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The Journal of Immunology, 2007, 178, 7259 -7266
Copyright © 2007 by The American Association of Immunologists, Inc.
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STAT1 Expression in Dendritic Cells, but Not T Cells, Is Required for Immunity to Leishmania major1

Leanne M. Johnson and Phillip Scott2

Department of Pathobiology, University of Pennsylvania, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The generation of Th1 responses is important for resistance to intracellular pathogens, including the parasite, Leishmania major. Although IFN-{gamma}R/STAT1 signaling promotes a Th1 response via the up-regulation of T-bet, the requirement for STAT1 in Th1 cell differentiation remains controversial. Although in some cases Th1 cells develop independently of STAT1, STAT1–/– mice fail to develop a Th1 response during L. major infection. However, the interpretation of this result is complicated by the role STAT1 plays in Ag presentation and, more importantly, in elimination of parasites by macrophages, because both defective Ag presentation and increased parasite burden can influence Th cell development. To resolve this issue, we assessed the ability of STAT1–/– T cells to become Th1 cells and protect mice against L. major following adoptive transfer into STAT1-sufficient mice. We found that whereas T-bet is critical for the differentiation of protective Th1 cells during L. major infection, IFN-{gamma}R and STAT1 are dispensable. Given that a STAT1-independent Th1 cell response was generated by STAT1-sufficient APCs, but not by STAT1–/– cells, we next addressed whether dendritic cells (DCs) require STAT1 signaling to effectively present Ag. We found that STAT1–/– DCs had impaired up-regulation of MHC and costimulatory molecules, and, as a consequence, the absence of STAT1 resulted in reduced Th1 cell priming. Taken together, these results demonstrate that T cell expression of STAT1 is not required for the development of Th1 cells protective against L. major and instead stress the importance of STAT1 signaling in DCs for the optimal induction of Th1 responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Defining the conditions that promote the development of effector Th1 and Th2 cells is critical for understanding how to manipulate the immune response, both for the development of vaccines and the identification of new immunotherapeutic approaches for controlling disease. Many factors, such as cytokines, chemokines, transcription factors, and Ag dose, influence the differentiation of T cells into Th cell subsets (reviewed in Ref. 1). Specifically, the development of Th1 cells appears to be strongly influenced by the expression of the transcription factors STAT4, activated by IL-12, and T-bet, activated via STAT1 and/or TCR engagement (1). The demonstration that IFN-{gamma} promotes Th1 cell development via STAT1 activation provided a key to understanding how the activation of NK cells and subsequent IFN-{gamma} production during the innate immune response might promote cell-mediated immunity (2). Consistent with these findings are several studies demonstrating the requirement for STAT1 in the development of Th1 cell responses in autoimmune diseases, such as ulcerative colitis, Crohn’s disease, celiac disease, and diabetes (3, 4, 5), as well as in viral and bacterial infections (6, 7, 8). In contrast, other studies suggest that the requirement for STAT1 in Th1 cell development might depend upon the conditions associated with T cell activation. For example, when IL-4 is blocked and IL-12 is added to STAT1-deficient T cells, they are able to produce IFN-{gamma}, albeit at lower levels than wild-type (WT)3 cells (9). Furthermore, STAT1 appears to be dispensable for Th1 cell development following infection with Toxoplasma gondii (10). Thus, the role of STAT1 in Th1 cell development remains somewhat unclear.

Experimental infections of mice with Leishmania major have been used extensively to define factors associated with Th cell development (11). The production of IFN-{gamma} by CD4+ Th1 cells activates macrophages, and subsequently leads to parasite killing. Susceptibility is associated with poor Th1 cell development and concomitant activation of Th2 cells (12, 13) and IL-10-producing cells (14). Thus, infections of mice lacking the cytokines or transcription factors required for Th1 cell development, such as IL-12, STAT4, and T-bet, invariably lead to a susceptible phenotype and a dominant Th2 response (15, 16, 17). Similarly, it was found that STAT1–/– mice fail to develop a Th1 response during L. major infection and are unable to control parasites (18). Although the lack of Th1 cell development during L. major infection is consistent with STAT1 being essential for the generation of Th1 responses, the inability of STAT1-deficient mice to control parasite replication would be expected, given that macrophage activation requires IFN-{gamma} acting via STAT1. Because infection of macrophages with Leishmania can induce the production of immunosuppressive cytokines, such as TGF-beta, PGE2, or IL-10 (14, 19, 20, 21, 22, 23), uncontrolled parasite growth also has the potential to influence Th1 cell development indirectly. Moreover, STAT1 expression may be important for efficient Ag presentation by dendritic cells (DCs) (24, 25), further complicating the interpretation of studies using mice where STAT1 is deficient in all cells.

