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*
Department of Oncology and Surgical Sciences, Padua, Italy;
Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program, Bethesda, MD 20814; and
Surgery Branch, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-irradiated or they will grow progressively, killing
the host. We observed that 23 of 75 (31%) human tumor lines and two
commonly used mouse tumor lines spontaneously produced GM-CSF. In mice,
chronic GM-CSF production by tumors suppressed Ag-specific
CD8+ T cell responses. Interestingly, an inhibitory
population of adherent CD11b(Mac-1)/Gr-1 double-positive cells caused
the observed impairment of CD8+ T cell function upon direct
cell-to-cell contact. The inhibitory cells were positive for some
markers associated with Ag presenting cells, like F4/80, but were
negative for markers associated with fully mature DC like DEC205, B7.2,
and MHC class II. We have previously reported that a similar or
identical population of inhibitory "immature" APC was elicited
after immunization with powerful recombinant immunogens. We show here
that these inhibitory cells can be elicited by the administration of
recombinant GM-CSF alone, and, furthermore, that they can be
differentiated ex vivo into "mature" APC by the addition of IL-4
and GM-CSF. Thus, tumors may be able to escape from immune detection by
producing "unopposed" GM-CSF, thereby disrupting the balance of
cytokines needed for the maturation of fully functional DC. Further,
CD11b/Gr-1 double-positive cells may function as "inhibitory" APC
under the influence of GM-CSF alone. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It now seems likely that tumors are not destroyed by immune cells
because the tumor microenvironment is a poor site for the initiation of
immune responses. Tumor cells generally do not express the requisite
costimulatory and cytokine molecules that signal an inflammatory state.
In this regard, tumors are like most normal tissues in the body.
Activation of the cellular immune response appears to be largely
effected by "professional" APC, including dendritic cells (DC)
(9, 10, 11, 12). Professional APC originate from granulocyte-monocyte lineage
progenitors that have the ability to capture Ag but lack the requisite
accessory signals for T cell activation (13, 14). Under the influence
of cytokines, such as GM-CSF, IL-4, and TNF-
, progenitor cells
undergo terminal differentiation to professional APC capable of
presenting Ags in the context of immunostimulatory molecules that
result in the activation T cell reactivities (9, 13, 15).
Local production of GM-CSF by -irradiated cDNA-transduced tumors is
thought to recruit and expand professional APCs that present Ags
released by apoptotic tumor cells (16, 17, 18). In the B16 mouse model, the
presentation of tumor Ags by professional APCs to naive lymphocytes can
result in memory T cell responses that prevent subsequent challenge
with the wild-type tumor (18). Gene modification of tumor cells with
GM-CSF in the absence of -irradiation, however, does not generally
change the growth rate or lethality of tumor cells in their
nonirradiated state. Thus, the induction of apoptosis by
-irradiation in GM-CSF gene-modified tumor cells might be required
to enhance the processing and presentation of TAA by DC (19, 20).
Indeed, the production of GM-CSF in the absence of an apoptotic
"danger signal" could have very different effects on DC recruitment
and maturation. It is perplexing that some murine tumor cells
spontaneously produce GM-CSF (21). Furthermore, the production of
GM-CSF has been correlated with the ability of mouse tumor cells to
spontaneously metastasize (22). Human prostate cancers (23) and human
melanoma cells (our unpublished data) have been found to spontaneously
produce GM-CSF.
