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The Journal of Immunology, 2007, 178: 7814-7821.
Copyright © 2007 by The American Association of Immunologists, Inc.
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Protein Kinase C{delta} Regulates Antigen Receptor-Induced Lytic Granule Polarization in Mouse CD8+ CTL1

Jennifer S. Y. Ma*, Ngozi Monu{ddagger}, David T. Shen*, Ingrid Mecklenbräuker§, Nadezda Radoja, Tarik F. Haydar{dagger}, Michael Leitges2,||, Alan B. Frey{ddagger}, Stanislav Vukmanovic* and Sasa Radoja3,*

* Center for Cancer and Immunology and {dagger} Center for Neuroscience, Children’s National Medical Center, Children’s Research Institute, Washington, DC 20010; {ddagger} Department of Cell Biology and Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016; § Laboratory of Lymphocyte Signaling, The Rockefeller University, New York, NY 10021; Developmental Skin Biology Unit, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892; and || Max Planck Institute of Experimental Endocrinology, Hannover, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lytic granule exocytosis is the major pathway used by CD8+ CTL to kill virally infected and tumor cells. Despite the obvious importance of this pathway in adaptive T cell immunity, the molecular identity of enzymes involved in the regulation of this process is poorly characterized. One signal known to be critical for the regulation of granule exocytosis-mediated cytotoxicity in CD8+ T cells is Ag receptor-induced activation of protein kinase C (PKC). However, it is not known which step of the process is regulated by PKC. In addition, it has not been determined to date which of the PKC family members is required for the regulation of lytic granule exocytosis. By combination of pharmacological inhibitors and use of mice with targeted gene deletions, we show that PKC{delta} is required for granule exocytosis-mediated lytic function in mouse CD8+ T cells. Our studies demonstrate that PKC{delta} is required for lytic granule exocytosis, but is dispensable for activation, cytokine production, and expression of cytolytic molecules in response to TCR stimulation. Importantly, defective lytic function in PKC{delta}-deficient cytotoxic lymphocytes is reversed by ectopic expression of PKC{delta}. Finally, we show that PKC{delta} is not involved in target cell-induced reorientation of the microtubule-organizing center, but is required for the subsequent exocytosis step, i.e., lytic granule polarization. Thus, our studies identify PKC{delta} as a novel and selective regulator of Ag receptor-induced lytic granule polarization in mouse CD8+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD8+ CTL play a central role in adaptive immunity to tumors and intracellular pathogens. They mediate the immune response by secreting cytokines, which can be cytotoxic and/or activate other immune cells, and by directly killing target cells using Fas-mediated or granule exocytosis-mediated cytotoxic mechanisms (1, 2). Granule exocytosis is the dominant pathway used by CTL to kill tumor or virally infected cells. Degranulation releases the pore-forming protein, perforin, and several serine proteases (or granzymes) that are stored in lytic granules (3). In mouse cytolytic cells, granzymes A, B, C, D, E, F, G, K, and M have been found, but granzymes A and B are the most abundant and are currently best characterized. Effector CTL granules can be characterized as secretory lysosomes because they, in addition to the cytolytic proteins, contain lysosomal proteins, such as cathepsins B and D, beta-hexosaminidase, and lysosome-associated membrane proteins (Lamp)4 1 (CD107a), Lamp-2 (CD107b), and Lamp-3 (CD63) (4).

Subcellular events that lead to granule exocytosis in CTL are clearly defined; TCR-mediated recognition of cognate Ag on target cells induces reorientation of the microtubule-organizing center (MTOC) to the plasma membrane at the point of contact with the target cell. Subsequently, lytic granules polarize toward the contact area by moving along microtubules, granules fuse with the CTL plasma membrane, and the granule contents are exposed directly to the target cell membrane (3). However, the signaling pathways and downstream effector molecules involved in the regulation of these events are not fully characterized.

TCR-induced activation of protein kinase C (PKC) and increased intracellular calcium are two signals that are known to be critical in lytic granule release (5, 6, 7, 8, 9, 10, 11, 12). In addition to these two events, there have been only a few signaling molecules whose role in the regulation of CTL granule exocytosis is well documented. Those include PI3K and MAPK/ERK (13, 14, 15, 16, 17, 18, 19). The extent to which each of these kinases contribute to the regulation of CD8+ T cell degranulation has not been established completely, and the order in which they make their contributions to the signaling cascade leading to CTL degranulation is not known.

Despite the major role of PKC in the regulation of CTL granule exocytosis, it is not known which step of this process is regulated by PKC. In addition, it is unknown exactly which PKC isoform(s) is involved in the regulation of this cytotoxic mechanism. The PKC family has been classified into three categories: conventional ({alpha}, betaI, betaII, and {gamma}), which are regulated by calcium, diacylglycerol, and phospholipids; novel ({delta}, {epsilon}, {eta}, and {theta}), which are regulated by diacylglycerol and phospholipids, and atypical ({zeta} and {lambda}), which are insensitive to both calcium and diacylglycerol (20). Therefore, we set out to determine which of the PKC isoforms is involved in the regulation of lytic granule exocytosis in CD8+ CTL, and then to determine which step of granule exocytosis is regulated by the identified isoform.

