Differential implication of protein kinase C isoforms in cytotoxic T lymphocyte degranulation and TCR-induced Fas ligand expression
Julián Pardo1,
Michel Buferne2,
María-José Martínez-Lorenzo3,
Javier Naval1,
Anne-Marie Schmitt-Verhulst2,
Claude Boyer2 and
Alberto Anel1
1 Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain 2 Centre dImmunologie de MarseilleLuminy INSERMCNRSUniversité de la Méditerranée, 13288 Marseille, France 3 Servicio de Inmunología, Hospital Clínico Universitario Lozano Blesa, Universidad de Zaragoza, 50009 Zaragoza, Spain
Correspondence to: A. Anel; E-mail: anel{at}posta.unizar.es
Transmitting editor: J. Borst
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Abstract
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CD8+ cytotoxic T lymphocyte (CTL) clones are able to exert both perforin- and Fas-dependent cytotoxicity. We show in the present work that phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 prevent TCR/CD3-induced functional Fas ligand (FasL) expression, but not perforin-dependent cytotoxicity. The specific inhibitor of classical protein kinase C (PKC) isoforms, Gö6976, completely inhibited perforin-dependent cytotoxicity and only affected slightly TCR/CD3-induced FasL expression, while the opposite was observed using rottlerin, an inhibitor with higher specificity for PKC
. To address further the dependence of FasL expression on PI3K, a luciferase reporter controlled by the FasL promoter was used. Reporter gene induction by anti-CD3 mAb was abolished in cells transfected with dominant-negative PI3K (PI3K-DN) and increased in cells transfected with constitutively active PI3K (PI3K*). Transfection with constitutively active mutants (A/E) of PKC
, and especially of PKC
, improved anti-CD3 mAb-induced reporter expression and completely abolished inhibition by wortmannin, while transfection with dominant-negative (K/R) PKC
prevented the induction of the reporter. Finally, transfection with PKC
A/E, but not with PKC
A/E, cooperated with ionomycin to induce degranulation in the CTL line 1.3E6SN. Altogether, the results suggest that TCR/CD3-induced FasL gene transcription is controlled by PI3K and PKC
activation, while this signaling pathway is not implicated in CTL degranulation, which is rather dependent on the activation of classical PKC isoforms.
Keywords: cellular activation, cytotoxicity, signal transduction, T lymphocyte
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Introduction
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T cell-mediated cytotoxicity in vitro has been shown to be mostly accounted for by two mechanismsperforin/granzyme- and Fas-dependent cytotoxicity (1). Perforin/granzyme-dependent cytotoxicity is essential in antiviral and antitumor immunity (24). Although also contributing to cell-mediated cytotoxicity, the main role of the FasFas ligand (FasL) system is the control of peripheral tolerance (5,6). Substantial advances have been made to determine the signaling pathways and transcription factors that control FasL expression in T cells. Initial studies using murine cytotoxic T lymphocyte (CTL) clones demonstrated that TCR/CD3-induced functional FasL expression was dependent on tyrosine kinase, calcineurin and phosphatidylinositol 3-kinase (PI3K) activation (7,8). In agreement with the described calcineurin implication, it was later demonstrated that FasL transcription was under the control of the NF-AT and Egr families of transcription factors (9,10), and was furthermore dependent on cooperation between NF-AT and FosJun (11). The transcription factor NF-
B has also been implicated in the control of FasL transcription (12,13).
It is as yet unknown, however, which signaling pathway downstream of PI3K activation leads from antigen receptor engagement to FasL transcription in T cells. One major pathway controlled by PI3K involves the activation of the serine/threonine kinase c-akt (also known as PKB) (14). This pathway has been implicated in cell survival through the maintenance of several anti-apoptotic mechanisms (15). The in vitro activation of novel (
and
) and atypical (
) protein kinase C (PKC) isoenzymes by lipid products of PI3K activity (16,17), and, more recently, the cellular activation of PKC
and PKC
through PDK1, the PIP3-dependent kinase (18), has been demonstrated. A role for PI3K in PKC
activation has also been described in platelet-derived growth factor-activated fibroblasts (19). PKC
, another member of the novel PKC family selectively expressed in the T cell lineage (20,21), has been implicated in phorbol myristate acetate (PMA)/ ionomycin-induced FasL transcription in T cells (22,23). More recently, it was shown that PKC
recruitment to the immunological synapse and its activation is dependent on PI3K, and not on the classical phospholipase C
pathway (24).
