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Regular Articles |
From the Departments of Clinical Physiopathology*
and Medical-Surgical Disciplines,
University
of Torino, Torino, Italy; and the Institute of Molecular Medicine and
Department of Dermatology and Cutaneous
Biology,
Thomas Jefferson University,
Philadelphia, Pennsylvania
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TGF-ß is a 25-kd dimeric cytokine with pleiotrophic effects on a wide spectrum of target cells. Three highly conserved isoforms of human TGF-ß (1–3) encoded by separate genes are known; the TGF-ß isoforms share considerable structural and sequence homology and exert similar effects when tested in biological systems.5 Aberrant expression of different TGF-ß isoforms is widespread among human tumors,6 including pancreatic carcinoma,7,8 breast carcinoma,9 glioma,10-12 and malignant melanoma.13-15 In support of a significant tumor-protective role of TGF-ß in vivo, transfection of TGF-ß1 into highly immunogenic mouse tumor cells has been shown to enable these cells to survive in immunocompetent syngeneic animals.16 Conversely, increased immunogenicity and decreased tumorigenicity in syngeneic animals was observed when TGF-ß production in rat gliobastoma cells was suppressed using antisense TGF-ß2.17 In addition, neutralizing antibodies to TGF-ß have been shown to inhibit the tumorigenicity of human breast cancer cells in nude mice; this effect was associated with increased NK cell activity.18 That tumor-derived TGF-ß may also contribute to immune evasion of human tumor cells is suggested by studies in glioma. An activity that inhibits proliferation of T cells and autologous lymphokine-activated killer (LAK) cells was isolated from conditioned medium (CM) of glioma cells19 and cyst fluid recovered from the tumor bed of subtotally resected glioblastomas20 ; this activity was shown to be TGF-ß2.
IL-10, like TGF-ß, is a cytokine that exerts multiple effects on the
immune system relating to antigen presentation, B- and T-cell
proliferation, cytokine production, and monocyte/macrophage function
(for a review see Ref. 21
). IL-10 prevents up-regulation of B-7
expression during macrophage activation22
and
down-modulates expression of a broad range of cytokines in peripheral
blood mononuclear cells (PBMC) preparations, including interferon-
(IFN-),23
IL-2,24
and tumor necrosis
factor-
(TNF-).25
IL-10 has been found to be commonly
expressed in human carcinoma cells26,27
and in
melanomas.28,29
In melanoma patients increased serum IL-10
levels have been described.30
Whether IL-10 production by
tumor cells is relevant to local and/or systemic antitumor immune
responses in cancer patients is currently unclear.
In this study, we have examined expression patterns and functional consequences of TGF-ß and IL-10 production by human pancreatic carcinoma cells for systemic T-cell responses in vitro and in vivo. To characterize the relative contributions of tumor-derived TGF-ß and IL-10 on systemic immune parameters in pancreatic cancer patients, we determined, in the presence and absence of specific neutralizing antibodies to these cytokines, the effects of CM from pancreatic carcinoma cells on proliferative potential, cytotoxic activity, and the Th1/Th2-like cytokine profiles upon activation of PBMC derived from normal donors. To assess the potential effects of tumor-derived TGF-ß and IL-10 in pancreatic cancer patients themselves, we also determined the Th1/Th2-like cytokine profiles of PBMC preparations obtained from these patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A group of 10 pancreatic carcinoma patients (seven men and three women, aged 46–71) who underwent surgical resections at the Department of Medical-Surgical Disciplines (University of Torino, Torino, Italy) were included in this study. All patients were affected with histopathologically confirmed primary pancreatic duct adenocarcinomas representing stage II (n = 2), stage III (n = 3), and stage IV (n = 5) pancreatic neoplasms according to the classification by Warshaw and Fernandez-del Castillo.31 Pancreatic cancer tissue samples and normal pancreatic tissue were frozen in liquid nitrogen immediately after surgical removal and before RNA extraction. Venous blood from pancreatic carcinoma patients was collected before anesthesia and surgery. PBMCs from patients and age- and sex-matched healthy donors were separated by Ficoll-Hypaque gradient centrifugation and used immediately for analysis. Donor and patient serum samples were frozen at -70°C until analysis.
