Listeria monocytogenes Infection Overcomes the Requirement for CD40 Ligand in Exogenous Antigen Presentation to CD8+ T Cells

Sara E. Hamilton, Amy R. Tvinnereim and John T. Harty
J Immunol November 15, 2001, 167 (10) 5603-5609; DOI: https://doi.org/10.4049/jimmunol.167.10.5603

Abstract

In vivo priming of CD8+ T lymphocytes against exogenously processed model Ags requires CD4+ T cell help, specifically interactions between CD40 ligand (CD40L) expressed by activated CD4+ T cells and CD40, which is present on professional APC such as dendritic cells (DCs). To address this issue in the context of bacterial infection, we examined CD40L-CD40 interactions in CD8+ T cell priming against an exogenously processed, nonsecreted bacterial Ag. CD40L interactions were blocked by in vivo treatment with anti-CD40L mAb MR-1, which inhibited germinal center formation and CD8+ T cell cross-priming against an exogenous model Ag, OVA. In contrast, MR-1 treatment did not interfere with CD8+ T cell priming against a nonsecreted or secreted recombinant Ag expressed by Listeria monocytogenes. Memory and secondary responses of CD8+ T cells against nonsecreted and secreted bacterial Ags were also largely unimpaired by transient MR-1 treatment. When MR-1-treated mice were concurrently immunized with L. monocytogenes and OVA-loaded splenocytes, cross-priming of OVA-specific naive CD8+ T cells occurred. No significant decline in cross-priming against OVA was measured when either TNF or IFN-γ was neutralized in L. monocytogenes-infected animals, demonstrating that multiple signals exist to overcome CD40L blockade of CD8+ T cell cross-priming during bacterial infection. These data support a model in which DCs can be stimulated in vivo through signals other than CD40, becoming APC that can effectively stimulate CD8+ T cell responses against exogenous Ags during infection.

Induction of CD8+ T cell responses against exogenously processed model Ags requires CD4+ T cell help (1, 2, 3). This has been demonstrated in systems using model Ags loaded into splenocytes or expressed in cells which are not APCs. In these systems CD8+ T cells are primed through an Ag-processing mechanism termed cross-presentation (1, 2, 3, 4). During cross-presentation, cell-associated Ag is taken up by bone marrow-derived APC and presented via exogenous MHC class I processing pathways (reviewed in Ref. 5). A model for CD8+ T cell cross-priming has emerged in which a CD4+ T cell recognizes peptide-MHC class II complexes on the surface of an APC causing activation of the T cell and up-regulation of CD40 ligand (CD40L)3 on its surface. This enables the T cell to signal through CD40 on an APC or dendritic cell (DC) to induce its maturation. Ab-mediated ligation of CD40 replaces the need for CD4+ T cell help in vivo for efficient priming of CD8+ T cells against model exogenous Ags (1, 2, 3). The factors required for efficient priming of naive CD8+ T cells by DCs are not known, but up-regulation of costimulatory molecules and/or the secretion of various proinflammatory cytokines may be required. CD40 ligation, LPS, and TNF-α can cause the up-regulation of B7.1, B7.2, and MHC class II as well as the secretion of IL-12 by DCs (6, 7).

Several studies have examined CD8+ T cell responses in CD40L−/− mice after viral infection. CD8+ T cell responses appear to be normal in response to many viruses including lymphocytic choriomeningitis virus (LCMV), suggesting that viral infection may in some instances bypass the requirement for CD40L in DC activation (8). Some deficiencies have been observed in primary CD8+ T cell responses after vesicular stomatitis virus (VSV) infection and memory CD8+ T cell responses after LCMV infection (9, 10). Early T cell independent inflammatory responses to infection with the intracellular bacterium L. monocytogenes (LM) and T cell-mediated protective immunity appear to be unaffected by the absence of CD40L (11). Recent evidence has also shown a minimal impact on the splenic CD8+ T cell response to secreted LM Ags in CD40−/− mice (12). However, these studies have measured responses generated through the endogenous MHC class I presentation pathway. Thus, it is not known whether CD40L-CD40 interactions are required for naive CD8+ T cell priming against exogenously processed Ags expressed by pathogens.

This study examines the role of CD40L-CD40 interactions in CD8+ T cell priming against LM Ags presented by either the endogenous or exogenous MHC class I presentation pathway. LM is a Gram-positive facultative intracellular bacterial pathogen that can enter and multiply within phagocytic and nonphagocytic cells. LM has the ability to escape from the membrane-bound phagosome after infection and enter the host cell cytoplasm (13). Secreted bacterial Ags can then be processed through the endogenous MHC class I presentation pathway, which involves proteolytic degradation by the proteasome and transport into the endoplasmic reticulum by the TAP complex. Nonsecreted bacterial Ags, although not accessible to the endogenous pathway, may become accessible to exogenous MHC class I presentation pathways after destruction of the organism (14). Consistent with this idea, infection of mice with recombinant LM strains expressing an H-2Ld-restricted epitope from the nucleoprotein of LCMV (NP118–126) as either a secreted or a nonsecreted fusion protein prime NP118–126-specific CD8+ T cell responses that differ in magnitude by only 3- to 4-fold (14).

To examine the role of CD40L-CD40 interactions in CD8+ T cell priming against exogenously processed bacterial Ags, CD8+ T cell responses were measured in mice treated with CD40L-blocking Abs and infected with recombinant LM. Our data demonstrate that although CD40L blocking Ab treatment prevented cross-priming of CD8+ T cells against a model Ag, it does not inhibit priming against LM Ags. In addition, we show that LM infection overcomes CD40L blockade to allow CD8+ T cell cross-priming against a model exogenous Ag.

Materials and Methods

Mice and bacteria

Six- to 8-wk-old female BALB/c (H-2d MHC) and CB6F1 (H-2bxd MHC) mice were purchased from National Cancer Institute (Frederick, MD). Virulent LM strains used in this study were recombinant strain XFL304 (LM-NPns) expressing a nonsecreted fusion protein containing the LCMV NP118–126 epitope and strain XFL303 (LM-NPs) expressing the same fusion protein in secreted form (14). Attenuated LM strain DP-L1942 (ActA− LM), which carries an in-frame deletion in the actA gene (15), and attenuated LM strain DP-L2161 (listeriolysin O (LLO)− LM), which carries an in-frame deletion in the hly gene (16), were also used. Growth and maintenance of all LM strains were described previously (17).

