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*
Department of Gynecologic Oncology, M. D. Anderson Cancer Center, Houston, TX 77030; and
Department of Microbiology and Immunology, Temple University, Philadelphia, PA 19140
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, or TNF-, and they
expressed CD14, CD16, and CD54, but not HLA-DR, CD80, CD86, CD11a,
CD11b, or CD25 cell surface Ags. Since this subset of monocytes could
affect the modulation of tumor immune responses in vivo, studies were
undertaken to determine their effect on the activation and
proliferation of autologous T cells from the peritoneal cavity of
patients with ovarian carcinoma. Expression of cytokine-specific
transcripts in T cells was determined by RT-PCR. Transcripts for the
following cytokines were detected in patient specimens that also
contained the IL-10-producing monocytes IL-2 (12 of 17 specimens),
GM-CSF (9 of 17 specimens), IFN-
(6 of 17 specimens), and TNF- (4
of 17 specimens). Cytokine production by T cells was determined by
intracellular flow cytometry and by ELISA. IL-2 and IFN- proteins,
unlike their transcripts, were detected only in specimens that lacked
IL-10-producing monocytes. IL-10-producing monocytes cocultured with
autologous T cells inhibited the proliferation of the T cells in
response to PHA. However, T cells cocultured with PEC from which the
IL-10-producing monocytes had been removed did not inhibit T cell
proliferation. Moreover, the inhibition of T cell proliferation by
IL-10-producing monocytes could be reversed by adding neutralizing Abs
to both IL-10R and TGF-ß2. These results suggest that this subset of
monocytes may modulate immune responses by inhibiting T cell
proliferation and cytokine protein production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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transcripts (5), and the presence of
early and late stage activation Ags (6). However, TIL in
the ascitic fluid of EOC patients are frequently exposed to molecules
produced in the tumor microenvironment that could inhibit a variety of
functions associated with activation. IL-10 (IL-10) is an 18-kDa inhibitory cytokine (7) that can be produced by human CD4+ T cells (8, 9, 10), CD8+ T cells (7, 11), macrophages, and monocytes, (12) and other cells such as keratinocytes (13), activated B cells (14), and Burkitt lymphoma cell lines (15). IL-10 has immunosuppressive functions, as shown by its inhibitory effects on T cell activation in vivo and in vitro (10, 16, 17, 18, 19). IL-10 transcripts have been identified in RNA preparations from peritoneal exudate cells (PEC) present in ascitic fluid and from solid tumor specimens from patients with EOC (5, 20). IL-10 protein has also been detected by IL-10-specific ELISA in ascitic fluid from EOC patients (21). However, IL-10 transcripts were not detected in RNA preparations from six ovarian tumor cell lines in our laboratory (5), suggesting that cells other than tumor cells are responsible for the production of IL-10 in ovarian cancer. Production of IL-10 at the tumor site may contribute to the inhibition or down-regulation of the antitumor immune response in ovarian carcinoma.
We report here the identification of a subset of peritoneal monocytes
that produce IL-10. These cells expressed the CD14, CD16, and CD54
differentiation Ags, but not the HLA-DR, CD80, CD86, CD11a, CD11b, or
CD25 cell surface Ags. The phenotype of this monocyte population is not
characteristic of APC (i.e., the cells lack costimulatory and
activation markers). However, since this cell population was detected
in most of the examined PEC specimens, these cells may have an
important role in regulating tumor immunity in patients with EOC. We
further found that these IL-10-producing monocytes inhibited both the
proliferation of autologous T cells in response to PHA and the
production of IFN- by autologous T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Heparinized specimens of malignant ascites were collected from 21 patients with a diagnosis of EOC who had not received chemotherapy. Of these patients, 15 patients had high grade papillary serous carcinoma of the ovary, 4 patients had a mixed pattern of serous and transitional cell carcinoma, 1 patient had borderline papillary serous carcinoma of the ovary, and 1 patient had grade 1 papillary transitional cell carcinoma.
