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The Journal of Immunology, 2008, 180, 5601 -5612
Copyright © 2008 by The American Association of Immunologists, Inc.
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*Listeria Infections

Protein Kinase C-{theta} Critically Regulates the Proliferation and Survival of Pathogen-Specific T Cells in Murine Listeriosis1

Monika Sakowicz-Burkiewicz*, Gopala Nishanth*, Ulrike Helmuth*, Katrin Drögemüller*, Dirk H. Busch{ddagger}, Olaf Utermöhlen§, Michael Naumann{dagger}, Martina Deckert and Dirk Schlüter2,*

* Institut für Medizinische Mikrobiologie and {dagger} Institut für Experimentelle Innere Medizin, Otto-von-Guericke Universität Magdeburg, Magdeburg; {ddagger} Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich; § Institut für Medizinische Mikrobiologie, Immunologie und Hygiene and Abteilung für Neuropathologie, Klinikum der Universität zu Köln, Cologne, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Protein kinase C-{theta} (PKC-{theta}) is essential for the activation of T cells in autoimmune disorders, but not in viral infections. To study the role of PKC-{theta} in bacterial infections, PKC-{theta}–/– and wild-type mice were infected with Listeria monocytogenes (LM). In primary and secondary listeriosis, the numbers of LM-specific CD8 and CD4 T cells were drastically reduced in PKC-{theta}–/– mice, resulting in increased CFUs in spleen and liver of both PKC-{theta}–/– C57BL/6 and BALB/c mice. Furthermore, immunization with peptide-loaded wild-type dendritic cells induced LM-specific CD4 and CD8 T cells in wild-type but not in PKC-{theta}–/– mice. In listeriosis, transfer of wild-type T cells into PKC-{theta}–/– mice resulted in a normal control of Listeria, and, additionally, a selective expression of PKC-{theta} in LM-specific T cells was sufficient to drive a normal proliferation and survival of these T cells in LM-infected PKC-{theta}–/– recipients, illustrating a cell-autonomous function of PKC-{theta} in LM-specific T cells. Conversely, adoptively transferred PKC-{theta}–/– T cells were partially rescued from cell death and proliferated in LM-infected wild-type recipients, demonstrating that a PKC-{theta} deficiency of LM-specific T cells can be partially compensated for by a wild-type environment. Additionally, in vitro experiments showed that only the addition of IL-2, but not an inhibition of caspase-3, induced proliferation and prevented death of PKC-{theta}–/– T cells stimulated with LM-infected wild-type dendritic cells, further demonstrating that the impaired proliferation and survival of PKC-{theta}–/– T cells in listeriosis is not intrinsically fixed and can be experimentally improved.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The protein kinase C (PKC)-{theta}3 is a serine/threonine kinase that is predominantly expressed in T cells, muscle cells, and platelets (1). Upon stimulation of the TCR, PKC-{theta} translocates to the immunological synapse, where it is phosphorylated by Lck and subsequently induces the activation of NF-{kappa}B, NFAT, and AP1 (2, 3, 4).

Studies in PKC-{theta}–/– mice have shown that PKC-{theta} is not required for the thymic development of T cells (5). However, in naive peripheral T cells, activation of PKC-{theta} via the TCR is required for IL-2 secretion, IL-2 receptor up-regulation, and clonal expansion of CD4 and CD8 T cells. Correspondingly, in noninfectious, T cell-mediated autoimmune diseases, expansion of PKC-{theta}–/– T cells is greatly diminished, resulting in an amelioration of the inflammatory response and the clinical disease (6, 7, 8, 9, 10). Additionally, PKC-{theta} is essential for the induction of Th2 responses in allergic and parasitic diseases (11, 12).

In sharp contrast, CD8 T cell and Th1 responses are normal in several viral and parasitic diseases of PKC-{theta}–/– mice, and in vitro studies suggest that strongly activated dendritic cells (DC) may compensate for the PKC-{theta} deficiency, resulting in a normal expansion, survival, and frequency of pathogen-specific CD8 T cells in viral infections (12, 13, 14, 15). The exact in vivo mechanism of how DCs compensate for a T cellular PKC-{theta} deficiency in viral infections is unknown, but it may include T cell activation by DC-derived proinflammatory cytokines including IL-1 and TNF, which can activate NF-{kappa}B in PKC-{theta}–/– T cells (5, 15). Additionally, it has recently been demonstrated that stimulation of TLRs on T cells can compensate for a T cellular PKC-{theta} deficiency in autoimmune myocarditis (6). Furthermore, PKC-{theta} has been reported to inhibit apoptosis of T cells stimulated via the TCR (16, 17, 18). In contrast, Fas-mediated apoptosis of peripheral T cells is PKC-{theta}-dependent (19). Thus, as reported for T cell activation and proliferation depending on the activating stimulus and the engaged receptors as well as the T cell subtype (CD4 vs CD8), PKC-{theta} has diverse and even in part opposing effects on T cell apoptosis.

At present it is unclear whether PKC-{theta} is required for the generation of protective T cell responses in bacterial infections. Immunity against the facultative intracellular bacterium Listeria monocytogenes (LM) is mainly mediated by MHC class Ia- and Ib-restricted CD8 T cells and, to a lesser extent, by CD4 T cells (20). We used the murine model of listeriosis to study the functional role of PKC-{theta} for LM-specific T cell responses and for the control of the bacterium. Our results demonstrate that PKC-{theta} expression of T cells is important to generate a normal LM-specific T cell response, including a normal proliferation and survival of the T cells, and to control the bacterium in vivo. In listeriosis, proliferation and survival of PKC-{theta}–/– T cells can be partially restored by a wild-type (WT) environment and IL-2, but not by inhibition of caspases.


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

Age- and sex-matched C57BL/6 and BALB/c WT mice, obtained from Harlan Sprague Dawley, as well as C57BL/6 PKC-{theta}–/– and BALB/c PKC-{theta}–/– mice (5) were used. OT-I and OT-II mice were crossed with C57BL/6 PKC-{theta}–/– mice in our animal facility. All animals were kept under conventional conditions in an isolation facility throughout the experiments. Experiments were approved and supervised by local governmental institutions.

