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*
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892;
Department of Microbiology and Immunology, University of Kentucky Medical Center, University of Kentucky, Lexington, KY 40536; and
Laboratory of Immunogenetics, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rockville, MD 20852
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF-
, and increased production of IL-6 and
IL-10. The GCS-induced changes in IL-12 and IL-10 occurred in
monocytes, and involved changes in IL-12 p40 and IL-10 mRNA expression.
Incubation with GCS also resulted in reduced expression of MHC class II
and of CD80/86 costimulatory molecules on monocytes. The
immunosuppressive effects were not the result of IL-6 or TGF-ß1 that
was detected in GCS. However, it was due to a factor(s) that is
resistant to pH extremes, differentially susceptible to temperature,
susceptible to trypsin, and has a minimum molecular mass of 40
kDa. Our findings show that glioblastoma-generated factors that are
known to suppress T cell responses alter the cytokine profiles of
monocytic APC that, in turn, inhibit T cell function. This model
indicates that monocytes can serve as an intermediate between
tumor-generated immune-suppressive factors and the T cell responses
that are suppressed in gliomas. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production and increases in
IL-4, IL-5, IL-6, and/or IL-10 production. Human tumors in which one or
more of these cytokine changes have been reported include Hodgkin’s
lymphomas (4), cervical (5, 6) and ovarian carcinomas (7), melanomas
(8), basal and squamous cell carcinomas (9), renal cell carcinomas
(10), non-small cell lung cancer (11), and gliomas (12).
Tumor-associated immune dysregulation can also be reflected in T cells
at the level of signal transduction, as defects in STAT5 have been
reported in a murine breast tumor model (13), and Janus kinase 3 (Jak3)
expression is down-regulated by a soluble factor from a human renal
cell carcinoma (14).
Human gliomas provide an interesting example of tumor-associated immune
dysfunction. The in vitro responses of T cells from patients who
present with primary gliomas are impaired in their ability to respond
in vitro to Ags and T cell mitogens by proliferation and IL-2
production (3, 15, 16). Surgical removal of the primary tumor can
result in restoration of systemic in vitro responses to T cell
mitogens, which again declines with recurrence of the tumor (17).
Glioma patients also frequently fail to elicit delayed skin reactions
(15), and patients’ T cells express reduced numbers of high affinity
IL-2R (18, 19). One of our laboratories recently reported that T cells
from glioma patients exhibit defects in tyrosine phosphorylation of
several proteins, reduced levels of phospholipase C1 and
p56lck, as well as reduced mobilization of
calcium (20). Other studies demonstrated that cultures of glioblastoma
cell lines produce a factor(s) that inhibits Ag- and mitogen-stimulated
proliferation and IL-2 production by T cells from healthy individuals
(21, 22). These findings suggest that one or more factors contained in
glioma culture supernatant
(GCS)3 exerts immunoregulatory
effects on systemic cellular immunity, as well as at the site of the
primary tumor.
Although Th cell proliferation and IL-2 production have been demonstrated to be defective in glioma patients and in cultures of PBMC exposed to GCS (21), the possibility that these T cell defects have their origin in APC has not been addressed. The production of IL-12 and/or IL-10 and the stimulatory and costimulatory molecules that are important for T cell activation are altered in certain infectious diseases such as leprosy (23) and AIDS (24, 25). Increased IL-10 production and mRNA expression have also been reported in several tumors including gliomas (5, 8–11, 26–29), and IL-12 has been used to inhibit the growth of murine tumors (30, 31). These findings raise the possibility that the depressed cellular immune condition associated with certain tumors contributes to their neoplastic disease and is linked to cytokine dysregulation (2).
In the present study, we investigated whether exposure of healthy human
blood donors’ PBMC and monocytes to GCS would result in decreased
IL-12 and increased IL-10 production and mRNA expression. Because MHC
class II and CD80/86 expression have been shown to be down-regulated by
IL-10 in human and murine in vitro models (32, 33), we also tested for
reduced expression of MHC class II and CD80/86. Finally, we tested
whether in vitro Th response would be generated by a mixture of
autologous T cells and GCS-exposed monocytes. Our results indicate that
GCS induces down-regulation of IL-12, MHC class II, and CD80/86, and
concomitant up-regulation of IL-10 in monocytes. We also observed that
proliferative responses to recall Ags were abrogated when monocytes
were exposed to GCS before mixing with autologous T cells. These
findings suggest that the defects seen in the Th of patients with
gliomas originated as defective APC function that, in turn, resulted in
aberrant signaling of T cells and subsequent down-regulation of IL-2
and IFN- production and gene expression. No single cytokine that we
detected in GCS has been demonstrated to induce all of these changes in
monocytes. The GCS activity was attributed to a factor(s) that is
resistant to pH extremes, differentially susceptible to temperature,
susceptible to trypsin, with a minimum molecular mass of approximately
40 kDa.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Samples of whole blood were provided for in vitro laboratory studies by the Transfusion Medicine Department (National Institutes of Health, Bethesda, MD), under a National Institute of Health Institutional Review Board-approved protocol. The PBMC were separated on Lymphocyte Separation Media (Organon Teknika, Rockville, MD), and resuspended at 1.5 x 106 cells/ml in RPMI 1640 (Life Technologies, Rockville, MD), supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µM HEPES buffer, and 2 µM glutamine (National Institutes of Health Media Unit, Bethesda, MD), and 5% human AB+ serum (Sigma, St. Louis, MO).
