Patricia Rayman

Sunitinib Mediates Reversal of Myeloid-Derived Suppressor Cell Accumulation in Renal Cell Carcinoma Patients

Purpose: Immune dysfunction reported in renal cell carcinoma (RCC) patients may contribute to tumor progression. Myeloid-derived suppressor cells (MDSC) represent one mechanism by which tumors induce T-cell suppression. Several factors pivotal to the accumulation of MDSC are targeted by the tyrosine kinase inhibitor, sunitinib. The effect of sunitinib on MDSC-mediated immunosuppression in RCC patients has been investigated. Experimental Design: Patient peripheral blood levels of MDSC and regulatory T-cell (Treg) and T-cell production of IFN-γ were evaluated before and after sunitinib treatment. Correlations between MDSC and Treg normalization as well as T-cell production of IFN-γ were examined. The in vitro effect of sunitinib on patient MDSC was evaluated. Results: Metastatic RCC patients had elevated levels of CD33+HLA-DR− and CD15+CD14− MDSC, and these were partially overlapping populations. Treatment with sunitinib resulted in significant reduction in MDSC measured by several criteria. Sunitinib-mediated reduction in MDSC was correlated with reversal of type 1 T-cell suppression, an effect that could be reproduced by the depletion of MDSC in vitro. MDSC reduction in response to sunitinib correlated with a reversal of CD3+CD4+CD25hiFoxp3+ Treg cell elevation. No correlation existed between a change in tumor burden and a change in MDSC, Treg, or T-cell production of IFN-γ. In vitro addition of sunitinib reduced MDSC viability and suppressive effect when used at ≥1.0 μg/mL. Sunitinib did not induce MDSC maturation in vitro. Conclusions: Sunitinib-based therapy has the potential to modulate antitumor immunity by reversing MDSC-mediated tumor-induced immunosuppression.

Sunitinib Reverses Type-1 Immune Suppression and Decreases T-Regulatory Cells in Renal Cell Carcinoma Patients

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-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 (IFNγ) 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 IFNγ-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.

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

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.

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

Activation of the transcription factor nuclear factor-kappaB (NFkappaB) 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 NFkappaB binding activity is downstream from the stimulus-induced degradation of the cytoplasmic factor IkappaBalpha. Tumor-derived soluble products from cultured RCC explants inhibit NFkappaB activity in T cells from healthy volunteers, despite a normal level of stimulus-induced IkappaBalpha 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 G(m1) and G(d1a), suppressed NFkappaB binding activity in T cells and reduced expression of the cytokines IL-2 and IFN-gamma. Taken together, our findings suggest that tumor-derived gangliosides may blunt antitumor immune responses in patients with RCCs.

Characterization of a human renal cell carcinoma specific cytotoxic CD8+ T cell line

Human renal cell carcinoma (RCC) is one of the tumors most sensitive to immunotherapy and in that regard it is similar to malignant melanoma. Clinical studies reporting responses to therapy suggested that a host immune response may be involved in the antitumor activity induced by immunotherapy in these tumors. Although detection of a specific T cell response to melanoma has been well documented, this has not been the case for RCC. The lytic response of interleukin-2 (IL-2) cultured tumor-infiltrating lymphocytes (TILs) from RCC has been nonspecific. However, in this report we describe a CD8+ TIL line derived from a primary RCC tumor that displays specificity for the autologous tumor. This line is lytic for autologous RCC but does not lyse autologous lymphoblasts, allogeneic RCC, or tumor cell lines of other histologic types. It also proliferates specifically to the autologous tumor in the absence of exogenous IL-2. However, the addition of low dose IL-2 to the cultures can significantly augment its proliferative response. When stimulated with autologous RCC but not allogeneic RCC the CD8+ line will produce interferon-gamma (IFN-gamma). It appears that recognition of RCC by this TIL line is through the TCR/CD3 complex because anti-CD3 antibody blocks the lytic activity, proliferation and IFN-gamma production of the line in response to the autologous tumor. Additional studies illustrate that cytotoxic T lymphocytes with apparent specificity for the autologous tumor are present in unseparated cultured TILs that can be detected by clonal analysis. Collectively these results suggest that there is a specific T cell response to human RCC.

Characterization of tumor-infiltrating lymphocyte subsets from human renal cell carcinoma: Specific reactivity defined by cytotoxicity, interferon-γ secretion, and proliferation

The detection of T cells with specificity for human renal cell carcinoma (RCC) has been difficult to document. In an attempt to improve our identification of RCC-reactive T cells, tumor-infiltrating lymphocytes (TIL) were expanded in interleukin-2/interleukin-4 (IL-2/IL-4) and then separated into CD4+ and CD8+ subsets using antibody-coated biomagnetic beads. TIL grown in IL-2/IL-4 expanded to greater numbers than TIL grown in IL-2 alone. From 16 patients in whom subset separation was performed, three CD4+ and three CD8+ TIL consistently had specificity for RCC that was detected by cytotoxicity, proliferation, or interferon-gamma (IFN-gamma) production. Four of the six lines were derived from the IL-2/IL-4 cultures. Two CD8+ TIL lines displayed specific lytic activity, lysing the autologous tumor but not allogeneic RCC or nonrenal tumors. Moreover, the lytic activity of these lines was blocked by anti-CD3 antibody, suggesting that tumor recognition was through the TCR/CD3 complex. Two additional TIL lines showed preferential lysis of RCC because they were cytotoxic for autologous tumor and one or more allogeneic RCC but not other tumor types. Two nonlytic CD4+ lines as well as the two CD8+ lines that were specifically lytic also produced IFN-gamma in response to the autologous tumor but not allogeneic RCC. Although these TIL lines produce IFN-gamma when stimulated with tumor alone, the addition of 5 U/ml of IL-2 significantly enhanced IFN-gamma secretion. The four TIL lines that showed specificity for RCC in terms of IFN-gamma production also had enhanced proliferation to the autologous RCC plus IL-2 but not to multiple allogeneic RCC plus IL-2. These studies demonstrate that TIL from RCC patients contain both CD4+ and CD8+ T cells that have specificity for RCC. In addition to cytotoxicity, specificity to RCC can be defined by IFN-gamma production and proliferation.

Glioblastomas Induce T-Lymphocyte Death by Two Distinct Pathways Involving Gangliosides and CD70

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 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.

GM2 Expression in Renal Cell Carcinoma: Potential Role in Tumor-Induced T-Cell Dysfunction

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 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.

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

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) γ] 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-γ 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-γ-producing type 1 effector cells. This represents a relevant mechanism by which renal cell carcinoma can inhibit protective antitumor immunity.

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

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 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.