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The Journal of Immunology, 2007, 179, 590-596
Copyright © 2007 by The American Association of Immunologists, Inc.
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NK Cells Enhance Dendritic Cell Response against Parasite Antigens via NKG2D Pathway1

Hongbing Guan*, Magali Moretto*,{dagger}, David J. Bzik{ddagger}, Jason Gigley{ddagger} and Imtiaz A. Khan2,*,{dagger}

* Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA 70112; {dagger} Department of Microbiology, Tropical Medicine and Immunology, George Washington University, Washington, DC 20037; and {ddagger} Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, NH 03756


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Recent studies have shown that NK-dendritic cell (DC) interaction plays an important role in the induction of immune response against tumors and certain viruses. Although the effect of this interaction is bidirectional, the mechanism or molecules involved in this cross-talk have not been identified. In this study, we report that coculture with NK cells causes several fold increase in IL-12 production by Toxoplasma gondii lysate Ag-pulsed DC. This interaction also leads to stronger priming of Ag-specific CD8+ T cell response by these cells. In vitro blockade of NKG2D, a molecule present on human and murine NK cells, neutralizes the NK cell-induced up-regulation of DC response. Moreover, treatment of infected animals with Ab to NKG2D receptor compromises the development of Ag-specific CD8+ T cell immunity and reduces their ability to clear parasites. These studies emphasize the critical role played by NKG2D in the NK-DC interaction, which apparently is important for the generation of robust CD8+ T cell immunity against intracellular pathogens. To the best of our knowledge, this is the first work that describes in vivo importance of NKG2D during natural infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The coordination of an efficient immune response requires the recognition of pathogens and subsequent induction of innate and adaptive immune responses, which are critical for the resolution of infection. Although adaptive immunity is essential for ultimate protection and memory against the pathogen, the innate immune system is critical for limiting the spread of infectious disease during the initial phase of infection (1). Moreover, recent studies have demonstrated that innate immune factors play a crucial role in the induction of adaptive immune response during microbial infection (2). NK cells and dendritic cells (DC)3 represent two distinct cell types of the innate immune system, both of which play an important role in combating early infection. Although the importance of NK cells lies in their ability to produce cytokines or lyse infected cells (3), DC function as APCs, essential for generating T cell immunity against the pathogen (4). As better understanding of the innate immune system is evolving, attention has focused on NK-DC interactions and the consequences of such communication or cross-talk during the immune response.

Several studies have shown evidence for NK-DC cross-talk that results in cellular activation, maturation, and cell death. Since early work by Fernandez et al. (5, 6), studies have supported an important role of NK-DC interactions in vivo during antitumor immunity and viral infection. In vitro, the cross-talk results in maturation, activation, and cytokine production by both cell types, including NK cell proliferation, cytotoxicity, and DC maturation (7, 8, 9, 10, 11). These studies also suggest that this cross-talk may be controlled by the interaction of specific receptors, including inhibitory and activating receptors on the surface of NK cells recognizing ligands present on DC (8, 12).

Although NK-DC interaction has been reported to be important for induction of protective immunity against tumors and viral infections (13, 14), the role of this interplay in parasite infection has not been evaluated. Moreover, studies investigating the mechanism of this process have not been performed. A surface receptor that plays a relevant role in NK-mediated cytolysis of some tumors is NKG2D, a homodimeric C-type lectin-like protein that is expressed by all NK cells, subsets of NKT cells, CD8+ T cells, and macrophages (15). The NKG2D receptor binds ligands that are poorly expressed on normal cells, but are up-regulated on infected, transformed, or stressed cells (15). Previously, we reported induction of a strong and prolonged NK cell response against Toxoplasma gondii, an obligate intracellular parasite, in CD4+ T cell-deficient mice (16). An elevated NK cell response in these CD4–/– mice was responsible for the generation of optimal CD8+ T cell immunity in the infected animals. In the current study, we have analyzed the role of NKG2D in the stimulation of NK-DC response to Toxoplasma Ags. Furthermore, we evaluate the role of this interaction in the induction of CD8+ T cell immunity against the parasite.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Five- to 8-wk-old C57BL/6, sex-matched IFN-{gamma}–/– mice on the same genetic background, and C57BL/6 x 129 mice were purchased from The Jackson Laboratory. Animals were housed in a specific pathogen-free environment in the Animal Care Facility at Louisiana State University Health Sciences Center, George Washington University, and Dartmouth Medical School. All animal procedures were performed according to National Institutes of Health guidelines under protocols approved by the Animal Care and Use Committee of the University.

