Patricia Rayman

Publications of Patricia Rayman

• Myeloid-derived suppressor cell accumulation and function in patients with newly diagnosed glioblastoma.

  Authors: Baisakhi Raychaudhuri, Patricia Rayman, Joanna Ireland, Jennifer Ko, Brian Rini, Ernest C Borden, Jorge Garcia, Michael A Vogelbaum, James Finke

  Neuro-oncology. 06/2011; 13(6):591-9.

  To assess the accumulation of myeloid-derived suppressor cells (MDSCs) in the peripheral blood of patients with glioma and to define their heterogeneity and their immunosuppressive function.To assess the accumulation of myeloid-derived suppressor cells (MDSCs) in the peripheral blood of patients with glioma and to define their heterogeneity and their immunosuppressive function. Peripheral blood mononuclear cells (PBMCs) from healthy control subjects and from patients with newly diagnosed glioma were stimulated with anti-CD3/anti-CD28 and T cells assessed for intracellular expression of interferon (IFN)-γ. Antibody staining of PBMCs from glioma patients and healthy donors (CD33, HLADR, CD15, and CD14) followed by 4-color flow cytometry analysis-defined MDSC levels in the peripheral blood. To assess the role of MDSCs in suppressing T cell IFNγ production, PBMCs were depleted of MDSCs using anti-CD33 and anti-CD15 antibody-coated beads prior to T cell stimulation. Enzyme-linked immunosorbent assays were used to assess plasma arginase activity and the level of granulocyte colony-stimulating factor (G-CSF). Patients with glioblastoma have increased MDSC counts (CD33+HLADR-) in their blood that are composed of neutrophilic (CD15(+); >60%), lineage-negative (CD15(-)CD14(-); 31%), and monocytic (CD14(+); 6%) subsets. After stimulation, T cells from patients with glioblastoma had suppressed IFN-γ production when compared with healthy, age-matched donor T cells. Removal of MDSCs from the PBMCs with anti-CD33/CD15-coated beads significantly restored T cell function. Significant increases in arginase activity and G-CSF levels were observed in plasma specimens obtained from patients with glioblastoma. The accumulation of MDSCs in peripheral blood in patients with glioma likely promotes T cell immune suppression that is observed in this patient population. Increased plasma levels of arginase and G-CSF may relate to MDSC suppressor function and MDSC expansion, respectively, in patients with glioma.

• Direct and differential suppression of myeloid-derived suppressor cell subsets by sunitinib is compartmentally constrained.

  Authors: Jennifer S Ko, Patricia Rayman, Joanna Ireland, Shadi Swaidani, Geqiang Li, Kevin D Bunting, Brian Rini, James H Finke, Peter A Cohen

  Cancer research. 05/2010; 70(9):3526-36.

  The antiangiogenic drug sunitinib is a receptor tyrosine kinase inhibitor with significant, yet not curative, therapeutic effects in metastatic renal cell carcinoma (RCC). Sunitinib is also anThe antiangiogenic drug sunitinib is a receptor tyrosine kinase inhibitor with significant, yet not curative, therapeutic effects in metastatic renal cell carcinoma (RCC). Sunitinib is also an immunomodulator, potently reversing myeloid-derived suppressor cell (MDSC) accumulation and T-cell inhibition in the blood even of nonresponder RCC patients. We observed that sunitinib similarly prevented MDSC accumulation and restored normal T-cell function to the spleens of tumor-bearing mice, independent of the capacity of sunitinib to inhibit tumor progression (RENCA>CT26>4T1). Both monocytic and neutrophilic splenic MDSC were highly repressible by sunitinib. In contrast, MDSC within the microenvironment of 4T1 tumors or human RCC tumors proved highly resistant to sunitinib and ambient T-cell function remained suppressed. Proteomic analyses comparing tumor to peripheral compartments showed that granulocyte macrophage colony-stimulating factor (GM-CSF) predicted sunitinib resistance and recombinant GM-CSF conferred sunitinib resistance to MDSC in vivo and in vitro. MDSC conditioning with GM-CSF uniquely inhibited signal transducers and activators of transcription (STAT3) and promoted STAT5 activation. STAT5ab(null/null) MDSC were rendered sensitive to sunitinib in the presence of GM-CSF in vitro. We conclude that compartment-dependent GM-CSF exposure in resistant tumors may account for the regionalized effect of sunitinib upon host MDSC modulation and hypothesize that ancillary strategies to decrease such regionalized escape will enhance the potency of sunitinib as an immunomodulator and a cancer therapy.

