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(Journal of Leukocyte Biology. 2001;69:803-814.)
© 2001 by Society for Leukocyte Biology

Phosphatidylinositol 3-kinase inhibitors prevent mouse cytotoxic T-cell development in vitro

Tommy Phu, S. M. Mansour Haeryfar, Bruce L. Musgrave and David W. Hoskin

Department of Microbiology & Immunology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada

Correspondence: David W. Hoskin, Department of Microbiology and Immunology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada. E-mail: dwhoskin{at}is.dal.ca


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ABSTRACT
 
To become competent killer cells, CD8+ T cells require stimulation through signal transduction pathways associated with the T-cell receptor, costimulatory molecules such as CD28, and cytokine receptors such as the interleukin (IL)-2 receptor. We used wortmannin and LY294002, two inhibitors of phosphatidylinositol 3-kinase (PI3-K), to study the role of PI3-K in mouse cytotoxic T-lymphocyte (CTL) induction in response to mitogenic anti-CD3 antibody. Anti-CD3-induced CD8+ T-cell proliferation and CTL development were inhibited dose dependently by both PI3-K inhibitors. IL-2 synthesis by anti-CD3-activated CD8+ T cells was also diminished by PI3-K inhibition. PI3-K inhibition resulted in a modest decrease in anti-CD3-induced CD4+ T-cell proliferation but failed to affect IL-2 expression by anti-CD3-activated CD4+ T cells. PI3-K inhibition during CTL induction resulted in decreased levels of mRNAs coding for granzyme B, perforin, and Fas ligand. In addition, CTL induced in the presence of PI3-K inhibitors failed to conjugate normally with P815 target cells. Exogenous IL-2 did not reverse the effects of PI3-K inhibition on CD8+ T-cell proliferation and CTL induction. These results support the conclusion that PI3-K activation is involved in T-cell receptor, CD28, and IL-2 receptor signaling of CD8+ T cells. PI3-K is, therefore, an important component of multiple signal transduction pathways involved in CTL generation.

Key Words: phosphoinositide 3-kinase • cytotoxic T lymphocyte • signal transduction • proliferation • gene expression • adhesion molecules • cytotoxic effector function


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INTRODUCTION
 
Resting CD8+ T lymphocytes are induced to proliferate and differentiate into cytotoxic T lymphocytes (CTLs) by a combination of signals resulting from antigen-specific triggering of the T-cell receptor–CD3 complex, ligation of costimulatory molecules such as CD28, and the interaction of critical cytokines such as interleukin (IL)-2 with the appropriate cytokine receptor [reviewed in ref. 1 and 2]. Costimulatory signaling through CD28, which occurs after ligation of CD28 by either CD80 or CD86 on antigen-presenting cells, synergizes with signals transduced by the T-cell receptor–CD3 complex upon engagement by antigens presented in the context of self major histocompatibility complex (MHC) proteins to up-regulate expression of gene products involved in T-cell activation, proliferation, and survival. These include the high-affinity IL-2 receptor {alpha} chain (CD25) and growth-promoting cytokines such as IL-2 [reviewed in ref. 3 ], as well as the Bcl-XL survival protein [4 ]. Engagement of the high-affinity IL-2 receptor by IL-2 results in signal transduction events which drive both T-cell proliferation and differentiation but, paradoxically, also serve to negatively regulate the growth and activation of both T and B lymphocytes [reviewed in ref. 5 ].

Phosphatidylinositol 3-kinase (PI3-K) is a lipid and protein serine kinase which is known to be a key component of many eukaryotic signaling pathways, including those involved in the activation of T lymphocytes [reviewed in ref. 6 ]. Class IA PI3-K found in T lymphocytes is a heterodimer composed of a regulatory 85-kDa subunit (p85) and a catalytic 110-kDa subunit (p110) [reviewed in ref. 7 ]. Interaction of src homology-2 (SH2) domains on the p85 subunit with phosphorylated tyrosine residues on receptor-associated tyrosine kinases, as well as nonreceptor tyrosine kinases and kinase substrates, recruits PI3-K to activated receptors where the p110 subunit phosphorylates cell membrane-associated phosphatidylinositols. This process results in the production of phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-biphosphate, and phosphatidylinositol 3,4,5-triphosphate, of which phosphatidylinositol 3,4,5-triphosphate is believed to be the major product in vivo. It is interesting that the role, if any, of the serine kinase activity of PI3-K during T-cell activation is not yet known [6 ].

Signaling through the T-cell antigen receptor–CD3 complex, CD28, and the IL-2 receptor has been demonstrated to involve PI3-K activation [8 9 10 ]. Although PI3-K binds directly to CD28 via the 173(p)YMNM motif [11 ], coupling of PI3-K to the T-cell antigen receptor and the IL-2 receptor appears to occur independently of a (p)YMNM motif since these signaling molecules lack this particular motif [6 ]. Instead, PI3-K associates with the CD3-{zeta} polypeptide chain of the T-cell antigen receptor–CD3 complex via the {zeta}A "Reth motif" [YXXL(X)7–8YXXL/I] [12 ]. Although the details of PI3-K coupling to the IL-2 receptor are not yet clear, PI3-K is activated in response to signals from the S region (amino acids 267–322) of the IL-2 receptor ß chain [13 ]. PI3-K lipid products are believed to function as "second messengers" during T-cell activation [6 ]. A recent report suggests that the tyrosine phosphatase SHP-1, which exhibits increased association with the p85 subunit of PI3-K in Jurkat T cells after T-cell receptor ligation, may be an important regulator of T-cell receptor-associated PI3-K signaling [14 ]. PTEN, another protein tyrosine phosphatase, has also been shown to function as a phosphatidylinositol 3-phosphatase in T lymphocytes and may, therefore, down-regulate antigen receptor-induced activation of T cells by counteracting the effects of PI3-K activation [15 ].