Antibodies
A neutralizing rat anti-human Ab to the IL-10 receptor (clone 3F9 at 11 mg/ml) was a kind gift from Dr. Kevin Moore (DNAX, Palo Alto, CA). Neutralizing Ab to TGF-ß was purchased from Chemicon Industries (Temecula, CA). Abs to cell-surface differentiation markers for HLA-DR, CD80, CD86, CD11a, CD11b, CD25, and CD54 were purchased from Becton Dickinson Immunocytometry Systems (San Jose, CA). Abs to CD14, CD16, and CD68 were purchased from Caltag Laboratories (Burlingame, CA).
Primers
RT-PCR primers were made in core facilities at the M.
D. Anderson Cancer Center for the following cytokine sequences as
previously published (5): IL-10, sense
5'-TGAAGGGATCAGCTGGACAAC3-', antisense 5'-TCGTTCACAGAGAAGCTCAG-3',
product size 351 bp; IL-2, sense 5'-ATGTACAGGATGCAACTCCTGTCTT-3',
antisense 5'-GTCAGTGTTGAGATGATGCTTTGAC-3',product size 458 bp;
IFN-, sense 5'-ATGAAATATACAAGTTATATCTTGGCTTT-3', antisense
5'-GATGCTCTTCGACCTCGAAACAGCAT-3', product size 494 bp; TGF-ß2,
sense 5'-AAATGGATACACGAACCCAA-3', antisense
5'-GCTGCATTTGCAAGACTTTAC-3', product size 247 bp; and ß-actin, sense
5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3', antisense
5'-CTAGAAGCATTGCGGTGGACGATGGAGGG-3', product size 661 bp.
Isolation of PEC
Heparinized specimens of malignant ascites were collected from patients with epithelial ovarian carcinoma who had not received chemotherapy and were processed as previously described (1). Briefly, RBC and granulocytes were depleted from ascites by layering the cell mixture on a Ficoll-Hypaque density cushion. The interface, consisting of mononuclear leukocytes, tumor cells, and nonmalignant cells was collected and washed twice in RPMI 1640 medium (Life Technologies, Grand Island, NY). Further separation of the PEC was performed to determine the presence of the IL-10 transcript in RNA extracts from isolated cell populations as follows.
T cells.
T cell isolation from PEC after Ficoll-Hypaque was performed using a
nylon wool column. The purity of the resulting T cell population was
determined by flow cytometry analysis for the expression of CD3 and
TCRß. Further purification using CD16-labeled magnetic beads for
depletion of NK cells was performed if the population contained less
than 90% CD3+,TCRß+
cells.
Leukocyte and non-leukocyte populations. At least 1 x 107 PEC after Ficoll-Hypaque were processed for the separation of leukocyte and non-leukocyte populations, to determine whether IL-10-producing cells were present in the leukocyte or non-leukocyte population. Cells were centrifuged at 1500 rpm and resuspended in 1 ml of serum-free RPMI 1640. Then, 200 µl of anti-CD45 mAb-coated magnetic beads were added, and the mixture was incubated for 30 min at 4°C. CD45-positive cells were separated from CD45-negative cells with a magnetic particle concentrator (Advanced Magnetics, Oslo, Norway)
Adherent macrophages. Adherent macrophages were separated by resuspending 5 x 106 PEC from the Ficoll-Hypaque interface in 5 ml of serum-free RPMI 1640. Cells were then transferred to a T25 flask and incubated for 1 h at 37°C. Nonadherent cells were decanted, and weakly adherent cells were washed off with RPMI 1640 media. Adherent macrophages remained attached to the plastic.
Monocytes. Further separations of cells of the monocyte lineage were performed using goat anti-mouse-labeled magnetic Dynabeads (Dynal, Oslo, Norway) and mouse anti-human Abs reactive against the cell surface molecules CD14, CD16, CD68, or HLA-DR. PEC (5 x 106) were suspended in 0.5 ml RPMI 1640, and 10 µg of the unlabeled primary Ab (mouse anti-human) was added. Cells were incubated 30 min at 4°C and then brought up in RPMI 1640 to a total volume of 5 ml. Then, 200 µl of stock concentration Dynabeads were added, and the cells were incubated for another 30 min at 4°C. At the end of this incubation, cells positive for the surface marker of interest were selected through the use of a magnetic particle concentrator (Dynal).