Toxoplasma lysate Ag (TLA) preparation

TLA was prepared from tachyzoites of the RH strain of T. gondii, as described before (17), and was subjected to testing for endotoxin contamination using the Limulus amebocyte lysate Complete 20 Test Kit (Cambrex BioScience). Based on this assay, it was determined that endotoxin levels in TLA were ≤0.03 EU/ml.

NK-DC coculture

DCs were isolated according to a previously described protocol (18). Briefly, spleens were digested with 1 mg/ml collagenase D (Roche Diagnostics) and 50 µg/ml DNase I (Sigma-Aldrich). Cell suspension was resuspended in NycoPrep medium (5 ml per spleen) (Axis-shield PoC), and centrifuged for 15 min. Positive selection of DC was performed according to the EasySep Biotin selection kit recommendations (StemCell Technologies). In brief, cells were labeled with biotin-labeled anti-CD11c Ab, and then positively selected by anti-biotin magnetic nanoparticles. The purity of isolated CD11c+ DC cells was 95–97% as determined by flow cytometric analysis.

NK cells were isolated from spleens by using a two-step magnetic selection (16). T and B cells were depleted by incubation with anti-CD3 and anti-CD19 biotin-conjugated Abs, followed by anti-biotin-coupled beads and magnetic depletion. The remaining cell suspension was labeled with biotin-conjugated anti-NK1.1 Ab, and then positively selected by anti-biotin magnetic nanoparticles according to the manual of EasySep Biotin selection kit. The percentage of NK1.1+ cells as evaluated by flow cytometry using a FACSCalibur was >95%. NK-DCs were cocultured at 1:1 ratio (2 x 105/well) in presence of TLA (20 µg/ml) in 96-well tissue culture plates. Cell culture supernatants were collected and stored at –20°C until analyzed for cytokine production.

IL-12 assay

NK-DC cocultures were established, as described above, and after 24-h incubation, supernatants were collected and assayed for IL-12 production using commercially available ELISA kits (Biolegend), as per manufacturer’s instructions. In some of the assays, Ab to NKG2D (clone C7; eBioscience) or hamster IgG control (Jackson ImmunoResearch Laboratories) was added to the culture (30 µg/ml) at the time of incubation.

CD8+ T cell proliferation and CTL assay

CD8+ T cells were purified by magnetic separation by previously described protocol (19) and added to 24-h NK-DC cocultures (5 x 104/well). In some of the assays, Abs to NKG2D (30 µg/ml) or IFN-{gamma} (10 µg/ml) or their respective isotype controls were added to the culture. After a 5-day incubation, [3H]thymidine was added to the wells and proliferative responses were measured by standard procedures (20). For cytotoxic assay, CD8+ T cell cultures stimulated with TLA were harvested and incubated with radiolabeled infected macrophages at various E:T ratios in 96-well U-bottom plates. After 4-h incubation, the supernatants were measured for radioactive release, and the percentage of lysed target cells was calculated (21).

IFN-{gamma} production

CD8+ T cells were added to the NK-DC coculture, as described above. After 72-h incubation, the cells were collected and the number of CD3+CD8+ T cells producing IFN-{gamma} was determined by intracellular staining using Cytofix/CytoPerm kit (BD Pharmingen), as previously described (16). Cells were labeled with FITC-conjugated anti-CD8{alpha} and CyChrome-conjugated anti-CD3 (eBioscience), fixed and permeablized with paraformaldehyde, and then stained with PE-conjugated anti-IFN-{gamma} (eBioscience). Labeled cells were analyzed by flow cytometry, and the results were evaluated using CellQuest software.