Cell Lines and CM
Human pancreatic carcinoma cell lines Capan2 (American Type Culture Collection (ATCC), Rockville, MD), PT45, and BxPC3 (kindly provided by Dr. M. F. DiRenzo, Department of Biomedical Sciences and Human Oncology, University of Torino, Torino, Italy) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (GIBCO, Grand Island, NY). All cell lines were routinely screened for Mycoplasma contamination, using the Hoechst dye H33258. To obtain serum-free CM, Capan2, PT45, and BxPC3 cells were trypsinized, extensively washed with phosphate-buffered saline (pH 7.3), and seeded at 3 x 105/ml in 5 ml of serum-free DMEM containing 0.25 vol% fatty acid-free bovine serum albumin fraction V (Boehringer Mannheim). After a 48-hour incubation in a humidified atmosphere containing 5% CO2, cell-free supernatants were collected after centrifugation, concentrated five-fold by filtration with Amicon Diaflo concentrators equipped with YM5 membranes (Danvers, MA), and stored at -70°C until use.
Antibodies and Reagents
The hybridoma-producing monoclonal antibody (mAb) OKT3 (anti-CD3) was obtained from the ATCC. Neutralizing anti-IL-10 goat and panspecific anti-TGF-ß rabbit polyclonal antibodies were from R&D Systems Europe (Abingdon, England). For immunohistochemistry, rabbit antisera reacting specifically with TGF-ß1, TGF-ß2, or TGF-ß3 (epitopes corresponding to amino acid sequences mapping at the carboxy terminus of the precursor forms of TGF-ß1, TGF-ß2, and TGF-ß3 of human origin, respectively) from Santa Cruz Biotechnology (Santa Cruz, CA) and mAbs to IL-10 (JES-9D7 and 12G8) from Pharmingen (San Diego, CA) were used. Recombinant human TGF-ß1, TGF-ß2, and TGF-ß3 isoforms were obtained from R&D Systems Europe. Staphylococcus aureus strain Cowan I (SAC) was from Calbiochem (La Jolla, CA) and was used at 1:10,000 final dilution.
Cytokine Mapping by Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA from normal and neoplastic pancreatic tissues and from the three pancreatic carcinoma cell lines included in this study was extracted with a commercially available kit based on the single-step RNAzol method (Cinna/Biotex, Houston, TX). Reverse transcription (RT) was performed at 37°C for 1 hour, using oligo-dT primer in a final reaction volume of 20 µl containing 20 U of MMLV reverse transcriptase, 1x reverse transcriptase buffer, 24 U of RNAse inhibitor, and 0.5 mmol/L dNTP mix. For each polymerase chain reaction (PCR), 10 µl of first-strand cDNA was added to 20 µl of PCR mix containing 100 ng each of 5' and 3' cytokine-specific primers and 1 U Taq polymerase. All PCR reagents were purchased from Life Technologies (Paisley, Scotland). Human IL-10-specific primers were 5'-ATGCCCCAAGCTGAGAACCAAGACCCA-3' (sense) and 5'-AAGTCTCAAGGGGCTGGGTCAGCTA-3' (antisense). PCR conditions were as follows: 3 minutes at 94°C, 20 seconds at 60°C, and 30 seconds at 72°C (32 cycles). The predicted size of IL-10 amplimers was 325 bp. Human TGF-ß1-specific primers were 5'-GCCCTGGACACCAACTATTGC-3' (sense) and 5'-GCACTTGCAGGAGCGCA-3' (antisense). PCR conditions were as follows: 4 minutes at 95°C, 1 minute at 58°C, and 35 seconds at 72°C (32 cycles). The predicted size of TGF-ß1 amplimers was 333 bp. Human TGF-ß2 and TGF-ß3-specific primers were 5'-AAATGGATACACGAACCCAA-3' (sense) and 5'-GCTGCATTTGCAAGACTTTAC-3' (antisense); 5'-AAGTGGGTCCATGAACCTAA-3' (sense) and 5'-GCTACATTTACAAGACTTCAC-3' (antisense), respectively. PCR conditions were as follows: 4 minutes at 94°C, 20 seconds at 51°C, and 25 seconds at 72°C (32 cycles). The predicted size of both TGF-ß2 and TGF-ß3 amplimers was 247 bp. Diagnostic restriction enzyme digestion of the PCR amplimers13 was used to confirm the specificity of the primers used for the TGF-ß isoforms targeted. Human ß-actin primers and amplification conditions have been described by us previously.32 PCR products were analyzed by size fractionation, using 2% agarose gels stained with ethidium bromide.