Due to the difficulty in ascribing a role for STAT1 in any particular cell type in the experiments that have been done with STAT1-deficient mice, we initiated studies to differentiate between a role for STAT1 in T cells and non-T cells. We confirmed that STAT1-deficient mice fail to develop a Th1 response, and by using an attenuated L. major parasite, found that this was independent of parasite replication. In contrast, we found that STAT1–/– Th1 cells developed in vivo and provided protection against L. major infection in Rag–/– mice, suggesting that T cells do not require STAT1 for immunity to L. major, whereas T cells from T-bet–/– mice were unable to protect Rag–/– mice from infection. Therefore, although a linear pathway in which STAT1 activates T-bet to induce IFN-{gamma} has been proposed (9, 26), our results suggest that during L. major infection, STAT1 and T-bet are differentially required for IFN-{gamma} production and protective immunity. In contrast, STAT1 expression in non-T cells was important for Th1 cell development. Thus, we found that STAT1-sufficient T cells were less able to develop into Th1 cells when primed with STAT1–/– DCs compared with WT DCs. Taken together, decreased Th1 cell development in STAT1–/– mice can, at least in part, be attributed to defective priming by STAT1–/– DCs. Overall, these results suggest that expression of STAT1 in DCs may be a more important factor in Th1 cell differentiation than STAT1 expression in T cells.


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

STAT1–/– mice were generated as previously described (27), and breeding pairs were provided by D. Levy (New York University, New York, NY). E. Pearce (University of Pennsylvania, Philadelphia, PA) provided mice transgenic for the OVA323–339-specific TCR (OT-II) on a C57BL/6 background. Sex- and age-matched WT C57BL/6 mice were used as controls (The Jackson Laboratory). All mice were used at 5–8 wk of age. Mice were maintained in a specific pathogen-free barrier at the University of Pennsylvania. Experiments were conducted in accordance with the guidelines of the University of Pennsylvania Institutional Animal Care and Use Committee.

Parasites and Ag

WT L. major (WHO/MHOM/IL/80/Friedlin) and thymidine auxotrophic L. major (E10-5A3, dihydrofolate reductase-thymidylate synthase– (dhfr-ts–)) (provided by S. Beverley, Washington University, St. Louis, MO) (28) were used for these studies. Parasites were grown to stationary phase in Grace’s insect culture medium (Invitrogen Life Technologies) supplemented with 20% heat-inactivated FBS (HyClone; ≤0.125 EU/ml), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine (Sigma-Aldrich). Thymidine (10 µg/ml; Sigma-Aldrich) was added to cultures of dhfr-ts– parasites. Previously described L. major parasites expressing OVA (Leish-OVA; Lmj8SrRNA:HASPB18OVA) (29) were grown in complete Schneider’s medium (Sigma-Aldrich) (20% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml/streptomycin). Stationary-phase promastigotes were harvested on day 7, washed three times with PBS, and used for infections. Soluble Leishmania Ag (SLA) was prepared, as described previously (30).

Infections

A total of 2 x 106 WT L. major, 10 x 106 dhfr-ts–, or 25 x 106 Leish-OVA stationary-phase parasites was injected into hind footpads of mice. Lesion size was determined by subtracting the thickness of the uninfected contralateral footpad from that of the infected footpad using digital calipers (Mitutoyo). For some studies, mice were treated with 25 µg of CpG (ODN 1826; Coley Pharmaceuticals) at the time of infection. Additionally, in some studies, mice were treated with 0.2 µg of IL-12 (Genetics Institute) intralesionally at the time of infection and on days 3, 6, and 9 postinfection. To enhance Th1 cell differentiation, IL-12-treated mice were also treated with 4 mg of anti-IL-4 (11B11) i.p. on days 0 and 9 postinfection. For ear infections, 1 x 106 stationary-phase L. major promastigotes were injected into the ear dermis. To quantify parasites in the lesions, single-cell suspensions were prepared and plated in 10-fold serial dilutions (initial dilution 1/100) in supplemented Schneider’s Drosophila medium (Invitrogen Life Technologies). Each sample was plated in triplicate, and the mean of the negative log parasite titer was determined on day 7 of culture at 26°C.

Cell culture and flow cytometry

For in vitro recall responses, single-cell suspensions were prepared from draining lymph nodes (dLN), draining the foot (popliteal) or the ear (retromaxillary) of L. major-infected mice at various time points postinfection. Cells were washed, and 4 x 106 cells were plated in 1 ml of RPMI medium supplemented with 10% heat-inactivated FBS, 25 mM HEPES, 5 x 10–5 2-ME, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin in 24-well plates for 4 days. Cells were stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (BFA) (10 µg/ml) for 4 h. Cells were harvested and stained with fluorochrome-conjugated mAbs against surface markers (CD4 (GK1.5); BD Pharmingen), then permeabilized and stained for intracellular cytokines (IFN-{gamma} (XMG1.2)) and IL-4 (11B11); eBioscience) (31). Data were acquired on a FACSCalibur flow cytometer (BD Pharmingen), and analysis was performed using FlowJo software (Tree Star).

In some experiments, cells were isolated from infected ears. Ears were washed with 70% ethanol and allowed to dry. The dorsal and ventral layers of the ear were separated using forceps, and then layers were incubated at 37°C in liberase:DMEM for 30 min. Afterward, ears were homogenized in Medicon grinders for 4 min in a Medimachine (BD Biosciences); cells were collected in complete RPMI medium and restimulated for 4 h with PMA, ionomycin, and BFA; and cytokines were assessed, as above.