RAE-1 and MULT-1 expression by DC

Splenic DC (106 cells) isolated and stimulated with TLA in culture for 24 h were fixed with 1% paraformaldehyde and stained with 0.25 µg of FITC-conjugated anti-I-A/I-E (BD Pharmingen). Subsequently, cells were labeled with 0.3 µg of biotin-conjugated anti-RAE-1 (R&D Systems; clone 186107) and 0.05 µg of PE-conjugated streptavidin (BD Pharmingen). For MULT-1 expression, cells were treated with anti-MULT-1 (0.3 µg) (R&D Systems; clone 237104) and PE-conjugated anti-rat IgG (GeneTex) (0.05 µg). The stained cells were evaluated by flow cytometry and data were analyzed using CellQuest software.

NKG2D neutralization

The CX5 Ab was obtained from L. Lanier (University of California, San Francisco, CA) and is specific for mouse NKG2D receptor (15, 22). Rat IgG control was purchased from Jackson ImmunoResearch Laboratories. C57BL/6 mice were treated with either 200 µg of anti-NKG2D Ab or isotype control via i.p. route starting 1 day before infection and continued on days 3, 5, and 7 postinfection (p.i.) thereafter.

T cell subset proliferation

CD4+ and CD8+ T cells from treated mice were purified from splenocytes by positive selection using microbeads (StemCell Technologies). The purity of cells exceeded 95%, as determined by FACS analysis. A total of 1 x 105 purified cells was incubated with 5 x 104 irradiated feeder cells in presence of TLA (20 µg/ml). Ag-specific proliferative response was assessed after 72 h of incubation by thymidine incorporation.

Parasite burden by quantitative real-time PCR

Mice were infected perorally with 20 cysts of Me49 strain of parasite. At day 10 p.i., infected animals were euthanized via CO2 overdose and tissues (gut, liver, spleen, and brain) were harvested and flash frozen in liquid nitrogen. DNA was extracted from entire organ using a Qiagen DNeasy Tissue Kit (Qiagen). Amplification of parasite DNA from 400 ng of purified tissue DNA was performed using primers specific for the T. gondii B1 gene (23) at 10 pMol each per reaction (Integrated DNA Technologies) and amplified by real-time fluorogenic PCR using SMartMix HM (Cepheid) on a Cepheid Smart Cycler. Each reaction contained one lyophilized SMartMix HM bead, SYBR Green I (Cambrex BioScience). Parasite DNA equivalents were used for a standard curve to which mouse tissue DNA samples were compared, and parasite numbers were then calculated via extrapolation from the standard curve.

Statistical analysis

Statistical analysis of the data was performed by using unpaired Student’s t test (24).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Enhanced IL-12 production by DCs with NK cell help

IL-12 is known to be critical for resolution of a number of infectious diseases, and DC have been identified as the primary source of this cytokine (25). In vitro studies have demonstrated a central role for DC-derived IL-12 in the induction of IFN-{gamma} production by NK cells in different systems (26). T. gondii is known to induce a potent IL-12-dependent cell-mediated immune response that shuts down the growth of the replicative tachyzoite stage, thus promoting host survival (27, 28, 29). Previous studies have demonstrated that DC are the major cell type responsible for IL-12 production during T. gondii infection, which is important for induction of primary Th1 immunity against the parasite (30). Using NK-DC coculture, we first examined whether NK cells modulate IL-12 production by DC pulsed with TLA. After coculture was established, supernatants were collected after 24-h incubation, and evaluated for IL-12 levels by ELISA. Consistent with previous studies (30), DC pulsed with TLA produced significant amount of IL-12. However, interestingly, the presence of NK cells in the culture significantly increased the ability of TLA-stimulated DC to secrete IL-12 (p = 0.00585) (Fig. 1). IL-12 was undetectable in the NK cell cultures alone stimulated with TLA.


Figure 1
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  		FIGURE 1. NK cells enhance IL-12 production in the TLA-primed DC. NK cells and DC from spleens of naive wild-type and IFN-{gamma}–/– mice were isolated, as described in Materials and Methods. NK and DC were cocultured at concentrations of 2 x 105 cells/well at 1:1 ratio. TLA (20 µg/ml) was added to the cultures and after 24 h, supernatants were collected and assayed for IL-12 production by ELISA. The results are mean ± SD. Data are representative of three independent experiments.