Infection with LM

Age- and sex-matched adult BALB/c or CB6F1 mice were infected by i.v. injection with 2 × 103 LM-NPns or LM-NPs, 1 × 106 ActA− LM, and 1 × 108 LLO− LM. Actual numbers of CFU injected were determined for each experiment by plate count.

Cross-priming with OVA-loaded splenocytes

Age- and sex-matched CB6F1 mice were immunized with OVA-loaded BALB/c splenocytes as previously described (18, 19). Briefly, RBC-depleted BALB/c splenocytes were incubated with freshly prepared OVA (10 mg/ml) in hypertonic medium (RPMI 1640, 0.5 M sucrose, 10% polyethylene glycol 1000, and 10 mM HEPES, pH 7.2) for 10 min at 37°C. Splenocytes were then diluted in warm hypotonic medium (60% HBSS and 40% H2O) and incubated for 2 min at 37°C. Cells were washed twice in HBSS and resuspended at 60–70 × 106 splenocytes/ml. CB6F1 mice received 0.5 ml/animal i.v. For coimmunization with LM and OVA-loaded splenocytes, i.v. infection with the indicated number of organisms was performed first followed 2–4 h later by injection of OVA-loaded splenocytes.

Cells lines and Abs

Hybridomas producing anti-CD40L mAb MR-1 (20) (a gift from Dr. T. Waldschmidt, University of Iowa, Iowa City, IA) and anti-TNF (XT22 and XT3 used in combination at a mass ratio of 1:1) (21) were grown in DMEM supplemented with 10% FCS, l-glutamine, and antibiotics. Hybridomas producing anti-IFN-γ (XMG1.2) (22) were grown in RPMI 1640 supplemented with 10% FCS, l-glutamine, and antibiotics (RP10) (23). Ab was purified from culture supernatant by affinity chromatography with protein G (Amersham Pharmacia, Piscataway, NJ). Each batch of Ab was concentrated to 2 mg/ml and assessed for endotoxin contamination by Limulus amebocyte lysate assay (Associates of Cape Cod, Falmouth, MA). All preparations contained <15 ng/ml endotoxin.

Anti-CD40L Ab MR-1 has been used in multiple studies to block CD40L in vivo (24). To determine whether our purification procedure was sufficient in yielding MR-1 that could effectively block CD40L in vivo, MR-1- or hamster Ig (hIg)-treated BALB/c mice were immunized with SRBC. Immunization results in CD40L-dependent germinal center formation within 4 days in control hIgG (Jackson ImmunoResearch, West Grove, PA)-treated animals or animals receiving no Ab treatment. In animals that received 1 mg MR-1 in total, given on days −2, 0, 2, and 4, surface staining revealed a 70–85% decrease in germinal center B cells (data not shown), a level below that of germinal center B cells found in naive animals housed in our animal facility. Similar data were obtained 7 days after SRBC immunization (data not shown).

Intracellular cytokine staining of splenocytes

The number of CD8+ T cells specific for OVA257–264 in the context of H-2Kb, NP118–126 in the context of H-2Ld, LLO91–99 in the context of H-2Kd, or p60217–225 in the context of H-2Kd was determined by intracellular cytokine staining for IFN-γ as previously described (25). RBC-depleted splenocytes from immunized mice were incubated for 5–6 h in 2 μl/ml brefeldin A at 37°C with or without synthetic peptides at 200 nM. For staining, cells were placed on ice, washed, and incubated with Ab directed against the FcγII/III receptors (2.4G2) and FITC-conjugated anti-CD8 mAb (BD PharMingen, San Diego, CA), fixed, permeabilized, and stained with PE-conjugated anti-IFN-γ mAb according to manufacturer’s instructions (BD PharMingen).

Data were acquired on FACScan flow cytometer (Becton Dickinson, Mountain View, CA) using Summit software. One hundred thousand events were collected for analysis with FlowJo software (Tree Star, San Carlos, CA). The gate for IFN-γ+ cells was selected so that the percentage of IFN-γ+ cells in the unstimulated sample for each mouse was 0.2% or less of CD8+ splenocytes. This value was subtracted from the peptide-stimulated values to determine the frequency of Ag-specific CD8+ T cells. Total numbers of epitope-specific CD8+ T cells/spleen were calculated using this frequency, the percentage of CD8+ T cells in each sample, and the total number of splenocytes per animal.

IFN-γ ELISPOT

Ag-specific CD8+ T cell responses were measured by IFN-γ ELISPOT analysis as previously described (26). Briefly, splenocytes (104–105/well) were cultured with medium alone or with 100 nM synthetic peptide (LLO91–99 or NP118–126) for 36–48 h in flat-bottom 96-well plates that had been previously coated with rat anti-mouse IFN-γ mAb (RA-6A2, BD PharMingen). Following washes with PBS-0.05% Tween, the plates were incubated for 3 h at room temperature with rabbit anti-IFN-γ antiserum (a gift from Dr. J. Cowdery, University of Iowa). After washing, the plates were incubated for 4–5 h with donkey anti-rabbit Ig conjugated to alkaline phosphatase (Jackson ImmunoResearch). Further washing was followed by addition of 1 mg/ml 5-bromo-4-chloro-3-indolylphosphate (Sigma, St. Louis, MO) buffer with 0.75% agarose. The reaction was developed for 0.5–1 h at 37°C and was stopped by storage at 4°C. Spots were counted using a dissecting microscope. The average frequency of responders from triplicate wells was multiplied by the total number of splenocytes in calculating the number of responders per spleen. Background (no peptide stimulation) was subtracted from this value to determine the total number of Ag-specific cells per spleen.

Results

Blocking CD40L in vivo inhibits CD8+ T cell priming against OVA-loaded splenocytes

Disruption of CD40L-CD40 interactions has been shown to prevent CD8+ T cell cross-priming against model Ags (1, 2). We first determined whether our preparation of MR-1 (CD40L blocking Ab) was capable of disrupting CD40L-CD40 interactions in vivo by measuring CD8+ T cell cross-priming against OVA-loaded splenocytes in animals treated with MR-1.

