Higher Levels Of IL-12p70 Research Articles

T cell subpopulations and cytokine levels in hemodialysis patients

HD patients have impaired adaptive immune responses, which might depend on the primary cause of chronic kidney disease (CKD). We analyzed percentages of T cells subpopulations with the expression of CD69, CD25, CD95, and HLA-DR antigens in HD patients to determine the status of T cell activation. Also, we determined serum levels of cytokines: IL12p70, TNF, IL-10, IL-6, IL-1β, IL-8. HD patients had increased percentages of CD4+CD25+, CD4+CD69+, CD4+HLA-DR+, CD8+CD69+, and CD8+HLA-DR+ cells compared to healthy people. Also, their IL-6 and IL-8 serum levels were higher. Changes in T cell subpopulations were seen in patients with diabetic nephropathy (DN) or ischemic nephropathy (IN) but not with glomerulonephritis (GN). HD patients dialyzed for more than six months had a lower percentage of CD4+CD69+, CD8+HLA-DR+, CD8+CD95+ cells, higher IL-12p70 levels, and lower IL-8 levels. Our results show that HD treatment and CKD cause influence T cell activation status.

Evaluation of cytokine response to extracorporeal membrane oxygenation.

Increased cytokine response is common in patients receiving extracorporeal life support and is often a poor prognostic indicator. There is interest in using adjunctive cytokine adsorption technologies to reduce inflammatory burden, However, it is debated whether extracorporeal membrane oxygenation (ECMO) itself provides therapeutic benefit beyond gas exchange. Thus, we sought to characterize the inflammatory profile of ECMO in the first 72-96h of and quantify its effect on cytokine levels in a case series of patients undergoing ECMO. Eight patients initiating ECMO were studied. Of these, we measured cytokines pre- and post-oxygenator over 96h. Comparisons of cytokine levels were made across the oxygenator and over time. The average age of patients was 64.3years with 62% being male. Centrally cannulated patients had higher IL-6 levels (820.43 vs. 6907.53pg/ml, p<0.03), whereas peripherally cannulated patients had higher IL-12p70 levels (7.73 vs. 2.59pg/ml, p<0.05). Cytokine levels on day one included IL-12p70 (4.17±2.56), IL-6 (4971.23±8569.88), TNF (undetected), IL-8 (346.68±670.18), IL-1B (undetected), and IL-10 (72.27±87.9). Cytokine levels increased over 96h; however, no significant differences were appreciated despite blood product transfusion. On day 3, IL-12p70 levels were significantly lower post-oxygenator (p<0.05). The inflammatory profile of ECMO does not change significantly over the early course of illness when accounting for transfusion. However, the decrease in IL-12p70 specifically at day 3 of ECMO may indicate adsorption of specific inflammatory markers by the oxygenator although the clinical significance of this is still unknown. Further investigation of the oxygenator on cytokine response is warranted.

Protective effect of fungal extracellular vesicles against murine candidiasis.

Extracellular vesicles (EVs) are lipid bilayered compartments released by virtually all living cells, including fungi. Among the diverse molecules carried by fungal EVs, a number of immunogens, virulence factors and regulators have been characterised. Within EVs, these components could potentially impact disease outcomes by interacting with the host. From this perspective, we previously demonstrated that EVs from Candida albicans could be taken up by and activate macrophages and dendritic cells to produce cytokines and express costimulatory molecules. Moreover, pre-treatment of Galleria mellonella larvae with fungal EVs protected the insects against a subsequent lethal infection with C. albicans yeasts. These data indicate that C. albicans EVs are multi-antigenic compartments that activate the innate immune system and could be exploited as vaccine formulations. Here, we investigated whether immunisation with C. albicans EVs induces a protective effect against murine candidiasis in immunosuppressed mice. Total and fungal antigen-specific serum IgG antibodies increased by 21 days after immunisation, confirming the efficacy of the protocol. Vaccination decreased fungal burden in the liver, spleen and kidney of mice challenged with C. albicans. Splenic levels of cytokines indicated a lower inflammatory response in mice immunised with EVs when compared with EVs + Freund's adjuvant (ADJ). Higher levels of IL-12p70, TNFα and IFNγ were detected in mice vaccinated with EVs + ADJ, while IL-12p70, TGFβ, IL-4 and IL-10 were increased when no adjuvants were added. Full protection of lethally challenged mice was observed when EVs were administered, regardless the presence of adjuvant. Physical properties of the EVs were also investigated and EVs produced by C. albicans were relatively stable after storage at 4, -20 or -80°C, keeping their ability to activate dendritic cells and to protect G. mellonella against a lethal candidiasis. Our data suggest that fungal EVs could be a safe source of antigens to be exploited in vaccine formulations.