In the present article, we attempt to delineate the role of GM-CSF production by tumors on the recruitment and maturation of APC. We studied a panel of human tumors for the production of GM-CSF and found that 31% of human tumors spontaneously secreted this cytokine. To characterize the immunological significance of this finding, GM-CSF-producing and -nonproducing murine tumors were grown in vivo. The production of GM-CSF by tumors was found to induce a population of cells that morphologically resembled granulocyte-monocyte progenitor cells and phenotypically expressed the granulocyte-monocyte markers C11b (Mac-1) and Gr-1 (Ly-6G). We have previously reported that these cells can cause apoptotic death of CD8+ T cells after immunization with powerful vaccinia virus-based immunogens. (24). We show here that these CD11b+/Gr-1+ cells could mature in vitro under the influence of GM-CSF and IL-4 to produce functional APCs that are capable of enhancing T cell responses in a mixed lymphocyte reaction. We explore the hypothesis that tumors may evade immune destruction by dysregulating the maturation of APC through the unopposed secretion of GM-CSF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CT26.WT, the ß-galactosidase (ß-gal)-expressing CT26.CL25
clone, EL4 thymoma, and its lacZ transfectants E22 have been
described (25, 26). The mouse mammary adenocarcinoma tumor, TS/A, was
kindly provided by Dr. Guido Forni (University of Turin, Orbassano,
Italy). MBL-2 and LSTRA are Moloney virus-induced lymphomas. Cell lines
were maintained in culture media (CM) consisting of RPMI 1640, 10%
heat-inactivated FBS (Biofluids, Rockville, MD), 0.03%
L-glutamine, 100 µg/ml streptomycin, 100 µg/ml
penicillin, and 50 µg/ml gentamicin sulfate (National Institutes of
Health Media Center, Bethesda, MD). The following human tumor cell
lines were obtained from American Type Culture Collection (Manassas,
VA): BT474, MCF-7, SKOV3, MB 361, BT549, MDA 231, MS 751, SIHA, CASKI,
IGROV-1, OVCAR-3, OV-1, WIDR, H508, COLO 205, SW480, LOVO, and SK23.
The following human cells were provided by Suzanne Topalian (National
Cancer Institute (NCI; Bethesda, MD): LNCaP, DU 145, PC-3, and 1542
CP3TX. The following human cell lines were generated in the
Surgery Branch, NCI: 1312, 1338, 501A, 1143, 537, 1353, 553B, 677 II,
1173, 1402, 1290-2, 1199, 888, 1287, 1011, 624, 1359, 1363, 1495,
1280-1, 697, 1362, 526, 1308, 1335, 586, 1182, 1479, 1102, 836, 938,
1379, 624, 883, 1286, 397, 1123, 1390, 1448 Y, and 1195. The following
human cell lines were generated in the Urologic Oncology Branch, NCI:
Ce-RCC, M-RCC, Gr-RCC, R-RCC, Ca-RCC, Gi-RCC, N-RCC, P-RCC, Ba-RCC,
Bu-RCC, and W-RCC. Cells were plated in duplicate in 96-well plates
(Costar, Cambridge, MA) at a concentration of 105
cells/well in 250 µl of CM. Values are expressed as pg/ml produced in
16 h by 105 cells, unless otherwise stated. Genetic
modification of B16 tumor cells to secrete GM-CSF was performed as
follows. The E86 packaging cell line carrying the LmGM-CSFSN
retroviral vector expressing the mouse GM-CSF was a gift of Mario
Colombo (Istituto Nazionale Tumori, Milan, Italy). B16 melanoma was
trans-infected with the supernatant of the packaging cell
line in the presence of Polybrene (8 µg/ml) and then subcloned.
Stable transfectants were screened for cytokine production after 2 wk
of continuous in vitro passages in the presence of G418 (0.2 mg/ml).
The subclones of the B16 tumor engineered with GM-CSF were plated at
106 cells in 1 ml of CM. ELISA kits for human IFN- and
mouse GM-CSF were purchased from Endogen (Cambridge, MA), and kits for
human GM-CSF were purchased from R&D Systems (Minneapolis, MN). All
values refer to duplicate samples.
Peptides, Abs, and cytokines
The following synthetic peptides were synthesized by Peptide
Technologies (Washington, DC) to a purity of >99% as determined by
HPLC and amino acid analyses: TPHPARIGL (amino acids 876–884 of
ß-gal, H-2Ld-restricted (27)), DAPIYTNV (amino acids
96–103 of ß-gal, H-2Kb-restricted (28)). Rat mAb
recognizing NLDC-145 (DEC-205) and F4/80 were purchased from BMA
Biomedicals (Augst, Switzerland). FITC- or PE-labeled mAb recognizing
mouse CD3, CD8, CD4, CD11b (Mac-1), CD45R/B220, CD11c, CD80 (B7-1),
CD86 (B7-2), Ly-6G (Gr-1), anti-H-2
I-Ed/I-Ad, Kd, and the
isotype-matched controls were purchased from PharMingen (San Diego,
CA). The mAb 24G.2 (CD16/CD23; PharMingen), which recognizes the
extracellular domain of the mouse Fc-RIII and RII, was used
to block the nonspecific mAb binding. Recombinant mouse GM-CSF and IL-4
were purchased from PreproTech (Rocky Hill, NJ) and resuspended in HBSS
containing 1% mouse serum (Sigma, St. Louis, MO).
