Protective Toxoplasma gondii-Specific T-Cell Responses Require T-Cell-Specific Expression of Protein...

INFECTION AND IMMUNITY, Aug. 2010, p. 3454–3464 Vol. 78, No. 80019-9567/10/$12.00 doi:10.1128/IAI.01407-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Protective Toxoplasma gondii-Specific T-Cell Responses RequireT-Cell-Specific Expression of Protein Kinase C-Theta�

Gopala Nishanth,1 Monika Sakowicz-Burkiewicz,1 Ulrike Handel,1 Stefanie Kliche,2 Xiaoqian Wang,2Michael Naumann,3† Martina Deckert,4† and Dirk Schluter1*

Institut fur Medizinische Mikrobiologie, OvG Universitat Magdeburg, Magdeburg, Germany1; Institut fur Molekulare undKlinische Immunologie, OvG Universitat Magdeburg, Magdeburg, Germany2; Institut fur Experimentelle Innere Medizin,

OvG Universitat Magdeburg, Magdeburg, Germany3; and Abteilung fur Neuropathologie,Universitatsklinikum Koln, Cologne, Germany4

Received 17 December 2009/Returned for modification 22 January 2010/Accepted 12 May 2010

Protein kinase C-theta (PKC-�) is important for the activation of autoreactive T cells but is thought to beof minor importance for T-cell responses in infectious diseases, suggesting that PKC-� may be a target for thetreatment of T-cell-mediated autoimmune diseases. To explore the function of PKC-� in a chronic persistinginfection in which T cells are crucial for pathogen control, we infected BALB/c PKC-��/� and PKC-��/�

wild-type mice with Toxoplasma gondii. The PKC-��/� mice succumbed to necrotizing Toxoplasma encephalitisdue 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-��/� mice, resulting in the impairedproduction of protective cytokines (gamma interferon, tumor necrosis factor) and antiparasitic effector mol-ecules (inducible nitric oxide, gamma interferon-induced GTPase) in the spleen and brain. In addition,Th2-cell numbers were reduced in infected the PKC-��/� mice, paralleled by the diminished GATA3 expres-sion of PKC-��/� 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-�B, AP-1, andMAPK pathways in T. gondii-infected PKC-��/� mice. Adoptive transfer of wild-type CD4 plus CD8 T cellssignificantly protected PKC-��/� mice from death by increasing the numbers of gamma interferon-producingT. gondii-specific CD4 and CD8 T cells, illustrating a cell-autonomous, protective function of PKC-� in T cells.These findings imply that PKC-� inhibition drastically impairs T. gondii-specific T-cell responses with fatalconsequences for intracerebral parasite control and survival.

Toxoplasma gondii is an obligate intracellular protozoan thatinfects about a third of the world’s population (30). In general,toxoplasmosis is either clinically asymptomatic or associatedwith only mild clinical symptoms. Nevertheless, the parasitepersists in the host central nervous system (CNS). However,immunocompromised individuals, including fetuses and AIDSpatients, may suffer from life-threatening toxoplasmosis due tothe inability to prevent parasite-induced tissue necrosis. Exper-imental studies with mice have revealed that control of T.gondii in both acute and chronic toxoplasmosis is criticallydependent on gamma interferon (IFN-�)-producing CD4 andCD8 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 responsesare dependent on several T-cell-intrinsic signaling molecules, in-cluding tumor progression locus 2, T-bet, signal transducer andactivator of transcription 4 (STAT4), STAT6, MyD88, Teckinases (Rlk, Itk), and nuclear factor (NF)-�B (6–8, 18, 22, 23,28, 37, 50). Experimental studies have revealed that severalNF-�B proteins critically regulate protective T-cell responses

in toxoplasmosis: RelB is important for the IFN-� productionof T cells (6), NF-�B2 inhibits T-cell apoptosis (7), and c-Relis crucial for T-cell activation, proliferation, and IFN-� pro-duction (28). However, the signaling pathways leading to theactivation of NF-�B in Toxoplasma-specific T cells are incom-pletely understood.

The serine/threonine-specific protein kinase C-theta(PKC-�) is predominantly expressed in T cells, muscle cells,and platelets. Upon activation of the T-cell receptor (TCR),PKC-� is recruited to the immunological synapse and contrib-utes to the activation of the transcription factors NF-�B, acti-vating protein 1 (AP-1), and extracellular-signal regulated ki-nase (ERK) (3, 44; reviewed in reference 15). Functionally,PKC-� is required for T-cell activation, proliferation, and sur-vival (25, 34, 44). Experimental studies with mice have un-equivocally demonstrated that PKC-� is crucial for the activa-tion of autoreactive T cells and induction of T-cell-mediatedautoimmune diseases (2, 16, 36, 48).

In contrast to autoimmune diseases, PKC-� is dispensablefor the induction of CD4 and CD8 T-cell responses in viralinfections caused by vaccinia virus, influenza virus, lymphocyticchoriomeningitis virus, and murine herpesvirus 68 (4, 13,26). In addition, development of Th1-cell responses againstthe intracellular protozoan Leishmania major is normal in bothPKC-��/� C57BL/6 and BALB/c mice (27). However, PKC-�plays a critical role in the development of Th2-cell immuneresponses after infection with Nippostrongylus brasiliensis (27).To address the role of PKC-� in bacterial infections, we re-

* Corresponding author. Mailing address: Institut fur MedizinischeMikrobiologie, OvG Universitat Magdeburg, Leipzigerstrasse 44, Magde-burg 39120, Germany. Phone: 49 391 67 13317. Fax: 49 391 67 290717.E-mail: [email protected].