CpG-PEG Conjugates and their Immune Modulating Effects after Systemic Administration.

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs were found to be able to target cells that express Toll-like receptor 9 to modulate innate and adaptive immune reactions. But their in vivo application in immunotherapy against cancer has not been successful. We attempted in this study to examine polyethylene-glycol (PEG) conjugated CpG ODNs and investigated their mechanism of immune modulation in anti-cancer therapy. CpG-PEG conjugates with different PEG lengths were synthesized. In vitro activity as well as in vivo pharmacokinetics and pharmacodynamics properties were evaluated. CpG-PEG20Ks were found to be able to persist longer in circulation and activate various downstream effector cells. After intravenous injection, they resulted in higher levels of IL-12p70 in the circulation and lower M-MDSC infiltrates in the tumor microenvironment. Such activities were different from those of CpG ODNs without PEGylation, suggesting different PK-PD profiles systemically and locally. Our data support the development of CpG-PEGs as a new therapeutic agent that can be systemically administered to modulate immune responses and the microenvironment in tumor tissues.

A subunit vaccine based on rH-NS induces protection against Mycobacterium tuberculosis infection by inducing the Th1 immune response and activating macrophages.

Mycobacterium tuberculosis (Mtb) is a Gram-positive pathogen which causes tuberculosis in both animals and humans. All tested rH-NS formulations induced a specific Th1 response, as indicated by increased production of interferon γ (IFN-γ) and interleukin 2 (IL-2) by lymphocytes in the spleen of mice which were immunized with rH-NS alone or with rH-NS and the adjuvant cyclic GMP-AMP (cGAMP). Serum from mice immunized with rH-NS with or without adjuvant also had higher levels of IL-12p40 and TNF-α, compared with those from control mice immunized with phosphate-buffered saline. Both vaccines increased protective efficacy in mice which were challenged with Mtb H37Rv, as measured by reduced relative CFU counts in the lungs. We found that rH-NS induced the production of TNF-α, IL-6, and IL-12p40, which relied on the activation of mitogen-activated protein kinases by stimulating the rapid phosphorylation of ERK1/2, p38, and JNK, and on the activation of transcription factor NF-κB in macrophages. Additionally, we also found that rH-NS could interact with TLR2 but not TLR4 in pull-down assays. The rH-NS-induced cytokine production from TLR2-silenced RAW264.7 cells was lower than that from BALB/c macrophages. Prolonged exposure (>24 h) of RAW264.7 cells to rH-NS resulted in a significant enhancement in IFN-γ-induced MHC II expression, which was not found in shTLR2-treated RAW264.7 cells. These results suggest that rH-NS is a TLR2 agonist which induces the production of cytokines by macrophages and up-regulates macrophage function.

