Recombinant Newcastle Disease Virus Immunotherapy Drives Oncolytic Effects and Durable Systemic Antitumor Immunity.

A recombinant Newcastle Disease Virus (NDV), encoding either a human (NDVhuGM-CSF, MEDI5395) or murine (NDVmuGM-CSF) GM-CSF transgene, combined broad oncolytic activity with the ability to significantly modulate genes related to immune functionality in human tumor cells. Replication in murine tumor lines was significantly diminished relative to human tumor cells. Nonetheless, intratumoral injection of NDVmuGM-CSF conferred antitumor effects in three syngeneic models in vivo; with efficacy further augmented by concomitant treatment with anti-PD-1/PD-L1 or T-cell agonists. Ex vivo immune profiling, including T-cell receptor sequencing, revealed profound immune-contexture changes consistent with priming and potentiation of adaptive immunity and tumor microenvironment (TME) reprogramming toward an immune-permissive state. CRISPR modifications rendered CT26 tumors significantly more permissive to NDV replication, and in this setting, NDVmuGM-CSF confers immune-mediated effects in the noninjected tumor in vivo Taken together, the data support the thesis that MEDI5395 primes and augments cell-mediated antitumor immunity and has significant utility as a combination partner with other immunomodulatory cancer treatments.

The Extrinsic and Intrinsic Roles of PD-L1 and Its Receptor PD-1: Implications for Immunotherapy Treatment.

Programmed death-ligand 1 (PD-L1) is an immune checkpoint inhibitor that binds to its receptor PD-1 expressed by T cells and other immune cells to regulate immune responses; ultimately preventing exacerbated activation and autoimmunity. Many tumors exploit this mechanism by overexpressing PD-L1 which often correlates with poor prognosis. Some tumors have also recently been shown to express PD-1. On tumors, PD-L1 binding to PD-1 on immune cells promotes immune evasion and tumor progression, primarily by inhibition of cytotoxic T lymphocyte effector function. PD-1/PD-L1-targeted therapy has revolutionized the cancer therapy landscape and has become the first-line treatment for some cancers, due to their ability to promote durable anti-tumor immune responses in select patients with advanced cancers. Despite this clinical success, some patients have shown to be unresponsive, hyperprogressive or develop resistance to PD-1/PD-L1-targeted therapy. The exact mechanisms for this are still unclear. This review will discuss the current status of PD-1/PD-L1-targeted therapy, oncogenic expression of PD-L1, the new and emerging tumor-intrinisic roles of PD-L1 and its receptor PD-1 and how they may contribute to tumor progression and immunotherapy responses as shown in different oncology models.

MicroRNA-155 is essential for the optimal proliferation and survival of plasmablast B cells.

A fast antibody response can be critical to contain rapidly dividing pathogens. This can be achieved by the expansion of antigen-specific B cells in response to T-cell help followed by differentiation into plasmablasts. MicroRNA-155 (miR-155) is required for optimal T-cell-dependent extrafollicular responses via regulation of PU.1, although the cellular processes underlying this defect are largely unknown. Here, we show that miR-155 regulates the early expansion of B-blasts and later on the survival and proliferation of plasmablasts in a B-cell-intrinsic manner, by tracking antigen-specific B cells in vivo since the onset of antigen stimulation. In agreement, comparative analysis of the transcriptome of miR-155-sufficient and miR-155-deficient plasmablasts at the peak of the response showed that the main processes regulated by miR-155 were DNA metabolic process, DNA replication, and cell cycle. Thus, miR-155 controls the extent of the extrafollicular response by regulating the survival and proliferation of B-blasts, plasmablasts and, consequently, antibody production.

Epigenomic Modifications Mediating Antibody Maturation.

