Cell Differentiation and Function Enhances Dendritic Cell Maturation ...

The Journal of Immunology

Cytoplasmic Entry of Listeria monocytogenes Enhances Dendritic Cell Maturation and T Cell Differentiation and Function Kristina L. Brzoza, Andrea B. Rockel, and Elizabeth M. Hiltbold1 Protective immunity to the intracellular bacterial pathogen, Listeria monocytogenes, is mediated by a vigorous T cell response. In particular, CD8ⴙ cytolytic T cells provide essential effector function in the clearance of bacterial infection. The cytoplasmic entry of Listeria facilitated by listeriolysin O is an essential feature not only of the bacteria’s virulence, but of the ability of the bacteria to elicit protective immunity in the host. To determine how cytoplasmic entry of Listeria regulates the development of protective immunity, we examined the effects of this process on the maturation of murine dendritic cells (DC) and on their ability to prime naive CD8ⴙ T cell responses. Costimulatory molecules (CD40, CD80, and CD86) were induced by listerial infection only when the bacteria invaded the cytoplasm. In addition, the production of IL-12, IL-10, IL-6, and TNF-␣ was most efficiently triggered by cytosolic Listeria. Naive T cells primed by peptide-loaded DC infected with either wild-type or nonhemolytic mutant Listeria proliferated equivalently, but a much larger proportion of those primed by wild-type Listeria monocytogenes produced IFN-␥. Costimulatory molecules induced by cytosolic entry regulated T cell proliferation and, as a result, the number of functional T cells generated. DC-produced cytokines (specifically IL-12 and IL-10) were the major factors determining the proportion of T cells producing IFN-␥. These data highlight the requirement for listerial cytoplasmic invasion for the optimal priming of T cell cytokine production and attest to the importance of this event to the development of protective CTL responses to this pathogen. The Journal of Immunology, 2004, 173: 2641–2651.

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rotective immunity to intracellular bacterial pathogens such as Listeria monocytogenes requires a robust adaptive immune response. The CD8⫹ T cell response to Listeria provides essential effector function in bacterial clearance (1, 2). Initiation of such CTL responses requires that naive CD8⫹ T cells be stimulated by APCs (most typically dendritic cells (DC))2 that express not only the appropriate MHC/peptide complexes, but also additional molecules such as costimulatory molecules (CD80, CD86, CD40) and cytokines (such as IL-12) that provide the necessary second and third signals (3–5). To become competent for activation of naive T cells, APCs must undergo a maturation process in which costimulatory molecules and cytokines are up-regulated, coupled with a reduction of endocytic activity. Maturation can be induced by a number of stimuli including ligation of CD40 (6), triggering of TLR by bacterial modulins such as LPS (7, 8), or inflammatory cytokines such as TNF-␣ or type I IFN (9, 10). In the absence of proper activation, presentation of Ag to T cells by immature DC results in the induction of a nonfunctional state known as anergy (reviewed in Ref. 11). Thus, the ability of DC to stimulate T cell proliferation and

Department of Microbiology and Immunology, School of Medicine, Wake Forest University, Winston-Salem, NC 27157 Received for publication February 12, 2004. Accepted for publication June 10, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Elizabeth M. Hiltbold, Department of Microbiology and Immunology, School of Medicine, Wake Forest University, 5112 Gray Building Medical Center Boulevard, Winston-Salem, NC 27157. E-mail address: [email protected] 2 Abbreviations used in this paper: DC, dendritic cell; BHI, brain heart infusion; DAPI, 4⬘,6⬘-diamidino-2-phenylindole; Hly, hemolysin; LLO, listeriolysin O; MOI, multiplicity of infection.

Copyright © 2004 by The American Association of Immunologists, Inc.

function depends on the type and strength of maturation stimulus that they encounter. L. monocytogenes is an intracellular bacterial pathogen that can cause serious infections in immunocompromised individuals and pregnant women. Listeria has an intracellular life cycle that involves its escape from the phagosome and entry into the cytoplasm of the host phagocyte (12). The bacteria can survive and multiply within the cytosol and spread from cell to cell by polymerizing the host cell actin into comet-like tails (13–15). Cytoplasmic invasion is mediated by the activity of the hemolytic toxin, listeriolysin O (LLO). Listeria express an array of virulence factors that enable the bacteria to survive and spread (16). However, LLO, the product of the hemolysin (hly) gene, is a pivotal virulence factor for not only the survival of the bacteria, but also for the development of protective immunity (17–21). Noninvasive (hly⫺) mutants of Listeria, which are unable to enter the cytoplasm, are killed by host phagocytes (19). Likewise, immunization with killed Listeria provides no substantial level of protective immunity (22–24). Interestingly, a recent study demonstrated that mice immunized with a high dose of killed Listeria, while not protected from virulent challenge, developed a substantial population of CD8⫹, Listeria-specific T cells (24). These cells displayed a central memory surface phenotype, but were nonfunctional as measured by cytolytic activity, IFN-␥ production, or ability to clear bacterial challenge. One potential explanation for the differential T cell responses induced by live vs killed or noninvasive Listeria is that DC infected with Listeria may prime T cell proliferation and/or function in a cytoplasmic invasion-dependent manner. Thus, to determine how cytoplasmic entry of Listeria affects DC function, we investigated the effect of listerial cytoplasmic invasion on DC maturation and on the ability of Listeria-infected DC to prime CD8⫹ T cell responses. To examine specifically the costimulatory function of DC and minimize the contribution of Ag 0022-1767/04/$02.00

2642 dose as a variable, we loaded the DC with defined concentrations of exogenous OVA peptide Ag. To evaluate the ability of Listeriainfected DC to prime naive T cells, we used an OVA-specific TCR transgenic model system. Immature, bone marrow-derived DC were infected with Listeria and concomitantly loaded with the OVA peptide recognized by the T cells. T cell proliferation and function were then monitored after 3 days of culture. We found that DC infected with wild-type (Hly⫹) Listeria underwent maturation as measured by vigorous expression of costimulatory molecules and production of cytokines. These DC also activated T cells to proliferate robustly and gain effector function in the form of IFN-␥ production. In contrast, T cells primed by DC infected with nonhemolytic Listeria (Hly⫺) proliferated equivalently, but developed only partial effector function. The ability of DC to prime T cell proliferation and function was attributable not to differential expression of costimulatory molecules, but to the balance of IL-12 and IL-10 induced by infection. These data demonstrate that maturation of DC through cytoplasmic entry of Listeria is a pivotal event in the initiation of CD8⫹ T cell responses to this pathogen.