CB6F1 (H-2bxd) animals were treated with MR-1 or hIg and injected i.v. with OVA-loaded BALB/c (H-2d) splenocytes. Priming of an H-2Kb-restricted CD8+ T cell response to the OVA257–264 epitope in this experiment requires cross-presentation, since the Ag is loaded into BALB/c splenocytes, which lack the necessary MHC class I-presenting molecule. OVA257–264-specific CD8+ T cell responses were quantitated 7 days after injection using intracellular IFN-γ staining of splenocytes to detect Ag-specific cells (25). Representative mice treated with hIg control Abs or MR-1 are shown in Fig. 1⇓A. In the absence of peptide stimulation, few CD8+ T cells make IFN-γ. However, when splenocytes from hIg-treated, OVA-immunized animals were incubated with the OVA257–264 peptide, approximately 0.75% of CD8+ T cells produced IFN-γ after subtraction of background. The total number of Ag-specific cells ranged from 0.4–1 × 105/spleen in this experiment and is representative of 12 independent experiments (Fig. 1⇓B). In contrast, CD8+ splenocytes from MR-1-treated, OVA-immunized animals failed to make a detectable IFN-γ response over background after stimulation with synthetic OVA257–264 (Fig. 1⇓, A and B). In total, 4 of 36 MR-1-treated mice and 33 of 36 hIg-treated mice had responses above the limit of detection. Similar results were reported with CD40L−/− and CD40−/− mice (2). Thus, MR-1 treatment effectively prevents detectable cross-priming of CD8+ T cells against a model exogenous Ag.

FIGURE 1.
FIGURE 1.

Cross-priming of OVA257–264-specific CD8+ T cells is inhibited by CD40L-blocking Ab. CB6F1 (H-2bxd) mice were immunized i.v. with 30–35 × 106 OVA-loaded BALB/c (H-2d) splenocytes. On days −2, 0, 2, and 4, mice received i.p. injections of 0.25 mg hIg control Ab (▪) or CD40L Ab MR-1 (□). On day 7 after OVA immunization intracellular staining for IFN-γ was performed on OVA257–264 peptide (H-2Kb-restricted)-stimulated splenocytes. A, Representative FACS profiles of splenocytes with and without peptide stimulation from both a mouse that received hIg control Ab treatment and a mouse that received MR-1 treatment. Both animals were also immunized with OVA-loaded BALB/c splenocytes. The frequency of Ag-specific CD8+ T cells in the spleen is indicated. α-, Anti-. B, Total number of OVA257–264-specific CD8+ T cells in the spleen. Data represent individual mice from one experiment and are representative of 12 independent experiments. LOD, limit of detection.

CD8+ T cell responses against secreted and nonsecreted LM Ags are not inhibited with CD40L blockade

We investigated the role of CD40L-CD40 interactions in CD8+ T cell priming against secreted (endogenously processed) and nonsecreted (exogenously processed) LM Ags. Mice treated with MR-1 or hIg control Abs were infected with 2 × 103 LM-NPns (∼0.1 LD50) that expresses a well-characterized CD8+ T cell epitope from the nucleoprotein of LCMV (NP118–126) as a nonsecreted fusion protein. As an internal control, CD8+ T cell priming against a secreted, endogenously processed epitope from the LM molecule LLO91–99 was also measured. Ag-specific CD8+ T cells were detected by IFN-γ intracellular staining 7 days after infection. Responses of approximately 3.3–3.9 × 105 LLO91–99-specific cells/spleen were detected from both groups of animals (Fig. 2⇓A). Similarly, the total number of NP118–126-specific CD8+ splenocytes from both MR-1- and hIg-treated animals ranged from 6 to 8 × 104 cells/spleen (Fig. 2⇓B). Splenocytes from uninfected mice did not produce IFN-γ after stimulation with either peptide (data not shown). As a control for the course of infection, liver CFU analysis was performed on days 3 and 7, as infections in the spleen and liver are generally parallel (J. Harty, unpublished observations). MR-1- and hIg control Ab-treated animals showed no difference in infection at these time points and did not have CFU in the liver above the limit of detection (∼150 organisms/g liver) on day 7 (data not shown). This demonstrates that in contrast to cross-priming against OVA, naive CD8+ T cell priming against nonsecreted or secreted Ags expressed by LM is not inhibited by MR-1 treatment.

FIGURE 2.
FIGURE 2.

Responses of CD8+ T cells to secreted and nonsecreted bacterial Ags under conditions of CD40L blockade. BALB/c mice were immunized i.v. with 2 × 103 CFU LM-NPns. On days −2, 0, 2, and 4 mice received i.p. injections of 0.25 mg hIg control Ab (▪) or anti-CD40L Ab MR-1 (□). On day 7 after infection intracellular staining for IFN-γ was performed on peptide-stimulated splenocytes. Total numbers of LLO91–99 (A)- and NP118–126 (B)-specific CD8+ T cells in the spleen were calculated. α-, Anti-. Data are the means ± SD of four independent experiments, each with three mice per group. Total numbers of LLO91–99 (C)- and NP118–126 (D)-specific CD8+ T cells in the spleen were also determined by IFN-γ ELISPOT at 28 days after infection and by intracellular staining for IFN-γ 5 days after challenge with 2 × 105 CFU LM-NPs (E and F). A representative experiment of two is shown. The limit of detection is 104 for A, B, E, and F and 103 for C and D. Data are the means ± SD of three mice per group. ∗, p < 0.05.

Impaired memory CD8+ T cell responses after LCMV infection have been reported in CD40L−/− mice (10). To investigate whether transient blockade of CD40L-CD40 interactions would impact CD8+ T cell memory responses, mice treated with MR-1 or control hIg were infected with 2 × 103 LM-NPns. Twenty-eight days after infection splenocytes were stimulated for 36 h with LLO91–99 or NP118–126 peptide, and Ag-specific cells were quantitated by IFN-γ ELISPOT (Fig. 2⇑, C and D). In a preliminary experiment memory CD8+ T cell responses to NP118–126 were at or below the level of detection using intracellular cytokine staining for IFN-γ. Therefore, the data shown in Fig. 2⇑, C and D, were determined using the ELISPOT assay, which allows detection of low frequencies of Ag-specific CD8+ T cells. A 2- to 3-fold decrease in the number of Ag-specific cells was observed in MR-1-treated mice compared with control animals, but this revealed statistical significance only for NP118–126-specific cells. However, since both LLO91–99- and NP118–126-specific cells were reduced in number, and a slightly larger spleen size was seen in the hIg control group, it is unlikely that this difference is biologically significant.