By combination of pharmacological inhibitors and use of mice with targeted gene deletions, we identified PKC{delta} as a positive regulator of granule exocytosis in CD8+ CTL. Our studies show that upon Ag receptor engagement, PKC{delta} selectively regulates the polarized movement of lytic granules toward the CTL/target cell synapse.


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

C57BL/6 and BALB/c mice were purchased from Taconic Farms. PKC{delta}-deficient mice on a C57BL/6 background were used for the studies. PKC{theta}-deficient mice (C57BL/6 background) used for the studies were obtained from Dr. D. Littman (New York University Medical Center, New York, NY). 2C11 hybridoma, P815, and L1210 cells (all from American Type Culture Collection) were cultured in RPMI 1640 containing 10% FBS. Experiments using mice were performed with the permission of the Children’s National Medical Center Institutional Animal Care and Use Committee, protocol number 133-03-22 (S. Radoja, principal investigator). All experiments on mice were performed in accordance with the institutional and national guidelines and regulations.

Abs and reagents

Splenocytes were stimulated in vitro with culture supernatants containing anti-CD3 Ab produced by 2C11 hybridoma. CD8+ spleen T cells were purified using the magnetic bead-coupled Ab MACS system (Miltenyi Biotec). Purified spleen T cells were stimulated with plate-bound anti-CD3 Ab, clone 2C11 (BD Pharmingen). Hamster IgG1{kappa} (BD Pharmingen) was used as an isotype control. The same Abs were used in a redirected chromium release assay. FITC-conjugated anti-CD107a Ab or the isotype-matched control, FITC-conjugated rat IgG2a, anti-mouse CD8-allophycocyanin, CD25-PE, CD44-PE, and CD69-PE (all from BD Pharmingen) were used for the cell surface staining, followed by flow cytometry. The following Abs were used for intracellular staining: PE-conjugated anti-human granzyme B and mouse IgG1-PE isotype control Ab (both from Caltag Laboratories), PE-anti-mouse IFN-{gamma} and mouse IgG1-PE isotype control Ab (both from BD Pharmingen), mouse monoclonal anti-beta-tubulin Ab (Boehringer Mannheim), and the secondary PE-conjugated anti-mouse IgG Ab (BD Pharmingen). The following Abs were used for immunoblotting: rabbit anti-mouse perforin Ab (eBioscence), HRP-conjugated anti-rabbit secondary Ab (Jackson ImmunoResearch Laboratories), mouse monoclonal anti-beta-tubulin Ab (Boehringer Mannheim), and HRP-conjugated anti-mouse IgG secondary Ab (Jackson ImmunoResearch Laboratories). The Lysotracker Red was used for the labeling of lysosomes in T cells, whereas Cell Tracker Blue (both from Molecular Probes) was used for labeling P815 cells. EGTA was obtained from Sigma-Aldrich. Chromium 51 radionuclide used in the redirected cytotoxic assay was from PerkinElmer.

In vitro T cell stimulation and MLR

To generate T cell blasts, 4 x 106 total splenocytes in 4 ml of complete RPMI 1640 per well of a 6-well plate were cultured in the presence of 1% (v/v) of supernatants containing anti-CD3 Ab produced by the 2C11 hybridoma. After indicated periods of time, splenocytes were either stained for CD8 cell surface expression in combination with the indicated cell surface markers and analyzed by flow cytometry, or CD8+ T cells were purified by magnetic immunobeading and used as described in Results. Purity of T cells was ≥95%, as determined by flow cytometry. To generate MLR, 5 x 106 total responder splenocytes (H-2b) were mixed with an equal number of irradiated stimulator splenocytes (H-2d) in 2 ml of complete RPMI 1640 per well of a 24-well plate and were cultured for 5 days. CD8+ T cells were then isolated from the responder population by magnetic immunobeading and used in chromium release assay.

Degranulation assays

Purified CD8+ T cells were incubated for the indicated periods of time with either anti-CD3 or hamster IgG Ab immobilized on plastic (10 µg/ml Ab in 100 µl of PBS/well of a 96-well plate was incubated for 60 min or longer at 37°C) and assayed for degranulation by beta-hexosaminidase release, granzyme A release, or Lamp-1 flow cytometric analysis. Cells (2 x 105/well) were plated in 100 µl of RPMI 1640 containing 10% FCS in 96-well plates and were incubated for 4 h at 37°C. For beta-hexosaminidase assay, 50 µl of the supernatant was mixed with 150 µl of 1 mM p-nitrophenyl N-acetyl-beta-D-glucosaminide (Sigma-Aldrich) in citrate phosphate buffer and incubated at 37°C. The reaction was stopped after 1 h by the addition of 100 µl of 1 M Na2CO3, and the absorbance at 405 nm was recorded using a spectrophotometer (Molecular Devices). For the granzyme A assay, 20 µl of the supernatant was mixed with 180 µl of substrate (PBS, 0.2 mM N-benzyloxy-carbonyl-L-lysine-thiobenzylester, 0.2 mM 5,5'-dithio-bis(2-nitrobenzoic acid)) for 30 min, and the absorbance was read at 415 nm. For both beta-hexosaminidase and granzyme A, maximal release from cells was determined by treatment of cells with 1% Triton X-100, whereas spontaneous release was determined from the supernatants of cells incubated with medium only. The supernatant activity for both enzymes was expressed as a percentage of the total enzyme in Triton X-100 cell lysates. For Lamp-1 cell surface translocation, 2 x 105 cells in 200 µl of complete RPMI 1640 were stimulated for 4 h at 37°C with the plate-bound anti-CD3 or hamster IgG isotype control Ab in the presence of 10 µM monensin and 5 µg/ml FITC-conjugated anti-Lamp-1 or FITC-conjugated rat IgG2a isotype control Ab, followed by flow cytometry analysis of Lamp-1 expression in the FACSCalibur flow cytometer (BD Biosciences).

