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Clinical Cancer Research Vol. 10, 6360S-6366S, September 15, 2004
© 2004 American Association for Cancer Research

Proceedings of the First International Conference

Effect of Renal Cell Carcinomas on the Development of Type 1 T-Cell Responses

Patricia Rayman1, Amy K. Wesa4, Amy L. Richmond1, Tanya Das5, Kaushik Biswas1, Gira Raval1, Walter J. Storkus4, Charles Tannenbaum1, Andrew Novick2, Ronald Bukowski3 and James Finke1,2,3

1 Department of Immunology in the Lerner Research Institute, 2 The Glickman Urological Institute, and 3 Experimental Therapeutics, The Cleveland Clinic Foundation, Cleveland, Ohio; 4 University of Pittsburgh School of Medicine, Hillman Cancer Center, Pittsburgh, Pennsylvania; and 5 Bose Institute Kolkata, India


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 OPEN DISCUSSION
 REFERENCES
 
Purpose: We reported that in renal cell carcinoma patients with active disease, T-cell reactions to the tumor-associated antigens MAGE-6 and EphA2 are highly skewed toward TH2-type cytokine responses [interleukin (IL) 5]. Herein, we determined whether tumor-derived products, including gangliosides isolated from renal cell carcinoma patients, participate in the down-regulation of type 1 T-cell responses.

Experimental Design: T cells from healthy volunteers or renal cell carcinoma patients were cultured in the presence and absence of supernatants derived from renal cell carcinoma explants or with gangliosides isolated from those tumor supernatants. T cells were stimulated or not with either autologous dendritic cells pulsed with superantigen (Staphylococcus enterotoxin B) or with phorbol 12-myristate 13-acetate and ionomycin and then were assessed for type 1 or type 2 responses (cytokine production and gene expression) and apoptosis.

Results: Tumor supernatants efficiently inhibited the TH1-type responses [interferon (IFN) {gamma}] of T cells stimulated with either S. enterotoxin B or phorbol 12-myristate 13-acetate and ionomycin but had no inhibitory effect on activated T-cell production of type 2 cytokines (IL-4, IL-5, and IL-10). Likewise, IFN-{gamma} mRNA and protein production were inhibited when T cells were cocultured with either renal cell carcinoma supernatant-derived gangliosides or a commercial source of purified GD1a. It was also determined that gangliosides impair type 1 responses by inducing apoptosis of activated T cells.

Conclusions: We propose that renal cell carcinoma-derived tumor products such as gangliosides can induce a type 2 bias in antitumor immunity by initiating apoptosis in the IFN-{gamma}-producing type 1 effector cells. This represents a relevant mechanism by which renal cell carcinoma can inhibit protective antitumor immunity.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 OPEN DISCUSSION
 REFERENCES
 
A number of studies indicate that a type 1 response (TH1/TC1) plays a critical role in the rejection of tumors (1, 2, 3, 4) . TH1-type CD4+ T cells secrete interferon (IFN) {gamma} and interleukin (IL) 2 that promote cellular immunity, in part by providing helper signals for the cytotoxic CD8+ T lymphocytes that also have the capacity to produce IFN-{gamma} in response to antigen (1, 2, 3, 4) . TH2-type cells produce IL-4 and IL-5 and typically promote a humoral immune response, whereas TH3/T-reg cells produce immunosuppressive cytokines (IL-10 and transforming growth factor ß) that can dampen both TH1- and TH2-type immune responses (5, 6, 7, 8) .

Several studies have examined the issue of type 1 versus type 2 polarization in renal cell carcinoma patients. Although one report suggests that a type 1 response predominates in renal cell carcinoma, most studies indicate that the cytokine profile observed is most consistent with a type 2 bias in situ (9, 10, 11, 12, 13, 14) . These studies mainly examined the cytokine profile of the infiltrating mononuclear cells, and none of the studies determined the type 1 and type 2 bias of T cells responding to tumor-associated antigens expressed on renal cell carcinoma.