† Both authors contributed equally to this work.� Published ahead of print on 24 May 2010.

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cently studied listeriosis in PKC-��/� and PKC-��/� wild-type(WT) C57BL/6 and BALB/c mice (35). In both strains of mice,PKC-� was required for the proliferation and survival of IFN-�-producing Listeria monocytogenes-specific CD4 and CD8 Tcells in primary and secondary listeriosis. Consequently, thediminished T-cell responses in the absence of PKC-� signalingresulted in the impaired control of Listeria. Collectively, thesecontrasting findings in various infectious diseases illustrate thatthe functional importance of PKC-� for the development ofpathogen-specific T cells is decisively determined by the un-derlying pathogen.

This view is strongly supported by the findings of the presentstudy, which analyzed the functional role of PKC-� in murinetoxoplasmosis. PKC-� was essential for the induction of pro-tective T. gondii-specific Th1 cells and IFN-�-producing CD8 Tcells in T. gondii-resistant BALB/c mice (45). In addition, thenumbers of IL-4-producing CD4 T cells were strongly reduced.Finally, the impaired T-cell responses resulted in a failure tocontrol T. gondii in the CNS and, thus, lethal Toxoplasmaencephalitis (TE) up to day 40 after infection. In contrast toBALB/c mice, PKC-��/� C57BL/6 mice survived the infection,illustrating that the functional role of PKC-� is dependent onthe genetic background of the host.

MATERIALS AND METHODS

Animals. C57BL/6 PKC-��/� 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 backgroundwith BALB/c mice obtained from Harlan-Winkelmann (Borchen, Germany).Age- and sex-matched mice BALB/c PKC-��/� and C57BL/6 PKC-��/� mice(35) as well as BALB/c and C57BL/6 PKC-��/� WT mice, both obtained fromHarlan-Winkelmann, were used for the experiments. All experimental mice wereallowed to adapt to the OvG Universitat Magdeburg animal facility for at least14 days and were kept under conventional conditions in an isolation facilitythroughout the experiments. The experiments were approved and supervised bylocal governmental institutions.

Parasites and T. gondii infection. Cysts of the T. gondii DX strain (a type IIstrain) (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 �l was administered orally by gavage to theexperimental animals.

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

Quantification of intracerebral T. gondii. The numbers of T. gondii parasiteswere determined microscopically on anti-T. gondii-immunostained brain sections bycounting at least 100 randomly selected high-power fields (magnification, �400) permouse. For each mouse, identical regions of the brain, including the cortex, basalganglia, and cerebellum, were analyzed.

Isolation of leukocytes from brain and spleen. Splenic leukocytes were isolatedfrom the dead mice by passing the spleens through a 70-�m-pore-size cellstrainer (BD Biosciences, Heidelberg, Germany), and erythrocytes were lysedwith ammonium chloride. Before isolation of cerebral leukocytes, the animalswere anesthetized with isoflurane (Baxter, Deerfield, IL) and intracardially per-fused with 0.9% NaCl to remove contaminating intravascular leukocytes fromthe brain. Thereafter, brain tissue was minced through a 100-�m-pore-size cellstrainer, 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 stainedwith (i) rat anti-mouse CD4-phycoerythrin (PE), rat anti-mouse CD8-fluoresceinisothiocyanate (FITC), and rat anti-mouse CD45-PE-Cy5; (ii) rat anti-mouseLy6G-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, ratanti-mouse B220-FITC, and rat anti-mouse CD45-PE-Cy5. CD8 T cells specificfor the MHC class I H-2Ld-restricted Gra6-HPGSVNEFDF (HF10) epitope (5)

were detected by DimerX (BD Biosciences) staining. In brief, the Gra6-HF10peptide (JPT, Berlin, Germany) was coupled to DimerX H-2Ld molecules andrat anti-mouse IgG1-PE by overnight incubation at 37°C. Subsequently, leuko-cytes 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 obtainedfrom BD Biosciences. Flow cytometry was performed on a FACSCalibur instru-ment (BD Biosciences), and the data were analyzed with WinMDI or CellQuestsoftware.

ELISPOT assay. The numbers of T. gondii-specific CD4 and CD8 T cells weredetermined by enzyme-linked immunospot (ELISPOT) assay, as described pre-viously (21). Before the ELISPOT assay, CD4 and CD8 T cells were isolatedfrom 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-�-coated or rat anti-mouse IL-4-coated (both from Invitrogen, CA) ELISPOT assay plates and coincubated withspleen cells from noninfected BALB/c WT mice (2 � 105/well), which werepreloaded with heat-killed Toxoplasma (HKT; three parasites per splenocyte).Controls included coincubation of isolated leukocytes with spleen cells withoutpeptide loading and incubation of leukocytes from noninfected mice with pep-tide-loaded spleen cells. All ELISPOT assay plates were incubated overnight anddeveloped with biotin-labeled rat anti-mouse IFN-� or biotin-labeled rat anti-IL-4 (BD Biosciences), peroxidase-conjugated streptavidin, and aminoethylcar-bazole dye solution (Sigma-Aldrich). The spots were counted microscopically,and the numbers of antigen (Ag)-specific CD4 and CD8 T cells per organ werecalculated from the number of spots in triplicate wells.