Immunohistochemical Detection of Cytokines
Sections from paraffin-embedded pancreatic carcinoma tissue samples of the patients studied here were stained with antibodies to IL-10 and TGF-ß as described previously.32 TGF-ß was detected using rabbit antisera reacting specifically with either TGF-ß1, TGF-ß2, or TGF-ß3. In the case of IL-10 a combination of two rat mAbs (JES3-9D7 and 12G8) was used as described.33 Specificity of the antibodies used was tested by preincubation with saturating amounts of the appropriate recombinant TGF-ß isoforms or with IL-10 followed by immunostaining of tissue sections.
Generation of LAK Cells and Cytotoxicity Assay
PBMCs isolated by Ficoll-Hypaque gradient separation were cultured for 6 days in RPMI 1640 medium supplemented with 100 U/ml of IL-2 (a gift from Glaxo, Geneva, Switzerland) in the presence and absence of 20 vol% of 5x concentrated CM of pancreatic carcinoma cell lines Capan2, PT45, and BxPC3. LAK activity of these PBMC preparations was evaluated using standard 4-hour 51Cr release assays,34 using the Burkitt lymphoma cell line Daudi as a target.
PBMC Proliferation Assay
The proliferative response of T cells in response to anti-CD3 mAb OKT3 was determined by measuring [3H]thymidine (TdR) uptake. PBMCs were cultured at 2 x 105 cells/well in OKT3-coated flat-bottom 96-well plates in the presence and absence of increasing concentrations of 5x concentrated CM of pancreatic carcinoma cell lines BxPC3, Capan2, and PT45. After 48 hours of incubation, the cells were pulsed for 6 hours with 1 µCi [3H]TdR (6.7 mCi/mmol; NEN-Dupont, Boston, MA) and harvested on glass fiber filters. [3H]TdR uptake was measured using a ß-scintillation counter. In selected experiments, cell line supernatants were preincubated for 1 hour at room temperature with neutralizing antisera to IL-10 (10 µl/ml) and TGF-ß (5 µl/ml) or control antibodies before addition to PBMC cultures.
Determination of Cytokine Concentrations in Sera and CM
IFN-, IL-10, and IL-4 protein concentrations were determined by
enzyme-linked immunosorbent assay (ELISA), using kits commercially
available from Biosource (Camarillo, CA). TGF-ß1 and TGF-ß2
concentrations were determined using ELISA kits available from Genzyme
(Cambridge, MA) and from R&D Systems (Minneapolis, MN), respectively.
The lower threshold of sensitivity of the IFN-, IL-10, and IL-4
assays was 5 pg/ml, whereas the TGF-ß1 and TGF-ß2 assays had a
sensitivity threshold of 50 pg/ml. IL-12 levels were determined using a
radioimmunoassay (RIA) specific for the p40 chain of IL-12 heterodimer,
as previously described.35
The lower threshold of detection
of this assay was 30 pg/ml.
Statistical Analysis
To assess statistically significant differences between data sets, Student's t-tests for independent samples were performed using SigmaPlot software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess IL-10 and TGF-ß production in pancreatic carcinoma
patients, we determined IL-10, TGF-ß1, and TGF-ß2 serum
concentrations in 10 pancreatic carcinoma patients. As compared to
normal age- and sex-matched control donors not affected by obvious
neoplastic or inflammatory diseases, significantly elevated mean serum
levels of all three cytokines were observed in the majority of
pancreatic cancer patients (Figure 1)
.