CD8+ T cell recall responses were also examined in animals that received transient CD40L blockade during primary infection with LM-NPns. For challenge, the mice were infected with LM-NPs, which is isogenic with LM-NPns except that LM-NPs secretes the NP118–126 fusion protein and primes a 3- to 4-fold higher NP118–126-specific response (14). On day 30 after primary infection, mice were challenged with 2 × 105 LM-NPs (∼10LD50 for naive mice; Fig. 2⇑, E and F). Both MR-1-treated and hIg-treated animals contained 3–5 × 106 LLO91–99-specific CD8+ T cells (Fig. 2⇑E) and 2.4–2.7 × 106 NP118–126-specific CD8+ T cells (Fig. 2⇑F) in the spleen as determined by IFN-γ intracellular staining five days after infection. Challenge of immune mice with LM-NPns also resulted in comparable levels of secondary Ag-specific CD8+ T cell responses between MR-1- and hIg-treated animals (data not shown). These experiments illustrate that MR-1 treatment during the primary exposure to Ag does not impact the ability of CD8+ T cells to respond to either secreted or nonsecreted Ags (Fig. 2⇑). Memory responses to NP118–126 were significantly decreased 2- to 3-fold compared with control animals, but this did not result in any deficiency in CD8+ T cell responses to a secondary challenge.

Infection with rLM overcomes CD40L blockade in cross-priming

We next asked whether LM infection could overcome the capacity of MR-1 to block CD8+ T cell cross-priming against OVA-loaded splenocytes. MR-1 treatment blocked the OVA257–264-specific CD8+ T cell response in CB6F1 animals immunized with OVA-loaded splenocytes (Figs. 1⇑ and 3⇓). In contrast, animals treated with MR-1, infected with 2 × 103 LM-NPns that lacks the OVA257–264 epitope, and immunized with OVA-loaded splenocytes, made a substantial OVA257–264 response of 3.3 × 104 CD8+ T cells/spleen (Fig. 3⇓). This response was not significantly different from OVA-immunized, uninfected animals treated with hIg control Abs. There was a significant increase in the number of OVA257–264-specific CD8+ T cells (∼6-fold) under conditions of infection and hIg treatment, suggesting that infection results in an enhanced environment for CD8+ T cell cross-priming. The experiment in Fig. 3⇓ demonstrates that LM infection is able to overcome CD40L blockade in cross-priming of naive CD8+ T cells against OVA-loaded splenocytes.

FIGURE 3.
FIGURE 3.

Infection with virulent LM overcomes CD40L blockade of CD8+ T cell cross-priming against OVA-loaded splenocytes. CB6F1 mice were given i.p. injections of 0.25 mg hIg control Ab (▪ and ▧) or MR-1 (below level of detection and ▨) on days −2, 0, 2, and 4. On day 0 mice were infected i.v. with 2 × 103 CFU LM-NPns (▨ and ▧) and injected with 30–35 × 106 OVA-loaded BALB/c splenocytes (all groups). Intracellular IFN-γ staining for peptide-specific CD8+ T cells was performed on splenocytes 7 days later. The total number of OVA257–264-specific CD8+ T cells in the spleen was calculated. Data are means ± SD of three independent experiments, each with three mice per group. α-, Anti-. LOD, limit of detection. ∗, p < 0.025 vs hIg control Ab plus LM-NPns.

Attenuated LM strains also overcome CD40L blockade of cross-priming

The mechanism by which LM infection overcomes CD40L blockade of cross-priming is unknown, but may relate to specific properties of the organism or the host response to LM. To address this issue, we asked whether infection with the attenuated LM strain DP-L1942 (ActA− LM) would also be able to overcome MR-1 treatment. ActA− LM has an in-frame deletion in the actA gene, which is required for intracellular spread of LM to neighboring host cells (15). Since this strain is highly attenuated, mice in this experiment were infected with 1 × 106 ActA− LM (∼0.1 LD50). CB6F1 animals were treated with MR-1 or hIg as described, infected with ActA− LM, and immunized with OVA-loaded splenocytes. Similar to the experiments with virulent LM, OVA257–264-specific CD8+ T cell responses were only found in MR-1-treated animals when they were also infected with LM (Figs. 3⇑ and 4⇓). The magnitude of the response in MR-1-treated, ActA− LM-infected animals was approximately 105 Ag specific cells/spleen and was equivalent to that seen in uninfected hIg-treated animals. These data show that infection with either virulent or attenuated LM enhances CD8+ T cell cross-priming against OVA-loaded splenocytes, overcoming CD40L blockade in MR-1-treated animals and increasing the Ag-specific response in hIg-treated animals.

FIGURE 4.
FIGURE 4.

Attenuated LM overcomes MR-1 blockade of CD8+ T cell cross-priming. A, CB6F1 mice were given i.p. injections of 0.25 mg hIg control Ab (▪ and ▧) or MR-1 (below level of detection and ▨) or on days −2, 0, 2, and 4. On day 0 mice were infected i.v. with 1 × 106 CFU ActA− LM (▨ and ▧). Mice were also immunized i.v. with 30–35 × 106 OVA-loaded BALB/c splenocytes. Intracellular IFN-γ staining was performed on splenocytes on day 7. The total number of OVA257–264-specific CD8+ T cells in the spleen was calculated. Data are the means ± SD of three independent experiments, each with three mice per group. B, The total numbers of OVA257–264- and p60217–225-specific CD8+ T cells in the spleen after infection with the indicated dosages of ActA− LM (alive or heat killed). C, CB6F1 mice were treated as described above, except LM infection was with 1 × 108 CFU LLO− LM. Individual mice are shown from four independent experiments. α-, Anti-. LOD, limit of detection. ∗, p < 0.025 vs hIg control ActA− LM.