Materials and Methods Mice C57BL/6 and OT-1 TCR transgenic mice specific for OVA (257–264) presented by Kb were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were maintained and bred in the animal facility at Wake Forest University School of Medicine. Bone marrow from CD40⫺/⫺ (25) and B7.1-B7.2⫺/⫺ (26) mice was a generous gift from R. Hodes (National Institutes of Health, National Institute on Aging, Bethesda, MD).

Reagents Cytokines. Mouse rGM-CSF was purchased from BioSource International (Camarillo, CA). Peptide. Synthetic OVA peptide 257–264 (SIINFEKL) was purchased from the Emory University School of Medicine Peptide Synthesis Facility (Atlanta, GA). Abs. Neutralizing Abs to murine IL-12, IL-10, IL-6, and TNF-␣ were purchased from BioSource International and were used at a final concentration of 10 ␮g/ml. Fluorescently labeled Abs against mouse cell surface Ags CD40 PE (clone 3/23), CD80 PE (clone 16-10A1), CD86 PE (clone GL1), I-Ab PE (clone AF6-120.1), and CD8 PE (clone Ly-2 53-6.7) were purchased from BD Pharmingen (San Diego, CA). Fluorescently tagged anti-IFN-␥ APC used for intracellular cytokine staining was also purchased from BD Pharmingen. The hybridoma producing the mAb, 25.D1, specific for OVA257–264 ⫹ Kb was the generous gift of R. Germain (National Institutes of Health, National Institute of Allergy and Infectious Diseases) (27). Culture supernatants from this hybridoma were used undiluted to stain DC loaded with OVA peptide. Anti-mouse Alexa 488 Ab (Molecular Probes, Eugene, OR) was used to detect 25.D1 staining. CFSE, Alexafluor488-labeled phalloidin, and 4⬘,6⬘-diamidino-2-phenylindole (DAPI) were purchased from Molecular Probes. Growth and preparation of Listeria (strain 10403S wild type, Hly⫹; or LLO deletion mutant, Hly⫺, strain DPL2161) were cultured to log-phase growth in brain heart infusion (BHI) broth overnight with vigorous shaking at 37°C. Bacteria were washed three times in ice-cold PBS by centrifugation (3000 rpm, 4°C). Bacteria were resuspended in PBS and quantitated by OD at 570 nm using a Genesys 10 UV (Thermo Spectronic, Rochester, NY) spectrophotometer. Bacterial numbers were confirmed subsequently by colony counts on BHI agar. Bacteria were diluted into antibiotic-free RPMI 1640 medium for infection of DC.

DC propagation Bone marrow-derived DC were generated, as previously described (28). Briefly, bone marrow was removed from the tibias and femurs of 8- to 10-wk-old C57BL/6 (or indicated) mice. Following red cell lysis and washing, progenitor cells (106/ml) were resuspended and plated in RPMI 1640 containing 10% FCS supplemented with 20 ng/ml GM-CSF (without antibiotics). DC were cultured for 6 days at 37°C in 5% C02 and were gently washed and fed with fresh medium and cytokine on days 2 and 4. On day 6, the DC were routinely 95% CD11c⫹ and displayed low levels of CD40,

CYTOPLASMIC ENTRY OF Listeria MATURES DC CD80, CD86, and MHC class II, characteristic of immature DC (data not shown).

Listerial infection of DC DC, cultured as above, were harvested and plated in 48-well plates at a density of 5 ⫻ 105/well (or on 12-mm coverslips for microscopic examination) and infected with either wild-type (Hly⫹) Listeria, strain 10403S, or an LLO deletion mutant (Hly⫺), strain DPL2161 (gifts of D. Portnoy, University of California, Berkeley, CA) at a multiplicity of infection (MOI) of 1 for 4 h in antibiotic-free medium. This infection period was designed to allow for cytoplasmic entry of the bacteria and to minimize differential bacterial growth. Infection was enhanced by a brief spin (5 min) of the bacteria onto the DC at 2000 rpm. The infection was then terminated with antibiotics (chloramphenicol, 10 ␮g/ml; Fisher Scientific, Fair Lawn, NJ), and the cells were cultured for an additional 18 h. To monitor the number of bacteria present in the cells and their entry into the cytoplasm, the cells on coverslips were fixed after infection with paraformaldehyde (2%) and stained with fluorescent phalloidin to identify polymerizing actin (an indicator of bacterial cytoplasmic entry) and with DAPI to stain host and bacterial DNA (to enumerate bacteria).

Measurement of costimulatory molecule and cytokine expression by DC following listerial infection DC were plated in 48-well plates at a density of 5 ⫻ 105 DC/ml in antibiotic-free medium. The cells were then infected with the indicated MOI for 4 h. Infection was enhanced by a brief spin (5 min) of the bacteria onto the DC at 2000 rpm. The infection was then terminated with antibiotics and the cells were cultured for an additional 18 h to elaborate cytokines and to up-regulate costimulatory molecules. Culture supernatants were then removed, and the presence of cytokines (IL-6, IL-10, IL-12, or TNF-␣) was measured by ELISA (OptiEIA kits; BD Pharmingen). The cells were then harvested and stained with PE-conjugated Abs to murine CD40, CD80, CD86, or I-Ab. Nonspecific background staining was assessed using isotype control Abs (rat IgG PE for CD40, CD80, and CD86, and mouse IgG PE for I-Ab).

T cell priming assay DC (105/ml) were plated in 48-well plates and treated with LPS (100 ng/ ml), left untreated, or infected with Hly⫹ (strain 10403S) or Hly⫺ (strain DPL2161) Listeria (MOI ⫽ 1). Infection was enhanced by a brief spin (5 min) of the bacteria onto the DC at 2000 rpm. The infection was terminated after 4 h by the addition of chloramphenicol (10 ␮g/ml). To examine specifically the effects of listerial infection on the costimulatory activities of DC while minimizing the contribution of Ag dose as a variable, DC were loaded with defined doses (1– 0.01 ng/ml) of preprocessed OVA (257–264) peptide. These low Ag concentrations were chosen to reveal differences in costimulatory efficiencies of DC. The cells were cultured for additional 18 h before the addition of T cells. Naive, OVA-specific CD8⫹ T cells were isolated from the spleens of OT-1 TCR transgenic mice by negative selection to remove MHC class II⫹ cells. T cells were stained with CFSE, according to manufacturer’s instructions, and were added to the DC at a ratio of 10 T cells per DC. T cells were harvested 72 h later, and the production of IFN-␥ in response to Ag was then measured by incubation with OVA (257–264) peptide (1 ␮g/ml) in the presence of Golgi Plug reagent for 5 h. The cells were then stained, fixed, and permeabilized for intracellular cytokine staining using the Cytofix/ Cytoperm kit from BD Pharmingen, according to manufacturer’s instructions. Flow cytometric data were acquired using a BD FACSCalibur (BD Biosciences, San Diego, CA). CFSE proliferation data were analyzed, as previously described (29), using the FLOJO program for flow cytometric data analysis (Tree Star, Ashland, OR). The division index represents the mean number of divisions that a cell present in the starting population undergoes. The proliferation index is the mean number of divisions of cells in cycle. Statistical significance was determined using a two-tailed, paired Student’s t test. Values of p ⬍ 0.05 were considered significant differences.