To better define the polarization status of CD4+ T cells and CD8+ T cells, we examined the immune response to the renal cell carcinoma-associated antigens MAGE-6 and EphA2 using an enzyme-linked immunospot assay and MHC-peptide tetramers (15, 16, 17) . MAGE-6, an antigen broadly expressed on different tumor types, is detected in >30% of renal cell carcinomas. Likewise, the tyrosine kinase receptor EphA2, an antigen that is overexpressed by a wide array of human tumor types, is also present in a high percentage of renal cell carcinomas (90%; ref. 18 ). As we reported previously, peripheral blood CD4+ T cells derived from HLA-DR4+ patients with active disease (stage III/IV) demonstrated an impaired TH1-type response. MAGE-6–specific IFN-{gamma}-producing CD4+ T cells were rarely detectable in the periphery, whereas IL-5 responses against that same antigen were frequently and readily apparent (15 , 16) . The CD4+ T-cell response to EphA2 epitopes was likewise skewed toward TH2-type reactivity in patients with more advanced disease (17) . Interestingly, in patients where the tumor had been removed and there was no evidence of remaining disease, the cytokine response was predominately type 1 (IFN-{gamma}), suggesting that the presence of tumor was responsible for the induction of a type 2 response (IL-5) in patients with active disease (15, 16, 17) . Additional findings now suggest that the type 2 bias detected in renal cell carcinoma patients with active disease may be attributable to an increased sensitivity of the MAGE-6 and EphA2 CD4+ T cells to apoptosis. This notion was supported by the use of HLA-DR4 tetramers associated with MAGE-6 or EphA2 peptides (in collaboration with William Kwok, Benaroya Research Institute, Seattle, WA). Detectable binding of T cells to HLA-DR4-tumor peptide tetramers was noted in the peripheral blood of renal cell carcinoma patients, although the frequency was less than that for CD4+ T cells binding to a viral peptide (FluM1; ref. 18 ). However, double staining with MHC tetramers and Annexin V demonstrated that a high percentage of the CD4+ T-cell population displayed early signs of apoptosis. This was not true when evaluating influenza-specific T cells, suggesting that tumor-specific T cells were selectively undergoing apoptosis (18) .

The finding that type 2 responses are characteristic of renal cell carcinoma patients with active disease, whereas type 1 responses are associated with disease-free status, indicates that the tumor environment may play a role in regulating the polarization of a T-cell response in those cancer patients (15, 16, 17) . Data presented here suggest that tumor supernatants from renal cell carcinoma explants can similarly skew T cells from even healthy volunteers toward TH2 responsiveness, thus mimicking the phenotype observed in patients with active disease. Additional experiments suggest that gangliosides present in the tumor supernatant participate in the skewing to a type 2 response via an apoptotic mechanism.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 OPEN DISCUSSION
 REFERENCES
 
T-Cell Isolation.
T cells were isolated from peripheral blood first with a Ficoll-Paque gradient (Amersham Pharmacia Biotech AB, Uppsala, Sweden), followed by negative selection using StemSep antibody mixture (StemCell Technologies, Vancouver, British Columbia, Canada). Blood and renal cell carcinoma tissue from patients as well as blood from normal volunteers was obtained through Institutional Review Board-approved protocols (CCF IRB4639 and 1382, and University of Pittsburgh IRB 020429)

Renal Cell Carcinoma Supernatants and Gangliosides Isolation.
Renal cell carcinoma supernatant extracts were produced by cutting renal cell carcinoma tumor tissue into 3 x 3-mm pieces, washing off blood overnight, and then placing the pieces in RPMI 1640 (15 mL/g of tissue) for 3 or 4 days at 37°C in 5% carbon dioxide and 95% air. The supernatants were then removed, passed through a Nitex mess filter, centrifuged at 3000 rpm for 30 minutes, filtered through a 0.22-µm membrane; and stored at –20°C. Ganglioside extracts were isolated using a solvent partition method developed by McKallip et al. (19)

TH1 and TH2 Cytokine Determination.
Isolated T cells were cultured with renal cell carcinoma supernatants or gangliosides for 48 hours with stimulation for 24 hours using phorbol 12-myristate 13-acetate (PMA; 10 ng/mL, Sigma, St. Louis, MO) and ionomycin (0.75 ng/mL, Sigma). Trypan blue exclusion and the terminal deoxynucleotide transferase-mediated nick end labeling (TUNEL) assay were used for determination of cell death. The T-cell supernatants were collected and tested for human IFN-{gamma}, IL-5, and IL-10 using ELISA assay (Pierce Biotechnology, Rockford, IL). These experiments were also set up so that total RNA was extracted from the T cells, and real-time PCR was performed as described previously (20) using 5.0 µg of total RNA and the Taqman Reverse Transcription Reagents (Applied Biosystems, Foster City, CA). cDNA was used in the subsequent PCR reaction using SYBR green PCR core reagents (Applied Biosystems) with primers for IFN-{gamma}, IL-5, and ß-glucuronidase (ß-GUS). The cycle threshold values obtained were then used to calculate the fold differences of cytokine expression (IFN{gamma} and IL-5) relative to the expression of the housekeeping gene (ß-GUS).

