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. Author manuscript; available in PMC: 2010 Jun 23.
Published in final edited form as: J Immunol. 2009 Oct 15;183(8):5050–5058. doi: 10.4049/jimmunol.0900259

Elevated Levels of Select Gangliosides In T Cells From Renal Cell Carcinoma Patients Is Associated With T-Cell Dysfunction

Soumika Biswas 1, Kaushik Biswas 1, Amy Richmond 2, Jennifer Ko 1, Sankar Ghosh 1, Matthew Simmons 3, Patricia Rayman 1, Brian Rini 3, Inderbir Gill 4, Charles S Tannenbaum 1, James H Finke 1,3
PMCID: PMC2890308  NIHMSID: NIHMS177731  PMID: 19801523

Abstract

Increased expression of gangliosides by different tumor types including renal cell carcinoma (RCC) is thought to contribute to the immune suppression observed in cancer patients. Here we report an increase in apoptotic T cells from RCC patients compared to T cells from normal donors that coincided with the detection of T cells staining positive for GM2 and that the apoptosis was predominantly observed in the GM2+ but not the GM2− T cell population. Ganglioside shedding from tumor rather than endogenous production accounts for GM2+ T cells since there was no detectable level of mRNA for GM2 synthase in RCC patient T cells and in T cells from normal healthy donors after incubation with either purified GM2 or supernatant from RCC cell lines despite their staining positive for GM2. Moreover, reactive oxygen species as well as activated caspase−3, −8, and −9 were predominantly elevated in GM2+ but not GM2− T cells. Similarly, increased staining for GD2 and GD3 but not GD1a was detected with patient T cells with elevated levels of apoptosis in the GD2+ and GD3+ cells. These findings suggest that GM2, GD2 and GD3 play a significant role in immune dysfunction observed in RCC patient T cells.

Introduction

Immune response are initiated to tumor associated antigens in cancer patients, however, these responses are relatively ineffective as evident from the continued tumor growth and disease progression (1, 2). Tumors have utilized various mechanisms to evade the immune system (1, 3). For example tumor cells can promote immune escape by causing destruction of immune T effector cells (4–7). Indeed a significant percentage of tumor-infiltrating lymphocytes (TILs) were found to be apoptotic in some tumor types, including renal cell carcinoma (RCC).

Tumor cells likely promote apoptosis of T cells in vivo since T lymphocytes undergo the same physiologic changes associated with apoptosis following in vitro culture with RCC cell lines (5, 6, 8). Various mechanisms are proposed by which tumors can induce T-cell apoptosis. Tumors express elevated levels of tumor necrosis factor (TNF)-related ligands (i.e., FasL, TNF-related apoptosis-inducing ligand, and CD70), which can induce T cell apoptosis in a receptor dependent manner (4, 5, 9). Expression of immunosuppressive costimulatory molecule like B7-H1 can induce T cell apoptosis (10) or suppress IFN-γ (Th1) response in T cells (10). T cells can also be suppressed in tumor bearing host by (CD4+CD25hi+Foxp3+) regulatory T cells (Tregs) (11). As a result of tumor-induced changes in myelopoiesis a heterogeneous population of myeloid cells, with suppressive activity are elevated in cancer patients (12). These myeloid cells are reported to inhibit T cell function directly as well as indirectly, via the induction of Treg formation (13, 14).

Overexpression of select gangliosides in different tumor types has been reported (6, 15–17). In addition to promoting tumor growth and metastasis (18), gangliosides produced by tumor cells including GM2 may enhance tumor growth indirectly by suppressing immune cell function. Several reports have suggested inhibition of multiple steps in cellular immune responses by gangliosides, including antigen processing and presentation, T cell proliferation, and production of cytokines, such as IFN-γ and IL4 (6, 19–22). Additional studies have shown that gangliosides derived from either RCC lines as well as from RCC tumor explants, can either sensitize T cells to activation-induced cell death (23) or induce T-cell apoptosis directly (6, 24). While it is recognized that some ganglioside can inhibit in vitro DC function and the production of a type-1 (IFNγ) T cell response it is also clear that some gangliosides can stimulate an immune response. Indeed, endogenous humoral immune responses to different gangliosides have been demonstrated in some cancer patients. Gangliosides such as GM2 have been targets for immunotherapy employing monoclonal antibodies or vaccine to stimulate anti-GM2 antibodies (25, 26). Clinical trials demonstrating induction of anti-GM2 antibodies was associated with better prognosis however the overall response rate was low (27). Thus, under the appropriate conditions select gangliosides can be both immune-stimulatory and immunosuppressive.