 
Because NK cells are known to secrete large amounts of IFN-{gamma} upon stimulation (31, 32), we evaluated whether this cytokine is involved in NK-DC cross-talk during the processing of T. gondii Ags. To examine this, we compared in vitro IL-12 production by DC-NK cell interaction in the wild-type vs IFN-{gamma}–/– mice. As shown in Fig. 1 (lanes 3 and 5), DCs from IFN-{gamma}–/– mice pulsed with TLA produced similar levels of IL-12 upon incubation with wild-type NK cells as wild-type DC. However, IL-12 production by the TLA-pulsed DC from both wild-type and IFN-{gamma}–/– mice was significantly reduced when cultured with NK cells from IFN-{gamma}–/– animals (Fig. 1, lanes 4 and 6) (p = 0.00462 and 0.00494, respectively). Nevertheless, IL-12 release from cocultures of both wild-type and IFN-{gamma}–/– DC with IFN-{gamma}–/– NK cells was still higher than DC cultured alone (lanes 4 and 6). These data demonstrate that IFN-{gamma} secretion by NK cells is at least partially involved in enhancing the IL-12 production by TLA-pulsed DC. In agreement with earlier findings by Scharton-Kersten et al. (33), IL-12 production by DC alone is independent of the IFN-{gamma} released by these cells (Fig. 1, lanes 1 and 2).

NK-DC interaction induces strong CD8+ T cell immunity

To determine whether the increased IL-12 production due to NK cell help enhances their ability to prime a CD8+ T cell response against the parasite, TLA-pulsed DC were cultured with NK cells at 1:1 ratio. Twenty-four hours later, syngeneic naive CD8+ T cells were added, and subsequently after 5-day incubation, proliferation was measured by [3H]thymidine incorporation assay. As shown in Fig. 2A, although CD8+ T cells proliferated when cultured with TLA-pulsed DC alone (Fig. 2A), their proliferative response was significantly increased (p = 0.005) in presence of NK-DC coculture. As expected, CD8+ T cells cultured alone in the absence of DC did not respond to antigenic stimulation (Fig. 2A).


Figure 2
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  		FIGURE 2. CD8+ T cells primed by NK-DC coculture exhibit enhanced Ag-specific responses. Purified CD8+ T cells were added to NK-DC cocultures (5 x 104/each cell population/well) stimulated with TLA 24 h earlier, and their proliferation, cytotoxic activity, and number of IFN-{gamma}-producing cells were measured. A, Proliferation: after 5-day incubation, [3H]thymidine was added to the culture, and proliferation of CD8+ T cells was measured. B, Cytotoxic activity: after 5-day culture, CD8+ T cells were collected and incubated with 51Cr-labeled Toxoplasma-infected macrophages at various E:T ratios. Four hours later, cytotoxic activities were measured by radioactive release. C, IFN-{gamma}-positive cells: after 72-h incubation in a NK-DC coculture, number of IFN-{gamma}-producing CD8+ T cells was assayed by intracellular staining. Data are expressed as mean ± SD and are representative of two independent experiments.

 
The enhanced induction of an Ag-specific CD8+ T cell response by the DC-NK coculture was further evaluated by using a CTL assay. Affinity-purified CD8+ T cells were incubated with DC or NK-DC coculture, and after 5-day incubation, their cytotoxic activity against 51Cr-labeled infected macrophages was measured. Similar to proliferation data, TLA-pulsed DCs induced a CTL response, which was significantly increased when CD8+ T cells were primed in a DC-NK coculture (Fig. 2B; p = 0.01).

To further establish the ability of NK cells to enhance DC-mediated priming of naive CD8+ T cells, the percentage of Ag-specific CD8+ T cells secreting IFN-{gamma} was evaluated by intracellular staining. Consistent with the results obtained with the proliferation and cytotoxicity assays, the percentage of IFN-{gamma}-secreting CD8+ T cells primed in a NK-DC coculture was significantly higher than those cultured with DC alone (Fig. 2C; p = 6.24 x 10–6). Collectively, these results demonstrate that NK cell can play a helper role during priming of CD8+ T cell effector response against T. gondii infection.

