Perforin and the serine protease granzymes are key effectors of CD8+ T cell granule-mediated cytotoxicity, but the requirements for their expression remain largely undefined. We show in this study that IL-2 increased the expression of perforin and granzyme A, B, and C mRNA; intracellular granzyme B protein levels; and cytolytic function in a dose-dependent manner during primary activation of murine CD8+ T cells in vitro. Two approaches showed that these responses were not a consequence of the effects of IL-2 on cell survival and proliferation. First, IL-2 enhancement of perforin and granzyme expression was equivalent in CD8+ T cells from wild-type and bcl-2 transgenic mice, although only the latter cells survived in low concentrations or the absence of added IL-2. This property of bcl-2 transgenic T cells also allowed the demonstration that induction of granzyme A, B, and C mRNA and granzyme B protein required exogenous IL-2, whereas induction of perforin and IFN-γ expression did not. Second, analysis of perforin and granzyme mRNA levels in cells separated according to division number using the dye CFSE showed that the effects of IL-2 were unrelated to division number. Together, these findings indicate that IL-2 can directly regulate perforin and granzyme gene expression in CD8+ T cells independently of its effects on cell survival and proliferation.

Granule-mediated cytotoxicity is one of the major mechanisms used by CD8+ T cells to eliminate harmful or foreign bodies, such as virus-infected cells, tumors, and allografts. After Ag recognition, activated CD8+ T cells release the contents of their cytotoxic granules into the extracellular space, where they are taken up by the target cell, and apoptosis is initiated (1). The cytotoxic granules contain a number of molecules, including the pore-forming protein, perforin, and serine proteases, known as granzymes. Perforin was originally thought to cause cell lysis by penetrating the target cell membrane (2), but recent work favors the theory that perforin functions by enabling the granzymes to escape from endosomes into the cytosol of the target cell (3, 4). Whatever its exact role, perforin is essential, because Ag-specific granule-mediated cytotoxicity is absent in perforin-deficient CD8+ T cells and NK cells (5).

Murine CD8+ T cells can express at least three granzymes, A, B, and C, which initiate distinct apoptotic pathways. Substrates for granzyme B include procaspase-3 (6) and Bid (7), whereas one of the primary targets of granzyme A is the SET complex (8). Although mice deficient for either granzyme A or B do not display the severity of phenotype observed in perforin-deficient mice (9, 10), a key role for these cytotoxic molecules is demonstrated by the impaired ability of mice deficient in both these granzymes to control poxvirus infections (11). Recombinant granzyme C has been reported to induce an apoptotic pathway that is distinct from the pathways activated by granzymes A and B (12). However, its enzymatic targets have yet to be determined, and its biological relevance in vivo is unknown. Nevertheless, granzyme C mRNA can be expressed at comparable levels as granzyme B mRNA in activated CD8+ T cells in vitro (13), and it has been suggested that this and other orphan granzymes may provide a fail-safe mechanism against immune evasion by pathogens (14).

Although the perforin and granzyme genes are known to be inducible, because a T cell must be activated before the cytolytic molecules are expressed at the mRNA or protein levels (13, 15), the signals responsible for regulating gene expression have yet to be identified. The exceptions are studies examining the role of the cytokine, IL-2. IL-2 has been shown to up-regulate perforin and granzymes A and B in human PBL (16), and binding sites for the IL-2-induced transcription factor, STAT-5, have been located in the perforin promoter region (17, 18). However, it is not known whether regulation of perforin and granzyme genes by IL-2 is direct or a consequence of its other properties. Although IL-2 can initiate the expression of effector genes, such as IFN-γ (19), it also controls T cell growth and survival (20, 21). The signaling cascades that lead to these effects are not distinct, but consist of a complex network of kinases and transcription factors (22). For example, IL-2 activation of STAT-5a/b leads to an increase in the expression of the antiapoptotic gene bcl-2 (23) as well as the expression of cyclins, which are essential for cell cycle progression (24).

It is possible that the responses of perforin and the granzymes to IL-2 are due to enhanced cell viability or proliferation and not to direct induction of these effector genes. To determine how IL-2 regulates cytolytic gene expression, this study examined the contributions of survival and proliferation to the transcription of perforin and granzymes A, B, and C in naive CD8+ T cells.

Specific pathogen-free female C57BL/6 mice were obtained from the Animal Resource Center and used at 6–9 wk of age. Bcl-2-36 mice (25) were provided by Dr. A. Harris (The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia) and were maintained on a C57BL/6 background at the Queensland Institute of Medical Research. All animal studies were approved by the Queensland Institute of Medical Research animal ethics committee.

