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Originally published online as doi:10.1189/jlb.0705395 on January 13, 2006

Published online before print January 13, 2006
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(Journal of Leukocyte Biology. 2006;79:586-595.)
© 2006 by Society for Leukocyte Biology

The immunoregulatory effects of gangliosides involve immune deviation favoring type-2 T cell responses

Fabian A. Crespo*, Xichun Sun*, James G. Cripps* and Rafael Fernandez-Botran*,{dagger},1

* Departments of Pathology & Laboratory Medicine and
{dagger} Microbiology & Immunology, School of Medicine, University of Louisville, Kentucky

1 Correspondence: Department of Pathology and Laboratory Medicine, School of Medicine, University of Louisville, Louisville, KY 40292. E-mail: rafael{at}louisville.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gangliosides, sialic acid-containing glycosphingolipids present in most cell membranes, are thought to participate in the maintenance of immune privilege and tumor-induced immunosuppression. However, the mechanisms responsible for their immunomodulatory activity remain poorly understood. The purpose of this study was to investigate whether gangliosides are able to modulate the balance of type-1/type-2 T cell responses and to characterize the cellular mechanisms involved. The effects of different gangliosides on anti-CD3-stimulated murine splenocytes and purified T cells were studied. The presence of gangliosides during T cell activation reduced the expression of interferon-{gamma} (IFN-{gamma}) and enhanced that of interleukin (IL)-4, suggesting a shift toward a type-2 response. Intracellular cytokine staining demonstrated that gangliosides inhibited IFN-{gamma} production in CD4+, CD8+, and natural killer (NK)1.1+ cell populations and enhanced IL-4 in CD4+ T cells. The ganglioside-mediated enhancement in IL-4 production was independent of changes in endogenous IFN-{gamma}, did not occur with cells from CD1d-deficient mice, and was partially inhibited by anti-CD1d antibodies. The inhibitory effects on IFN-{gamma} were independent of endogenous IL-4 or the presence of NKT cells and were unaffected by anti-CD1d antibodies. These results suggest that gangliosides may modify the immunological environment by promoting immune deviation in favor of type-2 T cell responses.

Key Words: immunoregulation • interferon-{gamma} • interleukin-4


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gangliosides constitute a large group of sialylated glycosphingolipids, which are expressed widely in mammalian tissues as components of the outer leaflet of plasma membranes [1 2 ]. Gangliosides are particularly abundant in the central nervous system (CNS) [2 ] and are also expressed at high levels on a variety of tumor cells, including neuroblastomas, gliomas, melanomas, and small cell lung carcinomas [2 3 4 5 ]. Indeed, certain gangliosides [e.g., disialoganglioside (GD)2 and GD3] have been used as tumor markers and as targets of immunologic therapies against tumors [6 ]. Gangliosides are involved in many physiological processes, including the regulation of membrane fluidity, cellular recognition, proliferation, differentiation, apoptosis, and signal transduction [2 7 8 ]. For example, gangliosides modulate signaling by several growth factor and cytokine receptors, including interleukin-2 receptor (IL-2R), by influencing the properties of membrane lipid rafts and/or the ability of different receptor subunits to interact with each other [7 8 9 10 ].

Gangliosides have a variety of regulatory functions on cells of the immune system, which are usually associated with immunosuppressive activities [11 12 13 14 15 16 ]. For instance, gangliosides inhibit the proliferative response of T lymphocytes to mitogens, antigens, and mixed lymphocyte cultures; induce cell cycle arrest and apoptosis in T cell lines and thymocytes; and inhibit IL-2-mediated T cell proliferation [14 17 18 ]. The effects of gangliosides extend to other cell types as well, including inhibition of the proliferation of hemopoietic precursor cells; reduced generation and function of dendritic cells; and decreased antigen presentation and production of proinflammatory cytokines by monocytes [13 19 20 21 22 ]. The fact that many of these effects have been observed with gangliosides purified from brain tissue and malignant cell sources suggests an immunosuppressive role for CNS- and tumor-derived gangliosides. Consistent with this idea, elevated levels of gangliosides in the serum of patients with a variety of malignancies have been correlated with immunosuppression [3 23 24 25 26 27 ].

Although the mechanisms responsible for the immunosuppressive effects of gangliosides have not been elucidated completely, evidence points to a preferential down-regulatory effect on inflammatory and type-1 responses. Indeed, gangliosides have a marked antagonistic effect on the production and activity of the type-1 cytokine, interferon-{gamma} (IFN-{gamma}). Irani and colleagues [13] reported that gangliosides inhibit the production of IL-2 and IFN-{gamma} but not IL-4 by T lymphocytes. In addition, Zou et al. [28 ] reported that incubation of peripheral blood mononuclear cells with supernatants derived from glioma cells results in inhibition of the production of IFN-{gamma}, IL-12, and tumor necrosis factor {alpha} and enhances secretion of IL-6 and IL-10. Recently, Rayman et al. [29 ] found that gangliosides isolated from renal carcinoma supernatants inhibited type-1 but not type-2 responses in activated human T cells. The antagonism between gangliosides and IFN-{gamma} production coupled to a potential alteration of T helper cell type 1 (Th1)/Th2 subset differentiation is consistent with many of the immune defects observed in cancer patients, which primarily affect their cellular immunity. Gangliosides may thus play an active role in the immunosuppressed status of these patients and consequently, in the progression of their disease.