Increased percentage of IFN-γ producing CD56+CD3+ cells in active tuberculosis patients upon CFP-10 stimulation of peripheral mononuclear cells

Aiming to identify a possible biomarker that distinguishes immune cellular response of active tuberculosis from latent infection. Peripheral blood mononuclear cells (PBMCs) of pulmonary tuberculosis patients (PTB), tuberculin positive household contacts (TST(+) HHC), and tuberculin negative non-household contacts (TST− Non HHC) were stimulated with PPD or CFP-10 and the percentage of CD69(+) cells, proliferating precursor and IFN-γ producing CD4(+), CD8(+), CD56(+)CD3(−) and CD56(+)CD3(+) cells were compared. IL-2, IL-12p70, IL-15, IL-18 and IL-10 were measured in culture supernatants. PTB and TST+ HHC presented higher percentages of CD69(+) cells, IFN-γ(+) and proliferating precursors in all subpopulations studied and higher IL-12p70 levels than TST- Non HHC. The increased percentage of IFN-γ producing CD56(+)CD3(+) cells in response to CFP-10 in PTB, compared with TST− Non HHC and the ratios between the percentage of CD56(+)CD3(+) cells/CD56(+)CD3(−) and CD8(+) cells producing IFN-γ suggest that these parameters may distinguish active TB from latently infected individuals.

Lymphopenia associated with highly virulent H5N1 virus infection due to plasmacytoid dendritic cell-mediated apoptosis of T cells.

Although lymphopenia is a hallmark of severe infection with highly pathogenic H5N1 and the newly emerged H7N9 influenza viruses in humans, the mechanism(s) by which lethal H5N1 viruses cause lymphopenia in mammalian hosts remains poorly understood. Because influenza-specific T cell responses are initiated in the lung draining lymph nodes (LNs), and lymphocytes subsequently traffic to the lungs or peripheral circulation, we compared the immune responses in the lung draining LNs postinfection with a lethal A/HK/483/97 or nonlethal A/HK/486/97 (H5N1) virus in a mouse model. We found that lethal H5N1, but not nonlethal H5N1, virus infection in mice enhances Fas ligand (FasL) expression on plasmacytoid dendritic cells (pDCs), resulting in apoptosis of influenza-specific CD8(+) T cells via a Fas-FasL-mediated pathway. We also found that pDCs, but not other DC subsets, preferentially accumulate in the lung draining LNs of lethal H5N1 virus-infected mice, and that the induction of FasL expression on pDCs correlates with high levels of IL-12p40 monomer/homodimer in the lung draining LNs. Our data suggest that one of the mechanisms of lymphopenia associated with lethal H5N1 virus infection involves a deleterious role for pDCs.

P55. Dendritic cell vaccination for postremission therapy in AML

Cellular immunotherapy is a highly effective treatment option for patients with acute myeloid leukaemia (AML) as shown by the low relapse rate after allogeneic stem cell transplantation. However, many patients are not eligible for this treatment. This has lead to the development of various immunotherapeutic approaches that aim at inducing autologous cellular and humoral immune responses against AML and specifically against residual leukaemic stem cells (LSCs). Dendritic cells (DCs) are important regulators of the human immune response. We have developed a three-day DC manufacturing protocol that starts with peripheral blood monocytes, e.g. from AML patients in remission following intensive chemotherapy. By using a cytokine cocktail containing a synthetic TLR7/8 agonist, the resulting DCs develop improved immunogenicity. For healthy donors as well as for AML patients, we were able to show that these DCs display a positive costimulatory profile, secrete high levels of IL-12p70, show chemotaxis to CCR7 ligands, and activate NK cells. After loading the DCs with mRNA, they effectively induce antigen-specific T cell responses with a strong type-1 polarization. Due to these properties, this DC type seems highly suitable for application in cancer immunotherapy. We have recently initiated a phase I/II clinical trial for the application of these DCs in the setting of AML postremission strategy. WT1 and PRAME were chosen as leukaemia-associated antigens due to their overexpression on leukaemic blasts and specifically on cells that are enriched for LSCs. DCs transfected with mRNA encoding CMV-pp65 are included into the vaccine as an adjuvant as well as a surrogate antigen. 20 patients with a non-favourable risk profile or with confirmed minimal residual disease (MRD), but who are not eligible for allogeneic stem cell transplantation, will be included. The primary objective of this study is to evaluate feasibility and safety of this immunotherapeutic approach. Important secondary endpoints are immune responses to the applied antigens and MRD control. First results of this study will be presented.

