The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Subauste, C. S.
Right arrow Articles by Wessendarp, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Subauste, C. S.
Right arrow Articles by Wessendarp, M.
The Journal of Immunology, 2000, 165: 1498-1505.
Copyright © 2000 by The American Association of Immunologists

Human Dendritic Cells Discriminate Between Viable and Killed Toxoplasma gondii Tachyzoites: Dendritic Cell Activation After Infection with Viable Parasites Results in CD28 and CD40 Ligand Signaling That Controls IL-12-Dependent and -Independent T Cell Production of IFN-{gamma}1

Carlos S. Subauste2 and Matthew Wessendarp

Division of Infectious Diseases, University of Cincinnati College of Medicine, Cincinnati, OH 45267


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We studied how the interaction between human dendritic cells (DC) and Toxoplasma gondii influences the generation of cell-mediated immunity against the parasite. We demonstrate that viable, but not killed, tachyzoites of T. gondii altered the phenotype of immature DC. DC infected with viable parasites up-regulated the expression of CD40, CD80, CD86, and HLA-DR and down-regulated expression of CD115. These changes are indicative of DC activation induced by T. gondii. Viable and killed tachyzoites had contrasting effects on cytokine production. DC infected with viable T. gondii rather than DC that phagocytosed killed parasites induced secretion of high amounts of IFN-{gamma} by T cells from T. gondii-seronegative donors. IFN-{gamma} production in response to DC infected with viable parasites required CD28 and CD40 ligand (CD40L) signaling. In addition, this IFN-{gamma} response was dependent in part on IL-12 secretion. Production of IL-12 p70 occurred after interaction between T cells and DC infected with viable T. gondii, but not after incubation of T cells with DC plus killed tachyzoites. IL-12 synthesis was inhibited by blockade of CD40L signaling. IL-12-independent IFN-{gamma} production required CD80/CD86-CD28 interaction and, to a lesser extent, CD40-CD40L signaling. Taken together, T. gondii-induced activation of human DC is associated with T cell production of IFN-{gamma} through CD40-CD40L-dependent release of IL-12 and through CD80/CD86-CD28 and CD40-CD40L signaling that mediate IFN-{gamma} secretion even in the absence of bioactive IL-12.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cell-mediated immunity that results in IL-12 and IFN-{gamma} secretion is required for control of intracellular pathogens (1). Identification of the events that regulate cytokine production during infection with these organisms is crucial to our understanding of the mechanisms that determine whether protective immunity is elicited. APC should receive special attention, since events pivotal to the induction of protection against intracellular pathogens are those that transpire during the interaction between APC and T cells. In this regard, we have demonstrated that CD28-CD80/CD86 signaling regulates IFN-{gamma} production, and CD40 ligand (CD40L)3-CD40 interaction regulates IL-12 and IFN-{gamma} secretion during the cross-talk between T cells and monocytes infected with the intracellular protozoan Toxoplasma gondii (2, 3).

Dendritic cells (DC) are considered the APC responsible for the generation of primary immune responses (4). DC originate in the bone marrow and reach peripheral tissues through the blood. After encountering Ags and in response to inflammatory mediators, DC undergo a maturation process characterized by increased expression of MHC and costimulatory molecules (5, 6, 7, 8). These changes are accompanied by migration to T cell-dependent areas of secondary lymphoid organs where mature DC stimulate naive T cells (5, 9, 10). However, there is less information regarding how this process influences the generation of cell-mediated immunity against intracellular pathogens.

We have demonstrated that unprimed human T cells secrete IFN-{gamma} in response to T. gondii-infected APC (2, 3, 11). Therefore, the in vitro model of T. gondii infection is well suited to study how the interaction between DC and an intracellular pathogen affects the generation of T cell-mediated immunity. We demonstrate that live, but not killed, tachyzoites of T. gondii up-regulate CD40, CD80, CD86, and MHC class II molecules on human DC. In turn, DC activation is associated with the production of high amounts of IFN-{gamma} by T cells from T. gondii-seronegative donors through CD40-CD40L-dependent IL-12 secretion and through CD80/CD86-CD28 and CD40-CD40L interactions that act via a mechanism that does not require IL-12.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and cytokines

The following mAbs were used for cell purifications: anti-CD2, anti-CD3, anti-CD8, anti-CD56 (all from Becton Dickinson, San Jose, CA), anti-CD11b (OKM1, American Type Culture Collection, Manassas, VA), anti-CD16 (Medarex, Annandale, NJ), anti-CD19 (Coulter, Hialeah, FL), and anti-glycophorin A (10F7 MN, gift from Rene de Waal Malefyt, DNAX Research Institute, Palo Alto, CA).

CTLA-4-Ig (gift from Bristol-Myers Squibb, Princeton, NJ) (12) and mAbs against CD40L (M90, gift from Immunex, Seattle, WA) were used in functional assays (all at 10 µg/ml). Isotype-matched mAbs and human IgG were obtained from PharMingen (San Diego, CA) and Sigma (St. Louis, MO), respectively. GM-CSF and IL-4 were purchased from PeproTech (Rocky Hill, NJ).

The following conjugated or unconjugated mAbs were used for flow cytometry (purchased from Becton Dickinson, except when indicated): FITC-anti-CD3, FITC-anti-CD14, FITC-anti-CD19 (Caltag, South San Francisco, CA), FITC-anti-CD40 (PharMingen), FITC-anti-CD56, FITC-anti-HLA-DR (Caltag), PE-anti-CD80, PE-anti-CD86 (PharMingen), PE-anti-HLA-DR (Caltag), unconjugated anti-CD80, unconjugated anti-CD83 (HB-15a, gift from Thomas Tedder, Duke University, Durham, NC) (13), and unconjugated rat anti-CD115 (M-CSF receptor, 2-4A5, Zymed, San Francisco, CA). FITC-F(ab')2 rabbit anti-mouse IgG (Serotec, Oxford, U.K.) and cyanin-5-F(ab')2 goat anti-rat IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA) were used as secondary Ab when necessary. Unrelated murine mAbs (PharMingen) and rat IgG2a (Zymed) were used as negative controls.

Cell purifications

Using centrifugation on Ficoll-Hypaque gradients (Pharmacia LKB Biotechnology, Piscataway, NJ), PBMC were isolated from buffy coats of heparinized blood of healthy volunteers donors obtained from the Hoxworth Blood Center (Cincinnati, OH). Serologic tests for detection of anti-T. gondii IgG and IgM were performed in all samples of blood. Unless otherwise sated, the samples used had no demonstrable T. gondii IgG or IgM Abs.

Monocyte-derived DC (md-DC) were obtained as described previously (14, 15). Briefly, purified monocytes (1 x 106/ml) isolated as previously described (2) were incubated in complete medium (CM) consisting of RPMI 1640 with 10% FBS (HyClone, Logan, UT) that contained 1000 U/ml GM-CSF and 500 U/ml IL-4. Cytokines were replenished every 3–4 days. Cells were used after 7 days of in vitro culture.