Epigenetic modifications, such as histone modifications, DNA methylation status, and non-coding RNAs (ncRNA), all contribute to antibody maturation during somatic hypermutation (SHM) and class-switch recombination (CSR). Histone modifications alter the chromatin landscape and, together with DNA primary and tertiary structures, they help recruit Activation-Induced Cytidine Deaminase (AID) to the immunoglobulin (Ig) locus. AID is a potent DNA mutator, which catalyzes cytosine-to-uracil deamination on single-stranded DNA to create U:G mismatches. It has been shown that alternate chromatin modifications, in concert with ncRNAs and potentially DNA methylation, regulate AID recruitment and stabilize DNA repair factors. We, hereby, assess the combination of these distinct modifications and discuss how they contribute to initiating differential DNA repair pathways at the Ig locus, which ultimately leads to enhanced antibody-antigen binding affinity (SHM) or antibody isotype switching (CSR). We will also highlight how misregulation of epigenomic regulation during DNA repair can compromise antibody development and lead to a number of immunological syndromes and cancer.

A Novel Murine GITR Ligand Fusion Protein Induces Antitumor Activity as a Monotherapy That Is Further Enhanced in Combination with an OX40 Agonist.

Purpose: To generate and characterize a murine GITR ligand fusion protein (mGITRL-FP) designed to maximize valency and the potential to agonize the GITR receptor for cancer immunotherapy.Experimental Design: The EC50 value of the mGITRL-FP was compared with an anti-GITR antibody in an in vitro agonistic cell-based reporter assay. We assessed the impact of dose, schedule, and Fc isotype on antitumor activity and T-cell modulation in the CT26 tumor model. The activity of the mGITRL-FP was compared with an agonistic murine OX40L-FP targeting OX40, in CT26 and B16F10-Luc2 tumor models. Combination of the mGITRL-FP with antibodies targeting PD-L1, PD-1, or CTLA-4 was analyzed in mice bearing CT26 tumors.Results: The mGITRL-FP had an almost 50-fold higher EC50 value compared with an anti-murine GITR antibody. Treatment of CT26 tumor-bearing mice with mGITRL-FP-mediated significant antitumor activity that was dependent on isotype, dose, and duration of exposure. The antitumor activity could be correlated with the increased proliferation of peripheral CD8+ and CD4+ T cells and a significant decrease in the frequency of intratumoral Tregs. The combination of mGITRL-FP with mOX40L-FP or checkpoint inhibitor antagonists enhanced antitumor immunity above that of monotherapy treatment.Conclusions: These results suggest that therapeutically targeting GITR represents a unique approach to cancer immunotherapy and suggests that a multimeric fusion protein may provide increased agonistic potential versus an antibody. In addition, these data provide, for the first time, early proof of concept for the potential combination of GITR targeting agents with OX40 agonists and PD-L1 antagonists. Clin Cancer Res; 23(13); 3416-27. ©2017 AACR.

Rational Selection of Syngeneic Preclinical Tumor Models for Immunotherapeutic Drug Discovery.

Murine syngeneic tumor models are critical to novel immuno-based therapy development, but the molecular and immunologic features of these models are still not clearly defined. The translational relevance of differences between the models is not fully understood, impeding appropriate preclinical model selection for target validation, and ultimately hindering drug development. Across a panel of commonly used murine syngeneic tumor models, we showed variable responsiveness to immunotherapies. We used array comparative genomic hybridization, whole-exome sequencing, exon microarray analysis, and flow cytometry to extensively characterize these models, which revealed striking differences that may underlie these contrasting response profiles. We identified strong differential gene expression in immune-related pathways and changes in immune cell-specific genes that suggested differences in tumor immune infiltrates between models. Further investigation using flow cytometry showed differences in both the composition and magnitude of the tumor immune infiltrates, identifying models that harbor "inflamed" and "non-inflamed" tumor immune infiltrate phenotypes. We also found that immunosuppressive cell types predominated in syngeneic mouse tumor models that did not respond to immune-checkpoint blockade, whereas cytotoxic effector immune cells were enriched in responsive models. A cytotoxic cell-rich tumor immune infiltrate has been correlated with increased efficacy of immunotherapies in the clinic, and these differences could underlie the varying response profiles to immunotherapy between the syngeneic models. This characterization highlighted the importance of extensive profiling and will enable investigators to select appropriate models to interrogate the activity of immunotherapies as well as combinations with targeted therapies in vivo Cancer Immunol Res; 5(1); 29-41. ©2016 AACR.