Abstract P4-13-06: Ligation of dectin-1 on tumor infiltrating dendritic cells promotes breast cancer rejection

Abstract Human breast cancer microenvironment displays features of T helper2 (Th2) immunity which promotes tumor development via IL-13/IL13 receptor pathway. This is driven by breast cancer cell-derived thymic stromal lymphopoietin (TSLP) which, by inducing OX40L expression on DCs, contributes to the Th2 immunity conducive to breast tumor development. In order to reprogram the inflammatory pro-tumor Th2 (iTh2) into anti-tumor Th1 microenvironment, we tested the impact of targeting the innate receptors on DCs to render them resistant to tumor environment. We show that intratumoral delivery of b-glucan, a natural ligand for dectin-1 expressed on DCs, blocks the generation of iTh2 cells leading to decreased IL-13 in the tumor microenvironment and prevents breast cancer development in vivo. β-glucan inhibits OX40L expression on tumor-associated DCs which is due to a block in STAT6 phosphorylation. β-glucan-treated DCs, when exposed to breast cancer supernatant, secrete higher levels of IL-12p70 and do not expand iTh2 cells thereby enabling the T helper 1 cells secreting IFN-γ. Importantly, β-glucan exposed DCs expand CD8+ T cells that express CD103, a ligand for E-cadherin. These CD8+ T cells, which produce higher IFN-γ, Granzyme A and Granzyme B, accumulate in the tumors leading to enhanced tumor necrosis in vivo. DC reprogramming by β-glucan is dependent upon dectin-1 engagement. The ligation of TLR7/8 (CL075) and TLR3 (polyI:C) show the same effect in Th2 response inhibition but no effect on the functions of CD8+ T cells. Taken together, our data demonstrate that exploiting pattern recognition receptors on tumor-infiltrating DCs enables cancer rejection. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-13-06.

Abstract 1434: Levels of IL-12 family cytokines in ascitic fluid are associated with extent and resectability of ovarian cancer and correlate with overall survival.

Abstract Background: Ovarian cancer (OC) is commonly associated with ascites and predominantly remains in the peritoneal cavity. Soluble factors present in ascites such as growth factors and cytokines likely play a key role in local tumor behavior including stage, invasiveness and resectability and survival. Members of IL-12 family (IL-12p70, IL-23, IL-27 and IL-35) play a critical role in the regulation of inflammatory response. However, their role as local factors in ovarian cancer growth has not been well studied. We hypothesized that that the level of these important immunomodulatory and pro-inflammatory cytokines are associated with the clinical behavior of OC. Methods: Ascites fluid was collected and analyzed from women undergoing maximal debulking surgery for presumed OC, and relevant clinical endpoints were abstracted. Concentrations of IL-12 related cytokines (IL-23, IL-27, IL-35, and IL-12p70) were assessed by ELISA. Cytokines were also assessed on matching pre-surgery plasma samples. Cytokine concentrations between matched ascites and plasma samples were examined using scatter plots and Spearman correlations. Associations between ascites concentrations and clinical characteristics were assessed using Wilcoxon rank-sum tests. Associations between ascites levels with overall survival in newly diagnosed invasive OC patients were assessed by Cox proportional hazards models using a log2 transformation. Results: Ascites fluid was analyzed from 20 patients with primary epithelial OC including 18 serous histology, 19 patients with stage III or IV disease: 16 (80%) were optimally debulked. We observed that higher levels of IL-12p70 were associated with locally invasive disease including higher rates of sub-optimal debulking (p=0.0074) and stage IV disease (p=0.012). For other IL-12 family members we observed similar associations between higher cytokine levels and suboptimal disease and stage IV disease including IL-23 (p=0.73, p=0.17), IL-27 (p=0.041, p=0.018), and IL35 (p=0.12, p=0.11), respectively. In addition, increase in IL-23 was associated with worse overall survival (log2 HR = 1.85, p=0.031). Conclusions: Higher levels of IL-12 family of cytokines in ascitic fluid are associated with several surrogates of biological aggressiveness in OC including stage, resectability and survival. Given the importance of local tumor environment on clinical behavior, a detailed understanding of IL-12 pathway may help in developing novel therapeutic strategies to treat OC. Citation Format: Eati Basal, Mathew S. Block, Matthew J. Maurer, Krista Goergen, Courtney L. Erskine, Marshall Behrens, Kimberly R. Kalli, Keith Knutson, William Cliby. Levels of IL-12 family cytokines in ascitic fluid are associated with extent and resectability of ovarian cancer and correlate with overall survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1434. doi:10.1158/1538-7445.AM2013-1434