• Quantification of Carbonic Anhydrase IX Expression in Serum and Tissue of Renal Cell Carcinoma Patients Using Enzyme-linked Immunosorbent Assay: Prognostic and Diagnostic Potentials.

  Authors: Grace X Zhou, Joanna Ireland, Patricia Rayman, James Finke, Ming Zhou

  Urology. 12/2009;

  OBJECTIVES: To validate enzyme-linked immunosorbent assay (ELISA) as an accurate and objective method to measure carbonic anhydrase IX (CAIX) expression in RCC tissue specimens and to alsoOBJECTIVES: To validate enzyme-linked immunosorbent assay (ELISA) as an accurate and objective method to measure carbonic anhydrase IX (CAIX) expression in RCC tissue specimens and to also investigate the diagnostic and prognostic utility of serum CAIX levels. Recent studies found protein levels of CAIX, quantified using immunohistochemistry, correlated with clinical outcomes and therapeutic response to immunotherapy in advanced stage clear cell renal cell carcinoma (CCRCC). MATERIALS AND METHODS: A CAIX ELISA kit was used to measure the CAIX levels in the serum and tissue of 21 CCRCCs and 19 non-CCRCCs. CAIX immunohistochemical stains were performed on the corresponding formalin fixed, paraffin embedded tumor tissues. RESULTS: Tissue CAIX levels measured by ELISA highly correlated with the CAIX levels detected by immunohistochemistry (Pearson correlation coefficient = .802, P <.001). The CCRCC tumor tissue had significantly higher CAIX levels than non-CCRCC tissue (77 023 vs 938 pg/mL, P <.001) detected by ELISA. Serum CAIX levels in CCRCC patients were significantly higher than in non-CCRCC patients (126.1 vs 2.1 pg/mL, P = .013). The serum CAIX levels detected by ELISA correlated with the tumor size in CCRCC patients (Pearson correlation coefficient = .498, P = .036). CONCLUSIONS: ELISA provides a quantitative and objective method to measure CAIX levels in renal tumors. The tissue CAIX levels measured with ELISA highly correlate with the results obtained with the standard immunohistochemistry. Serum CAIX levels are significantly higher in CCRCC than in non-CCRCC and may aid the differential diagnosis of RCC. Serum CAIX levels also correlate with the tumor size in CCRCC patients.

• Elevated levels of select gangliosides in T cells from renal cell carcinoma patients is associated with T cell dysfunction.

  Authors: Soumika Biswas, Kaushik Biswas, Amy Richmond, Jennifer Ko, Sankar Ghosh, Matthew Simmons, Patricia Rayman, Brian Rini, Inderbir Gill, Charles S Tannenbaum, James H Finke

  Journal of immunology (Baltimore, Md. : 1950). 10/2009; 183(8):5050-8.

  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. In this study, weIncreased expression of gangliosides by different tumor types including renal cell carcinoma (RCC) is thought to contribute to the immune suppression observed in cancer patients. In this study, we report an increase in apoptotic T cells from RCC patients compared with 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.

• Sunitinib reverses type-1 immune suppression and decreases T-regulatory cells in renal cell carcinoma patients.

  Authors: James H Finke, Brian Rini, Joanna Ireland, Patricia Rayman, Amy Richmond, Ali Golshayan, Laura Wood, Paul Elson, Jorge Garcia, Robert Dreicer, Ronald Bukowski

  Clinical cancer research : an official journal of the American Association for Cancer Research. 10/2008; 14(20):6674-82.