Cell suspensions were obtained by passing axillary, brachial, inguinal, and lumbar lymph nodes (LN)4 through stainless steel mesh, followed by Ficoll-Paque (Amersham Biosciences) separation. Cells were incubated with PE-conjugated anti-CD8α Ab (53.6) and FITC-conjugated anti-CD44 Ab (IM7.81; BD Pharmingen), then resuspended in balanced salt solution with 5% heat-inactivated FCS (CSL) and 1 μg/ml propidium iodide (Calbiochem). Viable cells were purified by FACS (MoFlo; DakoCytomation) based on CD8+ and CD44low (lowest 30%) expression; cells were >97% CD8+ on reanalysis. For some experiments, purified cells were labeled with CFSE (Molecular Probes), as described previously (26). CD8+ T cells were stimulated in an accessory cell-free system (27, 28). Briefly, 24-well plates (Falcon; BD Biosciences) were incubated overnight with protein G-purified hamster anti-CD3ε (145-2C11; 10 μg/ml), rat anti-CD8α (53.6; 10 μg/ml), and rat anti-CD11a (I21/7.7; 5 μg/ml) mAb. Plates were then washed three times in PBS. Purified CD8+ T cells (2 × 104) were cultured in 2 ml of growth medium (modified DMEM supplemented with 50 μM 2-ME, 216 mg/l l-glutamine, and 10% heat-inactivated FCS) (27) containing various concentrations of human rIL-2 (National Institutes of Health). In some experiments anti-murine IL-2 mAb (protein G purified from S4B6 supernatant) was added to growth medium at 10 μg/ml. Growth medium was changed every 24 h, which included rIL-2 and anti-murine IL-2 mAb where relevant. At 3 or 4 days of culture, cells were harvested, and viable cells were purified by Ficoll-Paque separation. Cells previously stained with CFSE were separated by FACS according to division peaks. ModFit (Verity Software House) was used to determine the number of cells in each division peak.

RNA was isolated from FACS-purified or Ficoll-purified samples of 1 × 104 cells by mixing with TRIzol reagent (Invitrogen Life Technologies) and freezing on dry ice, followed by chloroform extraction, isopropanol precipitation, and washing with ethanol according to the manufacturer’s instructions. Isolated RNA pellets were dissolved in 20 μl of H2O and incubated at 65°C for 2 min. cDNA synthesis mix was added to samples, giving the following final concentrations: buffer (50 mM KCl, 10 mM Tris-HCl (pH 8), and 2 mM MgCl2), 6 mM MgCl2, 2.4 nM oligo(dT) (Roche), 500 μM dNTPs (Promega), 1 mM DTT, 20 U of RNaseOUT (Invitrogen Life Technologies), and 5 U of avian myeloblastosis virus reverse transcriptase (Promega). Samples were incubated for 2 h at 42°C. Samples were then diluted to a final volume of 100 μl in H2O and stored at −20°C.

cDNA was quantified using real-time PCR analysis. PCR mix (10 μl) was added to cDNA samples (5 μl; equivalent to cDNA from 500 cells) to give the following final concentrations: 5 mM MgCl2; 200 μM dNTPs; 50 nM 5′ primer, 3′ primer, and internal probe; and 0.3 U of platinum Taq (Invitrogen Life Technologies). Primers and probes were designed using the VectorNTI program (InfoMax) or Primer3 online software (Whitehead Institute for Biomedical Research). PCR products spanned intron-exon junctions to avoid genomic DNA amplification (see Table I). All probes were labeled with Black Hole Quencher-1 at the 3′ end, and either FAM for IFN-γ, perforin, granzyme A, granzyme B, and granzyme C or 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein for β2-microglobulin (β2M) and CD3ε, at the 5′ end. β2M and CD3ε were coamplified with genes using FAM-labeled probes. Products were amplified using a Rotor-Gene 3000 (Corbett Research) under the following conditions: 95°C for 2 min and 95°C for 5 s, followed by 60°C for 30 s for 40 cycles. Known copy numbers of cloned cDNA were used to generate a standard curve for each gene. Absolute cDNA copy numbers for each gene were extrapolated from the respective standard curve and then expressed as a ratio to the number of β2M cDNA molecules detected in the same sample (β2M units = (target gene copy number/β2M copy number) × 1000). Triplicate cDNA samples were generated for each culture condition, and each cDNA sample was then assayed in duplicate. The results were reported in β2M units and represent the mean of all replicates.

Table I.