The purpose of these studies was to investigate whether gangliosides are able to modulate the balance of type-1/type-2 T cell responses and to characterize the cellular mechanisms involved. Our results indicate that gangliosides have the ability to induce immune deviation in favor of type-2 T cell responses by acting at different levels, including the inhibition of IFN-{gamma} production by several cell sources and an enhancement of IL-4-dependent differentiation of CD4+ T cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Female mice (4–6 weeks of age, 20–25 g) of the strains C57Bl/6, BALB/c, 129S, and C,129-Cdtm1Gru (CD1d-deficient) were purchased from The Jackson Laboratories (Bar Harbor, ME). Mice were housed under veterinary supervision at the Research Resources Center, University of Louisville Health Sciences Center (KY). All procedures involving animals were approved by an institutional committee and performed according to the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Institutes of Health, Publication No. 86-23.

Antibodies, cytokines, and reagents
The following monoclonal antibodies (mAb) were purchased from BD PharMingen (San Diego, CA): anti-mouse CD1d (1B1), anti-mouse CD3{epsilon} (145-2C11), anti-mouse CD28 (37.51), anti-mouse IL-4 (biotinylated BVD6-24G2), and anti-mouse IFN-{gamma} (R4-6A2 and biotinylated XMG1.2). An anti-mouse IL-4 antibody, 11B11, was a kind gift of Dr. Ellen Vitetta (University of Texas Southwestern Medical Center, Dallas). Fluorescent antibodies for intracellular cytokine staining were purchased from eBioscience (San Diego, CA). The recombinant murine cytokines IL-2 and IFN-{gamma} were purchased from BD PharMingen. Recombinant murine IL-4 was kindly provided by Immunex (Seattle, WA). Purified gangliosides monosialoganglioside (GM)1, GM2, GM3, GD1a, GD2, GD3, trisialoganglioside (GT)1b, and asialoganglioside (asialo)-GM1 were purchased from Matreya (Pleasant Gap, PA) or Sigma Chemical Co. (St. Louis, MO). Reagents for enzyme-linked immunosorbent assay (ELISA; avidin-alkaline phosphatase and p-nitrophenyl phosphate) or cell culture (10x penicillin/streptomycin solution, L-glutamine, RPMI-1640 medium, sodium pyruvate, and 2-mercaptoethanol) were purchased from Sigma Chemical Co. Minimal essential medium nonessential amino acids solution was from Gibco-BRL (Grand Island, NY), and fetal calf serum (FCS) was from HyClone (Logan, UT).

Splenic cell preparations
Mice were killed by cervical dislocation. Spleens were then removed aseptically, and cell suspensions were prepared by gently teasing the spleens with a pair of forceps in balanced salt solution containing 1% FCS (BSS-1% FCS). The single-cell suspension was aspirated, and the cells were washed twice with cold BSS-1% FCS. The cells were then resuspended in complete medium [RPMI 1640 containing 10% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), sodium pyruvate (1 mM), nonessential amino acids (0.1 mM), and 2-mercaptoethanol (50 µM)] and counted. The cells were cultured at this point (whole spleen cell cultures) or used in the T cell enrichment protocol.

T cell enrichment protocol
Whole spleen cell suspensions were prepared as described above and depleted of erythrocytes by incubation in red cell lysis buffer (0.14 M NH4Cl, 17 mM Tris HCl, pH 7.2) for 5 min at room temperature. The cell suspensions were then washed twice in BSS-1% FCS. Enrichment of T lymphocytes, CD4+ T cells, or CD8+ T cells was carried out using the appropriate mouse T cell/CD4+ T cell/CD8+ T cell enrichment columns (R&D Systems, Minneapolis, MN), according to the manufacturer’s protocol. After eluting the columns, the cells were counted and resuspended in complete medium.

Cell cultures
Splenocyte or T cell cultures were carried out at an initial cell density of 5 x 106 cells/ml in complete medium at 37°C/5% CO2. A 6-day protocol was used in which splenocytes or enriched T cells were first stimulated with anti-CD3 antibodies in soluble (0.5 µg/ml) or absorbed form, respectively. Cultures were supplemented with recombinant IL-2 (rIL-2; 5 ng/ml) and anti-CD28 antibodies (1 µg/ml). Different gangliosides (1–100 µg/ml; 0.5–50 µM), antibodies (5–10 µg/ml), or cytokines (5 ng/ml) were added to the cultures during this period. After 3 days, supernatants were collected, and the cells were washed and restimulated with plate-bound anti-CD3 antibodies and IL-2 (5 ng/ml) at a cell density of 2 x 106/ml. After 3 additional days of culture (Day 6), the supernatants were harvested and stored frozen at –20°C.