The findings presented here suggest that select gangliosides such as GM2, GD2 and GD3 possibly shed from tumors can bind to T cells and promote immune dysfunction. When compared to T cells from normal healthy donors a portion of T cells from RCC patients stained positive for GM2 without expressing detectable levels of the mRNA for the enzyme GM2 synthase. We also report that T cells from RCC patients display a greater level of apoptosis without cell culture than did T cells from healthy donors and that the majority of apoptosis was observed in the GM2+ (GD2+ or GD3+) T cell populations. These in vivo findings in RCC patients could be mimicked by culturing T cells from healthy donors with supernatant from RCC cell lines that contain shed gangliosides. We propose that the shedding of some ganglioside species in cancer patients may bind to and alter T cell viability.

Materials and methods

Reagents

A hamster anti-GM2 antibody (DMF10.167.4) was a gift from Corixa Corporation, Seattle, WA and Dr. Kenneth Rock (University of Massachusetts Medical School, Worcester, MA) (27). Mouse anti-human GM2 antibody (KM696) was also a gift from Kyowa Hakko Kogyo Corporation, Tokyo, Japan. Mouse anti-human GD2 and GD3 antibodies were purchased from BD Pharmingen San Jose, CA. Mouse anti-human GD1a antibody was purchased from Seikagaku Corporation, Tokyo, Japan (28). FITC conjugated rabbit anti-mouse IgM was bought from Zymed, San Fransisco, CA. Alexa Fluor 594 rabbit anti-mouse IgG and Alexa Fluor 488 goat anti-hamster IgG were obtained from Invitrogen, Eugene, Oregon. Bovine brain derived GM2 were purchased from Matreya, Pleasant Gap, PA. Normal anti-armenian hamster IgG and normal anti-mouse IgM were purchased from Santa Cruz Biotechnology, Santa Cruz, CA and BD Pharmingen, San Jose, CA respectively. Vectashield mounting media containing 4′-6′-diamidino-2-phenylindole (DAPI) was purchased from Vector Laboratories, Inc., Burlingame, CA. AnnexinV-PE, 7-AAD and CD3-FITC were obtained from BD Biosciences, San Jose, CA. HPLC grade methanol and analytical grade chloroform, isopropanol, diisopropyl ether and n-butanol were obtained from Fisher Scientific, Fair Lawn, NJ. DiOC6 was purchased from Molecular Probes, Eugene, OR. Complete media (RPMI 1640 Cleveland Clinic Media Core) consist of; 10% FBS (Hyclone, Logan, UT), 2 mM L-glutamine, 50 μg/liter gentamicin, 100 mM MEM sodium pyruvate solution and 10 mM MEM nonessential amino acid solution (Invitrogen, Carlsbad, CA).

Cell lines

RCC cell line SK-RC-26B was obtained from Dr. Neil Bander (The New York Hospital, Cornell University Medical College, New York, NY). The RCC cell line 0827LM was generated at the Cleveland Clinic from a metastatic lesion. These cells were maintained in complete RPMI 1640 (Biowhittaker, Walkerville, MD) medium at 37°C with 5% CO2 and were allowed to reach confluence in 150mm dishes before use in co-culture experiments with peripheral blood T lymphocytes. The normal kidney epithelial (NKE) cell line was established from the uninvolved kidney tissue of a patient with RCC and was immortalized by transfection with the gene for telomerase (Drs. Katerina Gurova and Andrei V. Gudkov, RPMI, Buffalo NY).

Isolation of Tumor Infiltrating Lymphocytes (TILs)

Tissue resected from primary renal cell carcinomas were provided by the Cooperative Human Tissue Network (funded by the National Cancer Institute). Informed consent (approved IRB) was obtained from all RCC patients. TILs (n=16) were prepared after a 2 hour digestion at 37 °C using collagenase type-II (3.5mg/ml; Sigma, St. Louis, MO) and egg white trypsin inhibitor (1mg/ml; Sigma) as already described (5). TILs were washed in RPMI 1640 and prepared for staining by Flow cytometry.

Isolation and storage of peripheral blood mononuclear cells (PBMCs)

Peripheral blood was obtained from normal healthy volunteers (n=35) and from clear cell RCC patients with either localized (n=9) or metastatic (n=22) disease. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque (Amersham Pharmacia Biotech AB, Uppsala, Sweden) gradient as previously described (5, 6) and then either directly stained or frozen at −80°C and then transferred to liquid nitrogen. T cells were purified by negative magnetic selection using microbeads coated with antibodies to remove macrophages, NK cells, B cells and RBCs (Stem Cell Technologies, Vancouver, Canada). The T-cell isolation procedure yielded cells that were more than 97% positive for CD3 as defined by immunocytometry.

Activated T cells maintained in recombinant IL-2 (Proleukin)(Chiron, Emeryville, CA) were used for most experiments since they are most sensitive to ganglioside mediated apoptosis. Moreover, primed T cells are most relevant to study since they are likely the ones responding to tumor cells. To generate primed T cells, peripheral blood mononuclear cells were added at a density of 1 × 106/ml to flasks pre-coated with 10 μg/ml anti-CD3 and 5 μg/ml anti-CD28 antibodies. After 3 days of activation T cells were transferred to flasks and expanded for 8–12 days in the presence of 200 U/ml IL-2 before use.