Although there is mounting evidence from studies with CD4+ T cells that PI3-K is an important element of signal transduction pathways associated with the T-cell receptor–CD3 complex, CD28, and the IL-2 receptor of T helper cells [6 ], the role of PI3-K activation during CTL development is not well understood. In this study we used two structurally different inhibitors of PI3-K, wortmannin and LY294002, to investigate the role of PI3-K in the signal transduction cascade leading to mouse CD8+ T-cell activation and differentiation into MHC-unrestricted CTL in response to mitogenic anti-CD3 monoclonal antibody (mAb). Wortmannin is a fungal metabolite that irreversibly blocks the catalytic activity of PI3-K [16 ]. Although wortmannin has been widely used to study the role of PI3-K in signal transduction [6 ], this cell-permeable inhibitor of PI3-K can also inhibit both phosphatidylinositol 4-kinase and phospholipase A2 at high nanomolar concentrations and is, therefore, a less selective inhibitor of PI3-K than LY294002 [17 , 18 ]. LY294002 is a highly selective, cell-permeable inhibitor of PI3-K which acts as a competitive antagonist for the ATP-binding site of the PI3-K p110 subunit [19 ]. Here we show that PI3-K inhibition during anti-CD3-activated CTL induction results in MHC-unrestricted effector cells with an impaired ability to bind to and kill P815 mastocytoma target cells. Our data are consistent with PI3-K as a key mediator of signaling through the T-cell receptor–CD3 complex, CD28, and the IL-2 receptor of CD8+ mouse T lymphocytes.


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MATERIALS AND METHODS
 
Mice
Female 6- to 8-week-old C57BL/6 mice (H-2b haplotype) were purchased from Charles River Canada (Lasalle, Quebec, Canada). Mice were maintained on standard laboratory chow and water supplied ad libitum in our animal care facilities.

Medium and reagents
RPMI 1640 medium (ICN Biomedicals Canada Ltd., Mississauga, Ontario, Canada), hereafter referred to as complete RPMI 1640 medium, was supplemented with 10 mM L-glutamine, 100 µg/mL of streptomycin, 100 U/mL of penicillin (all from ICN Biomedicals Canada), 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (pH 7.4) (Sigma Chemical Co., St. Louis, MO), and 5% heat-inactivated (at 56°C for 30 min) fetal calf serum (Life Technologies Ltd., Burlington, Ontario, Canada). Mouse recombinant IL-2 (rIL-2) was obtained from Genzyme Diagnostics (Cambridge, MA). Specific activity was expressed as units per milliliter where one unit is defined as the reciprocal of the dilution required to cause 50% stimulation of mouse CTLL-2 cells. The hybridoma (clone 145-2C11) that produces hamster anti-mouse CD3{varepsilon} mAb [20] was kindly provided by J. Bluestone (University of Chicago, Chicago, IL). Rat anti-mouse CD28 mAb, rat anti-mouse CD25 mAb (fluorescein isothiocyanate conjugated), and rabbit complement were from Cedarlane Laboratories (Hornby, Ontario, Canada). Rat anti-mouse CD4, rat anti-mouse CD8, rat anti-mouse CD11a, and rat anti-mouse CD54 mAbs were from hybridomas (GK1.5, 2.43, FD441.8, and YN1/1.7.4, respectively) obtained from the American Type Culture Collection (Manassas, VA). Fluorescein isothiocyanate-conjugated mouse anti-rat immunoglobulin G F(ab')2 and purified rat immunoglobulin G were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Anti-asialoGM1 rabbit polyclonal antiserum was from Wako Chemicals (Richmond, VA). Phorbol 12-myristate 13-acetate (PMA), ionomycin, and dimethyl sulfoxide (DMSO) were obtained from Sigma. Wortmannin and LY294002 were purchased from Research Biochemicals (Natick, MA). Stock solutions of wortmannin and LY294002, prepared by dissolving the reagents in DMSO, were stored at -20°C. P815 murine (H-2d) mastocytoma cells and CTLL-2 cells were obtained from American Type Culture Collection and maintained by in vitro passage in complete RPMI 1640 medium (plus 50 U/mL of IL-2 for CTLL-2 cells).

Generation of anti-CD3-activated T lymphocytes
C57BL/6 spleen cell preparations were depleted of erythrocytes by osmotic shock and passaged once through nylon wool columns (Cellular Products, Inc., Buffalo, NY) to remove most B cells and macrophages [21 ]. Nylon wool nonadherent spleen cells were depleted of natural killer cells and CD4+ T cells by sequential treatment with anti-asialoGM1 antiserum and rat anti-mouse CD4 mAb plus rabbit complement. The resulting CD8+ T-cell-enriched preparation [which was essentially devoid of CD4+ T and natural killer cells by flow-cytometric analysis] was adjusted to a concentration of 4 x 106 cells/mL in complete RPMI 1640 medium and seeded into wells of a 24-well flat-bottom tissue culture plate. CTLs were induced as previously described [22] by stimulating CD8+ T cells with soluble anti-CD3 mAb (1:20 dilution of hybridoma supernatant or ~5 µg/mL). Cultures were maintained for 48 h at 37°C and 5% CO2 in a 95% humidified atmosphere. Anti-CD3-activated CTLs were then collected for use. In some experiments, CD4+ T cells were obtained by sequential treatment of nylon wool nonadherent spleen cells with anti-asialoGM1 antiserum and rat anti-mouse CD8 mAb plus rabbit complement. The resulting CD4+ T cells were cultured and activated with anti-CD3 mAb, as described for CD8+ T cells.

Akt1/protein kinase B{alpha} assay
Akt1 activity in mouse T cells activated with anti-CD3 mAb in the absence or presence of PI3-K inhibitors was measured using an Akt1 immunoprecipitation kinase assay kit purchased from Upstate Biotechnology (Lake Placid, NY). Briefly, 107 T cells were stimulated with soluble anti-CD3 mAb (1:20 dilution of hybridoma supernatant) in the absence or presence of 100 nM wortmannin or 10 µM LY294002. Five minutes later, cell lysates were prepared from which Akt1 was subsequently isolated by immunoprecipitation with specific antibody. Immunoprecipitated Akt1 was incubated for 10 min at 30°C with 3 µg of Bad in the presence of 500 µM ATP and 75 mM MgCl2. Bad was then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with a 1:500 dilution of anti-phospho-Bad mAb to detect phosphorylated Bad. Individual phosphorylated Bad protein bands on the immunoblot were quantified by densitometric analysis.

