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Glycobiology Program, Center for Cancer and Transplantation Biology, Children’s National Medical Center, and Departments of Pediatrics and Biochemistry/Molecular Biology, George Washington University School of Medicine,Washington, DC 20010
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One proposed mechanism of tumor escape is the release, by tumor cells, of soluble factors into their microenvironment, leading to the suppression of the immune response (5, 6, 7). We have hypothesized that gangliosides, a class of biologically active cell surface molecules, may function as soluble modulators of the immune response (8). Evidence that gangliosides may be active in the suppression of the antitumor immune response include studies demonstrating that tumor cells synthesize and shed gangliosides into their microenvironment (5, 8, 9, 10, 11, 12), coupled with studies showing that gangliosides are highly immunosuppressive in vitro. Gangliosides inhibit multiple steps in the cellular immune response, including, for example, Ag processing and presentation (13, 14), lymphocyte proliferation (13, 15, 16, 17, 18, 19), and the generation of a cytotoxic response (20, 21, 22). A gap in our knowledge, however, is the fact that modulation of the immune response by gangliosides in a syngeneic tumor system in vivo has not been shown, leaving us with only indirect proof of this concept. Here we have addressed this issue.
The immune response to tumors is distinct from the responses to other stimuli such as polyclonal mitogens, soluble bacterial Ags, and allogeneic stimuli (23). Thus, although the effects of gangliosides on the response to several non-tumor-associated Ags have been well documented (16, 17, 18), the effects of gangliosides on the antitumor immune response have not been completely elucidated. To overcome these pitfalls, we performed a comprehensive examination of the critical aspects of ganglioside involvement in tumor cell escape from the immune system. This includes evaluating ganglioside synthesis, shedding, and immunosuppressive activity, in a physiologically relevant in vivo tumor model.
This study has taken advantage of the well-documented and fully characterized antitumor immune response developed in the FBL-3 tumor model (23). FBL-3, an erythroleukemia of B6 origin, expresses unique TSTA that can be recognized by the immune system. Priming syngeneic mice with irradiated FBL-3 tumor cells leads to the subsequent rejection of future live tumor challenges (23). Although FBL-3 is immunogenic, injection of live FBL-3 tumor cells into naive syngeneic mice results in tumor growth, suggesting that the FBL-3 tumor cells possess mechanisms of escaping immune recognition and destruction.
Using this model, we have specifically and comprehensively examined the impact of tumor gangliosides on the antitumor immune response. We were able to directly assess, in a single system, all aspects of the hypothesis (8) that tumor cells actively shed high concentrations of gangliosides into their microenvironment and that these shed gangliosides serve as a protective barrier, binding to host lymphocytes and preventing the host from mounting an efficient antitumor immune response. With an autochthonous system (C57BL/6 mice, syngeneic FBL-3 cells, and highly purified FBL-3 gangliosides), the metabolism (synthesis and shedding) and tumor-specific inhibition of the immune response in vitro and in vivo by the tumor gangliosides was demonstrated. This is the crux of the concept that gangliosides enhance tumor formation by an immunological mechanism. These results provide direct evidence that tumor gangliosides are highly effective inhibitors of the syngeneic antitumor immune response, thereby establishing ganglioside metabolism as an active factor in the process of tumor formation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FBL-3, a Friend virus-induced erythroleukemia of B6 origin, was kindly provided by Dr. Philip Greenberg. B78H1 melanoma, a variant of B16 melanoma, was kindly provided by Dr. Hyam Levitsky. Both cell lines were maintained in vitro by serial passage in RPMI 1640 medium supplemented with 10% FCS, 1% nonessential amino acids, sodium pyruvate, L-glutamine, penicillin 50 U/ml, streptomycin 50 µg/ml, and 10 mM HEPES buffer (BioWhittaker, Walkersville, MD).
Mice
Female 5–7 wk old C57BL/6 mice (H-2b Thy-1.2), to which the FBL-3 and B78H1 tumor cell lines are syngeneic, were obtained from Harlan Sprague Dawley.