Chromium release assay

The chromium release assay was performed as previously described (16). In redirected assays, anti-CD3 or hamster IgG isotype control Abs were added to the cells at a final concentration of 1 µg/ml and were present during the assay. In some assays, effectors (CTL) were preincubated for 30 min at 37°C with 1 mM sodium salt of EGTA (pH 7.5–8.0) and addition was maintained during the 4-h lysis assay.

Conjugate formation assay

CD8+ T cells were purified from the 5-day MLRs by magnetic immunobeading and then labeled with Lyostracker Red. Two x 105 of the labeled CD8+ T cells were mixed with 1 x 105 of Cell-Tracker Blue-labeled P815 target cells and spun at 16,000 x g for 20 s to promote conjugate formation. Cells were resuspended in 200 µl of complete RPMI 1640, incubated for 15 min at 37°C, and transferred to coverslips coated with poly-L-lysine. After 5 min of incubation at room temperature, the cells were fixed in 4% paraformaldehyde, and conjugates were enumerated by confocal microscopy by counting the number of CD8+ T cell/P815 cell conjugates per microscopic field and the total number of CD8+ T cells in the same field. In each experiment, 100 or more CD8+ T cells were scored for conjugate formation. Percent conjugates refer to the ratio of the number of CD8+ T cells that form conjugates with P815 cells to the total number of scored CD8+ T cells, multiplied by 100%. Lyostracker Red and Cell-Tracker Blue loading was performed as described in Intracellular staining and labeling and confocal microscopy and resulted in >99% labeling of CD8+ T cells or P815 cells, respectively, as determined by flow cytometry.

Generation of PKC{delta} expression vector

To create the PKC{delta}-internal ribosome entry site (IRES)-GFP expression vector, the PKC{delta} gene was amplified by PCR from cDNA generated from naive CD8+ splenocytes using the primers that introduced the HindIII restriction site at the 5' end and BamHI site after the stop codon at the 3' end of the gene. The PCR product was cloned into HindIII-BamHI sites of the bicistronic pIRES2-AcGFP-1 plasmid vector (BD Clontech). The identity and the absence of mutations in the cloned PKC{delta} genes was confirmed by sequencing.

Transfection

Total resting splenocytes were stimulated in the presence of anti-CD3 Ab for 36 h and then transfected with the plasmid DNA using a nucleofection kit for primary mouse T cells according to the manufacturer’s protocol (Amaxa Biosystems). After nucleofection, the cells were cultured in RPMI 1640 medium containing 10% FCS in the absence of anti-CD3 Ab and in the presence of IL-2 for an additional 16–24 h, and then were either analyzed by flow cytometry or CD8+ T cells were purified by magnetic immunobeading and used in chromium release assays.

Immunoblotting

CTL lysates with 1 x 107 CTL/ml were prepared in Nonidet P-40 buffer (20 mM Tris (pH 7.6), 157 mM NaCl, 10% glycerol, 1% Nonidet P-40, and 2 mM EDTA) containing complete protease inhibitors (Roche). The lysates were separated by 10% SDS-PAGE in reducing conditions and were transferred to polyvinylidene fluoride membrane (Amersham Biosciences). Membranes were incubated with blocking buffer (1x TBS, 0.1% Tween 20, 5% w/v ratio nonfat dry milk) for 60 min at room temperature. Primary and secondary Abs were individually diluted in blocking buffer and were incubated with the membrane at 4°C overnight and at room temperature for 60 min, respectively. Finally, membranes were rinsed with washing buffer for five 3-min washes and exposed to SuperSignal West Pico Chemiluminescent Substrate (Pierce) for 2 min.

Intracellular staining and labeling and confocal microscopy

Intracellular staining for granzyme B and IFN-{gamma} was performed using a BD Cytofix/Cytoperm kit (BD Pharmingen) according to the manufacturer’s protocol, followed by flow cytometry analyses. Lysotracker Red and Cell-Tracker Blue loading was performed by incubating CD8+ T cells or P815 cells, respectively, at 37°C for 60 min with the 60 nM dye. For the assessment of MTOC- or lytic granule polarization, 2 x 105 of CD8+ T cells were mixed with an equal number of P815 target cells in 200 µl of complete RPMI 1640 containing 1 µg/ml anti-CD3 Ab. The cells were spun at 5000 rpm for 30 s, incubated for 5- 15 min at 37°C, and transferred to coverslips coated with poly-L-lysine. After 5 min of incubation at room temperature, the cells were fixed in 4% paraformaldehyde, permeabilized with 0.01% saponin and 0.5% BSA, and stained intracellularly for beta-tubulin (indirect staining using mouse monoclonal anti-beta-tubulin Ab followed by PE-conjugated anti-mouse IgG Ab staining) or granzyme B (direct staining using PE conjugate anti-human granzyme B Ab) and analyzed by confocal microscopy (Zeiss LSM-510 META; Zeiss).