Isolation and depletion of Gr-1+ splenocytes
A panning technique employing flasks coated with mouse anti-rat Abs (T-25 AIS MicroCell; Applied Immune Sciences, Santa Clara, CA) was used to enrich or eliminate Gr-1+ splenocytes from either tumor-bearing mice or mice treated with recombinant GM-CSF. Briefly, spleens were depleted of RBC through ACK lysis buffer (Biofluids) and resuspended in HBSS containing 1 mM EDTA and 10% mouse serum (HBSS-EDTA-MS). Cells were transferred on ice, incubated for 30 min with anti-Gr-1 Ab at a concentration of 10 µg/107 cells, and washed with cold HBSS-EDTA. Cells were resuspended in ice-cold HBSS-EDTA-MS, transferred to flasks coated with the secondary Ab (anti-rat), and incubated for 1 h at 4°C. The nonadherent cells were then dislodged, and fluorescence labeling confirmed a >95% depletion. Cells that remained attached to the flasks were provided with CM and incubated for further studies. Adherent cells were usually recovered by gentle scraping in Versene solution (1:5000; Biofluids).
Maturation of CD11b+/Gr-1+ cells in vitro
Gr-1+ splenocytes were isolated as described above. Isolated cells were grown for 6 days in culture with 100 ng/ml GM-CSF, 100 ng/ml IL-4, a combination of both GM-CSF and IL-4, or in media alone. After 6 days of culture, the adherent cells were analyzed for phenotypic markers or added to a 6-day mixed lymphocyte stimulation. In the mixed lymphocyte reaction, BALB/c splenocytes (H-2d) at 3 x 106 were incubated with 3 x 106 irradiated C57BL/6n splenocytes (H-2b) in the presence or absence of 0.18 x 106 Gr-1+ splenocytes (H-2d). After 6 days of stimulation, the BALB/c lymphocytes were tested in a standard chromium release assay against syngeneic (CT26 or LSTRA) or allogeneic (MBL-2) targets.
Enrichment of spleen-derived DC
Spleens were collected aseptically, minced, and incubated for 30 min at room temperature in HBSS containing collagenase (1 mg/ml; Sigma). Spleens were depleted of RBC through ACK lysis buffer and washed. The single cell suspension was resuspended in CM and plated in a 150 x 25-mm tissue culture dish (Falcon, Cockeysville, MD). Splenocytes were incubated for 2 h at 37°C, and the nonadherent cells were removed by gentle washing. Adherent cells were further incubated for 18 h in CM containing 3 ng/ml mouse GM-CSF. At the end of the incubation, the supernatant was collected, and DC were finally enriched by centrifugation (1900 x g) over a 45% Percoll cushion. At this point, >80% of the cells were B7-2+, B220-, class II MHC +, CD11c+.
Evaluation of CD8+ T cell responses
Eight- to twelve-week-old female BALB/c or C57BL/6 mice (Animal
Production Colonies, Frederick Cancer Research Facility, National
Institutes of Health, Frederick, MD) were immunized with 5 x
106 PFU/mouse of the construct VJS6, a recombinant vaccinia
virus (rVV) expressing ß-gal (ß-gal-rVV) (25). Three weeks after
immunization, two to three mice per group were injected s.c. with 0.1
ml of HBSS containing 106 TS/A tumor cells. Two to five
weeks after tumor challenge, spleens were collected, separated into a
single cell suspension, and cultured in CM containing 1 mg/ml of the
immunodominant ß-gal peptides (27, 28). After 6 days, cultures were
tested for their ability to lyse ß-gal-positive targets in a 6-h
51Cr release assay using target cells previously incubated
with 200 µCi Na51CrO4 (51Cr) for
90 min (together with 1 µg/ml of peptide) (25).The amount of
51Cr released was determined by -counting, and the
percentage of specific lysis was calculated from triplicate samples
using the formula: [(experimental cpm - spontaneous
cpm)/(maximal cpm - spontaneous cpm)] x 100. In some
experiments, mice received a 3-day cycle of GM-CSF (5 µg/mouse, i.p.
twice daily) 3 wk following immunization with ß-gal rVV.