Ganglioside isolation
The cellular gangliosides were isolated from FBL-3 tumor cells which were pelleted, lyophilized, and extracted twice with chloroform-methanol (1:1). The total lipid extract was taken to dryness. Gangliosides were purified by partitioning the dried total lipids in diisopropylether/1-butanol/0.1% aqueous NaCl (24). The lyophilized final aqueous phase was dissolved in a small volume of water, after which Sephadex G-50 gel exclusion chromatography was used to remove salt and low m.w. impurities from the ganglioside fraction. Gangliosides were quantified as nanomols of lipid-bound sialic acid by a modified colorimetric resorcinol assay (25). Analysis of gangliosides was performed using 10- x 20-cm precoated Silica Gel 60 high performance TLC plates. The plates were developed in chloroform-methanol, 0.25% aqueous CaCl2·2H2O (60:40:9). Gangliosides were visualized as purple bands with resorcinol-HCl (26).
High performance liquid chromatographic purification of gangliosides
To obtain highly purified tumor cell gangliosides for the
immunological studies, in a final step the gangliosides were purified
by normal phase HPLC. Briefly, 
800 nmol lipid-bound sialic acid
(LBSA) of FBL-3 cell gangliosides in 100 µl HPLC water were
chromatographed with a Perkin-Elmer (Norwalk, CT) HPLC system, on a
LiChrosorb-NH2 column (250 mm long, 10 mm inside
diameter, Merck, Germany). The separation was conducted at ambient
temperature with a solvent gradient program described by Gazzotti et
al. (27). The eluting solvent system was composed of
acetonitrile-5 mM Sorensen’s phosphate buffer (83:17), pH 5.6 (solvent
A), and acetonitrile-20 mM Sorensen’s phosphate buffer (1:1), pH 5.6
(solvent B). The gradient elution program of 80 min, at a flow rate of
6.25 ml/min, was as follows: 100% of solvent A for 7 min, then a
linear gradient from 100% solvent A to solvent A-solvent B (66:34)
over 53 min, and then a linear gradient from solvent A-solvent B
(66:34) to solvent A-solvent B (36:64) over 20 min. The elution profile
was monitored by flow-through detection of UV absorbance at 215 nm. The
isolated gangliosides were lyophilized, dissolved in a small volume of
distilled water, desalted by Sephadex G-50 gel exclusion
chromatography, and lyophilized.
Mass spectrometric analysis
The carbohydrate structure of gangliosides was characterized by negative-ion fast atom bombardment mass spectrometry (FAB-MS). Ceramide structures were elucidated by negative ion FAB collisionally activated dissociation tandem MS (FAB CAD-MS/MS) without prior derivatization using linked scans. Approximately 1 µl of ganglioside-methanol solution was mixed with 2 µl of triethanolamine (matrix) on the FAB probe tip. Ions were formed by bombardment with a 6-keV beam of xenon atoms in a JEOL HX-110 double-focusing mass spectrometer. For analysis of the ceramide structure by FAB CAD-MS/MS, the [CerO]- fragment ion was selected as the precursor ion. Helium was used as the collision gas, and the helium pressure was adjusted to reduce the abundance of the precursor by 75%. A JEOL DA-500 data system generated the linked scans (28).
Ganglioside shedding
FBL-3 tumor cells were metabolically labeled by culture for 48 h in RPMI 1640 medium containing 10 µCi each of D-[1-14C]glucosamine hydrochloride and D-[1-14C]galactose, after which the cells were washed three times in RPMI medium and resuspended at 2.0 x 106 cells/ml in three 25-cm2 tissue culture flasks. The supernatant and the cells from the same flask were harvested 24 h after the cells were resuspended. The gangliosides present in the cells and in the supernatant were then purified by previously described methods (24). The radiolabeled gangliosides were quantified by ß-scintillation counting of the ganglioside-associated radioactivity, and equal aliquots of radiolabeled gangliosides were qualitatively analyzed by high performance TLC (HPTLC) autoradiography (29) with radiolabeled rat brain gangliosides as standards.
Proliferative responses
Mice were immunized by injecting 107
-irradiated (10,000 rad) FBL-3 tumor cells in 100 µl PBS i.p.
twice at 2-wk intervals (23). The spleens were removed
from these mice for use in the immunological studies 2 wk after the
second injection. Single-cell suspensions of splenocytes were prepared
with a laboratory homogenizer (Stomacher, Tekmar, Cincinnati, OH).
