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Purpose: Human interleukin-21 (IL-21) is a class I cytokine that mediates activation of CD8+ T cells, natural killer (NK) cells, and other cell types. We report final clinical and biological results of a phase II study of recombinant human IL-21 (rIL-21) in patients with metastatic melanoma.Experimental Design: Open-label, single-arm, two-stage trial. Eligibility criteria: unresectable metastatic melanoma, measurable disease by Response Evaluation Criteria in Solid Tumors, no prior systemic therapy (adjuvant IFN permitted), adequate major organ function, good performance status, no significant autoimmune disease, and life expectancy at least 4 months. Primary objective: antitumor efficacy (response rate). Secondary objectives: safety, blood biomarkers, and generation of anti-rIL-21 antibodies. rIL-21 (30 μg/kg/dose) was administered by intravenous bolus injection in 8-week cycles (5 dosing days followed by 9 days of rest for 6 weeks and then 2 weeks off treatment).Results: Stage I of the study comprised 14 patients. One confirmed complete response (CR) was observed, and as per protocol, 10 more patients were accrued to stage II (total n = 24: 10 female and 14 male). Best tumor response included one confirmed CR and one confirmed partial response, both with lung metastases. Treatment was overall well tolerated. Biomarker analyses showed increases in serum soluble CD25, frequencies of CD25+ NK and CD8+ T cells, and mRNA for IFN-γ, perforin, and granzyme B in CD8+ T and NK cells.Conclusions: rIL-21 administered at 30 μg/kg/d in 5-day cycles every second week is biologically active and well tolerated in patients with metastatic melanoma. Confirmed responses, including one CR, were observed.

Supplementary Figures S1-S3 from Clinical and Biological Efficacy of Recombinant Human Interleukin-21 in Patients with Stage IV Malignant Melanoma without Prior Treatment: A Phase IIa Trial

TNF‐blockade has shown clear therapeutic value in rheumatoid arthritis and other immune‐mediated inflammatory diseases, however its mechanism of action is not fully elucidated. We investigated the effects of TNF‐blockade on CD4+ T cell activation, maturation, and proliferation, and assessed whether TNF‐inhibitors confer regulatory potential to CD4+ T cells. CyTOF and flow cytometry analysis revealed that in vitro treatment of human CD4+ T cells with the anti‐TNF monoclonal antibody adalimumab promoted IL‐10 expression in CD4+ T cells, whilst decreasing cellular activation. In line with this, analysis of gene expression profiling datasets of anti‐TNF‐treated IL‐17 or IFN‐γ‐producing CD4+ T cells revealed changes in multiple pathways associated with cell cycle and proliferation. Kinetics experiments showed that anti‐TNF treatment led to delayed, rather than impaired T‐cell activation and maturation. Whilst anti‐TNF‐treated CD4+ T cells displayed some hyporesponsiveness upon restimulation, they did not acquire enhanced capacity to suppress T‐cell responses or modulate monocyte phenotype. These cells however displayed a reduced ability to induce IL‐6 and IL‐8 production by synovial fibroblasts. Together, these data indicate that anti‐TNF treatment delays human CD4+ T‐cell activation, maturation, and proliferation, and this reduced activation state may impair their ability to activate stromal cells., Through deep phenotyping using CyTOF and functional assays we have shown that TNF inhibition using adalimumab altered the phenotype and function of multiple populations of human CD4+ T‐cells, leading to an overall decreased pro‐inflammatory potential. These results provide insight into the anti‐TNF mechanism of action in human CD4+ T‐cells.

Histone lysine methylations have primarily been linked to selective recruitment of reader or effector proteins that subsequently modify chromatin regions and mediate genome functions. Here, we describe a divergent role for histone H4 lysine 20 mono-methylation (H4K20me1) and demonstrate that it directly facilitates chromatin openness and accessibility by disrupting chromatin folding. Thus, accumulation of H4K20me1 demarcates highly accessible chromatin at genes, and this is maintained throughout the cell cycle. In vitro, H4K20me1-containing nucleosomal arrays with nucleosome repeat lengths (NRL) of 187 and 197 are less compact than unmethylated (H4K20me0) or trimethylated (H4K20me3) arrays. Concordantly, and in contrast to trimethylated and unmethylated tails, solid-state NMR data shows that H4K20 mono-methylation changes the H4 conformational state and leads to more dynamic histone H4-tails. Notably, the increased chromatin accessibility mediated by H4K20me1 facilitates gene expression, particularly of housekeeping genes. Altogether, we show how the methylation state of a single histone H4 residue operates as a focal point in chromatin structure control. While H4K20me1 directly promotes chromatin openness at highly transcribed genes, it also serves as a stepping-stone for H4K20me3-dependent chromatin compaction., The effect of histone H4 lysine 20 methylation (H4K20me) on chromatin accessibility are not well established. Here the authors show how H4K20 methylation regulates chromatin structure and accessibility to ensure precise transcriptional outputs through the cell cycle using genome-wide approaches, in vitro biophysical assays, and NMR.

Visual Abstract, Highlights • The GLP-1RAs liraglutide and semaglutide reduce cardiovascular risk in type 2 diabetes patients. • In ApoE−/− mice and LDLr−/− mice, liraglutide and semaglutide treatment significantly attenuated plaque lesion development, in part independently of body weight and cholesterol lowering. • Semaglutide decreased levels of plasma markers of systemic inflammation in an acute inflammation model (lipopolysaccharide), and transcriptomic analysis of aortic atherosclerotic tissue revealed that multiple inflammatory pathways were down-regulated by semaglutide., Summary The glucagon-like peptide-1 receptor agonists (GLP-1RAs) liraglutide and semaglutide reduce cardiovascular risk in type 2 diabetes patients. The mode of action is suggested to occur through modified atherosclerotic progression. In this study, both of the compounds significantly attenuated plaque lesion development in apolipoprotein E-deficient (ApoE−/−) mice and low-density lipoprotein receptor-deficient (LDLr−/−) mice. This attenuation was partly independent of weight and cholesterol lowering. In aortic tissue, exposure to a Western diet alters expression of genes in pathways relevant to the pathogenesis of atherosclerosis, including leukocyte recruitment, leukocyte rolling, adhesion/extravasation, cholesterol metabolism, lipid-mediated signaling, extracellular matrix protein turnover, and plaque hemorrhage. Treatment with semaglutide significantly reversed these changes. These data suggest GLP-1RAs affect atherosclerosis through an anti-inflammatory mechanism.

