IL-2 Regulates Perforin and Granzyme Gene
Expression in CD8 + T Cells Independently of
Its Effects on Survival and Proliferation
This information is current as
of June 15, 2017.
Michelle L. Janas, Penny Groves, Norbert Kienzle and Anne
Kelso
J Immunol 2005; 175:8003-8010; ;
doi: 10.4049/jimmunol.175.12.8003
http://www.jimmunol.org/content/175/12/8003
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References
The Journal of Immunology
IL-2 Regulates Perforin and Granzyme Gene Expression in
CD8ⴙ T Cells Independently of Its Effects on Survival
and Proliferation1
Michelle L. Janas,2 Penny Groves, Norbert Kienzle, and Anne Kelso3
G
ranule-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
Cooperative Research Center for Vaccine Technology and Queensland Institute of
Medical Research, Brisbane, Australia
Received for publication January 18, 2005. Accepted for publication September
29, 2005.
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.
2
Current address: The Babraham Institute, Babraham Research Campus, Cambridge,
U.K. CB24AT.
3
Address correspondence and reprint requests to Dr. Anne Kelso, Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, Queensland 4029,
Australia. E-mail address: [email protected]
Copyright © 2005 by The American Association of Immunologists, Inc.
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-␥ (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.
0022-1767/05/$02.00
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Perforin and the serine protease granzymes are key effectors of CD8ⴙ 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 CD8ⴙ 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 CD8ⴙ 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-␥ 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 CD8ⴙ T cells independently of its effects on cell survival and proliferation. The Journal of Immunology, 2005,
175: 8003– 8010.
8004
PERFORIN AND GRANZYME REGULATION BY IL-2
Table I. Real-time PCR primer and probe sequences
Gene
Murine ␤2M
Murine CD3␧
Murine IFN-␥
Murine perforin
Murine granzyme A
Murine granzyme B
Murine granzyme C
Sequence
5⬘ Primer
3⬘ Primer
Probe
5⬘ Primer
3⬘ Primer
Probe
5⬘ Primer
3⬘ Primer
Probe
5⬘ Primer
3⬘ Primer
Probe
5⬘ Primer
3⬘ Primer
Probe
5⬘ Primer
3⬘ Primer
Probe
5⬘ Primer
3⬘ Primer
Probe
5⬘-TCTTTCTGGTGCTTGTCTCAC-3⬘
5⬘-GTTCGGCTTCCCATTCTC-3⬘
5⬘-JOE-CGGCCTGTATGCTATCCAGAAAACC-BHQ-1–3⬘
5⬘-AAGTAATGAGCTGGCTGC-3⬘
5⬘-CTACACTGGTTCCTGAGATG-3⬘
5⬘-JOE-CAGGACGATGCCGAGAACATT-BHQ-1–3⬘
5⬘-GGATGCATTCATGAGTATTGC-3⬘
5⬘-GTGGACCACTCGGATGAG-3⬘
5⬘-FAM-TGAGGTCAACAACCCACAGGTCC-BHQ-1–3⬘
5⬘-GAGAAGACCTATCAGGACCA-3⬘
5⬘-AGCCTGTGGTAAGCATG-3⬘
5⬘-FAM-AACCTCCACTCCACCTTGACTTCA-BHQ-1–3⬘
5⬘-TTTCATCCTGTAATTGGACTAA-3⬘
5⬘-GCGATCTCCACACTTCTC-3⬘
5⬘-FAM-CAGCCCTCTGCTATGTGATGGTATT-BHQ-1–3⬘
5⬘-CCTCCTGCTACTGCTGAC-3⬘
5⬘-GTCAGCACAAAGTCCTCTC-3⬘
5⬘-FAM-CCCTACATGGCCTTACTTTCGATCA-BHQ-1–3⬘
5⬘-TTCTCCTGACCCTACTTCTG-3⬘
5⬘-TGTTAGCACGAATTTGTCTC-3⬘
5⬘-FAM-ATGTTCTGCGGAGGCTTCCTG-BHQ-1–3⬘
Materials and Methods
Mice
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 preparation and culture
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-CD8␣ 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 ␮g/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-CD3⑀ (145-2C11; 10 ␮g/ml), rat anti-CD8␣ (53.6;
10 ␮g/ml), and rat anti-CD11a (I21/7.7; 5 ␮g/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 ␮M
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 ␮g/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.
Cytoplasmic RNA extraction and cDNA synthesis
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 ␮l of H2O and
4
Abbreviations used in this paper: LN, lymph node; ␤2M, ␤2-microglobulin; Tg,
transgenic; WT, wild type.
