The P2Y11 receptor of human M2 macrophages activates canonical and IL-1 receptor signaling to translate the extracellular danger signal ATP into anti-inflammatory and pro-angiogenic responses.

The cytoprotective ATP receptor P2Y11 is upregulated during M2 macrophage differentiation and contributes to the anti-inflammatory properties of this macrophage subset. Here, we studied P2Y11-induced reprogramming of human M2 macrophages at the level of mRNA and protein expression. Upregulation of IL-1 receptor (IL-1R) and its known downstream effectors VEGF, CCL20 and SOCS3 as well as downregulation of the ATP-degrading ecto-ATPase CD39 emerged as hallmarks of P2Y11 activation. The anti-inflammatory signature of the P2Y11 transcriptome was further characterized by the downregulation of P2RX7, toll-like receptors and inflammasome components. P2Y11-induced IL-1R upregulation formed the basis for reinforced IL-1 responsiveness of activated M2 macrophages, as IL-1α and IL-1ß each enhanced P2Y11-induced secretion of VEGF and CCL20 as well as the previously reported shedding of soluble tumor necrosis factor receptor 2 (sTNFR2). Raising intracellular cyclic AMP (cAMP) in M2 macrophages through phosphodiesterase 4 inhibition enhanced P2Y11-driven responses. The cAMP-binding effector, exchange protein activated by cAMP 1 (Epac1), which is known to induce SOCS3, differentially regulated the P2Y11/IL-1R response because pharmacological Epac1 inhibition enhanced sTNFR2 and CCL20 release, but had no effect on VEGF secretion. In addition to cAMP, calcium and protein kinase C participated in P2Y11 signaling. Our study reveals how P2Y11 harnesses canonical and IL-1R signaling to promote an anti-inflammatory and pro-angiogenic switch of human M2 macrophages, which may be controlled in part by an Epac1-SOCS3 axis.

Intratumoral Th2 predisposition combines with an increased Th1 functional phenotype in clinical response to intravesical BCG in bladder cancer.

Th1-type immunity is considered to be required for efficient response to BCG in bladder cancer, although Th2 predisposition of BCG responders has recently been reported. The aim was to evaluate the relationship of Th1 and Th2 components in 23 patients undergoing BCG treatment. Peripheral blood, serum and urine samples were prospectively collected at baseline, during and after BCG. Th1 (neopterin, tryptophan, kynurenine, kynurenine-to-tryptophan ratio (KTR), IL-12, IFN-γ, soluble TNF-R75 and IL-2Rα) and Th2 (IL-4, IL-10) biomarkers as well as CD4 expression in T helper (Th), effector and regulatory T cells were determined. Local immune cell subsets were measured on formalin-fixed, paraffin-embedded cancer tissue by immunohistochemistry to examine expression of transcription factors that control Th1 (T-bet) and Th2-type (GATA3) immunity. We confirmed a Th2 predisposition with a mean GATA3/T-bet ratio of 5.51. BCG responders showed significantly higher levels of urinary (p = 0.003) and serum neopterin (p = 0.012), kynurenine (p = 0.015), KTR (p = 0.005), IFN-γ (p = 0.005) and IL-12 (p = 0.003) during therapy, whereas levels of IL-10 decreased significantly (p < 0.001) compared to non-responders. GATA3/T-bet ratio correlated positively with serum neopterin (p = 0.008), IFN-γ (p = 0.013) and KTR (p = 0.018) after the first BCG instillation. We observed a significant increase in CD4 expression in the Th cell population (p < 0.05), with only a modest tendency toward higher frequency in responders compared to non-responders (p = 0.303). The combined assessment of GATA3/T-bet ratio, neopterin and KTR may be a useful biomarker in predicting BCG response. Th2-promoting factors such as GATA3 may trigger Th1-type immune responses and thus contribute to the BCG success.

Essential requirements of zoledronate-induced cytokine and γδ T cell proliferative responses.