BACKGROUND: Beneficial roles for glucagon-like peptide 1 (GLP-1)/GLP-1R signaling have recently been described in diseases, where low-grade inflammation is a common phenomenon. We investigated the effects of GLP-1 in Brunner's glands and duodenum with abundant expression of GLP-1 receptors, as well as GLP-1 effect on colonic inflammation.METHODS: RNA from Brunner's glands of GLP-1R knockout and wild-type mice were subjected to full transcriptome profiling. Array results were validated by quantitative reverse transcription polymerase chain reaction in wild-type mice and compared with samples from inflammatory bowel disease (IBD) patients and controls. In addition, we performed a detailed investigation of the effects of exogenous liraglutide dosing in a T-cell driven adoptive transfer (AdTr) colitis mouse model.RESULTS: Analyses of the Brunner's gland transcriptomes of GLP-1R knockout and wild-type mice identified 722 differentially expressed genes. Upregulated transcripts after GLP-1 dosing included IL-33, chemokine ligand 20 (CCL20), and mucin 5b. Biopsies from IBD patients and controls, as well as data from the AdTr model, showed deregulated expression of GLP-1R, CCL20, and IL-33 in colon. Circulating levels of GLP-1 were found to be increased in mice with colitis. Finally, the colonic cytokine levels and disease scores of the AdTr model indicated reduced levels of colonic inflammation in liraglutide-dosed animals.CONCLUSIONS: We demonstrate that IL-33, GLP-1R, and CCL20 are deregulated in human IBD, and that prophylactic treatment with 0.6 mg/kg liraglutide improves disease in AdTr colitis. In addition, GLP-1 receptor agonists upregulate IL-33, mucin 5b, and CCL20 in murine Brunner's glands. Taken together, our data indicate that GLP-1 receptor agonists affect gut homeostasis in both proximal and distal parts of the gut.

Objective Conflicting evidence exists regarding the suppressive capacity of Treg cells in the peripheral blood (PB) of patients with rheumatoid arthritis (RA). The aim of this study was to determine whether Treg cells are intrinsically defective in RA. Methods Using a range of assays on PB samples from patients with chronic RA and healthy controls, CD3+CD4+CD25+CD127low Treg cells from the CD45RO+ or CD45RA+ T cell compartments were analyzed for phenotype, cytokine expression (ex vivo and after in vitro stimulation), suppression of Teff cell proliferation and cytokine production, suppression of monocyte-derived cytokine/chemokine production, and gene expression profiles. Results No differences between RA patients and healthy controls were observed with regard to the frequency of Treg cells, ex vivo phenotype (CD4, CD25, CD127, CD39, or CD161), or proinflammatory cytokine profile (interleukin-17 [IL-17], interferon-γ [IFNγ], or tumor necrosis factor [TNF]). FoxP3 expression was slightly increased in Treg cells from RA patients. The ability of Treg cells to suppress the proliferation of T cells or the production of cytokines (IFNγ or TNF) upon coculture with autologous CD45RO+ Teff cells and monocytes was not significantly different between RA patients and healthy controls. In PB samples from some RA patients, CD45RO+ Treg cells showed an impaired ability to suppress the production of certain cytokines/chemokines (IL-1β, IL-1 receptor antagonist, IL-7, CCL3, or CCL4) by autologous lipopolysaccharide-activated monocytes. However, this was not observed in all patients, and other cytokines/chemokines (TNF, IL-6, IL-8, IL-12, IL-15, or CCL5) were generally suppressed. Finally, gene expression profiling of CD45RA+ or CD45RO+ Treg cells from the PB revealed no statistically significant differences between RA patients and healthy controls. Conclusion Our findings indicate that there is no global defect in either CD45RO+ or CD45RA+ Treg cells in the PB of patients with chronic RA.

Inflammatory β-cell failure contributes to type 1 and type 2 diabetes pathogenesis. Pro-inflammatory cytokines cause β-cell dysfunction and apoptosis, and lysine deacetylase inhibitors (KDACi) prevent β-cell failure in vitro and in vivo, in part by reducing NF-κB transcriptional activity. We investigated the hypothesis that the protective effect of KDACi involves transcriptional regulation of microRNAs (miRs), potential new targets in diabetes treatment. Insulin-producing INS1 cells were cultured with or without the broad-spectrum KDACi Givinostat, prior to exposure to the pro-inflammatory cytokines IL-1β and IFN-γ for 6 h or 24 h, and miR expression was profiled with miR array. Thirteen miRs (miR-7a-2-3p, miR-29c-3p, miR-96-5p, miR-101a-3p, miR-140-5p, miR-146a-5p, miR-146b-5p, miR-340-5p, miR-384-5p, miR-455-5p, miR-466b-2-3p, miR-652-5p, and miR-3584-5p) were regulated by both cytokines and Givinostat, and nine were examined by qRT-PCR. miR-146a-5p was strongly regulated by cytokines and KDACi and was analyzed further. miR-146a-5p expression was induced by cytokines in rat and human islets. Cytokine-induced miR-146a-5p expression was specific for INS1 and β-TC3 cells, whereas α-TC1 cells exhibited a higher basal expression. Transfection of INS1 cells with miR-146a-5p reduced cytokine signaling, including the activity of NF-κB and iNOS promoters, as well as NO production and protein levels of iNOS and its own direct targets TNF receptor associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1). miR-146a-5p was elevated in the pancreas of diabetes-prone BB-DP rats at diabetes onset, suggesting that miR-146a-5p could play a role in type 1 diabetes development. The miR array of cytokine-exposed INS1 cells rescued by KDACi revealed several other miRs potentially involved in cytokine-induced β-cell apoptosis, demonstrating the strength of this approach.