When infection with ActA- LM was titrated, injection of as few as 1 × 104 ActA− LM (0.001 LD50) overcame CD40L blockade of cross-priming (Fig. 4⇑B). A lower dose of 1 × 103 ActA− LM resulted in measurable priming against an endogenous, secreted LM epitope (p60217–225) that primes similar numbers of Ag-specific cells to OVA during LM infection (27). However, this dose of infection failed to overcome CD40L blockade of OVA257–264-specific CD8+ T cell priming. Immunization with 1 × 106 heat-killed ActA− LM also did not result in measurable CD8+ T cell priming against OVA or LM Ags. This result demonstrates that bacterial components alone, at least at the level tested here, are not sufficient to overcome CD40L blockade of CD8+ T cell cross-priming and that active infection must take place in the host.

A second attenuated strain, DP-L2161 (LLO− LM), was also used to infect CB6F1 animals treated with MR-1 or hIg and immunized with OVA-loaded splenocytes (Fig. 4⇑C). LLO− LM has an in-frame deletion in the hly gene, which encodes the LLO virulence factor (16). Loss of functional LLO results in organisms that are highly attenuated due to an inability to escape from the host cell phagosome (28, 29). A lower number of OVA257–264-specific CD8+ T cells was measured in mice infected with 1 × 108 (LD50 >109) LLO− LM and treated with hIg compared with hIg-treated animals infected with ActA− LM (Fig. 4⇑, compare A and C). Many MR-1 treated, LLO− LM-infected animals had no OVA257–264-specific response above the level of detection (7 of 12 animals). However, some animals did have measurable OVA257–264-specific responses ranging from approximately 1 × 104 to 1 × 105 CD8+ T cells/spleen. This indicates that infection with LLO− LM can overcome CD40L blockade of cross-priming, but does so less efficiently than virulent LM or ActA− LM.

Neutralization of TNF or IFN-γ does not prevent a response to OVA-loaded splenocytes during infection

During LM infection, various proinflammatory cytokines, such as IFN-γ and TNF, are generated that could serve as activation signals for DCs. TNF in particular stimulates the maturation of DCs and promotes cross-presentation of model Ags in vivo in the absence of CD40L (30, 31). To determine whether TNF or IFN-γ is important for the ability of LM infection to overcome CD40L blockade of cross-priming, CB6F1 animals were treated with MR-1 and TNF- or IFN-γ-neutralizing Abs. Cytokine-neutralizing Abs were used at a dosage previously determined to be effective for neutralization in vivo (32). Animals were then infected with 1 × 106 ActA− LM and immunized with OVA-loaded splenocytes. Animals treated with TNF-neutralizing Abs had enlarged spleens (3–4 × 108 splenocytes), and six of eight animals had organisms remaining in the liver 7 days after infection (data not shown). All other treatment groups had similar spleen sizes (∼2 × 108 splenocytes) and had cleared the infection by day 7. The total number of OVA257–264-specific CD8+ T cell was not significantly different in αTNF-α- or αIFN-γ-treated animals compared with control rat Ig-treated animals (Fig. 5⇓). However, the lack of statistical significance in TNF-depleted mice may be impacted by the increased level of infection in these animals. This experiment demonstrates that neutralization of these two cytokines under conditions of CD40L blockade and bacterial infection does not prevent naive CD8+ T cell priming against OVA-loaded splenocytes.

FIGURE 5.
FIGURE 5.

Neutralization of IFN-γ or TNF does not inhibit the ability of LM to overcome CD40L blockade of CD8+ T cell cross-priming. CB6F1 animals were given i.p. injections of 0.25 mg hIg (control) or MR-1 on days −2, 0, 2, and 4. On day −1 mice were given i. p. injections of α-TNF-α (XT22 and XT3), α-IFN-γ (XMG1.2), or rat Ig control Abs. On day 0 mice were infected i. v. with 1 × 106 CFU ActA− LM. All mice were also immunized i.v. with 30–35 × 106 OVA-loaded BALB/c splenocytes. Intracellular staining for IFN-γ was performed on splenocytes on day 7. A, The total number of OVA257–264-specific CD8+ T cells in the spleen was calculated. Data are the mean ± SD of three independent experiments. α-, Anti-. LOD, limit of detection.

FIGURE 11.
FIGURE 11.

Cross-priming of OVA257–264-specific CD8+ T cells is inhibited by CD40L-blocking Ab. CB6F1 (H-2bxd) mice were immunized i.v. with 30–35 × 106 OVA-loaded BALB/c (H-2d) splenocytes. On days −2, 0, 2, and 4, mice received i.p. injections of 0.25 mg hIg control Ab (▪) or CD40L Ab MR-1 (□). On day 7 after OVA immunization intracellular staining for IFN-γ was performed on OVA257–264 peptide (H-2Kb-restricted)-stimulated splenocytes. A, Representative FACS profiles of splenocytes with and without peptide stimulation from both a mouse that received hIg control Ab treatment and a mouse that received MR-1 treatment. Both animals were also immunized with OVA-loaded BALB/c splenocytes. The frequency of Ag-specific CD8+ T cells in the spleen is indicated. α-, Anti-. B, Total number of OVA257–264-specific CD8+ T cells in the spleen. Data represent individual mice from one experiment and are representative of 12 independent experiments. LOD, limit of detection.

FIGURE 21.
FIGURE 21.

Responses of CD8+ T cells to secreted and nonsecreted bacterial Ags under conditions of CD40L blockade. BALB/c mice were immunized i.v. with 2 × 103 CFU LM-NPns. On days (D) −2, 0, 2, and 4 mice received i.p. injections of 0.25 mg hIg control Ab (▪) or anti-CD40L Ab MR-1 (□). On day 7 after infection intracellular staining for IFN-γ was performed on peptide-stimulated splenocytes. Total numbers of LLO91–99 (A)- and NP118–126 (B)-specific CD8+ T cells in the spleen were calculated. α-, Anti-. Data are the means ± SD of four independent experiments, each with three mice per group. Total numbers of LLO91–99 (C)- and NP118–126 (D)-specific CD8+ T cells in the spleen were also determined by IFN-γ ELISPOT at 28 days after infection and by intracellular staining for IFN-γ 5 days after challenge with 2 × 105 CFU LM-NPs (E and F). A representative experiment of two is shown. The limit of detection is 104 for A, B, E, and F and 103 for C and D. Data are the means ± SD of three mice per group. ∗, p < 0.05.

FIGURE 31.
FIGURE 31.