RT-PCR
Preparation of RNA. RNA was extracted from the PEC or their subpopulations by a standard extraction method (5). RNA concentrations were determined using 1:100 diluted samples read at 260 nm and 280 nm on a Beckman DU-65 spectrophotometer (Fullerton, CA).
cDNA synthesis and RT-PCR. cDNA was synthesized using 200 U Superscript II reverse transcriptase (Life Technologies) as described (5). The resulting cDNA was analyzed for transcriptional activity in vivo by PCR amplification of 5–10% cDNA aliquots in 50 µl of master mix, consisting of nucleotides, buffer, 0.5 units Taq polymerase (Life Technologies), and primers for the DNA of interest. PCR amplification of the housekeeping gene, ß-actin, was performed simultaneously. The sequence of amplification involved an initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1.5 min in a Perkin-Elmer DNA Thermal Cycler (Branchburg, NJ). A commercially prepared 100 bp ladder was used as a m.w. marker. The RT-PCR products were visualized by ethidium bromide staining of a 1.6% agarose gel and photographed using a Spectroline CCD camera (Fisher Scientific).
Flow cytometry
Cell surface immunofluorescence.
PEC subpopulations were analyzed for the expression of surface markers
(HLA-DR, CD80, CD86, CD11a, CD11b, CD25, CD54, CD14, CD16, CD3, and
TCRß) and cytokine production (IL-10, IL-2, and IFN-) by flow
cytometry using the appropriate mAbs as described previously
(1). Fluorescence was read on a Coulter Epics Profile
Analyzer. Control Abs included isotype-matched
fluorescence-conjugated Igs.
Intracellular staining for cytokines.
Cells producing cytokines were identified by a flow cytometric assay
that had been modified to detect intracellular cytokines. First, cells
were incubated in brefeldin A (Sigma, St. Louis, MO), an inhibitor of
protein transport, at a concentration of 10 µg/ml for a minimum of
4 h at 37°C. Then, surface markers were stained with the
appropriate Abs, and cells were simultaneously fixed and permeabilized
in PBS containing 2% FCS, 0.02% sodium azide, 4% paraformaldehyde,
and 0.1% saponin for 15 min at 4°C. Cells then were washed and
stained with fluorescence-conjugated, cytokine-specific Ab to IL-10
(Caltag), IL-2, or IFN- (Becton Dickinson Immunocytometry Systems).
Cells were incubated 30 min at 4°C, washed, and analyzed on a Coulter
Epics Profile Analyzer (Miami, FL). In certain experiments, cells were
stained by triple color to determine proportions of
CD14+,HLA-DR-,IL-10+
cells.
ELISA
Cytokine-specific ELISAs for IL-10 or IFN- were conducted on
supernatants collected from PEC, T cells coincubated with the
IL-10-producing monocytes, and T cells alone. Supernatants collected
from the monocyte population at 72 h were also examined for the
presence of IL-10 or TGF-ß2. The samples were assayed for secreted
cytokine concentration using IL-10-, IFN-- (Biosource International,
Camarillo, CA), and TGF-ß2- (R&D Systems, Minneapolis, MN) specific
ELISA kits, according to the manufacturer’s directions.
MTT proliferation assay
The effect of the IL-10-producing monocytes on the proliferation response of autologous T cells to PHA was determined using an MTT proliferation assay. A population of monocytes depleted of IL-10 producers was used as a control. T cells were seeded onto a 96-well flat-bottom plate at a concentration of 5 x 106 cells/well in 100 µl of RPMI 1640 media. PHA was added to all wells at a concentration of 20 µg/ml. IL-10-producing monocytes were added to some wells at a concentration of 1 x 105 cells/well. A subpopulation of monocytes that had been depleted of the IL-10-producing monocytes was added to other wells at a concentration of 1 x 105 cells/well as a control. Unseparated PEC were added to the final third of the wells at 1 x 105 cells/well. To establish baseline values for the coincubated cell populations, each of the four cell populations (i.e., T cells, IL-10-producing monocytes, non-IL-10-producing monocytes, and unsorted PEC) was incubated alone, and control values were determined. In certain experiments, neutralizing Abs to IL-10R (10 µg/ml) or TGF-ß (500 ng/ml) were added to the cultures.