Enhanced IL-12 production during the NK-DC interaction is dependent on cell contact

Next, we determined whether NK cell help in enhancing IL-12 production by DC is contact dependent or mediated by soluble factors. Ag-pulsed DC and NK cells were cultured in a Transwell system, in which the two populations were separated by a porous membrane. As shown in Fig. 3, increased IL-12 release by DC was completely abrogated when they were separated from NK cells by a membrane. The amounts of IL-12 produced by DC-NK cocultures in a Transwell system were similar to the DC cultured alone. Although increased IL-12 release by NK-DC coculture was partially dependent on IFN-{gamma} produced by NK cells (Fig. 1), these cells needed to interact directly with DC to produce the cytokine. When stimulated with TLA in a Transwell, NK cells failed to release IFN-{gamma} (data not shown). As observed earlier, DC in direct contact with NK cells released significantly increased amounts of IL-12 (Fig. 3; p = 9.6 x 10–5).


Figure 3
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  		FIGURE 3. NK cell-mediated enhancement of IL-12 production by TLA-primed DC is contact dependent. Purified NK cells and DC were cultured in Transwell plates separated by a 0.4-µm pore-size polyester membrane in which DC were seeded in the lower well, whereas NK cells were placed on the top. TLA was added to both the wells, and cell supernatants in both the compartments were collected and assayed for IL-12 production by ELISA. The results are mean ± SD, and the graph represents one of the three experiments.

 
Blocking of NKG2D inhibits the NK and DC interaction

The above studies demonstrate that the NK-DC interaction resulting in enhanced IL-12 release requires direct cell-to-cell contact. It has been recently reported that human NK cell-dependent DC maturation is mediated by TNF-{alpha} and IFN-{gamma} released upon engagement of the NKp30-activating receptor on NK cells (10). A well-characterized NK cell receptor on both human and mouse NK cells is NKG2D, which has previously been implicated in antitumor immunity (13, 34). We treated NK-DC cell cultures with a neutralizing Ab to NKG2D and measured IL-12 release after 24 h. As shown in Fig. 4A, blocking of NKG2D significantly inhibited (p = 0.0882) the enhanced IL-12 production in the NK-DC cocultures. Conversely, treatment of NK-DC cocultures with a control Ab had no effect on the IL-12 production by DC (Fig. 4A). To determine whether blockade of NKG2D affects the CD8+ T cell response, affinity-purified CD8+ T cells were added to NK-DC cocultures and proliferation was measured. As shown in Fig. 4B, CD8+ T cell proliferation in the cultures treated with Ab to NKG2D was significantly reduced in comparison with the cultures treated with isotype controls (p = 0.082). Treatment of NK-DC cultures with anti-IFN-{gamma} Ab also reduced their ability to induce CD8+ T cell proliferation (p = 0.021), but the decrease was significantly less compared with NK-DC cocultures treated with anti-NKG2D Ab (p = 0.026) (Fig. 4B). These studies show that NKG2D plays an important role in NK-DC interaction, which is essential for optimal IL-12 release and CD8+ T cell response against the parasite.


Figure 4
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  		FIGURE 4. NK cell-mediated up-regulation of IL-12 release by DC is NKG2D dependent. A and B, Anti-NKG2D treatment blocks the increase of IL-12 release in NK-DC cocultures and the induction of CD8+ T cell response. Anti-NKG2D or an isotype control IgG (30 µg/ml) was added to NK-DC coculture 1 h before addition of TLA. Twenty-four hours later, the supernatants were collected and assayed for IL-12 production. The data are expressed as mean ± SD, and data are representative of two independent experiments (A). Treatment of DC-NK coculture with anti-NKG2D Ab inhibits their ability to prime CD8+ T cells. DC-NK cultures stimulated with TLA were treated with anti-NKG2D or anti-IFN-{gamma} Ab, as mentioned in Materials and Methods. Twenty-four hours later, affinity-purified CD8+ T cells (5 x 104 cells/well) were added to the cultures. After incubation for 72 h, proliferation was measured by thymidine incorporation assay (B). C–F, Expression of NKG2D ligands RAE-1 or MULT-1 on TLA-primed DC. A total of 2 x 105 purified DC was cultured with or without TLA. Twenty-four hours later, the cells were analyzed for expression of RAE-1 (B–D) or MULT-1 (C–E) by flow cytometry. The results are expressed as mean ± SD, and the graph represents one of at least two independent experiments.