The mean serum level of TGF-ß1 in pancreatic carcinoma patients was
320.4 ± 252 ng/ml (mean ± SD) as compared to 70.8 ±
27.4 in normal controls (P < 0.05; Student's
t-test for independent samples). The mean serum level of
TGF-ß2 in pancreatic carcinoma patients was 947 ± 620 pg/ml, as
compared to 380 ± 89 in normal controls (P
< 0.05). The mean serum level of IL-10 in cancer patients was
233.7 ± 13.6 pg/ml, as compared to 40.4 ± 17.2 in normal
controls (P < 0.05). In seven of the patients
tumor tissue was available for RT-PCR analysis of IL-10 and TGF-ß1,
-ß2, and -ß3 transcripts. Either IL-10 and/or TGF-ß amplimers of
the expected size were identified in several tumor tissue samples
(Figure 2)
. Whereas expression of
TGF-ß1, TGF-ß2, and IL-10 mRNAs was variable between different
tumors, TGF-ß3 amplimers were identified at comparably high levels in
all pancreatic carcinoma tissue samples. In normal pancreatic tissue,
RT-PCR products corresponding to TGF-ß3, but not TGF-ß1, TGF-ß2,
or IL-10, were identified. Immunohistochemical analysis of TGF-ß1,
-ß2, and -ß3 and IL-10 expression confirmed expression of these
cytokines by pancreatic carcinoma cells. Representative results are
shown in Figure 3
, revealing moderate to
strong staining for all cytokines in tumor cells.
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. To account for overall TGF-ß expression, we also
determined by RT-PCR expression of TGF-ß2 and TGF-ß3 mRNA in these
cells. Primers specific for TGF-ß2 and -ß3 yielded amplification
products of the predicted size in all three cell lines tested.
Consistent with the RT-PCR results, TGF-ß1 protein was secreted by
BxPC3 and PT45 but not Capan2 cells, and IL-10 protein was detected by
ELISA in CM of Capan2 and BxPC3 cells but not PT45 cells (Table 1)
. In summary, whereas IL-10 was
secreted by 2/3 pancreatic carcinoma cell lines, at least 2/3 known
TGF-ß isoforms were expressed by all three cell lines.
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To investigate functional consequences of IL-10 and TGF-ß
production by pancreatic carcinoma cells on immune parameters
potentially relevant to tumor development, we next determined the
effects of media conditioned by pancreatic carcinoma cell lines on
lymphokine-activated cytotoxicity and proliferative capacity of PBMC.
First, the effects of CM on LAK activity of PBMCs derived from two
normal donors was tested. To this end, PBMCs were incubated for 6 days
with 5x concentrated CM of pancreatic carcinoma cell lines added at 20
vol% to RPMI 1640 medium supplemented with IL-2. When LAK activity was
determined by 51Cr release assays using Daudi target cells
at effector-to-target ratios from 40:1 to 5:1, no significant change of
cytotoxic activity was observed when compared to medium controls (not
shown). Second, we determined the effects of pancreatic carcinoma CM on
anti-CD3-dependent proliferation of PBMC obtained from three normal
donors. This was based on previous reports that both TGF-ß and IL-10
affect proliferation of several types of immune cells, including T and
B lymphocytes36-40
and NK cells.36,41
The
addition of 5x concentrated pancreatic carcinoma CM to culture media
at concentrations ranging from 5 to 20 vol% resulted in dose-dependent
inhibition of [3H]TdR uptake in all three PBMC
preparations tested (Figure 4)
. To determine the relative contribution
of TGF-ß and IL-10 to this phenomenon, we used neutralizing
antibodies known to inhibit the biological activity of either all three
TGF-ß isoforms and/or IL-10. These experiments demonstrated that a
combination of the two neutralizing antibodies almost completely
(>90%) reversed the PBMC growth-inhibitory activity produced by the
three pancreatic carcinoma cell lines (Figure 5)
. As predicted by the
cytokine production patterns (see Table 1
and Figure 2
), the anti-IL-10 antibody had no significant effect
in the case of PT45 cells that do not produce IL-10. However, the
TGF-ß antibody was sufficient to neutralize the growth-inhibitory
activity present in PT45 conditioned medium. By contrast, in Capan2 and
BxPC3 cells, the TGF-ß antibody only partially reversed PBMC growth
inhibition, whereas the IL-10 antibody was more effective. In summary,
these results show that the PBMC antiproliferative activity present in
pancreatic carcinoma CM was primarily accounted for by the combined
action of IL-10 and TGF-ß.