Immunofluorescent microscopic analysis

GM2 (GD2, GD3, GD1) positive T cells were determined in two ways. In some experiments ganglioside positive cells were detected after culturing T cells from healthy donors with either media alone or with supernatants from RCC cell lines (SK-RC26B or 0827LM). In other experiments, purified CD3+ T cells from normal donors and RCC patients were immunostained to assess expression levels of GM2, GD2, GD3 and GD1a. In both cases 1×105 cells were adhered onto poly-L-Lysine coated slides (Electron Microscopy Sciences, Ft. Washington, PA) at 37°C for 1 hour. Prior to washing with 1X PBS, fixing with 3.7% paraformaldehyde (PFA) and blocking with 1% BSA, T cells were incubated at room temperature (25°C) for 2 hours with respective anti-ganglioside antibodies (1°Ab). Next cells were incubated with the appropriately labeled secondary antibodies. After 3 washes with 1X PBS, the cells were mounted with Vectashield mounting media containing DAPI to visualize the nuclei. A total of 200 cells were counted to asses the percentage of T cells staining positive for a given ganglioside.

Immunocytometric analysis of PBLs and TILs for GM2 expression and apoptosis

PBMCs from normal healthy donors and RCC patients (localized and metastatic) as well as TILs were isolated by methods described earlier. T cells were then analyzed for ganglioside positivity (GM2, GD2, GD3 and GD1a) and levels of apoptosis. 1×106 cells were stained with the hamster anti-human GM2 Ab (DMF10.167.4) antibody for 30min at 4°C. In a smaller subset of patients peripheral blood T cells were stained with mouse anti-human GD2, GD3 and GD1a antibodies (plus appropriate secondary antibodies). Cells were then washed twice with FACs buffer and stained with PE conjugated CD3 for an additional 30min at 4°C. T cells were then washed twice with FACs buffer and fixed with 1%PFA in 1X PBS. For detection of apoptosis in ganglioside positive and negative T cells, lymphocytes were then suspended in 1X annexinV binding buffer and stained with annexinV-PE and 7AAD for 15min at room temperature under constant shaking. 10,000 events were acquired on a FACS Calibur multivariable flow cytometer, and analyzed using CellQuest v3.3 software (BD, San Jose, CA).

mRNA analysis of GM2 synthase by RT-PCR in T cells staining positive for GM2

Total RNA was isolated from RCC line SK-RC26B and T cells using the RNAeasy minikit (Qiagen), following the manufacturer’s protocol. For RT-PCR analysis of GM2-synthase and GAPDH mRNA, the Superscript First-Strand Synthesis System (Ambion) was used to synthesize the respective cDNAs. PCR was conducted using 5′-CAT CGG ATC CTT TGC CGC TGC CTT AGA GCG TTA-3′ as the sense primer and 5′-CGA GCG GCC GCG ACA GCC AGT AGA GTG CTC ACA-3′ as the antisense primer, which gave 1734 basepair fragment of GM2-synthase. GAPDH primers were as follows: 5′-ACCTGGCCAAGGTCATCCAT-5′ (sense) and 5′-TCCACCACCCTGTTGCTGTA-3′ (antisense), giving an expected product of 506 basepair. The reaction was conducted for 35 cycles, using the following parameters 94°C for 30 sec, 58°C for 30 sec and 72°C for 45 sec. Electrophoresis of the samples were done on a 1.5% agarose gel and visualized by ethidium bromide staining.

Analysis of ROS and caspase activation in T cells

T cells were incubated in six well plates at 1×106 cells/ml in presence or absence of SK-RC-26B gangliosides for 18, 48 or 72 hours at 25μg/ml. Another aliquot of cells were also treated with 2μM H2O2 for 18 hours, which was used as a positive control (not shown). At the desired time points, cells were harvested and stained for active caspase- 3, 8 and 9, ROS, AnnexinV/7AAD and GM2. To study activation of caspases, cells were stained with Fluorochrome Inhibitors of Caspases (FLICA), from Immunochemistry Technologies, (LLC., Bloomington, MN, USA) for 1 hour at 37°C protected from light, followed by staining with hamster anti-human GM2 antibody. Induction of ROS was determined by staining the cells with CM-H2DCFDA dye from Invitrogen, (Eugene, Oregon, USA), for 4 hours at 37°C followed by staining for GM2. Apoptosis induction in SK-RC-26B ganglioside treated T cells was studied by surface staining with hamster anti-human GM2 antibody (or anti-GD2 or anti-GD3 antibodies) followed by staining with annexin V-PE and 7AAD.