Production of GCS from different glioblastoma cell lines
The SNB-19 and U251 glioma cell lines were used for production
of GCS. Two independently carried SNB-19 lines were studied: Both
originated from Dr. Paul Kornblith (University of Pittsburgh,
Pittsburgh, PA). One has been carried for several years by the
University of Kentucky laboratory (Lexington, KY); the other was
recently obtained as a cryopreserved sample from American Type Culture
Collection (ATCC, Manassas, VA). The U251 line (34) is carried in
National Cancer Institute laboratory. These cells were maintained in
RPMI 1640 culture media containing 5% FCS, 100 U/ml penicillin, 100
µg/ml streptomycin, and 10 µM HEPES buffer in a humidified, 37°C,
5% CO2 incubator. Cells were passaged at 4- to 7-day
intervals using 0.25% trypsin (Life Technologies, Grand Island, NY) in
PBS (pH 7.3–7.4). Supernatants were harvested from all of the
glioblastoma lines after 4 to 7 days of culture in 5% FCS/RPMI 1640
medium. GCS was also generated by culturing the U251 cell line for 3
days in conditioned Cellgro Complete Serum Free Media (Mediatech,
Herndon, VA) or FCS-free RPMI 1640 to obtain GCS samples for factor
purification studies. Culture supernatants of these glioblastoma lines
were tested directly for factor activity by production of IL-12, IL-10,
IFN-, and T cell proliferation (see below). Supernatants from one of
the SNB-19 cell lines grown to confluency were concentrated
(38–89-fold) on a Minitan tangential flow concentrator using 100-kDa
molecular mass cutoff, low protein binding, and regenerated cellulose
filters to collect GCS. Following concentration, the GCS was filtered
through 0.22-mm filters (Costar, Cambridge, MA) and stored at
-80°C until needed. In most experiments, GCS preparations were used
at a final 1/20 dilution. Supernatants from the other SNB-19 and the
U251 cell lines were either concentrated 20–40-fold using a
differential molecular mass cutoff Centricon Plus-80 Centrifugal Filter
Device (Millipore, Bedford, MA), or were tested without concentration.
Culture supernatants exhibited activity, irrespective of whether they
had been concentrated. We verified that different preparations of GCS
do not exert a toxic effect on PBMC cultured for 7 days.
As controls for the glioblastoma lines, we tested supernatants of ovarian carcinoma A2780, A2780/CP (35), and National Institute of Health-ovcar-3; the T2, U937, and K562 lymphoma lines; the prostate carcinoma PC-3 (National Institutes of Health-ovcar-3, T2, U973, K562, and PC-3 were obtained from ATCC); and two EBV-transformed lymphoma cell lines generated in our laboratory.
T cell function assay
Different preparations of GCS were tested for inhibition of T cell function by culturing 1.5 x 106 PBMC/ml, or 1 x 106 T cells/ml with 0.5 x 106 autologous monocytes/ml in 200 µl of culture media in 96-well flat-bottom culture plates (Costar) in a humidified, 37°C, 7% CO2 incubator. The cultures were either unstimulated, or were stimulated with PHA-M (1/80 dilution) (Life Technologies) or a pool of recall Ags consisting of: influenza A virus (FLU) (A/Bangkok/RX173, H3N2) (final dilution of 1/800); tetanus toxoid (TT) (Connaught Laboratories, Swiftwater, PA) (final dilution of 1/800); and Candida albicans Ag (CASTA) (Greer Laboratories, Lenoir, NC) (10 µg/ml). The cultures were pulsed with [3H]thymidine on day 2 for PHA and day 6 for recall Ags, harvested 20 h later using a Basic 96 Harvester (Skatron Instruments, Sterling, VA), and counted in a ß-spectrometer (Wallac, Gaithersburg, MD).