  PURPOSE: Immune dysfunction is well documented in renal cell carcinoma (RCC) patients and likely contributes to tumor evasion. This dysfunction includes a shift from a type-1 to a type-2 T-cellPURPOSE: Immune dysfunction is well documented in renal cell carcinoma (RCC) patients and likely contributes to tumor evasion. This dysfunction includes a shift from a type-1 to a type-2 T-cell cytokine response and enhanced T-regulatory (Treg) cell expression. Given the antitumor activity of select tyrosine kinase inhibitors such as sunitinib in metastatic RCC (mRCC) patients, it is relevant to assess their effect on the immune system. EXPERIMENTAL DESIGN: Type-1 (IFNgamma) and type-2 (interleukin-4) responses were assessed in T cells at baseline and day 28 of treatment with sunitinib (50 mg/d) by measuring intracellular cytokines after in vitro stimulation with anti-CD3/anti-CD28 antibodies. RESULTS: After one cycle of treatment, there was a significant increase in the percentage of IFNgamma-producing T cells (CD3(+), P < 0.001; CD3(+)CD4(+), P = 0.001), a reduction in interleukin-4 production (CD3(+) cells, P = 0.05), and a diminished type-2 bias (P = 0.005). The increase in type-1 response may be partly related to modulation of Treg cells. The increased percentage of Treg cells noted in mRCC patients over healthy donors (P = 0.001) was reduced after treatment, although not reaching statistical significance. There was, however, an inverse correlation between the increase in type-1 response after two cycles of treatment and a decrease in the percentage of Treg cells (r = -0.64, P = 0.01). In vitro studies suggest that the effects of sunitinib on Treg cells are indirect. CONCLUSIONS: The demonstration that sunitinib improved type-1 T-cell cytokine response in mRCC patients while reducing Treg function provides a basis for the rational combination of sunitinib and immunotherapy in mRCC.

• Determinants of cytokine induction by small interfering RNA in human peripheral blood mononuclear cells.

  Authors: Maryam Zamanian-Daryoush, Joao T Marques, Michael P Gantier, Mark A Behlke, Matthias John, Patricia Rayman, James Finke, Bryan R G Williams

  Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 05/2008; 28(4):221-33.

  Synthetic small interfering RNAs (siRNAs) can trigger a strong innate immune response in mammalian cells. This nonspecific side effect may hinder the application of siRNAs as tools in gene silencing.Synthetic small interfering RNAs (siRNAs) can trigger a strong innate immune response in mammalian cells. This nonspecific side effect may hinder the application of siRNAs as tools in gene silencing. Chemically synthesized siRNAs, including traditional 19-mers with 2-nt 3' overhangs, longer duplexes with blunt or 3' overhangs, and asymmetric duplexes with a blunt end and a 2-nt 3' overhang, can evoke strong dose-dependent interferon-alpha (IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) release in human peripheral blood mononuclear cells (PBMCs). This response is independent of retinoic acid-inducible gene I but may involve endosomal toll-like receptors (TLRs). The immunostimulatory effect of the siRNAs is directly related to either or both of the strands of the duplex in a sequence-dependent manner. However, although some single-stranded RNAs and siRNAs potently evoked both IFN-alpha and TNF-alpha induction, these responses were not always coupled. In accordance with this, specific chemical modifications differentially altered cytokine production, suggesting recruitment of different TLRs in a sequence-dependent manner.

• Renal cell carcinoma tumors induce T cell apoptosis through receptor-dependent and receptor-independent pathways.

  Authors: Tanya Das, Gaurisankar Sa, Ewa Paszkiewicz-Kozik, Cynthia Hilston, Luis Molto, Patricia Rayman, Daisuke Kudo, Kaushik Biswas, Ronald M Bukowski, James H Finke, Charles S Tannenbaum

  Journal of immunology (Baltimore, Md. : 1950). 05/2008; 180(7):4687-96.