ELISA assay to test the specificity of different anti-ganglioside antibodies

ELISA was performed to demonstrate the specificity of the hamster anti-human (DMF10.167.4) and mouse anti-human GM2 antibody (KM696) described earlier (28). Bovine brain derived gangliosides coated on a 96 well format flat bottom ELISA plate, were immunostained with 1μg/ml of the mouse anti-human GM2 antibody (KM696) followed by staining with HRP conjugated rabbit anti-mouse IgM (2°Ab).

Ganglioside isolation from the SK-RC-26B cell line

Gangliosides were isolated from tumor cells as described before (6) with minor modifications. Extraction of gangliosides was performed with chloroform-methanol (1:1) for 18h at 4°C followed by partitioning in 10ml Diisopropyl ether/1-Butanol/0.1% aqueous NaCl. The lyophilized, final aqueous phase was passed through a Sephadex G-25 column to remove the salts and small molecular weight impurities. The ganglioside profile was determined by HPLC (Waters) with on-line electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) as previously described (6).

Statistical analysis

p-value was calculated using student’s t-test from the standard error of means (SEM) for unpaired samples, using Microsoft Excel, v2003 software. All the experiments were done at least 3 times.

Results

The ganglioside GM2 that is expressed by many renal cell carcinomas is also detected on a portion of T cells from RCC patients but not those from normal healthy donors

To quantitate the percentage of T cells positive for GM2 staining, PBMCs from normal donors and PBMCs from RCC patients along with TILs were incubated with anti-GM2 and anti-CD3 antibodies followed by analysis using flow cytometry (Fig 1A). A significant percentage of T cells from the peripheral blood of RCC patients but not normal donors stained positive for GM2 (Mean 15% ± 3.51)(Fig 1A). Interestingly, TILs displayed the highest percentage of GM2 positive T cells (Mean 45.9% ± 4.9). Fig. 1B depicts a representative density plot analysis of T cells from normal donor blood, patient blood versus TILs. Similar flow cytometry results were obtained when patient T cells and those from normal donors were stained with a second mouse anti-human GM2 antibody (KM696) that is also specific for GM2. Likewise comparable results were observed when patient and normal donor T cells were stained with anti-GM2 antibody and examined using a fluorescent microscope. The intensity of GM2 staining per cell was measured from images captured with a Leica DMR Fluorescent microscope followed by quantitation using NIH Image J software (version 1.41), which showed that T cells from RCC patients displayed significant high intensity of GM2 specific fluorescence when compared with that of T cells from normal volunteers (Data not shown).

Figure 1.

Figure 1

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Ganglioside GM2 is detected on RCC patient T cells but not T cells isolated from normal donors. T cells were isolated from peripheral blood lymphocytes (PBLs) of patients and normal donors and subsequently stained for GM2 with mouse anti-human GM2 antibody (KM696). The relative expression of GM2 positive T cells was measured by flow cytometry (Fig. 1A & 1B). The percentage of GM2 positive T cells was determined by flow cytometric analysis of PBLs and TILs after staining cells with anti-CD3 and anti-GM2 antibody. As shown in Figure 1A, a significantly higher percentage of T cells (both localized and metastatic) stain positive for GM2 compared to normal donor T cells. Fig. 1B is a representative density plot showing that a higher percentage of T cells from the blood and tumor of RCC patients stain positive for GM2 as compared to normal PBLs. GM2+ T cells in both patient PBLs and TILs displayed significantly high Mean Fluorescence Intensity (MFI) as determined by flow cytometry (Figure 1C). Figure 1D is a representative histogram showing that both patient PBLs and TILs display high MFI by flow cytometry.

The GM2 detected on RCC patient T cells is not attributable to endogenous production but rather binding of exogenous ganglioside

To address the question of whether patient T cells synthesize and express GM2, total RNA was isolated from the RCC cell line SK-RC26B as a positive control and from three metastatic RCC patient T cells using RNAeasy minikit (Qiagen). Electrophoresis of the cDNA following RT-PCR reaction demonstrates no detectable message for GM2 synthase mRNA in any of the 3 metastatic samples. However, mRNA from SK-RC26B yielded a positive band with the appropriate base pair PCR product, indicating detectable GM2 synthase message (Fig. 2A). These findings suggest that patient T cells lack detectable expression of the message required for synthesizing the GM2 synthase enzyme. In the same experiments where mRNA was isolated immunofluorescent staining with mouse anti-human GM2 antibody (KM696) demonstrated T cells from all 3 metastatic patients cells stained positive for GM2, although to varying degrees, as did the SK-RC-26B cell line (Fig 2B). The inability to detect GM2 synthase mRNA in patient T cells does not appear to be attributable to the insensitivity of the RT-PCR assay. This was addressed by determining the number of GM2+ SK-RC-26B cells (5.6, 2.8, 1.4, 0.7 and 0.35 × 105 cells) that is required to detect GM2 synthase when these tumor cells are mixed with NKE cells (2×106) which do not express GM2 or GM2 synthase (Fig 2C). As seen in Figure 2C GM2 synthase mRNA could be detected even when the GM2+ RCC line represented only 3% (0.7×105) of the total cell population. However, when RNA was isolated from patient T cells (5×106) no GM2 synthase mRNA was detected even though 38% and 24% of T cells from 2 patients respectively, stained positive using anti-GM2 antibody (Fig 2B).