51Cr-release assay
MHC-unrestricted CTLs induced with anti-CD3 mAb were washed extensively with phosphate-buffered saline, resuspended in complete RPMI 1640 medium, and seeded into wells of a 96-well V-bottom microtiter plate in graded dilutions to obtain the desired effector cell/target cell ratio. P815 mastocytoma cells were labeled with 100 µCi of Na251CrO4 (ICN Biomedicals Canada) for 1 h at 37°C, washed three times, resuspended in complete RPMI 1640 medium, and added to the microtiter plate at a concentration of 5 x 103 cells/well. The microtiter plate was then incubated for 4 h at 37°C and 5% CO2 in a 95% humidified atmosphere. After centrifugation of the microtiter plate, 100 µL of supernatant were collected from each well and 51Cr-release (in cpm) was determined by {gamma}-counting. Percent lysis was determined by the following equation % lysis = (E - S)/(M - S) x 100, where E is the release from experimental samples, S is the spontaneous release, and M is the maximum release upon lysis with 10% sodium dodecyl sulfate.

T-cell proliferation assay
After 48 h of stimulation, 50-µL volumes of CD8+ or CD4+ T cells were transferred to quadruplicate wells of a 96-well round-bottom microtiter plate. The cultures were pulsed with 0.5 µCi of tritiated thymidine ([3H]TdR; specific activity, 65 Ci/mmol; ICN Canada) per well and maintained at 37°C in a 5% CO2 humidified atmosphere for 6 h to measure DNA synthesis. Cultures were harvested onto glass fiber mats (ICN Canada) using a Titer-Tek multiple sample harvester, and [3H]TdR incorporation was determined by liquid scintillation counting.

Enzyme-linked immunosorbent assay
IL-2 in supernatants from 24-h cultures of anti-CD3-activated CD8+ T cells were measured by sandwich enzyme-linked immunosorbent assay (ELISA) using paired mAb, recombinant IL-2, and protocols supplied by PharMingen (Mississauga, Ontario, Canada).

Conjugate formation assay
This procedure was performed as previously described [23].

Semi-quantitative reverse transcriptase-polymerase chain reaction
Total RNA was isolated from CTLs using TRIzol reagent as recommended by the manufacturer (Life Technologies) after 4 h (Fas ligand), 24 h (IL-2), or 48 h (granzyme B, perforin) of culture. Single-stranded complementary DNA (cDNA) was synthesized from 0.5 µg of RNA with 200 U of Moloney murine leukemia virus-derived reverse transcriptase (RT) (Life Technologies) in the presence of 0.2 mM deoxynucleotide triphosphates, 1 µg of random hexamers, and 10 mM dithiothreitol. Polymerase chain reaction (PCR) was conducted in an automatic DNA thermocycler (MJ Research, Inc., Watertown, MA). Each PCR used equal amounts of cDNA, 2.5 U of Taq DNA polymerase (Life Technologies), 0.2 mM deoxynucleotide triphosphates, and each primer pair (0.5 µM) in a 1:10 dilution of PCR buffer [2 M KCl, 1 M Tris-HCl (pH 8.4), 1 M MgCl2, 1 mg/mL of bovine serum albumin]. The following primer pairs were used for PCR (amplicon size is given after the reverse primer). "F" refers to forward primer and "R" refers to reverse primer. All primers were designed to bind intron-bridging exons of the respective gene: glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (F) 5'-ACTCACGGCAAATTCAACGGC-3' and GAPDH (R) 5'-ATCACAAACATGGGGGCATCG-3' (product size, 247 bp); perforin (F) 5'-TCAATAACGACTGGCGTGTGG-3' and perforin (R) 5'-GTGGAGCTGTTAAAGTTGCGG-3' (product size, 252 bp); granzyme B (GzmB) (F) 5'-GCCCACAACATCAAAGAACAG-3' and granzyme B (R) 5'-GAGAACACATCAGCAACTTGGG-3' (product size, 889 bp); Fas ligand (F) 5'-ATGGTTCTGGTGGCTCTGGT-3' and Fas ligand (R) 5'-GTTTAGGGGCTGGTTGTTGC-3' (product size, 362 bp); and IL-2 (F) 5'-TGATGGACCTACAGGAGCTCCTGAG-3' and IL-2 (R) 5'-GAGTCAAATCCAGAACATGCCGCAG-3' (product size, 170 bp).

PCR mixtures were overlaid with 100 µL of mineral oil. The amplification protocols for GAPDH (25 cycles), Gzm B (28 cycles), and perforin (30 cycles) were as follows: denaturation at 92°C for 30 s, annealing at 57°C for 30 s, and primer extension at 72°C for 1 min, with the exception of Gzm B, which was for 2 min. Fas ligand (35 cycles) and IL-2 (32 cycles) were amplified by denaturation at 94°C for 1 min and 30 s, respectively; annealing at 57°C for 1 min and 30 s, respectively; and primer extension at 72°C for 1.5 and 1 min, respectively. The numbers of PCR cycles chosen for IL-2, Fas ligand, perforin, Gzm B, and GAPDH amplifications were previously determined to generate PCR product during the exponential phase of amplification. RT-PCR performed under these conditions has been shown to be semiquantitative, providing reliable detection of twofold or greater differences in mRNA levels [24]. PCR products were visualized by electrophoresis across an ethidium bromide-stained 1.5% Tris-acetate buffer-agarose gel, and the detected PCR amplicon was compared with a 100-bp ladder (Promega Corp., Madison, WI). Relative levels of PCR products were quantified by densitometric analysis of gel photographs and normalization relative to steady-state expression of GAPDH.

Flow-cytometric analysis
The percentages of CD11a-, CD25-, and CD54-positive cells in 48 h cultures of anti-CD3-activated T cells were determined by flow-cytometric analysis using a standard protocol [22].

Statistical analysis
Statistical comparisons of data were performed using the Instat statistics program (GraphPad Software, Inc., San Diego, CA). Student’s t-test or one-way analysis of variance were used when appropriate. Values of P < 0.05 were considered to be statistically significant.