Background/Aims: Murine nephrotoxic nephritis (NTN) is a well-established model resembling chronic kidney disease. Investigating gene expression patterns separately in the glomerular and cortical tubulointerstitial structure could provide new knowledge about structure-specific changes in expression of genes in the NTN model. Methods: Glomerular, cortical tubulointerstitial and whole kidney tissues from mice subjected to nephrotoxic serum (NTS) or phosphate buffered saline (PBS) were collected on day 7, 21 and 42 using laser microdissection (LMD). Total RNA was extracted and subjected to nCounter NanoString. Histology, immunohistochemistry, in situ hybridization and/or quantitative real time PCR (qRT PCR) were performed to confirm regulation of selected genes. Results: LMD provided detailed information about genes that were regulated differently between structures over time. Some of the fibrotic and inflammatory genes (Col1a1, Col3a1 and Ccl2) were upregulated in both structures, whereas other genes such as Spp1 and Grem1 were differentially regulated suggesting spatial pathogenic mechanisms in the kidney. Downregulation of cortical tubulointerstitium genes involved in iron metabolism was detected along with iron accumulation. Conclusion: This study demonstrates several regulated genes in pathways important for the pathogenesis of the NTN model and that LMD identifies structure-specific changes in gene expression during disease development. Furthermore, this study shows the benefits of isolating glomeruli and cortical tubulointerstitium in order to identify gene regulation.

Background and Aim. Interleukin-21 (IL-21) is primarily a T cell-derived cytokine; it is upregulated in patients with Crohn’s Disease (CD) and could be a potential new therapeutic target in CD. Methods. In human material, IL-21 and IL-21R expression was investigated by in situ hybridization (ISH) and immunohistochemistry (IHC) in noninflammatory bowel disease (non-IBD) controls and patients with CD. The pathologic role of IL-21 was examined in murine models of T cell-dependent and T cell-independent colitis, either with a neutralizing monoclonal antibody against IL-21 or with the transfer of CD4+CD45RBhighIL-21R−/− T cells. Colonic pathology was examined by endoscopy, histopathology, IHC, ELISA, and Luminex. Results. In the human intestine, IL-21 and IL-21R mRNA and protein-expressing cells were observed in the mucosa, in lymphoid aggregates of submucosa in non-IBD controls, and in lymphoid aggregates of muscularis externa in patients with CD. IL-21 expression was most abundant in germinal centers (GCs) of the lymphoid aggregates, and IL-21R expression assessed semiquantitatively, was significantly higher in patients with CD compared to non-IBD controls. Following prophylactic and interventive anti-IL-21 mAb treatment in the adoptive transfer (AdTr) model, clinical and pathological parameters were significantly reduced. The most persistent finding was a reduction in colonic infiltrating neutrophils. As well, Rag2−/− mice receiving CD4+CD45RBhighIL-21R−/− T cells developed less severe colitis compared to Rag2−/− mice receiving CD4+CD45RBhighIL-21R+/+ T cells. No effect of reduced IL-21 signalling was observed in T cell-independent colitis. Conclusion. Our study shows that patients with CD have significant expression of IL-21 and IL-21R in the gut. As well, we show that neutralization of IL-21 in experimental T cell-driven colitis is associated with a reduction in clinical and pathological findings. This amelioration seems to be associated with a reduction in colon-infiltrating neutrophils.

The crystal structure of the full-length glucagon receptor in complex with a glucagon analogue NNC1702 reveals how the peptide ligand interacts with its target and shows the conformational changes required for receptor activation. The glucagon receptor is a class B G-protein-coupled receptor with an important role in glucose homeostasis. Activation of this receptor triggers the release of glucose, making it an important target for the treatment of type 2 diabetes. Beili Wu and colleagues now report the crystal structure of the full-length glucagon receptor bound to a glucagon analogue and a partial agonist, NNC1702. The 3.0 A resolution of the structure provides key insights into how the peptide ligand interacts with its target and the conformational changes involved in the initial stages of activation. The structural data is reinforced by using double electron–electron resonance (DEER) spectroscopy, which demonstrates the extent of rearrangement that is involved in accommodating the glucagon analogue. Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases1,2,3,4,5,6,7,8. Previous work9,10,11 has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved12,13,14. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resolution limitations, the specific molecular interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding inter-domain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 A-resolution crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analogue and partial agonist, NNC1702. This structure provides molecular details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR–NNC0640–mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual-binding-site trigger model for GCGR activation—which requires conformational changes of the stalk, first extracellular loop and TMD—that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs.

The relative contributions of the JNK subtypes in inflammatoryβ-cell failure and apoptosis are unclear. The JNK protein family consists of JNK1, JNK2, and JNK3 subtypes, encompassing many different isoforms. INS-1 cells express JNK1α1, JNK1α2, JNK1β1, JNK1β2, JNK2α1, JNK2α2, JNK3α1, and JNK3α2 mRNA isoform transcripts translating into 46 and 54 kDa isoform JNK proteins. Utilizing Lentiviral mediated expression of shRNAs against JNK1, JNK2, or JNK3 in insulin-producing INS-1 cells, we investigated the role of individual JNK subtypes in IL-1β-inducedβ-cell apoptosis. JNK1 knockdown prevented IL-1β-induced INS-1 cell apoptosis associated with decreased 46 kDa isoform JNK protein phosphorylation and attenuated Myc expression. Transient knockdown of Myc also prevented IL-1β-induced apoptosis as well as caspase 3 cleavage. JNK2 shRNA potentiated IL-1β-induced apoptosis and caspase 3 cleavage, whereas JNK3 shRNA did not affect IL-1β-inducedβ-cell death compared to nonsense shRNA expressing INS-1 cells. In conclusion, JNK1 mediates INS-1 cell death associated with increased Myc expression. These findings underline the importance of differentiated targeting of JNK subtypes in the development of inflammatoryβ-cell failure and destruction.

microRNA-155 (miR-155) has been implicated as a central regulator of the immune system, but its function during acute inflammatory responses is still poorly understood. Here we show that exposure of cultured macrophages and mice to lipopolysaccharide (LPS) leads to up-regulation of miR-155 and that the transcription factor c/ebp Beta is a direct target of miR-155. Interestingly, expression profiling of LPS-stimulated macrophages combined with overexpression and silencing of miR-155 in murine macrophages and human monocytic cells uncovered marked changes in the expression of granulocyte colony-stimulating factor (G-CSF), a central regulator of granulopoiesis during inflammatory responses. Consistent with these data, we show that silencing of miR-155 in LPS-treated mice by systemically administered LNA-antimiR results in derepression of the c/ebp Beta isoforms and down-regulation of G-CSF expression in mouse splenocytes. Finally, we report for the first time on miR-155 silencing in vivo in a mouse inflammation model, which underscores the potential of miR-155 antagonists in the development of novel therapeutics for treatment of chronic inflammatory diseases.