Bone marrow-derived macrophage and DC culture

DCs were grown from bone marrow for 8–10 days in complete RPMI supplemented with 20 ng/ml murine rGM-CSF (PeproTech), as described (32). Purities of CD11c+ were >85% and similar between WT and STAT1–/– DCs. For the generation of macrophages, bone marrow was grown in complete RPMI supplemented with 30% L929 cell-conditioned medium, as described previously (33). For maturation profiles, macrophages or DCs were cultured overnight with 1 µg/ml CpG; stained with Abs, CD80 (16-10A1), CD86 (PO3.1), CD40 (HM40-3), and MHC II (M5/114.15.2); and assessed by flow cytometry.

T cell purification and adoptive transfer

T cells were purified from WT or various knockout mice, as described above. In some experiments, mice were depleted of CD8+ T cells by injection with 250 µg of anti-CD8 mAb (H35) 1 and 3 days before they were sacrificed, then enriched for T cells to obtain a pure population (>90%) of CD4+ T cells. A total of 5 x 106 purified T cells was transferred via the retro-orbital plexus into Rag–/– recipients. Mice were challenged with L. major 24 h later, as described above. Protection was assessed until 12 wk after challenge. Data are representative of three to four mice per group. For in vivo priming of OTII cells, 2 x 106 CFSE-labeled T cells (34) were transferred into either WT or STAT1–/– mice. One day later, mice were immunized with 100 µg of OVA protein and 25 µg of CpG in the hind footpad. Five days later, dLN were isolated and restimulated for 4 h with PMA, ionomycin, and BFA, followed by intracellular cytokine staining and flow cytometry. For some experiments, CFSE-labeled OTII T cells were transferred into Thy1.1 WT mice that were then immunized in the footpad with 5 x 105 OVA-pulsed DCs. Five days later, dLN cells were isolated and restimulated with PMA, ionomycin, and BFA in FBS-free medium (supplemented with 3% normal mouse serum).

Real-time PCR

Total RNA was extracted from homogenized dLN tissue with RNeasy columns, following the manufacturer’s instructions (Qiagen). The cDNA was generated using SuperScript II reagents (Invitrogen Life Technologies). Real-time PCR for IL-12p40 was performed using SYBR Green primers (Qiagen). beta-actin was used as an endogenous control for normalization.

Statistical analysis

Two-tailed Student’s t test was used to compare means of lesion sizes and parasite burdens from different groups of mice. Differences were considered significant at p ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Failure to induce Th1 cells in STAT1–/– mice with attenuated parasites

As previously reported, we found that STAT1–/– mice develop large lesions with an overwhelming parasite burden (Fig. 1, A and B) (18) following L. major infection. STAT1 is required for IFN-{gamma} signaling to induce the NO required to kill L. major, and thus, these results are predictable. Because a high parasite burden has been associated with enhanced production of Th1-suppressive factors, such as TGF-beta, IL-10, and PGE2 (14, 19, 20, 21, 22, 23), we asked whether infection with a nonreplicating parasite that might fail to induce such immunosuppressive cytokines would promote a Th1 response in STAT1–/– mice. These parasites lack dhfr-ts, and thus replicate poorly in vivo, and after several weeks are eliminated (28). C57BL/6 WT or STAT1–/– mice were infected with dhfr-ts– parasites, and after 2 wk cells from the dLN were collected and stimulated with SLA. Although WT T cells produced IFN-{gamma} and IL-4, STAT1–/– mice failed to generate an Ag-specific Th1 response during dhfr-ts– infection, consistent with results using WT L. major parasites (Fig. 1C) (18). These data suggest that the inability of STAT1–/– macrophages to control parasite replication is not responsible for the failure of STAT1–/– T cells to differentiate into Th1 cells.


Figure 1
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  		FIGURE 1. STAT1–/– mice fail to develop Th1 cells during L. major infection. A, WT and STAT1–/– mice were infected with 2 x 106 L. major parasites in the footpad. Lesion size was followed over time by subtracting thickness of uninfected footpad from infected footpad. Significant difference (**) between WT and STAT1–/– (p < 0.01). B, Parasites were measured in infected footpads 4.5 wk postinfection using limiting dilution analysis. Significant difference (***) between WT and STAT1–/– (p < 0.001). C, WT and STAT1–/– mice were infected with 2 x 106 L. major or 10 x 106 dhfr-ts– parasites, and 2 wk later, dLN cells were restimulated in vitro with 50 µg/ml SLA and intracellular staining was performed. Plots are gated on CD4+ T cells, and percentages indicate frequencies of IFN-{gamma}+ or IL-4+ cells. Data represent two to three experiments with two to three mice per group. D, Mice were infected with dhfr-ts–, treated with 25 µg of CpG, and restimulated as in C. WT CD4+ T cells are shown in the upper panels, whereas STAT1–/– CD4+ T cells are shown in the lower panels. Similar results were obtained in three independent experiments using two to three mice per group.