Real-time PCR primer and probe sequences

GeneOligonucleotideSequence
Murine β2M 5′ Primer 5′-TCTTTCTGGTGCTTGTCTCAC-3′ 
 3′ Primer 5′-GTTCGGCTTCCCATTCTC-3′ 
 Probe 5′-JOE-CGGCCTGTATGCTATCCAGAAAACC-BHQ-1–3′ 
Murine CD3ε 5′ Primer 5′-AAGTAATGAGCTGGCTGC-3′ 
 3′ Primer 5′-CTACACTGGTTCCTGAGATG-3′ 
 Probe 5′-JOE-CAGGACGATGCCGAGAACATT-BHQ-1–3′ 
Murine IFN-γ 5′ Primer 5′-GGATGCATTCATGAGTATTGC-3′ 
 3′ Primer 5′-GTGGACCACTCGGATGAG-3′ 
 Probe 5′-FAM-TGAGGTCAACAACCCACAGGTCC-BHQ-1–3′ 
Murine perforin 5′ Primer 5′-GAGAAGACCTATCAGGACCA-3′ 
 3′ Primer 5′-AGCCTGTGGTAAGCATG-3′ 
 Probe 5′-FAM-AACCTCCACTCCACCTTGACTTCA-BHQ-1–3′ 
Murine granzyme A 5′ Primer 5′-TTTCATCCTGTAATTGGACTAA-3′ 
 3′ Primer 5′-GCGATCTCCACACTTCTC-3′ 
 Probe 5′-FAM-CAGCCCTCTGCTATGTGATGGTATT-BHQ-1–3′ 
Murine granzyme B 5′ Primer 5′-CCTCCTGCTACTGCTGAC-3′ 
 3′ Primer 5′-GTCAGCACAAAGTCCTCTC-3′ 
 Probe 5′-FAM-CCCTACATGGCCTTACTTTCGATCA-BHQ-1–3′ 
Murine granzyme C 5′ Primer 5′-TTCTCCTGACCCTACTTCTG-3′ 
 3′ Primer 5′-TGTTAGCACGAATTTGTCTC-3′ 
 Probe 5′-FAM-ATGTTCTGCGGAGGCTTCCTG-BHQ-1–3′ 
GeneOligonucleotideSequence
Murine β2M 5′ Primer 5′-TCTTTCTGGTGCTTGTCTCAC-3′ 
 3′ Primer 5′-GTTCGGCTTCCCATTCTC-3′ 
 Probe 5′-JOE-CGGCCTGTATGCTATCCAGAAAACC-BHQ-1–3′ 
Murine CD3ε 5′ Primer 5′-AAGTAATGAGCTGGCTGC-3′ 
 3′ Primer 5′-CTACACTGGTTCCTGAGATG-3′ 
 Probe 5′-JOE-CAGGACGATGCCGAGAACATT-BHQ-1–3′ 
Murine IFN-γ 5′ Primer 5′-GGATGCATTCATGAGTATTGC-3′ 
 3′ Primer 5′-GTGGACCACTCGGATGAG-3′ 
 Probe 5′-FAM-TGAGGTCAACAACCCACAGGTCC-BHQ-1–3′ 
Murine perforin 5′ Primer 5′-GAGAAGACCTATCAGGACCA-3′ 
 3′ Primer 5′-AGCCTGTGGTAAGCATG-3′ 
 Probe 5′-FAM-AACCTCCACTCCACCTTGACTTCA-BHQ-1–3′ 
Murine granzyme A 5′ Primer 5′-TTTCATCCTGTAATTGGACTAA-3′ 
 3′ Primer 5′-GCGATCTCCACACTTCTC-3′ 
 Probe 5′-FAM-CAGCCCTCTGCTATGTGATGGTATT-BHQ-1–3′ 
Murine granzyme B 5′ Primer 5′-CCTCCTGCTACTGCTGAC-3′ 
 3′ Primer 5′-GTCAGCACAAAGTCCTCTC-3′ 
 Probe 5′-FAM-CCCTACATGGCCTTACTTTCGATCA-BHQ-1–3′ 
Murine granzyme C 5′ Primer 5′-TTCTCCTGACCCTACTTCTG-3′ 
 3′ Primer 5′-TGTTAGCACGAATTTGTCTC-3′ 
 Probe 5′-FAM-ATGTTCTGCGGAGGCTTCCTG-BHQ-1–3′ 

Cells were washed with PBS and incubated in 0.2% saponin in PBS on ice for 10 min, then with a previously optimized concentration of R-PE-conjugated mouse anti-human granzyme B Ab GB12 or control R-PE-conjugated murine IgG1 (Caltag Laboratories) for an additional 50 min on ice. Cells were then washed, resuspended in balanced salt solution with 2% heat-inactivated FCS, and analyzed using a FACSCalibur and CellQuest Pro version 5.1.1 software (BD Biosciences) with forward and side scatter gates set to include both small lymphocytes and blasts.