Enrichment of monocytes and T cells
Enriched monocytes and T cells were obtained from elutriated lymphocyte-depleted and monocyte-depleted populations isolated from PBMC of healthy blood donors. Remixing experiments were performed using autologous depleted and enriched cell populations.
To obtain enriched monocytes, lymphocyte-depleted PBMC were incubated on ice for 30 min with an Ab mixture consisting of mouse anti-human CD3, CD16, and CD19 mAb (IgG) (PharMingen, San Diego, CA), at 5 µg of each mAb per 10 x 106 cells in 100 µl PBS containing 10% FBS (PBS/FBS). The cells were washed three times in PBS/FBS, the cell pellet was resuspended in PBS/FBS in the presence of Dynabeads M280 sheep anti-mouse IgG (Dynal, Oslo, Norway) (10 beads/cell), and the mixture was incubated on ice for 30 min. The cell-bead mixture was exposed to a magnet through three cycles of magnetic separation and washing of the unattached cells. This procedure resulted in enrichment of monocytes to greater than 90% CD14+ cells, determined by flow cytometry. The enriched monocytes were tested by flow cytometry for the presence of CD83+ cells, a marker of mature dendritic cells (36), and none were detected.
To obtain enriched T cells, monocyte-depleted cells were incubated with the Lympho-Quik-T Isolation Reagent (One Lambda, Canoga Park, CA), which depletes of all cell types except T cells by Ab-mediated, complement-dependent lysis (37).
Cytokine production and detection
The production of cytokines by PBMC, enriched monocytes, or monocytes plus autologous T cells was assessed by culturing 3 x 106 PBMC, 1 x 106 monocytes, or 1 x 106 monocytes plus 2 x 106 T cells in 2 ml of culture media in 24-well plates (Costar), respectively, in a humidified, 37°C, 7% CO2 incubator. Cells were either unstimulated or were stimulated with Staphylococcus aureus Cowan strain 1 (SAC) (0.01%) (Pansorbin, Calbiochem-Behring, La Jolla, CA). Culture supernatants were harvested after 24 h and stored at -80°C.
The IL-12 p70 heterodimer production was assessed by ELISA from R&D
(Minneapolis, MN). Total IL-12 p40, IL-2, IL-4, and IL-6 productions
were detected by ELISA from Genzyme (Cambridge, MA). IL-10, IL-5,
IFN-, and TNF- productions were assessed in the supernatants of
24-h SAC-stimulated cultures, using PharMingen capture and detection
Abs, as previously described (37). The limit for detection of these
cytokines was in the range of 5–20 pg/ml.
Detection of IL-12 and IL-10 mRNA
Expression of hypoxanthine phosphoribosyltransferase (HPRT), IL-10, and IL-12 p40 mRNA was assessed on PBMC stimulated with SAC for 6 h, using a semiquantitative RT-PCR protocol, as previously described (37).
Detection of intracellular cytokines
PBMC were cultured for 6 h with or without stimulation in
Teflon vials (Pierce Chemicals, Rockford, IL) in a 37°C, 7%
CO2 incubator; then Brefeldin A (Sigma) (5 µg/ml) was
added for an additional 18 h of incubation. In some experiments,
PBMC were cultured without or with 10 µg/ml Brefeldin A in 5%
human AB+ serum, RPMI 1640 medium for 1 day. Cell viability
was tested by trypan blue exclusion or propidium iodide staining. The
cells also were analyzed by FACS with anti-CD3 and CD14 staining.
No differences were obtained in cell viability (>90%) in either the
CD3+ or CD14+ populations in the presence or
absence of Brefeldin A. Cells were harvested and washed in a staining
buffer (PBS containing 1% FBS and 0.1% w/v sodium azide),
preincubated with human IgG at 4°C for 30 min to block FcR. The cells
were then stained at 4°C for 30 min with fluorochrome-conjugated mAb
specific for a cell surface Ag such as CD14 and CD3 (PharMingen, San
Diego, CA). The cells were subsequently washed with staining buffer,
pelleted by centrifugation, and fixed in 500 µl of fixing buffer (4%
w/v paraformaldehyde in PBS) at 4°C for 30 min or overnight. The
cells were washed in the staining buffer, pelleted by centrifugation,
and resuspended in 100 µl of permeabilization buffer (PBS containing
1% FBS, 0.1% w/v sodium azide, 0.1% w/v saponin). The cells were
incubated for 30 min at 4°C with 0.5 µg fluorochrome-conjugated
anti-cytokine Abs (anti-IL-12 p40 and p70, IL-10, IL-6,
IFN-, and TNF- from PharMingen). The cells were then washed twice
in permeabilization buffer, resuspended in staining buffer, and
analyzed by flow cytometry using a FACScan (Becton Dickinson, San Jose,
CA). The cells were gated on monocytes or lymphocytes based on forward
and side light scatter. In some experiments, the binding of
fluorochrome-conjugated anti-cytokine mAb was blocked by
preincubation of the conjugated mAb with excess recombinant cytokine
(IL-12 p40 and IL-10; PharMingen).