 
We next asked whether inclusion of adjuvants associated with Th1 cell development might overcome the defect in Th1 cell polarization in STAT1–/– mice during L. major infection. Because immunostimulatory CpG oligodeoxynucleotides have been shown to enhance Leishmania-specific Th1 cell responses (35, 36, 37, 38), we administered CpG with the attenuated parasites in an attempt to boost the immune response in STAT1–/– mice. When given at the time of infection, CpG treatment enhanced IFN-{gamma} production by WT lymph node (LN) cells after Ag stimulation, but was unable to restore IFN-{gamma} production by STAT1–/– cells (Fig. 1D). However, despite its inability to boost an IFN-{gamma} response in STAT1–/– mice, CpG treatment markedly decreased IL-4 production by both WT and STAT1–/– cells (Fig. 1D), in accordance with recent evidence that suggests that TLR ligands suppress Th2 responses (39).

STAT1-independent Th1 cell polarization and control of L. major infection

Our results demonstrate that in the absence of an overwhelming parasite burden, STAT1-deficient mice are still unable to mount a Th1 response. To determine whether STAT1–/– T cells could become Th1 cells in the presence of STAT1-sufficient APCs, adoptive transfers into Rag–/– mice were used. These mice, although lacking B and T cells, have an intact innate immune system, and thus the role of STAT1 in T cells could be assessed independently of its role in Ag presentation or parasite killing. Purified CD3+ T cells from WT and STAT1–/– were adoptively transferred into Rag–/– mice, and 1 day later mice were infected with L. major. Eight weeks postinfection, cells were isolated from the dLN and the site of infection, and IFN-{gamma} production was assessed by intracellular cytokine staining. T cells from WT mice developed into IFN-{gamma}-producing cells with very few IL-4+ cells, either in the LN or at the site of infection (Fig. 2, A and B, left panels). Surprisingly, when transferred into Rag–/– mice, STAT1–/– IFN-{gamma}-producing T cells were detected in both the dLN and at the site of infection, albeit at somewhat lower levels than that observed with WT T cells (Fig. 2, A and B, right panels). These results suggest that in the presence of an intact innate immune system, STAT1 may not be required for Th1 development during L. major infection.


Figure 2
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  		FIGURE 2. STAT1–/– T cells become Th1 cells that provide protection in Rag–/– mice. A and B, Rag–/– mice were reconstituted with 5 x 106 CD3+ T cells purified from either WT or STAT1–/– mice. After 1 day, mice were infected with 1 x 106 L. major parasites in the ear. Eight weeks postinfection, cells from the dLN (A) and lesion (B) were isolated and restimulated for 4 h with PMA, ionomycin, and BFA, and IFN-{gamma} production was measured by flow cytometry. Plots are gated on CD4+ T cells. Numbers indicate the percentage of cytokine-positive cells in each quadrant; the frequency for the lower right quadrant (IFN-{gamma}+IL-4–) is indicated slightly above the quadrant. Data are representative of three independent experiments with two to three mice per group. C, Rag–/– mice were reconstituted with 5 x 106 CD3+ T cells purified from either WT or STAT1–/– mice. One day later, Rag–/– mice were infected with 2 x 106 L. major parasites. Lesion size was assessed as in Fig. 1. Significant difference (*) between WT or STAT1–/– CD3+ T cell transfers compared with no transferred cell controls (p < 0.05). D, Ten to 12 wk postinfection, parasite load in the lesions was measured by limiting dilution analysis. ***, Significant difference between T cell transfers and no T cells (p < 0.001). E, A total of 5 x 106 purified CD4+ T cells from WT or STAT1–/– mice was adoptively transferred into Rag–/– mice, followed by infection, as in C. Lesion size was monitored over time. Significant difference (*) between WT or STAT1–/– CD4+ T cell transfers compared with no transferred cell controls (p < 0.05). F, Eight to 11 wk postinfection, parasite burden in the footpad was quantified. ***, Significant difference between T cell transfers and no T cells (p < 0.001). Data are representative of three experiments with three mice per group.

 
Because STAT1–/– T cells could become IFN-{gamma}-producing cells in Rag–/– mice, we wanted to determine whether these cells would protect normally susceptible Rag–/– mice from infection. Rag–/– mice were reconstituted with CD3+ T cells and infected, and the course of infection was monitored over time. Although lesion sizes of Rag–/– mice reconstituted with STAT1–/– T cells were slightly larger than mice reconstituted with WT T cells, when compared with Rag–/– mice that received no cells, a significant decrease in lesion size was observed (Fig. 2C). In accordance with lesion size, parasite titers of Rag–/– with STAT1–/– T cells were higher than with WT T cells, but were significantly reduced compared with Rag–/– mice with no cells (Fig. 2D). Furthermore, adoptive transfer of purified CD4+ T cells from either WT or STAT1–/– mice was sufficient for protection against L. major, as assessed by both decreased lesion size (Fig. 2E) and parasite burden (Fig. 2F) compared with unreconstituted mice. Taken together, these data imply that T cells do not require STAT1 to provide protection against L. major infection when non-T cells express STAT1.