Contaminating erythrocytes were lysed by resuspending the splenocytes
in 5 ml lysing buffer, ACK (BioWhittaker) for 1 min and then washing
three times in HB104 medium. After the third wash, the splenocytes were
adjusted to 4 x 106 cells/ml in HB104
medium containing 2% FCS and 1% HEPES buffer. This suspension was
aliquoted (25 µl) into 96-well A/2 flat-bottom plates. FBL-3 cell
gangliosides in 10 µl HB104 medium were added to each well to achieve
a final concentration of 0–20 µM. Finally, the cultures were
stimulated by addition of 104 irradiated (10,000
rad) syngeneic FBL-3 tumor cells per well. The splenocyte proliferative
response was determined 4 days later by adding 0.5 µCi
[3H]thymidine (DuPont NEN, Wilmington, DE) to
each well for 4 h. [3H]Thymidine uptake
was determined by ß-scintillation counting. The possibility that the
gangliosides were directly toxic to the cells was addressed by
determining the number and viability of the splenocytes present at the
end of the culture period by trypan blue dye exclusion.
CTL response
Splenocytes (4 x 106 cells/well)
from mice preimmunized against FBL-3 tumor cells were prepared as
described above and then cultured in the presence or absence of 20 µM
FBL-3 gangliosides in 24-well plates in a final volume of 2 ml for
4 h before restimulation with 4 x 105
-irradiated (10,000 rad) FBL-3 tumor cells. The splenocytes were
harvested 4 days later and washed three times in HB104 medium to remove
unbound gangliosides. The number of splenocytes added was adjusted to
achieve the desired E:T ratios and aliquoted (100 µl) to 96
round-bottom plates. Tumor targets were labeled by incubating
106 tumor cells with 100 µCi sodium
[51Cr]chromate (DuPont NEN) for 1 h in
RPMI medium containing 10% FCS. The tumor target cells were then
washed three times in HB104 medium, adjusted to
105 cells/ml, and added (5 x
103 cells/50 µl/well) to 96-well round-bottom
plates. Lysis of the 51Cr-labeled tumor cells was
assessed in a standard 4-h chromium release assay (22).
51Cr-labeled EL-4 tumor cells were used as
nonspecific control targets. Supernatants were harvested via the
Skatron supernatant collection system (Skatron, Sterling, VA).
Spontaneous release of label was measured in parallel wells containing
target but not effector cells. Maximum release of label was quantified
by addition of 50 µl of 1% Triton X-100 to wells containing target
cells alone. Samples were counted in a gamma counter (Cobra II, Hewlett
Packard, Meriden, CT). Percent lysis was determined by the formula
lysis (%) = [(experimental release - spontaneous release)
(total release - spontaneous release)] x 100.
In vivo primary immune response
To assess the effects of gangliosides on the primary antitumor immune response, naïve C57BL/6 mice were immunized by injecting 103 irradiated (10,000 rad) FBL-3 tumor cells in the presence or absence of 10 nmol FBL-3 gangliosides s.c. into their left rear footpads. Two weeks later, the effect of gangliosides on the primary immunization was assessed by determining the response to a secondary tumor challenge, measured with a modified version of the popliteal lymph node (PLN) assay (30, 31). Here, mice were rechallenged by injecting them in their left rear footpad with 105 irradiated (10,000 rad) FBL-3 tumor cells in 50 µl PBS. The immune response was measured 5 days later by determining the changes in PLN mass, cell number, and [3H]thymidine incorporation, and comparing them with those of unstimulated lymph nodes isolated from the right rear footpad. The cell number was determined after preparing a single-cell suspension of the lymph nodes with a laboratory homogenizer. The lymph node cell suspensions were adjusted to 106/ml in RPMI medium containing 10% FCS, and 100 µl were added to triplicate wells in a 96-well flat-bottom plate, pulsed for 18 h with 0.5 µCi [3H]thymidine, and harvested, and [3H]thymidine uptake was determined by ß-scintillation counting.
In vivo secondary immune response
Mice were immunized by injecting 107
-irradiated (10,000 rad) FBL-3 tumor cells in 100 µl PBS i.p.
twice at 2-wk intervals (23) and were used for the
immunological studies 2 wk after the second injection. The immunized
mice received a secondary challenge by injecting them in their left
rear footpad with 2.5 x 106 irradiated
(10,000 rad) FBL-3 tumor cells in 50 µl PBS, admixed with or without
10 nmol of purified FBL-3 tumor cell gangliosides. As a positive
control of inhibition, immunized mice were injected into their left
footpad with irradiated FBL-3 tumor cells and concomitantly received
cyclosporin, 24 mg/kg i.p. daily) (32). As a control for
specificity of the response, other mice received 2.5 x
106 irradiated unrelated (B78H1) tumor cells.