Purpose: Human interleukin-21 (IL-21) is a class I cytokine that mediates activation of CD8+ T cells, natural killer (NK) cells, and other cell types. We report final clinical and biological results of a phase II study of recombinant human IL-21 (rIL-21) in patients with metastatic melanoma.Experimental Design: Open-label, single-arm, two-stage trial. Eligibility criteria: unresectable metastatic melanoma, measurable disease by Response Evaluation Criteria in Solid Tumors, no prior systemic therapy (adjuvant IFN permitted), adequate major organ function, good performance status, no significant autoimmune disease, and life expectancy at least 4 months. Primary objective: antitumor efficacy (response rate). Secondary objectives: safety, blood biomarkers, and generation of anti-rIL-21 antibodies. rIL-21 (30 μg/kg/dose) was administered by intravenous bolus injection in 8-week cycles (5 dosing days followed by 9 days of rest for 6 weeks and then 2 weeks off treatment).Results: Stage I of the study comprised 14 patients. One confirmed complete response (CR) was observed, and as per protocol, 10 more patients were accrued to stage II (total n = 24: 10 female and 14 male). Best tumor response included one confirmed CR and one confirmed partial response, both with lung metastases. Treatment was overall well tolerated. Biomarker analyses showed increases in serum soluble CD25, frequencies of CD25+ NK and CD8+ T cells, and mRNA for IFN-γ, perforin, and granzyme B in CD8+ T and NK cells.Conclusions: rIL-21 administered at 30 μg/kg/d in 5-day cycles every second week is biologically active and well tolerated in patients with metastatic melanoma. Confirmed responses, including one CR, were observed.

Recombinant interleukin-21 (rIL-21) is an immune stimulating cytokine recently tested in two Phase 1 trials for immune responsive cancers. A secondary objective of these trials was to characterize pharmacodynamic responses to rIL-21 in patients. Here, we report the effects of systemic rIL-21 on serum markers of immune stimulation.Recombinant IL-21 was administered by intravenous bolus injection at dose levels from 1 to 100 microg/kg using two distinct treatment regimens: thrice weekly ('3/w') for 6 weeks; or once daily for five consecutive days followed by nine dose-free days ('5 + 9'). In the absence of dose limiting toxicity, additional cycles of dosing were initiated immediately following the nine dose-free days. An array of 70 different proteins was profiled in subject serum samples from several time points during the course of the study. Hierarchical clustering analysis was performed on a normalized subset of these data.Systemic administration of rIL-21 affected the serum levels of several cytokines, chemokines, acute-phase proteins and cell adhesion proteins. The magnitude and duration of response were dose dependent for a subset of these biomarkers. The 5 + 9 dosing regimen generally produced cyclic changes that were of greater magnitude, as compared to a more chronic stimulation with the 3/w dosing regimen. Despite these differences, rIL-21 effects on many analytes were similar between regimens when averaged over the time of treatment. Based on similar temporal, between-subject and dose response changes, groups of analytes were identified that exhibited distinct components of the rIL-21-mediated immune activation. Biomarkers indicative of lymphocyte activation (increased IL-16, decreased RANTES), acute phase response (increased CRP, ferritin), myeloid activation (increased MDC, MIP-1 alpha), and leukocyte chemotaxis/trafficking (increased sCAMs, MCP-1) were strongly modulated in subjects treated with rIL-21.Administration of rIL-21 resulted in activation of multiple cell types and immune response pathways. The changes observed in serum proteins were consistent with coincident processes of lymphoid and myeloid cell activation and trafficking, and acute phase response.

Purpose Human interleukin-21 (IL-21) is a class I cytokine previously reported in clinical studies on immune responsive cancers. Here we report the effects of systemic IL-21 therapy on the immune system in two phase 1 trials with this novel cytokine. Experimental design Recombinant IL-21 was administered by intravenous bolus injection at dose levels from 1 to 100 μg/kg using two planned treatment regimens: thrice weekly for 6 weeks (3/week); or once daily for five consecutive days followed by nine dose-free days (5 + 9). The following biomarkers were studied in peripheral blood mononuclear cells (PBMC) during treatment: phosphorylation of STAT3, alterations in the composition of leukocyte subsets, ex vivo cytotoxicity, expression of effector molecules in enriched CD8+ T cells and CD56+ NK cells by quantitative RT-PCR, and gene array profiling of CD8+ T cells. Results Effects of IL-21 were observed at all dose levels. In the 5 + 9 regimen IL-21 induced a dose dependent decrease in circulating NK cells and T cells followed by a return to baseline in resting periods. In both CD8+ T cells and CD56+ NK cells we found up-regulation of perforin and granzyme B mRNA. In addition, full transcriptome analysis of CD8+ T cells displayed changes in several transcripts associated with increased cell cycle progression, cellular motility, and immune activation. Finally, cytotoxicity assays showed that IL-21 enhanced the ability of NK cells to kill sensitive targets ex vivo. Conclusions IL-21 was biologically active at all dose levels administered with evidence of in vivo NK cell and CD8+ T cell activation. Electronic supplementary material The online version of this article (doi:10.1007/s00262-008-0479-4) contains supplementary material, which is available to authorized users.