Blood DC (b-DC) were obtained following a modification of a previously described protocol (16). PBMC were incubated with neuraminidase-treated SRBC. Nonrosetting cells were treated with anti-CD3, anti-CD8, anti-CD11b, anti-CD16, anti-CD19, and anti-glycophorin A mAb followed by incubation with magnetic beads coated with anti-mouse IgG (PerSeptive Biosystems, Framingham, MA). Populations obtained after removal of rosetting cells with a magnet contained >90% b-DC as defined by previously established criteria (HLA-DR+, CD3- CD14-/low CD19- CD56- cells) (16) and contained <4% monocytes (CD14high). Resting T cells (>99% CD3+) were obtained as previously described (2). Cells were cultured in RPMI 1640 with 10% dye test-negative human AB serum (Gemini Bio-Products, Calabasas, CA). b-DC were not used in experiments that addressed T. gondii-induced DC activation and the effects of different parasite preparations on cytokine secretion, because b-DC spontaneously undergo maturation in vitro.

T. gondii and infection

Tachyzoites of the RH strain were obtained from infected monolayers of human foreskin fibroblasts as well as from peritoneal fluid of mice (17). DC were infected with T. gondii and cultured in Teflon vessels as previously described (2). The dose of tachyzoites per DC was 0.5/1 when studying cytokine production and 2/1 for phenotypic analysis. Neither uninfected human foreskin fibroblasts nor tachyzoite-free peritoneal lavage fluids from infected mice (after passage through a 0.45-µm pore size filter) mediated changes in the expression of the surface molecules tested. In certain experiments tachyzoites were killed by incubation in 1% paraformaldehyde in PBS (17). T. gondii lysate Ag (TLA) was prepared as described previously (2) and used at 10 µg/ml. Antigenic preparations were devoid of detectable levels of endotoxin (<0.015 EU/ml) using a Limulus amebocyte lysate assay (Sigma). The percentage of cells with intracellular tachyzoites was determined by light microscopy (2).

Flow cytometry

Cells were incubated for 30 min with 250 µg/ml human IgG (Sigma) to block Fc receptors. This was followed by 30-min incubation at 4°C with unconjugated mAb or isotype control Ab in PBS containing 1% FBS and 0.1% sodium azide. Cells were washed and counterstained with the appropriate conjugated secondary Ab. After blocking with mouse IgG, cells were stained with conjugated mAbs. Cells were fixed in 1% paraformaldehyde and analyzed using a FACSCalibur (Becton Dickinson). Corrected mean fluorescence intensity (MFI) was calculated by subtracting the MFI of the appropriate isotype control mAb from the MFI of each specific mAb. Sorting of DC into CD115- CD86high and CD115+ CD86int was performed after 18-h incubation with T. gondii.

Cytokine assays

Purified resting peripheral blood T cells were incubated in 96-well plates with either uninfected or T. gondii-infected DC. Concentrations of T cells and DC were 1 x 106/ml and 2.5 x 105/ml, respectively, when studying IFN-{gamma} production and 2 x 106/ml and 5 x 105/ml, respectively, for assays of IL-12 secretion. Abs were added to DC 30 min before incubation with T cells. Concentrations of IL-12 (p40 or p70; R&D Systems, Minneapolis, MN) and IFN-{gamma} (Endogen, Cambridge, MA) were measured by ELISA in supernatants collected at 24 and 72 h, respectively. The lower limit of detection was 39 pg/ml for IFN-{gamma} and IL-12 p40, and 0.6 pg/ml for IL-12 p70. The data in the figures are presented as the mean of triplicate wells ± SEM. In addition, the percent inhibition of cytokine production was calculated in each experiment that examined the effects of neutralizing Ab. The mean percent inhibition ± SEM of all comparable experiments are shown in the text.

Statistical analysis

Statistical significance was assessed by ANOVA and Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Viable, but not killed, T. gondii tachyzoites activate human DC

DC generated after culturing monocytes with GM-CSF and IL-4 (md-DC) exhibit a phenotype characteristic of immature DC (CD115+ CD80low CD86int CD83-) (18, 19). These cells were used to determine whether T. gondii alters the phenotype of human DC. Incubation with viable tachyzoites resulted in the appearance of a subpopulation of md-DC that exhibited down-regulation of the macrophage marker CD115 (Fig. 1Go). Loss of CD115 expression was accompanied by up-regulation of CD80 and CD86. On the average, CD80high CD115- and CD86high CD115- md-DC had 2.6 ± 0.4 times higher expression of CD80 (MFI, 42.4 ± 4.3 vs 17.4 ± 3.5) and 1.7 ± 0.2 times higher expression of CD86 (MFI, 576.4 ± 32.3 vs 335.3 ± 56.9) than control md-DC (n = 10). Simultaneous staining with anti-CD80, anti-CD86, and anti-CD115 mAbs indicated that CD80high CD115- were also CD86high (data not shown).



View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 1. T. gondii up-regulates DC expression of CD80 and CD86. After 48 h of in vitro culture, md-DC were subjected to two-color FACS analysis using anti-CD115 mAb plus cyanin-5-goat anti-rat (GAR) and either PE-anti-CD80 or PE-anti-CD86 mAbs. The box within the contour graph and the percentage indicate either CD115- CD80high or CD115- CD86high DC. The results of one representative experiment of 10 are shown.

 
Because CD40 and HLA-DR are required for optimal T cell responses against T. gondii (3, 11), we analyzed the expression of these surface molecules on md-DC incubated with or without T. gondii. To this end, DC were stained with anti-CD115 plus anti-CD86 mAbs and either anti-CD40 or anti-HLA-DR mAbs. As shown in Table IGo, CD115- CD86high md-DC induced by incubation with viable tachyzoites up-regulated HLA-DR and CD40 (n = 7).


View this table:
[in this window]
[in a new window]
  		Table I. Viable T. gondii up-regulates CD40 and HLA-DR on md-DCa

 
Next, we studied whether viability of tachyzoites was required for the induction of phenotypic changes on DC. In contrast to results obtained with viable tachyzoites, incubation of md-DC with killed T. gondii failed to alter the expression of CD80, CD86, and CD115 (Fig. 2Go). Similarly, killed tachyzoites did not up-regulate DC expression of CD40 and HLA-DR (Table IGo). These results were not caused by an inability of md-DC to internalize killed tachyzoites, because the percentages of DC with intracellular tachyzoites after 2-h incubation with either viable or killed parasites were 40 ± 4 and 24 ± 1%, respectively (n = 3). Increasing the dose of killed T. gondii 2-fold failed to alter the md-DC phenotype. Therefore, the changes in expression of these surface molecules appeared unlikely to be due simply to phagocytosis of T. gondii. Taken together, incubation with viable, but not killed, tachyzoites induces DC activation.



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 2. Viable, but not killed, T. gondii tachyzoites up-regulate CD80 and CD86 on DC. The md-DC were incubated with viable tachyzoites of T. gondii (Tg), killed T. gondii (kTg), or CM alone (Control). The percentage of CD115- CD80high and CD115- CD86high md-DC were determined by flow cytometry. Killed T. gondii failed to alter DC phenotype even when using a concentration of parasites 2-fold higher than that of viable tachyzoites. The results shown represent data pooled from four independent experiments.

 
DC maturation is associated with induction of CD83 (13, 19, 20). Therefore, md-DC were stained with anti-CD83, anti-CD86, and anti-CD115 mAbs to determine whether T. gondii up-regulates CD83. Although T. gondii-induced CD115- CD86high md-DC could acquire CD83 expression (19.3 ± 3.7% CD83+ compared with 1.9 ± 0.4% CD83+ cells among CD115+ CD86int DC), induction of CD83 occurred in only three of six independent experiments (data not shown).