Adoptive transfer of T cells. Polyclonal CD4, CD8, and CD4 plus CD8 T cellswere isolated from the spleens of noninfected WT and PKC-��/� mice by MACSusing CD4, CD8, and pan-T-cell isolation kits, respectively (Miltenyi). Isolated Tcells (1 � 106) were injected intravenously (i.v.) into PKC-��/� mice 24 h beforeinfection with T. gondii.

Western blotting (WB). CD4 and CD8 T cells were isolated from the spleensof WT and PKC-��/� mice by MACS as described above and resuspended inlysis 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; 1mM phenylmethylsulfonyl fluoride;1 mM NaF; 1 mM Na4O7P2; 1 mM Na3VO4;and aprotinin, leupeptin, and pepstatin (1 �g/ml each). Equal amounts of pro-teins were separated on 10% SDS-polyacrylamide gels and transferred to polyvinyl-idene fluoride membranes, followed by incubation with anti-PKC-�, anti-phos-pho-PKC-� (p-PKC-�), p-IKK1/2, p-p65, anti-GAPDH, and anti-caspase 3 (allfrom Cell Signaling Technology, MA) and with p-c-Fos, p-Jun, p-ERK, andGATA3 (all from Santa Cruz Biotechnology, CA). Blots were developed usingan ECL Plus kit (GE Healthcare).

RT-PCR. For reverse transcription-PCR (RT-PCR), mRNA was isolated fromthe brains and spleens of uninfected and T. gondii-infected mice (RNAeasy kit;Qiagen, Hilden, Germany). mRNA was transcribed into cDNA by use of theSuperScript reverse transcriptase kit with oligo(dT) primers (Invitrogen). Quan-titative RT-PCR for IFN-�, tumor necrosis factor (TNF), IL-10, inducible nitricoxide synthase (iNOS), and hypoxanthine phosphoribosyltransferase (HPRT)was performed with cDNA from BALB/c WT and BALB/c PKC-��/� mice witha GeneAmp 5700 sequence detection system (Applied Biosystems, Weiterstadt,Germany). Quantitation was performed with the sequence detector softwareSDS (version 2.1; Applied Biosystems), according to the ��CT threshold cycle(CT) method with HPRT as the housekeeping gene. Data are expressed as theincrease in the level of mRNA expression in infected mice over that in nonin-fected controls of the respective mouse strain. All primers and probes wereobtained from Applied Biosystems.

Statistics. Statistical significance was determined using the two-tailed Studentt test or nonparametric Mann-Whitney rank sum test (Statistica 5; StatSoft). Allexperiments were performed at least twice. P values of 0.05 were consideredsignificant.

RESULTS

Infection with T. gondii induces sustained phosphorylationof PKC-� in CD4 and CD8 T cells. Since PKC-� plays animportant role in the TCR-mediated activation and prolifera-tion of T cells, we analyzed PKC-� expression and phosphory-lation in the CD4 and CD8 T cells of BALB/c WT mice afterinfection with T. gondii. WB analysis showed that (nonacti-

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vated) PKC-� was expressed in CD4 as well as CD8 T cells ofboth noninfected (day 0) and T. gondii-infected mice (Fig. 1).In uninfected mice, activated PKC-� was weakly expressed inCD4 and CD8 T cells (Fig. 1). Upon infection, PKC-� wasstrongly activated in both CD4 and CD8 T cells at days 7, 14,and 21 postinfection (p.i.) (Fig. 1). These findings illustrate thesustained activation of PKC-� in CD4 and CD8 T cells of T.gondii-infected mice and provide the basis for analysis of thefunctional role of PKC-� in toxoplasmosis.

BALB/c PKC-��/� mice succumb to necrotizing TE. To ana-lyze whether PKC-� plays a critical role for survival of toxo-plasmosis, BALB/c PKC-��/� and WT mice were infected withT. gondii cysts. Whereas 92% of the PKC-��/� mice suc-cumbed to the infection up to day 40 p.i., only 8% of WT micedied up to day 60 p.i. (Fig. 2A). In contrast, both C57BL/6PKC-��/� and WT mice survived the infection up to day60 p.i. (Fig. 2B). A detailed histopathological examinationrevealed that BALB/c PKC-��/� mice died of a necrotizingTE with huge amounts of intracerebral parasites (Fig. 2C). Incontrast, BALB/c WT mice harbored only low numbers ofparasites in the brain without tissue necrosis. Quantification ofthe intracerebral toxoplasms revealed that the number of par-asites was significantly increased in the brains of BALB/c PKC-��/� mice compared to the number in the WT controls at day21 p.i. (Fig. 2D). These findings demonstrate that PKC-� isessential for intracerebral parasite control and survival of tox-oplasmosis in BALB/c mice. In the following experiments,BALB/c PKC-��/� and WT mice were used to analyze the roleof PKC-� in toxoplasmosis.