Live imaging

Two x 105 of Lysotracker Red-labeled CD8+ CTL were mixed with an equal number of Cell-Tracker Blue-labeled target P815 cells in 200 µl of complete RPMI 1640. Anti-CD3 Ab was added to the cells at 1 µg/ml and the cells were placed in a temperature-controlled chamber (heated open superfusion chamber (RC-25F; Warner Instruments)). Preheated serum-free medium was pumped over the slices for the length of the imaging experiment and the chamber and perfusate temperature was maintained at 37°C. Sequential confocal images were acquired every 4 s for 10–15 min with a Zeiss LSM 510 META NLO system equipped with an Axiovert 200M microscope (Zeiss), with 488-nm epifluorescence and Nomarski differential interference contrast for the transmitted light. The images were processed with Zeiss LSM version 3.2 software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PKC{delta} is required for granule exocytosis-mediated cytotoxicity in CD8+ T cells

We tested the effects of Go6976, a pharmacological inhibitor of PKC{alpha} and beta isoforms, and rottlerin, a pharmacological inhibitor of PKC{delta} and PKC{theta} isoforms, on cytolytic function of mouse alloreactive CD8+ CTL. As shown in Fig. 1A, rottlerin inhibited alloantigen-specific cytolytic activity by 50% at a concentration between 3 and 10 µM, which is close to the IC50 of this inhibitor for PKC{delta} and PKC{theta} isoforms (IC50 = 3–6 µM). In contrast, Go6976 did not inhibit lytic activity even at 30 µM, which is well above the IC50 of this inhibitor for PKC{alpha} (IC50 = 2.3 nM) or PKCbeta (IC50 = 6.2 nM). These experiments implied that PKC{delta} and/or PKC{theta} isoforms are involved in the regulation of CD8+ T cell cytotoxicity.


Figure 1
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  		FIGURE 1. PKC{delta} is required for granule exocytosis-mediated cytotoxicity of mouse CD8+ CTL. A, C57BL/6 spleen cells (H-2b) were stimulated in vitro with irradiated BALB/c splenocytes (H-2d) for 5 days. CD8+ T cells (effectors) were isolated from the responder population by magnetic immunobeading mixed with 51Cr-labeled target P815 cells (H-2d) at different E:T ratios and assessed for the ability to lyse the targets after 4 h of coculture in the presence or absence of increasing concentrations of indicated inhibitors. Each E:T ratio was assayed in quadruplicate samples. For simplicity of the data interpretation, only the E:T of 10:1 is shown. This is a representative of four independent experiment yielding similar results. Error bars, SD. B and C, Cytolytic activity of in vitro-generated alloantigen-specific WT (H-2b) and (B) PKC{delta}-deficient (H-2b) or (C) PKC{theta}-deficient CTL (H-2b) against P815 cells, in the presence or absence of 1 mM EGTA, was tested in a 4-h chromium release assay at the indicated E:T ratios. Each E:T ratio was done in quadruplicate samples. B, In addition to P815 cells, Fas-resistant L1210 cells (H-2d) were used as targets. Data shown in B and C are representatives of five independent experiments yielding similar results. Error bars, SD. D, Quantification of conjugate formation of in vitro-generated alloantigen-specific WT (H-2b) and PKC{delta}-deficient (H-2b) CTL with P815 cells, as described in Materials and Methods. The mean values of three independent experiments are shown. KO, Knockout.

 
To validate the pharmacological inhibitor data and to determine whether both or only one of the two PKC isoforms is required for CTL cytotoxicity, we assessed alloantigen-specific cytolytic function of CTL from either PKC{delta}- or PKC{theta}-deficient mice. The chromium release assay results consistently showed that PKC{delta}-deficient CTL have significantly reduced cytolytic activity compared with wild-type (WT) CTL (Fig. 1B), while the cytotoxicity of PKC{theta}-deficient CTL was comparable to the activity of the control, WT CTL (Fig. 1C). Lytic activity of both WT and PKC{delta}-deficient CTL was almost completely inhibited by EGTA (Fig. 1B), a selective inhibitor of granule exocytosis-mediated cytotoxicity (21). Similar results were obtained if concanamycin A, another selective inhibitor of perforin-mediated cytotoxicity, was used in the cytotoxic assays (data not shown). When Fas-resistant L1210 cells were used as targets in the chromium release assay, cytotoxic activity of both PKC{delta}-deficient and WT CTL was further diminished compared with the lytic activity against the Fas-sensitive P815 cells, indicating that Fas ligand-mediated cytotoxicity is not defective in PKC{delta}-deficient CTL (Fig. 1B). Collectively, these experiments demonstrated that PKC{delta} but not PKC{theta} is required for lytic granule exocytosis-mediated cytotoxicity in mouse CD8+ CTL.

