Depletion of immunosuppressive tumor-associated macrophages (TAM) or reprogramming towards a pro-inflammatory activation state represent different strategies to therapeutically target this frequent myeloid population. Here we report that inhibition of colony-stimulating factor-1 receptor (CSF-1R) signaling sensitizes TAM to profound reprogramming in the presence of a CD40 agonist prior to their depletion. Despite the short-lived nature of macrophage hyperactivation, combined CSF-1R/CD40 stimulation of macrophages is sufficient to trigger a productive and durable T cell response in various mouse cancer models. The central role of macrophages in regulating T cell-dependent tumor rejections was substantiated by depletion experiments and transcriptomic analysis of ex vivo sorted TAM. Since CD40 expression on human TAM varies between different tumor types, co-expression of human CSF-1R and CD40 in colorectal adenocarcinoma and mesothelioma can serve as criteria to select these tumor types for clinical development Overall design: Female C57BL/6N mice (6-8 weeks in age, obtained from Charles River) were inoculated with 106 MC38 colorectal adenocarcinoma tumor cells subcutaneously. Tumor growth curves were monitored by caliper measurement and once tumor size reached 250 mm3 in average, groups were allocated for antibody treatment. Ten mice/group were treated with 30 mg/kg IgG1 isotype control antibody clone MOPC-21 (BioXCell), 4 mg/kg anti-CD40 rat IgG2a antibody clone FGK45 (BioXCell), 30mg/kg anti-CSF-1R antibody clone 2G2, 4 mg/kg rat IgG2a control clone 2A3 (BioXCell). For depletion experiments 4mg/kg mouse anti-CD4 antibody clone GK1.5 (Biolegend), 4mg/kg anti-NK1.1 antibody clone PK136 (BioXCell) and 4mg/kg anti-CD8a antibody clone 53-6.7 (BioXCell) were administered when tumor size reached 190mm3 in average. Antibodies were given every second day for four times. In between doses two and three of the depleting antibodies, animals were further treated with vehicle control (0,9% sodium saline), MOPC21, FGK45, 2G2 or combination of FGK45 and 2G2. The anti-CSF-1R antibody or respective IgG1 control antibody were administered weekly until tumors regressed completely or animals reached termination criteria, while the anti-CD40 antibody was only administered once at day 11 simultaneously with the anti-CSF-1R antibody. All antibodies were given intraperitoneally. All procedures were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and European Union directives and guidelines.
Rapid activation of tumor-associated macrophages boosts preexisting tumor immunity.
Specimen part, Treatment, SubjectView Samples
In order to understand the transcriptional effects of CD44s expression in a cell line that does not express CD44 in its native form we transfected CD44s into HEK cells and measured the transcriptional chances compared to native HEK cells
CD44 Isoform Status Predicts Response to Treatment with Anti-CD44 Antibody in Cancer Patients.
No sample metadata fieldsView Samples
Hemophagocytes are activated macrophages seen morphologically to have engulfed other hematopoietic cells. Their function is unknown. Attempts to induce these cells in vitro or purify them ex vivo have been unsuccessful.
Brief report: alternative activation of laser-captured murine hemophagocytes.
Sex, Age, Specimen partView Samples
microRNAs (miRNAs) are essential components of gene regulation, but identification of miRNA targets remains a major challenge. Most target prediction and discovery relies on perfect complementarity of the miRNA seed to the 3 untranslated region (UTR). However, it is unclear to what extent miRNAs target sites without seed matches. Here, we performed a transcriptome-wide identification of the endogenous targets of a single miRNAmiR-155in a genetically controlled manner. We found that approximately forty percent of miR-155-dependent Argonaute binding occurs at sites without perfect seed matches. The majority of these non-canonical sites feature extensive complementarity to the miRNA seed with one mismatch. These non-canonical sites confer regulation of gene expression albeit less potently than canonical sites. Thus, non-canonical miRNA binding sites are widespread, often contain seed-like motifs, and can regulate gene expression, generating a continuum of targeting and regulation.
Transcriptome-wide miR-155 binding map reveals widespread noncanonical microRNA targeting.