Bacterial and viral infection of mice

WT LM (EGD strain), recombinant OVA-expressing LM (LMova), and recombinant LM expressing the gp33–41 epitope derived from the glycoprotein of lymphocytic choriomeningitis virus (LCMV) (LMgp, strain XFL703) (21) were grown in tryptose soy broth and aliquots of log-phase cultures were stored at –80°C. For each experiment, the respective strain of LM was thawed and diluted appropriately in sterile pyrogen-free PBS (pH 7.4) and i.p. administered at the indicated concentration. Mice were i.p. infected with 1 x 104 WT LM, 5 x 104 LMgp33, or 5 x 104 LMova for primary infection and 1 x 106 WT LM or 5 x 106 LMova for secondary infection. For each experiment, the bacterial dose used for infection was controlled by plating an inoculum on tryptose soy agar and counting colonies after incubation at 37°C for 24 h. To determine CFUs in spleens and livers of LM-infected mice, organs were dissected and homogenized with sterile tissue grinders. Ten-fold serial dilutions of the homogenates were plated on tryptose soy agar. Bacterial colonies were counted microscopically after incubation at 37°C for 24 h.

LCMV (strain WE) was generated and titrated on L929 cells as PFUs, and mice were i.v. infected with 1 x 105 PFU (22).

Isolation of leukocytes from liver, mesenteric lymph node, blood, and spleen

Before isolation of leukocytes, animals were anesthetized with isoflurane (Baxter). Blood was obtained by puncture of the heart with a 25-gauge needle attached to a 1-ml syringe. Isolated blood was mixed with heparin/PBS to prevent clotting of the blood. Splenic and MLN leukocytes were isolated from sacrificed mice by passing these lymphatic organs through a 70-µm cell strainer (BD Biosciences). Erythrocytes in blood, spleen, and MLN were lysed with ammonium chloride. Before isolation of hepatic leukocytes, mice were intracardially perfused with 0.9% NaCl to remove contaminating intravascular leukocytes from the liver. Thereafter, liver tissue was minced through a 100-µm cell strainer, and leukocytes were separated by Percoll gradient centrifugation (GE Healthcare) followed by lysis of erythrocytes with ammonium chloride.

ELISPOT

The number of LM- and LCMV-specific CD4 and CD8 T cells was determined by an IFN-{gamma} specific ELISPOT as described previously (23). Triplicates of isolated splenic leukocytes (2 x 105, 2 x 104, and 2 x 103 cells/well) from infected mice on C57BL/6 background were added to rat anti-mouse IFN-{gamma}- (BioSource International) coated ELISPOT plates and coincubated with spleen cells from noninfected WT C57BL/6 mice (2 x 105/well), which were preloaded with listeriolysin O (LLO)190–201 (10–6 M), the LCMV-derived epitope gp61–80 (10–6 M), OVA257–264 (10–8 M), and gp33–41 (10–8 M) peptide, respectively. Isolated splenic and hepatic leukocytes from infected BALB/c were coincubated with spleen cells from noninfected BALB/c WT mice (2 x 105/well), which were loaded with LLO189–200 (10–6 M) or LLO91–99 (10–8 M) peptide. 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 plates were incubated overnight and developed with biotin-labeled rat anti-mouse IFN-{gamma} (BD Pharingen), peroxidase-conjugated streptavidin, and amino-ethylcarbazole dye solution (Sigma-Aldrich). The spots were counted microscopically, and the number of Ag-specific cells per organ was calculated from the number of spots in triplicate wells and the number of leukocytes per organ.

Western blot

T cells were isolated from the spleen by MACS Pan T cell isolation kit (Miltenyi Biotec) 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% Na-deoxycholate, 1 mM PMSF, 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, transferred to polyvinylidene fluoride membranes, and followed by incubation with anti-PKC-{theta}, anti-phospho-PKC-{theta} (Thr538), and anti-GAPDH Abs (Cell Signaling Technology). Blots were developed using an ECL Plus kit (GE Healthcare).

Immunization with peptide-coated bone marrow-derived DCs (BMDC)

BMDCs were isolated, cultivated, and matured with LPS as described by Inaba et al. (24). Two days after adding LPS (500 ng/ml), OVA257–264 (10–5 M) and LLO190–201 (10–4 M) peptides were added to cultures 4 h before harvest. Based on the percentage of CD11c+ cells as determined by flow cytometry, 2.5 x 105 mature BMDCs were injected i.v. per mouse. Control mice were treated with nonpeptide-loaded BMDCs.

Adoptive transfer of T cells

Polyclonal T cells were isolated from the spleen of noninfected C57BL/6 WT or PKC-{theta}–/– mice by MACS using a Pan T cell isolation kit. T cells (2 x 107) were injected i.v. into C57BL/6 PKC-{theta}–/– mice 24 h before infection with WT LM. CD8 and CD4 T cells were isolated from spleens of noninfected WT and PKC-{theta}–/– OT-I and OT-II mice using CD8 and CD4 T cell isolation kits (Miltenyi), respectively. Purified OT-I or OT-II T cells (5 x 106) were injected i.v. into recipient mice 24 h before infection.

Detection of Ag-specific CD8 T cells by flow cytometry

Splenic Ag-specific CD8 T cells were detected by staining with PE-conjugated MHC class Ia SIINFEKL (OVA257–264) and PE-conjugated KAVYNFATM (LCMVgp33–41) pentamers (both from ProImmune) and PE-conjugated MHC class Ib fMIGWII tetramers (25), respectively, in combination with anti-CD8-FITC (clone 53-6.7). Control staining was performed with isotype-matched control Abs.

T cell proliferation and activation

CD8 T cells from OT-I WT and OT-I PKC-{theta}–/– or CD4 T cells from OT-II WT and OT-II PKC-{theta}–/– were purified using CD4+ and CD8+ isolation kits (Miltenyi Biotec), respectively. Purified cells were washed in cold PBS, resuspended at 5 x 106 cells/ml, and labeled with CSFE (Invitrogen) at a final concentration of 0.5 µM at room temperature for 10 min. The reaction was terminated with PBS containing 5% FCS. The purity of CD4+ and CD8+ T cells was 85–95% as determined by FACS staining. CSFE-labeled T cells (5 x 106) of the mouse strains were injected separately into the lateral tail veil of recipient mice 1 day before infection with LMova. Proliferation and activation of CSFE-labeled cells were characterized by staining with anti-CD8– PECy5 (clone 53-6.7) or anti-CD4-PECy5 (clone RM4-5), in combination with anti-V{alpha}2-PE (clone B20.1), anti-CD44-PE (clone IM7), and anti-CD69-PE (clone H1.2F3), respectively, followed by flow cytometry.

Active caspase-3 staining

Active caspase-3 was detected by flow cytometry using the Active Caspase-3 PE mAb Apoptosis kit (BD Pharmingen), in combination with anti-CD8-APC.