T-bet, but not IFN-{gamma}R, is required for protective immunity to L. major

To address the factors involved in the activation of STAT1 and in the downstream consequences of STAT1, adoptive cell transfer experiments into Rag–/– mice using IFN-{gamma}R–/– and T-bet–/– T cells were performed. Reconstitution of Rag–/– mice with IFN-{gamma}R–/– T cells resulted in a similar course of infection and parasite burden to mice receiving WT T cells (Fig. 3, A and B), indicating that T cell-specific IFN-{gamma}R is not required for control of L. major. Of note, the protection obtained with IFN-{gamma}R-deficient T cells was greater than that obtained with STAT1-deficient cells (Fig. 2, C and D), suggesting that the inability of STAT1–/– T cells to provide complete protection compared with WT cells may not be due to the absence of IFN-{gamma} signaling. In contrast, Rag–/– mice reconstituted with T-bet–/– T cells developed progressive lesions with high parasite burdens (Fig. 3, C and D), suggesting that Th1 cell lineage commitment is required to control infection. Therefore, protective immunity to L. major can be achieved in the absence of T cell expression of the IFN-{gamma}R and STAT1, but not T-bet.


Figure 3
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  		FIGURE 3. Expression of T-bet, but not IFN-{gamma}R, in T cells is required for protection against L. major. A, A total of 5 x 106 purified T cells from WT and IFN-{gamma}R–/– mice was transferred into Rag–/– mice that were then infected with 2 x 106 L. major parasites 1 day later. Lesion progression was determined over time, as in Fig. 1. B, Parasites in lesions were enumerated 8 wk postinfection by limiting dilution analysis. ***, Significant differences between T cell-reconstituted mice and mice receiving no cells (p < 0.001). C, Rag–/– mice were reconstituted with 5 x 106 WT and T-bet–/– T cells, and lesion size was monitored over time. D, Seven weeks postinfection, parasites were isolated and quantified from lesions. ***, Significant differences between WT T cell-reconstituted mice and unreconstituted mice (p < 0.001). Data are representative of two similar experiments with three mice per group.

 
Impaired function of STAT1–/– APCs

Our data demonstrate that STAT1–/– T cells have the ability to become protective Th1 cells in a STAT1-sufficient, but not a STAT1-deficient environment, raising questions of the importance of STAT1 signaling in APCs for the initiation of Th1 cell development. To examine the capacity of STAT1–/– APCs to prime Th1 cell responses, T cells were purified from OVA-transgenic OTII mice, CFSE labeled, and adoptively transferred into either WT or STAT1–/– mice. One day later, mice were immunized with OVA + CpG, and 5 days later, donor cell proliferation and IFN-{gamma} production were measured in the dLN. Although proliferation of OTII T cells in response to Ag was comparable in WT and STAT1–/– mice, there was a marked decrease in the frequency of IFN-{gamma}-producing cells in the STAT1–/– mice (Fig. 4A). These results suggest that APCs require STAT1 signaling to optimally promote Th1 cell development.


Figure 4
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  		FIGURE 4. Impaired function of STAT1–/– APCs. A, A total of 1–2 x 106 CFSE-labeled T cells purified from OTII mice was adoptively transferred into WT (left panel) or STAT1–/– (right panel) mice. One day later, mice were immunized with 100 µg of OVA protein and 25 µg of CpG. Five days later, dLN cells were stained for intracellular IFN-{gamma}. Plots are gated on CD4+ donor cells and are representative of two independent experiments with three mice per group. B, Bone marrow-derived DCs were stimulated overnight with 1 µg/ml CpG. Cells were then stained for CD11c and maturation markers, and flow cytometry was performed. Plots are gated on CD11c+ DCs. WT DCs (bold line) and STAT1–/– DCs (dotted line) are compared with unstimulated WT DCs (shaded). Histograms of unstimulated STAT1–/– DCs overlapped unstimulated WT controls (data not shown). The mean fluorescence intensities for each parameter are presented in the accompanying table. Similar results were obtained in four independent experiments. C, Bone marrow-derived macrophages were stimulated as in B, followed by staining for CD11b, CD40, and CD80. Plots are gated on CD11b+ cells. WT macrophages (bold line) and STAT1–/– (dotted line) are compared to unstimulated WT macrophages (shaded).

 
To efficiently present Ag to naive T cells, DCs must mature and up-regulate costimulatory molecules. Previous studies, using LPS to induce DC maturation, found that whereas STAT1–/– DCs exhibit compromised up-regulation of CD40, they are able to express MHC II comparably to WT DCs (24). We sought to determine whether CpG stimulation induced a similar maturation pattern. Bone marrow-derived DCs from WT and STAT1–/– mice were stimulated overnight with CpG, and then surface expression of CD11c, MHC II, CD80, CD40, and CD86 was assessed by flow cytometry. We found an impairment in CD40 up-regulation in STAT1–/– DC similar to published results (Fig. 4B) (24), and expression of CD80, CD86, and MHC II was also diminished in STAT1–/– DCs in response to CpG (Fig. 4B). Additionally, the maturation of macrophages in response to CpG was assessed. Macrophages were grown from WT and STAT1–/– bone marrow for 7 days. Cells were stimulated overnight with CpG, and maturation profiles were evaluated, as in Fig. 4B. Although no up-regulation of CD86 or MHC II was observed in either WT or STAT1–/– DCs (data not shown), CpG stimulation induced CD40 expression and to a lesser extent CD80 expression on macrophages. Thus, comparable to the results with DCs, STAT1 appears to be required for the CpG-induced up-regulation of CD40 and CD80 by macrophages (Fig. 4C). These results further support the hypothesis that STAT1–/– DCs and macrophages have impaired Ag-presenting capacity.