 
To further understand the involvement of NKG2D in the DC-NK cell interaction, we examined whether RAE-1 or MULT-1, ligands for NKG2D in mice (35, 36), were present on DC pulsed with T. gondii Ags. After overnight incubation with TLA, the DC were stained with anti RAE-1 and MULT-1 Abs, and the expression of these molecules was determined by FACS analysis. Incubation with TLA caused a significant increase in percentage of DC-expressing RAE-1 (Fig. 4, C and E; p = 4.42 x 10–3) or MULT-1 (Fig. 4, D and F; p = 1.32 x 10–7) ligands.

Neutralization of NKG2D reduces the protection against T. gondii infection

To evaluate the importance of NKG2D molecule in an in vivo infection, mice infected perorally with 20 T. gondii cysts were treated with neutralizing Ab against NKG2D. Although animals treated with anti-NKG2D appeared to be sicker and had lower body weight than those administered with isotype control Ab, none of them succumbed to infection (data not shown). However, analysis of parasite load in the tissues (gut, spleen, liver, and brain) of infected animals demonstrated higher parasite burden in the anti-NKG2D-treated animals (Fig. 5A). Except for the liver, anti-NKG2D treatment led to significant increase in the parasite load in the rest of the tissues analyzed (gut, spleen, and brain) (Fig. 5A; p = 0.0011, 0.03, and 0.003, respectively). Some of these animals (four mice per/group) were sacrificed 1 mo postinfection, and the number of brain cysts was enumerated. Compared with control Ab-treated mice, significantly increased number of brain cysts was observed in the animals administered anti-NKG2D Ab (355 ± 88 vs 1022 ± 152, p = 4.13 x 10–5). Moreover, although anti-NKG2D-treated mice did not exhibit any mortality when challenged with 20 cysts, >80% mortality (five of six mice) was observed when C57BL/6 x 129 mice, administered with anti-NKG2D Ab, were challenged with 50 cysts (data not shown). Similar to C57BL/6 mice, no mortality in the anti-NKG2D-treated animals was observed when B6 x 129 animals were infected perorally with 20 cysts (data not shown). These findings are similar to our earlier report, which demonstrated that NK cell-depleted animals survived a challenge dose of 20 cysts, but succumbed to infection when it was increased to 50 cysts/animal (32).


Figure 5
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  		FIGURE 5. Neutralization of NKG2D receptor reduces the CD8+ T cell immunity against T. gondii infection. C57BL/6 mice were infected via oral route with 20 T. gondii cysts. A group of infected mice was treated with anti-NKG2D Ab, as described in Materials and Methods. A, Parasite load in the organs of anti-NKG2D-treated mice. At day 10 p.i., mice were euthanized, and organs (gut, liver, spleen, and brain) were harvested and flash frozen in liquid nitrogen. DNA was extracted from entire organ and PCR was performed, as described in Materials and Methods. There were five to six animals per group, and the experiment was performed twice with similar results. B and C, Ag-specific response of CD4+ and CD8+ T cells from mice treated with anti-NKG2D Ab. T. gondii-infected and uninfected control animals treated with either anti-NKG2D or isotype control Ab were sacrificed at day 14 p.i. Spleens were pooled (three mice/group), and CD8+ (B) and CD4+ T cells (C) were isolated by affinity purification. A total of 1 x 105 purified cells was cultured with syngeneic naive, irradiated splenocytes (5 x 104) in presence of 15 µg/ml TLA. After 72-h incubation, lymphoproliferation was assayed by [3H]thymidine incorporation. The experiment was performed twice with similar results, and data are representative of one experiment. D and E, Analysis of IFN-{gamma}-producing NK, CD4+, and CD8+ T cells from anti-NKG2D-treated mice. Spleens from Ab-treated mice were pooled (three mice per group), harvested, and cultured in vitro with PMA, ionomycin, and monensin for 4 h. The cultured cells were labeled for CD8 (D), CD4, and NK1.1 (E) before intracellular staining for IFN-{gamma}. Data are presented as percentage (mean ± SD) of cells positive for IFN-{gamma} and are pooled from two different experiments.