Cytokine neutralization To neutralize the activity of specific cytokines (IL-6, IL-10, IL-12, or TNF-␣) in the priming cultures, neutralizing Abs (10 ␮g/ml; BioSource International) were added to the cultures at the time of infection and were maintained throughout the T cell priming assay.


Results The level of costimulatory molecule expression by Listeriainfected DC is augmented by cytoplasmic entry of the bacteria To address the effect of cytoplasmic entry of Listeria on the expression of costimulatory molecules by DC, the expression of CD80, CD86, CD40, and MHC class II was measured by flow cytometry following infection (Fig. 1). DC generated from the bone marrow of C57BL/6 mice were infected with Hly⫹ or Hly⫺ Listeria, as indicated (MOI designated by arrows on histograms), and the infection was terminated after 4 h. Eighteen hours later, the cells were stained and analyzed by flow cytometry. DC infected with increasing numbers of nonhemolytic (Hly⫺) Listeria displayed no increased expression of CD86 over untreated DC (Fig. 1A). In contrast, DC infected with wild-type (Hly⫹) Listeria displayed a dose-dependent increase in the expression of CD86, as depicted by the histograms in Fig. 1B. We also observed bacterial dose-dependent increases in the expression of CD40, CD80, and I-Ab (indicated by increases in geometric mean fluorescence; Fig. 1, C–F) by DC infected with increasing numbers of Hly⫹ Listeria. In contrast, we observed almost no change in the level of expression of CD80, CD86, or CD40, and only a slight increase in MHC class II expression induced by infection with nonhemolytic (Hly⫺) mutant Listeria. These findings confirm and extend the results of Shedlock et al. (30) by demonstrating the role of cytoplasmic entry of the bacteria in eliciting DC maturation. These results also suggest that the cytoplasmic entry-dependent induction of costimulatory molecule expression may mediate the development of protective immunity to this pathogen. To determine whether Hly⫺ Listeria were capable of inducing DC maturation at higher MOI, we infected DC with 10 or 100 Hly⫺ L. monocytogenes per cell. Under these conditions, we observed substantially higher costimulatory molecule expression by DC, comparable to that induced by Hly⫹ Listeria at an MOI of 1 (data not shown). However, under these conditions, we also observed breakthrough of the Hly⫺ bacteria into the cytosol, detected by actin tail formation. This LLO-independent cytoplasmic inva-

2643 sion was most likely facilitated by the phospholipases expressed by the bacteria, which are sufficient to disrupt the phagosome at high MOI. Although we were unable to compare phagosomal vs cytosolic bacteria under these conditions, these findings support the hypothesis that cytoplasmic entry of Listeria, regardless of the mechanism, enhances DC maturation. Heat-killed Listeria has also been reported to stimulate DC maturation (30). Therefore, we wanted to determine the efficiency of DC maturation induced by killed vs live bacteria. We found that DC treated with heat-killed Listeria demonstrated augmented expression of CD40, CD80, and CD86, but only upon addition of 100 killed bacteria per cell (data not shown). Thus, DC maturation is induced over 100 times more efficiently by live, Hly⫹ Listeria than by heat-killed Listeria. Cytokine production by DC is preferentially induced by listerial cytoplasmic entry To address the role of cytoplasmic entry of Listeria on the production of cytokines by infected DC, the secretion of IL-6, IL-10, IL-12, and TNF-␣ was measured following infection with Hly⫹ or Hly⫺ L. monocytogenes. DC were infected with the indicated listerial strains (MOI ⫽ 0.01–1) for 4 h, or were treated with LPS (10 ng/ml-1 ␮g/ml) or left untreated, antibiotics were added, and the cells were incubated additional 18 h. Supernatants were then tested for the presence of cytokines by ELISA. All of the cytokines measured were induced very efficiently by infection with Hly⫹ Listeria (Fig. 2). In contrast, the only cytokines induced by infection with Hly⫺ L. monocytogenes were IL-12 and IL-6, and these were induced only at the highest MOI, 1. In fact, no detectable cytokines were produced by DC infected with Hly⫺ Listeria below an MOI of 1. Yet, IL-12 (p40) and IL-6 production by Hly⫹ L. monocytogenes-infected DC was evident even at very low MOI (0.01 and 0.1) (Fig. 2, A and D). Thus, the efficiency of IL-12 and IL-6 production was enhanced nearly 100fold by cytoplasmic entry of the bacteria. IL-10 is known to be induced in vivo by listerial infection (31), although its production by DC has not been characterized. We

FIGURE 1. Costimulatory molecule expression by DC following listerial infection. A, Representative CD86 expression by untreated DC and by DC infected with increasing numbers of Hly⫺ L. monocytogenes. Bacterial MOI indicated by arrows. B, Representative CD86 expression by cells infected with increasing numbers of Hly⫹ Listeria. MOI indicated by arrows. C–F, Expression of costimulatory molecules or MHC class II indicated by geometric mean fluorescence intensity. Geometric mean fluorescence of the total live population was determined using CellQuest flow cytometry data analysis program. Data shown are representative of seven independent experiments. C, CD80; D, CD86; E, CD40; and F, I-Ab.