  Tumors can promote their own progressive growth by inducing T cell apoptosis. Though previous studies suggested that tumor-mediated T cell killing is receptor dependent, we recently showed that tumorTumors can promote their own progressive growth by inducing T cell apoptosis. Though previous studies suggested that tumor-mediated T cell killing is receptor dependent, we recently showed that tumor gangliosides also participate, a notion consistent with reports indicating that, in some cell types, gangliosides can activate the intrinsic apoptotic pathway by stimulating reactive oxygen species production, cytochrome c release, and caspase-9 activation. In this study, we used normal peripheral blood T cells, as well as caspase-8-, caspase-9-, and Fas-associated death domain protein-deficient Jurkat cells, to assess whether the death ligands and gangliosides overexpressed by the renal cell carcinoma (RCC) cell line SK-RC-45 can independently stimulate T cell apoptosis as a mechanism of immune escape. Anti-FasL Abs and the glycosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP) each partially inhibited the ability of SK-RC-45 to kill cocultured activated T cells; together, as purified molecules, RCC gangliosides and rFasL induced a more extensive mitochondrial permeability transition and greater levels of apoptosis than either agent alone, equivalent to that induced by the FasL- and ganglioside-expressing RCC line itself. rFasL-mediated apoptosis was completely inhibited in caspase-8- and Fas-associated death domain protein-negative Jurkat cells, though apoptosis induced by purified gangliosides remained intact, findings that correlate with the observed partial inhibition of SK-RC-45-induced apoptosis in the Jurkat lines with defective death receptor signaling. Western blot analysis performed on lysates made from wild-type and mutant Jurkat cells cocultured with SK-RC-45 revealed caspase activation patterns and other biochemical correlates which additionally supported the concept that tumor-associated gangliosides and FasL independently activate the caspase cascade in T cells through the intrinsic and extrinsic pathways, respectively.

• GM1 and tumor necrosis factor-alpha, overexpressed in renal cell carcinoma, synergize to induce T-cell apoptosis.

  Authors: Tanya Das, Gaurisankar Sa, Cynthia Hilston, Daisuke Kudo, Patricia Rayman, Kaushik Biswas, Luis Molto, Ronald Bukowski, Brian Rini, James H Finke, Charles Tannenbaum

  Cancer research. 03/2008; 68(6):2014-23.

  The ability to induce T-cell apoptosis is one mechanism by which tumors evade the immune system, although the molecules involved remain controversial. We found that renal cell carcinoma (RCC)-inducedThe ability to induce T-cell apoptosis is one mechanism by which tumors evade the immune system, although the molecules involved remain controversial. We found that renal cell carcinoma (RCC)-induced T-cell apoptosis was inhibited by >50% when cocultures were performed with ganglioside-depleted tumor cells, caspase-8-negative lymphocytes, or anti-tumor necrosis factor-alpha (TNFalpha) antibodies, suggesting that tumor gangliosides synergize with signals delivered through TNFalpha death receptors to mediate T-cell killing. The synergy between tumor-derived TNFalpha and the RCC-overexpressed ganglioside GM1 for killing resting T cells is corroborated by studies using purified GM1 and rTNF alpha, which indicate that a 48-hour pretreatment with the ganglioside optimally sensitizes the lymphocytes to a TNFalpha-induced apoptotic death. However, activated T cells, which synthesize TNFalpha themselves, can be killed by exogenous GM1 alone. RelA-overexpressing lymphocytes are protected from GM1 plus TNFalpha-mediated apoptosis, a finding consistent with our previous studies indicating that gangliosides inhibit nuclear factor-kappaB activation. These results are clinically relevant because, similar to T-cells cocultured with GM1-overexpressing RCC lines, T cells isolated from the peripheral blood of patients with metastatic RCC are also heavily coated with that tumor-shed ganglioside. This population of patient cells, unlike T cells isolated from normal donors, is highly susceptible to apoptosis induced by rTNF alpha or by metastatic patient sera, which contain elevated levels of the cytokine. This report thus extends our previous studies by demonstrating that tumor-derived TNFalpha enhances RCC apoptogenicity not only by inducing ganglioside synthesis but also by initiating receptor-dependent apoptosis in T cells in which the nuclear factor-kappaB activation pathway has been inhibited by GM1.