TCR-induced degranulation is defective in PKC{delta}-deficient CTL

PKC{delta}-deficient CTL showed a normal ability to form conjugates with target cells in vitro (Fig. 1D), which implied that the observed dysfunction in cytolytic activity is due to the inability of PKC{delta}-deficient CTL to release lytic granules. To test this, we used two TCR-induced degranulation assays: beta-hexosaminidase release and increase in Lamp-1 cell surface translocation (22, 23). For this purpose, we used in vitro-activated spleen T cell blasts as CTL. In this system, in vitro culture of total spleen cells in the presence of anti-CD3 Ab results in >99% activation of CD8+ T cells from both WT and PKC{delta}-deficient mice, as evidenced by their forward and side scatter and their cell surface phenotype (Fig. 2A). We have previously shown that virtually all CD8+ T cells activated in this manner rapidly acquire granule exocytosis-mediated cytotoxicity (24), which facilitates the quantification of degranulation events in the given population of CTL. We used this system to compare the degranulating ability of PKC{delta}-deficient and WT CTL. Activated CD8+ T cells were stimulated with plate-bound anti-CD3 Ab and assessed for both beta-hexosaminidase release and increase in Lamp-1 cell surface translocation. As expected, these experiments demonstrated that PKC{delta}-deficient CTL are not able to degranulate in response to TCR ligation (Fig. 2, B and C). Also, TCR-induced exocytosis of lytic granules, as determined by granzyme A release, is defective in PKC{delta}-deficient CTL (Fig. 2D). This defect is specific, because the extent of production of IFN-{gamma}, a major CD8+ T cell effector cytokine, in response to secondary TCR stimulation, was similar in PKC{delta}-deficient and WT CD8+ T cells (Fig. 2E).


Figure 2
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  		FIGURE 2. TCR-induced degranulation is defective in PKC{delta}-deficient CD8+ T cells. Total splenocytes from WT or PKC{delta}-deficient mice were cultured in the presence of anti-CD3 Ab. A, After 24 h, cell surface activation marker expression on CD8+ T cells was assessed by flow cytometry. B, Purified 2-day in vitro-activated CD8+ T cells were stimulated for 4 h with plate-bound anti-CD3 (tracing) or isotype-matched control Ab (filled histogram) in the presence of 10 µM monensin and 5 µg/ml FITC-conjugated anti-Lamp-1 Ab, followed by flow cytometry analysis of Lamp-1 expression. Staining of the cells with the Lamp-1 isotype-matched control Ab resulted in the fluorescence intensity equivalent to the one observed in the unstained cells. This experiment was repeated four times, giving similar results. C and D, After 2 days of activation, CD8+ T cells were purified by magnetic immunobeading, stimulated for 4 h with the plate-bound anti-CD3- or isotype-matched control Ab and were assayed for beta-hexosaminidase (C) or granzyme A (D) release in quadruplicate samples. Error bars, SD. C and D, A representative of three independent experiments is shown. In all degranulation experiments, no significant death of CD8+ T cells upon degranulation was observed, as determined by trypan blue exclusion and propidium iodide staining followed by flow cytometry. E, After 2 days of activation, CD8+ T cells were purified by magnetic immunobeading and stimulated for 4 h with plate-bound anti-CD3- or isotype-matched control Ab in the presence of brefeldin A, followed by intracellular staining for IFN-{gamma} and flow cytometry analyses. Staining of the cells with the isotype-matched control Ab for IFN-{gamma} resulted in the fluorescence intensity equivalent to the one observed in the unstained cells. The numbers in the flow cytometry dot plots refer to the percentage of cells in a given quadrant. A representative of three independent experiments giving similar results is shown.

 
TCR-induced expression of lytic molecules is not inhibited in PKC{delta}-deficient CTL

The ability of the PKC{delta} inhibitor rottlerin to block lytic granule exocytosis demonstrates that PKC{delta} regulates the execution of this process in CD8+ CTL. However, this did not exclude the possibility that PKC{delta} may also be involved in the regulation of CTL maturation. To address this question, we assessed the levels of expression of cytolytic molecules in in vitro-generated PKC{delta}-deficient CD8+ CTL. Expression levels of both intracellular granzyme B (Fig. 3A) and perforin (Fig. 3B) were comparable between the PKC{delta} deficient and WT CTL, as determined by flow cytometry and immunoblotting, respectively, indicating that PKC{delta} is not required for the regulation of expression of cytolytic molecules during CTL maturation. Collectively, these results showed that, in contrast to lytic granule exocytosis, other TCR-mediated responses (activation marker cell surface expression, cytokine production, and expression of lytic molecules) are not inhibited in PKC{delta}-deficient CD8+ T cells.


Figure 3
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  		FIGURE 3. PKC{delta}-deficient CD8+ T cells express cytolytic molecules in response to TCR stimulation. A, Purified resting or 2-day in vitro-activated WT or PKC{delta}-deficient CD8+ T cells were stained intracellularly for granzyme B followed by flow cytometry. Staining of the cells with the granzyme B isotype-matched control Ab resulted in a fluorescence intensity equivalent to the one observed in the unstained cells. MFI, Mean fluorescent intensity. A representative of three independent experiments giving similar results is shown. B, Purified 2-day in vitro-activated WT or PKC{delta}-deficient CD8+ T cells were lysed and subjected to SDS-PAGE, followed by immunoblotting using Abs specific for mouse perforin or beta-actin (loading control). A representative of three independent experiments giving similar results is shown.