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RESULTS
 
Role of PI3-K in anti-CD3-induced CD8+ T-cell proliferation and cytotoxicity
We first determined whether PI3-K activation is required for mouse CD8+ T lymphocytes to proliferate in response to triggering through the T-cell receptor–CD3 complex. CD8+ T cells (prepared by anti-CD4 mAb plus complement treatment of asialoGM1- nylon wool column-passaged spleen cells from C57BL/6 mice) were stimulated with anti-CD3 mAb in the absence or presence of the PI3-K inhibitors wortmannin or LY294002 and, after 48 h of culture, T-lymphocyte proliferation was measured by [3H]TdR incorporation. Inhibition of anti-CD3-induced activation of PI3-K by wortmannin and LY294002 was confirmed by measuring Akt1/protein kinase B{alpha} activity in T cells activated in the absence or presence of the PI3-K inhibitors. Akt1/protein kinase B{alpha} is a serine/threonine kinase which is activated in a PI3-K-dependent manner [25 ]. Anti-CD3-induced Akt1 kinase activity was reduced by ~40% in the presence of 100 nM wortmannin and by ~60% in the presence of 10 µM LY294002 (data not shown), indicating that both wortmannin and LY294002 inhibit the activation of PI3-K in T cells stimulated with anti-CD3 mAb.

As shown in Figure 1 , anti-CD3-induced CD8+ T-cell proliferation was inhibited in a dose-dependent fashion by both wortmannin and LY294002 with approximate 50% inhibitory concentrations (IC50s) of 7.5 nM and 1.5 µM, respectively. These data indicated a role for PI3-K in signaling the entry of CD8+ T lymphocytes into the cell cycle. We have previously shown that mouse T cells activated with soluble anti-CD3 mAb develop potent MHC-unrestricted cytolytic activity against a range of tumor target cells, including P815 mastocytoma cells [26 ]. To determine the effect of PI3-K inhibition on MHC-unrestricted CTL development, we stimulated CD8+ T cells with anti-CD3 mAb in the absence or presence of various concentrations of wortmannin or LY294002. After 48 h of culture, viable T cells were collected and counted, and cytotoxicity on a per-cell basis was assessed in a 51Cr-release assay against radiolabeled P815 target cells.



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  		Figure 1. PI3-K inhibitors cause reduced CD8+ T-cell proliferation in response to anti-CD3 mAb. Highly enriched CD8+ T cells were stimulated with anti-CD3 mAb in the presence of medium alone, DMSO (the drug vehicle for PI3-K inhibitors), or the indicated concentrations of (A) wortmannin or (B) LY294002. After 48 h of culture, proliferation was measured by [3H]TdR incorporation. Results from a representative experiment (n =3) are expressed as mean cpm ± SD of quadruplicate samples. Statistical significance of results was determined by one-way analysis of variance to be P < 0.0001.

Figure 2 shows a dose-dependent inhibitory effect of both PI3-K inhibitors on the development of anti-CD3-induced MHC-unrestricted CTLs, indicating that this process is PI3-K dependent. T-lymphocyte proliferation and the generation of cytolytic activity were unaffected by the DMSO vehicle. Neither wortmannin nor LY294002 was toxic to CD8+ T cells at concentrations in excess of 25 nM and 2.5 µM, respectively, as determined by staining with trypan blue at the 48-h time point (data not shown). These drug concentrations were, therefore, unless otherwise noted, selected for use in subsequent experiments.



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  		Figure 2. Development of cytotoxic effector function by anti-CD3-activated CD8+ T cells is impaired in the presence of PI3-K inhibitors. Highly enriched CD8+ T cells were stimulated with anti-CD3 mAb in the presence of DMSO (the drug vehicle for PI3-K inhibitors) or the indicated concentrations of (A) wortmannin or (B) LY294002. After 48 h of culture, cytolytic activity against P815 target cells at the indicated effector cell/target cell ratio (E:T) was determined by 51Cr-release assay. Results from a representative experiment (n =3) are expressed as mean percent lysis of P815 target cells ± SD of triplicate samples. Statistical significance of results was determined by one-way analysis of variance to be P < 0.0001.

Effect of PI3-K inhibitors on CD4+ T-cell proliferation and IL-2 synthesis in response to anti-CD3 antibody
Since CTL development is normally enhanced by cytokines produced by activated CD4+ T cells [27 ], we decided to determine the effect of PI3-K inhibition by wortmannin or LY294002 on the proliferative response of CD4+ T cells (prepared by anti-CD8 mAb plus complement treatment of asialoGM1- nylon wool column-passaged spleen cells from C57BL/6 mice) stimulated with anti-CD3 mAb. Figure 3 shows that anti-CD3-induced CD4+ T-cell proliferation, as determined by [3H]TdR incorporation, was inhibited in a dose-dependent fashion by both wortmannin (Fig. 3A) and LY294002 (Fig. 3B) with approximate IC50s of 50 nM and 2.5 µM, respectively. However, it is noteworthy that, in comparison to CD8+ T cells (Fig. 1) , the proliferative response of anti-CD3-activated CD4+ T cells was somewhat resistant to the effects of PI3-K inhibition. We also examined the effect of PI3-K inhibition on IL-2 gene expression by anti-CD3-activated CD4+ T cells. As shown in Figure 4 , semiquantitative RT-PCR analysis revealed that there was little or no inhibitory effect on IL-2 mRNA expression at 24 h of culture when CD4+ T cells were activated with anti-CD3 mAb in the presence of wortmannin (50 nM) or LY294002 (5 µM). IL-2 gene transcription by anti-CD3-activated CD4+ T cells was, therefore, not dependent on PI3-K activation.



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  		Figure 3. PI3-K inhibitors cause reduced CD4+ T cell proliferation in response to anti-CD3 mAb. Highly enriched CD4+ T cells were stimulated with anti-CD3 mAb in the presence of an appropriate concentration of DMSO (the drug vehicle for PI3-K inhibitors), or the indicated concentrations of (A) wortmannin or (B) LY294002. After 48 h of culture, proliferation was measured by [3H]TdR incorporation. Results from one of two independent experiments are expressed as mean cpm ± SD of quadruplicate samples. Statistical significance of results was determined by one-way analysis of variance to be P < 0.0001.