Infection with virulent LM overcomes CD40L blockade of CD8+ T cell cross-priming against OVA-loaded splenocytes. CB6F1 mice were given i.p. injections of 0.25 mg hIg control Ab (▪ and ▧) or MR-1 (below level of detection and ▨) on days −2, 0, 2, and 4. On day 0 mice were infected i.v. with 2 × 103 CFU LM-NPns (▨, ▧) and injected with 30–35 × 106 OVA-loaded BALB/c splenocytes (all groups). Intracellular IFN-γ staining for peptide-specific CD8+ T cells was performed on splenocytes 7 days later. The total number of OVA257–264-specific CD8+ T cells in the spleen was calculated. Data are means ± SD of three independent experiments, each with three mice per group. α-, Anti-. LOD, limit of detection. ∗, p < 0.025 vs hIg control Ab and LM-NPns.

FIGURE 41.
FIGURE 41.

Attenuated LM overcomes MR-1 blockade of CD8+ T cell cross-priming. A, CB6F1 mice were given i.p. injections of 0.25 mg hIg control Ab (▪ and ▧) or MR-1 (below level of detection and ▨) or on days −2, 0, 2, and 4. On day 0 mice were infected i.v. with 1 × 106 CFU ActA− LM (▨, ▧). Mice were also immunized i.v. with 30–35 × 106 OVA-loaded BALB/c splenocytes. Intracellular IFN-γ staining was performed on splenocytes on day 7. The total number of OVA257–264-specific CD8+ T cells in the spleen was calculated. Data are the means ± SD of three independent experiments, each with three mice per group. B, The total numbers of OVA257–264- and p60217–225-specific CD8+ T cells in the spleen after infection with the indicated dosages of ActA− LM (alive or heat killed). C, CB6F1 mice were treated as described above, except LM infection was with 1 × 108 CFU LLO− LM. Individual mice are shown from four independent experiments. α-, Anti-. LOD, limit of detection. ∗, p < 0.025 vs hIg control ActA− LM.

FIGURE 51.
FIGURE 51.

Neutralization of IFN-γ or TNF does not inhibit the ability of LM to overcome CD40L blockade of CD8+ T cell cross-priming. CB6F1 animals were given i.p. injections of 0.25 mg hIg (control) or MR-1 on days −2, 0, 2, and 4. On day −1 mice were given i. p. injections of TNF-α (XT22 and XT3), IFN-γ/α (XMG1.2), or recombinant Ig control Abs. On day 0 mice were infected i. v. with 1 × 106 CFU ActA− LM. All mice were also immunized i.v. with 30–35 × 106 OVA-loaded BALB/c splenocytes. Intracellular staining for IFN-γ was performed on splenocytes on day 7. A, The total number of OVA257–264-specific CD8+ T cells in the spleen was calculated. Data are the mean ± SD of three independent experiments. α-, Anti-. LOD, limit of detection.

Discussion

Here we examine the influence of CD40L-CD40 interactions on CD8+ T cell priming against exogenous Ags during LM infection. Although it is accepted that the maturation of DCs is required for priming of CD8+ T cells against exogenous Ags, the signals required for this maturation in vivo are unknown. LPS, bacterial DNA, CD40 stimulation, and TNF-α have all been shown to result in the maturation of DCs in vitro, and anti-CD40 Abs have been shown to promote CD8+ T cell priming against exogenous model Ags in vivo in the absence of CD4+ T cells (3, 7).

Although MR-1 treatment prevented CD8+ T cell cross-priming against OVA-loaded splenocytes, it did not affect CD8+ T cell priming against nonsecreted or secreted LM Ags. It has been shown that CD4+ T cells or CD40L are not required for CD8+ T cell priming under conditions of viral infection and that there is minimal impact on the splenic CD8+ T cell response to secreted LM Ags in CD40−/− mice (8, 12). However, these studies did not address the issue of priming against Ags that do not have access to endogenous MHC class I processing pathways. It has additionally been demonstrated that memory CD8+ T cell responses decline in the absence of CD40L. Although we saw a significant decline in the CD8+ T cell memory response to NP118–126, secondary responses to both NP118–126 and LLO91–99 were unaffected in MR-1-treated mice in our system. Since our MR-1 treatment was only performed transiently during the initial exposure to Ag, it is unknown whether constant blockade of CD40L in these animals would affect primary or memory CD8+ T cell responses after LM infection.

Consistent with the idea that immunization with model Ags may not reflect the host response to infection, we found that both virulent and attenuated LM could overcome CD40L blockade of CD8+ T cell cross-priming. In addition, it was consistently seen that hIg-treated animals exhibited increased CD8+ T cell responses in the presence of infection ( Figs. 3–5⇑⇑⇑). Cross-presentation requires bone marrow-derived DCs, which were recently reported to be of the CD8α+ or lymphoid lineage (33). An interesting hypothesis is that the increase in CD8+ T cell priming against OVA in the presence of infection is due to an increase in the number of cells or types of cells (i.e., CD8α−) able to participate in cross-priming as effective APCs. Alternatively, when both CD40L and microbial signals are present, DCs show augmented IL-12 production in vivo, a situation that could result in increased T cell responses (34).

In contrast, LLO− LM, which have a deletion in the hly gene, overcame CD40L blockade less efficiently in the majority of MR-1-treated animals. It is likely that the host response to LLO− LM is different from the response to other strains of LM. Infection with LLO− LM elicits lower IL-12 production compared with virulent LM, which may result in a cytokine environment that is not optimal for CD8+ T cell cross-priming in the presence of MR-1 (35, 36). Previous experiments show that IL-12 is required in at least some models of exogenous Ag presentation, although the requirement for this cytokine in cross-presentation has not been examined (37). Recent evidence also demonstrated that CD40-mediated, CD8+ T cell priming against exogenously processed heat-killed LM was dependent on endogenous IL-12 production (38).