Measurement of apoptosis

Apoptosis was analyzed by quantifying phosphatidylserine residues exposed on the external cell membrane. In brief, cells were resuspended in annexin binding buffer (10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) and stained with annexin V-PE and 7-aminoactinomycin D (7-AAD) for 15 min at room temperature, in combination with anti-CD8-APC or anti-CD4-APC. Stained cells were analyzed by flow cytometry within 1 h.

In vitro infection of DCs with LM

LMova was grown in brain heart infusion medium, washed in antibiotic-free RPMI 1640, and added to BMDCs at a multiplicity of infection of 100 in RPMI 1640 medium with 10% FCS and 50 µM 2-ME without antibiotics. After 1 h of infection, 50 µg/ml gentamicin was added to kill extracellular bacteria for an additional 3 h. Thereafter, infected BMDCs were washed in PBS, resuspended in RPMI 1640 medium supplemented with 10 µg/ml gentamicin, and cocultured with T cells.

In vitro proliferation of T cells

Noninfected and LMova-infected BMDCs were added to 96-well round-bottom plates at 1–2 x 104 per well. MACS-purified primary, CSFE-labeled OT-I WT, and OT-I PKC-{theta}–/– T cells (2 x 105 cells/well), respectively, were added to WT BMDCs and cultured in 200 µl RPMI 1640 medium with 10% FCS, 50 µM 2-ME, 50 U/ml penicillin/streptomycin, and 10 µg/ml gentamicin. In some experiments, BMDCs-T cell cultures were supplemented with murine rIL-2 (15 ng/ml; PeproTech), 40 µM N-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) (pan-caspase) inhibitor (Bachem), and 100 µM caspase-3 inhibitor VII (Calbiochem), respectively. After 48 h, CSFE profiles of T cells were analyzed by flow cytometry.

Statistics

Statistical significance was determined using the t test (Statistica 5, StatSoft). p of <0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Reduced numbers of Listeria-specific, but not of LCMV-specific, CD4 and CD8 T cells in PKC-{theta}–/– mice

In good agreement with previously published data (13), the number of LCMVgp33–41-specific CD8 T cells was identical in spleens of LCMV-infected WT and PKC-{theta}–/– (Fig. 1A) as revealed by pentamer staining. However, the number of CD8 T cells specific for the MHC class I OVA257–264 epitope was significantly reduced in PKC-{theta}–/– mice as compared with WT animals after infection with LMova (Fig. 1A). Importantly, the number of gp33–41-specific CD8 T cells was also reduced in PKC-{theta}-deficient mice after infection with transgenic LM expressing the LCMV gp33–41 CD8 T cell epitope (Fig. 1A). Additional ELISPOT assays confirmed that numbers of pathogen-specific CD8 T cells were normal in LCMV infection, but not in listeriosis of PKC-{theta}–/– mice (data not shown). In parallel to CD8 T cells, the number of cells specific for the LCMV gp61–80 MHC class II epitope was unaffected by PKC-{theta}-deficiency in LCMV infection, whereas the number of cells specific for the LM-derived LLO190–201 MHC class II epitope was significantly reduced in both LMova- and LMgp-infected PKC-{theta}–/– mice (Fig. 1B).


Figure 1
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  		FIGURE 1. Reduced numbers of pathogen-specific CD8 and CD4 T cells in Listeria, but not LCMV infection of PKC-{theta}–/– mice. WT and PKC{theta}–/– C57BL/6 mice were infected with 1 x 105 PFU of LCMV, 1 x 105 LMgp (transgenic for the LCMV gp33–41 epitope) and 1 x 105 LMova, respectively. A, The number of splenic gp33–41- and OVA257–267-specific CD8 T cells was determined in LCMV, LMgp, and LMova infected (day 8 p.i.) as well as noninfected WT and PKC-{theta}–/– mice by staining with anti-CD8-FITC in combination with OVA257–264 (SIINKFEKL)- or gp33–41 (KAVYNFATM)-PE-conjugated pentamers, and analyzed by flow cytometry. Data are means ± SD of three mice per experimental groups. *, p ≤ 0.02 for PKC-{theta}–/– vs WT mice. B, The number of gp61–80- and LLO190–201-specific cells was determined in spleen cells of noninfected and infected (day 8 p.i.) WT and PKC-{theta}–/– mice (day 8 p.i.) by an IFN-{gamma} ELISPOT. Data are means ± SD of 3 mice per experimental group. n.d. indicates not detectable. *, p ≤ 0.001 for PKC-{theta}–/– vs WT mice.

 
Collectively, PKC-{theta} plays an important role for the normal generation of LM-specific CD4 and CD8 T cells. Importantly, the reduction of LM-specific T cells in PKC-{theta}–/– mice depends on the pathogen rather than on the epitope, because PKC-{theta}–/– mice mounted a reduced CD8 T cell response against the gp33–41 epitope upon infection with LMgp, but not after infection with LCMV.

Reduced numbers of MHC class Ia-, MHC class Ib-, and MHC class II-restricted LM-specific T cells in primary and secondary listeriosis of PKC-{theta}–/– mice

To analyze whether PKC-{theta} regulates the kinetics of LM-specific T cells, we infected C57BL/6 WT and PKC-{theta}–/– mice with LMova and quantified the number of LMova-specific T cells at different time points after primary and secondary infections. The number of OVA257–264-specific MHC class Ia-restricted CD8T cells was significantly reduced in PKC-{theta}–/– mice (Fig. 2A) at all time points between days 5 and 50 after primary infection. Additionally, the number of fMIGWII-specific MHC class Ib-restricted CD8 T cells was significantly reduced in acute listeriosis of PKC-{theta}–/– mice (Fig. 2B). In parallel to CD8 T cells, the number of LM-specific IFN-{gamma}-producing CD4 T cells was significantly reduced in listeriosis of PKC-{theta}–/– mice at days 7 and 9 postinfection (p.i.) (Fig. 2C).


Figure 2
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  		FIGURE 2. Kinetics of LM-specific T cells in WT and PKC-{theta}–/– mice. The number of OVA257–264-specific (A and D) and fMIGWII-specific (B) CD8 T cells was determined in the spleen of C57BL/6 WT and PKC-{theta}–/– mice by staining with OVA257–264-PE pentamer (A and D) and fMIGWII-specific tetramer (B) in combination with anti-CD8-FITC after primary (A and B) and secondary (D) infection with LMova. C and E, The frequency of LLO190–201-specific splenic CD4 T cells was determined by an IFN-{gamma} ELISPOT after primary (C) and secondary (E) infection with LMova. Data represent the means ± SD of 3–4 mice per experimental group. n.d. indicates not detectable. *, p ≤ 0.05 and **, p ≤ 0.001 for PKC-{theta}–/– vs WT mice.