• TNF-alpha induction of GM2 expression on renal cell carcinomas promotes T cell dysfunction.

  Authors: Gira Raval, Soumika Biswas, Patricia Rayman, Kaushik Biswas, Gaurisankar Sa, Sankar Ghosh, Mark Thornton, Cynthia Hilston, Tanya Das, Ronald Bukowski, James Finke, Charles S Tannenbaum

  Journal of immunology (Baltimore, Md. : 1950). 06/2007; 178(10):6642-52.

  Previous studies from our laboratory demonstrated the role of tumor-derived gangliosides as important mediators of T cell apoptosis, and hence, as one mechanism by which tumors evade immunePrevious studies from our laboratory demonstrated the role of tumor-derived gangliosides as important mediators of T cell apoptosis, and hence, as one mechanism by which tumors evade immune destruction. In this study, we report that TNF-alpha secreted by infiltrating inflammatory cells and/or genetically modified tumors augments tumor-associated GM2 levels, which leads to T cell death and immune dysfunction. The conversion of weakly apoptogenic renal cell carcinoma (RCC) clones to lines that can induce T cell death requires 3-5 days of TNF-alpha pretreatment, a time frame paralleling that needed for TNF-alpha to stimulate GM2 accumulation by SK-RC-45, SK-RC-54, and SK-RC-13. RCC tumor cell lines permanently transfected with the TNF-alpha transgene are similarly toxic for T lymphocytes, which correlates with their constitutively elevated levels of GM2. TNF-alpha increases GM2 ganglioside expression by enhancing the mRNA levels encoding its synthetic enzyme, GM2 synthase, as demonstrated by both RT-PCR and Southern analysis. The contribution of GM2 gangliosides to tumor-induced T cell death was supported by the finding that anti-GM2 Abs significantly blocked T cell apoptosis mediated by TNF-alpha-treated tumor cells, and by the observation that small interfering RNA directed against TNF-alpha abrogated GM2 synthase expression by TNF-transfected SK-RC-45, diminished its GM2 accumulation, and inhibited its apoptogenicity for T lymphocytes. Our results indicate that TNF-alpha signaling promotes RCC-induced killing of T cells by stimulating the acquisition of a distinct ganglioside assembly in RCC tumor cells.

• GM2 expression in renal cell carcinoma: potential role in tumor-induced T-cell dysfunction.

  Authors: Kaushik Biswas, Amy Richmond, Patricia Rayman, Soumika Biswas, Mark Thornton, Gaurisankar Sa, Tanya Das, Renliang Zhang, Ali Chahlavi, Charles S Tannenbaum, Andrew Novick, Ronald Bukowski, James H Finke

  Cancer research. 08/2006; 66(13):6816-25.

  Multiple mechanisms have been proposed to account for immune escape by tumors. Although gangliosides have long been known to suppress T-cell immunity, few studies have examined the effect of humanMultiple mechanisms have been proposed to account for immune escape by tumors. Although gangliosides have long been known to suppress T-cell immunity, few studies have examined the effect of human tumor-derived gangliosides on immune responses. Here, we show that gangliosides isolated from renal cell carcinoma (RCC) cell lines and clear cell tumor tissue can induce apoptosis in peripheral blood T cells. The RCC tissue-derived gangliosides also suppressed IFN-gamma and, in many cases, interleukin-4 production by CD4+ T cells at concentrations (1 ng/mL-100 pg/mL) well below those that induce any detectable T-cell death (4-20 microg/mL). Additional findings show that GM2 expressed by RCC plays a significant role in promoting T-cell dysfunction. This is supported by the demonstration that all RCC cell lines examined (n = 5) expressed GM2 as did the majority of tumors (15 of 18) derived from patients with clear cell RCC. Furthermore, an antibody specific for GM2 (DMF10.167.4) partially blocked (50-60%) T-cell apoptosis induced by coculturing lymphocytes with RCC cell lines or with RCC tissue-derived gangliosides. DMF10.167.4 also partially blocked the suppression of IFN-gamma production induced by RCC tissue-derived gangliosides, suggesting that GM2 plays a role in down-regulating cytokine production by CD4+ T cells.