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In certain parasitic diseases such as infection with
Leishmania major, different patterns of cytokine production
have been distinguished that correspond to the induction of
functionally diverse T-cell subsets and seem to be associated with
different clinical outcomes.42,43
Generally,
predominance of the Th1 cytokine pattern (IFN- and IL-12) is
associated with a vigorous cytotoxic T-cell response and favorable
outcome, whereas the Th2 cytokine pattern (IL-4, IL-5, IL-10) is
associated with chronic disease or disease progression. Upon antigenic
stimulation, both IL-1044
and TGF-ß45,46
have
been shown to favor development of a Th2-like cytokine pattern and,
thus, are likely to affect immune responses in vivo. Based
on these findings, we asked whether IL-10 and TGF-ß present in
pancreatic carcinoma CM affected the patterns of Th1/Th2 cytokines
produced by PBMC obtained from normal donors. Specifically, we tested
whether pancreatic carcinoma cell CM down-modulated the production of
the Th1-associated cytokines IFN- and IL-12 by PBMCs upon activation
by either anti-CD3 antibody or SAC. As determined by
cytokine-specific ELISA or RIA, CM from all three pancreatic carcinoma
cell lines significantly (P < 0.05) reduced
production of IFN- (Table 2)
and the
p40 chain of IL-12 (Table 3)
by PBMCs
from three normal donors. A combination of neutralizing antibodies to
IL-10 and TGF-ß abolished the inhibitory effect of all three
pancreatic carcinoma CMs on both IFN- and IL-12p40 production in the
three PBMC preparations, except in two PBMC preparations pretreated
with PT45 CM (Figure 6)
. Optimal
neutralization of the effect of pancreatic carcinoma CM on IFN-
production was observed with a combination of anti-IL-10 and
anti-TGF-ß antibodies. The antibody to IL-10 had a comparatively
stronger effect in neutralizing the IL-12 suppressive activity
contained in Capan2 and BxPC3 CM and a weaker effect on PT45 CM. It
should be noted that the anti-IL-10 antibody effectively reduced the
effect of PT45 CM on IFN- production in 3/3 and on IL-12 production
in 2/3 PBMC preparations, although PT45 did not produce IL-10 (see
Table 1
). In contrast to the anti-IL-10 antibody, the anti-TGF-ß
antibody only moderately antagonized down-regulation of IFN- and
IL-12 production by Capan2 and BxPC3 CM. However, it was comparatively
more effective in the case of PT45 CM, which produces TGF-ß but not
IL-10. These results are consistent with the view that production of
either IL-10 alone or a combination of IL-10 and TGF-ß by pancreatic
carcinoma cells contributes to the inhibition of Th1-like responses in
naive PBMCs.
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To address whether elevated TGF-ß and IL-10 serum levels in
pancreatic carcinoma patients may similarly affect the Th1/Th2-balance
in vivo, we tested production of Th1 (IFN-, IL-12) and
Th2 (IL-4) cytokines in PBMC preparations of six pancreatic carcinoma
patients upon stimulation with either anti-CD3 antibody or SAC (Table 4)
. We observed a clear preference for
production of the Th2 cytokine IL-4 but not the Th1 cytokines IFN-
and IL-12 when compared to the cytokine patterns obtained when using
PBMCs from six normal donors. As compared to normal controls, PBMC
preparations from pancreatic carcinoma cells produced, on average,
5.4-fold higher levels of IL-4 upon stimulation with anti-CD3 antibody.