 
Upon secondary infection at day 50 after primary infection, numbers of OVA257–264-specific CD8 T cells were reduced at any time point between days 1 and 21 after reinfection with LMova (Fig. 2D). Moreover, numbers of LLO190–201-specific CD4 T cells were also reduced in PKC-{theta}–/– mice, although differences were only significant at day 5 after reinfection (Fig. 2E). In summary, these findings illustrate that PKC-{theta} is important for an optimal expansion of both LM-specific CD8 and CD4 T cells in primary and secondary listeriosis.

Impaired control of LM in PKC-{theta}–/– mice

To study whether the reduced T cell responses in PKC-{theta}–/– mice correspond to an impaired pathogen control, we determined CFUs in liver and spleen. After primary infection with WT LM, PKC-{theta}–/– mice harbored significantly increased numbers of LM in the liver and spleen starting at day 5 p.i. (Fig. 3, A and B). Thus, the inability of PKC-{theta}–/– mice to control LM became evident at the same time point when stronger T cell responses were detectable in WT mice. Upon secondary infection, PKC-{theta}–/– mice had also increased numbers of LM in liver and spleen, and, as observed in primary infection, the differences became significant in the liver earlier than in the spleen (Fig. 3, C and D). These data indicate that PKC-{theta} is functionally relevant for control of LM in primary and secondary infection in vivo.


Figure 3
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  		FIGURE 3. Impaired control of LM in PKC-{theta}–/– mice during primary and secondary infection. CFUs were determined in liver and spleen of C57BL/6 WT and PKC-{theta}–/– mice after primary infection with 1 x 104 (A and B) and secondary infection with 1 x 106 (C and D) WT LM. CFUs were determined in 4 mice per experimental group at the indicated time points p.i. Data represent the mean + SD. n.d. indicates not detectable. *, p ≤ 0.05 and **, p ≤ 0.005 for PKC-{theta}–/– vs WT mice.

 
LM-specific CD8 and CD4 T cells are generally reduced in lymphoid organs and liver of PKC-{theta}–/– mice

To analyze whether the reduction of LM-specific CD8 and CD4 T cells is a general finding or due to altered distribution or trafficking among different organs, we determined the frequencies and numbers of LM-specific CD8 and CD4 T cells in spleen, liver, MLN, and blood of LMova-infected C57BL/6 WT and PKC-{theta}–/– mice at day 9 after primary infection. The percentage of OVA257–264-specific CD8 T cells was reduced in spleen, liver, MLN, and blood of PKC-{theta}–/– mice (Fig. 4A). Analysis of the absolute numbers of OVA257–264-specific CD8 T cells revealed that they were significantly reduced in all organs analyzed (Fig. 4B). In parallel to CD8 T cells, numbers of LM-specific IFN-{gamma}-producing CD4 T cells also were significantly reduced in spleen, liver, MLN, and blood of PKC-{theta}–/– mice (Fig. 4C). Thus, the reduction of LM-specific T cells in the spleen of PKC-{theta}–/– mice (Figs. 1 and 2) is not caused by altered distribution of T cells among the lymphoid organs and liver in these mice.


Figure 4
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  		FIGURE 4. Reduced numbers of LM-specific CD8 and CD4 T cells in organs of PKC-{theta}–/– mice. The number of OVA257–264-specific (A and B) CD8 T cells and LLO190–201-specific CD4 T cells (C) was determined in the spleen, liver, MLN, and blood of C57BL/6 WT and PKC-{theta}–/– mice at day 9 after primary infection with 5 x 104 LMova. A, Leukocytes were isolated from the various organs of 6 mice per experimental group, and the leukocytes of each organ were pooled and stained with OVA257–264-PE pentamer in combination with anti-CD8-FITC. Dot plots of OVA257–264-PE pentamer and CD8-FITC are shown after gating on CD8+ cells. B, The absolute number of OVA257–264-specific CD8 T cells per organ was calculated from the percentage of OVA257–264-specific CD8 T cells (see A) and total leukocyte counts in the organ samples of individual mice. Data show the means ± SD of 6 mice per organ and experimental group. C, The number of LLO190–201-specific splenic CD4 T cells was determined by an IFN-{gamma} ELISPOT. Data represent the mean ± SD of 6 mice per experimental group. *, p ≤ 0.05 and **, p ≤ 0.001 for PKC-{theta}–/– vs WT mice.

 
The role of PKC-{theta} for the generation of LM-specific T cells and bacterial control is independent of the host genetic background

Immunity to LM is partially dependent on the host genetic background (26). To assess whether the role of PKC-{theta} for the generation of LM-specific cells and control of the bacterium is influenced by the host genetic background, we additionally infected BALB/c WT and PKC-{theta}–/– mice with WT LM (Fig. 5). As observed for mice on the C57BL/6 background (Fig. 3), BALB/c PKC-{theta}–/– mice harbored significantly more LM in spleen and liver as compared with WT animals on day 9 p.i. (Fig. 5A). Additionally, the number of LM-specific CD8 and CD4 T cells was significantly reduced in spleen and liver of BALB/c PKC-{theta}–/– as compared with the corresponding WT mice (Fig. 5, B and C). These findings illustrate that the important function of PKC-{theta} for an optimal T cell response and pathogen control in listeriosis is independent of the host genetic background.


Figure 5
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  		FIGURE 5. The immune response against LM is also severely impaired in PKC-{theta}–/– mice of the BALB/c background. BALB/c WT and PKC-{theta}–/– mice were i.p infected with 1 x 104 WT LM and analyzed at day 9 after infection. A, CFUs were determined in spleen and liver of 5 mice per group (mean ± SD, *, p < 0.01). B, The number of LLO91–99 and (C) LLO189–200-specific cells was determined in spleen and liver of 5 mice per group by IFN-{gamma} ELISPOT assays (mean ± SD, *, p < 0.01, **, p < 0.005).

 
Adoptive transfer of WT T cells compensates for PKC-{theta} deficiency in listeriosis

To validate that the impaired control of LM in PKC-{theta}–/– mice was caused by an insufficient T cell response, we adoptively transferred purified polyclonal WT T cells into PKC-{theta}–/– mice before infection with LMova and monitored the bacterial load in these animals. In fact, CFUs were significantly reduced in spleen (Fig. 6A) and liver (Fig. 6B) of PKC-{theta}–/– mice supplemented with WT T cells as compared with PKC-{theta}–/– supplemented either with PKC-{theta}–/– T cells or without adoptive transfer. Importantly, the bacterial load in PKC-{theta}–/– mice supplemented with WT T cells was as low as in infected WT control mice (Fig. 6, A and B).