In vitro Stimulation and Intracellular Cytokine Detection.
Peripheral blood was isolated from renal cell carcinoma patients, with dendritic cells (DCs) subsequently generated from adherent mononuclear cells in granulocyte-macrophage colony-stimulating factor (Amgen, Thousand Oaks, CA) and IL-4 as described previously (18) and then matured in the presence of IFN-{gamma} (Endogen, Rockford, IL) on day 5 and lipopolysaccharide (Sigma) on day 6. Day 7 DCs were pulsed with Staphylococcus enterotoxin B (1 ng/mL) for 1 to 2 hours at room temperature and washed and cultured with autologous CD4+ T cells isolated via StemSep negative selection from previously frozen nonadherent mononuclear cells. DCs and T cells were cocultured in complete medium (RPMI plus 10% fetal bovine serum; Invitrogen, Carlsbad, CA) for 7 days in the presence or absence of various concentrations of renal cell carcinoma tumor supernatant. On day 7, cells were harvested, counted using trypan blue exclusion of dead cells, and restimulated with PMA (10 ng/mL, Sigma) and ionomycin (5 ng/mL, Sigma) in the presence of brefeldin A (10 µg/mL, Sigma) in 96-well V-bottomed plates for 18 hours. Unstimulated cells cultured in the presence of brefeldin A were used as a control. Cells were then fixed in 4% paraformaldehyde and washed with 0.1% saponin in FACScan buffer (0.2% bovine serum albumin and 0.02% NaN3 in PBS). After blocking with 1 mg/mL of human IgG (Sigma), cells were incubated with anti-IL-4 phycoerythrin-conjugated monoclonal antibody and anti-IFN-{gamma} fluorescein isothiocyanate-conjugated monoclonal antibody (BD-PharMingen, San Diego, CA) or isotype control monoclonal antibodies. Cells were resuspended in FACScan buffer and used for flow cytometry (Epics-XL, Beckman Coulter, Fullerton, CA), and data were analyzed with WinMDI software (The Scripps Research Institute, La Jolla, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 OPEN DISCUSSION
 REFERENCES
 
Supernatants from Renal Cell Carcinoma Explants Inhibit Type 1 T-Cell Responses.
To determine the effects of renal cell carcinoma supernatants on the autologous DC-mediated induction of TH1 responses, CD4+ T cells from renal cell carcinoma patients were cultured with S. enterotoxin B-pulsed DCs in the presence or absence of various concentrations (0% to 25% v/v) of the renal cell carcinoma supernatants. On day 7, the resulting CD4+ T cells were restimulated with PMA and ionomycin, and the TH cytokine profiles were assessed by intracellular cytokine flow cytometry. As shown in Fig. 1Citation , the presence of renal cell carcinoma supernatant at concentrations exceeding 5% (v/v) reduced the absolute numbers of CD4+ T cells producing the TH1-type cytokine IFN-{gamma} but had little or no effect on the absolute number of CD4+ responder T cells producing the TH2-type cytokine IL-4. Thus, the supernatants from renal cell carcinoma explants appear to preferentially suppress the induction of type 1 CD4+ T-cell responses (versus type 2) against antigen-loaded DCs.



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  		Fig. 1. Renal cell carcinoma (RCC) supernatants preferentially suppress type 1 CD4+ T-cell responses against antigen-loaded DCs in vitro. CD4+ T cells isolated from the blood of 2 RCC patients were primed with superantigen S. enterotoxin B-loaded autologous DCs. After 7 days, T cells were stimulated with PMA and ionomycin. The frequency of cytokine-producing cells was measured by intracellular cytokine staining monitored by flow cytometry. The absolute numbers of CD4+ T cells producing cytokines (IFN-{gamma} or IL-4) was determined. The tumor supernatants inhibited a TH1-type response but had a minimal effect on TH2-type responses. Note that the TH2-type response of one of the patients T cells was low even in the absence of RCC supernatant.

 
The inhibition of a type 1 response by renal cell carcinoma supernatants was also observed with peripheral blood T cells, containing a mixture of CD4+ and CD8+ cells, suggesting that both TH1 and TC1 are affected. As seen in Fig. 2Citation , supernatants derived from multiple renal cell carcinoma explants (clear cell tumors) all inhibited IFN-{gamma} production in response to PMA and ionomycin. Although not shown, we have also found that the renal cell carcinoma supernatants similarly inhibit the stimulus-dependent production of IL-2 by T lymphocytes. Interestingly, these supernatants did not inhibit the production of the suppressive cytokine, IL-10, by the activated T cells but rather caused a modest increase in IL-10 expression as determined by ELISA.



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  		Fig. 2. Supernatants of renal cell carcinoma (RCC) explants are capable of producing a type 2 bias in normal T cells. Peripheral blood T cells from healthy individuals were cultured with or without RCC supernatants for 24 hours and then were stimulated with PMA and ionomycin for 24 hours. The T-cell supernatants were then collected to measure protein production of the type 1 cytokine, IFN-{gamma}, and type 2 cytokine, IL-10, by ELISA. These findings show that RCC supernatants preferentially inhibited IFN-{gamma} but not IL-10 protein expression.

 
Gangliosides within the Supernatants from Renal Cell Carcinoma Explants Promote TH2-Type Biased T-Cell Responses.
To define the potential of shed tumor gangliosides to inhibit type 1 T-cell responses, we isolated gangliosides from tumor supernatants by a standard protocol and added them to peripheral blood T cells. After 48 hours of coculture, T cells were stimulated with PMA and ionomycin for 18 hours before isolating RNA and performing real-time PCR. There was minimal to no T-cell expression of mRNA for either IFN-{gamma} or IL-5 in the absence of stimulation. The addition of gangliosides to the T-cell cultures did not inhibit the PMA and ionomycin-induced expression of IL-5 mRNA, although the isolated gangliosides caused almost complete inhibition of IFN-{gamma} mRNA expression (Fig. 3)Citation . Similar results were observed with gangliosides isolated from an unrelated renal cell carcinoma supernatant (data not shown). Thus, these findings show that gangliosides present in the tumor supernatant induce type 2 biased immunity, similar to that promoted by the crude supernatant.