Type II-Activated Murine Macrophages Produce IL-4

BackgroundType II activation of macrophages is known to support Th2 responses development; however, the role of Th2 cytokines (esp. IL-4) on type II activation is unknown. To assess whether the central Th2 cytokine IL-4 can alter type II activation of macrophages, we compared the ability of bone marrow-derived macrophages from wild type (WT) and IL-4Rα-deficient mice to be classically or type II-activated in vitro.ResultsWe found that although both WT and IL-4Rα-deficient macrophages could be classically activated by LPS or type II activated by immune complexes plus LPS, IL-4Rα-deficient macrophages consistently produced much higher levels of IL-12p40 and IL-10 than WT macrophages. Additionally, we discovered that type II macrophages from both strains were capable of producing IL-4; however, this IL-4 was not responsible for the reduced IL-12p40 and IL-10 levels produced by WT mice. Instead, we found that derivation culture conditions (GM-CSF plus IL-3 versus M-CSF) could explain the different responses of BALB/c and IL-4Rα−/− macrophages, and these cytokines shaped the ensuing macrophage such that GM-CSF plus IL-3 promoted more IL-12 and IL-4 while M-CSF led to higher IL-10 production. Finally, we found that enhanced IL-4 production is characteristic of the type II activation state as other type II-activating products showed similar results.ConclusionsTaken together, these results implicate type II activated macrophages as an important innate immune source of IL-4 that may play an important role in shaping adaptive immune responses.

Single Nucleotide Polymorphism in the Interleukin 12B Gene is Associated with Risk for Breast Cancer Development

We analysed the association of a single nucleotide polymorphism (SNP) in the gene encoding the IL-12 subunit p40 (IL12B, rs3212227, A>C) with breast cancer. The SNPs allelic and genotypic frequencies were compared between patients (n = 191) and healthy (n = 194) women in a case-control study from Croatia. The major allele (A) was associated with susceptibility to breast cancer (P = 0.003; OR = 1.67; 95% CI: 1.17-2.38). Likewise, the minor allele (C) was significantly correlated with protection (P = 0.003; OR = 0.60; 95% CI: 0.42-0.86). At the genotype level, AA homozygosity was significantly associated with predisposition to disease (P = 0.013; OR = 1.68, 95% CI: 1.09-2.59), whereas the minor allele homozygosity (CC) was correlated with protection to disease (P = 0.020, OR = 0.28, 95% CI: 0.09-0.91). The heterozygous genotype showed no significant correlation with disease. The product of the IL12B gene (IL-12 p40) can either form a homodimeric cytokine or be part of two pro-inflammatory (IL-12 and IL-23) cytokines. It is presently unclear whether the major allele is associated with higher or lower protein levels of IL-12 p40 and IL-12 p70, which are critical in inflammation and adaptive immune responses. However, as the A allele is high producer of IL12B (p40) mRNA, these results might imply that higher levels of IL-12 p40 (either as homodimers or joined with one or both of the other two subunits) predispose to breast cancer.