In addition to minimal up-regulation of MHC and costimulatory molecules, STAT1–/– DCs have also been shown to have diminished IL-12 production following TLR ligation (25). Decreased levels of IL-12 can have profound influences on Th1 cell development during infection, because IL-12–/– mice are highly susceptible to L. major and fail to mount a Th1 response (15). Thus, we assessed IL-12 mRNA in either WT or STAT1–/– dLN following infection with L. major. As shown in Fig. 5A, STAT1–/– mice have significantly less IL-12p40 mRNA than WT mice, indicating that the expression of IL-12 during infection is compromised in the absence of STAT1. We next addressed whether the addition of exogenous IL-12 at the time of L. major infection could restore Th1 cell development in STAT1–/– mice. STAT1–/– mice were infected and simultaneously treated with rIL-12, and then boosted every 3 days with IL-12. Mice were also treated with IL-4-neutralizing Abs on days 0 and 9 to favor the development of Th1 cells. After 2 wk of infection, dLN cells were stimulated in vitro with SLA, and after 4 days, IFN-{gamma} levels were measured by ELISA and intracellular cytokine staining. In accordance with Fig. 1, untreated STAT1–/– cells do not produce IFN-{gamma} (Fig. 5, B and C). However, when STAT1–/– mice are treated with IL-12, Ag-specific IFN-{gamma} production is substantially enhanced (Fig. 5, B and C). Therefore, in the absence of STAT1, IL-12 is able to promote STAT1-independent IFN-{gamma} responses. These results are consistent with in vitro studies in which the addition of IL-12 and neutralization of IL-4 resulted in significant IFN-{gamma} production by STAT1–/– T cells (9) (data not shown).


Figure 5
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  		FIGURE 5. Diminished IL-12 responses in STAT1–/– mice. A, WT and STAT1–/– mice were infected with L. major, and after 7 days, total RNA was isolated from dLN cells and reverse transcribed, and then real-time quantitative PCR was performed with primers specific for IL-12p40. Data are normalized using beta-actin and presented as fold increase over respective naive controls, which are arbitrarily set at a value of 1. Data represent three experiments with two to four mice per group. B, STAT1–/– mice were infected as in Fig. 1A and treated with rIL-12 intralesionally (0.2 µg) on days 0, 3, 6, and 9. Mice were also treated with 4 mg of anti-IL-4 Ab on days 0 and 9. At 2 wk postinfection, cells were restimulated with SLA and supernatants were assayed for IFN-{gamma} production by ELISA. C, STAT1–/– mice were infected and treated with IL-12, as in B, and following restimulation with SLA, stained for CD4 and intracellular IFN-{gamma}. The percentage of CD4+ T cells that are positive for IFN-{gamma} is shown. Data are representative of two independent experiments with two to three mice per group.

 
Reduced Th1 responses elicited by STAT1–/– DCs

Our results, in conjunction with others (25), imply that Ag presentation and cytokine production are impaired in STAT1–/– mice. Therefore, we next addressed whether these defects could influence polarization of STAT1-sufficient T cells during L. major infection. CFSE-labeled OTII T cells were transferred into WT or STAT1–/– mice, followed the next day by infection with Leish-OVA. After 5 days of infection, dLN were isolated and analyzed by flow cytometry for proliferation by monitoring CFSE dilution. Although infection with Leish-OVA parasites induced proliferation of OTII T cells in WT mice, T cell proliferation was substantially less in STAT1–/– mice infected with Leish-OVA (Fig. 6A). When Th1 cell polarization was assessed in Leish-OVA-infected mice, again STAT1 deficiency resulted in diminished T cell IFN-{gamma} production. Thus, of the OTII cells that responded to the infection by proliferating, 13% of them produced IFN-{gamma} in WT mice, whereas only 5% produced IFN-{gamma} in STAT1–/– mice (Fig. 6A). The deficit in T cell proliferation seen in STAT1–/– mice was not observed following immunization with OVA + CpG (Fig. 4A), suggesting that possibly due to less TLR signaling, L. major is more dependent upon STAT1 for Ag-presenting function. Finally, to directly test whether STAT1–/– DCs are unable to prime efficient Th1 responses, IFN-{gamma} production by OTII T cells was measured after DC immunizations. OTII T cells were CFSE labeled and transferred into WT recipients, which were then injected with OVA-pulsed WT or STAT1–/– DCs in the footpads. After 5 days, dLN were isolated and proliferation and IFN-{gamma} production were measured by flow cytometry. As shown in Fig. 6B (top panel), WT DCs induced both proliferation and IFN-{gamma} production by donor T cells. In contrast, although STAT1–/– DCs induced proliferation of OTII cells, the frequency of IFN-{gamma}-producing cells was significantly reduced (Fig. 6B, bottom panel), indicating that STAT1–/– DCs induce suboptimal Th1 cell responses. Taken together, these results indicate that in the absence of STAT1 signaling in APCs, Th1 cell responses are compromised.