Cytokine assays
The levels of IFN-{gamma} and IL-4 in culture supernatants were assayed by two-site ELISAs, using pairs of anticytokine mAb as described [30 ]. Concentrations were calculated by comparison with recombinant cytokine standards.

Cytokine expression at the mRNA level
Expression of IFN-{gamma} and IL-4 at the mRNA level was analyzed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR. Purified T cells were stimulated with anti-CD3 antibodies in the presence or absence of gangliosides as indicated previously. At different times, the cells were harvested by centrifugation and lysed using a guanidinium-phenol extraction reagent (RNAwiz, Ambion, Austin, TX). Total RNA was purified by chloroform extraction followed by precipitation in isopropanol/ethanol. The samples were then reconstituted in RNase-free water, quantitated spectrophotometrically, and stored at –80°C until use. RT and PCR amplification were carried out using SuperScript One Step RT-PCR with Platinum Taq (Invitrogen, Carlsbad, CA) and the appropriate ß-actin-, IFN-{gamma}-, or IL-4-specific primers (BioSource, Camarillo, CA). PCR products were separated by polyacrylamide gel electrophoresis, stained with ethidium bromide, and quantitated by scanning densitometry and comparison with the control gene (ß-actin). For real-time PCR, cDNA was first synthesized using TaqMan RT reagents (Applied Biosystems, Foster City, CA) following the manufacturer’s protocol. Reactions in which the enzyme or RNA was omitted were used as negative controls. Real-time PCR was performed with a Prism 7500 sequence detection system and SYBR Green I dye reagents (Applied Biosystems). The specific primers were designed for mouse glyceraldehyde 3-phosphate dehydrogenase (mGAPDH), IFN-{gamma}, and IL-4 using the Primer3 software program [31 ]: mGAPDH-right primer (RP): 5'-TCTTCTGGGTGGCAGTGATG-3', mGAPDH-left primer (LP): 5'-TTGTGGAAGGGCTCATGACC-3', mIFN-{gamma}-RP: 5'-GCTGATGGCCTGATTGTCTT-3', mIFN-{gamma}-LP: 5'-GCTTTGCAGCTCTTCCTCAT-3', mIL-4-RP: 5'-CGAAAAGCCCGAAAGAGTC-3', mIL-4-LP: 5'-CCTCACAGCAACGAAGAACA-3'. The parameter threshold cycle (Ct) was defined as the fraction cycle number at which the fluorescence passed the threshold. The relative gene expression of IFN-{gamma} and IL-4 was analyzed using the 2–{Delta}{Delta}Ct method by normalizing with GAPDH gene expression in all the experiments.

Intracellular cytokine staining
The expression of IFN-{gamma} and IL-4 by different subpopulations of cells was analyzed by immunofluorescent, intracytoplasmic staining. Splenocytes or enriched T cells were stimulated with anti-CD3 antibodies and rIL-2 in the presence or absence of GT1b (10–75 µg/ml) as described above. Monensin (3 µM) was added for the last 4 h of culture at 37°C. The cells were then harvested by centrifugation, resuspended in ice-cold staining buffer [phosphate-buffered saline (PBS)-1% FCS-10 mM NaN3], and counted. Aliquots of cells (1x106cells/100 µl) were then surface-stained for 30 min on ice with phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-labeled antibodies to mouse CD4, CD8, or natural killer (NK)1.1 molecules. The cells were then washed twice with staining buffer, resuspended with 100 µl fixation buffer (eBioscience), and incubated at room temperature for 20 min. After the incubation, the cells were exposed for 5 min/room temperature to permeabilization buffer (PBS–0.1% saponin–0.1% bovine serum albumin–0.1% NaN3) and stained with PE- or FITC-conjugated antibodies to mouse IFN-{gamma}, IL-4, or the appropriate isotype-matched controls for 20 min at room temperature. After the last incubation, the cells were washed once with 1 ml permeabilization buffer and resuspended in 0.5 ml staining buffer. Analysis was performed in a FACScan flow cytometer (Becton Dickinson, San Jose, CA).