Immunoprecipitation
To remove IL-6, TGF-ß, and CGRP, GCS was diluted sevenfold in PBS. Anti-IL-6 (clone MQ2-13A5 rat IgG1, final concentration of 5 µg/ml), anti-TGF-ß mAb (mouse IgG1, final concentration of 10 µg/ml), and rabbit anti-human CGRP serum (final 1/120 dilution) were added singly or in combination. The GCS and Ab mixture was incubated overnight at 4°C under rotating conditions. An excess of GammaBind G Sepharose (Pharmacia Biotech, Piscataway, NJ) was added for 10 h, and the mixture was centrifuged for 10 min at 2000 x g. An excess of protein A-Sepharose (Pharmacia) was added to the supernatant, which was incubated overnight under rotating conditions at 4°C. The mixture was centrifuged again, and the supernatant was sterilized by passing through a 0.22-µm filter, then tested for ability to suppress the Th function and induce the cytokine changes seen with the original GCS. We verified by specific ELISA or enzyme immunoassay (EIA) that the immunoprecipitation removed all detectable IL-6, TGF-ß, and CGRP.
Ion exchange and gel filtration columns
The supernatants were harvested from the U251 glioblastoma line
after 3 days of culture in conditioned FCS-free RPMI 1640 media. The
GCS samples for factor purification studies were tested for binding to
CM, Q, SP, and DEAE Sepharose Fast Flow Columns (Pharmacia Biotech).
The unbound fraction and eluted fractions were tested for GCS activity.
The bound fractions that contained GCS activity were fractionated on
the Superdex 75 and Superdex 200 columns (Pharmacia Biotech). To
determine the molecular mass of the active factor(s), 25–50
fractions were each tested for GCS activity by analysis of IL-12,
IL-10, IFN- production, and PHA-stimulation response of PBMC, and
compared with the unfractionated GCS, as described above.
Reagents
The additional following reagents were used in this study: anti-human IL-10 neutralizing mAb (clone JES 319 F11; DNAX, Palo Alto, CA); anti-human IL-10R mAb (clone 37607.11; R&D); anti-human IL-6 neutralizing mAb (clone MQ2-13A5; PharMingen); paraformaldehyde and saponin (Sigma); TGF-ß1 human ELISA kit and PGE2 EIA kit (Biotrak, Amersham, Arlington Heights, IL); Ultrapure natural TGF-ß1, human rTGF-ß2, and mouse monoclonal anti-human TGF-ß1, TGF-ß2, TGF-ß3 neutralizing Ab (Genzyme); human CGRP, rabbit anti-human CGRP serum, and high sensitivity EIA kit (Pennisula Laboratories Europe, Belmont, CA); and insoluble trypsin (Sigma).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test whether the preparations of GCS generated by the SNB-19
glioblastoma cell lines inhibited in vitro T cell responses to a
mitogen and recall Ags, PBMC from healthy individuals were stimulated
with PHA (Fig. 1
A) or with a
mixture of FLU, TT, and CASTA (Fig. 1B) in the absence or
presence of GCS. The results indicate that GCS inhibited proliferative
responses to both stimuli in a dose-dependent manner at dilution
ranging from 1/20 to 1/20,000. Therefore, the GCS produced by the tumor
cell line strongly inhibited T lymphocyte responses to a T cell mitogen
and to Th-dependent recall Ags that require intact APC function. As
negative controls, we found that culture supernatants from three of
seven tumor lines and the two laboratory-generated EBV-transformed cell
lines did not inhibit T cell proliferation or induce changes in IL-12
and IL-10 production when added to PBMC (data not shown).
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Because SAC is a strong stimulator of IL-12 and IL-10 production
by monocytes, we tested different dilutions of GCS on SAC-stimulated
IL-12 and IL-10 production in 24-h cultures of PBMC. The data in Fig. 2 demonstrate that GCS decreased IL-12
and increased IL-10 in a dose-dependent way.
