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. 2010 May 24;78(8):3454–3464. doi: 10.1128/IAI.01407-09

Protective Toxoplasma gondii-Specific T-Cell Responses Require T-Cell-Specific Expression of Protein Kinase C-Theta▿

Gopala Nishanth 1, Monika Sakowicz-Burkiewicz 1, Ulrike Händel 1, Stefanie Kliche 2, Xiaoqian Wang 2, Michael Naumann 3,†, Martina Deckert 4,†, Dirk Schlüter 1,*
PMCID: PMC2916261  PMID: 20498263

Abstract

Protein kinase C-theta (PKC-θ) is important for the activation of autoreactive T cells but is thought to be of minor importance for T-cell responses in infectious diseases, suggesting that PKC-θ may be a target for the treatment of T-cell-mediated autoimmune diseases. To explore the function of PKC-θ in a chronic persisting infection in which T cells are crucial for pathogen control, we infected BALB/c PKC-θ−/− and PKC-θ+/+ wild-type mice with Toxoplasma gondii. The PKC-θ−/− mice succumbed to necrotizing Toxoplasma encephalitis due to an insufficient parasite control up to day 40, whereas the wild-type mice survived. The number of T. gondii-specific CD4 and CD8 T cells was significantly reduced in the PKC-θ−/− mice, resulting in the impaired production of protective cytokines (gamma interferon, tumor necrosis factor) and antiparasitic effector molecules (inducible nitric oxide, gamma interferon-induced GTPase) in the spleen and brain. In addition, Th2-cell numbers were reduced in infected the PKC-θ−/− mice, paralleled by the diminished GATA3 expression of PKC-θ−/− CD4 T cells and reduced T. gondii-specific IgG production in serum and cerebrospinal fluid. Western blot analysis of splenic CD4 and CD8 T cells revealed an impaired activation of the NF-κB, AP-1, and MAPK pathways in T. gondii-infected PKC-θ−/− mice. Adoptive transfer of wild-type CD4 plus CD8 T cells significantly protected PKC-θ−/− mice from death by increasing the numbers of gamma interferon-producing T. gondii-specific CD4 and CD8 T cells, illustrating a cell-autonomous, protective function of PKC-θ in T cells. These findings imply that PKC-θ inhibition drastically impairs T. gondii-specific T-cell responses with fatal consequences for intracerebral parasite control and survival.

Toxoplasma gondii is an obligate intracellular protozoan that infects about a third of the world's population (30). In general, toxoplasmosis is either clinically asymptomatic or associated with only mild clinical symptoms. Nevertheless, the parasite persists in the host central nervous system (CNS). However, immunocompromised individuals, including fetuses and AIDS patients, may suffer from life-threatening toxoplasmosis due to the inability to prevent parasite-induced tissue necrosis. Experimental studies with mice have revealed that control of T. gondii in both acute and chronic toxoplasmosis is critically dependent on gamma interferon (IFN-γ)-producing CD4 and CD8 T cells (10, 46). In addition, interleukin-4 (IL-4), B cells, and antibodies contribute to the control of T. gondii in the CNS (20, 47).

In toxoplasmosis, protective pathogen-specific T-cell responses are dependent on several T-cell-intrinsic signaling molecules, including tumor progression locus 2, T-bet, signal transducer and activator of transcription 4 (STAT4), STAT6, MyD88, Tec kinases (Rlk, Itk), and nuclear factor (NF)-κB (6-8, 18, 22, 23, 28, 37, 50). Experimental studies have revealed that several NF-κB proteins critically regulate protective T-cell responses in toxoplasmosis: RelB is important for the IFN-γ production of T cells (6), NF-κB2 inhibits T-cell apoptosis (7), and c-Rel is crucial for T-cell activation, proliferation, and IFN-γ production (28). However, the signaling pathways leading to the activation of NF-κB in Toxoplasma-specific T cells are incompletely understood.

The serine/threonine-specific protein kinase C-theta (PKC-θ) is predominantly expressed in T cells, muscle cells, and platelets. Upon activation of the T-cell receptor (TCR), PKC-θ is recruited to the immunological synapse and contributes to the activation of the transcription factors NF-κB, activating protein 1 (AP-1), and extracellular-signal regulated kinase (ERK) (3, 44; reviewed in reference 15). Functionally, PKC-θ is required for T-cell activation, proliferation, and survival (25, 34, 44). Experimental studies with mice have unequivocally demonstrated that PKC-θ is crucial for the activation of autoreactive T cells and induction of T-cell-mediated autoimmune diseases (2, 16, 36, 48).

In contrast to autoimmune diseases, PKC-θ is dispensable for the induction of CD4 and CD8 T-cell responses in viral infections caused by vaccinia virus, influenza virus, lymphocytic choriomeningitis virus, and murine herpesvirus 68 (4, 13, 26). In addition, development of Th1-cell responses against the intracellular protozoan Leishmania major is normal in both PKC-θ−/− C57BL/6 and BALB/c mice (27). However, PKC-θ plays a critical role in the development of Th2-cell immune responses after infection with Nippostrongylus brasiliensis (27). To address the role of PKC-θ in bacterial infections, we recently studied listeriosis in PKC-θ−/− and PKC-θ+/+ wild-type (WT) C57BL/6 and BALB/c mice (35). In both strains of mice, PKC-θ was required for the proliferation and survival of IFN-γ-producing Listeria monocytogenes-specific CD4 and CD8 T cells in primary and secondary listeriosis. Consequently, the diminished T-cell responses in the absence of PKC-θ signaling resulted in the impaired control of Listeria. Collectively, these contrasting findings in various infectious diseases illustrate that the functional importance of PKC-θ for the development of pathogen-specific T cells is decisively determined by the underlying pathogen.