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  		Figure 4. PI3-K inhibitors failed to substantially affect IL-2 mRNA expression by anti-CD3-activated CD4+ T cells. Highly enriched CD4+ T cells were stimulated with anti-CD3 mAb in the absence (lane 1) or presence (lane 2) of 50 nM wortmannin or 5 µM LY294002 (lane 3). After 24 h of culture, total RNA was isolated, and IL-2 mRNA levels were determined by semiquantitative RT-PCR. GADPH mRNA levels were also determined to control for equal loading of amplicons which were resolved by gel electrophoresis and visualized by ethidium bromide staining. Data are from one experiment and are representative of two independent experiments.

Effect of PI3-K inhibition on cytolytic effector molecule expression in anti-CD3-activated CD8+ T-cell cultures
To further document the effect of PI3-K inhibitors on CTL induction, we used semiquantitative RT-PCR analysis to investigate the effect of wortmannin and LY294002 on granzyme B, perforin, and Fas ligand mRNA expression in anti-CD3-activated CD8+ T-cell cultures. Previous work has established that, after CD8+ T-cell activation with anti-CD3 mAb, the levels of granzyme B and perforin mRNA peak at 48 h of culture, while Fas ligand mRNA levels are maximal after 4 h of culture [22 ]. As shown in Figure 5 , expression of all three cytolytic effector molecule mRNAs was dramatically reduced when CTLs were induced in the presence of wortmannin or LY294002, suggesting the involvement of PI3-K in the signaling processes which lead to granzyme B, perforin, and Fas ligand gene transcription.



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  		Figure 5. PI3-K inhibitors prevent expression of granzyme B, perforin, and Fas ligand mRNAs by anti-CD3-activated CD8+ T cells. Highly enriched CD8+ T cells were stimulated with anti-CD3 mAb in the absence (lane 1) or presence (lane 2) of 25 nM wortmannin or 2.5 µM LY294002 (lane 3). After 4 h (Fas ligand) or 48 h (granzyme B, perforin) of culture, total RNA was isolated and (A) perforin and granzyme B or (B) Fas ligand mRNA levels were determined by semiquantitative RT-PCR. GADPH mRNA levels were also determined to control for equal loading of cDNA per reaction, which were resolved by gel electrophoresis and visualized by ethidium bromide staining. Data are from one experiment and are representative of three independent experiments.

Effect of PI3-K inhibitors on IL-2 production and receptor expression in anti-CD3-activated CD8+ T-cell cultures
Since CD8+ T cells stimulated with anti-CD3 mAb can synthesize significant quantities of IL-2 [28 ], we next determined the effect of PI3-K inhibition by wortmannin or LY294002 on IL-2 synthesis by anti-CD3-activated CD8+ T lymphocytes after 24 h of culture. Previous studies have established that IL-2 levels peak in anti-CD3-activated T-cell cultures at this time point [28 ]. The data tabulated in Table 1 indicated that PI3-K inhibition had a profound inhibitory effect (~80% suppression) on IL-2 synthesis by anti-CD3-activated CD8+ T cells, as measured by ELISA. These results were confirmed by semiquantitative RT-PCR analysis of IL-2 mRNA expression by anti-CD3-activated CD8+ T cells, which revealed a similar decrease in IL-2 mRNA levels of CD8+ T cells activated in the presence of 25 nM wortmannin or 5 µM LY294002 (data not shown). PI3-K activation is, therefore, necessary for IL-2 production by CD8+ T lymphocytes after activation via the T-cell receptor/CD3 complex. Moreover, activation in the presence of PI3-K inhibitors resulted in a modest but consistent decrease in the percentage of CD8+ T cells expressing the high-affinity IL-2 receptor (CD25; see Fig. 6 ), as measured by flow-cytometric analysis. We conclude that the ability of CD8+ T lymphocytes to utilize IL-2 may be compromised in the absence of PI3-K activation.


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  		Table 1. PI3-K Inhibitors Suppress IL-2 Synthesis by Anti-CD3-Activated CD8+ T Cells



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  		Figure 6. CD11a, CD25, and CD54 expression by CD8+ T cells activated in the presence of PI3-K inhibitors. Highly enriched CD8+ T cells were stimulated with anti-CD3 mAb in the presence of the PI3-K inhibitor wortmannin (25 nM) or LY294002 (2.5 µM) or of DMSO, the drug vehicle. After 48 h of culture, the percentage of positive cells and the level of receptor expression were determined by flow cytometry as described in Materials and Methods. Cytofluorimetric profiles for unstained isotype controls (filled peaks) and for CD8+ T cells stained with specific mAb (open peaks) are shown. Data are from one experiment and are representative of four independent experiments.

Role of PI3-K in adhesion molecule expression by anti-CD3-activated CD8+ T cells
We have previously demonstrated that the ß2 integrin lymphocyte function-associated antigen (LFA)-1 plays a primary role in the conjugation of anti-CD3-activated CTLs to P815 target cells [29 ]. To determine whether PI3-K inhibition might alter the LFA-1-dependent adherence of CD8+ T cells to tumor target cells, we tested the ability of CD8+ T lymphocytes which had been activated with anti-CD3 mAb in the presence of wortmannin or LY294002 to conjugate with P815 mastocytoma cells. The data summarized in Table 2 indicated that CTLs induced under conditions of PI3-K inhibition were defective in their ability to conjugate with P815 target cells (~50% reduction). In addition, flow-cytometric analysis revealed that CD11a (the {alpha}L chain of LFA-1) was expressed at substantially lower levels on CD8+ T cells activated by anti-CD3 mAb in the presence of PI3-K inhibitors, whereas the percentage of CD11a-bearing CD8+ T cells was also reduced (Fig. 6) . The percentage of anti-CD3-activated CD8+ T cells expressing the activation marker and LFA-1 ligand intercellular adhesion molecule (ICAM)-1 (CD54) was also decreased in the absence of PI3-K activation, as was the level of CD54 expression on individual T cells. Taken together, these data indicate that PI3-K is a critical intracellular mediator involved in adhesion molecule up-regulation after CD8+ T-cell activation via the T-cell receptor/CD3 complex.