After an incubation period of 96 h, MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added at a concentration of 130 µg/ml per well, and the plates were incubated for 3 h at 37°C. One hundred microliters of solubilization buffer (50% dimethyl formamide and 20% SDS) was added, and the plates were incubated overnight at 37°C and then read on a Microplate AutoReader (Bio-Tek Instruments, Winooski, VT) at 570 nm to quantify absorbance. Since MTT is converted to formazan only in the mitochondria of viable cells, the relative amounts of proliferation could be determined by comparing control and coincubated cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify the cells responsible for IL-10 production at the
tumor site, mononuclear leukocytes from the malignant ascites of
patients with ovarian carcinoma were first separated into T cells,
adherent macrophages, leukocytes (CD45+), and
non-leukocytes (CD45-). RNA was prepared from
each separated cell population and examined by RT-PCR for the presence
of IL-10 transcripts. Representative results from 12 separate
experiments are shown in Fig. 1
. IL-10
transcripts were detected in the total leukocyte population
(CD45+), but not in the T cell or adherent
macrophage cell populations. IL-10 transcripts were not detected in the
non-leukocyte population (CD45-), which
comprised mainly tumor cells, fibroblasts, and mesothelial cells.
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). The cells were then double-stained
with anti-CD14 and anti-IL-10 mAbs and analyzed by flow
cytometry. IL-10 production was detected initially in 5% of the total
CD14+ population.
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A), which stained
positively with a combination of anti-CD14 and anti-IL-10 mAbs.
In Fig. 3B, we show that this procedure improved the yield
of monocytes producing IL-10 from 5% to 20%.
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A). Table I shows the numbers of mononuclear
leukocytes and the proportions of
CD14+/HLA-DR-/IL-10+
and of
CD14+/HLA-DR+/IL-10-
cells in patients with malignant ascites.
CD14+/HLA-DR-/IL-10+
cells accounted for 5.52 ± 1.26% of the total CD14-positive cell
population. Table II shows that
CD14+/HLA-DR-/IL-10+
cells were not detected in the peripheral blood lymphocytes of ovarian
cancer patients (n = 21) or of normal donors
(n = 12). In contrast,
CD14+/HLA-DR+/IL-10-
cells were detected in peripheral blood lymphocytes of ovarian cancer
patients, with a mean proportion of 34.9 ± 5.9%, and a range in
values from 21.4% to 44.2%, and
CD14+/HLA-DR+/IL-10- cells were detected in PBL
of normal donors, with a mean proportion of 32.1 ± 4.3% and a
range in values from 25.5% to 38.1%.
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B). Adherence and HLA-DR selections were
also performed on peripheral blood specimens from ovarian cancer
patients. In contrast to the findings in the PEC, IL-10 transcripts
were not detected by RT-PCR (Fig. 4C), and the
IL-10-producing cells were not detected by intracellular FACS (Fig. 4D). Activation and differentiation markers on IL-10-producing cells
CD14+, HLA-DR-
IL-10-producing cells were examined for the presence of other cell
surface markers of differentiation and activation. The presence of
IL-10 protein was verified in the cytoplasm of these cells using
intracellular flow cytometry. The presence or absence of cell surface
Ags associated with differentiation or activation on the
IL-10-producing cell was determined using a double staining method.
Cells were stained with an anti-IL-10 mAb and mAbs to each one of
the following surface Ags: CD80, CD86, CD25, CD16, CD68, CD11a, CD11b,
or CD54. Of these Ags, only the CD16 and CD54 surface markers were
detected on the IL-10-producing
CD14+/HLA-DR- cell
population (Table III).
