (South San Francisco, CA) or Cells were harvested after 3 or 5 days in culture and stained with PE- or PharMingen. After staining, cells were washed and fixed in 1% parafor- FITC-conjugated Abs specific for the markers indicated (gray) or an iso- maldehyde before analysis on a Becton Dickinson (Mountain View, CA) type control Ab (outline) and examined by flow cytometry. The results FacsScan or FacsCa1ibur Flow Cytometer. Gating was on large granular shown were obtained from a single experiment with a single donor and are cells, and 2000–5000 gated events were collected from each sample. Data representative of two to four similar experiments that gave similar results. 2446 MATURATION OF DENDRITIC CELLS BY MICROBIAL LIPOPEPTIDES

adhesion, which facilitate Ag recognition and cell-cell interactions while expressing low levels of molecules associated with Ag capture. To further characterize lipopeptide-induced DC maturation, the modulation of various cell surface molecules in response to synthetic and microbial lipopeptides was examined by ﬂow cytometry. Similar to CD83, levels of the Ag-presenting molecule MHC-II, costimulatory molecules CD80 and CD86, and adhesion molecules CD54 and CD58 were ϳ1.5–10 times higher on DC

cultured with PAM3CysSerLys4 or a synthetic 19-kDa lipopeptide from M. tuberculosis, relative to unstimulated cells (Fig. 3). In contrast, levels of Fc␥RII (CD32), which mediates uptake of Ag-Ab complexes, was 2-fold lower on DC matured with lipopeptides. In addition, IL-12 could be detected in the culture supernatant ﬂuids of lipopeptide-stimulated DC (data not shown). Stimu- lation with the T. pallidum 47-kDa lipopeptide (Fig. 4; data not shown) and LPS yielded similar results, although the absolute fold increase of MFI observed showed some donor variability. Overall, these data support the notion that lipopeptides and LPS have a

similar biological effect on the activation and maturation of DC. Downloaded from Immature DC are efﬁcient at Ag capture and have a high level of endocytosis. Upon maturation and concomitant with an increase in Ag presenting function, DC have a reduced capacity for Ag capture via endocytic activity. Such activity can be quantitated using ﬂow cytometry to monitor the uptake of F-Dx. To determine

whether mechanisms of Ag capture could also be modulated by http://www.jimmunol.org/ lipopeptides, endocytic activity was measured in immature as well as lipopeptide- and LPS-matured DC. Similar to LPS-stimulated FIGURE 2. Dose-dependent induction of DC maturation by lipopep- cells, DC cultured in the presence of lipopeptide took up lower tide. Adherent cells from peripheral blood were cultured in vitro with 200 U/ml GM-CSF and 100 U/ml IL-4 for a total of 5 days. The lipopeptide levels of F-Dx relative to immature DC, resulting in cells with a MFI ϳ8-fold lower than untreated cells (Fig. 5). This finding pro- Pam3CysSerLys4 was added at the concentration indicated for the last 40–48 h of incubation. Cells were stained with a PE-conjugated Ab spe- vides further evidence that microbial lipopeptides can drive DC cific for CD83 (gray) or an isotype-matched control Ab (outline). A, His- maturation. tograms showing the level of CD83 expression. The value indicated on each histogram is the MFI of the marker-specific Ab. B, Plot of the MFI vs The lipid portion of lipopeptides is required for DC by guest on September 28, 2021 concentration of lipopeptide. The results shown were obtained from a sin- responsiveness gle experiment with a single donor and are representative of three similar experiments that gave similar results. Previous work describing the biological properties of lipopeptides has revealed that the lipid portion of the molecule is required for activity (11, 13, 14, 17, 19). To determine whether microbial li- shown). This finding suggests that lipopeptides can induce DC popeptide-induced DC maturation was dependent upon the lipid maturation. moiety of the molecule, synthetic 19-kDa lipopeptide and synthetic Mature DC are potent APCs, expressing high levels of cell sur- 47-kDa lipopeptide were compared with unlipidated peptides with face molecules involved in Ag presentation, costimulation, and the same amino acid sequences. DC matured for 2 days in the