 
Ectopic expression of PKC{delta} reverses the lytic defect in PKC{delta}-deficient CD8+ T cells

It has been suggested that the retention of selectable marker cassettes in targeted loci can cause unexpected phenotypes in knockout mice due to the disruption of expression of neighboring genes within a locus (25). It was therefore plausible that the expression of a gene (or genes) required for lytic granule exocytosis is adversely affected in the PKC{delta}-deficient mice. To address these possibilities, we reconstituted PKC{delta} gene expression in PKC{delta}-deficient CTL. For this purpose, we cloned the full-length PKC{delta} gene into a bicistronic plasmid vector containing GFP under the control of IRES, positioned downstream of the CMV-driven PKC{delta} gene. To introduce the PKC{delta} gene into mature CTL, we modified nucleofection, an electroporation-based transfection method, such that we consistently achieved a high frequency of ectopic gene expression in polyclonally activated T cells. The PKC{delta} expression vector or the control vector was transfected into activated PKC{delta}-deficient or WT splenic T cells, and 16 h posttransfection, CD8+ T cells were purified and assessed for lytic function. Viability of transfected cells was ~70% that of control, non-nucleofected splenocytes, as determined by scatter analysis (Fig. 4A), trypan blue exclusion, and propidium iodide staining (data not shown). In this system, virtually the entire population of viable nucleofected T cells expressed exogenous gene, as determined by FACS analysis of GFP+ cells (Fig. 4B). As shown in Fig. 4C, the ectopic expression of PKC{delta} reversed defective cytolysis in PKC{delta}-deficient CD8+ CTL. Thus, these results confirmed that PKC{delta} is required for the execution of granule exocytosis-mediated cytotoxicity in CD8+ T cells.


Figure 4
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  		FIGURE 4. Ectopic expression of PKC{delta} restores cytolytic activity in PKC{delta}-deficient CTL. Total resting splenocytes from PKC{delta}-deficient mice were stimulated in the presence of anti-CD3 Ab for 36 h. A, The cells were either left untreated (untransfected), or nucleofected with the PKC{delta}-IRES-GFP expression vector. Sixteen to 24 h posttransfection, the cells were analyzed by flow cytometry. Numbers in the gates in forward vs side scatter plots indicate the percentage of total cells. B, The cells were nucleofected with control, non-GFP coding, pcDNA3.1 plasmid vector (thin lines) or with PKC{delta}-IRES-GFP expression vector (thick lines). Sixteen to 24 h posttransfection, the cells were analyzed by flow cytometry. The gated cells (A) were analyzed for GFP expression. Identical results were obtained if splenocytes from WT C57BL/6 mice were nucleofected. Representative of four independent experiments giving similar results is shown. C, WT or PKC{delta}-deficient splenocytes were stimulated with anti-CD3 for 36 h and then either left untreated (untransfected) or nucleofected with pIRES2-Ac-GFP1 vector (empty vector) or with PKC{delta}-IRES-GFP vector (PKC{delta}). After 16 h, CD8+ T cells were purified by magnetic immunobeading followed by a redirected 4-h chromium release assay against P815 cells. Each E:T ratio was done in triplicate samples. A representative of three independent experiments giving similar results is shown. Error bars, SD. KO, Knockout.

 
PKC{delta} regulates Ag receptor-induced lytic granule polarization in CD8+ T cells

To determine exactly which step of lytic granule exocytosis is regulated by PKC{delta}, we assessed MTOC reorientation and lytic granule polarization in PKC{delta}-deficient CTL after target cell recognition. WT- or PKC{delta} -deficient allospecific CTL were allowed to form conjugates with target cells, followed by intracellular staining for beta-tubulin or granzyme B, which enabled the visualization of MTOC and lytic granules, respectively. Target cell-induced polarization of MTOC or lytic granules was analyzed by confocal microscopy. We consistently observed that PKC{delta}-deficient CTL efficiently reorient MTOC in response to target cell recognition (Fig. 5). In a marked difference, PKC{delta}-deficient CTL were not able to polarize lytic granules toward the contact site with target cells (Fig. 5). The inability of PKC{delta}-deficient CTL to polarize lytic granules was confirmed by live imaging experiments in which the target cell recognition-induced polarization of lysosomal granules in the WT- or PKC{delta}-deficient CTL was monitored by two-photon live cell microscopy (supplemental video 1: lysosomal granule polarization in live WT CTL responding to target cell recognition and supplemental video 2: lysosomal granule polarization in live PKC{delta}-deficient CTL responding to target cell recognition). 5 Collectively, these results demonstrated that PKC {delta} is dispensable for Ag receptor induced MTOC reorientation, but is required for the subsequent lytic granule exocytosis step that is lytic granule polarization.