• Glioblastomas induce T-lymphocyte death by two distinct pathways involving gangliosides and CD70.

  Authors: Ali Chahlavi, Patricia Rayman, Amy L Richmond, Kaushik Biswas, Renliang Zhang, Michael Vogelbaum, Charles Tannenbaum, Gene Barnett, James H Finke

  Cancer research. 07/2005; 65(12):5428-38.

  Here we report that glioblastoma multiforme (GBM) mediates immunosuppression by promoting T-cell death via tumor-associated CD70 and gangliosides that act through receptor-dependent andHere we report that glioblastoma multiforme (GBM) mediates immunosuppression by promoting T-cell death via tumor-associated CD70 and gangliosides that act through receptor-dependent and receptor-independent pathways, respectively. GBM lines cocultured with T cells induced lymphocyte death. The GBM lines were characterized for their expression of CD70, Fas ligand (FasL), and tumor necrosis factor-alpha (TNF-alpha), and the possible participation of those molecules in T-cell killing was assessed by doing GBM/T cell cocultures in the presence of anti-CD70 antibodies, Fas fusion proteins, or anti-TNF-alpha antibodies. CD70 but not TNF-alpha or FasL is responsible for initiating T-cell death via the receptor-dependent pathway. Of the four GBM cell lines that induced T-cell death, three highly expressed CD70. Two nonapoptogenic GBM lines (CCF3 and U138), on the other hand, had only minimally detectable CD70 expression. Blocking experiments with the anti-CD70 antibody confirmed that elevated CD70 levels were involved in the apoptogenicity of the three GBM lines expressing that molecule. Gangliosides were found to participate in the induction of T-cell apoptosis, because the glucosylceramide synthase inhibitor (PPPP) significantly reduced the abilities of all four apoptogenic lines to kill the lymphocytes. High-performance liquid chromatography (HPLC) and mass spectroscopy revealed that GM2, GM2-like gangliosides, and GD1a were synthesized in abundance by all four apoptogenic GBM lines but not by the two GBMs lacking activity. Furthermore, gangliosides isolated from GBM lines as well as HPLC fractions containing GM2 and GD1a were directly apoptogenic for T cells. Our results indicate that CD70 and gangliosides are both products synthesized by GBMs that may be key mediators of T-cell apoptosis and likely contribute to the T-cell dysfunction observed within the tumor microenvironment.

• Effect of renal cell carcinomas on the development of type 1 T-cell responses.

  Authors: Patricia Rayman, Amy K Wesa, Amy L Richmond, Tanya Das, Kaushik Biswas, Gira Raval, Walter J Storkus, Charles Tannenbaum, Andrew Novick, Ronald Bukowski, James Finke

  Clinical cancer research : an official journal of the American Association for Cancer Research. 10/2004; 10(18 Pt 2):6360S-6S.

  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 responsesPURPOSE: 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.

• Degradation of NF-kappa B in T cells by gangliosides expressed on renal cell carcinomas.

  Authors: Mark V Thornton, Daisuke Kudo, Patricia Rayman, Claudine Horton, Luis Molto, Martha K Cathcart, Christopher Ng, Ewa Paszkiewicz-Kozik, Ronald Bukowski, Ithaar Derweesh, Charles S Tannenbaum, James H Finke

  Journal of immunology (Baltimore, Md. : 1950). 04/2004; 172(6):3480-90.