Reduced numbers of CD4 and CD8 T cells in spleens andbrains of T. gondii-infected PKC-��/� mice. To determinewhether PKC-� plays a role in the recruitment of leukocytes tothe brain and affects leukocyte numbers in the spleen in toxo-plasmosis, intracerebral and splenic leukocytes were isolatedfrom uninfected as well as T. gondii-infected mice at day 21 andday 42 p.i. Uninfected PKC-��/� and WT mice harbored equalnumbers of CD4 and CD8 T cells, B cells, macrophages, andgranulocytes in their brains and spleens (Fig. 3A and B). Uponinfection with T. gondii, the numbers of these leukocyte pop-ulations increased in both strains of mice (Fig. 3A and B).However, the numbers of CD4 and CD8 T cells were signifi-cantly increased in the spleens and brains of WT animals at day21 and day 42 p.i. In addition, the numbers of B cells wereincreased in the spleens and brains of WT mice compared tothe numbers in PKC-��/� mice. The numbers of splenic and

FIG. 2. Reduced survival and impaired parasite control of T. gondii-infected BALB/c PKC-��/� mice. BALB/c and C57BL/6 WT and PKC-��/� mice were orally infected with T. gondii. (A) Significantly moreBALB/c PKC-��/� mice than BALB/c WT animals succumbed to theinfection (P 0.005) up to day 60 p.i. Ten mice of each mouse strain wereanalyzed. (B) Both C57BL/6 PKC-��/� and C57BL/6 WT mice survivetoxoplasmosis up to day 60 p.i. (C) A histopathological analysis revealedthat BALB/c PKC-��/� mice had developed a severe necrotizing TE withhuge amounts of intracerebral parasites at day 21 p.i. In contrast, BALB/cWT mice harbored only a few parasitic cysts (arrow) at this stage ofinfection. Anti-T. gondii immunostaining, slight counterstaining withhemalum. Magnification, �100. (D) The numbers of T. gondii in 100high-power fields (HPF) per mouse brain were determined microscopi-cally. Four mice per group were analyzed at days 14 and 21 p.i., and datarepresent the means � standard deviations (**, P 0.01).

FIG. 1. Sustained activation of PKC-� in CD4 and CD8 T cells intoxoplasmosis. WB analysis of PKC-� expression and phosphorylationin 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 cellswere selectively isolated by MACS from four mice per time point.Representative blots are shown.

3456 NISHANTH ET AL. INFECT. IMMUN.

intracerebral granulocytes and macrophages did not differ be-tween PKC-��/� and WT mice at either time point after in-fection (Fig. 3A and B). Since PKC-� is predominantly ex-pressed in T cells and the numbers of CD4 and CD8 T cellswere strongly reduced in the spleens and brains of T. gondii-infected PKC-��/� mice, we further focused on the impact ofPKC-� on T. gondii-specific T cells.

Diminished T. gondii-specific CD4 and CD8 T-cell responsesin PKC-��/� mice. To analyze whether PKC-� regulates T.gondii-specific CD8 T cells, we determined the frequency andnumber of T. gondii-specific Gra6-HF10-specific CD8 T cells inthe spleens and brains of mice by DimerX staining at days 7,14, and 21 p.i. (Fig. 4A and B). Flow cytometry revealed thatboth the percentage (Fig. 4A) and the number (Fig. 4B) ofGra6-HF10-specific CD8 T cells increased in the spleens ofWT mice and reached a maximum at day 21 p.i., which is inaccordance with data from Blanchard et al. (5). In parallel, the

percentage and absolute numbers of Gra6-HF10-specific CD8T cells increased in the brains of WT animals. Although thepercentage and the absolute number of Gra6-HF10-specificCD8 T cells increased gradually in PKC-��/� mice over time,the values for both parameters remained significantly reducedcompared to the values for the WT animals at all time points.

Additional ELISPOT experiments showed that the numbersof T. gondii-specific IFN-�-producing CD4 and CD8 T cellswere also significantly reduced in the spleens and brains ofPKC-��/� mice (Fig. 4C and D). Since IFN-� production byCD4 and CD8 T cells is crucial to control T. gondii in the CNS,these findings imply that the insufficient generation of IFN-�-producing T cells in the absence of PKC-� accounts for thelethal necrotizing TE in PKC-��/� mice.

Since it has been reported that PKC-� is important for thedevelopment of Th2 cells upon infection with Nippostrongylusbrasiliensis and Leishmania major (27), we studied the effect ofPKC-� on Th2 responses in toxoplasmosis by IL-4 ELISPOTassay of CD4 T cells. The numbers of T. gondii-specific IL-4-producing CD4 T cells were significantly reduced in PKC-��/�

mice at all time points after infection (Fig. 4E). In parallel, theexpression of the transcription factor GATA3, which inducesTh2 responses, was strongly upregulated in CD4 T cells of WTanimals, whereas the level of upregulation of GATA3 expres-sion was lower in the CD4 T cells of T. gondii-infected PKC-��/� mice at days 7, 14, and 21 p.i. (Fig. 4F). Thus, T. gondii-specific Th2 responses were reduced in the absence of PKC-�.