Experiments were conducted to further explore the role of T. gondii infection on DC activation. The md-DC incubated with viable T. gondii were sorted by FACS into CD115- CD86high and CD115+ CD86int cells. Microscopic examination of these cells revealed that whereas 51 ± 0.7% of CD86high CD115- md-DC had intracellular tachyzoites, only 1 ± 0.2% of CD86int CD115+ md-DC contained intracellular T. gondii (n = 3). Thus, infection with viable T. gondii leads to DC activation.

Infection of DC with viable tachyzoites is required to trigger optimal IFN-{gamma} production by T cells from T. gondii-seronegative individuals

We studied whether the contrasting effects of viable and killed parasite preparations on DC activation were associated with differences in cytokine production. Whereas T cells from T. gondii-seronegative individuals secreted high amounts of IFN-{gamma} when incubated with DC infected with viable T. gondii, stimulation of T cells with DC plus killed tachyzoites resulted in the production of markedly lower concentrations of IFN-{gamma} (Fig. 3GoA, 2,847 ± 1,042 vs 331 ± 142 pg/ml, respectively; p < 0.01; n = 7). These differences in IFN-{gamma} production were not caused by dissimilarities in the percentages of DC with intracellular tachyzoites. The aforementioned experiments as well as all studies of cytokine production described below were conducted with a concentration of killed parasites that was 2-fold higher than that of viable tachyzoites. Thus, the percentages of DC with intracellular tachyzoites were 5.5 ± 0.4 and 4.7 ± 0.2 for DC incubated with viable or killed T. gondii, respectively. Moreover, the marked differences in IFN-{gamma} secretion did not appear to be due to an inability of T cells to recognize DC plus killed parasites, since in the presence of exogenous IL-12, T cells stimulated with DC plus either viable or killed tachyzoites produced high amounts of IFN-{gamma} (Fig. 3GoB). Finally, in contrast to T cells from T. gondii-seronegative donors, T cells from healthy individuals chronically infected with T. gondii secreted high amounts of IFN-{gamma} in response to DC plus either viable or killed tachyzoites (13,373 ± 1,202 and 11,962 ± 868 pg/ml, respectively; n = 3; data not shown).



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 3. T cells secrete optimal amounts of IFN-{gamma} after stimulation with DC incubated with viable, but not killed, T. gondii tachyzoites. A, Purified resting T cells (1 x 106/ml) from T. gondii-seronegative donors were incubated with md-DC (2.5 x 105/ml) with or without viable (Tg) or killed (kTg) tachyzoites of T. gondii. The concentration of killed T. gondii was twice that of viable T. gondii, so that percentages of DC with intracellular tachyzoites were equivalent in both groups. B, T cells and md-DC were cultured as described in A with or without rIL-12 (2.5 ng/ml). Supernatants were collected after 72 h and were used to measure concentrations of IFN-{gamma} by ELISA. The results shown represent one of three (B) to seven (A) independent experiments.

 
IL-12 p70 is secreted only after interaction between T cells and DC infected with viable T. gondii tachyzoites

The studies shown in Fig. 3GoB raised the possibility that differences in IFN-{gamma} secretion after T cells were stimulated with DC plus either viable or killed tachyzoites might be caused by differences in the production of bioactive IL-12. To begin to explore this possibility, we studied the effects of a neutralizing anti-IL-12 mAb on IFN-{gamma} production. Fig. 4Go shows that whereas anti-IL-12 mAb induced a significant inhibition of IFN-{gamma} secretion in response to md-DC infected with viable T. gondii (55.9 ± 2.0% inhibition; p < 0.01; n = 12; Fig. 4GoA), in parallel experiments this mAb did not affect the low level IFN-{gamma} production triggered by md-DC and killed tachyzoites (Fig. 4GoB). At the concentration of mAb used in these studies, anti-IL-12 mAb neutralized induction of IFN-{gamma} secretion mediated by addition of 1 ng of rIL-12 to T cells stimulated with either md-DC plus killed tachyzoites or monocytes plus TLA (94.8 ± 1.2% inhibition; p < 0.0001; n = 3; Fig. 4GoC). The lack of effect of anti-IL-12 mAb on IFN-{gamma} production caused by md-DC plus killed parasites was not due to a general inability to modulate cytokine secretion, because blockade of the CD28 and CD40L pathways abrogated IFN-{gamma} secretion (Fig. 4GoB). Thus, whereas IFN-{gamma} production in response to DC plus viable T. gondii is partially dependent on IL-12 secretion, IFN-{gamma} production triggered by DC plus killed tachyzoites is largely independent of bioactive IL-12 production.



View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 4. T cell production of IFN-{gamma} in response to DC infected with viable T. gondii is partially dependent on IL-12 secretion. A, T cells (1 x 106/ml) were incubated with md-DC (2.5 x 105/ml) that were either uninfected or infected with viable T. gondii. Neutralizing anti-IL-12 mAb or isotype control mAb were used at 10 µg/ml. B, T cells were incubated with uninfected md-DC or md-DC that phagocytosed killed T. gondii (kTg) in the presence of anti-IL-12 mAb, anti-CD40L mAb, CTLA-4-Ig, or mouse or human IgG. All Ab were used at 10 µg/ml. Samples with undetectable IFN-{gamma} are shown as having a cytokine concentration half the lower limit of detection of the ELISA. C, T cells were incubated with md-DC plus killed T. gondii with or without rIL-12 (1 ng/ml) in the presence of anti-IL-12 or isotype control mAbs. The results of one representative experiment of three (B and C) or 12 (A) are shown.

 
Next, we determined whether incubations with viable and killed tachyzoites have differential effects on the secretion of bioactive IL-12. Stimulation of T cells with md-DC infected with viable T. gondii resulted in IL-12 p70 production (Fig. 5Go). In contrast, no IL-12 p70 was detected after T cell stimulation with md-DC incubated either alone or with killed T. gondii. IL-12 p70 production required T cells, because md-DC incubated with viable parasites failed to release IL-12 p70 in the absence of T cells. Taken together, interaction between T cells and md-DC infected with viable T. gondii is required for IL-12 p70 secretion.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 5. IL-12 p70 is secreted during interaction between T cells and DC infected with viable T. gondii. The md-DC (5 x 105/ml)) incubated with or without viable (Tg) or killed tachyzoites (kTg) of T. gondii were cultured in the presence or the absence of T cells (2 x 106/ml). Supernatants were collected after 24 h and were used to measure concentrations of IL-12 p70 by ELISA. Samples with undetectable IL-12 p70 are shown as having a cytokine concentration half the lower limit of detection of the ELISA. The results shown represent one of three independent experiments.

 
CD28 and CD40L signaling regulate IFN-{gamma} production triggered by DC and T. gondii

We have previously demonstrated that the CD28 and CD40L pathways are crucial for IFN-{gamma} secretion by presumably unprimed T cells stimulated with T. gondii-infected monocytes (2, 3). The results described above indicate that T cell-APC cognate interaction also regulates IFN-{gamma} production by T cells stimulated with DC plus killed T. gondii. Therefore, we determined whether CD28 and CD40L signaling control IFN-{gamma} production in response to DC infected with viable T. gondii. As shown in Fig. 6GoA, IFN-{gamma} secretion by T cells from T. gondii-seronegative individuals was significantly inhibited by either anti-CD40L mAb (56.4 ± 3.1% inhibition; p < 0.03; n = 5), or CTLA-4-Ig (63.0 ± 7.2% inhibition; p < 0.03; n = 5). Moreover, simultaneous addition of these two molecules resulted in further inhibition of IFN-{gamma} secretion (83.5 ± 4.2% inhibition; p < 0.01; n = 5).