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FIGURE 2. Cytokine production by DC is enhanced by cytoplasmic entry of Listeria. Cytokines IL-12 (A), IL-10 (B), TNF-␣ (C), or IL-6 (D) were detected in the culture supernatants by ELISA following infection with the indicated listerial strain (Hly⫹ or Hly⫺) or treatment with LPS. Data are mean with SD of three independent experiments.

found that IL-10 was produced by DC infected with wild-type Listeria, although a relatively high MOI was required to induce this cytokine (Fig. 2B). Although we could detect no IL-10 produced by DC infected with Hly⫺ L. monocytogenes at or below an MOI of 1, IL-10 production was induced by infection with larger (MOI ⫽ 10 or 100) numbers of Hly⫺ Listeria (data not shown). Therefore, Listeria that enter the cytosol stimulate DC to produce more IL-10 compared with Listeria which are retained in the phagosome. Similarly, TNF-␣ was induced efficiently by infection with wild-type L. monocytogenes at an MOI of 0.1 (Fig. 2C). In contrast, nonhemolytic L. monocytogenes infection failed to induce TNF-␣ production at any MOI tested. Thus, TNF-␣ production is induced only by Listeria that enter the cytoplasm. These data support the hypothesis that Listeria that penetrate the DC cytosol induce inflammatory cytokine production more efficiently than Listeria that are retained within the phagosome. DC contain comparable numbers of bacteria One potential explanation for the differences in DC maturation induced by Hly⫹ vs Hly⫺ L. monocytogenes is that the DC contain significantly different numbers of bacteria, biasing the results toward the more abundant strain. Therefore, to monitor the number of bacteria present in the cells and their entry into the cytoplasm, the DC were plated on coverslips, fixed after infection, and stained with fluorescent phalloidin to identify polymerizing actin (an indicator of bacterial cytoplasmic entry) and with DAPI to stain host and bacterial DNA (to enumerate bacteria; Fig. 3). Following 4 h of infection with Hly⫹ L. monocytogenes at an MOI of 1, 77% of the DC were infected and contained an average of 5 ⫾ 4 bacteria detectable by DAPI staining (representative image, Fig. 3, top panels). Of 50 microscopic fields examined, approximately one-third of the DAPI-detectable bacteria displayed actin nucleation, indicating cytoplasmic entry. The 3 cells depicted in the upper panels of Fig. 3 contain a total of 20 DAPI-detectable bacteria, with 14 displaying actin nucleation. Those indicated with open arrows have visible actin nucleation, and those highlighted with gray arrows have no detectable actin nucleation.

CYTOPLASMIC ENTRY OF Listeria MATURES DC

FIGURE 3. Numbers of Hly⫹ vs Hly⫺ Listeria in DC following 4-h infection. DC were infected with Listeria (strain indicated to left of picture) at an MOI of 1. DC were then fixed and stained with phalloidin (left panels) to detect actin nucleation around cytosolic bacteria and DAPI (center panels) to enumerate bacteria. Open arrows indicate bacteria detectable by both DAPI and actin nucleation. Gray arrows indicate bacteria detectable only by DAPI staining.

Similarly, 70% of the cells receiving Hly⫺ L. monocytogenes became infected. The infected cells harbored an average of 3 bacteria (⫾2) detectable by DAPI staining at the end of the 4-h infection period (Fig. 3, bottom panels). As expected, we did not detect actin nucleation in DC infected with Hly⫺ Listeria under these conditions. The two DC depicted in the bottom panels of Fig. 3 contain a total of 11 DAPI-detectable bacteria with no detectable actin nucleation (highlighted by gray arrows). To determine the number of viable bacteria in the cells, the DC were lysed and plated on BHI agar plates. We found that at 4 h postinfection, there was ⬃2-fold more Hly⫹ than Hly⫺ L. monocytogenes in the cells (data not shown). Thus, DC infected with Hly⫹ Listeria contained only 2-fold higher numbers of bacteria on average than Hly⫺ L. monocytogenes-infected cells, and cytoplasmic entry occurred in these cells in an LLO-dependent manner. Yet, as depicted in Fig. 2, the induction of cytokines by DC infected with Hly⫺ L. monocytogenes required 100 times more bacteria than Hly⫹. Thus, the enhanced DC maturation induced upon cytoplasmic entry of the bacteria cannot be accounted for by differences in bacterial numbers. Cytoplasmic entry of Listeria in DC has a minimal effect on T cell proliferation Two essential features of the protective CTL response to Listeria are clonal expansion (proliferation) and acquisition of effector function (IFN-␥ production and cytolytic activity). To identify the cellular and molecular mechanisms through which cytoplasmic entry of Listeria stimulates the development of protective immunity in vivo, we performed a series of in vitro T cell priming assays. For these analyses, T cell priming was measured by proliferation and IFN-␥ production. The histograms in Fig. 4A illustrate the proliferative profiles (CFSE staining) of OT-1 T cells cultured with DC loaded with 1– 0.01 ng/ml OVA (257–264) peptide. The cells shown were gated on viable, CD8⫹ cells. As the Ag dose was decreased, we observed incremental decreases in the percentage of cells recruited into cell cycle in both Hly⫹ and Hly⫺ L. monocytogenes-infected DC-primed populations. This is indicated by the percentage of cells in the original population dividing (corrected for division, as


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FIGURE 4. T cell proliferation primed by Listeria-infected DC is only minimally affected by the entry of Listeria into DC cytoplasm. DC were infected with Listeria (Hly⫹ or Hly⫺, MOI ⫽ 1) and loaded with OVA peptide. OT-1 T cells were stained with CFSE and added to the DC cultures. Seventy-two hours later, the T cells were harvested, and their proliferation was quantitated by flow cytometry. A, Histograms of CFSE proliferation. Peptide Ag dose is indicated across top of plots, and bacterial strain with which DC were infected is indicated to left of plots. The percentage of cells in each population that divided is indicated in the upper left of each panel. B, Division indices (mean number of divisions of entire population) calculated from CFSE profiles of four independent experiments, as described in Materials and Methods. C, Total viable cells recovered from priming cultures after 3 days. D, Density of OVA (257–264)-Kb complexes by DC infected or treated, as indicated. DC were treated or infected and pulsed with OVA peptide (10 ␮g/ml) for 18 h. OVA-Kb complexes were detected using the 25.D1 mAb. Inset numbers, Indicate mean fluorescence intensity of 25.D1 staining. Data shown are averaged from four independent experiments.