Statistics
Results are expressed as mean values ± SD. The data were analyzed by ANOVA and Bonferroni’s Multiple Comparisons Test. Statistical significance was established at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of gangliosides on the production of IFN-{gamma} and IL-4 by anti-CD3-stimulated splenocytes
To investigate whether the presence of gangliosides influences the cytokine response of activated T cells, murine splenocytes were stimulated with anti-CD3 antibodies/rIL-2 along with varying concentrations of gangliosides of the 1-series, which are normally enriched in the CNS (GM1, GD1a, GT1b, and asioalo-GM1). After 3 days, culture supernatants were collected, and the cells were restimulated with plate-bound anti-CD3/rIL-2 for 3 additional days in the absence of gangliosides. Supernatants collected at the end of the primary (Day 3) and restimulated cultures (Day 6) were assayed for IFN-{gamma} and IL-4 by ELISA. As shown in Figure 1A 1B 1C 1D , addition of gangliosides to the splenocytes during the initial stimulation resulted in a dose-dependent decrease in the levels of IFN-{gamma} and a concomitant increase in levels of IL-4 in primary (Fig. 1A and 1B) and restimulated (Fig. 1C and 1D) supernatants. Asialo-GM1 did not have any effect, suggesting that sialic acid residues in the polar head of gangliosides were necessary for this effect. Indeed, the potency of gangliosides of the 1-series correlated with their sialic acid content (GT1b>GD1a>GM1). Nevertheless, the inhibition of IFN-{gamma} and increase in IL-4 secretion were not exclusive to gangliosides of the 1-series, inasmuch as gangliosides of the 2 (GM2, GD2)- and 3 (GM3, GD3)-series, some of which are found in a variety of malignant cells, showed similar effects (Fig. 1E and 1F) . Consistent with the previous experiment, the sialic acid content of these gangliosides correlated with their potency, and GD2 and GD3 showed greater activity than the respective GM2 and GM3. Although the presence of gangliosides during the initial T cell stimulation was required, their presence during restimulation had no further effects on the levels of IFN-{gamma} and IL-4. Costimulation with anti-CD28 antibodies enhanced the amount of secreted cytokines but did not alter the cytokine profile or the effect of gangliosides.


Figure 1
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  		Figure 1. Effects of gangliosides on the production of IFN-{gamma} (A, C, E) and IL-4 (B, D, F) by anti-CD3-stimulated splenocytes from BALB/c mice. Cells were cultured at a density of 5 x 106 cells/ml in the presence of soluble anti-CD3 antibodies (0.5 µg/ml), rIL-2 (5 ng/ml), and different concentrations of purified gangliosides (0–75 µg/ml). Following a 3-day culture, the supernatants were collected, and the cells were washed and restimulated (in the absence of gangliosides) with plate-bound anti-CD3 antibodies and IL-2 for 3 additional days. Supernatants were also collected at the end of this period. Primary (Day 3, A and B) and restimulated (Day 6, C and D) supernatants were assayed for IFN-{gamma} and IL-4 by ELISA. (E and F) Effect of gangliosides of the 2- and 3-series on the production of IFN-{gamma} (E) and IL-4 (F) by anti-CD3-stimulated splenocytes. Results show the concentrations of IFN-{gamma} and IL-4 on supernatants of restimulated (Day 6) cultures. Means and SD from three different experiments are shown. AGM1, Asialo-GM1.

 
Comparison of the effects on gangliosides on C57Bl/6 and BALB/c splenocytes
To determine whether the effect of gangliosides varied in different mouse strains according to their propensity to mount preferential type-1 or type-2 responses, cultures of C57Bl/6 and BALB/c splenocytes were compared. As expected, the baseline levels of IL-4 in the supernatants of BALB/c mice were higher than those of C57Bl/6 mice; however, the presence of two gangliosides, GT1b and GD3, increased IL-4 and decreased IFN-{gamma} production in both strains (Fig. 2 ).


Figure 2
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  		Figure 2. Comparison of the effects of gangliosides on BALB/c and C57Bl/6 splenocytes. Spleen cell suspensions were prepared from BALB/c (B/c) and C57Bl/6 (C57) mice and cultured in the presence of anti-CD3 antibodies, rIL-2 and GT1b or GD3 gangliosides (50 µg/ml), as described in Figure 1 . Results show the concentrations of IFN-{gamma} (A) and IL-4 (B) on supernatants of primary (3d) and restimulated (6d) cultures. Means and SD from three different experiments are shown. Note different scales for IL-4 levels in BALB/c and C57Bl/6 cultures.