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, IL-6, and
TNF-. PBMC were also preincubated with GCS for 1 h, the GCS was
washed out, and the treated PBMC were stimulated with SAC for 3, 6, 12,
and 24 h (Fig. 3). The kinetics of
the response of control cultures, either unstimulated or stimulated but
not incubated with GCS, was also followed. The production of IL-12 p70
and p40, as well as IFN- was greatly reduced by addition of GCS to
SAC-stimulated PBMC (Fig. 3, A, B, and
D). In contrast, GCS increased SAC-stimulated IL-10
production (Fig. 3C). IL-6 production was appreciably
increased by GCS or SAC alone, and the combination of GCS and SAC
induced an additional increase (Fig. 3E). SAC-stimulated
TNF- production was reduced approximately twofold by GCS (Fig. 3F). Data similar to those shown in Fig. 3 were obtained in
six independent experiments. We also observed that 48-h cultures of GCS
and SAC yielded results indistinguishable for the 24-h cultures (data
not shown). These results indicate that GCS can rapidly induce a
decrease in the production of IL-12 and IFN- and a concomitant
increase in IL-6 and IL-10 production. The 1-h preincubation of PBMC
with GCS before SAC stimulation induced changes in cytokine profiles
that were similar to those observed when PBMC were exposed to GCS and
SAC simultaneously.
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, and increased IL-10 production, and also abolished
PHA-stimulated T cell proliferation (Table I). Culture supernatants from four of
seven control tumor cell lines exhibited weak GCS-like activity (A2780,
National Institute of Health-ovcar-3, T2, PC-3); as noted above,
supernatants from the three other tumor lines did not show any GCS-like
activity (A2780/CP, U937, K562).
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.
Incubation of GCS with PBMC for as little as 3 min resulted in
SAC-stimulated decreased IL-12 p40 and IFN- and increased IL-10
production. Preincubation with GCS for 1 h was as effective as
maintaining GCS in the cultures with SAC for 24 h.
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To identify immunoregulatory factors that might be contained in
GCS, we tested six different lots of supernatants collected from one of
the SNB-19 glioblastoma cell lines that had been shown to inhibit in
vitro T cell proliferation. The supernatants were found to contain
IL-6, TGF-ß1, CGRP (38), and very low levels (below 7 pg/ml) of
PGE2 (Table III), but not
detectable levels of IL-4, IL-10, IL-12, TNF-, or IFN- (data not
shown). The levels of PGE2 detected were below those
reported to reduce IL-12 production by human PBMC or dendritic cells
(39, 40). Based on these data, three types of experiments were
performed to determine whether the changes in cytokine production
induced by GCS could be attributed to any of these immunosuppressive
factors.
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production and to increase IL-10 production by PBMC. Third,
we immunoprecipitated IL-6, TGF-ß, and CGRP, either singly or in
combination, with the same Abs used for inhibition. The GCS treated in
this way did not lose any of its ability to decrease IL-12 production.
Only slight reductions were observed in ability to increase IL-10 and
decrease IFN- by combination immunoprecipitation of IL-6 and TGF-ß
(Table IV). Taken together, these results
do not support the conclusion that the cytokine dysregulation induced
by GCS is the result of IL-6, TGF-ß, or CGRP, acting singly or in
combination.
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To determine whether GCS also affected IL-12 and IL-10 mRNA,
expression of IL-12 p40 and IL-10 mRNA was analyzed in PBMC that were
incubated with SAC, GCS, and SAC + GCS. The results of one
representative of four independent experiments are shown in Fig. 4 for IL-12 p40 and IL-10. Incubation
with GCS resulted in a modest increase in IL-12 p40 mRNA, and in a
large increase in IL-10 mRNA. As previously reported (25), SAC
stimulation alone resulted in expression of IL-12 p40 and of IL-10
mRNA. Incubation with GCS + SAC increased expression of IL-10 message
above SAC alone, and decreased IL-12 p40 expression compared with SAC
alone. These data are in agreement with the regulation of IL-12 and
IL-10 production by GCS.
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To determine whether monocytes contained in the PBMC were
responsible for the production of IL-12 and IL-10, 24-h SAC-stimulated
PBMC were gated for CD14+ and CD3+ cells and
stained with anti-IL-12 and anti-IL-10 Abs. IL-12 and IL-10
were detected only in the CD14+ population. PBMC incubated
alone or with GCS, SAC, or SAC + GCS were used for isotype control
staining (data not shown). The data presented in Fig. 5 illustrate the IL-12 and IL-10
intracellular staining patterns using the same conditions. Incubation
of PBMC with GCS indicated that 3.4% of the monocytes stained for
intracellular IL-10, but only 0.3% stained for IL-12. Stimulation of
PBMC with SAC alone resulted in the staining of 8.1% for IL-10, 5.7%
for IL-12, and 1.5% for both. The PBMC incubated with SAC + GCS
resulted in a skewing toward IL-10-producing cells, as 13% stained for
IL-10, 1.9% stained for IL-12, and 1.6% stained for both cytokines.