Tu1966 Inhibition of CD4+ T-Helper Cells in Crohn's Disease by Imipramine is Mediated via Acid Sphingomyelinase Blockade

G A A b st ra ct s increase of CD11c+ cells in the lamina propria of which are 90% CD11b+ and 40% β2-AR positive. Conclusion: Stimulation of the β2-AR expressed by BMDC results in a significant anti-inflammatory cytokine profile compared to the inflammatory phenotype with high levels of IL-12p70, IL-6, TNFα and low IL-10 of vehicle treated BMDC. In T cell dependant colitis there is a significant increase of CD11c+CD11b+ DC in the lamina propria which express the β2-AR. Treating IBD patients with β2-AR agonists might have the same effect on inflammatory DC as in BMDC and could potentially ameliorate disease activity.

Tu1964 Upregulation of Melanin-Concentrating Hormone Receptor in Lamina Propria CD4+ Cells From Patients With Inflammatory Bowel Disease

G A A b st ra ct s increase of CD11c+ cells in the lamina propria of which are 90% CD11b+ and 40% β2-AR positive. Conclusion: Stimulation of the β2-AR expressed by BMDC results in a significant anti-inflammatory cytokine profile compared to the inflammatory phenotype with high levels of IL-12p70, IL-6, TNFα and low IL-10 of vehicle treated BMDC. In T cell dependant colitis there is a significant increase of CD11c+CD11b+ DC in the lamina propria which express the β2-AR. Treating IBD patients with β2-AR agonists might have the same effect on inflammatory DC as in BMDC and could potentially ameliorate disease activity.

Priming CD8+ T cells with dendritic cells matured using TLR4 and TLR7/8 ligands together enhances generation of CD8+ T cells retaining CD28

AbstractTLRs expressed on dendritic cells (DCs) differentially activate DCs when activated alone or in combination, inducing distinct cytokines and costimulatory molecules that influence T-cell responses. Defining the requirements of DCs to program T cells during priming to become memory rather than effector cells could enhance vaccine development. We used an in vitro system to assess the influence of DC maturation signals on priming naive human CD8+ T cells. Maturation of DCs with lipopolysaccharide (LPS; TLR4) concurrently with R848 (TLR7/8) induced a heterogeneous population of DCs that produced high levels of IL12 p70. Compared with DCs matured with LPS or R848 alone, the DC population matured with both adjuvants primed CD8+ T-cell responses containing an increased proportion of antigen-specific T cells retaining CD28 expression. Priming with a homogenous subpopulation of LPS/R848–matured DCs that were CD83Hi/CD80+/CD86+ reduced this CD28+ subpopulation and induced T cells with an effector cytokine signature, whereas priming with the less mature subpopulations of DCs resulted in minimal T-cell expansion. These results suggest that TLR4 and TLR7/8 signals together induce DCs with fully mature and less mature phenotypes that are both required to more efficiently prime CD8+ T cells with qualities associated with memory T cells.

Abstract 756: Genetically enhanced DC vaccines for cancer: Bringing the ex vivo, in vivo