Cells of the FcR+ mastocytoma line P815 were labeled with Na51CrO4 (Amersham Biosciences) for 60 min at 37°C and washed twice. Labeled target cells (2–5 × 103) were incubated for 4–5 h at 37°C with serial dilutions of T cells and 1 μg/ml anti-CD3 Ab (redirected assay) in 200 μl of growth medium in round-bottom, 96-well plates. Harvested supernatants were dried onto 96-well solid Lumaplates (Packard), and radioactivity was counted in a TopCount microplate scintillation counter (Packard). Spontaneous lysis of target cells was typically <10%, and differences in sample release, performed in triplicate, were within 5%. Total 51Cr release from target cells was obtained by lysis in 1% SDS. The percent specific lysis was calculated by the following formula: 100 × ((sample cpm − spontaneous release cpm)/(total release cpm − spontaneous release cpm)).

Previous studies examining the responses of perforin and the granzyme genes to IL-2 have used semiquantitative Northern blot analysis, often with cell lines or heterogeneous leukocyte populations (16, 18, 29). To investigate whether IL-2 can induce perforin and granzyme gene expression in naive lymphocytes, CD8+ LN cells of naive (CD44low) phenotype were cultured in an accessory cell-free culture system with immobilized mAb to CD3, CD8, and CD11a and varying concentrations of human rIL-2. This well-defined culture system was used to limit the influence of other cytokines and costimulatory molecules on cytolytic gene expression. Culture medium was replaced daily to reduce the accumulation of potentially stimulatory endogenous products, and IL-2 was supplemented daily to overcome IL-2 exhaustion in cultures where the cytokine concentration was limiting. On days 3 and 4, live cells were analyzed for the expression of a panel of genes by real-time PCR under conditions that allowed reliable quantification of one cDNA copy per cell and detection of one cDNA copy per 10 cells. Identity of the PCR products was confirmed using fluorescent probes, which was particularly important for the granzyme genes because they share a high degree of sequence similarity at the cDNA level (14).

Dose-response curves for perforin and granzymes A, B, and C are shown in Fig. 1,A. Quantification was achieved by comparison with known copy numbers of cloned standards and normalization against the housekeeping gene, β2M. To ensure that β2M was a suitable reference gene, results were compared with a second gene, CD3ε. Fig. 1 shows that CD3ε expression levels were constant at both time points and all IL-2 concentrations. IFN-γ gene expression was used as a positive control in this study, because IL-2 is a known regulator of IFN-γ in cytolytic T cells (19, 30). IFN-γ expression levels increased >100-fold across the IL-2 concentration gradient, and the dose-response curves for both time points were similar.

FIGURE 1.

Perforin and granzyme A, B, and C genes are responsive to IL-2 in a dose-dependent manner. A, CD8+CD44low LN cells were cultured with immobilized mAb to CD3, CD8, and CD11a and varying concentrations of rIL-2 for 3 (○) or 4 (▪) days before cDNA isolation. Gene expression was assayed by real-time PCR. The threshold of assay detection is indicated by the broken line. ND, not detected. Due to low cell viability, no data were obtained for cells grown in the absence of IL-2. B, Cells cultured in 20 (▪), 2 (▵), or 0.2 (•) U/ml IL-2 for 4 days were assayed for cytolytic activity against anti-CD3 mAb-coated, 51Cr-labeled P815 target cells. Cytolytic activity in the absence of the bridging Ab was at background levels (data not shown). C, The number of viable cells harvested on days 3 (○) and 4 (▪) was determined using trypan blue exclusion. Data represent the average from two to six pooled culture wells.

FIGURE 1.

Perforin and granzyme A, B, and C genes are responsive to IL-2 in a dose-dependent manner. A, CD8+CD44low LN cells were cultured with immobilized mAb to CD3, CD8, and CD11a and varying concentrations of rIL-2 for 3 (○) or 4 (▪) days before cDNA isolation. Gene expression was assayed by real-time PCR. The threshold of assay detection is indicated by the broken line. ND, not detected. Due to low cell viability, no data were obtained for cells grown in the absence of IL-2. B, Cells cultured in 20 (▪), 2 (▵), or 0.2 (•) U/ml IL-2 for 4 days were assayed for cytolytic activity against anti-CD3 mAb-coated, 51Cr-labeled P815 target cells. Cytolytic activity in the absence of the bridging Ab was at background levels (data not shown). C, The number of viable cells harvested on days 3 (○) and 4 (▪) was determined using trypan blue exclusion. Data represent the average from two to six pooled culture wells.