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 6. T cell production of IFN-{gamma} in response to T. gondii-infected DC is dependent on CD40-CD40L and CD80/CD86-CD28 signaling. T cells (1 x 106/ml) were incubated with md-DC (A) or b-DC (B and C; both at 2.5 x 105/ml) that were either uninfected or infected with viable T. gondii. Anti-IL-12 mAb, anti-CD40L mAb, CTLA-4-Ig, or mouse or human IgG was added as indicated. The results of one representative experiment of four (C) or five (A and B) are shown.

 
To confirm the relevance of CD28 and CD40L signaling for regulation of IFN-{gamma} secretion, experiments similar to those described above were performed using b-DC. Fig. 6GoB shows that IFN-{gamma} production in response to T. gondii-infected b-DC was significantly inhibited by either anti-CD40L mAb (52.3 ± 6.4% inhibition; p < 0.03; n = 5) or CTLA-4-Ig (69.1 ± 6.1% inhibition; p < 0.01; n = 5). Combination of these two molecules resulted in further inhibition of IFN-{gamma} secretion (80.5 ± 5.6% inhibition; p < 0.01; n = 5). In addition, similar to md-DC, IFN-{gamma} production in response to T. gondii-infected b-DC was significantly inhibited by anti-IL-12 mAb (50.5 ± 2.1% inhibition; p < 0.01; n = 4; Fig. 6GoC). Taken together, these data indicate that IFN-{gamma} secretion in response to T. gondii-infected DC is largely dependent on the CD28 and CD40L pathways.

Next, we examined whether CD28 and CD40L signaling modulate IL-12-independent IFN-{gamma} production triggered by DC plus viable T. gondii. Fig. 7Go shows that after neutralization of IL-12 (incubation with anti-IL-12 mAb), addition of anti-CD40L mAb to T cells stimulated with md-DC and viable tachyzoites resulted in a moderate inhibition of IFN-{gamma} production (30.9 ± 1.5% inhibition; p < 0.01; n = 4). Blockade of the CD80/CD86-CD28 interaction with CTLA-4-Ig induced marked inhibition of IFN-{gamma} secretion (65.3 ± 3.9% inhibition; p < 0.005; n = 4). Simultaneous incubation with anti-CD40L mAb and CTLA-4-Ig further impaired IFN-{gamma} production (80.1 ± 2.5; p < 0.001; n = 4). Thus, CD28 and, to a lesser extent, CD40L signaling control IL-12-independent IFN-{gamma} secretion in response to T. gondii-infected DC.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 7. The CD80/CD86-CD28 and CD40-CD40L pathways regulate IL-12-independent T cell production of IFN-{gamma} in response to T. gondii-infected DC. T cells (1 x 106/ml) were stimulated with either uninfected or T. gondii-infected md-DC (2.5 x 105/ml). Anti-IL-12, anti-CD40L mAbs, CTLA-4-Ig, and mouse or human IgG were added as indicated. The results shown represent one of four independent experiments.

 
IL-12 production during T cell-T. gondii-infected DC interaction is dependent on CD40L

In experiments parallel to those shown in Fig. 7Go, anti-CD40L mAb more prominently inhibited IFN-{gamma} production if IL-12 was not neutralized (61.7 ± 1.3% inhibition; n = 4; see Fig. 6GoA). These results suggested that CD40-CD40L signaling during cognate interaction between T cells and T. gondii-infected DC may also influence IFN-{gamma} secretion through regulation of IL-12 secretion. Therefore, we examined whether blockade of the CD40-CD40L pathway impairs IL-12 secretion triggered by T. gondii. Incubation of T cells with T. gondii-infected b-DC resulted in IL-12 p40 production (Fig. 8GoA). Addition of anti-CD40L mAb to these cells significantly inhibited IL-12 p40 secretion (70.4 ± 9.8% inhibition; p < 0.03; n = 3). To further confirm the role of CD40L signaling on IL-12 production, we studied the effects of anti-CD40L mAb on IL-12 p70 synthesis. Addition of anti-CD40L mAb to T cells incubated with md-DC infected with viable T. gondii remarkably inhibited IL-12 p70 secretion (86.6 ± 8.1% inhibition; p < 0.01; n = 2; Fig. 8GoB). Thus, T cell-dependent IL-12 production by T. gondii-infected DC is controlled by CD40-CD40L signaling.



View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 8. The CD40-CD40L pathway regulates IL-12 production during the interaction between T cells and T. gondii-infected DC. Uninfected or T. gondii-infected b-DC (A) or md-DC (B; all at 5 x 105/ml) were incubated either alone or in the presence of T cells (2 x 106/ml). Supernatants were collected after 24 h and were used to measure concentrations of either IL-12 p40 (A) or IL-12 p70 (B) by ELISA. Samples with undetectable IL-12 are shown as having a cytokine concentration half the lower limit of detection of the ELISA. Results of one representative experiment of two (B) or three (A) are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Inasmuch as DC appear to be the APC mainly responsible for generation of primary T cell responses (4), we considered it important to study how the interaction between DC and the intracellular protozoan T. gondii affects the generation of cell-mediated immunity against this pathogen. We have established that infection with viable tachyzoites, but not phagocytosis of killed parasites, induced activation of human DC. This process was accompanied by a strong T cell production of IFN-{gamma}, a cytokine crucial for protection against T. gondii (21, 22). Our studies indicate that T cell-T. gondii-infected DC cognate interaction is pivotal for IFN-{gamma} production in response to the parasite. Secretion of this cytokine is regulated by CD40-CD40L-dependent IL-12 production. In addition, CD80/CD86-CD28 and CD40-CD40L interactions control IFN-{gamma} synthesis through a mechanism that is operative even after neutralization of bioactive IL-12.

We report that incubation of immature DC with viable, but not killed, tachyzoites induced the appearance of CD115- CD40high CD80high CD86high HLA-DRhigh DC, a phenotype indicative of DC activation. Only 51% of activated md-DC contained intracellular tachyzoites, which were at different stages of degeneration. It remains to be determined whether human md-DC kill intracellular T. gondii. Such a finding would explain why not all activated dendritic cells contain intracellular parasites 18 h after infection. Of relevance to the T. gondii-induced md-DC activation is our demonstration that T. gondii-infected human monocytes also up-regulate the expression of CD40, CD80, CD86, and HLA-DR (2, 3). Certain microbes (Gram-positive and Gram-negative bacteria, mycobacteria, and measles virus) as well as LPS, CD40L, and cytokines such as TNF-{alpha} and IL-1ß can induce DC maturation (7, 23, 24, 25, 26, 27, 28, 29). Although T. gondii up-regulates costimulatory ligands and HLA-DR expression on DC, the parasite may be unable to optimally induce the maturation marker CD83. However, full DC maturation (as defined by CD83 expression) may be achieved during T cell-DC interaction (30). Nevertheless, the capacity of human DC to up-regulate costimulatory ligands and MHC molecules after encountering viable, but not killed, preparations of T. gondii suggests that the parasite-DC interaction may influence the nature of the ensuing immune response.