Figure 6
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  		FIGURE 6. Decreased Th1 cell priming in the absence of STAT1. A, A total of 1 x 106 CFSE-labeled T cells purified from OTII mice (Thy1.1) was adoptively transferred into WT (top panel) or STAT1–/– (bottom panel) mice. One day later, mice were infected with 25 x 106 L. major parasites expressing OVA (29 ). Five days later, dLN cells were isolated. CFSE dilution of Ag-specific donor cells was analyzed. Plots are gated on CD4+, Thy1.1+ donor cells. The number in the corner represents the percentage of donor cells that were CFSEdim in the dLN. Similar results were obtained in two independent experiments with two to four mice per group. B, A total of 1–2 x 106 CFSE-labeled T cells purified from OTII mice was adoptively transferred into congenic Thy1.1 B6 mice. One day later, mice were immunized in footpads with 5 x 105 OVA-pulsed WT (top panel) or STAT1–/– (bottom panel) DCs. Five days later, dLN cells were isolated and intracellular IFN-{gamma} staining was performed. Plots are gated on CD4+, Thy1.2+ donor cells. Similar results were obtained in two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The ability to generate a CD4+ Th1 cell response is critical for resistance to numerous pathogens. Many cytokines and transcription factors involved in the transition of a naive T cell to a Th1 cell have been identified. Notably, the transcription factor, T-bet, has been shown to be a critical factor for IFN-{gamma} production by CD4+ T cells (17). Previous reports have revealed that T-bet can be activated by IFN-{gamma} and IL-27 signaling via STAT1, leading to up-regulation of IL-12Rbeta2 and enhanced functional responsiveness to IL-12 (9, 26, 40). Therefore, it was proposed that STAT1 is essential for the generation of Th1 cells. However, studies from infectious disease models have yielded conflicting results; consistent with the requirement for STAT1 in Th1 cell polarization, STAT1–/– mice fail to generate a Th1 response when infected with L. major (18), but an adequate Th1 response develops when STAT1–/– mice are infected with T. gondii (10). Therefore, the exact requirement for STAT1 expression in CD4+ T cell remains unclear. In this study, we show that T cells deficient in either IFN-{gamma}R or STAT1 differentiate into Th1 cells, albeit in the case of STAT1 cells, at slightly lower frequencies than WT T cells, and are sufficient for protection against L. major in Rag–/– hosts. In contrast, T-bet–/– T cells are unable to become Th1 cells and fail to provide protection against L. major. Thus, the data presented in this work demonstrate that although T-bet is vital for the generation of Th1 cells, IFN-{gamma} and STAT1 play a limited role in Th1 cell differentiation during L. major infection.

Although our results reinforce the requirement for T-bet in Th1 lineage commitment, they challenge the current model of IFN-{gamma}- and STAT1-dependent activation of T-bet. The molecular mechanism underlying the ability of STAT1–/– T cells, but not T-bet–/– T cells, to produce IFN-{gamma} during L. major infection is unknown. Disparate responses between STAT1–/– and T-bet–/– mice have been observed in other systems, particularly during experimental autoimmune encephalomyelitis; STAT1–/– mice developed severe disease, whereas T-bet–/– mice were resistant to experimental autoimmune encephalomyelitis (41). The current model of Th1 differentiation proposes that IFN-{gamma} signaling via STAT1 is required for T-bet expression (9, 26). T-bet then serves to induce the up-regulation of the beta2 chain of the IL-12R to constitute a functional IL-12R, and thus precedes IL-12-mediated selection of Th1 cells (9, 42). However, several studies have shown that some T-bet expression can occur independently of STAT1 (9, 10, 43). In fact, TCR engagement in the absence of exogenous cytokines can induce T-bet expression (44), and provides a likely mechanism for STAT1-independent T-bet induction. Therefore, low levels of T-bet expressed in STAT1–/– T cells may be sufficient for the up-regulation of IL-12Rbeta2 and enhanced IL-12 responsiveness. Along these lines, our results indicate that IL-12 signaling is, at least partially, intact in STAT1–/– T cells, given that exogenous IL-12 can restore IFN-{gamma} production of STAT1–/– T cells both in vitro and in vivo (Fig. 4C) (9), especially when IL-4 is simultaneously neutralized. Furthermore, during T. gondii infection, which induces a strong, systemic IL-12 response, STAT1 was dispensable for Th1 cell development (10).

Similar to STAT1, multiple factors involved in the polarization of Th1 cells have been shown to be conditionally required for Th1 cell development. For example, under certain conditions, IFN-{gamma} production by CD4+ T cells can be independent of IL-12 (45, 46, 47, 48, 49, 50), STAT4 (51), IFN-{gamma}R (52, 53), and T-bet (54, 55). Additionally, we have found previously that IL-27 signaling is required to promote IFN-{gamma} production in the presence, but not the absence, of IL-4 (56). Likewise, IFN-{gamma} seems to be required by CD4+ T cells only in the presence of IL-4 for the up-regulation of IL-12Rbeta2 and subsequent responsiveness to IL-12 (57). Along these lines, T-bet has recently been shown to play a crucial role in the maintenance of STAT4 expression and IL-12 responsiveness by negatively regulating GATA3 (43). Even in the presence of IL-12, T-bet–/– T cells became Th2 cells due to an increased level of GATA3 that impairs IL-12Rbeta2 expression and STAT4 signaling (43, 58). In accordance with these in vitro results, we and others (data not shown) (17) found that T-bet–/– T cells become Th2 cells during L. major infection. Thus, a likely explanation for the Th2 response generated in infected T-bet–/– mice is that they are unable to down-regulate the GATA3 induced by the early burst of IL-4 elicited by L. major parasites.