Concerning perforin-dependent cytotoxicity, granule exocytosis could be triggered by a combination of a Ca2+ ionophore and the pharmacological agent PMA, a PKC activator (25,26). Perforin-mediated lysis of target cells additionally requires a close adhesion contact between effector and target, and the polarization of the microtubule organizing center towards the site of contact with the target cell (25,27). PKC-mediated phosphorylation of microtubule-associated proteins or of kinesin or dynamin motors could be related to its role in CTL degranulation (28). More recently, the implication of Rab27a in the final degranulation events, such as fusion with the plasma membrane of polarized granules, has been characterized using mutant ashen mice (29,30). The implication of PMA-sensitive PKC isoforms in this exocytosis process was demonstrated by the fact that their depletion by prolonged PMA exposure rendered CTL clones unable to degranulate upon antigenic or PMA/ionomycin stimulation, although the treated cells conserved other functional and biochemical responses intact (3133). However, both classical (
, ß and
) and novel (
,
and
) PKC isoforms are sensitive to PMA activation and down-modulation, perhaps to different degrees, and these studies do not clarify the role of these families of PKC isoforms in CTL degranulation.
In the present work, we have investigated the possible implication of different signaling pathways downstream of PI3K in FasL expression, especially that of different types of PKC isoforms (classical, novel or atypical). In parallel, we have studied the implication of those different types of PKC isoforms in CTL degranulation, needed for the execution of perforin/granzyme-dependent cytotoxicity.
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Methods
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Cells, mAb and reagents
KB5.C20 and BM3.3, two murine CTL clones of B10.BR and CBA/J origin respectively, both specific for the H-2Kb alloantigen, were maintained in long-term culture as previously described (7). The target cells used included L1210.3, a lymphocytic leukemia of DBA/2 (H-2d) origin that expressed undetectable levels of the Fas antigen; L1210Fas, cells transfected with the Fas antigen cDNA and expressing high levels of the Fas antigen (34); and the H-2Kb-expressing T lymphoma EL4.F15, which is completely resistant to Fas-induced apoptosis (35). The human T cell line Jurkat and the murine CTL line 1.3E6SN were used for transfection experiments. 1.3E6SN is a CTL line of C57BL/6 origin, which lost its original anti-H-Y specificity and killed the H-2d-expressing P815 mastocytoma (36). The cell lines were cultured in RPMI 1640 supplemented with 5% FCS, 50 µM 2-mercaptoethanol, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 µg/ml streptomycin. The clones and the CTL line were cultured in the same medium supplemented with a 10% supernatant from PMA-stimulated EL4-C16 cells (final murine IL2 concentration 510 U/ml), and the CTL clones were re-stimulated weekly with irradiated alloantigenic spleen cells from C57BL/6 mice.
mAb used in this study were the anti-clonotypic mAb Désiré-1, specific for KB5.C20 TCR, the anti-murine CD3-
mAb 145.2C11 (a gift from J. Bluestone) and the anti-human CD3-
mAb UCHT1 (a gift from Dr Marisa Toribio, Centro de Biología Molecular, Madrid, Spain). Anti-PKC
,
,
and
rabbit polyclonal antibodies were a kind gift from Dr Peter J. Parker (Imperial Cancer Research Fund, London, UK).
PMA, ionomycin, wortmannin, cycloheximide and actinomycin D were purchased from Sigma (Madrid, Spain). LY294002, Ro31-8220, Gö6976, rapamycin and rottlerin were from Calbiochem (La Jolla, CA). The PKC
inhibitory peptide coupled to antennapedia was a kind gift from Dr D. Mochly-Rosen (Stanford University, CA).
Plasmids
The FasL reporter construct consisted of 486 bp of genomic DNA immediately upstream 5' of the translational start site of FasL cloned upstream of the firefly luciferase gene (9) (a kind gift from Dr Pierre Henkart, National Cancer Institute, Bethesda, MD). The FasLluciferase signal was normalized using a control reporter construct containing the thymidine kinase (TK) promoter upstream the Renilla luciferase gene (kindly provided by Dr Manuel Izquierdo, Universidad de Valladolid, Spain). The dominant-negative (PI3K-DN) and constitutively active p110
PI3K mutants (PI3K*) are chimeric cDNAs attached to the extracellular and transmembrane regions of rat CD2 (a gift from Dr Doreen Cantrell, Imperial Cancer Research Fund, London, UK). The membrane targeting of p110
generates a constitutively active PI3K, while in the case of PI3K-DN, a R/P point mutation was introduced in position 916 of the molecule, which renders p110
inactive. The PI3K chimeras were assembled by subcloning the mutated cDNAs into the pcDNA3 vector (Invitrogen) (37). The pMT-2 vector was used to express PKC
and PKC
mutants, and the pCO2 vector was used to express the mutant PKC
. The constitutively active PKC clones are full-length cDNAs with a single point mutation in their inhibitory pseudosubstrate sequences. Mutants were generated by substitution of an E position by an A (A/E), in position 25 for PKC
, position 159 for PKC
and position 119 for PKC
(38). These A/E PKC mutants were a kind gift from Dr Peter J. Parker. The cDNA encoding the constitutively active and dominant-negative mutants of PKC