This view is strongly supported by the findings of the present study, which analyzed the functional role of PKC-θ in murine toxoplasmosis. PKC-θ was essential for the induction of protective T. gondii-specific Th1 cells and IFN-γ-producing CD8 T cells in T. gondii-resistant BALB/c mice (45). In addition, the numbers of IL-4-producing CD4 T cells were strongly reduced. Finally, the impaired T-cell responses resulted in a failure to control T. gondii in the CNS and, thus, lethal Toxoplasma encephalitis (TE) up to day 40 after infection. In contrast to BALB/c mice, PKC-θ−/− C57BL/6 mice survived the infection, illustrating that the functional role of PKC-θ is dependent on the genetic background of the host.

MATERIALS AND METHODS

Animals.

C57BL/6 PKC-θ−/− were originally obtained from Dan Littman (Skirball Institute of Biomolecular Medicine, New York University, New York, NY [44]) and backcrossed for more than 8 generations on a BALB/c background with BALB/c mice obtained from Harlan-Winkelmann (Borchen, Germany). Age- and sex-matched mice BALB/c PKC-θ−/− and C57BL/6 PKC-θ−/− mice (35) as well as BALB/c and C57BL/6 PKC-θ+/+ WT mice, both obtained from Harlan-Winkelmann, were used for the experiments. All experimental mice were allowed to adapt to the OvG Universität Magdeburg animal facility for at least 14 days and were kept under conventional conditions in an isolation facility throughout the experiments. The experiments were approved and supervised by local governmental institutions.

Parasites and T. gondii infection.

Cysts of the T. gondii DX strain (a type II strain) (9) were harvested from the brains of chronically infected NMRI mice. Parasites were adjusted to a concentration of 10 cysts/ml in 0.1 M phosphate-buffered saline (PBS), and 500 μl was administered orally by gavage to the experimental animals.

Histology.

For immunohistochemistry on frozen sections, mice were perfused intracardially with 0.9% NaCl while they were under methoxyflurane anesthesia. The brains were processed, and immunohistochemistry for T. gondii was performed with rabbit anti-Toxoplasma polyclonal antibody (Ab) (DCS, Hamburg, Germany), as described previously (42).

Quantification of intracerebral T. gondii.

The numbers of T. gondii parasites were determined microscopically on anti-T. gondii-immunostained brain sections by counting at least 100 randomly selected high-power fields (magnification, ×400) per mouse. For each mouse, identical regions of the brain, including the cortex, basal ganglia, and cerebellum, were analyzed.

Isolation of leukocytes from brain and spleen.

Splenic leukocytes were isolated from the dead mice by passing the spleens through a 70-μm-pore-size cell strainer (BD Biosciences, Heidelberg, Germany), and erythrocytes were lysed with ammonium chloride. Before isolation of cerebral leukocytes, the animals were anesthetized with isoflurane (Baxter, Deerfield, IL) and intracardially perfused with 0.9% NaCl to remove contaminating intravascular leukocytes from the brain. Thereafter, brain tissue was minced through a 100-μm-pore-size cell strainer, and leukocytes were separated by Percoll (GE Healthcare, Freiburg, Germany) density gradient centrifugation, as described before (40).

Flow cytometry.

Leukocytes isolated from the brain and spleen were stained with (i) rat anti-mouse CD4-phycoerythrin (PE), rat anti-mouse CD8-fluorescein isothiocyanate (FITC), and rat anti-mouse CD45-PE-Cy5; (ii) rat anti-mouse Ly6G-PE, rat anti-mouse CD11b-FITC, and rat anti-mouse CD45-PE-Cy5; and (iii) rat anti-mouse major histocompatibility complex (MHC) class II PE, rat anti-mouse B220-FITC, and rat anti-mouse CD45-PE-Cy5. CD8 T cells specific for the MHC class I H-2Ld-restricted Gra6-HPGSVNEFDF (HF10) epitope (5) were detected by DimerX (BD Biosciences) staining. In brief, the Gra6-HF10 peptide (JPT, Berlin, Germany) was coupled to DimerX H-2Ld molecules and rat anti-mouse IgG1-PE by overnight incubation at 37°C. Subsequently, leukocytes were stained with DimerX-Gra6-HF10-PE, rat anti-mouse CD62L-FITC, and rat anti-mouse CD8 PE-Cy5. Controls included staining with isotype-matched control Abs and an irrelevant control peptide. All Abs were obtained from BD Biosciences. Flow cytometry was performed on a FACSCalibur instrument (BD Biosciences), and the data were analyzed with WinMDI or CellQuest software.

ELISPOT assay.

The numbers of T. gondii-specific CD4 and CD8 T cells were determined by enzyme-linked immunospot (ELISPOT) assay, as described previously (21). Before the ELISPOT assay, CD4 and CD8 T cells were isolated from the spleens with CD4 and CD8 T-cell isolation kits, respectively (Miltenyi, Bergisch-Gladbach, Germany). Triplicates of magnetic-activated cell sorting (MACS)-isolated splenic CD4 and CD8 T cells (2 × 105, 2 × 104, and 2 × 103 cells/well) were added to rat anti-mouse IFN-γ-coated or rat anti-mouse IL-4-coated (both from Invitrogen, CA) ELISPOT assay plates and coincubated with spleen cells from noninfected BALB/c WT mice (2 × 105/well), which were preloaded with heat-killed Toxoplasma (HKT; three parasites per splenocyte). Controls included coincubation of isolated leukocytes with spleen cells without peptide loading and incubation of leukocytes from noninfected mice with peptide-loaded spleen cells. All ELISPOT assay plates were incubated overnight and developed with biotin-labeled rat anti-mouse IFN-γ or biotin-labeled rat anti-IL-4 (BD Biosciences), peroxidase-conjugated streptavidin, and aminoethylcarbazole dye solution (Sigma-Aldrich). The spots were counted microscopically, and the numbers of antigen (Ag)-specific CD4 and CD8 T cells per organ were calculated from the number of spots in triplicate wells.