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 ␮M 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 ␮l in H2O and stored
at ⫺20°C.
Real-time PCR analysis
cDNA was quantified using real-time PCR analysis. PCR mix (10 ␮l) was
added to cDNA samples (5 ␮l; equivalent to cDNA from 500 cells) to give
the following final concentrations: 5 mM MgCl2; 200 ␮M 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-␥, perforin, granzyme A, granzyme B, and granzyme C or 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein for ␤2-microglobulin (␤2M) and
CD3⑀, at the 5⬘ end. ␤2M and CD3⑀ 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
␤2M cDNA molecules detected in the same sample (␤2M units ⫽ (target
gene copy number/␤2M 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 ␤2M units and
represent the mean of all replicates.
Intracellular granzyme B detection
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.
51
Cr release assay
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 ␮g/ml anti-CD3 Ab (redirected assay) in 200 ␮l
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Oligonucleotide
The Journal of Immunology
8005
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)).
Results
Perforin and granzyme A, B, and C genes are responsive to IL2 in a dose-dependent manner
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 (E) or 4 (f) 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 (f), 2 (‚), or 0.2 (F) 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 (E) and 4 (f) was determined using trypan blue exclusion. Data
represent the average from two to six pooled culture wells.
terpretation of these results is confounded by the poor survival and
proliferation of cells cultured in low IL-2 concentrations. Fig. 1C
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).
Regulation of perforin and granzyme genes by IL-2 is
independent of IL-2-induced survival
Effects of IL-2 on cell viability were overcome by using T cells
transgenic (Tg) for bcl-2, one of the anti-apoptotic molecules upregulated by signaling through IL-2R␤ (31, 32). T cells overexpressing Bcl-2 are factor independent in vitro (33). Fig. 2A shows
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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 cellfree culture system with immobilized mAb to CD3, CD8, and
CD11a and varying concentrations of human rIL-2. This welldefined 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. 1A. Quantification was achieved by comparison with
known copy numbers of cloned standards and normalization against
the housekeeping gene, ␤2M. To ensure that ␤2M was a suitable
reference gene, results were compared with a second gene, CD3⑀. Fig.
1 shows that CD3⑀ expression levels were constant at both time points
and all IL-2 concentrations. IFN-␥ gene expression was used as a
positive control in this study, because IL-2 is a known regulator of
IFN-␥ in cytolytic T cells (19, 30). IFN-␥ expression levels increased
⬎100-fold across the IL-2 concentration gradient, and the dose-response curves for both time points were similar.
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. 1B 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, in-
8006
PERFORIN AND GRANZYME REGULATION BY IL-2
Regulation of perforin and the granzyme genes by IL-2 is
independent of IL-2-induced proliferation
FIGURE 2. The bcl-2 Tg T cells have enhanced survival, but not enhanced proliferation, in limiting IL-2 concentrations. A, WT (E) and bcl-2
Tg (f) 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.
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.
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. 2B). 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
ⱖ2 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⫹CD44low bcl-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
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. 2B, 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, CD3⑀, 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.
Discussion
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
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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-␥ 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.
The Journal of Immunology
8007
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-2mediated 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-␥ 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
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FIGURE 3. The regulation of perforin and the granzyme genes by IL-2 is independent of IL-2-induced survival. WT (E) and bcl-2 Tg (f) 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.
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-␥ 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-2R␤ activates the
8008
PERFORIN AND GRANZYME REGULATION BY IL-2
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-␥, and IL-2R␣, directly after IL-2 stimulation (18, 37, 38). In addition, IL-2 activation of NF-␬B 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-2R␤ (22). Recently, the novel transcription
factor, eomesodermin, was identified in activated CD8⫹ T cells
and was shown to drive perforin, granzyme B, and IFN-␥ 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 ␥-chain with the IL-2R,
particularly IL-15, whose receptor also shares the IL-2R ␤-chain;
several of these cytokines have been shown to enhance or contrib-
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 (E) and division cycles 5– 8 in day 4 cultures (f).
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.
ute to CTL responses under some conditions in vitro and in vivo
(46 – 48). However, although the common ␥-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 –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 (53–55). However, although leukocyte numbers and phenotypes are usually measured,
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
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.
The Journal of Immunology
Acknowledgments
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.
Disclosures
The authors have no financial conflict of interest.
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PERFORIN AND GRANZYME REGULATION BY IL-2