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  		Table 2. CD8+ T Cells Activated in the Presence of PI3-K Inhibitors Exhibit Reduced Conjugation with Target Cells

Effect of exogenous IL-2 on CTL development and CD8+ T-cell proliferation in the presence of PI3-K inhibitors
Since PI3-K inhibition had a dramatic inhibitory effect on IL-2 synthesis by anti-CD3-activated CD8+ T cells and IL-2 is known to be required for CTL development [27 ], we next determined whether the addition of exogenous IL-2 (100 U/mL) at initiation of culture could ablate or at least diminish the inhibitory action of PI3-K inhibitors on anti-CD3-induced CTL generation. The amount of IL-2 added to culture was twice that normally found in 48-h cultures of anti-CD3-activated T cells [28 ]. The data summarized in Table 3 demonstrate that wortmannin and LY294002 had an equivalent inhibitory effect on anti-CD3-induced CTL development, regardless of whether exogenous IL-2 had been added at the start of culture. Similar results were obtained when we examined the proliferative response of CD8+ T cells activated in the absence or presence of either PI3-K inhibitor with or without exogenous IL-2 (Table 4 ).These data are consistent with the involvement of PI3-K in signal transduction through the IL-2 receptor, as well as signaling through the T-cell receptor–CD3 complex and CD28.


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  		Table 3. Exogenous IL-2 Fails to Restore Cytotoxic Effector Function to CD8+ T Cells Activated in the Presence of PI3-K Inhibitors


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  		Table 4. Exogenous IL-2 Fails to Restore the Proliferative Response of CD8+ T Cells Stimulated with Anti-CD3 mAb in the Presence of PI3-K Inhibitors

Contribution of PI3-K in signal transduction associated with the T-cell receptor–CD3 complex, CD28, and the IL-2 receptor
Protein kinase C activation and Ca2+ mobilization are key events in T-cell activation [1 ]. If PI3-K acts upstream of protein kinase C and the release of Ca2+ from intracellular stores during CD8+ T-cell activation, PI3-K inhibitors should fail to inhibit CD8+ T-lymphocyte proliferation induced by ionomycin, a Ca2+ ionophore, in combination with PMA, a protein kinase C activator. Indeed, at concentrations which strongly inhibited CD8+ T-cell proliferation and cytotoxicity, neither wortmannin nor LY294002 significantly inhibited PMA and ionomycin-induced proliferation of CD8+ T cells (Table 5 ).


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  		Table 5. PI3-K Inhibitors Fail To Affect CD8+ T-Cell Proliferation in Response to PMA and Ionomycin

We next sought to determine the relative importance of PI3-K in signaling through the T-cell receptor–CD3 complex, CD28, and the IL-2 receptor of CD8+ T lymphocytes. To examine the role of PI3-K in T-cell receptor signal transduction, highly purified CD8+ T cells were stimulated with immobilized anti-CD3 mAb plus PMA in the presence or absence of PI3-K inhibitors (25 nM wortmannin or 2.5 µM LY294002), and proliferation was measured after 48 h of culture. As shown in (Fig. 7A ), CD8+ T-cell proliferation in response to stimulation through the T-cell receptor was virtually abrogated (>90% inhibition) when either wortmannin or LY294002 was present. PI3-K involvement in CD28 signaling was studied by stimulating highly purified CD8+ T cells with cross-linking anti-CD28 mAb plus PMA in the presence or absence of PI3-K inhibitors. Proliferation was markedly impaired (56% and 65% suppression, respectively) after 48 h of culture in the presence of either wortmannin or LY294002 (Fig. 7B) . Finally, we examined PI3-K participation in IL-2 receptor signaling by culturing IL-2-dependent mouse CD8+ CTLL-2 cells with or without PI3-K inhibitors. Figure 7C shows that IL-2-dependent CTLL-2 proliferation after 24 h in the presence of wortmannin or LY294002 was inhibited by 29 or 42%, respectively. Figure 7D depicts the mean percent inhibition by LY294002 (2.5 µM) of CD8+ T-cell proliferation in response to T-cell receptor, CD28, and IL-2 receptor signaling in three independent experiments. Taken together, these data suggest that, although PI3-K is involved in signal transduction through the T-cell receptor–CD3 complex, CD28, and the IL-2 receptor of CD8+ T lymphocytes, the relative importance of PI3-K is different in each signal transduction pathway, with T-cell receptor > CD28 > IL-2 receptor.



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  		Figure 7. Differential effects of PI3-K inhibition on T-cell receptor, IL-2 receptor, and CD28 signaling. Highly enriched CD8+ T cells were stimulated with (A) immobilized anti-CD3 mAb plus PMA (15 ng/mL) or (B) anti-CD28 mAb (10 µg/mL) plus PMA (15 ng/mL) in the absence or presence of wortmannin (25 nM) or LY294002 (2.5 µM). After 48 h of culture, proliferation was measured by [3H]TdR incorporation. (C) CTLL-2 CD8+ T cells were cultured in the presence of IL-2 (100 U/mL) with or without wortmannin (25 nM) or LY294002 (2.5 µM). After 24 h of culture, proliferation was measured by [3H]TdR incorporation. Results from a representative experiment (n =3) are expressed as mean cpm ± SD of triplicate (A) or quadruplicate (B and C) samples. Statistical significance in comparison with the control was determined by Student’s t-test. (D) Mean percent inhibition (±SD; n =3) by LY294002 (2.5 µM) of T-cell proliferation in response to T-cell receptor (TCR), IL-2 receptor (IL-2R), or CD28 signaling.


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DISCUSSION
 
PI3-K has been implicated in a host of receptor-initiated cellular events and biochemical responses in different eukaryotic cell systems, including membrane ruffling [31 ], cytoskeletal rearrangement [32 ], p70 S6 kinase activation [33 ], and protection against apoptosis [34 ]. Recent studies have linked PI3-K activation in T lymphocytes with T-cell receptor-induced proliferative responses [35 ]; regulated on activation, normal T expressed and secreted (RANTES)-dependent chemotaxis and polarization [36 ], CD2-associated regulation of ß1 integrin function [37 ], and IL-2 gene transcription [38 ]. In the present investigation, we have demonstrated, for the first time, that PI3-K also plays a critical role in the process by which precursor CTLs develop into competent effector cells.