In contrast, production of IFN- and IL-12 was 2.7- and 9.5-fold
lower, respectively, than in controls; the differences in cytokine
levels were all statistically significant (P <
0.05). These data show a preference for the development of a Th2-like
response in PBMCs obtained from pancreatic carcinoma patients.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Earlier studies in syngeneic mouse tumor model systems have shown that both TGF-ß and IL-10, when overexpressed by tumor cells, modulate the antitumor immune response with significant consequences for survival and growth of the tumor cells in the host. However, whereas TGF-ß is generally considered to exert immunosuppressive effects enabling tumor cells to survive in the host, the immunomodulatory role of IL-10 as it relates to the antitumor immune response is less clear. Specifically, transfection of ultraviolet-induced mouse tumor cells with TGF-ß1 has been shown to reduce the capacity of these cells to stimulate cytolytic T-cell responses and renders them tumorigenic in vivo.16 In contrast, high-level IL-10 expression in mouse mammary adenocarcinoma cells induced by transfection of an IL-10 transgene appears to augment tumor rejection.48,49 In vitro, TGF-ß inhibits the growth of NK cells,34,41,50 T lymphocytes,38,51,52 and B lymphocytes,53 although TGF-ß has also been shown to stimulate the growth and differentiation of activated T lymphocytes.54 IL-10 has been shown to inhibit the proliferation of T39 and B lymphocytes55 in certain experimental in vitro settings. However, the effect of tumor-derived IL-10 alone and the combined effect of IL-10 and TGF-ß on human PBMC proliferation is poorly defined at present. We demonstrate that pancreatic carcinoma-derived TGF-ß and IL-10 cooperatively inhibited the proliferation of PBMCs derived from normal donors. Although the contribution of either cytokine to the overall inhibition of DNA synthesis varied depending on the pattern of cytokine production by the three pancreatic carcinoma cell lines investigated, either IL-10 alone or IL-10 in combination with TGF-ß was the main cytokine responsible for the PBMC growth-inhibitory activity present in pancreatic carcinoma CM. This conclusion is supported by the capacity of neutralizing antibodies to these two cytokines to reverse growth inhibition of PBMCs by pancreatic carcinoma CM. In contrast to their antiproliferative activity, we observed no significant effects of pancreatic carcinoma CM on LAK activity generated in PBMCs from normal donors. This result is consistent with earlier observations that TGF-ß134 and IL-1025 only weakly inhibit the cytotoxic activity of LAK cells. Taken together, these results suggested that IL-10 and TGF-ß produced by pancreatic carcinoma cells can influence the nature of the antitumor immune response by inhibiting the expansion of immune effector cells.
In addition to suppression of PBMC proliferation, we observed
inhibitory effects of pancreatic carcinoma CM on PBMC cytokine
production upon unspecific antigenic stimulation. We focused this
investigation on the modulation of IL-12 and IFN- production, based
on the relevance of these two cytokines to the development of
T-lymphocyte-mediated adaptive immunity, which is thought to be
important for immunological tumor rejection. As in the case of PBMC
proliferation, we observed that TGF-ß and IL-10 contained in Capan2
and BxPC3 CM cooperatively inhibited IL-12 and IFN- production
induced by either SAC or anti-CD3 antibody. The contribution of TGF-ß
to the suppression of IL-12 production was marginal, as demonstrated by
the comparably small effects of neutralizing anti-TGF-ß antibody,
whereas the anti-IL-10 antibody alone was sufficient to neutralize the
inhibitory effect of Capan2 and BxPC3 CM on IL-12 production by PBMCs.
Interestingly, anti-IL-10 treatment also reversed some of the effect of
PT45 CM on IL-12 production by PBMCs, although PT45 cells did not
express IL-10 mRNA or secreted IL-10 in measurable quantities. We
consider the effect of the anti-IL-10 antibody in the absence of
tumor-derived IL-10 to reflect the neutralization of PBMC-derived
IL-10. This interpretation is supported by the finding that the
anti-IL-10 antibody in the absence of pancreatic carcinoma CM increased
IL-12 production four-fold, presumably by neutralizing IL-10 derived
from PBMCs themselves. Note that treatment with the anti-IL-10 antibody
did not completely restore IL-12 production to control levels in 2/3