Abstract Background and Objectives: Dendritic Cells (DCs) are potent antigen presenting cells, capable of activating naïve T-cells to recognize and kill tumor cells. Since DCs are also able to reverse the tumor-induced dormancy of killer and helper T-cells, DC vaccines have been developed to induce anti-tumor immune responses. To improve DC vaccine efficacy, we have focused on augmenting DC function using an inducible MyD88/CD40 signaling switch (iMC) capable of stimulating DC activation in a Th1-polarizing manner. iMC abrogates the need for more traditional, toxic DC activators such as LPS and allows for spatio-temporal control of DC activation, further increasing vaccine efficacy. In ex vivo pre-established tumor studies, DCs pulsed with tumor-specific antigen (TSA) and manipulated to express iMC significantly decreased tumor growth in mice relative to DCs pulsed with TSA alone. While potentially efficacious, ex vivo modification and re-delivery of DCs requires specialized and expensive methods, which limit their utility as primary cancer-fighting agents. Therefore, we have focused on developing a strategy capable of TSA and iMC delivery to in vivo DCs using adenovectors. Treatment of tumor-bearing mice with adenovectors expressing only TSA was effective in significantly decreasing tumor growth. Combining this therapy with adenovectors expressing iMC resulted in only modest therapeutic enhancement. We hypothesize that reliance on simultaneous transgene expression by two separate vectors in the same cell may prevent more potent immunogenicity. Our lab has shown that such double expression occurs in only about 20-50% of relevant cells. Therefore, we have developed a bicistronic adenovector (BAV), encoding both TSA and iMC. Methods: To test dual transgene expression by BAV, DCs were transduced and both β-galactosidase activity (the TSA) and IL-12 secretion (by iMC) were assessed using X-gal staining and IL-12p70 ELISA, respectively. To evaluate anti-tumor response, mice bearing pre-established B16-LacZ tumors were treated via footpad injection of BAV followed by two days of dimerizer drug. Results: DCs transduced with BAV were shown to express the TSA and secrete high levels of IL-12p70 when compared to empty vector or LPS-treated DCs. Treatment of B16-LacZ tumor-bearing mice with BAV resulted in significantly enhanced tumor reduction versus Ad-LacZ alone. Conclusions: In order to further enhance BAV's ability to induce anti-tumor immune responses, we are adapting a method of targeting adenovectors to CD40+ DCs using bi-functional molecules (CFm40L), described by Pereboev et al. (Molecular Therapy, 2004). Our preliminary results have shown that adenoviral modification with CFm40L significantly increases DC transduction efficiency. This technology should further increase the therapeutic efficacy of BAV by specifically targeting it to DCs while at the same time reducing total viral dosage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 756. doi:10.1158/1538-7445.AM2011-756

Abstract 3828: Uncarinic acid C plus IFN-γ generates monocyte-derived dendritic cells and induces a potent Th1 polarization with capacity to migrate

Abstract Uncarinic acid C (URC) is triterpene isolated from Uncaria rhynchophylla and modulates human DC function in a fashion that favors Th1 cell polarization depending on TLR4 signaling. The induction of dendritic cells (DC) is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. Monocyte-derived DCs are used as adjuvant cells in cancer immunotherapy and have shown promising results. We studied the effect of interferons (IFN-α and IFN-γ) and TNF-α on phenotypic and functional maturation, and cytokine production of URC-primed DC in vitro. Human monocytes were exposed to URC alone, or in combination with TNF-α, IFN-α and IFN-γ. All three cytokines induced phenotypically mature DC. The expression levels of CD38 and CCR7 on URC-primed DC were enhanced by IFN-γ and IFN-γ augmented the T cell stimulatory capacity in allo MLR to URC-primed DC. DC matured with URC plus IFN-γ produce high levels of IL-12p70 upon CD40 triggering. IFN-γ secretion from naïve T cells co-cultured with URC-primed DC also augmented by IFN-γ. In addition, DC maturated with URC plus IFN-γ has an intermediate migratory capacity towards CCL19 and CCL21. In contrast, surface molecule up-regulation and function of URC-primed DC were slightly enhanced by TNF-α and IFN-α. These data support that DC differentiated with URC in combination with IFN-γ might be used on DC-based vaccine for cancer immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3828.

Combined use of toll-like receptor agonists and prostaglandin E 2 in the FastDC model: Rapid generation of human monocyte-derived dendritic cells capable of migration and IL-12p70 production

Phenotypical maturation, IL-12p70 production and migration upon chemokine receptor CCR7 ligation are currently proposed as requirements for the use of human monocyte-derived dendritic cells (DC) in antitumoral vaccination. We have previously described a short-term protocol for DC generation from monocytes including stimulation with TNF-α, IL-1β and PGE 2 ( FastDC). These “conventional” FastDC are mature, migrate in response to CCR7 ligation and effectively stimulate autologeous T cells in vitro, but are deficient in IL-12p70 production. Here, conventional FastDC were compared to FastDC activated with different TLR ligands. High levels of IL-12p70 were induced by combined activation of FastDC with TLR4 and TLR7/8 ligands. IL-12 secretion could be maximized by additional T cell-derived stimulation. However, TLR-stimulated FastDC failed to migrate upon CCR7 ligation, independent of additional activation with CD40 ligand and IFN-γ. The presence of PGE 2 during TLR ligation fully restored migratory capacity of FastDC, but left IL-12p70 production and activation of tumor antigen-specific cytotoxic T cells unaffected, challenging previous findings obtained with standard 7-day monocyte-derived DC. The FastDC model thus not only represents an effective tool for antitumoral vaccination, but may also provide novel insights into human DC biology.