IL-12 is a cytokine pivotal for control of T. gondii infection (31, 32, 33). We demonstrated that human DC secrete IL-12 p70 in response to viable, but not killed, T. gondii tachyzoites. However, IL-12 p70 was produced only after T cells were added to T. gondii-infected DC. Pertinent to these results are the reports that cognate interaction between human T cells and T. gondii-infected APC regulates IL-12 p40 secretion through CD40-CD40L signaling (3, 34). In contrast to studies in humans, experiments performed in mice indicate that in vitro production of IL-12 p40 by spleen cells can occur in the absence of T cells, and that i.v. administration of T. gondii-soluble Ags to mice results in a CD40L-independent transient in vivo production of IL-12 by DC (35). These contrasting results may be caused by host-related differences in the immune response to T. gondii.

We have demonstrated that IL-12 p70 production takes place after interaction between T. gondii-infected activated DC and T cells. Although it has been reported that only mature DC secrete IL-12 p70 (24), recent studies indicate that DC maturation does not necessarily imply that these cells will produce IL-12 (36). DC induced to mature in the presence of PGE2 show impaired production of IL-12 p70 and promote the secretion of Th2-type cytokines by naive Th cells (36). Thus, it has been proposed that rather than maturation per se, the types of events that lead to DC maturation determine the polarizing capacity of DC (36). The nature of the signals triggered by T. gondii infection that cause DC activation remains to be characterized. Identification of the mechanisms through which T. gondii induces DC activation is likely to explain at least in part why this pathogen is a potent inducer of IL-12/IFN-{gamma} secretion.

Although IL-12 is crucial for regulation of IFN-{gamma} secretion, our studies indicate that the IFN-{gamma} secretion by T cells stimulated with T. gondii-infected DC is in part independent of IL-12. These results are unlikely to be due to partial neutralization of IL-12. In parallel experiments, the anti-IL-12 mAb used in these studies neutralized the effect of 1 ng of rIL-12, a concentration far greater than the amount of IL-12 p70 secreted during T cell-T. gondii-infected DC interaction. Of relevance is the report that mice infected with the ts-4 strain of T. gondii secrete IFN-{gamma} in an IL-12-independent manner (37). Moreover, this study suggested that class II-restricted T cells are involved in the IL-12-independent secretion of IFN-{gamma} (37). It is interesting to point out that the in vitro reactivity to T. gondii of unprimed human {alpha}ß T cells lies within the CD4+ subset, and that this response requires MHC class II molecules (11). Thus, these similarities suggest an in vivo correlate to our results. IL-12-independent pathways for IFN-{gamma} and Th1-type cytokine response have also been reported in murine models of viral infections (38, 39).

T. gondii mediates up-regulation of CD80, CD86, and CD40 on DC, a phenomenon that is likely to have important implications for the initiation of a T cell response against the pathogen. Indeed, blockade of the CD28 and CD40L pathways inhibits IFN-{gamma} production in response to T. gondii-infected DC. Thus, our results suggest that these signaling pathways play an important role in the generation of protective immunity against the parasite in humans. Studies in CD28 and in CD40L knockout mice indicate that these animals are resistant to acute infection with the ME49 strain of T. gondii (40). It remains to be determined whether these results represent the development of compensatory mechanisms in the form of costimulation provided by alternative signaling pathways. Nevertheless, splenocytes from T. gondii-infected CD28 knockout mice exhibit impaired secretion of IFN-{gamma} in response to T. gondii, and these animals show increased susceptibility to rechallenge with a virulent strain of T. gondii (40). In contrast to animal studies, data in humans reveal that patients with hyper-IgM syndrome, an immunodeficiency caused by lack of functional CD40L, exhibit impaired IL-12 and IFN-{gamma} production in response to T. gondii (3). Moreover, these patients are at risk for the development of toxoplasmic encephalitis and disseminated toxoplasmosis (41, 42, 43). Therefore, the association between hyper-IgM syndrome and toxoplasmosis supports the idea that events that occur during the cognate interaction between T cells and APC (at least in the form of CD40-CD40L signaling) are important for the control of T. gondii infection in humans.

The demonstration that T cell production of IFN-{gamma} in response to T. gondii-infected DC is regulated by both IL-12 and CD28 signaling is in agreement with the roles of these molecules in IFN-{gamma} production in response to PHA (44) and the synergistic effects of IL-12 and CD28 stimulation on T cell cytokine secretion and proliferation (45, 46). Our studies revealed that CD28 costimulation also regulates IL-12-independent T cell secretion of IFN-{gamma} in response to T. gondii (stimulation with DC plus viable T. gondii in the presence of anti-IL-12 mAb and stimulation with DC plus killed T. gondii). These results suggest that CD28 directly mediates T cell production of IFN-{gamma} in response to T. gondii. Indeed, after neutralization of IL-12, a stimulatory anti-CD28 mAb increases IFN-{gamma} secretion by PBMC incubated with PHA (44). Moreover, increased CD28-mediated costimulation can influence T cell cytokine production by preferentially promoting IFN-{gamma} over IL-4 secretion (47).

CD40-CD40L interaction is not only involved in cytokine secretion by APC (48, 49, 50), but also modulates T cell function (51, 52, 53, 54). Thus, the pleiotropic nature of this signaling pathway raised the possibility that CD40-CD40L interaction regulates IFN-{gamma} secretion in response to T. gondii-infected DC through multiple mechanisms. Our results indicate that during the cross-talk between presumably unprimed human T cells and T. gondii-infected DC, CD40-CD40L signaling controls IFN-{gamma} production through induction of IL-12 secretion. Of relevance is the demonstration that blockade of this pathway impairs IL-12 secretion when PBMC are incubated with T. gondii and when T cells from chronically infected individuals are stimulated with DC plus T. gondii (3, 34). However, our studies also suggest that CD40-CD40L interaction regulates IFN-{gamma} secretion in response to DC plus T. gondii through an IL-12-independent mechanism. In this regard, CD40 can provide a costimulatory signal to T cells that results in increased IFN-{gamma} and IL-2 production and enhanced T cell proliferation (51).

Collectively, our studies provide evidence of the importance of T cell-APC cognate interaction for the generation of IL-12/IFN-{gamma}-dependent immunity against T. gondii. CD40-CD40L signaling triggers IL-12 secretion by T. gondii-infected DC, which, in turn, mediates T cell production of IFN-{gamma}. In addition, CD80/CD86-CD28 and CD40-CD40L signaling controls IFN-{gamma} production even in the absence of bioactive IL-12. These results suggest that CD80/CD86-CD28 and CD40-CD40L interactions also regulate IFN-{gamma} production through direct T cell costimulation. It could be proposed that the reasons for the lower IFN-{gamma} production after T cell stimulation with DC plus killed parasites are the absence of IL-12 p70 secretion as a result of the lack of activation of the CD40-CD40L pathway, and the lower levels of CD28- and CD40L-dependent costimulation (lower DC expression of CD40, CD80, and CD86).