 
Direct effects of gangliosides on T cells
We further investigated whether the effects of gangliosides were mediated by direct action on the T cells or indirectly through actions on accessory cells. T lymphocytes (CD3+) were purified from splenic cell suspensions and compared with whole splenocyte cultures after stimulation with plate-bound anti-CD3 antibodies. As shown in Figure 3A and 3B , the effects of GT1b, particularly the enhancement of IL-4 production, were actually of greater magnitude when performed with cultures of purified T cells rather than with whole splenocytes. Although potential effects of gangliosides on accessory cells cannot be disregarded, these results argued in favor of a direct effect on T lymphocytes. Consistent with such an idea, addition of ganglioside-pretreated, splenic, adherent cells to anti-CD3-stimulated T lymphocytes failed to alter their cytokine secretion pattern (results not shown). Similar experiments carried out using enriched CD4+ and CD8+ T cell cultures demonstrated that gangliosides affect both cell T cell subsets. As shown in Figure 3C , the presence of gangliosides significantly decreased IFN-{gamma} production in cultures of anti-CD3-stimulated CD4+ or CD8+ T cell populations, even when the latter produced approximately four times as much IFN-{gamma} on a per-cell basis. Consistent with the increase in IL-4 production by cultures of splenic cells or enriched T cells, elevated levels of IL-4 were seen in the CD4+ T cell cultures exposed to gangliosides (Fig. 3D) . As expected, little if any IL-4 was detected in cultures of CD8+ T cells.


Figure 3
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  		Figure 3. Comparison of the effects of gangliosides on splenocytes and enriched total T cells, CD4+ T cells, and CD8+ T cells. Spleen cell suspensions were prepared from C57Bl/6 mice and T lymphocytes enriched by passage through mouse T cell enrichment columns as described in Materials and Methods. Whole spleen or enriched T cells were then cultured for 3 days at a cell density of 5 x 106 cells/ml in the presence of plate-bound anti-CD3 antibodies, rIL-2 (5 ng/ml), and different concentrations of ganglioside GT1b (0–75 µg/ml). The cells were then harvested, washed, and restimulated in the absence of gangliosides with anti-CD3 antibodies as described. Graphs show concentrations of IFN-{gamma} (A, C) and IL-4 (B, D) on supernatants of restimulated (6-day) cultures of splenocyte and enriched T cell cultures (A, B), and primary cultures of CD4+ and CD8+ T cells (C, D). Means and SD from three different experiments are shown.

 
Intracellular cytokine staining of lymphocytes after stimulation with anti-CD3 in the presence and absence of gangliosides
To determine whether the ganglioside-induced changes in the supernatant levels of IFN-{gamma} and IL-4 correlated with expression at the cell level, cultures of enriched T cells were stimulated with anti-CD3 (with or without gangliosides) and stained for surface CD4, CD8, or NK1.1 expression and intracellular IFN-{gamma} or IL-4. Stains performed 24 h after stimulation showed an ~2% content of NK1.1+ cells and substantial IFN-{gamma} expression on NK1.1– and NK1.1+ cells. Exposure to GT1b resulted in an ~40% reduction in the numbers of IFN-{gamma}-expressing cells on both populations (Fig. 4A and 4B ). Expression of IL-4, however, was undetectable at this time-point on either population (NK1.1–/NK1.1+; Fig. 4C and 4D ). Stains for CD4/CD8 and cytokines performed 72 h after anti-CD3 stimulation showed IFN-{gamma} expression on CD4+ and CD8+ T cells. Consistent with the ELISA results, exposure to GT1b resulted in reduced numbers of IFN-{gamma}+ cells on both T cell subpopulations (Fig. 4E and 4F) . IL-4 expression in these cultures, although low, was confined to CD4+ cells and was enhanced in the presence of GT1b (from 0.18% to 0.5%, Fig. 4 G and 4H ). As shown in Figure 4I and 4J , the enhancement in the expression of IL-4 by CD4+ cells was more evident when T cells from BALB/c mice were used (as opposed to C57Bl/6 in the previous experiment). Intracellular staining conducted on cultures of enriched CD4+ and CD8+ T cells corroborated these results, showing that exposure to GT1b decreased IFN-{gamma} production in both T cell subpopulations and increased IL-4 production by the CD4+ subset (data not shown).


Figure 4
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  		Figure 4. Intracellular cytokine staining of enriched T cell cultures from C57Bl/6 (A–H) or BALB/c mice (I, J) stimulated with plate-bound anti-CD3 and rIL-2 in the presence of medium alone (left panels) or GT1b (50 µg/ml, right panels). Twenty-four-hour cultures were stained with a FITC-labeled anti-NK1.1 antibody and PE-labeled anti-IFN-{gamma} (A, B) or anti-IL-4 (C, D) antibodies. Seventy-two-hour cultures were stained with a FITC-labeled anti-CD4 antibody and PE-labeled anti-IFN-{gamma} (E, F) or anti-IL-4 (G, H) antibodies. In the case of BALB/c cultures, the antibodies were a PE-labeled anti-CD4 and FITC-labeled anti-IL-4 (I, J).