Adoptive transfer of T cells.

Polyclonal CD4, CD8, and CD4 plus CD8 T cells were isolated from the spleens of noninfected WT and PKC-θ−/− mice by MACS using CD4, CD8, and pan-T-cell isolation kits, respectively (Miltenyi). Isolated T cells (1 × 106) were injected intravenously (i.v.) into PKC-θ−/− mice 24 h before infection with T. gondii.

Western blotting (WB).

CD4 and CD8 T cells were isolated from the spleens of WT and PKC-θ−/− mice by MACS as described above and resuspended in lysis buffer containing 50 mM Tris-HCl (pH 7.4); 5 mM EDTA; 100 mM NaCl; 1% Triton X-100; 10% glycerol; 10 mM KH2PO4; 0.5% sodium deoxycholate; 1 mM phenylmethylsulfonyl fluoride;1 mM NaF; 1 mM Na4O7P2; 1 mM Na3VO4; and aprotinin, leupeptin, and pepstatin (1 μg/ml each). Equal amounts of proteins were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes, followed by incubation with anti-PKC-θ, anti-phospho-PKC-θ (p-PKC-θ), p-IKK1/2, p-p65, anti-GAPDH, and anti-caspase 3 (all from Cell Signaling Technology, MA) and with p-c-Fos, p-Jun, p-ERK, and GATA3 (all from Santa Cruz Biotechnology, CA). Blots were developed using an ECL Plus kit (GE Healthcare).

RT-PCR.

For reverse transcription-PCR (RT-PCR), mRNA was isolated from the brains and spleens of uninfected and T. gondii-infected mice (RNAeasy kit; Qiagen, Hilden, Germany). mRNA was transcribed into cDNA by use of the SuperScript reverse transcriptase kit with oligo(dT) primers (Invitrogen). Quantitative RT-PCR for IFN-γ, tumor necrosis factor (TNF), IL-10, inducible nitric oxide synthase (iNOS), and hypoxanthine phosphoribosyltransferase (HPRT) was performed with cDNA from BALB/c WT and BALB/c PKC-θ−/− mice with a GeneAmp 5700 sequence detection system (Applied Biosystems, Weiterstadt, Germany). Quantitation was performed with the sequence detector software SDS (version 2.1; Applied Biosystems), according to the ΔΔCT threshold cycle (CT) method with HPRT as the housekeeping gene. Data are expressed as the increase in the level of mRNA expression in infected mice over that in noninfected controls of the respective mouse strain. All primers and probes were obtained from Applied Biosystems.

Statistics.

Statistical significance was determined using the two-tailed Student t test or nonparametric Mann-Whitney rank sum test (Statistica 5; StatSoft). All experiments were performed at least twice. P values of <0.05 were considered significant.

RESULTS

Infection with T. gondii induces sustained phosphorylation of PKC-θ in CD4 and CD8 T cells.

Since PKC-θ plays an important role in the TCR-mediated activation and proliferation of T cells, we analyzed PKC-θ expression and phosphorylation in the CD4 and CD8 T cells of BALB/c WT mice after infection with T. gondii. WB analysis showed that (nonactivated) PKC-θ was expressed in CD4 as well as CD8 T cells of both noninfected (day 0) and T. gondii-infected mice (Fig. 1). In uninfected mice, activated PKC-θ was weakly expressed in CD4 and CD8 T cells (Fig. 1). Upon infection, PKC-θ was strongly activated in both CD4 and CD8 T cells at days 7, 14, and 21 postinfection (p.i.) (Fig. 1). These findings illustrate the sustained activation of PKC-θ in CD4 and CD8 T cells of T. gondii-infected mice and provide the basis for analysis of the functional role of PKC-θ in toxoplasmosis.

FIG. 1.

FIG. 1.

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Sustained activation of PKC-θ in CD4 and CD8 T cells in toxoplasmosis. WB analysis of PKC-θ expression and phosphorylation in CD4 and CD8 T cells of noninfected as well as T. gondii-infected (days 7, 14, and 21 p.i.) WT mice was performed. CD4 and CD8 T cells were selectively isolated by MACS from four mice per time point. Representative blots are shown.

BALB/c PKC-θ−/− mice succumb to necrotizing TE.

To analyze whether PKC-θ plays a critical role for survival of toxoplasmosis, BALB/c PKC-θ−/− and WT mice were infected with T. gondii cysts. Whereas 92% of the PKC-θ−/− mice succumbed to the infection up to day 40 p.i., only 8% of WT mice died up to day 60 p.i. (Fig. 2A). In contrast, both C57BL/6 PKC-θ−/− and WT mice survived the infection up to day 60 p.i. (Fig. 2B). A detailed histopathological examination revealed that BALB/c PKC-θ−/− mice died of a necrotizing TE with huge amounts of intracerebral parasites (Fig. 2C). In contrast, BALB/c WT mice harbored only low numbers of parasites in the brain without tissue necrosis. Quantification of the intracerebral toxoplasms revealed that the number of parasites was significantly increased in the brains of BALB/c PKC-θ−/− mice compared to the number in the WT controls at day 21 p.i. (Fig. 2D). These findings demonstrate that PKC-θ is essential for intracerebral parasite control and survival of toxoplasmosis in BALB/c mice. In the following experiments, BALB/c PKC-θ−/− and WT mice were used to analyze the role of PKC-θ in toxoplasmosis.