Phenotypic screening reveals TNFR2 as a promising target for cancer immunotherapy.

Antibodies that target cell-surface molecules on T cells can enhance anti-tumor immune responses, resulting in sustained immune-mediated control of cancer. We set out to find new cancer immunotherapy targets by phenotypic screening on human regulatory T (Treg) cells and report the discovery of novel activators of tumor necrosis factor receptor 2 (TNFR2) and a potential role for this target in immunotherapy. A diverse phage display library was screened to find antibody mimetics with preferential binding to Treg cells, the most Treg-selective of which were all, without exception, found to bind specifically to TNFR2. A subset of these TNFR2 binders were found to agonise the receptor, inducing iκ-B degradation and NF-κB pathway signalling in vitro. TNFR2 was found to be expressed by tumor-infiltrating Treg cells, and to a lesser extent Teff cells, from three lung cancer patients, and a similar pattern was also observed in mice implanted with CT26 syngeneic tumors. In such animals, TNFR2-specific agonists inhibited tumor growth, enhanced tumor infiltration by CD8+ T cells and increased CD8+ T cell IFN-γ synthesis. Together, these data indicate a novel mechanism for TNF-α-independent TNFR2 agonism in cancer immunotherapy, and demonstrate the utility of target-agnostic screening in highlighting important targets during drug discovery.

A survey of Lab Tests Online-UK users: a key resource for patients to empower and help them understand their laboratory test results.

Background Lab Tests Online-UK celebrated its 10th anniversary in 2014 and to mark the occasion the first comprehensive survey of website users was undertaken. Methods A pop-up box with a link to Survey Monkey was used to offer website users the chance to participate in the survey, which was live from 4 March 2014 to 11 April 2014. Results Six hundred and sixty-one participants started the questionnaire and 338 completed all of the demographic questions. Although the website is designed and aimed at patients and the public, a significant number of respondents were health-care professionals (47%). The majority of survey participants found the Lab Tests Online-UK website via a search engine and were visiting the site for themselves. The majority of participants found what they were looking for on the website and found the information very easy or fairly easy to understand. The patient respondents were keen to see their laboratory test results (87%), but the majority did not have access (60%) at the time of the survey. Conclusions This survey provides good evidence that the Lab Tests Online-UK website is a useful resource for patients and health-care professionals alike. It comes at a poignant time as the release of results direct to patients starts with access to their medical records. The Lab Tests Online-UK website has a key role in enabling patients to understand their lab test results, and therefore empowering them to take an interest and engage in their own healthcare.

MicroRNA-155 controls affinity-based selection by protecting c-MYC+ B cells from apoptosis.

The production of high-affinity antibodies by B cells is essential for pathogen clearance. Antibody affinity for antigen is increased through the affinity maturation in germinal centers (GCs). This is an iterative process in which B cells cycle between proliferation coupled with the acquisition of mutations and antigen-based positive selection, resulting in retention of the highest-affinity B cell clones. The posttranscriptional regulator microRNA-155 (miR-155) is critical for efficient affinity maturation and the maintenance of the GCs; however, the cellular and molecular mechanism by which miR-155 regulates GC responses is not well understood. Here, we utilized a miR-155 reporter mouse strain and showed that miR-155 is coexpressed with the proto-oncogene encoding c-MYC in positively selected B cells. Functionally, miR-155 protected positively selected c-MYC+ B cells from apoptosis, allowing clonal expansion of this population, providing an explanation as to why Mir155 deletion impairs affinity maturation and promotes the premature collapse of GCs. We determined that miR-155 directly inhibits the Jumonji family member JARID2, which enhances B cell apoptosis when overexpressed, and thereby promotes GC B cell survival. Our findings also suggest that there is cooperation between c-MYC and miR-155 during the normal GC response, a cooperation that may explain how c-MYC and miR-155 can collaboratively function as oncogenes.