described (29)) shown in the upper left of each histogram. However, at each Ag dose, Hly⫺ L. monocytogenes-infected DC induced a slightly lower percentage of cells to proliferate than Hly⫹ L. monocytogenes-infected DC. The division index, which represents the mean number of divisions of the entire population, provides a quantitative indicator of the overall proliferative outcome of T cell priming (Fig. 4B). At the highest Ag dose, 1 ng/ml, we observed a somewhat weaker response in T cells primed by DC infected with Hly⫺ Listeria vs Hly⫹ L. monocytogenes-infected or LPS-treated DC. The division index of these Hly⫺ L. monocytogenes-primed T cells was significantly lower than T cells primed by Hly⫹ L. monocytogenes-infected DC ( p ⬍ 0.05), but not untreated (NT) DC. This difference was attributable to the percentage of cells entering division, not to the average number of divisions that dividing cells underwent, which was not significantly different for any treatment (data not shown). At the intermediate and low Ag doses, 0.1 and 0.01 ng/ml, we detected fewer cells entering division in all populations. Although there was a minor difference between T cells primed by Hly⫹ vs Hly⫺ L. monocytogenes-infected DC in the percentage of cells dividing, there was no significant difference in the division index. These data are supported by the total viable cell numbers recovered from the cultures on day 3 (Fig. 4C). The only significant difference observed in T cell numbers primed by Hly⫹ vs Hly⫺ Listeria was at the highest Ag dose. Therefore, cytoplasmic entry of Listeria does not significantly augment T cell proliferation under conditions of limiting Ag concentration.

To rule out differences in Ag dose as a potential variable between the differently treated DC populations, we wanted to determine the density of Ag presented by each DC population. To quantitate the density of Ag present on the DC, we measured the relative amount of MHC-peptide complexes present on the DC after treatment or infection using the 25.D1 mAb specific for the OVA257–264-Kb complex (a gift from R. Germain, National Institutes of Health). The open histograms shown in Fig. 4D indicate the level of OVA-Kb expression detected on each DC population loaded with 10 ␮g/ml peptide. The level of staining observed on DC loaded with no peptide is indicated by the filled histograms. The mean fluorescence intensity of each population is shown in the upper left corner of each panel. The very low doses of Ag detected by the T cells (i.e., 1 ng/ml and less) were not detectable (above background) by the Ab in this assay due to its lower degree of sensitivity (data not shown). However, it is likely that any differences in the expression of the OVA-Kb complex detected at this higher peptide dose (and the other doses measured, 1 and 50 ␮g/ml; data not shown) would be proportional to changes in the presentation of this complex at lower Ag doses. The results of this analysis indicate that there is very little difference in the mean fluorescence intensity of OVA-Kb staining on DC under the conditions tested. CTL production of IFN-␥ is enhanced by cytoplasmic entry of Listeria in DC To assess T cell function, we measured the Ag-dependent production of IFN-␥ by intracellular cytokine staining. First, we wanted to confirm the naive phenotype of the OT-1 transgenic T cells.

2646 Because the level of CD44 expression is indicative of the naive (CD44low) or activated (CD44high) phenotype, we determined the level of CD44 staining on the OT-1 T cells vs an age-matched nontransgenic C57BL/6 control (Fig. 5A). Splenocytes were isolated from these mice and stained with a fluorescently labeled Kb-OVA-specific tetramer (a generous gift from J. Grayson, Wake Forest University School of Medicine, Winston-Salem, NC), as well as Abs to CD8 and CD44. The dot plots in Fig. 5A depict the level of tetramer staining and CD44 expression on live, CD8⫹ T cells. These data demonstrate that 97.2% of the CD8⫹ T cells expressed the OVA-specific TCR, and that 93% of the total CD8⫹ T cells (corresponding to 96% of the tetramer⫹ cells) expressed a low level of CD44 staining and, thus, naive phenotype. In contrast, 21% of the CD8⫹ T cells from the control animal were CD44 high, indicating activation. Additionally, we detected only a small background staining (0.4%) with the tetramer in these nontransgenic animals, confirming the specificity of this reagent.

CYTOPLASMIC ENTRY OF Listeria MATURES DC The dot plots in Fig. 5B were gated on CD8⫹ cells and depict the production of IFN-␥ as a function of proliferation. CD8⫹ T cells were primed by DC infected with Hly⫹ or Hly⫺ Listeria and pulsed with the indicated dose of OVA peptide. In the absence of peptide in the priming culture (Fig. 5B, upper right corner), the T cells remained CFSE high, indicating that they had not divided, and a small nonspecific level of IFN-␥ production was detected in 0.3% of the cells. T cells cultured with DC loaded with the high peptide concentration (1 ng/ml) and infected with Hly⫹ L. monocytogenes induced ⬃85% of the T cells to produce IFN-␥, while DC infected with Hly⫺ L. monocytogenes induced only 55% of the T cells to produce cytokine (Fig. 5, B and C). We also detected differences in the amount of cytokine produced by each cell (indicated by the arrows in the upper left, denoting mean fluorescence intensity of cytokine-positive cells) along the y-axis. In fact, cytoplasmic entry of Listeria increased the amount of IFN-␥ produced by each cell

FIGURE 5. T cell IFN-␥ production primed by Listeria-infected DC is augmented by cytoplasmic entry of Listeria. A, Expression of activation marker, CD44, by OT-1 T cells vs age-matched nontransgenic C57BL/6 mice. Splenocytes were stained with Kb-OVA tetramer APC, CD8 PE, and CD44 FITC. Live, CD8⫹ T cells are shown in dot plots. Percentage of cells in each quadrant appears in the corners of each panel. B, Dot plots reveal IFN-␥ production (y-axis) as a function of proliferation (x-axis). DC were infected with Hly⫹ or Hly⫺ Listeria, as indicated, and loaded with the OVA peptide concentrations listed across the top of plots. Gate shown was drawn based on the level of cytokine production detected in the absence of peptide in the stimulation for intracellular cytokine staining. Cells within this gate were considered positive for IFN production. Mean fluorescence of IFN-␥ is indicated by the arrows in each panel. C, Percentage of total T cells expressing IFN-␥. Percentage was determined based on the cells included or excluded from the gate in B. Mean and SD of four independent experiments are indicated by error bars. D, Percentage of T cells in each division that express IFN-␥. Treatment of DC indicated in inset. Mean and SD of four independent experiments are indicated by error bars.