Figure 2. GM2 synthase message is absent in RCC metastatic patient T cells, though they stain positive for GM2.

Figure 2

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mRNA was isolated from metastatic patient T cells and also from the cell line SK-RC26B by methods described earlier. RT-PCR was done to detect the levels of GM2 synthase mRNA. GAPDH was used as the housekeeping gene (Fig. 2A). Both SK-RC-26B cells and RCC patient T cells were subsequently stained with mouse anti-human GM2 antibody to detect the presence of GM2 by fluorescent microscopy (Fig. 2B). NKE (normal kidney epithelial) cells were mixed with varying percentage (14% – 1.6%) of the RCC cell line, SK-RC-26B. The percentage of GM2+ SK-RC-26B cells (as calculated from flow cytometry analysis, data not shown) is shown in Figure 2C. mRNA was isolated from the NKE/SK-RC-26B cell mixture using Qiagen RNeasy kit as described earlier. RT PCR was done to detect levels of GM2 synthase mRNA. β-actin was used as the endogenous control. Data demonstrates that GM2 synthase message can be detected from a population containing as low as 3% GM2 positive cells (Fig. 2C).

To test whether exogenous GM2, possibly from tumor cells is binding to T lymphocytes, CD3/CD28 activated T cells were co-cultured with purified bovine brain derived GM2 (80μg/ml) for varying time points (24–96 hours) in the presence of IL2 as shown in Fig. 3. T cells were then immunostained with mouse anti-human GM2 antibodies (KM696 and DMF10.163.4) to detect GM2. SK-RC26B cells were also immunostained for GM2 in parallel as a positive control (Fig. 3A). A time dependent increase in GM2 positivity of the T cells after incubation with purified bovine brain derived GM2 (Fig. 3A) was observed. RT-PCR analysis of the RNA isolated from these T cells shows no detectable message for GM2 synthase in comparison to that from the SK-RC26B line (Fig. 3B). We also tested whether T cells from RCC patients and healthy donors when incubated with purified GM2 would have similar ability to bind GM2. Using T cells isolated from two RCC patients and two healthy donors we did not observe an appreciable difference in the percentage of GM2 staining cells (data not shown).

Figure 3. Peripheral blood T cells from healthy donors do not express the message for GM2 synthase, though they stain positively for GM2 when incubated with either purified bovine brain derived GM2 or with supernatants derived from RCC cell lines.

Figure 3

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CD3/CD28 activated T cells were cultured with 100μgs of purified bovine brain derived GM2 for 24–96 hrs. Cells were stained with mouse anti-human GM2 (KM696) antibody for immunofluorescent detection of GM2 by microscopy (Fig. 3A), and mRNA was isolated from these T cells to assess GM2 synthase mRNA by RT-PCR (Fig. 3B). Results indicate detectable GM2 staining by microscopy (Fig. 3A), though no GM2 synthase message was found to be present in the T cells treated with GM2 (Fig. 3B). Similarly, T cells which were cultured with conditioned media from RCC cell lines SK-RC-26B and 0827LM show significant GM2 staining (as indicated by green fluorescence) by microscopy (Fig. 3C), no detectable GM2 synthase was found in RNA isolated from the tumor conditioned media treated T cells (Fig. 3D).

Additionally, immunofluorescent staining using the mouse anti-human GM2 antibody (KM696) demonstrates significant levels of GM2 positive T cells (Fig. 3C) after culture with supernatants from either 0827LM (24%) or SK-RC26B (33%) cell lines when compared to media control T cells (0%), indicating transfer of GM2 from RCC cell line derived supernatants. Data from RT-PCR analysis shows no detectable message for GM2 synthase in T cells treated with/without RCC supernatants (Fig. 3D) confirming, that GM2 detected on the T cells is transferred from conditioned RCC supernatants during co-culture.