Interleukin (IL)-21 is a recently discovered cytokine in early clinical development, which has shown anti-tumor activity in various animal models. In the present study, we examine the anti-tumor activity of IL-21 protein therapy in two syngeneic tumor models and its effect on the density of tumor infiltrating T cells. We treated mice bearing established subcutaneous B16 melanomas or RenCa renal cell carcinomas with intraperitoneal (i.p.) or subcutaneous (s.c.) IL-21 protein therapy and subsequently scored the densities of tumor infiltrating CD4(+) and CD8(+) T cells by immunohistochemistry. Whereas both routes of IL-21 administration significantly inhibited growth of small, established RenCa and B16 tumors, only s.c. therapy significantly inhibited the growth of large, established tumors. We found a greater bioavailability and significant drainage of IL-21 to regional lymph nodes following s.c. administration, which could account for the apparent increase in anti-tumor activity. Specific depletion of CD8(+) T cells with monoclonal antibodies completely abrogated the anti-tumor activity, whereas NK1.1(+) cell depletion did not affect tumor growth. In accordance, both routes of IL-21 administration significantly increased the density of tumor infiltrating CD8(+) T cells in both B16 and RenCa tumors; and in the RenCa model s.c. administration of IL-21 led to a significantly higher density of tumor infiltrating CD8(+) T cells compared to i.p. administration. The densities of CD4(+) T cells were unchanged following IL-21 treatments. Taken together, these data demonstrate that IL-21 protein has anti-tumor activity in established syngeneic tumors, and we show that IL-21 therapy markedly increases the density of tumor infiltrating CD8(+) T cells.

Background Inflammatory bowel disease (IBD) is a complex multi-factorial inflammatory disease with Crohn’s disease (CD) and ulcerative colitis (UC) being the two most common forms. A number of transcriptional profiling studies have provided compelling evidence that describe the role of protein-coding genes and microRNAs in modulating the immune responses in IBD. Methods In the present study, we performed a genome-wide transcriptome profiling of lncRNAs and protein-coding genes in 96 colon pinch biopsies (inflamed and non-inflamed) extracted from multiple colonic locations from 45 patients (CD = 13, UC = 20, controls = 12) using an expression microarray platform. Results In our study, we identified widespread dysregulation of lncRNAs and protein-coding genes in both inflamed and non-inflamed CD and UC compared to the healthy controls. In cases of inflamed CD and UC, we identified 438 and 745 differentially expressed lncRNAs, respectively, while in cases of the non-inflamed CD and UC, we identified 12 and 19 differentially expressed lncRNAs, respectively. We also observed significant enrichment (P-value

Aims/hypothesis Type 2 diabetes is characterised by progressive loss of pancreatic beta cell mass and function. Therefore, it is of therapeutic interest to identify factors with the potential to improve beta cell proliferation and insulin secretion. Bone morphogenetic protein 4 (BMP4) expression is increased in diabetic animals and BMP4 reduces glucose-stimulated insulin secretion (GSIS). Here, we investigate the molecular mechanism behind this inhibition. Methods BMP4-mediated inhibition of GSIS was investigated in detail using single cell electrophysiological measurements and live cell Ca2+ imaging. BMP4-mediated gene expression changes were investigated by microarray profiling, quantitative PCR and western blotting. Results Prolonged exposure to BMP4 reduced GSIS from rodent pancreatic islets. This inhibition was associated with decreased exocytosis due to a reduced Ca2+ current through voltage-dependent Ca2+ channels. To identify proteins involved in the inhibition of GSIS, we investigated global gene expression changes induced by BMP4 in neonatal rat pancreatic islets. Expression of the Ca2+-binding protein calbindin1 was significantly induced by BMP4. Overexpression of calbindin1 in primary islet cells reduced GSIS, and the effect of BMP4 on GSIS was lost in islets from calbindin1 (Calb1) knockout mice. Conclusions/interpretation We found BMP4 treatment to markedly inhibit GSIS from rodent pancreatic islets in a calbindin1-dependent manner. Calbindin1 is suggested to mediate the effect of BMP4 by buffering Ca2+ and decreasing Ca2+ channel activity, resulting in diminished insulin exocytosis. Both BMP4 and calbindin1 are potential pharmacological targets for the treatment of beta cell dysfunction.

Using a known dual PPARα/γ activator (5) as a structural template, SAR evaluations led to the identification of triple PPARα/γ/δ activators (18–20) with equal potency and efficacy on all three receptors. These compounds could become useful tools for studying the combined biological effects of PPARα/γ/δ activation.

IL-17+ CD4+ T (Th17) cells contribute to the pathogenesis of several human inflammatory diseases. Here we demonstrate that TNF inhibitor (TNFi) drugs induce the anti-inflammatory cytokine IL-10 in CD4+ T cells including IL-17+ CD4+ T cells. TNFi-mediated induction of IL-10 in IL-17+ CD4+ T cells is Treg-/Foxp3-independent, requires IL-10 and is overcome by IL-1β. TNFi-exposed IL-17+ CD4+ T cells are molecularly and functionally distinct, with a unique gene signature characterized by expression of IL10 and IKZF3 (encoding Aiolos). We show that Aiolos binds conserved regions in the IL10 locus in IL-17+ CD4+ T cells. Furthermore, IKZF3 and IL10 expression levels correlate in primary CD4+ T cells and Aiolos overexpression is sufficient to drive IL10 in these cells. Our data demonstrate that TNF-α blockade induces IL-10 in CD4+ T cells including Th17 cells and suggest a role for the transcription factor Aiolos in the regulation of IL-10 in CD4+ T cells.

Gene expression signatures can provide an unbiased view into the molecular changes underlying biologically and medically interesting phenotypes. We therefore initiated this study to identify signatures that would be of utility in studying rheumatoid arthritis (RA). We used microarray profiling of peripheral blood mononuclear cells (PBMCs) in 30 RA patients to assess the effect of different biologic agent (biologics) treatments and to quantify the degree of a type-I interferon (IFN) signature in these patients. A numeric score was derived for the quantification step and applied to patients with RA. To further characterize the IFN response in our cohort, we employed type-I IFN treatment of PBMCs in vitro and in reporter assays. Profiling identified a subset of RA patients with upregulation of type-I IFN-regulated transcripts, thereby corroborating previous reports showing RA to be heterogeneous for an IFN component. A comparison of individuals currently untreated with a biologic with those treated with infliximab, tocilizumab, or abatacept suggested that each biologic induces a specific gene signature in PBMCs. It is possible to observe signs of type-I IFN pathway activation in a subset of clinically active RA patients without C-reactive protein elevation. Furthermore, biologics-specific gene signatures in patients with RA indicate that looking for a biologic-specific response pattern may be a potential future tool for predicting individual patient response.

