Project description:C57BL/6 mouse pancreas cells were infected with a CRISPR library targeting TSGs. Total HLA and SIINFEKL presentation was measured by flow and cells were sorted for sequencing
Project description:To study the effect of stress on macrophages due to Toxoplasma, we stimulated murine bone marrow-derived macrophages (BMDMs) with IFN-γ (no-stimulate control) and infected them with the apicomplexan parasite Toxoplasma gondii. scRNA-Seq (10X Chromium genomics ) was performed to understand the changes in the immune cells and study the impact of the parasite.
Project description:Pathophysiology of Duchenne Muscular Dystrophy (DMD) is still elusive. Although progressive damage to muscle fibres is a cause of muscle deterioration leading to premature death, there is a growing body of evidence indicating that the triggering effects of DMD mutation are present at the very early stage of muscle development. To study this, we have performed an RNA-seq experiment to compare the transcriptional profile of early stage myoblasts from dystrophic mice compared to WT mice. We have used a myoblast cell line carrying the mdx mutant allele (SC5), which results in a premature stop codon in the Dystrophin gene, leading to a dystrophic phenotype. The WT cell line is the IMO myoblast cell line.
Project description:The precise timing and pathway of memory CD8+ T cells differentiation from naïve T cells have remained undetermined. We found the smaller cell-size and slower cell cycling cells were segregated from the proliferative larger cell-size activated T cell pool at the peak of infection. Gene signature of the smaller cell-size slower cycling cells and the large cell-size proliferative cells was compared to the signature of naïve, effector, central and effector memory CD8+ T cells. Total RNA samples were prepared from sorted populations of larger or smaller cell-sized cells from spleens of influenza virus PR8-OVA-infected mice on day 7 p.i. or from in vitro 7 days culture after stimulation with plate-bound anti-CD3ε (1.0 μg ml−1) and anti-CD28 mAb (0.5 μg ml−1). Effector T-cell control samples were prepared from SIINFEKL (100 ng ml−1) stimulated OT-I cells after 4 days of in vitro culture with rIL-2 (10 ng/ml) and sorted as CD8+CD44hiCD62Llo. Control bona fide effector memory and central memory T cells were sorted from the spleens of PR8-OVA-infected mice on day 42 p.i. Naive cells were sorted as CD8+CD44loCD62Lhi cells from uninfected C57BL/6 mice.
Project description:The interaction of Cryptococcus neoformans with phagocytic cells of the innate immune system is a key step in disseminated disease leading to meningoencephalitis in immunocompromised individuals. Transcriptional profiling of cryptococcal cells harvested from cell culture medium or from macrophages found differential expression of metabolic and other functions during fungal adaptation to the intracellular environment by SAGE analysis. We focused on the ACL1 gene for ATP-citrate lyase, which converts citrate to acetyl-CoA, because this gene showed elevated transcript levels in macrophages and because of the importance of acetyl-CoA as a central metabolite. Mutants lacking ACL1 showed delayed growth on medium containing glucose, reduced cellular levels of acetyl-CoA, defective production of virulence factors, increased susceptibility to the antifungal drug fluconazole and decreased survival within macrophages. Importantly, acl1 mutants were unabl e to cause disease in a murine inhalation model, a phenotype that was more extreme than other mutants with defects in acetyl-CoA production (e.g., an acetyl-CoA synthetase mutant). Loss of virulence is likely due to perturbation of critical physiological interconnections between virulence factor expression and metabolism in C. neoformans. Phylogenetic analysis and structural modeling of cryptococcal Acl1 identified three indels unique to fungal protein sequences; these differences may provide opportunities for the development of pathogen-specific inhibitors. SAGE analysis of C. neoformans cells isolated from macrophages The murine macrophage-like cell line J774A.1 was maintained at 37M-BM-0C in 10% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FBS), 1% nonessential amino acids, 100 M-BM-5g mL-1 penicillin-streptomycin, and 4 mM L-glutamine (Invitrogen). Cryptococcus cells were opsonized with monoclonal antibody 18B7 against capsule (1 M-BM-5g mL-1), and macrophages were treated with recombinant