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  		Fig. 3. Gangliosides isolated from supernatants of renal cell carcinoma (RCC) explants are capable of producing a type 2 bias in normal T cells. Peripheral blood T cells from healthy individuals were cultured with or without RCC-isolated gangliosides for 48 hours and then were stimulated with PMA and ionomycin for 18 hours. RNA was then extracted and subjected to reverse transcription-PCR to assess the relative expression of the type 1 cytokine, IFN-{gamma}, and type 2 cytokine, IL-5, when compared with the control gene huGUS. These findings show that RCC-derived gangliosides preferentially inhibited IFN-{gamma} but not IL-5 mRNA expression. huGUS, human ß-glucronidase.

 
Prior studies by others using TLC have shown that when compared with normal adjacent tissue, renal cell carcinomas demonstrate increased expression of the gangliosides GM1, GM2, and GD1a (21) . We have recently obtained similar results by using high-performance liquid chromatography to profile 12 different clear cell tumors and comparing those findings to the profile of gangliosides isolated from normal adjacent tissues.6 High-performance liquid chromatography analysis of gangliosides isolated from an renal cell carcinoma supernatant demonstrated the presence of multiple gangliosides, including those with retention times similar to the standard gangliosides GM1 and GD1a. Given that GD1a was increased in expression in renal cell carcinoma, we asked whether the purified bovine brain-derived ganglioside GD1a would alter the expression of type 1 and type 2 cytokines in T cells stimulated with PMA and ionomycin. As seen in Fig. 4Citation , GD1a caused a significant inhibition in the induction of IFN-{gamma} gene expression, while actually augmenting the expression of IL-5 induced by PMA and ionomycin. The concentration of GD1a used for these experiments was 200 µg/ml. The concentration of the mixture of gangliosides isolated from the tumor supernatants was estimated by quantifying the amount of lipid-bound sialic acid per milliliter of solution using a method published previously (22) . With this assay the concentration of ganglioside (lipid-bound sialic acid) was between 4 and 14 µg/mL for the four different gangliosides preparations examined.



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  		Fig. 4. Standard bovine brain-derived ganglioside, GD1a, is capable of producing a type 2 bias in normal T cells. Peripheral blood T cells from healthy individuals were cultured with or without GD1a for 48 hours and then were stimulated with PMA and ionomycin for 18 hours. RNA was then extracted and subjected to reverse transcription-PCR to assess the relative expression of the type 1 cytokine, IFN-{gamma}, and type 2 cytokine, IL-5, when compared with the control gene huGUS. These findings show that GD1a, a ganglioside shown to be overexpressed in renal cell carcinoma, preferentially inhibited IFN-{gamma} but not IL-5 mRNA expression. huGUS, human ß-glucronidase.

 
Gangliosides Promote Type 2 Biased Immunity by Inducing Type 1 T-Cell Apoptosis.
We reported previously that gangliosides isolated from some renal cell carcinoma supernatants can induce T cells to undergo apoptosis (23) . Here we examined the ability of tumor supernatant-derived gangliosides to inhibit IFN-{gamma} production via the induced apoptotic death of type 1 T cells. This ganglioside preparation caused a significant inhibition in the production of IFN-{gamma} protein, as demonstrated by ELISA, within 24 hours of stimulation with PMA and ionomycin (Fig. 5)Citation . The inhibition of IFN-{gamma} expression coincided with the induction of T-cell death, which was also mediated by the tumor-derived ganglioside. Although activation with PMA and ionomycin alone did not induce any significant apoptosis, the presence of gangliosides induced 50% of the T cells to die. Although this cell death was assessed by the uptake of trypan blue, we also showed that this ganglioside preparation and others could induce DNA breaks as determined by the TUNEL assay (Fig. 6)Citation . The data in Fig. 6Citation also indicate that gangliosides appear to be most effective at inducing apoptosis in activated T cells. Although the ganglioside alone induced 35% of the resting T cells to undergo apoptosis, the number of apoptotic cells increased to >80% when activated with PMA and ionomycin.



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  		Fig. 5. Gangliosides isolated from supernatants of renal cell carcinoma explants are capable of inhibiting production of IFN-{gamma} and inducing apoptosis in normal T cells. Peripheral blood T cells from healthy individuals were cultured with or without renal cell carcinoma-isolated gangliosides for 48 hours and were stimulated with PMA and ionomycin for the last 18 hours. The T-cell supernatants were then collected to measure protein production of the type 1 cytokine, IFN-{gamma}, by ELISA (top) and the T cells counted with trypan blue exclusion to measure apoptosis (bottom). These findings show that renal cell carcinoma-derived gangliosides inhibited IFN-{gamma} protein production and induce apoptosis in normal T cells.