The potent nitrogen-containing bisphosphonate zoledronate inhibits farnesyl pyrophosphate synthase, a key enzyme of the mevalonate pathway that is often hyperactive in malignant cells. Zoledronate activates human Vγ9Vδ2 T cells, which are immune sentinels of cell stress and tumors, through upstream accumulation of the cognate Ag isopentenyl pyrophosphate. IL-18 was shown to enhance zoledronate-induced γδ T cell activation. Although monocytes have been considered important accessory cells that provide the Ag isopentenyl pyrophosphate, CD56(bright)CD11c(+) NK cells were postulated to mediate the costimulatory effects of IL-18. We report in this article that downstream depletion of geranylgeranyl pyrophosphate (GGPP), which is required for protein prenylation, caused cell stress in monocytes, followed by caspase-1-mediated maturation and release of IL-18, which, in turn, induced γδ T cell CCL2. Likewise, zoledronate caused a substantial delay in γδ T cell expansion, which could be skipped by GGPP supplementation. Moreover, repletion of GGPP, which prevented acute zoledronate toxicity, and supplementation with IL-18, which strongly upregulated IL-2Rα (CD25) and favored the central memory phenotype, were sufficient to enable zoledronate-induced expansion of highly purified γδ T cells, even when starting cell numbers were as low as 10(4) γδ T cells. Our study reveals essential components of γδ T cell activation and indicates that exogenous IL-18, which can directly costimulate γδ T cells, eliminates the need for any accessory cells. Our findings will facilitate the generation of robust γδ T cells from small blood or tissue samples for cancer immunotherapy and immune-monitoring purposes.

Quality of life during dendritic cell vaccination against metastatic renal cell carcinoma.

Patients with metastatic renal cell carcinoma (RCC) undergoing cytokine or targeted therapies may show a remarkable decline in quality of life (QoL). We wanted to evaluate QoL in patients with metastatic RCC undergoing therapeutic vaccination with dendritic cells (DCs). In a cross-sectional analysis, QoL was therefore assessed in RCC patients participating in three consecutive clinical trials of DC vaccination. Before the first and after the third vaccination with DCs, patients completed a QoL questionnaire (EORTC QLQ-C30, version 3). Data were transformed into scale scores and analysed using SPSS 12.0 software. Mean values of the resulting scores obtained before and after DC vaccination were compared using students t test and Wilcoxon rank-sum test. P < 0.05 was considered statistically significant. The questionnaire was completed by 55 of 71 patients (compliance rate, 77.5%) who had a median age of 58.7 years (from 30 to 75 years). No significant reductions in functioning scales including physical, emotional and social criteria as well as symptom scores, which assess typical symptoms of tumour therapies, were observed indicating that QoL remained high during DC vaccination. Significant correlations were found between overall survival and functional as well as symptom scores. Our data indicate that DC vaccination, which is a personalised treatment modality, maintains QoL and thus represents an attractive nontoxic treatment option for patients with metastatic RCC. It will be important to identify the most effective conditions of DC vaccination including combinations with other therapeutics to maximise clinical efficacy while still preserving QoL.

CD56+ human blood dendritic cells effectively promote TH1-type gammadelta T-cell responses.

CD56+ human dendritic cells (DCs) have recently been shown to differentiate from monocytes in response to GM-CSF and type 1 interferon in vitro. We show here that CD56+ cells freshly isolated from human peripheral blood contain a substantial subset of CD14+CD86+HLA-DR+ cells, which have the appearance of intermediate-sized lymphocytes but spontaneously differentiate into enlarged DC-like cells with substantially increased HLA-DR and CD86 expression or into fully mature CD83+ DCs in response to appropriate cytokines. Stimulation of CD56+ cells containing both DCs and abundant gammadelta T cells with zoledronate and interleukin-2 (IL-2) resulted in the rapid expansion of gammadelta T cells as well as in IFN-gamma, TNF-alpha, and IL-1beta but not in IL-4, IL-10, or IL-17 production. IFN-gamma, TNF-alpha, and IL-1beta production were almost completely abolished by depleting CD14+ cells from the CD56+ subset before stimulation. Likewise, depletion of CD14+ cells dramatically impaired gammadelta T-cell expansion. IFN-gamma production could also be blocked by neutralizing the effects of endogenous IL-1beta and TNF-alpha. Conversely, addition of recombinant IL-1beta, TNF-alpha, or both further enhanced IFN-gamma production and strongly up-regulated IL-6 production. Our data indicate that CD56+ DCs from human blood are capable of stimulating CD56+ gammadelta T cells, which may be harnessed for immunotherapy.