mouse gamma interferon (IFN-gamma) (50 U mL-1) and lipopolysaccharide (LPS) (0.3 M-BM-5g mL-1) prior to co-incubation at a multiplicity of infection (MOI) of 1:1. Macrophages were inoculated with H99 cells and washed after 1 h of inoculation to remove unattached, extracellular fungal cells. After 6 h of incubation, sterile, ice-cold distilled H2O was applied to each well to lyse the macrophages, and the fungal cells (4 x 107) were harvested by centrifugation. H99 control cells were prepared by growth under the same condition but without macrophages, and 108 cell s were harvested. SAGE library construction, sequencing and analysis SAGE library construction, sequencing and analysis were as previously described (Hu et al., 2007; Hu et al., 2008; Steen et al., 2002; Steen et al., 2003). Briefly, RNA was isolated from lyophilized cells by vortexing with glass beads (3.0 mm, acid-washed and RNase-free) for 15 min in 15 mL of TRIZOL extraction buffer (Invitrogen, Carlsbad, CA, USA). The mixture was incubated for 15 min at room temperature and total RNA was isolated according to the manufacturer's instructions. Total RNA was used directly for SAGE library construction as described by Velculescu et al. (1995) using the I-Long SAGE kit (Invitrogen). The tagging enzyme for cDNA digestion was NlaIII and 29 PCR cycles were performed to amplify the ditags during library construction. Colonies were screened by PCR (M13F and M13R primers) to assess the average clone insert size and the percentage of recombinants. Clones from the libraries were sequenced by BigDye primer cycle sequencing on an ABI PRISM 3700 DNA analy zer. Sequence chromatograms were processed using PHRED (Ewing et al., 1998), and vector sequence was detected using Cross_match (Gordon et al., 1998). Fourteen-base-pair tags were extracted from the vector-clipped sequence, and an overall quality score for each tag was derived based on the cumulative PHRED score. Duplicate ditags and linker sequences were removed as described previously (Steen et al., 2002). Only tags with a predicted accuracy of 99% were used, and statistical differences between tag abundance in different libraries were determined as described (Audic and Claverie, 1997). The libraries yielded 23,904 tags from cells grown in media without macrophages for 6hr and 38,5444 tags from cells grown in media with macrophages for 6hr. An overview of the abundance classes for both SAGE libraries is presented in Table S1 in the supplementary material, with both the number of different tag sequences and the total number of tags present in each class for the cells from th e 6h infection and the control. All libraries were normalized to 23,904 to allow direct comparisons, and the tags that appeared less than once in any given library were removed. The EST database available for strain H99 at the University of Oklahoma's Advanced Center for Genome Technology http://www.genome.ou.edu/cneo.html) was used for the preliminary assignment of tags to genes. When an EST sequence could not be identified for a particular tag, the genomic sequence for H99 at the Duke University Center for Genome Technology (http://cgt.genetics.duke.edu/data/index.html) and the Broad Institute (http://cneo.genetics.duke.edu/) was used to identify contigs with unambiguous tag assignments. Note that a limitation of the SAGE approach is that some transcripts are not detected because of low abundance and/or the absence of an NlaIII site for transcript processing. The control (fungal cells alone) and cryptococcal-macrophage interaction SAGE datasets were deposited to the NCBI under accession number GSM986206 and GSM986207 respectively.
Project description:The cytokine interleukin-12 (IL-12) is known to play a central role in adaptive and innate immunity. We employed microarray analysis of IL-12 induced gene expression to provide further insights into its effects on immune response. Experiment Overall Design: PBMC's were isolated from 7 healthy NIH blood bank donors. For each donor, expression levels were measured for PBMCs stimulated with IL-12 and for unstimulated controls, using the Affymetrix Human Genome HG-U133A and HG-U133B GeneChips.
Project description:Using a newly-developed workflow for quantitative newly synthesized proteome analysis (QuaNPA), featuring automated sample processing and multiplexed DIA (plexDIA) analysis, changes in the newly synthesized proteome of IFN-gamma treated Hela cells were monitored over time.