 


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  		Fig. 6. Gangliosides isolated from renal cell carcinoma supernatants can induce apoptosis in normal T cells. T cells from healthy volunteers were incubated with gangliosides isolated from renal cell carcinoma supernatants, with or without stimulation with PMA and ionomycin. The T cells were then assessed for apoptosis by TUNEL analysis. The isolated gangliosides from renal cell carcinoma did induce apoptosis of T cells after 48 hours of coculture, and the level of ganglioside-induced apoptosis was increased by simultaneous stimulation with PMA and ionomycin.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
OPEN DISCUSSION
 REFERENCES
 
There is evidence that immune responses to renal cell carcinoma protect patients against progression of the disease and in limited cases mediate tumor regression. This notion is supported by the infrequent although detectable occurrences of spontaneous regression in renal cell carcinoma patients and by the higher incidence of renal cell tumors in patients who are immunosuppressed after receiving kidney allografts (24, 25, 26) . There is also a significant infiltrate of T lymphocytes in primary renal tumors (27) , and in some patients, tumor-specific T-cell lines and clones have been expanded from their tumors (28, 29, 30) . Furthermore, clonal expansion of T-cell receptor {alpha}/ß T cells has been reported in renal cell tumors, most notably in those that are regressing (9 , 31) . Our previous findings show that CD4+ and CD8+ T cells specific for MAGE-6 and EphA2 antigens that are expressed on renal cell carcinoma are detectable in the peripheral blood and can mediate type 1 responses in patients who have no current evidence of disease. However, it is also evident that in patients with active renal cell carcinoma, the development of an effective type 1 T-cell response is impaired (15, 16, 17) . A TH2-type CD4+ T cell bias to MAGE-6 antigen is not unique to renal cell carcinoma patients, because defective CD4+ IFN{gamma} response to MAGE-6 was reported in patients with melanoma (15 , 16) . Our findings of a TH1-type CD4+ deficiency to tumor-specific antigens is in agreement with a study that examined IL-4 and IFN{gamma} production by T cells from renal cell carcinoma patients in response to polyclonal activation, which showed that the proportion of CD4+ cells producing IL-4 was significantly higher and the Th1:Th2 ratio lower in patients with high-stage disease (32) . Animal studies using the B16 melanoma model have demonstrated that there is a shift from a mixed Th1:Th2 type CD4+ T-cell response to a Th2:Tr-type dominated response during tumor progression and that administration of antibodies to IL-4, IL-10, and transforming growth factor-B1 blocked tumor-induced Th 2 bias (33, 34, 35) . Whether the administration of cytokines, such as IFN-{alpha}, that have some clinical activity in renal cell carcinoma patients can cause a shift from a Th2 response to a Th1-type response is not known but is under investigation.

Our findings suggest that soluble mediators present in the tumor microenvironment represent one possible mechanism to explain the type 2 bias observed in renal cell carcinoma patients with progressing disease. It is known that tumors exhibit augmented synthesis of select gangliosides, which are shed into the tumor microenvironment (36 , 37) . Malignant melanomas and neuroblastomas overexpress GD3, GD2, and GM2 (38) , whereas renal cell carcinoma demonstrates increased expression of GD1a, GM1, and GM2 compared with normal kidney tissue (21) . Gangliosides isolated from different tumors are known to inhibit immune responses (39 , 40) . The in vitro data presented herein show that gangliosides present in supernatants from renal cell carcinoma explants can polarize T-cell responses toward a dominant type 2 functional phenotype after mitogenic activation with either the superantigen S. enterotoxin B or PMA and ionomycin. Our findings are consistent with those of others (41) , which demonstrated that a mixture of bovine brain-derived gangliosides inhibited IFN-{gamma} production but not IL-4 after the T-cell stimulation. Herein, we show that GD1a (bovine derived), a ganglioside overexpressed in renal cell carcinoma, can also inhibit the development of type 1 T-cell responses. It is currently not known which of the gangliosides expressed by renal cell carcinoma and present in supernatants from renal cell carcinoma explants are responsible for the selective suppression of the type 1 response, although this is an active area of our ongoing investigations. It is also possible that the impaired Th1 CD4+ response to tumor-associated antigens in renal cell carcinoma patients is due in part to circulating gangliosides derived from the tumor. Others have shown that GD2 levels are detectable in the peripheral blood of neuroblastoma patients and that rapid disease progress and low survival rate was associated with high circulation of GD2 in these patients (42 , 43) . We are currently testing whether renal cell carcinoma patients display elevated levels of gangliosides in their sera and whether gangliosides levels correlate with the reported Th2 bias.