Figure 6
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  		FIGURE 6. WT T cells compensate for PKC-{theta} deficiency in listeriosis, but peptide-loaded WT DCs do not induce a normal expansion of peptide-specific T cells in PKC-{theta}–/– mice. A and B, On day 8 after infection with WT LM, CFUs were determined in spleen (A) and liver (B) of 1) C57BL/6 WT mice, 2) PKC-{theta}–/– mice, 3) PKC-{theta}–/– mice supplemented with 2 x 107 polyclonal WT T cells, and 4) PKC-{theta}–/– mice supplemented with 2 x 107 polyclonal PKC-{theta}–/– T cells. Adoptive T cell transfers were performed 1 day before infection. CFUs of individual mice are shown and the horizontal bar represents the mean value. *, p ≤ 0.05; **, p ≤ 0.004. C, Western blot analysis of PKC-{theta}, phospho-PKC-{theta}, and GAPDH expression in MACS-isolated splenic T cells of noninfected WT mice and WT LM-infected WT, PKC-{theta}–/–, and PKC-{theta}–/– mice supplemented with either polyclonal WT or PKC-{theta}–/– T cells. T cells were isolated from infected mice at day 8 p.i. Results of 1 representative mouse per group from two independent experiments with 3 mice each are shown. D and E, WT and PKC-{theta}–/– mice were immunized with OVA257–264 and LLO190–201 peptide-loaded BMDCs or with nonpeptide-loaded DCs. The number of OVA257–264-specific CD8 T cells was determined by OVA257–264-PE pentamer staining (D), and the number of LLO190–201-specific cells was determined by an IFN-{gamma}-ELISPOT (E) at day 7 after immunization. Data show the means ± SD of 5 mice per experimental group. *, p ≤ 0.01 and **, p ≤ 0.006 for PKC-{theta}–/– vs WT mice.

 
Additional Western blot analysis showed that PKC-{theta} was present in spleen cells of noninfected and infected WT mice as well as in LMova-infected PKC-{theta}–/– mice supplemented with WT T cells, whereas it was, as expected, absent in PKC-{theta}–/– mice (Fig. 6C). Moreover, phosphorylated PKC-{theta}, already detectable in noninfected WT spleens at varying signal intensities (compare Exp.1 and Exp. 2), was enhanced in infected WT controls as well as in PKC-{theta}–/– mice supplemented with WT T cells (Fig. 6C).

Since LM secretes virulence factors, in particular LLO, which may affect the Ag-presenting capacity of DCs and may reduce T cell responsiveness upon contact with APCs (21), we studied whether an immunization with WT DCs loaded with OVA257–264 and LLO190–201 peptides induces normal T cell responses in PKC-{theta}–/– mice. In good agreement with data from Hamilton and Harty (27), WT mice generated an OVA257–264-specific CD8 T cell and LLO190–201-specific CD4 T cell response (Fig. 6, D and E). However, numbers of OVA257–264-specific CD8 and LLO190–201-specific CD4 T cells were significantly reduced in PKC-{theta}–/– mice (Fig. 6, D and E).

These findings indicate that the impaired control of LM in PKC-{theta}–/– mice is due to an intrinsic defect of T cells and can be restored by transfer of WT T cells. In contrast, an immunization with peptide-loaded, mature activated WT DCs does not compensate for lack of T cellular PKC-{theta}-expression for the induction of OVA257–264-specific CD8 and LLO190–20-specific CD4 T cells.

PKC-{theta} is important for proliferation and survival of LM-specific CD8 T cells, but its function can be partially compensated by neighboring WT cells

To determine the impact of PKC-{theta} on proliferation, apoptosis, and activation of LM-specific T cells, we crossed PKC-{theta}–/– mice with OVA257–264-specific TCR transgenic OT-I mice and adoptively transferred MACS-purified CSFE-labeled PKC-{theta}–/– or WT OT-I CD8 T cells into WT (Fig. 7A) or PKC-{theta}–/– (Fig. 7B) recipients before infection with LMova. In noninfected mice, only small numbers of transferred WT and PKC-{theta}–/– OT-I T cells proliferated in both WT and PKC-{theta}–/– recipients (Fig. 7, A and B). Upon infection with LMova, 84.5% of transferred WT OT-I T cells proliferated in WT recipients (Fig. 7A), and a comparable percentage (79.9%) of transferred WT OT-I T cells proliferated in LMova-infected PKC-{theta}–/– recipients (Fig. 7B). In contrast, proliferation of PKC-{theta}–/– OT-I CD8 T cells was reduced in LMova-infected recipients: only 55.6% of adoptively transferred PKC-{theta}–/– OT-I CD8 T cells proliferated in LMova-infected WT mice (Fig. 7A), and PKC-{theta}–/– OT-I CD8 T cells did not proliferate in LMova-infected PKC-{theta}–/– recipients (Fig. 7B).


Figure 7
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  		FIGURE 7. Decreased proliferation and survival of PKC-{theta}–/– OT-I CD8 T cells in LMova-infected mice is partially rescued in WT hosts. MACS-purified, CSFE-labeled WT and PKC-{theta}–/– OT-I CD8 T cells were adoptively transferred into WT (A) or PKC-{theta}–/– (B) recipients. Recipients were left uninfected or were infected with 5 x 104 LMova 1 day after adoptive transfer. Representative histograms show CSFE profiles of adoptively transferred CD8 T cells 48 h after infection, and the percentage of proliferating cells is presented. Dot plots show results for annexin V/7-AAD staining of proliferating and nonproliferating cells as indicated by arrows. The percentage of annexin V and 7-AAD positive or negative cells is presented. Three to four recipient mice were analyzed per group, and representative data are shown. C, The activation of CSFE+ CD8 T cells adoptively transferred into WT mice was analyzed by staining with anti-CD44, anti-CD69, and anti-V{alpha}2 Abs, respectively, followed by flow cytometry at day 2 after infection with 5 x 104 LMova. Three to 4 recipient mice were analyzed per group, and the mean ± SD is shown.