 
Effects of gangliosides on IFN-{gamma} and IL-4 expression at the mRNA level
To investigate whether the effects of gangliosides on the production of IFN-{gamma} and IL-4 took place at the transcriptional level, semiquantitative RT-PCR and real-time PCR analysis were conducted on cultures of enriched T lymphocytes stimulated with anti-CD3 antibodies in the presence and absence of gangliosides. Both techniques (Fig. 5A and 5B ) demonstrated a reduction in the steady-state levels of IFN-{gamma}-specific and a concomitant increase in IL-4-specific transcripts in ganglioside-exposed T cells compared with control cultures. Although such an effect was already evident as early as 12 h after stimulation (not shown), it became more pronounced upon further ganglioside exposure (24–72 h), reflecting the progressive shift to a type-2 response. Similar results were obtained in BALB/c and C57Bl/6 mice.


Figure 5
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  		Figure 5. Effects of gangliosides on IFN-{gamma} and IL-4 mRNA expression. T cells were enriched from C57Bl/6 spleen cell suspensions and cultured in the presence of plate-bound anti-CD3 antibodies, rIL-2, and different concentrations of ganglioside GT1b (0–75 µg/ml), as described in Figure 1 . After 72 h, the cells were harvested and lysed, and total RNA was isolated. (A) RT-PCR: Ratio of the expression of IFN-{gamma} or IL-4 to that of ß-actin. (B) Real-time PCR: The relative expression of IFN-{gamma} and IL-4 were analyzed using the 2–{Delta}{Delta}Ct method by normalizing with GAPDH expression. Data are represented as mean ± SEM of four different experiments.

 
Role of endogenous cytokines on the modulatory effects of gangliosides
The possibility was investigated that the observed effects of gangliosides were related indirectly to alterations in the activity of endogenous cytokines, particularly in view of previous reports of the inhibition of IFN-{gamma} activity by brain-derived gangliosides [13 ]. To this end, we tested whether the effects of inhibition or supplementation of endogenous IFN-{gamma} or IL-4 affected the cytokine secretion pattern of T cells exposed to gangliosides. Inhibition of endogenous IL-4 activity with anti-IL-4 or anti-IL-4R antibodies could not prevent the inhibitory effect of GT1b on the production of IFN-{gamma} in restimulated cultures, thus suggesting that it was not mediated indirectly by increased endogenous levels of IL-4 (Fig. 6A ). Moreover, anti-IL-10 or anti-transforming growth factor-ß mAb were also unable to reverse the ganglioside-mediated decrease in IFN-{gamma} levels (results not shown), indicating that their effects were also independent of these cytokines. In contrast, supplementation of primary cultures with rIFN-{gamma} partially reduced the extent of the inhibitory effect of GT1b on IFN-{gamma} secretion by restimulated cultures. Blocking of endogenous IL-4 activity, however, completely prevented the production of IL-4 in restimulated cultures, whether in the presence or absence of GT1b, indicating that endogenous IL-4 was essential for the development of type-2 responses and that gangliosides could not substitute for it (Fig. 6B) . Neither the blocking of endogenous IFN-{gamma} nor supplementation of the primary cultures with rIFN-{gamma} had any effect in the ganglioside-mediated enhancement of IL-4, suggesting that such an effect was independent of endogenous IFN-{gamma} activity. Results of these experiments were comparable in BALB/c and C57Bl/6 mice.


Figure 6
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  		Figure 6. Role of endogenous cytokines on the effects of gangliosides. Enriched T lymphocytes from C57Bl/6 mice were cultured for 3 days in the presence of plate-bound anti-CD3 antibodies, rIL-2, and medium (control) or ganglioside GT1b (50 µg/ml) as described in Figure 1 . Antibodies directed against the IL-4R, IL-4, and IFN-{gamma} (5 µg/ml) or rIFN-{gamma} (5 ng/ml) were added to different wells. Medium alone was used as control. The cells were then harvested, washed, and restimulated with anti-CD3 and rIL-2 in the absence of gangliosides, antibodies, or cytokines for an additional 3 days. At Day 6, the supernatants were harvested and assayed for IFN-{gamma} (A) and IL-4 (B) by ELISA. Results depict a representative experiment of three performed. *, Significantly different from medium (P<0.05).

