Blockade of the PD-1 axis has modest efficacy in diffuse large B-cell lymphoma (DLBCL), but data regarding LAG3 are sparse. The impact of LAG3 digital gene expression was tested in 309 patients with DLBCL treated with standard chemoimmunotherapy. Cellular distribution of LAG3 protein was determined by immunohistochemistry and flow cytometry. In tumor-infiltrating lymphocytes (TILs), LAG3 expression was highest on CD4+ regulatory T cells (Tregs) and was also highly expressed on CD8+ T cells compared with CD4+ non-Tregs (both P = .008). LAG3high TILs were enriched in PD-1 and TIM-3. LAG3 was also expressed on a proportion of malignant B cells, and these patients had significantly higher LAG3 messenger RNA in their biopsies (P = .03). LAG3high gene expression was associated with inferior survival in discovery/validation cohorts, independent of cell of origin and the international prognostic index. Patients who were PD-L1high were fivefold more likely to be LAG3high (P < .0001). Patients who were LAG3high/PD-L1high had an inferior progression-free survival (P = .011) and overall survival (P = .005) compared with patients who were LAG3low/PD-L1high. Digital spatial protein analysis confirms LAG3 expression on T cells and, surprisingly, tumor-associated macrophages (TAMs) at higher levels than found on CD20+ B cells in the tumor microenvironment. LAG3 is frequently expressed on CD4+ Tregs and CD8+ TILs, typically with other immune checkpoints, and is also present in a proportion of malignant B cells in DLBCL and in areas enriched for TAMs. LAG3high expression is associated with poor outcome independent of conventional prognosticators.

Cystic fibrosis (CF) lung disease is characterized by chronic and exaggerated inflammation in the airways. Despite recent developments to therapeutically overcome the underlying functional defect in the cystic fibrosis transmembrane conductance regulator, there is still an unmet need to also normalize the inflammatory response. The prolonged and heightened inflammatory response in CF is, in part, mediated by a lack of intrinsic down-regulation of the proinflammatory NF-κB pathway. We have previously identified reduced expression of the NF-κB down-regulator A20 in CF as a key target to normalize the inflammatory response. Here, we have used publicly available gene array expression data together with a statistically significant connections' map (sscMap) to successfully predict drugs already licensed for the use in humans to induce A20 mRNA and protein expression and thereby reduce inflammation. The effect of the predicted drugs on A20 and NF-κB(p65) expression (mRNA) as well as proinflammatory cytokine release (IL-8) in the presence and absence of bacterial LPS was shown in bronchial epithelial cells lines (16HBE14o−, CFBE41o−) and in primary nasal epithelial cells from patients with CF (Phe508del homozygous) and non-CF controls. Additionally, the specificity of the drug action on A20 was confirmed using cell lines with tnfαip3 (A20) knockdown (siRNA). We also show that the A20-inducing effect of ikarugamycin and quercetin is lower in CF-derived airway epithelial cells than in non-CF cells.A20 | NF-κB | connectivity mapping | drug repositioning | CF airway inflammation

This article reports a novel mechanism by which Epstein-Barr virus (EBV) microRNA (miRNA) plays a role to fine-tune the expression of LMP1-driven amplification of inhibitory checkpoint programmed death ligand 1 (PD-L1) and PD-L2 in EBV+ diffuse large B-cell lymphoma. Identification and understanding of the immune checkpoint regulation via miRNA may enable potential novel RNA-based therapies to emerge.

MicroRNA (miRNA)s are dysregulated in Diffuse large B-cell lymphoma (DLBCL), where they reflect the malignant B-cells and the immune infiltrate within the tumor microenvironment. There remains a paucity of data in DLBCL regarding cell-free (c-f) miRNA as disease response biomarkers. Immunosuppressive monocyte/macrophages, which are enriched in DLBCL, are disease response markers in DLBCL, with miRNA key regulators of their immunosuppressive function. Our aim was to determine whether plasma miRNA that reflect the activity of the malignant B-cell and/or immunosuppressive monocytes/macrophages, have value as minimally-invasive disease response biomarkers in DLBCL.Quantification of 99 DLBCL tissues, to select miRNA implicated in immunosuppressive monocytes/macrophage biology, found miR-494 differentially elevated. In a discovery cohort (22 patients), pre-therapy c-f miR-494 and miR-21 but not miR-155 were raised relative to healthy plasma. Both miR-494 and miR-21 levels 3-6 months reduced post immuno-chemotherapy. The validation cohort (56 patients) was from a prospective clinical trial. Interestingly, in sequential samples both miRNAs decreased in patients becoming Positron Emission Tomography/Computerized Tomography (PET/CT)-ve, but not in those remaining interim-PET/CT+. Patient monocytes were phenotypically and functionally immunosuppressive with ex-vivo monocyte depletion enhancing T-cell proliferation in patient but not healthy samples. Pre-therapy monocytes showed an immunosuppressive transcriptome and raised levels of miR-494. MiR-494 was present in all c-f nanoparticle fractions but was most readily detectable in unfractionated plasma.Circulating c-f miR-494 and miR-21 are disease response biomarkers with differential response stratified by interim-PET/CT in patients with DLBCL. Further studies are required to explore their manipulation as potential therapeutic targets.