The mechanism by which LM infection overcomes CD40L blockade of cross-priming is still unknown. However, there is a clear requirement for active infection, since the injection of large numbers of heat-killed bacteria (Fig. 4⇑B) failed to stimulate measurable CD8+ T cell priming against OVA-loaded splenocytes or LM Ags. This indicates that the presence of bacterial products such as the cell wall or bacterial DNA was not sufficient to overcome CD40L blockade at least at the levels tested. The ability of LM infection to overcome CD40L blockade was not inhibited by treatment with mAbs that efficiently neutralize IFN-γ or TNF, illustrating that multiple signals generated during bacterial infection may stimulate the maturation of DCs. The depletion of TNF or IFN-γ also does not prevent naive CD8+ T cell priming against secreted or nonsecreted LM Ags in the presence or the absence of MR-1 (S. E. Hamilton and J. T. Harty, unpublished observations). Evidence has accumulated demonstrating the stimulatory properties of members of the TNF ligand superfamily (4-1BBL, OX40L, TNF-related apoptosis-inducing ligand, and TNF-related activation-induced cytokine) on DCs (39). 4-1BBL can influence CD4+ T cell and CD8+ T cell responses, particularly under suboptimal antigenic stimulation (40). Based on our results, it would be of interest to examine the effect of CD40L blockade on cross-priming in 4-1BBL−/− mice in the presence or the absence of LM infection and/or OVA-loaded splenocyte immunization. LM can also infect DCs themselves, suggesting that this could also be route to induce maturation during infection (41). This may be particularly relevant in experiments in which TNF was neutralized in mice resulting in increased levels of infection. Alternatively, another soluble molecule, such as IL-1, could be important for the maturation of dendritic cells during bacterial infection.

How cross-presentation results in either priming or tolerance of CD8+ T cells is still under debate. Cross-presentation is a critical mechanism both for the induction of peripheral tolerance and for immune responses to Ags without obvious access to the cytosol of APCs (reviewed in Ref. 5). Our results support a model in which tolerance is the likely result when the APC environment is lacking in inflammatory stimuli. In this situation the APC would take up and present Ag, but would lack the costimulatory molecules required to effectively stimulate naive T cells. However, under conditions of infection or inflammation, changes in the APC environment, such as cytokine secretion or pathogen products, would promote maturation of DCs for effective stimulation of naive T cells. This poses a potential problem for the host, since the uptake of peripheral self Ags would presumably continue in a T cell stimulatory environment and increase the likelihood of autoimmunity. This is consistent with models of autoimmunity in which conditions often worsen during concurrent infections (42). How tolerance to self Ags can be maintained during infection requires further investigation.

Acknowledgments

We thank J. Cowdery, T. Waldschmidt, and R. Lynch for reagents. We also thank S. Perlman for critical reading of the manuscript.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grants AI42767, AI46653, and AI50073 (to J.T.H.). S.E.H. was supported by Predoctoral Training Program in Immunology Grant 5T32A107485.

  • ↵2 Address correspondence and reprint requests to Dr. J. T. Harty, 3-512 Bowen Science Building, Department of Microbiology, University of Iowa, Iowa City, IA 52242. E-mail address: john-harty{at}uiowa.edu

  • ↵3 Abbreviations used in this paper: CD40L, CD40 ligand; DC, dendritic cell; hIg, hamster Ig; LCMV, lymphocytic choriomeningitis virus; LLO, listeriolysin O; LM, Listeria monocytogenes; NP, nucleoprotein.

  • Received July 6, 2001.
  • Accepted September 10, 2001.