 
Effects of gangliosides on cells from CD1d-deficient mice and potential involvement of CD1 molecules
Taking into account the requirement for endogenous IL-4 in the enhancing effect of gangliosides on type-2 T cell responses and the important role of NKT cells as the initial sources of endogenous IL-4 reported in some models [32 33 ], we investigated the effects of gangliosides on splenic cell cultures derived from CD1d-deficient mice (which also lack NKT cells). As shown in Figure 7A , stimulation with anti-CD3 antibodies in the presence of GT1b resulted in similar decreases in IFN-{gamma} production in CD1d–/–-deficient and control BALB/c and 129S mice, indicating that the inhibitory effects of gangliosides on IFN-{gamma} production were not dependent on the presence of NKT cells and/or endogenous IL-4. However, whereas GT1b enhanced the production of IL-4 in BALB/c and 129S strains, it failed to induce any IL-4 production in the CD1d–/– cultures (Fig. 7B) . These results are consistent with the requirement for endogenous IL-4 indicated by the previous experiment (Fig. 6) and raise the possibility for a potential involvement of CD1d molecules and/or NKT cells in the promoting effects of gangliosides on type-2 T cell responses. Inasmuch as CD1d–/– mice lack expression of CD1 molecules, and these have been reported to play a role in the recognition of glycolipids by NKT cells [33 34 35 36 ], we explored a potential connection between CD1d molecules and the ability of gangliosides to enhance type-2 responses. To this end, the effects of 1B1, an anti-mouse CD1d mAb, were examined. As shown in Figure 7C and D , although addition of the 1B1 antibody to cultures of anti-CD3-stimulated splenocytes did not have any effect on the GT1b-mediated decrease in IFN-{gamma} levels, it partially reversed the GT1b-mediated enhancement of IL-4 production in the cultures, suggesting a potential role for CD1d molecules or CD1d-expressing cells in the potentiation of type-2 responses by gangliosides. Results of these experiments were comparable in BALB/c and C57Bl/6 mice as well.


Figure 7
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  		Figure 7. Effects of gangliosides on cells from CD1d-deficient mice (A, B). Splenocytes were obtained from CD1–/–, BALB/c, and 129S mice and cultured for 3 days in the presence of anti-CD3 antibodies, rIL-2, and medium alone or ganglioside GT1b (50 µg/ml). Culture supernatants were harvested and assayed for IFN-{gamma} (A) and IL-4 (B) by ELISA. Mean and SD from three different experiments are shown. Effect of anti-CD1d antibodies (1B1) on the effect of gangliosides (C, D). Purified T lymphocytes were obtained from C57Bl/6 mice and cultured for 3 days in the presence of anti-CD3 (5 µg/ml), rIL-2, GT1b (0–75 µg/ml), and an anti-1B1 antibody or an isotype-matched control (10 µg/ml). Culture supernatants were harvested and assayed for IFN-{gamma} (C) and IL-4 (D) by ELISA. Results depict a representative experiment of three performed. *, Significantly different from control (P<0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although gangliosides have long been associated with a wide variety of suppressive effects on different cells and aspects of immune responses, their mechanisms of action have not been elucidated clearly. The idea that gangliosides may have important roles as tumor-derived immunosuppressive molecules has been supported by a number of recent observations [11 24 25 26 29 ] and thus, the importance of understanding the bases for their immunoregulatory activities. Based on evidence suggesting that gangliosides may preferentially down-regulate inflammatory and type-1 T cell responses [13 ], we hypothesized that a potential mechanism of action may involve immune deviation in favor of type-2 T cell responses.

Results from our studies are consistent with the above hypothesis and furthermore, suggest a complex scenario, with gangliosides acting at multiple regulatory points. Although the expression and production of IFN-{gamma} were reduced, and that of IL-4 was enhanced by the presence of gangliosides after T cell stimulation, suggesting a potential modulation of the differentiation of Th cells, the effects on the production of these two cytokines appeared to be independent of each other. Moreover, inhibition of IFN-{gamma} production took place, not only on CD4+ T cells but also on CD8+ T cells and NK1.1+ cells, as suggested by intracellular cytokine staining. The inhibitory effect on IFN-{gamma} production was not dependent on endogenous IL-4, as blocking its activity with anti-IL-4 or anti-IL-4R antibodies did not prevent gangliosides from decreasing IFN-{gamma} levels. It appeared, however, that the inhibitory effect on the production of IFN-{gamma} after restimulation of enriched T cells was attenuated partially by supplementation of the primary cultures with rIFN-{gamma}, suggesting that gangliosides may antagonize an IFN-{gamma}-mediated signal or activity. In contrast, the effects of gangliosides on the type-2 pathway were not dependent on alterations in endogenous IFN-{gamma} activity. For example, inhibition of endogenous IFN-{gamma} was unable to mimic the effect of gangliosides on the production of IL-4, and addition of rIFN-{gamma} could not prevent their effects. Rather, the enhancing effects of gangliosides on the type-2 responses were absolutely dependent on endogenous IL-4 production, inasmuch as blocking its activity completely inhibited the generation of IL-4-producing cells, even in the presence of gangliosides.