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Minimal up-regulation of perforin gene expression by IL-2 was observed on day 3. However, after 4 days of culture, perforin displayed a 10-fold increase in expression levels across the IL-2 gradient, consistent with previous reports that demonstrated sensitivity of perforin to IL-2 (16, 18). All three granzyme genes responded to a rise in IL-2 concentration, particularly granzyme B, whose expression increased 10,000-fold. Granzyme B was previously shown to reach maximum expression levels by day 3 in this culture system, whereas the induction of granzymes A and C was delayed (13). This result was reproduced in this study, with granzyme B expression rising 5- to 10-fold from days 3 to 4, whereas granzyme A and C expression levels rose 50- to 100-fold. To assess the functional effects of varying IL-2 concentrations, CD8+ T cells were tested for their cytolytic activity using a redirected 51Cr release assay. Fig. 1,B shows that although T cells cultured in 20 U/ml IL-2 demonstrated significant cytolytic activity, cells cultured in lower IL-2 concentrations had poor or no detectable lytic ability. Together, the results demonstrate that the perforin and granzyme A, B, and C genes in naive CD8+ T cells all respond to IL-2 in a dose-dependent manner, and that these responses are associated with marked effects on cytolytic activity. However, interpretation of these results is confounded by the poor survival and proliferation of cells cultured in low IL-2 concentrations. Fig. 1 C shows that the numbers of recovered cells were significantly lower at IL-2 concentrations <2 U/ml, particularly by day 4. These cultures contained a high proportion of dying cells, and the remaining live cells were small in size (data not shown).

Effects of IL-2 on cell viability were overcome by using T cells transgenic (Tg) for bcl-2, one of the anti-apoptotic molecules up-regulated by signaling through IL-2Rβ (31, 32). T cells overexpressing Bcl-2 are factor independent in vitro (33). Fig. 2 A shows that although the viability of wild-type (WT) T cells was <50% by day 4 in cultures with <0.6 U/ml IL-2, 80–90% of bcl-2 Tg T cells remained viable. At higher IL-2 concentrations, the viabilities of WT and bcl-2 Tg cells were comparable.

FIGURE 2.

The bcl-2 Tg T cells have enhanced survival, but not enhanced proliferation, in limiting IL-2 concentrations. A, WT (○) and bcl-2 Tg (▪) T cells were cultured as described in Fig. 1. Cell viability was measured on days 3 and 4 by propidium iodide uptake. B, WT and bcl-2 Tg T cells were stained with CFSE, and the percentage of cells in each division peak was determined on days 3 and 4. The broken lines indicate the major division peak of Bcl-2 Tg T cells cultured in the absence of IL-2.

FIGURE 2.

The bcl-2 Tg T cells have enhanced survival, but not enhanced proliferation, in limiting IL-2 concentrations. A, WT (○) and bcl-2 Tg (▪) T cells were cultured as described in Fig. 1. Cell viability was measured on days 3 and 4 by propidium iodide uptake. B, WT and bcl-2 Tg T cells were stained with CFSE, and the percentage of cells in each division peak was determined on days 3 and 4. The broken lines indicate the major division peak of Bcl-2 Tg T cells cultured in the absence of IL-2.

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To confirm that the bcl-2 transgene only enhanced survival and did not affect proliferation, T cells were stained with CFSE, and their division profile was measured after culture with varying IL-2 concentrations (Fig. 2 B). Only the bcl-2 Tg T cells were cultured in the absence of IL-2 due to the inability of WT cells to survive. In the presence of IL-2, the proliferative rate of bcl-2 Tg T cells was equivalent to that of WT cells. Increasing IL-2 concentrations increased the proliferative rate for bcl-2 Tg and WT T cells, although these differences were minimal at IL-2 concentrations ≥2 U/ml.

To determine whether the perforin and granzyme genes in bcl-2 Tg T cells displayed the same dose-dependent response to IL-2 as WT cells, CD8+CD44lowbcl-2 Tg and WT T cells were cultured as described above. An anti-murine IL-2 mAb (S4B6) was added to some cultures to inhibit the effects of endogenous IL-2 secretion. The poor viability of WT cells grown in limiting IL-2 concentrations was exacerbated by the anti-murine IL-2 mAb, presumably because it inhibited the small amount of IL-2 secreted by these cells. Therefore, no data were obtained for WT cells cultured in 0.2 U/ml IL-2 with the anti-murine IL-2 mAb.