 
Because in vitro data suggested that NK-DC interaction is important for the induction of optimal CD8+ T cell immunity, next, we determined whether in vivo anti-NKG2D treatment affects this response in the infected animals. CD8+ T cells from the anti-NKG2D- or control Ab-treated animals were isolated at day 14 p.i., and proliferative response to antigenic stimulation was measured. As shown in Fig. 5B, CD8+ T cells isolated from anti-NKG2D-treated animals exhibited significantly reduced proliferative response to TLA in comparison with the cells isolated from mice injected with control Ab (p = 0.0063). Conversely, anti-NKG2D treatment had no effect on the mitogenic response of CD8+ T cells from naive or infected mice (data not shown). The effect of NKG2D blockade on the CD8+ T cell response during T. gondii infection was further evaluated by determining the level of IFN-{gamma} production by these cells. Treatment of T. gondii-infected mice with anti-NKG2D significantly reduced the number of IFN-{gamma}-positive CD8+ T cells in these animals (Fig. 5D; p = 0.01).

Next, we determined the development of CD4+ T cell and NK response in anti-NKG2D-treated mice. CD4+ T cells from the mice treated with anti-NKG2D or isotype control were isolated, and day 14 p.i. and Ag-specific proliferation was determined. As shown in Fig. 5C, CD4+ T cells from the animals administered anti-NKG2D Ab exhibited significantly reduced (p = 0.00284) proliferative response to TLA. Similarly, significantly lower IFN-{gamma}-producing CD4+ T cell response was generated in mice treated with anti-NKG2D-treated animals. As compared with mice injected with control Ab, anti-NKG2D treatment also led to a significant reduction in the IFN-{gamma} production by NK cells (p = 0.01) (Fig. 5E).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We demonstrate that interaction with NK cell leads to elevated IL-12 production by DC and also increases their ability to prime CD8+ T cell response against the parasite. The efficient priming of an in vitro CD8+ T cell response is demonstrated by higher Ag-specific proliferation, cytotoxicity, and IFN-{gamma} production. Increased IL-12 production due to DC-NK interaction is dependent on direct contact and also is partly mediated by IFN-{gamma} released by NK cells. In the absence of direct interaction with DC, NK cells lose their ability to produce IFN-{gamma} in response to antigenic stimulation, because the cytokine was undetectable when they were cultured in a Transwell (data not shown). These findings are in agreement with earlier studies that demonstrate that IFN-{gamma} production by NK cells is dependent on cell contact with myeloid DC (11).

Increased IL-12 production by TLA-pulsed DC is mediated by NKG2D, an activating receptor on human and murine NK cells (37), because Ab to this molecule significantly reduces the level of cytokine production. Moreover, upon priming with TLA, increased expression of NKG2D ligands, RAE-1 and MULT-1, on the DC was observed. Importance of NKG2D during T. gondii infection is further demonstrated by in vivo neutralization studies. T. gondii-infected animals treated with Ab to NKG2D exhibited higher parasite burden in the tissues, and at higher challenge doses they succumb to infection. Treatment with neutralizing Ab to NKG2D led to reduced CD8+ T cell response against the parasite. The reduction in Ag-specific response was not restricted to CD8+ T cell subset, as CD4+ T cell proliferation and IFN-{gamma} production were down-regulated in these animals. This is the first work that describes the induction of NKG2D receptors on DC by parasite Ag and also demonstrates the importance of this interaction in the generation of optimal CD8+ T cell immunity in the infected host.