PBMC preparations exposed to PT45 CM, suggesting the presence of yet
another activity unrelated to either IL-10 or TGF-ß that reduces
IL-12 production by PBMCs. This activity is not likely to be
prostaglandin E2 (PGE2), which has recently
been reported to be produced by tumor cells56
and to induce
Th2 cytokine patterns,57
as the pancreatic carcinoma CM
used contained very low PGE2 levels (<25 pg/ml). At this
concentration range PGE2 reportedly has no effect on
cytokine production by PBMCs56
or CD4+ T
cells.57
The strong inhibitory effects of pancreatic carcinoma-derived IL-10 on
the production of the Th1 cytokines IL-12 and IFN- in naive PBMCs
suggested that elevated serum levels of IL-10 in pancreatic carcinoma
patients could also affect T-lymphocyte responses in PBMCs derived from
these patients. As expected, we found a predominant Th2-like phenotype
that develops upon stimulation of PBMCs from pancreatic carcinoma
patients with either anti-CD3 antibody or SAC. Specifically, production
of IL-12 and IFN- was significantly lower in all PBMC preparations
from pancreatic carcinoma patients when compared to controls. This
effect was most obvious in the case of IL-12 in that IL-12 levels
amounted to less than 10% of controls in 5/6 PBMC preparations from
tumor patients. Furthermore, the low levels of Th1 cytokine production
in all six tumor patients were associated with significantly higher
levels of the Th2 cytokine IL-4. These results demonstrate that PBMC
preparations of pancreatic carcinoma patients are primed to develop a
Th2-like rather than a Th1-like response upon antigenic stimulation. A
preponderance of Th2 cytokine production has commonly been observed in
mouse tumor models46
and has also recently been described
in association with human tumors, including colorectal
carcinoma,58
and in biopsy specimens of human basal cell
carcinoma but not benign hyperproliferative lesions of the
epidermis.59
Our findings are consistent with the
conclusion that, in pancreatic carcinoma patients, tumor-derived
TGF-ß and IL-10 similarly contribute to systemic Th2-type immune
responses. Experiments are currently under way to determine whether
this observation extends to specific T-cell responses to recall
antigens.
The functional consequences of a predominant Th2-like cytokine profile for antitumor immunity in pancreatic carcinoma patients are not known. In experimental models both Th1 (IL-2, IL-12) and Th2 (IL-4, IL-10) cytokines, when administered as vaccine adjuvants, have been shown to induce protective immune responses to malignant tumors.60-63 These results support the conclusion that both Th1 and Th2 cells participate in generating effective antitumor immunity. However, at least one study suggests that an excessive, vaccine-induced Th2 cytokine pattern is associated with poor protection against malignant tumors.64 In this study, the efficacy of different vaccine preparations containing a peptide sequence within the mammary mucin MUC1 were explored in a mouse tumor model consisting of human mucin (MUC1) expressed in murine BALB/c 3T3 cells. The capacity of vaccine preparations containing MUC1 expressed on tumor cells, MUC1-containing synthetic peptides, MUC1 fusion proteins, or natural mucin (HMFG) to induce protective immunity was tested. Immunization with whole cells expressing MUC1 induced predominantly Th1 immune responses associated with protective immunity. In contrast, vaccine preparations containing soluble synthetic or native materials provided poor protection associated with the development of prominent Th2 responses. It remains to be investigated whether aberrant Th2 immune responses induced by tumor-derived IL-10 and TGF-ß in patients serve to protect tumor cells from effective antitumor immunity.
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Acknowledgements |
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Footnotes |
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Supported in part by a grant from the Italian Association for Cancer Research to G.B. and National Institutes of Health grant (CA 25874) to U.R.
Accepted for publication April 11, 1999.
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References |
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 R. Falk, M. Hacham, A. Nyska, J. F. Foley, A. J. Domb, and I. Polacheck Induction of interleukin-1{beta}, tumour necrosis factor-{alpha} and apoptosis in mouse organs by amphotericin B is neutralized by conjugation with arabinogalactan J. Antimicrob. Chemother., May 1, 2005; 55(5): 713 - 720. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