Our results reveal that human DC discriminate between viable and nonviable preparations of T. gondii. The data indicate that the interaction between T. gondii and APC induces changes in infected APC, which, in turn, are pivotal for influencing cytokine response. This pathogen-APC-T cell interaction results in IL-12/IFN-{gamma} production in situations where an IL-12/IFN-{gamma}-dependent cell-mediated response would be appropriate (infection with viable tachyzoites), whereas no such response is triggered when encountering nonviable parasites. These data are reminiscent of the "danger" model, where distinction between noxious and harmless stimuli is made by APC through up-regulation of costimulatory ligands (55). Our results suggest that the immune system is capable of distinguishing between viable and killed T. gondii tachyzoites through modulation of CD28 and CD40L signaling as a result of the interaction between viable T. gondii and DC.

A recent report indicates that mice immunized with DC pulsed with TLA were protected against challenge with T. gondii tissue cysts (56). Resistance to infection was accompanied by ex vivo IFN-{gamma} secretion in response to TLA (56). However, these studies were performed using splenic DC after overnight incubation in complete medium. Since such culture conditions result in DC maturation, data from these animals studies do not conflict with our results. The fact that infection with viable T. gondii bradyzoites, rather than immunization with TLA, is necessary for acquisition of resistance to tachyzoites of a virulent strain of the parasite (57) suggests that our results may be relevant to the in vivo immune response to the parasite.

Since our studies were performed using presumably unprimed T cells (T. gondii-seronegative donors), the results indicate that human DC are likely to be important for polarization of the T cell response against T. gondii. Indeed, DC appear to be the initial source of IL-12 in mice exposed in vivo to T. gondii Ags (35). Our data support the hypothesis that signals provided during the interaction between microbial organisms and DC influence the generation of a primary immune response. In the case of T. gondii, these events may explain why this pathogen is a potent inducer of IL-12/IFN-{gamma} secretion. In contrast, by inhibiting DC maturation and impairing the capacity of these cells to process Ag (58, 59, 60), other pathogens may have evolved mechanisms of defense based on inhibition of recognition by the immune system. Further studies of the pathogen-APC-T cell interaction may unravel mechanisms that determine the nature of the immune response elicited.


   Acknowledgments
 
We express our appreciation to Jack Remington for performing T. gondii serology. We thank William Fanslow, Elaine Thomas, Rene de Waal Malefyt, Thomas Tedder, Giorgio Trinchieri, Peter Linsley, and Stanley Wolf for providing reagents.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI37936 and a grant from the American Foundation for AIDS Research. Back

2 Address correspondence and reprint requests to Dr. Carlos S. Subauste, Division of Infectious Diseases, Department of Medicine, University of Cincinnati College of Medicine, P.O. Box 670560, Cincinnati, OH 45267-0560. Back

3 Abbreviations used in this paper: CD40L, CD40 ligand; b-DC, blood dendritic cell; CM, complete medium; MFI, mean fluorescence intensity; DC, dendritic cell; md-DC, monocyte-derived DC; TLA, T. gondii lysate Ags. Back