Five days after the secondary challenge, the immune response was
determined by aseptically removing the draining lymph nodes and
quantifying the increase in lymph node mass, cell number, and
lymphocyte DNA synthesis compared with those of unstimulated lymph
nodes isolated from the right rear footpad, as described above.
Flow cytometric analysis
Cells (5 x 105) isolated from the PLNs of C57BL/6 mice were resuspended in cold PBS-0.5% BSA and incubated with fluorescently labeled mAb. The following mAb (PharMingen, San Diego, CA) were used: PE-conjugated hamster IgG isotype standard (anti-keyhole limpet hemocyanin); PE-conjugated anti-CD8; PE-conjugated anti-CD69; PE-conjugated anti-CD25; PE-conjugated anti-CD69; FITC-conjugated anti-CD3; FITC-conjugated anti-CD4; FITC-conjugated anti-CD19; and FITC-conjugated anti-Mac-3. The cells were stained with the mAbs for 30 min on ice, washed twice in PBS-0.5% BSA, and analyzed with a FACStarPlus flow cytometer (Becton Dickinson, Mountain View, CA).
Statistical analysis
Student’s unpaired t test (two-tailed) was used to determine the significance of differences between experimental and control data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FBL-3 gangliosides were isolated (24), quantified
(25), and initially analyzed by HPTLC (27).
FBL-3 tumor cells contained 12 nmol
gangliosides/108 cells. HPTLC analysis of the
FBL-3 ganglioside migration pattern (Fig. 1
) revealed the FBL-3 ganglioside
fraction to contain one major ganglioside doublet. It migrated with
HPTLC mobility between that of the human brain ganglioside standards
GM1 and GD1a, similar to
the migration of a ganglioside observed previously in several other
mouse tumors and identified as GM1b (28, 29).
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). They correspond to two molecular
species of the ganglioside GM1b. Fig. 2 shows the negative-ion mass spectrum of
one of the molecular species of GM1b. The peak at
m/z 1626 represents the molecular ion, [M -
H]-. In addition, there are peaks in the mass
spectrum that resulted from the loss of N-acetylneuraminic
acid, galactose, N-acetylgalactosamine and a second
galactose. Specifically, the molecular ion [M - H
]- at m/z 1626 yielded the peak at
m/z 1335.6 by the loss of N-acetylneuraminic
acid, and the peak at m/z 1173.6 by the loss of
N-acetylneuraminic acid and galactose. The loss of
N-acetylneuraminic acid, galactose, and
N-acetylgalactosamine gave the peak at m/z 970.8,
whereas the loss of N-acetylneuraminic acid, galactose,
N-acetylgalactosamine, and a second galactose resulted in
the peak at m/z 808.7. The corresponding
[CerO]- was also detected, at m/z
646.6.
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). With this same approach, the ceramide structure of the second
molecular species of GM1b was identified. Thus,
the analysis revealed that the GM1b ganglioside
had two ceramide structures, d18:1-C16:0 and d18:1-C24:1 (Table I) and
led to the conclusion that two molecular species of
GM1b, differing in their ceramide structures, are
the major gangliosides of FBL-3 tumor cells. This latter finding is
consistent with the general concept, shown in a number of other
studies, that tumor-derived gangliosides frequently express
heterogeneous ceramide structures (28, 33, 34, 35). FBL-3 cells shed gangliosides
Significant shedding of gangliosides by tumor cells into the
cellular microenvironment is thought to be a critical element in
satisfying the hypothesis that tumor gangliosides interfere with the
antitumor immune response, because to interact with host leukocytes,
these molecules should be released by the tumor cells. We quantified
ganglioside shedding by the FBL-3 tumor cells and found that 17% of
cellular gangliosides were shed into the microenvironment in a 24-h
period. This translates into a shedding rate of 90
pmol/108 cells/h, indicating that FBL-3 tumor
cells shed gangliosides at a substantial rate. Qualitative analysis of
the shed gangliosides by TLC/autoradiography revealed that
GM1b is both the major cellular and the major
shed ganglioside (Fig. 3). This is
consistent with previous observations and with the generally accepted
concept that the major ganglioside species present on the cell membrane
are also the species most prominent as shed molecules in the cell
culture supernatant (8, 12, 28).
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As a first step in determining the effects of tumor-derived
gangliosides on the antitumor immune response, the effect of FBL-3
gangliosides on the secondary proliferative response of splenocytes to
syngeneic tumor cells was examined. In these experiments, splenocytes
from preimmunized mice were cultured in the presence or absence of
FBL-3 gangliosides and restimulated with irradiated FBL-3 tumor cells.