Genome-wide association studies (GWAS) have heralded a new era in susceptibility locus discovery in complex diseases. For type 1 diabetes, >40 susceptibility loci have been discovered. However, GWAS do not inevitably lead to identification of the gene or genes in a given locus associated with disease, and they do not typically inform the broader context in which the disease genes operate. Here, we integrated type 1 diabetes GWAS data with protein-protein interactions to construct biological networks of relevance for disease. A total of 17 networks were identified. To prioritize and substantiate these networks, we performed expressional profiling in human pancreatic islets exposed to proinflammatory cytokines. Three networks were significantly enriched for cytokine-regulated genes and, thus, likely to play an important role for type 1 diabetes in pancreatic islets. Eight of the regulated genes (CD83, IFNGR1, IL17RD, TRAF3IP2, IL27RA, PLCG2, MYO1B, and CXCR7) in these networks also harbored single nucleotide polymorphisms nominally associated with type 1 diabetes. Finally, the expression and cytokine regulation of these new candidate genes were confirmed in insulin-secreting INS-1 β-cells. Our results provide novel insight to the mechanisms behind type 1 diabetes pathogenesis and, thus, may provide the basis for the design of novel treatment strategies.

The pituitary hormone prolactin (PRL) plays an important role in mammary gland development. It was also suggested to contribute to breast cancer progression. In vivo data strongly supported a crucial role of PRL in promoting tumour growth; however, PRL demonstrated only a weak, if any, pro-proliferative effect on cancer cells in vitro. Several recent studies indicated that PRL action in vivo may be influenced by the hormonal milieu, e.g. other growth factors such as 17β-oestradiol (E2). Here, we explored the potential interplay between PRL and E2 in regulation of gene expression and cell growth. PRL alone induced either a weak or no proliferative response of T47D and BT-483 cells respectively, while it drastically enhanced cell proliferation in E2-stimulated cultures. Affymetrix microarray analysis revealed 12 genes to be regulated by E2, while 57 genes were regulated by PRL in T47D cells. Most of the PRL-regulated genes (42/57) were not previously described as PRL target genes, e.g. WT1 and IER3. One hundred and five genes were found to be regulated upon PRL/E2 co-treatment: highest up-regulation was found for EGR3, RUNX2, EGR1, MAFF, GLIPR1, IER3, SOCS3, WT1 and AREG. PRL and E2 synergised to regulate EGR3, while multiple genes were regulated additively. These data show a novel interplay between PRL and E2 to modulate gene regulation in breast cancer cells.

Interleukin-21 (IL-21) is a class I cytokine with antitumor properties due to enhanced proliferation and effector function of CD8 + T cells and natural killer (NK) cells. Here we have explored the magnitude and time-course of cytostatics-induced lymphopenia in mice and investigated whether treatment with cytostatics influences the antitumor effect of IL-21 in mouse tumor models. We show that pegylated liposomal doxorubicin (PLD), irinotecan and oxaliplatin induced transient lymphopenia, whereas 5-fluorouracil (5-FU) transiently increased lymphocyte counts. B cells were more sensitive than T cells towards irinotecan and oxaliplatin. Additive antitumor effects were observed after combining IL-21 with PLD, oxaliplatin and to less extent 5-FU but not irinotecan, and larger effect was observed when IL-21 administration was postponed relative to chemotherapy, suggesting that these agents may transiently impair immune function. However, the chemotherapies did not significantly alter the levels of circulating regulatory T cells and only marginally affected the ability of CD8 + T cells to respond to IL-21 measured as increased granzyme B mRNA. Our results show that IL-21 therapy can be successfully combined with agents from different chemotherapeutic drug classes, i.e. topoisomerase II inhibitors (PLD), anti-metabolites (5-FU) and platinum analogs (oxaliplatin) provided that IL-21 therapy is delayed relative to chemotherapy.

Interleukin (IL)-21 is a novel cytokine that has been shown to enhance proliferation and activation of CD8+ T cells, enhance natural killer (NK) cell activity and costimulate anti-CD40-driven B-cell proliferation in mice. Several studies have furthermore demonstrated antitumour effects of IL-21 administration in mouse models. In this study we have investigated how IL-21 affects the survival and cytotoxicity of human NK cells and modulates their expression of surface receptors and of the effector molecules granzyme B and perforin. In contrast to murine NK cells, where IL-21 alone cannot sustain survival, IL-21 and IL-2 were equally efficient in sustaining survival of human NK cells. In the absence of other cytokines, IL-21 had little effect on expression of a panel of surface receptors on human NK cells. However, IL-21 synergized with IL-2 to up-regulate several surface receptors, including NKG2A, CD25, CD86 and CD69. The CD25+ CD86+ NK cells were CD56(bright) and were large and granular. Expression of the effector molecules perforin and granzyme A and B was up-regulated by IL-21 at both mRNA and protein levels. Furthermore, IL-21 increased the cytotoxicity of NK cells against K562 target cells. These findings suggest that IL-21 modulates NK cell activity through induction of intracellular effector molecules as well as modulation of surface receptor expression.

A large number of databases are currently being implemented within toxicology aiming to integrate diverse biological data, such as clinical chemistry, expression, and other types of data. However, for these endeavors to be successful, tools for integration, visualization, and interpretation are needed. This paper presents a method for data integration using a hierarchical model based on either principal component analysis or partial least squares discriminant analysis of clinical chemistry, expression, and nuclear magnetic resonance data using a toxicological study as case. The study includes the three toxicants alpha-naphthyl-isothiocyanate, dimethylnitrosamine, and N-methylformamide administered to rats. Improved predictive ability of the different classes is seen, suggesting that this approach is a suitable method for data integration and visualization of biological data. Furthermore, the method allows for correlation of biological parameters between the different data types, which could lead to an improvement in biological interpretation.