The calculation of LU was performed because it conveys the activity of the culture more effectively than simply showing a single E:T ratio. Calculations of LU per 106 effector cells were performed as follows: divide 106 by the number of cells giving 30% lysis of specific target cells (we used 2 x 103 ß-gal peptide-pulsed EL4 cells). There was no lysis of unpulsed target cells in this assay.
In cell separation experiments, splenocytes were cultured at the same cell concentration in 24-well plates (Costar) containing a culture chamber insert with 0.4-µm pores (Millipore, Bedford, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GM-CSF secretion by tumor lines has been sporadically described in
the literature (29, 30), but to our knowledge, the prevalence of GM-CSF
production by tumors has not been systematically studied. Thus, we
evaluated a panel of 75 human tumor cell lines (Table I
) and found that 23 produced levels of
GM-CSF higher than the background (>10 pg/ml/105 cells),
especially those lines originating from kidney and prostate cancers.
Moreover, IFN- release during the same assay was not above the
detection limit (12 pg/ml), indicating the specific release of GM-CSF.
Thus, our finding that human tumors spontaneously produce GM-CSF is
consistent with previous reports.
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To test the immune consequences of spontaneous GM-CSF production
by tumors on the maturation of APC, two experimental tumor lines were
grown in vivo. The colon adenocarcinoma, CT26.WT, and the mammary
adenocarcinoma, TS/A, spontaneously produced significant levels of
GM-CSF (average 50 and 250 pg/ml/106 cells/day,
respectively). Mice bearing unmanipulated s.c. TS/A tumors 
1 cm in
diameter were splenectomized. A representative cytofluorometric
analysis of spleen cells (shown in Fig. 1A) demonstrates an increase
in the numbers of CD11b+/Gr-1+ cells. These
cells comprised a mean of 22.3 ± 1.83% of all splenocytes in
five independent experiments, compared with an average of 2.4 ±
0.43% in mice with a small but palpable tumor. Evaluation of
splenocytes from tumor-bearing mice did not reveal substantial changes
in the percentages of lymphocytes with T or B cell markers (31).
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B, control). However, mice bearing large
tumors failed to mount an anamnestic, ß-gal-specific CD8+
T cell response.
To determine whether or not the Gr-1+ population that was
so prevalent in mice bearing large tumors was responsible for the
observed inhibition of Ag-specific CD8+ T cell responses, a
fraction of the splenocytes from mice bearing large tumors was depleted
with a Gr-1-specific Ab, or an isotype-controlled (IgG2b)
Ab before restimulation. The simple depletion of Gr-1+
cells normalized CD8+ T cell function. Indeed, the lytic
levels observed were nearly as great as those seen in ß-gal-immune
mice bearing small, but palpable, tumors (Fig. 1B, compare
anti-Gr-1 and control). Isotype-matched mAb did not normalize T
cell function (Fig. 1B, IgG2b). Experiments with
the CT26 tumor cell line, which also naturally produces GM-CSF, yielded
similar results (data not shown). Our results with mice bearing CT26
are consistent with those previously published by G. Nabel and
colleagues (32) in which mice bearing this tumor were found to have
large numbers of CD11b+ cells in their spleens and
inhibition of allospecific responses. Therefore, the newly induced
population of CD11b+/Gr-1+ cells appeared to be
responsible for the abrogation of CD8+ T cell function.
We have previously shown that purified CD8+ T lymphocytes obtained from splenic preparations of mice with high levels of CD11b (Mac-1)+/Gr-1+ suppressor cells are normally responsive (24). Thus, the inhibition observed is not a quality of the CD8+ T cells, but is solely a quality of the CD11b/Gr-1 double-positive population.
GM-CSF, not the tumor-bearing state, drives the production of CD11b+/Gr-1+ cells
To determine whether or not the
CD11b+/Gr-1+ cells were induced by GM-CSF or
some other stimulus from the tumor, the murine melanoma, B16, which
does not normally produce GM-CSF, was retrovirally transduced with the
cDNA for mouse GM-CSF. Immediately following transduction, transduced
B16 were cloned. To minimize clonotypic variability, several clones
were isolated that released comparable amounts of the cytokine
(600–900 pg/ml; Table II).