The proliferative response of CD8+ mouse T lymphocytes stimulated with anti-CD3 mAb in the presence of wortmannin or LY294002 was dramatically reduced in comparison with controls. This effect can be attributed to a combination of diminished IL-2 production, a lower percentage of T cells expressing the high-affinity IL-2 receptor, and impaired IL-2 receptor signaling in the presence of PI3-K inhibitors. Anti-CD3-induced proliferation of CD4+ T cells was also inhibited in the presence of PI3-K inhibitors, although the inhibition was considerably less potent than that observed with CD8+ T cells. Surprisingly, in contrast to the marked inhibition of IL-2 synthesis observed when CD8+ T cells were activated in the presence of PI3-K inhibitors, neither wortmannin nor LY294002 was able to inhibit IL-2 expression by CD4+ T cells. This finding is consistent with an earlier report that wortmannin fails to prevent anti-CD3 mAb-induced IL-2 synthesis by CD4+ T cells from DO11.10 T-cell receptor-{alpha}ß-transgenic mice, even though IL-2 production in response to antigen was markedly diminished by the PI3-K inhibitor [39 ]. Shi et al. [39 ] suggest that the differential sensitivity of anti-CD3- and antigen-activated CD4+ T lymphocytes to wortmannin might be due to either an effect of PI3-K inhibition on the ability of antigen but not anti-CD3 mAb to engage and trigger the T-cell receptor or to major qualitative differences in the signals elicited by the interaction of anti-CD3 mAb and antigen with the T-cell receptor. The first possibility seems unlikely in light of the finding that anti-CD3-induced CD4+ T-cell proliferation is effectively suppressed by PI3-K inhibitors. Moreover, with regard to the second possibility, our results indicate that differences in the signals delivered by mAb-mediated perturbation of the T-cell receptor and those elicited by antigen are apparent only if IL-2 gene induction is used as a readout. In addition, our findings imply the existence of qualitative differences between T-cell receptor-associated signaling pathways involved in the up-regulation of IL-2 gene expression by CD4+ and CD8+ T cells. In other words, anti-CD3 mAb may activate additional signaling pathways in CD4+ but not CD8+ T cells that circumvent the need for PI3-K activation as a prerequisite for IL-2 gene transcription. Our data are also consistent with the recent finding that PI3-K activity is required for IL-2 gene expression by normal murine lymph node T cells stimulated with immobilized anti-CD3 mAb [38 ], since lymph node cells are a mixture of CD4+ and CD8+ T cells which, although exhibiting differential sensitivity to PI3-K inhibition, are both capable of synthesizing IL-2 [28 ].

IL-2 gene expression requires coordinate signaling through both the T-cell receptor and costimulatory molecules such as CD28 [1 ]. The T-cell receptor–CD3 complex-associated signal transduction pathway of CD8+ T cells is particularly sensitive to PI3-K inhibition since CD8+ T-cell proliferation induced by immobilized anti-CD3 mAb in combination with PMA was virtually abrogated in the presence of wortmannin or LY294002. A similar inhibitory effect on IL-2 production in CD8+ T-cell cultures stimulated with PMA and immobilized anti-CD3 mAb was also observed [T. Phu and D. Hoskin, unpublished results]. PI3-K is known to function downstream of protein tyrosine kinases activated as a result of T-cell receptor ligation [35 , 38 ]. T-cell receptor-associated tyrosine kinase activity is coupled to the phosphorylation and activation of phospholipase C{gamma}1, which generates inositol phosphates and diacylglycerols by the hydrolysis of inositol phospholipids. These second messengers bring about the activation of protein kinase C family members and the mobilization of Ca++ from intracellular stores, which in turn activates the phosphatase calcineurin responsible for nuclear translocation of the transcription factor nuclear factor of activated T cells (NF-AT) [1 , 2 ]. Neither wortmannin nor LY294002 affected CD8+ T-cell proliferation induced by PMA, a protein kinase C activator, in combination with ionomycin, a Ca++ ionophore, leading us to conclude that PI3-K acts upstream of protein kinase C and calcineurin activation in CTL precursors. Our data are, therefore, in line with the finding that wortmannin prevents the activation of NF-AT and activator protein (AP)-1 transcription factors in lymph node T cells by interfering with T-cell receptor signaling at the level of the Ras/extracellular signal-related kinase pathway [38 ].

Although PI3-K is known to be associated with and enzymatically activated after CD28 ligation [9 ], the role of PI3-K in CD28-mediated costimulation of CTL precursors remains controversial [40 ]. Studies with CD8+ T cells from mice that are transgenic for a T-cell receptor specific for the simian virus 40 large-T antigen presented in the context of H2-K have revealed that B7-CD28 costimulation is required for antigen-driven IL-2 production by CD8+ T lymphocytes [41 ]. However, B7-1-dependent proliferation of CD8+ T cells from normal mice has been reported to be unaffected by wortmannin, suggesting that PI3-K activation is not required in the CD28-mediated signaling involved in costimulating precursor CTLs to proliferate [42 ]. In contrast, we have demonstrated that both wortmannin and LY294002 cause an intermediate inhibition of CD8+ T-cell proliferation in response to PMA in combination with anti-CD28 mAb, implying a major role for PI3-K in the CD28-dependent costimulation of precursor CTL proliferation. Recent data from our laboratory indicating that B7-2, rather than B7-1, is the primary ligand for CD28 during mouse CTL induction by anti-CD3 mAb suggest a potential explanation for these apparently conflicting findings [43 ]. It is possible that ligation of CD28 by B7-1 activates additional signaling pathways which render PI3-K redundant, whereas CD28 engagement by B7-2, as occurs in our experimental system, results in only PI3-K-dependent signal transduction.