As mentioned above, NK cells and DC are central components of the innate immune system (1, 8), and recently, attention has been focused on the interaction between these two cell types (38). Initial studies suggested that this effect is unidirectional; NK cell effector functions are stimulated through direct contact with activated DC (12). Subsequently, it has been reported that this interaction is bidirectional and could result in maturation of DC (6, 39). In humans, the NK cell-activating receptor NKp30 plays an important role in DC maturation or apoptosis induced by NK cells (8). Furthermore, recent studies by Borg et al. (9) have reported that NK cells and DC form a stimulatory synapse, which allows polarized secretion of a preassembled stock of IL-12 by DC. Moreover, activated NK cells induced a 100-fold enhanced IL-12 production by DC. Similar to these findings, our data show that IL-12 production by TLA-pulsed DC increased significantly when incubated with NK cells. The increased IL-12 production was apparently due to TLA-mediated up-regulation of NKG2D receptors such as MULT-1 and RAE-1 on DC, and addition of anti-NKG2D to the cultures inhibited the increased cytokine release by these cells. Although IL-12 is critical for polarization of CD4+ T cell response, our laboratory has reported the importance of this cytokine in the induction of CD8+ T cell immunity against T. gondii infection (19). Moreover, recent studies have demonstrated the importance of IL-12 in conditioning of CD8+ T cells for long-term immune response against infection (4, 40). Based on our earlier report mentioned above, reduced CD8+ T cell response in anti-NKG2D-treated animals may be due to decrease in IL-12 production by DC in these animals. However, central focus of our current observation is that NK-DC interaction via NKG2D-dependent mechanism is essential for the induction of robust CD8+ T cell immunity against the parasite.

Similar to our current observations, studies with Mycobacterium tuberculosis have demonstrated that interaction between NKG2D receptors and their ligands plays a role in the lysis of mononuclear phagocytes infected with the bacilli (41). Also, in a very recent study using an in vitro model of infection, it has been suggested that IL-15-mediated enhancement of CD8+ T cell effectors against M. tuberculosis is dependent on NKG2D receptor (42). Likewise, in the current findings, we noted that NK-DC interaction led to increase in the priming of Ag-specific CD8+ T cell response against T. gondii. However, because most of these studies have been conducted in vitro, in vivo importance of NK-DC interaction in general or NKG2D molecule in particular during natural infection has not been well established. Novelty of our findings is that it is the first study that demonstrates the role of NKG2D in the elicitation of optimal immune response in an in vivo model of infection. Neutralization of NKG2D molecule in T. gondii-infected mice led to subdued primary CD8+ T cell response and defect in the parasite clearance in these animals.

Previous studies from our laboratory have reported that mice lacking CD4+ T cells elicit a normal primary CD8+ T cell response against T. gondii that is due to strong NK cell induction in these animals (16). Recent studies by Adam et al. (38) have reported that the interplay between DC and NK cell can completely replace CD4+ T cell help in the induction of CD8+ CTLs against tumors. Based on these observations, it can be postulated that in the absence of CD4+ T cells, NK cells can induce optimal CD8+ T cell response. Thus, T. gondii may allow a unique opportunity for NK-DC interaction, which accentuates DC responses and has the ability to bypass the requirement of CD4+ T cell help in the induction of primary CD8+ T cell immunity. The precise Toxoplasma Ags involved in the up-regulation of NKG2D ligands and signaling events following this interaction need to be identified. Nevertheless, our findings have far-reaching implications in immunosuppressive conditions in which optimal CD8+ T cell immunity against viruses or tumors in the absence of appropriate CD4+ T cell help cannot be evoked. These findings also have strong implication in diseases such as HIV infection, in which poor CD4+ T cell response leads to down-regulation of CD8+ T cell immunity (43), resulting in the reactivation of latent T. gondii infection (44). A strategy to accentuate NK cell response under these conditions may prove beneficial for maintaining robust CD8+ T cell immunity in the host.


   Acknowledgments
 
We are grateful to Drs. Lewis Lanier and Jessica Hammerman, Department of Microbiology and Immunology, Cancer Research Institute (San Francisco, CA), for providing Abs against NKG2D receptor and suggestions during the preparation of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI33325 (to I.A.K.) and AI41930 (to D.J.B.). Back

2 Address correspondence and reprint requests to Dr. Imtiaz A. Khan, Department of Microbiology, Immunology, and Tropical Medicine, George Washington University Medical Center, Ross Hall, 2300 I Street, Washington, DC 20037. E-mail address: mtmixk{at}gwumc.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; p.i., postinfection; TLA, Toxoplasma lysate Ag. Back

Received for publication April 2, 2007. Accepted for publication April 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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