  T cells from cancer patients are often functionally impaired, which imposes a barrier to effective immunotherapy. Most pronounced are the alterations characterizing tumor-infiltrating T cells, whichT cells from cancer patients are often functionally impaired, which imposes a barrier to effective immunotherapy. Most pronounced are the alterations characterizing tumor-infiltrating T cells, which in renal cell carcinomas includes defective NF-kappaB activation and a heightened sensitivity to apoptosis. Coculture experiments revealed that renal tumor cell lines induced a time-dependent decrease in RelA(p65) and p50 protein levels within both Jurkat T cells and peripheral blood T lymphocytes that coincided with the onset of apoptosis. The degradation of RelA/p50 is critical for SK-RC-45-induced apoptosis because overexpression of RelA in Jurkat cells protects against cell death. The loss of RelA/p50 coincided with a decrease in expression of the NF-kappaB regulated antiapoptotic protein Bcl-xL at both the protein and mRNA level. The disappearance of RelA/p50 protein was mediated by a caspase-dependent pathway because pretreatment of T lymphocytes with a pan caspase inhibitor before coculture with SK-RC-45 blocked RelA and p50 degradation. SK-RC-45 gangliosides appear to mediate this degradative pathway, as blocking ganglioside synthesis in SK-RC-45 cells with the glucosylceramide synthase inhibitor, PPPP, protected T cells from tumor cell-induced RelA degradation and apoptosis. The ability of the Bcl-2 transgene to protect Jurkat cells from RelA degradation, caspase activation, and apoptosis implicates the mitochondria in these SK-RC-45 ganglioside-mediated effects.

• Gangliosides expressed by the renal cell carcinoma cell line SK-RC-45 are involved in tumor-induced apoptosis of T cells.

  Authors: Daisuke Kudo, Patricia Rayman, Claudine Horton, Martha K Cathcart, Ronald M Bukowski, Mark Thornton, Charles Tannenbaum, James H Finke

  Cancer research. 05/2003; 63(7):1676-83.

  It is now understood that the genetic plasticity of cancer cells can lead to alterations that confer selective growth advantages to the tumor, some of which play a role in immune escape. A number ofIt is now understood that the genetic plasticity of cancer cells can lead to alterations that confer selective growth advantages to the tumor, some of which play a role in immune escape. A number of mutations veiling tumor cells from host immune defenses have been well characterized but more recent studies suggest that a variety of tumors can also express products that are actually toxic for the immune effectors. A component of this tumor-induced T-cell death has been attributed to receptor-mediated apoptosis. Some tumors, however, synthesize soluble factors that mediate similar effects. In this regard, we previously showed that supernatants from explanted renal cell carcinoma (RCC) tumors sensitized normal T cells to activation induced cell death, and the responsible products had the features of gangliosides. We have also shown that renal tumor lines, including SK-RC-45, induce apoptosis of both Jurkat cells and normal T lymphocytes. Here, we used the ganglioside synthesis inhibitor PPPP to define the role of gangliosides in RCC cell line (SK-RC-45)-mediated T cell and Jurkat cell apoptosis and to elucidate the proapoptotic molecular events by which the glycosphingolipids produce their effects. The ganglioside-synthesizing SK-RC-45 line stimulated the TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labeling) positivity of cocultured T cells by a mechanism that involved decreasing lymphocyte expression levels of Bcl-2 and Bcl-(XL), inducing cytochrome c release from their mitochondria and activating caspases 9 and 3. These proapoptotic events were partially or completely abrogated when tumor cells were pretreated with PPPP for 5 days before the SK-RC-45/T lymphocyte coincubation, a regimen that reduced tumor-associated ganglioside levels by 70-80%. Our results suggest that gangliosides may be key mediators of RCC-induced T-cell apoptosis and imply that they contribute to the T-cell dysfunction in the tumor microenvironment.