Ganglioside concentrations as low as 5 µM caused an 80% decrease in
the proliferative response, in comparison with the secondary
proliferative response of control splenocytes cultured in the absence
of added gangliosides. Splenocytes exposed to 20 µM gangliosides
showed almost 100% inhibition of proliferation (Fig. 4A). These results demonstrate
that tumor-derived gangliosides can inhibit the tumor-specific
secondary proliferative response.
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B); whereas in the presence of
20 µM FBL-3 gangliosides, the proliferative response never achieved a
level greater than 15% of the control response on any day. Thus, the
addition of tumor-derived gangliosides completely inhibited the
proliferative response without altering the kinetics of the
response. We excluded the possibility that the immunosuppressive activity of the FBL-3 gangliosides was the result of a nonspecific direct cytotoxic effect on the splenocytes. We exposed splenocytes from preimmunized mice to FBL-3 gangliosides for 4 days and then determined the viable cell counts. The cell counts of splenocytes incubated in 20 µM FBL-3 gangliosides were nearly equal to those of control cells (2.28 and 2.40 x 106/ml, respectively, representing a cell recovery of 57 and 60% of the initial 4 x 106 cells/ml plated). Therefore, the inhibition of the proliferative response caused by exposure of splenocytes to FBL-3 gangliosides was not the result of a direct effect on cell viability. Further supporting this conclusion is the fact that mitogen-induced lymphoproliferative responses are relatively unaffected even at ganglioside concentrations which cause marked inhibition of Ag-induced responses (36).
FBL-3 gangliosides inhibit the generation of CTL specific for syngeneic FBL-3 tumor cells
The ability of the immune system to develop T cells capable of
recognizing and lysing specific tumor targets is critical for tumor
elimination in vivo (37). To examine the effects of FBL-3
gangliosides on the CTL response, splenocytes (4 x
106/well) from preimmunized mice were cultured in
the presence or absence (control) of 20 µM FBL-3 gangliosides and
restimulated with 4 x 105 irradiated FBL-3
tumor cells. After 4 days of culture, the nonadherent cells were
harvested and redirected against 51Cr-labeled
FBL-3 tumor targets at E:T ratios ranging from 100:1 to 3:1. A vigorous
specific CTL response was observed; at an E:T ratio of 100:1, there was
72% specific lysis of FBL-3 targets and no significant lysis of the
unrelated target, EL-4. FBL-3 gangliosides (20 µM) had a significant
inhibitory effect on the CTL response, as shown in Fig. 5. At an E:T ratio of 100:1, for example,
target cell lysis was reduced by 97%. These results clearly show that
FBL-3 gangliosides inhibit the generation of tumor-specific
CTLs.
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). Thus, the inhibitory effect of the tumor
gangliosides on the antitumor CTL response reflects a significant
inhibitory effect on the generation phase combined with only a minimal
effect on the effector phase of the response. C57BL/6 mice develop a measurable and specific antitumor immune response to FBL-3 cells
The first step in the establishment of an in vivo tumor system in which the antitumor immune response could be measured was to develop a mouse model that is capable of recognizing and lysing the specific tumor cell, FBL-3. To accomplish this, C57BL/6 mice were immunized with FBL-3 tumor cells as described by Kern et al. (23). Two weeks after the second immunization, we determined whether or not the immunization was successful by injecting immunized as well as naive mice (i.e., not immunized with FBL-3 cells) with a live tumor cell challenge (5 x 106 FBL-3 cells) and observing them for survival. All three of three immunized mice rejected the tumor challenge, whereas all naive mice rapidly developed tumors.
To determine whether an immune response to the FBL-3 tumor cells could be detected in vivo, we used a modified PLN assay (30, 31). Previously immunized mice were challenged in their left rear footpads with 2.5 x 106 irradiated FBL-3 tumor cells or B78H1 tumor cells. Five days after the injection, the immune response was quantified by harvesting the stimulated draining PLNs and comparing their mass and cell number to those of unstimulated PLNs. FBL-3-stimulated lymph nodes showed an increase in mass of 1.9 mg and an increase in cell number of 1.7 x 106/node, compared with unstimulated lymph nodes, demonstrating that there was a vigorous and quantifiable immune response to the syngeneic FBL-3 tumor cells. In contrast, mice preimmunized with irradiated FBL-3 and challenged with unrelated tumor cells (B78H1) did not produce a significant response (0.3 ± 0.2 mg increase in mass and no detectable increase in cell number), demonstrating that the immune response was specific for the FBL-3 tumor cells. These results confirm previous studies (38) showing that C57BL/6 mice immunized with FBL-3 cells can develop an effective and specific immune response to an FBL-3 tumor challenge and demonstrate that this model can be used to directly measure the effects of tumor gangliosides on the antitumor immune response to syngeneic tumor cells.