The Journal of Immunology by 1.7- or 1.9-fold (at the high and intermediate Ag dose, respectively) over Hly⫺ L. monocytogenes. At the intermediate Ag dose, 0.1 ng/ml, we observed a nearly 2-fold difference in the percentage of IFN-␥-producing cells dependent on the ability of Listeria to enter the cytosol (Fig. 5, B and C; Hly⫹ 55%, Hly⫺ 29%). These data demonstrate that under conditions in which T cell proliferation is similar between the two populations, there is a more substantial difference in the percentage of T cells producing cytokine in response to Ag. At the lowest Ag dose (0.01 ng/ml), we observed a more remarkable, 8-fold difference in the percentage of T cells producing IFN-␥ dependent on cytoplasmic entry of Listeria (Hly⫹ 12%, Hly⫺ 1.5%; Fig. 5, B and C). These data indicate that the hemolytic activity of Listeria is required in DC primarily for the induction of cytokine production in T cells and not for stimulation of proliferation. Interestingly, cytoplasmic entry of Listeria enhanced T cell IFN-␥ production progressively as the cells entered later divisions. As depicted in Fig. 5D, at the intermediate Ag dose, the T cell population primed by Hly⫹ L. monocytogenes-infected DC included a high frequency of functional cells upon the first division, and as the cells progressed through later divisions, higher percentages of cells were functional. In contrast, the percentage of IFNproducing cells primed by Hly⫺ L. monocytogenes-infected or untreated DC was largely determined upon the first division and did not increase significantly with subsequent divisions. Interestingly, LPS-treated DC induced a lower frequency of T cells to produce IFN-␥ than Hly⫹ L. monocytogenes-infected DC, and the percentage of functional cells did not increase beyond the second cell division. These data reveal some novel insights about T cell proliferation and cytokine production. As Ag dose decreases, T cell function is down-regulated before T cell proliferation. In addition, cytoplasmic entry of Listeria in DC determines the ability of these cells to prime functional vs anergic T cells. Because we observed a differential effect of cytoplasmic entry on T cell proliferation vs function at the intermediate Ag concentration, 0.1 ng/ml, we will focus our subsequent experiments on this Ag dose in the following studies. Costimulatory molecules B7.1 and/or B7.2 induced by listerial cytoplasmic entry are required for T cell proliferation and cytokine production To examine the role of costimulatory molecules on listerial cytoplasmic entry-dependent T cell activation, we acquired bone marrow from mice lacking both B7.1 and B7.2 (B7.1, 2⫺/⫺), or lacking CD40 (CD40⫺/⫺) (a gift of R. Hodes). We then generated DC from these bone marrow samples with GM-CSF, as above. DC were infected with Listeria or treated with LPS and used as APC in our T cell priming assay. On day 3, the proliferative and functional profile of the T cells primed by these DC was measured. The dot plots depicting proliferation vs IFN-␥ production at the intermediate Ag dose (0.01 ng/ml) are shown in Fig. 6A. In wild-type DC, as in previous experiments, we observed a ⬃2-fold enhancement of functional activity generated by infection with Hly⫹ L. monocytogenes over Hly⫺ L. monocytogenes and no significant difference in the proliferative response (Fig. 6). No significant difference was observed in proliferation or cytokine production in the absence of CD40, compared with wild-type DC (Fig. 6, B and D). However, in the absence of B7.1 and B7.2, the functional response induced by infection with Hly⫹ L. monocytogenes was decreased to the level stimulated by Hly⫺ L. monocytogenes in wild-type DC. More notably, IFN-␥ production primed by Hly⫺ L. monocytogenes-infected DC was nearly abolished in the absence of B7.1 and B7.2 (Fig. 6, A and B). Thus, even though

2647 Hly⫺ L. monocytogenes induced little or no up-regulation of B7.1 and B7.2 in wild-type DC, the low level expressed by these cells was sufficient to induce a moderate level of T cell function, which was lost in cells completely lacking these molecules. The requirement for B7.1 and B7.2 was also evident in the proliferative response of the T cells. The lack of B7.1 and B7.2 resulted in a reduction in the mean number of divisions of cells dividing (Fig. 6D), and a less significant decrease in the percentage of cells entering cycle (indicated by the numbers in the upper left of each panel, Fig. 6C). These data indicate that B7.1 and/or B7.2 are essential not only for the development of effector function at limiting Ag concentrations, but also for completing the proliferative program. However, we did not observe substantial differences in proliferation primed by Hly⫹ vs Hly⫺ L. monocytogenes-infected DC. Thus, the differential expression of costimulatory molecules by these cells is unlikely to explain our findings. Therefore, the low level of costimulatory molecules expressed by DC infected with Hly⫺ L. monocytogenes must be sufficient to prime T cell proliferation comparable to that primed by DC infected with Hly⫹ L. monocytogenes. Because production of IFN-␥ by T cells is dependent on proliferation (32, 33), it is likely that T cell function is also regulated by B7.1 and B7.2 at the level of proliferation. Interestingly, the loss of CD40 expression had no significant effect on T cell function or proliferation primed by DC infected with either bacterial strain. The cytokines IL-12 and IL-10 modulate the degree of T cell function, but do not alter the level of T cell proliferation To identify the specific roles of the cytokines induced by listerial cytoplasmic entry in the priming of CD8⫹ T cells, we used Abs to neutralize these cytokines in the cultures. For these studies, we infected DC as above with Hly⫹ or Hly⫺ Listeria (MOI ⫽ 1), loaded the DC with 0.1 ng/ml OVA peptide, and added either 10 ␮g/ml neutralizing Abs, or the same dose of the appropriate Ab isotype control. The dot plots shown in Fig. 7A illustrate the proliferation and IFN-␥ production of T cells primed in the presence or absence of Abs neutralizing IL-10 or IL-12 at the intermediate Ag dose, 0.1 ng/ml. T cells primed by Hly⫹ L. monocytogenes-infected DC (and treated with isotype control Ab) showed robust proliferation and function. As observed previously, T cells primed by DC infected with Hly⫺ L. monocytogenes demonstrated a similar proliferative response, but reduced function. The removal of IL-10 had an enhancing effect on the percentage of functional T cells primed by Hly⫹ L. monocytogenes-infected DC (Fig. 7, A and C). The removal of IL-10 enhanced T cell function primed by Hly⫺ L. monocytogenes-infected DC approximating the level of function induced by DC infected with Hly⫹ L. monocytogenes. These data suggest that although it was not detectable in Hly⫺ priming cultures by ELISA, IL-10 was present in these cultures in amounts sufficient to inhibit T cell function. IL-10 neutralization also augmented the function of T cells primed by Hly⫹ L. monocytogenesinfected DC, indicating its inhibitory role in these cultures as well. Importantly, IL-10 neutralization had no effect on the proliferative profile of T cells (Fig. 7B, division index indicated in upper left of each panel). In contrast to IL-10, the neutralization of IL-12 in the priming cultures substantially inhibited the function of T cells primed by DC infected with either Hly⫹ or Hly⫺ Listeria. In fact, IL-12 neutralization reduced the percentage of functional cells primed by Hly⫹ L. monocytogenes-infected DC even below the level of that induced by Hly⫺ L. monocytogenes-infected DC (Fig. 7C). Importantly, the neutralization of IL-12, like IL-10, had no significant