A comparison of the bcl-2 Tg and WT dose-response curves showed an equivalent response for all genes (Fig. 3). Results from the bcl-2 Tg cultures show that IL-2 is not necessary for the expression of either IFN-γ or perforin, because significant cDNA levels were detectable when cells were stimulated in the absence of IL-2, consistent with previous findings (18, 34). However, granzymes A, B, and C all required IL-2 for gene induction, because cDNA was undetectable in T cells cultured without IL-2. The addition of the anti-murine IL-2 mAb further highlighted the sensitivity of the granzyme genes to IL-2. In these cultures, granzyme mRNA from bcl-2 Tg T cells was not detectable in the presence of 0.2 U/ml IL-2, presumably because the Ab lowered levels of endogenous cytokine below the threshold concentration required to induce gene expression. Similar results were obtained when intracellular granzyme B protein expression was measured by flow cytometry (Fig. 4). Both the frequency of granzyme B-containing WT and bcl-2 Tg T cells and the average granzyme B level per positive cell declined with decreasing IL-2 concentration. Because perforin and granzyme expression was no higher in bcl-2 Tg T cells than in WT T cells at limiting IL-2 concentrations, we conclude that the up-regulation of perforin and granzyme expression by IL-2 is not due its anti-apoptotic effects.

FIGURE 3.

The regulation of perforin and the granzyme genes by IL-2 is independent of IL-2-induced survival. WT (○) and bcl-2 Tg (▪) T cells were cultured as described in Fig. 1. Anti-murine IL-2 mAb (S4B6) was added to the indicated cultures. On days 3 and 4, live cells were harvested, and cDNA was isolated and analyzed by real-time PCR. The threshold of assay detection is indicated by the dashed line. ND, not detected. Due to low cell viability, no data were obtained for WT cells that had been cultured in 0.2 U/ml IL-2 with the anti-murine IL-2 mAb.

FIGURE 3.

The regulation of perforin and the granzyme genes by IL-2 is independent of IL-2-induced survival. WT (○) and bcl-2 Tg (▪) T cells were cultured as described in Fig. 1. Anti-murine IL-2 mAb (S4B6) was added to the indicated cultures. On days 3 and 4, live cells were harvested, and cDNA was isolated and analyzed by real-time PCR. The threshold of assay detection is indicated by the dashed line. ND, not detected. Due to low cell viability, no data were obtained for WT cells that had been cultured in 0.2 U/ml IL-2 with the anti-murine IL-2 mAb.

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FIGURE 4.

IL-2 enhances the expression of granzyme B protein in WT and bcl-2 Tg T cells. CD8+CD44low LN T cells from WT (left panels) and bcl-2 Tg (right panels) mice were cultured as described in Fig. 1. After 4 days, cells were harvested, permeabilized, incubated with anti-granzyme B Ab (shaded histograms) or isotype control Ab (open histograms), and analyzed by FACS. The percentage of granzyme B-positive cells is indicated within each panel. Due to low cell viability, no data were obtained for WT cells cultured in the absence of IL-2.

FIGURE 4.

IL-2 enhances the expression of granzyme B protein in WT and bcl-2 Tg T cells. CD8+CD44low LN T cells from WT (left panels) and bcl-2 Tg (right panels) mice were cultured as described in Fig. 1. After 4 days, cells were harvested, permeabilized, incubated with anti-granzyme B Ab (shaded histograms) or isotype control Ab (open histograms), and analyzed by FACS. The percentage of granzyme B-positive cells is indicated within each panel. Due to low cell viability, no data were obtained for WT cells cultured in the absence of IL-2.

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It has been reported that there is a direct relationship between cell cycle progression and the initiation of cytokine gene transcription in T cells (35, 36). It is therefore possible that IL-2 regulation of the perforin and granzyme genes is directly linked to IL-2-induced proliferation. The relationship between gene expression and division number was examined by labeling cells with CFSE before culture and then purifying them on the basis of division number before RNA isolation (Fig. 5). The proliferation profiles of these cultures were similar to those shown in Fig. 2,B, with an increase in the IL-2 concentration correlating to an increase in the proliferative rate (data not shown). With the exception of the reference gene, CD3ε, the average expression level of each gene increased with the rise in IL-2 concentration, as shown in Fig. 1. However, these increases in expression levels did not correlate with division progression. Instead, the expression levels of some genes, particularly granzyme C, increased with time. One exception is the division-dependent decline in the low levels of granzyme C mRNA seen on day 4 in cultures with 0.2 U/ml IL-2; this result is likely to have reflected impaired viability in the most rapidly dividing cells at this limiting IL-2 concentration. Overall, these results demonstrate that the responses of perforin and the granzyme genes to IL-2 were not due to differences in division rates between cultures.