FBL-3 tumor gangliosides inhibit the in vivo primary antitumor immune response to FBL-3 tumor cells
The primary immune response to a tumor challenge most closely
mirrors the likely initial interaction between the tumor cell and the
immune system. Unsuccessful attempts to directly assess this response
suggest that the FBL-3 tumor cells are not sufficiently immunogenic to
produce a measurable primary immune response in the PLN assay.
Therefore, to measure the effects of tumor gangliosides on the primary
antitumor immune response in vivo, 10 nmol of FBL-3 gangliosides were
coinjected with the primary FBL-3 cell immunization into naive mice.
The modulatory effects of the tumor gangliosides on the primary immune
response were assessed by determining how this influenced the response
to a subsequent (secondary) challenge with 105
irradiated FBL-3 cells alone. This challenge was administered 2 wk
later, and the response was quantified 5 days after the challenge by
determining the changes in PLN mass, cell number, and
[3H]thymidine uptake, compared with
unstimulated control nodes. Mice given a primary FBL-3 immunization in
the absence of gangliosides had a significant secondary anti-FBL-3
immune response; the PLN mass increased by 2.8 ± 0.3 mg (Fig. 6A), the cell number increased
by 2.7 x 106 cells/node (Fig. 6B), and the thymidine uptake increased by 6.8 x
103 dpm (Fig. 6C), compared with
unstimulated control lymph nodes. However, when the immunizing FBL-3
tumor cells were admixed with 10 nmol FBL-3 gangliosides, there was a
significant (p < 0.001) reduction in the
secondary immune response (70, 69, and 72% reduction of the FBL-3
cell-induced increase in PLN mass, cell number, and thymidine
incorporation, respectively), which directly reflects a reduction of
the efficiency of the primary immune response. These results
demonstrate that tumor gangliosides significantly inhibit the primary
antitumor immune response in vivo.
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In addition to examining the effects of tumor gangliosides on the
primary antitumor immune response, using the same assay system, the
modified PLN assay, we quantified the effects of FBL-3 gangliosides on
the in vivo secondary antitumor immune response. The first parameter
measured was the increase in PLN mass in response to syngeneic FBL-3
tumor cell challenge. When previously immunized mice were stimulated
with FBL-3 tumor cells admixed with FBL-3 gangliosides (10 nmol), there
was a marked reduction in the increase in PLN mass. Whereas the mean
increase in lymph node mass in mice stimulated with FBL-3 tumor cells
was 1.85 mg, the coinjection of purified FBL-3 gangliosides with FBL-3
tumor cells resulted in only a 0.72-mg increase in lymph node mass.
This 61% inhibition of the response demonstrates that FBL-3
gangliosides significantly (p 
0.02) suppress
the increase in lymph node mass caused by syngeneic tumor challenge in
vivo (Fig. 7A), and in fact to
a degree similar to that caused by treatment of mice with cyclosporin
(24 mg/kg i.p. daily for 4 days), a standard system for suppression of
the cellular immune response (32).
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The effect of FBL-3 gangliosides on the in vivo secondary
lymphoproliferative response to syngeneic tumor challenge was directly
examined in two ways: by determination of the increase in PLN cell
number; and by quantification of the spontaneous proliferative
response. Unstimulated lymph nodes contained 0.19 x
106 cells/node. Lymph nodes stimulated with
-irradiated FBL-3 cells contained 1.83 x
106 cells/node, which represents an almost
10-fold increase in cell number. However, when the FBL-3 cell challenge
was injected together with FBL-3 gangliosides, the draining lymph nodes
harvested 5 days later contained only 0.73 x
106 cells, indicating a marked inhibition by
gangliosides of the increase in cell number (Fig. 7B). FBL-3
gangliosides also significantly (p = 0.006)
reduced the spontaneous proliferative response, as measured by
[3H]thymidine uptake, by 41%, from 1.06
x 103 dpm/105 cells to
0.63 x 103 dpm/105
cells (Fig. 7C). Once again these results indicated a
similar effect to that achieved by the repeated injection of
cyclosporin (Fig. 7, B and C). These in vivo
studies provide strong evidence that FBL-3 gangliosides significantly
suppress the antitumor immune response in vivo.