The human G protein-coupled ATP receptor P2Y11 is a target for anti-inflammatory strategies.

BACKGROUND AND PURPOSE: The ATP receptor P2Y11 , which couples to Gq and Gs proteins, senses cell stress and promotes cytoprotective responses. P2Y11 receptors are upregulated during differentiation of M2 macrophages. However, it is unclear whether and how P2Y11 receptors contribute to the anti-inflammatory properties of M2 macrophages. EXPERIMENTAL APPROACH: Transcriptome and secretome profiling of ectopic P2Y11 receptors was used to analyse their signalling and function. Findings were validated in human monocyte-derived M2 macrophages. The suramin analogue NF340 and P2Y11 receptor-knockout cells confirmed that agonist-mediated responses were specific to P2Y11 receptor stimulation. KEY RESULTS: Temporal transcriptome profiling of P2Y11 receptor stimulation showed a strong and tightly controlled response of IL-1 receptors, including activation of the IL-1 receptor target genes, IL6 and IL8. Secretome profiling confirmed the presence of IL-6 and IL-8 proteins and additionally identified soluble tumour necrosis factor receptor 1 and 2 (sTNFR1 and sTNFR2) as targets of P2Y11 receptor activation. Raised levels of intracellular cAMP in M2 macrophages, after inhibition of phosphodiesterases (PDE), especially PDE4, strongly increased P2Y11 receptor-induced release of sTNFR2 through ectodomain shedding mediated by TNF-α converting enzyme (TACE/ADAM17). Both IL-1α and IL-1ß synergistically enhanced P2Y11 receptor- induced IL-6 and IL-8 secretion and release of sTNFR2. During lipopolysaccharide-induced activation of TLR4, which shares the downstream signalling pathway with IL-1 receptors, P2Y11 receptors specifically prevented secretion of TNF-α. CONCLUSIONS AND IMPLICATIONS: Targeting P2Y11 receptors activates IL-1 receptor signalling to promote sTNFR2 release and suppress TLR4 signalling to prevent TNF-α secretion, thus facilitating resolution of inflammation.

Serum IgG against Candida predict survival in patients with metastatic renal cell carcinoma.

BACKGROUND AND AIM: In contrast to hematologic malignancies, little is known about the role of fungi in the development and progression of solid tumors. This prompted us to analyze and correlate serum levels of different fungal IgG with survival of patients with metastatic renal cell carcinoma. PATIENTS AND METHODS: Serum IgG to Candida sp., Saccharomyces cerevisiae and Aspergillus fumigatus were measured in a cross-sectional study in 64 patients with advanced disease. Univariate and multivariate analyses were chosen to study serum IgG as prognostic indicators. RESULTS: Median follow-up was 29.0 months (range 0.3-122). Median overall survival of patients, which tested negative for Candida IgG, was significantly increased (median not reached, >29 months) compared with Candida IgG positive patients (17.8 months, P = 0.002). Median survival of Saccharomyces IgG negative patients was 33.1 months as opposed to 19.4 months in Saccharomyces IgG positive patients, although this difference was not significant (P = 0.281). No difference in overall survival was found between Aspergillus IgG positive patients (28.0 months) and Aspergillus IgG negative patients (29.1 months) (P = 0.181). Cox backward-stepwise regression confirmed Candida IgG as the strongest predictor of survival in metastatic renal cell carcinoma patients (risk ratio 3.27, P = 0.001, [95% CI 1.86-5.73]). CONCLUSION: Our findings suggest that IgG antibodies directed against yeast fungi and particularly against Candida but not against mold fungi have prognostic relevance.

The Human G Protein-Coupled ATP Receptor P2Y11 Is Associated With IL-10 Driven Macrophage Differentiation.