References

  1. ↵
    Schoenberger, S. P., R. E. M. Toes, E. I. H. van der Voort, R. Offringa, C. J. M. Melief. 1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393: 480
    OpenUrlCrossRefPubMed
  2. ↵
    Bennett, S. R. M., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. A. P. Miller, W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393: 478
    OpenUrlCrossRefPubMed
  3. ↵
    Ridge, J. P., F. Di Rosa, P. Matzinger. 1998. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 393: 474
    OpenUrlCrossRefPubMed
  4. ↵
    Bevan, M. J.. 1976. Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross react in the cytotoxic assay. J. Exp. Med. 143: 1283
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Heath, W. R., F. R. Carbone. 2001. Cross-presentation, dendritic cells, tolerance and immunity. Annu. Rev. Immunol. 19: 47
    OpenUrlCrossRefPubMed
  6. ↵
    Caux, C., C. Massacrier, B. Vanbervliet, B. Dubois, C. Van Kooten, I. Durand, J. Banchereau. 1994. Activation of human dendritic cells through CD40 cross-linking. J. Exp. Med. 180: 1263
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Banchereau, J., R. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392: 245
    OpenUrlCrossRefPubMed
  8. ↵
    Whitmire, J. K., R. A. Flavell, I. S. Grewal, C. P. Larsen, T. C. Pearson, R. Ahmed. 1999. CD40-CD40 ligand costimulation is required for generating antiviral CD4 T cell responses but is dispensable for CD8 T cell responses. J. Immunol. 163: 3194
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Andreasen, S. O., J. E. Christensen, O. Marker, A. R. Thomsen. 2000. Role of CD40 ligand and CD28 in induction and maintenance of antiviral CD8+ effector T cell responses. J. Immunol. 164: 3689
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Borrow, P., A. Tishon, S. Lee, J. Xu, I. S. Grewal, M. B. A. Oldstone, R. A. Flavell. 1996. CD40L-deficient mice show deficits in antiviral immunity and have an impaired memory CD8+ CTL response. J. Exp. Med. 183: 2129
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Grewal, I. S., P. Borrow, E. G. Pamer, M. B. A. Oldstone, R. A. Flavell. 1997. The CD40-CD154 system in anti-infective host defense. Curr. Opin. Immunol. 9: 491
    OpenUrlCrossRefPubMed
  12. ↵
    Pope, C., S. Kim, A. Marzo, K. Williams, J. Jiang, H. Shen, L. Lefrancois. 2001. Organ-specific regulation of the CD8 T cell response to Listeria monocytogenes infection. J. Immunol. 166: 3402
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Cossart, P., M. F. Vicente, J. Mengaud, F. Baquero, D. J. C. Perez, P. Berche. 1989. Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation. Infect. Immun. 57: 3629
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Shen, H., J. F. Miller, X. Fan, D. Kolwyck, R. Ahmed, J. T. Harty. 1998. Compartmentalization of bacterial antigens: differential effects on priming of CD8 T cells and protective immunity. Cell 92: 535
    OpenUrlCrossRefPubMed
  15. ↵
    Brundage, R. A., G. A. Smith, A. Camilli, J. A. Theriot, D. A. Portnoy. 1993. Expression and phosphorylation of the Listeria monocytogenes ActA protein in mammalian cells. Proc. Natl. Acad. Sci. USA 90: 11890
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Jones, S., D. Portnoy. 1994. Characterization of Listeria monocytogenes pathogenesis in a strain expression perfringolysin O in place of listeriolysin O. Infect. Immun. 62: 5608
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Harty, J. T., M. J. Bevan. 1995. Specific immunity to Listeria monocytogenes in the absence of IFNγ. Immunity 3: 107
    OpenUrl
  18. ↵
    Moore, M. W., F. R. Carbone, M. J. Bevan. 1988. Introduction of soluble protein into the class I pathway of antigen processing and presentation. Cell 54: 777
    OpenUrlCrossRefPubMed
  19. ↵
    Tvinnereim, A. R., J. T. Harty. 2000. CD8+ T cell priming against a non-secreted Listeria monocytogenes antigen is independent of the anti-microbial activities of IFN-γ. Infect. Immun. 68: 2196
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Noelle, R. J., M. Roy, D. M. Shepherd, I. Stamenkovic, J. A. Ledbetter, A. Arrufo. 1992. A novel ligand on activated T helper cells binds CD40 and transduces the signal for the cognate activation of B cells. Proc. Natl. Acad. Sci. USA 89: 6550
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Abrams, J., M. Roncarolo, H. Yssel, U. Andersson, G. Gleich, J. Silver. 1992. Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples. Immunol. Rev. 127: 5
    OpenUrlCrossRefPubMed
  22. ↵
    Cherwinski, H., J. Schumacher, K. Brown, T. Mosmann. 1987. Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies. J. Exp. Med. 166: 1229
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Harty, J. T., M. J. Bevan. 1992. CD8+ T cells specific for a single nonamer epitope of Listeria monocytogenes are protective in vivo. J. Exp. Med. 175: 1531
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Foy, T. M., J. D. Laman, J. A. Ledbetter, A. Aruffo, E. Claassen, R. J. Noelle. 1994. gp39-CD40 interactions are essential for germinal center formation and the development of B cell memory. J. Exp. Med. 180: 157
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Badovinac, V. P., J. T. Harty. 2000. Intracellular staining for TNF and IFN-γ detects different frequencies of antigen-specific CD8+ T cells. J. Immunol. Methods 238: 107
    OpenUrlCrossRefPubMed
  26. ↵
    White, D., A. MacNeil, D. Busch, I. Pilip, E. Pamer, J. Harty. 1999. Perforin-deficient CD8+ T cells: in vivo priming and antigen specific immunity against Listeria monocytogenes. J. Immunol. 162: 980
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Badovinac, V. P., A. R. Tvinnereim, J. T. Harty. 2000. Regulation of antigen-specific CD8+ T cell homeostasis by Perforin and IFN-γ. Science 290: 1354
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Gaillard, J., P. Berche, J. Mounier, S. Richard, P. Sansonetti. 1987. In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2. Infect. Immun. 55: 2822
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Portnoy, D., P. Jacks, D. Hinrichs. 1988. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J. Exp. Med. 167: 1459
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Sallusto, F., A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor α. J. Exp. Med. 179: 1109
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Green, E. A., F. S. Wong, K. Eshima, C. Mora, R. A. Flavell. 2000. Neonatal tumor necrosis factor α promotes diabetes in nonobese diabetic mice by CD154-independent antigen presentation to CD8+ T cells. J. Exp. Med. 191: 225
    OpenUrlAbstract/FREE Full Text
  32. ↵
    White, D. W., J. T. Harty. 1998. Perforin-deficient CD8+ T cells provide immunity to Listeria monocytogenes by a mechanism that is independent of CD95 and IFN-γ but requires TNF-α. J. Immun. 160: 898
    OpenUrlAbstract/FREE Full Text
  33. ↵
    den Haan, J. M. M., S. M. Lehar, M. J. Bevan. 2000. CD8+ but not CD8− dendritic cells cross-prime cytotoxic T cells in vivo. J. Exp. Med. 192: 1685
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Schulz, O., A. D. Edwards, M. Schito, J. Aliberti, S. Manickasingham, A. Sher, C. Reis e Sousa. 2000. CD40 triggering of heterodimeric IL-12 p70 production by dendritic cells in vivo requires a microbial priming signal. Immunity 13: 453
    OpenUrlCrossRefPubMed
  35. ↵
    Rudnicka, W., M. Kaczmarek, J. Szeliga, T. Germann, M. Wieckowska, B. Rozalska. 1997. The host response to Listeria monocytogenes mutants defective in genes encoding phospholipases C (plcA, plcB) and actin assembly (actA). Microbiol. Immunol. 41: 847
    OpenUrlCrossRefPubMed
  36. ↵
    Locksley, R. M.. 1993. Interleukin-12 in host defense against microbial pathogens. Proc. Natl. Acad. Sci. USA 90: 5879
    OpenUrlFREE Full Text
  37. ↵
    Wild, J., M. Grusby, R. Schirmbeck, J. Reimann. 1999. Priming MHC-I-restricted cytotoxic T lymphocyte responses to exogenous hepatitis B surface antigen is CD4+ T cell dependent. J. Immunol. 163: 1880
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Rolph, M. S., S. H. E. Kaufmann. 2001. CD40 signaling converts a minimally immunogenic antigen into a potent vaccine against the intracellular pathogen Listeria monocytogenes. J. Immunol. 166: 5115
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Anderson, D. M., E. Maraskovsky, W. L. Billingsley, W. C. Dougall, M. E. Tometsko, E. R. Roux, M. C. Teepe, R. F. DuBose, D. Cosman, L. Galibert. 1997. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390: 175
    OpenUrlCrossRefPubMed
  40. ↵
    Cannons, J., P. Lau, B. Ghumman, M. DeBenedette, H. Yagita, K. Okumura, T. Watts. 2001. 4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy. J. Immunol. 167: 1313
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Guzman, C., M. Rohde, T. Chakraborty, E. Domann, M. Hudel, J. Wheland, K. Timmis. 1995. Interaction of Listeria monocytogenes with mouse dendritic cells. Infect. Immun. 63: 3665
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Noseworthy, J. H., C. Lucchinetti, M. Rodriguez, B. G. Weinshenker. 2000. Multiple sclerosis. N. Engl. J. Med. 343: 938
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section