FIG. 2.

FIG. 2.

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Reduced survival and impaired parasite control of T. gondii-infected BALB/c PKC-θ−/− mice. BALB/c and C57BL/6 WT and PKC-θ−/− mice were orally infected with T. gondii. (A) Significantly more BALB/c PKC-θ−/− mice than BALB/c WT animals succumbed to the infection (P < 0.005) up to day 60 p.i. Ten mice of each mouse strain were analyzed. (B) Both C57BL/6 PKC-θ−/− and C57BL/6 WT mice survive toxoplasmosis up to day 60 p.i. (C) A histopathological analysis revealed that BALB/c PKC-θ−/− mice had developed a severe necrotizing TE with huge amounts of intracerebral parasites at day 21 p.i. In contrast, BALB/c WT mice harbored only a few parasitic cysts (arrow) at this stage of infection. Anti-T. gondii immunostaining, slight counterstaining with hemalum. Magnification, ×100. (D) The numbers of T. gondii in 100 high-power fields (HPF) per mouse brain were determined microscopically. Four mice per group were analyzed at days 14 and 21 p.i., and data represent the means + standard deviations (**, P < 0.01).

Reduced numbers of CD4 and CD8 T cells in spleens and brains of T. gondii-infected PKC-θ−/− mice.

To determine whether PKC-θ plays a role in the recruitment of leukocytes to the brain and affects leukocyte numbers in the spleen in toxoplasmosis, intracerebral and splenic leukocytes were isolated from uninfected as well as T. gondii-infected mice at day 21 and day 42 p.i. Uninfected PKC-θ−/− and WT mice harbored equal numbers of CD4 and CD8 T cells, B cells, macrophages, and granulocytes in their brains and spleens (Fig. 3 A and B). Upon infection with T. gondii, the numbers of these leukocyte populations increased in both strains of mice (Fig. 3A and B). However, the numbers of CD4 and CD8 T cells were significantly increased in the spleens and brains of WT animals at day 21 and day 42 p.i. In addition, the numbers of B cells were increased in the spleens and brains of WT mice compared to the numbers in PKC-θ−/− mice. The numbers of splenic and intracerebral granulocytes and macrophages did not differ between PKC-θ−/− and WT mice at either time point after infection (Fig. 3A and B). Since PKC-θ is predominantly expressed in T cells and the numbers of CD4 and CD8 T cells were strongly reduced in the spleens and brains of T. gondii-infected PKC-θ−/− mice, we further focused on the impact of PKC-θ on T. gondii-specific T cells.

FIG. 3.

FIG. 3.

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Reduced numbers of CD4 and CD8 T cells in the spleens and brains of T. gondii-infected PKC-θ−/− mice. The leukocyte populations in the spleens (A) and brains (B) of uninfected and T. gondii-infected WT and PKC-θ−/− mice were phenotyped by flow cytometry at the indicated time points. Six mice per group and time point were analyzed. Data represent the means + standard deviations for each cell population (*, P < 0.05; **, P < 0.01).

Diminished T. gondii-specific CD4 and CD8 T-cell responses in PKC-θ−/− mice.

To analyze whether PKC-θ regulates T. gondii-specific CD8 T cells, we determined the frequency and number of T. gondii-specific Gra6-HF10-specific CD8 T cells in the spleens and brains of mice by DimerX staining at days 7, 14, and 21 p.i. (Fig. 4A and B). Flow cytometry revealed that both the percentage (Fig. 4A) and the number (Fig. 4B) of Gra6-HF10-specific CD8 T cells increased in the spleens of WT mice and reached a maximum at day 21 p.i., which is in accordance with data from Blanchard et al. (5). In parallel, the percentage and absolute numbers of Gra6-HF10-specific CD8 T cells increased in the brains of WT animals. Although the percentage and the absolute number of Gra6-HF10-specific CD8 T cells increased gradually in PKC-θ−/− mice over time, the values for both parameters remained significantly reduced compared to the values for the WT animals at all time points.

FIG. 4.

FIG. 4.

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Impaired T. gondii-specific T-cell responses in PKC-θ−/− mice. (A) Splenic and intracerebral leukocytes were isolated and stained with Gra6-HF10 DimerX and anti-CD62L in combination with anti-CD8 at the indicated time points (d, day). Leukocytes were gated on CD8 T cells, and dot plots show Gra6-HF10- and CD62L-stained CD8+ T cells. The percentage of Gra6-HF10-positive (Gra6-HF10+) CD62L-negative (CD62L−) cells is indicated in each dot plot. Six mice were analyzed per time point and experimental group, and representative dot plots are shown. (B) The mean numbers + standard deviations of T. gondii-specific CD8 T cells in the spleen and brain of WT and PKC-θ−/− mice were calculated from the percentage of Gra6-HF10+ CD62L− CD8 T cells and the absolute number of CD8 T cells. Six mice were analyzed per group and time point (*, P < 0.05; **, P < 0.01). (C and D) The frequencies of IFN-γ-producing splenic (C) and intracerebral (D) CD4 and CD8T cells were determined by an ELISPOT assay. Splenic and intracerebral CD4 and CD8 T cells were isolated by MACS before analysis. Intracerebral leukocytes were analyzed at day 21 p.i. Data show the means + standard deviations of six mice per group (*, P < 0.05; **, P < 0.01). (E) The frequency of IL-4-producing T. gondii-specific, MACS-isolated splenic CD4 T cells was determined by an IL-4 ELISPOT assay at the indicated time points. Four mice were analyzed per experimental group and time point. Data show the means + standard deviations (*, P < 0.05). (F) Proteins were isolated from MACS-isolated splenic CD4 T cells of WT and PKC-θ−/− mice and stained for GATA3 and GAPDH by WB. Representative blots are shown. (G) CD4 and CD8 T cells were isolated from the spleens of three to five mice per experimental group by MACS at the indicated time points. WB analysis for caspase 3 and active caspase 3 was performed, and representative data are shown.