Anti-CD3 activation in the presence of wortmannin or LY294002 resulted in a modest decrease in the percentage of CD8+ T cells expressing CD25, the {alpha} chain of the high-affinity IL-2 receptor. This is most likely a consequence of deficient T-cell receptor signaling due to the inhibition of receptor-associated PI3-K, since a similar decrease in the percentage of CD25+ T cells was noted when mouse T lymphocytes were activated in the presence of 1 µM cyclosporin A [29 ]. Cyclosporin A is known to prevent T-cell activation by inhibiting the action of calcineurin associated with the T-cell receptor signal transduction pathway [44 ], without affecting signaling through the CD28 pathway [45 ]. Fewer CD8+ T cells expressing the high-affinity IL-2 receptor would lead to fewer CD8+ T lymphocytes responding to the reduced amount of IL-2 synthesized by T cells activated in the presence of PI3-K inhibitors. Moreover, the IL-2-dependent proliferation of CD8+ mouse CTLL-2 cells was suppressed in the presence of wortmannin or LY294002, implying that PI3-K is involved in IL-2-regulated signal transduction pathways. Our data are, therefore, in good agreement with the recent finding that PI3-K couples the IL-2 receptor with p70 S6 kinase and the serine/threonine protein kinase B [10 ]. PI3-K linked with the IL-2 receptor has also been shown to promote activation of the cell cycle regulator E2F involved in activating the cell cycle machinery [46 ]. However, it must be emphasized that IL-2 receptor signal transduction was far less sensitive to PI3-K inhibition than was T-cell receptor or CD28 signaling in mouse CD8+ T lymphocytes, suggesting the existence of PI3-K-independent signaling processes linked with the IL-2 receptor of this T-lymphocyte subset.

The inhibitory effect of wortmannin and LY294002 on anti-CD3-induced CD8+ T-lymphocyte proliferation was paralleled by a potent suppressive effect on the development of MHC-unrestricted cytotoxic activity. In fact, nonspecific CTLs induced with anti-CD3 mAb in the presence of either PI3-K inhibitor exhibited a dramatic reduction in mRNA species coding for the cytolytic effector molecules granzyme B, perforin, and Fas ligand. Fas ligand expression is largely dependent on T-cell receptor signaling [47 ] and does not require CD28 costimulation, since blockade of CD28-B7 interactions does not alter Fas ligand mRNA expression [43 ]. The nearly absolute requirement for PI3-K in T-cell receptor signaling, therefore, accounts for the virtual absence of Fas ligand mRNA expression by CD8+ T cells activated in the presence of either PI3-K inhibitor. Since CTL expression of both granzyme B and perforin is inducible by IL-2 [48 ], sharply reduced IL-2 synthesis in cultures of CD8+ T cells activated in the presence of wortmannin or LY294002 likely explains the profound reductions observed in perforin and granzyme B gene expression. It is not surprising that the addition of exogenous IL-2 to anti-CD3-activated CD8+ T-cell cultures failed to overcome the inhibitory effect of wortmannin or LY294002 on CD8+ T-cell proliferation and CTL development, given that PI3-K inhibition affects both high-affinity IL-2 receptor expression and signaling through the IL-2 receptor. Moreover, this observation implies that even in the presence of anti-CD3-activated CD4+ T cells that are able to produce IL-2 without a requirement for PI3-K activation, the anti-CD3-induced differentiation of CD8+ T cells into cytotoxic effector cells will be impaired by PI3-K inhibitors.

Reduced cytolytic effector molecule expression in the presence of PI3-K inhibitors is no doubt a major reason for the reduced effector function of MHC-unrestricted CTLs generated under these conditions. However, decreased expression of key T-cell adhesion molecules is also likely to be an important contributing factor. Conjugation of anti-CD3-activated CTLs to P815 mastocytoma cells is mediated by LFA-1/ICAM-1 interactions between the effector cell and target cell [30 ]. Although the role of PI3-K in ß2 integrin function has not been well studied, the T-lymphocyte integrin regulators CD2 and CD28 have been shown to use PI3-K in the signaling process, which results in the up-regulation of ß1 integrin adhesiveness after CD2 or CD28 ligation [37 , 49 ]. We have shown here that nonspecific CTLs induced in the presence of PI3-K inhibitors conjugated with reduced efficiency to P815 target cells. Moreover, decreased effector-target cell conjugation was associated with a substantial reduction in surface LFA-1 expression by anti-CD3-activated CD8+ T cells, suggesting the PI3-K is involved in up-regulating ß2 integrin expression after CD8+ T-cell activation. PI3-K inhibition also reduced the percentage of T cells expressing ICAM-1, which is indicative of impaired T-cell activation [50 ]. Lower numbers of ICAM-1-bearing CD8+ T cells after wortmannin or LY294002 treatment may also be a factor in reduced conjugate formation, since P815 mastocytoma cells are known to express LFA-1 [51 ].

In summary, we report that PI3-K inhibition with wortmannin or LY294002 during CD8+ T-lymphocyte activation with anti-CD3 mAb strongly inhibited the development of MHC-unrestricted cytolytic effector cells and impaired subsequent effector cell binding to tumor target cells. Fuller and colleagues have recently reported that PI3-K inhibition with wortmannin abrogates perforin/granzyme granule exocytosis by influenza virus-specific CTL clones, thereby inhibiting the effector phase of cytolysis [52 ]. It is interesting that the Fas/Fas ligand pathway of cytolysis was unaffected by even very high concentrations of wortmannin, suggesting that granule-independent killing does not depend on PI3-K activation. We have also noted that PI3-K inhibition during the effector phase of cytolysis failed to affect Fas ligand-dependent killing of P815 tumor cells (rendered Fas+ by pretreatment with etoposide and performed in the presence of Ca2+ chelator as previously described in ref. 53 ) by MHC-unrestricted CTLs induced with anti-CD3 mAb [M. Haeryfar and D. Hoskin, unpublished results]. However, in contrast to the antigen-specific CTL described by Fuller et al., PI3-K inhibition had only a modest inhibitory effect on granule-mediated cytolysis of Fas- P815 tumor cells by MHC-unrestricted CTL induced with anti-CD3 mAb. Nevertheless, PI3-K is clearly an essential component of signal transduction pathways involved in CTL development, as well as some aspects of effector function.


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ACKNOWLEDGEMENTS
 
This work was supported by a grant (OGP0046295) to D.H. from the Natural Sciences and Engineering Research Council of Canada (NSERC). S.M.M.H. and B.L.M. are recipients of postgraduate scholarships from NSERC and the Killam Trust.

Received May 8, 2000; revised November 5, 2000; accepted December 27, 2000.


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