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). Two weeks after tumor inoculation, we were able to detect
increased serum levels of GM-CSF in mice bearing gene-modified tumors
but not in mice bearing parental tumor, indicating that cytokine
production had not been silenced in vivo (Table II). As seen with CT26
and TS/A, cytofluorometric analysis of spleens from mice bearing
GM-CSF-transduced tumors demonstrated an increase in the percentage of
the CD11b+ cells (Table II). The lytic activity against ß-gal after in vitro stimulation of the spleens with the Kb-restricted ß-gal peptide in control mice were similar to those found in mice bearing the B16 wild-type tumor (B16 wt). In sharp contrast, growth of all the subclones producing GM-CSF (GM15-47) resulted in the inhibition of the anamnestic CD8+ T cell responses, as indicated by the reduction of the lytic units in the peptide-stimulated cultures. These data suggest that GM-CSF production by tumors cells can inhibit CD8+ T cell responses.
Recombinant GM-CSF administered alone induces CD11b+/Gr-1+ cells and inhibits CD8+ T cell function
To further test the hypothesis that GM-CSF was directly
responsible for the increase in CD11b+/Gr-1+
cells, a 3-day cycle of recombinant mouse GM-CSF was administered twice
daily to ß-gal immune mice and naive controls. The administration of
GM-CSF caused an increase in CD11b+/Gr-1+ cells
in the spleens of treated mice (Fig. 2A) and inhibited the immune
responses observed after stimulation with ß-gal peptide (Fig. 2, B and C). These data demonstrated that a exposure
to GM-CSF was sufficient to induce both the appearance of a distinct
population of CD11b+/Gr-1+ cells and to induce
CD8+ T cell inhibition.
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To characterize the inhibitory population of CD11b+
cells in greater detail, splenocytes were positively selected from the
spleens of TS/A-bearing mice with anti-Gr-1 mAb and were cultured
in vitro in standard medium. After 7 days of culture, a homogenous
population of weakly adherent cells was isolated that retained its
ability to inhibit T lymphocyte function (see Fig. 4A, "no
cytokine"). We sought to characterize the phenotypes of these cells
in more detail. For comparison, we simultaneously prepared a population
of "classical" DC from naive mice using an established protocol
(compare Fig. 3A with
granulocyte-monocyte progenitor cells, Fig. 3B).
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M chain and Ly-40) (Fig. 3B). The expression
of CD11c (integrin 
chain) in the purified inhibitory
cell was low, and cytofluorometric quantification of these cells varied
somewhat from experiment to experiment with CD11cdim cells
comprising between 25 and 60% (compare with Fig. 4B, "no cytokine").
Interestingly, expression of Gr-1 (Ly-6G), another granulocyte-monocyte
lineage marker, was present early but was consistently lost both when
the cells were prepared from tumor-bearing mice or from mice that
received a short course of GM-CSF.
Clearly, these inhibitory cells differed from "classical" mature DC
and activated monocytes and macrophages most significantly by their
lack of uniform expression of the costimulatory molecule B7-2
(CD86/B70/Ly-58). They also lacked the expression of DEC 205
(NLDC-145), a protein involved in carbohydrate endocytosis and Ag
presentation (Fig. 3A vs 3B) (35).
"Classical" DC, and activated macrophages for that matter, express
high levels of MHC class II, whereas the inhibitory cells were
consistently negative. While the cell surface marker CD16 (FcRIII)
is found in low quantities on DC and is a developmental marker on
monocytes and macrophages, the inhibitory cells were negative. Normal
levels of MHC class I were present, and there was no expression of T
and B lymphocyte markers (data not shown), excluding the possibility
that these CD11b+/Gr-1+ were CD8a+
as recently described for a subpopulation of DC with a suppressive
function (36, 37). Cells with similar granulocyte-monocyte progenitor
markers were found in C57BL/6n and BALB/c after induction, in vivo,
with twice daily regimen of GM-CSF alone given for 3 days (data not
shown).
Inhibitory cells prevent CD8+ T cell generation through a contact-dependent mechanism
We next investigated the mechanism used by CD11b+/Gr-1+ cells to inhibit CD8+ cell function. To determine whether soluble factors were responsible for the suppression, we did experiments in which inhibitory APC were cultured either together with, or in close proximity to, an allogeneic MLC. The anti-H-2b MLC was generated by admixing BALB/c (H-2d) splenocytes with irradiated C57BL/6n (H-2b) splenocytes. Inhibitory cells, which were enriched from mice bearing TS/A tumors, were allowed to differentiate in vitro in medium devoid of exogenously added cytokines. These inhibitory cells were then added to MLC, either in the same well, or in a transwell. The effectors were then tested in a microcytotoxicity assay against syngeneic (LSTRA) or allogeneic (MBL-2) targets.