were cloned in the eukaryotic expression vector pEFneo. The constitutively active PKC
mutant was obtained following a similar protocol as that described for the other PKC isoforms, by a substitution of an E for an A in its inhibitory pseudosubstrate sequences, position 148, while the dominant-negative mutant was obtained by the substitution of a R for a K in position 409, generating a kinase-inactive mutant (22). The NF-
Bluciferase reporter contains several copies of the consensus NF-
B binding site in the Ig
promoter cloned upstream of the firefly luciferase gene (39). PKC
A/E, PKC
K/R and NF-
Bluciferase constructs were a gifts from Drs Martín Villalba and Amnon Altman (La Jolla Institute for Allergy and Immunology, San Diego, CA). The truncated human CD2 (hCD2) construct used to select transfected 1.3E6SN cells lacks its cytoplasmic domain due to the introduction of two stop codons at amino acid 253 (40) and does not transduce activation signals. This construct was a gift from Dr Sylvie Guerder (Centre dImmunologie de Marseille-Luminy)
CTL stimulation and cytotoxicity tests
For testing functional FasL expression in CTL clones, effector cells (KB5.C20 or BM3.3) were pre-incubated with one of the following stimuli during 3 h: a combination of 10 ng/ml PMA and 600 nM ionomycin, the alloantigen-bearing target cell RMA or the anti-KB5.C20 TCR mAb Désiré-1 at 20 µg/ml. After this incubation, cells were used for cytotoxicity assays on L1210Fas cells in the presence of 1 mM EGTA and 1.5 mM MgCl2 to chelate extracellular Ca2+ and ensure Mg2+ excess. The inhibitors used were added 1015 min before the stimuli, were present during the 3-h pre-incubation time and were washed out before the cytotoxicity tests. In some experiments, the inhibitors were included in the cytotoxicity test on L1210Fas after the 3-h pre-stimulation period and, as previously reported for wortmannin (8), were without effect on apoptosis induction (data not shown).
Perforin-mediated cytotoxicity was tested by anti-CD3 mAb-redirected lysis of Fc receptor-expressing, Fas L1210.3 cells (in the presence of 3 µg/ml of 145-2C11 anti-CD3
mAb) or by alloantigenic lysis of H-2Kb-expressing EL4.F15 cells. These cells, although expressing low amounts of Fas at the plasma membrane, are resistant to Fas-induced apoptosis induced by anti-Fas mAb or by PMA/ionomycin-stimulated d12S cells (35). In this case, effectors were not pre-stimulated, inhibitors were added 1015 min before mixing effector and target cells, and were present throughout the assay. 51Cr-release cytotoxicity assays were then performed for 4 h as described elsewhere (7).
Transfections and luciferase assays
The induction of FasL gene transcription and NF
B activation in Jurkat cells were determined by transfection of constructions with firefly luciferase reporter genes. Jurkat cells (10 x 106) in log phase were collected, washed twice with PBS and electroporated (280 mV, 975 µF) in 500 µl of PBS + 10 mM MgCl2 containing 20 or 10 µg of FasLluciferase or NF-
Bluciferase construction respectively, and 1 µg of the control TKRenilla luciferase construct. In these conditions, transfection time was always 2025 ms and cell viability was
50%. If co-transfections with mutant PKC or PI3K constructions were performed, a 2:1 ratio PKC/PI3K:reporter construction was used to ensure that cells transfected with the reporter also contained the mutant construct. Total DNA amount in the transfections was normalized using a control empty vector. After electroporation, cells were incubated for 10 min on ice and resuspended in 5 ml of complete medium. After 24 h (NF-
B activation) or 48 h (FasL expression) of culture, cells were collected and activated (1 x 106 cells/ml) for 3 h with immobilized anti-CD3 mAb (UCHT1, 10 µg/ml) or ionomycin (600 nM) in the presence or absence of the corresponding inhibitors (100 nM wortmannin, 50 µM LY294002 or 20 µM rottlerin). After activation, cells were washed twice with PBS and lysed (3 x 107 cells/ml) in a buffer containing 1% of Triton X-100 for 30 min at 4°C. Lysates were centrifuged for 15 min at 14,000 r.p.m., 4°C, and supernatants were analyzed for firefly and Renilla luciferase activities in a MicroLumat Plus LB 96V from Berthold (Bad Wildbad, Germany), using a Dual-Luciferase Assay System from Promega (Barcelona, Spain). Transfection with the PKC mutants used was controlled in parallel by immunoblot with specific antibodies of vector- or PKC-transfected cells (data not shown).
Degranulation measurements
1.3E6SN cells (40 x 106) were transfected with 40 µg of the truncated hCD2 construct described above, and 80 µg of either empty vector or constitutively active mutants of PKC
or
(A/E). After 24 or 48 h in culture, transfected cells were purified by MACS sorting using anti-hCD2 antibodies and they were left untreated, or were stimulated for 3 h with either 600 nM ionomycin or 10 ng/ml PMA alone, or in combination. Aliquots were taken from the supernatants and secreted granzyme A activity was determined by a colorimetric method, based on the specific proteolysis of H-D-Pro-Phe-Arg-p-nitroanylide (H-D-PFR-pNA; Sigma). Results were expressed as the granzyme A activity released into the medium as a percentage of the total activity present in the same transfected cells before stimulation.
