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B Activation1
*
Immune and Gene Therapy Laboratory, Department of Oncology and Pathology, Cancer Center Karolinska,
Center for Genomic Research, and
Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden; and
CALAB Research/NOVA Medical Flow Cytometry Laboratory, Stockholm, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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release occurred earlier and at lower doses of exogenously
added H2O2 than required to induce apoptosis.
This suggests that there is a dose window of oxidative stress leading
to T cell unresponsiveness in the absence of apoptosis. The reduction
of Th1 cytokines, induced by H2O2, was
predominantly observed in memory/effector (CD45RO+) T cells
and correlated with a block in NF-
B activation. IL-10 production was
more profoundly influenced by low doses of H2O2
than IFN-, TNF-
, and IL-2. The influence of
H2O2 on production of IL-10 was not
significantly different between memory/activated and naive T cells.
These observations suggest that Th1 and Th2 cytokines are differently
regulated under conditions of oxidative stress. Taken together, these
findings may explain why Ag-experienced, CD45RO+, T cells
found in the tumor milieu are functionally
suppressed. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression and loss of Ag-specific T cell responses
(6, 7, 8). Monocytes recovered from human PBMC can inhibit
autologous NK cell-mediated cytotoxicity via secretion of
H2O2, leading to induction
of apoptosis (9). In addition, macrophage-derived NO
reduces the phosphorylation and activation of Janus kinase 3/STAT5
signal transduction proteins, thus inhibiting the proliferative
responses of T cells to IL-2 (10). The above findings lead
to the hypothesis that local
H2O2 or NO secretion by
activated macrophages may be one mechanism behind tumor-induced immune
suppression, as indicated by decreased signal transduction capacity,
poor effector functions, and apoptosis of T and NK cells infiltrating
the tumor lesion in cancer patients. To further investigate the role of oxidative stress in relation to tumor immunology, we have examined how oxidative stress affected different subsets of T cells, with a particular focus on the cytokine production by cells expressing the memory/activation marker CD45RO, as this marker is commonly expressed by T cells infiltrating into tumors (11, 12, 13). We have also attempted to delineate whether loss of T cell function after H2O2 exposure is a predecessor to apoptosis of T cells or an early consequence following the initiation of the apoptotic process.
Adding to the complexity,
H2O2 has been described to
act as a second messenger leading to activation of NF-B in T cells
(14, 15, 16). NF-B is activated by >150 different stimuli
and regulates the transcription of >150 target genes, among which 27
are cytokines including IL-2, IFN-, and TNF- (17).
Block of NF-B activation has been associated with T cell tolerance
and decreased capacity to produce effector cytokines such as IFN-,
IL-2, and IL-4 (18, 19). Thus, NF-B is an important
regulator of the human immune response. However, much of the evidence
supporting H2O2 as an
activator of NF-B stems from studies in a particular T cell line
(Jurkat Wurzburg), and the notion of ROS as an activator of NF-B has
been questioned by several recent reports (4, 5). Still,
it remains a central question whether hydrogen peroxide acts as an
activator or inhibitor of NF-B in freshly isolated human T cells and
what potential implications this has for the activity of the immune
system.
In this study, we demonstrate that a dose window of oxidative stress
exists where a state of unresponsiveness of T cells can be induced that
is not followed by apoptosis. We also show that IL-10 production is
much more sensitive to oxidative stress than the production of IL-2,
IFN-, and TNF-. Strikingly, the production of these Th1 cytokines
was more efficiently targeted in CD45RO+
(activated/memory) T cells, correlating with a more pronounced block of
NF-B activation, as opposed to CD45RO-
(naive) T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were obtained from buffy coats from healthy blood donors admitted to the blood bank at the Karolinska Hospital by Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden) gradient centrifugation for 20 min. CD3+ T cells were negatively selected using a MiniMACS kit (Miltenyi Biotec, Göteborg, Sweden). Selected cells were 95% T cells, as defined by FACS staining using a mAb against CD3. In some experiments, CD45RO+ cells were isolated using an anti-CD45RO MiniMACS kit (Miltenyi Biotec). The positive fraction always contained 95% CD45RO+ cells, and the negative fraction contained <15% CD45RO+ cells. Cells were resuspended at 1 x 106/ml in AIM-V (Life Technologies, Auckland, NZ) and then subjected to H2O2 exposure for 10 min. After exposure, cells were washed once and resuspended at 1 x 106 cells/ml before they were stimulated and tested in the various assays described below.
Electrophoretic mobility shift assay
PBMC were exposed to hydrogen peroxide, washed, and stimulated
with OKT-3 for 4 or 12 h. After two washes, nuclear extracts
(5–10 µg) were prepared and preincubated with 1 µg poly(dl-dC) in
binding buffer (10 mM Tris, 50 mM NaCl, 20% glycerol, 1 M DTT, and 0.5
mM EDTA) for 5 min at room temperature. Approximately 20,000 cpm of
32P-labeled DNA probe of the class I MHC NF-B
site was then added and allowed to bind for 20 min. The complexes were
separated by 5% PAGE and detected by autoradiography.
Apoptosis analysis
Apoptosis measurements were performed using a flow cytometry-based method according to the manufacturer’s protocol (Nexin Research, Kattendijke, The Netherlands). Cells were stained for annexin V and the vital die 7-amino actinomycin D (7-AAD) (Sigma, Stockholm, Sweden). Live cells were defined as double negative for these markers.
IFN- ELISPOT
IFN- ELISPOT was performed as previously described
(20). Briefly, 96-well plates (MAIPS4510; Millipore,
Molsheim, France) were coated with 2 µg/ml capturing Ab and
anti-human IFN- (mAb 1-D1K; Mabtech, Stockholm, Sweden) at 4°C
for 18 h and then blocked with AIM-V 2% human albumin
(Baxter Medical, Stockholm, Sweden) for 1 h at 37°C. PBMC
from healthy donors were then added to wells using 20,000 cells/well
for OKT-3 (DAKO, Glostrup, Denmark) treatment and 7 x
105 cells/well for specific responses against the
influenza virus matrix-derived peptide (MP58–66,
GILGFVFTL). OKT-3 was added at 25 ng/ml, and peptide was added at 2
µg/ml. Cells were incubated for 4 h in 37°C, 10%
CO2. After six washes with 0.05% PBS-Tween 20
(Merck, Haar, Germany), anti-human IFN- (mAb
7B6-1-biotin; Mabtech) was added at 0.75 µg/ml and left for 2 h
for incubation at 22°C. Another round of washing was done before
streptavidin-alkaline phosphatase was added for a 1-h
incubation. Spots were developed by addition of substrate (nitroblue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; Life
Technologies) and counted using an ELISPOT reader (KS ELISPOT; Zeiss,
Oberkochen, Germany).
Lymphocyte activation for the cytokine-staining experiments
Cells exposed to H2O2 were washed, and the lymphocyte concentration was adjusted to 1 x 106 cells/ml. The cellular viability was in all experiments >97%, as determined with 7-AAD (Sigma). The lymphocytes were stimulated for 4 h at 37°C with PMA and ionomycin (Sigma) in the presence of 10 µg/ml brefeldin A (Sigma) (21).
FACS analysis
A four-color method was used. The cells were first stained for
surface Ags (30 min at 4°C) with anti-CD45RO-FITC (DAKO),
anti-CD8-PerCP, and anti-CD3-allophycocyanin (BD
Biosciences, Stockholm, Sweden). Thereafter, the lymphocytes were
permeabilized with FACS-lysing solution and with FACS-permeabilizing
solution (BD Biosciences) and stained for 10 min at room temperature in
the dark with PE-conjugated mAbs directed to IL-2, IFN-, IL-10, and
TNF- (BD Biosciences). The staining protocol included isotype
controls for both surface and cytoplasmic staining. The stimulation and
permeabilization procedures were checked by cytoplasmic staining for
CD69 (BDIS Biosciences) and vimentin (Serotec, Oslo, Norway). After
staining, the cells were fixed with CellFix (BD Biosciences).
Acquisition was performed in less than 2 h. The flow cytometric
measurements were performed on a FACSCalibur (BD Biosciences). The
instrument performance was checked daily with bead calibrators: QC
Windows, CaliBRITE, and Q1000, and monthly with both beads and cells,
as previously described (22). Data of at least 10,000
cells per sample were collected. Data analysis was done with CellQuest
software (BD Biosciences), according to a standardized
pattern-protocol. Gates were applied on all lymphocytes (light scatter
cytogram) and all CD3+ lymphocytes (forward
scatter vs Fl4), followed by subsequent gating on
CD8+ and CD8- subsets
(forward scatter vs Fl3). Each subset was further divided in two
subpopulations: CD8+CD45RO+ and
CD8+CD45RO-, or
CD8-CD45RO+ and
CD8-CD45RO-, respectively (Fl1 vs Fl3). The
background fluorescence was determined with markers applied on the
isotype control cytograms, and was in all cases <1%. As all analyzed
cells were T cells (CD3+), the CD8-
lymphocytes were considered CD4+, and the
CD45RO- lymphocytes were considered
CD45RA+.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We developed a model to study the functional and molecular
consequences of exposing T cells to oxidative stress.
H2O2 dose and the exposure
time needed for suppression of specific and nonspecific T cell
responses was determined. A 10-min preincubation of PBMC to
H2O2 at concentrations
spanning from 25 to 100 µM was enough to significantly reduce
peptide-specific and nonspecific (CD3 cross-linking) responses (Fig. 1
A), as measured by an ELISPOT
assay for the Th1 cytokine IFN- (p < 0.03).
The presence of catalase during the
H2O2 exposure completely
abrogated the loss of function induced by
H2O2 (Fig. 1B).
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A). Therefore, we conclude that a short exposure of PBMC to low concentrations of H2O2 severely impaired Ag-specific and nonspecific TCR-triggered cytokine production.
Loss of cytokine production is associated with block of NF-B
activation
Because IFN- and also many other Th1 cytokines, including IL-2
and TNF-, are regulated by NF-B, we were interested in studying
the effects of oxidative stress on NF-B in our model. Our results
indicate that pretreatment of PBMC (data not shown) or of purified T
cells with H2O2 at
concentrations of 25 µM or more reduced the anti-CD3-induced
NF-B activation (Fig. 2), correlating
with the dose needed to suppress cytokine production in experiments
performed in parallel and with cells from the same donor (Fig. 1). Due
to experimental variation, suppression of NF-B was sometimes seen
only at higher doses of
H2O2, but always
correlating with the dose needed to achieve down-modulation of IFN-
production. These results are compatible with the interpretation that
the inhibitory effect of micromolar levels of
H2O2 on cytokine production
is related to inhibition of NF-B activation.
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We next analyzed the possibility that the loss of function and
block of NF-B activation seen after treatment with
H2O2 were consequences of
apoptosis and cell death. To this end, the percentage of live cells,
defined as double negative for annexin V and 7-AAD after exposure to
various concentrations of
H2O2 and subsequent stimuli
with OKT-3, was measured. These experiments revealed that T cells
(CD3+) died in a dose- and time-dependent manner
following exposure to H2O2
(Fig. 3A). At 4 h after
exposure of up to 100 µM
H2O2, the majority of T
cells did not show signs of apoptosis/cell death (Fig. 3A).
When incubated for 15 or 60 h after
H2O2 treatment, the
majority of the cells exposed to 100 µM were apoptotic or dead. Loss
of cytokine production was seen already at 4 h (Figs. 1 and 3B), when no signs of apoptosis were apparent. Importantly,
most cells exposed to levels of
H2O2 in the range of 25–50
µM, which effectively inhibited cytokine production following TCR
stimulation, were still alive without signs of apoptosis even after
60 h (Fig. 3, A and B). In Fig. 3B, this is illustrated by calculating how many cells that
were capable of producing IFN- per a fixed number of viable cells at
60 h. In conclusion, exposure to low doses of
H2O2 resulted in a loss of
T cell function without significant apoptosis, providing evidence that
there is a dose window of exposure to oxidative stress in which cells
enter an anergic rather than a preapoptotic state.
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To characterize how
H2O2 affects the spectra of
cytokine responses in T cells, an intracellular staining protocol was
used. In confirmation of, and extending the ELISPOT data shown in Fig. 1, this assay demonstrated how PMA/ionomycin-induced IFN-, IL-2,
TNF-, and IL-10 release from T cells was lost when cells were
pre-exposed to H2O2 at
micromolar concentrations (Fig. 4A). However, it is notable
that the production of the Th2 cytokine IL-10 was significantly more
sensitive to low levels (12.5–25 µM) of
H2O2 than was the
production of IFN-, IL-2, and TNF- (Fig. 4A)
(p < 0.03). Confirming the intracellular
staining results, IL-10- and IFN--specific ELISPOT assays also
demonstrated a more pronounced decrease in the number of T cells
producing IL-10 as compared with IFN- in
H2O2-exposed and
OKT-3-triggered T cells (Fig. 4B). In conclusion, the
production of the Th2 cytokine IL-10 seems to be more sensitive to
oxidative stress than that of Th1 cytokines.
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, TNF-, and IL-2 predominantly occurs in CD45RO+
cells
The phenotype of the cells that lost their capacity to respond to
activation, when pre-exposed to
H2O2, was measured by a
protocol for intracellular staining of cytokines. We found that T cells
with an activated/memory phenotype, as defined by CD45RO expression,
lost their capacity to produce IFN-, TNF-, and IL-2 when
pre-exposed to 50 µM H2O2
before stimulation with PMA/ionomycin (Fig. 5, A–C)
(p < 0.05 for all cytokines and subsets except
CD8+CD45RO+ cells producing
IL-2). In contrast, no significant changes in the number of naive T
cells, as defined by CD45RO negativity, were observed for the
production of any of the three tested Th1 cytokines (Fig. 5, A–C) (p > 0.2). This indicates
that the mechanisms responsible for production of IFN-, TNF-, and
IL-2 are more sensitive to oxidative stress in activated/memory T
cells, as compared with naive T cells. However, the difference between
CD45RO+ and CD45RO- cells
regarding production of IFN-, although statistically significant,
remains somewhat unclear, because production of this cytokine was very
limited by CD45RO- cells (Fig. 5C).
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D). Importantly, these differences were not due to selective cell death of particular T cell subsets, as the percentage of cells belonging to different subsets remained stable throughout the experiment, with increasing concentration of H2O2 (data not shown).
Blockade of NF-B activation is more pronounced in
CD45RO+ cells
Next, we analyzed the molecular explanation for the selective
targeting of CD45RO+ cells by oxidative stress
with respect to production of IFN-, TNF-, and IL-2.
CD45RO-positive and CD45RO-negative cells were isolated by positive
selection of CD45RO+ cells before exposure to
H2O2 and stimulation, and
were then subjected to analysis of NF-B activation. We observed that
NF-B was more strongly activated in CD45RO+
cells than in CD45RO- cells (Fig. 6A, lane 6 compared
with lane 1), possibly reflecting the fact that there are
higher numbers of Th1 cytokine-producing cells within this subset (Fig. 5, A–C). Also, lower doses of
H2O2 (25 µM) were needed
to substantially inhibit the NF-B activation in
CD45RO+ cells compared with
CD45RO- cells (Fig. 6A, lane
9 compared with lane 4). A densitometry of this
experiment is shown in Fig. 6B. In parallel with the NF-B
analysis, IFN- ELISPOT assays were performed to confirm activation
of T cells and inhibitory effects of
H2O2. The loss of NF-B
always correlated with the loss of cytokine production (data not
shown). In conclusion, these data are compatible with an effect of
H2O2 on primarily
CD45RO+ cells due to targeting of NF-B in this
subset.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A).
However, there exists a lower-dose threshold at which cells become
unresponsive to stimuli without progressing to apoptosis up to 60
h postexposure (Fig. 3B).
A novel aspect of this study is the observed differential sensitivity
to oxidative stress of Th1 cytokines vs IL-10 production. Although
production of all the cytokines assayed (IL-2, IFN-, TNF-, and
IL-10) was generally reduced after a short exposure to
H2O2, production of IL-10
was significantly more affected by this treatment (Fig. 4, A
and B). T cell tolerance is usually associated with
decreased IL-2 and IFN- production and enhanced production of IL-10
(23, 24, 25), which is why our data indicate that exposure of
T cells to low levels of oxidative stress leads to a nonresponsiveness
different from classical T cell tolerance. Although IL-10 is generally
regarded as an immune-suppressive cytokine, this cytokine was also
reported to promote T cell functions (26, 27, 28, 29), which is
why the functional consequences of decreased IL-10 production by T
cells remain unclear.
The effect of H2O2 on
NF-B in T cells, in terms of activation or inhibition, remains
controversial (for review, see Refs. 30, 31). Studies
in a subclone of Jurkat (Wurzburg) showed that
H2O2 at micromolar
concentrations activated NF-B, an effect that could be blocked by
antioxidants (14, 15, 16). However, in a more recent study
(5), no activation of NF-B in human PBL could be
observed in response to hydrogen peroxide. Our results are compatible
with studies on human PBL because NF-B activation was inhibited in
response to hydrogen peroxide (3, 4, 5). We speculate that
the effect of H2O2 on
NF-B may be cell type related and depend on differences between
human T cells and Jurkat cells. Previous studies on Jurkat cells have
reported that activation of NF-B following exposure to
H2O2 results in enhanced
transcription of various genes, such as Fas ligand (32).
Also, H2O2-induced
apoptosis has been reported to result from activation of NF-B and
increased transcription of apoptosis-inducing genes (33).
If NF-B is inhibited by oxidative stress, as indicated by our data,
alternative mechanisms for activation of apoptotic genes not affected
by NF-B may be responsible.
It is known that NF-B regulates IL-2, TNF-, and
IFN-, but not IL-10 (reviewed in Ref. 17). In this
study, the block of NF-B activation was almost complete at doses in
which changes in IFN-, IL-2, and TNF- began to appear (25–50
µM) (Figs. 1 and 2 and 4, A and B). This
indicates that loss of NF-B activation might be the underlying
mechanism for the impaired production of these cytokines. In contrast,
IL-10 production was decreased already at doses of
H2O2 below those needed to
suppress NF-B activation, suggesting that the loss of IL-10
production was not a result of impaired NF-B activation. This
observation is in line with recent reports, showing that induction of
IL-10 production is regulated by Stat 3, and not by NF-B (17, 34). In this study, OKT-3 treatment did not lead to activation
of Stat 3 (data not shown), which is why the effect of
H2O2 on this signal
transduction pathway could not be examined.
The capacity to produce cytokines under conditions of oxidative stress
was studied in CD45RO+ (activated/memory) cells
and naive, CD45RO- cells within the CD4 and CD8
compartments. Two phenomena of particular interest were found. First,
we noticed that the CD45RO+ subset lost the
capacity to produce IFN-, TNF-, and IL-2 following exposure to
H2O2 (Fig. 5, A–C), whereas CD45RO- cells
producing these cytokines were not affected by oxidative stress.
Second, IL-10 production followed a completely different pattern; with
all subsets being equally sensitive to
H2O2 (Fig. 5D).
The finding of activated/memory cells, in contrast to naive cells,
being more sensitive to
H2O2 with respect to the
production of cytokines regulated by NF-B, suggests that NF-B
activation might be more sensitive to oxidative stress in this
subpopulation of T cells. Analyses of NF-B contents in the nuclear
extracts from these populations after activation revealed that
CD45RO+ cells had more pronounced levels of
activation of NF-B, and also that this activation was blocked at
lower H2O2 concentrations
than in CD45RO- cells (Fig. 6, A and
B).
The explanation to this differential sensitivity of memory/activated vs naive T cells to oxidative stress observed on a functional as well as molecular level remains to be established, but this observation may have important biological implications and may constitute a mechanism by which the immune system can modulate the activity of T cells after specific or nonspecific activation.
Finally, we would like to interpret our results in the context of the
observed immune suppression taking place in cancer patients. Patients
with cancer in advanced stages have a poorly functioning immune system
(35), characterized by diminished responses to recall Ags
(36), decreased T cell proliferation (37, 38), loss of cytokine production (39), defective
signal transduction, and loss of transcription factor activity in T and
NK cells (40, 41, 42, 43, 44, 45). These alterations also correlate with
severity of the disease and with poor survival (40, 46, 47). Furthermore, there is evidence for increased apoptosis
among CD8+ T cells in PBL from cancer patients
(48, 49, 50) and mice with experimental tumors
(49, 50). Mechanismsthat may account for these
immune-suppressive effects in tumor-bearing individuals
include Fas-Fas ligand interaction leading to T cell apoptosis, shown
to involve caspase 3-mediated cleavage of CD3 (51), as
well as tumor-derived gangliosides inducing defective NF-B
activation in renal cell carcinoma lines (52). Also,
release of H2O2 from
activated macrophages derived from tumor lesions can result in
loss of Ag-specific T cell functions and decreased expression of the
CD3 molecule in T and NK cells (7, 8).
The data presented in this work suggest that increased amounts of ROS
might be one mechanism explaining why activated/memory
(CD45RO+) T cells recruited to inflammatory
sites, including cancer lesions and other pathological conditions,
often exhibit anergic properties similar to the ones found in this
study. We speculate that oxidative stress might be one possible
mechanism behind the defective NF-B activation in T cells from
tumor-bearing mice and cancer patients (44, 53). Thus,
treatments aiming at reversing immune suppression, therefore, may
target the altered redox status in cancer patients, and allow
endogenous effector cells to function as desired.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Karl-Johan Malmberg, Immune and Gene Therapy Laboratory, Department of Oncology and Pathology, Cancer Center Karolinska, R8:01, Karolinska Hospital, S-17176 Stockholm, Sweden. E-mail address: kalle.malmberg{at}cck.ki.se
3 Abbreviations used in this paper: ROS, reactive oxygen species; 7-AAD, 7-amino actinomycin D.
Received for publication March 19, 2001. Accepted for publication July 3, 2001.
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K.-J. Malmberg, R. Lenkei, M. Petersson, T. Ohlum, F. Ichihara, B. Glimelius, J.-E. Frodin, G. Masucci, and R. Kiessling A Short-Term Dietary Supplementation of High Doses of Vitamin E Increases T Helper 1 Cytokine Production in Patients with Advanced Colorectal Cancer Clin. Cancer Res., June 1, 2002; 8(6): 1772 - 1778. [Abstract] [Full Text] [PDF]
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