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To determine whether other cytokines were produced by the
IL-10-producing monocytes, cDNAs were prepared from RNA extracts of
HLA-DR-,CD14+
IL-10-producing cells and examined by RT-PCR for the presence of the
transcripts for the following cytokines: IL-12, TNF-, IL-1, and
TGF-ß2. TGF-ß2 transcripts were detected in
CD14+ IL-10-producing cells from all patients,
whereas TGF-ß2 transcripts were not detected in specimens that
produced no IL-10. IL-12 and IL-1 transcripts were not detected in
any of the specimens, and TNF- transcripts were detected in only two
specimens that also included the IL-10 transcripts (Table IV). TGF-ß2 protein and IL-10 protein
were detected by ELISA in the supernatants of the isolated
IL-10-producing monocyte population after 72 h in culture (Table V). TGF-ß2 has been detected in the
peritoneal fluid of patients with peritoneal carcinomatosis by ELISA,
and in certain mononuclear leukocytes by immunohistochemical staining
of PEC (R. S. Freedman, unpublished data).
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To determine cytokine transcript expression by peritoneal T cells,
T cells from freshly obtained malignant ascites from patients with EOC
were isolated using nylon wool columns. cDNA was synthesized, and the
presence of the cytokine transcripts IL-2, IL-4, IL-10, GM-CSF,
IFN-, and TNF- was examined by RT-PCR. Transcripts for IL-10 were
not detected by RT-PCR in any of the 17 specimens. In contrast,
transcripts for IL-2 were detected in 12/17 specimens, followed by
GM-CSF with 9/17. Transcripts for TNF- and IFN- were less
frequently detected in 4/17 and 6/17 specimens, respectively. Only 1 of
17 specimens exhibited IL-4 transcripts. IFN-, TNF-, IL-2,
GM-CSF, IL-4, and IL-10 were not detected in 4/17 specimens (Table VI). Since these peritoneal T cells were
obtained from patients who also had IL-10-producing monocytes, these
results suggest that ascitic T cells can make certain cytokine
transcripts, and are therefore at least partially activated, even in
the presence of IL-10-producing monocytes.
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Cytokine protein was detected in T cells and monocytes using an
intracellular flow cytometric assay after a 72-h coculture of
autologous cell populations. Fluorescence-conjugated mAb to either
IFN- or IL-2 was used for detection of those cytokines in
CD3+ cells. Fluorescence-conjugated Ab to IL-10
was used for detection of IL-10 produced by the
HLA-DR- monocytes. IFN- and IL-2 were not
detected by flow cytometry in T cells from malignant ascites isolated
from patients that contained IL-10-producing monocytes (Fig. 5A). However, IFN- and IL-2
proteins were detected in the T cells of certain specimens that did not
contain IL-10-producing monocytes (Fig. 5B).
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, although IL-10 was detected in these cocultures (Table VII). In contrast, supernatants from T
cells coincubated with autologous monocytes that did not produce IL-10
(CD14+,HLA-DR+ cells)
contained detectable levels of IFN- (Table VII). Inhibition of T
cell IFN- production by IL-10-producing monocytes could be
neutralized by the addition of anti-IL-10R Ab alone or in
combination with anti-TGF-ß Ab, but not by anti-TGF-ß Ab
alone (Table VIII).
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T cells isolated from PEC were cocultured with autologous
IL-10-producing monocytes, with peritoneal monocytes that did not
produce IL-10, and with the unseparated PEC population. The effects of
the IL-10-producing cells on T cell proliferation can be seen in Fig. 6. T cells coincubated with the
IL-10-producing monocytes exhibit inhibition of proliferation, whereas
T cells coincubated with cells depleted of the IL-10-producing
monocytes were stimulated to proliferate. The effect of the
IL-10-producing monocytes can be seen in the total cell population as
an inhibition of proliferation.
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). Either Ab alone was not sufficient to
reverse the inhibition, although greater effect was seen with the
anti-TGF-ß alone than with the anti-IL-10R alone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The subpopulation of monocytes described here expressed CD16 and CD54
surface Ags, the presence of which might be useful in determining the
functions of these cells. CD16 (FcRIII) is involved in the
phagocytosis of Ab-coated particles. CD54 (ICAM-1) is an accessory
molecule that is preferentially expressed on activated leukocytes.
Since this subset of monocytes did not express CD11a, which is
typically present on T cells and IFN--activated monocytes, it
suggests that these cells may have been activated via an
IFN--independent mechanism.