The reduced absolute numbers of CD4 and CD8 T cells aswell as parasite-specific CD4 and CD8 T cells may be caused bythe increased apoptosis of PKC-�-deficient T cells. Therefore,we isolated CD4 and CD8 T cells from both strains of mice andanalyzed the expression of active caspase 3. Both WT andPKC-��/� mice expressed equally low levels of activatedcaspase 3 at day 14 p.i. At all other time points, active caspase3 was not detectable by WB. However, further in vitro analysisof T-cell proliferation and apoptosis was precluded by theextremely low number of parasite-specific CD4 and CD8 Tcells in PKC-��/� mice.

Reduced cytokine responses in PKC-��/� mice. To study theimpact of the reduced number of IFN-�-producing T. gondii-specific T cells on cytokine production, a quantitative RT-PCRanalysis was performed at day 21 p.i. The level of IFN-�mRNA was significantly reduced in the spleens and brains of T.gondii-infected PKC-��/� mice (Fig. 5A). In addition, the levelof expression of TNF mRNA was reduced in PKC-��/� mice(Fig. 5B). Interestingly, IL-10 mRNA, which is mainly pro-duced by conventional Foxp3-negative (Foxp3�) Th1 cells intoxoplasmosis (17), was also significantly reduced in thespleens and brains of PKC-��/� mice (Fig. 5C). Furthermore,the levels of iNOS and gamma interferon-induced GTPase(IGTP), two important IFN-�-regulated antiparasitic effectormolecules, were significantly reduced in PKC-��/� mice (Fig.5C and D). In conclusion, the levels of production of themRNA of essentially protective cytokines and IFN-�-regulatedantiparasitic effector molecules were significantly diminishedin PKC-��/� mice.

Impaired activation of NF-�B, AP-1, and ERK in PKC-��/�

mice. The activation and regulation of the transcription factorsNF-�B and AP-1 as well as the kinase ERK were studied byWB of the T cells of T. gondii-infected mice. In these experi-

FIG. 3. Reduced numbers of CD4 and CD8 T cells in the spleensand brains of T. gondii-infected PKC-��/� mice. The leukocyte popu-lations in the spleens (A) and brains (B) of uninfected and T. gondii-infected WT and PKC-��/� mice were phenotyped by flow cytometryat the indicated time points. Six mice per group and time point wereanalyzed. Data represent the means � standard deviations for eachcell population (*, P 0.05; **, P 0.01).


FIG. 4. Impaired T. gondii-specific T-cell responses in PKC-��/� mice. (A) Splenic and intracerebral leukocytes were isolated and stained withGra6-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 areshown. (B) The mean numbers � standard deviations of T. gondii-specific CD8 T cells in the spleen and brain of WT and PKC-��/� mice werecalculated from the percentage of Gra6-HF10� CD62L� CD8 T cells and the absolute number of CD8 T cells. Six mice were analyzed per groupand time point (*, P 0.05; **, P 0.01). (C and D) The frequencies of IFN-�-producing splenic (C) and intracerebral (D) CD4 and CD8T cellswere determined by an ELISPOT assay. Splenic and intracerebral CD4 and CD8 T cells were isolated by MACS before analysis. Intracerebral


ments, we focused on bulk CD4 and CD8 T cells, since thenumber of parasite-specific CD4 and CD8 T cells, especially inPKC-��/� mice, was too low to allow a direct ex vivo analysisby WB. As shown in Fig. 6, the CD4 and CD8 T cells ofPKC-��/� mice showed an impaired activation of NF-�B, asindicated by the reduced phosphorylation of IKK1/2 and p65.In addition, the level of phosphorylation of the AP-1 transcrip-tion factor c-Jun, but not that of c-Fos, was reduced in CD4and CD8 T cells of PKC-��/� mice compared to the level in

WT mice. Furthermore, PKC-��/� mice showed a slight re-duction in the level of ERK activation in both CD4 and CD8Tcells compared to that in WT animals. Taken together, thesedata indicate that in the absence of PKC-�, the activation ofseveral important signaling pathways, including NF-�B, AP-1,and ERK, was impaired in CD4 and CD8 T cells.

Reduced T. gondii-specific IgG production in serum andCSF of PKC-��/� mice. B cells play a protective role in toxo-plasmosis, and T. gondii-specific Ab responses are partiallyregulated by CD4 T cells (20, 24). Therefore, we evaluated theT. 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-��/� and WT mice (Fig. 7A and B). In contrast,the levels of T. gondii-specific IgG were reduced in the serumand CSF of PKC-��/� mice (Fig. 7C and D). These resultsillustrate that T. gondii-specific IgG but not IgM productionwas partially dependent on PKC-�.

WT T cells compensate for PKC-� deficiency in toxoplas-mosis. To validate that the lethal course of toxoplasmosis inPKC-��/� mice was caused by an insufficient T-cell response,we adoptively transferred purified polyclonal WT T cells intoPKC-��/� mice before infection with T. gondii and monitoredthe survival of these animals. As shown in Fig. 8A, 68% ofPKC-��/� mice reconstituted with WT T cells survived theinfection, whereas 100% of both PKC-��/� mice without T-celltransfer or with transfer of PKC-��/� T cells succumbed to theinfection. As shown before, all WT mice (i.e., without T-celltransfer) survived the infection. Compared to the numbers inWT animals, the surviving PKC-��/� mice with adoptive WTT-cell transfer harbored equally low numbers of T. gondii cystsin their brains at day 60 p.i. (P 0.05) (Fig. 8B).