In addition, other soluble mediators besides gangliosides are likely to contribute to the induction of a TH2-type T-cell response bias in renal cell carcinoma patients. We noted previously in a subset of patients with stage IV disease (3 of 8) that transforming growth factor ß but not IL-10 was produced in vitro after EphA2 peptide stimulation of responder CD4+ T cells. Interestingly, these same patients displayed both weak TH1 (IFN-{gamma}) and weak type 2 (IL-5) CD4+ T-cell reactivities against EphA2 peptides (17) . Thus, it is possible that TH3/T-reg CD4+ T cells may play a role in inhibiting the development of TH1-type T-cell responses against the EphA2 tumor-associated antigen in at least some renal cell carcinoma patients.

Our data also suggest that the induction of apoptosis in the type 1 T cells is a major mechanism that potentially explains the type 2 bias that is observed in peripheral blood T cells responding to tumor antigens (MAGE-6 and EphA2) in vivo and to mitogens in vitro. Furthermore, tumor-derived products, including gangliosides, may be involved in promoting the apoptosis of type 1 T cells. This idea is supported by our observations that gangliosides isolated from tumor supernatants that are apoptogenic for T cells can also activate the death sequence. We have demonstrated that gangliosides are most effective at inducing apoptosis in T cells that are in an activated state. Activated T cells would be the most sensitive and likely target population susceptible to ganglioside-induced apoptosis in cancer patients, because these lymphocytes are continuously exposed to tumor antigens (either directly or via cross-presentation on antigen-presenting cells). Indeed, several groups have shown that tumor-infiltrating T cells and a subset of peripheral blood T cells from cancer patients are more sensitive to apoptosis than T cells from healthy individuals (44 , 45) . Our data indicate that gangliosides are likely involved in mediating apoptosis of T cells within the tumor microenvironment. It is also possible that shed gangliosides present in the serum can promote a TH2-type bias in renal cell carcinoma patient T cells via an apoptogenic effect, although this notion remains to be formally tested. We propose that the inhibition of type 1 T-cell responses represents a relevant mechanism by which renal cell carcinoma can inhibit protective antitumor immunity and, thus, promote tumor survival and progression.


   OPEN DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 OPEN DISCUSSION
REFERENCES
 
Dr. James Yang: The peripheral blood shows the TH2 bias, and some of the apoptotic changes are not found in fluid but rather in the tumor. Is there any way to know if that might occur because of the tumor microenvironment? Have you been able to identify cells with unrelated reactivities in the tumor microenvironment that are suppressed? In other words, if you look for flu in a vaccination setting, would you find flu cells in the tumor environment that were apoptotic?

Dr. James Finke: We’ve mostly done this in the peripheral blood, so now we’re moving this system to tumor infiltrating lymphocytes.

Dr. Yang: Are you trying to generate a response from tumor infiltrating lymphocytes? Are those also suppressed? A flu reaction cannot be affected, so you can postulate that the specificity exists because it’s generated in the tumor microenvironment. So, there will be passenger lymphocytes in the tumor microenvironment. You can then test cells against alloantigens or even flu and potentially show that T cells were apoptotic or suppressed and not in the blood.

Dr Finke: Right.

Dr. Michael Atkins: Have you looked at the {zeta} chain as well?

Dr. Finke: We have not looked at the {zeta} chain in these patients. We haven’t planned to look at the {zeta} chain, but we have planned to look at TH2/TH1 response in additional patients and compare the type of helper response generated to the levels of expression of gangliosides in the tumor and in the serum. In terms of the {zeta} chain depletion, it is possible that it is due to elevated arginase levels in patients, but there is also evidence that the {zeta} chain is actually a target of caspases in apoptotic cells.


   FOOTNOTES
 
Presented at the First International Conference on Innovations and Challenges in Renal Cancer, March 19–20, 2004, Cambridge, Massachusetts.

Grant support: NIH CA56937, CA/AI90995, CA57840 (W. J. Storkus), and the Senior Investigator Award of the Kidney Cancer Association.

Requests for reprints: James Finke, Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: 216-444-5186; E-mail: finkej{at}ccf.org

6 K. Biswas, P. Rayman, G. Raval, et al., unpublished observations. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 OPEN DISCUSSION
 REFERENCES
 