Figure 5
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  		FIGURE 5. PKC{delta} regulates lytic granule polarization in CTL responding to target cell recognition. A, CD8+ T cells purified by magnetic immunobeading from WT or PKC{delta}-deficient mouse splenocytes activated in vitro with anti-CD3 Ab for 2 days were allowed to form conjugates with P815 cells for 15 min at 37°C in the presence of anti-CD3 Ab. The cells were transferred to coverslips, stained intracellularly for beta- tubulin or granzyme B as described in Materials and Methods, and analyzed by confocal microscopy. Arrowheads, CTL/target cells synapse. A representative from three independent experiments is shown. B, Quantification of MTOC and lytic granule polarization frequencies in WT or PKC{delta}-deficient CTL responding to target cell recognition. Percent polarization refers to the ratio of the number of CTL that polarize MTOC/lytic granules to the total number of scored conjugates, multiplied by 100%. The mean values of four independent experiments are shown. In each experiment, 40 or more CTL target cell conjugates were scored for the polarization of MTOC or lytic granules toward the contact sites.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
There are several important findings resulting from this study. First, we identified PKC{delta} as a novel regulator of lytic granule exocytosis in mouse CD8+ CTL. Second, we showed that PKC{delta} is a specific and selective inducer of this effector function in T cells. (This is a first report of selective regulation of the cytotoxic effector function in CTL by a kinase.) Third, before our work, PKC{delta} was shown only to be a negative regulator of Ag receptor-mediated responses in immune cells. We now demonstrate that PKC{delta} is a positive regulator of an effector function in T lymphocytes.

Before our work, only two molecules (Rab27a and {alpha} subunit of Rab geranylgeranyl transferase (RGGTA)) had been reported to specifically regulate lytic granule exocytosis in mouse CD8+ CTL. Rab27a regulates the distal stages of TCR-induced lytic granule exocytosis and lytic granule movement from microtubules to the plasma membrane following granule polarization toward the target cell contact site (22, 26). Similar to what we found with PKC{delta}, it was demonstrated that Rab27a is not required for other TCR-mediated functions in mouse T cells, including cytokine production, lytic molecule expression, and Fas ligand expression. Phenotypes of Rab27a and PKC{delta}-deficient CTL, however, show that the two molecules regulate temporally and spatially distinct stages of lytic granule exocytosis. This is based on the fact that Rab27a regulates granule exocytosis at the stage of the detachment of lytic granules from microtubules, after lytic granule polarization had occurred. Our data clearly show that PKC{delta} regulates lytic granule polarization, a step that precedes the one regulated by Rab27a. Thus, this implies that PKC{delta} is placed upstream of Rab27a in the process of the regulation of lytic granule exocytosis. However, one cannot exclude the possibility that PKC{delta} regulates more than one stage of this process. PKC{delta} could potentially interact with Rab27a and/or modulate its function (e.g., recruitment of other molecule(s) involved in regulation of granule exocytosis).

The function of RGGTA is prenylation of a subset of Rab proteins, a posttranslational modification that allows the targeting of Rab proteins to intracellular membrane compartments, including lysosomal granules (27). Consequently, CTL from mice with diminished RGGTA activity (gunmetal mice) have defective granule exocytosis due to the inability of lytic granules to polarize toward the target cell contact site (26). Therefore, RGGTA and PKC{delta} regulate the same step of the process of lytic granule exocytosis. The roles of the two molecules in the regulation of this stage of granule exocytosis are, however, likely to be different. The role of RGGTA is probably indirect, as it was proposed that it prenylates a yet-unidentified Rab molecule(s) involved in regulation of lytic granule polarization (26).

Mutations in the Lyst gene, found in patients with Chediak-Higashi syndrome and its mouse model, the beige mouse, also result in defective granule exocytosis-mediated cytotoxicity (28, 29). LYST protein is not directly involved in granule exocytosis, but it regulates lysosomal biogenesis/membrane fusion. As a consequence, LYST-deficient cells have abnormally enlarged lysosomal granules. It has been shown that CTL from Chediak-Higashi syndrome patients have defective cytotoxicity, due to the inability to release lysosomal granules upon TCR engagement (30). Mouse CTL lacking LYST probably have the same phenotype, but detailed phenotypic analyses of CTL from the beige mice have not been reported to date. Based on the function of LYST protein, it is likely that LYST deficiency results specifically in defective granule exocytosis and that it does not affect the other TCR-induced responses. However, there is currently no published data to support this assumption.

Other molecules that are established to have a role in the regulation of granule exocytosis-mediated cytotoxicity in mouse CD8+ T cells are ERK1/2 and PI3K (13, 14, 15, 16, 17, 18, 19). These kinases are components of general TCR-mediated signaling cascade and are, in addition to the regulation of lytic function, involved in control of the other aspects of T cell function, including activation, proliferation, differentiation, and cytokine production (31). Previous studies (32, 33) and our work presented in this study indicate that PKC{delta} does not have a major role in positive regulation of these functions. In the present work, we show that PKC{delta} is required for Ag receptor-induced granule exocytosis-mediated cytotoxicity in CD8+ T cells. Thus, PKC{delta} is the first kinase demonstrated to specifically regulate lytic function in CTL. This further suggests that PKC{delta} might be a component of the TCR-induced signaling pathway(s) that selectively regulates CTL cytotoxicity. Indications that the cytotoxicity-specific TCR pathway exists initially came from the studies describing split anergy in CD8+ T cells, characterized by the retention of lytic function, despite an inability to proliferate and produce cytokines in response to TCR stimulation (34, 35). However, molecular constituents of such a pathway remained uncharacterized to date. Identification of upstream and/or downstream PKC{delta} effectors in the future studies might allow the characterization of TCR signaling pathway(s) that selectively regulates granule exocytosis-mediated cytotoxicity in CD8+ T cells.