Additional ELISPOT experiments showed that the numbers of T. gondii-specific IFN-γ-producing CD4 and CD8 T cells were also significantly reduced in the spleens and brains of PKC-θ−/− mice (Fig. 4C and D). Since IFN-γ production by CD4 and CD8 T cells is crucial to control T. gondii in the CNS, these findings imply that the insufficient generation of IFN-γ-producing T cells in the absence of PKC-θ accounts for the lethal necrotizing TE in PKC-θ−/− mice.

Since it has been reported that PKC-θ is important for the development of Th2 cells upon infection with Nippostrongylus brasiliensis and Leishmania major (27), we studied the effect of PKC-θ on Th2 responses in toxoplasmosis by IL-4 ELISPOT assay of CD4 T cells. The numbers of T. gondii-specific IL-4-producing CD4 T cells were significantly reduced in PKC-θ−/− mice at all time points after infection (Fig. 4E). In parallel, the expression of the transcription factor GATA3, which induces Th2 responses, was strongly upregulated in CD4 T cells of WT animals, whereas the level of upregulation of GATA3 expression was lower in the CD4 T cells of T. gondii-infected PKC-θ−/− mice at days 7, 14, and 21 p.i. (Fig. 4F). Thus, T. gondii-specific Th2 responses were reduced in the absence of PKC-θ.

The reduced absolute numbers of CD4 and CD8 T cells as well as parasite-specific CD4 and CD8 T cells may be caused by the increased apoptosis of PKC-θ-deficient T cells. Therefore, we isolated CD4 and CD8 T cells from both strains of mice and analyzed the expression of active caspase 3. Both WT and PKC-θ−/− mice expressed equally low levels of activated caspase 3 at day 14 p.i. At all other time points, active caspase 3 was not detectable by WB. However, further in vitro analysis of T-cell proliferation and apoptosis was precluded by the extremely low number of parasite-specific CD4 and CD8 T cells in PKC-θ−/− mice.

Reduced cytokine responses in PKC-θ−/− mice.

To study the impact of the reduced number of IFN-γ-producing T. gondii-specific T cells on cytokine production, a quantitative RT-PCR analysis was performed at day 21 p.i. The level of IFN-γ mRNA was significantly reduced in the spleens and brains of T. gondii-infected PKC-θ−/− mice (Fig. 5A). In addition, the level of expression of TNF mRNA was reduced in PKC-θ−/− mice (Fig. 5B). Interestingly, IL-10 mRNA, which is mainly produced by conventional Foxp3-negative (Foxp3−) Th1 cells in toxoplasmosis (17), was also significantly reduced in the spleens and brains of PKC-θ−/− mice (Fig. 5C). Furthermore, the levels of iNOS and gamma interferon-induced GTPase (IGTP), two important IFN-γ-regulated antiparasitic effector molecules, were significantly reduced in PKC-θ−/− mice (Fig. 5C and D). In conclusion, the levels of production of the mRNA of essentially protective cytokines and IFN-γ-regulated antiparasitic effector molecules were significantly diminished in PKC-θ−/− mice.

FIG. 5.

FIG. 5.

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Reduced production of cytokines and antiparasitic effector molecules in T. gondii-infected PKC-θ−/− mice. A quantitative RT-PCR analysis of IFN-γ (A), TNF (B), IL-10 (C), iNOS (D), and IGTP (E) mRNA expression in the spleens and brains of WT and PKC-θ−/− mice was performed. Data are expressed as the increase in the respective levels of mRNA from infected (day 21 p.i.) over uninfected mice and were normalized to the level of HPRT expression. Data represent the means + standard deviations of three to five mice per group and time point (*, P < 0.05; **, P < 0.01; d0, day 0).

Impaired activation of NF-κB, AP-1, and ERK in PKC-θ−/− mice.

The activation and regulation of the transcription factors NF-κB and AP-1 as well as the kinase ERK were studied by WB of the T cells of T. gondii-infected mice. In these experiments, we focused on bulk CD4 and CD8 T cells, since the number of parasite-specific CD4 and CD8 T cells, especially in PKC-θ−/− mice, was too low to allow a direct ex vivo analysis by WB. As shown in Fig. 6, the CD4 and CD8 T cells of PKC-θ−/− mice showed an impaired activation of NF-κB, as indicated by the reduced phosphorylation of IKK1/2 and p65. In addition, the level of phosphorylation of the AP-1 transcription factor c-Jun, but not that of c-Fos, was reduced in CD4 and CD8 T cells of PKC-θ−/− mice compared to the level in WT mice. Furthermore, PKC-θ−/− mice showed a slight reduction in the level of ERK activation in both CD4 and CD8T cells compared to that in WT animals. Taken together, these data indicate that in the absence of PKC-θ, the activation of several important signaling pathways, including NF-κB, AP-1, and ERK, was impaired in CD4 and CD8 T cells.

FIG. 6.

FIG. 6.

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WB analysis of the NFκB, AP-1, and ERK pathways in CD4 and CD8 T cells. CD4 and CD8 T cells were isolated by MACS from the spleens of three to five WT and PKC-θ−/− mice at the indicated time points. After isolation and blotting, proteins were stained for p-IKK1/2, p-p65, p-c-Fos, anti-p-c-Jun, p-ERK, and GAPDH.