Similar results were obtained in four repetitive experiments. In
contrast to the cells gated for lymphocytes, no T cells were found that
produced IL-10 or IL-12 after a 24-h incubation with GCS, SAC, or SAC +
GCS (data not shown).
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To determine whether incubation with GCS also affected IL-12 p70,
p40, and IL-10 produced by enriched monocytes, the following
experiments were performed. The monocytes were enriched from PBMC by
elutriation, followed by negative selection of T, B, and NK cells. The
enriched monocytes (shown >90% CD14+, but undetectable
<1% CD3+, CD19+, CD1a+, and
CD16+) were stimulated with SAC for 24 h in the
absence or presence of GCS. The results indicate that GCS abrogated
IL-12 production (Fig. 6A) and
increased IL-10 production (Fig. 6B). GCS also decreased
SAC-stimulated IL-12 p40 production by twofold (data not shown). These
results indicate that GCS can also affect IL-12 and IL-10 production by
enriched monocytes in the absence of other cell types.
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A). Addition of autologous T cells increased IL-10
production by twofold above monocytes alone, and addition of GCS
further increased IL-10 production (Fig. 6B). We also
assessed SAC-stimulated IFN- production in the same cultures (Fig. 6C), and demonstrated that without T cells, this cytokine
was not produced. Addition of T cells resulted in IFN- production,
which was reduced approximately fourfold by GCS.
To determine whether the observed GCS-induced changes in monokine
production would be reflected in Th cell function, a mixture of
autologous monocytes and T cells was stimulated with the recall Ag
mixture of FLU, TT, and CASTA, and thymidine incorporation measured 6
days later. Similar to the data obtained for IL-12 and IL-10
production, addition of GCS to cultures of monocytes during Ag
stimulation, or preincubation of monocytes with GCS for 1 h before
mixing with T cells and Ag stimulation, abrogated the Ag-stimulated
proliferative response (Fig. 7).
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, and anti-IL-10R on IL-12,
IL-10, and IFN- production
Enriched monocytes were incubated with anti-IL-10 mAb (10
µg/ml) or with rIFN- (10 U/ml) during the 24-h incubation with GCS
and/or SAC. SAC-stimulated IL-12 p40 production was appreciably
increased by anti-IL-10 and even more by IFN-. In the
combination, GCS partially decreased the enhancing effects of
anti-IL-10 mAb and rIFN- on SAC-stimulated IL-12 p40 production
(Fig. 8). Our finding that anti-IL-10
did not completely block the GCS-induced reduction of IL-12 p40
suggests that GCS affects IL-12 production by a partly
IL-10-independent mechanism(s).
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). Incubation of PBMC with
anti-IL-10R raised IL-12 p70, p40, and IFN- production, but had
no effect on IL-10 production. In the presence of anti-IL-10R mAb
(20 µg/ml), GCS still partially reduced p70, p40, and IFN-
production, confirming that the inhibitory effect of GCS on IL-12
production is not mediated exclusively via IL-10 production.
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To determine whether exposure to GCS would affect expression
of MHC class II and CD80/86 (B7.1 and B7.2) expression, PBMC were
preincubated for 1 h with GCS and then incubated with medium in
the absence or presence of rIFN- for 24 h (Table V). The cultured cells were analyzed by
flow cytometry by gating on CD14+ population of monocytes
to determine class I, class II, CD80, and CD86 expression. The results
indicate that MHC class II, but not class I expression was reduced by
approximately twofold. Expression of CD80 and CD86 was similarly
reduced. Although rIFN- enhanced class I, class II, and CD80
expression on monocytes, GCS reduced the mean channel fluorescence
increases by approximately twofold for class II and CD80, but not for
class I.
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The mitogen and Ag-specific responses of T cells obtained from
patients with glioma are severely impaired (15). To determine whether
PBMC from glioma patients would also exhibit changes in IL-12 and IL-10
production similar to that shown above using GCS in vitro, PBMC from
primary glioma patients who had not received steroids or phenytoin and
healthy control blood donors were stimulated with SAC to initiate IL-12
and IL-10 synthesis. The production of IL-10 was increased, whereas
production of IL-12 p70 and p40 as well as IFN- were all decreased
compared with controls, as shown for one patient in Fig. 10. Similar data were obtained using
PBMC from three additional glioma patients. These preliminary results
resemble the IL-12/IL-10 shifts generated in our in vitro model of
GCS-exposed PBMC and monocytes from healthy blood donors.