Before our study, PKC{delta} was considered to be a negative regulator of Ag receptor-mediated responses in immune cells (20). In B cells, PKC{delta} regulates negative feedback important for immune homeostasis and prevention of autoimmune disease (33, 36). In mast cells, it inhibits Ag-receptor induced degranulation (37). Previous work suggested that PKC{delta} plays a role in the negative regulation of IL-2 cytokine production and proliferation in T cells (32). The work presented in this study demonstrates for the first time a role of PKC{delta} in the positive regulation of an Ag receptor-mediated function, that is lytic granule exocytosis, in immune cells. In contrast, positive regulation by PKC{delta} of stimulus-induced granule secretion has been described in nonimmune cell types. PKC{delta} was proposed to have a role in the induction of dense granule and insulin secretion in platelets and pancreatic beta cells, respectively (38, 39). Interestingly, depending on the type of stimulus, PKC serves either as a positive or as a negative regulator of dense granule secretion in platelets. Similarly, in T cells, PKC{delta} attenuates proliferation and IL-2 secretion, but induces lytic granule exocytosis upon TCR engagement. Thus, PKC{delta} can act either as a positive or as a negative regulator of cellular responses, depending on the cell type and/or its selective interaction with distinct effectors within the same cell.

Another interesting aspect of our study is that PKC{delta} and PKC{theta}, two closely related members of the same subfamily of calcium-independent PKC, have specific and nonredundant functions in T cells. In direct contrast to PKC{delta}, PKC{theta} has been shown to have an important role in T cell activation, including the proliferation and IL-2 production in T cells (40). Recently, contradictory findings have been reported concerning the involvement of PKC{theta} in in vitro cytotoxic function of human and mouse CD8+ T cell clones/lines (10, 11, 12). Our results now unequivocally show that (in contrast to PKC{delta}-deficient CD8+ T cells) primary CD8+ CTL from PKC{theta}-deficient mice do not have defective granule exocytosis-mediated cytotoxicity in vitro.

Identification of PKC{delta} as a selective inducer of lytic granule exocytosis significantly advances our knowledge regarding the regulation of this T cell effector function. Determination of the exact mechanism of action of PKC{delta} and identification of PKC{delta} effectors in the future studies will further help the delineation of the TCR-induced regulatory pathway of lytic granule exocytosis. The knowledge gained by these studies might potentially allow selective manipulation of cytolytic activity by specifically modifying the function of PKC{delta} and/or its molecular partners. This, in turn, could enable the selective tuning of granule exocytosis-mediated cytotoxicity of CD8+ T cells in disease.

There has been increasing evidence in the literature that the dysregulation of granule exocytosis-mediated cytotoxicity in CD8+ CTL has a crucial role in initiation of familial hemophagocytic lymphohistiocytosis (FHL), a rare, fatal pediatric immune disorder (41). To date, mutations in the perforin (42), Munc 13-4 (43), and syntaxin 11 (44) genes have been shown to be associated with FHL2, FHL3, and FHL4 subtypes, respectively. Genetic defects responsible for the pathogenesis of the remaining FHL (some estimated 45–50% of the cases, excluding FLH4, for which the corresponding data are not yet available) have not be determined to date (45).

Thus, discovery of novel genes that regulate lytic granule release might potentially lead to the identification of the gene(s) responsible for pathogenesis of the remaining FLH subtype(s). The use of mouse models for such studies is of particular importance because the majority of the genes identified so far to be required for lytic granule exocytosis in mouse CTL have been demonstrated to have equivalent functions in human CTL. Furthermore, mutations in some of these genes (i.e., perforin, Rab27a, and Lyst) result in hemophagocytic lymphohistiocytosis or hemophagocytic lymphohistiocytosis-like syndromes in humans (4, 41). Therefore, it is tempting to speculate that PKC{delta} might be involved in regulation of FHL. Future studies concerning the requirement of PKC{delta} for lytic function in human CD8+ CTL and/or assessment of the functional status of PKC{delta} in CTL from FHL patients will address this possibility.


   Acknowledgments
 
We thank Dr. Dan Littman, New York University Medical Center, for providing PKC{theta}-deficient mice. We also thank Daniela Pappini for the assistance with flow cytometry analyses.


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


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

1 This work was supported by National Institutes of Health Grant R01 CA108573 (to A.B.F.), National Institutes of Health Grant F32CA101449-02 Award and Research Advisory Council Grant (to S.R.), and National Institutes of Health Grants R01AI48837 and R01AI41573 (to S.V.). Back

2 Requests for PKC{delta}-deficient mice should be sent to michael.leitges@biotek.uio.no Back

3 Address correspondence and reprint requests to Dr. Sasa Radoja, Center for Cancer and Immunology, Children’s Research Institute, Children’s National Medical Center, 111 Michigan Avenue, NW Washington, DC 20010. E-mail address: sradoja{at}cnmc.org Back

4 Abbreviations used in this paper: MTOC, microtubule-organizing center; PKC, protein kinase C; IRES, internal ribosome entry site; WT, wild type; RGGTA, Rab geranylgeranyl transferase; FHL, familial hemophagocytic lymphohistiocytosis. Back

5 The online version of this article contains supplemental material. Back

Received for publication January 30, 2007. Accepted for publication April 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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