FIGURE 5.

The regulation of perforin and the granzyme genes is independent of IL-2-induced proliferation. CD8+CD44low LN cells were stained with CFSE and then cultured under the conditions described in Fig. 1. Cells were purified from CFSE peaks corresponding to division cycles 2–5 in day 3 cultures (○) and division cycles 5–8 in day 4 cultures (▪). cDNA was isolated from the purified cells and analyzed by real-time PCR. The threshold of assay detection is indicated by the dashed line. ND, not detected. Due to insufficient cell numbers, no data were obtained for cells that had undergone five divisions from day 3 cultures with 0.2 U/ml IL-2.

FIGURE 5.

The regulation of perforin and the granzyme genes is independent of IL-2-induced proliferation. CD8+CD44low LN cells were stained with CFSE and then cultured under the conditions described in Fig. 1. Cells were purified from CFSE peaks corresponding to division cycles 2–5 in day 3 cultures (○) and division cycles 5–8 in day 4 cultures (▪). cDNA was isolated from the purified cells and analyzed by real-time PCR. The threshold of assay detection is indicated by the dashed line. ND, not detected. Due to insufficient cell numbers, no data were obtained for cells that had undergone five divisions from day 3 cultures with 0.2 U/ml IL-2.

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We show in this study that IL-2 is a potent regulator of perforin and the granzyme genes during primary activation of CD8+ T cells. Previously, the pleiotropic properties of IL-2 made it difficult to dissociate the effects of IL-2 on T cell survival and proliferation from its effects on the expression of genes for cytolytic mediators and other products. This issue was addressed in the present study in two ways, demonstrating that the IL-2 signaling pathway responsible for the regulation of these genes is distinct from the pathways responsible for cell cycle progression and protection from apoptosis.

First, we used T cells from the bcl-2 Tg mouse strain. Despite their lack of dependence on IL-2 for survival in vitro, these Tg T cells exhibited marked IL-2 dose-dependent increases in the expression of perforin and granzyme A, B, and C mRNA and granzyme B protein comparable to those observed in WT cells, indicating that cytolytic gene induction is independent of IL-2-mediated cell survival. The use of bcl-2 Tg T cells also revealed that the addition of IL-2 was essential for the induction of detectable levels of granzyme A, B, and C mRNA, whereas TCR-mediated signaling alone was sufficient for perforin and IFN-γ expression. Effects on granzyme B were particularly notable; although undetectable in the absence of IL-2, granzyme B mRNA reached levels 106-fold above the detection threshold at optimal IL-2 concentrations. Strong enhancement of cytolytic activity was seen in assays with the perforin/granzyme B-sensitive target cell, P815.

Second, using the dye CFSE to separate cells on the basis of division number, we found that the enhancement of perforin and granzyme expression by IL-2 was unrelated to cell proliferation after the first division. Other work in our laboratory has shown that most of the mRNA species assayed in this study are detected in both undivided and divided cells by day 2 in CD8+ T cells activated in this culture system, indicating that the onset of expression does not require division; by comparison, the IL-4-induced induction of IL-4 expression is highly division dependent as described by others (35, 36) (our unpublished observations). The division independence of perforin, granzyme, and IFN-γ expression in the present study, therefore, mainly reflects the early induction of these genes under the strong activation conditions used in this study. The data also show that once these genes are induced, the enhancement of their expression by IL-2 does not depend on other division-linked events.

The upstream regulatory sequences of perforin and granzyme B contain a number of sites that could mediate regulation by IL-2 in CD8+ T cells directly rather than as an indirect consequence of survival or proliferation. Phosphorylation of IL-2Rβ activates the transcription factors STAT5a/b, for which binding sites have been identified upstream of the human and mouse perforin genes (17, 18); IL-2-activated Stat5 has been shown to induce the expression of a number of genes, including perforin, IFN-γ, and IL-2Rα, directly after IL-2 stimulation (18, 37, 38). In addition, IL-2 activation of NF-κB leads to binding of this transcription factor to the upstream enhancer of the perforin promoter in NK cells (39), and both the perforin and granzyme B promoters contain sequences that can bind AP-1 (40, 41), which is also phosphorylated upon signaling through IL-2Rβ (22). Recently, the novel transcription factor, eomesodermin, was identified in activated CD8+ T cells and was shown to drive perforin, granzyme B, and IFN-γ expression when ectopically expressed in Th2 CD4+ cells; dominant negative eomesodermin impaired granzyme B expression and cytolytic function in CD8+ T cells (42). It will be important to assess whether IL-2 acts upstream or downstream of this proposed master regulator of CD8+ effector T cell differentiation.