The secretion of soluble factors, such as inhibitory cytokines, by the
CD11b+/Gr-1+ population may be expected to
inhibit the allo-MLC reaction when the inhibitory cells were placed in
diffusion chambers. However, as shown in Fig. 4, no such inhibition was
observed, and the MLC functioned normally when the inhibitory cells
were in transwells. Indeed, it appeared from this experiment that
cell-cell contact was required for the immunosuppressive activity of
the inhibitory cells, since suppression of allospecific cytolysis was
observed only when the Gr-1+-derived population was not
separated by a membrane from the population responsive to stimulation
with allogeneic cells.
Inhibitory cells can be differentiated into fully functional APC when GM-CSF is given in combination with IL-4
Based on their phenotype, we hypothesized that these cells were immature APC in the granulocyte-monocyte lineage, developmentally blocked in their ability to mature into function APC capable of activating T lymphocytes. One way of measuring the T cell stimulatory capacity of APC is the use of a MLR (9, 38).
To test the hypothesis that these inhibitory cells were APC
progenitors, arrested in their development, we did an experiment in
which we isolated cells previously shown to be inhibitory (see Fig. 2),
as described above, from TS/A-bearing mice. These inhibitory cells
were then exposed to either no cytokines, GM-CSF alone, IL-4 alone, or
to a combination of GM-CSF and IL-4. After 6 days of differentiation,
these cells were tested for their function and phenotype. The cells
were cytofluorometrically analyzed for three markers (MHC class II,
B7-2, and CD11c) of "mature" APC and simultaneously added to a MLC
to test their function. Specifically, these cytokine treated cells
comprised 3% of the total cells in a MLC in which BALB/c splenocytes
(H-2d) were mixed with -irradiated C57BL/6n splenocytes
(H-2b).
Phenotypically, cells partially differentiated under the influence of
GM-CSF alone had an up-regulation of B7-2 expression (Fig. 5B). Functionally, these cells
retained their ability to inhibit T cells, although some variability
was observed (Fig. 5A). Addition of IL-4 alone resulted in a
high level of CD11c expression but a low level of B7-2. Again, these
cells remained inhibitory. But when the cells were differentiated with
a combination of GM-CSF and IL-4, we observed a significant
up-regulation of B7-2 and CD11c, but no significant MHC class II was
induced (I-Ad/I-Ed). More importantly, cells
differentiated under the combined effects of GM-CSF and IL-4 resulted
in an enhanced anti-H-2b response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-irradiated B16, gene-modified to produce GM-CSF, can
induce long-lasting CD8+ T cell responses against the
parental tumor (data not shown).
It is puzzling that the growth of B16 clones secreting GM-CSF inhibited
CD8+ T cell function while the same tumor cells, irradiated
and administered s.c., protected from a challenge with the parental
B16. One of several possibilities is that when tumor cells are lethally
irradiated, their GM-CSF production may be limited in duration compared
with nonirradiated tumors that grow progressively. Short-term, local
expression of GM-CSF might allow for the terminal differentiation of
dermal DC (Langerhans cells) (39), whereas, the more sustained or
systemic presence of GM-CSF alone may cause the egress of inhibitory
granulocyte-monocyte progenitors from the bone marrow. Indeed, our data
demonstrate that a GM-CSF-producing tumor must grow for 2–5 wk in vivo
before CD11b+/Gr-1+ cells appear in the spleen
(see Fig. 1A). There is also likely to be a threshold serum
level at which GM-CSF acts in this fashion. Furthermore,
-irradiation of cells has been linked with the induction of
caspase-dependent apoptotic death (40, 41, 42). Apoptotic death of tumor
cells may elicit the production of inflammatory cytokines by resident
macrophages. IL-1ß, in particular, is activated by caspase-1, also
known as ICE (IL-1ß converting enzyme), the homologue of CED-3 (43).
Together with other inflammatory cytokines, the microenvironment of the
dying GM-CSF gene-modified tumor may stimulate the maturation of APC
(15). In addition, apoptotic death may result in the selective uptake
of fragmented cells by mature DC and other activating APC (44).
The main reason why we should be concerned about the ectopic release of GM-CSF by tumors relates to our current efforts to develop effective therapeutic cancer vaccines (45). Although these efforts have met with some real success in the clinic, most patients do not respond to our attempts at intervention (3).