2648

CYTOPLASMIC ENTRY OF Listeria MATURES DC

FIGURE 6. Costimulatory molecules B7.1 and B7.2, but not CD40, mediate T cell proliferation and cytokine production induced by listerial cytoplasmic entry. A, DC were generated from bone marrow of wild-type (WT) mice or from mice lacking either CD40 (CD40⫺/⫺) or B7.1 and B7.2 (B7.1, 2⫺/⫺). These cells were infected with Hly⫹ or Hly⫺ Listeria and loaded with 0.1 ng/ml OVA peptide. T cell proliferation and IFN-␥ production are indicated by dot plots. B, Percentage of IFN-␥-expressing cells for the entire T cell population. C, Histograms of T cell proliferation indicated by CFSE peaks. T cells were primed by DC from indicated mouse strains infected with Hly⫹ or Hly⫺ L. monocytogenes. The percentage of T cells in the original population dividing is indicated in upper left corner of histogram. D, Proliferation indices (mean number of cycles of dividing cells) of T cells primed by DC lacking either CD40 or B7.1 and B7.2 vs wild-type DC.

effect on the proliferative profile of the T cell response, as has been reported by others (34, 35). Neutralization of TNF-␣ also resulted in decreased priming of T cell function. This cytokine is known to enhance DC maturation, and therefore, may act at the level of the DC to enhance T cell function (9). Interestingly, the neutralization of IL-6 had no effect on the proliferation or cytokine production of the primed T cells. Cytoplasmic invasion of Listeria mediated by LLO has been demonstrated to trigger a signaling cascade, resulting in the expression of several inflammatory cytokines, including type I IFNs (36, 37). Type I IFNs have many functions, including a recently described potent ability to activate or mature DC (10). Cytoplasmic entry of Listeria may therefore represent an important component of the bacteria’s ability to activate DC and eventually initiate a robust T cell response. Based on these reports, we also evaluated the effect of these cytokines on T cell priming in vitro. Surprisingly, we found that the neutralization of IFN-␣ had no significant effect on cytokine production, and neutralization of IFN-␤ had only a modest inhibitory effect on this function (Fig. 7D). Therefore, these data suggest that type I IFNs do not directly regulate the T cell priming capability of DC. Thus, our findings suggest that the cytokines induced by cytoplasmic entry of Listeria contribute to the functional outcome of T cell priming, but have little to no effect on the proliferative response.

Discussion With this study, we demonstrate for the first time that cytoplasmic entry of Listeria is required for maximal maturation of bone marrow-derived DC by Listeria and for the optimal priming of T cell proliferation and cytokine production by these cells. In this work, we have quantitatively examined the effects of two important signals to T cell activation in the presence of defined Ag concentrations: costimulatory molecules and cytokines. We have also determined the relative contribution of each of these molecules in regulating T cell proliferation and function. Under conditions of defined Ag dose, the outcome of T cell priming can be dramatically affected by the strength of stimulus encountered by the DC. Cytoplasmic entry of Listeria provides a robust stimulus that induces costimulatory molecule expression and cytokine production, both of which mediate the activation of naive T cells in specific ways. Our results also offer insights into how Ag dose (strength of signal 1) regulates T cell proliferation and function primed by Listeria-infected DC. As Ag dose was decreased, the percentage of cells recruited to proliferate diminished in all populations (Fig. 4A). Although the percentage of cells dividing was modulated by the strength of APC stimulus, the mean number of divisions for dividing cells did not change. Therefore, changes in the magnitude


2649

FIGURE 7. Cytokines IL-12 and IL-10 modulate the function of T cells, but do not alter proliferation. A, T cells primed by DC infected with Hly⫹ or Hly⫺ L. monocytogenes and loaded with 0.1 ng/ml OVA peptide in the presence of Abs neutralizing the activity of IL-12 or IL-10. Dot plots indicate IFN-␥ production as a function of proliferation. Murine IgG isotype control for neutralizing Ab treatment. B, Histograms of T cell proliferation primed by DC infected with Hly⫹ or Hly⫺ L. monocytogenes in the presence of the indicated neutralizing Ab. Division indices (mean number of divisions for entire population) for each treatment are indicated in upper left corner of histograms. C, Percentage of T cells producing IFN-␥ in the presence of the indicated cytokine-neutralizing Ab in the priming culture. D, Percentage of T cells producing IFN-␥ in the presence of IFN type I neutralizing Abs. Results are representative of three independent experiments.

of the T cell response were predominantly regulated by the number of cells entering cell cycle. Interestingly, we detected no significant difference in the proliferative profile induced by DC infected with Hly⫹ vs Hly⫺ L. monocytogenes, except at the highest Ag dose. The proportion of T cells producing IFN-␥ resulting from priming by L. monocytogenes-infected DC was also dependent on the Ag dose. As Ag dose decreased, the percentage of functional cells decreased in all populations, as did the amount of cytokine detected in each cell. Interestingly, at the intermediate Ag dose, we observed almost no difference in the division index of T cells primed by Hly⫹ vs Hly⫺ L. monocytogenes-infected DC (Fig. 4B), yet there was a 2-fold difference in the percentage of functional cells depending on the ability of the bacteria to invade the cytoplasm (Fig. 5C). These data suggest that under conditions of limiting Ag dose, T cell function is lost before proliferation. These conditions may in part represent those elicited by immunization of mice with killed or noninvasive Listeria, in which T cell anergy is detected. The recruitment of T cells into cell cycle and the subsequent rate of division have been reported to be enhanced by costimulatory molecules such as B7.1 and B7.2 (29, 38). Our data support these findings by demonstrating that in the absence of costimulatory molecules, in particular B7.1 and/or B7.2, both the percentage of T cells dividing and the number of cell divisions detected were