Purpose: Human interleukin-21 (IL-21) is a pleiotropic class I cytokine that activates CD8+ T cells and natural killer cells. We report a phase 1 study of recombinant human IL-21 in patients with surgically incurable metastatic melanoma. The primary objective was to investigate safety and tolerability by determining dose-limiting toxicity (DLT). The secondary objectives were to identify a dose response for various biomarkers in the peripheral blood, estimate the minimum biologically effective dose, determine the pharmacokinetics of IL-21, determine if anti-IL-21 antibodies were induced during therapy, and measure effects on tumor size according to Response Evaluation Criteria in Solid Tumors.Experimental Design: Open-label, two-arm, dose escalation trial of IL-21 administered by i.v. bolus injection at dose levels from 1 to 100 μg/kg using two parallel treatment regimens: thrice weekly for 6 weeks (3/wk) or three cycles of daily dosing for 5 days followed by 9 days of rest (5+9).Results: Twenty-nine patients entered the study. IL-21 was generally well tolerated and no DLTs were observed at the 1, 3, and 10 μg/kg dose levels. In the 3/wk regimen, DLTs were increased in alanine aminotransferase, neutropenia, and lightheadedness with fever and rigors. DLTs in the 5+9 regimen were increased in aspartate aminotransferase and alanine aminotransferase, neutropenia, fatigue, and thrombocytopenia. The maximum tolerated dose was declared to be 30 μg/kg for both regimens. Effects on biomarkers were observed at all dose levels, including increased levels of soluble CD25 and up-regulation of perforin and granzyme B mRNA in CD8+ cells. One partial tumor response observed after treatment with IL-21 for 2 × 6 weeks (3/wk) became complete 3 months later.Conclusions: IL-21 is biologically active at all dose levels administered and is generally well tolerated, and phase 2 studies have commenced using 30 μg/kg in the 5+9 regimen.

To understand the molecular mechanisms regulating pancreatic endocrine development and function, pancreatic gene expression was compared between Ngn3-deficient mice and littermate controls on embryonic days 13 and 15. Microarray analysis identified 504 genes with significant differences in expression. Fifty-two of these showed at least twofold reduction in Ngn3 knockouts compared to controls. Many of them were previously described to be involved in endocrine development and function. Among the genes not previously characterized were Rhomboid veinlet-like 4, genes involved in tetrahydrobiopterin biosynthesis and the Iroquois-type homeobox gene Irx1, the latter was selected for further investigation. In situ hybridisation demonstrated that two Iroquois genes, Irx1 and Irx2, were expressed in pancreatic endoderm of wild-type, but not Ngn3 mutant embryos. Furthermore, ectopic Ngn3 induced prominent Irx2 expression in chicken endoderm. Co-labelling established that Irx1 and Irx2 mRNA is located to glucagon-, but not insulin- or somatostatin-producing cells in mice and chicken. These data suggest that Irx1 and Irx2 serve an evolutionary conserved role in the regulation of α-cell-specific gene expression.

Peroxisome proliferator-activated receptor (PPAR)-alpha controls the transcription of a variety of genes involved in lipid metabolism and is the target receptor for the hypolipidemic drug class of fibrates. In the present study, the molecular and physiological effects of seven different PPAR-activating drugs have been examined in a rodent model of dyslipidemia. The drugs examined were selected to display varying potencies and efficacies toward PPAR-alpha. To help elucidate the link between the gene regulation elicited by PPAR-alpha ligands and the concomitant physiological changes, we have used cDNA microarray analysis to identify smaller gene sets that are predictive of the function of these ligands. A number of genes showed strong correlations to the relative PPAR-alpha efficacy of the drugs. Furthermore, using multivariate analysis, a strong relationship between the drug-induced triglyceride lowering and the transcriptional profiles of the different drugs could be found.

Two potent nonselective, but PPARalpha-preferring, PPAR agonists 5 and 6 were designed and synthesized in high yields. The concept of dimeric ligands in transcription factors was investigated by synthesizing and testing the corresponding dimers 7, 8a, and 8b in PPAR transactivation assays. The three dimeric ligands all showed agonist activity on all three PPAR receptor subtypes, but with different profiles compared to the monomers 5 and 6. Despite breaking all the "rule of five" criteria, the dimers had excellent oral bioavailability and pharmacokinetic properties, resulting in good in vivo efficacy in db/db mice. X-ray crystal structure and modeling experiments suggested that the dimers interacted with the AF-2 helix as well as with amino acid residues in the lipophilic pocket close to the receptor surface.

Peroxisome proliferator activated receptor (PPAR)-alpha controls the expression of multiple genes involved in lipid metabolism, and activators of PPAR-alpha, such as fibrates, are commonly used drugs in the treatment of hypertriglyceridemia and other dyslipidemic states. Recent data have also suggested a role for PPAR-alpha in insulin resistance and glucose homeostasis. In the present study, we have assessed the transcriptional and physiological responses to PPAR-alpha activation in a diet-induced rat model of insulin resistance. The two PPAR-alpha activators, fenofibrate and Wy-14643, were dosed at different concentrations in high-fat fed Sprague-Dawley rats, and the transcriptional responses were examined in liver using cDNA microarrays. In these analyses, 98 genes were identified as being regulated by both compounds. From this pool of genes, 27 correlated to the observed effect on plasma insulin, including PPAR-alpha itself and the leukocyte antigen-related protein tyrosine phosphatase (PTP-LAR). PTP-LAR was downregulated by both compounds, and showed upregulation as a result of the high-fat feeding. This regulation was also observed at the protein level. Furthermore, downregulation of PTP-LAR by fenofibric acid was demonstrated in rat FaO hepatoma cells in vitro, indicating that the observed regulation of PTP-LAR by fenofibrate and Wy-14643 in vivo is mediated as a direct effect of the PPAR agonists on the hepatocytes. PTP-LAR is one of the first genes involved in insulin receptor signaling to be shown to be regulated by PPAR-alpha agonists. These data suggest that factors apart from skeletal muscle lipid supply may influence PPAR-alpha-mediated amelioration of insulin resistance.

Thiazolidinediones (TZDs) are a new class of compounds that improve insulin sensitivity in type 2 diabetic patients as well as in rodent models of this disease. These compounds act as ligands for a member of the nuclear hormone receptor superfamily, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), which is highly expressed in adipose tissue and, moreover, has been shown to play an important role in adipocyte differentiation. The strong correlation between the antidiabetic activity of TZDs and their ability to activate PPAR-gamma suggests that PPAR-gamma, through downstream-regulated genes, mediates the effects of TZDs. In this report, we present the isolation and characterization of 81 genes, encoding proteins of known function, differentially expressed during TZD-stimulated differentiation of 3T3-L1 cells. By the use of different reverse- Northern blot techniques, the differential expression of 50 of these genes could be verified, and 21 genes were specifically regulated by a potent TZD during the course of adipocyte differentiation, whereas no effect of a PPAR-gamma antagonist could be observed in mature adipocytes. The differential expression of a large fraction of the isolated genes was also shown to occur in white adipose tissue of ob/ob mice treated with rosiglitazone; combined, our results suggest that an important effect of rosiglitazone in adipose tissue is based on activation of PPAR-gamma in preexisting preadipocytes found among the mature adipocytes, resulting in subsequent adipocyte differentiation.