GM-CSF can act effectively in concert with other cytokines to induce the maturation of APC (9). However, we found that GM-CSF given alone appears to elicit granulocyte-monocyte progenitor cells from the bone marrow that fail to develop into functional APC in the periphery. Other reports are consistent with these observations. The daily administration of GM-CSF (at doses similar to those used in this study) had virtually no effect on the splenocytes with a DC phenotype (46). In another study, transgenic mice overproducing GM-CSF exhibited only a modest increase in the number of DC in the thymus and spleen (47). Bone marrow cell cultures stimulated with GM-CSF alone do not induce mature DC, but induce a population of what Starzl and colleagues (48) call "DC-progenitors." These bone marrow cells, phenotypically characterized as class IIlow, B7-1dim, B7-2 negative, functionally induce hyporesponsiveness in allogeneic T cells (48). Further, these cells were able to significantly prolong cardiac allograft survival when transferred to graft recipients (49). Finally, the addition of IL-4 to the GM-CSF regimen corrected the defects in class II and costimulatory molecule expression and eliminated the anergizing capability of these "dendritic precursor" cells (48). Thus, GM-CSF appears to have an important, but not singular influence on APC homeostasis and differentiation.
Other cytokines produced by tumors may act in concert with GM-CSF to dysregulate the maturation of APCs and inhibit T cell activity. TGF-ß interferes with the development and function of DC (9). IL-10 can suppress DC function and differentiation in addition to shifting the balance of a Th1 response toward the Th2 type response (50, 51, 52, 53). Vascular endothelial growth factor (VEGF) has also been shown to impair the generation of mature DC from granulocyte-monocyte progenitor cells (53, 54). TS/A tumor cells used in this study have been shown to produce VEGF and TGF-ß, in addition to various CSF (55, 56). In addition, tumor cells have been shown to evade immune recognition by a variety of mechanisms that include poor Ag processing, loss of ß2-microglobulin, loss of Ag, and production of immunoinhibitory cytokines (57, 58, 59). The expression of Fas ligand by tumor cells is still controversial (60, 61). In any case, tumor cells may use one or more of these mechanisms to subvert immune responses, although the administration GM-CSF by itself was sufficient to abrogate the development of mature APC.
The mechanism used by the granulocyte-monocyte progenitor cells to inhibit the effector function of CD8+ T cells is unknown. However, we are currently investigating several potential mechanisms. It has been reported that inhibition of T lymphocyte reactivity could be induced by a subclass of CD8a+ lymphoid-derived DC. This inhibition could be reversed with exogenous IL-2 (36). Recently, these authors went on to demonstrate that the apoptotic death of CD4+ T cells induced by this subpopulation of DC was mediated by Fas (CD95/Apo-1) ligand (37). The cells described in this report are phenotypically quite different from the CD8a+ DC described above, but they have a striking resemblance to the bone marrow-derived "DC-progenitors" that develop under the influence of GM-CSF (48, 49). The T cell defects induced by these previously reported "DC-progenitors" could be reversed by the addition of an anti-CD28 mAb or with exogenous IL-2 (48). Thus, the unresponsiveness of the CD8+ T lymphocytes upon stimulation through their clonotypic receptors may result from a deficit in costimulation and cytokine signaling.
The beneficial effect of GM-CSF administration in immunocompromised patients after bone marrow or stem cell transplantation has been documented since the late 1980s (62). This effect may be in apparent conflict with our results. However, GM-CSF is thought to accelerate the recovery of mature granulocytes, a component of the innate immune system. Granulocytes and other phagocytes are the first line of defense in the nonspecific neutralization of infectious agents. Our results suggest that GM-CSF may inhibit the adaptive immune system, including CD8+ T cell function, while favoring the function of the innate immune system.
On the basis of our findings, we propose that the alterations in the immune response induced by some malignancies are a consequence of the induction of granulocyte-monocyte progenitor cells that are unable to mature into functional APC due to the unopposed secretion of GM-CSF. In the absence of costimulation and the appropriate cytokine mileu provided by mature APC, it may not be possible to initiate or sustain immune reactivity at the tumor site. Additional experiments are needed to evaluate whether or not these CD11b+/Gr-1+ cells are recruited when GM-CSF is employed as immune or hemopoietic adjuvant, especially when large doses of this cytokine are administered systemically. The induction and isolation of granulocyte-monocyte progenitor cells may represent a powerful therapeutic tool to control the unrestrained immune reactivity associated with autoimmune diseases and transplant rejections.