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Results
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Functional FasL expression in murine CTL clones KB5.C20 and BM3.3 is dependent on transcription and de novo protein synthesis
In previous studies, we demonstrated that functional FasL expression could be induced in the murine CTL clone KB5.C20 by either TCR or PMA/ionomycin stimulation, correlating with a substantial increase in FasL mRNA levels and suggesting that the expression of the death ligand was subjected to transcriptional control in these cells (7,8). We have analyzed the effect of the macromolecular synthesis inhibitors cycloheximide and actinomycin D on the induction of functional FasL expression in the CTL clone KB5.C20. As shown in Fig. 1, both inhibitors substantially prevented functional FasL expression induced by PMA/ionomycin or by the anti-TCR mAb Désiré. This indicates that functional FasL expression in this CTL clone can be used to analyze the signals implicated in the transcriptional control of FasL gene expression.

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Fig. 1. Macromolecular synthesis inhibitors prevent TCR/CD3- and PMA/ionomycin-induced FasL expression in murine CTL clones. CTL clones KB5.C20 (KB) and BM3.3 (BM) were tested against L1210.3 (black bars) or L1210Fas target cells at a fixed 5:1 E:T ratio. Four-hour cytotoxicity tests were performed in the presence of 1 mM EGTA and 1.5 mM MgCl2. Effectors were pretreated with 10 ng/ml PMA plus 600 nM ionomycin (PI), with 20 µg/ml of the anti-clonotypic mAb Désiré (Dés) or with alloantigen-bearing RMA cells (RMA) during 3 h. Pre-incubations were performed in the absence (white bars) or in the presence of 10 µg/ml cycloheximide (pointed bars, CHX) or 0.1 µg/ml actinomycin D (hatched bars, Act D). Results are the means of triplicate determinations, with SEM values always <5% of the mean. The experiment shown is representative of 10 different experiments.
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In the case of the CTL clone BM3.3, although it was described that PMA/ionomycin stimulation did not induce FasL expression, correlating with the defect in the expression of some PKC isoforms, the subclone used in the present work has reverted this phenotype, and expresses the same PKC isoforms as clone KB5.C20 and normal murine spleen cells [
, ßI,
,
,
and
; (8) and data not shown]. We have observed that antigen- and PMA/ionomycin-induced FasL expression in BM3.3 cells is also dependent on transcription and de novo protein synthesis (Fig. 1). The extent of FasL expression in this CTL clone is always lower than in clone KB5.C20, while the reverse is true for the execution of perforin-dependent cytotoxicity [see (8) and Fig. 1]. This is why in the present work KB5.C20 results are shown for Fas-dependent cytotoxicity and BM3.3 results for perforin-dependent cytotoxicity, although both clones are able to exert both types of cytotoxicity.
Differential sensitivity of Fas- or perforin-dependent cytotoxicity exerted by murine CTL clones to PI3K or PKC inhibitors
In a previous study, it was demonstrated that TCR/CD3-induced functional FasL expression in murine CTL clones was inhibited by low doses (30100 nM) of the PI3K inhibitor wortmannin, while induction by PMA/ionomycin was not affected (8). We have confirmed the effect of wortmannin on KB5.C20 cells stimulated with the anti-clonotypic mAb Désiré (Fig. 2a) and have obtained a similar inhibition by a different PI3K inhibitor, LY294002 (41) (Fig. 2b). No effect of these inhibitors was observed on PMA/ionomycin-induced FasL expression (data not shown). The effect of these inhibitors on perforin-dependent cytotoxicity in the absence of FasL contribution was studied in two different cellular settings: alloantigen-restricted lysis of Fas-resistant EL4.F15 cells and anti-CD3 mAb-redirected lysis of Fas L1210.3 cells. No effect of the higher wortmannin dose used (100 nM) was observed in both types of experiments (Fig. 2c and d). In agreement with this result, LY294002 at 25 µM, which completely inhibited functional FasL expression, also had no effect on BM3.3-mediated lysis of EL4.F15 or L1210.3 cells (Fig. 2c and 2d). On the other hand, LY294002 at 50 µM consistently inhibited perforin-dependent cytotoxicity, especially the redirected lysis of L1210.3 cells (Fig. 2d). The latter result could suggest some implication of PI3K in the execution of perforin-dependent cytotoxicity or could be attributed to non-specific effects of high doses of the inhibitor, which has been described to inhibit casein kinase at the same concentration (41). In any event, the present results clearly indicate a main role of PI3K activation in TCR/CD3-induced FasL expression, but not in CTL degranulation.