• Renal cell carcinoma–derived gangliosides suppress nuclear factor-κB activation in T cells

  Authors: Robert G Uzzo, Patricia Rayman, Vladimir Kolenko, Peter E. Clark, Martha K Cathcart, Tracy Bloom, Andrew C Novick, Ronald M Bukowski, Thomas Hamilton, James H Finke

  Activation of the transcription factor nuclear factor-κB (NFκB) is impaired in T cells from patients with renal cell carcinomas (RCCs). In circulating T cells from a subset of patients with RCCs, theActivation of the transcription factor nuclear factor-κB (NFκB) is impaired in T cells from patients with renal cell carcinomas (RCCs). In circulating T cells from a subset of patients with RCCs, the suppression of NFκB binding activity is downstream from the stimulus-induced degradation of the cytoplasmic factor IκBα. Tumor-derived soluble products from cultured RCC explants inhibit NFκB activity in T cells from healthy volunteers, despite a normal level of stimulus-induced IκBα degradation in these cells. The inhibitory agent has several features characteristic of a ganglioside, including sensitivity to neuraminidase but not protease treatment; hydrophobicity; and molecular weight less than 3 kDa. Indeed, we detected gangliosides in supernatants from RCC explants and not from adjacent normal kidney tissue. Gangliosides prepared from RCC supernatants, as well as the purified bovine gangliosides Gm1 and Gd1a, suppressed NFκB binding activity in T cells and reduced expression of the cytokines IL-2 and IFN-γ. Taken together, our findings suggest that tumor-derived gangliosides may blunt antitumor immune responses in patients with RCCs.

• Quantification of Carbonic Anhydrase IX Expression in Serum and Tissue of Renal Cell Carcinoma Patients Using Enzyme-linked Immunosorbent Assay: Prognostic and Diagnostic Potentials

  Authors: Grace X. Zhou, Joanna Ireland, Patricia Rayman, James Finke, Ming Zhou

  Urology.

  ObjectivesTo validate enzyme-linked immunosorbent assay (ELISA) as an accurate and objective method to measure carbonic anhydrase IX (CAIX) expression in RCC tissue specimens and to also investigateObjectivesTo validate enzyme-linked immunosorbent assay (ELISA) as an accurate and objective method to measure carbonic anhydrase IX (CAIX) expression in RCC tissue specimens and to also investigate the diagnostic and prognostic utility of serum CAIX levels. Recent studies found protein levels of CAIX, quantified using immunohistochemistry, correlated with clinical outcomes and therapeutic response to immunotherapy in advanced stage clear cell renal cell carcinoma (CCRCC).Materials and MethodsA CAIX ELISA kit was used to measure the CAIX levels in the serum and tissue of 21 CCRCCs and 19 non-CCRCCs. CAIX immunohistochemical stains were performed on the corresponding formalin fixed, paraffin embedded tumor tissues.ResultsTissue CAIX levels measured by ELISA highly correlated with the CAIX levels detected by immunohistochemistry (Pearson correlation coefficient = .802, P <.001). The CCRCC tumor tissue had significantly higher CAIX levels than non-CCRCC tissue (77 023 vs 938 pg/mL, P <.001) detected by ELISA. Serum CAIX levels in CCRCC patients were significantly higher than in non-CCRCC patients (126.1 vs 2.1 pg/mL, P = .013). The serum CAIX levels detected by ELISA correlated with the tumor size in CCRCC patients (Pearson correlation coefficient = .498, P = .036).ConclusionsELISA provides a quantitative and objective method to measure CAIX levels in renal tumors. The tissue CAIX levels measured with ELISA highly correlate with the results obtained with the standard immunohistochemistry. Serum CAIX levels are significantly higher in CCRCC than in non-CCRCC and may aid the differential diagnosis of RCC. Serum CAIX levels also correlate with the tumor size in CCRCC patients.

• Alterations in NF B Activation in T Lymphocytes of Patients With Renal Cell Carcinoma

  Authors: Robert G Uzzo, Peter E. Clark, Patricia Rayman, Tracy Bloom, Lisa Rybicki, Andrew C Novick, Ronald M Bukowski, James H Finke