Multicentre study of investigation and management of inpatient hyponatraemia in the UK.

PURPOSE: Hyponatraemia is associated with significant morbidity and mortality. The objectives of this study were to evaluate the investigation and management of hyponatraemia and to assess the use of different therapeutic modalities and their effectiveness in routine practice. STUDY DESIGN: This multicentre, retrospective, observational study was conducted at three acute NHS Trusts in March 2013. A retrospective chart review was performed on the first 100 inpatients with serum sodium (sNa) ≤128 mmol/L during hospitalisation. RESULTS: One hundred patients (47 male, 53 female) with a mean±SD age of 71.3±15.4 years and nadir sNa of 123.4±4.3 mmol/L were included. Only 23/100 (23%) had measurements of paired serum and urine osmolality and sodium, while 31% had an assessment of adrenal reserve. The aetiology of hyponatraemia was unrecorded in 58% of cases. The mean length of hospital stay was 17.5 days with an inpatient mortality rate of 16%. At hospital discharge, 53/84 (63.1%) patients had persistent hyponatraemia, including 20/84 (23.8%) with sNa <130 mmol/L. Overall 37/100 (37%) patients did not have any treatment for hyponatraemia. Among 76 therapeutic episodes, the most commonly used treatment modalities were isotonic saline in 38/76 cases (50%) and fluid restriction in 16/76 (21.1%). Fluid restriction failed to increase sNa by >1 mmol/L/day in 8/10 (80%) cases compared with 4/26 (15.4%) for isotonic saline. CONCLUSIONS: Underinvestigation and undertreatment of hyponatraemia is a common occurrence in UK clinical practice. Therefore, development of UK guidelines and introduction of electronic alerts for hyponatraemia should be considered to improve clinical practice.

The miR-155-PU.1 axis acts on Pax5 to enable efficient terminal B cell differentiation.

A single microRNA (miRNA) can regulate the expression of many genes, though the level of repression imparted on any given target is generally low. How then is the selective pressure for a single miRNA/target interaction maintained across long evolutionary distances? We addressed this problem by disrupting in vivo the interaction between miR-155 and PU.1 in mice. Remarkably, this interaction proved to be key to promoting optimal T cell-dependent B cell responses, a previously unrecognized role for PU.1. Mechanistically, miR-155 inhibits PU.1 expression, leading to Pax5 down-regulation and the initiation of the plasma cell differentiation pathway. Additional PU.1 targets include a network of genes whose products are involved in adhesion, with direct links to B-T cell interactions. We conclude that the evolutionary adaptive selection of the miR-155-PU.1 interaction is exercised through the effectiveness of terminal B cell differentiation.

miR-155: an ancient regulator of the immune system.

MicroRNAs (miRNAs) are a newly recognized class of regulatory genes which repress the expression of protein-coding genes. Numerous studies have uncovered a complex role for miRNAs regulating many aspects of a variety of cellular processes including cell growth, differentiation, and lineage commitment. In the immune system, miR-155 is unique in its ability to shape the transcriptome of activated myeloid and lymphoid cells controlling diverse biological functions ranging from inflammation to immunological memory. Not surprisingly, a tight control of miR-155 expression is required to avoid malignant transformation, as evidenced by miR-155 overexpression in many cancers of B-cell origin. In this review, we discuss the potential of miR-155 as a molecular target for therapeutic intervention and discuss the function of miR-155 in the context of protective immunity. We first look back into the emergence of miR-155 in evolution, which is coincidental with the emergence of the ancestors of the antigen receptors. We then summarize what we have learned about the role of miR-155 in the regulation of lymphoid subsets at the cellular and molecular level in the context of recent progress in this field.