The G protein-coupled P2Y11 receptor is known to sense extracellular ATP during inflammatory and immune responses. The dinucleotide NAD+ has also been proposed to be a P2Y11 receptor ligand but its role is less clear. Here, we have examined for the first time human P2Y11 receptor protein levels and show that the receptor was upregulated during polarization of M2 macrophages. IL-10 reinforced P2Y11 receptor expression during differentiation of M2c macrophages expressing CD163, CD16, and CD274 (PD-L1). Nutlin-3a mediated p53 stabilization further increased P2Y11 receptor, CD16, and PD-L1 expression. AMP-activated kinase (AMPK), which mediates anti-inflammatory effects of IL-10, and nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway, which is under the control of AMPK, were also required for P2Y11 receptor expression. The P2Y11 receptor agonist ATPγS and NAD+ could independently stimulate the production of IL-8 in M2 macrophages, however, only the ATPγS-induced response was mediated by P2Y11 receptor. Both in a recombinant system and in macrophages, P2Y11 receptor-driven IL-8 production predominantly depended on IkB kinase (IKK), and extracellular signal-regulated kinase (ERK). In conclusion, our data indicate that an AMPK-NAMPT-NAD+ signaling axis promotes P2Y11 receptor expression during M2 polarization of human macrophages in response to IL-10. PD-L1 expressing M2c macrophages that secrete the cancer-promoting chemokine IL-8 in response to P2Y11 receptor stimulation may represent an important target in checkpoint blockade immunotherapy.

The cyclopentenone prostaglandin PGA2 costimulates the maturation of human dendritic cells.

OBJECTIVE: Dendritic cells (DCs), also referred to as the sentinels of the immune system, induce and coordinate important functions of immune surveillance. Prostaglandin E2 (PGE2), a member of the eicosanoid family of arachidonic acid derivatives, is widely used to enhance the TNF-alpha-driven maturation of human monocyte-derived DCs (moDCs) both in basic research and in clinical settings. However, PGE2 is known to rapidly undergo nonenzymatic dehydration to produce PGA2, a member of the cyclopentenone PGs, which have been implicated in anti-inflammatory processes. METHODS: In a side-by-side analysis we therefore compared the influence of PGE2 and PGA2 on the TNF-alpha-induced maturation of human moDCs. Phenotypic changes, migratory responses towards MIP-3beta, and T-cell responses induced by the differentially matured moDCs were assessed. RESULTS: We found that PGA2 is nearly as potent as PGE2 in costimulating the TNF-alpha-induced phenotypic maturation of human moDCs. Both PGE2 and PGA2 further enhanced the migratory and T-cell-stimulatory capacity of TNF-alpha-treated moDCs. Maturation of moDCs with either PGE2 or PGA2 resulted in enhanced IFN-gamma, TNF-alpha, and IL-5 production and repressed IL-10 production in allogeneic mixed leukocyte cultures. PGE2 was always more potent than PGA2. CONCLUSIONS: Our data suggest that some of the effects attributed to PGE2 may in fact be mediated by its degradation product PGA2. This work also demonstrates that cyclopentenone PGs may have pro-inflammatory properties and that both PGE2 and PGA2 can contribute to the development of Th1-type immune responses.

Ecto-ATPase CD39 Inactivates Isoprenoid-Derived Vγ9Vδ2 T Cell Phosphoantigens.

In humans, Vγ9Vδ2 T cells respond to self and pathogen-associated, diphosphate-containing isoprenoids, also known as phosphoantigens (pAgs). However, activation and homeostasis of Vγ9Vδ2 T cells remain incompletely understood. Here, we show that pAgs induced expression of the ecto-ATPase CD39, which, however, not only hydrolyzed ATP but also abrogated the γδ T cell receptor (TCR) agonistic activity of self and microbial pAgs (C5 to C15). Only mevalonate-derived geranylgeranyl diphosphate (GGPP, C20) resisted CD39-mediated hydrolysis and acted as a regulator of CD39 expression and activity. GGPP enhanced macrophage differentiation in response to the tissue stress cytokine interleukin-15. In addition, GGPP-imprinted macrophage-like cells displayed increased capacity to produce IL-1ß as well as the chemokine CCL2 and preferentially activated CD161-expressing CD4(+) T cells in an innate-like manner. Our studies reveal a previously unrecognized immunoregulatory function of CD39 and highlight a particular role of GGPP among pAgs.

Generation of clinical-grade monocyte-derived dendritic cells using the CliniMACS system.

We describe the generation of monocyte-derived dendritic cells (MoDC) from leukapheresis products using the CliniMACS system from Miltenyi Biotec. In a clinical setting, the method turned out to be feasible for the generation of clinical-grade MoDC from patients with metastatic renal-cell carcinoma. MoDC generated with this system exhibited a fully mature phenotype as well as high migratory and T-cell stimulatory capacity.