 
To determine the impact of PKC-{theta} on the survival of LM-specific CD8 T cells, we stained adoptively transferred PKC-{theta}–/– and WT OT-I T cells with 7-AAD and annexin V. In noninfected mice, the vast majority of both WT and PKC-{theta}–/– OT-I T cells died independently of PKC-{theta} expression of the host within 2 days after adoptive transfer (Fig. 7, A and B). In contrast, ~80% of proliferating WT OT-I T cells survived (annexin V–/7-AAD–) in both LMova-infected WT and PKC-{theta} recipients 2 days after infection. Proliferating PKC-{theta}–/– OT-I T cells were also partially rescued from death (62.1% annexin V–/7-AAD–) in LMova-infected WT recipients (Fig. 7A), which is in contrast to the few proliferating PKC-{theta}–/– T cells in LMova-infected PKC-{theta}–/– recipients (Fig. 7B). Nonproliferating PKC-{theta}–/– and WT OT-I T cells did not survive in LMova-infected mice independent of the PKC-{theta} expression of the recipient.

Additionally, both proliferating WT and PKC-{theta}–/– OT-I T cells equally up-regulated CD44 and CD69 activation markers and down-regulated the V{alpha}2 TCR in LMova-infected WT recipients (Fig. 7C).

In parallel, we also performed adoptive transfer experiments with OVA-specific WT and PKC-{theta}–/– OT-II CD4 T cells, which provided essentially the same results with respect to proliferation, survival, and activation as demonstrated for OT-I CD8 T cells (data not shown).

These findings illustrate 1) that a WT milieu partially rescues proliferation and survival of PKC-{theta}–/– LM-specific T cells, and 2) that a cell-autonomous expression of PKC-{theta} by LM-specific T cells is sufficient for proliferation and survival of these T cells in a PKC-{theta}-deficient host.

Externally supplemented IL-2, but not inhibition of caspases, partially restores proliferation of PKC-{theta}–/– T cells in vitro

To identify parameters that might mediate the compensatory effect of a WT milieu on PKC-{theta}–/– T cell proliferation and survival, we used an in vitro system composed of LMova-infected WT BMDCs and MACS-purified WT or PKC-{theta}–/– OT-I T cells.

After coincubation with noninfected WT BMDCs, only a very small number of CSFE-labeled WT or PKC-{theta}–/– OT-I cells proliferated (Fig. 8A). In contrast, stimulation of WT OT-I T cells with LMova-infected BMDCs resulted in proliferation of >50% of the WT T cells, whereas PKC-{theta}–/– OT-I cells still did not proliferate (Fig. 8A). However, addition of IL-2 induced proliferation of 40% of PKC-{theta}–/– OT-I cells stimulated with LMova-infected WT BMDCs. The proliferation of WT OT-I cells did not further increase after addition of IL-2.


Figure 8
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  		FIGURE 8. IL-2, but not caspase inhibitors, enhances proliferation of PKC-{theta}–/– OT-I CD8 T cells after stimulation with LMova-infected WT BMDCs in vitro. A, Uninfected and LMova-infected WT BMDCs were coincubated with MACS-purified, CSFE-labeled WT or PKC-{theta}–/– OT-I CD8 T cells. Cultures were left untreated or were supplemented with either IL-2, a caspase-3 inhibitor, or the pan-caspase inhibitor Z-VAD-FMK. Triplicate wells were analyzed, and representative histograms show CSFE profiles of OT-I CD8 T cells 48 h after infection. The percentage of proliferating cells is presented. Dot plots show results for annexin V/7-AAD staining of CSFE+ CD8 T cells, and the percentage of cells in each quadrant is presented. B, MACS-purified, CSFE-labeled WT and PKC-{theta}–/– OT-I T cells were adoptively transferred into WT recipients. Recipients were left uninfected or were infected with 5 x 104 LMova. Forty-eight hours after infection, spleen cells were stained for active caspase-3 and CD8. Controls included staining with isotype-matched control Abs. Dot plots show the CSFE profile of transferred CD8 T cells in combination with anti-caspase-3 staining or control staining. The percentage of cells per quadrant is shown. Three recipients per group were analyzed, and a representative dot plot is shown.

 
As observed for noninfected recipients in vivo (Fig. 7), only small numbers of WT and PKC-{theta}–/– OT-I cells coincubated with noninfected BMDCs survived as revealed by annexin V/7-AAD staining (Fig. 8A). Stimulation with LMova-infected BMDCs resulted in proliferation and an increased survival of OT-I T cells (34.5% annexin V–/7-AAD–), whereas the number of annexin V–/7-AAD– PKC-{theta}–/– remained unchanged (14.6%) after WT BMDC-LMova stimulation. However, the addition of IL-2 to PKC-{theta}–/– OT-I T cells stimulated with LMova-infected BMDC resulted in an increased survival rate of the T cells (25.3%, Fig. 8A). The addition of IL-2 did not modify the survival rate of WT OT-I cells stimulated with LMova-infected BMDCs.

These findings illustrate 1) that LMova-infected WT BMDCs only induce the proliferation and survival of WT OT-I cells, but they are not sufficient to compensate for a T cellular PKC-{theta} deficiency, and 2) that IL-2 partially restores proliferation and survival of LM-specific PKC-{theta}–/– T cells stimulated with LMova-infected WT BMDCs.

Our data presented in Fig. 7 indicate that PKC-{theta} prevents the apoptosis of activated LM-specific T cells. To further analyze the impact of PKC-{theta} on apoptosis of LM-specific T cells, we determined the presence of active caspase-3 in WT and PKC-{theta}–/– OT-I T cells after adoptive transfer to WT recipients. In noninfected WT recipients, ~70% of transferred WT and PKC-{theta}–/– OT-I T cells were positive for active caspase-3 (Fig. 8B), which corresponds to the percentage of annexin V/7-AAD-positive cells (Fig. 7). Upon infection with LMova, 82.9% of WT OT-I cells proliferated and only about one-fifth of the proliferating cells were positive for active caspase-3 (Fig. 8B). In contrast and as observed before (Fig. 7), only 48% of PKC-{theta}–/– OT-I cells proliferated in LMova-infected WT recipients and ~50% of these proliferating cells were active caspase-3+ (Fig. 8B).

To study the functional role of caspases, we treated cocultures of LMova-infected BMDC and OT-I cells with either a caspase-3 inhibitor or the pan-caspase inhibitor Z-VAD-FMK (Fig. 8A). Caspase-3 inhibition had no influence on proliferation of WT OT-I cells, although it strongly increased the number of surviving annexin V–/7-AAD– OT-I cells. In contrast, the pan-caspase inhibitor strongly inhibited the proliferation of WT OT-I T cells by >50% (52.6% proliferation without and 22.9% proliferation with Z-VAD-FMK). Additionally, the pan-caspase inhibitor decreased the survival rate of WT OT-I cells. In PKC-{theta}–/– OT-I T cells, neither the pan-caspase nor the caspase-3 inhibitor induced proliferation or prevented the death of these T cells.