Reduced T. gondii-specific IgG production in serum and CSF of PKC-θ−/− mice.

B cells play a protective role in toxoplasmosis, and T. gondii-specific Ab responses are partially regulated by CD4 T cells (20, 24). Therefore, we evaluated the T. gondii-specific Ab responses in T. gondii-infected PKC 0−/− and WT mice. At day 21 p.i., equal amounts of T. gondii-specific IgM were present in the serum and cerebrospinal fluid (CSF) of PKC-θ−/− and WT mice (Fig. 7A and B). In contrast, the levels of T. gondii-specific IgG were reduced in the serum and CSF of PKC-θ−/− mice (Fig. 7C and D). These results illustrate that T. gondii-specific IgG but not IgM production was partially dependent on PKC-θ.

FIG. 7.

FIG. 7.

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Reduced levels of IgG but normal levels of T. gondii-specific IgM production in PKC-θ−/− mice. (A to D) T. gondii-specific IgM (A and B) and IgG (C and D) Abs were determined in serum (A and C) and CSF (B and D) of WT and PKC-θ−/− mice by enzyme-linked immunosorbent assay at day 21 p.i. The serum and CSF were serially diluted, and the highest positive dilution is shown. Serum and CSF were isolated from five to six mice per group and time point. Data show the means + standard deviations of Ab titers in serum (*, P < 0.05). The CSF from mice of each experimental group was pooled to obtain a sufficient amount of CSF, and the means of duplicates are shown.

WT T cells compensate for PKC-θ deficiency in toxoplasmosis.

To validate that the lethal course of toxoplasmosis in PKC-θ−/− mice was caused by an insufficient T-cell response, we adoptively transferred purified polyclonal WT T cells into PKC-θ−/− mice before infection with T. gondii and monitored the survival of these animals. As shown in Fig. 8A, 68% of PKC-θ−/− mice reconstituted with WT T cells survived the infection, whereas 100% of both PKC-θ−/− mice without T-cell transfer or with transfer of PKC-θ−/− T cells succumbed to the infection. As shown before, all WT mice (i.e., without T-cell transfer) survived the infection. Compared to the numbers in WT animals, the surviving PKC-θ−/− mice with adoptive WT T-cell transfer harbored equally low numbers of T. gondii cysts in their brains at day 60 p.i. (P > 0.05) (Fig. 8B).

FIG. 8.

FIG. 8.

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WT T cells compensate for PKC-θ deficiency in toxoplasmosis. MACS-purified WT and PKC-θ−/− T cells were adoptively transferred into PKC-θ−/− recipients. Recipients as well as control WT and PKC-θ−/− mice were orally infected with T. gondii. (A) The survival of six mice per experimental group was monitored until day 60 p.i. (B) The numbers of T. gondii cysts in WT and PKC-θ−/− mice with adoptively transferred WT T cells were determined at day 60 p.i. Data represent the means + standard deviations of six surviving WT and four surviving PKC-θ−/− mice with transfer of WT T cells. (C and D) The numbers of T. gondii-specific Gra6-HF10-positive and CD62L-negative CD8 T cells in the spleen (C) and brains (D) of the indicated groups of mice were determined by flow cytometry at day 21 p.i. (E) The frequency of IFN-γ-producing splenocytes was determined by an IFN-γ ELISPOT assay at day 21 p.i. (C and D) The data represent the means + standard deviations of five mice per group. (F) MACS-purified WT CD4, WT CD8, and WT CD4 plus CD8 mouse T cells as well as PKC-θ−/− mouse CD4 and CD8 T cells were adoptively transferred into PKC-θ−/− recipients. Recipients as well as control WT and PKC-θ−/− mice were orally infected with T. gondii, and the survival of six mice per experimental group was monitored until day 60 p.i. (A to F) P < 0.05 (*) and P < 0.01 (**) for each group versus the results for PKC-θ−/− mice.

To further analyze whether the adoptive transfer of WT T cells into PKC-θ−/− mice improved the parasite-specific T-cell responses in PKC-θ−/− mice, the number of Gra6-HF10-specific CD8 T cells was determined. In both the spleens (Fig. 8C) and the brains (Fig. 8D), the number of Gra6-HF10-specific CD8 T cells was significantly increased in PKC-θ−/− mice with WT T-cell transfer compared to the number in PKC-θ−/− mice without WT T-cell transfer (P < 0.05 for both organs). As shown before, the numbers of Gra6-HF10-specific CD8 T cells were also significantly increased in the spleens and brains of WT mice compared to the numbers in PKC-θ−/− animals. In addition, the numbers of T. gondii-specific IFN-γ-producing T cells were significantly increased in the spleens of both PKC-θ−/− mice with WT T-cell transfer and WT mice compared to the numbers in PKC-θ−/− without T-cell transfer (P < 0.01) (Fig. 8E). These findings demonstrate that WT T cells can significantly protect PKC-θ−/− mice from lethal toxoplasmosis.

To determine whether adoptively transferred CD4 and/or CD8 WT T cells confer protection against toxoplasmosis in PKC-θ−/− mice, CD4, CD8, and CD4 plus CD8 T cells were adaptively transferred to PKC-θ−/− mice before infection with T. gondii. As illustrated in Fig. 8F, only transfer of both T-cell populations significantly protected PKC-θ−/− mice from lethal toxoplasmosis. Although transfer of either CD4 or CD8 WT T cells prolonged the survival times of PKC-θ−/− mice by a maximum of 10 days, all of these animals succumbed to the infection. Thus, the combined action of both CD4 and CD8 WT T cells was required to compensate efficiently for the PKC-θ deficiency in BALB/c mice with toxoplasmosis.