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Studies are in progress to isolate, identify, and purify the
factor responsible for the biologic activity contained in GCS. To
characterize the factor, GCS was generated using the U251 glioblastoma
cultured in protein-free conditioned RPMI 1640 medium. We observed that
heating GCS to 56°C for 30 min did not affect its ability to modulate
cytokine production (IL-12, IL-10, or IFN-), but did reduce its
ability to abrogate PHA-stimulated T cell proliferation (Table VI). Heating GCS to 100°C for 30 min
abrogated the activity responsible for modulating cytokine production.
Exposure to pH extremes of 2 and 11 for 15 min resulted in retention of
40–60% of the GCS activity for altering cytokine production, but had
no affect on its ability to abolish PHA-stimulated T cell
proliferation. All functional activity of GCS was lost by exposure to
insoluble trypsin for 30 min at room temperature. The activity of GCS
for all of the above parameters was retained by anion-exchange columns
(Q and DEAE Sepharose), but not by cation-exchange columns (SP and CM
Sepharose). Preliminary characterization by Superdex 200 gel filtration
column experiments of the anion-exchange-retained fractions indicates a
minimum molecular mass of approximately 40 kDa, although GCS functional
activity for changes in all three cytokines and T cell proliferation
was observed over a wide molecular mass range up to approximately 150
kDa (Table VI). This range of activity suggests various states of
aggregate formation, and is consistent with molecular sizing filter
experiments in which functional activity was present, but reduced by a
100-kDa filter (data not shown). Each condition that decreased IL-12
also decreased IFN- and increased IL-10 (Table VI).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(decreased), and TNF- (decreased). The
demonstration that exposure of monocytes to GCS before mixing with
autologous T cells and Ag abolished proliferative responses indicated
that the changes in monocyte IL-12, IL-10, MHC class II, and/or CD80/86
expression affected Th function. Importantly, the data also show that
PBMC from glioma patients exhibit a similar shift in IL-12 and IL-10
production.
It has been suggested that the immunosuppressive effect of glioma
culture supernatant is the result from the synthesis of TGF-ß (41, 42). However, our mAb inhibition and immunoprecipitation data do not
support the hypothesis that the factor responsible for rapidly inducing
shifts in IL-12 and IL-10 expression/production and down-regulation of
MHC class II and CD80/86 was TGF-ß, IL-6, CGRP, or a combination of
these immunoregulatory molecules. It remains to be determined whether
this glioma-generated factor, which appears to be a protein that has a
minimum molecular mass of approximately 40 kDa, is a known
immunoregulatory molecule or represents a new immune modulator that
affects monokine expression. Other tumor-generated molecules
have been reported to inhibit in vitro T cell function. For example,
adenocarcinoma-associated MUC1 mucin inhibits T cell proliferation that
is reversible by IL-2 (43). In contrast, addition of rIL-2 to our
GCS-treated culture did not restore T cell function (data not shown).
As controls for the GCS, we tested the supernatants of seven other
tumor lines of different origins, and found that three had no effect
when cultured with PBMC. In contrast, supernatants from four other
tumor lines moderately decreased IL-12 and IFN- production,
increased IL-10 production, and reduced PHA-stimulated T cell
proliferation. However, these effects were not as dramatic as those
induced by GCS. Therefore, factors that exert immunologic effects on
monocytes and T lymphocytes similar to those we report in this work for
GCS may be produced by some but not all cell lines from other types of
tumor.
Our observations that anti-IL-10 and anti-IL-10R Abs did not completely neutralize the GCS-induced reduction in IL-12 indicate that part of the GCS effect on IL-12 was independent of IL-10. Although glioma cell lines have been reported to produce IL-10 (44), the SNB-19 glioblastomas that we studied did not produce IL-10. Instead, our experiments demonstrate the induction of IL-10 gene expression and production in monocytes exposed to factor(s) contained in the SNB-19 culture supernatants.