Our finding that IL-2 is required for granzyme A, B, and C expression under defined conditions in vitro does not rule out the possibility that other molecules can serve this function in vitro and in vivo. Mice deficient in IL-2 retain the ability to raise CTL responses against many viruses, tumors, and allografts (43, 44), although impairment has been reported in certain conditions (45). Candidates to compensate for the absence of IL-2 include cytokines whose receptors share the common γ-chain with the IL-2R, particularly IL-15, whose receptor also shares the IL-2R β-chain; several of these cytokines have been shown to enhance or contribute to CTL responses under some conditions in vitro and in vivo (46, 47, 48). However, although the common γ-chain receptor-sharing cytokine IL-4 can enhance CTL activity in some circumstances (47), its effects on perforin and granzyme gene expression in the system used in this study are distinct from those of IL-2. We found that activation of naive CD8+ T cells in the presence of IL-2 and IL-4 leads to generation of poorly cytolytic CD8low effectors in which levels of perforin, granzyme B, and granzyme C mRNA and perforin and granzyme B protein are markedly lower than in cells activated without IL-4 (49) (our unpublished observations).

Many studies have demonstrated beneficial effects of IL-2 on CTL responses in vivo, for example, in mouse models of virus or intracellular bacterial infection where CTL play roles in direct elimination of infected cells and control of pathogen spread between cells (50, 51, 52). In humans, IL-2 has been used extensively with positive results as an immunotherapeutic agent in the treatment of malignancy and some infections by both direct administration and expression in cellular vaccines (53, 54, 55). However, although leukocyte numbers and phenotypes are usually measured, few human trials assess the effects of IL-2 on CTL activity. Even when CTL are assayed, it is difficult in both human and animal studies to distinguish direct effects of IL-2 on cytolytic function from its effects on lymphocyte expansion. This is particularly pertinent to studies of IL-2 treatment in HIV infection. Because IL-2 is generally used to reconstitute the immune system, most reports from clinical trials in HIV infection focus on its effect on CD4+ T cell numbers (56, 57). However, CD8+ T cells are activated in this infection, and Lieberman et al. (58) have reported that CD8+ T cells kill HIV-infected lymphocytes predominantly by granule-mediated cytotoxicity. It has also been reported that although freshly isolated CD8+ T cells were poorly cytolytic against HIV-infected targets, overnight exposure to IL-2 restored cytolytic function to a level that could not be explained by clonal expansion of virus-specific T cells (59). Our data raise the possibility that this response reflected a direct effect of IL-2 on the expression of cytolytic mediator genes.

We have shown in this study that IL-2 regulates perforin and granzyme gene expression directly and independently of its effects on CD8+ T cell survival and proliferation. Although it is well documented that IL-2 enhances cytolytic function and the expression of some cytolytic mediators, many previous studies could not determine whether these effects were simply the consequence of T cell dependence on IL-2 for survival or expansion. Our data indicate that IL-2 may mediate its immunotherapeutic role not only by expanding activated CD8+ and CD4+ T cell numbers, but also by directly increasing perforin and granzyme gene expression in cytolytic CD8+ T cells. The ability of IL-2 to enhance perforin and granzyme expression directly may be physiologically important in circumstances where other signals counteract the effects of IL-2 on lymphocyte expansion or would otherwise down-regulate cytolytic gene expression.

We thank Paula Hall and Grace Chojnowski for their skillful assistance with FACS, Dr. Alan Harris (Walter and Eliza Hall Institute of Medical Research) and Dr. Christian Engwerda (Queensland Institute of Medical Research) for generously providing Bcl-2-36 mice, and the National Institutes of Health AIDS Research and Reference Reagent Program for the gift of rIL-2.

The authors have no financial conflict of interest.

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 the National Health and Medical Research Council of Australia and the Australian Government’s Cooperative Research Centers Program.

4

Abbreviations used in this paper: LN, lymph node; β2M, β2-microglobulin; Tg, transgenic; WT, wild type.

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