Gangliosides shed from the SK-RC-26B cell line induce ROS, activate caspases and apoptosis predominantly in the GM2+ T cell population

Previously we had reported that RCC lines express gangliosides including GM2 and that co-culture of T cells with the RCC lines, like SK-RC-26B, induced lymphocyte apoptosis after 48–72 hours (6). However, apoptosis could be reduced in these co-cultures by 50% when anti-GM2 antibody was added, suggesting that GM2 was one ganglioside capable of promoting T cell apopoptosis. Here we determined whether gangliosides (15μg/ml) isolated from SK-RC-26B tumor cells would induce in a time dependent manner T cell death via a mechanism that involves ROS formation, caspase activation resulting in annexinV and 7AAD staining. As can be seen in Fig 4A (top left panel) incubation of T cells with tumor derived ganglioside induced ROS formation that doubled over background within 18 hours and that increased thereafter for 72 hours. SK-RC-26B gangliosides also induced activation of caspases-3, −8 and −9 that was detectable at 18 hrs and reached maximum activity at 48 hours (top right panel Fig 4A). By double staining T cells with anti-GM2 antibody plus Fluorochrome Inhibitors of Caspases to detect active caspases we noted that the majority of the caspase activity was present in GM2+ T cells rather than the GM2-T cells (Fig 4B, bottom left panel). Similar results were observed when T cells were assessed for apoptosis following staining with annexinV, 7AAD and GM2 antibody. When T cells were cultured with SK-RC-26B gangliosides, apoptosis increased to 49% compared to T cells cultured in media alone (17%). Analysis of annexinV/7AAD staining in both the GM2+ and GM2− T cells revealed that a high percentage of GM2+ T cells (69%) were apoptotic compared to the GM2− T cells (30%, Fig 4B, bottom right panel). Similar results were observed in two additional experiments. These findings are consistent with the idea that gangliosides shed from the tumor microenvironment can bind to T cells and promote apoptosis.

Figure 4. Gangliosides shed from the SK-RC-26B cell line induce ROS, activate caspases and apoptosis predominantly in the GM2+ T cell population.

Figure 4

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T cells were incubated with 15μg/ml of SK-RC-26B gangliosides for 18, 48 and 72 hrs and stained with H2DCFDA (Fig. 4A, left panel), a dye that stains for ROS induction and Fluorochrome Inhibitors for Caspases 3, 8 and 9 (FLICA) (Fig. 4A, right panel). Results indicate induction of ROS in T cells treated with SK-RC-26B gangliosides, as indicated by the increased MFI of H2DCFDA, which doubled in 18hrs over background and then increased thereafter till 72hrs (Fig. 4A, top left panel). Caspases 3, 8 and 9 were also increased at 18hrs, which reached maximum activity at 48hrs (Fig. 4B, top right panel). Double staining of the T cells treated with SK-RC-26B gangliosides with anti-GM2 antibody and FLICA demonstrate that caspase activity was predominantly present in GM2+ T cells over GM2− T cells (Fig. 4B, bottom left panel). Similar results were observed when T cells were assessed for apoptosis following triple staining of the cells with anti-GM2 Ab, annexinV and 7AAD, showing increased apoptosis mainly in the GM2+ T cells population rather than the GM2− T cells (Fig. 4B, bottom right panel).

GM2 positive T cells from RCC patients display heightened level of apoptosis

In order to determine whether exogenous GM2 bound to patient T-cells in vivo is associated with T cell death, PBMCs isolated from the peripheral blood of RCC patients and normal donors along with TILs were immunostained for CD3, GM2, annexinV, and 7AAD. Flow cytometric analysis revealed that peripheral blood T cells from RCC patients demonstrated a higher level of apoptosis (Mean 21% ± 3.02) when compared to that of normal donor T cells (Mean 5% ± 0.83) (Fig. 5A). Interestingly, TILs demonstrate an even more heightened level of apoptosis as indicated by the percentage of CD3+/annexin V+/7AAD+ lymphocytes (Mean 53% ± 4.9) (Fig. 5A). Density plots from representative experiments illustrate the increased level of apoptosis in T cells derived from TIL and patients peripheral blood (Fig 5B). We also noted that the percentage of apoptotic T cells in the peripheral blood of patients was significantly higher in the GM2+ (Mean 61% ± 5.9) than the GM2− (Mean 16% ± 2.4) population (Fig. 5C). A similar trend was observed for TILs. A significantly higher percentage of GM2+ TILs (Mean 77% ± 4.6) were apoptotic compared to GM2− TILs (Mean 36% ± 6.3)(Fig 5C). However, a significant level of apoptosis was found in the GM2− TILs suggesting that other tumor-derived cells or products play a role in T cell death within the tumor microenvironment. Representative density plots showing apoptosis of GM2+ and GM2− T cells (from peripheral blood and tumor) are shown in Fig 5D.

Figure 5. GM2+ T cells from RCC patients display heightened level of apoptosis.

