Identification of an IL-10-Producing HLA-DR-Negative Monocyte Subset in the Malignant Ascites of Patients with Ovarian Carcinoma That Inhibits Cytokine Protein Expression and Proliferation of Autologous T Cells

Amy E. Loercher, Micheal A. Nash, John J. Kavanagh, Chris D. Platsoucas and Ralph S. Freedman
J Immunol December 1, 1999, 163 (11) 6251-6260;

Abstract

A newly described subset of monocytes has been identified in peritoneal exudate cells (PEC) from the malignant ascites from patients with ovarian cancer. These cells were characterized by the production of IL-10 and TGF-β2, but not IL-12, IL-1α, or TNF-α, 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-γ (6 of 17 specimens), and TNF-α (4 of 17 specimens). Cytokine production by T cells was determined by intracellular flow cytometry and by ELISA. IL-2 and IFN-γ 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-β2. These results suggest that this subset of monocytes may modulate immune responses by inhibiting T cell proliferation and cytokine protein production.

Tcell activation is usually associated with clonal expansion of Ag-specific T cells and production of cytokines or cell-mediated cytotoxicity by the activated effector cells. T cells isolated from the peritoneal cavity of patients diagnosed with epithelial ovarian carcinoma (EOC)3 can be stimulated to grow in culture in the presence of low concentrations of rIL-2 (1). Certain rIL-2-expanded T cells exhibit preferential autologous tumor cell killing (2) and cytokine production (3). Tumor-infiltrating lymphocytes (TIL) in patients with EOC contain activated T cells, as characterized by the oligoclonal expansion of the TCR (4), production of IL-2 and IFN-γ 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-γ by autologous T cells.

Materials and Methods

Patient specimens

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-β 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-γ, sense 5′-ATGAAATATACAAGTTATATCTTGGCTTT-3′, antisense 5′-GATGCTCTTCGACCTCGAAACAGCAT-3′, product size 494 bp; TGF-β2, sense 5′-AAATGGATACACGAACCCAA-3′, antisense 5′-GCTGCATTTGCAAGACTTTAC-3′, product size 247 bp; and β-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 TCRαβ. Further purification using CD16-labeled magnetic beads for depletion of NK cells was performed if the population contained less than 90% CD3+,TCRαβ+ cells.

Leukocyte and non-leukocyte populations.

At least 1 × 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 × 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 × 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, β-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 TCRαβ) and cytokine production (IL-10, IL-2, and IFN-γ) 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-γ (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-γ 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-β2. The samples were assayed for secreted cytokine concentration using IL-10-, IFN-γ- (Biosource International, Camarillo, CA), and TGF-β2- (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 × 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 × 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 × 105 cells/well as a control. Unseparated PEC were added to the final third of the wells at 1 × 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-β (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.

Results

Weakly adherent, HLA-DR-negative monocytes spontaneously produce IL-10

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.

FIGURE 1.
FIGURE 1.

IL-10 localization in PEC by RT-PCR. PEC obtained from malignant ascites were separated into cell populations based on surface marker expression or physical characteristics as follows: T cells, adherent macrophages (Ad.), leukocytes (CD45+), and tumor, fibroblast, and mesothelial cells (CD45−). RNA extracted from each cell population was examined by RT-PCR to determine the presence or absence of the IL-10 transcript. NIH-3T3 murine fibroblasts transfected with the human IL-10 gene under constitutive promoter were examined as a positive control (Con) for IL-10. The m.w. marker (MW) was a 100 bp ladder. β-actin transcripts are shown for each cell population. IL-10 transcripts were detected in the CD45+ population and in the total cell population. This gel is representative of 12 experiments. Mφ, macrophage.

These results suggested that the IL-10-producing cells were present within the leukocyte population, although they were not detected among T cells or adherent macrophages. Further characterization of the IL-10-producing population by magnetic bead separation on the basis of the CD14 surface marker expression revealed that IL-10 transcripts were present in CD14+ cells, but not in CD14− cells (Fig. 2⇓). 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.

FIGURE 2.
FIGURE 2.

IL-10 transcripts detected in CD14+ population. CD45+ cells were separated into CD14-positive (CD14+) and CD14-negative (CD14−) cell populations by first incubating with a mouse anti-human CD14, then selecting the CD14+ cells using goat anti-mouse-coated magnetic beads. RNA was extracted, and RT-PCR was utilized to detect the IL-10 transcript. β-actin transcripts were detected in all specimens. IL-10 transcripts were present in the total and CD14+ cell populations. This gel is a representative result from 12 experiments.

To increase the proportion of CD14+ cells that produced IL-10, we utilized the differential adherence properties of these cells. It has been previously demonstrated that monocyte populations could be separated based on their adherence properties (22). In our experiments, the cells were separated into nonadherent, weakly adherent, and adherent subsets. RNA extracts were prepared from the three different adherence populations, and RT-PCR was performed to detect the presence of the IL-10 transcript. IL-10 transcripts were detected only in the weakly adherent monocyte subset (Fig. 3⇓A), which stained positively with a combination of anti-CD14 and anti-IL-10 mAbs. In Fig. 3⇓B, we show that this procedure improved the yield of monocytes producing IL-10 from 5% to 20%.