BackgroundGene expression connectivity mapping has gained much popularity in recent years with a number of successful applications in biomedical research testifying its utility and promise. A major application of connectivity mapping is the identification of small molecule compounds capable of inhibiting a disease state. In this study, we are additionally interested in small molecule compounds that may enhance a disease state or increase the risk of developing that disease. Using breast cancer as a case study, we aim to develop and test a methodology for identifying commonly prescribed drugs that may have a suppressing or inducing effect on the target disease (breast cancer).ResultsWe obtained from public data repositories a collection of breast cancer gene expression datasets with over 7000 patients. An integrated meta-analysis approach to gene expression connectivity mapping was developed, which involved unified processing and normalization of raw gene expression data, systematic removal of batch effects, and multiple runs of balanced sampling for differential expression analysis. Differentially expressed genes stringently selected were used to construct multiple non-joint gene signatures representing the same biological state. Remarkably these non-joint gene signatures retrieved from connectivity mapping separate lists of candidate drugs with significant overlaps, providing high confidence in their predicted effects on breast cancers. Of particular note, among the top 26 compounds identified as inversely connected to the breast cancer gene signatures, 14 of them are known anti-cancer drugs.ConclusionsA few candidate drugs with potential to enhance breast cancer or increase the risk of the disease were also identified; further investigation on a large population is required to firmly establish their effects on breast cancer risks. This work thus provides a novel approach and an applicable example for identifying medications with potential to alter cancer risks through gene expression connectivity mapping.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-017-1989-x) contains supplementary material, which is available to authorized users.

Objectives Viral respiratory infections cause considerable morbidity and economic loss. While rhinoviruses (RV) typically cause little more than the common cold, they can produce severe infections and disease exacerbations in susceptible individuals, such as those with asthma. Variations in the regulation of key antiviral cytokines, particularly type I interferon (IFN‐α and IFN‐β), may contribute to RV susceptibility. To understand this variability, we compared the transcriptomes of high and low type I IFN producers. Methods Blood mononuclear cells from 238 individuals with or without asthma were cultured in the presence or absence of RV. Those samples demonstrating high or low RV‐stimulated IFN‐α production (N = 75) underwent RNA‐sequencing. Results Gene expression patterns were similar in samples from healthy participants and those with asthma. At baseline, the high IFN‐α producer group showed higher expression of genes associated with plasmacytoid dendritic cells, the innate immune response and vitamin D activation, but lower expression of oxidative stress pathways than the low IFN‐α producer group. After RV stimulation, the high IFN‐α producer group showed higher expression of genes found in immune response biological pathways and lower expression of genes linked to developmental and catabolic processes when compared to the low IFN‐α producer group. Conclusions These differences suggest that the high IFN‐α group has a higher level of immune system readiness, resulting in a more intense and perhaps more focussed pathogen‐specific immune response. These results contribute to a better understanding of the variability in type I IFN production between individuals.

An early symptom of Alzheimer’s disease (AD) is an impaired sense of smell, for which the molecular basis remains elusive. Here, we generated human olfactory neurosphere-derived (ONS) cells from people with AD and mild cognitive impairment (MCI), and performed global RNA sequencing to determine gene expression changes. ONS cells expressed markers of neuroglial differentiation, providing a unique cellular model to explore changes of early AD-associated pathways. Our transcriptomics data from ONS cells revealed differentially expressed genes (DEGs) associated with cognitive processes in AD cells compared to MCI, or matched healthy controls (HC). A-Kinase Anchoring Protein 6 (AKAP6) was the most significantly altered gene in AD compared to both MCI and HC, and has been linked to cognitive function. The greatest change in gene expression of all DEGs occurred between AD and MCI. Gene pathway analysis revealed defects in multiple cellular processes with aging, intellectual deficiency and alternative splicing being the most significantly dysregulated in AD ONS cells. Our results demonstrate that ONS cells can provide a cellular model for AD that recapitulates disease-associated differences. We have revealed potential novel genes, including AKAP6 that may have a role in AD, particularly MCI to AD transition, and should be further examined.

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