IL-2 costimulation enables statin-mediated activation of human NK cells, preferentially through a mechanism involving CD56+ dendritic cells.

Statins are inhibitors of cholesterol biosynthesis and protein prenylation that also have been studied in cancer therapy and chemoprevention. With regard to natural killer (NK) cells, only inhibitory effects of statins such as suppression of granule exocytosis have been reported so far. In this study, we show that statins can cooperate with IL-2 to potently induce the activation of CD56(dim) NK cells in a synergistic, time- and dose-dependent fashion. Supplementation experiments revealed that the statin effect was specific to inhibition of their target hydroxymethylglutaryl coenzyme A reductase and that downstream depletion of geranylgeranyl pyrophosphate was responsible for cooperating with IL-2 in NK cell activation. Mechanistic studies revealed that CD56(+)HLA-DR(+)CD14(+) dendritic cell (DC)-like accessory cells mediated the ability of statin to activate NK cells. In contrast, BDCA-1(+) (CD1c(+)) myeloid DCs, which partially expressed CD56, were somewhat less potent. Conventional blood monocytes, which lack CD56, exhibited the lowest accessory cell capacity. NK cell IFN-γ production was IL-12 independent but required endogenous IL-18, IL-1ß, and caspase-1 activity. Statins directly induced apoptosis in human cancer cell lines and cooperated with NK cell-derived IFN-γ to generate potent cytotoxic antitumor effects in vitro even in the presence of statin-mediated inhibitory effects on granule exocytosis. Our work reveals novel and unexpected immunomodulatory properties of statins, which might be harnessed for the treatment of cancer.

Serum antibodies against Saccharomyces cerevisiae: a new prognostic indicator in metastatic renal-cell carcinoma.

PURPOSE: A recent study reported that a diet rich in bread and refined cereals might have an unfavorable role in the development of renal cell carcinoma (RCC). To test whether an underlying intolerance of bread ingredients is responsible for the unfavorable influence of bread on RCC, we examined patient sera for the presence of food-specific IgG. EXPERIMENTAL DESIGN: A commercial test was used to detect food-specific IgG directed against a panel of 113 food antigens in sera of 54 patients with metastatic RCC. Kaplan-Meier estimates were used for univariate survival analysis, and differences in survival curves were assessed with the log-rank test. Multivariate survival analysis was done using a Cox regression model. RESULTS: We found that RCC patients with elevated serum levels of IgG antibodies against S. cerevisiae, commonly known as baker's yeast and yet another bread component, have an unfavorable clinical course. Median survival of patients with high levels of S. cerevisiae IgG was only 17.8 months, whereas median survival of patients with low S. cerevisiae IgG was 43.8 months (P = 0.0022; log-rank). Multivariate survival analysis identified high levels of S. cerevisiae IgG as a strong and independent prognostic risk factor (risk ratio 4.6, P = 0.001; 95% CI 1.61-13.08). CONCLUSIONS: Our findings indicate that serum levels of IgG against S. cerevisiae may predict survival in patients with metastatic RCC. The data suggest not cereals but baker's yeast being the critical component of bread that may cause immune deviation and impaired immunosurveillance in predisposed RCC patients.

Antigen-independent immune responses after dendritic cell vaccination.

The ability of cultured, antigen-loaded dendritic cells (DCs) to induce antigen-specific T cell immunity in vivo has previously been demonstrated and confirmed. Immune monitoring naturally focuses on immunity against vaccine antigens and may thus ignore other effects of DC vaccination. Here we therefore focused on antigen-independent responses induced by DC vaccination of renal cell carcinoma patients. In addition to the anticipated response against the vaccine antigen KLH, vaccination with CD83(+) monocyte-derived DCs resulted in a strong increase in the ex vivo proliferative and cytokine responses of PBMCs stimulated with LPS or BCG. In addition, LPS strongly enhanced the KLH-induced proliferative and cytokine response of PBMCs. Moreover, proliferative and cytokine responses of PBMCs stimulated with the homeostatic cytokines IL-7 and IL-15 were also clearly enhanced after DC vaccination. In contrast to LPS induced proliferation, which is well known to depend on monocytes, IL-7 induced proliferation was substantially enhanced after monocyte depletion indicating that monocytes limit IL-7 induced lymphocyte expansion. Our data indicate that DC vaccination leads to an increase in the ex vivo responsiveness of patient PBMCs consistent with a DC vaccination induced enhancement of T cell memory. Our findings also suggest that incorporation of bacterial components and homeostatic cytokines into immunotherapy protocols may be useful in order to enhance the efficacy of DC vaccination and that monocytes may limit DC vaccination induced immunity.