Figure 5

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PBMCs isolated from RCC patients and normal donors along with TILs were immunostained for CD3, GM2, annexinV and 7AAD (Fig. 5). Fig. 5A shows that peripheral blood T cells and TILs demonstrate heightened level of apoptosis, as indicated by annexinV+/7AAD+ T cell population when compared to normal donor T cells. Fig. 5B represents density plots from a single experiment illustrating increased levels of apoptosis in T cells derived from TILs and patient peripheral blood. Analysis of the GM2+ vs GM2− T cell populations demonstrate significantly more GM2+ apoptotic T cells as indicated by annexinV+/7AAD+ T cells compared to the GM2− counterpart (Fig. 5C). Representative density plots showing apoptosis in GM2+ and GM2− T cells are shown in Fig. 5D.

Other gangliosides beside GM2 promote T cell apoptosis in RCC patients

We determined whether other gangliosides might also be increased in expression on RCC patient T cells compared to T cells isolated from healthy donors. As seen in Figure 6A approximately 50% of T cells from normal donors stained positive for GD1a and the percentage of GD1a+ T cells was not significantly different when compared to the staining of RCC patient T cells. However, RCC patient T cells did show a dramatic increase in the levels of both GD2+ (n=10) and GD3+ T cells (n=14) (Fig. 6A) relative to normal donor T cells (n=16). Very similar data was obtained with immunofluorescence staining of T cells attached to poly-L-lysine slides and counting 200 cells to assess the percentage of ganglioside positive cells (Fig 6B).

Figure 6. Gangliosides other than GM2 also promote T cell apoptosis in RCC patients.

Figure 6

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RCC patient and normal donor T cells were stained with mouse anti-human GD1a, GD2 and GD3 antibodies followed by FACs analysis to measure expression of different gangliosides on T cells (Fig. 6A). Results indicate that high levels of GD1a expression in both patient and normal T cells, however, RCC patient T cells show a dramatic increase in the levels of both GD2+ and GD3+ T cells relative to normal donor T cells. Similar data obtained by immunofluorescence microscopy confirms the results obtained by flow cytometry, illustrating comparable expression of GD1a (Fig. 6B, bottom left panel) but significantly increased levels of GD2 and GD3 (Fig. 6B, bottom right panel) in RCC patient T cells when compared to normal donor T cells.

To assess whether GD2 and GD3 positivity in T cells of RCC patients is correlated with apoptosis, four color staining (CD3, GD2 or GD3, annexinV and 7AAD) was performed (n=11). Gating within the CD3+ population for GD2+ and GD2− cells revealed that GD2+ T cells are significantly more apoptotic (Mean 57% ± 16.2 ) than GD2− (Mean 16% ± 3.9) cells (Fig. 7A). Likewise, significantly higher percentage of GD3+ T cells (Mean 58% ± 15.1) stained positive for annexinV/7AAD relative to the GD3− cell population (Mean 19% ± 5.7 ) (Fig. 7B). These findings suggest that GM2, GD2 and GD3 but not GD1a can contribute to T cell apoptosis observed in RCC patients.

Figure 7. GD2 and GD3 positive T cells from RCC patient T cells display heightened level of apoptosis.

Figure 7

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Four color flow cytometry analysis was performed with RCC patient T cells for CD3, GD2, GD3, annexinV and 7AAD to find out the levels of T cell apoptosis in respective ganglioside positive populations (Fig. 7). Fig. 7A demonstrates increased levels of T cell apoptosis in GD2+ cells relative to the GD2− cells as indicated by the increased number of annexinV+/7AAD+ T cells. Similar results were obtained with the GD3+ vs GD3− population (Fig. 7B), indicating that CD3+/GD3+ T cells stained significantly more positive for annexinV/7AAD when compared to CD3+/GD3− cells.

Discussion

Over expression of various gangliosides has been reported in different histological tumor types including RCC (15–17, 29, 30), likely resulting from modifications in the expression of key enzymes regulating the rate determining step of ganglioside biosynthesis (30, 31). Tumor-derived gangliosides have been shown to inhibit development of anti-tumor immune responses in vivo in several different murine models (32). Additionally, studies in vitro with bovine brain derived GM2 demonstrated suppressed dendritic cell function (33) and in a separate study, GM2 induced apoptosis of a cytotoxic T cell line (34). The first report suggesting the immunosuppressive role of tumor-derived GM2 in RCC patients came from our lab, which showed that GM2 present in RCC tissues and cell lines not only induced T cell apoptosis in vitro, but also suppressed stimulus-induced production of IFN-γ in T cells at lower concentrations (100ng/ml) (6). Here we provide in vivo data indicating the immunosuppressive role of this ganglioside in tumor bearing host. Our findings suggest that GM2 which is expressed on many RCCs, can be shed from the tumor with subsequent binding to patient T lymphocytes resulting in apoptosis.