Received for publication March 22, 2000. Accepted for publication May 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kaufmann, S. H. E.. 1993. Immunity to intracellular bacteria. Annu. Rev. Immunol. 11:129.[Medline]
  2. Subauste, C. S., R. de Waal Malefyt, F. Fuh. 1998. Role of CD80 (B7.1) and CD86 (B7.2) in the immune response to an intracellular pathogen. J. Immunol. 160:1831.[Abstract/Free Full Text]
  3. Subauste, C. S., M. Wessendarp, R. U. Sorensen, L. Leiva. 1999. CD40-CD40 ligand interaction is central to cell-mediated immunity against Toxoplasma gondii: patients with hyper IgM syndrome have a defective type-1 immune response which can be restored by soluble CD40L trimer. J. Immunol. 162:6690.[Abstract/Free Full Text]
  4. Steinman, R. M.. 1991. The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9:271.[Medline]
  5. Larsen, C. P., R. M. Steinman, M. D. Witmer-Pack, D. F. Hankins, P. J. Morris, J. M. Austyn. 1990. Migration and maturation of Langerhans cells in skin transplants and explants. J. Exp. Med. 172:1483.[Abstract/Free Full Text]
  6. Streilein, J. W., S. F. Grammer, T. Yoshikawa, A. Demidem, M. Vermeer. 1990. Functional dichotomy between Langerhans cells that present antigen to naive and memory/effector T lymphocytes. Immunol. Rev. 117:159.[Medline]
  7. Sallusto, F., M. Cella, C. Danielli, A. Lanzavecchia. 1995. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J. Exp. Med. 182:389.[Abstract/Free Full Text]
  8. Cella, M., F. Sallusto, A. Lanzavecchia. 1997. Origin, maturation and antigen presenting function of dendritic cells. Curr. Opin. Immunol. 9:10.[Medline]
  9. Austyn, J. M.. 1992. Antigen uptake and presentation by dendritic leukocytes. Semin. Immunol. 4:227.[Medline]
  10. Inaba, K., J. P. Metlay, M. T. Crowley, R. M. Steinman. 1990. Dendritic cells pulsed with protein antigens in vitro can prime antigen-specific, MHC-restricted T cells in situ. J. Exp. Med. 172:631.[Abstract/Free Full Text]
  11. Subauste, C. S., F. Fuh, R. de Waal Malfyt, J. S. Remington. 1998. {alpha}ß T cell response to Toxoplasma gondii in previously unexposed individuals. J. Immunol. 160:3403.[Abstract/Free Full Text]
  12. Linsley, P. S., W. Brady, M. Urnes, L. S. Grosmaire, N. K. Damle, J. A. Ledbetter. 1991. CTLA-4 is a second receptor for the B cell activation antigen B7. J. Exp. Med. 174:561.[Abstract/Free Full Text]
  13. Zhou, L. J., T. F. Tedder. 1995. Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily. J. Immunol. 154:3821.[Abstract]
  14. Romani, N., S. Gruner, D. Brang, E. Kampgen, A. Lenz, B. Trockenbacher, G. Konwalinka, P. O. Fritsch, R. M. Steinman, G. Schuler. 1994. Proliferating dendritic cell progenitors in human blood. J. Exp. Med. 180:83.[Abstract/Free Full Text]
  15. 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 {alpha}. J. Exp. Med. 179:1109.[Abstract/Free Full Text]
  16. O’Doherty, U., M. Peng, G. Gezelter, W. J. Swiggard, M. Betjes, N. Bhardwaj, R. M. Steinman. 1994. Human blood contains two subsets of dendritic cells, one immunologically mature and other immature. Immunology 82:487.[Medline]
  17. Subauste, C. S., J. Y. Chung, D. Do, A. H. Koniaris, C. A. Hunter, J. G. Montoya, S. Porcelli, J. S. Remington. 1995. Preferential activation and expansion of human peripheral blood {gamma}{delta} T cells in response to Toxoplasma gondii in vitro and their cytokine production and cytotoxic activity against T. gondii-infected cells. J. Clin. Invest. 96:610.
  18. Romani, N., D. Reider, M. Heuer, S. Ebner, E. Kampgen, B. Eibl, D. Niederwieser, G. Schuler. 1996. Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. J. Immunol. Methods 196:137.[Medline]
  19. Bender, A., M. Sapp, G. Schuler, R. M. Steinman, N. Bhardwaj. 1996. Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood. J. Immunol. Methods 196:121.[Medline]
  20. Zhou, L. J., T. F. Tedder. 1996. CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells. Proc. Natl. Acad. Sci. USA 93:2588.[Abstract/Free Full Text]
  21. Suzuki, Y., M. A. Orellana, R. D. Schreiber, J. S. Remington. 1988. Interferon-{gamma}: the major mediator of resistance against Toxoplasma gondii. Science 240:516.[Abstract/Free Full Text]
  22. Gazzinelli, R. T., F. T. Hakim, S. Hieny, G. M. Shearer, A. Sher. 1991. Synergistic role of CD4+ and CD8+ T lymphocytes in IFN-{gamma} production and protective immunity induced by an attenuated Toxoplasma gondii vaccine. J. Immunol. 146:286.[Abstract]
  23. Rescigno, M., S. Citterio, C. Thery, M. Rittig, D. Medaglini, G. Pozzi, S. Amigorena, P. Ricciardi-Castagnoli. 1998. Bacteria-induced neo-biosynthesis, stabilization, and surface expression of functional class I molecules in mouse dendritic cells. Proc. Natl. Acad. Sci. USA 95:5229.[Abstract/Free Full Text]
  24. Winzler, C., P. Rovere, M. Rescigno, F. Granucci, G. Penna, L. Adorini, V. S. Zimmermann, J. Davoust, P. Ricciardi-Castagnoli. 1997. Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. J. Exp. Med. 185:317.[Abstract/Free Full Text]
  25. Ojcius, D. M., Y. Bravo de Alba, J. M. Kanellopoulus, R. A. Hawkins, K. A. Kelly, R. G. Rank, A. Dautry-Varsat. 1998. Internalization of Chlamydia by dendritic cells and stimulation of Chlamydia-specific T cells. J. Immunol. 160:1297.[Abstract/Free Full Text]
  26. Thurnher, M., R. Ramoner, G. Gastl, C. Radmayr, G. Bock, M. Herold, H. Klocker, G. Bartsch. 1997. Bacillus Calmette-Guérin mycobacteria stimulate human blood dendritic cells. Int. J. Cancer 70:128.[Medline]
  27. Kim, K. D., H. G. Lee, J. K. Kim, S. N. Park, I. S. Choe, Y. K. Choe, S. J. Kim, E. Lee, J. S. Lim. 1999. Enhanced antigen-presenting activity and tumour necrosis factor-{alpha}-independent activation of dendritic cells following treatment with Mycobacterium bovis bacillus Calmette-Guérin. Immunology 97:626.[Medline]
  28. Henderson, R. A., S. C. Watkins, J. L. Flynn. 1997. Activation of human dendritic cells following infection with Mycobacterium tuberculosis. J. Immunol. 159:635.[Abstract]
  29. Schnorr, J. J., S. Xanthakos, P. Keikavoussi, E. Kampgen, V. ter Meulen, S. Schneider-Schaulies. 1997. Induction of maturation of human blood dendritic cell precursors by measles virus is associated with immunosuppression. Proc. Natl. Acad. Sci. USA 94:5326.[Abstract/Free Full Text]
  30. Caux, C., C. Massacrier, B. Banbervliet, B. Dubois, C. van Kooten, I. Durand, J. Banchereau. 1994. Activation of human dendritic cells through CD40 cross-linking. J. Exp. Med. 180:1263.[Abstract/Free Full Text]
  31. Gazzinelli, R. T., S. Hieny, T. A. Wynn, S. Wolf, A. Sher. 1993. Interleukin 12 is required for the T-lymphocyte-independent induction of interferon {gamma} by an intracellular parasite and induces resistance in T-cell-deficient hosts. Proc. Natl. Acad. Sci. USA 90:6115.[Abstract/Free Full Text]
  32. Khan, I. A., T. Matsuura, L. H. Kasper. 1994. Interleukin-12 enhances murine survival against acute toxoplasmosis. Infect. Immun. 62:1639.[Abstract/Free Full Text]
  33. Hunter, C. A., C. S. Candolfi, V. van Cleave Subauste, J. S. Remington. 1995. Studies on the role of interleukin-12 in acute murine toxoplasmosis. Immunology 84:16.[Medline]
  34. Seguin, R., L. H. Kasper. 1999. Sensitized lymphocytes and CD40 ligation augment interleukin-12 production by human dendritic cells in response to Toxoplasma gondii. J. Infect. Dis. 179:467.[Medline]
  35. Reis e Sousa, C., S. Hieny, T. Scharton-Kersten, D. Jankovic, H. Charest, R. N. Germain, A. Sher. 1997. In vivo microbial stimulation induces rapid CD40 ligand-independent production of interleukin 12 by dendritic cells and their redistribution to T cell areas. J. Exp. Med. 186:1819.[Abstract/Free Full Text]
  36. Kalinski, P., J. H. N. Schuitemaker, C. M. U. Hilkens, M. L. Kapsenberg. 1998. Prostaglandin E2 induces the final maturation of IL-12-deficient CD1a+ CD83+ dendritic cell: the levels of IL-12 are determined during the final dendritic cell maturation and are resistant to further modulation. J. Immunol. 161:2804.[Abstract/Free Full Text]
  37. Scharton-Kersten, T., P. Caspar, A. Sher, E. Y. Denkers. 1996. Toxoplasma gondii: evidence for interleukin 12-dependent and -independent pathways of interferon-{gamma} production induced by an attenuated parasite strain. Exp. Parasitol. 84:102.[Medline]