Bee venom secretory phospholipase A2 and phosphatidylinositol-homologues cooperatively disrupt membrane integrity, abrogate signal transduction and inhibit proliferation of renal cancer cells.

Bee venom secretory phospholipase A2 (bv-sPLA2) and phosphatidylinositol-(3,4)-bisphosphate (PtdIns(3,4)P2) act synergistically to induce cell death in tumour cells of various origins with concomitant stimulation of the immune system. Here, we investigated the mechanisms involved in such actions and examined structural requirements of PtdIns-homologues to inhibit tumour cells in combination with bv-sPLA2. Renal cancer cells were treated with bv-sPLA2 alone or in combination with PtdIns-homologues. Inhibitory effects on [(3)H] thymidine incorporation and intracellular signal transduction pathways were tested. Reaction products generated by bv-sPLA2 interaction with PtdIns(3,4)P2 were identified by mass spectrometry. Among the tested PtdIns-homologues those with a phosphate esterified to position 3 of the inositol head group, were most efficient in cooperating with bv-sPLA2 to block tumour cell proliferation. Growth inhibition induced by the combined action of bv-sPLA2 with either PtdIns(3,4)bisphosphate or PtdIns(3,4,5)trisphosphate were synergistic and accompanied by potent cell lysis. In contrast, PtdIns, which lacked the phosphate group at position 3, failed to promote synergistic growth inhibition. The combined administration of PtdIns(3,4)P2 and bv-sPLA2 abrogated signal transduction mediated by extracellular signal regulated kinase 1 and 2 and prevented transduction of survival signals mediated by protein kinase B. Surface expression of the epidermal growth factor (EGF)-receptor was reduced after PtdIns(3,4)P2-bv-sPLA2 administration and associated with a blockade of EGF-induced signalling. In addition, mass spectroscopy revealed that bv-sPLA2 cleaves PtdIns(3,4)P2 to generate lyso-PtdIns(3,4)P2. In conclusion, we suggest that the cytotoxic activity mediated by PtdIns(3,4)P2 and bv-sPLA2 is due to cell death that results from disruption of membrane integrity, abrogation of signal transduction and the generation of cytotoxic lyso-PtdIns(3,4)P2.

Antitumor action and immune activation through cooperation of bee venom secretory phospholipase A2 and phosphatidylinositol-(3,4)-bisphosphate.

We evaluated tumor cell growth modulation by bee venom secretory phospholipase A2 (bv-sPLA2) and phosphatidylinositol-(3,4)-bisphosphate as well as potential cooperative effects. In addition, the immunomodulatory impact of tumor cell treatment was examined by monitoring changes in phenotype and function of monocyte-derived dendritic cells (moDCs) cocultured with pretreated tumor cells. Bv-sPLA2 or phosphatidylinositol-(3,4)-bisphosphate alone displayed moderate effects on the proliferation of A498 renal cell carcinoma cells, T-47D breast cancer cells, DU145 prostate cancer cells and BEAS-2B transformed lung cells. However, when bv-sPLA2 was coadministered with phosphatidylinositol-(3,4)-bisphosphate a potent inhibition of [3H] thymidine incorporation into all tested cell lines occurred. This inhibition was due to massive cell lysis that reduced the number of cells with proliferative capacity. Importantly, tumor cell lysates generated with bv-sPLA2 plus phosphatidylinositol-(3,4)-bisphosphate induced maturation of human moDCs demonstrated by enhanced expression of CD83 and improved stimulation in allogeneic mixed leukocyte reactions. Our data demonstrate that bv-sPLA2 and phosphatidylinositol-(3,4)-bisphosphate synergistically generate tumor lysates which enhance the maturation of immunostimulatory human monocyte-derived dendritic cells. Such tumor lysates which represent complex mixtures of tumor antigens and simultaneously display potent adjuvant properties meet all requirements of a tumor vaccine.