FBL-3 gangliosides inhibit the tumor-induced activation and expansion of lymph node cell populations in vivo
Changes in the PLN populations induced by gangliosides were
further delineated by determining the expression of cell surface
markers specific for cell lineage and activation status. Lymph node
cells from mice injected in the footpad with irradiated FBL-3 cells
admixed with FBL-3 gangliosides (10 nmol) or injected with irradiated
FBL-3 cells alone (control) were stained with fluorescence-conjugated
mAbs for lineage-specific markers including CD3 (T cells), CD4 (Th
cell), CD8 (CTL), CD19 (B cells), and Mac-3 (dendritic
cells/macrophage) as well as activation markers CD69 (early
activation marker), and CD25 (IL-2 receptor) and analyzed by FACS. This
allowed us to determine changes in both the total number and activation
status of cells from the various cell populations involved in the
antitumor immune response in vivo. Comparing the cell populations of
lymph nodes draining the site of FBL-3 cell injection with the cell
populations of unstimulated lymph nodes, it was apparent that s.c.
injection of the FBL-3 cells caused a significant increase in all cell
populations examined (Fig. 8A). This increase was
suppressed in the lymph nodes of mice also receiving the FBL-3
gangliosides. For example, the T cell population
(CD3+) increased from 2.2 x
105 cells/node in the unstimulated lymph node to
11.1 x 105 cells/node in the stimulated,
control lymph nodes, but to only 5.5 x 105
cells/node in the stimulated lymph nodes of ganglioside-treated mice
(63% decrease). These ganglioside-induced changes were evident in the
T cell subpopulations, in which there was 57 and 65% inhibition of the
CTL and T helper cell populations, as well as in the B cell and
dendritic cell/macrophage populations, which were decreased by 74 and
69%, respectively. The expression of activation markers was also
affected; coinjection of FBL-3 tumor gangliosides with FBL-3 cells led
to a significantly reduced number of lymph node cells expressing CD69
and CD25 (60 and 71%, respectively). Overall, ganglioside-induced
inhibition of each lymph node cell subpopulation studied was
statistically significant (p 0.04), whereas
the distribution of the cell populations showed that the inhibition was
not specific for any single population examined (Fig. 8B).
Thus, the in vivo administration of the tumor gangliosides caused a
generalized suppression of the observed increase of cell populations
associated with in the antitumor immune response in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study has taken advantage of the well documented antitumor immune response developed in the FBL-3 tumor model, allowing us to comprehensively examine the impact of tumor gangliosides on the antitumor immune response. Here we directly tested the hypothesis that tumor cells actively shed biologically active gangliosides into their microenvironment, where they serve as a protective barrier, binding to host lymphocytes and preventing them from developing an efficient antitumor immune response. We found that FBL-3 tumor cells shed gangliosides at a high rate, creating a tumor microenvironment rich in tumor-derived gangliosides. Using the highly purified FBL-3 gangliosides, we showed in vitro inhibition of the tumor-specific secondary proliferative response and of the generation of tumor-specific CTLs. In vivo, we observed inhibition of both the primary and secondary antitumor immune responses. These studies provide the first direct evidence that tumor gangliosides are highly effective inhibitors of the syngeneic antitumor immune response and thereby establish tumor cell ganglioside metabolism as an active factor in the process of tumor formation.
Immunosuppressive activity is a general biological property of gangliosides that is not specifically limited by the source of the gangliosides but that is influenced by their molecular structure. In fact, gangliosides other than the disialoganglioside GM1b studied here, such as those isolated from LAN-5 neuroblastoma cells (which are mainly GD2 and GM2) (43), also are effective inhibitors of the immune response to FBL-3 tumor cells (unpublished data). Variations in either the carbohydrate or the ceramide structure can lead to differing degrees of activity. For example, in comparing a series of highly purified normal human brain gangliosides, those with a terminal sialic acid linked to a compact neutral oligosaccharide had the greatest immunosuppressive activity (36). Other studies examining the relationship between structure and activity revealed that tumor gangliosides were frequently more immunosuppressive than corresponding normal human brain gangliosides of identical carbohydrate structure. These differences were inversely related to the length of the ceramide fatty acyl chain (34). Thus, immunosuppressive activity is quantitatively affected by ganglioside structure.