  1. Janssen EM, Lemmens EE, Wolfe T, Christen U, von Herrath MG, Schoenberger SP CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature 2003;421:852-6.[CrossRef][Medline]
  2. Gajewski TF, Fallarino F Rational development of tumour antigen-specific immunization in melanoma. Ther Immunol 1995;2:211-25.[Medline]
  3. Matsui S, Ahlers JD, Vortmeyer AO, et al A model for CD8+ CTL tumor immunosurveillance and regulation of tumor escape by CD4 T cells through an effect on quality of CTL. J Immunol 1999;163:184-93.[Abstract/Free Full Text]
  4. Ossendorp F, Mengede E, Camps M, Filius R, Melief CJ Specific T helper cell requirement for optimal induction of cytotoxic T lymphocytes against major histocompatibility complex class II negative tumors. J Exp Med 1998;187:693-702.[Abstract/Free Full Text]
  5. Abbas AK, Murphy KM, Sher A Functional diversity of helper T lymphocytes. Nature 1996;383:787-93.[CrossRef][Medline]
  6. Constant SL, Bottomly K Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu Rev Immunol 1997;15:297-322.[CrossRef][Medline]
  7. Levings MK, Bacchetta R, Schulz U, Roncarolo MG The role of IL-10 and TGF-beta in the differentiation and effector function of T regulatory cells. Int Arch Allergy Immunol 2002;129:263-76.[CrossRef][Medline]
  8. Chatenoud L, Salomon B, Bluestone JA Suppressor T cells-they’re back and critical for regulation of autoimmunity!. Immunol Rev 2001;182:149-63.[CrossRef][Medline]
  9. Angevin E, Kremer F, Gaudin C, Hercend T, Triebel F Analysis of T-cell immune response in renal cell carcinoma: polarization to type 1-like differentiation pattern, clonal T-cell expansion and tumor-specific cytotoxicity. Int J Cancer 1997;72:431-40.[CrossRef][Medline]
  10. Schoof DD, Terashima Y, Peoples GE, et al CD4+ T cell clones isolated from human renal cell carcinoma possess the functional characteristics of Th2 helper cells. Cell Immunol 1993;150:114-23.[CrossRef][Medline]
  11. Goldman M, Druet P The TH1/TH2 concept and its relevance to renal disorders and transplantation immunity. Nephrol Dial Transplant 1995;10:1282-4.[Free Full Text]
  12. Elsasser-Beile U, Kolble N, Grussenmeyer T, et al Th1 and Th2 cytokine response patterns in leukocyte cultures of patients with urinary bladder, renal cell and prostate carcinomas. Tumour Biol 1998;19:470-6.[CrossRef][Medline]
  13. Fridman WH, Tartour E Macrophage- and lymphocyte-produced Th1 and Th2 cytokines in the tumour microenvironment. Res Immunol 1998;149:651-3.[CrossRef][Medline]
  14. Wang Q, Redovan C, Tubbs R, et al Selective cytokine gene expression in renal cell carcinoma tumor cells and tumor-infiltrating lymphocytes. Int J Cancer 1995;61:780-5.[Medline]
  15. Tatsumi T, Kierstead LS, Ranieri E, et al Disease-associated bias in T helper type 1 (Th1)/Th2 CD4(+) T cell responses against MAGE-6 in HLA-DRB10401(+) patients with renal cell carcinoma or melanoma. J Exp Med 2002;196:619-28.[Abstract/Free Full Text]
  16. Tatsumi T, Kierstead LS, Ranieri E, et al MAGE-6 encodes HLA-DRbeta1*0401-presented epitopes recognized by CD4+ T cells from patients with melanoma or renal cell carcinoma. Clin Cancer Res 2003;9:947-54.[Abstract/Free Full Text]
  17. Tatsumi T, Herrem CJ, Olson WC, et al Disease stage variation in CD4+ and CD8+ T-cell reactivity to the receptor tyrosine kinase EphA2 in patients with renal cell carcinoma. Cancer Res 2003;63:4481-9.[Abstract/Free Full Text]
  18. Tatsumi T, Wesa A, Finke J, Bukowski R, Storkus W CD4 T cell-mediated immunity to cancer Finke J Bukowski R eds. . Cancer immunotherapy at the crossroads: how tumors evade immunity and what can be done 200467-86. Humana Press Totowa, NJ
  19. McKallip R, Li R, Ladisch S Tumor gangliosides inhibit the tumor-specific immune response. J Immunol 1999;163:3718-26.[Abstract/Free Full Text]
  20. Thornton MV, Kudo D, Rayman P, et al Degradation of NF-kappaB in T cells by gangliosides expressed on renal cell carcinomas. J Immunol 2004;172:3480-90.[Abstract/Free Full Text]
  21. Hoon DS, Okun E, Neuwirth H, Morton DL, Irie RF Aberrant expression of gangliosides in human renal cell carcinomas. J Urol 1993;150:2013-8.[Medline]
  22. Raval GN, Parekh LJ, Patel MM, et al Role of sialic acid and alkaline DNase in breast cancer. Int J Biol Markers 1997;12:61-7.[Medline]
  23. Finke JH, Rayman P, George R, et al Tumor-induced sensitivity to apoptosis in T cells from patients with renal cell carcinoma: role of nuclear factor-kappaB suppression. Clin Cancer Res 2001;7:940s-6s.