Specimen partView Samples
The import of nuclear transcribed RNAs into mitochondria is an emerging area that presents tremendous opportunity to develop human metabolic therapeutics. However, our knowledge base is quite limited. Much remains to be discovered regarding specific RNA localization and mechanisms of import. In order to identify novel RNAs imported into mitochondria, all RNAs within the mitochondria were characterized using next generation sequencing technology. Several nuclear transcribed RNAs were found within mitochondrial RNA samples, including nuclear ribosomal RNAs, gamma satellite RNA and VL30 retroelement RNA. The presence of these RNAs within mitochondria coupled with RNA sequencing data (RNAseq) from other laboratories investigating mitochondrial RNA processing, lead us to hypothesize that nuclease treatment of mitoplasts is insufficient for removing contaminating cytoplasmic RNAs. In contrast to traditional methodology, mitochondrial import was evaluated by qRT-PCR after stepwise removal of the outer mitochondrial membrane and subsequent lysis of mitochondria. This allowed identification of RNAs lost from the mitochondria with the same kinetics as mtDNA-transcribed RNAs. This approach provided an improved evaluation of nuclear RNA enrichment within mitochondrial membranes in order to characterize nuclease protection and mitochondrial import and identify false-positive detection errors. qRT-PCR results confirmed the presence of VL30 retroelement RNA within mitochondria and question the hypothesis that the RNA component of RNase P is imported. These results illustrate a reliable approach for evaluating the presence of RNAs within mitochondria and open new avenues of investigation relating to mitochondrial RNA biology and in targeting mitochondrial based therapeutics. Overall design: RNA isolated from purified mitoplasts was sequenced on an Illumina Genome Analyzer IIx
Mitochondrially-imported RNA in drug discovery.
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Single-cell RNA sequencing has generated the first catalogs of transcriptionally defined neuronal subtypes of the brain. However, the cellular processes that contribute to neuronal subtype specification and transcriptional heterogeneity remain unclear. By comparing the gene expression profiles of a subset of single layer 6 corticothalamic neurons in somatosensory cortex, we show that transcriptional subtypes primarily reflect axonal projection pattern, laminar position within the cortex, and neuronal activity state. Pseudotemporal ordering of 1023 cellular responses to sensory manipulation demonstrates that changes in expression of activity-induced genes both reinforced cell-type identity and contributed to increased transcriptional heterogeneity within each cell type. This is due to cell-type biased choices of transcriptional states following manipulation of neuronal activity. These results reveal that axonal projection pattern, laminar position, and activity state define significant axes of variation that contribute both to the transcriptional identity of individual neurons and to the transcriptional heterogeneity within each neuronal subtype. Overall design: 1023 single cell RNA-Seq and 6 bulk RNA-seq
Variation in Activity State, Axonal Projection, and Position Define the Transcriptional Identity of Individual Neocortical Projection Neurons.
Sex, Specimen part, Cell line, SubjectView Samples
Human genome-wide Affymetrix GeneChip arrays were used to compare the levels of gene expression in the peripheral blood mononuclear cells (PMBCs) of male patients with post-viral chronic fatigue (n=8) and male healthy control subjects (n=7). Patients and healthy subjects differed significantly in the level of expression of 366 genes. Analysis of the differentially expressed genes indicated functional implications in immune modulation, oxidative stress and apoptosis. Prototype biomarkers were identified on the basis of differential levels of gene expression and possible biological significance. Differential expression of key genes identified in this study offer an insight into the possible mechanism of chronic fatigue following infection. The representative biomarkers identified in this research appear promising as potential biomarkers for diagnosis and treatment.
A gene signature for post-infectious chronic fatigue syndrome.
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Previous studies identified a role for latent herpesvirus infection in cross-protection to infection and exacerbation of chronic inflammatory diseases. Here, we compared the gene expression signature from livers, spleens and brains of mice infected with wild-type gammaherpesvirus 68 (MHV68), a mutant virus defective in the establishment of latency (ORF73.stop) or mockulum. We identified over 600 genes differentially expressed in organs of mice latently infected with MHV68 and found distinct sets of genes linked to different pathways were altered in spleen compared to liver. Several of the most differentially expressed latency-specific genes (e.g. IFN, Cxcl9, Ccl5) are associated with known latency-specific phenotypes.
Latent gammaherpesvirus 68 infection induces distinct transcriptional changes in different organs.
Specimen partView Samples
Genetic variation modulating risk of sporadic Parkinson's disease (PD) has been primarily explored through genome wide association studies (GWAS). However, like many other common genetic diseases, the impacted genes remain largely unknown. Here, we used single-cell RNA-seq to characterize dopaminergic (DA) neuron populations in the mouse brain at embryonic and early postnatal timepoints. These data facilitated unbiased identification of DA neuron subpopulations through their unique transcriptional profiles, including a novel postnatal neuroblast population and substantia nigra (SN) DA neurons. We use these population-specific data to develop a scoring system to prioritize candidate genes in all 49 GWAS intervals implicated in PD risk, including known PD genes and many with extensive supporting literature. As proof of principle, we confirm that the nigrostriatal pathway is compromised in Cplx1 null mice. Ultimately, this systematic approach establishes biologically pertinent candidates and testable hypotheses for sporadic PD, informing a new era of PD genetic research. Overall design: 473 single cell RNA-Seq samples from sorted mouse Th-eGFP+ dopaminergic neurons collected at two timepoints from three distinct brain regions.
Single-Cell RNA-Seq of Mouse Dopaminergic Neurons Informs Candidate Gene Selection for Sporadic Parkinson Disease.
Specimen part, SubjectView Samples