FIGURE 3. Modulation of cell surface expression of various markers during lipopeptide-induced DC maturation. Three- day DC were cultured for an additional 24 h in the absence of stimuli, or with the li- ␮ popeptide Pam3CysSerLys4 (5 g/ml), a synthetic 19-kDa lipopeptide (5 ␮g/ml), or LPS (20 ng/ml). Cells were stained with PE- or FITC-conjugated Abs speciﬁc for the marker indicated (gray) or an isotype- matched control Ab (outline). The value indicated on the histogram is the MFI of the cells stained with the marker-speciﬁc Ab. The results shown were obtained from a single experiment with a single donor and are representative of 3–10 similar experiments that gave similar results. The Journal of Immunology 2447

TLR2 mediates lipopeptide-induced DC maturation TLR2 has previously been shown to mediate responses to lipopeptides in cells of the monocyte lineage (11, 13, 17, 18). Conse- quently, the role of TLR2 in mediating lipopeptide-induced DC maturation was tested. Immature DC were preincubated with anti- TLR2 Ab or an IgG1 isotype control Ab for 30 min before the addition of suboptimal concentrations of lipopeptide and subse- quent maturation. Preincubation of DC with anti-TLR2 before the addition of lipopeptide blocked the up-regulation of CD80 and

CD86 induced by the 19-kDa lipopeptide or PAM3CysSerLys4 by 90–100% (Fig. 6A), relative to untreated cells or cells treated with IgG1 alone. In some cultures, the IgG1 Ab was slightly stimulatory and enhanced expression of CD80 and CD86, as indicated by increases in MFI. Anti-TLR2 also reduced the absolute percentage of cells positive for CD80, resulting in ϳ7-fold fewer CD80-positive cells compared with cells cultured with lipopeptide alone (Fig. 6B). However, the relative percentage of CD86-positive cells did not change significantly (Fig. 6B), as nearly all immature and mature Downloaded from FIGURE 4. The lipid moiety of the lipopeptide is required for induction DC expressed CD86. Anti-TLR2 also blocked the decrease in en- of DC maturation. Three-day DC were cultured for an additional 2 days in the absence of stimuli, with a synthetic 19-kDa lipopeptide (10 ␮g/ml), the docytic activity observed in lipopeptide-matured DC (Fig. 6C). 47-kDa lipopeptide (10 ␮g/ml), or unlipidated control peptides (10 ␮g/ml). Additionally, IL-12 was not detected in culture supernatant fluids Cells were stained with PE- or FITC-conjugated Abs specific for the of cells incubated with the anti-TLR Ab (data not shown) as we marker indicated (gray) or an isotype-matched control Ab (outline). The have previously shown (11). In summary, anti-TLR2 blocked the value indicated on the histogram is the MFI of the cells stained with the lipopeptide-induced increase in CD80 MFI and the increase in the http://www.jimmunol.org/ marker-specific Ab. The results shown were obtained from a single exper- percentage of cells positive for CD80. Anti-TLR2 blocked in- iment with a single donor and are representative of two similar experiments creases in the CD86 MFI, but not the percentage of cells positive that gave similar results. for CD86. In whole, these results indicate that lipopeptide activation of immature DC via TLR2 leads to increases of cell surface markers associated with mature DC and decreases in endocytic presence of the lipidated 19-kDa or 47-kDa peptides showed in- function, suggesting that TLR2 regulates multiple factors associ- creased levels of CD83, CD80, CD86, and MHC-II expression, ated with lipopeptide-induced DC maturation. whereas unlipidated control peptides induced only small or no in- by guest on September 28, 2021 creases in levels of expression of these molecules relative to un- Lipopeptide-matured DC have increased stimulatory potential in treated cells (Fig. 4). Together, these results confirm that the lipid MLRs portion of the lipopeptide is required for the induction of DC We observed that lipopeptide-matured DC expressed increased maturation. levels of Ag-presenting and costimulatory molecules. To determine whether, as a result of these phenotypic changes, lipopeptide- matured DC had enhanced functional properties, we compared the capacity of immature and lipopeptide-matured DC to stimulate T