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2 Address correspondence and reprint requests to Dr. Nicholas P. Restifo, Building 10, Room 2B42, Bethesda, MD 20892-1502. E-mail address:
3 Abbreviations used in this paper: TAA, tumor-associated Ags; DC, dendritic cell; ß-gal, ß-galactosidase; CM, culture medium; rVV, recombinant vaccinia virus.
Received for publication December 3, 1998. Accepted for publication February 18, 1999.
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L. I. Terrazas, K. L. Walsh, D. Piskorska, E. McGuire, and D. A. Harn Jr. The Schistosome Oligosaccharide Lacto-N-neotetraose Expands Gr1+ Cells That Secrete Anti-inflammatory Cytokines and Inhibit Proliferation of Naive CD4+ Cells: A Potential Mechanism for Immune Polarization in Helminth Infections J. Immunol., November 1, 2001; 167(9): 5294 - 5303. [Abstract] [Full Text] [PDF]
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O. Atochina, T. Daly-Engel, D. Piskorska, E. McGuire, and D. A. Harn A Schistosome-Expressed Immunomodulatory Glycoconjugate Expands Peritoneal Gr1+ Macrophages That Suppress Naive CD4+ T Cell Proliferation Via an IFN-{gamma} and Nitric Oxide-Dependent Mechanism J. Immunol., October 15, 2001; 167(8): 4293 - 4302. [Abstract] [Full Text] [PDF]
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A. Mann, K. Breuhahn, P. Schirmacher, A. Wilhelmi, C. Beyer, A. Rosenau, S. Özbek, S. Rose-John, and M. Blessing Up- and Down-Regulation of Granulocyte/Macrophage-Colony Stimulating Factor Activity in Murine Skin Increase Susceptibility to Skin Carcinogenesis by Independent Mechanisms Cancer Res., March 1, 2001; 61(5): 2311 - 2319. [Abstract] [Full Text]
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E. Apolloni, V. Bronte, A. Mazzoni, P. Serafini, A. Cabrelle, D. M. Segal, H. A. Young, and P. Zanovello Immortalized Myeloid Suppressor Cells Trigger Apoptosis in Antigen-Activated T Lymphocytes J. Immunol., December 15, 2000; 165(12): 6723 - 6730. [Abstract] [Full Text] [PDF]
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V. Bronte, E. Apolloni, A. Cabrelle, R. Ronca, P. Serafini, P. Zamboni, N. P. Restifo, and P. Zanovello Identification of a CD11b+/Gr-1+/CD31+ myeloid progenitor capable of activating or suppressing CD8+ T cells Blood, December 1, 2000; 96(12): 3838 - 3846. [Abstract] [Full Text] [PDF]
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S. A. Kusmartsev, Y. Li, and S.-H. Chen Gr-1+ Myeloid Cells Derived from Tumor-Bearing Mice Inhibit Primary T Cell Activation Induced Through CD3/CD28 Costimulation J. Immunol., July 15, 2000; 165(2): 779 - 785. [Abstract] [Full Text] [PDF]
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E. Daro, B. Pulendran, K. Brasel, M. Teepe, D. Pettit, D. H. Lynch, D. Vremec, L. Robb, K. Shortman, H. J. McKenna, et al. Polyethylene Glycol-Modified GM-CSF Expands CD11bhighCD11chigh But Not CD11blowCD11chigh Murine Dendritic Cells In Vivo: A Comparative Analysis with Flt3 Ligand J. Immunol., July 1, 2000; 165(1): 49 - 58. [Abstract] [Full Text] [PDF]
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M. Graner, A. Raymond, D. Romney, L. He, L. Whitesell, and E. Katsanis Immunoprotective Activities of Multiple Chaperone Proteins Isolated from Murine B-Cell Leukemia/Lymphoma Clin. Cancer Res., March 1, 2000; 6(3): 909 - 915. [Abstract] [Full Text]
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S. Salvadori, G. Martinelli, and K. Zier Resection of Solid Tumors Reverses T Cell Defects and Restores Protective Immunity J. Immunol., February 15, 2000; 164(4): 2214 - 2220. [Abstract] [Full Text] [PDF]
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