reduced (Fig. 6C). Because IFN-␥ production is cell cycle dependent (32, 33), to become functional, T cells must proliferate. Therefore, B7.1 and/or B7.2 represent the second required signal for T cell activation regulating both proliferation and function due to the interrelatedness of these two phenomena. It will be interesting in future studies to determine the specific contribution of either B7.1 or B7.2 to T cell priming, but for now it is not clear which molecule is most important for CD8⫹ T cell priming in our system. As previously mentioned, we observed little difference in the proliferative profiles, but a 2-fold difference in function primed by DC infected with Hly⫹ vs Hly⫺ L. monocytogenes-infected DC at the intermediate Ag dose (Fig. 5). Because there was no significant difference in proliferation, the enhanced expression of costimulatory molecules induced by Hly⫹ L. monocytogenes infection is not likely to account for the observed difference in function. Therefore, the functional difference observed under these conditions is most likely dependent on the different levels of cytokines present, particularly IL-12. This hypothesis is supported by the data in Fig. 7 demonstrating that when IL-12 was neutralized, the percentage of functional T cells primed by Hly⫹ L. monocytogenes-infected DC is dramatically reduced. Our findings are in agreement with others that IL-12 modulates T cell function, but does not significantly affect T cell proliferation (39). Thus, the ability to elicit full T cell

2650 function is dependent not only on the presence of Ag and costimulatory molecules, but also on the presence of stimulatory cytokines such as IL-12 to prevent the induction of anergy. We therefore postulate that cytoplasmic entry of Listeria promotes the development of protective CTL responses by inducing maximal DC activation in the form of inflammatory cytokines. One surprising result of our cytokine neutralization analysis was that IL-10 neutralization had an enhancing effect on T cell function primed by Hly⫺ L. monocytogenes-infected DC. Although IL-10 was not detectable in the supernatants of DC infected with an MOI of 1 bacteria/cell of Hly⫺ L. monocytogenes, neutralization of this cytokine dramatically enhanced T cell function primed by these DC (Fig. 7, A and C). Therefore, it must be present in the cultures at levels sufficient to inhibit T cell priming. This cytokine may be produced at such low levels by the DC that it is rapidly consumed or bound to cell surface receptors and is not detectable as secreted cytokine in the ELISA. It is also interesting that IL-10, which was produced by DC infected with Hly⫹ Listeria at substantially higher levels than by DC infected with Hly⫺ Listeria, only partially inhibited T cell function primed by Hly⫹ L. monocytogenesinfected cells. Therefore, the ability to inhibit or enhance T cell priming must be due to the balance of cytokines induced by infection. For example, low levels of IL-10 may be very inhibitory in the absence of a strong enhancing IL-12 signal (i.e., Hly⫺ L. monocytogenes-infected DC) and likewise, a higher level of IL-10 may be less inhibitory in the presence of a strong IL-12 response (i.e., Hly⫹ L. monocytogenes-infected DC). Based on our data, however, it is not clear whether the T cell or DC is predominantly targeted by the cytokines examined. Therefore, our observed effects could be mediated by cytokines acting directly on the T cell or indirectly through maturation of the DC. To more closely map the molecular targets of these molecules, further study using cells deficient in specific cytokine receptors will be required. The cytosolic surveillance pathway, in which type I IFNs are induced, has been previously shown to be activated by the presence of bacteria in the cytoplasm (36, 37). This surveillance mechanism was activated by Listeria (expressing LLO) and was induced only upon bacterial cytoplasmic penetration. Induction of type I IFN was shown to depend not on the hemolysin molecule itself, but on its activity because it was induced even in the absence of LLO expression in permissive cells. In these cells, listerial cytoplasmic entry is mediated by phospholipases. Based on these reports, we also evaluated the effect of these cytokines on T cell priming in vitro. Surprisingly, we found that the removal of either IFN-␣ or IFN-␤ with neutralizing Abs had no effect on T cell proliferation and only a modest inhibitory effect on cytokine production (Fig. 7D). To more closely examine the role of these molecules in T cell priming, further study will be required using T cells or DC lacking receptors for these cytokines. The molecular basis for the differences observed in the presence or absence of listerial cytoplasmic entry is most likely due to a combination of signals mediated through TLR on the cell surface or in phagosomes, and by cytoplasmic detector molecules such as NOD2. Insights into these molecular mechanisms have recently emerged through the use of mice lacking MyD88, an adapter molecule mediating signals from TLR as well as IL-1R and IL-18R. One such report demonstrates that cytoplasmic entry of Listeria is required for the production of MCP-1, a chemokine required for recruitment of DC to the spleen during listerial infection in vivo, yet MCP-1 production was independent of MyD88 (40). These data suggest that a cytoplasmic surveillance mechanism recognizes bacterial structures, resulting in the induction of MCP-1 and, subsequently, migration of DC to the spleen. In contrast, the same study demonstrated that monocyte activation (as measured by

CYTOPLASMIC ENTRY OF Listeria MATURES DC TNF-␣ and IL-12 production) is MyD88 dependent. It will be important, with future studies, to evaluate the relative contribution of signaling through MyD88 vs signaling through the cytosolic surveillance pathway to the DC response to listerial infection. It is surprising that we do not observe a more robust stimulation by infection with Hly⫺ listerial infection given their expression of several TLR agonists. This may be due to one of two potential explanations. First, the bacterial TLR agonists such as peptidoglycan may be less accessible for TLR binding on a live, intact bacterium than a killed or fractionated preparation. Alternatively, the live bacteria may produce factors that inhibit the stimulation or maturation of DC. To determine whether Hly⫺ L. monocytogenes inhibit DC maturation, or simply fail to induce maturation, we will need to ascertain whether the bacteria can inhibit maturation induced by known agonists such as LPS. Our findings suggest that cytoplasmic entry of Listeria mediates the development of protective immunity in vivo by inducing vigorous DC maturation, resulting in optimal CTL priming. These studies also explain in part why killed listerial vaccines are unable to elicit protective immunity. Without the ability to enter the cytoplasm, Listeria are poor inducers of DC maturation. However, with these and future studies exploring the molecular mechanisms through which cytoplasmic bacteria trigger DC maturation, we will be able to design more effective vaccines to target intracellular pathogens such as Listeria.

Acknowledgments We thank Dr. Richard Hodes, Dr. Joy Williams, and Dr. JoAnne Lumsden for the donation and preparation of bone marrow from CD40⫺/⫺ and B7.1B7.2⫺/⫺ mice. We also thank Dr. Ronald Germain for the donation of the 25.D1 Ab. We are grateful for the donation of the Kb-OVA-specific tetramer from Dr. Jason Grayson. We are also grateful to Dr. Martha Alexander-Miller and Dr. Jason Grayson for helpful discussions and critical reading of this manuscript.

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