  38. Schijns, V. E. C. J., B. L. Haagmans, C. M. H. Wierda, B. Kruithof, I. A. F. M. Heijen, G. Alber, M. C. Horzinek. 1988. Mice lacking IL-12 develop polarized Th1 cells during viral infection. J. Immunol. 160:3958.[Abstract/Free Full Text]
  39. Oxenius, A., U. Karrer, R. M. Zinkernagel, H. Hengartner. 1999. IL-12 is not required for induction of type 1 cytokine responses in viral infections. J. Immunol. 162:965.[Abstract/Free Full Text]
  40. Villegas, E. N., M. M. Elloso, G. Reichmann, R. Peach, C. A. Hunter. 1999. Role of CD28 in the generation of effector and memory responses required for resistance to Toxoplasma gondii. J. Immunol. 163:3344.[Abstract/Free Full Text]
  41. Levy, J., T. Espanol-Boren, C. Thomas, A. Fischer, P. Tove, P. Bordigoni, I. Resnick, A. Fasth, M. Baer, L. Gomez, et al 1997. Clinical spectrum of X-linked hyper-IgM syndrome. J. Pediatr. 131:47.[Medline]
  42. Leiva, L. E., J. Junprasert, D. Hollenbaugh, R. U. Sorensen. 1998. Central nervous system toxoplasmosis with an increased proportion of circulating {gamma}{delta} T cells in a patient with hyper IgM syndrome. J. Clin. Immunol. 18:283.[Medline]
  43. Tsuge, I., H. Matsuoka, A. Nakagawa, Y. Kamachi, K. Aso, T. Negoro, M. Ito, S. Torii, K. Watanabe. 1998. Necrotizing toxoplasmic encephalitis in a child with the X-linked hyper-IgM syndrome. Eur. J. Pediatr. 157:735.[Medline]
  44. McDyer, J. F., T. J. Goletz, E. Thomas, C. H. June, R. A. Seder. 1998. CD40 ligand/CD40 stimulation regulates the production of IFN-{gamma} from human peripheral blood mononuclear cells in an IL-12- and/or CD28-dependent manner. J. Immunol. 160:1701.[Abstract/Free Full Text]
  45. Murphy, E. E., G. Terres, S. E. Macatonia, C. Hsieh, J. Mattson, L. Lanier, M. Wysocka, G. Trinchieri, K. Murphy, A. O’Garra. 1994. B7 and interleukin-12 cooperate for proliferation and interferon {gamma} production by mouse T helper clones that are unresponsive to B7 costimulation. J. Exp. Med. 180:223.[Abstract/Free Full Text]
  46. Kubin, M., M. Kamoun, G. Trinchieri. 1994. Interleukin-12 synergizes with B7/CD28 interaction in inducing efficient proliferation and cytokine production of human T cells. J. Exp. Med. 180:211.[Abstract/Free Full Text]
  47. Murtaza, A., V. K. Kuchroo, G. J. Freeman. 1999. Changes in the strength of co-stimulation through the B7/CD28 pathway alter functional T cell responses to altered peptide ligands. Int. Immunol. 11:407.[Abstract/Free Full Text]
  48. Shu, U., M. Kiniwa, C. Y. Wu, C. Malistewski, N. Vezzio, J. Hakimi, M. Gately, G. Delespesse. 1995. Activated T cells induce interleukin-12 production by monocytes via CD40-CD40 ligand interaction. Eur. J. Immunol. 25:1125.[Medline]
  49. Kennedy, M. K., K. S. Picha, W. C. Fanslow, K. H. Grabstein, K. R. Alderson, K. N. Clifford, W. A. Chin, K. M. Mohler. 1996. CD40/CD40 ligand interactions are required for T cell-dependent production of interleukin-12 by mouse macrophages. Eur. J. Immunol. 26:370.[Medline]
  50. Cella, M., D. Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, G. Alber. 1996. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J. Exp. Med. 184:747.[Abstract/Free Full Text]
  51. Cayabyab, M. H., H. J. Phillips, L. L. Lanier. 1994. CD40 preferentially costimulates activation of CD4+ T lymphocytes. J. Immunol. 152:1523.[Abstract]
  52. 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.[Medline]
  53. 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.[Medline]
  54. 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.[Medline]
  55. Matzinger, P.. 1994. Tolerance, danger, and the extended family. Annu. Rev. Immunol. 12:991.[Medline]
  56. Bourguin, I., M. Moser, D. Buzoni-Gatel, F. Tielemans, D. Bout, J. Urbain, O. Leo. 1998. Murine dendritic cells pulsed in vitro with Toxoplasma gondii antigens induce protective immunity in vivo. Infect. Immun. 66:4867.[Abstract/Free Full Text]
  57. Nagasawa, H., T. Manabe, Y. Maekawa, M. Oka, K. Himeno. 1991. Role of L3T4+ and Lyt-2+ T cell subsets in protective immune responses of mice against infection with a low or high virulent strain of Toxoplasma gondii. Microbiol. Immunol. 35:215.[Medline]
  58. Urban, B. C., D. J. P. Ferguson, A. Pain, N. Willcox, M. Plebanski, J. M. Austyn, D. J. Roberts. 1999. Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells. Nature 400:73.[Medline]
  59. van Overtvelt, L., N. Vanderheyde, V. Verhasselt, J. Ismaili, L. de Vos, M. Goldman, F. Willems, B. Vray. 1999. Trypanosoma cruzi infects human dendritic cells and prevents their maturation: Inhibition of cytokines, HLA-DR, and costimulatory molecules. Infect. Immun. 67:4033.[Abstract/Free Full Text]
  60. Engelmayer, J., M. Larsson, M. Subklewe, A. Chahroudi, W. I. Cox, R. M. Steinman, N. Bhardwaj. 1999. Vaccinia virus inhibits the maturation of human dendritic cells: a novel mechanism of immune evasion. J. Immunol. 163:6762.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
J. P. Gigley, B. A. Fox, and D. J. Bzik
Cell-Mediated Immunity to Toxoplasma gondii Develops Primarily by Local Th1 Host Immune Responses in the Absence of Parasite Replication
J. Immunol., January 15, 2009; 182(2): 1069 - 1078.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. S. Subauste, A. Subauste, and M. Wessendarp
Role of CD40-Dependent Down-Regulation of CD154 in Impaired Induction of CD154 in CD4+ T Cells from HIV-1-Infected Patients
J. Immunol., February 1, 2007; 178(3): 1645 - 1653.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
A. Ben Nasr, J. Haithcoat, J. E. Masterson, J. S. Gunn, T. Eaves-Pyles, and G. R. Klimpel
Critical role for serum opsonins and complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in phagocytosis of Francisella tularensis by human dendritic cells (DC): uptake of Francisella leads to activation of immature DC and intracellular survival of the bacteria
J. Leukoc. Biol., October 1, 2006; 80(4): 774 - 786.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
N. Courret, S. Darche, P. Sonigo, G. Milon, D. Buzoni-Gatel, and I. Tardieux
CD11c- and CD11b-expressing mouse leukocytes transport single Toxoplasma gondii tachyzoites to the brain
Blood, January 1, 2006; 107(1): 309 - 316.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. Giese, S. Stuhlsatz, W. Daubener, and C. R. MacKenzie
Inhibition of the Growth of Toxoplasma gondii in Immature Human Dendritic Cells Is Dependent on the Expression of TNF-{alpha} Receptor 2
J. Immunol., September 1, 2004; 173(5): 3366 - 3374.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
A. Vecchiarelli, D. Pietrella, P. Lupo, F. Bistoni, D. C. McFadden, and A. Casadevall
The polysaccharide capsule of Cryptococcus neoformans interferes with human dendritic cell maturation and activation
J. Leukoc. Biol., September 1, 2003; 74(3): 370 - 378.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
D. Chaussabel, R. T. Semnani, M. A. McDowell, D. Sacks, A. Sher, and T. B. Nutman
Unique gene expression profiles of human macrophages and dendritic cells to phylogenetically distinct parasites
Blood, July 15, 2003; 102(2): 672 - 681.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
U. Wille, E. N. Villegas, L. Craig, R. Peach, and C. A. Hunter
Contribution of Interleukin-12 (IL-12) and the CD28/B7 and CD40/CD40 Ligand Pathways to the Development of a Pathological T-Cell Response in IL-10-Deficient Mice
Infect. Immun., December 1, 2002; 70(12): 6940 - 6947.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
S. Wei, F. Marches, J. Borvak, W. Zou, J. Channon, M. White, J. Radke, M.-F. Cesbron-Delauw, and T. J. Curiel
Toxoplasma gondii-Infected Human Myeloid Dendritic Cells Induce T-Lymphocyte Dysfunction and Contact-Dependent Apoptosis
Infect. Immun., April 1, 2002; 70(4): 1750 - 1760.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
M. F. Lipscomb and B. J. Masten
Dendritic Cells: Immune Regulators in Health and Disease
Physiol Rev, January 1, 2002; 82(1): 97 - 130.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Subauste, C. S.
Right arrow Articles by Wessendarp, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Subauste, C. S.
Right arrow Articles by Wessendarp, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS