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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1634-1642
Interleukin-10-Treated Human Dendritic Cells Induce a
Melanoma-Antigen-Specific Anergy in CD8+ T Cells
Resulting in a Failure to Lyse Tumor Cells
By
Kerstin Steinbrink,
Helmut Jonuleit,
Gabriele Müller,
Gerold Schuler,
Jürgen Knop, and
Alexander H. Enk
From the Department of Dermatology, University of Mainz, Mainz,
Germany; and the Department of Dermatology, University of Erlangen,
Erlangen, Germany.
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ABSTRACT |
Dendritic cells (DC) are critically involved in the initiation of
primary immune processes, including tumor rejection. In our study, we
investigated the effect of interleukin-10 (IL-10)-treated human DC on
the properties of CD8+ T cells that are known to be
essential for the destruction of tumor cells. We show that
IL-10-pretreatment of DC not only reduces their allostimulatory
capacity, but also induces a state of alloantigen-specific anergy in
both primed and naive (CD45RA+) CD8+ T
cells. To investigate the influence of IL-10-treated DC on melanoma-associated antigen-specific T cells, we generated a
tyrosinase-specific CD8+ T-cell line by several rounds of
stimulation with the specific antigen. After coculture with
IL-10-treated DC, restimulation of the T-cell line with untreated,
antigen-pulsed DC demonstrated peptide-specific anergy in the
tyrosinase-specific T cells. Addition of IL-2 to the anergic T cells
reversed the state of both alloantigen- or peptide-specific anergy. In
contrast to optimally stimulated CD8+ T cells, anergic
tyrosinase-specific CD8+ T cells, after coculture with
peptide-pulsed IL-10-treated DC, failed to lyse an HLA-A2-positive
and tyrosinase-expressing melanoma cell line. Thus, our data
demonstrate that IL-10-treated DC induce an antigen-specific anergy in
cytotoxic CD8+ T cells, a process that might be a
mechanism of tumors to inhibit immune surveillance by converting DC
into tolerogenic antigen-presenting cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DENDRITIC CELLS (DC) are highly
specialized antigen-presenting cells (APC) of the immune
system.1 Their strategic positioning in nonlymphoid tissue
and their ability to circulate via blood and lymph to lymphoid organs
after antigen stimulation demonstrate their important role in the
induction of immune responses against invading
pathogens.2,3 During their migration, DC such as Langerhans
cells (LC) are thought to undergo characteristic modulations of
function and phenotype.4,5 Locally produced inflammatory
cytokines and the encounter with an antigen promote the maturation and
migration of DC to regional lymph nodes. During this process DC undergo
a differentiation from a processing to a presenting functional cell
type, characterized by the expression of costimulatory molecules,
cytokine production, and a typical morphology.5-7 In
contrast to other types of APC, fully mature DC are potent activators
of naive T cells and are regarded as important initiators of primary
specific immune responses.1
Tumors use various strategies for escape from immunologic recognition
or destruction.8 One of the potential escape mechanism is
the production of the immunosuppressive cytokine interleukin-10 (IL-10)
by the tumor cells themselves or the induction of such factors in
tumor-infiltrating cells. The release of IL-10 from many tumors, such
as human renal, colon, ovarian, lung, and basal cell carcinomas, brain
neoplasms, and Epstein-Barr virus-transformed B
lymphomas, has been reported.9-14 Furthermore, in malignant melanoma cells, the expression of IL-10 mRNA or protein was
demonstrated.10,15-17 Studies in progressing tumors showed
a correlation of an advanced course of the disease with elevated IL-10
serum levels, a preferential detection of IL-10 secreting tumor cells
in metastatic lesions, and a higher release of IL-10 by metastatic
cells from patients with progressive melanoma metastases compared with
metastases responding to therapy.18-20
The immunosuppressive properties of IL-10 have been well documented in
several studies. An inhibitory effect on the function of APC and T
cells has been described. The inhibitory influence of IL-10 on the APC
function of DC and macrophages is due to the downregulation of major
histocompatibility complex (MHC) class II and several
costimulatory molecules and the reduction of a variety of secreted
inflammatory cytokines.21-28 With regard to tumor
rejection, it was demonstrated that IL-10 inhibits tumor antigen
presentation by epidermal APC in the murine system.29 More
importantly, it was shown that IL-10-treated LC induce an antigen-specific tolerance in Th1 cells, but not in Th2 cell
clones.30
Recently, we demonstrated that IL-10-treated human DC, generated from
peripheral blood, induce a state of antigen-specific anergy in various
populations of CD4+ T cells.31
In the present study, we investigated the effect of IL-10-treated DC
on the function of CD8+ T cells to evaluate a potential
mechanism of tumor cells to inhibit the cytotoxic capacity of T cells
by anergy induction. We demonstrate that IL-10-treated DC show a
reduced capacity to stimulate the proliferation of both primed and
naive CD8+ T cells in allogeneic MLR and anti-CD3 assays.
More interestingly, IL-10-treated DC induce a state of
alloantigen-specific anergy in CD8+ T cells. To investigate
the influence on melanoma-associated antigen-specific T cells, the
effect of IL-10-treated DC on a tyrosinase-specific T-cell line was
observed. Restimulation of the specific T cells with untreated,
antigen-pulsed DC demonstrated a peptide-specific anergy in the
tyrosinase-specific CD8+ T cells. In contrast to optimally
stimulated CD8+ T cells, tyrosinase-specific anergic
CD8+ T cells failed to lyse an HLA-A2+,
tyrosinase-expressing melanoma cell line. In conclusion, production of
IL-10 by tumor cells may reflect an escape mechanism from immune surveillance by converting DC into tolerogenic APC.
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MATERIALS AND METHODS |
Preparation of DC.
Blood-derived DC were prepared according to a modified protocol
originally described by Romani et al.32 Briefly, whole
blood was heparinized and separated by a Ficoll gradient. Peripheral blood mononuclear cell fractions were then depleted of T
and B cells using immunomagnetic beads coated with anti-CD2 and
anti-CD19 monoclonal antibody (MoAb; Dynal, Oslo, Norway). The
remaining cells were cultured in X-VIVO 15 (Biowhittaker, Walkersville, MD) in 6-well-plates (Costar, Cambridge, MA) for 7 days.
Cultures were supplemented with 1,000 U/mL human IL-4 (hIL-4; PBH,
Hannover, Germany), 800 U/mL human granulocyte-macrophage
colony-stimulating factor (hGM-CSF; Leukomax; Sandoz, Basel,
Switzerland), and 1% autologous plasma. Cells were fed with fresh
medium every 2 days. On day 7, nonadherent cells were rinsed off the
plates and resuspended in fresh complete medium with GM-CSF and IL-4
and additionally stimulated with IL-1 (10 ng/mL), tumor necrosis
factor- (TNF-; 10 ng/mL; PBH), IL-6 (1,000 U/mL; R&D Systems,
Wiesbaden, Germany), and prostaglandin E2
(PGE2; 1 µg/mL; Sigma, München,
Germany) to induce and stabilize the maturation of DC. DC were
harvested for fluorescence-activated cell sorting (FACS) analysis or
functional tests 3 to 5 days after resuspension and stimulation.
Simultaneously to the addition of the stimulating mixture of cytokines
and PGE2, IL-10 (40 ng/mL; DNAX, Palo Alto, CA) was added
to the culture for the last 2 days of culture. For the study of
kinetics, IL-10 was added at various time points (as indicated).
T-cell purification, allogeneic proliferation, and anti-CD3 assay.
T cells were prepared from human blood using Ficoll gradients and
subsequent purification by antibody-coated immunomagnetic beads (MACS
Systems; Miltenyl, Bergisch Gladbach, Germany) according to standard
protocols (purity of >95% CD8+ T cells and >90%
CD45RA+ T cells). In some experiments, cord blood was used
as the source of naive CD8+ T cells. Purity was tested
using FACS analysis.
DC were prepared as described above and cocultured with 2 × 105 T cells per well in 96-well plates (Costar). For
anti-CD3 assays anti-CD3 MoAb (OKT3, ATCC, CRL 8001) was used at a
titrated dilution of a hybredoma supernatant (1:20). After 2 days
(anti-CD3 assay) or 4 days (allogeneic MLR), the cells were pulsed with
1 µCi of [3H] TdR
([methyl-3H]thymidine)/well for 16 hours, harvested, and
counted. Tests were performed in triplicate, and results were expressed
as the mean cpm ± standard deviation (SD).
Tyrosinase-specific T-cell lines (CTL).
Naive CD8+ T cells (2 × 105) from
HLA-A2+ donors (purity of >95% CD8+ T cells,
generated as described above) were cultured in X-vivo 20 (Biowhittaker)
and stimulated with mature, HLA-A2+, autologous DC (2 × 104) pulsed with the specific tyrosinase peptide
(YMDGTMSQV; 20 µg/mL). After several restimulations (5 to 8 times)
every 7 days and expansion of the cell number by addition of IL-2 (10 U/mL), the peptide-specific proliferation was tested using specific
(tyrosinase) and unspecific (MART-1 [EAAGIGLTV], MAGE-1
[EADPTGHSY]) melanoma-associated antigens. Before their use in
experiments, T cells were rested 7 to 8 days after the last addition of
antigen and stimulator cells. Three tyrosinase-specific
CD8+ T-cell lines from 2 unrelated HLA.A2+
donors were generated and used for the experiments. MART-1-specific T-cell lines served as controls.
Anergy assay.
Allogeneic CD8+ T cells or tyrosinase-specific
CD8+ T cells were prepared as described above. T cells were
cocultured during the first incubation at a density of 2 × 105 (allogeneic CD8+) or 2 × 104 (tyrosinase-specific CD8+ T cells) with 1 × 104 or 1 × 103 DC, pretreated with
IL-10 (40 ng/mL), or untreated. In some experiments, anti-CD3 MoAb was
added at a titrated dilution of a supernatant (as described above).
Thirty-six hours later, T cells were separated by Histopaque and rested
for 1 to 7 days in culture medium containing 2 U/mL IL-2. Subsequently,
T cells were restimulated with DC generated from the same donor as used
for the first culture in experiments with CD8+ T cells or
with DC generated from an HLA-A2+ donor. Proliferation was
measured 48 hours later by thymidine incorporation. Tests were carried
out in triplicates, and results were expressed as mean cpm ± SD.
Additionally, cytokine production was measured by enzyme-linked
immunosorbent assay (ELISA) in supernatants of restimulated cultures 48 hours after the beginning of culture.
Cytotoxicity assay.
Cytotoxic activity was measured in a standard 4-hour assay using the
51Cr-labeled tyrosinase-expressing and HLA.A2+
melanoma cell line SK-MEL 28 (provided by Dr T. Wölfel, Mainz, Germany) as targets. Briefly, 3 × 103
51Cr-labeled tumor cells were cultured with
tyrosinase-specific, HLA.A2+ CD8+ CTL,
precultured with mature or IL-10-treated DC, in effector:target ratios
as indicated for 4 hours at 37°C. Tyrosinase-specific
CD8+ T cells cocultured with the unrelated peptide MART-1
and untreated or IL-10-treated DC or HLA-mismatched DC
(HLA.A1+) during the primary culture were used as controls.
Additional control experiments were performed using DC
(HLA.A2+) as target cells pulsed with various peptides:
tyrosinase (HLA.A2-restricted presentation), MART-1 (HLA.A2-restricted
presentation), or MAGE-1 (HLA.A1-restricted presentation). Similar to
the experimental setting described above, these
51Cr-labeled target cells were cocultured with the
tyrosinase-specific, HLA.A2+ CD8+
CTL, precultured with mature or IL-10-treated DC, for 4 hours at 37°C.
The percentage of specific lysis was calculated from the average of
triplicates as 100 × (51Cr-release into supernatant  spontaneous release)/(total release in detergent spontaneous release). All synthetic petides were tested for nonspecific
lysis of target cells in the absence of cytotoxic T lymphocytes.
Cytokine analysis.
For assessment of cytokine production, supernatants were collected 48 hours after restimulation of allogeneic-specific/tyrosinase-specific CD8+ T cells with mature, untreated DC and stored at
70°C. Amounts of IL-2, IL-4, IL-10, transforming growth
factor-(TGF-), and interferon- (IFN-) were measured by
ELISA using commercially available antibodies and standards according
to the manufacturer's protocols (Pharmingen, Hamburg, Germany).
| |
RESULTS |
Inhibition of the alloantigen-induced or anti-CD3-induced
proliferation of CD8+ T cells after coculture with
IL-10-treated DC.
To evaluate the effect of IL-10 on the stimulatory capacity of DC,
precursors of DC, generated from peripheral blood, were cultured for 7 days as described above and subsequently stimulated for the last 2 days
with the defined cytokine cocktail (IL-1, TNF-, and IL-6) and
PGE2 alone or additionally treated with IL-10 (40 ng/mL)
and used as APC in variable numbers in alloantigen-induced or
anti-CD3-induced proliferation assays (Fig
1A and B). Human allogenic CD8+ T cells, purified from
peripheral blood or in some experiments from cord blood, were used as
responder cells. A significant inhibition of the proliferation was
demonstrated in all T-cell:DC ratios, if IL-10-treated DC were set in
as APC both in alloantigen-induced and in anti-CD3-induced
proliferation assays. The reduced proliferation was demonstrated, if
naive CD8+/CD45RA+ T cells (Fig 1A and B) or
activated CD8+/CD45RO+ T cells (data not shown)
were cocultured with IL-10-treated DC.
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| Fig 1.
Inhibitory effects of IL-10-pretreated DC on
alloantigen-or anti-CD3-induced proliferation of naive
CD8+ T cells: dependence on the state of maturation of
DC. DC were generated from peripheral progenitors as described above,
resuspended at day 7, and stimulated with IL-1, IL-6, TNF- , and
PGE2 to induce and stabilize the maturation of the DC.
IL-10 was added for the last 2 days of culture at various time points
after the stimulation: simultaneous additon at day 7 and harvested of
the DC at day 9 (A and B) or addition at a later stage of maturation
(day 10) and subsequently harvest at day 12 (C and D) . Control and
IL-10-treated DC were cocultured with naive CD45
RA+/CD8+ T cells (2 × 105) in
allogeneic MLR (A and C) or anti-CD3 assays (B and D). Proliferation
was measured by [3H] TdR uptake. The results are
representative of five experiments.
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The effect of IL-10 on the DC was inhibited by the simultaneous
addition of an anti-IL-10 antibody (10 µg/mL; R&D Systems, Weisbaden, Germany) to the culture (data not shown).
Mature DC are resistant to the effect of IL-10.
Prior experiments had shown that fully mature DC were generated after
11 to 14 days of culture after stimulation with IL1-, IL-6, TNF-,
and PGE2 at day 7 and that these cells induced the most
effective proliferative T-cell response.33 In our
experiments, IL-10 was added at various time points after the
stimulation of the DC to determine whether IL-10 would affect the
function of DC at all stages of their differentiation. The addition of
IL-10 was most effective if DC at days 7 to 9 of culture were used, whereas fully mature DC at days 10 to 13 of culture were completely resistant to the effect of IL-10 (Fig 1). As an example, no inhibition of proliferation was observed if DC at day 12 of culture, after treatment with IL-10 at day 10, were cocultured with CD8+ T
cells both in alloantigen-induced or anti-CD3-induced proliferation assays (Fig 1C and D).
Induction of alloantigen-specific anergy in CD8+ T
cells by IL-10-treated DC.
To test whether IL-10-treated DC would induce a state of
alloantigen-specific anergy in CD8+ T cells, we performed a
two-step anergy assay. In these experiments, allogeneic
CD8+ T cells were cocultured with untreated or
IL-10-treated DC. After the first coculture, T cells were rescued,
cultured for 36 hours in the presence of IL-2 (10 U/mL), and
subsequently restimulated with untreated, fully mature DC.
CD8+ T cells, cultured with untreated DC during the first
coculture, showed a vigorous proliferation to restimulation with mature
DC during the second coculture (Fig 2). In
contrast, CD8+ T cells cocultured with IL-10-treated DC
were unresponsive to further stimulation with APC, if these cells were
generated from the same donor as used in the first coculture (Fig 2).
To determine whether this induction of anergy was antigen-specific, DC
from a second, unrelated donor were used for restimulation. In these experiments, an unrestricted proliferation of the CD8+ T
cells was observed independently of a pretreatment with IL-10 (Fig 2).
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| Fig 2.
Induction of alloantigen-specific anergy in
CD8+ T cells by IL-10-treated DC. Purified naive
CD8+ T cells were cultured in medium alone; with
untreated, control DC; or with DC pretreated with IL-10 (first
coculture). After 36 hours, T cells were rescued, cultured for 1 to 7 days in medium containing low levels of IL-2 (2 U/mL), and subsequently
restimulated (second coculture) with untreated, mature DC generated
from the same donor ( ) or a second unrelated donor ( ).
[3H] TdR incorporation was determinated after 48 hours.
The left bars show the antigen-specific response and the right bars
show the T-cell response to IL-2 (100 U/mL).
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All experimental groups of T cells responded vigorously to IL-2, a
cytokine that has been described to overcome the state of anergy (Fig
2).30
To assess the kinetics of the induction of this T-cell anergy,
CD8+ T cells rescued after the first coculture were
cultured for various time periods (up to 7 days) in the presence of
IL-2 (10 U/mL) before restimulation with untreated mature DC. In all
experiments performed, the T cells showed a markedly reduced
proliferation if IL-10-treated DC were used in the primary culture and
the APC for both cultures were derived from the same donor (data not shown).
These experiments demonstrate that IL-10 converts the function of DC to
anergy-inducing cells and that this state of T-cell anergy is
alloantigen-specific.
Inhibition of peptide-specific proliferation of a tyrosinase-specific
cytotoxic CD8+ T-cell line.
Cytotoxic CD8+ T cells are important for the lysis and
elimination of tumors cells. To analyze the effect of IL-10-treated DC
on the function of cytotoxic, melanoma-associated antigen-specific CD8+ T cells, we established three
tyrosinase-peptide-specific CD8+ T-cell lines from two
unrelated HLA.A2+ donors. Cytotoxic
CD8+ T cells were used after 5 to 8 weekly restimulations
with peptide-pulsed autologous DC in the presence of low levels of IL-2
(10 U/mL).
We assessed the proliferation of the tyrosinase-specific
CD8+ T-cell line after coculture with untreated versus
IL-10-treated tyrosinase peptide-pulsed DC
(Fig 3). Strong peptide-specific proliferation was observed in cultures stimulated with untreated tyrosinase-peptide pulsed DC. In contrast, DC generated in the presence
of IL-10 induced a markedly impaired proliferation of the T-cell line
(Fig 3).
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| Fig 3.
Inhibition of the proliferation of cytotoxic
tyrosinase-specific CD8+ T cells by IL-10-treated DC.
Tyrosinase-specific, cytotoxic CD8+ T cells were cultured
with the specific tyrosinase peptide, either in combination with
HLA.A2+ DC pretreated with IL-10 or untreated. In control
experiments, the unspecific peptide MART-1 was added to the culture.
Proliferation was measured by incorporation of [3H] TdR
after 3 days of culture. The results represent one of three
experiments.
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No proliferation was observed if a second unrelated melanoma-associated
antigen MART-1 was added to the culture medium independently of the
pretreatment of the DC with IL-10, indicating a peptide-specific proliferation of the CD8+ T-cell line.
Induction of peptide-specific anergy in a tyrosinase-specific
CD8+ T-cell line.
In the following experiments we evaluated whether tyrosinase-specific
CD8+ T-cell lines were anergized by the stimulation with
IL-10-pretreated DC. In a first coculture, the peptide-specific
cytotoxic CD8+ T cells were cultured with autologous,
IL-10-treated, or untreated (HLA.A2+) DC in combination
with the specific tyrosinase peptide. Subsequently, T cells were
rescued, cultured for 1 day (in some kinetic experiments up to 7 days;
data not shown), and restimulated with mature, untreated DC in the
presence of specific tyrosinase peptide in a second coculture
(Fig 4).
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| Fig 4.
Induction of melanoma antigen-specific anergy in
tyrosinase-specific, cytotoxic CD8+ T cells. In a first
coculture, tyrosinase-specific HLA.A2+ CD8+
T cells were used as responder cells for HLA.A2+ DC
additionally pretreated with IL-10 or untreated (cultured in complete
medium alone). Cultures were set up in the presence of the specific
tyrosinase peptide or an unspecific control peptide (MART-1).
Subsequently, the T cells were rescued, cultured for 1 to 7 days in the
presence of low levels of IL-2 (2 U/mL), and restimulated with mature
HLA.A2+ DC during the second coculture. After 2 days, the
proliferation was measured by [3H]TdR incorporation. The
left bars show the specific response and the right bars show the T-cell
response to IL-2 (100 U/mL). The results are representative for three
experiments.
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After restimulation, a vigorous proliferative response was observed in
CD8+ T cells precultured with untreated DC in the presence
of the tyrosinase peptide. In contrast, coculture with
IL-10-pretreated DC in the presence of the specific antigen induced a
significant inhibition of the T-cell proliferation, indicating a
tyrosinase-specific (melanoma-associated antigen-specific) anergy.
To show that the induction of anergy is antigen-specific, we stimulated
the tyrosinase-specific CD8+ T-cell line during the first
coculture with the unrelated peptide MART-1. After the second
coculture, an unrestricted T-cell proliferation was observed,
independent of a primary stimulation with untreated or IL-10-treated
DC (Fig 4).
Additional control experiments were performed using untreated and
IL-10-treated HLA-mismatched DC (HLA.A1+) stimulated with
the tyrosinase peptide during the first coculture. After restimulation,
an unhibited proliferation of the CD8+ tyrosinase-specific
T cells was demonstrated, indicating an HLA-restricted antigen
presentation and anergy induction (data not shown).
The addition of IL-2 (100 U/mL) to the second coculture reversed the
state of peptide-specific anergy in the CD8+ T
cells (Fig 4).
To analyze the cytokine pattern of the anergic, peptide-specific
CD8+ T cells after restimulation, cytokines in the
supernatants were detected by ELISA. The anergic T cells showed a
markedly reduced secretion of the cytokines IL-2 and IFN-, but no
production of IL-4 or IL-10. These data indicate a block in Tc1
cytokine production but no shift to a Tc2 pattern in the anergic T
cells (Table 1).
Anergic CD8+ T cells fail to lyse melanoma cells.
The release of immunosuppressive factors such as IL-10
has been described for many tumors, including malignant
melanoma.9-19 To test whether this process might be one
possible escape mechanism of tumor cells by inhibiting the stimulatory
function of DC, we analyzed the cytotoxic function of the anergic,
tyrosinase-specific CD8+ T cells after coculture with
untreated or IL-10-treated DC. The cytotoxic activity was measured
using the 51Cr-labeled tyrosinase-expressing and
HLA.A2+ melanoma cells SK-MEL 28 as target cells.
Tyrosinase-specific control and anergic CD8+ T cells were
cocultured with the melanoma cells for 4 hours in effector-target
ratios, as indicated (Fig 5).
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| Fig 5.
Anergic tyrosinase-specific CTL fail to lyse tumor cells.
Control (precultured with untreated DC) and anergic (precultured with
IL-10-treated DC) tyrosinase-specific HLA.A2+
CD8+ T cells were cocultured with the
HLA.A2+ tyrosinase-expressing melanoma cell line SK-MEL
28 in a 51Cr-release assay for 4 hours. Experiments with
specific CD8+ T cells cocultured with MART-1 during the
primary culture served as controls. Various effector:T-cell ratios were
used in the experimental setting. The percentage of specific lysis was
calculated from the average of triplicates as 100 × (51Cr-release into supernatant spontaneous
release)/(total release in detergent spontaneous release). All
synthetic peptides were tested for nonspecific lysis of target cells in
the absence of CTL.
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The tyrosinase-specific CD8+ T cells, precultured with
untreated DC, showed a strong cytotoxic activity at all effector:target ratios used, with up to 85% lysis of the target tumor
cells (Fig 5). In contrast, after prior coculture with IL-10-treated
DC, tyrosinase-specific anergic CD8+ T cells induced a
significantly decreased lysis of the melanoma cells.
Antigen specifity was demonstrated by control experiments. No reduction
of the cytotoxicity was shown if the unrelated peptide MART-1 was added
to the tyrosinase-specific CD8+ T cells during the primary
culture, independent of a coculture with untreated or IL-10-treated DC
(Fig 5).
To show the HLA-dependent restriction of the anergy induction, control
experiments with HLA-mismatched (HLA.A1+) untreated and
IL-10-treated DC pulsed with the tyrosinase peptide during the primary
culture were performed. The stimulated tyrosinase-specific CD8+ T cells demonstrated an unhibited lysis of the
tyrosinase-expressing, HLA.A2+ melanoma cells, independent
of the use of untreated or IL-10-treated, HLA.A1+ DC (data
not shown).
Similar results were observed if peptide-pulsed mature DC were used as
target cells. Coculture of anergic tyrosinase-specific CD8+
T cells with tyrosinase-pulsed DC in a 51Cr-release assay
led to a markedly reduced lysis of the target cells compared with
control tyrosinase-specific CD8+ T cells. Peptide-specifity
was demonstrated by the use of DC pulsed with the unrelated peptide
MART-1 or MAGE-1 (data not shown).
| |
DISCUSSION |
The release of immunosuppressive factors such as IL-10 has been
described for many tumors, including malignant
melanoma.9-19 This immunologic process might be a mechanism
of tumor cells to inhibit immune surveillance by converting DC from
potent stimulatory cells of the immune system to tolerogenic APC.
In the present study, we investigated the effect of human
IL-10-treated DC on the properties of cytotoxic CD8+ T
cells that are known to be involved in tumor rejection. We demonstrate
that IL-10-treated DC induce an alloantigen-specific anergy in naive
and activated CD8+ T cells and a tyrosinase-specific anergy
in cytotoxic CD8+ T cells, resulting in a failure to lyse
melanoma cells.
The escape mechanism of tumor cells regulated by the secretion of IL-10
may be due either to inhibition of recognition by immune cells or to
inhibition of destruction of tumor cells by effector cells. In support
of the first possibility, it was shown that IL-10 dowregulates MHC
class I expression by tumor cells and prevents their lysis by cytotoxic
T cells.34,35 Although in these models a decreased
expression of MHC class I molecules is described, significant residual
levels of class I antigens that remain on the tumor targets argue
against this alteration of tumor cells being the sole mechanism
responsible for resistance to CTL lysis.34,35
Alternatively, IL-10 might inhibit the antigen-presenting function of
APC-like DC or macrophages and thus prevent a T-cell-mediated antitumor response. Experiments have shown that IL-10 downregulates the
stimulatory capacity of APC such as macrophages and monocytes, but not
that of B cells. This inhibitory influence of IL-10 is due to the
downregulation of MHC class II molecules and the costimulatory molecules B7-1/-2 and intercellular adhesion molecule
(ICAM-1).21,22,24,25 Furthermore, IL-10 reduces the release
of a variety of inflammatory cytokines by monocytes/macrophages,
including IL-1, IL-6, IL-8, TNF-, and GM-CSF.23,26-28
We recently demonstrated that IL-10-treated human DC induce an
alloantigen- or peptide-specific anergy in CD4+ T cells, if
IL-10 was added to immature DC.31 The pretreatment with
IL-10 results in a reduced expression of the costimulatory molecules
CD58 and CD86 and MHC class II molecules.31 The induction of anergy in various populations of T cells in this system might be due
to a lack of costimulatory molecules and can be partially overcome by
stimulatory CD28 MoAbs. Additionally, analysis of the supernatants of
the IL-10-treated DC demonstrated an inhibited production of the
proinflammatory cytokines IL-1, IL-6, and TNF- and a lack of
IL-12 production by the DC.
In our present study, we demonstrate that IL-10-treated human DC are
able to induce a state of antigen-specific anergy also in
CD8+ T cells. In the case of the tyrosinase-specific
CD8+ T cells, a significantly decreased lysis of melanoma
cells was observed after coculture with the anergic CD8+ T
cells and the tumor cells in a 51Cr-release assay. Analysis
of the cytokine pattern of the anergic T cells showed a diminished
secretion of IL-2 and IFN-. Because IL-10-treated DC are known to
have a reduced expression of costimulatory molecules such as CD86 and
show a reduced production of IL-12, this might result in a defect to
activate effector function of CTL characterized by the inhibition of
IL-2 and IFN- production.
As a consequence, our findings support earlier data showing that
production of IL-10 by tumor and/or tumor-infiltrating lymphoid cells might serve as a mechanism for tumor-induced anergy. This induction of anergy was described in various tumor models, including malignant melanoma.20,36 In these systems, IL-10 can act
directly on the properties of cytotoxic CD8+ T cells as
shown as a significantly reduced lysis of murine lymphoma cells in an
in vitro model.36 On the other hand, IL-10 is able to
modulate the stimulatory characteristics of the APC by altering the
surface expression of various MHC class I/II, costimulatory molecules,
or the pattern of the cytokines secreted.21-28
Investigations of human DC of responding or progressing melanoma
metastases demonstrated a markedly increased production of IL-10 in
tumor cells of progressively growing melanomas.20
Furthermore, in a costimulation-dependent anti-CD3 tolerance assay, DC
of the progressive tumor cells but not of the responding cells induce a
state of antigen-specific anergy in cocultured T
lymphocytes.20
Additional studies have shown that IL-10 inhibits tumor antigen
presentation by epidermal antigen-presenting cells in a murine squamous
cell carcinoma model,29 and in a murine plasmocytoma model
it was demonstrated that, in the initiation but not in the effector
phase of the immune response, IL-10 prevents DC accumulation in the
tumor and inhibits rejection of the tumor if IL-10 was simultaneously
expressed in the GM-CSF-transfected tumor cells.37 Furthermore, IL-10 transgenic mice, in which the expression of IL-10
was expressed under the control of the IL-2 promotor, were unable to
limit the growth of Lewis lung tumor cells.38 The injection
of anti-IL-10 antibodies significantly reduced the tumor progression,
demonstrating a direct effect of IL-10 transgene to IL-10
action.38
Paradoxically, in some experimental models, IL-10 has an
immunostimulatory activity. Systemic injection of IL-10, transfection of tumor cells with IL-10, or the high physiological expression of
IL-10 in certain tumors resulted in an increased immunogenicity and
rejection of the tumor cells.39-43
These contrasting results might be due to the different tumor models
used, the varying amounts of IL-10 used, and the different forms of
IL-10 (virus IL-10 v cellular IL-10) applied. It was demonstrated that the antitumor effect of IL-10 was dose-dependent and
that only very high levels of IL-10 were effective in tumor rejection.40-43 Furthermore, the amount of IL-10 required
for immunosuppression may change for different tumors.
In our model, we demonstrate that a pretreatment of human DC with IL-10
induces a state of antigen-specific anergy in cytotoxic CD8+ T cells. When a tyrosinase-specific CD8+
T-cell line was used, the anergic T cells failed to lyse melanoma cells. Therefore, the secretion of IL-10 in the environment of tumor
cells might be one mechanism of tumors to inhibit immune surveillance
by reversing the properties of human DC from potent stimulatory cells
of the immune system to tolerance of inducing cells.
| |
ACKNOWLEDGMENT |
The authors thank L. Paragnik for excellent technical assistance and Dr
T. Tüting for critical reading of the manuscript.
| |
FOOTNOTES |
Submitted July 24, 1998; accepted October 19, 1998.
Supported by the DFG and the BMBF. K.S. was supported by a fellowship
of the Deutsche Forschungsgemeinschaft.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Kerstin Steinbrink, MD,
Department of Dermatology, University of Mainz, Langenbeckstr. 1, D-55131 Mainz, Germany; e-mail:
steinbrink{at}hautklinik.klinik.uni-mainz.de.
| |
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Top
Abstract
Introduction