cells in an MLR. DC were treated with PAM3CysSerLys4 or 19- kDa lipopeptide for 2 days before coculture with T cells from an unrelated donor. Lipopeptide-matured DC were more effective at stimulating a MLR, as observed by a 1.5- to 3-fold increase in T cell-proliferative responses compared with untreated DC (Fig. 7A). Again, treatment of DC with an unlipidated peptide did not result in enhanced T cell proliferation. We also measured the ability of these DC to stimulate a MLR by measuring the production of IFN-␥ by T cells. Lipopeptide-matured DC stimulated 3- to 11- fold greater levels of IFN-␥ production relative to control cultures (Fig. 7B). Together, these data demonstrate that lipopeptide-matured DC have greater T cell-stimulatory activity than immature DC. The addition of anti-TLR2 Ab before the maturation of DC with lipopeptides abrogated the ability of the DC to stimulate enhanced T cell proliferation and IFN-␥ production in a MLR (Fig. FIGURE 5. Maturation of DC with lipopeptides down-regulates endo- 7, C and D). In summary, these results demonstrate that lipopep- cytic activity. Three-day DC cultured for an additional 2 days with the tide-matured DC have enhanced T cell-stimulatory activity, and stimuli indicated were analyzed for endocytic activity by uptake of F-Dx. induction of this activity is dependent upon TLR2. Cells were incubated with F-Dx (1 mg/ml) for1hat37°C (gray) or at 4°C (outline) and were analyzed by flow cytometry to measure specific uptake and nonspecific binding, respectively. The value indicated on the histogram Discussion is the MFI of the cells cultured at 37°C. The results shown were obtained DC play an integral role in host defense in that they are the only from a single experiment with a single donor and represent three similar APC capable of activating naive lymphocytes, resulting in the ini- experiments that gave similar results. tiation of protective immune responses. Critical to this function is 2448 MATURATION OF DENDRITIC CELLS BY MICROBIAL LIPOPEPTIDES

biological activity (11, 13, 14, 17, 19). Functionally, lipopeptide- matured DC have enhanced T cell-stimulatory activity in MLRs. Most importantly, we demonstrate for the ﬁrst time that activation via human TLR2 mediates both phenotypic and functional maturation events, hereby providing a mechanism by which DC maturation is induced by microbial products. During the immune response to infectious agents, DC concentrate microbial ligands and Ags and mature into highly effective APCs. Previous studies have demonstrated that stimulation with LPS or live bacteria induces DC maturation. In these studies, culture of immature DC with LPS from Escherichia coli (2) induced increases in a number of cell surface markers, including MHC-II, CD80, CD40, CD54, and CD58, whereas expression of CD14, CD32, and endocytic activity was reduced. Additionally, infection of human DC with live bacillus Calmette-Gue´rin (20), M. tuberculosis (21), Listeria monocytogenes (9), Streptococcus gordonii (22), or Leishmania major (10) results in an increase in MHC and costimulatory molecule expression and enhanced T cell-stimula-

tory activity (20). Similar results were obtained with murine DC; Downloaded from these cells also demonstrated an enhanced ability to induce T cell responses in mice (23). In our studies, we found that the 19-kDa lipoprotein from M. tuberculosis, as well as synthetic lipopeptides, induced DC maturation. The resulting mature DC had increased cell surface expression of MHC-II, CD80, CD83, CD86, CD54,