Immunostaining of human tissue with a monoclonal antibody to GM2 (KM696) (35) demonstrated that a wide variety of epithelial malignancies express this ganglioside. GM2 is also expressed on normal epithelial tissue at secretary borders as well as the brain but is not expressed on normal connective tissue or immune tissue such as spleen and lymph nodes (35). Here we confirm that GM2 is not detected on T cells from the peripheral blood of healthy donors. However, our findings did show that a significant percentage of peripheral blood T cells from RCC patients (Mean 15% ± 3.5) stained positive for GM2 and the percentage was even greater when staining tumor infiltrating T cells (Mean 46% ± 4.98)(Figure 1). However, the GM2 positive T cells present in the peripheral blood of RCC patients did not appear to synthesize their own GM2 since RT-PCR analysis failed to detect message for GM2 synthase, the key enzyme that regulates GM2 synthesis, even though mRNA for this enzyme was readily detected in the SK-RC-26B cell line (Fig. 2A).

The presence of GM2+ T cells from RCC patients in the absence of any detectable levels of mRNA for GM2 synthase leaves open the possibility that GM2 was shed from patient tumors and then taken up by T cells as visualized by immunostaining. This hypothesis is supported by in vitro studies showing that when T cells were incubated with either purified bovine brain derived GM2 (Fig. 3A) or with conditioned supernatants from SK-RC26B or 0827 LM cell lines (Fig. 3C) a significant number of T cells that were initially negative for GM2 stained positive for GM2 starting at 24 hours after initiation of cultures. Moreover, in both the experiments, the message for GM2 synthase was absent from the lymphocytes thereby confirming the notion that GM2 detected on the T cells is likely shed from RCC tumors (Fig 3B and D). As shown in Fig 3A the vast majority of the SK-RC-26B cells (and 0827LM, not shown) stain positive for GM2 and additional studies have shown that the supernatants obtained from 4 day cultures of the RCC lines (Sk-RC-26B and 0827LM) contained GM2 as detected by HPLC-mass spectrometry (data not shown).

Several groups have earlier reported significantly higher levels of gangliosides in the plasma and serum of patients with different cancers (16, 36). Therefore it is possible that GM2 is elevated in the serum of RCC patients which could then bind to T cells. We are currently testing whether serum from RCC pts can transfer GM2 to peripheral blood T cells. We have recently found that exogenous purified bovine brain derived GD3 and RCC-derived gangliosides are most readily internalized in activated T cells when compare to naïve resting T lymphocytes (Sa G. et al Cancer Research in press). These findings may explain why not all of the T cells stain positive for GM2 in either the peripheral blood or tumor of RCC patients.

Prior work from our laboratory previously showed that GM2 expressing RCC cell lines along with tumor supernatants induced apoptosis in peripheral blood T cells from healthy donors and that addition of neutralizing antibody partially blocked apoptosis by greater than 50% (6). Here we show that incubating T cells from normal donor with purified RCC cell derived gangliosides can directly induce ROS formation, caspase activation and the induction of apoptosis. As seen in Fig 4 the majority of the apoptosis was confined to the GM2+ stained T cells and not the GM2− population. Data presented here also shows that without cell culturing, patient T cells freshly isolated from the blood or tumor demonstrated a higher levels of apoptosis relative to T cells from healthy donors. Our finding in RCC patients are consistent with those of others that reported increased apoptotic activity of T cells from patients with squameous cell carcinoma of the head and neck as well as gliomas (37). Furthermore, analysis of our four color flow cytometry data revealed that a significantly higher percentage of the GM2+ T were apoptotic compared to the GM2− population (Fig. 5B) thereby implying GM2’s potential role in T cell apoptosis observed in RCC patients.

Our studies also identified two additional gangliosides that may be possible mediators of T cell apoptosis observed in RCC patients. Both immunofluorescent staining and flow analysis showed that a higher percentage of RCC patient T cells stain positive for GD3 and GD2 when compared to T cells from normal donors (Fig. 6). Moreover GD3 is reported to induce apoptosis in various cell types (38). Interestingly enhanced expression of GD3 has been observed in different tumors in both human and animal models (39). Likewise GD2 is expressed by different tumor types and has been implicated in immune dysfunction (33). Four color analysis of our flow cytometry data demonstrates much higher percentage of apoptotic T cells in the CD3+/GD2+ as well as CD3+/GD3+ populations relative to that of CD3+/GD2− and CD3+/GD3− cells (Fig. 7), clearly indicating that GD2 and GD3 expression on patient T cells is associated with T cell death. Overall, our findings suggest that binding of tumor derived gangliosides to T cells resulting in apoptosis, represents a means by which tumor cells may escape the immune system. Thus, targeting gangliosides for immune therapy in an attempt to reverse immune suppression in cancer, combined with dendritic cell based vaccines pulsed with tumor derived peptides may be a viable therapeutic approach.

Acknowledgments

This work was supported by; NIH grants RO1-CA56937 (JHF), CA116255 (JHF), CA111917 (CST) and the Frank Rudy Fund for Cancer Research.

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