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Fig. 2. Effect of PI3K inhibitors on Fas- or perforin-dependent cytotoxicity. (a and b) CTL clone KB5.C20 was tested against L1210Fas target cells. Four-hour cytotoxicity tests were performed in the presence of 1 mM EGTA and 1.5 mM MgCl2. Effectors were pretreated with 20 µg/ml of the anti-clonotypic mAb Désiré during 3 h. Pre-incubations were performed in the absence (open circles) or in the presence of either 25 (open diamonds), 50 (open squares) or 100 nM (open triangles) wortmannin (a) or 15 (closed diamonds), 25 (closed squares) or 50 µM (closed triangles) LY294002 (b). (c and d) CTL clone BM3.3 was tested against EL4.F15 (c) or L1210.3 (d) target cells. In (d), assays were performed in the presence of 3 µg/ml of 145-2C11 anti-CD3 mAb. Four-hour cytotoxicity tests were performed in the absence (open circles) or in the presence of either 100 nM wortmannin (open triangles), or 25 (closed squares) or 50 µM (closed triangles) LY294002. Results are the means of triplicate determinations, with SEM values always <5% of the mean. The experiments shown are representative of at least six different experiments for each experimental condition.
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The next step in our research was to characterize the molecules downstream of PI3K and implicated in the signal transduction pathway that leads to FasL gene transcription. One major pathway controlled by PI3K involves the activation of the serine/threonine kinase c-akt (also known as PKB), leading, together with the mammalian target of rapamycin (mTOR), to the activation of p70S6 kinase, which regulates some translation and transcription processes (42). We did not, however, observe any inhibition of TCR- or PMA/ionomycin-induced functional FasL expression in CTL clones by rapamycin at the relevant inhibitory concentrations (data not shown), in agreement with a previous report (43). Alternatively, PI3K has been implicated, at least in B cells, in intracellular calcium increase through the activation of Btk (44). However, no effect of wortmannin or LY294002 was observed on the intracellular calcium increase induced by phytohemagglutinin on Jurkat cells or by cross-linked mAb Désiré on KB5.C20 cells, measured by Fura-2 fluorimetry (data not shown).
A third possibility would be that PI3K products were implicated in activation of specific PKC isoforms. It has been reported that PIP3 activates in vitro the atypical PKC
isoform (16), and also members of the family of novel PKC isoforms, such as PKC
and
(17). On the other hand, it has been shown that PKC
, belonging to the novel PKC isoform family, is translocated to the immunological synapse in a process dependent on PI3K activity (24) and is implicated in PMA/ionomycin-induced FasL expression (22,23). To analyze this possibility, we tested the effect of PKC inhibitors with different specificity on TCR/CD3- or PMA/ionomycin-induced functional FasL expression in CTL, comparing also the effect on perforin-dependent cytotoxicity. As shown in Fig. 3(a), the general PKC inhibitor Ro31-8220 (4 µM) (45), and rottlerin (20 µM), with specificity at least for PKC
(22), greatly inhibited Désiré-induced functional FasL expression in KB5.C20 cells. However, Gö6976 (4 µM), a specific inhibitor of classical PKC isoforms (
, ßI, ßII and
) (46), only moderately affected TCR-induced FasL expression. This same inhibitor did not affect at all PMA/ionomycin-induced FasL expression, while Ro31-8220 completely prevented it (Fig. 3b). Rottlerin inhibition was also observed in PMA/ionomycin-induced FasL expression (Fig. 3b). Since no other inhibitor with specificity for PKC
was available, we also used a cell permeable PKC
antagonist peptide coupled to antennapedia as a carrier (47). As shown in Fig. 3(c), the PKC
antagonist peptide partially prevented TCR/CD3-induced FasL expression. If combined with 10 µM rottlerin, which also had a partial effect, the inhibition was complete (Fig. 3c). The effect of the PKC inhibitors on perforin-dependent cytotoxicity exerted by BM3.3 cells on EL4.F15 was completely different. Both Ro31-8220 and Gö6976 completely inhibited perforin-dependent cytotoxicity, while rottlerin was without effect (Fig. 3d). No effect of the PKC
antagonist peptide was observed on perforin-dependent lysis (data not shown). Similar results were obtained for anti-CD3 mAb-redirected lysis of L1210.3 cells (data not shown). These results indicate that while TCR/CD3-induced functional FasL expression in CTL clones is dependent on the activation of novel PKC isoforms (
and/or
), classical PKC isoform activation is required for perforin-dependent cytotoxicity.