The dependence on endogenous IL-4 may explain results of the experiments showing that the enhancing effects of gangliosides on type-2 responses did not take place in cells from CD1d-deficient mice. These mice lack NKT cells, which are responsible for the initial production of IL-4 in several systems [32 33 ]. Thus, the absence of NKT cells would have resulted in deficiency of endogenous IL-4 upon initial anti-CD3 stimulation and therefore, in the lack of effect of gangliosides on Th2 differentiation. As for the targets of ganglioside action, several possibilities exist. It is possible that gangliosides may enhance the production of endogenous IL-4 by NKT cells. Although the inability to detect IL-4+ cells among the NK1.1+ cells within 24–72 h of stimulation would argue against this explanation, limitations in the sensitivity of intracellular staining and low numbers of IL-4+ cells, particularly in C57Bl/6 mice, preclude discounting this possibility. Nevertheless, measurements of IL-4 levels in 24 h cultures (after addition of an anti-IL-4R antibody to prevent absorption to cells) indicated that the presence of gangliosides did not increase early endogenous levels of IL-4. Other explanations include a ganglioside-mediated potentiation of the effects of endogenous IL-4 on (CD4+) Th cell precursors, promoting differentiation toward Th2 effector cells, and as distinct apoptotic pathways operate in Th1 versus Th2 cells [37 38 ], gangliosides may also promote selective apoptosis in Th1 cells, resulting in the preferential survival of Th2 cells [29 ]. In this regard, we observed greater apoptosis in cultures of CD8+ T cells exposed to gangliosides as compared with CD4+ T cells, suggesting that gangliosides may also alter the balance between CD4+ and CD8+ T cells, which in turn, may lead to an environment that favors deviation toward type-2 responses.

The reasons for the partial reversal of the enhancement of IL-4 production by anti-CD1d antibodies remain unclear. It is known that CD1 molecules are a family of antigen-presenting molecules, which are capable of presenting nonpeptide lipid and glycolipid antigens, including gangliosides, to NKT and some T cells [35 36 ]. However, the fact that enhanced IL-4 production was only observed upon anti-CD3 stimulation and not with gangliosides alone (results not shown) suggested that most of the IL-4 may actually originate from the anti-CD3-activated T cells.

Besides their relatively high content in the CNS, increased or "abnormal" ganglioside expression often occurs in malignant cells. Several types of tumors are known to express high levels of gangliosides, not usually expressed by normal cells, including GM2, GD2, and GD3. Moreover, some tumors actively synthesize and shed gangliosides into their local environment, resulting in high levels of soluble gangliosides in the serum of tumor-harboring patients [3 9 23 24 25 26 ]. Thus, tumor-derived gangliosides may contribute to the immunosuppression observed in cancer patients. Indeed, evidence suggests that gangliosides may play a role in tumorigenicity and metastasis. For instance, alterations in the expression of gangliosides in small cell lung cancer cells by transfection with GD3-synthase cDNA (thereby increasing GD2 and GD3 expression) resulted in an enhancement of cell proliferation and invasiveness in vitro [39 ]. In a separate study, Deng and Ladisch [16 ] reported that although inhibition of ganglioside expression in a melanoma cell line by means of a glucosylceramide synthase inhibitor did not affect cell proliferation, it did reduce tumorigenicity and pulmonary metastases in mice, thus suggesting that gangliosides may indeed be involved in suppressing anti-tumor immune responses. Whether differences exist in the immunosuppressive potential of normal tissue versus tumor cell-derived gangliosides is not particularly clear. Differences in the carbohydrate portion (on which ganglioside nomenclature is based) may not be the only factor. Ladisch et al. [40 ] reported that differences in the lipophilic moiety determined not only the shedding rate of gangliosides but also immunosuppressive properties, and ceramides contained shorter fatty acids (more abundant in malignant cell sources) and were the most immunosuppressive and more easily shed.

Although our results appear in contrast to those of Kanda and Watanabe [41 ], who reported that gangliosides, like GT1b, enhanced IFN-{gamma} production and suppressed IL-4 production, several differences between the two systems may account for the apparent discrepancies; for example, differences in the cellular models used (e.g., phytohemagglutinin-stimulated human peripheral blood T cells vs. anti-CD3-stimulated murine T cells), culture times (24 h vs. 3–6 days), and the concentration of gangliosides (1–1000 nM vs. 1–50 µM). The higher concentrations used in our studies may be more representative of the high levels of gangliosides found in the tumor microenvironment.

Although the evidence linking gangliosides with immunosuppresive activities is relatively strong, the role of tumor-derived gangliosides in the immunologic dysfunctions of cancer patients has not been established clearly. Results of this study provide evidence for the ability of gangliosides to modulate the phenotype of T cell responses in vitro and thus, suggest that tumor-derived gangliosides may promote similar type-2 effects in the tumor microenvironment. Understanding of the mechanisms involved may lead to the identification of potential strategies to prevent or reverse immune dysfunction in cancer patients.


   ACKNOWLEDGEMENTS
 
We are indebted to Samuel Wellhousen, Ph.D. (J. Graham Brown Cancer Center, University of Louisville), for his help with the fluorescent-activated cell sorter analysis and to Silvia Uriarte, Ph.D. (Department of Medicine, University of Louisville), for her assistance in the design of PCR primers.

Received July 18, 2005; revised October 28, 2005; accepted November 25, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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