and CD58, suggesting that the lipopeptide alone is sufficient to http://www.jimmunol.org/ induce maturation events. Lipopeptide-matured DC were also more potent than immature DC in stimulating T cells in a MLR. Together, these findings provide a mechanism by which cells of the innate immune system can recognize and be activated by microbial products, leading to the initiation of an adaptive immune response. Many studies with LPS-matured DC give an indication of the relevance of such maturation events as we have described herein. Recent work has revealed that LPS treatment of DC enhances pep- by guest on September 28, 2021 tide-MHC-II complex formation and its trafficking to the cell surface (24). This trafficking also results in the clustering of peptide- MHC-II/costimulatory molecules on the surface of DC, which enhances the T cell-stimulatory capacity of these cells. Further- FIGURE 6. Lipopeptide-induced maturation of DC can be blocked with more, surface MHC-II molecules on LPS-matured DC have a anti-TLR2 Ab. Three-day DC were left untreated (open columns), prein- longer half-life than that of immature DC, and mature DC maintain cubated with anti-TLR2 (filled columns) or an IgG1 isotype control Ab their in vitro T cell-stimulatory capacity for several days longer in (hatched columns) for 30 min before the addition of the 19-kDa lipopeptide culture compared with immature cells (25). Because of the func- ␮ ␮ (5 g/ml) or the lipopeptide Pam3CysSerLys4 (0.3125 g/ml). After two tional similarity of LPS and lipopeptides, we predict that lipopep- additional days in culture, cells were stained with PE-conjugated Abs spe- tides will have a similar effect on peptide loading and MHC-II cific for the marker indicated and analyzed by flow cytometry. The MFI (A) stability, although this remains to be determined. and percentage of cells staining positive (B, % Positive) for each marker Before this work, the mechanism by which microbe-induced DC were determined from histogram analysis. Results in A and B were obtained from a single donor in a single experiment and are representative of maturation occurs had not been identified. However, the recent four similar experiments with three different donors. C, Histograms show- identification of TLRs as CD14-associated signaling molecules has ing F-Dx uptake by immature DC, or DC matured with 19-kDa lipopeptide shed new light on the mechanisms by which cells of the immune (5 ␮g/ml) following pretreatment with an IgG1 isotype control or anti- system respond to microbial products. TLRs are a family of trans- TLR2 blocking Ab. The value indicated on the histogram is the MFI of the membrane proteins that are evolutionarily conserved in species cells cultured at 37°C and represents two similar experiments. ranging from insects to mammals (26). In Drosophila, Toll is involved in dorsal-ventral patterning, as well as induction of innate immune responses to microbial pathogens (27). Humans have at a program of maturation induced by microbial products that en- least 10 different TLRs that are expressed primarily on cells of hances the Ag-presenting and costimulatory functions of DC. We myeloid origin, but TLRs have also been found on epithelial cells have demonstrated that culture of immature DC with lipopeptides (26). Of these receptors, TLR2 and TLR4 have been shown to increases cell surface expression of MHC-II, CD83, CD80, CD86, mediate cellular responses to LPS from Gram-negative bacteria CD54, and CD58, while decreasing endocytic activity and expres- (16, 28), lipopeptides from mycobacteria (13), as well as pepti- sion of CD32, resulting in cells with a phenotype characterized by doglycans and lipoteichoic acids from Gram-positive bacteria (29). the efficient Ag-presenting and costimulatory capacity of mature The result of such activation includes induction of the NF-␬B sig- DC. The lipid moiety of synthetic lipopeptides was found to be naling pathway and the production of cytokines (13, 16, 30, 31). essential for the maturation process, consistent with previous stud- Here we have described a role for TLR2 in mediating DC mat- ies that have demonstrated the necessity of the lipid moiety for uration, thereby providing a mechanism by which lipopeptides act The Journal of Immunology 2449 Downloaded from http://www.jimmunol.org/

FIGURE 7. Lipopeptide-matured DC have enhanced T cell-stimulatory activity in a MLR, a function that can be blocked with anti-TLR2. Three-day DC were further matured for 2 days in the presence or absence of lipopeptides and used as stimulating cells in a MLR. Puriﬁed T cells were added to the

DC at the indicated DC:T cell ratio (A) or a 1:20 DC:T cell ratio only (B–D) and incubated for an additional 5 days before the addition of T cells. by guest on September 28, 2021 ␥ ␮ Proliferation of T cells (A) and IFN- in culture supernatant ﬂuids (B) in a MLR using DC matured with 10 g/ml Pam3CysSerLys4, 19-kDa lipopeptide, or 19-kDa control peptide. Data shown in A and B represent one of three experiments that gave similar results. Error bars represent the SD of triplicate determinations. C and D, Three-day DC were left untreated (e), or preincubated with anti-TLR2 (f) or an IgG1 isotype control Ab (o) for 30 min before the addition of suboptimal concentrations of the 19-kDa lipopeptide or Pam3CysSerLys4. DC were matured for an additional 2 days with lipopeptide before the addition of T cells. C, Proliferation of T cells in MLR as assessed by [3H]thymidine uptake (cpm). Background proliferation (cpm) of wells containing T cells alone was subtracted from cpm obtained in wells containing DC plus T cells. The concentrations of 19-kDa lipopeptide (5 ␮g/ml) and ␮ ␥ Pam3CysSerLys4 (0.5 g/ml) were optimal for demonstrating a block in proliferative responses. D, IFN- in culture supernatant ﬂuids from a MLR; 19 ␮ ␮ ␥ kDa lipopeptide (2 g/ml) and Pam3CysSerLys4 (0.2 g/ml) were used at optimal concentrations for demonstrating a block in IFN- production. The results shown in C and D were obtained from one experiment, but were consistent with two different stimuli and for two different readouts. Error bars represent SE (C)orSD(D) of triplicate determinations.

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