Previous findings have suggested that like T-bet, STAT1 is also required for the generation of Th1 cells during L. major infection (18). Our ability to generate Leishmania-specific STAT1–/– Th1 cells seemingly contradicts these previous findings. However, in the experiments of Rosas et al. (18), total LN cells were used for Ag restimulations, and thus, not only T cells, but also APCs, lacked STAT1. In light of the data presented in this work, the discrepancies could be explained by the fact that STAT1–/– APCs are unable to provide the signals necessary to generate Th1 cells. Along these lines, we found that STAT1–/– DCs fail to stimulate equivalent Th1 cell polarization to WT DCs in vivo (Fig. 6).

IFN signaling has been associated with maturation and cytokine production of DCs (reviewed in Ref. 59). In this study, we additionally show that the downstream signaling molecule, STAT1, is required for the up-regulation of MHC, CD40, CD80, and CD86 (Fig. 4). The variations between our results and those of Jackson et al. (24), who found that STAT1 was not required for LPS-induced up-regulation of MHC, may be due to differences in culture conditions. Moreover, the receptors for LPS and CpG have distinct expression patterns and signaling cascades that can further contribute to the differences in maturation observed (reviewed in Ref. 60). Once matured, DCs secrete cytokines, including IL-12, which play an important role in the polarization of naive T cells. Recently, IFN-{alpha}beta and STAT1 have been found to be key regulators of IL-12p70 production by DCs following TLR ligation (25), and thus, in the absence of STAT1, IL-12 production is limited. In this study, we show that during L. major infection, IL-12 mRNA expression is also significantly reduced in STAT1–/– mice compared with WT mice (Fig. 5). The inability to secrete adequate IL-12, as well as decreased maturation, correlates with the failure to induce optimal Ag-specific T cell proliferation and IFN-{gamma} production, as seen in the studies presented in this work (Fig. 6). In addition, differences in DC trafficking could contribute to the defects in Th1 cell priming in STAT1–/– mice. Along these lines, STAT1–/– mice failed to establish Leishmania donovani infection due to proposed defects in STAT1-deficient macrophage trafficking to the liver (61). Taken together, our data support a requirement for STAT1 in DCs to provide the necessary factors, both Ag presentation machinery and cytokines, to initiate Th1 cell development. The inability of STAT1–/– DCs to prime optimal Th1 cell responses is consistent with the phenotype of IFN-{gamma}R–/– and T-bet–/– DCs, which also induce diminished Th1 cell differentiation (62, 63). Additionally, IFN-{gamma}R–/– DCs, similar to STAT1–/– DCs, produced decreased IL-12 after stimulation (62), stressing the importance of IFN-{gamma} signaling in DC maturation and cytokine secretion.

In summary, our data uncover distinct requirements for IFN-{gamma} signaling in T cells and APCs. Although IFN-{gamma}R signaling through STAT1 is superfluous for the generation of Th1 cells in our system, STAT1 expression in DCs is important for the induction of Th1 cell development. These results imply that the ability of a pathogen to induce or suppress STAT1 activation in DCs may influence the type of Th cell response elicited. Along these lines, Leishmania parasites have been shown to interfere with IFN-{gamma} signaling by both suppressing expression of the IFN-{gamma}R and inducing proteasome-mediated degradation of STAT1 in macrophages (64, 65). Thus, vaccination strategies designed to promote or maintain STAT1 activation in DCs may be beneficial for enhancing DC maturation and cytokine production and subsequent generation of protective Th1 responses during infection.


   Acknowledgments
 
We thank Dr. Steven Beverley for providing the dhfr-ts– mutant parasites. We thank Dr. David Levy for providing the STAT1–/– mice and Dr. Edward Pearce for providing the OTII mice for these experiments. We also thank the members of the Department of Pathobiology for useful discussions and Dr. Steve Reiner for critical reading of this 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 Grant 35914 (to P.S.). Back

2 Address correspondence and reprint requests to Dr. Phillip Scott, Department of Pathobiology, School of Veterinary Medicine, 380 South University Avenue-Hill Pavilion 310B, Philadelphia, PA 19104. E-mail address: pscott{at}vet.upenn.edu Back

3 Abbreviations used in this paper: WT, wild type; BFA, brefeldin A; DC, dendritic cell; dhfr-ts–, dihydrofolate reductase-thymidylate synthase; dLN, draining lymph node; Leish-OVA, L. major parasites expressing OVA; LN, lymph node; SLA, soluble Leishmania Ag. Back

Received for publication October 26, 2006. Accepted for publication March 22, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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