MicroRNA-155 is required for Mycobacterium bovis BCG-mediated apoptosis of macrophages.

Pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium bovis, cause significant morbidity and mortality worldwide. However, the vaccine strain Mycobacterium bovis BCG, unlike virulent strains, triggers extensive apoptosis of infected macrophages, a step necessary for the elicitation of robust protective immunity. We here demonstrate that M. bovis BCG triggers Toll-like receptor 2 (TLR2)-dependent microRNA-155 (miR-155) expression, which involves signaling cross talk among phosphatidylinositol 3-kinase (PI3K), protein kinase Cδ (PKCδ), and mitogen-activated protein kinases (MAPKs) and recruitment of NF-κB and c-ETS to miR-155 promoter. Genetic and signaling perturbations presented the evidence that miR-155 regulates PKA signaling by directly targeting a negative regulator of PKA, protein kinase inhibitor alpha (PKI-α). Enhanced activation of PKA signaling resulted in the generation of PKA C-α; phosphorylation of MSK1, cyclic AMP response element binding protein (CREB), and histone H3; and recruitment of phospho-CREB to the apoptotic gene promoters. The miR-155-triggered activation of caspase-3, BAK1, and cytochrome c translocation involved signaling integration of MAPKs and epigenetic or posttranslational modification of histones or CREB. Importantly, M. bovis BCG infection-induced apoptosis was severely compromised in macrophages derived from miR-155 knockout mice. Gain-of-function and loss-of-function studies validated the requirement of miR-155 for M. bovis BCG's ability to trigger apoptosis. Overall, M. bovis BCG-driven miR-155 dictates cell fate decisions of infected macrophages, strongly implicating a novel role for miR-155 in orchestrating cellular reprogramming during immune responses to mycobacterial infection.

Characterisation of 5-HT3C, 5-HT3D and 5-HT3E receptor subunits: evolution, distribution and function.

The 5-HT(3) receptor is a member of the 'Cys-loop' family of ligand-gated ion channels that mediate fast excitatory and inhibitory transmission in the nervous system. Current evidence points towards native 5-HT(3) receptors originating from homomeric assemblies of 5-HT(3A) or heteromeric assembly of 5-HT(3A) and 5-HT(3B). Novel genes encoding 5-HT(3C), 5-HT(3D), and 5-HT(3E) have recently been described but the functional importance of these proteins is unknown. In the present study, in silico analysis (confirmed by partial cloning) indicated that 5-HT(3C), 5-HT(3D), and 5-HT(3E) are not human-specific as previously reported: they are conserved in multiple mammalian species but are absent in rodents. Expression profiles of the novel human genes indicated high levels in the gastrointestinal tract but also in the brain, Dorsal Root Ganglion (DRG) and other tissues. Following the demonstration that these subunits are expressed at the cell membrane, the functional properties of the recombinant human subunits were investigated using patch clamp electrophysiology. 5-HT(3C), 5-HT(3D), and 5-HT(3E) were all non-functional when expressed alone. Co-transfection studies to determine potential novel heteromeric receptor interactions with 5-HT(3A) demonstrated that the expression or function of the receptor was modified by 5-HT(3C) and 5-HT(3E), but not 5-HT(3D). The lack of distinct effects on current rectification, kinetics or pharmacology of 5-HT(3A) receptors does not however provide unequivocal evidence to support a direct contribution of 5-HT(3C) or 5-HT(3E) to the lining of the ion channel pore of novel heteromeric receptors. The functional and pharmacological contributions of these novel subunits to human biology and diseases such as irritable bowel syndrome for which 5-HT(3) receptor antagonists have major clinical usage, therefore remains to be fully determined.