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Fig. 3. Effect of PKC inhibitors on Fas- or perforin-dependent cytotoxicity. (ac) CTL clone KB5.C20 was tested against L1210Fas target cells. Four-hour cytotoxicity tests were performed in the presence of 1 mM EGTA and 1.5 mM MgCl2. Effectors were pretreated with 20 µg/ml of the anti-clonotypic mAb Désiré (a and c) or with 10 ng/ml PMA plus 600 nM ionomycin (b) during 3 h. Pre-incubations were performed in the absence (open circles) or in the presence of either 4 µM Ro31-8220 (open diamonds), 4 µM Gö6976 (open squares) or 20 µM rottlerin (open triangles) (a and b), or in the presence of either 500 nM of a cell-permeable PKC antagonist peptide (closed diamonds), 10 µM rottlerin (open triangles) or a combination of both (closed triangles) (c). (d) CTL clone BM3.3 was tested against EL4.F15 target cells. Four-hour cytotoxicity tests were performed in the absence (open circles) or in the presence of either 4 µM Ro31-8220 (open diamonds), 4 µM Gö6976 (open squares) or 20 µM rottlerin (open triangles). Results are the means of triplicate determinations, with SEM values always <5% of the mean. The experiments shown are representative of at least five different experiments for each experimental condition.
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Analysis of TCR/CD3-induced FasL transcription by FasLluciferase transfection in Jurkat cells: effect of inhibitors and co-transfection with mutant PI3K and PKC isoforms
The described activation of novel PKC isoforms by products of PI3K activity would support a role for one or several of these PKC as the downstream PI3K molecules in the signaling pathway leading to FasL expression. To analyze the role of individual PKC isoforms in the transcriptional activation of the FasL gene we performed transfection experiments with constitutively active or dominant-negative mutants of individual enzymes. Taking into account the low transfection efficiency observed in normal CTL clones, the readout system should be restricted as much as possible to the transfected cells. For that, we have used a construction containing the promoter region of human FasL coupled to the luciferase gene [FasLluciferase (9)], detecting luciferase expression by luminometry, and transient co-transfection experiments with constitutively active mutants of PI3K (37), PKC
,
,
(38) or
(22), and also with dominant-negative mutants of PI3K (37) or PKC
(22). In all cases, the amount of the mutant constructions was 2- or 3-fold that of the FasLluciferase, to assure that cells detected by luminometry also contained the mutant constructs. In normal CTL clones, although FasLluciferase was induced by TCR engagement and PI3K inhibitors prevented it, none of the co-transfected molecules had any effect on the induction of FasL expression (data not shown). In these experiments, no increase in the expression of the transfected PKC isoforms was detected by western blot relative to vector-transfected CTL clones (data not shown). This could be due to their extremely low transfection efficiencies (from undetectable to 3%, estimated by lucifer yellow staining in the same electroporation conditions).
Hence, we chose to perform these transfection experiments using the human T cell line Jurkat. This cell line has been extensively used to study FasL transcriptional control by performing transient and stable transfections, its transfection efficiency being higher (2025%, determined with lucifer yellow). We have observed a high basal FasLluciferase level in transfected Jurkat cells (data not shown), in agreement with the described expression of preformed FasL protein in these cells (48). However, 3-h stimulation with immobilized anti-CD3 mAb UCHT1 was able to induce substantial increases in FasL transcription detected by this method (between 40 and 100% of increase; see Fig. 4). In agreement with the results obtained with the CTL clones, this increase, but not the basal level, was completely inhibited by 100 nM wortmannin and substantially by 20 µM rottlerin (Fig. 4A). Consistent with this result, the increase in FasLluciferase expression induced by TCR/CD3 ligation was improved in Jurkat cells co-transfected with PI3K* and PI3K* cooperated with ionomycin to induce the highest levels of FasLluciferase expression (Fig. 4A). In addition, co-transfection with PI3K-DN abolished TCR/CD3-induced FasLluciferase expression, without affecting the low-level induction by ionomycin alone (Fig. 4A). Similar results were obtained in four of four transfection experiments. The parallelism with the results obtained using normal CTL clones indicates that the Jurkat model can be used to study FasL transcription in spite of the basal expression of FasL.

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Fig. 4. Effect of kinase inhibitors and co-transfection with PI3K or PKC mutants on the induction of FasLluciferase in transfected Jurkat cells. (A) Jurkat cells (10 x 106) were transfected with 20 µg FasLluciferase together with 40 µg of either empty vector, PI3K* or PI3K-DN mutants. After 48 h in culture, transfected cells were left untreated or were stimulated for 3 h with either immobilized anti-CD3 mAb UCHT1 (+ -CD3) or 600 nM ionomycin (+Iono) in the presence or absence, as indicated, of either 100 nM wortmannin (+ -CD3+Wort) or 20 µM rottlerin (+ -CD3+Rotl). (B and C) Jurkat cells (10 x 106) were transfected with 20 µg FasLluciferase together with 40 µg of either empty vector, constitutively active mutants (A/E) of PKC and/or - or a dominant-negative mutant (K/R) of PKC . After 48 h in culture, transfected cells were left untreated or were stimulated for 3 h with either immobilized anti-CD3 mAb UCHT1 in the absence (+ -CD3) or in the presence of 100 nM wortmannin (+ -CD3+Wort), or with 600 nM ionomycin (+Iono). Results are shown as percentage of RLU increase with respect to the corresponding control for each transfection and are the means of triplicate determinations, with SEM values always <15% of the mean. The experiments shown are representative of four experiments in (A), 10 experiments for PKC A/E and six experiments for PKC A/E mutants, as indicated in the text. (D) Analysis of PKC or PKC expression by immunoblot in vector- or in PKC A/E- or PKC A/E transfected Jurkat cells, as indicated. The blots shown correspond to the experiment shown in (C).