P. Monti, B. E. Leone, A. Zerbi, G. Balzano, S. Cainarca, V. Sordi, M. Pontillo, A. Mercalli, V. Di Carlo, P. Allavena, et al. Tumor-Derived MUC1 Mucins Interact with Differentiating Monocytes and Induce IL-10highIL-12low Regulatory Dendritic Cell J. Immunol., June 15, 2004; 172(12): 7341 - 7349. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. E. Warrino, W. C. Olson, W. T. Knapp, M. I. Scarrow, L. J. D'Ambrosio-Brennan, R. S. Guido, R. P. Edwards, W. M. Kast, and W. J. Storkus Disease-Stage Variance in Functional CD4+ T-Cell Responses Against Novel Pan-Human Leukocyte Antigen-D Region Presented Human Papillomavirus-16 E7 Epitopes Clin. Cancer Res., May 15, 2004; 10(10): 3301 - 3308. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-C. Yang, S. Hillinger, K. Riedl, L. Zhang, L. Zhu, M. Huang, K. Atianzar, B. Y. Kuo, B. Gardner, R. K. Batra, et al. Intratumoral Administration of Dendritic Cells Overexpressing CCL21 Generates Systemic Antitumor Responses and Confers Tumor Immunity Clin. Cancer Res., April 15, 2004; 10(8): 2891 - 2901. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hillinger, S.-C. Yang, L. Zhu, M. Huang, R. Duckett, K. Atianzar, R. K. Batra, R. M. Strieter, S. M. Dubinett, and S. Sharma EBV-Induced Molecule 1 Ligand Chemokine (ELC/CCL19) Promotes IFN-{gamma}-Dependent Antitumor Responses in a Lung Cancer Model J. Immunol., December 15, 2003; 171(12): 6457 - 6465. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. A. Holly, C. A. Eberle, and P. M. Bracci Prior History of Allergies and Pancreatic Cancer in the San Francisco Bay Area Am. J. Epidemiol., September 1, 2003; 158(5): 432 - 441. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. P. Smyth, P. P. Stapleton, C. B. Barden, J. R. Mestre, T. A. Freeman, M. D. Duff, S. Maddali, Z. Yan, and J. M. Daly Renal Cell Carcinoma Induces Prostaglandin E2 and T-Helper Type 2 Cytokine Production in Peripheral Blood Mononuclear Cells Ann. Surg. Oncol., May 1, 2003; 10(4): 455 - 462. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Tatsumi, L. S. Kierstead, E. Ranieri, L. Gesualdo, F. P. Schena, J. H. Finke, R. M. Bukowski, J. Mueller-Berghaus, J. M. Kirkwood, W. W. Kwok, et al. Disease-associated Bias in T Helper Type 1 (Th1)/Th2 CD4+ T Cell Responses Against MAGE-6 in HLA-DRB10401+ Patients With Renal Cell Carcinoma or Melanoma J. Exp. Med., September 2, 2002; 196(5): 619 - 628. [Abstract] [Full Text] [PDF]
|
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S. Sharma, M. Stolina, L. Zhu, Y. Lin, R. Batra, M. Huang, R. Strieter, and S. M. Dubinett Secondary Lymphoid Organ Chemokine Reduces Pulmonary Tumor Burden in Spontaneous Murine Bronchoalveolar Cell Carcinoma Cancer Res., September 1, 2001; 61(17): 6406 - 6412. [Abstract] [Full Text] [PDF]
|
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B.-C. Sheu, R.-H. Lin, H.-C. Lien, H.-N. Ho, S.-M. Hsu, and S.-C. Huang Predominant Th2/Tc2 Polarity of Tumor-Infiltrating Lymphocytes in Human Cervical Cancer J. Immunol., September 1, 2001; 167(5): 2972 - 2978. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Wang, R. J. Homer, Q. Chen, and J. A. Elias Endogenous and Exogenous IL-6 Inhibit Aeroallergen-Induced Th2 Inflammation J. Immunol., October 1, 2000; 165(7): 4051 - 4061. [Abstract] [Full Text] [PDF]
|
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S. Sharma, M. Stolina, J. Luo, R. M. Strieter, M. Burdick, L. X. Zhu, R. K. Batra, and S. M. Dubinett Secondary Lymphoid Tissue Chemokine Mediates T Cell-Dependent Antitumor Responses In Vivo J. Immunol., May 1, 2000; 164(9): 4558 - 4563. [Abstract] [Full Text] [PDF]
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) shows
proliferation in the presence of BxPC3 CM; and column 3
(
) shows proliferation
in the presence of PT45 CM. Each column shows the mean ± SD of
three independent experiments using three different normal donors.
Results are expressed as a percentage of [3H]TdR uptake
relative to that of cells grown in the absence of either neutralizing
antibodies or CM. No effect was observed in the presence of a nonimmune
rabbit antiserum used as the control (data not
shown). Asterisks indicate statistically
significant differences (P < 0.05 in Student's
t-tests) of data sets when compared
to controls in the presence of anti-IL-10 and anti-TGF-ß neutralizing
antibodies.
).