To further analyze whether the adoptive transfer of WT Tcells into PKC-��/� mice improved the parasite-specific T-cell

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) Thefrequency of IL-4-producing T. gondii-specific, MACS-isolated splenic CD4 T cells was determined by an IL-4 ELISPOT assay at the indicated timepoints. Four mice were analyzed per experimental group and time point. Data show the means � standard deviations (*, P 0.05). (F) Proteinswere isolated from MACS-isolated splenic CD4 T cells of WT and PKC-��/� mice and stained for GATA3 and GAPDH by WB. Representativeblots 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 indicatedtime points. WB analysis for caspase 3 and active caspase 3 was performed, and representative data are shown.

FIG. 5. Reduced production of cytokines and antiparasitic effectormolecules in T. gondii-infected PKC-��/� mice. A quantitative RT-PCR analysis of IFN-� (A), TNF (B), IL-10 (C), iNOS (D), and IGTP(E) mRNA expression in the spleens and brains of WT and PKC-��/�

mice was performed. Data are expressed as the increase in the respec-tive levels of mRNA from infected (day 21 p.i.) over uninfected miceand were normalized to the level of HPRT expression. Data representthe means � standard deviations of three to five mice per group andtime point (*, P 0.05; **, P 0.01; d0, day 0).

FIG. 6. WB analysis of the NF�B, AP-1, and ERK pathways inCD4 and CD8 T cells. CD4 and CD8 T cells were isolated by MACSfrom the spleens of three to five WT and PKC-��/� mice at theindicated time points. After isolation and blotting, proteins were stainedfor p-IKK1/2, p-p65, p-c-Fos, anti-p-c-Jun, p-ERK, and GAPDH.


responses in PKC-��/� mice, the number of Gra6-HF10-spe-cific CD8 T cells was determined. In both the spleens (Fig. 8C)and the brains (Fig. 8D), the number of Gra6-HF10-specificCD8 T cells was significantly increased in PKC-��/� mice withWT T-cell transfer compared to the number in PKC-��/� micewithout WT T-cell transfer (P 0.05 for both organs). Asshown before, the numbers of Gra6-HF10-specific CD8 T cellswere also significantly increased in the spleens and brains ofWT mice compared to the numbers in PKC-��/� animals. Inaddition, the numbers of T. gondii-specific IFN-�-producing Tcells were significantly increased in the spleens of both PKC-��/� mice with WT T-cell transfer and WT mice compared tothe numbers in PKC-��/� without T-cell transfer (P 0.01)(Fig. 8E). These findings demonstrate that WT T cells cansignificantly protect PKC-��/� mice from lethal toxoplasmosis.

To determine whether adoptively transferred CD4 and/orCD8 WT T cells confer protection against toxoplasmosis inPKC-��/� mice, CD4, CD8, and CD4 plus CD8 T cells wereadaptively transferred to PKC-��/� mice before infection withT. gondii. As illustrated in Fig. 8F, only transfer of both T-cellpopulations significantly protected PKC-��/� mice from lethaltoxoplasmosis. Although transfer of either CD4 or CD8 WT Tcells prolonged the survival times of PKC-��/� mice by amaximum of 10 days, all of these animals succumbed to theinfection. Thus, the combined action of both CD4 and CD8WT T cells was required to compensate efficiently for thePKC-� deficiency in BALB/c mice with toxoplasmosis.

DISCUSSION

The present study demonstrates that PKC-� is absolutelyrequired for protective T. gondii-specific T-cell responses, ef-ficient parasite control in the brain, and survival from cerebraltoxoplasmosis in BALB/c mice. Upon infection with T. gondii,CD4 and CD8 T cells upregulated p-PKC-� as early as day 7p.i., when the levels of T. gondii-specific T cells were stillbelow the detection limit of DimerX staining and theELISPOT assay. At the time of the maximum T-cell response, i.e.,at day 21 p.i., CD4 and CD8 T cells still contained activatedPKC-�, indicating its functional importance for activation, pro-liferation, 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-��/� mice throughout the study without a significant increaseover time of infection.

The impaired splenic and intracerebral CD4 and CD8 T-cellresponses of T. gondii-infected PKC-��/� mice was reflectedby the reduced numbers of both T-cell populations, the re-duced numbers of Gra6-HF10-specific CD8 T cells, as well asthe reduced numbers of IFN-�-producing CD4 and CD8 Tcells. Since IFN-�-producing CD4 and CD8 T cells are abso-lutely required for the control of T. gondii (10), the diminishedT-cell response of PKC-��/� mice consequently resulted in alethal necrotizing TE. The protective function of IFN-� ispartially mediated by the induction of other protective cyto-kines, including TNF, as well as the induction of antiparasiticeffector molecules, including iNOS and IGTP (11, 38, 39, 41,49). This explains (i) why the strong reduction of intracerebraland splenic IFN-� mRNA levels was accompanied by the mas-sive reduction of TNF, iNOS, and IGTP levels in infectedPKC-��/� mice compared to the levels in WT mice and (ii)why PKC-��/� mice were unable to restrict T. gondii prolifer-ation in the brain. Interestingly, the level of IL-10, which isrequired to prevent a lethal IFN-�-mediated immunopathol-ogy in toxoplasmosis (12, 33), was also reduced in PKC-��/�

mice. Since IL-10 is mainly produced by conventional Foxp3�

Th1 cells in toxoplasmosis (17), the reduced level of IL-10mRNA production is most probably caused by the impaireddevelopment of T. gondii-specific Th1 cells in PKC-��/� mice.Of note, in mice of the same T. gondii-resistant BALB/c back-ground, IL-33 receptor-mediated downregulation of Th1 re-sponse is important to prevent intracerebral immunopathologyand to control parasite growth in the brain (19). Thus, inBALB/c mice both the PKC-�-dependent activation of protec-tive Th1 and Th2 responses (see below) and the IL-33-depen-dent limitation of Th1 responses are required for effectiveparasite control and prevention of an exacerbated encephalitis.