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We have also performed co-transfection experiments with constitutively active mutants of PKC
,
,
and
in Jurkat cells. In all the experiments performed, neither PKC
A/E nor PKC
A/E had any effect on TCR/CD3- or ionomycin-induced FasLluciferase nor affected wortmannin inhibition (data not shown). However, in 10 of 12 experiments, two of which are shown in Fig. 4(B and C), transfection with PKC
A/E reduced the inhibitory effect of wortmannin on TCR-induced FasLluciferase expression. In all the experiments performed (six of six), transfection with PKC
A/E alone improved both TCR/CD3- and ionomycin-induced FasLluciferase (Fig. 4B and 4C). In addition, wortmannin inhibition of TCR/CD3-induced FasLluciferase in PKC
A/E transfected Jurkat cells was partial (two of six, Fig. 4B) or non-existent (four of six, Fig. 4C). In all the experiments performed (six of six), co-transfection with PKC
A/E and PKC
A/E together completely abolished wortmannin inhibition of TCR/CD3-induced FasLluciferase (Fig. 4B and C). Similar results were obtained for inhibition with 25 µM LY294002 (data not shown). In the same experiments, transfection with PKC
A/E or PKC
A/E clearly increased the amount of these proteins, as detected by western blot (Fig. 4D). In addition, transfection with a dominant-negative mutant of PKC
(PKC
K/R) abolished TCR/CD3- and ionomycin-induced FasLluciferase in Jurkat cells (Fig. 4B and 4C). These results clearly indicate that PKC
and, especially, PKC
are the molecules downstream PI3K in the signal transduction pathway leading from TCR engagement to FasL transcription and expression.
1.3E6SN degranulation experiments
While novel PKC isoforms are implicated in TCR/CD3-induced FasL expression, data shown in Fig. 3 indicate that perforin-based cytotoxicity, dependent on CTL degranulation, rather involves classical PKC isoform activation. Since normal CTL clones have a very low transfection efficiency, we chose the murine CTL line 1.3E6SN, for which the transfection efficiency is consistently
5%. We transfected these cells with a truncated hCD2 construct together with PKC
and PKC
A/E, purified transfected cells by MACS sorting of hCD2+ cells, and analyzed degranulation induced by ionomycin. As shown in Fig. 5, we compared the degranulation induced in hCD2+ cells and also in hCD2 cells as a control for each sample. Ionomycin induced low-level degranulation in all hCD2 cells and also in vector or PKC
A/E transfected hCD2+ cells. However, ionomycin induced
70% degranulation in PKC
A/E transfected, hCD2+ cells. It should be noted that in the same experiment, PMA plus ionomycin or anti-CD3 stimulation induced no more than 55% degranulation in hCD2 cells (data not shown). This result indicates that PKC
A/E, but not PKC
A/E, is able to cooperate with ionomycin to induce CTL degranulation.
 |
Discussion
|
---|
The present study demonstrates a differential usage of PKC isoforms in T cells mediating Fas- versus perforin-dependent cytotoxicity. While the induction of FasL after antigen receptor ligation is dependent on the activation of PKC
and especially PKC
, through a PI3K-dependent pathway, degranulation is rather dependent on the activation of classical PKC isoforms such as PKC
. This does not exclude the implication of other classical PKC isoforms, such as PKCßI, which is also expressed by CTL clones (8). The induction of FasL by the pharmacological combination of PMA plus ionomycin is also dependent on novel PKC isoforms, especially PKC
, as previously demonstrated (22,23), and not on classical PKC isoforms.
The results obtained in Fig. 2 on the effect of PI3K inhibitors on Fas- or perforin-dependent cytotoxicity contrast with a previous report (49). In this connection, a recent study has demonstrated an additional role of PI3K during CTL effector function: the TCR/CD3-induced CD8 adhesion to MHC class I (50). In the latter study it is also shown that PI3K inhibitors prevent lysis exerted by CD8-dependent CTL clones, but not by CD8-independent ones. BM3.3 is a CD8-independent CTL and this could explain the differences with the results obtained by Fuller et al. (49). The lack of inhibition by wortmannin observed by Fuller et al. for Fas-dependent cytotoxicity could be explained if their particular CTL clones expressed preformed FasL, as has been described for other murine (51) and human T cells (48,52). However, as shown in Fig. 1, this is not the case of the CTL clones used in the present study. Also, in the CTL clones used by Fuller et al., it has been demonstrated that the signals needed to activate Fas-dependent cytotoxicity are of lesser magnitude than those needed to activate perforin-dependent cytotoxicity (53), while no such differences have been observed in the CTL clones used in the present study.