614 SERUM GROWTH FACTORS AND APOPTOSIS MARKERS REFLECT INFLAMMATORY ACTIVITY IN CHRONIC HEPATITIS C

R-848 (TLR7/8L), c. serum from a patient chronically infected by HBV (as control), with high viraemia levels (−9 log10), d. serum from a patient chronically infected by HCV, with high viraemia levels (−7 log10), e. supernatant from 2215 hepatocytes (HBV progeny), f. various concentrations of ribavirin. Pulsing with all these stimuli took place either with or without the presence of CD40L-expressing cells as additional pDC activation stimulus. The aim was to explore the effect of the stimuli used, to the pDC Th1skewing capacity and type I IFN production (i.e. secretion of IFN-a, IL-6 and IL-12p40, in cell-culture supernatants, measured with commercially available ELISA kits) and to compare the effect of known TLR ligands (CpG ODN and R-848) to the effect of HCV and also the effect of a component of the currently established antiviral treatment (Ribavirin). Results: Our data demonstrate: a. stimulation of IL-6 production by CpG ODN and HBV serum/2215 S/N at comparable levels, stimulation of IL-6 production by R-848 at levels 3−4 times higher than the ones elicited by HCV serum, b. IL-12p40 production stimulation by R-848 at levels 3−4 times higher than the ones observed after incubation with HCV serum and also very high levels of IL-12p40 after stimulation with Ribavirin, c. IFNa production stimulated by CpG ODN and R-848 at levels comparable to the ones observed with HBV and HCV serum pulsing, respectively. Conclusions: It appears that: (a) the Th-1 skewing capacity of pDC is partially affected by HCV infection, (b) Ribavirin possibly exerts an immunomodulatory role by triggering IL-12 production (Th1 skewing).

SOCS1 silencing can break high-dose dendritic cell immunotherapy-induced immune tolerance

Dendritic cells (DCs) play a pivotal role in T cell-mediated immunity and have been shown to induce strong anti-tumor immune responses. As of yet, only a limited number of objective tumor regressions have been observed in clinical studies using a DC vaccine. Suppressor of cytokine signaling-1 (SOCS1) is a key negative regulator of the JAK/STAT signal pathway and plays an essential role in suppressing systemic autoimmunity that is mediated by DCs. The aim of this study was to investigate whether SOCS1-silenced DCs can break the vaccine-induced immune tolerance stimulated by high-dose DC, thereby enhancing anti-tumor activity. In the mouse melanoma model, we found that a 2x106 TRP2-pulsed DC vaccine was able to induce immune tolerance, while a 2x106 SOCS1-silenced DC/TRP2 vaccine prevented immune tolerance. Further experiments revealed that activation-induced T cell death (AICD) through the Fas/Fas-L pathway may play a crucial role in immune tolerance induced by 2x106 TRP2-pulsed DC. SOCS1-silencing in DCs could prevent immune tolerance by inhibiting Fas and Fas-L expression, induced by an increase in IL-12p70 and IL-6 production. In addition, in 2x106 SOCS1-silenced DC/TRP2 immunized mice, higher levels of IL-12p70 and IFN-γ and lower IL-17 production may inhibit tumor angiogenesis and therefore assist in breaking immune tolerance. In conclusion, high-doses of DCs can inhibit the vaccine-induced AICD of T cells and cytokine regulation in tumor angiogenesis. These results indicate that SOCS1-silenced DC vaccines may greatly enhance anti-tumor activity by breaking self-tolerance.