Particularly noteworthy is our finding that GCS decreased both MHC class II and CD80/86 costimulatory molecule expression, but did not affect MHC class I. Earlier reports indicated that IL-10 reduced Ag-stimulated human T cell proliferation by decreasing MHC class II expression on monocytes and induced T cell anergy (32). In a murine model, IL-10 inhibited Con A-induced T cell proliferation when APC function was provided by macrophages, but not when provided by B cells (33). This inhibitory effect was associated with reduced costimulatory activity and B7 expression (33). Thus, in the human Ag-stimulated model, down-regulation of class II was adequate for loss of APC function leading to T cell anergy; in the murine mitogen-stimulated model, down-regulation of costimulatory molecules was sufficient for loss of APC function required for T cell activation. It is also known that IL-10 can down-regulate IL-12 (25, 45), and therefore is likely to contribute to the decreased IL-12 production observed in the GCS-exposed monocytes.
Astrocytes and microglial cells can be induced to become
macrophage-like cells in the CNS. MHC molecules detected on microglial
cells are down-regulated under normal conditions, but can be induced to
express MHC class II Ags under pathologic conditions or upon exposure
to IFN- (46). Up-regulation of MHC molecules can transform
astrocytes into APC (47). Furthermore, increased expression of IL-12
and CD80/86 has been reported in multiple sclerosis lesions (48).
Although external stimuli can increase MHC expression in potential APC
in the CNS, reduced or absent expression of MHC is often considered to
be the normal situation. Our finding that GCS contains a factor(s) that
down-regulates class II expression and counters the up-regulatory
effects of IFN- on class II expression raises the possibility that
reduced or absent MHC expression by astrocytes and glial cells is an
active process. This process could involve localized autocrine and/or
paracrine production of a GCS-like factor that would prevent damaging
CNS inflammation and neuroimmunologic reactions. The potential for
functional APC activity in the CNS would be further inhibited by
factor-initiated reduced expression of the CD80/86 costimulatory
molecule. Thus, there could be two mechanistically distinct but related
sites at which a glioma-generated factor prevents APC in the CNS from
stimulating T cells: one interfering with T cell activation via
APC class II 
TCR interaction, and the other reducing
costimulatory function via CD80/86 CD28 interaction.
Glioma patients exhibit a number of immunologic abnormalities that resemble those seen in AIDS patients (49), including decreases in the absolute number and percentage of CD3+ and CD4+, but not of CD8+ T lymphocytes (50). Furthermore, the monocyte cytokine and surface molecule expression pattern seen in AIDS and in our GCS-exposed monocytes are similar (24, 25, 51, 52, 53). It is possible that HIV-infected and/or exposure to HIV-1 protein (for example, Tat, nef, gp120, or gp41) or to tumor-derived factors such as GCS induce mechanistically similar changes in IL-12, IL-10, MHC class II, and CD80/86 expression by monocytes (54). These modified APC could then initiate anergic rather than stimulatory signals to Th cells that would contribute to apoptotic-mediated depletion of the T cell repertoire reported in AIDS patients (55, 56), as well as to the loss of tumor-specific T cells in cancer patients. Such factor-induced aberrant APC may activate common immunologic dysregulatory signals in the Th of patients with AIDS or immunosuppressive tumors. It is noteworthy that escape of malignant melanoma from T cell surveillance has been suggested to be due to absence of expression of CD80/86 on the tumor cells (57). Tumor cell lines that are inoculated into cancer patients for increasing tumor-specific immunity (58) should be tested for expression of IL-10, IL-12, class II, and CD80/86. If these cells function to present tumor Ags, but exhibit an APC profile similar to that described in this work after GCS exposure, negative rather than positive signals could be transmitted to the tumor-specific T cells, resulting in anergy and clonal depletion. Thus, an appreciation and understanding of the negative immunoregulatory signals initiated by suppressive factor-altered monocytic APC could be important in designing optimal therapeutic strategies aimed at enhancing tumor-specific immunity by dendritic cell immunization (59, 60). It will be important to determine whether dendritic cells are more resistant than monocytes to tumor-derived immune dysregulatory-inducing factors such as GCS, and whether Ag presentation via dendritic cells can effectively counteract and/or circumvent the potential negative signals induced by monocytes that express increased IL-10 and decreased IL-12, MHC class II, and CD80/86.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gene M. Shearer, Experimental Immunology Branch, Building 10, Room 4B-36, NCI, National Institutes of Health, Bethesda, MD 20892-1360. E-mail address:
3 Abbreviations used in this paper: GCS, glioblastoma culture supernatant; CASTA, Candida albicans Ag; CGRP, calcitonin gene-related peptide; CNS, central nervous system; EIA, enzyme immunoassay; FLU, influenza A virus; SAC, Staphylococcus aureus Cowan strain 1; TT, tetanus toxoid.
Received for publication July 30, 1998. Accepted for publication January 19, 1999.
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