FIGURE 3.
FIGURE 3.

IL-10 localization in weakly adherent (Ad.) cells from malignant ascites. A, Cells from malignant ascites were separated into three populations on the basis of their adherence properties. β-actin transcripts are shown for each cell population. The IL-10 transcript was present in the weakly adherent cell population (cells that were washed off after 1 h incubation). This gel is representative of the results from 12 separate experiments. B, The percentage of IL-10-producing cells as detected by intracellular FACS increased from 5% to 20% after selecting the weakly adherent cell population. MW, m.w. marker.

Because HLA-DR+ macrophages are required for Ag-induced T cell activation, whereas HLA-DR− macrophages seem to suppress T cell activation (23), we used magnetic beads to separate CD14+ cells into two populations on the basis of HLA-DR expression as described in Materials and Methods. RNA was prepared from the HLA-DR-positive and HLA-DR-negative cell populations. cDNAs were amplified by RT-PCR using IL-10-specific primers to determine the presence of IL-10 transcripts. IL-10 transcripts were detected in the HLA-DR-negative cells, but not in the HLA-DR-positive cells (Fig. 4⇓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%.

FIGURE 4.
FIGURE 4.

IL-10 detected in HLA-DR-negative cells. A, Cells from malignant ascites were separated into HLA-DR-positive (HLA-DR+) and HLA-DR-negative (HLA-DR−) populations using Ab-labeled magnetic beads. RNA was extracted from each population, and RT-PCR was utilized to identify the IL-10 transcript in the HLA-DR− population. Transcripts for β-actin are shown for each population. This gel is representative of 10 experiments. B, After adherence and HLA-DR selection, the IL-10-producing cells were increased to 80% as detected by intracellular flow cytometry, as compared with 5% in the unselected CD14+ cells. C, IL-10 transcripts were not detected in RNA extracts of PBMC collected from five patients with ovarian cancer. β-actin transcripts are shown for each specimen. D, FACS analysis shows that IL-10-producing cells were not detected in PBMC collected from ovarian cancer patients either before or after HLA-DR selection.

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Table I.

Numbers and percentages of CD14+/HLA-DR−/IL-10+ cells and CD14+/HLA-DR+/IL-10− cells in PEC from patients with ovarian cancer

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Table II.

Numbers and percentages of CD14+/HLA-DR−/IL-10+ and CD14+/HLA-DR+/IL-10− cells in PBMC of patients with ovarian cancer and normal donors

Next, the weakly adherent, HLA-DR-negative cells were examined with anti-CD14 and anti-IL-10 mAbs in a two-color staining procedure and analyzed by flow cytometry for intracellular production of IL-10. The percentage of monocytes producing IL-10 in this selected population was 70–80% (Fig. 4⇑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. 4⇑C), and the IL-10-producing cells were not detected by intracellular FACS (Fig. 4⇑D).

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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Table III.

Proportions of IL-10+ monocytes that express leukocyte cell surface Ag by immunofluorescence

TGF-β2 production by IL-10-producing monocytes

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-α, IL-1α, and TGF-β2. TGF-β2 transcripts were detected in CD14+ IL-10-producing cells from all patients, whereas TGF-β2 transcripts were not detected in specimens that produced no IL-10. IL-12 and IL-1α transcripts were not detected in any of the specimens, and TNF-α transcripts were detected in only two specimens that also included the IL-10 transcripts (Table IV⇓). TGF-β2 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-β2 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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Table IV.

Expression of cytokine transcripts in CD14+/HLA-DR− monocytes isolated from malignant ascites of patients with ovarian cancer

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Table V.

Determination by ELISA of IL-10 and TGF-β2 proteins present in the supernatant of HLA-DR-negative monocytes

Monocyte-derived IL-10 did not inhibit cytokine transcript expression by T cells

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-γ, and TNF-α 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-α and IFN-γ were less frequently detected in 4/17 and 6/17 specimens, respectively. Only 1 of 17 specimens exhibited IL-4 transcripts. IFN-γ, TNF-α, 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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Table VI.

Cytokine transcript expression by purified T cells from malignant ascites of patients with ovarian cancer

Monocyte-derived IL-10 inhibits cytokine protein production by T cells

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-γ 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-γ 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. 5⇓A). However, IFN-γ and IL-2 proteins were detected in the T cells of certain specimens that did not contain IL-10-producing monocytes (Fig. 5⇓B).

FIGURE 5.
FIGURE 5.