These in vitro findings illustrate 1) that the reduced proliferation and survival of PKC-{theta}–/– T cells is caspase-independent, and 2) that caspases other than caspase-3 are required to allow a normal proliferation and survival of WT T cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our data reveal that T cell-specific expression of PKC-{theta} is required for the generation of protective pathogen-specific T cells and for the effective control of the pathogen in murine listeriosis. In good agreement with previous studies in murine LCMV infection (13), PKC-{theta} was in contrast dispensable for the generation of LCMV-specific CD4 and CD8 T cells. The observation that PKC-{theta}–/– mice mounted a normal CD8 T cell response against the LCMV-derived gp33–41 MHC class I epitope after infection with LCMV, but not after infection with gp33–41 transgenic LM, directly demonstrates that the necessity of PKC-{theta} for pathogen-specific T cell responses is strongly dependent on the pathogen, but not on the epitope itself.

The more general importance of PKC-{theta} for the development of LM-specific T cell responses is further documented by the finding that PKC-{theta}–/– mice with different genetic backgrounds (i.e., C57BL/6 (H-2b) and BALB/c (H-2d)) had significantly reduced numbers of LM-specific CD4 and CD8 T cells and a significantly increased bacterial burden after infection with LM. Thus, the genetic background of the host, which determines the susceptibility to listeriosis (26), does not affect the critical function of PKC-{theta} for the induction of LM-specific T cell responses. Additionally, numbers of LM-specific CD4 and CD8 T cells were reduced in various lymphoid organs and liver of PKC-{theta}–/– mice, further demonstrating that PKC-{theta} is of general importance for LM-specific T cell responses and that this role is not restricted to a specific organ.

In both primary and secondary listeriosis of C57BL/6 mice, numbers of MHC class Ia-restricted LM-specific CD8 T cells were significantly reduced in the absence of PKC-{theta} at all time points, which is in marked contrast to several viral infections in which PKC-{theta} is dispensable for a normal MHC class Ia-restricted CD8 T cell response (12, 13, 14, 15). The observation that LM-specific CD8 T cells were reduced at the peak of the primary immune response (day 9 p.i.), late after primary infection (day 50 p.i.) and early after secondary infection (day 1 after reinfection), indicates that both effector and memory CD8 T cells are PKC-{theta}-dependent in listeriosis. Furthermore, our data also provide the first evidence that the development of MHC class Ib-restricted CD8 T cells, which play a protective role in primary, but not secondary listeriosis (25, 28), is PKC-{theta}-dependent. Additionally, the number of IFN-{gamma}-producing pathogen-specific CD4 T cells was also reduced in both primary and secondary listeriosis of PKC-{theta}–/– mice, which is in contrast to infection with influenza virus, LCMV, and Leishmania (12, 15). However, numbers of LM-specific CD4 T cells did not differ late after primary infection (day 14 to day 50 p.i.) and after the peak of the secondary LM-specific CD4 T cell response (days 9 and 21 p.i.), indicating that PKC-{theta} may play a less important role for the maintenance of LM-specific CD4 T cells as compared with LM-specific CD8 T cells.

Murine listeriosis is characterized by the development of a Th1 and the absence of a Th2 CD4 T cell response (29). Early after infection with LM, CD4 NK T cells transiently produce IL-4 (30), which is a major promoter of Th2 development. However, this IL-4 production is rapidly down-regulated by endogenous IL-12 produced by macrophages after infection (31), which fosters LM-specific Th1 and abrogates Th2 CD4 T cell responses. Therefore, the important function of PKC-{theta} for the development of Th2 responses in infectious diseases (12) does not affect Th1/Th2 differentiation in listeriosis.

In contrast to T cell responses in viral infections, PKC-{theta} plays an important role for the induction of autoimmune T cells in various models of autoimmune disorders (6, 7, 8). This suggests that during viral infectious diseases the strong stimulation of T cells by the inflammatory milieu and highly activated DCs is sufficient to compensate for a T cellular PKC-{theta} deficiency, whereas, in general, in autoimmune disorders T cell stimulation remains below this critical level of T cell activation. By analogy, in listeriosis, the stimulation of pathogen-specific T cells by the inflammatory milieu and DCs may be weaker than in viral infections, preventing normal activation and proliferation of T cells in the absence of PKC-{theta}. The assumption that external factors including the inflammatory milieu and the activation status of DCs partially determine the functional importance of PKC-{theta} for the activation and proliferation of Ag-specific T cells is further corroborated by the observation that infection with LCMV, but not immunization with DCs loaded with LCMV-specific peptides, induced proliferation of LCMV-specific T cells in PKC-{theta}–/– mice (13). Also in our experiments, an immunization with OVA257–264 and LLO190–201 peptide-loaded WT DCs was insufficient to induce normal numbers of OVA-specific CD8 and LLO-specific CD4 T cells in PKC-{theta}–/– mice.

Importantly, the reduced proliferative capacity of PKC-{theta}–/– T cells is not fixed, but it can be overcome by a stronger activation of the Ag-specific T cells, for example, by stimulation with peptide-loaded DCs hyperactivated with TLR9 ligand (15) or by a stimulation of TLR9 on PKC-{theta}–/– T cells (6).

To analyze under which experimental conditions PKC-{theta}–/– mice develop an improved LM-specific T cell response and control LM more efficiently, we performed additional in vivo and in vitro experiments. The observation that PKC-{theta}–/– mice reconstituted with polyclonal WT T cells controlled LM as efficiently as did WT animals illustrates that LM-specific WT T cells are efficiently activated and are protective in a PKC-{theta}–/– host. This finding is further supported by the adoptive transfer experiments with TCR transgenic T cells, which also demonstrated that WT OT-I T cells proliferated normally in PKC-{theta}–/– recipients and were protected from cell death upon infection with LMova. Thus, in listeriosis the expression of PKC-{theta} in Ag-specific T cells was sufficient to drive a normal proliferation and survival of these T cells independent of PKC-{theta} expression by any other cell type, including DCs, which are assumed to play a role for the induction of LM-specific T cells and bacterial dissemination (32, 33, 34).