In the present study, we wanted to determine whether efficient gene delivery using an epidermal growth factor (EGF)/DNA polyplex could be accomplished in small cell lung cancer (SCLC) cell lines expressing low EGF receptor (EGFR) levels. EGFR expression levels and transduction efficiencies with polyplexes were examined in five SCLC cell lines and two controls. EGFR expression was examined by binding assays and demonstrated low EGFR levels ranging from 3.6 to 87.4 fmol/mg protein. The SCLC cell lines exhibited high sensitivity to adenovirus infection, which was an important determinant for transduction efficiency when adenovirus was used as an endosomolytic agent. The transduction efficiencies with EGF/DNA polyplexes ranged from 41% +/- 3.5% to 73% +/- 4.6% in the EGFR-positive SCLC cell lines. In the controls lacking EGFRs, only 5% +/- 1.0% and 8% +/- 1.8% of the cells were transduced. Furthermore, the transduction efficiency could be reduced from 50% +/- 4.9% to 18% +/- 1.1% when excess EGF was added to compete with the EGF/DNA polyplexes. In the present study, receptor-targeted gene delivery to SCLC cell lines has been demonstrated for the first time. Our results indicate that even low receptor expression levels in the target cells are sufficient for efficient and specific in vitro gene delivery with EGF/DNA polyplexes.

Background Upregulation of mRNA IFN-alpha activity (IFN signature) has been demonstrated in some patients with systemic lupus erythematosus (SLE). Based on this observation anti-IFN-alpha mAbs are currently being developed for the treatment of SLE. Interestingly naturally occurring antibodies towards IFN-alpha have previously been demonstrated in a small fraction of healthy individuals, in therapeutic IgG preparations and in patients with inflammatory disorders. Objectives Recently naturally occurring antibodies against a single IFN alpha subtype were observed in SLE patients. A high level of such anti-IFN antibodies could theoretically affect the IFN gene signature and clinical disease activity. Therefore we carried out a longitudinal study of IFN activity, anti-IFN antibodies (frequency, titre, neutralizing capacity, specificity) and disease activity in a cohort of SLE patients. Methods Peripheral blood mononuclear cells (PBMC) and plasma samples were collected over an average of 6 visits (range 2-12) from 23 SLE patients (median SLEDAI score at initiation 6 (range 0-35) and 5 healthy donors. Patients were followed from 197 to 812 days. Plasma levels of autoantibodies towards a mixture of IFN-alpha 1, 2- and 8 or the individual subtypes were measured by radioimmunoassay, and 44 other autoantibodies were evaluated in a multiplex luminex system by Rules-Based-Medicine. The mRNA IFN signature was deduced from full transciptome analyses in PBMCs using Human Genome U133 Plus 2.0 arrays and RT-PCR. Results In healthy donors no autoantibodies towards IFN-alpha were detected; in alignment with historic data where the frequency is 3-5/1.000 individuals. In contrast, we detected specific anti-IFN-alpha antibodies in 4/23 (17%) of the patients. In three of these, the autoantibodies were present throughout the study while the last patient developed autoantibodies between visits 5 and 6. In one of the patients very high titres of antibodies to the IFN mixture and against the individual IFN subtypes were demonstrated while the other patients had lower titres. The antibodies in the highly positive patient demonstrated increasing neutralizing capacity throughout the study. The four anti-IFN-alpha-positive patients did not demonstrate an increased level of other autoantibodies when compared to anti-IFN antibody negative patients. A composite score of 12 well characterized IFN-alpha inducible genes (IFN signature) was defined. A 1.2 to 4-fold fluctuation in the IFN-score was observed over time in the individual patients. The patient with high levels of neutralizing anti-IFN antibodies showed significantly reduced IFN signature activity during the study. Conclusions Anti-IFN autoantibodies are present in a subset of SLE patients. An association of anti-IFN alpha 4 antibodies and disease activity has previously been reported, and our study confirms this for additional IFN-alpha subtypes. Naturally occurring autoantibodies against IFN-alpha could affect treatment outcome following anti-IFN mAb therapy. Disclosure of Interest C. Ross Shareholder of: Novo Nordisk, Employee of: Novo Nordisk, D. Lundsgaard Shareholder of: Novo Nordisk, Employee of: Novo Nordisk, J. Fleckner Shareholder of: Novo Nordisk, Employee of: Novo Nordisk, M. Olferiev: None Declared, K. Kirou: None Declared, C. Wiberg Shareholder of: Novo Nordisk, Employee of: Novo Nordisk, K. Bendtzen: None Declared, K. Frederiksen Shareholder of: Novo Nordisk, Employee of: Novo Nordisk, M. Crow: None Declared

Results Ex vivo analysis of patients with RA on TNFi therapy revealed an enrichment of Th17 cells in peripheral blood compared to those on disease-modifying anti-rheumatic drugs or healthy controls. However, we also found an increase in IL-10-producing CD4+ T-cells. The enrichment in IL-17+ and IL-10+ CD4+ T-cells, including IL17+IL-10+ co-expressing CD4+ T-cells, was recapitulated in vitro by the addition of TNFi drugs (adalimumab, infliximab, etanercept, and certolizumab) to human monocyte/CD4+ T-cell co-cultures. IL-10 induction was independent of FcgR binding, IL-10 and CD4+CD25+ Tregs. TNFi-induced Th17 cells were functionally distinct as shown by an ability to modulate CD14+ monocytes in an IL-10-dependent manner. We report the identification of a transcription factor that is strongly associated with IL-10 expression in TNFi-induced IL-17+ CD4+ T-cells, and show that overexpression of this transcription factor drives IL-10 expression in primary CD4+ T-cells.