The absence of certain cell surface molecules could also be useful for characterizing these cells. The absence of detectable CD11b and CD68 on these cells suggested that they could represent a population of monocytes that are at an earlier stage of maturation, rather than a fully differentiated tissue macrophage. Further absence of costimulatory molecules, such as CD80 and CD86, and of MHC class II molecules, such as HLA-DR, suggested that these cells are unlikely to perform the functions of APCs. The high affinity IL-2R (CD25), which is associated with response to low levels of IL-2, typical of the ovarian tumor microenvironment, was also not detected on the IL-10+ monocytes.
The monocytes in this study may influence T cell responses through the
production of IL-10, as well as through the expression, or lack of
expression, of certain cell surface markers. Since tumor-specific
immunity is mediated through T cell responses, it is important to
examine T cell activation in the tumor environment. Results from
experiments by others indicate that TIL may be anergic at the tumor
site (27, 28), and it is reasonable to hypothesize that
the production of IL-10 could be involved. Previous studies have
determined that IL-10 produced by human monocytes could have strong
down-regulatory effects on Ag-specific T cell activation at several
levels (16). These include down-regulation of HLA class I
expression on tumor cells and HLA class II expression on monocytes, as
well as inhibition of the expression of costimulatory molecules such as
B7.1, B7.2, and ICAM on APCs (29, 30, 31). The ICAM adhesion
molecule is necessary for the activation of resting T cells, whereas
the B7 molecules are important in the stimulation of Ag-activated T
cells. Exogenous IL-10 has also been shown to influence T cell
activation by decreasing the stability of the mRNA transcripts of other
cytokines produced by T cells (32, 33). In this study, we
examined the expression of cytokine transcripts present in T cells
isolated from the tumor environment. Transcripts for Th1-like
cytokines, including IL-2 and, less frequently, IFN-, have been
detected in T cells from the peritoneal cavity of patients with ovarian
cancer (5); however, transcripts for Th2-like cytokines,
like IL-4 and IL-10, were not detected in these same T cells. These
results would suggest that cell-mediated T cell responses were being
initiated, if not completed.
The absence of IL-2 and IFN- in cultures including the
IL-10-producing phenotype (shown in Fig. 6) indicated that these cells
may be affecting cytokine production at the translational level. The
observation that IL-2 and IFN- protein were present in cultures from
which the IL-10-producing monocytes were removed suggested that the
IL-10 present in the tumor environment may also inhibit T cell
activation passively through decreased expression of costimulatory
molecules.
These results seemed to indicate a partial block of T cell activation that may not be due entirely to IL-10 production in the tumor environment. TGF-ß2, which has inhibitory effects on T cell activation, especially on proliferation (34), was also produced by this subset of monocytes. In the proliferation experiments described here, Abs to both IL-10R and TGF-ß were needed to restore full proliferative activity to the T cells incubated with the IL-10-producing monocytes.
The production of IL-10 by a subset of monocytes may be one mechanism by which tumor progression occurs. IL-10 production by monocytes inhibited Th1 responses by autologous T cells, as well as T cell proliferation. The presence of TGF-ß2 transcripts and the lack of costimulatory molecules in this population, or in dendritic cells isolated from the peritoneum (35), would also contribute to an overall attenuation of cell-mediated immune responses. The effects of these monocytes are of considerable importance in the context of malignant tumors, since a concomitant reduction in immune function is associated with tumor growth and progression. Efforts to inhibit the immunosuppressive effects of these monocytes may result in improved therapeutic approaches to immunogenic cancers.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. R. S. Freedman, University of Texas M. D. Anderson Cancer Center, Department of Gynecologic Oncology, 1515 Holcombe Boulevard, Box 67, Houston, TX 77030.
3 Abbreviations used in this paper: EOC, epithelial ovarian carcinoma; PEC, peritoneal exudate cells; TIL, tumor-infiltrating lymphocytes.
Received for publication March 24, 1999. Accepted for publication September 10, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and granulocyte-macrophage colony stimulating factor in ovarian cancer biopsies. Proc. Natl. Acad, Sci. USA 89:7708.This article has been cited by other articles:
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 A. S. Yang and E. C. Lattime Tumor-induced Interleukin 10 Suppresses the Ability of Splenic Dendritic Cells to Stimulate CD4 and CD8 T-Cell Responses Cancer Res., |
, macrophage.