DISCUSSION

The present study demonstrates that PKC-θ is absolutely required for protective T. gondii-specific T-cell responses, efficient parasite control in the brain, and survival from cerebral toxoplasmosis in BALB/c mice. Upon infection with T. gondii, CD4 and CD8 T cells upregulated p-PKC-θ as early as day 7 p.i., when the levels of T. gondii-specific T cells were still below the detection limit of DimerX staining and the ELISPOT assay. At the time of the maximum T-cell response, i.e., at day 21 p.i., CD4 and CD8 T cells still contained activated PKC-θ, indicating its functional importance for activation, proliferation, and maintenance of T. gondii-specific T cells. In fact, the absolute numbers of splenic and intracerebral T. gondii-specific CD4 and CD8 T cells remained extremely low in PKC-θ−/− mice throughout the study without a significant increase over time of infection.

The impaired splenic and intracerebral CD4 and CD8 T-cell responses of T. gondii-infected PKC-θ−/− mice was reflected by the reduced numbers of both T-cell populations, the reduced numbers of Gra6-HF10-specific CD8 T cells, as well as the reduced numbers of IFN-γ-producing CD4 and CD8 T cells. Since IFN-γ-producing CD4 and CD8 T cells are absolutely required for the control of T. gondii (10), the diminished T-cell response of PKC-θ−/− mice consequently resulted in a lethal necrotizing TE. The protective function of IFN-γ is partially mediated by the induction of other protective cytokines, including TNF, as well as the induction of antiparasitic effector molecules, including iNOS and IGTP (11, 38, 39, 41, 49). This explains (i) why the strong reduction of intracerebral and splenic IFN-γ mRNA levels was accompanied by the massive reduction of TNF, iNOS, and IGTP levels in infected PKC-θ−/− mice compared to the levels in WT mice and (ii) why PKC-θ−/− mice were unable to restrict T. gondii proliferation in the brain. Interestingly, the level of IL-10, which is required to prevent a lethal IFN-γ-mediated immunopathology in toxoplasmosis (12, 33), was also reduced in PKC-θ−/− mice. Since IL-10 is mainly produced by conventional Foxp3− Th1 cells in toxoplasmosis (17), the reduced level of IL-10 mRNA production is most probably caused by the impaired development of T. gondii-specific Th1 cells in PKC-θ−/− mice. Of note, in mice of the same T. gondii-resistant BALB/c background, IL-33 receptor-mediated downregulation of Th1 response is important to prevent intracerebral immunopathology and to control parasite growth in the brain (19). Thus, in BALB/c mice both the PKC-θ-dependent activation of protective Th1 and Th2 responses (see below) and the IL-33-dependent limitation of Th1 responses are required for effective parasite control and prevention of an exacerbated encephalitis.

The transfer of WT T cells improved intracerebral parasite control, increased parasite-specific T-cell responses close to WT levels, and significantly protected T. gondii-infected PKC-θ−/− mice from death. Interestingly, the combined action of WT CD4 and CD8 T cells was required to protect PKC-θ−/− mice from fatal toxoplasmosis, since the adoptive transfer of either WT CD4 or CD8 T cells did not prevent the death of T. gondii-infected PKC-θ−/− mice. These data illustrate that the T-cell-autonomous expression of PKC-θ is sufficient to compensate for PKC-θ deficiency. In addition, these observations illustrate that the deficiency of PKC-θ in T cells but not in other cell populations, including NK cells, is decisive for the fatal course of chronic TE in PKC-θ−/− mice. These data extend the findings of previous studies of murine listeriosis, in which PKC-θ was also required for normal pathogen-specific CD4 and CD8 T-cell responses (35).

A direct ex vivo WB analysis revealed that CD4 and CD8 T cells required PKC-θ for normal activation of the NF-κB pathway at all time points of the infection, as indicated by the reduced activities of IKK1/2 and p65 in the CD4 and CD8 T cells of PKC-θ−/− mice. IKK1/2 and p65 are part of the canonical NF-κB pathway, and PKC-θ regulates this pathway directly (reviewed in reference 15). The assumption that the NF-κB pathway is critical for T. gondii-specific T-cell responses is substantiated by the importance of the NF-κB molecules c-Rel and RelB for the development of protective T. gondii-specific T-cell responses (6, 28). In addition, the reduced phosphorylation of c-Jun in CD4 and CD8 T cells of PKC-θ−/− mice illustrates the impaired activation of AP-1 in T. gondii-infected PKC-θ−/− mice, although it is at present unclear to what extent T-cell responses are AP-1 dependent in toxoplasmosis. Furthermore, activation of ERK was slightly reduced in PKC-θ−/− CD4 and CD8 T cells, especially at day 21 p.i. in CD4 T cells. Collectively, these data illustrate that the normal activation of several major signaling pathways in T cells of T. gondii-infected mice was PKC-θ dependent. It should be stressed that this analysis was performed with bulk T cells, i.e., T. gondii-specific and bystander T cells, and that the observed differences in the activation of signaling molecules are due to the direct effect of PKC-θ on signaling pathways as well as the reduced levels of cytokine production in PKC-θ−/− mice. Thus, these ex vivo data sum up the direct and indirect effects of PKC-θ on the major signaling pathways in T cells and thereby extend previous in vitro findings on the role of PKC-θ for the activation of NF-κB, AP-1, and ERK in T cells upon stimulation of the TCR (3, 44; reviewed in reference 15).