A, Lack of T cell cytokine production following coculture with IL-10+ monocytes. IL-10-producing monocytes, selected by adherence and absence of HLA-DR expression, were examined in the presence of T cells by intracellular flow cytometry for the production of the IL-10 protein. IL-10 protein was detected by intracellular flow cytometry in the CD14+ population. A live gate was placed over the lymphocyte population to determine the production of the IL-2 and IFN-γ proteins by these cells. IL-2 and IFN-γ proteins were not detected in CD3+ cells that had been cocultured with the IL-10-producing cells. B, T cell cytokine production detected following coculture with IL-10-negative monocytes. The IL-10 protein was not detected in a monocyte population depleted of the IL-10-producing cells. IL-2 and IFN-γ proteins were detected in CD3+ cells that had been coincubated with the IL-10-negative monocytes.

Secreted cytokine protein was detected in certain patients by cytokine-specific ELISA. Supernatants from T cells coincubated with autologous IL-10-producing monocytes contained no detectable levels of IFN-γ, 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-γ (Table VII⇓). Inhibition of T cell IFN-γ production by IL-10-producing monocytes could be neutralized by the addition of anti-IL-10R Ab alone or in combination with anti-TGF-β Ab, but not by anti-TGF-β Ab alone (Table VIII⇓).

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Table VII.

Effects on the production of IFN-γ and IL-10 following the coculture of peritoneal T cells either with IL-10-producing or with non-IL-10-producing monocytes

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Table VIII.

T cells cocultured with IL-10-producing monocytes produce IFN-γ with the addition of neutralizing Ab to IL-10R as detected by ELISA

IL-10-producing cells inhibit T cell proliferation

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.

FIGURE 6.
FIGURE 6.

PHA-induced proliferation of peritoneal T cells was inhibited following coculture with IL-10-producing monocytes. T cell proliferation was measured using the colorimetric MTT assay after a 96-h incubation. T cells were stimulated with 20 μg/ml PHA and coincubated with either the HLA-DR− IL-10-producing cells, the HLA-DR+,IL-10-negative cells, or both cell populations. The HLA-DR− IL-10-producing cells were able to inhibit PHA-induced T cell proliferation, even in the presence of the HLA-DR+,IL-10-negative cells (as shown in the coincubation with total PEC). Only the T cells coincubated with the HLA-DR+,IL-10-negative cells demonstrated mitogen-induced proliferation. Unstimulated T cells were used as a control.

Inhibition of T cell proliferation to PHA could be reversed with the addition of neutralizing Abs to both TGF-β and IL-10R (Fig. 7⇓). Either Ab alone was not sufficient to reverse the inhibition, although greater effect was seen with the anti-TGF-β alone than with the anti-IL-10R alone.

FIGURE 7.
FIGURE 7.

Neutralizing Abs to IL-10R and TGF-β reverse inhibition of T cell proliferation following coculture with IL-10+ monocytes. T cell proliferation was measured using the colorimetric MTT assay after a 96 h incubation. T cells were stimulated with 20 μg/ml PHA and coincubated with HLA-DR−, IL-10-producing cells. Neutralizing Ab to TGF-β (500 ng/ml), IL-10R (10 μg/ml), or both Abs were added to certain cultures. Inhibition of T cell proliferation could be fully reversed by the addition of neutralizing Ab to both IL-10R and TGF-β, but not by either Ab alone, although the reversal of inhibition was greater with the neutralizing α-TGF-β alone than with the α-IL-10R alone.

Discussion

Human monocyte populations are heterogeneous in size and morphology, and also in their function. In this study, we have identified, for the first time, a subset of monocytes from the peritoneal cavity of patients with ovarian cancer that were able to suppress T cell responses. These cells, which were CD14 positive, did not express molecules that are associated with activation or costimulation, such as HLA-DR, CD80, or CD86. These monocytes were therefore unlikely to have Ag presenting functions but could represent a population of cells with immunosuppressive functions. The IL-10-producing cells were detected only in the PEC of ovarian cancer patients and were not detected in the peripheral blood of these patients. However, the CD14+/HLA-DR−/IL-10+ cells were detected after in vitro stimulation of PBMC from patients with ovarian cancer utilizing rGM-CSF and rIL-2 (24). Suppressor monocyte populations have been identified in the microenvironment of other tumor systems, notably fibrosarcomas and lung carcinomas (25, 26). In these studies, tumor growth was correlated with the presence of active suppressor populations of cells that expressed monocyte surface markers and that seemed to be stimulated by the tumor.

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 (FcγRIII) 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-γ-activated monocytes, it suggests that these cells may have been activated via an IFN-γ-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-γ, 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-γ 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-γ 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-β2, 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-β 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-β2 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.

Footnotes

  • ↵1 This paper was supported in part by National Institutes of Health Grants RR-02558 and CA-64943.

  • ↵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 March 24, 1999.
  • Accepted September 10, 1999.

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The Journal of Immunology: 163 (11)
The Journal of Immunology
Vol. 163, Issue 11
1 Dec 1999
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