In direct extension, our results show that PKC-{theta} deficiency of LM-specific T cells can be partially compensated for by a WT environment in vivo. An adoptive transfer of PKC-{theta}–/– OT-I T cells before infection with LMova resulted in proliferation of PKC-{theta}–/– OT-I T cells, and the proliferating cells were largely rescued from death. However, proliferation of PKC-{theta}–/– T cells was still reduced by ~40% in a WT host as compared with WT OT-I T cells, indicating that the expression of PKC-{theta} in the Ag-specific T cell is required for a maximal expansion of the T cell. The additional in vitro observation that LMova-infected WT DCs did not induce proliferation of PKC-{theta}–/– OT-I T cells, whereas the addition of IL-2 resulted in proliferation, indicates that non-DC-derived factors, including IL-2 produced by T cells, may partially compensate for a T cellular PKC-{theta} deficiency in listeriosis. However, the proliferation rate of PKC-{theta}–/– OT-I T cells was still reduced by 20% as compared with WT T cells after addition of IL-2. The observation that WT T cells and IL-2 can partially restore proliferation and survival of PKC-{theta}–/– T cells stimulated with peptide-loaded DC in vitro has also been made by Barouch-Bentov et al. (16), although the exact molecular mechanisms of how IL-2 partially compensates for PKC-{theta}-deficiency are at present unresolved.

In listeriosis, the reduced proliferation rate of PKC-{theta}–/– T cells even under optimal in vivo and in vitro conditions may be caused by a critical function of PKC-{theta} for proliferation or inhibition of apoptosis, which can only be partially substituted by other factors. In fact, previous experiments have shown that PKC-{theta} has an antiapoptotic function (16, 17, 18), although PKC-{theta} may also be proapoptotic depending on the apoptosis-inducing stimulus (19). Noteworthy, our in vivo adoptive transfer experiments revealed that proliferation of both WT and PKC-{theta}–/– T cells was intimately linked with their activation (i.e., up-regulation of CD44 and CD69 and down-regulation of V{alpha}2 TCR) as well as survival (Fig. 6). However, the possibility remained that the reduced proliferation rate of PKC-{theta}–/– T cells in vivo in WT or PKC-{theta}–/– recipients or in vitro after stimulation with LMova-infected WT DCs was caused by the lacking antiapoptotic function of PKC-{theta}. In fact, caspase-3-dependent apoptosis of T cells has been demonstrated in listeriosis, which, however, largely affected bystander T cells in WT mice (35, 36). To further analyze the function of caspase-3 in PKC-{theta}–/– T cells, we determined the expression of active caspase-3 in adoptively transferred WT and PKC-{theta}–/– OT-I T cells. In noninfected WT recipients, 70% of both adoptively transferred WT and PKC-{theta}–/– OT-I T cells were active caspase-3+, indicating that death of these cells was mediated by caspases. Upon infection, only 34% of adoptively transferred WT T cells were still caspase-3+, whereas nearly 60% of PKC-{theta}–/– OT-I T cells remained caspase-3+. These findings raise the possibility that the reduced proliferation of LM-specific PKC-{theta}–/– T cells was at least in part caused by an increased caspase-mediated apoptosis. To analyze whether an inhibition of caspases resulted in an increased survival and proliferation of PKC-{theta}–/– T cells, we treated cocultures of LMova-infected WT DCs and WT or PKC-{theta}–/– OT-I T cells with caspase inhibitors. Treatment with a caspase-3 inhibitor increased the number of annexin V–/7-AAD– live WT OT-I T cells from 34 to 50%, which is compatible with some role of caspase-3 in the induction of T cell apoptosis under these in vitro conditions. However, inhibition of caspase-3 had no influence on the percentage of proliferating WT T cells. Thus, the number of live WT T cells was not directly linked to the number of proliferating cells under these conditions.

In contrast to caspase-3 inhibition, treatment with a pan-caspase inhibitor strongly reduced proliferation of WT OT-I T cells. The latter finding is completely compatible with a function of caspase-8, which is inhibited by the pan-caspase inhibitor, for the activation and proliferation of T cells (37, 38). In activated T cells, the active caspase-8/c-FLIP-long form complex migrates to lipid-rafts and colocalizes with the NF-{kappa}B signaling molecules receptor interacting protein 1, TNFR-associated factor (TRAF) 2, and TRAF6, as well as upstream NF-{kappa}B regulators PKC-{theta}, caspase-recruitment domain membrane-associated guanylate kinase protein 1, Bcl-10, and MALT1, which connect to the TCR (39). The function of caspase-8 for the activation and proliferation of LM-specific T cells is currently unknown, but based on its central position in the TCR-mediated activation of T cells and on our preliminary results, it is likely that caspase-8, in contrast to caspase-3 (40), is crucial for the activation of LM-specific T cells.

In PKC-{theta}–/– OT-I T cells, inhibition of caspase-3 improved neither survival or proliferation of T cells, indicating that the impaired survival and proliferation of LM-specific PKC-{theta}–/– T cells is not mediated by an increased caspase-3 activity and that death of PKC-{theta}–/– T cells is also caused by caspase-3-independent pathways. The latter finding is compatible with findings indicating caspase-3-dependent and -independent apoptosis of LM-specific T cells (35).

In conclusion, the present study demonstrates that the functional importance of PKC-{theta} signal transduction in pathogen-specific T cells in vivo and in vitro is critically dependent on the pathogen. The specific mechanisms by which PKC-{theta} differentially influences the T cell response in viral and bacterial infections as well as in autoimmune diseases still remain to be unraveled. Their identification might provide the means to selectively manipulate Ag-specific T cell responses in infectious and autoimmune diseases.


   Acknowledgments
 
We gratefully acknowledge the expert technical assistance of Annette Sohnekind and Nadja Schlüter. We thank D. Littman for providing PKC-{theta}–/– mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts 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 in part by grants from the Bundesministerium für Bildung und Forschung (NBL3/2, 01ZZ0407) and the Deutsche Forschungsgemeinschaft (GRK 1167, De 486/6-3, Schl 392/6-1, SFB 575 TP8). Back

2 Address correspondence and reprint requests to Dr. Dirk Schlüter, Institut für Medizinische Mikrobiologie, Otto-von-Guericke Universität Magdeburg, Leipzigerstrasse 44, 39120 Magdeburg, Germany. E-mail address: dirk.schlueter{at}medizin.uni-magdeburg.de Back

3 Abbreviations used in this paper: PKC-{theta}, protein kinase C-{theta}; BMDC, bone marrow-derived DC; DC, dendritic cell; LCMV, lymphocytic choriomeningitis virus; LM, Listeria monocytogenes; LMgp, recombinant LM expressing glycoprotein of LCMV; LMova, recombinant OVA-expressing LM; LLO, listeriolysin O; MLN, mesenteric lymph node; p.i., postinfection; WT, wild type; Z-VAD-FMK, Val-Ala-Asp-fluoromethylketone. Back

Received for publication September 20, 2007. Accepted for publication February 9, 2008.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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