The transfer of WT T cells improved intracerebral parasitecontrol, increased parasite-specific T-cell responses close toWT levels, and significantly protected T. gondii-infected PKC-��/� mice from death. Interestingly, the combined action ofWT CD4 and CD8 T cells was required to protect PKC-��/�

mice from fatal toxoplasmosis, since the adoptive transfer ofeither WT CD4 or CD8 T cells did not prevent the death of T.gondii-infected PKC-��/� mice. These data illustrate that theT-cell-autonomous expression of PKC-� is sufficient to com-pensate for PKC-� deficiency. In addition, these observationsillustrate that the deficiency of PKC-� in T cells but not in

FIG. 7. Reduced levels of IgG but normal levels of T. gondii-spe-cific IgM production in PKC-��/� mice. (A to D) T. gondii-specific IgM(A and B) and IgG (C and D) Abs were determined in serum (A andC) and CSF (B and D) of WT and PKC-��/� mice by enzyme-linkedimmunosorbent assay at day 21 p.i. The serum and CSF were seriallydiluted, and the highest positive dilution is shown. Serum and CSFwere isolated from five to six mice per group and time point. Datashow the means � standard deviations of Ab titers in serum (*, P 0.05). The CSF from mice of each experimental group was pooled toobtain a sufficient amount of CSF, and the means of duplicates areshown.


FIG. 8. WT T cells compensate for PKC-� deficiency in toxoplasmosis. MACS-purified WT and PKC-��/� T cells were adoptively transferredinto PKC-��/� recipients. Recipients as well as control WT and PKC-��/� mice were orally infected with T. gondii. (A) The survival of six miceper experimental group was monitored until day 60 p.i. (B) The numbers of T. gondii cysts in WT and PKC-��/� mice with adoptively transferredWT T cells were determined at day 60 p.i. Data represent the means � standard deviations of six surviving WT and four surviving PKC-��/� micewith 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-�-producingsplenocytes was determined by an IFN-� ELISPOT assay at day 21 p.i. (C and D) The data represent the means � standard deviations of five miceper group. (F) MACS-purified WT CD4, WT CD8, and WT CD4 plus CD8 mouse T cells as well as PKC-��/� mouse CD4 and CD8 T cells wereadoptively transferred into PKC-��/� recipients. Recipients as well as control WT and PKC-��/� mice were orally infected with T. gondii, and thesurvival 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 resultsfor PKC-��/� mice.


other cell populations, including NK cells, is decisive for thefatal course of chronic TE in PKC-��/� mice. These dataextend the findings of previous studies of murine listeriosis, inwhich PKC-� was also required for normal pathogen-specificCD4 and CD8 T-cell responses (35).

A direct ex vivo WB analysis revealed that CD4 and CD8 Tcells required PKC-� for normal activation of the NF-�B path-way at all time points of the infection, as indicated by thereduced activities of IKK1/2 and p65 in the CD4 and CD8 Tcells of PKC-��/� mice. IKK1/2 and p65 are part of the ca-nonical NF-�B pathway, and PKC-� regulates this pathwaydirectly (reviewed in reference 15). The assumption that theNF-�B pathway is critical for T. gondii-specific T-cell responsesis substantiated by the importance of the NF-�B moleculesc-Rel and RelB for the development of protective T. gondii-specific T-cell responses (6, 28). In addition, the reduced phos-phorylation of c-Jun in CD4 and CD8 T cells of PKC-��/�

mice illustrates the impaired activation of AP-1 in T. gondii-infected PKC-��/� mice, although it is at present unclear towhat extent T-cell responses are AP-1 dependent in toxoplas-mosis. Furthermore, activation of ERK was slightly reduced inPKC-��/� CD4 and CD8 T cells, especially at day 21 p.i. inCD4 T cells. Collectively, these data illustrate that the normalactivation of several major signaling pathways in T cells of T.gondii-infected mice was PKC-� dependent. It should bestressed that this analysis was performed with bulk T cells, i.e.,T. gondii-specific and bystander T cells, and that the observeddifferences in the activation of signaling molecules are due tothe direct effect of PKC-� on signaling pathways as well as thereduced levels of cytokine production in PKC-��/� mice. Thus,these ex vivo data sum up the direct and indirect effects ofPKC-� on the major signaling pathways in T cells and therebyextend previous in vitro findings on the role of PKC-� for theactivation of NF-�B, AP-1, and ERK in T cells upon stimula-tion of the TCR (3, 44; reviewed in reference 15).