  24. Edwards MJ, Anderson JA, Angel JR, Harty JI Spontaneous regression of primary and metastatic renal cell carcinoma. J Urol 1996;155:1385[CrossRef][Medline]
  25. Kliem V, Kolditz M, Behrend M, et al Risk of renal cell carcinoma after kidney transplantation. Clin Transplant 1997;11:255-8.[Medline]
  26. Kunisch-Hoppe M, Hoppe M, Bohle RM, et al Metastatic RCC arising in a transplant kidney. Eur Radiol 1998;8:1441-3.[CrossRef][Medline]
  27. Finke JH, Rayman P, Hart L, et al Characterization of tumor-infiltrating lymphocyte subsets from human renal cell carcinoma: specific reactivity defined by cytotoxicity, interferon-gamma secretion, and proliferation. J Immunother Emphasis Tumor Immunol 1994;15:91-104.[Medline]
  28. Finke JH, Rayman P, Alexander J, et al Characterization of the cytolytic activity of CD4+ and CD8+ tumor-infiltrating lymphocytes in human renal cell carcinoma. Cancer Res 1990;50:2363-70.[Abstract/Free Full Text]
  29. Belldegrun A, Kasid A, Uppenkamp M, Rosenberg SA Lymphokine mRNA profile and functional analysis of a human CD4+ clone with unique antitumor specificity isolated from renal cell carcinoma ascitic fluid. Cancer Immunol Immunother 1990;31:1-10.[CrossRef][Medline]
  30. Schendel DJ, Gansbacher B, Oberneder R, et al Tumor-specific lysis of human renal cell carcinomas by tumor-infiltrating lymphocytes. I. HLA-A2-restricted recognition of autologous and allogeneic tumor lines. J Immunol 1993;151:4209-20.[Abstract]
  31. Puisieux I, Bain C, Merrouche Y, et al Restriction of the T-cell repertoire in tumor-infiltrating lymphocytes from nine patients with renal-cell carcinoma. Relevance of the CDR3 length analysis for the identification of in situ clonal T-cell expansions. Int J Cancer 1996;66:201-8.[CrossRef][Medline]
  32. Onishi T, Ohishi Y, Goto H, Tomita M, Abe K An assessment of the immunological status of patients with renal cell carcinoma based on the relative abundance of T-helper 1- and -2 cytokine-producing CD4+ cells in peripheral blood. BJU Int 2001;87:755-9.[CrossRef][Medline]
  33. Nagai H, Hara I, Horikawa T, Oka M, Kamidono S, Ichihashi M Elimination of CD4(+) T cells enhances anti-tumor effect of locally secreted interleukin-12 on B16 mouse melanoma and induces vitiligo-like coat color alteration. J Invest Dermatol 2000;115:1059-64.[CrossRef][Medline]
  34. Seo N, Hayakawa S, Takigawa M, Tokura Y Interleukin-10 expressed at early tumour sites induces subsequent generation of CD4(+) T-regulatory cells and systemic collapse of antitumour immunity. Immunology 2001;103:449-57.[CrossRef][Medline]
  35. Nebl T, Pestonjamasp KN, Leszyk JD, Crowley JL, Oh SW, Luna EJ Proteomic analysis of a detergent-resistant membrane skeleton from neutrophil plasma membranes. J Biol Chem 2002;277:43399-409.[Abstract/Free Full Text]
  36. Ladisch S, Gillard B, Wong C, Ulsh L Shedding and immunoregulatory activity of YAC-1 lymphoma cell gangliosides. Cancer Res 1983;43:3808-13.[Abstract/Free Full Text]
  37. Black PH Shedding from the cell surface of normal and cancer cells. Adv Cancer Res 1980;32:75-199.[Medline]
  38. Ritter G, Livingston PO Ganglioside antigens expressed by human cancer cells. Semin Cancer Biol 1991;2:401-9.[Medline]
  39. Li R, Villacreses N, Ladisch S Human tumor gangliosides inhibit murine immune responses in vivo. Cancer Res 1995;55:211-4.[Abstract/Free Full Text]
  40. Ladisch S, Li R, Olson E Ceramide structure predicts tumor ganglioside immunosuppressive activity. Proc Natl Acad Sci USA 1994;91:1974-8.[Abstract/Free Full Text]
  41. Irani DN, Lin KI, Griffin DE Brain-derived gangliosides regulate the cytokine production and proliferation of activated T cells. J Immunol 1996;157:4333-40.[Abstract]
  42. Schulz G, Cheresh DA, Varki NM, Yu A, Staffileno LK, Reisfeld RA Detection of ganglioside GD2 in tumor tissues and sera of neuroblastoma patients. Cancer Res 1984;44:5914-20.[Medline]
  43. Valentino L, Moss T, Olson E, Wang HJ, Elashoff R, Ladisch S Shed tumor gangliosides and progression of human neuroblastoma. Blood 1990;75:1564-7.[Abstract/Free Full Text]
  44. Uzzo RG, Rayman P, Kolenko V, et al Mechanisms of apoptosis in T cells from patients with renal cell carcinoma. Clin Cancer Res 1999;5:1219-29.[Abstract/Free Full Text]
  45. Saito T, Dworacki G, Gooding W, Lotze MT, Whiteside TL Spontaneous apoptosis of CD8+ T lymphocytes in peripheral blood of patients with advanced melanoma. Clin Cancer Res 2000;6:1351-64.[Abstract/Free Full Text]



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