IL-4 inhibits the TNF-alpha induced proliferation of renal cell carcinoma (RCC) and cooperates with TNF-alpha to induce apoptotic and cytokine responses by RCC: implications for antitumor immune responses.

OBJECTIVE: While previous reports clearly demonstrated antiproliferative effects of IL-4 on renal cell carcinoma (RCC) in vitro, the administration of IL-4 to patients with metastatic RCC in clinical trials could not recapitulate the promising preclinical results. In the present study we wanted to examine the context of IL-4 action and to establish conditions of enhanced IL-4 efficacy. METHODS: Primary and permanent human RCC cells were cultured in either serum-supplemented or chemically defined, serum-free culture medium in the presence or absence of cytokines. Cell proliferation was assessed as [(3)H]-thymidine incorporation. Cell apoptosis was measured using the fluorescent DNA intercalator 7-aminoactinomycin D and flow cytometry. In addition, culture media conditioned by RCC were subjected to cytokine antibody array and cytokine multiplex analysis. RESULTS: Our results indicate that the previously reported antiproliferative effects of IL-4 are serum-dependent. Under serum-free conditions, IL-4 failed to exhibit growth-inhibitory effects or was even growth-stimulatory. In a chemically defined, serum-free medium (AIM-V), however, IL-4 inhibited the TNF-alpha induced proliferation of RCC. IL-4 and TNF-alpha synergistically induced apoptosis of RCC as well as a complex cytokine response by RCC, which included the synergistic upregulation of RANTES and MCP-1. CONCLUSIONS: IL-4 alone has little effect on the spontaneous proliferation of RCC but can prevent the enhancement of proliferation induced by growth promoters like FBS and TNF-alpha. The concomitant growth inhibitory, apoptosis-inducing, and cytokine-enhancing effects of IL-4 in combination with TNF-alpha on RCC support the view that Th2 cytokines may be required for productive immune responses against RCC.

Dendritic-cell activation by secretory phospholipase A2.

Dendritic cells (DCs), also referred to as the sentinels of the immune system, induce and coordinate important functions of immune surveillance. DCs acquire immunity-initiating capacity only after a process of maturation usually induced by ligands that bind to members of the tumor necrosis factor (TNF) or toll-like receptor families. Secretory phospholipase A2 (sPLA2), which hydrolyzes the sn-2 ester bond of glycerophospholipids, regulates a variety of cellular functions including migration of endothelial cells and neurite outgrowth. In the present study we investigated the role of sPLA2 in DC biology. We report that human monocyte-derived DC cultures lack sPLA2 activity but respond to exogenous sPLA2. sPLA2 alone and in cooperation with TNF-alpha and interleukin 1 beta (IL-1beta) induced fatty acid release from DC membranes, which was accompanied by upregulation of surface markers and by an increase in the migratory and immunostimulatory capacity of the DCs. Our findings indicate that secreted enzymes such as sPLA2 can contribute to DC maturation and emphasize the role of lipid mediators in the regulation of immune responses. This observation may also have implications for DC-based vaccine development.

Monitoring of CD4+ and CD8+ T-cell responses after dendritic cell-based immunotherapy using CFSE dye dilution analysis.

CFSE dye dilution analysis and [3H] thymidine incorporation were used side by side to assess proliferative responses of peripheral blood mononuclear cells (PBMCs) after vaccination of renal cell carcinoma patients (n=6) with antigen-loaded dendritic cells. Immune responses against the control antigen keyhole limpet hemocyanin (KLH) were induced in all patients. While [3H] thymidine incorporation revealed a 4 to 977-fold increase in KLH-induced proliferation (mean: 209-fold), CFSE-labeling experiments demonstrated that the KLH-responsive population of postvaccination PBMCs represented 7-53% (mean: 23%). Combining CFSE-labeling with T-cell subset analysis confirmed the presence of CD4+ KLH-reactive T cells but also revealed a substantial population of CD8+ KLH-reactive T cells in one patient as well as minor populations of CD8+ KLH-reactive T cells in three other patients. Our data indicate that CFSE dye dilution analysis is a valuable tool for immune monitoring after dendritic cell vaccination.