Although toxoplasmosis is characterized by a strong protec-tive Th1 response, additional IL-4-producing Th2 cells developand contribute to the optimal control of the parasite and sur-vival in toxoplasmosis (47). In our experiments, the T. gondii-specific Th2 response was also PKC-� dependent, since in theabsence of PKC-�, the frequency of IL-4-producing CD4 Tcells was greatly diminished. Interestingly, the level of GATA3,which is of key importance for the Th2 differentiation of CD4T cells (31), was also reduced in the CD4 T cells of PKC-��/�

mice. These data extend previous in vitro observations foranti-CD3/CD28-stimulated CD4 T cells, which also revealedthat PKC-� is involved in the upregulation of GATA3 (43). Inaddition, Th2 differentiation of Leishmania and Nippostrongy-lus brasiliensis-specific CD4 T cells is PKC-� dependent (27).However, in contrast to T. gondii-specific Th1 cells, the devel-opment of Leishmania-specific Th1 responses was PKC-� in-dependent (27).

In discussing the importance of PKC-� for T-cell differenti-ation, it should be stressed that only low numbers of T. gondii-specific Th1 and Th2 CD4 as well as CD8 T cells were detect-able in PKC-��/� mice. Thus, the reduction of the levels of allT. gondii-specific T-cell subsets may be caused by either theimpaired proliferation or survival of T cells. Both the survivaland proliferation of T cells are critically regulated by PKC-�after TCR stimulation in vitro as well as in listeriosis (25, 34, 35,

44). A lack of increased activation of caspase 3 in splenic CD4and CD8 T cells of PKC-��/� may imply that an increased rateof apoptosis was not responsible for the reduced numbers of T.gondii-specific CD4 and CD8 T cells. However, the very lownumbers of T. gondii-specific T cells in PKC-��/� mice pre-vented a further in vivo functional analysis of T-cell prolifera-tion and apoptosis.

Recently, Blanchard et al. (5) identified an H-2Ld-restrictedCD8 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 andreach maximal numbers in the spleen as late as day 21 p.i. (5),which perfectly fits with our previous data obtained with �-ga-lactosidase transgenic toxoplasms (21). In the present study,we extend these data and show that (i) intracerebral CD8 Tcells follow the same kinetics, (ii) 31% of the intracerebal CD8T 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 CD62Lnegativity. Thus, both the splenic and intracerebral CD8 T-cellresponses of H-2d BALB/c mice focuses very much on theGra6-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-��/� mice than in the WTanimals. Correspondingly, the T. gondii-specific IgG levelswere significantly reduced in the serum and also in the CSF ofPKC-��/� mice, whereas the IgM levels did not differ betweenthe two experimental groups. These results imply that PKC-�supports the development of T. gondii-specific IgG but not IgMresponses. Since PKC-� is not expressed in B cells, this effect ismost probably mediated indirectly by insufficient T-cell re-sponses in PKC-��/� mice. Although B cells are protective inmurine toxoplasmosis (20) and, thus, the death of PKC-��/�

mice may be partially caused by the reduced IgG response, it isthought to play a minor role in comparison to the stronglydiminished T-cell responses of PKC-��/� mice.

It should be stressed that the present study was performedwith BALB/c mice, which are resistant to T. gondii infectionand which develop a chronic nonlethal TE (45). Interestingly,and in sharp contrast to the results for BALB/c mice, PKC-��/� mice in a C57BL/6 mouse background survived the in-fection with the same kinetics as C57BL/6 WT mice up to day60 p.i. Thus, the functional role of PKC-� in toxoplasmosisappears to be determined by the host genetic background. Thisis in sharp contrast to listeriosis, in which the importance ofPKC-� for T-cell responses and pathogen control was indepen-dent of host genetics (35). At present, it is unclear why the hostgenetic background has such a strong impact on the role ofPKC-� in toxoplasmosis, but it adds another layer of complex-ity to the functional role of PKC-� in infectious diseases.

At present, it is not definitely clarified why PKC-� plays animportant role in the development of pathogen-specific T-cellresponses in listeriosis and toxoplasmosis, a limited role inT-cell responses in leishmaniasis, and even no role in antiviralT-cell responses. It has been suggested that the strength ofdendritic cell (DC) activation determines the importance ofT-cellular PKC-� for T-cell activation (26). Since both T. gondiiand Listeria monocytogenes infect DCs and suppress the acti-vation of these target cells (1, 14, 29, 32), the influence of thesepathogens on DCs may impact the role of PKC-� on the acti-


vation of pathogen-specific T cells. Opposite the contrastingrole of PKC-� in infectious diseases, PKC-� is unequivocallyrequired to induce disease in T-cell-mediated autoimmunedisorders. Therefore, it has been suggested that PKC-� may bean interesting therapeutic target in T-cell-mediated autoim-mune diseases after expansion of the analysis of the effects ofPKC-� deletion to additional models of immunologically rele-vant diseases (15). In fact, the important protective role ofPKC-� in some widely distributed infectious diseases such aschronic persisting TE provides a challenge to define the clinicalsettings for the safe treatment of autoimmune disorders usingPKC-� inhibitors.

ACKNOWLEDGMENTS

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

The expert technical assistance of Elena Fischer, Annette Sohne-kind, Nadja Schluter, and Dana Zabler is gratefully acknowledged.

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Editor: J. H. Adams


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