Another factor influencing activity is presence of these molecules in the tumor microenvironment. Because tumor cells shed gangliosides at a high rate, it was predicted that the concentration of gangliosides in the tumor microenvironment would be very high. This was supported by recent findings in an in vitro system in which the shedding rate and microenvironmental ganglioside concentrations could be estimated (39, 40). Therefore, in contrast to lower concentrations in the peripheral circulation, the tumor cell microenvironment may contain high concentrations of highly active gangliosides, leading to maximal inhibition of the antitumor immune response at the site of tumor formation and progression.
The mechanisms of ganglioside-induced immunosuppression are most likely
multiple and remain to be fully elucidated. However, it is clear that
both APC and T cells are affected by tumor gangliosides. Some of the
known mechanisms are reversible inhibition of APC function (13, 14), inhibition of IL-2-induced T cell proliferation
(44), and down-regulation of CD4 expression
(45, 46, 47). Most recently, it has been shown that exposure
of mouse splenocytes to gangliosides results in reduced gene
transcription of the Th1-associated cytokines, IL-2 and IFN-, while
leaving gene transcription of the Th2-associated cytokines, IL-4 and
IL-10, unaffected (48). Others have reported a ganglioside-induced
increase in T cell IL-10 production (49). Together, these findings
suggest that shed tumor gangliosides may shift the balance of the
antitumor immune response from the normally predominant Th1 response
toward the Th2 response, possibly leading to a reduction in the
cellular antitumor immune response critical for tumor elimination.
Because the earliest stage of the cellular immune response known to be
affected by exposure to gangliosides is Ag presentation (13, 14), it is
tempting to speculate that the initial step in tumor
ganglioside-induced immunosuppression in vivo is interference with the
functions of APC, which in the FBL-3 tumor model would predominantly be
dendritic cells.
Notwithstanding gaps in knowledge regarding the mechanism of immunosuppression by tumor gangliosides, the conclusion supported by the present work, that gangliosides shed by tumor cells can suppress the ability of the immune system to recognize and eliminate tumor cells, may have direct relevance to human cancer. Corroborating evidence includes the observations that aggressive neuroblastomas shed significant quantities of gangliosides in vivo and that the level of shed gangliosides was directly related to the incidence of tumor recurrence and the rapidity of progression (50). It has been proposed that clinically it may be possible to boost the immune response, specifically or nonspecifically, to enhance tumor cell recognition and elimination (51, 52, 53). However, to date such attempts have had only limited success. This may stem in part from the ability of tumor cells to escape immune recognition by various mechanisms, one being the shedding of tumor gangliosides into the microenvironment. Thus, a potentially therapeutic enhancement of the immune response could be negated by the particular immunosuppressive activity of the tumor. Approaches designed to inhibit such tumor cell escape mechanisms would be desirable. Recently, it was shown that metabolic depletion of tumor gangliosides is possible by treating cells with inhibitors of the synthesis of glucosylceramide, a metabolic precursor of gangliosides (40, 43, 54, 55). With the recognition of a causative role of gangliosides in tumor-associated immunosuppression and a facilitative role in tumor progression, the use of such inhibitors of ganglioside synthesis might enhance the effectiveness of immunotherapeutic approaches to cancer.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Stephan Ladisch, MD, Center for Cancer and Transplantation Biology, Children’s Research Institute, 111 Michigan Avenue, NW, Washington, DC 20010.
3 Abbreviations used in this paper: TSTA, tumor-specific transplantation Ags; FAB-MS; fast atom bombardment mass spectrometry; FAB CAD-MS/MS, FAB collisionally activated dissociation tandem MS; HPTLC, high performance TLC; PLN, popliteal lymph node.
Received for publication February 1, 1999. Accepted for publication July 22, 1999.
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) of 20 µM
FBL-3 cell gangliosides and determining the proliferative response on
days 3–7 by ß-scintillation counting. Data are means ± SE of
triplicate determinations. The proliferative responses of splenocytes
treated with 5–20 µM FBL-3 gangliosides were significantly different
from that of control splenocytes, p 
). The SE of the triplicates was routinely <10% of
the total release.
), were compared
with control unstimulated lymph node cell populations (
).
A, Absolute number of cells expressing the indicated
markers. B, percent of the total lymph node cell
population expressing the same markers. Data represent the mean ±
SE of the results of three separate experiments.