Although toxoplasmosis is characterized by a strong protective Th1 response, additional IL-4-producing Th2 cells develop and contribute to the optimal control of the parasite and survival in toxoplasmosis (47). In our experiments, the T. gondii-specific Th2 response was also PKC-θ dependent, since in the absence of PKC-θ, the frequency of IL-4-producing CD4 T cells was greatly diminished. Interestingly, the level of GATA3, which is of key importance for the Th2 differentiation of CD4 T cells (31), was also reduced in the CD4 T cells of PKC-θ−/− mice. These data extend previous in vitro observations for anti-CD3/CD28-stimulated CD4 T cells, which also revealed that PKC-θ is involved in the upregulation of GATA3 (43). In addition, Th2 differentiation of Leishmania and Nippostrongylus brasiliensis-specific CD4 T cells is PKC-θ dependent (27). However, in contrast to T. gondii-specific Th1 cells, the development of Leishmania-specific Th1 responses was PKC-θ independent (27).

In discussing the importance of PKC-θ for T-cell differentiation, it should be stressed that only low numbers of T. gondii-specific Th1 and Th2 CD4 as well as CD8 T cells were detectable in PKC-θ−/− mice. Thus, the reduction of the levels of all T. gondii-specific T-cell subsets may be caused by either the impaired proliferation or survival of T cells. Both the survival and proliferation of T cells are critically regulated by PKC-θ after TCR stimulation in vitro as well as in listeriosis (25, 34, 35, 44). A lack of increased activation of caspase 3 in splenic CD4 and CD8 T cells of PKC-θ−/− may imply that an increased rate of apoptosis was not responsible for the reduced numbers of T. gondii-specific CD4 and CD8 T cells. However, the very low numbers of T. gondii-specific T cells in PKC-θ−/− mice prevented a further in vivo functional analysis of T-cell proliferation and apoptosis.

Recently, Blanchard et al. (5) identified an H-2Ld-restricted CD8 T-cell epitope of T. gondii, the Gra6-HF10 decamer. DimerX staining with this peptide showed that the levels of T. gondii-specific CD8 T cells gradually increase over time and reach maximal numbers in the spleen as late as day 21 p.i. (5), which perfectly fits with our previous data obtained with β-galactosidase transgenic toxoplasms (21). In the present study, we extend these data and show that (i) intracerebral CD8 T cells follow the same kinetics, (ii) 31% of the intracerebal CD8 T cells are specific for this single epitope at day 21 p.i., and (iii) all of these CD8 T cells are activated because of their CD62L negativity. Thus, both the splenic and intracerebral CD8 T-cell responses of H-2d BALB/c mice focuses very much on the Gra6-HF10 epitope, and this may also explain why the H-2Ld gene confers resistance to toxoplasmosis (45).

In addition to T cells, the numbers of splenic B cells were significantly lower in infected PKC-θ−/− mice than in the WT animals. Correspondingly, the T. gondii-specific IgG levels were significantly reduced in the serum and also in the CSF of PKC-θ−/− mice, whereas the IgM levels did not differ between the two experimental groups. These results imply that PKC-θ supports the development of T. gondii-specific IgG but not IgM responses. Since PKC-θ is not expressed in B cells, this effect is most probably mediated indirectly by insufficient T-cell responses in PKC-θ−/− mice. Although B cells are protective in murine toxoplasmosis (20) and, thus, the death of PKC-θ−/− mice may be partially caused by the reduced IgG response, it is thought to play a minor role in comparison to the strongly diminished T-cell responses of PKC-θ−/− mice.

It should be stressed that the present study was performed with BALB/c mice, which are resistant to T. gondii infection and which develop a chronic nonlethal TE (45). Interestingly, and in sharp contrast to the results for BALB/c mice, PKC-θ−/− mice in a C57BL/6 mouse background survived the infection with the same kinetics as C57BL/6 WT mice up to day 60 p.i. Thus, the functional role of PKC-θ in toxoplasmosis appears to be determined by the host genetic background. This is in sharp contrast to listeriosis, in which the importance of PKC-θ for T-cell responses and pathogen control was independent of host genetics (35). At present, it is unclear why the host genetic background has such a strong impact on the role of PKC-θ in toxoplasmosis, but it adds another layer of complexity to the functional role of PKC-θ in infectious diseases.

At present, it is not definitely clarified why PKC-θ plays an important role in the development of pathogen-specific T-cell responses in listeriosis and toxoplasmosis, a limited role in T-cell responses in leishmaniasis, and even no role in antiviral T-cell responses. It has been suggested that the strength of dendritic cell (DC) activation determines the importance of T-cellular PKC-θ for T-cell activation (26). Since both T. gondii and Listeria monocytogenes infect DCs and suppress the activation of these target cells (1, 14, 29, 32), the influence of these pathogens on DCs may impact the role of PKC-θ on the activation of pathogen-specific T cells. Opposite the contrasting role of PKC-θ in infectious diseases, PKC-θ is unequivocally required to induce disease in T-cell-mediated autoimmune disorders. Therefore, it has been suggested that PKC-θ may be an interesting therapeutic target in T-cell-mediated autoimmune diseases after expansion of the analysis of the effects of PKC-θ deletion to additional models of immunologically relevant diseases (15). In fact, the important protective role of PKC-θ in some widely distributed infectious diseases such as chronic persisting TE provides a challenge to define the clinical settings for the safe treatment of autoimmune disorders using PKC-θ inhibitors.

Acknowledgments

This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (grants GRK 1167 and SFB 854-TP5).

The expert technical assistance of Elena Fischer, Annette Sohnekind, Nadja Schlüter, and Dana Zabler is gratefully acknowledged.

Editor: J. H. Adams

Footnotes

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Published ahead of print on 24 May 2010.

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