The use of Jurkat cells in studies in which PI3K is involved has been questioned recently (54), because, as a consequence of the lack of expression of the phosphoinositide phosphatase PTEN, the PI3K pathway is overactivated (55). In agreement with that, we have observed a high basal FasLluciferase level in transfected Jurkat cells, correlating with the described expression of preformed FasL protein in these cells (48). However, FasL is induced in Jurkat cells after anti-CD3 mAb stimulation and this induction has a similar sensitivity to PI3K or PKC inhibitors as functional FasL expression in normal murine CTL clones (Fig. 4), indicating that the model can be used to study the regulation of de novo FasL gene transcription. On the other hand, it has been described that the high basal FasL transcription level in Jurkat cells, as in Sertoli cells, is rather due to constitutive overactivation of the Sp1 transcription factor, while FasL induction after TCR ligation is dependent on the activation of NF-AT and/or other inducible transcription factors (56).
The implication of PKC
as the main PI3K downstream molecule in the signaling pathway leading from TCR engagement to FasL transcription complements the recent observation on the role of PI3K in PKC
recruitment to the immunological synapse, which seems independent of phospholipase C
-mediated diacylglycerol production (24). PKC
implication in PMA/ionomycin-induced FasL transcription had been also reported (22,23), although in this case PKC is directly activated by phorbol ester, and is independent of TCR signaling and PI3K activation (8).
The main signaling role of PKC
in T cells seems to be NF-
B activation (39), as established in PKC
knockout mice (57). However, a more recent PKC
knockout indicates that it is implicated in NF-AT activation, at least in primary mouse T cells (58). PKC
is also implicated in NF-
B activation (38), especially in mature T cells (59). On the other hand, both NF-AT and NF-
B are crucially implicated in the control of FasL transcription (9,12,13). We have observed the inhibition of NF-
B activation by PI3K-DN transfection in Jurkat cells (data not shown), but we have not analyzed its possible role in NF-AT activation.
The present results indicate that TCR/CD3-induced FasL gene transcription is controlled by PI3K and PKC
activation, while this signaling pathway is not implicated in CTL degranulation, which is rather dependent on the activation of classical PKC isoforms. The molecular details controlling CTL degranulation are not well known, nor the exact effector molecules downstream of classical PKC activation or the intracellular Ca+2 increase. It has been suggested that PKC-mediated phosphorylation of microtubule-associated proteins or of kinesin or dynamin motors could be related to its role in CTL degranulation (28), but further molecular characterization is needed.
The observed differences in PKC isoform implication in CTL degranulation versus FasL expression could be exploited for the specific inhibition of these immune effector mechanisms. For example, in the case of transplant rejection, the inhibition of CTL and NK degranulation could be of great benefit, without affecting immune regulation through the FasFasL pathway. Such a selective inhibition would also be relevant in those autoimmune diseases where the uncontrolled perforin/granzyme pathway was the main contributor to tissue damage. On the contrary, the specific inhibition of FasL expression could be of benefit if this is the main effector pathway to tissue damage, such as in specific autoimmune diseases and fulminant hepatitis, without causing a general immunodeficiency by the use of non-specific immunosuppressors.
 |
Acknowledgements
|
---|
We gratefully acknowledge Dr Pierre Henkart for FasLluciferase, Dr Doreen Cantrell for PI3K* and PI3K-DN; Dr Peter J. Parker for PKC
,
and
A/E, Drs Amnon Altman and Martín Villalba for PKC
A/E and K/R, NF-
Bluciferase and critical reading of the manuscript, Dr Sylvie Guerder for the human CD2 construct; Dr D. Mochly-Rosen for the cell-permeable PKC
inhibitory peptide, and Dr Pierre Golstein for critical reading of the manuscript. This work was supported by HF97-1 SpanishFrench Integrated Action, by Institutional Grants from Institut de la Santé et de la Recherche Medical (France) and Centre National de la Recherche Scientifique (France), by a grant from Association pour la Recherche sur le Cancer (France), and by grants 99/1250 from Fondo de Investigaciones Sanitarias (Spain), SAF2001-1774 from Dirección General de Investigación (Spain) and P24/2000 from Diputación General de Aragón/Fondo Social Europeo.
 |
Abbreviations
|
---|
CTLcytotoxic T lymphocyte
FasLFas ligand
hCD2human CD2
PI3Kphosphatidylinositol 3-kinase
PI3K-DNdominant-negative phosphatidylinositol 3-kinase
PI3K*constitutively active phosphatidylinositol 3-kinase
PKCprotein kinase C
PMAphorbol myristate acetate
pNAp-nitroanylide
 |
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